katherine gillette-browne thesis
TRANSCRIPT
β-ADRENOCEPTOR DETERMINANTS OF CONTRACTILITY IN THE HUMAN HEART: THE
ROLE OF PHOSPHODIESTERASE ENZYMES
Katherine Tegan Gillette-Browne BPharm BSci (Hons)
06126979
Submitted in [partial] fulfilment of the requirements for the degree of
Doctor of Philosophy
School of Biomedical Sciences
Faculty of Health
Queensland University of Technology
[May 2015]
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes i
Keywords
adrenaline, affinity, arrhythmia, β-adrenoceptor, β-blocker, cardiac, carvedilol,
cilostamide, contractility, enantiomer, esmolol, heart failure, inotropy, lusitropy,
metoprolol, noradrenaline, PDE, PDE3, PDE4, pharmacology, phosphodiesterase
enzyme, phosphodiesterase inhibitor, rolipram, ryanodine, RyR2
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes ii
Abstract
The β-blockers carvedilol and metoprolol provide important therapeutic strategies for
the management of heart failure. While short-term treatment with phosphodiesterase
(PDE) inhibitors has proven beneficial in acute settings, long-term therapy in heart
failure patients results in increased mortality. It is unknown whether PDE3 and PDE4
mediate their inotropic and lusitropic effects through β1- and/or β2-adrenoceptors in
human myocardium. These studies investigated whether the PDE3-selective
inhibitor cilostamide (0.3 µM) or PDE4-selective inhibitor rolipram (1-10 µM)
modified the positive inotropic and lusitropic effects of catecholamines in ventricular
myocardium from heart failure patients treated with or without metoprolol or
carvedilol. Ventricular trabeculae from freshly explanted hearts of 5 non-β-blocked
patients, 15 metoprolol-treated patients, and 9 carvedilol-treated patients with
terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline,
mediated through β1-adrenoceptors (β2-adrenoceptors blocked with ICI 118,551), and
(-)-adrenaline, mediated through β2-adrenoceptors (β1-adrenoceptors blocked with
CGP 20712A), were assessed in the absence and presence of the PDE inhibitors.
The inotropic potency, estimated from shifts of concentration-effect curves,
increased 4-fold for (-)-noradrenaline and 5-fold for (-)-adrenaline in metoprolol-
treated but did not change for (-)-noradrenaline and decreased 16-fold for (-)-
adrenaline in right ventricular trabeculae from carvedilol-treated, compared to non-
β-blocked patients. The positive inotropic and lusitropic effects of (-)-noradrenaline
were potentiated (2-3-fold) by cilostamide in metoprolol-treated but not in non-β-
blocker-treated and carvedilol-treated patients. Cilostamide caused marginal, 3-5-
fold and 19-35 fold potentiation of the inotropic and lusitropic effects of (-)-
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes iii
adrenaline, in non-β-blocker-treated, metoprolol-treated and carvedilol-treated
patients respectively. Results from left ventricle were similar to those of right
ventricle. Rolipram did not affect the inotropic and lusitropic potencies of (-)-
noradrenaline or (-)-adrenaline, suggesting negligible control by PDE4.
Carvedilol, and to a lesser extent metoprolol, induce control by PDE3 of the
inotropic and lusitropic effects mediated through β2-adrenoceptors, plausibly by
changing the feedback between receptor activity and PDE3. Treatment of heart
failure patients with carvedilol induces PDE3 to selectively control the positive
inotropic and lusitropic effects mediated through ventricular β2-adrenoceptors
compared to β1-adrenoceptors. This property of carvedilol may provide protection
against β2-adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor
treated patients. PDE4 does not control β1- or β2-adrenoceptor-mediated inotropic
and lusitropic effects in metoprolol or carvedilol -treated patients.
β-blockers all share a common racemic structure and in general, the antagonistic
efficacies of the S-enantiomers are much more potent (up to 500-fold) than those of
the R-enantiomers. The pharmacodynamics and pharmacokinetics of the racemates
also differ from one another, which may lead to adverse effects from one racemate
while the other provides the majority of the clinical benefits. With very few
exceptions, the clinically used β-blockers are only available as racemic mixtures,
despite the fact that enantiomerically pure preparations may increase clinical efficacy
while reducing the incidence of adverse effects. In this work, the effects of R-(+) and
S-(-) esmolol on human right atrial contractility, β1L- and β3-adrenoceptors, and their
affinities at human β1- and β2-adrenoceptors was investigated. S-(-) esmolol was a
competitive antagonist at β1-adrenoceptors with a β1/β2-adrenoceptor selectivity ratio
of ~ 4.3. The enantiomers of esmolol showed stereoselectivity for blockade of β1-
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes iv
and β2-adrenoceptors. S-(-) esmolol had ~80 fold higher affinity for β1-adrenoceptors
than R-(+) esmolol. The stereoselectivity requirements at β2-adrenoceptors were
lower where S-(-) esmolol had ~13-fold higher affinity for β2-adrenoceptors than R-
(+) esmolol.
As for the majority of β-blockers, the S-(-) enantiomer of esmolol has higher affinity
for blocking both β1- and β2-adrenoceptors than the R-(+) enantiomer. However,
because the β2-adrenoceptor has a much lower stereoselectivity, this could indicate a
role of R-(+) esmolol in the mediation of inotropic and/or lusitropic effects via the
β2-adrenoceptor in vivo and in the clinical setting.
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes v
Table of Contents
Keywords ..................................................................................................................... i Abstract ...................................................................................................................... ii Table of Contents ........................................................................................................ v List of Figures .......................................................................................................... viii List of Tables ............................................................................................................. xi List of Abbreviations ............................................................................................... xiii Statement of Original Authorship ............................................................................. xv Publications .............................................................................................................. xvi Acknowledgements ................................................................................................. xvii CHAPTER 1: LITERATURE REVIEW ............................................................... 1 1.1 Background ........................................................................................................... 1 1.2 The β-adrenoceptor System: Healthy vs. Failing Heart ........................................ 2 1.2.1 The β-adrenoceptor System in the Healthy Heart .................................. 2 1.2.2 The β-adrenoceptor System in Heart Failure ......................................... 5 1.3 The Ryanodine Receptor ....................................................................................... 6 1.3.1 Ryanodine Receptor Structure: The Healthy Heart ............................... 6 1.3.2 Mediation of ECC in the Healthy Heart ................................................ 8 1.3.3 The Ryanodine Receptor in the Failing Heart ....................................... 9 1.4 The Clinical Use of β-Blockers for the Treatment of Heart Failure ................... 11 1.4.1 The Development of β-Blockers .......................................................... 11 1.4.2 The Evolution of the β-Blocker ........................................................... 12 1.4.3 Efficacy of β-Blockers Clinically Used for Heart Failure ................... 13 1.4.4 Signaling Bias of β-Blockers ............................................................... 18 1.4.5 Dynamic Bias ....................................................................................... 21 1.5 The Enantiomers of β-Blockers .......................................................................... 22 1.5.1 Racemic Mixtures ................................................................................ 22 1.5.2 Esmolol ................................................................................................ 28 1.5.3 The Clinical Relevance of Enantiomerically Pure β-Blockers ............ 32 1.6 Phosphodiesterase Enzymes: Healthy vs. Failing Hearts ................................... 33 1.6.1 Phosphodiesterases in the Healthy Heart ............................................. 33 1.6.2 Phosphodiesterase Compartmentalization ........................................... 36 1.6.3 Roles of the PDE Subfamilies in Healthy and Failing Hearts ............. 36 1.7 Hypothesis, Summary, and Implications ............................................................ 44 CHAPTER 2: MATERIALS AND METHODS ...................................................47 2.1 Ventricular Experiments ..................................................................................... 47 2.1.1 Research Design Summary ...................................................................47
2.1.2 Materials and Methods ......................................................................... 47 2.1.3 Analysis of Contractile Data ................................................................ 52 2.1.4 Drugs ................................................................................................... 52 2.1.5 Participants ........................................................................................... 53 2.2 Right Atrial Experiments .................................................................................... 55
2.2.1 Research Design Summary .................................................................. 55 2.2.2 Explantation and Transport ................................................................. 55 2.2.3 Dissection and Set-Up of Trabeculae .................................................. 55
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes vi
2.2.4 Specific Activation of β1- and β2-Adrenoceptors ................................ 56 2.2.5 Analysis of Contractile Data ................................................................ 57 2.2.6 Drugs .................................................................................................... 57 2.2.7 Participants ........................................................................................... 58 CHAPTER 3: THE EFFECTS OF METOPROLOL ON PDE3 AND PDE4 CONTROL OF β1- AND β2-ADRENOCEPTOR MEDIATED INOTROPY AND LUSITROPY IN HUMAN FAILING VENTRICLE ................................. 59 3.1 Background and Purpose .................................................................................... 59 3.2 Methodology and Research Design .................................................................... 59
3.2.1 Research Design ................................................................................ 59 3.3 Materials and Methods ........................................................................................ 60
3.3.1 Participants ........................................................................................... 60 3.3.2 In vitro Human Heart Trabeculae Experiments ................................. 61 3.3.3 Isolated Ventricular Trabeculae from Heart Transplant Patients ........ 62 3.3.4 Specific Activation of β1- and β2-Adrenoceptors ................................ 62 3.3.5 Analysis of Contractile Data ................................................................ 63 3.3.6 Drugs .................................................................................................... 64
3.4 Results ................................................................................................................ 64 3.4.1 Chronic Metoprolol Treatment Increases the Inotropic Potencies of Catecholamines ....................................................................................... 64 3.4.2 Cilostamide Fails to Potentiate the Inotropic Effects of
Catecholamines in Right Ventricular Trabeculae from Non- β-Blocker-Treated Patients ..................................................................... 68
3.4.3 Cilostamide Potentiates the Effects Mediated Through β2- Adrenoceptors More than β1-Adrenoceptors in Ventricular Trabeculae from Metoprolol-Treated Patients .............................................................70
3.4.4 Rolipram Does Not Modify Inotropic or Lusitropic Potencies of (-)-Noradrenaline or (-)-Adrenaline ..........,..............................................77
3.5 Discussion ........................................................................................................... 80 3.5.1 Control by PDE3 of β1- and β2-Adrenoceptors in Heart Failure
Patients Treated with Metoprolol .............................................................. 80 3.5.2 PDE4 Inhibition Does Not Affect the Inotropic or Lusitropic Effects
of Catecholamines ..................................................................................... 83 3.6 Clinical Implications ........................................................................................... 86 3.7 Conclusions ......................................................................................................... 87 CHAPTER 4: THE EFFECTS OF CARVEDILOL ON PDE3 AND PDE4 CONTROL OF β1- AND β2-ADRENOCEPTOR MEDIATED INOTROPY AND LUSITROPY IN HUMAN FAILING VENTRICLE ................................. 88 4.1 Background and Purpose .................................................................................... 88 4.2 Methodology and Research Design .................................................................... 88 4.2.1 Heart Transplant Patients ........................................................................... 88 4.2.2 Isolated Right Ventricular Trabeculae from Heart Transplant Patients ..... 89 4.2.3 Specific Activation of β1- and β2-Adrenoceptors ...................................... 90 4.2.4 Analysis and Statistics ............................................................................... 91 4.3 Results ................................................................................................................. 92 4.3.1 Decrease of Inotropic and Lusitropic Potencies for (-)-Adrenaline by Chronic Treatment with Carvedilol ........................................................... 92 4.3.2 Cilostamide Potentiates the Effects of (-)-Adrenaline More Than
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes vii
(-)-Noradrenaline in Trabeculae From Carvedilol-Treated Patients ......... 99 4.3.3 Rolipram Does Not Modify Inotropic or Lusitropic Potencies of (-)-Noradrenaline or (-)-Adrenaline in Trabeculae From Patients Treated With Carvedilol ....................................................................................... 102 4.4 Discussion ..........................................................................................................102 4.4.1 Carvedilol Treatment Reduces Responses Through β2-Adrenoceptors
But Not β1-Adrenoceptors ...........................................................................103 4.4.2 Carvedilol Facilitates the Control by PDE3 of β2- More Than β1- Adrenoceptor-Mediated Effects ...............................................................104 4.4.3 Quantitative Comparison of β1- and β2-Adrenoceptor Blockade by Carvedilol in Combination with PDE3 Inhibition ................................... 107 4.4.4 PDE4 Does Not Control the Inotropic or Lusitropic Effects of Catecholamines in Carvedilol-Treated Patients with Heart Failure .........108 4.5 Conclusions ....................................................................................................... 109 CHAPTER 5: ENANTIOMERS OF ESMOLOL .............................................. 112 5.1 Background and Purpose ................................................................................. 112 5.2 Methods ............................................................................................................. 112
5.2.1 Participants ..........................................................................................112 5.2.2 Preparation of Right Atrial Trabeculae .............................................. 113 5.2.3 Acute Effects of S-(-) Esmolol on Human Right Atrium .................. 114 5.2.4 Effect of S-(-) Esmolol in the Presence of 3-isobutyl-1-
methylxanthine and Nadolol .............................................................. 114 5.2.5 Determination of the Affinity of S-(-) and R-(+) Esmolol at
Human Atrial β1- and β2-Adrenoceptors ............................................ 114 5.3 Results ................................................................................................................115
5.3.1 Acute Effects of S-(-) Esmolol on Human Right Atrium .................. 115 5.3.2 Effect of S-(-) Esmolol in the Presence of 3-isobutyl-1-
methylxanthine and Nadolol .............................................................. 117 5.3.3 Determination of the Affinity of S-(-) and R-(+) Esmolol at Human
Atrial β1- and β2-Adrenoceptors ........................................................ 118 5.4 Discussion ......................................................................................................... 122
5.4.1 Patient Population ..........,................................................................... 122 5.4.2 Acute Effects of S-(-) Esmolol on Human Right Atrium .................. 122 5.4.3 Lack of Agonist Activity of S-(-) Esmolol on β1L-Adrenoceptors .... 122 5.4.4 Affinity of S-(-) and R-(+) Esmolol at Human Atrial β1- and β2-Adrenoceptors ............................................................................... 123
5.5 Conclusions and Future Directions ................................................................... 123 CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS ..................... 125 6.1 Role of PDE3 and PDE4 on Contractility of the Human Heart ....................... 125 6.2 The Future of β-Blockers .................................................................................. 129 6.2.1 Biased β-Blockers .............................................................................. 129 6.2.2 Enantiomerically Pure β-Blocker Preparations .................................. 132 6.3 The Future of Phosphodiesterase Inhibitors ..................................................... 133 BIBLIOGRAPHY ................................................................................................. 136 APPENDICES ....................................................................................................... 177 Appendix A: British Journal of Pharmacology Publication ................................... 177 Appendix B: Naunyn-Schmeideberg's Archives of Pharmacology Publication ..... 188
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes viii
List of Figures
Figure 1.1. The ryanodine receptor macromolecular signaling complex ......... 7 Figure 1.2. RYR2 mediates excitation-contraction coupling in the healthy heart ....................................................................................... 9
Figure 1.3. Example of ligand-biased signaling for the β2-adrenoceptor ....... 20 Figure 1.4. Chemical structures of β-blockers ................................................ 25 Figure 1.5. Catecholamine-induced activation of β-adrenoceptors leads to a cAMP-PKA-mediated phosphorylation of the RyR2 .................... 34 Figure 3.1. Effects of chronic administration of metoprolol compared with no- β-blocker on inotropic effects of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from failing hearts ................................................................................................. 65 Figure 3.2. Effects of chronic administration of metoprolol compared with no β-blocker on lusitropic effects of (-)- noradrenaline and (-)-adrenaline in right ventricular trabeculae from failing hearts ..... 67 Figure 3.3. Lack of effect of cilostamide on the inotropic responses of (-)- noradrenaline and (-)-adrenaline in right ventricular trabeculae from patients with heart failure not treated with a β-blocker ........... 68 Figure 3.4. Effect of cilostamide on the lusitropic responses of (-)- noradrenaline and (-)-adrenaline in right ventricular trabeculae from patients with heart failure not treated with a β-blocker ........... 69 Figure 3.5. Representative experiment carried out on right ventricular trabeculae obtained from a 48-year-old male patient with IHD, chronically administered metoprolol ....................................... 72 Figure 3.6. Potentiation of the inotropic effects of (-)-adrenaline by cilostamide in right ventricular trabeculae from seven patients from Brisbane/Dresden with heart failure chronically administered metoprolol ................................................................... 73 Figure 3.7. Cilostamide, but not rolipram, potentiates the lusitropic effects of (-)-adrenaline and (-)-noradrenaline in right ventricular trabeculae from seven patients with heart failure chronically administered metoprolol ................................................................... 74 Figure 3.8. Cilostamide potentiates the inotropic effects of (-)-adrenaline in left ventricular trabeculae from patients with heart failure chronically administered metoprolol ................................................ 75
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes ix
Figure 3.9. Cilostamide, but not rolipram, potentiates the lusitropic effects of (-)-noradrenaline and (-)-adrenaline in left ventricular trabeculae from patients with heart failure chronically administered metoprolol ................................................................... 76 Figure 3.10. Effects of the combination of cilostamide and rolipram on the inotropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from three patients with heart failure chronically administered metoprolol ................................................ 78 Figure 3.11. Effects of the combination of cilostamide and rolipram on the lusitropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from 3 patients with heart failure chronically administered metoprolol ................................................ 79
Figure 4.1. Marked reductions in potency for inotropic effects of (-)- adrenaline but not for (-)-noradrenaline in right ventricular trabeculae .......................................................................................... 93 Figure 4.2. Effects of chronic administration of carvedilol compared to no β-blocker on lusitropic effects of (-)-noradrenaline and (-)- adrenaline in right ventricular trabeculae from failing hearts .......... 95 Figure 4.3. Potentiation of inotropic effects of both (-)-noradrenaline and (-)- adrenaline in the presence of cilostamide but not rolipram ....... 97 Figure 4.4. Marked potentiation of the inotropic effects of (-)-adrenaline vs. (-)-noradrenaline by cilostamide in right ventricular trabeculae from three hearts chronically treated with carvedilol ................................. 98 Figure 4.5. Marked potentiation of lusitropic effects of (-)-noradrenaline and (-)-adrenaline by cilostamide in right ventricular trabeculae from three hearts chronically treated with carvedilol ....................... 99 Figure 4.6. Cilostamide potentiates the inotropic effects of (-)-adrenaline more than (-)-noradrenaline in right ventricular trabeculae from heart failure patients chronically administered carvedilol .............. 100 Figure 4.7. Cilostamide, but not rolipram, potentiates lusitropic effects of (-)-adrenaline and (-)-noradrenaline in right ventricular trabeculae from patients with heart failure chronically administered carvedilol ........................................................................................ 101
Figure 5.1. Effect of S-(-) esmolol on force of contraction and relaxation, TPF, and t50 ....................................................................................... 116 Figure 5.2. Lack of agonist activity of S-(-) esmolol at β1L-adrenoceptors .. 117
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes x
Figure 5.3. Determination of the affinity of S-(-) and R-(+) esmolol at β1- adrenoceptors in human right atrium ................................................. 119 Figure 5.4. Determination of the affinity of S-(-) and R-(+) esmolol at β2- adrenoceptors in human right atrium ................................................. 121
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xi
List of Tables
Table 1.1. Generations of β-blockers .........................................................................13 Table 1.2. Multi-center, double-blind, placebo-controlled studies on survival benefits of β-blockers ....................................................................................14 Table 1.3. β-adrenoceptor ligands and their efficacy patterns towards adenylyl cyclase and the β-arrestin/ERK1/2 pathways of the β1-and β2-adrenoceptors ..........................................................................................21 Table 1.4 Families, subfamilies, substrates, and tissue expression of
phosphodiesterase enzymes ...........................................................................35 Table 3.1. Summary of Brisbane/Dresden patients that were not taking a β–blocker or chronically administered metoprolol prior to heart transplantation ...............................................................................................61 Table 3.2. Summary of Oslo patients chronically administered metoprolol prior to heart transplantation .........................................................................61 Table 3.3. Inotropic potencies of (-)-noradrenaline and (-)-adrenaline acting through ventricular β1- and β2-adrenoceptors, respectively ..........................66 Table 3.4. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through right ventricular β1- and β2-adrenoceptors, respectively .....70 Table 3.5. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through left ventricular β1- and β2-adrenoceptors of metoprolol-treated patients ..........................................................................................................77 Table 3.6. Reduction of inotropic and lusitropic responses as well as protection against arrhythmias, mediated through myocardial β1- and β2- adrenoceptors, by PDE3 and PDE4 in different species ...............................85 Table 4.1.A. Patients that were not taking a β–blocker prior to heart
transplantation ................................................................................................89 Table 4.1.B Summary of patients chronically administered carvedilol prior to heart transplantation ......................................................................................89 Table 4.2. ΔpEC50 obtained in the presence or absence of PDE inhibitor and calculated from mean values for each patient ..............................................94 Table 4.3. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through right ventricular β1- and β2-adrenoceptors, respectively, from patients chronically treated with carvedilol ..................................................96
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xii
Table 5.1. Summary of patient details .....................................................................113 Table 5.2. pKB values of S-(-) esmolol and R-(+) esmolol determined at human β1- and β2-adrenoceptors .............................................................................118
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xiii
List of Abbreviations
AC adenylyl cyclase ADR adrenaline AF atrial fibrillation AKAP A kinase anchoring proteins AMP adenosine 5’-monophosphate ATP adenosine 5’-triphosphate β1L-adrenoceptor low affinity β1-adrenoceptor β1H-adrenoceptor high affinity β1-adrenoceptor β-blocker β-adrenoceptor blocking agent Ca2+ calcium CAMKII calmodulin-dependent protein kinase cAMP cyclic 3’-5’ adenosine monophosphate CGP 12177 (±)-CGP-12177 hydrochloride, 4-[3-[(1,1-
Dimethylethyl)amino]-2-hydroxypropoxy]-1, 3-dihydro-2H-benzimidazol-2-one hydrochloride
CGP20712A (±)-2-Hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4
(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl] amino]ethoxy]-benzamide
CICR calcium-induced calcium release GPCR G-protein coupled receptor GTP guanosine 5-triphosphate Gi inhibitory G protein Gs stimulatory G protein IBMX 3-isobutyl-1-methylxanthine ICaL L-type Ca2+ channel
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xiv
ICI 118,551 (±)-1-[2,3-(Dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride
KB dissociation constant at equilibrium Lmax length/tension required to produce a maximal contraction mAKAP muscle-selective A-kinase anchoring protein PBZ phenoxybenzamine PDE phosphodiesterase enzyme pEC50 -log molar concentration of agonist required to produce half
maximum response PKA cAMP-dependent protein kinase A PKB protein kinase B pKB -log molar concentration of antagonist which occupies 50% of
receptors PO open probability NA noradrenaline RA right atrium/ right atrial RV right ventricle/ right ventricular RyR2 ryanodine receptor/channel S.E.M standard error of the mean SERCA sarcoplasmic reticulum Ca2+-ATPase SR sarcoplasmic reticulum t ½ half-life TPF time to peak force t50 time to 50% relaxation
QUT Verified Signature
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xvi
Publications and Communications
Molenaar, P.M., Christ, T., Hussain, R.I., Engel, A., Berk, E., Gillette, K.T., Chen,
L., Galindo-Tovar, A., Krobert, K.A., Ravens, U., Levy, F.A., & Kaumann, A.J.
(2013). PDE3, but not PDE4, reduces β1- and β2- adrenoceptor-mediated inotropic
and lusitropic effects in failing ventricle from metoprolol-treated patients. British
Journal of Pharmacology, 169, 528-538.
Molenaar, P.M., Christ, T., Berk, E., Engel, A., Gillette, K.T., Galindo-Tovar, A.,
Ravens, U., & Kaumann, A.J. (2014). Carvedilol induces greater control of β2- than
β1-adrenoceptor-mediated inotropic and lusitropic effects by PDE3, while PDE4 has
no effect in human failing myocardium. Naunyn-Schmeideberg’s Archives of
Pharmacology, 387, 629-640.
Molenaar, P., Christ, T., Hussain, R.I., Engel, A., Berk, E., Gillette, K.T., Chen, L.,
Galindo-Tovar, A., Krobert, K.A., Ravens, U., Levy, F.O., & Kaumann, A.J. (2012).
Carvedilol induces greater control by PDE3 of β2- than β1-adrenoceptor-mediated
inotropic effects in human failing myocardium while PDE4 has no effect. ASCEPT-
APSA Sydney.
Molenaar, P., Christ, T., Gillette, K.T., Chen, L., & Kaumann, A.J. (2012).
Carvedilol induces greater control by PDE3 of β2- than β1-adrenoceptor-mediated
inotropic effects in human failing myocardium while PDE4 has no effect. The Prince
Charles Hospital Research Forum Brisbane.
Molenaar, P., & Gillette, K.T. (2013). β1- and β2-adrenoceptor – phosphodiesterase
control over human heart contractility. ASCEPT-SEAWP Melbourne.
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xvii
Acknowledgements
Firstly, I would like to acknowledge that these experiments are the result of a
collaboration between the groups here in Brisbane, Australia: Peter Molenaar and
myself, in Dresden, Germany: Alberto Kaumann, Alejandro Galindo-Tovar, Torsten
Christ, Andreas Engle, Emanuel Berk, and Ursula Ravens, and in Oslo, Norway:
Rizwan Hussain, Kurt Krobert, and Finn Olav Levy. The members of these groups
have all collaborated for the experiments, the data analysis, and the writing of results
for publication. I myself contributed to all three stages, including tissue retrieval,
experiment completion, data compilation, statistical analysis, writing, table
formulation, and proofreading. This work has already resulted in two publications,
which are part of the appendix of this thesis.
I would like to thank QUT for allowing me this amazing opportunity and for their
financial support, which made completion of this endeavour possible. I would also
like to thank Professor Malcolm West and Associate Professor Ian Yang of the
University of Queensland School of Medicine for allowing me to work in the in vitro
Human Heart Laboratory. Special thanks also goes to the surgeons of The Prince
Charles Hospital, Brisbane, and the Gustav Carus Hospital, Dresden, for providing
the tissues for experiments and to the Transplant Coordinators for gaining patient
consents.
I would thank my supervisors Associate Professor Peter Molenaar and Dr. Annalese
Semmler for their guidance and support. A very heartfelt and humbled thank you
goes to Peter Molenaar, who has helped and encouraged me through every step of
this challenging adventure, and whose patience with tired PhD candidates and
unflagging dedication to his field deserve special commendation.
Lastly but certainly not least, I would like to thank my family for their constant
support and encouragement; my Husband Lucas for being my wailing wall and
β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xviii
biggest cheerleader, and my Mom for supplying her little science nerd with
everything from microscopes to buckets for adopted salamanders.
CHAPTER 1 Literature Review 1
1. Literature Review
1.1 BACKGROUND
Heart failure and arrhythmic disease are major causes of death in Australia, ranked 8
in females and 11 in males in 2012 (Australian Bureau of Statistics, 2012). Ischemic
heart disease, which commonly progresses to heart failure, is ranked as the leading
cause of death in both males and females. In terms of rankings, no progress appears
to have been made since 1998 (Australian Bureau of Statistics, 2012). The
sympathetic nerve- β-adrenoceptor system contributes to the progression of heart
failure, morbidity and mortality (Molenaar & Parsonage, 2005).
Heart failure can clinically be defined as a syndrome “in which patients have typical
symptoms and signs resulting from an abnormality of cardiac structure or function”
(ESC Guidelines, 2012), and 50% of patients who are diagnosed with heart failure
will die within five years (Nguyen et al., 2014). Sudden cardiac death accounts for
approximately 30-50% of these deaths (Zipes et al., 2009; Adamson & Gilbert,
2006). Patients who have died suddenly whilst having a Holter monitor recording to
detect the presence of arrhythmias have primarily shown complex ventricular activity
and ventricular tachyarrhythmias most often degenerating into ventricular
fibrillation. Structural changes and remodeling associated with heart failure
combined with increased sympathetic nerve β-adrenoceptor activity is largely
responsible for ventricular arrhythmias leading to sudden death (Adamson & Gilbert,
2006).
CHAPTER 1 Literature Review 2
1.2 THE β-ADRENOCEPTOR SYSTEM: HEALTHY VS. FAILING HEART
1.2.1 The β-adrenoceptor system in the Healthy Heart
There are two stimulatory β-adrenoceptors in the human heart, the β1-adrenoceptor
and the β2-adrenoceptor (Brodde, 1991; Molenaar et al., 2007). Noradrenaline,
released from sympathetic nerves in response to physiological demands including
exercise or low blood pressure, or pathologic stimuli including heart failure, activates
β1-adrenoceptors on heart muscle cells to cause an increase in heart rate (positive
chronotropy) and an increase in force of contraction (positive inotropy). This causes
an increase in heart function with consequent increase in cardiac output to meet
physiological demand. Adrenaline, released from the adrenal medulla in response to
stress, circulates in the blood and upon reaching the heart, activates both β1- and β2-
adrenoceptors on heart muscle cells to increase heart rate and force of contraction.
Human heart β1-adrenoceptors are associated with heart failure, coronary artery
disease, and arrhythmias. The role of β2-adrenoceptors in pathology is less clear. The
harmful effects of the sympathetic nerve β-adrenoceptor system have largely been
attributed to the β1-adrenoceptor. In contrast a ‘protective’ role of β2-adrenoceptors
has been advocated leading to proposals to administer β2-adrenoceptor agonists for
the management and treatment of human heart failure (Zheng, Zhu, Han, & Xiao,
2005). Based on observations of differences between β1- and β2-adrenoceptors made
in animal species a ‘current consensus’ has been described in which “β1-adrenoceptor
stimulation serves as a causal factor of congestive heart failure, while β2-
adrenoceptor activation may actually be protective for the failing heart” (Zheng et
al., 2005). Furthermore a rationale for a “combination of β1-adrenoceptor blockade
with β2-adrenoceptor activation as a new prevention and intervention strategy for the
CHAPTER 1 Literature Review 3
treatment of congestive heart failure” has been proposed (Zheng et al., 2005).
However, based on other work (Kaumann et al., 1999; Molenaar et al., 2000;
Molenaar et al., 2007) the proposal for implementation of β2-adrenoceptor activation
for the management of human heart failure is likely to contribute to fatal ventricular
arrhythmias and sudden death.
There are fundamental differences between β2-adrenoceptor signalling in rodent and
human hearts. Release of noradrenaline and adrenaline from sympathetic nerves and
the adrenal medulla causes cardiostimulation through both β1- and β2-adrenoceptors
in the human heart. In the human heart, but not necessarily in other mammals,
activation of β1- and β2-adrenoceptors causes similar increases of cAMP, cAMP-
dependent protein kinase (PKA), and comparable PKA-catalyzed phosphorylation of
target proteins including phospholamban, troponin I, and myosin binding protein C
(Kaumann et al., 1999; Molenaar et al., 2000; Molenaar et al., 2007). As a result of
these biochemical effects, β1- and β2-adrenoceptors also mediate similar positive
inotropic and lusitropic effects (Kaumann et al., 1999; Molenaar et al., 2000;
Molenaar et al., 2007). Therefore, activation of β2-adrenoceptors could also be
responsible for the occurrence of arrhythmias, including fatal ventricular
arrhythmias.
In the human atrium and ventricle, activation of β1- and β2-adrenoceptors causes an
increase in contractile force and a hastening of relaxation, (positive inotropy and
lusitropy, respectively) (Kaumann et al., 1999; Molenaar et al., 2000; Molenaar et al.,
2002; Sarsero et al., 2003; Molenaar et al., 2006; Molenaar et al., 2007). Activation
of β1-adrenoceptors in the human heart occurs as a physiological response to heart
failure, enabling the heart to maintain adequate haemodynamic function and
CHAPTER 1 Literature Review 4
perfusion pressure in organs. The β1-adrenoceptor has at least two binding sites,
which can both be activated to cause cardiostimulation. The first, termed the β1H-
adrenoceptor (high affinity site of β1-adrenoceptor) is activated by noradrenaline and
adrenaline and blocked by relatively low concentrations of β-blockers including
metoprolol, bisoprolol and carvedilol (Kaumann & Molenaar, 2008). The other,
termed the β1L-adrenoceptor (low affinity site of β1-adrenoceptor) has lower affinity
for noradrenaline and adrenaline and is activated by some β-blockers including CGP
12177 and pindolol, both at higher concentrations than those required to block the
receptor (Kaumann & Molenaar, 2008). Activation of β2-adrenoceptors located on
pre-junctional sympathetic nerve terminals facilitates the release of noradrenaline to
increase contractility, while activation of β2-adrenoceptors located directly on human
heart muscle causes maximal or near maximal increases in contractile force in the
human atrium (Molenaar et al., 2007) and ventricle (Kaumann et al., 1999; Molenaar
et al., 2000).
Human atrial and ventricular β1-adrenoceptors (β1H- and β1L-adrenoceptors) and β2-
adrenoceptors couple to the stimulatory Gsα-protein → adenylyl cyclase → cyclic
AMP → cyclic AMP dependent protein kinase (PKA) pathway. Evidence of
activation of PKA includes PKA dependent phosphorylation of Ser16-
phospholamban, C-protein and troponin I for both the β1H-adrenoceptor and the β2-
adrenoceptor (Kaumann et al., 1999; Molenaar et al., 2000; Molenaar et al., 2007),
which is consistent with increases in contractile force and hastening of relaxation.
Activation of the β1L-adrenoceptor, like that of β1H-adrenoceptor, also causes an
increase in contractile force and hastening of relaxation. β1L-adrenoceptor responses
are enhanced in the presence of non-selective blockade of phosphodiesterase
CHAPTER 1 Literature Review 5
enzymes with isobutylmethylxanthine (IBMX). In the presence of IBMX,
accumulation of cyclic AMP and activation of PKA can be demonstrated in human
right atrium (Kaumann & Molenaar 2008; Sarsero et al., 2003).
1.2.2 The β-adrenoceptor System in Heart Failure
As the human heart fails, the body uses a variety of mechanisms to compensate for
decreased cardiac output and organ perfusion (Lehnart et al., 2005). One such
compensatory mechanism is an increase in sympathetic signaling, primarily through
the activation of the β1-adrenoceptor (Marks, 2001; Molenaar & Parsonage, 2005).
Acute sympathetic activation (activation that lasts for minutes to days) serves an
important function in the healthy heart in response to events such as trauma, blood
loss, or exercise; it allows the heart to increase its hemodynamic output significantly
within a matter of seconds (Port & Bristow, 2001). In contrast, chronic sympathetic
activation in the failing heart leads to adverse changes mediated through α1- β1- and
β2-adrenoceptors (Packer et al., 1998), and sympathetic activation is inversely
proportional to survival rate (Cohn, 1984). Sustained increases in circulating
noradrenaline leads to oxidative stress and apoptosis through both α1- and β1-
adrenoceptors (Packer et al., 1998; Port & Bristow, 2001). Activation of α1-
adrenoceptors impairs the ability of the kidneys to excrete both salt and water; this
leads to ventricular hypertrophy and increased intravascular volume, and ultimately
in increased oxygen demand by cardiomyocytes (Packer et al., 1998). Over time, the
chronic adrenergic activation of the failing heart leads to cardiac malfunction, and
more specifically to altered Ca2+ mediated excitation-contraction coupling
(Andersson & Marks, 2010; Blayney & Lai, 2009). The desensitization of β1- and β2-
adrenoceptors by chronic stimulation also leads to the uncoupling of the β-
CHAPTER 1 Literature Review 6
adrenoceptors to Gsα, ultimately leading to a reduction in the amount of cAMP
normally accumulated by their activation; the higher the increase in adrenergic
activation, the higher the level of desensitization and consequent cardiomyocyte
damage (Port & Bristow, 2001).
In the healthy human heart, the ratio of β1- to β2-adrenoceptors is approximately
70:30. In heart failure, where the β1-adrenoceptor is down-regulated, this ratio is
closer to 50:50 (Port & Bristow, 2001; Barrese & Taglialatela, 2013). Both β1- and
β2-adrenoceptors are decoupled from their signaling pathways, in large part due to an
upregulation of the phosphorylating G-protein coupled receptor kinase GRK2, or
βARK1 (Lohse et al., 2003; Port & Bristow, 2001). Giα subunits, which antagonize
β-adrenoceptor signaling, are up-regulated (Neumann et al., 1988). Taken together,
these alterations in cardiac signaling effectively limit the heart’s ability to contract
normally and retain adequate systemic organ perfusion (Lohse et al., 2003).
1.3 THE RYANODINE RECEPTOR
1.3.1 Ryanodine Receptor Structure: The Healthy Heart
The ryanodine receptor, or RyR2, is a large homotetrameric Ca2+ channel within the
lipid bilayer of the sarcoplasmic reticulum (SR) in cardiac and skeletal muscle cells
(Marks, 1996; Dulhunty, Beard, Pouliquin, & Casarotto, 2007). On its cytoplasmic
terminus, the RyR2 associates with Protein Kinase A (PKA) and PDE4D3 (Blainey
& Lai, 2009). The phosphatases PP1 and PP2A also associate with the cytoplasmic
portion of the RyR2, and all of these cytosolic accessory compounds help to
physically and temporally regulate phosphorylation and dephosphorylation of the
RyR2 (Blayney & Lai 2009; Dulhunty et al., 2007; Marx et al., 2000). (Figure 1.1)
CHAPTER 1 Literature Review 7
Phosphorylation of the RyR2 activates the channel by increasing its open probability
(PO) and dephosphorylation results in channel stabilisation and closing (Fill &
Copello, 2002; Wehrens & Marks, 2003). (Figure 1.1)
Figure 1.1. The ryanodine receptor macromolecular signaling complex. Four RyR2 monomers contribute to the tetrameric Ca2+ release channel macromolecular complex. Regulatory proteins and enzymes associate with the large cytoplasmic RyR2 domains protruding into the cytosolic space. Calmodulin (CaM), and FKBP12.6 (calstabin2) are thought to bind directly to the RyR2 monomers, whereas binding of other subunits is mediated by specific targeting proteins. Triadin and junctin interact with RyR2 on the luminal side of the channel complex. PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; CAMKII, calmodulin-dependent kinase II; PDE, phosphodiesterase enzyme; PKA, protein kinase A; Ser, serine; mAKAP, muscle-A kinase anchoring protein. (Figure from Mohler & Wehrens, 2007).
CHAPTER 1 Literature Review 8
1.3.2 Mediation of ECC in the Healthy Heart
The RyR2 channels serve to mediate excitation–contraction coupling (ECC) in the
heart by controlling Ca2+ release from the luminal sarcoplasmic reticulum (SR)
stores. Upon depolarisation, the L-Type Calcium Channel (ICaL) allows an influx of
Ca2+ into the cytosolic space between the sarcolemma and the SR known as the
dyadic cleft. This Ca2+ influx results in the RyR2 channel opening, releasing a much
larger amount of Ca2+ into the cleft in a process called calcium-induced calcium
release (CICR) (Wehrens & Marks, 2003; Marks, 1996). Activation of β1- and β2-
adrenoceptors causes dissociation of the Gsα subunit, which in turn activates
adenylate cyclase to make cAMP. cAMP then activates Protein Kinase A (PKA)
which phosphorylates the RyR2, further regulating the opening of the RyR2 (Marx et
al., 2000). The Ca2+ released by the RyR2 into the cytosol then binds to troponin C, a
regulatory protein of the myofilaments; once this occurs, the myofilaments actin and
myosin are able to interact. The activation of actin and myosin leads to a shortening
of the sarcomere, causing contraction of the muscle, or systole (Andersson & Marks,
2010). (Figure 1.2)
Phosphorylation of the RyR2 by PKA increases the PO of the channel by dissociating
calstabin2 from the macromolecular complex. The removal of calstabin2 causes
increased sensitivity of the channel to Ca2+ influx via the ICaL (Wehrens & Marks,
2003; Marx et al., 2000; Dulhunty et al., 2007; Brillantes et al., 1994). During
diastole the RyR2 closes, which stops the flow of Ca2+ into the dyadic cleft; Ca2+
diffuses away from troponin C and is pumped into the SR by the sarcoplasmic
reticulum Ca2+ ATPase pump, SERCA2A. This allows the sarcomeres to lengthen,
CHAPTER 1 Literature Review 9
the muscle to relax, and ultimately allows the heart to fill. (Figure 1.2) SERCA2A is
under the control of phospholamban, and phosphorylation of phospholamban by
PKA at Ser16 results in decreased inhibition of SERCA2A (Simmerman et al., 1986;
Koss & Kranias, 1996; Kaumann et al., 1999).
Figure 1.2. The ryanodine receptor (RyR2) mediates excitation-contraction coupling in the healthy heart. CICR, calcium-induced calcium release; SR, sarcoplasmic reticulum; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca2+-ATPase; NCX, Na+/Ca2+ exchange pump; SL, sarcolemma; AP, action potential; LCC, L-type Ca2+ channel. Figure from Blayney & Lai (2009).
1.3.3 The Ryanodine Receptor in the Failing Heart
The increased adrenergic activation during heart failure results in increased cAMP
levels in cardiac myocytes, higher localised PKA levels, hyperphosphorylation of
RyR2, dissociation of calstabin2, and an increase in the channel’s PO (Marks, 2001;
Lehnart et al., 2005). Without calstabin2, the RyR becomes unstable during diastole,
allowing Ca2+ to leak into the dyadic cleft. Dissociation of calstabin2 has been shown
CHAPTER 1 Literature Review 10
to cause a four-fold increase in transient Ca2+ releases from the RyR2, termed Ca2+
sparks (Marks 2001; Marx et al., 2000; Brillantes et al., 1994).
An arrhythmic contraction is orchestrated through the untimely release of calcium
from the RyR2. Delayed after depolarizations, or DADs, can be caused by an
inappropriately timed, spontaneous efflux of Ca2+ escaping from the sarcoplasmic
reticulum via the RyR2; DADs are a characteristic of heart failure (Blayney & Lai,
2009). RyR2 is phosphorylated by PKA (Witcher, Kovacs, Schulman, & Cefali,
1991; Marx et al., 2000; Rodriguez, Bfhogal, & Colyer, 2003), and by
Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Witcher et al., 1991;
Wehrens, Lehnart, Reiken, & Marks, 2004). PKA phosphorylation of RyR2 causes
increased sensitivity to Ca2+ induced activation, increases the probability of channel
opening and destabilizes RyR2, which may lead to arrhythmias (Blayney & Lai,
2009; Marx et al., 2000).
A further consequence of hyperphosphorylation of the RyR2 is depletion of luminal
SR Ca2+ stores; as more Ca2+ is leaked from the lumen, the less is available for the
next contraction, resulting in smaller contractions and a longer time to decay (Marks,
2001; Dulhunty et al., 2007; Wehrens & Marks, 2003). The RyR2 has also been
shown to be down-regulated along with SERCA2a in failing human hearts, which
leads to further disruption of effective ECC (Wehrens & Marks, 2003; Marks, 1996;
Blayney & Lai, 2009).
CHAPTER 1 Literature Review 11
1.4 THE CLINICAL USE OF β-BLOCKERS FOR THE TREATMENT OF HEART FAILURE
1.4.1 Development of the β-blockers
Noradrenaline and adrenaline first bind to key amino acids of β1- and β2-
adrenoceptors to enable them to activate the receptors. The binding of noradrenaline
and adrenaline to the active site of β1- and β2-adrenoceptors is highly specific; β-
blockers competitively reduce the ability of noradrenaline and adrenaline to bind to
the active site of both of the β-adrenoceptors (Bristow, 1993). Treating heart failure
requires a complex approach to target the signaling pathways which lead to both
functional and structural remodeling of cardiomyocytes (Javed & Deedwania, 2009).
Considering that heart failure causes a reduction in cardiac output, inotropy, and
lusitropy, using an agent which blocks the positive effects of catecholamines that
normally serve to increase cardiac output may seem counter-intuitive; however,
multiple studies after their discovery in the 1960’s showed a clear survival benefit of
β-blockers in both coronary artery disease and heart failure (Black et al., 1965;
Packer et al., 1998; Waagstein, Hjalmarson, Varnauskas, & Wallentin, 1975;
Barrrese & Taglialatela, 2013).
The survival benefit of the β-blockers was put down to two different mechanisms of
action: their ability to block the harmful chronic effects of catecholamines, and their
ability to re-sensitize down-regulated β1-adrenoceptors to acute increases in
catecholamines (Nguyen et al., 2014). When first administered, β-blockers do in fact
reduce cardiac output; however, low-dose initiation with subsequent titration to
target dosing does minimize adverse effects, and long-term use of bisoprolol (CIBIS-
II, 1997; Lechat et al., 1997), metoprolol (Waagstein et al., 1993; MERIT-HF, 1993)
and carvedilol (Colucci et al., 1996; Packer et al., 1996; Packer et al., 2001) have all
CHAPTER 1 Literature Review 12
shown improvements in left ventricular ejection fraction (Packer et al., 1998; ESC
Guidelines, 2012) and reductions in hospitalizations and overall mortality (ESC
Guidelines, 2012). These long-term clinical improvements indicate that the chronic
administration of β-blockers is able to restore cardiac function sufficiently enough to
overcome any short-term cardiodepressant effects (Packer et al., 1998).
1.4.2 The Evolution of the β-blocker
There are three generations of β-blockers, each distinguished by their affinity for
adrenergic receptors (Bristow, 2000). The first generation included the non-β-
adrenoceptor-selective propranolol, timolol, and pindolol; second-generation were
designed to be β1-adrenoceptor selective in order to avoid complications in patients
with concomitant respiratory diseases such as COPD (Black et al., 2005), and include
atenolol, metoprolol, and bisoprolol. This generation does, however, block β2-
adrenoceptors at higher doses (Javed & Deedwania, 2009). Third generation β-
blockers are also non-selective, but mediate vasodilation in addition to β-blockade;
this class includes carvedilol and bucindolol (Javed & Deedwania, 2009; Mansoor &
Kaul, 2009). (Table 1.1) Both bucindolol and carvedilol mediate their vasodilatory
actions via the α1-adrenoceptor, and improve insulin sensitivity versus the first
generation β-blockers (Javed & Deedwania, 2009). Interestingly, many of the first
generation β-blockers, which were developed to be β1-adrenoceptor selective,
actually have poor β1-adrenoceptor selectivity in intact cells (Baker, 2005).
Furthermore, many of the β-blockers are not merely antagonists, but at differing
concentrations can also act as partial agonists (such as pindolol) and inverse agonists
(such as bucindolol) at β-adrenoceptors (Baker, 2005; Barrese & Taglialatela, 2013).
_______________________________________________________________
CHAPTER 1 Literature Review 13
Generation Selectivity Drugs _________________________________________________________________ 1st non-selective propranolol, timolol, pindolol, no vasodilation nadolol, sotalol 2nd β1 –selctive atenolol, bisoprolol, metoprolol no vasodilation β1-selective acebutolol with vasodilation 3rd non-selective carvedilol, bucindolol, nebivolol with vasodilation __________________________________________________________________
Table 1.1: Generations of β-blockers, adapted from Mansoor & Kaul (2009), and Bristow (2000b).
1.4.3 Efficacy of β-Blockers Clinically Used for Heart Failure
There have been many well-designed, multi-center, double blind, placebo-controlled
trials that have shown clear survival benefits for bisoprolol (CIBIS-II, 1999),
metoprolol (MERIT-HF Study Group, 1999; COMET, 2003), carvedilol
(COPERNICUS, 2002; COMET, 2003), and nebivolol (SENIORS, 2005) in heart
failure patients. In fact, most of the trials were stopped after one year due to vast
improvements in morbidity and mortality in the treatment groups versus the placebo
groups (Nguyen et al., 2014; Barrese & Taglialatela, 2013). (Table 1.2) The survival
benefit of β-blockers has been demonstrated so effectively that they are now
recommended to be initiated as soon as signs and symptoms of heart failure appear
(Nguyen et al., 2014); moreover, of the four oral medications currently used (the
other three being ACE inhibitors, anti-platelet therapy, and statins) to ameliorate
cardiovascular morbidity and mortality, β-blockers have been shown to have the
CHAPTER 1 Literature Review 14
highest survival benefit (Mansoor & Kaul, 2009; Javed & Deedwania, 2009; ESC
guidelines, 2012).
TRIAL n DOSE LVEF RESULTS
US Carvedilol HF Study; 1996
1094 6.25-‐50mg/bd 28%
Carvedilol lowered risk of death by 65%
(p<0.001)
CIBIS II; 1999 2647 1.25-‐10mg/ day
27.5% Bisoprolol significantly improved survival
(p<0.0001)
MERIT-‐HF (Metoprolol CR/XL Randomized Intervention Trial in Congestive Heart Failure); 1999
3991 200mg/ day 28%
Metoprolol CR/XL significantly improved survival (p=0.00009)
COPERNICUS (Carvedilol Prospective
Randomized Cumulative Survival
Trial); 2002
2289 3.15-‐25mg/ day
19.9% Carvedilol significantly improved survival
(p=0.0014)
COMET (Carvedilol or Metoprolol European
Trial); 2003 3029
Carv: 25mg/bd,
Met: 50mg/bd
26%
Carvedilol significantly improved survival vs. metoprolol TARTRATE
(p=0.0017)
SENIORS; 2005 2128 1.25-‐10mg/ day
36% Nebivolol significantly reduced morbitity and mortality (p=0.039)
Table 1.2: Multi-center, double-blind, placebo-controlled studies on survival benefits of β-blockers; modified from Nguyen et al. (2014).
Metoprolol was one of the first β-blockers to be studied extensively for the treatment
of systolic heart failure, and in 1999 the MERIT-HF trial found a 34% reduction in
overall mortality, with a 39% reduction in myocardial infarction of metoprolol versus
placebo (MERIT-HF, 1999). Bisoprolol was studied around the same time, and a
similar survival benefit was shown in a large double-blind trial (CIBIS-II, 1999). The
CHAPTER 1 Literature Review 15
fact that α1-, β1- and β2-adrenoceptors all mediate harmful effects of chronic
adrenergic stimulation in the heart, together with the fact that β-blockers are not all in
fact solely antagonists at β-adrenergic receptors (Barrese & Taglialatela, 2013), led
researchers to question the possibility of a non-selective β-blocker improving
morbidity and mortality in patients with heart failure versus a selective β1-
adrenoceptor blocker (Packer 1998). In addition, the intolerability of some patients
with severe heart failure to the initial cardiodepressant effects of the first-generation
β-blockers makes an agent that blocks both β1- and β2-adrenoceptors, as well as
having vasodilatory effects, in theory, a better therapeutic alternative (Packer, 1998;
Book & Hott, 2003). Carvedilol is the obvious stand-out for these additional
properties, as it is orally administered, blocks α1-, β1-, and β2-adrenoceptors,
ameliorates insulin sensitivity, and decreases smooth muscle cell proliferation
(Barrese & Taglialatela, 2013).
In 1999, three separate studies reported a higher increase in left ventricular ejection
fraction in carvedilol versus metoprolol-treated patients. However, the studies were
small, some not blinded, and only showed significant benefits of carvedilol when
patients were followed for more than six months (Metra et al., 2000; Di Lenarda et
al., 1999; Kukin et al., 1999; Sanderson et al., 1999). In 2000, Metra and colleagues
studied the effects of metoprolol tartrate in a larger, randomized, double blind, head-
to-head trial versus carvedilol. They found that carvedilol had a greater benefit in left
ventricular ejection fraction and stroke volume during exercise, but that the two
drugs had similar benefits in overall symptom improvement and quality of life.
Unfortunately, they did not use survival as a primary endpoint, and still only had a
small cohort of patients. The first large, (n=3029) multi-center, head-to-head trial of
CHAPTER 1 Literature Review 16
carvedilol versus immediate release metoprolol tartrate with survival as a primary
endpoint found a significant survival benefit of carvedilol over metoprolol, although
the researchers could not positively identify any of carvedilol’s additional adrenergic
effects as the reason for the reported increased survival benefit (COMET, 2003).
Despite the significant benefit of carvedilol versus metoprolol in the 2003 COMET
trial, questions emerged regarding the suitability of both the dose and dosage form of
metoprolol used in the study (Bristow et al., 2003). Metoprolol is orally available in
two formulations: as an immediate release metoprolol tartrate, which is dosed twice
daily, and as a controlled release metoprolol succinate (metoprolol XL/CR), which
only requires once daily dosing. Critics of the COMET study argued that the
metoprolol dosing used during the trial was unable to achieve adequate β1-
adrenoceptor blockade when compared to the carvedilol dosing; this was due to both
the use of the wrong formulation of metoprolol and administration of sub-therapeutic
maintenance doses. Immediate release metoprolol was not approved for the treatment
of heart failure due to wide fluctuations in plasma concentrations, leading to both
loss of β1-adrenoceptor selectivity at high concentrations and loss of β1-adrenoceptor
blockade at low concentrations (Wikstrand, 2000). In addition, the average
maintenance dose of metoprolol for the COMET trial was only 85 mg daily, which is
lower than the dose of metoprolol used in the Metoprolol in Dilated Cardiomyopathy
Trial, upon which COMET based its dosing (Waagstein et al., 1993; Bristow, 2003).
Critics of the findings from COMET argue that these oversights in the metoprolol
dosing are what led to the significant survival benefit of carvedilol (Bristow et al.,
2003).
Several groups have sought to answer the carvedilol-versus-metoprolol question
since the COMET results were published in 2003, with either original research or
CHAPTER 1 Literature Review 17
meta-analyses of existing trials. Unfortunately, results have remained conflicting
(Barrese & Taglialatela, 2013). In 2012, Shore and colleagues released a 10-year
study including over 3,700 patients with either ischemic or non-ischemic heart
failure treated with either carvedilol or the extended release metoprolol succinate.
They found that the type of heart failure significantly modified patient response; the
ischemic heart failure group showed greater survival when treated with metoprolol
succinate, whereas the non-ischemic group had greater survival when treated with
carvedilol. However, both extended release metoprolol and carvedilol showed similar
reductions in hospitalizations (Shore et al., 2012).
In 2013, a meta-analysis of trials studying atenolol, bisoprolol, bucindolol,
carvedilol, metoprolol succinate, or nebivolol was conducted to see if any of the
included β-blockers had a clearly significant benefit in survival endpoints over the
others (Chatterjee et al., 2013). While the investigators declared that survival
benefits and improvements in left ventricular ejection fraction appeared to be a class
effect and not significantly different between β-blockers, they did suggest that a
definitive answer as to any β-blocker’s superiority was not possible with their
available data, and that more head-to-head trials would need to be conducted, as
carvedilol did demonstrate the lowest (albeit not significantly lower) mortality
(Chatterjee et al., 2013). Another study was put forth by Pasternak et al. (2014)
which followed 11,664 Danish heart failure patients who were treated with either
carvedilol or metoprolol succinate over 3 years. Similar to the Chatterjee group, the
Pasternak study did not find any significant difference in mortality of carvedilol
versus slow-release metoprolol (Pasternak et al., 2014). In contrast, a meta-analysis
of 11 randomized trials spanning more than 5,000 patients found a significant benefit
CHAPTER 1 Literature Review 18
of carvedilol on overall survival versus atenolol, bisoprolol, metoprolol, and
nebivolol (DiNicolantonio et al., 2013). Again, the group was unable to pinpoint the
reasons for carvedilol’s improved survival rates; they speculated the benefits were
due to carvedilol’s additional α1-mediated vasodilation and β2-adrenoceptor
blockade, as well as its anti-arrhythmic effects (DiNicolantonio et al., 2013). These
studies demonstrate a clear need for more head-to-head trials not only investigating
the survival benefits of the β-blockers, but also how the β-blockers ameliorate their
beneficial effects in heart failure patients.
1.4.4 Signaling Bias of β-blockers
The studies conducted on the clinically available β-blockers have indicated that these
drugs should not just be grouped as a single class, or even into distinct generations.
β-blockers can be full, partial, or inverse agonists/antagonists of either the β1-and/or
the β2-adrenoceptor, and their differing effects on disease states such as heart failure
and asthma indicate that there is no “class effect” of β-blockers, and that in fact β-
blockers are not just simply β-blockers at all (Thanawala et al., 2014). Recent studies
have found a variety of ligands, including the β-blockers, which intrinsically activate
specific downstream signaling cascades of GPCRs rather than activating the
receptor’s full repertoires, by directly stabilizing a distinct receptor conformation;
this ability has been termed ‘ligand bias’ (Galandrin & Bouvier, 2006; DeWire &
Violin, 2011).
CHAPTER 1 Literature Review 19
β1-Adrenoceptor Signaling Bias
In addition to activating the cAMP pathway, β1-adrenoceptors have also been shown
to activate the EKR1/2 pathway via the scaffolding protein β-arrestin; in mice,
studies found this pathway to protect against catecholamine-induced apoptosis
(DeWire & Violin, 2011). Theoretically, biased ligands which could selectively
activate the ERK1/2 pathway may prove more cardioprotective than those which
block both the cAMP and ERK1/2 pathways. In a study which investigated the
potential ligand bias of 20 β-blockers, carvedilol was the only one approved for
clinical use in heart failure which activated this non-canonical ERK1/2 pathway,
which could be one explanation for why carvedilol has demonstrated an increase of
survival over the other β-blockers in certain studies (Kim et al., 2008; Barrese &
Taglialatela, 2013).
β2-Adrenoceptor Signaling Bias
The β2-adrenoceptor has at least two distinct signaling pathways: the adenylyl
cyclase pathway which ultimately results in the accumulation of cAMP, and the
mitogen-activated protein kinase (MAPK) pathway that activates ERK1 and ERK2
via the scaffolding protein β-arrestin (Audet & Bouvier, 2008; Thanawala et al.,
2014). (Figure 1.3)
CHAPTER 1 Literature Review 20
Figure 1.3. Example of ligand-biased signaling for the β2-adrenoceptor. Three of the signaling effectors (Gs, Gi and β-arrestin) that mediate β2-adrenoceptor–promoted activation of AC and MAPK are illustrated. From Audet & Bouvier, 2008.
Each β-blocker has shown distinct affinities and efficacies for each pathway; this
selective activation of distinct ligand-directed signaling is termed ‘biased signaling’
(Thanawala et al., 2014). Carvedilol and propranolol are both inverse agonists of the
cAMP signaling pathway, but partial agonists of the ERK1/2 pathway. Adrenaline
and formoterol are both full agonists of the β2-adrenoceptor for both pathways,
whereas nadolol and ICI 118,551 are inverse agonists for both pathways (Galandrin
& Bouvier, 2006; Thanawala et al., 2014). For the efficacy profiles of common
ligands of β-adrenoceptors, please refer to Table 1.3, below.
CHAPTER 1 Literature Review 21
Ligand β1-Adrenoceptor β2-Adrenoceptor AC ERK1/2 AC ERK1/2 Isoproterenol AGO AGO AGO AGO Labetalol AGO NEUT AGO AGO Bucindolol AGO AGO NEUT AGO Carvedilol AGO AGO NEUT AGO Propranolol INV AGO INV AGO Metoprolol INV NEUT INV INV Bisoprolol INV NEUT INV INV Atenolol INV NEUT INV INV
Table 1.3. β-adrenoceptor ligands and their efficacy patterns towards adenylyl cyclase (AC) and the β-arrestin/ERK1/2 pathways of the β1- and β2-adrenoceptors. AGO, partial or full agonist; NEUT, neutral agonist; INV, inverse agonist. Modified from Galandrin & Bouvier (2006). 1.4.5 Dynamic Bias
Building on the theory of ligand and signaling bias is the theory of dynamic bias; the
idea that both progressive disease states and chronic therapy with certain drugs can
lead to a dynamic shift in signaling bias in vivo, which is not seen in static, in vitro
studies (Michel, Seifert, & Bond, 2014). Michel and colleagues suggest that this
dynamic shifting in signaling bias may be what accounts for the seemingly
paradoxical benefit of β-blockers reducing mortality in heart failure, and may also
explain why despite acute worsening of cardiac function and output, long-term
CHAPTER 1 Literature Review 22
administration of β-blockers demonstrates a clear therapeutic benefit (Michel et al.,
2014).
As stated previously, the pharmacology of the clinically used β-blockers is
quite varied in terms of β1- versus β2-adrenoceptor affinities from generation to
generation. As even un-occupied β-adrenoceptors have intrinsic activity, β-blockers
are termed not just as antagonists but also as inverse agonists, due to their ability to
go beyond simply blocking the β-adrenoceptors and further reduce their resting
intrinsic activity (Maack et al., 2003). Pharmacodynamics also vary widely between
the β-blockers; for example, carvedilol is able to bind to the α1- and β1/ β2-
adrenoceptors for a longer duration than the other β-blockers, and actually exerts its
antagonistic effects even at concentrations too low to be detected in plasma (Maack
et al., 2003). In contrast, the β-blocker esmolol has such a rapid onset and short
duration of action that it is used peri-and post-operatively when rapid titration of
cardiac output is required (Zangrillo et al., 2009; Wiest, 1995; Byrd et al., 1984).
1.5 THE ENANTIOMERS OF β-BLOCKERS
1.5.1 Racemic Mixtures
For a drug to exist as an enantiomer, it must have a chiral center, around which the
molecules can rotate so as to create two distinct structural isoforms of the compound,
which are mirror images of each other (Mehvar & Brocks, 2010; Patil & Kothekar,
2006). The isomers are then classed as being “R” or “S” depending on the actual
arrangement of atoms, as well as being either dextro (+) or levo (-), which indicates
how polarized light is rotated around them (clockwise or anti-clockwise,
respectively) (Stoschitzky, Zernig, & Linder, 1998; Patil & Kothekar, 2006).
CHAPTER 1 Literature Review 23
Compounds that do not have a chiral centre and therefore do not exist as racemates
are termed ‘enantiomerically’ or ‘optically’ pure (Patil & Kothekar, 2006).
In order for a drug to interact and bind with its receptor(s), its molecular structure
must “fit,” or meet a stereospecific conformation unique to that receptor. Therefore,
different enantiomers of the same drug may not possess the same affinity or efficacy
within the human body, due to different binding conformations for their target
receptor(s) (Mehvar & Brocks, 2001). The pharmacokinetics of enantiomeric
compounds can be markedly different between racemates, especially in regards to
their protein binding profiles and metabolism, where receptor stereoselectivity is
more common (Caldwell, 1995; Lowenthal et al., 1985). The presence of racemates
within a compound can lead to clinical implications such as differences in efficacy,
bioavailability, volume of distribution, and rate of elimination (or subsequent
accumulation of one racemate) (Patil & Kothekar, 2006). This is especially true for
racemic mixtures in which one racemate either binds to receptors other than the
target receptor (such as R-amlodipine resulting in peripheral edema), has different
activity at the target receptor, (for example, (-)-pindolol activates β1L-adrenoceptors
at higher concentrations required to block the receptor (Kaumann & Lobnig, 1986),
or is metabolized into a molecule which accumulates or causes toxicity, such as R-
omeprazole leading to gastric carcinoids in poor CYP2C19 and CYP3A4
metabolizers (Patil & Kothekar, 2006).
It is estimated that as many as 60% of therapeutic agents contain chiral centers
(Tucker & Lennard, 1985) and that up to 20% of these agents are only clinically
available as racemic mixtures (Caldwell, 1995; Caner, Groner, & Levy, 2004). The
CHAPTER 1 Literature Review 24
administration of racemic mixtures may increase the incidence of toxicity or adverse
effects, especially when those unwanted effects are a product of the “inactive”
racemate (Caldwell, 1995; Mehvar & Brocks, 2001). By performing a “chiral
switch,” or removing the inactive or less-active racemate completely, many
compounds can be made more efficacious and better tolerated, especially for those
compounds in which the inactive form has different absorption and elimination rates
than its active isomer (Mehvar & Brocks, 2001; Patil & Kuthekar, 2006).
As β-blockers are synthetically derived from isoprenaline, a β-adrenoceptor agonist,
they all share a common racemic structure, the pharmacophore of which is an
aryloxyamino alcohol (Peter & Gyeresi, 2011; Davies, 1990; Stoschitzky et al.,
1998). (Figure 1.4) Common features of noradrenaline, adrenaline and all clinically
used β-blockers is the presence of a protonated nitrogen atom, which forms a strong
hydrogen bond with the receptor, and a chiral carbon atom (Kaumann & Molenaar,
2008). The presence of a chiral carbon in β-blockers means that they exist as
enantiomers which differ by the arrangement of hydrogen and hydroxyl groups
around the chiral carbon.
CHAPTER 1 Literature Review 25
Figure 1.4. Chemical structures of β-blockers. The asterisk denotes the chiral carbon. From Mehvar & Brocks. (2001)
With the exception of sotalol, the S-enantiomer of which has very limited
antagonistic effect at both β1-and β2-adrenoceptors (Johnston et al., 1985; Gomoll &
Bartek, 1986; Waldo et al., 1996), the efficacies of the S-enantiomers of the β-
blockers are much more potent (up to 500-fold) than those of the R-enantiomers, and
their pharmacokinetics and pharmacodynamics often differ from one another as well
(Stoschitzky et al., 1998; Agustiana et al., 2010; Peter & Gyeresi, 2011; Bekhradnia
& Ebrahimzadeh, 2012). The differences of the pharmacokinetics of the R- isomers
versus the S-isomers of many β-blockers are especially marked in genetically poor
metabolisers due to polymorphisms of both CYP1A and CYP2D6, and in patients
under exercise-induced stress (Benny & Adithan, 2001; Mehvar & Brocks, 2001).
CHAPTER 1 Literature Review 26
The differences in pharmacokinetics between racemates of the β-blockers are due
largely to the high stereoselectivity of the β-adrenoceptor (Stoschitzky et al., 1998).
Absorption appears not to differ between the racemates, as they are passively
absorbed through the gastrointestinal tract (Wetterich et al., 1996). The distribution
between racemates depends on their binding to plasma proteins; for example, (-)-
propranolol has a higher unbound fraction in plasma, and reaches higher
concentrations in certain tissues, including the heart (Mehvar & Brocks, 2001).
Metabolism and excretion of the β-blockers occurs both through hepatic and renal
pathways, and each pathway can show stereoselectivity for one racemate over
another; for example, S-metoprolol is favored by α-hydroxylation, whereas R-
metoprolol is favored by O-demethylation (Murthy et al., 1990).
The physicochemical and pharmacological properties of the enantiomers of the β-
blockers can also differ significantly (Kaumann & Molenaar, 2008). For example, (-
)-pindolol blocks both β1- and β2-adrenoceptors at sub-nanomolar concentrations
with moderate selectivity for β2-adrenoceptors (Kaumann & Lobnig, 1986), while
higher concentrations (~ 2½ - 3 log units) activate β1-adrenoceptors though the low
affinity binding site (Kaumann & Lobnig, 1986; Joseph et al., 2003; Kaumann &
Molenaar, 2008). (+)-Pindolol blocks β1- and β2-adrenoceptors at higher (~ 2 log
units) concentrations than those required for (-)-pindolol (Walter, Lemoine, &
Kaumann, 1984; Kaumann & Lobnig, 1986; Joseph et al., 2003). The steric
requirements for pindolol are higher at β1-adrenoceptors than β2-adrenoceptors
(Kaumann & Lobnig, 1986). Finally, (+)-pindolol causes a positive chronotropic
effect in guinea-pig atrium, predominantly through activation of β2-adrenoceptors
(Walter et al., 1984).
CHAPTER 1 Literature Review 27
Despite demonstrated differences in pharmacology, the clinically available β-
blockers are still, for the most part, only available as racemic mixtures; this has
resulted in an increased demand for enantiomerically pure preparations, as they will
allow for more predictable pharmacokinetic and pharmacodynamic profiles in
patients (Caldwell, 1995; Agustiana et al., 2010; Peter & Gyeresi, 2011; Patil &
Kuthekar, 2006). With more powerful separation technologies available now
compared to when many β-blockers were first marketed, studies investigating each
enantiomer’s distinct pharmacokinetics and pharmacodynamics are becoming more
available and critical (Gorczynski et al., 1984; Lowenthal et al., 1985; Tucker &
Lennard, 1990; Wang et al., 2011; Tang et al., 2012).
In order to study the effects of enantiomerically pure preparations of β-blockers in an
in vitro setting, it is desirable to utilise one which rapidly diffuses through tissue and
therefore displays a rapid onset of effect, so that contractility is not affected by long
incubation times. Esmolol, a parenterally available β-blocker, has a 60-second onset
and a very short half-life (Zuppa, Shi, & Adamson, 2003; Iskandrian et al., 1986).
CHAPTER 1 Literature Review 28
1.5.2 Esmolol
ASK-8052, or esmolol, is an ultra-short acting, β1-adrenoceptor-selective antagonist
with a half-life reported from 2 minutes to 19 minutes (Zangrillo et al., 2009),
although it is generally reported to have a 9 minute half-life in human plasma (Zuppa
et al., 1986; Kirshenbaum, Kloner, McGowan, & Antman, 1988; Spahn et al., 1993).
It has a rapid onset of 60 seconds, 90% β-blockade within 5 minutes, a maximum
effect at 6-10 minutes, and a duration of action ranging from 10-20 minutes,
(Zangrillo et al., 2009; Marik & Varon, 2009; Wiest, 1995). Esmolol levels drop to
undetectable concentrations in as little as 20 minutes after infusion, and complete β-
adrenoceptor recovery occurs in approximately 18 minutes (Wiest, 1995). While
esmolol has a similar structure to the other second-generation, β1-selective blockers
such as atenolol and metoprolol, the presence of an ester on its phenyl ring allows
esmolol to bind more selectively to the β1-adrenoceptors in the heart versus the β2-
adrenoceptors located in the bronchial tissue and peripheral blood vessels
(Lowenthal, Porter, Saris, Bies, Slegowski, & Stuadacher, 1985; Wiest, 1995; Pringle
& Riddel, 1989; Mansoor & Kaur, 2009). The selectivity of the β-blockers for
cardiac β1- or bronchial/peripheral β2-adrenoceptors depends largely on the
configuration of its aryloxypropanolamine group, and more specifically on the
placement of the nitrogen and oxygen atoms within this aryloxypropanolamine
group; different formations allow for stronger affinity to distinct β-adrenoceptors
(Bekhradnia & Ebrahimzadeh, 2012).
Esmolol is rapidly hydrolyzed in vivo; its acid metabolite only has 0.3% the activity
of the active metabolite and its metabolism by erythrocyte esterases within the
cytosol of red blood cells rather than by plasma cholinesterases give esmolol its
ultra-short duration of action (Gorczynski, 1985; Lowenthal et al., 1985; Zuppa et al.,
CHAPTER 1 Literature Review 29
2003; Tang, He, Yao, & Zeng, 2004, Tang, Wang, Hu, Yao, & Zeng, 2012; Marik &
Varon, 2009). The acid metabolite is almost entirely eliminated through the urine,
with only 2% being excreted unchanged (Lowenthal et al., 1985).
Esmolol was synthesized and first studied in canine models in 1982 when it was
suggested that a shorter-acting, rapidly titratable β-blocker would have improved
safety over the longer-acting preparations for acute peri-and post-operative
hypertensive and arrhythmic complications, which often precipitated symptoms of
CHF (Erhardt, Woo, Anderson, & Gorczynski, 1982a, 1982b; Zaroslinksi et al.,
1982; Turlapaty et al., 1987). Studies in human patients with supraventricular
arrhythmias soon followed in 1984, and esmolol demonstrated cardioselectivity and
efficacy at controlling ventricular rates in these patients, due to its ability to slow
signal conduction through the AV node (Byrd, Sung, Mark, & Parmley, 1984;
Gorczynski, 1985). Left untreated, tachyarrhythmias and hypertension can lead to
mortality or major morbidities, including myocardial infarction, stroke, and
irreversible organ damage (Turlapaty et al., 1987; Garnock-Jones, 2012). Esmolol
has been shown to reduce heart rate, ejection fraction, and cardiac output by reducing
oxygen demand, contractility, and shear stress by increasing the sinus and AV node
conduction and refractory times (Bakker et al., 2011; Wiest, 1995) in patients with
preserved left ventricular function (Iskandrian et al., 1986). These effects have also
been demonstrated in patients with unstable angina and history of myocardial
infarction and in patients with moderate left ventricular dysfunction or acute
myocardial infarction (Kirshenbaum et al., 1988). In canine reperfusion models,
esmolol showed an improvement in regional myocardial function and a reduction in
infarct size, although a total cardioprotective effect was not demonstrated (Spahn et
CHAPTER 1 Literature Review 30
al., 1993). A reduction in myocardial infarct size and oedema has also been
demonstrated in humans (Geissler et al., 2000).
Due to its cardioselectivity, rapid onset and washout times regardless of hepatic or
renal function (Marik & Varon, 2009), esmolol is used clinically when rapid titration
is required (Byrd et al., 1984; Kirshenbaum et al., 1988). It is used perioperatively
(Lowenthal et al., 1985; Kirshenbaum et al., 1988) and postoperatively (Marik &
Varon, 2009) to prevent and control hypertension, ventricular fibrillation and
tachycardia (Kirshenbaum et al., 1988). The short t ½ also allows for rapid reversal if
hypotension or bradycardia develops (Zuppa et al., 2003). The specific
pharmacokinetic profile of esmolol allows for rapid titration in vivo simply by
controlling infusion rate (Fang, Bykowski-Jurkiewicz, Sarver, & Erhardt, 2010).
Orally administered β-blockers half-lives which range from 2-14 hours, which makes
them much more difficult to titrate should adverse effects occur (Esmolol Research
Group, 1986).
Atrial fibrillation and sinus tachycardia occur in 25% of coronary artery bypass graft
and valve replacement patients, and is yet more common in patients who have had β-
blocker therapy ceased pre-operatively (Gray, Bateman, Czer, Conklin, & Matloff,
1985; Garnock-Jones, 2012). In these patients, postoperatively administered esmolol
effectively restores sinus rhythm and controls heart rate (Tang et al., 2012; Gray et
al., 1985). Esmolol is used both peri- and postoperatively in patients with acute
ischaemic heart disease (Iskandrian et al., 1986; Bakker et al., 2011; Garnock-Jones,
2012), atrial fibrillation/flutter (Tang et al., 2004; 2012), and for supraventricular
tachyarrythmias in patients with mild CHF, COPD, and valvular disease (Geissler et
al., 2011; Gray et al., 1985; Byrd et al., 1984; Yamakage, Iwasaki, Jeong, Sato, &
CHAPTER 1 Literature Review 31
Namiki, 2009) and in low doses in those with severe left ventricular dysfunction
(Iskandrian et al., 1986) and severe postoperative hypertension (Marik & Varon,
2009). Esmolol has demonstrated a reduction in perioperative mortality, (Bakker et
al., 2011) and in postoperative AF/MI following CABG surgery (Zangrillo et al.,
2009). However, esmolol can increase the incidence of peri-and post-operative
bradycardia and hypotension, and its cardioprotective efficacy has yet to be
effectively demonstrated (Bakker et al., 2011, Zangrillo et al., 2009; Garnock-Jones,
2012).
The most common adverse effect of esmolol is hypotension (incidence up to 50%),
due to decreased inotropy and chronotropy, and possibly due to a decrease in
peripheral vascular resistance (Byrd et al., 1984; Wiest, 1995), however it is usually
rapidly reversible (Garnock-Jones, 2012). Its tolerability is greatly due to its
cardioselectivity; esmolol is 18 times more selective for cardiac β1-adrenoceptors
versus bronchial β2-adrenoceptors than metoprolol, and 50 times more β1-selective
than propranolol (Gorczynski et al., 1983; Garnock-Jones, 2012).
Esmolol, like most β-blockers, is currently only marketed as a racemic mixture,
although its antagonistic effects are believed to reside solely in the S-(-) enantiomer
(Zuppa et al., 2003; Fang et al., 2010; Tang et al., 2012), with the R-(+) enantiomer
having been demonstrated as being inactive (Lowenthal et al., 1985). The red blood
cell esterases responsible for the majority of its metabolism have not shown
stereoselectivity in previous human studies (Zuppa et al., 2003; Tang et al., 2012).
However, plasma esterases demonstrate a high level of stereoselectivity for the R-(+)
enantiomer, due to a significant difference in protein bound fractions between the
enantiomers (Tang et al., 2012).
CHAPTER 1 Literature Review 32
1.5.3 The Clinical Relevance Of Enantiomerically Pure β-Blockers
Enantiomerically pure compounds are in greater demand, with the FDA now calling
for the justification of racemic mixtures being marketed for clinical use (Agustiana et
al., 2010). In both the United States and the European Union, all drug development
studies must now include a thorough investigation of any chiral structure, how the
enantiomers may be separated, the relative contributions of the separate enantiomers
to the compound’s overall efficacy, and how the racemates will be marketed; these
guidelines, introduced in the early 1990’s, have led to a world-wide reduction in the
marketing of racemic mixtures (Caner et al., 2004). This is not true for the β-
blockers, however, as timolol is the only one of its class which is available world-
wide as an enantiomerically pure preparation; S-atenolol and S-metoprolol were
introduced in India in 2003, and are still being trialled (Mehvar & Brocks, 2001;
Dasbiswas & Dasbiswas, 2010). The S-isomers of both atenolol and metoprolol have
been isolated with the intent of improving cardioselectivity at higher doses; the
racemic mixtures lose β1-selectivity as dose increases, and this is believed to be due
to the R-isomers relative β2-selectivity (Mehvar & Brocks, 2001; Patil & Kuthekar,
2006). R-metoprolol also causes interactions with commonly prescribed drugs such
as cimetidine, paroxetine, verapamil, and some antibiotics, due to competitive
binding for metabolising enzymes such as CYP2D6 (Stout et al., 2011); its removal
should also eliminate potentiation by co-administration with these compounds (Patil
& Kuthekar, 2006).
CHAPTER 1 Literature Review 33
1.6 PHOSPHODIESTERASE ENZYMES: HEALTHY VS. FAILING HEARTS
1.6.1 Phosphodiesterase Enzymes in the Healthy Heart
Phosphodiesterase (PDE) enzymes are 3’, 5’ cyclic nucleotide phosphatases that are
responsible for degrading the second messengers cAMP and cGMP by hydrolyzing
their phosphodiester bond (Bischoff, 2004). PDEs accomplish this degradation in
both spatially and temporally specific patterns due to highly regulated
compartmentation (Omori & Kotera, 2007; Lenhart & Marks, 2006). cAMP is a
second messenger of the β1- and β2-adrenoceptor pathways, and activates cAMP-
dependent protein kinase a (PKA) which is responsible for the phosphorylation of
many proteins orchestrating the ECC, including troponin I, PLB, and the L-type Ca2+
channel and the RyR2 complex (Guellich et al., 2014). Over-stimulation of these
proteins by chronic β-adrenoceptor activation leads to hypertrophy, fibrosis, altered
Ca2+ handling and arrhythmias, and eventually leads to ventricular dysfunction
(Guellich et al., 2014). The degradation of cAMP reduces the phosphorylation of
target proteins by PKA, and protects the heart against overstimulation by Ca2+ via
RyR2-mediated ECC (Omori & Kotera, 2007; Lenhart & Marks, 2006; Levy, 2103).
(Figure 1.5)
CHAPTER 1 Literature Review 34
Figure 1.5. Catecholamine-induced activation of β1 and β2-adrenoceptors leads to a cAMP-PKA-mediated phosphorylation of the RyR2.
There are 11 PDE families that are separated into 60 isoforms based on sequence,
structure, substrate specificity, and sensitivity to both physiological and
pharmacological inhibitors (Bischoff, 2004; Lenhart & Marks, 2006; Mika, Leroy,
Vandecasteele, & Fischmeister, 2011; Omori & Kotera, 2007). PDEs 1-5, 8 and 9
have been found in the mammalian heart, although PDE9 does not appear to regulate
cardiac physiology or pathology (Knight & Yan, 2012). All of the PDEs regulate
cAMP and/or cGMP signaling by hydrolyzing their phosphodiester bond, which
effectively reduces both cyclic nucleotides to their inactive forms. However, each
family is localized to different tissues in the human body (Bischoff, 2004; De
Courcelles, De Loore, Freyne, & Janssen, 1992), and consequently, selectively
inhibiting PDE enzymes leads to tissue-specific responses (De Courcelles et al.,
1992). (Table 1.4)
RyR2
CHAPTER 1 Literature Review 35
FAMILY ISOFORM(S) SUBSTRATE(S)/ REGULATION TISSUE EXPRESSION
PDE1 PDE1A,C cAMP/cGMP: Ca2+/CaM-stimulated
cardiac myocytes, vascular myocytes, neurons, lymphoid cells, myeloid cells, testes
PDE2 PDE2A cAMP/cGMP: cGMP-stimulated
brain, cardiac myocytes, liver, adrenal cortex, endothelium, platelets
PDE3 PDE3A,B cAMP: cGMP-inhibited
cardiac myocytes, vascular myocytes, brain, liver, adipose, pancreas, endothelium, epithelium, oocytes, platelets
PDE4 PDE4A-D cAMP cardiovascular myocytes, neurons, immune system, inflammatory system
PDE5 PDE5A cGMP vascular myocytes, diseased cardiac myocytes, lung, brain, platelets, kidney, gastrointestinal tissues, penis
PDE6 PDE6A,B,C cGMP photoreceptors, pineal gland
PDE7 PDE7A,B cAMP spleen, brain, lung, kidney, lymphoid cells, myeloid cells
PDE8 PDE8A,B cAMP testes, thyroid
PDE9 PDE9A cAMP spleen, brain, intestinal cells
PDE10 PDE10A cAMP brain, testes
PDE11 PDE11 cGMP prostate, testes, salivary gland, pituitary gland
Table 1.4. Families, subfamilies, substrates, and tissue expression of phosphodiesterase enzymes. Table adapted from Maurice et al., 2014 and Miller & Yan, 2010.
CHAPTER 1 Literature Review 36
1.6.2 Phosphodiesterase Compartmentalization
PDEs have very specific spatial localizations, not just within specific tissue types but
also within subcellular compartments, which allows their effects on signaling
cascades to be very tightly regulated (Ahmad et al., 2012). The effects of PDEs are
not only spatially regulated, but also functionally and temporally regulated by means
of their targeting other effectors such as anchoring proteins, kinases, cyclases, PKA,
PKG, ion channels, and Epacs, (Beca et al., 2013; Ahmad et al., 2012; Knight &
Yan, 2012). They are incorporated into a wide variety of macromolecular complexes,
which allows them even further specificity and compartmentation, and any change in
their activation or degradation can upset this delicate balance of regulation, leading
to a disruption of homeostasis of cyclic nucleotides and, eventually, to progression of
disease (Guellich et al., 2014; Knight & Yan, 2012).
1.6.3 Roles of the PDE Subfamilies in Healthy and Failing Hearts
PDE expression and activity can be either up-regulated or down-regulated in patients
with heart failure, depending on the PDE family (Mehel et al., 2013; Moltzau et al.,
2014; Lee & Kass, 2012; Lenhart & Marks, 2006). The failing heart has decreased
levels of cAMP, and this decrease is thought to be a protective mechanism against
the harmful effects of chronic β-adrenergic stimulation. The aim of the
administration of PDE inhibitors is to restore cAMP levels by blocking the enzyme
responsible for its degradation (Amsallem et al., 2013). In doing so, PDE inhibitors
increase the uptake of Ca2+ into the sarcoplasmic reticulum, which results in positive
inotropy (Arnold et al., 1993). Unfortunately, along with positive inotropy, broad
PDE inhibition also leads to increased energy expenditure, the development of
arrhythmias, and cardiomyocyte apoptosis (Eschenhagen, 2013).
CHAPTER 1 Literature Review 37
Role of Phosphodiesterase 1
PDE1 is encoded by three different genes (A, B, and C), is activated by CAMK-II,
and is found in the hearts of many species, including humans (Conti & Beavo, 2007).
While PDE1C was known to be the dominant isoform expressed in the human heart
(Conti & Beavo, 2007; Lee & Kass, 2012; Omori & Kotera, 2007), it was originally
thought not to be present in myocytes; however, PDE1C has since been found in the
cytosol of cardiomyocytes (Guellich et al., 2014), particularly along the Z- and M-
lines (Vandeput et al., 2007). PDE1 hydrolyses both cAMP and cGMP and is
activated by Ca2+, calmodulin, and PDE1A. It is up-regulated in heart failure, and
may contribute to hypertrophy and maladaptive remodeling (Guellich et al., 2104).
However, without a readily available PDE1-selective inhibitor, its possible functions
in the human heart remain unknown (Fischmeister et al., 2006; Guellich et al., 2014).
Role of Phosphodiesterase 2
PDE2 has also been identified in human myocytes, where it hydrolyzes both cAMP
and cGMP. It is activated by cGMP in a negative-feedback mechanism, which
protects the heart when cGMP is activated by increased levels of nitric oxide (Mehel
et al., 2013; Guellich et al., 2014). When cGMP levels are increased, PDE2 is
activated and stimulates the hydrolysis of cAMP in the vicinity of the L-type Ca2+
channel by up to 30-fold, attenuating the effects of β-adrenoceptor stimulation
(Hartzell & Fischmeister, 1986; Mehel et al., 2013). In heart failure, PDE2 is up-
regulated approximately 2-fold; this up-regulation leads to desensitisation of the
myocytes to acute β-adrenoceptor activation, and may be a protective mechanism of
the failing heart to stress and noradrenaline-induced hypertrophy (Mehel et al., 2013;
Moltzau et al., 2014). The inhibition of PDE2 may also restore human cardiac
CHAPTER 1 Literature Review 38
function, however this has not yet been established in clinical studies (Mehel et al.,
2013).
Role of Phosphodiesterase 3
PDE3 has been identified in the cytosol of both healthy and failing human hearts, and
has similar affinity for both cAMP and cGMP; high cGMP levels inhibits its ability
to bind to and hydrolyze cAMP (Knight & Yan, 2012). Cyclic AMP in the human
heart is mainly hydrolyzed by PDE3 (Bender & Beavo, 2006; Movsesian et al.
1991), thereby controlling atrial (Christ et al., 2006; Kaumann, Semmler, &
Molenaar, 2007) and ventricular (Omori & Kotera, 2007) β1- and β2-adrenoceptor
mediated positive inotropic and lusitropic effects.
PDE3 has two subfamilies, PDE3A and PDE3B. Both hydrolyze cAMP and cGMP;
PDE3B is expressed in hepatocytes, adipocytes, and the β-cells of the pancreas,
whereas PDE3A is highly expressed in vascular smooth muscle cells and the heart
(Ahmed et al., 2012). In both rat and human studies, the PDE3A subfamily regulates
cardiac contractility by associating with SERCA and PLB to regulate uptake of Ca2+
into the sarcoplasmic reticulum, whereas PDE3B appears to have minimal effects on
basal cardiac contractility (Ahmad et al., 2012; Beca et al., 2013). PDE3A knockout
mice have altered expressions of both SERCA and RyR2, which leads to impaired
contractility (Beca et al., 2013). In addition, in vitro studies show that chronic
inhibition of PDE3A leads to cardiomyocyte apoptosis (Guellich et al., 2014). In
transgenic mice with myocardial overexpression of the enzyme, PDE3A1 has been
shown to negatively regulate β-adrenoceptor signaling, attenuating catecholamine-
induced increases of inotropy, chronotropy, and lusitropy, and to prevent apoptosis
CHAPTER 1 Literature Review 39
and ischemic reperfusion injury (Oikawa et al., 2103). In contrast, it has been
suggested that PDE3B may play a role in cardioprotection in times of cardiac stress,
such as during exercise, surgery, or in chronic heart failure (Beca et al., 2013).
PDE3B is activated by PKA, PKB, and PKC-mediated phosphorylation, whereas
PDE3A is only activated by PKA and PKB-mediated phosphorylation (Omori &
Kotera, 2007). Both the PDE3A and PDE3B families have multiple and distinct
phosphorylation sites in their N-terminus domains, which preferentially interact with
PKA, PKB, or PKC (Movsesian et al., 2011).
PDE3 inhibitors compete with cAMP to bind to PDE3, competitively inhibiting the
enzyme’s ability to phosphorylate cAMP and allowing it to accumulate within the
cell (Amsallem et al., 2013). The increased intracellular concentration of cAMP
results in increased ECC, and therefore positive inotropy and chronotropy. Acute
administration of PDE3 inhibitors has been shown to be helpful in restoring the ECC
in heart failure, however chronic administration of PDE3 inhibitors leads to
dysregulation of Ca2+ signaling and an increase in mortality (Packer et al., 1991; De
Courcelles et al., 1992; Lenhart & Marks, 2006; Levy, 2013). The role of PDE3
remains unclear in human heart failure, with downregulation reported by some
studies, (Silver et al., 1990; Ding et al., 2005a, 2005b), but normal function reported
in others (Movsesian et al., 1991; Von Der Leyen et al., 1991).
PDE3 inhibitors have been used clinically to restore hemodynamics in patients with
heart failure in an acute setting (De Courcelles et al., 1992), although several large-
scale studies investigating the chronic administration of milrinone, a PDE3 inhibitor,
found that its clinical use leads to worsening of heart failure and an increase in
CHAPTER 1 Literature Review 40
overall mortality (Packer et al., 1993; Shakur et al., 2002; Lenhart & Marks, 2006;
Yan, Miller, & Abe, 2007). The reason for the increase in mortality is still unknown,
with researchers indicating excessive accumulation of cAMP, a direct increase in
arrhythmias, and possible interaction with concomitant administration with
vasodilators as possible mechanisms (Shakur et al., 2002; Amsallem et al., 2013).
Role of Phosphodiesterase 4
PDE4 is the largest of the PDE families (Guellich et al., 2014), is activated by PKA
(Conti & Beavo, 2007), and is cAMP specific (Fischmeister et al., 2006). PDE4 is
encoded by four genes (A-D) and has more than 25 isotypes; however PDE4A,
PDE4B, and PDE4D are the only ones expressed in the human heart, with PDE4D
being the dominant gene expressed (Richter et al., 2011). β1-adrenoceptors interact
preferentially with PDE4D8, while β2-adrenoceptors and the RyR2 interact
preferentially with PDE4D5 and PDE4D3, respectively (Richter et al., 2011). PDE4
is also responsible for controlling the PKA-mediated regulation of ICaL, troponin I,
and the PLB/SERCA complex associated with the RyR2 (Richter et al., 2011).
PDE4 is a well-known regulator of chronotropic and inotropic effects in animal
hearts (Heaslip, Buckley, Sickels, & Grimes, 1991; Herzer, Thomas, Carcillo,
Tofovic, & Jackson, 1998; Shakur et al., 2002; Kaumann et al., 2009; Richter et al.,
2011). PDE4B is highly localized to the sarcolemma in mouse cardiomyocytes, and
modulates β1, but not β2-adrenoceptor-mediated excitation-contraction coupling with
the RyR2 and the L-type Ca2+ channel (Mika et al., 2014). The reason for the β1-
adrenoceptor specific activation may be due to the fact that β1-adrenoceptors are
widely distributed throughout cardiomyocytes, whereas β2-adrenoceptors are
CHAPTER 1 Literature Review 41
spatially restricted to very specific subcellular domains (Nikolaev et al., 2006; Mika
et al., 2014).
In the rat heart, PDE4D3 mediates cardiac hypertrophy resulting from chronic β-
adrenoceptor activation, which leads to progressive cardiomyopathy and arrhythmias
in PDE4D3-knockout mice (Kaumann, 2011; Ahmad et al., 2012). PDE4 protects
against β1-adrenoceptor mediated arrhythmias in rodent ventricle (Lenhart et al.,
2005; Galindo-Tovar & Kaumann, 2008) and human atrium (Molina et al., 2012).
In rat myocytes, agonist stimulation of the β2-adrenoceptor by isoprenaline recruits
PDE4D5, along with β-arrestin, which results in a desensitization of the β2-
adrenoceptor to both the cAMP-PKA and ERK1 signaling cascades by competitively
inhibiting Gα-activation of adenylyl cyclase and increasing local cAMP hydrolysis
(Houslay et al., 2007).
PDE4D9 associates with the β2-adrenoceptor in mice at rest, and becomes
dissociated upon β-adrenoceptor stimulation, when PDE4D5 and PDE4D8 are
recruited to the β2-adrenoceptor, likely via binding to β2-adrenoceptor/β-arrestin
complexes (De Arcangelis et al., 2009). This highly localized and specific
recruitment of PDE4 isoforms by the β2-adrenoceptor suggests a very tightly
controlled spatial and temporal regulation of cAMP signaling in response to
catecholamine-induced β2-adrenoceptor activation, which suggests an important role
of the β2-adrenoceptor in the mediation of cardiac contractility.
The effects of PDE4 in both healthy and failing human hearts are largely unknown
(Shakur et al., 2002; Richter et al., 2011), and there is still controversy regarding the
CHAPTER 1 Literature Review 42
role of PDE4 in human heart, with some studies showing no effect on inotropy and
chronotropy in human ventricle (Movsesian et al., 1991; Christ et al., 2006) while
another study showed a significant potentiation of both β1 and β2 – mediated
arrhythmias in human atrium (Molina et al., 2012; Eschenhagen, 2013). Due to its
presence in the RyR2 complex, PDE4 may in theory mediate catecholamine-induced
arrhythmias via the β-adrenoceptors, however PDE4 does not seem to limit the
positive inotropic effects of (-)-noradrenaline and (-)-adrenaline through β1- and β2-
adrenoceptors respectively, in non-failing human atrium obtained from patients
treated with or without β-blockers (Christ et al., 2006; Kaumann et al., 2007). The
disparity between these studies could be due to the fact that PDE4 is activated at
higher localized cAMP levels than PDE3; thus, PDE4 inhibitors may only mediate
inotropy and lusitropy when cAMP levels are increased by either PDE3 inhibition or
the administration of isoprenaline, or in tissues where resting adenylyl cyclase
activity is higher, such as the human atrium (Molina et al., 2012; Guellich et al.,
2014).
PDE4 activity is reduced in heart failure (Levy, 2013), specifically PDE4D3 which
helps to regulate Ca2+ release from the RyR2 (Lenhart et al., 2005; Lenhart & Marks,
2006). PDE4D3 is an integral part of the RyR2 complex, and is also reduced in
failing human hearts (Lenhart et al., 2005), but its role in adrenergic stimulation of
contractility via β1- and β2-adrenoceptors in the human ventricle is not currently
known (Christ, Galindo-Tovar, Thoms, Ravens, & Kaumann, 2008; Galido-Tovar &
Kaumann, 2008; Nikolaev, Bunemann, Schmitteckert, Lohse, & Engelhardt, 2006;
Rochais et al., 2006).
CHAPTER 1 Literature Review 43
Role of Phosphodiesterase 5
PDE5A is found in human heart, lung, and smooth muscle, and PDE5A inhibitors
were first trialed in humans with the aim of treating coronary artery disease and
hypertension (Omori & Kotera, 2007; Lee & Kass, 2012). In mice, PDE5 has proven
to be an integral component of cardiac remodeling after myocardial infarction, with
its inhibition resulting in reduced hypertrophy and cardiomyocyte apoptosis
(Guellich et al., 2014). Although there appears to be minimal expression of PDE5A
in the healthy human heart, its expression is increased in hypertrophic and failing
hearts, which has led to new trials showing significant effects of PDE5A inhibition in
the failing heart (Lee & Kass, 2012). In the acute setting, PDE5 inhibition may offer
ischemic preconditioning (Kukreja et al., 2005; Das, Salloum, Xi, Rao, & Kukreja,
2009), and chronic administration may reverse hypertrophy (Fisher, Salloum, Das,
Hyder, & Kukreja, 2005) and reduce the morbidity and mortality of cardiomyopathy
due to doxorubicin toxicity (Koka & Kukreja, 2010). Despite all these findings,
PDE5 inhibition in the human failing heart has not been shown to improve clinical
outcomes (Redfield et al., 2013).
Role of Phosphodiesterase 8
Western blots have shown that PDE8 is expressed in human cardiac tissue, and a
single study in knockout mice has shown that PDE8A controls at least some
localization of cAMP in mouse cardiac myocytes, as the removal of the gene leads to
a higher incidents of Ca2+ sparks through the RyR2 and increased Ca2+ transients
upon β-adrenoceptor activation (Petrucco et al., 2010). However, the role of PDE8 in
the human heart has yet to be elucidated.
CHAPTER 1 Literature Review 44
1.7 HYPOTHESIS, SUMMARY, AND IMPLICATIONS While PDE3 and PDE4 subtypes have been extensively studied in knockout mice,
their roles in the mediation of human heart contractility have not yet been clarified.
Murine and human hearts have stark differences: where PDE4 is the dominant
isoform controlling catecholamine-induced inotropy and chronotropy in murine
hearts, accounting for approximately 30% of overall cardiac PDE activity, it only
accounts for approximately 10% of PDE activity in the human heart, and its
localization to areas of the heart associated with the mediation of contractility has
been conflicting between human heart studies (Eschenhagen, 2013). In murine,
rabbit, dog, and human heart failure studies, both PDE3 and PDE4 are down-
regulated, whereas PDE1, PDE2, and PDE5 are up-regulated (Guellich et al., 2014).
The main question that still remains to be answered is whether downregulation of
PDE3 and PDE4 is simply a product of the failing heart, or if they are an integral part
of the maladaptive remodeling which leads to heart failure - and if so, which
subfamilies need to be targeted in order to improve mortality in these patients?
While PDE3 and/or PDE4 appear to control ventricular effects of catecholamines
through the β1- and the β2-adrenoceptor in several species, their relative effects in
failing human ventricle are still unknown. These studies investigate whether the
PDE3-selective inhibitor cilostamide (300 nM–1 µM) or PDE4 inhibitor rolipram (1–
10 µM) modify the positive inotropic and lusitropic effects of catecholamines in
human failing myocardium of patients chronically treated with either metoprolol or
carvedilol versus no β-blocker therapy. This study seeks to fill the current literature
gap in the roles of PDE3 and PDE4 in human heart failure, specifically their effects
on β-adrenoceptor mediated inotropy and lusitropy in failing human ventricles.
CHAPTER 1 Literature Review 45
The aim of Chapter 3 is to investigate whether PDE3 and/or PDE4 enzymes play a
role in the mediation of positive inotropy and lusitropy through ventricular β1- or β2-
adrenoceptors in patients with terminal heart failure in response to the administration
of catecholamines, and whether or not chronic administration of metoprolol modifies
the effects of PDE inhibition. The aim of Chapter 4 is to investigate whether the
inotropic and lusitropic effects of catecholamines, mediated through ventricular β1-
or β2-adrenoceptors from patients with terminal heart failure, are enhanced by
selective PDE3 and/or PDE4 inhibition, and whether or not chronic administration of
carvedilol modifies the effects of PDE inhibition. I predict that both PDE3 and PDE4
control catecholamine-induced effects of β1- and β2-adrenoceptor mediated positive
inotropy and lusitropy in human failing ventricle.
The aim of Chapter 5 is to investigate the affinities of R-(+) and S-(-) esmolol at
human β1- and β2-adrenoceptors and their effects on human right atrial contractility
and β1L- and β3-adrenoceptors, in order to determine whether or not an
enantiomerically pure preparation of S-(-) esmolol would be beneficial in a clinical
setting. I predict that S-(-) esmolol will have higher affinity at β1H,- β1L,- and β2-
adrenoceptors, and that R-(+) esmolol will have no significant affinity or efficacy in
mediating contractility through any of the investigated β-adrenoceptors.
CHAPTER 2 Methods & Research Design 47
2. Methods & Research Design
2.1 VENTRICULAR EXPERIMENTS
2.1.1 Research Design Summary
Ventricular trabeculae from freshly explanted hearts of 5 non-β-blocked, 15
metoprolol-treated, and 9 carvedilol-treated patients with terminal heart failure were
paced to contract at 1 Hz. The inotropic and lusitropic effects of (-)-noradrenaline,
mediated through β1-adrenoceptors and (-)-adrenaline, mediated through β2-
adrenoceptors, were assessed in the absence and presence of the PDE3-selective
inhibitor cilostamide (0.3 nM – 1 µM) or PDE4 inhibitor rolipram (1-10 µM). The
inotropic potencies of (-)-noradrenaline and (-)-adrenaline were then determined by
fitting a Hill function with variable slopes to concentration-effect curves from
individual experiments, and then estimating the catecholamine concentrations
producing a half maximum response, –LogEC50M (pEC50).
2.1.2 MATERIALS AND METHODS
Explantation and Transport
Trabeculae from right or left ventricle from explanted hearts with terminal heart
failure were obtained immediately upon explantation and handed directly from the
surgeon to the researcher. The time between full removal of the heart and handover
to the experimenters was carefully monitored (< 1 minute) in order to ensure a
minimum of tissue damage due to lapse in oxygenation. Upon its receipt, the entire
organ was placed directly into ice-cold Krebs solution containing (mM; Na+ 125, K+
Chapter 2: Methods & Research Design 48
5, Ca2+ 2.25, Mg2+ 0.5, Cl- 98.5, SO42- 0.5, HCO3
- 32, HPO42- 1, EDTA 0.04) for
Brisbane experiments and into Tyrode’s solution (mM): Na+ 149, K+ 5.4, Ca2+ 1.8,
Mg2+ 1.05, Cl- 137.8, HCO3- 22, HPO4
2- 0.42, EDTA 0.04, ascorbate 0.2, glucose 5.5,
and equilibrated with 95% O2/5% CO2 for Dresden experiments (Kaumann et al.,
1996; 1999). Krebs and Tyrode’s solutions were oxygenated in advance, so as to
further minimize the possibility of damage due to loss of tissue oxygenation. Once
the organs were in the solutions, they were immediately placed back onto ice for the
brief (less than two minute) transport to the laboratory.
Dissection and Set-Up of Trabeculae
Trabeculae were dissected in ice-cold Krebs or Tyrode’s solution which were
continuously oxygenated with 95% O2/5% CO2. Trabeculae chosen for dissection
were approximately 1 mm or smaller in thickness, in order to maximize the diffusion
of gases, nutrients, and drugs across the tissue throughout the experiment. Each was
carefully removed using fine surgical scissors in order to avoid any damage to the
endocardium, as it can lead to a reduction in contractility. A fine silk thread was
attached to one end of each trabeculum and knotted around the smallest amount of
tissue possible, again to minimize any interference with basal contractility. The other
ends of the sutures were tied into a loop, which were then attached to Swema SG4-45
strain gauge transducers. The non-looped ends of the trabeculae were then clamped
to Blinks tissue electrode blocks (Blinks, 1965), care being taken to clamp the
smallest portion of the trabeculae as possible, while still ensuring a secure enough
attachment to prevent dislodgement during either contraction or length-tension
testing. The design of the electrode blocks described by Blinks (1965) allow
trabeculae to be securely clamped without sacrificing oxygen flow; rather than
Chapter 2: Methods & Research Design 49
oxygen being bubbled directly at tissues, the solution itself (Krebs or Tyrode’s) is
briskly oxygenated and flowed past tissues. The thinness of the trabeculum
preparations and means of clamping allow for high oxygen perfusion and access to
all surfaces (with the exception of the small clamped portion) of the tissues.
The tissue electrode blocks were housed within 50 ml organ baths, large enough to
accommodate 2 trabeculae on their respective clamps in the oxygenated solution
(above) in each bath. Within the organ baths, the Krebs or Tyrode’s solution was
supplemented with (mM): Na+ 15, fumarate 5, pyruvate 5, L-glutamate 5, and
glucose 10. This solution provided metabolic support to the tissues throughout the
duration of the experiment (Kaumann et al., 1999). Up to 10 separate organ baths (20
trabeculae) were then lowered into a water bath held at 37°C by heaters which
continuously cycled and monitored water temperature, ensuring that temperatures
were held as closely to physiological conditions as possible (Kaumann et al., 1996;
1999).
Once dissected, clamped, and brought to 37°C, trabeculae were driven with
squarewave pulses of 5 ms duration to contract at 1 Hz. Currents were individually
adjusted for each trabeculum in order to establish a rhythmic contraction, and were
then held just above threshold current for the duration of the experiment (Kaumann
et al., 1999; Molenaar et al., 2006). Once contracting, each tissue was stretched
slowly and the resulting change in force of contractions monitored, in order to
establish a length tension curve for each individual trabeculum; the Lmax, or stretched
length producing maximal contractions, was established and maintained for the
duration of the experiment (Gille et al., 1985; Kaumann et al., 1996; 1999; Molenaar
Chapter 2: Methods & Research Design 50
et al., 2006).
Specific Activation of β1- and β2-Adrenoceptors
Optimized conditions were used to selectively activate β1- or β2-adrenoceptors. For
both conditions, α-adrenoceptors, neuronal and extraneuronal uptake were
irreversibly blocked by 5 µM phenoxybenzamine, which was incubated with the
tissues for 90 minutes prior to the addition of catecholamines. After 90 minutes, the
incubation solution was exchanged to remove unbound phenoxybenzamine and
supplemented with ascorbic acid (0.2 mM) to prevent catecholamine oxidation. (Kaumann et al., 1987; Molenaar et al., 2000; Christ et al., 2006).
ICI 118,551 has up to 300-fold higher affinity for β2- than β1-adrenoceptors (Bilski et
al., 1983; Lemoine et al., 1985a; Kaumann & Lemoine, 1987). ICI 118,551 at a
concentration of 50 nM antagonizes adrenaline-induced increases in contractile force
(Kaumann & Lemoine, 1987), and the KD of ICI, 118,551 at the β2-adrenoceptor is
0.5-1.5 nM (Lemoine et al., 1985a; Kaumann & Lemoine, 1987; Hall, Kaumann, &
Brown, 1990). Therefore, to selectively activate β1-adrenoceptors, (-)-noradrenaline
was used in the presence of the β2-adrenoceptor selective blocker ICI 118,551 50 nM
(Gille et al., 1985; Kaumann et al., 1999; Molenaar et al., 2000; 2006; Christ et al.,
2006).
CGP20712A has a 10,000-fold higher affinity for β1- versus β2-adrenoceptors
(Kaumann & Lemoine, 1987; Dooley & Bittiger, 1984; Lemoine et al., 1985b). The
KD of CGP 20712A at β1-adrenoceptors is 0.3-0.1 nM (Kaumann & Lemoine, 1987;
Hall et al., 1990; Lemoine et al., 1985a). Therefore, to selectively activate β2-
Chapter 2: Methods & Research Design 51
adrenoceptors, (-)-adrenaline was used in the presence of 300 nM CGP 20712A
(Kaumann et al., 1989; 1999; Molenaar et al., 2000; 2006; Christ et al., 2006).
To determine the effect of PDE enzymes on effects mediated through activation of
β1- or β2-adrenoceptors, PDE3-selective inhibition was obtained with cilostamide
(300 nM) and PDE4-selective inhibition obtained with rolipram (1 µM) pre-
incubated with trabeculae for 30-45 minutes prior to incubation with catecholamines.
The IC50 of cilostamide at PDE3 is approximately 50 nM (Beavo, 1995; Sudo et al.,
2000; Vargas et al., 2006); therefore, 300 nM of cilostamide blocks 86% of PDE3
but less than 0.4% of PDE4 (Vargas et al., 2006; Galindo-Tovar et al., 2009). The
IC50 of rolipram at PDE4 is approximately 1 µM; therefore 1 µM of rolipram will
block approximately 50% of PDE4 and only 0.4% of PDE3 (Vargas et al., 2006).
At the completion of concentration-effect curves to catecholamines on ventricular
trabeculae, the maximal response was recorded for each trabeculae by incubating
tissues with 200 µM (-)-isoprenaline, which surmounts blockade by both ICI
188,551 and CGP 20712A, therefore activating both β1- and β2-adrenoceptors
(Kaumann et al., 1987). This allowed all force readings of the concentration-effect
curves to be calculated as a % maximal response for each tissue, as all were different
sizes and had individual basal contraction forces. Since up to 20 contracting
trabeculae were obtained from the same heart, (upper limit of 20 was due to total
number of available clamps within organ baths, not due to limit of suitable trabeculae
per heart) it was often possible to compare the influence of the PDE inhibitors on
responses mediated through both β1 and β2-adrenoceptors. Experiments were
concluded with the addition of Ca2+ to raise the final bath concentration to 9.25 mM
Chapter 2: Methods & Research Design 52
(Molenaar et al., 2000).
2.1.3 Analysis of Contractile Data
A complete single concentration-effect curve for (-)-noradrenaline or (-)-adrenaline
was obtained in the absence or presence of cilostamide (300 nM) or rolipram (1 µM).
Responses to the catecholamines were expressed as a percentage of each tissue’s
maximal response to (-)-isoprenaline (200 µM). The catecholamine concentrations
producing a half maximum response, or –LogEC50M (pEC50), were estimated from
fitting a Hill function with variable slopes to concentration-effect curves from
individual experiments. The data are expressed as mean ± S.E.M. of n = number of
patients or trabeculae (as indicated).
Contractile force (F), time to peak force (TPF) and time to half-relaxation (t0.5) were
recorded through PowerLab amplifiers on a Chart for Windows, Version 5.0
recording program (ADInstruments Pty Ltd., Castle Hill, Australia) or on Graphtec 8
and 12 channel recorders. Measurements taken from the Graphtec channel recorders
were then measured by hand.
Significance of differences between means were assessed with the use of either
Student’s t test or ANOVA followed by Tukey-Kramer Multiple comparisons ad hoc
test at P<0.05 using Instat software (GraphPad Software Inc., San Diego, CA USA).
2.1.4 Drugs
(-)-Adrenaline (+)-bitartrate salt and (-)-noradrenaline bitartrate salt (hydrate) were
purchased from Sigma-Aldrich (St. Louis, MO, USA or Castle Hill, Australia).
Chapter 2: Methods & Research Design 53
Rolipram, cilostamide, CGP 20712A (2- hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-
4-(trifluorometyl)-1H-imidazol-2yl] phenoxy]propyl]amino]ethoxy]-benzamide) and
ICI 118,551 (1-[2,3-dihydro-7-methyl-1H-inden-4-yl]oxy- 3-[(1 methylethyl)amino]-
2-butanol) were purchased from Tocris Bioscience (Bristol, UK) or Sigma-Aldrich
(St. Louis, MO, USA or Castle Hill, Australia). Stock solutions were prepared in
purified water and kept at -20°C to avoid oxidation. Further dilutions of the drugs
were made fresh daily and kept cool (0–4°C) and dark. Repetitive experiments
showed that drug solutions treated in these ways are stable.
2.1.5 Participants
All participants had terminal heart failure and underwent cardiac transplant surgery
at The Prince Charles Hospital, Brisbane, ethics approval numbers EC9876,
HREC10/QPCH/184 or Gustav Carus Hospital, Dresden Technological University
ethics committee (Document EK 1140 82202). The studies conformed with the
World Medical Association Declaration of Helsinki Ethical Principles for Medical
Research Involving Human Subjects (Rickham, 1964).
Written informed consent was obtained from all patients. Each patient was
approached by the Transplant Coordinator of their hospital and provided with
layman-appropriate written information about the study being performed, the
reasoning and purpose behind it, and how their information, tissue, and the results
from their tissue would be utilised both during and after the study, included
experiments, and any communication or publication regarding the study. Participants
were invited to ask further questions of both experimenters and surgeons, and care
was taken to ensure that all patients had adequate time to make their decision, were
Chapter 2: Methods & Research Design 54
comfortable with the parameters of the study, and were able to provide the
coordinator with informed and confident decisions to participate. Any patient who
was either not confident with their agreement or uncomfortable with any aspect of
the study was not included. Participants were also informed they were able to
withdraw at any time, for any reason.
Patients were administered with metoprolol, carvedilol, or no β-blocker prior to
transplantation, up to and including the day of transplantation and tissue collection.
Other medications administered included aspirin, warfarin, clopidogrel, digoxin,
amiodarone, nitrates, thyroid hormones, ACE inhibitors, (lisinopril, perindopril,
ramipril, enalapril) diuretics, (amiloride, frusemide) L-type calcium channel
blockers, (amlodipine, diltiazem, nifedipine) angiotensin-II receptor antagonists,
(candesartan, telmisartan, irbesartan, losartan) proton pump inhibitors (omeprazole,
esomeprazole, lansoprazole), statins (atorvastatin, rosuvastatin) and others, such as
antibiotics or nicotine replacement therapy. All medications were administered up to
transplantation, with the exception of aspirin, clopidogrel, and warfarin, which were
ceased up to 7 days prior to surgery. Reasons for transplantation included
cardiomyopathy, dilated cardiomyopathy, idiopathic dilated cardiomyopathy,
congestive heart failure, ischemic heart disease, and sick sinus syndrome. For
quantitative patient details and statistics, please see patient tables in Chapters 3 and
4.
Chapter 2: Methods & Research Design 55
2.2 RIGHT ATRIAL EXPERIMENTS
2.2.1 Research Design Summary
Right atrial appendages from 39 patients were obtained from patients undergoing
coronary artery bypass surgery were paced to contract at 1 Hz. The affinities of S-(-)
and R-(+) esmolol at β1-adrenoceptors were assessed in the presence of S-(-) esmolol
(1-100 µM) or R-(+) esmolol (100 µM) and concentration-effect curves to
noradrenaline established; The affinities of S-(-) and R-(+) esmolol at β2-
adrenoceptors were assessed in the presence of S-(-) esmolol (10 –100 µM) or R-(+)
esmolol (100 µM) and concentration-effect curves to adrenaline established.
2.2.2 Explantation and Transport
The right atrial appendages from non-failing hearts of patients undergoing coronary
artery bypass graft surgery were obtained at time of aortic cannulation and placed
immediately into ice-cold pre-oxygenated Krebs solution, as described above.
Appendages were then placed immediately back onto ice and were transported (less
than two minutes) to the laboratory.
2.2.3 Dissection and Set-Up of Trabeculae
Right atrial trabeculae were dissected under continuous oxygenation with 95%
O2/5% CO2, and set up in pairs onto Blinks tissue electrode blocks, as described
above. Trabeculae were driven with squarewave pulses of 5 ms duration to contract
at 1 Hz, and currents adjusted for individual trabeculae, as above. After establishing
a length-tension curve for each trabeculum, the length was set to obtain 50% of the
resting tension associated with maximum developed force.
Chapter 2: Methods & Research Design 56
2.2.4 Specific Activation of β1- and β2-Adrenoceptors
Optimized conditions were used to selectively activate β1- or β2-adrenoceptors, as
described above. To determine the acute effects of S-(-) esmolol, S-(-) esmolol (1 -
100 µM) was added to the tissue bath after stabilization of trabeculae and effects on
contractility observed for 30 min. To determine whether S-(-) esmolol activates β1L-
adrenoceptors (Sarsero et al 2003; Christ et al 2011), trabeculae were incubated with
nadolol (200 nM) for 30 min followed by 3-isobutyl-1- methylxanthine (IBMX, 10
µM) for approximately 20 min until equilibrium was obtained. S-(-) esmolol (100
µM) was then added and observed for 30 min.
For the determination of the equilibrium dissociation constant (KB) of S-(-) and R-(+)
esmolol at β1-adrenoceptors, trabeculae were incubated with S-(-) esmolol (1 - 100
µM) or R-(+) esmolol (100 µM) and 50 nM ICI 118,551 for at least 90 minutes and
then concentration-effect curves to (-)-noradrenaline established. For the
determination of the equilibrium dissociation constant (KB) of S-(-) and R-(+)
esmolol at β2-adrenoceptors, trabeculae were incubated with S-(-) esmolol (10 – 100
µM) or R-(+) esmolol (100 µM) and 300 nM CGP 20712A for at least 90 minutes
and then concentration-effect curves to (-)-adrenaline established. Concentration-
effect curves to (-)-noradrenaline and (-)-adrenaline were followed by a single
concentration of (-)-isoprenaline (200 µM) and calcium (7 mM, to final bath
concentration 9.25 mM).
Chapter 2: Methods & Research Design 57
2.2.5Analysis of Contractile Data
A complete single concentration-effect curve for (-)-noradrenaline or (-)-adrenaline
was obtained in the absence or presence of S-(-) esmolol (1 – 100 µM) or R-(+)
esmolol (100 µM). Responses to the catecholamines were expressed as a percentage
of each tissue’s maximal response to (-)-isoprenaline (200 µM). The catecholamine
concentrations producing a half maximum response, or –LogEC50M (pEC50), were
estimated from fitting a Hill function with variable slopes to concentration-effect
curves from individual experiments. The data are expressed as mean ± S.E.M. of n =
number of patients or trabeculae (as indicated).
Contractile force (F), time to peak force (TPF) and time to half-relaxation (t0.5) were
recorded and measured as described above.
Significance of differences between means were assessed with the use of either
Student’s t test or ANOVA followed by Tukey-Kramer Multiple comparisons ad hoc
test at P < 0.05 using Instat software (GraphPad Software Inc., San Diego, CA USA).
2.2.6 Drugs
(-)-Adrenaline (+)-bitartrate salt and (-)-noradrenaline bitartrate salt (hydrate) were
purchased from Sigma-Aldrich (St. Louis, MO, USA or Castle Hill, Australia). S-(-)
and R-(+) enantiomers of esmolol were provided by Baxter Healthcare, Old
Toongabbie, NSW, Australia.
Chapter 2: Methods & Research Design 58
2.2.7 Participants
Ethics approval was obtained from the University of Queensland Human Research
Ethics Committee (Ethics approval 2011001285) and The Prince Charles Hospital,
Metro North Health Service District (Ethics Approval HREC/11/QPCH/148). The
studies conformed with the World Medical Association Declaration of Helsinki
Ethical Principles for Medical Research Involving Human Subjects (Rickham, 1964).
Written informed consent was obtained from all patients prior to surgery, as above,
with information provided to the patient directly from the researcher. Participants
were informed they were able to withdraw at any time, for any reason. All patients
had been treated with a β-blocker prior to surgery. Other medications administered
included aspirin, clopidogrel, nitrates, ACE inhibitors, (lisinopril, perindopril,
ramipril, enalapril) diuretics, (amiloride, frusemide) L-type calcium channel
blockers, (amlodipine, diltiazem, nifedipine) angiotensin-II receptor antagonists,
(candesartan, telmisartan, irbesartan, losartan) statins (atorvastatin, rosuvastatin), and
hypoglycemic agents (metformin, gliclazide, insulin). All medications were
administered up to transplantation, with the exception of aspirin and clopidogrel,
which were ceased up to 7 days prior to surgery.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 59
3. The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle
3.1 BACKGROUND AND PURPOSE
Phosphodiesterases PDE3 and/or PDE4 control ventricular effects of catecholamines
in several animal species but their relative effects in failing human ventricle are as
yet unknown. The aim of this study is to investigate whether or not the PDE3-
selective inhibitor cilostamide (0.3-1µM) or the PDE4 inhibitor rolipram (1-10µM)
modifies the positive inotropic and lusitropic effects of catecholamines in human
failing myocardium in patients chronically administered metoprolol versus non non-
β-blocker-treated patients.
3.2 METHODOLOGY AND RESEARCH DESIGN
3.2.1 Research Design Ventricular trabeculae from freshly explanted hearts of 5 non-β-blocked and 15
metoprolol-treated (7 from Brisbane/Dresden group, 8 from Oslo group) patients
with terminal heart failure were paced to contract at 1Hz. The inotropic and
lusitropic effects of (-)-noradrenaline, mediated through β1-adrenoceptors and (-)-
adrenaline, mediated through β2-adrenoceptors, were assessed in the absence and
presence of the PDE3-selective inhibitor cilostamide (300 nM-1 µM) or PDE4
inhibitor rolipram (1-10 µM). The inotropic and lusitropic potencies of (-)-
noradrenaline and (-)-adrenaline were then estimated from the half maximal effect,
or pEC50 from non-linear curve fitting.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 60
3.3 MATERIALS AND METHODS
3.3.1 Participants
Written informed consent was obtained from all patients. All patients had terminal
heart failure and underwent cardiac transplant surgery at The Prince Charles
Hospital, Brisbane, ethics approval numbers EC9876, HREC10/QPCH/184 and
Gustav Carus Hospital, Dresden Technological University ethics committee
(Document EK 1140 82202). The study conformed with the World Medical
Association Declaration of Helsinki Ethical Principles for Medical Research
Involving Human Subjects. Clinical data from metoprolol and non-β-blocker treated
Brisbane & Dresden patients are shown on Table 3.1. Clinical data from metoprolol-
treated Oslo patients are shown on Table 3.2.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 61
n Aetiology n Age Sex n EF Medication n ________ 5 CHF 3 47 ± 3.2 M 5 26 ± 1.8 A5,B0,C4,D1,E4,F1,G0,H4,I2,J0,M4 DCM 1 F 0 DCM/SSS 1 Table 3.1.A. Summary of Brisbane/Dresden patients that were not taking a β–blocker prior to heart transplantation n Aetiology n Age Sex n EF Dose Medication n_______ 7 ICM 3 53 ± 1.1 M 6 23 ± 1.8 115 ± 26 A4,B1,C4,D2,E3, DCM 3 F 1 F1,G2,H1,I0,J0,M2 IHD 1 Table 3.1.B. Summary of Brisbane/Dresden patients chronically administered metoprolol prior to heart transplantation. n Aetiology n Age Sex n EF Dose Medication n_______ 8 CAD 6 52 ± 4 M 7 21.5 ± 1. 92 ± 26 A4,B3,C7,D0,E6,F0 CMP 2 F 1 G3,H5,I1,J1,K7,L1,M8 Table 3.2. Summary of Oslo patients chronically administered metoprolol prior to heart transplantation n, number of patients; Aetiology, CMP (cardiomyopathy), DCM (dilated cardiomyopathy), ICM (idiopathic cardiomyopathy), CHF (congestive heart failure), IHD (ischemic heart disease), SSS (sick sinus syndrome); Age (years); Sex, Male (M), Female (F); Left ventricular ejection fraction (EF); Dose (mg per day); Medication: A angiotensin converting enzyme inhibitor, B angiotensin receptor blocker, C diuretic, D digoxin, E warfarin, F nitrate, G statin, H amiodarone, I thyroid hormone, J clopidogrel, K aspirin, L aldosterone antagonist, M other.
3.3.2 In vitro human heart trabeculae experiments
Trabeculae from right ventricle from explanted hearts with terminal heart failure
were obtained immediately upon explantation, and placed directly into ice-cold pre-
oxygenated modified Krebs solution containing (mM; Na+ 125, K+ 5, Ca2+ 2.25,
Mg2+ 0.5, Cl- 98.5, SO42- 0.5, HCO3
- 32, HPO42- 1, EDTA 0.04), sealed and
transported to the laboratory.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 62
3.3.3 Isolated Ventricular Trabeculae From Heart Transplant Patients
Trabeculae were dissected in ice-cold Kreb’s solution with 95% O2/5% CO2 and
prepared for electrical stimulation by attaching a fine silk thread loop to one end of
each trabeculum. Trabeculae were then clamped to an electrode block and the thread-
looped ends attached to Swema SG4-45 strain gauge transducers. Blinks tissue
electrode blocks were housed within 50 ml organ baths, to accommodate 2 trabeculae
in the oxygenated solution (above) supplemented with (mM): Na+ 15, fumarate 5,
pyruvate 5, L-glutamate 5, glucose 10 at 37°C (Kaumann et al., 1999). Trabeculae
were driven with squarewave pulses of 5 ms duration just above threshold current to
contract at 1 Hz. A length tension curve was established to determine Lmax, the
length producing maximal contractions, and maintained (Kaumann et al., 1999;
Molenaar et al., 2006).
3.3.4 Specific Activation of β1- and β2-Adrenoceptors
Optimized conditions were used to selectively activate β1- or β2-adrenoceptors. For
both conditions, α-adrenoceptors, neuronal and extraneuronal uptake were
irreversibly blocked by 5 µM phenoxybenzamine (Christ et al., 2006). To selectively
activate β1-adrenoceptor, (-)-noradrenaline was used in the presence of the β2-
adrenoceptor selective blocker ICI 118,551 50 nM (Kaumann et al., 1999; Molenaar
et al., 2000; 2006; Christ et al., 2006). To selectively activate β2-adrenoceptors, (-)-
adrenaline was used in the presence of the β1-adrenoceptor selective blocker 300 nM
CGP 20712A (Kaumann et al., 1999; Molenaar et al., 2000; 2006; Christ et al., 2006;
Alexander et al., 2011). To determine the effect of PDE enzymes on effects mediated
through activation of β1- or β2-adrenoceptors, PDE3-selective inhibition was
obtained with cilostamide (300 nM) and PDE4-selective inhibition obtained with
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 63
rolipram (1-10 µM) pre-incubated with trabeculae for 30 minutes prior to incubation
with catecholamines. At the completion of concentration-effect curves to
catecholamines on right ventricular trabeculae, the effects of a maximal
concentration of (-)-isoprenaline (200 µM) were determined. Since up to 20
contracting trabeculae were obtained from the same heart, it was often possible to
compare the influence of the PDE inhibitors on responses mediated through both β1
and β2-adrenoceptors.
3.3.5 Analysis Of Contractile Data
A complete single concentration-effect curve for (-)-noradrenaline or (-)-adrenaline
was obtained in the absence or presence of cilostamide (300 nM) or rolipram (1-10
µM). Responses to the catecholamines were expressed as a percentage of the
response to (-)-isoprenaline (200 µM). The catecholamine concentrations producing a
half maximum response, –LogEC50M (pEC50), were estimated from fitting a Hill
function with variable slopes to concentration-effect curves from individual
experiments. The data are expressed as mean ± S.E.M. of n = number of patients or
trabeculae (as indicated).
Contractile force, time to peak force (TPF) and time to half-relaxation (t0.5) were
recorded through PowerLab amplifiers on a Chart for Windows, Version 5.0
recording program (ADInstruments Pty Ltd., Castle Hill, Australia) or on Graphtec 8
and 12 channel recorders.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 64
Significance of differences between means were assessed with the use of either
Student’s t test or ANOVA followed by Tukey-Kramer Multiple comparisons ad hoc
test at P<0.05 using Instat software (GraphPad Software Inc., San Diego, CA USA).
3.3.6 Drugs
(-)-Adrenaline (+)-bitartrate salt, (-)-noradrenaline bitartrate salt (hydrate), prazosin
hydrochloride, and atropine sulphate were purchased from Sigma-Aldrich (St. Louis,
MO, USA or Castle Hill, Australia). Rolipram, cilostamide, CGP 20712A (2-
hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluorometyl)-1H-imidazol-2yl]
phenoxy]propyl]amino]ethoxy]-benzamide) and ICI 118,551 (1-[2,3-dihydro-7-
methyl-1H-inden-4-yl]oxy- 3-[(1 methylethyl)amino]-2-butanol) were from Tocris
Bioscience (Bristol, UK) or Sigma-Aldrich (St. Louis, MO, USA or Castle Hill,
Australia). Stock solutions were prepared in purified water and kept at -20°C to
avoid oxidation. Further dilutions of the drugs were made fresh daily and kept cool
(0–4°C) and dark. Repetitive experiments showed that drug solutions treated in these
ways are stable.
3.4 RESULTS
3.4.1 Chronic Metoprolol Treatment Increases the Inotropic Potencies of Catecholamines
Chronic treatment of patients with metoprolol sensitized right ventricular trabeculae
to the inotropic effects of (-)- noradrenaline and (-)-adrenaline. The inotropic
potencies of (-)-noradrenaline and (-)-adrenaline were increased fourfold and
fivefold, respectively, in metoprolol-treated (P < 0.05) compared with non-β-blocker
treated patients (Figure 3.1.A and B, Table 3.3.). The lusitropic effects (TPF, t50) of
(-) noradrenaline, mediated through β1-adrenoceptors, were not significantly
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 65
enhanced but the t50-abbreviating potency of (-)-adrenaline increased sevenfold (P <
0.001) by treatment of patients with metoprolol (Figure 3.2). These results are
consistent with the up-regulation of the β1-adrenoceptor density and enhanced
inotropic responses through these receptors in metoprolol treated patients (Heilbrunn,
Shah, Bristow, Valantine, Ginsburg, Fowler, 1989; Sigmund, Jacob, Becker,
Hanrath, Schumacher, & Eschenhagen, 1996).
Figure 3.1. Effects of chronic administration of metoprolol compared with no β-blocker on inotropic effects of (-)-noradrenaline through activation of β1-adrenoceptors (a) and (-)-adrenaline through activation of β2-adrenoceptors (b) in right ventricular trabeculae from failing hearts. Note the increased potency of (-)-noradrenaline and (-)-adrenaline for inotropic effects in metoprolol-treated patients. β-adrenoceptor blockade did not significantly increase basal force [P = 0.07 for (-)-noradrenaline, P = 0.095 for (-)-adrenaline] and maximum force [P = 0.10 for (-)-noradrenaline, P = 0.054 for (-)-adrenaline]. Data is from four [(-)-noradrenaline experiments] or five [(-)-adrenaline experiments] patients with heart failure not treated with a β-blocker and seven patients with heart failure treated with metoprolol. See text and Table 3.3 for further detail.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 66
(-)-Noradrenaline (-)-Adrenaline ____________________________________________________________________ Non-βB Metoprolol Non-βB Metoprolol pEC50 (n) pEC50 (n) pEC50 (n) pEC50 (n) RV Control 5.65 ± 0.15 (11/4) 6.25 ± 0.13 (18/7)* 5.70 ± 0.27 (14/5) 6.40 ± 0.11 (18/7)* Cilostamide 5.89 ± 0.24 (10/4) 6.75 ± 0.17 (17/7) 6.19 ± 0.27 (12/5) 7.11 ± 0.16 (13/7)† Rolipram - 6.19 ± 0.15 (15/7) - 6.50 ± 0.17 (12/7) LV Control - 6.34 ± 0.16 (7/6) - 6.26 ± 0.16 (10/7) Cilostamide - 6.77 ± 0.19 (7/6) - 6.93 ± 0.12 (8/7)†† Rolipram - 6.25 ± 0.10 (7/6) - 6.29 ± 0.17 (8/7)
Table 3.3. Inotropic potencies of (-)-noradrenaline and (-)-adrenaline acting through ventricular β1- and β2-adrenoceptors, respectively. Effects of cilostamide (300 nM right ventricle, 1 µM left ventricle) and rolipram (1 µM right ventricle, 10 µM left ventricle) and chronic metoprolol treatment. Non-βB: not treated with β-blockers; LV, left ventricle; RV, right ventricle; *P < 0.05 versus non- βB; †P < 0.001 paired Student’s t-test for comparison between cilostamide and control (no PDE inhibitor); ††P < 0.05 versus control, one-way ANOVA with Bonferroni adjustment for multiple a priori comparisons for comparison between cilostamide, rolipram and control. Numbers in parentheses are (trabeculae/patients).
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 67
Figure 3.2. Effects of chronic administration of metoprolol compared with no β-blocker on lusitropic effects [time to peak force and time to 50% relaxation (t50)] of (-)-noradrenaline through activation of β1-adrenoceptors (a,c) and (-)-adrenaline through activation of β2-adrenoceptors (b,d) in right ventricular trabeculae from failing hearts. Data from four [(-)-noradrenaline experiments] or five [(-)- adrenaline experiments] patients with heart failure not treated with a β-blocker and seven patients with heart failure treated with metoprolol. See text and Table 3.4 for further detail.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 68
3.4.2 Cilostamide Fails to Potentiate the Inotropic Effects of
Catecholamines in Right Ventricular Trabeculae from Non-β-Blocker-Treated Patients
Cilostamide (300 nM) did not significantly increase contractile force or hasten
relaxation in the presence of ICI 118,551 or CGP 20712A in trabeculae from non β-
blocker-treated patients. Cilostamide did not potentiate the positive inotropic
effects of (-)-noradrenaline or (-)-adrenaline (Figure 3.3, Table 3.3). Cilostamide did
not affect the lusitropic effects of (-)-noradrenaline (Figure 3.4 A and C, Table 3.4)
but potentiated the (-)-adrenaline-evoked shortening of t50 (Figure 3.4 D, Table 3.4).
Figure 3.3. Lack of effect of cilostamide on the inotropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from four [(-)-noradrenaline experiments] or five [(-)-adrenaline experiments] patients with heart failure not treated with a β-blocker. Shown are concentration–effect curves to (-)-noradrenaline (a) and (-)-adrenaline (b) in the absence or presence of cilostamide (300 nM). Cilostamide did not significantly increase basal force (P = 0.36 for the noradrenaline group, P = 0.46 for the adrenaline group) or enhance the maximum force caused by (-)-noradrenaline (P = 0.41) or (-)-adrenaline (P = 0.13). See text and Table 3.3 for further detail.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 69
Figure 3.4. Effect of cilostamide on the lusitropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from four [(-)-noradrenaline experiments] or five [(-)-adrenaline experiments] patients with heart failure not treated with a β-blocker. Shown are concentration–effect curves to (-)-noradrenaline (a,c) and (-)-adrenaline (b,d) in the absence or presence of cilostamide (300 nM). Cilostamide potentiated the (-)-adrenaline-evoked hastening of relaxation (shortening of t50). See text and Table 3.4 for further detail.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 70
(-)-Noradrenaline (-)-Adrenaline ____________________________________________________________________ Non-βB Metoprolol Non-βB Metoprolol pEC50 (n) pEC50 (n) pEC50 (n) pEC50 (n) TPF Control 6.45 ± 0.20 (11/4) 6.41 ± 0.13 (17/7) 6.33 ± 0.28 (13/5) 6.62 ± 0.10 (13/7) Cilostamide 6.49 ± 0.26 (9/4) 6.80 ± 0.12 (13/7) 6.80 ± 0.28 (12/5) 7.06 ± 0.12 (13/7)† Rolipram - 6.67 ± 0.21 (11/7) T50 Control 6.21 ± 0.19 (11/4) 6.61 ± 0.13 (16/7) 5.96 ± 0.26 (13/5) 6.79 ± 0.16 (15/7)* Cilostamide 6.30 ± 0.24 (9/4) 7.12 ± 0.12 (16/7)† 6.84 ± 0.28 (12/5) 7.41 ± 0.14 (13/7)† Rolipram - 6.64 ± 0.11 (15/7) - 6.59 ± 0.22 (12/7)
Table 3.4. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through right ventricular β1- and β2-adrenoceptors, respectively. Effects of cilostamide (300 nM) and rolipram (1µM) in non- β-blocker (Non-βB) treated and chronic metoprolol-treated patients. * P < 0.01, vs. non-βB †P < 0.05 Paired Student’s t-test for comparison between Cilostamide and Control (no phosphodiesterase inhibitor) Numbers in parentheses are (trabeculae/patients).
3.4.3 Cilostamide Potentiates the Effects Mediated Through β2- Adrenoceptors More Than β1-Adrenoceptors in Ventricular Trabeculae from Metoprolol-Treated Patients
Cilostamide (300 nM) alone did not significantly change contractile force in the
presence of ICI 118,551 or CGP 20712A on right ventricular trabeculae. Cilostamide
caused leftward shifts of the inotropic concentration–effect curves of (-)-
noradrenaline and (-)-adrenaline as shown in the representative experiment in Figure
3.5. Inotropic results from right ventricular trabeculae of seven patients are shown in
Figure 3.6. Cilostamide almost significantly increased the inotropic potency of (-)-
noradrenaline (P = 0.06) (Figure 3.6 A, Table 3.3). When data from right ventricular
trabeculae of two additional metoprolol-treated Oslo patients (results not shown)
were pooled with the data from seven Brisbane–Dresden patients, cilostamide
significantly (P < 0.02, n = 9) potentiated the inotropic effects of (-)-noradrenaline.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 71
Cilostamide (300 nM) potentiated the effects of (-)-adrenaline on force (fivefold, P <
0.05, Figure 3.6 B, Table 3.3). Cilostamide potentiated threefold the effects of (-)-
noradrenaline on t50 (P < 0.05) but not time to peak force (TPF) (Figure 3.7 A and C,
Table 3.4). Cilostamide potentiated the effects of (-)-adrenaline on TPF (threefold)
and t50 (fourfold) respectively (both P < 0.05, Figure 3.7 B and D, Table 3.4).
Cilostamide (300 nM) caused a non-significant (P < 0.07) leftward shift of the
concentration–effect curve for the inotropic effects of (-)-noradrenaline on left
ventricular trabeculae (P < 0.07, Figure 3.8 A, Table 3.3), but potentiated the
inotropic effects of (-)-adrenaline fivefold (P < 0.05, Figure 3.8 B, Table 3.3).
Cilostamide did not potentiate the TPF effects of (-)- noradrenaline or (-)-adrenaline
on left ventricular trabeculae but potentiated the effects on time to reach 80%
relaxation fourfold and fivefold respectively (both P < 0.05, Figure 3.9, Table 3.5).
Cilostamide caused a non-significant trend of greater potentiation of the inotropic
effects of (-)-adrenaline through β2-adrenoceptors (0.80 ± 0.11 log units, n = 9) than
(-)-noradrenaline through β1-adrenoceptors in right ventricular trabeculae from
metoprolol-treated patients (0.48 ± 0.18 log units, n = 9, Brisbane/Dresden/Oslo
hearts) (P = 0.14, paired Student’s t-test). However, when all right and left
ventricular inotropic data from metoprolol-treated patients were pooled, cilostamide
(0.3–1 µM) potentiated significantly more the β2-adrenoceptor-mediated effects of (-
)-adrenaline (0.78 ± 0.12 log units, n = 15) than the β1-adrenoceptor-mediated effects
of (-)-noradrenaline (0.47 ± 0.12 log units, n = 15) (P = 0.037). These results suggest
that PDE3 limits the inotropic responses through β2-adrenoceptors more than β1-
adrenoceptors.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 72
Figure 3.5. Representative experiment carried out on right ventricular trabeculae obtained from a 48-year-old male patient with ischaemic heart disease, left ventricular ejection fraction 25 %, chronically administered metoprolol 142.5 mg daily. Shown are original traces for (-)-noradrenaline and (-)-adrenaline in the absence or presence of cilostamide (Cil, 300 nM), rolipram (Rol, 1 µM), or Cil + Rol, followed by (-)-isoprenaline (ISO, 200 µM). The bottom panels show the corresponding graphical representation with non-linear fits. Note the clear potentiation of inotropic effects of both (-)-noradrenaline and (-)-adrenaline in the presence of cilostamide but the lack of potentiation by rolipram.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 73
Figure 3.6. Potentiation of the inotropic effects of (-)-adrenaline by cilostamide (P < 0.05) in right ventricular trabeculae from seven patients from Brisbane/Dresden with heart failure chronically administered metoprolol (b). In the same hearts, cilostamide caused a leftward shift of the inotropic effects of (-)-noradrenaline (a), which was not quite significant (P = 0.06). Rolipram had no effect on the inotropic effects of (-)-noradrenaline or (-)-adrenaline. See text for further explanation. Shown are concentration–effect curves to (-)-noradrenaline (a) and (-)-adrenaline (b) in the absence or presence of cilostamide (300 nM) or rolipram (1 µM).
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 74
Figure 3.7. Cilostamide, but not rolipram, potentiates the lusitropic effects of (-)-adrenaline and (-)-noradrenaline in right ventricular trabeculae from seven patients with heart failure chronically administered metoprolol. Shown are concentration-effect curves to (-)-noradrenaline (a,c) and (-)-adrenaline (b,d) in the absence or presence of cilostamide (300 nM) or rolipram (1 µM).
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 75
Figure 3.8. Cilostamide potentiates the inotropic effects of (-)-adrenaline in left ventricular trabeculae from seven [(-)-noradrenaline experiments] or eight Oslo patients [(-)-adrenaline experiments] with heart failure chronically administered metoprolol. Shown are concentration–effect curves to (-)-noradrenaline (a) and (-)-adrenaline (b) in the absence or presence of cilostamide (1 µM) or rolipram (10 µM). Inotropic data are normalized as a percentage of the maximal response to (-)-noradrenaline or (-)-adrenaline.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 76
Figure 3.9. Cilostamide, but not rolipram, potentiates the relaxant effects of (-)-noradrenaline and (-)-adrenaline in left ventricular trabeculae from seven ((-)-noradrenaline experiments), or eight Oslo patients ((-)-adrenaline experiments) with heart failure chronically administered metoprolol. Shown are concentration-effect curves to (-)-noradrenaline (a,c) and (-)-adrenaline (b,d) in the absence or presence of cilostamide (1 µM) or rolipram (10 µM). See Table 3.5 for analysis.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 77
(-)-Noradrenaline (-)-Adrenaline pEC50 (n) pEC50 (n) TPF Control 6.54 ± 0.16 (7/6) 6.56 ± 0.31 (8/7) Cilostamide 7.00 ± 0.20 (7/6) 7.13 ± 0.17 (8/7) Rolipram 6.30 ± 0.11 (7/6) 6.76 ± 0.19 (8/7) t50 Control 6.64 ± 0.15 (7/6) 6.59 ± 0.19 (10/7) Cilostamide 7.41 ± 0.16 (7/6)† 7.33 ± 0.13 (8/7)† Rolipram 6.57 ± 0.13 (7/6) 6.58 ± 0.17 (8/7)
Table 3.5. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through left ventricular β1- and β2-adrenoceptors of metoprolol-treated patients. Effects of cilostamide (1 µM) and rolipram (10 µM). Ventricular trabeculae were treated with rolipram (10 µM), cilostamide (1 µM) or untreated (control) for ~15-30 min prior to administration of increasing concentrations of either (-)-noradrenaline or (-)-adrenaline as described in Methods. Values in parentheses are (trabeculae/patients). Data are mean ± S.E.M. † P < 0.05 vs. control, Oneway ANOVA with Bonferroni adjustment for multiple a-priori comparisons. TPF, time to peak force; t50, ime to 50% relaxation
3.4.4 Rolipram Does Not Modify Inotropic or Lusitropic Potencies of (-)-Noradrenaline and (-)-Adrenaline
Rolipram did not significantly modify force, TPF, t50 or t80 in right and left
ventricular trabeculae incubated with ICI 118,551 or CGP 20712A. The inotropic
and lusitropic effects of (-)-noradrenaline and (-)-adrenaline were not significantly
changed by rolipram (1 µM) in right ventricular trabeculae (inotropic: Figures 3.5
and 3.6, Table 3.3; lusitropic: Figure 3.7, Table 3.4) or rolipram (10 µM) in left
ventricular trabeculae (inotropic: Figure 3.8, Table 3.3; lusitropic: Figure 3.9, Table
3.5). The effects of the combination of cilostamide (300 nM) and rolipram (10 µM)
on the inotropic and lusitropic potencies of (-)-noradrenaline and (-)-adrenaline were
investigated in three metoprolol-treated patients. Cilostamide + rolipram potentiated
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 78
the inotropic [(-)-noradrenaline P < 0.05; (-)- adrenaline P < 0.05] and lusitropic [(-)-
noradrenaline TPF, t50 both P < 0.05; (-)-adrenaline TPF, t50 both P < 0.05] effects of
both (-)-noradrenaline and (-)-adrenaline, but the degree of potentiation did not
significantly (P > 0.05) differ from the potentiation caused by cilostamide alone
(inotropic: Figure 3.10; lusitropic: Figure 3.11).
Figure 3.10. Effects of the combination of cilostamide and rolipram on the inotropic responses of (-)-noradrenaline (a) and (-)-adrenaline (b) in right ventricular trabeculae from three patients with heart failure chronically administered metoprolol. While the combination of cilostamide and rolipram potentiated the inotropic responses of (-)-noradrenaline and (-)-adrenaline, the degree of potentiation did not differ from that caused by cilostamide alone.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 79
Figure 3.11. Effects of the combination of cilostamide (300 nM) and rolipram (10 µM) on the lusitropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from 3 patients with heart failure chronically administered metoprolol. Whilst the combination of cilostamide and rolipram potentiated the lusitropic responses of (-)-noradrenaline and (-)-adrenaline, the degree of potentiation did not differ from that caused by cilostamide alone.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 80
3.5 DISCUSSION
This work revealed two important aspects of the control by PDEs of the inotropic
effects of catecholamines. Chronic treatment of heart failure patients with metoprolol
induced PDE3 to reduce the inotropic responses more through β2-adrenoceptors than
β1-adrenoceptors. PDE4 appears not to be involved in the inotropic and lusitropic
through either β1- or β2-adrenoceptors.
3.5.1 Control by PDE3 of the Function of β1- and β2-Adrenoceptors in Heart Failure Patients Treated with Metoprolol
PDE3 activity is stimulated by activation of β1-adrenoceptors and β2-adrenoceptors,
which in turn causes a negative feedback by hydrolyzing cAMP and thereby
reducing inotropic and lusitropic effects. Atonic receptor activation by endogenous
catecholamines increases cAMP and PKA activity in a compartment that allows the
latter to phosphorylate and activate PDE3 (Gettys, Blackmore, Redmon, Beebe, &
Corbin, 1987), which in turn hydrolyses cAMP. This effect is likely to be more
important for β2-adrenoceptors, at least in human heart, because these receptors are
more efficient than β1-adrenoceptors at activating Gs and stimulating ventricular
adenylyl cyclase (Kaumann & Lemoine, 1987), as verified with recombinant
receptors (Levy, Zhu, Kaumann, & Birnbaumer, 1993). Therefore, inhibition of
PDE3 may potentiate β2-adrenoceptor-mediated responses more than β1-
adrenoceptor-mediated responses, as shown here for human failing ventricle and
previously for non-failing atrial myocardium from patients without heart failure
(Christ et al., 2006).
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 81
A reduction in the expression and activity of PDE3 has been reported in heart failure
patients (Silver et al., 1990; Ding et al., 2005a, 2005b). Treatment with isoprenaline
causes sustained down-regulation of PDE3A (Ding et al., 2005b), as also observed in
human heart failure and animal heart failure models (Ya & Abe, 2007), presumably
due to the high catecholamine plasma levels. A down-regulation of PDE3A would be
expected to increase cAMP levels in heart failure so that inhibition of the enzyme
would conceivably affect the effects of catecholamines less. The lack of significant
potentiation by cilostamide of the inotropic and lusitropic effects of both (-)-
noradrenaline through β1-adrenoceptors and marginal potentiation of the effects of
adrenaline through β2-adrenoceptors in this study’s five non-β-blocked patients is
consistent with this expectation. In contrast, chronic treatment of heart failure
patients with metoprolol revealed robust potentiation of the inotropic and lusitropic
effects of the catecholamines through β1- and β2-adrenoceptors. The ability of
chronically administrated metoprolol to potentiate positive inotropy and lusitropy of
catecholamines may be due to chronic β-adrenoceptor blockade. In a failing heart,
high circulating levels of catecholamines would normally suppress PDE3 activity.
When the β-adrenoceptors are blocked, these higher levels of catecholamines are
prevented from suppressing PDE3 activity; this allows cAMP concentrations to
increase and more PKA phosphorylation and subsequent activation of RyR2 to
occur, ultimately resulting in increased inotropic and lusitropic responses to
circulating catecholamines. The increased β2-adrenoceptor-mediated ventricular
inotropic and lusitropic effects in ventricular trabeculae caused by metoprolol
treatment of heart failure patients agree with a similar (six-fold) enhancement of the
β2-adrenoceptor mediated inotropic potency of (-)-adrenaline in human atria obtained
from patients without heart failure chronically treated with atenolol (Hall, Kaumann,
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 82
& Brown, 1990). The increased cardiac responsiveness to adrenaline through β2-
adrenoceptors appears to be the result of chronic β1-adrenoceptor blockade.
Experimental long-lasting exposure to catecholamines elicits up-regulation of Gia
(Eschenhagen et al., 1992). A similar situation occurs in heart failure in which the
sympathetic nervous system is hyperactive (Cohn, 1989), plasma noradrenaline
levels are increased, (Thomas & Marks, 1978) and ventricular Giα is increased
(Neumann, Schmitz, Scholz, von Meyerinck, Döring, & Kalmar, 1988). Human β2-
adrenoceptors can couple to and activate Giα, in addition to Gsα, when they are
stimulated by a very high isoprenaline concentration in human atrium (Kilts et al.,
2000). Through chronic β1-adrenoceptor blockade of patients by treatment with
metoprolol or possibly atenolol, the noradrenaline-induced elevation of Giα ceases,
Giα levels are reduced (Sigmund et al., 1996), conceivably thereby favoring coupling
of the β2-adrenoceptor to Gsα to allow enhanced inotropic and lusitropic effects of
adrenaline through β2-adrenoceptors. This hypothesis requires future research.
The results presented here suggest that chronic β-adrenoceptor blockade facilitates
the control by PDE3s of catecholamine effects, particularly through β2-
adrenoceptors. However, the generality of this argument has to be restricted to heart
failure, because in atrial myocardium obtained from patients without heart failure it
has been reported that cilostamide potentiated the effects of adrenaline, mediated
through β2-adrenoceptors, more than the effects of noradrenaline, mediated through
β1-adrenoceptors, regardless of whether or not patients had been treated with β1-
adrenoceptor-selective blockers (Christ et al., 2006). Changes of ventricular β2-
adrenoceptor function in heart failure (Nikolaev et al., 2010) and profound
anatomical differences between ventricle and atrium (Bootman, Smyrnias, Thul,
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 83
Coombes, & Roderick, 2011) may be relevant to account for the different
consequences of PDE3 control in the two tissues with respect to β1-adrenoceptor and
β2-adrenoceptor function after chronic β-adrenoceptor blockade.
The lusitropic (Figures 3.7, 3.9, 3.11, Tables 3.4 and 3.5) effects mediated through
β1- and β2-adrenoceptors were usually potentiated by cilostamide to a similar extent
as the corresponding inotropic effects in trabeculae from β-blocker treated patients
(Figures 3.6, 3.8, 3.10, Table 3.3). PDE3 activity in human ventricle is associated
with membrane vesicle-derived t-tubules and junctional sarcoplasmic reticulum (SR),
causing hydrolysis of cAMP in the vicinity of phospholamban (PLB) (Movsesian et
al., 1991; Lugnier, Muller, Le Bec, Beaudry, & Rousseau, 1993). In ventricular
myocardium from failing hearts, noradrenaline and adrenaline produce similar
increases in PKA-catalyzed phosphorylation of the proteins mediating myocardial
relaxation, PLB (at Ser16), troponin-I (TnI) and cardiac myosin-binding protein-C
(Kaumann et al., 1999). These lusitropic results are consistent with an increased
phosphorylation of PLB, TnI and myosin-binding protein-C by isoprenaline in the
presence of the PDE3 inhibitor pimobendan in human failing myocardium (Bartel et
al., 1996).
3.5.2 PDE4 Inhibition Does not Affect the Inotropic or Lusitropic Effects of Catecholamines
PDE4 isoenzymes, their subtypes and splicing variants, are equally expressed in
rodent and human ventricle but murine hearts have a considerably higher PDE4
activity than human hearts (Richter et al., 2011). Inhibition of PDE4 causes
potentiation of the positive inotropic effects mediated through rodent β1-
adrenoceptors (Kaumann, 2011). In contrast, these results from human failing
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 84
ventricle demonstrate that inhibition of PDE4 with rolipram did not potentiate the
positive inotropic and lusitropic effects of (-)-noradrenaline or (-)-adrenaline,
mediated through β1-and β2-adrenoceptors, respectively. It could be argued that this
study was unable to demonstrate a potentiating effect of rolipram because PDEs,
including PDE4s, are down-regulated in heart failure (Ding et al., 2005a, 2005b;
Lehnart et al., 2005). However, studies have also reported for human atrial
myocardium, obtained from non-failing hearts, that rolipram failed to potentiate the
positive inotropic effects of (-)-noradrenaline or (-)-adrenaline mediated through β1-
and β2-adrenoceptors (Christ et al., 2006; Kaumann et al., 2007). These findings are
consistent with an early report demonstrating that cilostamide but not rolipram
inhibited SR-associated PDE activity in human ventricle from heart failure patients
(Movsesian et al., 1991).
Taken together, the present results and a critical appraisal of the literature make it
unlikely that PDE4s modulate human inotropic and lusitropic effects of
catecholamines, mediated through either β1- or β2-adrenoceptors in non-failing and
failing hearts. Moreover, extrapolation of results from the PDE4 function in mouse
and rat hearts to human inotropic and lusitropic effects of physiological
catecholamines can actually be misleading. However, PDE4s can reduce the
occurrence of catecholamine-evoked arrhythmias in murine ventricle (Galindo-Tovar
& Kaumann, 2008; Lehnart et al., 2005) and apparently in human atrium (Molina et
al., 2012). However, clinical trials with a PDE4 inhibitor, roflumilast, have not
provided evidence for cardiovascular side effects in approximately 1500 roflumilast-
treated patients compared with 1500 placebo patients (Calverley, Rabe, Goehring,
Kristiansen, Fabri, & Martinez, 2009). A comparison between human and other
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 85
species of the control of β1-adrenoceptor and β2-adrenoceptor-mediated inotropy and
lusitropy by PDE3 and PDE4, as well as protection against arrhythmias, is
summarised in Table 3.6.
________________________________________________________________________
Function Species PDE βAR References
Inotropism Human PDE3 β1&β2 Christ et al., 2006; Kaumann et al., 2007; This study Lusitropism PDE3 β1&β2 This study
Arrhythmias PDE3 β1&β2 Molina et al., 2012 &PDE4
Inotropism Mouse PDE4 β1 Galindo-Tovar & Kaumann, 2008
Lusitropism PDE4 β1 Beca et al., 2011
Arrhythmias PDE4 β1 Galindo-Tovar and Kaumann, 2008; Leroy et al., 2011
Inotropism Rat PDE4 β1&β2 Christ et al., 2009 ; Afzal et al., 2011
Lusitropism PDE4 β1&β2 Christ et al., 2009 ; Afzal et al., 2011
Arrhythmias PDE4 β1 Boer et al., 2011
Inotropism Rabbit PDE3 β1 Kaumann et al., 2009 &PDE4 Arrhythmias PDE4 β1 Holbrook & Coker, 1991 ________________________________________________________________________ Table 3.6. Reduction of inotropic and lusitropic responses as well as protection against arrhythmias, mediated through myocardial β1-adrenoceptor and β2-adrenoceptor, by PDE3 and PDE4 in different species.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 86
3.6 CLINICAL IMPLICATIONS Although this study did not detect a direct inotropic change with cilostamide, this
PDE3 inhibitor potentiated the inotropic effects of the endogenous catecholamines
mediated through ventricular β1-and β2-adrenoceptors of metoprolol-treated patients,
consistent with PDE3 inhibition. The induction of PDE3 activity in metoprolol-
treated patients could further reduce cardiostimulation by endogenous
catecholamines. In human atrium, metoprolol blocks the effects of catecholamines
through β1-adrenoceptors only by 2.5-fold more than β2-adrenoceptors (Figure 3.12).
It is therefore logical to predict that heart failure patients under therapy with a PDE3
inhibitor + metoprolol could be at risk of not being protected against adverse stress-
induced surges of adrenaline, acting through β2-adrenoceptors. From simple
competitive inhibition, the concentration ratio (CR) of a catecholamine in the
presence and absence of metoprolol can be calculated from CR = 1 + ([metoprolol] x
KB-1). The therapeutic plasma level of 100 ng·mL-1 (310 nM) metoprolol
(Kindermann et al., 2004) which hardly binds to plasma proteins, using KB values of
40 nM for β1-adrenoceptors and 98 nM for β2-adrenoceptors (Figure 3.12), would
produce CR values of 8.8 for β1-adrenoceptors and 4.2 for β2-adrenoceptors. The
fivefold potentiation of the inotropic effects of (-)-adrenaline by cilostamide suggests
that endogenous increases in plasma (-)-adrenaline could conceivably surmount the
β2-adrenoceptor blockade caused by metoprolol in patients also treated with a PDE3
inhibitor.
CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 87
3.7 CONCLUSIONS Treatment with metoprolol induces the control by PDE3 of the ventricular inotropic
and lusitropic effects of (-)-noradrenaline and (-)-adrenaline through β1-
adrenoceptors and β2-adrenoceptors, respectively, plausibly by restoring the
decreased activity and expression of PDE3 in heart failure. Quantitative
considerations, based on differences in the affinity profile of metoprolol for β1- and
β2-adrenoceptors, suggest that treatment with a PDE3-selective inhibitor could
potentially facilitate adverse stress-induced adrenaline effects through β2-
adrenoceptors in patients treated with metoprolol. PDE4 does not control the
inotropic and lusitropic effects mediated through β1- and β2-adrenoceptors in human
heart.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 88
4. The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle
4.1 BACKGROUND AND PURPOSE
Both metoprolol and carvedilol provide important therapeutic benefits for heart
failure patients. The previous chapter concluded that chronic administration of
metoprolol facilitates the control by phosphodiesterase PDE3, but not by PDE4, of
inotropic effects of catecholamines in failing human ventricle. However, it is not
known whether carvedilol has the same effect. This chapter investigates whether the
PDE3-selective inhibitor cilostamide (0.3 µM) or PDE4-selective inhibitor rolipram
(1 µM) modifies the positive inotropic and lusitropic effects of catecholamines in
ventricular myocardium of heart failure patients chronically administered carvedilol.
4.2 METHODOLOGY AND RESEARCH DESIGN
4.2.1 Heart Transplant Patients Written informed consent was obtained from all patients. Patients with terminal heart
failure underwent heart transplant surgery at The Prince Charles Hospital, Brisbane,
ethics approval EC9876, HREC10/QPCH/184 and Gustav Carus Hospital, Dresden
Technological University ethics committee (Document EK 1140 82202). Clinical
data for patients are is shown in Table 4.1. The use of human myocardium conforms
with the principles outlined in the Declaration of Helsinki (Rickham, 1964).
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 89
n Aetiology n Age Sex n EF Medication n ________ 5 CHF 3 47 ± 3.2 M 5 26 ± 1.8 A5,B0,C4,D1,E4,F1,G0,H4,I2,J0,M4 DCM 1 F 0 DCM/SSS 1 Table 4.1.A. patients that were not taking a β–blocker prior to heart transplantation. (Please note: this is the same control group utilised in Chapter 3).
n Aetiology n Age (year) Sex n LVEF Dose (mg/day) Medication 9 IDCM 3 44±2.9 M5 20±0.5 43±9 A7, B1, C8, D4, DCM 4 F4 E4, F2, G6, H3, IHD 2 I2, J1
Table 4.1.B Summary of patients chronically administered with carvedilol prior to heart transplantation. n, number of patients; DCM, dilated cardiomyopathy; IDCM, idiopathic dilated cardiomyopathy; IHD, ischaemic heart disease; M, male; F, female, LVEF, left ventricular ejection fraction; A, angiotensin-converting enzyme inhibitor; B, angiotensin-receptor blocker; C, diuretic; D, digoxin; E, warfarin; F, nitrate; G, statin; H, amiodarone; I, thyroid hormone; J, clopidogrel. M other.
4.2.2 Isolated Right Ventricular Trabeculae from Heart Transplant Patients
Hearts from heart transplantation surgery were placed immediately into ice-cold pre-
oxygenated modified Krebs solution containing (mM; Na+ 125, K+ 5, Ca2+ 2.25,
Mg2+ 0.5, Cl- 98.5, SO4 2- 0.5, HCO3 - 32, HPO4 2- 1, EDTA 0.04), sealed and
transported to the laboratory where right ventricular trabeculae were dissected under
continuous oxygenation with 95% O2/5% CO2. Dresden experiments were carried
out in Tyrode’s solution (mM): Na+ 149, K+ 5.4, Ca2+ 1.8, Mg2+ 1.05, Cl- 137.8,
HCO3- 22, HPO4
2- 0.42, EDTA 0.04, ascorbate 0.2, glucose 5.5, and equilibrated
with 95% O2/5% CO2. Trabeculae were clamped to an electrode block and the other
end attached to Swema SG4-45 strain gauge transducers. Electrode blocks were
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 90
housed within 50 ml organ baths, to accommodate 2 trabeculae in the oxygenated
solution (above) supplemented with (mM): Na+ 15, fumarate 5, pyruvate 5, L-
glutamate 5, glucose 10 at 37°C (Kaumann et al., 1999). Trabeculae were driven with
squarewave pulses of 5ms duration just above threshold current to contract at 1 Hz.
Contractile force, time to peak force (TPF) and time to half-relaxation (t0.5) were
recorded through PowerLab amplifiers on a Chart for Windows, Version 5.0
recording programme (ADInstruments Pty Ltd., Castle Hill, Australia) or on
Graphtec 8 and 12 channel recorders (Kaumann et al., 1999; Molenaar et al., 2006).
After determination of a length-tension curve, the length of each trabeculum was set
to obtain a resting tension associated with maximum developed force. To irreversibly
block tissue uptake of catecholamines and α-adrenoceptors, trabeculae were
incubated for 90 minutes with phenoxybenzamine (5 µM) followed by washout
(Kaumann et al., 1999; Molenaar et al., 2006).
4.2.3 Specific Activation of β1- and β2-Adrenoceptors
To determine the effects of β1-adrenoceptor-selective activation, concentration-effect
curves for (-)-noradrenaline were obtained in the presence of ICI 118,551 (50 nM) to
selectively block β2-adrenoceptors (Kaumann et al., 1999; Molenaar et al., 2006). To
determine the effects of β2-adrenoceptor-selective activation, concentration-effect
curves for (-)-adrenaline were determined in the presence of CGP 20712A (300 nM)
to selectively block β1-adrenoceptors (Kaumann et al., 1999; Molenaar et al., 2006).
To assess the influence of the PDE3- selective inhibitor cilostamide (300 nM) and
the PDE4-specific inhibitor rolipram (1 µM) on the effects of the catecholamines, a
single concentration-effect curve for a catecholamine was obtained in the absence or
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 91
presence of a PDE inhibitor. Trabeculae were incubated with PDE inhibitors for 30–
45 minutes prior to commencement of catecholamine concentration-effect curves. At
the completion of concentration-effect curves to catecholamines on right ventricular
trabeculae, the effects of a maximal concentration of (-)-isoprenaline (200 µM) were
determined. Since up to 20 contracting trabeculae were obtained from the same heart,
it was often possible to compare the influence of the PDE inhibitors on responses
mediated through both β1 and β2-adrenoceptors.
4.2.4 Analysis and Statistics A single concentration-effect curve for (-)-noradrenaline or (-)-adrenaline was
obtained in the absence or presence of cilostamide (300 nM) or rolipram (1 µM).
Responses to the catecholamines were expressed as a percentage of the response to
(-)-isoprenaline (200 µM). The catecholamine concentrations producing a half
maximum response, –LogEC50M (pEC50), were estimated from fitting a Hill function
with variable slopes to concentration-effect curves from individual experiments. The
data are expressed as mean ± S.E.M. of n = number of patients or trabeculae as
indicated. Significance of differences between means were assessed with the use of
either Student’s t test or ANOVA followed by Tukey-Kramer Multiple comparisons
ad hoc test at P<0.05 using Instat software (GraphPad Software Inc., San Diego,
CA).
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 92
4.3 RESULTS
4.3.1 Decrease of Inotropic and Lusitropic Potencies for (-)-Adrenaline by Chronic Treatment with Carvedilol
The potencies of (-)-adrenaline through activation of β2-adrenoceptors were reduced
(inotropic 16-49– fold, P < 0.0001), (lusitropic 13-35– fold, P < 0.0001) in carvedilol
treated patients compared to non-β-blocker treated patients from previous studies
(Kaumann et al., 1999; Molenaar et al., 2006; previous chapter) (inotropic, Figure
4.1, Table 4.2; lusitropic Figure 4.2, Table 4.3). In contrast, the inotropic and
lusitropic potencies of (-)-noradrenaline for activation of β1-adrenoceptors in
carvedilol treated patients were only slightly (less than 4-fold) or not reduced
compared to non-β-blocker treated patients (Kaumann et al. 1999; Molenaar et al.
2006; previous chapter). (Figure 4.1, Table 4.2, Figure 4.2, Table 4.3). In trabeculae
obtained from 3 carvedilol-treated patients, the responses to (-)-adrenaline were
greatly depressed and almost absent, preventing the estimation of meaningful pEC50
values. Inotropic and lusitropic results from these 3 patients are therefore shown
separately (Figures 4.3, 4.4, and 4.5). There was no difference with the daily dose of
carvedilol administered to the two groups of patients (Figure 4.1, 47.9 ± 12.3 mg, n =
6; Figure 4.4, 33.3 ±1 1.0 mg, n = 3, P = 0.5).
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 93
Figure 4.1. Marked reductions in potency for inotropic effects of (-)-adrenaline (b) through β2-adrenoceptors, but not for (-)-noradrenaline (a) through β1-adrenoceptors, in right ventricular trabeculae from six ((-)-adrenaline experiments) or nine carvedilol-treated patients ((-)-noradrenaline experiments), compared to five non-β-blocker-treated patients from the previous chapter.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 94
Carvedilol-treated patients (-)-Noradrenaline (-)-Adrenaline pEC50 (n) pEC50 (n) Control 5.68±0.10 (24/9) 4.50±0.10 (18/6) Cilostamide 5.89±0.14 (23/9) 5.51±0.25 (18/6) ΔpEC50 0.285±0.127 (9) 1.462±0.408 (6) P1 0.0533 0.0491 P2 0.006 Rolipram 5.72±0.16 (13/7) 4.34±0.13 (10/5) ΔpEC50 0.021±0.080 (7) 0.4995±0.25 (5) P1 0.9374 0.3039 Historical data from carvedilol and non-β-blocker treated patients (-)-Noradrenaline (-)-Adrenaline Non-β-blocker Carvedilol Non-β-blocker Carvedilol pEC50 (n) pEC50 (n) pEC50 (n) pEC50 (n) Historical Dataa 5.93±0.13 (18/11) 5.68±0.16 (19/11) Historical Datab 6.21±0.11 (18/9) 5.62±0.12 (31/15) 6.19±0.13 (21/10) 4.63±0.12 (41/15) Historical Datac 5.65±0.15 (11/4) 5.70±0.27 (14/5) Table 4.2. ΔpEC50 of (-)-noradrenaline and (-)-adrenaline in the absence or presence of PDE3 and PDE4 inhibitor. ΔpEC50is the difference in pEC50 values for (-)-noradrenaline or (-)-adrenaline obtained in the presence of phosphodiesterase inhibitor (cilostamide or rolipram) and without phosphodiesterase inhibitor (control) calculated from mean values for each patient. Values in parentheses are (trabeculae/patients) or (patients). P1 are P values obtained from paired Student’s t test for comparisons between phosphodiesterase inhibitor (cilostamide or rolipram) and control (no phosphodiesterase inhibitor). P2 are comparisons of the effects of (-)-noradrenaline and (-)-adrenaline. P2 is the value obtained from Student’s t test for comparisons of ΔpEC50 between (-)-noradrenaline and (-)-adrenaline. Values calculated for effects of cilostamide only. aKaumann et al., 1999; bMolenaar et al., 2006; cPrevious chapter.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 95
Figure 4.2. Effects of chronic administration of carvedilol compared to no β-blocker on lusitropic effects (time to peak force and time to 50 % relaxation (t50)) of (-)-noradrenaline through activation of β1-adrenoceptors (a, c) and (-)-adrenaline through activation of β2-adrenoceptors (b, d) in right ventricular trabeculae from failing hearts. Data from four ((-)-noradrenaline experiments) or five ((-)-adrenaline experiments) patients with heart failure not treated with a β-blocker (from previous chapter) and nine ((-)-noradrenaline experiments) or six ((-)- adrenaline experiments) patients with heart failure treated with carvedilol. Carvedilol-treated patients showed marked reductions in potency for lusitropic effects of (-)-adrenaline (b, d). See Table 4.3 for more details.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 96
(-)-Noradrenaline (-)-Adrenaline pEC50 (n) pEC50 (n) TPF Control 6.16±0.11 (24/9) 4.96±0.20 (18/6) Cilostamide 6.49±0.12 (23/9) 6.40±0.17 (16/6) ΔpEC50 0.423±0.126 (9) 1.27±0.18 (6) P1 0.0086 0.0294 P2 0.0016 Rolipram 6.26±0.20 (12/7) 5.07±0.16 (10/5) ΔpEC50 0.396±0.226 (7) 0.301±0.166 (5) P1 0.1614 0.2051 t50
Control 6.18±0.10 (24/9) 4.84±0.14 (16/6) Cilostamide 6.52±0.17 (22/9) 6.38±0.18 (16/6) ΔpEC50 0.418±0.24 (9) 1.54±0.277 (6) P1 0.1181 0.0051 P2 0.0098 Rolipram 6.31±0.08 (12/7) 4.90±0.11 (10/5) ΔpEC50 0.216±0.11 (7) 0.0879±0.304 (5) P1 0.22518 0.7905 Historical Data from non-β-blocker-treated patients (-)-Noradrenaline (-)-Adrenaline Non-β-blocker Non-β-blocker pEC50 (n) pEC50 (n) TPF Historical Dataa 6.18±0.19 (18/11) 6.51±0.11 (19/11) Historical Datab 6.45±0.20 (11/4) 6.33±0.28 (13/5) t50 Historical Dataa 6.43±0.17 (18/11) 6.24±0.15 (19/11) Historical Datab 6.21±0.19 (11/4) 5.96±0.26 (13/5) Table 4.3. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through right ventricular β1- and β2-adrenoceptors, respectively, from patients chronically treated with carvedilol. Effects of cilostamide (300 nM) and rolipram (1 µM). Historical data is given for non-β-blocked patients. ΔpEC50 is the difference in pEC50 values for (-)-noradrenaline or (-)-adrenaline obtained in the presence of phosphodiesterase inhibitor (cilostamide or rolipram) and without phosphodiesterase inhibitor (control) calculated from mean values for each patient. Values in parentheses are (trabeculae/patients) or (patients). P1 are P values obtained from paired Student’s t test for comparisons between phosphodiesterase inhibitor (cilostamide or rolipram) and control (no phosphodiesterase inhibitor). P2 is the value obtained from Student’s t test for comparisons of ΔpEC50 between (-)-noradrenaline and (-)-adrenaline. Values calculated for effects of cilostamide only aKaumann et al., 1999; bPrevious chapter.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 97
Figure 4.3. Potentiation of inotropic effects of both (-)-noradrenaline and (-)- adrenaline in the presence of cilostamide (Cil, 300 nM) but not rolipram (Rol, 1 µM). Representative experiment carried out on right ventricular trabeculae obtained from a 38-year-old male patient with DCM, left ventricular ejection fraction of 20 %, and chronically administered carvedilol 37.5 mg daily. Shown are original traces for (-)-noradrenaline and (-)-adrenaline followed by (-)-isoprenaline (ISO, 200 µM). The bottom panels show the corresponding graphical representation of the concentration-effect curves to the catecholamines with non-linear fits.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 98
Figure 4.4. Marked potentiation of the inotropic effects of (-)-adrenaline (b) versus (-)-noradrenaline (a) by cilostamide in right ventricular trabeculae from three hearts chronically treated with carvedilol. The hearts were notable for poor responsiveness to (-)-adrenaline in the absence of cilostamide. (Note: S.E.M. are not displayed for rolipram data in order to allow better clarity of the graph)
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 99
Figure 4.5. Marked potentiation of lusitropic effects of (-)-noradrenaline (a) and (-)-adrenaline (b) by cilostamide in right ventricular trabeculae from three hearts chronically treated with carvedilol. The hearts were notable for poor responsiveness to (-)-adrenaline in the absence of cilostamide; shown are the concentration-effect curves to (-)-noradrenaline and (-)-adrenaline in the absence or presence of cilostamide (300nM) or a combination of both cilostamide (300 nM) and rolipram (1 µM). Rolipram on its own (not shown) did not have an effect on either (-)-adrenaline or (-)-noradrenaline-mediated lusitropic effects.
4.3.2 Cilostamide Potentiates the Effects of (-)-Adrenaline More Than (-)-Noradrenaline in Trabeculae from Carvedilol-Treated Patients
Cilostamide (300 nM) caused a small, not quite significant 1.9-fold increase of the
inotropic potency of (-)-noradrenaline (P = 0.053) (Figure 4.6, Table 4.2) but
significantly increased the potency of (-)-noradrenaline to shorten TPF by 2.6-fold
(P<0.01) (Figure 4.7, Table 4.3). Cilostamide did not significantly alter (-)-
noradrenaline-evoked shortening of t0.5 (P = 0.12) in trabeculae from 9 carvedilol-
treated patients (Figure 4.7, Table 4.3). Cilostamide significantly potentiated the
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 100
inotropic effects of (-)-adrenaline, 10.2-fold in trabeculae from patients treated with
carvedilol (P<0.05) (Figure 4.6, Table 4.2). The lusitropic effects of (-)-adrenaline
were potentiated by cilostamide (28-fold for TPF, P<0.03; 35-fold for t0.5, P<0.01)
(Figure 4.7, Table 4.3). The effects of cilostamide (300 nM) were also investigated
on additional trabeculae from the 3 carvedilol-treated patients that were poor
responders to (-)-adrenaline (Figures 4.3, 4.5). In these patients, cilostamide partially
restored the inotropic responses to (-)-adrenaline, allowing estimates of inotropic
potentiation. The average log concentration-ratios of (-)-adrenaline in the presence
and absence of cilostamide at the 10% and 20% of maximum response levels to
isoprenaline were approximately 2.0 and 1.7 log units, i.e. equivalent to a 100-fold
and 50-fold potentiation, respectively. The responses to (-)-noradrenaline of this
group tended to be potentiated and were included in the group displayed in Figure
4.6. Cilostamide also potentiated the lusitropic responses of (-)-adrenaline by 0.93 ±
0.12 log units (TPF) and 1.47 ± 0.64 log units (t50) in these 3 patients (Figure 4.5).
Figure 4.6. Cilostamide (300 nM) potentiates the inotropic effects of (-)-adrenaline (b, n = 6) more than (-)-noradrenaline (a, n = 9) in right ventricular trabeculae from heart failure patients chronically administered carvedilol. Rolipram did not potentiate the effects from either (-)-noradrenaline or (-)-adrenaline.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 101
Figure 4.7. Cilostamide, but not rolipram, potentiates lusitropic effects of (-)-adrenaline (TPF P<0.03; t50 P<0.01) and (-)-noradrenaline (TPF P<0.01, but not t50
P=0.12) in right ventricular trabeculae from 6 (-)-adrenaline experiments and nine (-)-noradrenaline experiments of patients with heart failure chronically administered carvedilol. Shown are the concentration-effect curves to (-)-noradrenaline (a, c) and (-)-adrenaline (b, d) in the absence or presence of cilostamide (300 nM) or rolipram (1 µM). (Note: S.E.M. are not displayed for rolipram experiments in order to allow better clarity of the graph)
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 102
4.3.3 Rolipram Does Not Modify Inotropic or Lusitropic Potencies of (-)-Noradrenaline and (-)- Adrenaline in Trabeculae from Patients Treated with Carvedilol
Rolipram (1 µM) did not significantly modify force, TPF or t50, in trabeculae from
carvedilol treated patients. The inotropic (Figures 4.6, 4.3, 4.4, 4.5, Table 4.2) and
lusitropic potencies (Figures 4.7, 4.5, Table 4.3) of (-)-noradrenaline and (-)-
adrenaline were not significantly changed by rolipram (1 µM) in trabeculae from
carvedilol-treated patients.
The effects of the combination of cilostamide (300 nM) and rolipram (1 µM) on the
inotropic and lusitropic potencies of (-)-noradrenaline and (-)-adrenaline were
investigated in 3 carvedilol-treated patients of Figures 4.3 and 4.4. Cilostamide +
rolipram potentiated the inotropic and lusitropic effects of both (-)-noradrenaline
(inotropic and TPF P < 0.003, but not t50, P = 0.07) and (-)-adrenaline (P<0.01), but
the degree of potentiation did not significantly (P>0.07) differ from the potentiation
caused by cilostamide alone (Figures 4.3, 4.4, and 4.5).
4.4 DISCUSSION This work confirms that chronic treatment of heart failure patients with carvedilol
reduces the inotropic and lusitropic effects and potencies of (-)-adrenaline.
Carvedilol therapy induced marked potentiation by cilostamide of the inotropic and
lusitropic effects of (-)-adrenaline mediated through β2-adrenoceptors. In contrast,
cilostamide had markedly less ability to increase the potency of (-)-noradrenaline for
β1-adrenoceptor mediated inotropic and lusitropic effects. PDE4 did not affect the
inotropic and lusitropic responses through β1- or β2-adrenoceptors.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 103
4.4.1 Carvedilol-treatment Reduces Responses Through β2- Adrenoceptors but Not β1-Adrenoceptors
The β-adrenoceptor blocking effects of carvedilol partially persist after removal of
tissues from the heart in isolated atrial and ventricular trabeculae (Kindermann et al.,
2004; Molenaar et al., 2006). Studies have previously shown that persistent β2-
adrenoceptor blockade is considerably greater than the residual β1-adrenoceptor
blockade after removal of tissue from the failing hearts of carvedilol-treated patients
(Molenaar et al., 2006). Furthermore, this work demonstrates with exogenously
administered antagonist, that carvedilol has a 13-fold higher affinity for human atrial
β2- than β1-adrenoceptors (Molenaar et al., 2006). The 16-fold reduction of the
inotropic potency of (-)-adrenaline, mediated through β2-adrenoceptors, found in
right ventricular trabeculae of carvedilol-treated patients compared to non β-blocker-
treated patients, is consistent with a 25-fold potency reduction reported previously
(Molenaar et al., 2006). In contrast, the inotropic and lusitropic potency of (-)-
noradrenaline was not changed by the chronic treatment of patients with carvedilol,
consistent with a mere 1.8-fold decrease of (-)-noradrenaline inotropic potency
(Molenaar et al., 2006).
Therapy of heart failure patients with the β-blocker metoprolol increases β1-
adrenoceptor density and function (Heilbrunn et al., 1989; Gilbert et al., 1996;
Sigmund et al., 1996). The unchanged noradrenaline potency in trabeculae from
carvedilol-treated patients could be the result of an actual upregulation of β1-
adrenoceptor density combined with partial β1-adrenoceptor occupancy with
carvedilol firmly bound to the receptors. Residual binding of carvedilol to β1-
adrenoceptors would also explain the lack of increase of β1-adrenoceptor receptor
density reported in heart failure patients treated with carvedilol (Gilbert et al., 1996).
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 104
4.4.2 Carvedilol Facilitates the Control by PDE3 of β2- More than β1- Adrenoceptor-Mediated Effects
Cilostamide produced a small potentiation of the positive inotropic and lusitropic
effects of (-)-noradrenaline, mediated through β1-adrenoceptors, but marked
potentiation of the effects of (-)-adrenaline mediated through β2-adrenoceptors in
ventricular trabeculae from carvedilol treated patients.
These data indicate that chronic administration of carvedilol to patients with severe
heart failure differentially modifies the control of PDE3 metabolism of cAMP
accumulated by activation of β1- and β2-adrenoceptors. The existence of spatial
microdomains incorporating β1- or β2-adrenoceptors, G proteins, cAMP, protein
kinases, PDEs and AKAP scaffolding proteins has been described in mouse and rat
hearts (Nikolaev et al., 2006, 2010; Perera & Nikolaev, 2013). In rat and mouse non-
failing ventricular cardiomyocytes, β2-adrenoceptor signalling was reported to be
localized to deep transverse tubules, whereas β1-adrenoceptor signalling was
distributed across the whole cell surface (Nikolaev et al., 2010). However, in a rat
model of chronic heart failure induced by myocardial infarction, β2-adrenoceptor
signalling in ventricular cardiomyocytes became diffuse and was distributed across
the whole cell surface (Nikolaev et al., 2010). The demonstration of organized
signalling structures and their ability, at least for β2-adrenoceptor signalling, to re-
organize in rat failing heart may shed light on the current findings. It may be possible
that the chronic administration of carvedilol in patients with human heart failure
adjusts the spatial alignment of PDE3 with β2- more than β1-adrenoceptor signalling
so that cAMP accumulated by activation of β2-adrenoceptors is more efficiently
metabolized by PDE3. This remains to be verified by high resolution imaging
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 105
techniques (Nikolaev et al., 2010), particularly in human myocardium.
Following β-adrenoceptor-mediated increases in cAMP levels and PKA activity,
PDE3 is phosphorylated (Gettys et al., 1987) and in turn hydrolyzes cAMP, thereby
reducing the positive inotropic and lusitropic effects of endogenous catecholamines.
This effect is likely to be more important for β2-adrenoceptors, at least in human
heart, because these receptors are more efficient than β1-adrenoceptors at activating
Gs protein and stimulating atrial and ventricular adenylyl cyclase from non-failing
human hearts (Gille, Lemoine, Ehle, & Kaumann, 1985; Kaumann & Lemoine,
1987), as later verified with recombinant receptors (Green, Holt, & Liggett, 1992;
Levy et al., 1993). Therefore, inhibition of PDE3 may potentiate responses more
through β2- than β1-adrenoceptors. β2-adrenoceptor selective potentiation of the
inotropic and lusitropic effects of (-)-adrenaline was also observed in human atrial
myocardium obtained from patients without heart failure, treated or not treated with
β-blockers (Christ et al., 2006) and in the previous chapter with ventricular
trabeculae from metoprolol-treated patients with heart failure. Interestingly, however,
these results from carvedilol-treated patients with heart failure reveal an even more
pronounced control of PDE3 of β2-adrenoceptor-mediated effects than in the
previous studies of Christ et al. (2006). Although β2-adrenoceptors of non-failing
human ventricle couple tightly to Gs (Kaumann & Lemoine, 1987) they appear
partially uncoupled from the Gs-cAMP pathway in chronic heart failure (Bristow et
al., 1989). Chronic treatment with carvedilol may improve the coupling of β2-
adrenoceptors to the Gs-cAMP pathway, thereby facilitating PKA-catalyzed
phosphorylation and activation of PDE3.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 106
The α subunits of Gi protein, Giα, are up-regulated in heart failure (Neumann et al.,
1988) and β2-adrenoceptors can couple to and activate Gi, in addition to Gs, when
stimulated by isoprenaline in human atrium (Kilts et al., 2000). A 3-month treatment
of heart failure patients with metoprolol causes a 74% decrease in Giα (Sigmund et
al., 1996). This reduction of Giα would allow a greater coupling of β2-adrenoceptors
to Gs, thereby facilitating inotropic and lusitropic effects mediated through these
receptors. I speculate that a decrease of Gi activity may also have occurred in chronic
carvedilol-treated patients, as observed with metoprolol (Sigmund et al., 1996),
thereby facilitating coupling of β2-adrenoceptors to Gs.
When the persistent β2-adrenoceptor blockade by carvedilol was surmounted with (-
)- adrenaline, improved coupling to Gs protein may occur and lead therefore to
greater PKA-catalyzed phosphorylation and activation of PDE3. Inhibition of PDE3
by cilostamide may then cause potentiation of the effects of (-)-adrenaline through
two plausible mechanisms, selective coupling of β2-adrenoceptors to Gs and
decreased Gi coupling. The potentiation by PDE3 inhibition of (-)-noradrenaline
effects is smaller than corresponding effects of (-)-adrenaline through β2-
adrenoceptors, apparently because β1-adrenoceptors couple less tightly to Gs than β2-
adrenoceptors and they do not couple to Gi.
As found with the inotropic effects, cilostamide potentiated the lusitropic effects
mediated through β2- more than β1-adrenoceptors in trabeculae from carvedilol-
treated patients (Figures 4.7 and 4.5, Table 4.3). This was expected from similar
increases in PKA-catalyzed phosphorylation of the proteins mediating myocardial
relaxation, phospholamban (at Ser16), troponin-I and cardiac myosin-binding protein
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 107
C through β1- and β2-adrenoceptors in ventricular myocardium from failing hearts
(Kaumann et al., 1999). These lusitropic results are consistent with the localization of
PDE3 in the vicinity of phospholamban (Movsesian et al., 1991; Lugnier et al.,
1993).
4.4.3 Quantitative Comparison of β1- and β2-Adrenoceptor Blockade by Carvedilol in Combination with PDE3 Inhibition
Following the oral administration of the therapeutically used concentration of 25 mg
carvedilol, peak plasma levels of 100 ng.ml-1 (246 nM) carvedilol have been
measured in heart failure patients (Tenero et al., 2000). Since approximately 98% of
carvedilol is bound to plasma protein (Martindale, 2002) the β-adrenoceptors would
be expected to be in contact with approximately only 4.9 nM of plasma carvedilol.
The concentrations of endogenous noradrenaline and adrenaline needed to surmount
the carvedilol-induced blockade of β1- and β2-adrenoceptors can be estimated from
the surmountable antagonism against noradrenaline and adrenaline respectively, and
the corresponding dissociation equilibrium constants KB (Molenaar et al., 2006).
From simple competitive inhibition, the concentration-ratio (CR) of a catecholamine
in the presence and absence of carvedilol can be calculated from CR = 1 +
([carvedilol] x KB-1). Using KB values of carvedilol of 0.95 nM for the β1-
adrenoceptor and 0.074 nM for the β2-adrenoceptor (Molenaar et al. 2006), 4.9 nM
carvedilol would produce CR values of 6.2 and 67 for β1- and β2-adrenoceptors,
respectively. Therefore, due to the β2-adrenoceptor-selective affinity of carvedilol,
endogenous adrenaline would hardly be expected to cause cardiostimulation through
β2-adrenoceptors in carvedilol-treated patients treated with a PDE3 inhibitor, despite
the 10-35-fold potentiation of the adrenaline effects.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 108
The estimates of blocking potency from plasma concentrations of carvedilol may
actually not represent the concentration of carvedilol at the β1- and β2-adrenoceptors
because carvedilol still causes substantial β-adrenoceptor blockade at plasma levels
below the carvedilol detection level of 1 ng.ml-1 (Kindermann et al., 2004). It is
likely that under these conditions carvedilol stored in cardiac tissues is slowly
released and diffuses across the cardiomyocytes thereby causing some occupancy of
β1-adrenoceptors and greater occupancy of β2-adrenoceptors due to its higher affinity
for the latter (Molenaar et al., 2006). The cardiac capture and storage of carvedilol is
supported by evidence demonstrating that the cardiac concentrations of active S-
enantiomer of carvedilol exceed the plasma concentrations by 7-fold (Stahl,
Mutschler, Baumgartner, & Spahn-Langguth, 1993). The ex-vivo experiments,
demonstrating consistently greater persisting blockade of β2- than β1-adrenoceptors
in atrial and ventricular tissues from heart failure patients treated with carvedilol
(Molenaar et al., 2006 confirmed in this work), are consistent with these
mechanisms.
4.4.4 PDE4 Does Not Control the Inotropic or Lusitropic Effects of Catecholamines in Carvedilol Treated Patients with Heart Failure
Rolipram did not potentiate the positive inotropic and lusitropic effects mediated
through β1- or β2-adrenoceptors in trabeculae from carvedilol-treated patients with
heart failure. Furthermore, rolipram did not enhance the potentiation caused by
cilostamide (Figure 4.4). A similar failure of rolipram to potentiate catecholamine
effects was reported previously for human non-failing atrium (Christ et al., 2006;
Kaumann et al., 2007) and failing ventricle from metoprolol-treated patients
(Previous Chapter). This evidence, taken together, allows the conclusion that under
these conditions PDE4 does not control human atrial and ventricular inotropic and
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 109
lusitropic effects, mediated through β1- and β2-adrenoceptors. However, it has been
reported that PDE4 reduces the incidence of arrhythmias mediated through human
atrial β1- and β2-adrenoceptors (Molina et al., 2012). Furthermore, although PDE4
protects against β1-adrenoceptor-mediated ventricular arrhythmias in mice (Lehnhart
et al., 2005; Galindo-Tovar & Kaumann, 2008), it is unknown whether this happens
in failing human ventricle.
4.5 CONCLUSIONS By preventing cAMP hydrolysis, PDE3 inhibitors (e.g. milrinone and enoximone)
enhance cardiac contractility through activation of cAMP-dependent pathways.
Short-lasting infusions or low-dose oral treatment with PDE3 inhibitors have been
shown to improve systolic function in chronic heart failure (Anderson, 1991; Van
Tassel, Radwanski, Movsesian, & Munger, 2008). It is plausible that the potentiation
of the effects of catecholamines contributes to the positive inotropic effects of PDE3
inhibitors in the clinic (Previous chapter and this study). It could be argued that the
consequences of the suppression by cilostamide of the PDE3-induced control of the
inotropic and lusitropic responses mediated through β1- and β2-adrenoceptors are
transient because they were only observed in a time frame of less than 2 hours.
Conceivably, at a later stage compensatory PDEs could be activated through PKA-
catalyzed phosphorylation by the cAMP accumulated during PDE3 inhibition.
Preliminary results reveal, however, that cilostamide reduces the fade of the inotropic
responses to noradrenaline up to 21 hours after cilostamide administration
(Unpublished experiments of Galindo-Tovar & Kaumann), inconsistent with
intervention of other PDEs.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 110
High-dose chronic treatment with PDE3 inhibitors worsens heart failure and
increases mortality, particularly through sudden death (Packer et al., 1991;
Amsallem, Kasparian, Haddour, Boissel, & Nony, 2005), presumably due to
ventricular fibrillation. Arrhythmias, elicited by endogenous catecholamines, may be
facilitated by PDE3 inhibitors. (-)-Noradrenaline and (-)-adrenaline produce a similar
incidence of arrhythmic contractions, mediated through β1- and β2-adrenoceptors
respectively, in human atrial myocardium from patients without heart failure
(Kaumann & Sanders, 1993), despite the lower density of β2-adrenoceptors
(Kaumann, Lynham, Sanders, Brown, & Molenaar, 1995), likely related to the tighter
coupling of human β2-adrenoceptors to Gs protein. Elevations of endogenous plasma
adrenaline levels during stress (Wortsman, 2002), stress cardiomyopathy (Wittstein
et al., 2005) and cardiopulmonary bypass surgery (Reves et al., 1981) are
considerably higher than increases in noradrenaline. Human ventricle from failing
hearts also shows pro-arrhythmic β2-adrenoceptor-mediated after-contractions and
after-transients and increases in Ca2+ transient amplitude, SR load, and twitch [Ca2+]
decline rate (De Santiago et al., 2008).
This study indicates that the joint treatment of heart failure patients with a PDE3
inhibitor and the slightly β1-adrenoceptor-selective blocker metoprolol could
facilitate adverse stress-induced adrenaline effects such as arrhythmias mediated
through β2-adrenoceptors (Previous Chapter). In contrast, due to the selective
blockage of β2-adrenoceptors, the quantitative assessment (see above) allows the
prediction that carvedilol also protects the heart against adverse effects of adrenaline
in heart failure patients treated together with a PDE3 inhibitor.
Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 111
Carvedilol appears to produce more benefit than metoprolol in heart failure as shown
in the COMET trial (Poole-Wilson et al., 2003). The greater benefit could be
partially related to the β2-adrenoceptor-selective (Molenaar et al., 2006) blockage.
Carvedilol, but not metoprolol, would prevent the appearance of β2-adrenoceptor-
mediated arrhythmias (Remme et al., 2007; Di Lenarda et al., 1999) by endogenous
adrenaline, which was even shown to decrease the incidence of arrhythmias in heart
failure patients carrying antibodies with agonist activity through β2-adrenoceptors
(Chiale et al., 1995; Li, Wang, Zhao, Zhou, & Zhu, 2010). However, experimental
evidence demonstrating β2-adrenoceptor-mediated arrhythmias, facilitated by PDE3
inhibition, is still pending for human myocardium.
CHAPTER 5: Enantiomers of Esmolol 112
5. Enantiomers of Esmolol
5.1. BACKGROUND AND PURPOSE
All β-blockers share a common racemic core structure, and the antagonistic
efficacies of the S-enantiomers are generally more potent than those of the R-
enantiomers. As both the pharmacodynamics and pharmacokinetics of the racemates
also commonly differ, it is desirable to know which racemates produce either clinical
benefits or unwanted side effects. Most β-blockers used clinically are only available
as racemic mixtures, including esmolol, which is commonly used in acute and
surgical settings.
The β1-adrenoceptor has two separate affinity states: the β1H-adrenoceptor, which is
called the “high affinity” receptor due to the high affinity of β-blockers to block its
activity, is activated by noradrenaline. Conversely, the β-blockers have low affinity
for the blockade of the β1L-adrenoceptor, which is activated by (–)-CGP 12177 and
(–)-pindolol (Molenaar et al., 1997; Kaumann, 1989; Sarsero et al., 1999). As β–
blockers can have differing affinities for these two physiological states of the β-
adrenoceptor, it is therefore possible that the separate racemates of β-blockers may
be responsible for the disparity in activity at these two sites. This chapter will
investigate the effects of R-(+) and S-(-) esmolol on human right atrial contractility,
β1L- and β3-adrenoceptors, and their affinities at human β1- and β2-adrenoceptors.
5.2 METHODS
5.2.1 Participants
Right atrial appendages were obtained from patients undergoing coronary artery
bypass surgery at The Prince Charles Hospital. Ethics approval was obtained from
Chapter 5: Enantiomers of Esmolol 113
the University of Queensland Human Research Ethics Committee (Ethics approval
2011001285) and The Prince Charles Hospital, Metro North Health Service District
(Ethics Approval HREC/11/QPCH/148). Written informed consent was obtained
from all patients prior to surgery. Patient characteristics are given in Table 5.1.
____________________________________________________________________ n Age Sex EF β-blocker, daily dose, mg (n) Other medication ____________________________________________________________________ 39 61.7±1.3 35M/4F 56.2±1.6 Aten 43.9±4.9 (9) Bisop 8.3±1.7(3) A34,B23,C5 D37 Carv 50 (1) Met 62.4±7.9 (26) E11,F32,J8 ____________________________________________________________________ Table 5.1. Summary of patient details. A, Angiotensin converting enzyme inhibitor/AT1 receptor antagonist; B, nitrate; C, diuretic, D, hypolipidemic; E, L-type Ca2+channel antagonist; F, Aspirin/Clopidogrel; J, hypoglycemic agent.
5.2.2 Preparation of Right Atrial Trabeculae
After surgical removal, right atrial tissues were placed immediately into ice-cold pre-
oxygenated modified Krebs solution containing (mM; Na+ 125, K+ 5, Ca2+ 2.25,
Mg2+ 0.5, Cl- 98.5, SO42- 0.5, HCO3- 32, HPO42- 1, EDTA 0.04) and transported
to the laboratory (within ~5 min of surgical removal) where atrial strips containing
intact trabeculae were dissected under continuous oxygenation with 95% O2/5%
CO2, set up, on occasion in pairs, and driven with square-wave pulses of 5 ms
duration just above threshold voltage to contract at 1 Hz in an apparatus with a 50 ml
organ bath in the solution above supplemented with (mM): Na+ 15, fumarate 5,
pyruvate 5, L-glutamate 5, glucose 10 at 37°C (Molenaar et al., 2007). The tissues
were attached to Swema SG4-45 strain gauge transducers and force recorded through
PowerLab amplifiers on a Chart for Windows, Version 7.0 recording programme
(ADInstruments Pty Ltd., Castle Hill, Australia) or on Graphtec 8 and 12 channel
recorders (Molenaar et al., 2007). After determination of a length-tension curve, the
Chapter 5: Enantiomers of Esmolol 114
length of each trabeculum was set to obtain 50% of the resting tension associated
with maximum developed force.
5.2.3 Acute effects of S-(-) Esmolol on Human Right Atrium
Upon stabilization of trabeculae, S-(-) esmolol (1 - 100 µM) was added to the tissue
bath and effects on contractility observed for 30 min.
5.2.4 Effect of S-(-) Esmolol in the Presence of 3-isobutyl-1- methylxanthine and Nadolol
All clinically used β-blockers block β1-adrenoceptors and some, including pindolol,
also activate β1L-adrenoceptors (Kaumann & Molenaar, 2008). To determine whether
S-(-) esmolol activates β1L-adrenoceptors (Sarsero et al., 2003; Christ et al., 2011),
trabeculae were incubated with nadolol (200 nM) for 30 min followed by 3-isobutyl-
1- methylxanthine (IBMX, 10 µM) for approximately 20 min until equilibrium was
obtained. S-(-) esmolol (100 µM) was then added and observed for 30 min.
5.2.5 Determination of the Affinity of S-(-) and R-(+) Esmolol at Human Atrial β1- and β2-Adrenoceptors
Tissues were incubated with 5 µM phenoxybenzamine to block α-adrenoceptors and
neuronal and extraneuronal uptake of catecholamines and either 50 nM ICI 118,551
to block β2-adrenoceptors and/or 300 nM CGP 20712A to block β1-adrenoceptors
(Gille et al., 1985; Kaumann et al., 1999). For the determination of the equilibrium
dissociation constant (KB) of S-(-) and R-(+) esmolol at β1-adrenoceptors, trabeculae
were incubated with S-(-) esmolol (1 - 100 µM) or R-(+) esmolol (100 µM) and 50
nM ICI 118,551 for at least 90 minutes and then concentration-effect curves to (-)-
noradrenaline established. For the determination of the equilibrium dissociation
Chapter 5: Enantiomers of Esmolol 115
constant (KB) of S-(-) and R-(+) esmolol at β2-adrenoceptors, trabeculae were
incubated with S-(-) esmolol (10 – 100 µM) or R-(+) esmolol (100 µM) and 300 nM
CGP 20712A for at least 90 minutes and then concentration-effect curves to (-)-
adrenaline established. Concentration-effect curves to (-)-noradrenaline and (-)-
adrenaline were followed by a single concentration of (-)-isoprenaline (200 µM) and
calcium (7 mM, final concentration 9.25 mM).
5.3 RESULTS
5.3.1 Acute Effects of S-(-) Esmolol on Human Right Atrium
S-(-) Esmolol caused time and concentration (1 – 100 µM) dependent reductions in
contractile force of electrically stimulated human right atrial trabeculae (Figure 5.1
a), most likely due in part to blockade of (-)-noradrenaline. There was no effect on
the duration of contraction, time to peak force (Figure 5.1 b) or time to 50%
relaxation (Figure 5.1 c).
Chapter 5: Enantiomers of Esmolol 116
Figure 5.1. Effect of S-(-) esmolol on force of contraction (a) and relaxation, time to peak force (b) and time to 50% relaxation. S-(-) esmolol caused a concentration and time dependent reduction in force of contraction, but did not affect the duration of the contraction. Force is expressed as a percentage of force at time 0 min.
Chapter 5: Enantiomers of Esmolol 117
5.3.2 Effect of S-(-) esmolol in the Presence of 3-isobutyl-1-methylxanthine (IBMX) and Nadolol
The addition of S-(-) esmolol (100 µM) to trabeculae equilibrated with nadolol (200
nM) and IBMX (10 µM), conditions designed to detect agonist activity at β1L-
adrenoceptors (Christ, Molenaar, Klenowski, Ravens, & Kaumann, 2011), caused a
time dependent reduction in contractile force (Figure 5.2). No agonist activity at β1L-
adrenoceptors was detected.
Figure 5.2. Lack of agonist activity of S-(-) esmolol at β1L-adrenoceptors. Right atrial trabeculae were equilibrated with nadolol (200 nM) and 3-isobutyl-1- methylxanthine (IBMX, 10 µM) and incubated with or without S-(-) esmolol (100 µM). Force is expressed as a percentage of initial force immediately before addition of nadolol or IBMX. Numbers in parentheses are (trabeculae/patients).
Chapter 5: Enantiomers of Esmolol 118
5.3.3 Determination of the Affinity of S-(-) and R-(+) Esmolol at Human Atrial β1- and β2-Adrenoceptors
β1-adrenoceptors
(-)-Noradrenaline caused concentration dependent increases in contractile force in
the absence and presence of S-(-) and R-(+) esmolol through activation of β1-
adrenoceptors (Figure 5.3 a). The pKB (-logKB) for S-(-) esmolol was 6.48 ± 0.12, n
= 33. The slope of the Schild plot was not different to unity (Figure 5.3 c). The
affinity of R-(+) esmolol, determined with one concentration of R-(+) esmolol (100
µM) at β1-adrenoceptors (pKB 4.57 ± 0.31, n = 7, Figure 7b) was ~ 80-fold less than
S-(-) esmolol.
__________________________________________________________ β1-adrenoceptors β2-adrenoceptors ________________________________________ S-(-) Esmolol 6.48 ± 0.12 (33/10) 5.85 ± 0.13 (20/9) R-(+) Esmolol 4.57 ± 0.31 (7/4) 4.74 ± 0.42 (5/3) __________________________________________________________
Table 5.2. pKB values of S-(-) Esmolol and R-(+) Esmolol determined at human atrial β1- and β2-adrenoceptors. S-(-) Esmolol, the slope of the Schild plot for experiments at β1-adrenoceptors was not different to unity. The slope of the Schild plot for experiments at β2-adrenoceptors was different to unity and therefore the pKB value is an estimate. R-(+) Esmolol pKB values determined with the use of a 100 µM concentration only. Numbers in parentheses are (trabeculae-patients).
Chapter 5: Enantiomers of Esmolol 119
Figure 5.3. Determination of the affinity of S-(-) and R-(+) esmolol at β1-adrenoceptors in human right atrium. Shown are cumulative concentration effect curves for (-)-noradrenaline at β1-adrenoceptors in the absence or presence of S-(-) (a) or R- (+) esmolol (b). The corresponding Schild plots are shown in (c). Numbers in parentheses are (trabeculae/patients).
!
Chapter 5: Enantiomers of Esmolol 120
β2-adrenoceptors
(-)-Adrenaline caused concentration dependent increases in contractile force in the
absence and presence of S-(-) and R-(+) esmolol through activation of β2-
adrenoceptors (Figure 5.4 a). The pKB (-logKB) for S-(-) esmolol was 5.85 ± 0.11, n
= 20. The slope of the Schild plot was different to unity (Figure 5.4 c) and therefore
the affinity value, pKB 5.85 is an estimate. The affinity of R-(+) esmolol, determined
with one concentration of R-(+) esmolol (100 µM) at β2-adrenoceptors (pKB 4.74 ±
0.94, n = 5, Figure 5.4 b) was ~ 13-fold less than S-(-) esmolol.
Chapter 5: Enantiomers of Esmolol 121
Figure 5.4. Determination of the affinity of S-(-) and R-(+) esmolol at β2-adrenoceptors in human right atrium. Shown are cumulative concentration effect curves for (-)-adrenaline at β2-adrenoceptors in the absence or presence of S-(-) (a) or R-(+) esmolol (b). The corresponding Schild plots are shown in (c). Numbers in parentheses are (trabeculae/patients).
A β2AR
Chapter 5: Enantiomers of Esmolol 122
5.4. DISCUSSION
5.4.1 Patient Population
This study was carried out in a relatively homogenous population of patients
undergoing open chested heart surgery at The Prince Charles Hospital. Right atrium
was obtained from patients, less than 75 years of age undergoing coronary artery
bypass surgery and who were chronically administered a β-blocker. Responses to (-)-
noradrenaline and (-)-adrenaline through activation of β1- and β2-adrenoceptors
respectively, differ in patients chronically administered a β-blocker or not taking a β-
blocker (Molenaar et al., 2007). Some patients that present for coronary artery bypass
surgery do not receive chronic administration of β-blockers.
5.4.2 Acute Effects of S-(-) Esmolol on Human Right Atrium
S-(-) esmolol caused a concentration and time dependent reduction in contractile
force of electrically stimulated right atrial trabeculae. This is most likely due to
blockade of β1-adrenoceptors activated by small amounts of (-)-noradrenaline
released upon nerve stimulation. There was no difference in the duration of
contraction, suggesting no alteration in phosphorylation of phospholamban or
troponin I, key proteins associated with relaxation in human atrium (Molenaar et al.,
2007).
5.4.3 Lack of Agonist Activity of S-Esmolol on β1L-Adrenoceptors
The β1-adrenoceptor is activated by (-)-noradrenaline and inhibited by all clinically
used β-blockers. Some β-blockers, for example (-)-pindolol, also activate β1-
adrenoceptors at higher concentrations (~2-3 orders of magnitude) than those
required to block it (Kaumann & Molenaar, 2008) through activation of a low
Chapter 5: Enantiomers of Esmolol 123
affinity binding site, the β1L-adrenoceptor. Using conditions optimized to detect
agonist activity of β-blockers at β1L-adrenoceptors, in the presence of nadolol and
IBMX (Christ et al., 2011), S-(-) esmolol 100 µM had no agonist activity in human
atrial trabeculae. On the other hand, S-(-) esmolol 100 µM caused cardiodepression.
5.4.4 Affinity of S-(-) and R-(+) Esmolol at Human Atrial β1- and β2- Adrenoceptors
S-(-) esmolol was a competitive antagonist at β1-adrenoceptors with a pKB of 6.48.
An estimated pKB value at β2-adrenoceptors was determined to be 5.84. This
represents a β1/β2-adrenoceptor selectivity ratio of ~ 4.3. The enantiomers of esmolol
showed stereoselectivity for blockade of β1- and β2-adrenoceptors. S-(-) esmolol had
~80 fold higher affinity for β1-adrenoceptors than R-(+) esmolol. The
stereoselectivity requirements at β2-adrenoceptors were lower where S-(-) esmolol
had ~13-fold higher affinity for β2-adrenoceptors than R-(+) esmolol.
5.5 CONLCUSIONS AND FUTURE DIRECTIONS
These results agree with previous findings that the main activity of β-blockers resides
mostly in the activity of the S-(-) enantiomer versus the R-(+) enantiomer (Kaumann
& Lobnig, 1986; Joseph et al., 2003; Kaumann & Molenaar, 2008). Interestingly, and
consistent with previous studies on the racemates of pindolol (Walter et al., 1984),
the β2-adrenoceptor displayed much lower stereoselectivity requirements, which may
indicate a role of R-(+) esmolol in the mediation of inotropic and/or lusitropic effects
via the β2-adrenoceptor in vivo. This may be another reason behind adverse effects
seen from racemic mixtures β-blockers; rather than just a product of altered
pharmacokinetics such as metabolism or excretion, the R-(+) enantiomers may
Chapter 5: Enantiomers of Esmolol 124
actually be mediating unwanted effects through the β2-adrenoceptor due to its
apparently less rigid stereoselectivity.
Timolol is already available in certain countries as an enantiomerically pure mixture,
however all of the other clinically available β-blockers are still only marketed as
racemic mixtures (Mehvar & Brocks, 2001). The affinities and efficacies of the
individual racemates of clinically important β-blockers such as metoprolol, atenolol,
bisoprolol, bucindolol, and carvedilol should be investigated in order to determine
the possible clinical benefit of their enantiomerically pure preparations. The
determination of the affinity values for both S- and R- enantiomers of β-blockers at
cloned human β1- and β2-adrenoceptors is recommended for the confirmation of in
vitro efficacy findings, and is also recommended for the confirmation of the findings
of this study (Kaumann et al., 2007; Kaumann & Molenaar, 2008).
Chapter 6: Conclusions and Future Directions 125
6. Conclusions and Future Directions
6.1 ROLE OF PDE3 AND PDE4 ON CONTRACTILITY OF THE HUMAN HEART
Ischemic heart disease, which leads to heart failure, is still the largest killer of both
men and women in all first-world countries, despite decades of research and
corresponding clinical advancements (Australian Bureau of Statistics, 2012).
Approximately 30-50% of the deaths from patients with heart failure are due to
sudden death, most likely due to the development ventricular arrhythmias (Adamson
& Gilbert, 2006; Zipes et al., 2009), which arise as a result of remodeling, apoptosis,
and altered Ca2+ signaling from chronic β1- and β2-adrenoceptor activation (Packer et
al., 1998; Bristow, 2001). PDE3 and PDE4 inactivate cAMP, effectively inhibiting
its accumulation and the subsequent increases in Ca2+ release and positive inotropy
and lusitropy that result from local increases in cAMP levels (Omori & Kotera, 2007;
Lenhart & Marks, 2006; Levy, 2103).
This work was carried out in order to determine whether PDE3 and/or PDE4 mediate
inotropic and lusitropic effects through β1- and/or β2-adrenoceptors in the failing
heart, and whether or not chronic administration of metoprolol or carvedilol alters
their effects on contractility. Previous studies have investigated the roles of PDE3
and PDE4 in animal models, however their roles in the human heart was largely
unknown. While PDE3 inhibition has been shown previously to increase the
inotropic potency of isoprenaline in failing and non-failing (Von Der Leyen et al.,
1991) and dobutamine (Metra et al., 2002) in failing human hearts. However, the role
of β1- and/or β2-adrenoceptors was not investigated. Metra and colleagues (2002)
Chapter 6: Conclusions and Future Directions 126
investigated the effect of chronic administration of metoprolol and carvedilol on the
inotropic responses to the PDE3 inhibitor enoximone in vivo, and found that neither
β-blocker had any effect on the inotropic response to its administration (Metra et al.,
2002).
Even less is known about the possible role of PDE4 in human ventricular
myocardium, despite the downregulation of certain subfamilies of PDE4 in human
heart failure (Lehnart et al., 2005). This work was able to directly investigate the
roles of PDE3 and PDE4 in the failing human ventricle, utilising a long-standing
model of live in vitro trabecular setup to compare not only β-blocked to non-β-
blocked human tissues, but also to compare β1-selective to non β-selective blockade
on inotropic and lusitropic responses to catecholamines.
These findings revealed that PDE3, but not PDE4, helps to control β1-and β2-
adrenoceptor-mediated increases in inotropy and lusitropy in the failing human
ventricle. Metoprolol, a β1-adrenoceptor selective antagonist, moderately induced (3-
5-fold) control by PDE3 of the positive inotropy and lusitropy mediated by β2-
adrenoceptors. In contrast, cilostamide had markedly less ability to increase the
potency of (-)-noradrenaline for β1-adrenoceptor mediated inotropic and lusitropic
effects. Chronic administration of metoprolol increased the inotropic potency 4-fold
for (-)-noradrenaline via β1-adrenoceptors and 5-fold for (-)-adrenaline via β2-
adrenoceptors in the absence of PDE inhibition. The ability of metoprolol
administration to potentiate the effects of PDE3 inhibition may be attributed to a
variety of mechanisms: firstly, by blocking β1-adrenoceptors, metoprolol also
reduces the negative feedback mechanism that circulating catecholamines would
normally exert on PDE3 enzymes. With the suppression of PDE3 lifted, cAMP is
Chapter 6: Conclusions and Future Directions 127
able to accumulate, increasing PKA phosphorylation and ultimately increasing
inotropic and lusitropic responses to noradrenaline via β1-adrenoceptors, and
adrenaline via β2-adrenoceptors. Secondly, metoprolol may restore the activity and
expression of PDE3 in failing hearts, in which PDE3 expression is down-regulated.
Thirdly, metoprolol administration may stop the catecholamine-induced increased
Giα levels, allowing β2-adrenoceptors to bind more tightly to the stimulatory Gsα
pathway, again increasing inotropic and lusitropic responses to adrenaline. This is
likely why metoprolol induces the effects of adrenaline, acting through β2-
adrenoceptors, more than it does the effects of noradrenaline via β1-adrenoceptors.
Carvedilol, a non-selective β-adrenoceptor and α-adrenoceptor antagonist, strongly
induced (19-35 fold) control by PDE3 of the positive inotropic and lusitropic effects
mediated by β2-adrenoceptors, likely due to its higher affinity for β2-adrenoceptors
over β1-adrenoceptors. Carvedilol may have induced positive inotropy and lusitropy
through a variety of mechanisms. Firstly, carvedilol administration may reduce the
activity of Giα which is normally seen in the failing heart, allowing improved
coupling of the β2-adrenoceptors to the stimulatory Gsα pathway. In addition,
carvedilol may help to restore the spatial localization of the β2-adrenoceptors, which
has been shown to be de-localised from stimulatory pathway proteins in failing
murine hearts. This re-localization would allow more efficient cAMP metabolism by
PDE3, and therefore a greater control over inotropic and lusitropic responses to
catecholamines. Chronic administration of carvedilol did not alter the inotropic
potency of (-)-noradrenaline via β1-adrenoceptors, but decreased the inotropic
potency of (-)-adrenaline via β2-adrenoceptors 16-fold in the absence of PDE
inhibition.
Chapter 6: Conclusions and Future Directions 128
PDE4 did not control β1- or β2-adrenoceptor-mediated inotropic and lusitropic effects
in metoprolol- or carvedilol-treated patients, in contrast with previous animal heart
failure model studies.
Role of Carvedilol in the Prevention of Arrhythmias
Carvedilol has a 13-fold higher affinity for β2-adrenoceptors in human atrium, and
the residual blockade of β2-adrenoceptors after removal of tissue from patients
chronically administered carvedilol continues for longer than the residual β1-
adrenoceptor blockade (Molenaar et al., 2006). Carvedilol strongly induced PDE3’s
control of the inotropic and lusitropic effects mediated through β2-adrenoceptors,
plausibly by changing the feedback between PDE3 and β-adrenoceptor activity.
Chronic administration of carvedilol in heart failure patients induces PDE3 to
selectively control the positive inotropic and lusitropic effects mediated through
ventricular β2-adrenoceptors more than β1-adrenoceptors. This selective potentiation
of the inotropic and lusitropic responses of β2-adrenoceptors by PDE3 may have
occurred due to the ability of the β2-adrenoceptors to activate the Gs protein pathway,
which leads to local increases of cAMP levels, more efficiently than the β1-
adrenoceptors (Gille et al., 1985; Kaumann & Lemoine, 1987).
High-dose, chronic treatment with PDE3 inhibitors such as milrinone leads to
worsening of heart failure and increased mortality, particularly through sudden death
(Packer et al., 1991; Amsallem et al., 2005). As both β1- and β2-adrenoceptors
activate the stimulatory Gs pathway (Kaumann & Lemoine, 1987), they may also
both mediate arrhythmias in the human heart. Therefore, chronic administration with
Chapter 6: Conclusions and Future Directions 129
the β1-adrenoceptor selective metoprolol may actually facilitate the development of
arrhythmias in response to stress- or surgery-induced increases of adrenaline. The
ability of carvedilol to selectively block β2-adrenoceptor-mediated arrhythmias
(Molenaar et al., 2006) may be the reason behind its apparent survival benefit versus
metoprolol (Poole-Wilson et al., 2003), and may provide protection against β2-
adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor treated
patients. However, experimental evidence demonstrating β2-adrenoceptor-mediated
arrhythmias, facilitated by PDE3 inhibition, is still pending for human myocardium;
determining the role of PDE3 inhibition on the development of β2-adrenoceptor-
mediated arrhythmias in failing human hearts is the next step for these experiments.
Limitations of this Work
The data for these studies was collected from three different research sites: Oslo,
Dresden, and Brisbane lab teams have collaborated and pooled data in order to obtain
tissue numbers necessary for statistical power. While the Oslo team worked
primarily with left ventricular trabeculae (and was therefore not pooled with the
other lab’s data), the Dresden and Brisbane labs data were pooled together. While
patient statistics were similar across groups and collection/experimental/analytical
protocol was kept as identical as possible, the presence of multiple researchers may
always introduce an element of inconsistency.
This work also did not utilize chemical assays to investigate the expression of PDE3
or PDE4 in the trabeculae that were included. As certain subfamilies become up or
down regulated as a result of human heart failure, each heart may have had unique
levels of PDE 3 and 4 expression, which may also have had an effect on inotropic
Chapter 6: Conclusions and Future Directions 130
and lusitropic responses.
6.2 THE FUTURE OF β-BLOCKERS
6.2.1 Biased β-blockers
Biased ligands, or those with the ability to selectively activate a variety of signaling
pathways opens up an entirely new field of possible GPCR-based targets, where the
drug does not merely act as an ‘agonist’ or ‘antagonist’, simply activating or
blocking all of the targeted pathways of a specific GPCR, but is designed to be
selectively biased towards activating or inactivating a specific pathway in its
particular GPCR target’s repertoire (DeWire & Violin, 2011). Ligand-biased
targeting would, in theory, minimize unwanted side-effects. For example, having a
“β-blocker” which could block the effects of chronic β1-and β2-adrenoceptor
activation in the heart without having any risk of worsening concomitant disease
states such as asthma would be extremely useful.
Recent studies in mice have found another signaling pathway of the β1-adrenoceptor
through the β-arrestin–ERK1/2 pathway, which has anti-apoptotic and
cardioprotective properties in mice when selectively activated (DeWire & Violin,
2011). Biased ligands which selectively activate the ERK1/2 pathway of the β1-
adrenoceptor may prove to have more cardioprotective benefits than classical, non-
biased β1-adrenoceptor antagonists. The β2-adrenoceptor is also linked to this ‘non-
canonical’ ERK1/2 signaling pathway (Audet & Bouvier, 2008), and could also
potentially mediate cardioprotective effects in response to chronic adrenergic
activation in heart failure. Interestingly, carvedilol is the only clinically approved β-
blocker for heart failure that can activate the β-arrestin-ERK1/2 pathway via both β1-
Chapter 6: Conclusions and Future Directions 131
and β2-adrenoceptors (DeWire & Violin, 2011; Thanawala et al., 2014). This biased
activation of theoretically cardioprotective signaling pathways may explain the
increase in survival in carvedilol-treated patients seen in multiple studies (COMET,
2003; DiNicolantonio et al., 2013).
Yet another reason behind carvedilol’s apparent survival benefits, and furthermore a
possible explanation behind the ostensibly paradoxical benefits of adding a β-blocker
to an already β-adrenergic down-regulated and desensitized heart in a failing state, is
the theory of dynamic signaling put forth by Michel et al. (2014). They propose that
both chronic administration of drugs and progressive disease states further regulate
the efficacy profiles and signaling bias of ligands, which would explain why chronic
administration of β-blockers demonstrates clear therapeutic benefits, despite an
initial worsening of heart function at the beginning of therapy. It may also explain
why, over time, PDE3 inhibitors show a worsening of mortality, in contrast to their
clinical benefits upon acute administration (Packer et al., 1993).
The ligand-biased efficacy of the β-blockers is certainly a mechanism of action
worth investigating further, for both refining the use of the existing β-blockers, and
in the development of newer, possibly more strongly biased β-adrenoceptor targeting
ligands. Investigating the efficacy profiles of the existing β-blockers will help create
a link between signaling bias and the possible therapeutic indications for drugs with
pluridimensional signaling efficacies (Galandrin & Bouvier, 2006). In addition to
investigating the existing β-adrenoceptor modifiers, more strongly biased ligands
need to be designed so that signaling bias and its translation to clinical effects can be
further elucidated.
Chapter 6: Conclusions and Future Directions 132
6.2.2 Enantiomerically Pure β-Blocker Preparations
Yet another possible avenue for capitalizing on the β-blocker’s ability to increase
survival while potentially minimizing current unwanted side effects is to use
enantiomerically pure preparations. Previous studies have shown that the both the
affinities and the efficacies of the racemates of β-blockers can differ markedly in
vitro, (Kaumann & Lobnig, 1986; Mehvar & Brocks, 2001; Kaumann & Molenaar,
2008) and the findings with esmolol presented here confirm these pharmacodynamic
differences.
With new separation techniques available, it is now easier and more cost-effective to
prepare single racemate solutions than when β-blockers first became clinically
available (Wang et al., 2011; Tang et al., 2012). β-blockers commonly used for the
treatment of heart failure such as metoprolol, bisoprolol, and carvedilol should be
separated and tested in vitro to assess the potential for possible clinical benefits over
their mixed counterparts. In fact, S-metoprolol has already been released in India and
is still undergoing clinical trials; as the R- enantiomer displays higher β2-
adrenoceptor selectivity, investigators are hoping to see fewer adverse effects
associated with β2-adrenoceptor activation with the new S-metoprolol (Mehvar &
Brocks, 2001; Patil & Kuthekar, 2006). The findings from S-metoprolol along with
the FDA’s push for enantiomerically pure preparations will likely prompt many
researchers to investigate the potential benefits of purified β-blocker preparations
(Agustiana et al., 2010).
Chapter 6: Conclusions and Future Directions 133
6.3 THE FUTURE OF PHOSPHODIESTERASE INHIBITORS
Clinically, PDE inhibitors are already mainstays in the treatment of pulmonary
hypertension, erectile dysfunction, and chronic obstructive pulmonary disease
(Francis et al., 2011). However, their role in the treatment of heart failure remains
controversial. Despite the previously demonstrated increases in mortality rates, the
use of PDE3 inhibitors may still have a place in the future of long-term heart failure
therapy. Rather than chronic administration, PDE3 inhibitors may be used repeatedly
in patients, but as a discontinuation therapy where the PDE inhibitor is used only
during acute worsening of cardiac function, and, once restored, removed once more
from therapy (Amsallem et al., 2005). This treatment strategy has not yet been
assessed in clinical trials, and would be a logical next step in the research of heart
failure combination therapies.
The level of intricacy and specificity of the structures, functions, expression,
distribution, (on the tissue, cellular, and sub-cellular levels) signaling, and regulation
of each of the PDE3/4 subfamilies is one of the reasons they are still being
investigated; despite more than 50 years of information gleaned from hundreds of
studies, not much is known regarding how PDE3 and PDE4 mediate contractility in
the human heart. Each new discovery leads to yet more important questions, and
there is a vast area of knowledge yet to fill; in future investigations, these questions
need to be answered one subfamily at a time (Guellich et al., 2014). As total
inhibition of PDE3 and/or PDE4 has initially proven harmful, it is possible that
inhibiting a specific subfamily or a specific subcellular localization of PDEs may
prove beneficial in the treatment of heart failure.
Chapter 6: Conclusions and Future Directions 134
While PDE3A seems to control basal heart function in rat studies, PDE3B has been
suggested as a cardioprotective subtype against stress (Beca et al., 2013). The
worsening of heart failure in patients chronically treated with PDE3 inhibitors (such
as milrinone in Packer et al., 1993) might not have been a consequence of PDE3
inhibition in general, but of inhibition of PDE3B specifically; trials investigating the
effects of targeted PDE3A inhibition will be required to determine whether this is the
case. Firstly, however, the roles of PDE3A and PDE3B will need to be determined in
both healthy and failing human hearts, as Molenaar et al. (2000; 2007) have shown
that what is demonstrated in murine hearts is not necessarily able to be extrapolated
to the human heart. In addition, the “PDE3 inhibitor” milrinone actually has similar
selectivity for both PDE3 and PDE4 (Eschenhagen, 2013). Therefore, the increase in
mortality in patients who received long-term therapy with milrinone, demonstrated in
multiple studies including the large Packer et al. study in 1993, may actually be the
result of a combination of the inhibition of certain PDE4 subtypes in addition to
PDE3.
The highly specific recruitment of distinct PDE4D isoforms in mouse heart by the β2-
adreoceptor demonstrated by De Arcangelis et al. (2009) may be a further
explanation as to why PDE4 inhibition in this study did not result in changes of
inotropic or lusitropic response: certain subtypes may be recruited upon stimulation,
whereas others may control basal cAMP levels. In future studies, the specific
inhibition of distinct PDE4 subfamilies and/or isoforms may uncover a role of PDE4,
and therefore subtype-specific PDE4 inhibition, in the treatment of heart failure.
Chapter 6: Conclusions and Future Directions 135
While the results of the studies researching the effects of PDE3 and PDE4 in both
murine and human hearts may be contradictory, a common theme does emerge from
their collective data: firstly, that different subfamilies and isoforms of the PDE3 and
PDE4 enzymes have highly specific roles in the regulation of β-adrenoceptor-
mediated inotropic and lusitropic responses to catecholamines, which demonstrates
the need for subtype-specific PDE inhibition in order to effectively determine the
roles of these enzymes. Secondly, that results from animal hearts cannot simply be
extrapolated to the human heart, as the human myocardium has striking differences
in both the expression of PDE families and the roles they play in basic cardiac
function and response. Moving forward, the inhibition of specific subtypes of both
PDE3 and PDE4 need to be carried out in both healthy and failing human ventricle
and atria to determine their role (or lack thereof) in human cardiac contractility; once
this has been achieved, the place of PDE3 inhibitors in the management of heart
failure may be determined.
136
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APPENDIX A: British Journal of Pharmacology Publication 177
A. APPENDIX A: British Journal of Pharmacology Publication
APPENDIX B: Naunyn-Schmeideberg’s Archives of Pharmacology Publication 188
B. APPENDIX B: NAUNYN-SCHMEIDEBERG’S ARCHIVES OF PHARMACOLOGY PUBLICATION