catecholamines and basal metabolism the myocardium€¦ · catecholamines and basal metaboh in the...
TRANSCRIPT
CATECHOLAMINES AND BASAL METABOLISM IN THE MYOCARDIUM
by
Sonya A. Baik
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology
University of Toronto
O Copyright by Sonya A. Baik 1998
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ABSTRACT
Catecholamines and Basal M e t a b o h in the Myocanliurn
Sonya A. Baik, M.Sc.. 1998
Graduate Department of Physiology
University of Toronto
Blood-based cardioplegia used to arrest the heart for cardiac surgery has elevated
levels of endogenous catecholamines. However, rnyocardial exposure to catecholarnines
in cardioplegia may increase basal metabolism, or oxygen consumption (MVO?). in the
arrested myocardium and contribute to poor post-ischemic functional recovery. Isolated
rabbit hearts (n=5) were perfusion-arrested at 37OC with 20 m M K+ Krebs-Henseleit
buffer and exposed to 25 nM isoproterenol (Iso), which resulted in a significant increase
in basal MVOz (0.054I0.006 to 0.088M.009 mL 02/rnin/g DW: p=0.0009). Hearts
exposed to 25 nM Iso in cardioplegia followed by 60 min of normothermic t 37°C)
ischernia and then reperfusion, demonstrated a significant increase in diastolic pressure
compared to control (-2 1 - 8 s .O mmHg vs. - l2.5fl.O r n d g : p=0.02). Hypothermia
(20°C) during ischernia prevented the deleterious effects of Iso. Furthemore. çxposure
of the beating rnyocardium to catecholarnines prior to arrest was shown to be deleterious
to post-ischemic functional recovery of systolic, diastolic, and developed pressures.
which was again prevented by hypothermia during ischernia (pe0.01). End-reperfusion
myocardial ADP, ATP, and total adenine nucleotide concentrations were significantly
(p10.0002) better preserved in the hypotherrnia group.
I thank Dr. Ivan Rebeyka, my primary supervisor, very much for al1 the invaluable
guidance, support, and insight he provided, which began even before my graduate
studies. 1 also thank Dr. Carin Wittnich very much for providing much guidance and
counsel as my CO-supervisor. The time and patience of my supervisors have been greatly
appreciated. 1 thank Dr. Peter Backx, my comrnittee member. for his invaluable help as
weli. 1 thank Dr. Uwe Ackemiann for taking the time to act as the chair of the defense. as
well as for sharing his humour and interesting assortment of jokes.
A big thanks to al1 the members of the laboratories of my program committee.
From Dr. Rebeykars lab, the support of Andrea Konig, Iill Waddell. and especidiy Dr-
Yoshi Saiki, was tremendous. 1 greatly appreciated al1 the time and advice provided by
the members of Dr. Wittnich's laboratory (Karim Bandeli, Mike Belanger. Cathy
Boscarino, Shona Torrance, and Jack Wailen) as well as by members of Dr. Backx's
Iaboratory . Finally, thanks to Claire Coulber, Joan Jowlabar, and Susy Taylor at the Division
of Cardiovascular Research at the Hospital for Sick Children for their constant support
and help.
TABLE OF CONTENTS
LIST OF FIGURES AND TABLES ............................................................... viii
1 INTRODUCTION 1
Overview of Cardiac Muscle Physiology .......... .. ............. ..... ........................... 1 3 Cardiac Action Potential ............................................... - ..............-.- .. .................... -
Mechanism of Excitation-Contraction Coupling ................... ......... ..--.-... ............. 4
Catecholamines and Beta-Adrenergic Receptor Stimulation ................................ 4
Excessive Catecholamine Stimulation and Myocardial Ischemia ......................... 6
Catecholamine Damage and Ischemic Damage.. . .. . ... . . . .. .. . . .. . ... . . . . . . . . . . . -. . -. . . . . . . . . . . 8
Mitigation of the Effects of Catecholamine Cardiotoxicity and Isc hemic Damage ... .. . .. . . . .. . .. . .. .. .. .. . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0
Excessive Catecholamine Stimulation of the Arrested Myocardium .................. 1 1
Catecholarnines and Myocardial Oxygen Consumption ................ ... ..... ..... . . . . 12
Basal Myocardid Oxygen Consumption ....................................................... 12
Cardioplegic Arrest Using Hyperkalemia ....,. . . . .... . .. .... .. .. . . - . . .. . . . . . . . . . . . . . . 1 3
Measurement of Basal Myocardial Oxygen Consumption .............................. 14
Catecholamines and Basal Myocardial Oxygen Consumption ........................... 18
Possible Role for Ca'+ in Mediating Catecholamine Effect on Basal MV0' ............... .. .............. .... ........................................................ 10
A Possible Link Between the Effect of Catecholamines on Basal Metabolism and Myocardial Functional Recovery Following Peri- Ischemic Excessive Catecholamine Stimulation: Formulation of
33 Hypotheses. .. .. .. .. ... . . . .... . . . . .. . ... . . . . . . . . . . . . . . . . . .. . .. . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -- i) Inhibition of Catecholamine Effect on Basal MVO .......................... 33
Beta-Antagonism of Catecholamine Effect on Basal MVO? ..................... .... ................................... 23
Esmolol, A Short-Acting Beta-Adrenoceptor Antagonist ...... ... 23
Ca2+ Modulation of Catecholamine Effect on Basal MV02 .................................................................... 25
Effects of BDM on Cardiac Muscle ............ .... .... ....... . .,... . . 26
ii) Catecholamine Effect . . on Basal MVOz Under ....................................................................... Ischemic Conditions 27
Protective Effect of Hypothermia ............................................... 28
The Hypotheses ................................................................................................. 30 ..................................................................................... Overview of Experiments 30
2 MATERIALS AND METHODS 33
2.1 Preparation of Perfusates ..................................................................... 33
............................................................. 2.2 Langendorff Perfusion S ystem 35
2.3 Basal Metabolism Under Varying Arrest Conditions .......................... 37
2.3.1 Heart Preparation ............................................................. 37
2.3 2 Experimental Protocol ........................................................ 37
2.3.3 Measurement of Oxygen Consumption ................... .. ..... 38
2.3.4 Dosage Studies ................................................................... 39
2.4 Peri-Ischernic Catecholarnine S timdation and Myocardial Functional Recovery ...................................................... 39
2.4.1 Heart Preparation ................................................................ 39
..................................... 2.4.2 Experimental Protocol .......... .... 40
2.4.2a Post-Ischemic Func tional Recovery Follow ing Catecholamine Stimulation of Arrested Myocardium ................................................. U)
2.4.2b Post-Ischemic Functional Recovery FoIIowing Pre-Ischemic Catecholamine Stimulation of
........................... Beating Myocardium ........... .. .... .. 43
2.4.3 Evaluation of Myocardial Function .................................... 43
2 -4.4 HPLC Analysis For Post-Isc hemic Myocardial Adenine Nucleotide Concentrations .......................... 44
2.5 S tatistical Andysis ............................................................................... 45
......... 2.5.1 Basal Metabolism Under Varying Arrest Conditions 42
2.5.2 Peri-Ischemic Catecholamine Stimulation and .................................... Myocardial Functional Recovery 47
3 RESULTS 48
3.1 Basal Metaboiism Under Varying Arrest Conditions ........................... 48
3.1.1 Perfusion Arrest with Catecholamine Stimulation ............. 48
3.1.2 Perfusion Arrest with Catecholarnine Stimulation .................................. and Beta-Adrenoceptor Antagonist 48
3.1.3 Perfusion Arrest with Catecholamine Stimulation and Ca'+-Modulator ...................................................... 52
.............. 3.1.4 Summary of Results for Basal Metabolism Study 54
3.2 Peri-Ischemic Catecholamine S tirnulation and ...................................................... Myocardial Functiond Recovery 55
3.2.1 Post-Ischemic Functional Recovery Following Catecholamine Stimulation of
............................ .............. Arrested Myocardium .... 5 5
....................... .......*... 3.2. l a Normothermic Ischernia Study ..... 55
i) Change in Systolic Pressure After ............................................ Normothermic Isc hemia.. 5 5
ii) Change in Diastolic Pressure After ............................................ Normothermic Isc hemia. 56
iii) Change in Developed Pressure After ............................................ Normothermic Isc hernia 57
iv) End-ReperFusion Myocardial Adenine Nucleotide Concentrations After Normothermic Ischemia ............................................ 58
.......................................... 3.2.1 b Hypothennic Ischemia Study 59
i) Change in Systolic Pressure After Hypothermie Ischemia .................. .. ..................... 59
ii) Change in Diastolic Pressure After Hypothermic Ischemia.. .................................... 59
iii) Change in Developed Pressure After Hypothermie Ischemia .............................................. 60
iv) End-Reperfusion Myocardial Adenine Nucleotide Concentrations After Hypothermie Ischemia .......................................... 60
3.2.2 Post-Ischemic Functional Recovery Following Pre-Ischemic Catecholamine Stimulation of
..................................................... Beating Myocardium.. 62
3.2.2a Ischernia Temperature Effect on Change in ........................................................... Systolic Pressure 62
3.2.2b Ischemia Temperature Effect on Change in Diastolic Pressure ......................................................... 63
3.2 .2~ Ischemia Temperature Effect on Change in Developed Pressure .............. ....... ............................ 64
3.2.2d Ischernia Temperature Effect on End-Reperfusion Myocardid Adenine Nucleotide Concentrations.. ..... ... 64
3.2.3 Ischemia Temperature Effect on Change in Diastolic Pressure in Control and Cp-Iso Groups ............ 65
3.2.4 Surnmary of Ischemia Smdy Results .................................. 66
4.1 Basal Metabolism Under Varying Arrest Conditions ........................... 68
4.1.1 Reasons for not Using Epinephrine as the Catecholamine ................................................. 68
4.1.2 Catecholamine Stimulation of Arrested Myocardiurn. .. . ... . 73
4.2 Peri-Ischemic Catecholamine Stimulation and Myocardial Functional Recovery .................................................... 77
4.3 Reversal of Catecholamine Effect on Basal MVO ............................... 81
8 APPENDICES 90
8.1 Appendix A: Effect of Epinephrine on MVO, .......... ............ .. ....... .. 90
8.2 Appendix B: Effect of 25 nM Isoproterenol on Beating Heart MVO, ................................................................... 93
8.3 Appendix C: Effect of EsmoloI on Beating Heart MVO? .................... 94
8.4 Appendix D: Coronary Flow and Heart Weight Data ......................... 96
vii
LIST OF FIGURES AND TABLES
FIGURES
Figure 2.1 Figure 2.2
Figure 2.3
Figure 2.4 Figure 3.1
Figure 3.2 Figure 3.3 Figure 3.4
Figure 3.5 Figure 3.6
Figure 3.7 Figure 3.8 Figure 3.9
Figure 3.10
Figure 3.11
Figure 3.12 Figure 3.13
Figure 3.14
Figure 3.15
Figure 3.16
Figure 3.17
Figure 3.18
Figure A.l
Figure A 3 Figure A 3
Langendorff perfusion system ............................................................. 36
Post-ischernic hinctiond recovery following catecholarnine stimulation of arrested myocardium ........................................... 41
Ischemia temperature effect on post-ischemic functionai recovery following pre-ischemic catecholamine stimulation of beating myocardium ...................................................................... 12
HPLC sample trace of end-reperfusion myocardiai tissue ................... 16 ..................................... Effect of 25 nM isoproterenol on basal MVOZ 49
Effect of 2 mg/L esmolol on basal MV02 ...................... .............. . . . 50
Effect of esmolol on basal MVO, in the presence of isoproterenol ..... 51
Effect of 30 m M BDM on basal MV02 ............................................... 53
Effect of BDM on basal MVOz in the presence of isoproterenol ......... 54
Post-normothermic ischemia change (A) in systolic pressure .............. 56
Post-normothermic ischemia change (A) in diastolic pressure ............ 57
Post-nonnothermic ischemia change (A) in developed pressure ......... 57
Post-normothennic ischemia myocardiai adenine nucleotide .................................................................................... concentrations 58
Post-hypothermie ischemia change (A) in systolic pressure ................. 59
Post-hypothermic ischemia change (A) in diastolic pressure ............... 60
Post-hypothermie ischemia change (A) in developed pressure ............ 61
Post-hypothermic ischemia myocardial adenine nucleotide concentrations .................................................................................... 61
Effect of ischemia temperature on change (A) in ................................................................................. systolic pressure 63
Effect of ischemia temperature on change (A) in ............................................................................... diastolic pressure 63
Effect of ischemia temperature on change (A) in ............................................................................. developed pressure 6.1
Ischemia temperature effect on end-reperfusion myocardial adenine nucleotide concentrations .............................. 65
Effect of ischemic temperature on post-ischemic change (A) in didiaslic pressure following catecholamine stimulation during arrest ....................................... 66
....................................... Effect of 2 n M epinephrine on basal MVO , 90
............... Effect of 2 nM epinephrine on beating, non-working MVO, 91
Effect of 25 nM epinephnne on beating. non-working MVO? ............. 91
Figure A.4 Effect of 50 nM epinephrine on beating. non-working MVOZ ............. 92
Figure A 5 Effect of 25 nM epinephnne and 1 ph4 prazosin on beating . non-working MVO. ................................. ... ....................................... 92
........... Figure B.l Effect of 25 nM isoproterenol on beating. non-working MVO 93
.................. Figure C.1 Effect of 2 mg/L esrnolol on beating. non-working MVO, 94
................ Figure C.2 Effect of 10 mgfL esmolol on beating. non-working MV02 95
.............. Figure C.3 Effect of 100 m& esmolol on beating. non-working M V 0 2 95
Table I Beating and arrested state myocardial oxygen consumption (MVO. ) values from the literature ..................................................... 15
Table II Experimental protocol for basal MVO. studies .................................... 37
Table III ................................................................ Pre-isc hemic baseline values 55 ................................................................ Table IV Pre-ischernic baseline values 62
...................... Table V(a) Coronary flow and heart weight data for MVOz study .. 96
Table V(b) Coronary fiow and heart weight data for MVO? study ........................ 97
LIST OF ABBREVIATIONS
ADP AMP ANOVA
AP
atm
ATP
ATPase
BDM OC
Cal+ CAMP C1-
cm
CO
coi, Cp-Iso
CRC
d DevP
DP DW
Ed EK E m Epi
Esm
g HzO HPLC
I K ~
IKS IK 1
If* 1
402
adenosine diphosphate
adenosine monophosphate
analysis of variance
action poten fiai
atmosphere
adenosine triphosphate
adenosine triphosphatase
2,3-butanedione 2-monoxime
degree Celsius
calcium ion
cyclic adenosine monophosphate
chloride ion
centimetre
cardiac output
carbon dioxide
treatment with 25 nM Iso in cardioplegiu
Ca" releease channel
da y
developed pressure
diastoiic pressure
dry weight
equilibrium potential of Cl- equilibrium potential of K+ membrane potential
epinephrine
esmolol
gram
water
high performance Iiquid chromatography
rapidly activated component of delayed rectifier K+ curren t
d o wly activated component of delayed rectifier K+ curren t
inward rectifier K+ current
Ca'+-dependent transient outward K+ current
voltage-dependent transient outward K+ current
i.m. i.v-
Iso
K K+
nm
nM
O2
PFK
Pg pi PK Prz
rE'm SEM SP SR TAN Tn-C
Tn-1 Tn-T
u
intravenous
isoproterenol
20 mM K+KHB solution (cardioplegia)
potassium ion
kilogram
Krebs-Henseleit bufler
litre
mil ligram
minute
millilitre
millimoleL
millimetre of mercury
millivolt
myocardial oxygen consurnption
sodium ion
n icorinam ide dinucleotide (oxidized)
nicotinamide dinucleo tide ( reduced)
nanometre
nanomole/Z
inorganic phosphate
protein kinase
p razosin
revolutions per minute
standard error of the mean
systoiic pressure
sarcoplasmic reticulum
total adenine nucleotides
Troponin C Troponin I
Troponin T unit
micro litre
micrometre
micrornoleL
wet weight
1 INTRODUCTION
Oventiew of Curdiac Muscle Physiology
Cardiac muscle is comprised of thick filaments, made of myosin. and thin
filaments, made of actin ~ a m e l l et al., 1990; Katz, 19921. Myosin molecules comprising
the thick filaments have a long, rod-like tail on one end and two globular heads at the other
end. There are two flexible joints, called hinges, dong the junction of the head and tail
regions. Within the myosin filament, the myosin molecules are arranged laterally and anti-
paraliel to each other, with the globular heads projecting out from the thick filament at
regular intervals. The actin-stimulated ATP-ase activity is contained in the myosin heads
which project from the thick filaments to form cross-bridges with adjacent actin thin
filaments Parnell et al., 1990; Katz, 19921.
The actin thin filament is cornposed of a double helix of F-actin which is
polymerized globular actin (G-actin) [DarneIl et al., 1990; Katz, 19923. Associated w ith
the actin are four proteins which mediate the regulatory role of C s + in contraction.
Filamentous tropomyosin molecules lie in one groove of the actin double helix and have
specific binding sites on the actin filament. Three troponin proteins cailed uoponin T (Tn-
T), uoponin 1 (Tn-1), and troponin C (Tn-C), are bound to specific sites on each
tropomyosin molecule. In the absence of Ca'+, troponin and tropomyosin inhibit rnyosin
ATPase activity by inhibiting the interaction of the myosin heads with actin. Tn-I and
tropomyosin, together, cause a conformational change in the actin. such that weak binding
with myosin heads occurs, thereby preventing the myosin ATPase activity. When Tn-C
binds Ca'+, the myosin heads bind to the exposed actin, thus activating myosin ATPase
[Darnell et al., 1990; Katz, 19921.
Each cardiac muscle fibre is surrounded by rnitochondria which are used for
generation of ATP [Dameil et al., 1990: Katz, 19921. The myosin-catalysis of ATP to
ADP and Pi acts as the fuel for muscle contraction. The first step in the cyclic process of
muscle contraction c m be considered to be the time when ATP binding to myosin weakens
binding of myosin heads to actin, thereby relaxing the muscle fibres [Darnell et al.. 19901.
The hydrolysis of myosin-bound ATP occurs with little change in free energy as the ADP
and Pi hydrolysis products remain bound to myosin. producing an "energized" state. The
release of these bound products from myosin are strongly exergonic steps. however. and
the free energy released is used to power the pivoting movement of the myosin head. The
myosin pivots via its hinges to be perpendicular to the actin filament. and then binds to the
adjacent actin if the intemal Cal+ concentration is high enough and. concomitantly. P, is
released. Once attached to actin, and with the release of ADP. the rnyosin head pivots
again on its hinge, thereby moving the actin filament relative to the fixed myosin and
resulting in contraction. The product of this contraction step is called the "rigor cornplex"
because the actin-myosin Linkage is inflexible and the two filaments cannot move past each
other. Subsequent binding of ATP to the rnyosin head releases the rnyosin head from the
actin, relaxing the muscle and reîomrnencing the cycle of contraction. Thus. as long as
the intracellular CP+ concentration is suffciently high and ATP is present. the myosin-
action cross-bridges will cycle continuously and the muscle will contract [DarneIl el d..
1990; Katz, 19921.
Cardiac Action Potentiul
The high intracellular CaZ+ concentration necessary for contraction is achieved
through the cardiac action potential (AP) [Damell et al., 1990; Katz. 1992: Weiss. 19971.
Briefly, using the Purkinje fibre ce11 as an example, Phase O is the AP upstroke. which is
carried predominantly by the Na+ current via voltage-dependent Na+ channels. The inward
Na+ current must be large enough to depolarize the adjacent cells to the threshold of their
Na+ channels (-60 mV), ensuring rapid propagation of the cardiac impulse [Katz. 19921.
Rapid inactivation ensues to minimize unnecessary Na+ influx because the removal of Na+
depends primarily on the energy-dependent Na+K+-ATPase pump. Furthemore. when
depolarization reaches the Ca2+ current threshold (-35 mV), the voltage-dependent L-type
Ca" channels open to generate a second depolarking current [Katz. 19921. After the AP
reaches its peak amplitude in Phase O (+30 mV), activation of the Ca2+-dependent ( I,,, ) and
voltage-dependent (Im2) components of the transient outward K+ current occur. This is
Phase 1, or early rapid repolarization [Katz, 1992; Weiss. 19971.
Slow repolarization, however, follows in Phase 2 because the Ca?+ current activates
and inactivates more slowly than the Na+ current, and thus maintains the membrane in a
depolarized state Weiss, 19971. This effect creates the plateau in the profile of the cardix
action potential. Early in the plateau phase. the slow inward Ca'+ current is high but.
toward the end of the plateau, as the Cazc channels inactivate, the delayed rectifier currents
open and are largely responsible for repolarkation. The AP plateau of Phase 2 is therefore
determined by inactivation of the L-type Ca2+ current and activation of delayed rectifier K+
currents. 1, and Ib. 1, activates rapidly and also inactivates. whereas 1, activates slowly
with depolarization and does not inactivate during rnaintained depolarization. The delayed
rectifier currents determine the duration of Phase 2 and contribute to Phase 3 [Weiss.
19971.
Towards the end of the plateau phase, the rate of repohrization accelerates.
primady due to a rapid increase in 1, and IK, [Weiss, 19971. As the membrane potential
further repolarizes duhg Phase 3, or late rapid repolarization, outward current through I,,
and IKI increases under positive feedback. I,, controls the initial onset of the rapid
depolarization phase and IK, controls the latter portion. I,, is the background strong inward
rectifier K+ current which stabilizes the resting membrane potential near EK due to increased
membrane conductance when E, is near EK. I,, is inactivated during depolarization and
prevents hyperpolarization by currents associated with the Na+/K+-ATPase pump [Weiss.
19973.
Following rapid repolarization to the maximum diastolic potential (-90 mV in
hirkinje cells), two time-dependent currents change to contribute to the 1, , -maintained
Phase 4, or diastolic depolarization [Katz, 1992; Weiss, 19971. An inward Na+lCa2+
exchange current progressively decays as the intracellular Caf+ concentration declines.
promoting hyperpolarization. In opposition to this is the gradua1 decay of the delayed
rectifier K+ current, which promotes depolarization [Weiss, 19971.
Mechanism of ficitution- Contraction Coupling
The slow inward Ca2+ current of Phase 2 is not, in itseif, sufficient to generate
contraction throughout the heart. Rather, the Cal+ influx through the plasma membrane
induces an even greater release of Cal+ stored intracellularly in the sarcoplasrnic reticulum
(SR), which serves as a reservoir of Ca'+ ions sequestered from the ce11 cytosol and
myofibrils [Darnell et al., 19901. Structural evidence suggests a direct interaction between
the Ca2+-release channels (CRCs) of the SR and the L-type CaLc channels located in
invaginations, cailed T-tubules, of the plasma membrane [Franzini-Armstrong, 1 9801. The
physical interaction between these two types of Gaz+ c h a ~ e l s is believed to account for the
mechanism of excitation-contraction coupling [Rios and Bmm, 19871. Depolarization of
the plasma membrane induces a conformational change in the voltagedependent L-type
Ca2+ channels which is transrnitted to the CRCs. thereby triggering "Ca=+-induced Ca2+
release" from the SR [Rios and Brurn, 1987; Darnell et al., 19901.
Carecholamines and Beta-Adrenergic Receptor Stimulation
Catecholamines, or adrenergic agonists, play an integral role in the regulation of
myocardiai contractility and metabolism. They elicit their effects through alpha-receptors.
which mediate a vasoconstrictive response, and through beta-receptors. which mediate a
vasodilatory and cardiomyocyte stimulatory response. Although the coronary vvascular bed
is predominantly characterized by alpha-recepton, the myocardiurn is predominantly a beta-
adrenergic receptor tissue [Katz, 1992; Lindemann and Watanabe, 19951. In fact. in most
mammalian species such as humans Brodde et al., 19861, dogs [Manalan et al.. 198 11.
cats [Kaumann and Lemoine, 19851, and rabbits [Tenner et al.. 19891. cardiac beta-
adrenergic receptors are of the beta,-receptor subtype [Bilezikian. 1987: Hie ble and
Ruffolo, 199 11. The subcellular events following beta-adrenergic receptor stimulation are
mediated by cyclic AMP (CAMP) which initiates a CAMP-dependent protein kinase (PK)
signalling cascade wobishaw and Foster, 19891. A number of sarcolemmal ion channels
appear to be regulated by the phosphorylation activity of CAMP-dependent PK. These
include the K+ (slowly activating component of delayed rectifier) [Walsh and Kass. 19881.
Na+ [Schubert et al., 1990; Matsuda et al., 19921, Cl- [Harvey et al.. 19901 ion channels.
and most notably, the Ca2+ ion channel vrautwein and Hescheler, 19901. Phosphorylation
of the sarcolemmal Cal+ channel increases the number of open channels at a given moment.
thereby increasing the rate of net Cal+ influx and inward currents [Akera. 19901. Direct
coupling between beta-adrenergic receptor-associated guanine nucleotide stimulatory
protein and Na+ [Schubert et al., 19891, K+ [Yatani et al.. 19871, and Cal+ [Yatani et rd. .
198ïb, but refuted by Hartzeli et aL, 199 11 channels have also been reported.
Cyclic AMP-dependent PK also phosphorylates the SR membrane protein
phospholamban which upregulates Ca2+ sequestration by the SR Ca2+-ATPase pumps
[Kirchberger and Tada, 1976; Lhidemann et al., 19831. B y significantly increasing Ca2-
uptake, catecholamines can increase the Ca'+ content of the SR, which can lead to greater
Ca2+ release from the SR and increased activation of the contractile proteins [Hess et (11..
1968: Shineboume et al., 1969; Shineboume and White, 19701.
Another target for CAMP-dependent PK-mediated phosphorylation is the contractile
regulatory protein troponin 1 which appears to decrease the Ca'+ sensitivity of the
actomyosin ATPase activity due to an increased rate of Cal+ dissociation from the Ca2--
specific binding site on troponin C [Robertson et al., 19821. As a result of this decreased
Ca2+ sensitivity as weil as the increased Ca'+ uptake by the SR, the contractile proteins gain
an increased ability to relax [Shineboume and White, 19701. Moreover, the decreased CaL+
sensitivity suggests that more of this ion must be released to achieve a given increase in
force, thereby demanding more Caf+ to be cycled with each beat [Chiu et rd.. 19891.
Consequently, with the increased intracellular Ca2+ levels and the increased rate of Ca2+
cycling, catecholamines appear to be able to influence the myofilament cross-bridge cycle
by increasing the number of cross-bridges activated per unit time [Hasenfuss et al.. 19941.
and by decreasing the average force-tirne integral of the individual cross-bridge cycle due to
a decrease in the attachment time and an increase in cycling rate [Hoh et (11.. 1988:
Hasenfuss et al., 19941. Therefore, the increased Ca'+ cycling. the stimulation of
contractile protein interaction, and the enhanced relaxant effects mediated by beta-
adrenergic stimulation can account for the positive inotropic and chronotropic effects of
catecholamines.
Excessive Catecholamine Stimulation and Myocardial Ischemia
Normdy, catecholamine stimulation is important for the upregulation of rnyocardial
hnction under conditions of increased stress, such as exercise or trauma, to meet the
increased circulatory dernands. However, in some situations, the very intensity of the
catecholamine response may unwittingly produce a harmful result. In fact. excessive
release or administration of catecholamines has k e n associated with cardiotoxicity [Rona et
al., 1959; Rona et al., 1975; Mosinger et al., 1977; Mosinger et al.. 1978: Yeager and
Iams, 198 1 ; Steen et al,, 1982; Muntz et al., 1984; Noronha et al., 1984; Noronha et al..
19851. Caspi et al. [ 19931 demonstrated that excessive epinephrine administration to
piglets yielded rupnired sarcolemrna, mitochondnai sweiling. and intramitochondrid dense
granules, confiirming earlier reports that the charactenstic lesion of catecholarnine injury
was an exaggerated contraction and contraction band formation with mitochondrial granular
densities ploorn and Canciiia, 1969; Csapo et al., 19721. Furthemore, Rona et al. [1959]
discovered that the synthetic catecholamine isoproterenol can produce "infarct-like"
rnyocardial necrosis in expenmental animals. where the higher the dose, the more severe
the injury [Chappel et al., 19591.
It has k e n suggested from some studies that the cellular damage resulting from
excess catecholamine stimulation is due to myocardial Ca2+ overload secondary to increased
sarcolemmal permeability [Rona et al., 19751, whilst others have demonstrated that
myocardial Cal+ overload is crucial in the pathogenesis of catecholamine-induced
myocardial necrosis [Bloom and Davis, 1972; Reckenstein. 19731. Fleckenstein [ 19731
documented that isoproterenol administration is followed by increased transport of Ca2+
into the cardiomyocyte. Upon fùrther investigation, Fleckenstein [1983] concluded that the
increased myocardial Ca2+ content causes myofilament overstimulation. resulting in
increased force and oxygen demand. and deleterious breakdown of high energy phosphate
fractions. The exhaustion of high energy phosphates was proposed to determine the
catecholamine-induced myocardial necrosis that is not due to isc hemia [Flecke nstei n.
19831. In addition, Ca2+-pump mechanisms at the sarcoIemmal and sarcoplasmic reticulu
membranes are altered in hearts treated with high doses of catecholamines [Dhalla et d..
1983: Makino et al., 1985; Panagia et al.. 19851, suggesting a possible contribution to the
elevation of the intracelIu1a.r Ca" concentration through membrane leakage.
Yet, catechoiamine-induced ce11 damage is not limited to the non-ischernic model.
Catecholamines appear to exacerbate the darnaging effects of ischemia by accelerating
injury in ischemic or hypoxic tissue [Maroko et al.. 197 1; Maroko et al., 1973: Vatner et
al., 1973; Karlsberg et al., 1979; Muntz, et al., 1984; Yoshida and Iimura. 19893. In
isolated rat hearts, Waldenstrom et al. [ 19781 found that norepinephrine facilitated the
spread of ischemic necrosis, while others have found that after 2040 min of ischemia. the
isc hemia itself induces norepinephrine release from my ocardial nerve terminais [Sc homig e r
al., 19841. This release can reach the micromolar range. which is 100- 1000 times the
normal plasma concentration and enough to promote tissue injury [Muntz et al.. 1984:
Schomig et al., 19841. Thus, in a vicious cycle, the damaging effect of catecholamines on
ischemic myocardiurn can worsen with M e r catecholamine stimulation
Catecholamote Damuge and Ischemic Damage
The ultimate damage effected by excessive catecholamine stimulation mirnics the
damage mediated by ischemia. Thus, an understanding of the ischemic process may shed
some understanding of the catecholamine-induced myocardial lesion. The two processes.
however. are no: mutually exclusive since prolonged ischemia is characterized by an
increase in endogenous catecholamines [Muntz et al., 1984; Schomig et al.. 19841. In
ischemia, lack or cessation of coronary blood flow and the consequent lack of oxygen that
the myocardium experiences induce a cascade of events affecting the energetic. metabolic.
and morphologic aspects of the myocardium. Immediately foilowing the onset of global
ischemia, the heart's contractile activity is greatly reduced [Jennings et al.. 19691. Lack of
coronary artery perfusion foilowed by metabolic dysfunction. contribute to such functiond
detenoration [Hearse. 1979; Katz, 19921. The available oxygen that is dissolved in the
cytoplasm of the myocytes is expended within the first few seconds after ischemic onset.
upon which anaerobic conditions develop within the cell [Katz. 19921.
Anaerobic glycolysis replaces aerobic glycolysis as the main source of energy for
the globally ischemic heart [Katz, 19921. Both humoral and biochemical rnechanisms are
involved in the transient acceleration of anaerobic ATP production. GIycolysis is
acceierated as an increased level of ADP allosterically stimulates the rate-limiting
phosphofructokinase (PFK) reaction [Darnell et al., 19901. Eventually. however.
glycolysis is inhibited as the accumulation of reduced NADH and the lack of oxidized
NAD+ inhibit PFK and the glyceraldehyde-3-phosphate reduction steps of glycolysis. As
lack of oxygen prevents pymvate entiy into the Kreb's cycle, the pyruvate build-up leads to
an accumulation of lactate which graduaiiy contributes to acidosis and inhibition of several
glycolytic enzymes, including PFK [Darnell et al., 19901. Thus. anaerobic glycolysis is
only a temporary energy source. Furthemore, this anaerobic metabolic pathway has a very
low energy yield of only two moles, versus 36 for the aerobic pathway, of ATP per
glucose Parnell et al., 19901. Consequently, an energy deficit is generated as the ischemic
stress continues and as the heart is unable to meet energy demands satisfactorily by a means
other than oxidative phosphorylation.
Under ischernia, the demand for energy exceeds supply and ATP is broken down
evennidly to adenosine which cm freely difise out of the ce11 unlike its phosphorylated
moieties [Wiedrneier et al., 1972; Snow et al., 19731. Such a loss may contribute to a
delay in the repletion of high energy phosphates which eventually may affect functionai
recovery. In fact, the decline in ATP levels greatly affects the contractility of the
myocardium [Kubler and Spieckermann. 197 1 ; Kubler and Katz. 1977: Hearse. 19791.
suggesting a criticd role in contractile dysfunction. in the initial stages of ischemia. the
activation of ATP-dependent sarcolemrnal and intraceilular Ca'+-channels that normally
accelerate Ca2+ e n 0 into the cell becomes attenuated, thereby contributing to the negative
inotropic effect of ischemia [Katz, 19921. Conversely, the ATP-dependent sarcolemmal
and SR Car+-pumps which are responsible for the efficient efflux of Ca2+ from the cytosol
are inhibited, resulting in impaired relaxation. The importance of ATP to ce11 viability is
compounded by the fact that ATP confen dosteric effects on the ion pumps. channels. and
actin-myosin interactions which regulate contraction and relaxation [Katz. 19971. Thus.
attenuation of the modulatory role of ATP in Ca'+ movement could even reduce contractility
and relaxation without having ATP levels necessarily undergoing severe depletion [Kubler
and Katz, 19771.
The balance among the Gaz+ fluxes that relax the heart and those that initiate systole
is precarious and favours contraction [Katz, 19921 since activation is less energy-requiring
than relaxation. Thus, as relaxation becornes compromised, contracture sets in. Actin-
myosin rigor complexes form, which cannot disassemble without ATP, as discussed
above. Furthermore, after prolonged ischemia, sarcolemrnal damage [Y ano et al., 19871
can M e r disnipt the intraceiiular environment [Jennings et al., 1969: Korb and Totovic.
1969; Kmg, 1970; Kloner et al., 19741. Excessive Ca'+ entry occurs. activating the
contracde proteins even more. Thus, with the progression of the ischemic period, many of
the affected myocytes become irrevenibly injured and will die even if the ischemia is
eliminated.
Mitigation of the Effects of Catecholamine Cnrdiotoxicity and Ischemic D m c t g e
The deleterious effects of catecholamines via beta-adrenergic activation are
consistent with the observed block of these changes by beta-adrenergic antagonists. Be ta-
antagonists like propranolol prevent the CAMP accumulation and the structural injury
induced by epinephrine in the rat heart. Lubbe et al. [1978] used the beta-adrenergic
antagonist atenolol to inhibit the increase in CAMP and ventricular fibrillation caused by
high doses of epinephrine. Kako [1966] reported that propranolol improved ATP levels in
hearts that were stimulated with excessive isoproterenol. Such data suggest that the
deleterious effects of excess catecholarnine stimulation is mediated by beta-adrenergic
receptor activation with subsequent depletion of energy.
Beta-adrenoceptor antagonists have also been used to attenuate the deleterious
effects of adrenergic cirive often associated with acute myocardial ischemia and infarction
and other cardiovascular disease States [Frishman and Silverman, 1979; McDevitt. 19791.
Experimental and clinical studies c o n f i that beta-adrenergic antagonists have beneficial
effects during myocardial ischemia. Beta-antagonists have been shown to lirnit the size of
experirnentally-produced infarcts kibby et al., 1973; Group, 1984: Roberts et cd.. L 9841.
while direct infusion of propranolol into an ischemic area of myocardium preserved high-
energy phosphate levels [Goodlett et al.. 19801. Propranolol has also been shown to
reduce the ultrastructural changes that occur dunng the first few hours of coronary
occlusion such as rnitochondrial swelling and microvascular injury [Kloner er cil.. 19771.
Thus, beta-antagonisis are cardioprotective as a result of an energy-sparing efiect upon
inhibition of the catecholamine response, whereupon tissue integrity is preserved.
Excessive Catecholamine Stimulation of the Arresred Myocardiwn
The relevance of, and interest in, excessive catecholamine stimulation can be
associated with the clinical situation where patients with congestive heart Mure are
characterized by elevated levels of circulating catecholamines [Ross er cil.. 19871 and where
diseased hearts of patients undergoing surgery will be subject to excessive catecholamine
stimulation induced by the surgery itself [Reves et al., 19821. In addition. exogenous
catecholamines are often adrninistered peri-operatively to support compromised myocardial
function, which rnay contribute to poor post-operative recovery.
Experiments reflecting the clinical scenario have also demonstrated poor post-
ischemic myocardial functional recovery. Takla et al. [ 19891 subjected hearts to ei ther
dopamine or dobutamine stimulation pnor to K+-arrest and 25 min normothermic global
ischemia. Recovery of cardiac output ranged from 47% to 66% in the inotropically
stimulated hearts, compared to the control recovery of 80%. Furthemore. Komai et cri.
[199 1 ] demonstrated that pre-ischemic administration of isoproterenol to working rat hearts
severely depressed functional recovery that was attributed to a possible catecholamine-
potentiated ischemic-repemision injury due to aggravated Ca'+ overload. As discussed
above, catecholamines may directly induce Ca'+ overload which can initiate high energy
phosphate breakdown, the "marker" of non-ischemic myocardial necrosis of excessive
catecholamines Feckenstein, 1973; Heckenstein, 19831.
In some cases, global myocardial ischemia for surgery is induced with a blood-
based cardioplegia of which the blood component is extracted from the patient upon
institution of the cardiopulmonary bypass circuit. Yet, at this time of surgery. Reves rr ni.
[ 19821 have demonstrated that plasma catecholamines rise markedl y. Basal plasma
epinephrine levels in normal. resting man have been measured to range from 0.05 nM-O. 14
nM [Kopin, 19861. If an average value of 0.1 nM is used, then comparison with
measurernents of plasma epinephrine levels upon the institution of bypass show an increase
ranging from 0.7 n M [Reves et al., 19821 to 3.1 1 nM [Hine et al., 19761, indicating
approximately a seven- to 30-fold nse. In fact, Rebeyka (unpublished data) found that
plasma epinephnne levels had elevated to 2.8 n M from basal levels of less than 0.8 nM in
blood destined for blood-based cardioplegia. This raises the question of whether the
elevated levels of catecholamines presented to the hem upon cardioplegic infusion could
affect the basal state, or basal metabolism, of the arrested heart and whether that e ffect may
be deleterious to pst-ischemic functional recovery.
Carecholarnines and Myocardial Oxygen Conswnption
The characteristic catecholamine-induced upregulaiion of Ca2+ cycling and
contractile protein activity affect the conîractile state [Somenbkk et al.. 1965: Graham et
al., 19671, tension development [Hasenfuss et al., 19891, hem rate. and activation [Suga
et al., 1983: Nozawa et al., 19881 of the heart, ail of which are energy-requinng processes.
Since the heart is primarily an aerobic organ that can only afford a small oxygen debt
[Harden et al., 19791 and which obtins 90% of its ATP energy from mitochondrial
oxidative phosphorylation [Crompton. 19901, the rate of myocardid oxygen consurnption
(MVO,) is quite an accurate measurement of the heart's total metabolism. Therefore. the
increase in MVOz accompanying catecholamine stimulation can be considered a retlection
of the effect catecholamines have on myocardial metabolism [Eckstein et al.. 1950: Fisher
and Williamson, 196 1; Klocke et al., 1965; Sonnenblick et al., 1965; Gibbs et cd.. 1967:
Coleman et al., 197 1; Suga et of., 19831. If total energy expenditure of the heart is divided
into basal and beating components, then an increase in energy demand in either component
is refiected by an increase in MVO,, which, in turn, has basal and beating components.
Basal Myocardial Oxygen Consumption
Basal myocardial oxygen consumption is considered to be the eneqy required for
the regenerative processes necessary to maintain structurai and functional muscle integrity
and for the processes necessary to maintain ionic and electrical homeostasis [Gibbs. 1978:
Suga, 19901. Thus, by definition, basal metabolism studies require rendering the heart
inactive in order to eliminate the active component of MVO, and isolate the basal activity of
the myocardium. This is done by either witholding electrical stimulation of muscle
preparations or cardioplegically arresting whole h e m .
Cardioplegic Arrest Using HyperMemia
The negative transmembrane potential of the resting cardiac ce11 represents the
activity gradient and membrane permeabilities of the various intracellular and extracellular
ions in the non-excited state. The resting membrane potential value is attributable primarily
to the K+ ion gradient and is closely related to the electrof hemical gradient for K+ across the
plasma membrane, because the resting plasma membrane is most permeable to K+ relative
to the other ions [Kako, 1966: Weiss, 19971. Thus, any variation in the extracellular K-
concentration directly influences the resthg potentiai and the formulation of the cardioplegic
solution exploits this fact. The hyperkalemic property of the cardioplegic solution
decreases the K+ gradient across the plasma membrane, thereby decreasing the resting
membrane potential by depolarizing the cell to a new, more positive potential value.
Because depolarization not only opens (activates) Na+ channels, but also. eventually. closes
(inactivates) them in a voltage-dependent manner, the partial depoluization induced by
hyperkalemia ultimately inactivates the Na+ channels [Katz, 19921. In the process of
becorning inactivated the channels that can be activated c m o d y generate a siowly rising
AP because there is less of a potentiai difference and less channels available to conuibute to
the upstroke. With the more slowly rising action potential, more Nat channels have time to
inactivate. This contributes M e r to reducing the rate and extent of the depolarizing Na+
current, und fmdy, ail channels are inactivated.
Repolarization of the myocyte to its original resting membrane potential allows for
the voltage-dependent Na+ channels to recover from inactivation and thereby, re-open. or
re-activate [Katz, 19921. However, with cardioplegia, al1 the Na+ channels become and
remain inactivated as the new, increased extracellular K+ concentration reduces the rest ing
membrane potential and deten any outward K+ current needed to repolarize the cell. Thus.
the myocyte cannot be stimulated to contract and remains in diastoiic arrest.
The diastolic arrest induced by the hyperkalemic cardioplegic solution is rapid and
by being so, preserves high energy phosphate reserves important for post-ischemic
recovery [Hearse, 1980; Hearse et al., 19741. Potassium-induced membrane-
depolarization, however, is not a perfect solution for myocardial protection from ischernia.
Similar to its effect on the Nr channeis. the partial depolarization of the membrane by
hyperkalemia produces calcium influx through the voltage-dependent Ca'+ channels.
thereby requiring energy-dependent rernoval of Ca?+. Specifically. the Cal+. or slow-
inward. current is activated at more depolarized membrane potentials than the Na+ current
(-35 mV vs. -60 mV) and. it activates and inactivates much more slowly than the Na-
current [Katz. 19921. Although the normal inward Cal+ current that accompanies normal
APs is inhibited, the hyperkalemia-induced depolarization predisposes the myocardium to
accumulation of intracellular Na+ and Ca2+ via activation of voltage-dependent "window
currents" (currents defined by the voltage range over which the steady-state activation and
inactivation curves overlap) [Weiss, 1997; Lopez et al.. 19961. Thus. with some
imperfection, hyperkalemic cardioplegia electrically and mechanically arrests the hem in
diastole, maintainhg the heart at a level of basal metabolism.
Memurement of Basal Myocardial Oxygen Conrwnption
Basal MVO, values tend to vary depending of the technique and animal species
used to measure its value. In general, however, basal MVO, has been cdculated to range
between 0.01-0.03 mUO-g wet weight, which represents as little as 10% of the estimated
0.08-0.1 rnL/02/g vaiue for the MVO, of the beating, working heart [Gibbs, 19951. Table
1 is a surnmary of a few basal MVO, values obtained from the literature. As shown, basal
MV02 values can range (in conside~g ody the wet weight values) from as low as 0.0049
mL 021min/g in the rabbit [Gibbs and Kotsanas, 19861 to as high as 0.03 mL OJrninIg in
the rat [Burkhoff et al., 19901. Some working and beating heart MVO, vaiues are also
given with their corresponding basal M V 4 vaiues to provide a relative comparison.
Animal Contractile State of Heart
Working Empty-beating
K+-arrested Ernpty-beating
K+-arrested Working
Empty-beating K+-arrested
Empty-beating K+-arrested
Empty-beating K+-mes ted K+-arrested
1 K;-kested - 0.0 130 Rabbit K+-arrested 0.0084
0.103 0.034 0.02
0.0083 0.0055 0.09 17 0.038 0.0 174 0.0524 0.0 147 0.0395 0.0096 0.02
Piglet
I U
1 Working 1 0.50/g DW
Workmg Empty-beating
Guinea Pig
Y
Ernpty -beating OSIg DW Rat K+-arrested 0.1361~ DW
0.0669 0.03 19
1 1 Working 1 0.0844
K+-arres ted K+-mested
Reference
O. 0049 0.0283
[McKeever et cri.. 19581 1 [Klocke et al.. 19651
[Gibbs et al.. 19801
[Sum et al.. 19831 1
[Nozawa et cil.. 19881 '
rf [Gibbs and Kotsanas.
[Hauge and Ove. l966bl 1 Y
[Challoner. 19681
[Lochner et al.. 19681
[Penpargkul and Scheuer. s [Sternbergh etal.. 1989)
[Burkhoff et al.. 19901 . Table 1. Bearing and arrested state myocardial o q g e n consumption (MVO,) values front rhe literurrire. The mean values for the working, empty-beating, and arrested States of the har t Vary arnong species and. to a lesser degree, within species. Values are expressed as mL OJmidg (wet weight). DW indicates a dry weight value.
Not only does basal MVO, vary from species to species [Loiselle and Gibbs.
19791, but it can also Vary with the arrest conditions [Penpargkul and Scheuer. 196%
Stembergh et al., 1989; Burkhoff et al., 19901, the status of the heart prior to arrest
Lochner et nL, 19681, and with the metabolic substrate used in the cardioplegic perfusate
[Chapman and Gibbs, 1974; Burns and Reddy, 1978; Gibbs and Kotsanas, 1986: Loiselle.
19871. Hearts subjected to depolarized arrest using hyperkalemia have demonstrated a
higher basal MVO, than hearts subjected to hyperpolarized arrest using the fast sodium
channel blocker, tetrodotoxin [Stembergh et al., 19891. The lower basal MVOZ of the latter
type of amst was attributed to a lower ionic flux during arrest. Hyperkalernic mest MVO,
has also k e n compared to acalcemic arrest MVO, but considerable discrepancy exists as
some have found depolarized arrest to be lower [Penpargkul and Scheuer. 1969: Gibbs and
Kotsanas, 19861, some have found acaicernic arrest to be lower [Burkhoff ar al.. 19901.
whiie others have found no difference [Kohn and Szymanski. 19631. In addition. Lochner
et al. [1968] found that basal MVOz of hearts arrested with hyperkalemia depended on the
MVO, of the beating state pnor to arrest. Hearts that had a higher beating MVO, induced
by a higher pefision pressure demonstrated a higher basal MV02 upon arrest, even though
the perfusion pressure had been equalized among ail groups for the arrest period [Lochner
et al., 19681.
Studies have also revealed that metabolic substrates affect basal MVO, . Pyruvate.
lactate, and acetate have aii k e n shown to increase basal metabolism [Chapman and Gibbs.
1974; Gibbs and Kotsanas, 19861. Amino acid mixtures administered to isolated cardiac
myocytes or whole h e m have demonstrated increases in basal MV02 [Burns and Reddy.
1978; Loiselle, 19871. These studies, however, could not conclude whether the increase in
MVO, evolved fiom increased protein synthesis or increased oxidative metabolism via the
tricarboxylic acid cycle. However, Kira et al. [1984] showed that protein synthesis could
occur during cardiac arrest without affecting basal MVO, and likewise. others have
obtained data suggesting the negligible contribution of protein synthesis to basal MVO,
[Loiselie, 1985; Schreiber et al., 19861-
The unique nature of the cardiac muscle. in contrast to its skeletal counterpart. is
evident in its basal activity state. Basal MVOz has been found to be approximately five
times greater in cardiac (papillary) than in skeletal (soleus) muscle of the nt [Gibbs. 1978:
Loiselle, 1987; Suga, 19901. Furthemore, the basal metabolism of the striated muscle is
only about 2-3% of its active metabolism whereas basai metabolism of the cardiac muscle is
about 2530% of its active metabolism (see Table I) boiselle. 19871.
Various investigaton have examined the nature of the unusually high basal MVOr
and have tried to estimate what ceiiular functions of the myocyte would require substantial
amounts of energy despite the resting state of the heart. One function that was betieved to
be a large component of basai metabolism was the membrane-bound ion pumping
mechanism which would have to work against the passive leakage of ions (primarily Na+.
Kt, and Ca2+) across the membrane during myocardial arrest. Yet, Gibbs and Chapman
[1979] could only account for about 10% of basai MVOl based on data of Na- flux. In
contrast. Gibbs [1983] suggested a value of as much as 25% which was in accord with
estimations made by Ponce-Homos [ 19901.
Ca2+-ATPase purnps have also been examined for their role in establishing the basal
MVOz value. Based on biochemicai data of the sarolemmal Calr-purnp, Ponce-Hornos
[1990] estimated that this pump comprises less than 1% of the basal metabolism of rabbit
and dog ventricles. and less than 0.1% of that of rat ventricles. In contrast, the SR Ca'+-
ATPase pump energy consumption is estirnated to be more substantial. at about 28% of
basal MVO? ponce-Hornos, 19901. This cortelates with the fact that the total membrane
area of the SR is much greater than that of the sarcolemma and, consequently. the SR Ca2+-
ATPase pumps are much greater in number and importance with respect to intracellular
Ca'+ removai [Katz, 1 9921.
Clearly, basal metabolism of the myocardium and the processes which may account
for its activity have yet to be M y understood. One aspect of basal metabolisrn that has not
been fully elucidated is the effect of catecholamines on oxidative phosphorylation in the
amsted myocardium, and therefore on basal MV02. The fact that basal MVO, is not fixed
and can be influenced by certain factors, as mentioned above, suggesis a possibility for an
effect by catecholamines.
Catecholamines and Basal Myocardial Oxygen Comumption
The increase in mechanical work of the myocardiurn in response to beta-adreneqic
stimulation is an energy-requiring process and thus, there is an accompanying metabolic
demand for an increased supply of energy. Cyclic AMP-dependent PK appears to also
regulate an increase in glycogen breakdown with a concomitant decrease in glycogen
synthesis [Namm, 19711. However, Mayer et al. [1963] and Williamson [1964] have
shown that catecholamine-induced augmentation of cardiac contractile force can be
dissociated from their effect on the phosphorylase enzyme. Altematively. the increase in
Cal+ influx induced by beta-adrenergic stimulation may upregulate oxidative metabolism
[Denton and McCormack, 1980a; Denton and McCormack, 1980b; Denton et al.. 1980:
Denton et al., 19881, thereby providing a mechanism of coordinating increased mechanical
performance with energy metabolism. Specificaily, Ca" appean to be able to stimulate
three intramitochondrial enzymes: pyruvate dehydrogenase [Denton et al.. 1980: Rutter et
al., 19891, oxoglutarate dehydrogenase [McCormack and Denton, 1979; Denton et c d . .
19801, and NAD-linked isocitrate dehydrogenase [Rutter and Denton, 19881, al1 of whic h
can also be stimulated by high levels of ADP or NADH. In this scenario. Ca?+ can act as a
communication link between the extramitochondnal increase in mechanical activity and the
intramitochondrial energy "factory" of the myocardium. Thus, as catecholamines may
increase Ca2+ rnobilization in the arrested myocardium, this increased level of Ca'+ could
upregulate mitochondrial metabolic activity.
Numerous studies have been conducted to determine whether catec holamines can
directly affect basal MVO, by administering catecholamines to the arrested heart. In
various studies using papillary muscles Lee and Yu. 1964; Chandler et al.. 1968: Coleman
et al., 197 11, catecholarnines did not affect basal MVO,. in cross-circulated. K+-arrested
dog hearts infused with dobutamine [Nozawa et al., 19881 or epinephrine [Suga et al..
19831, basal MV02 did not change. In contrast are reports of catecholamine effects on
basal MVO,. McKeever et al. [1958] found that in canine hearts. infusion of
norepinephrine raised basal MVOz of vagally arrested hearts from 3.3 m . 02/min/ 100 g to
6.4 mL OJmin/100 g. Dramatic augmentation of the resting metabolic rate by epinephrine
or norepinephrine was also observed in cat papillary muscle [Whalen. 19571 and K+-
arrested canine [Berne, 19581 and rat [Challoner and Steinberg, 1965: Hauge and Oye.
L966b; Hauge and Oye, 1966al hearts.
Klocke et al. [1965] also demonstrated a catecholamine effect on basal MVO: in
isolated canine hearts. This group compared the effect catecholamine stimulation had on
the beating state of the heart with the effect catecholamine stimulation had on the arres ted.
basal state of the same hem. With the highest dose of catecholamine administered ( 10 pg
bolus injection), the increase in basal M V O was 9% and 15% in hearts treated with
epinephrine and norepinephrine, respectively, and as much as 32% in hearts treated with
isoproterenol. However, for any given dose of epinephrine. norepinephrine. or
isoproterenol, the percent increase in basai MVO, induced by catecholamine stimulation
was aiways greater in the beating than non-beating States. Kiocke et al. [1965] concluded
that, in cornparison to their effect in the beating heart, catecholamines appeared to have a
minimal effect on basal MVO,.
Due to the disparity of the available data, evidence for direct effects of
catecholamines on oxidative phosphorylation is inconclusive and it is stU not clear whether
the increase in MVO, seen with increased chronotropy and inotropy under catecholamine
stimulation is directly proportional and tightly linked, or whether there is a disproportionate
increase in MVO,, signifying either uncoupling of or a direct effect on oxidative
metabolism. Chandler et al. [1968] addressed this issue by correlating chernical energy use
(CP and ATP) with mechanicd function. They found that norepinephrine-treated papillary
muscles used 115% of the chernical energy of control muscles in only 50% as rnany
contractions while performing only 874 as much work. They concluded that an "oxygen-
wasting" effect of norepinephrine resulted from a disproportionately increased use of
energy associated with an increased contractile state. However. earlier. Century [1954]
had found that epinephrine did not alter the rate of ATPase activity in rat heart homogenates
or slices, nor was there any increase in MVO,. Century [1954] conciuded that the
epinephrine effect on increased MVO, was indirect and resulted from its effect on muscle
work rather than a direct effect on oxygen rnetabolism or ATP breakdown.
As with Chandler et al. [1968], though, Eckstein [1950] observed that oxygen
consumption of the beating heart increased disproportionately to an increase in cardiac
output (CO). A sizable increase in MVOz occurred despite reduction of CO in the presence
of sympathetic nerve stimulation, thereby suggesting a catecholamine effect on non-
mechanical-related MVO, . Weisfeldt and Giirnore [ 19641 dso documented a dissociation
of the inotropic and oxygen consumption effecü of norepinephrine. where low doses of
norepinephnne produced changes in contractility with minimal change in MVO, . whereas
with higher doses, contractility did not continue to increase despite a markedly augmented
M V O . These authors [Weisfeldt and Gilmore, 19641 suggested that the dissociation may
have occurred due to the use of a dose which exceeded that required for near maximal
inotropic effect, whereupon a direct effect of catecholamines on oxidative metabolism could
be distinguished.
Possible Role for Ca'+ in Mediaring Catecholamine Effect on Basal MVO,
A possible mechanism through which catecholamine action in the arrested
myocardium may mediate increased basal MVO, may be a type of "diastolic" Ca?+ overload
through beta-adrenergic receptor stimulation [Lappé and Lakatta. 1980: Lakatta and Lappé.
198 11. That is, since beta-adrenoceptor stimulation is known to upregulate the L-type Ca2-
channel current [Trautwein and Hescheler, 19901, it may increase Ca2+ window currents
during hyperkalemic arrest. The increased level of cytosolic Ca'+ would not be as high as
that seen under contractile states. nor would it induce contraction. but it would be enough
to stimulate extra energy expenditure to maintain Ca2+ homeostasis. In fact. HHany and
Loiselle [1992] have demonstrated that basal MVO, is sensitive to the intracellular Ca2+
concentration despite the absence of mechanical function. In the presence of ouabain. the
Na+K+-ATPase inhibitor, the basal M V 4 of K+-arrested. guinea pig hearts was augmented
from 7f 1 m o i Oz/min/g DW to 2 4 s pmol OJmin/g DW. which was the same value of
oxygen consumption when the sarne hearts were in the empty-beating state. HanIey and
Loiselle [ 19921 proposed that Na+/K+-ATPase inhibition induced Na+ accumulation.
thereby promoting Na+-dependent Ca2+ influx which could be stimulating metabolism at a
subcellular level. This was tested by perfûsing hearts with Ca?+ -free solution which could
reverse the the NalCa'+ exchanger to perform Ca2+ efflux. As predicted. basal MVO,
remained low when the K+-arrested, ouabain-treated hearts were perfused with Ca'--free
solution, indicating that the basal rate of energy expenditure is sensitive to intracellular Ca'-
concentration. Under such conditions of high intracellular Ca?+, ATP eneqy perhaps may
even be wasted if the increased levei of Ca3 induces a low level of actornyosin ATPase
activity, but not enough to be manifested as a contraction [Solaro et cil.. 19741. Thus. if
excessive catecholamine stimulation can exhaust energy stores of the arrested myocardium
and the energy level of the myocardium at the end of an ischernic period is a determinant of
myocardid functiond recovery WoHenberger and Krause, 1968: Hearse et al.. 19741. then
post-ischemic myocardial hinctional recovery would be compromised.
A Possible Link Beiween the Effecr of Catecholamines on Basal Merabolism and
Myocardial Functional Recovery Following Peri-Ischemic Excessive Cateckokiminr
Stimulation: Formulation of Hypotheses
In sumrnary, conclusions about any catecholamine effect on basal MVO, are
difficult to establish based on the available conflicting reports. Some groups have
demonstrated that catecholamines increase basal M V O [Hauge and Oye. 1 9 6 6 ~ Hauge and
Oye, 1966b: Challoner and Steinberg, 1965; Whaien, 1957; McKeever et cil.. 1958: Berne.
19581 while others have found no effect of catecholarnines on basal MVO, [Lee and Yu.
1964; Chandler et al., 1968; Coleman et al., 1971; Nozawa et al.. 1988: Suga et cil.. 19831.
Furthemore, evidence of darnaging effects of catecholamines in ischemic tissues have
mostly used models of regional ischernia and/or low-flow ischemia [Maroko et al.. 197 1:
Neely et al., 1973; Karlsberg et al., 1979; Muntz et al.. 1984: Yoshida and Iimura. 19893
where the heart continued to bat. Catecholamine activity under beating heart conditions is
better understood than under conditions of myocardial arrest. Even the negative effects of
pre-arrest catecholamine stimulation on post-ischemic function, demonstrated by Komai et
al. [1991] and Takla et al. [1989], were done by exposing the beating heart to
catecholamines prior to arrest.
In the case of blood-based cardioplegia, however, the mested myocardium is
exposed to excessive catecholarnines, as discussed above (see pp. 1 1-12). Therefore. one
purpose of this study was to determine the effect of catecholamines in the arrested
myocardium. It was hypothesized that basal MVO, would increase upon exposure to
excessive catecholamine stimulation. The mechanism of this effect could be explained by
beta-adrenergic receptor stimulation and consequent increase in Ca'+ mobilization via
window currents. Increased diastoiic ictracellular Ca2+ concentration may affect myocardial
ion channel pumps, mitochondrial metabolisrn, and contractile protein interaction. despite
the arrested state of the heart. If the catecholamine effect on basal MVO, is mediated by
beta-adrenergic receptors and/or Ca2+, then interference with either should be able to inhibit
the effect. Specifically, the beta-adrenergic antagonist, esmolol, and the Ca'+-modulator.
BDM. were used as the dmg interventions to inhibit the catecholamine effect on basal
MVO, of the arrested myocardium.
i) Inhibition of Catecholamine Effect on Basai MVO,
Beta-Antagonism of Catechoiamine Ef/ect on Basni MVO,
As discussed above, beta-adrenergic antagonists have been shown to rnitigate the
effects of catecholamine stimulation by preventing CAMP accumulation [Lubbe et id..
19781. reducing structural inhibition [Kloner et al., 19771. preserving ATP levels [Kako.
19661, and limiting ischemic areas [Group, 1984; Libby et ai-, 1973: Roberts et al.. 19841.
If excessive catecholamine stimulation cm affect basai MVOt of the arrested myocardium
by increasing Gaz+ mobilization and increasing ATP breakdown. then beta-antagonism
should be able to reverse the effect. The beta-adrenoceptor antagonist chosen for this study
was esmolol because of its short-acting properties which could be easily controlled within
the time Iirnits of the expenment and which, from a clinical standpoint. would not have
long-lasting cardiodepressive effects. In the clinical situation, however. patients due for
cardiac surgery and its accompanying period of cardiac ischemic arrest. already have
increased levels of circulating catecholamines, as much as 20 times basal serum levels
(based on calculation frorn data of [Kopin. 19861 and [Hine et al., 19741). Thus. to
simulate the clinical situation in these experirnents, esmolol was added after the arrested
myocardium was exposed to catecholamines to determine whether the catecholamine effect
on basal MVOz could be reversed.
Esmolol. A Short-Acting Beta-Adrenoceptor Antagonist
Despite evidence supporting the beneficial use of beta-antagonists. the relatively
long duration of action of most available beta-antagonists, with elirnination hdf-lives
ranging from 2 to 6 hours, limits their use [Ritschel, 19801. The cardiodepressive effects
of the long-ac ting beta-adrenergic antagonis ts may linger and clinicali y, patients w ith acu te
myocardial infarction or those undergoing cardiac surgery are at nsk of adverse effects
such as cardiac failure, bradycardia, hypotension, or AV block.
In 1982, Zarosiinski et al. 119821 introduced the concept of ultrashort-acting beta-
adrenergic antagonists, envisioning a compound that is extensively and rapidiy metabolized
to inactive products in a sirnilar fashion to catecholamines or nitroglycerin. Such a
compound could therefore be administered by constant i.v. infusion to provide for
controlled levels of beta-antagonism that could be titrated and quickly altered if necessary.
An ultrashort-acting beta-antagonist was expected to be much safer to use in critical care
situations. If undesirable hernodynarnic effects or cardiac failure resulted from beta-
antagonism with an ultrashort-acting beta-antagonist, rapid recovery of hinction could be
achieved within minutes by reducing or eliminating its administration.
Consequently, Zaroslinski et al. [1982] developed the ultrashort acting beta-
adrenergic antagonist dmg esmolol, originaily identified as ASL-8052. The ultrashort beta-
antagonistic nature of esmolol is a consequence of the rapid and extensive metabolism of
esmolol in whole blood. In both dog and human blood, hydrolysis of esmolol's methyl
ester to yield methanol and the primary acid metaboiite. ASL-8 123, occurs in the cytosol of
the red blood cell as w e l as in the liver [Surn et al., 19821. Clinical studies have s hown
that the half life of elimination of esmolol in humans is about 9 min and the duration of
action of esmolol is very brief, with no trace of beta-antagonism activity 30 min after
cessation of infusion of even very high doses of esmolol (400 pg/kg/rnin) [Sum rr d.
19831.
Various experiments have been conducted to further charactenze the nature of
esmolol. Studies were done to compare the action of esmolol with that of propranolol. a
long-acting beta-antagonist, on anesthetized dogs given a submaximal bolus dose of
isoproterenol [Gorczynski et al., 19831. Steady-state levels of beta-antagonism were
achieved within 10 to 20 minutes of each beta-antagonist infusion. Esmolol caused a dose-
dependent decrease in tachycardia responses. Blockade with propranoioi, however.
progressively increased throughout most of the infusion period. Twenty minutes after
termination of esmolol infusion. no significant beta-antagonism was detectable regardless
of dose, whereas only minimal recovery from beta-antagonism was observed after
propranolol inhision was ceased.
The effect of esmolol on experimental myocardial infarct size has also been
investigated. In two separate models of coronary occlusion. esmolol reduced myocardial
infarct size and prevented early hinctional deterioration after coronary artery reperfusion.
Zaroslinski et al. [1982] showed that esmolol reduced rnyocardial infarct size when
coronary occlusion was maintained for one hou followed by one day of repemision. In an
extension of this study, Lange et al. [1983] treated dogs with a continuous infusion of
esmolol begun 15 min after coronary occlusion, which lasted for three hours and was
followed by three hours of repemision. In control animals, 73M% of the ischemic area at
risk became necrotic. while in treated animals only 48k7% became necrotic (pc0.035).
The untreated anirnals also showed a decrease in cardiac function in the early phases of
reperfusion. unlike the esmolol-treated group which showed no change in fùnction.
Therefore. esmolol presents itself as a possible candidate that cm potentially reverse the
increase in basal M V O induced by excessive catecholamine stimulation of the arrested
heart.
Ca'+- Modulation of Catecholamine Eflect on Baral MVO?
To M e r investigate the nature of the catecholamine effect on basal MVO? of the
arrested myocardium, another substance, BDM was chosen for its effects on myofibril
interaction with Ca2+. Ifcatecholamines can mobilize Ca'+ in the arrested myocardiurn and
thereby increase basal MVO,, then any substance that can negatively modulate the rffects of
Cat+ may consequently reduce basal MV02. To parallel the protocol with esmolol. the
administration of BDM occurred after the arrested myocardium was exposed to
catecholamines, to determine whether BDM could reverse the catecholamine e ffec t,
Effects of BDM on Cardiac Muscle
BDM, or 2.3-butanedione-2-monoxime, is a rapidly-acting, revenible. contractile
inhibitor which has been shown to decrease contractility in both cardiac [Li et cd.. 1985:
Gwathmey et al., 199 1; Marijic et ai., 199 1: Watkins et al.. 1992: Boban er al.. 1993:
O'Brien et al., 1993; Bach et al., 19941 and skeletai [Li et al., 1985: Higuchi and
Takemori. 1989; Higuchi et al., 19891 muscles. Similar to the quick elirnination of esmolol
following cessation of infusion, the effects of BDM are rapidly revened on washout [Sada
et al., 19851. The precise mechanism of action of BDM remains to be elucidated but
evidence strongly suggests a predominant effect on the contractile proteins. ln vitm
experirnents on rabbit skeletal muscle wguchi and Takemori, 1989: Higuchi et al.. 19893
indicated that BDM acts dkctly on the rnyosin molecule, suppressing ATPase activity and
energeticaily stabilizing the unattached state of the myosin molecule. Consequently. the
nurnber of force-generating rnyosin molecules is reduced as the rate of myosin binding to
actin is reduced. In support of this theory, studies in rat cardiac muscle have shown that
maximal force, twitch force, the rise of force development, and twitch duration were al1
diminished in the presence of BDM in a dose-dependent manner packx et al.. 19941.
BDM also appears to decrease myofilament sensitivity to Ca2+ [Gwathrney et al..
199 1; Backx et al., 19941. Not only does this contribute to a decrease in rnaximd force but
also, more Ca?+ must bind to the troponin-tropomyosin complex for cross-bridge
attachent to occur in the presence of BDM compared to control. However. troponin and
tropomyosin studies have indicated that BDM does not directly affect these regulatory
proteins, further suggesting BDM activity at the level of the contractile proteins (Higuchi
and Takemori, 1989; Higuchi et al., 19891.
Other experirnents show that BDM may also be implicated in the inhibition of Ca'+
influx via the slow inward channel mergey. 1978; Wiggins et al., 1978; Wiggins et cd . .
1980; Coulombe et al., 1990; Gwathmey et al., 19911. The decrease in transsarcolemmal
Ca2+ flux would weaken the stimulation for Ca2+-induced Ca2+ release and lessen SR Ca2+
loading, ultimately reducing the amount of Gaz+ available for force generation
A consequence of such Ca2+-modulatory activity suggests that BDM may attenuate
the effects of Ca2+ overload characteristic of excessive catec holamine stimulation
[Fleckenstein, 1973; Fleckenstein. 19831. Moreover. BDM has been shown to
significantly reduce the Cal+ gained during repemision after 30 min of ischernia in isolated
perfbsed rat hearts Elz and Nayler, 19881. Addition of 30 mM BDM to the reperfusate
during Ca'+ reperfusion afforded significant protection against Ca'+ paradox-induced Ca?+
gain in isolated rat hearts [Daly et al., 19871. Studies have even shown the preservation of
ATP after reperfusion with BDM [Elz and Nayler. 1988; Nayler et al.. 19881 and in hems
stored with 30 mM BDM cardioplegic solution [Stringham et al.. 19931. Studies on
myocaràial s d n g of canine hearts [O'Brien et al., 19931 and global ischernia of isolated
rabbit hearts [Stringham et al., 1992: Stringham et al.. 19931 have shown that BDM greatly
improved post-ischemic recovery compared with control hearts. In reflection of the
aforementioned discussion on BDM, O'Brien et al. [1993] have suggested that the
mechanisms of the beneficial action of BDM on the post-ischemic reperfused myocardium
could involve preservation of myocardial ATP levels and a decrease in Ca'+ overload
through effects on the sarcoplasmic reticulum or contractile proteins.
Ouest a The energy-sparing effects resulting from the Ca'+ modulation by BDM su,,
potential for myocardial protection from pen-ischernic excessive catecholarnine stimulation.
If catecholarnines can unnecessarily increase basal MVOz by increasing Ca2+ mobilization.
then. in the arrested heart, BDM may preserve basal MVOz through inhibition of the Ca'-
overload effect.
ii) Catecholamine Eflect on Basal MVa Under Ischemic Conditions
The effect of excessive catecholamine stimulation on the arrested myocardium has
clinical relevance to blood-based cardioplegia. However. in the clinical setting.
cardioplegia is associated with an ischemic period. In consideration of this. it was
proposed that the excessive catecholamine-induced increase in basal MVO, may contribute
to poor post-ischernic functional recovery. The excessive catecholamines may be
increasing Ca2+ mobilization and, as discussed above, this may affect myocardial ion
channel pumps, mitochondrial metabolism. and contractile protein interaction. Al1 of these
activities can affect ATP levels in the myocardium, which will influence post-isc hemic
recovery wollenberger and Krause, 1968; Hearse et al., 19741. In further consideration
of the clinical situation. hypothermia is often used during cardioplegic arrest and
cardiopulmonary bypass to improve myocardial protection and this effect may also protect
the hem from any catecholamine effect.
Proteciive Effect of Hypothemia
Hypothermia is used in conjunction with cardioplegic arrest to provide additional
myocardial protection during ischemia. The additional protective effect of using lower
temperatures during ischemic arrest has been well documented. Numerous studies show
how hypothermie arrest slows down ATP depletion dunng ischemia and improves
hinctional recovery during reperhision [Hearse et al., 1974; Tyers et al.. 1977: Jones et (il..
19821. The protective effect of hypothermia is pnmarily attributed to the temperature
dependenc y of metabolic rate, leading to energy conservation [Bigelow et cd. . 1954: Blair.
19651. Arrested hearts at 20°C have been demonstrated to have MVO, values that are about
50% of those at 37°C [Blair, 19651. Hypothermia slows down the ATP-requiring
enzymatic processes and reduces transrnembrane Ca2+ fluxes, including those at the
rnitochondrial level [Ferrari et al., 19901. By reducing tissue and rnitochondrial Ca:+
accumulation, hypothermia can consequently reduce the amount of energy-dependent
processes necessary to maintain cell homeostasis and preserve the ATP-producing function
of mitochondria. Hypothermia contributes to the goal of optimal myocardial protection
which aims to induce imrnediate eletromechanical arrest and minirnize metabolic needs
during ischemia.
The cardioprotective benefits of hypothennia during ischemia may not only afford
protection of the myocardium against catecholamuie stimulation during arrest. but may also
be protective of hearts challenged with catecholarnines pnor to arrest. It has already been
demonstrated that hearts exposed to catecholamines in the beating state prior to arrest have
compromised post-ischemic functionai recovery [Takia et ai., 198% Komai et c d . . 1 99 1 1.
but the effect of lowenng the ischernic temperature was not examined. Improved recovery
may be retlective of improved preservation of high energy stores by hypothermia during
ischernia.
Thus, it was hypothesized that hypothennia could also attenuate the deleterious
effects on post-ischemic myocardial functional recovery of hearts stimulated with
catecholamines in the beating state pnor to arrest.
î l e Hypotheses:
In summary, the hypotheses were as foilows:
Excessive catecholamine stimulation of the arrested myocardium will increase
basal MV02.
The catecholamine-induced increase in basal MVO? of the arrested
myocardium c m be reversed by either a beta-adrenoceptor antagonist or by a
Cal+-modulator.
The catecholarnine-induced increase in basal MVOz of the arrested
myocardium WU be deleterious to post-ischemic functional recovery and c m
be attenuated by hypothermia during ischemia
Poor pst-ischemic functional recovery following catecholamine stimulation of
basal MVO, is associated with decreased levels of myocardial adenine
nucleotides.
The detrimental effect of pre-ischemic catecholamine stimulation of the beating
heart on pst-ischemic functional recovery will be attenuated by hypothermia
during ischemia. Hypothennia will contribute to improved preservation of
myocardial adenine nucleotide concentrations.
Overview of Experiments
As a preliminary study. and to serve as the basis of the ischemia studirs.
experiments were done to determine basal metabolkm of the heart under varying arrest
conditions . This part of the study was designated under the title, "Basal Metabolism
Under Varying Arrest Conditions." Myocardial oxygen consumption was used as the
parameter to define the energy requirements of the heart and to observe how that energy
requirement would be affected by various arrest conditions, such as in the presence of
isoproterenol, esmolol, andor BDM. Arterial and venous oxygen contents and coronary
flow, ail of which are necessary to calculate MVOz, had to be obtained from the inflow and
the outflow of the heart and thus, the MVO, studies required constant perfusion of the
myocardium even under arrest conditions.
Upon detemiining whether catecholamines could affect basal MV02, in contrast to
their hemodynamic/mechanicd effect on total MVOz. m e r studies were done in ischemic
conditions to simulate the clinical situation. Thus, normothermic and hypothemic ischemia
conditions were used. This part of the study was designated under the title. "Peri-Ischemic
Catecholamine Stimulation and Myocardial Functional Recovery." In the first part of the
ischemia studies, the purpose was to determine whether the effect of catec holamines on the
metabolic rate of the arrested, ischemic myocardium could be deleterious to post-ischemic
myocardial func tional recovery. This section was entitled. "Post-Ischemic Functional
Recovery Following Catecholamine Stimulation of Arrested Myocardium."
The second part of the ischernia studies was based on previous findings described
in the Literature, where hearts stimulated with catecholamines in the beating state pnor to
arrest have decreased post-ischemic function [Takla et al.. 1989; Kornai et al.. 199 1 1. To
determine whether hypothermia could improve recovery, two additional groups of hearts
were studied. This part of the study was entitled, "Post-Ischemic Functional Recovery
FoiIowing Pre-Ischemic Catecholamine Stimulation of Beating Myocardium."
Ali experiments were conducted using isolated. Langendorff-perfused rabbit hearts.
Modified, crystalloid Krebs-Henseleit buffer (KHB) was used as the coronary perfusate.
In the MVO, studies, hearts were subjected to perfusion arrest with 20 rnM K+ KHB
("Ku). Isoproterenol was used as the catecholamine, while esmolol and BDM were used as
the beta-blocker and contractile inhibitor, respectively. Either isoproterenol, esmolol. or
BDM, or a combination of the inotropic agonist with an antagonist was added to the arrest
perfusate dunng certain portions of the entire arrest period. Myocardial oxygen
consumption was evaluated under these varying conditions to determine any change in
basal metabolic rate of the arrested heart.
The first part of the ischernia studies was divided into two temperature groups:
37°C normothermia for 60 min or 20°C hypothermia for 120 min. Two groups of hearts
were tested at each ischemic temperature. The control group of hearts (Control) was not
given any catecholamine stimulation. Group Cp-Iso was challenged with catecholamines
within the cardioplegic solution. All hearts undenuent the same basic protocol of
equilibration, cardioplegic arrest, ischemia. and reperhision. upon w hich recoveries of
sy stolic and diastolic function, and developed pressure were evaluated.
In the second part of the ischemia studies, one group of hearts subjected to
normotherrnic ischernia conditions (Group Re-Iso/37) was compared to another which was
subjected to hypothermie ischemia conditions (Group Pre-Iso/20). These hearts followed
the sarne basic protocol as described above, except that both groups were challenged with
catecholamines in the beating state just prior to arrest.
Myocardial adenine nucleotide concentrations were also measured from biopsies
taken at the end of each ischemia experiment. Functional recovery could then be correlated
to the biochemical energy statu of the myocardium and therefore, the effects of excessive
catecholamine stimulation of ischemic myocardium could possibly be correlated to a
decreased level of high energy phosphates.
2.1 Preparation of Perfusates
Modified Krebs-Henseleit control buffer ( W B ) [Rebeyka et al.. 19901 was
prepared with the following composition: 1 18.4 mM NaCl, 25.0 mM NaHCOi. 1 1.1 mM
D-(+)-glucose, 4.7 mM KCl, 1 .Z7 m M CaCl? H20, 1.2 mM KH,PO,. 1.2 mM MgSO,
(anhydrous). 2 U L insulin (bovine-porcine, Novopharm, ), 0.06 mM EDTA. then filtered
with a 5 prn filter. The perfusate was filtered once again (5 prn filter) in-line of the
Langendorff petfusion system. Any modifications made to the control buffer were made
after the fmt filtration.
Modifications made to the KHB perfusate are designated by the following
abbreviations. Concentrations used were either as specified below or within the
Experirnental Protocol sections if various concentrations were used.
a) K: addition of 15.3 mM K+ to bring total concentration of K+ to 20 mM
b) Iso: addition of 25 nM isoproterenol (isoproterenol hydrochloride injection. 0.7
mg/m.L; Sabex, Inc., Canada). In the beating heart, 30 min of pefision with KHB
and this dose of Iso was found to increase hem rate (by visual inspection) as well
as increase beating heart MVOz by over 50% (Figure B.1. Appendix B. p.93).
without having a toxic effect as MV02 retumed to its pre-stimulated values upon
reperfusion with just KHB.
Lnitidy, epinephrine (Epi) was used as the candidate catecholamine but pi10 t
experiments testing various doses of Epi on beating h e m MVOz did not yield
expected results of increased MVO2 [Challoner and Steinberg, 1965; Vasu et r d . .
19781. Starting with a dose of 2 nM Epi to mimic the clinical situation (Rebeyka.
unpublished data, see p. 12)- doses of 25 nM and 50 RM Epi were also tested
separately in beating hearts (Appendix A, Figures A.2. A.3. and A.4, respectively.
pp. 9 1-92). None of the three doses demonstrated the expected increase in beating
h e m MV02. The possible reasons for the lack of an effect are discussed in the
Discussion section. In this study, however, the focus was on the beta effect of
catecholamines and whether that effect can affect basal M V 0 2 . Iso clearly
demonstrated a beta-effect with increased MVO, (Figure B. 1, Appendix B. p.93).
and thus, was used instead.
c) Esm: addition of esmolol hydrochloride (Brevibloc. Zeneca Pharma. Canada).
Esmolol was chosen as the candidate beta-adrenoceptor antagonist for this study
due to its cardioselectivity and short-acting properties. Initially. 2 mg/L waï
chosen. based on doses administered to patients [Sum et al.. 19831. This dose had
no effect in the catecholamine-stirnulated, arrested heart (Figure 3.2, p.50). As i t
was uncertain whether the lack of an effect was tmly due to an inability to reverse
the catecholamine-effect or because this dose is ineffective in the model. 2 mg/L
Esm as well as 10 mgL and LOO mg/L were tested in pilot studies in the beating.
non-working heart. to study activity (Appendix C. pp.94-95). The dose of 10
mg/L appeared to decrease MV02 without any lingering effects on washout. The
highest dosage of 100 mg/L demonstrated a marked decrease in MVO. that
remained upon washout. Thus, to ensure an effect without cardiotoxicity. an
intermediate dose of 25 mg/L Esm (which is about l ( r M) was used in the MVO,
study. These studies on Esm are elaborated upon in the Discussion.
d) BDM: addition of 30 rnM 2,3-butanedione-2-monoxime (BDM) (Sigma Chernicd
Company, Canada). This dose has been previously demonstrated to be effective in
affording myocardial protection without cardiotoxicity [Daly er al., 1987: Stringham
et al., 1992; Hebisch et al.. 1993; Suingharn et al., 19931
e) Epi: addition of epinephnne (adrenaiine chloride injection U.S.P. 1: 1000. Parke-
Davis, Canada). Epinephrine was hitially used as the catecholamine challenge but
then, was replaced by isoproterenol (see (b), above). The reasons for this switch
and the preliminary results using Epi are given in the Discussion and Appendix A.
Marerials mtd Methodi
f) Prz: addition of 1 pM prazosin hydrochloride (Sigma Chernical Company. Canada).
This dose has been previously demonstrated to be effective in potentiating the beta-
receptor-rnediated inotropic response by inhibiting alpha-adrenergic stimulation
[Youngson and Talesnik, 19851.
2 .2 Langendorff Perfusion System
The isolated rabbit heart in Langendorff mode [Langendorff. 1895; Doring and
Dehnert, 19881 was used for ail experiments. A schematic diagram of this perfusion
system is given in Figure 2.1. Tygon tubing from the main (primary) reservoir ran through
a peristaltic pump (Dungey Incorporated Piper Pump, Canada), through a small. in-line
filter, up to a secondary, air-tight reservoir which was set at 75 cm H 2 0 to provide a
perfùsion pressure of 55 rnmHg. Tubing leading out of the secondary reservoir then ran
down to a heating coil. Tubing between the heating coil and the aortic cannula was
accommodated with a T-joint. with a stopcock, to d o w for "artenai" MVO, measurements
or cardioplegia infusion. The perfusion pressure was kept constant by continuous
replenishment of the secondary reservoir from the prirnary reservoir. The perfusate was
not recirculated and was oxygenated with 95% 0Z:5% CO2 using a bubble oxygenator
placed in the primary reservoir.
The glassware (Radnoti Glassware, Canada) was water-jacketed and connected to a
water bath with a circulating pump (Haake 001-3954, Berlin). The system was primed
prior to every experhent to ensure absence of air bubbles. After ailowing the water bath to
w m up, the temperature of the perfusate exiting the aortic cannula was tested to ensure a
constant temperature of 37S°C. A secondary water bah with circulating pump was kept at
20°C and connected to a separate heart chamber for use in the ischemia study.
Materials und Methods
Figure 2.1. Langendotffpe@sion ?stem. The Langendorff. isolated rabbit heart system was used to conduct experirnents in both the basal metabolism and ischemia studies. This d i a m briefly outlines the main features of the system used. See Materials and Methods section for details. A: Perfusate reservoir. B: RoIler pump. C: In-line filter. D: Secondary. air-tight reservoir set at 75 cm H 20. E: Heating coil. F: Side-arm for obtaining "arterial" samples or for infusing cardioplepic sohtion. G: Isolated, rabbit heart. H: Heart chamber. 1: Coronary effluent. J: Myocardial temperature probe monitor with attiiched probe.
Marerials and Merhods
2.3 Basal Metabolism Under Varying Arrest Conditions
2.3 .1 H e a ~ Preparatim
Al1 animals received hurnane care in accordance with guidelines of the Canadian
Council on Animal Care. In each experiment. New Zealand white nbbits weighing 600-
1200 g (30 d-45 d old) were pre-anesthetized with 50 mgkg ketamine (i-m.). heparinized
(300 U.S.P. unitskg, i.v.), and then anesthetized with sodium pentobarbital (40 mgkg.
i.v.). Bilateral stemotomy was performed and the inferior and superior vena cavae were
ligated near their insertions into the right atrium. Hearts were excised and irnmediately
cannulated via the aorta to be pemised in LangendorFf mode. Pemision occurred within
10- 15 seconds of excision. The pulmonary artery was cannulated for coronary effluent
collection and this cannula, fined with a stopcock on the end for sample extraction. was
propped up for support in order to minimize disturbance of the heart. A small temperature
probe inserted into the myocardium was used to ensure that the water-jacketed apparatus
and perfusate maintained a constant myocardial temperature of 37S°C. The hems were left
to k a t spontaneously in ail experirnents and were randornly assigned to the experiments.
2.3.2 Experimental Protocol
t Ex~erirnental f rotocoi
TabIe IL Experimentul protocol for busal MVO, studies. Schematic outline of chronology of perfusion intervals for each experiment. "Perf. "=Perfusion. Time denotes length of perfusion interval. Experiment name describes arrest conditions and results can be found in correspondhg Figure.
Figure 3.1
3.2
3 -3
3.4
3.5
A 30 min
KHB
C 15 min K+ko
K+Esm ,
K+Iso+Esm
, K+BDM
K+Iso+BDM
B 15 min
K
K
K+Iso
K
K+Iso
PetJInterval Experiment
K+ 25 n M Iso
K+ 25 mg/L Esm
K+ '
25 nM Iso+ 25 mg/L Esm
K+ 30 m M BDM
K+ 25 nM Iso+
30 mM BDM
1
B 15 min K
K
' K+Iso
K
K+Iso
A 30 min
KHB
C 15 min K+Iso
K+Esm
K+Iso+Esm
K+BDM
KiIso+BDM
Al1 MVOt experiments followed the same general protocol pattern, as described in
Table LI. After isolation and attachrnent to the Langendorff system, the hearts were
stabilized for 30 min with ECHB pemision before king perfused for 15 min with the high
potassium arrest solution, "K" (i.e. KHB + 20 mM K+). This "control arrest" period waï
then followed by a 15 min interval of perfbsion arrest with an added dmg. Aftenvards. the
hearts were re-introduced to 15 min perfusion with the original arrest perfusate. K.
followed by perfusion with the h g added to K again. Thus, the protocol followed an "A
B C B C A" pattern of perfusion, where A, B, and C would each represent a different
perfusatelperfusion interval, and A would always be KHB. When more that one dnig ( e . g
Iso and Esm) was added, the fmt dnig was added to the arrest perfusate immediately after
the 30 min equilibration period, while the second was added in the next perfusion interval.
Less than two minutes before the end of each perfusion interval. arterial and venous
samples were taken and coronary flow for one minute was recorded before switching to the
next perfusion interval.
2.3 -3 Measurement of Oxygen Consumption
Myocardial oxygen consumption (MVO?) was calculated as follows:
MVO, (mL OJminIg) = arterial - venous 0, tension h n k k l 760 rnmHg/aun
X (solubility of O?)
X coron- Bow ImUmin) weight of heart (g)
where the solubility of oxygen in water at 37.S°C is: 0.02370 mL O? mL solution/atrn
"Artenal" samples were drawn, by a syringe, from a short tube connected to the T-
joint leading to the aortic cannula. "Venous" samples were drawn. by a syringe. from a
stopcock in-line with the cannulated pulmonary artery. Both arterial and venous samples
were collected under air-tight conditions, capped, and placed on ice. Oxygen tension
measurements were then immediately obtained on a Radiometer Copenhagen ABL 330
blood-gas machine. Coronary effluent was collected over a one minute intervai to obtain
measurement of the rate of coronary flow. Samples for MVO caiculation were taken at the
end of every perfusion interval. At the end of the expenments. hearts were blotted. then
weighed to obtain wet weight (WW) values and then freeze-dried overnighr to obtain dry
weight (DW) values.
2.3 -4 Dosage Studies
The protocol used to determine the effect of a certain dosage of dmg on the beüting.
non-working heart foiiowed similar protocol to that used for the basal MVO? study. After a
30 min stabilization period with KHB perfusion (and MVOZ measurement). hearts were
exposed to the dmg (Epi, Iso, or Esm) for 30 minutes. MVO, data was collected at the 15
min and the 30 min time points. Hearts were then repemised with ECHB for a final 30 min.
at the end of which MVO, was measured. As above, coronary flow and h e m weight were
also recorded as required.
2 - 4 Peri-Ischemic Catecholamine Stimulation and Myocardial
Functional Recovery
2.4.1 Heurt Preparation
In each experiment, New Zealand white rabbits weighing 600- 1200 g (30 d-45 d
old) were pre-anesthetized with 50 mglkg ketamine (i.m.). heparinized (300 U.S.P.
unitskg, i.v.), and then anesthetized with sodium pentobarbital(40 mgkg, i.v.). Bilaterai
stemotomy was performed and the inferior and superior vena cavae were ligated near thrir
insertions into the right atrium. Hearts were excised and cannulated via the aorta ont0 the
Langendorff perfusion system, as descnbed above. A smali incision was made in the
pulmonary artery to relieve pressure in the right ventricle and to allow for collection of
coronary flow.
Myocardial function was evaluated by using a balloon in the left ventncle. To
prevent air from entering the left ventricle during balloon insertion, the heart was irnmersed
Mate riais and Methocls
in a w a m water bath before a small incision was made in the left atrium. As air is
compressible. any air pockets between the ventricle and the balloon. or within the balloon.
would give an inaccurate measure of pressure. A latex intraventricular balloon. to record
left ventricular pressure, was attached to the end of polyethylene tubing and was filled with
water to elirninate any air. After minimizing its volume, the intraventricular balloon was
inserted through the left atrium into the left ventncle and secured with a suture around the
remaining lefi auial tissue. The balioon and tubing were comected to a pressure transducer
which was linked to a Harvard Apparatus Universal Oscillograph for the recording of
pressure. Balloon volume was set during the stabilization period to provide a pre-ischemic
baseline end-diastolic pressure of 10 rnmHg.
A small temperature probe inserted into the myocardium was used to ensure that the
water-jacketed apparatus maintained a constant myocardiai temperature of 37S°C. The
hearts were lefi to k a t spontaneously in ail experiments and were randomly assigned to the
groups.
2.4.2 Erperimental Protocol
2.4.2a Post-lschernic Functional Recovery Following CateclzolrmUze
Stimularion of A rresred Myocardium
A schematic of the chronology of the experimental protocol is given in Figure 2.2.
Bnefly, hearts in the control group, Control (Figure 2.2a), had an initial equilibration
period of 30 min with KHB, followed by infusion with 20 mM K+ KHB cardioplegic
solution (K) to initiate ischernic arrest after perfusion through the aortic cannula was
clamped off. Ali hearts were given 20 m . of K to induce arrest. This volume was found
to be adequate for the experimental rnodel. The cardioplegia was infused by a syringe
pump (Syringe Infusion Pump 22, Model221/W, Harvard Apparatus Canada) at a rate of 6
W m i n through the side-am of the T-joint just above the aortic cannula. The temperature
of the cardioplegia was 37OC for h e m to be subjected to normothermic ischernia and ZO°C
for hearts to be subjected to hypothermie ischemia. That is, each of the two groups were
Materials and Merhods
a. CONTROL (Control Group) lschcmia
30 min Stabilization (60 min at 37°C or 120 min at W C ) JO min Reperfusion
4 Cardioplegia
b. CARDIOPLEGIC CATECHOLAMINE CHALLENGE (Group Cp-Iso)
Figure 2.2. POSI-ischetrric jitncrioriul recovery fi)lloivitig cnteclrulaniitie stitrtrtltr~ioti i f tirrestecl
~~~O~f l rd i i t111 . Control Group: Aficr a 30 rtiin stubilirition pcriod, hcuris wcrc subjected to cardioplcgic arrcsi and nonnothcrmic or hypotheniiic ischcriiiu. Hcuris wcrc ihcii reprî'uscd for 30 min. Change in systoliç, diastolic, und dcvclopcd pressures wcrc iiicüsurcd prc- und post-ischcmicully (bluck urrows) to cvüluuic functionul recavcry. Group Cp-lso hcarts diffcrsd frorii Contrnl only in thai ihcy wcre cxposed to 25 nM Iso wiihin ihe curdioplegiu (shudcd box). Tissue biopsies wcrc iukcn ut the end 01' rcpctrî'usion (*) t» riicüsurc myocnrdiul adcninc nucleoiidc conccniruiions.
a. Group Pre-Iso/37
15 niin Norniothermic lschemia (60 min ut 37°C)
Cardioplegia
b. Group Pre-1~0120
15 min Stabilization 1
Hypothermie lschemia (120 min at 20°C)
*
JO min Reperfusion
I 9 Cardioplegia
Figure 2.3. Isciieniirr terrrpercttrrre t~jii~ct oti post-ischetrric fiitictiorirtl rec-overy firllowitig pre-isclienric ctrreclrulu~rritre srirrirrlutist~ of beurirll: niyocicrrtlirrni . Aftcr 15 min of siabiliuiion, beuiing heurts were siimuluicd with 25 nM Iso for 15 min jus1 prior to orrcst with çurdioplcgiu und ischcmia. Group Pre- lso137 hearts were subjccted to norinothcriiiic ischcinia ut 37°C for 60 inin. Group Prc-lso/30 heüris were subjccted io hypoihcrmiç ischeiiiia at 20°C frir 120 niin. Heurts wcre thcn repcrfused for 30 niin. Systolic, diastolic, and dcvclopçd prcssurcs were iiieasured pre- and posi-ischciiiicully (hluck urrows) io cvnluute funçtionul rccovery. Tissuc biopsics wcre tuken ui the end 01' rcpcrfusion (*) io riieusure myoçurdiul adcninc nucleiiiidc conccntriltions.
studied at two different temperatures of ischemia. In the normothermic ischemia study.
hearts were subjected to cardioplegic arrest and global ischemia at 37°C for 60 min. In the
hypothermic ischemia study, hearts were subjected to cardioplegic arrest and global
ischemia at 20aC for 120 min. After ischemia, hearts were reperfbsed for 30 min.
The effect of excessive catecholamine stimulation during arrest was detemined by
sub~ecting some hearts to catecholamine stimulation within the cardioplegia (Group Cp-
Iso), where 25 nM Iso was added to the cardioplegic solution (Figure 2.2b). Equilibration.
ischemia and reperfusion were identical to the control (Control).
2.4.2b Post-lschemic Functionaf Recovery Foilowing Pre-Ischrmic
Cutecholamine Stimulation of Beating Myocardir un
A schematic of the chronology of the expenmental protocol is given in Figure 2.3.
The basic structure of these experiments was identical to the arrest study (Section 7.4.W.
except that 25 nM Iso was admùùstered to beating hearts in the 15 min prior to arrest (Le.
last half of stabilization period). Groups were then divided according to the temperature of
the ischemic period. H e m were subjected to either normothermic ischernia at 37°C for 60
min (Group Pre-1~0137, Figure 2.3a) or hypothermic ischemia at 20°C for 120 min (Group
Pre-1~0120, Figure 2.3b). After ischemia., heans were reperfused for 30 min. The effect of
ischemia temperature on excessive catecholamine stimulation of the beating hem pnor to
arrest was de termined in Group Pre-Iso/37 and Group Pre-Iso120.
2.4.3 Evaluution of Myocardial Function
Pre-ischemic (baseline) values of systolic pressure (SP) and diastolic pressure ( DP)
were recorded at the end of the stabilization penod. Post-ischemic values of these sarne
parameters of cardiac function were recorded at the end of the reperfusion period.
Developed pressure (DevP) was calculated as the difference between SP and DP. Post-
ischemic functional recovery was calcuiated as the absolute change (A) in systolic pressure.
diastolic pressure, and developed pressure from baseline to post-ischemic values.
Materials und Met/zuds
2.4.4 HPLC Ana &sis For Post- Ischemic Myocardial Adeninr
Nucleoiide Concentrations
The HPLC procedure used is sirnilar to that described by Weisel et cd . [ 19891.
Briefly, at the end of each experiment, a biopsy from the left ventricular free wall was
immediately taken and immersed in liquid nitrogen, to be freeze-dried ovemight at -50°C.
The freeze-dried muscle was stored in a -70°C freezer for subsequent HPLC analysis of
adenine nucleotides and their degradation products. The myocardial tissue was cleaned of
connective tissue. The muscle was homogenized for 10 minutes on ice with 20 pL 0.5 M
perchloric acidlmg tissue. An intemal standard, 2'-O-methyladenosine. was added üt a
known concentration to each sample. After a 10 min centrifugation at 2500 rpm. the
supernatant was neutralized with 2 M potassium hydroxide to pH 7.6. then reacidified with
0.1 M perchloric acid to pH 6.8. The sample was centrifuged again for 10 min at 1500
rpm, after which the supernatant was frozen in Liquid nitrogen to be freeze-dned ovemight.
The freeze-dried samples were stored at -70°C until al1 heart biopsies were ready to be
analyzed on the HPLC machine.
A modification of the step-gradient technique described by Hull-Ryde et al. [ 19861
was used to measure the levels of adenine nucleotides and their degradation products by
high performance liquid chromatography [Weisel et al., 19891. Sarnples were re-
suspended in LOO mM ammonium phosphate buffer (pH 5.7) just prior to analysis.
Sarnple injection was done by an auto-injector (Model 700 Satellite WIS. Waters
Associates, Mississauga, Canada). A reciprocating pump (Models 50 1 and 5 [O. Waters
Associates) performed step-gradient solvent delivery. The chromatographie column. a
Radial-Pak Resolve Cl8 Column (Waters Associates) with a 5 pM particle size. was
operated in a 175 bar-radial compression module (Model RCM LOO, Waters Associates).
Using a programmable multiwavelength detector (Model 490, Waters Associates), the
system rneasured uric acid, adenosine triphosphate (ATP), inosine monophosphate (MP).
adenosine diphosphate (ADP), hypoxanthine (HXN), xanthine (XN), adenosine
monophosphate (AMP). and inosine at a peak absorbance of 254 nm. Creatinine
phosphate was measured at a peak absorbance of 229 nrn. The total adenine nucleotide
(TAN) concentration was calculated as the surn of the concentrations for AMP, ADP. and
ATP. The results are expressed as pnoYg DW myocardial muscle. A sarnple trace from
the HPLC analysis of myocardiai tissue is shown in Figure 2.4.
2.5 Statistical Anafysis
2.5.1 Basal Metabolim Under Varying Arrest Conditions
The perfusion arrest expenments addressed changes in MVO, of the arrested state
of the hem. After the conuol arrest period. a drug was added to the arrested hem to
detennine its effect on basal MVO,. These two intervals were then repeated in sequence.
Thus, for each study, two-tailed, paired t-tests were used to test for any significant change
in basal MVO, of the arrested heart from the control arrest penod to the following "drug-
added" period. A two-tailed, paired t-test was also used to test for any significant
difference between the two control arrest MVO, values. A significance level of ~ ~ 0 . 0 5
was used. The equilibrium and reperfhsion penods where the heart was in a beating state
were considered as control periods only, to ensure that the heart was functional.
Experiments testing the effect of a certain d m g on the beating heart
addressed changes in MV02 of the beating heart. Afier recording MVOz of the control.
beating period, the dmg was administered and MVO, was recorded every 15 min for 30
min before retuming to the control perfbsate. Thus, ANOVA for repeated measures.
foilowed by post-hoc Bonferroni t-tests to isolate significant differences between any two
groups, was used to test for any significant changes arnong the initial control. beating
period MVOz and the two M V 4 values of the "dnig-added" period. A significmce level of
p 4 . 0 5 was used. Also, a two-tailed, paired t-test was used to test for any significant
Materials and Methods
difference between the initial and final control penods, using a significance level of
pc0.05.
Statistical analyses were performed on StatView 4.5 1 (Abacus Concepts. 1995) and
Instat 1.12 (GraphPad Software, 1992).
2.5.2 Pen-lschemic Cutecholamine Stimulation and Myocurdial
Functional Recovery
Baseline values and post-ischernic functional recovery values were compared by
two-tailed, unpaired t-tests to test for any significant differences (significance level of
p 4 . 0 5 ) between Groups Control and Cp-Iso, for each temperature study (Le.
normothermia and hypothermia). Parameters. compared to evaluate post-ischemic change in
functioo between the two groups were: change in systolic pressure. change in diastolic
pressure, change in developed pressure. The concentration of each adenine nucleotide was
also compared between each group (Control vs. Cp-Iso) for each separate temperature
study (normothemiic ischemia and hypothemiic ischemia).
The same method of statistical analysis was used io compare functional and adenine
nucleotide differences between Groups Pre-Iso/37 and Pre-Iso/2O.
In an additional analysis of data, the ischemia temperature e ffect on post-ischernic
change in diastolic pressure was compared in the Control and Cp-Iso groups (e.g change
in DP for normothermia Control vs. change in DP for hypothermia Control). Two-tailed.
unpaired t-tests were used with a significance of peO.05.
Statistical analyses were performed on StatView 4.5 1 (Abacus Concepts. 1995) and
instat 1.12 (GraphPad Software. 1992).
3 RESULTS
3.1 Basal Metabolkm Under Varying Arrest Conditions
Ail values are given as me-+SEM and statisticai significance. as described in
Materials and Methods, is indicated as a significant difference with p<0.05 using two-
tailed, paired t-tests.
3.1.1 P e m i o n Arrest With Catecholamine Stimulation
The effect of catecholamines on basal metabolism of the myocardium was evduated
by exposing perfusion-arrested hearts to isoproterenol (Figure 3.1 ). Hearts (n=5) exposed
to 25 n M Iso in the arrested state (Le. 25 n M Iso added to K) demonstrated a significant
(p=0.0009) increase in basal MVO, from perfusion arrest without Iso (Figure 3.1 ).
Specificaliy. the change was from 0.054+0.006 m . Oz/rnin/g DW to 0.088M.009 mL
OJminlg DW. Subsequent repetition of these two intervals (second Intervais B-C. Figure
3.1) appeared to demonstrate a sirnilar increase (0.034k0.004 to 0.063M.008 mL
OL/rnin/g DW) but the change was not statistically significant (p=0.07). However. the
MV02 of the second control arrest penod (second Interval B, Figure 3.1 ) was statistically
different frorn the MVOt of the initial control arrest penod (first Interval B. Figure 3.1 )
(pd.02). Recovery of the beating state is shown in the shaded area of the gnph (Figure
3.1), where pre-arrest MVOz was 0.342M.048 mL 02/min/g DW and post-arrest MVO:
was 0.35(WO.052 rnL OZ/min/g DW.
3.1.2 Perfusion Arrest with Catecholamine Stimulation rind Berci-
Adrenoceptor Antugonist
Since an increase in basal metabolism was observed dunng perfusion arrest with
isoproterenol, the ability of the beta-adrenoceptor antagonist, esmolol. to reverse the effect
was questioned. Initially, however, experirnents were done to evaluate the effect of
esmoloi alone in the perfusion-arrested heart (Figure 3.2). The MVOt of h e m (n= 13)
arrested with K (0.06m.004 mL 02/min/g DW) did not significantly change with the
addition of 2 m a esmolol to the arrest perfusate (0.052M.005 mL O?/min/g DW:
p=0.08). Repetition of these two perfbsion intervais yielded similar results (0.0Sm.005
T i e (min)
Figure 3.1. Eflecf of 25 nM isoproterenol on basal MVO? Basal MVO: significantly increased upon the addition of 25 nM isoproterenol to the arrest perfusate. The shaded area shows the recovery of the beating state MV02. VaIues are mearifSEM (n=5i. Perfusion intervais: A , KHB; B, K; C, K + 25 nM ïso. *p=0.0009 vs. prïor conrrol arrest intervai (B); 'p=0.02 vs. initial Interval B; two-tailed, paired t-test.
to 0.05 1M.004 mL 02/min/g DW; second Intervals B-C, Figure 3.2; pc0.8). The shaded
A
area of the graph (Figure 3.2) shows recovery of the beating heart MVO, from a pre-arrest
B C B C Perfusion Intervai
value of 0.246I0.033 rnL O,/min/g DW to a post-arrest value of 0.218M.029 rnL
A
O J d g DW.
Because the lack of any effect of 2 mg/L esmolol may have been due to an
ineffective dosage, the effects of 2, IO, and 100 m g L esmolol in the beating hem were
detennined in pilot snidies (Appendix C, pp.94-95). Whereas 10 mg/L esmolol tended to
decrease MVO, of the beating hem (Figure C.2. Appendix C, p.94). 1 0 mgR esmolol
I v I I
O 30 60 90 120
Time (min)
Figure 3.2. Effecr of 2 m g L esmolol on basal MVO,. Basal MVO, did not
significantly change upon the addition of 2 mgL csmolol to the arrest perfusate. The shaded area shows the recovery of the beating state MVO:. Values arc mean+SEM (n=13). Perfusion intervals: A. KHB; B. K; C, K + 2 mce/L Esm.
markedly reduced contractile function (visual inspection) and MV02, both of which tended
A
to remain depressed afier discontinuation of esmolol perfusion (Figure C.3. Appendix C.
p.95). Thus, an intermediate dose of 25 mg/L esmolol was chosen for use in subsequent
B C Perfusion Interval
expenments .
C A B
Expenments were then done with a dose of 25 mgL Esm to determine whether
esmolol could reverse, that is, eliminate, the effect Iso had on basal MVOz (Figure 3.3).
Mer equilibration with KHB for 30 minutes, hearts (n=ll) were arrested with K + Iso.
T h e (min)
Figure 3.3. Effecr of esmolol on basal MVO, in the presence of isoprorerenol. Basal MVOz sûrnulated with 25 n M isoproterenol did not significantly change upon the addition of 25 mg/L esmolol to the arrest perfusate. The shaded area shows the recovery of the beating state MVOP Values are r n e d E M (n= 1 1 ). Perfusion intervals: A, W B : B. K + 25 nM Iso; C, K + î5 n M Lso + 25 mg/L Esm.
After 15 minutes of arrest in the presence of Iso, mean MVO, of the heans was
A Perfusion Interval
0.089H.010 mL 02/min/g DW. MVO, did not significantly change after 25 mg/L Esm
was added to the peifusate of K + Iso (O.08SM.O 14 mL 02/min/g DW; p=0.8). Sirnilarly.
A
r e m to the original arrest pemisate of K + Iso to repeat the arrest conditions without and
then with 25 mg/L Esm demonstrated no significant change (0.07 lM.004 to 0.063H.009
mL 02/min/g DW: p=0.3). Recovery of M V 4 of the beating state is shown in the shaded
area of the graph (Figure 3.3) where pre-arrest MV02 was 0.42 1M.0 14 rnL Ojrnidg DW
and post-arrest MVOt was 0.387M.027 mL O,/min/g DW.
3.1.3 Pe fusion Arrest With Catecholamine Stimtrlution und Cd + -
Modulator
Studies were also done using BDM to determine whether this negative inotrope
codd reverse the change in MVOz induced by isoproterenol in the arrested myocardium.
As with the esmolol studies, experiments were initiaily done to determine the effect of
BDM alone in the arrested heart (Figure 3.4). The MVO, of hearts (n=6) arrested with K
did not significantiy change with the addition of 30 mM BDM to the hyperkalemic perfusate
(0.076+0.020 to 0.045M.006 mL Odmidg DW: p=O.l). Likewise. a subsequent
repetition of these two intervals of arrest (without and then with BDM) demonstrated no
change in basal MVO, ( 0 . 0 6 ~ . 0 1 0 to 0.057I0.013 rnL O,/min/g DW: p=0.9). The
shaded area of Figure 3.4 shows the recovery of the beating hem MVO, (pre-arrest MVO,:
0.378M.040 rnL Ojminlg DW; post-arrest MVO,: 0.361kO.W mL O,/min/g DW).
Time (min)
Figure 3.4. Effect of 30 m M BDM on bacal MW,. Basal MVO? did not signitïcnntly change upon the addition of 30 m M BDM ro the arrest perfusate. The shaded area shows the recovery of the beating stare MVO,. Values are rnea&SEM (n=61 Perfusion intervals: A , KHB; B. K; C, K + 30 mM BDM.
Following the protocol oudined for K + 25 nM Iso + 30 mM BDM in Table LI. the
C A .
effect of BDM during arrest in the presence of catecholarnines was determined (Figure
Perfusion in te mal
3.5). After equilibration with KHB for 30 min, h e m (n=9) were arrested with K+Iso. 15
B A
min afier which MVO, was 0.079W.018 mL OJminIg DW. No significant change in
C B
MVO, occurred with the addition of 30 mM BDM to the perfusate (0.058H.008 mL
Odmin/g DW) (Figure 3.5), despite an apparent pattern of decrease in MVO, (p=0.3).
Subsequent repetition of these two intervais of arrest (without and then with BDM. both in
the presence of Iso) demonstrated no change in basal MVO? (0.08W.O 14 to 0 . 0 5 0 . 0 12
mL OJrnin/g DW) (@.2). Recovexy of MVO, of the beating state is shown in the shaded
region of the graph (Figure 3.5; pre-arrest MV02: 0.37H.022 rnL OJmin/g DW: post-
arrest MVO,: 0.40 lM.024 mL O,/min/g DW).
Figure 3.5. Effecr of BDM on basal MVO, in the presence of isoprotereriui. Baal MVOZ stimulated with 25 nM isoproterenol did not significantly change upon the addition of 30 rnM BDM to the arrest perfusate. The shaded rireri shows the recovery of the beating state MVO:. Values are meankSEM (n=9). Perfusion intervais: A, KHB; B, K + 25 nM Iso; C, K + 25 n M Iso + 30 rnM BDM.
3.1 -4 Sumrnary of Reszilts for Basal Meta bolism Stridy
A
In this model, catecholamine stimulation significantly increased basal MVO,.
C
However, the addition of either esmolol or BDM did not significantly reverse this
catecholamine effect on basal MVO?.
B Perfusion Interval B A C
3.2 Peri-Ischemic Catecholamine Stimulation and Myocardial
Functional Recovery
3.2.1 Post-Ischemic Functional Recovery Following Cc~techof~mirrt?
Stimulation of Arresred Myocardium
As described in Materials and Methods. hearts were randomly assigned to one of
two groups: 1) Control Group: control hearts with no excessive catecholamine stimulation
during cardioplegic arrest and global ischemia: 2) Group Cp-Iso: hearts given cardioplegia
with 25 n M Iso (i.e. 20 mM K+ KHB + 25 nM Iso). The two groups in each of the
nomothermic and hypothennic ischemia studies did not differ in their baseline values for
systolic pressure. diastolic pressure. and developed pressure (Table III). Al1 values are
given as mean+SEM and statistical significance, as described in Materials and Methods. is
indicated as a significant ciifference with @.O5 using a two-tailed, unpaired t-test.
Developed Pressure. Al1 values are rne&EM, mmHg; numbers in brackets indicate sample sizes.
Ischemia 37°C
20°C
3.2.1 a Normothennic Ischemia Study
Groups in the nomothermia study had sample sizes of: Control= 1 1 : Cp-Iso= 12.
Normothermic ischemia was designated as an ischemic period of 60 minutes at 37°C.
Details of the experimentai protocol are in Materiais and Methods. and are depicted in
Figure 2.2 (p.41).
i) Change in Systolic Pressure Afer Normothennic Ischem ia
Recovery of systolic fimction was evaluated as the absolute change (A) in systolic
pressure of each group, from baseline values just before arrest to systolic pressure values
after 30 min repemision following 60 min normothermic ischemia (Figure 3.6). Hearts
which had 25 nM Iso added to the cardioplegia (Group Cp-Iso) had a -2 1 h S . 5 mmHg
Table III: Pre-ischemic basefine values. SP: Systolic Pressure; DP: Diastolic Pressure; DevP:
Group Control ( I l ) Cp-ISO ( 12) Control(8) Cp-ISO ( 10)
S Y 1 16.243.0 1 17.113.2 1 1 8.4k2.4 1 14.0f4.0
9.9k0.3 106.3t2.8 9.6+0.2 107.5k3.1 1
9.M0.5 1 09.4I2 -4 9.120.5 104.9&4.0
change in systolic pressure after 60 minutes of normothennic ischemia. This was not
significantly different (p=0.6) from the Control Group which was not subjected to
excessive catecholamine stimulation by Iso (- 19.7fl.7 mmHg).
Contml ~ m u p
Group Cp-ho
Figure 3.6. Post-nonnothermic ischemia change (A ) in qstolic pressure. Hearts exposd to 25 nM Iso (Group Cplso, n=12) pnor to arrest and global ischemia ar 37°C for 60 min dernonsmted no difference in recovery of systolic function compared to the Control Group (n= l 1 ). Values are meanfSEM.
ii) Change in Diastolic Pressure Afer Nomoihem ic Ischem ici
Recovery of diastolic fimction was evaluated as the absolute change (A) in diastolic
pressure of each group, from baseline values just before arrest to diastolic pressure values
afier 30 min reperfusion following 60 min normothermic ischemia (Figure 3.7). Group Cp-
Iso demonstrated decreased diastolic function reflected by a significant increase in diastolic
pressure (p=0.02) from its baseline value compared to the mean increase of 12.5S.0
mmHg in the Control Group. Specifically, Group Cp-Iso expenenced an increase of
2 1 -8k3.0 mmHg as diastolic pressure of hearts in this group changed from a mean baseline
value of 9.6M.2 d g prior to normothermic ischemia to 3 1.313.0 mmHg afterwards
(Figure 3.7).
Contrai ~ r o u p
Group Cp-lso
Figure 3.7. Posr-nomorhennic ischemia change ( A ) in diastolic pressure. Hearts stimulated with 25 nM Iso in the cardioplegia (Group Cp-Iso. n=l2) dernonstrated compromised port-ischemic diastolic function compared to the Control Group c n=l 1 1. Global ischemia was at 37OC for 60 min. Values are rneadEM. *p=0.02 vs. Control Group; two-tailed, unpaired t-test.
iii) Change in Developed Pressure Af er Normotherm ic Ischenz ici
Recovery of developed pressure was evaluated as the absolute change ( A ) in
developed pressure of each group, from baseline values just before arrest. to developed
pressure values after 30 min reperîusion following 60 min normothermic ischernia (Figure
3.8). The Control Group experienced a -32.3S.8 mmHg change in developed pressure
and Group Cp-Iso did not signifïcantly differ fp=O.l) from the Control Group. with a
recovery of -43.3k5.2 d g .
0 '
L 2 V1
-10-
L g 3 -20- control Group
Gmup Cp-lso
u 4
Figure 3.8. Post-normothennic ischemia change (A) in developed pressure. Administration of 25 nM Iso within the cardioplegia (Group CpIso. n=12) did not have any signiticantly detrimental effect on recovery of developed pressure, compared to Control Group (n= 1 1 ). Global ischemia was at 37OC for 60 min. Values are mean4SEM.
iv) End-ReperJhion Myocardial Adenine Nucelotide Concert trcitions
Afer Nonnothermic Ischemia
Biopsies of the myocardium were taken after the 30 min reperfusion period
foilowing normothermic ischemia to evaluate adenine nucleotide concentrations (Figure
3.9). Concentrations of AMP, ADP, and ATP were measured. and total rnyocardial
adenine oucleotides (TAN) were calculated. Cornparisons were made for each adenine
nucleotide between the two groups. Control Group (n=4) concentrations for AMP. ADP.
ATP, and TAN were 0.29M. 10, 3.25B.55, 1 1.18fl.78, and L4.73t 1.22 pmoVg DW.
respectively. The corresponding values for Group Cp-Iso (n=6) were not significantly
different (0.26M.08, p=0.8; 2.4M.34, p=0.2: 8.68k1.5 1. p=0.2: and 1 1.34I1.82.
p=0.2: p o V g DW, respectively).
Control Cp-Iso
AMP
ADP
ATP TAN
Figure 3.9. Post-normothermic ischemia myocardial adenine nueleoride concentrations. The presence of 25 nM Iso in the cardioplegia (Group Cp-Iso. n=6) did not affect end-reperfusion myoçardial adenine nucleotide levels. Global normothermic ischemia was at 37°C for 60 min. n=4 for Control Group. AMP: adenosine monophosphate; ADP: adenosine diphosphate; ATP: adenosine triphosphate; TAN: total adenine nudeotides. Values are m e d E M .
3.2. l b Hypothennic Ischemia Study
Groups in the hypothermia study had sample sizes of: Control=8: Cp-Iso=iO.
Hypothermic ischemia was designated as an ischemic period at 20°C for 120 minutes.
Details of the experimental protocols are in Materials and Methods and shown in Figure 2.2
(p.4 1 )-
i) Change in Systolic Pressure Afier Hypothemic Ischemiu
Recovery of systolic hnction was evaluated as the absolute change (A) in systolic
pressure of each group, from baseline values just before artest, to systolic pressure values
after 30 min reperfusion following 120 min hypothermic ischemia (Figure 3.10). The
Control Group experienced a decrease of 12.8325 rnmHg from its original systolic
pressure. Group Cp-Iso had similar results with a change of -15.M3.3 mmHg (Figure
3.10). Thus, there was no difference (p=0.6) in recovery of systolic function between the
two groups following hypothermic ischernia.
Control Group
Group Cp-Iso
Figure 3.10. Posr-hypothennic ischemia change ( A ) in sysrolic pressure. Herins stimulated with Iso within the cardioplegia (Group Cp-Iso, n=lO) regained systolic function, after 120 min of 20°C ischemia comparable to the control hearts. Control Group (n=8). Values are meanfSEM.
ii) Change in Diasrolic Pressure Afer Hypothermic Ischemia
Recovery of diastolic function was evaluated as the absolute change (A) in diastolic
pressure of each group, from baseline values just before arrest, to diastolic pressure values
after 30 min repehsion following 120 min hypothermic ischemia (Figure 3.1 1 1. In the
Control Group, diastolic function fully recovered, reflected by a mean average change of
-0.6k1.5 rnrnHg. Similarly, Group Cp-Iso had a mean change of O. I f 1.3 mmHg in
diastolic pressure (pa .7) .
Control Group
Group Cp-Iso
-21 1 -2.5
Figure 3.11. Post-hypothermie ischemia change ( A ) in diastolic pressure. Both Conuoi (n=8) and Group Cp-Iso (n=lO) hearts rnaintained their original diastolic tone folIowing 120 min ischemia at 20°C. Values are meanfSEM.
iii) Change in Developed Pressure Afer Hypothemic Ischmia
Recovery of developed pressure was evaluated as the absolute change ( A ) in
baseline developed pressure of each group. from baseline values just before arrest. to
developed pressure values after 60 min repemision following 120 min hypothermic
ischernia (Figure 3.12). Group Cp-Iso did not significantly differ from the Control Group
(p=0.6). Group Cp-Iso experienced a decrease of 13.9S.2 mrnHg compared to the
12.1e.3 mmHg decrease in Control Group hearts.
iv) End-Reperfusion Myocardial Adenine Nucelotide Concrnrrntions
Afer Hypothermie Ischemia
AMP, ADP, ATP, and total adenine nucleotide (TAN) concentrations in the
myocardium afier 30 min repemision foiiowing 120 min hypothennic ischernia of Group
CpIso were not signifiicantly Merent from their corresponding values in the control group
(p=0.4,0.5, 0.7, and 0.6, respectively; Figure 3.13). The values for the Control Group
Cwtrol Group Group Cp-Iso
Figure 3.12. Post-hypothermie ischemia change ( A ) in developed pressirre. Administration of 25 nM Iso within the cvdioplegia (Group CpIso. n= 10) had no effect on recovery of developed pressure compared to the Control Group (n=8). rifter 120 min hypothermie ischemia at 20°C. Values are meanfSEM.
0 Ai iP
I ADp ATP TAN
Control Cp-Iso
Figure 3.13. Post-hypothermie ischemia myocardial adenine rrriclrotidu concentrations. The presence of 25 nM Iso in the cardioplegia did not affect snd- reperfusion myocardial adenine nucleotide levels. Global normothermic ischemia was at 20°C for 120 min. n=8 for Control Group. AMP: adenosine monophosphate; ADP: adenosine diphosphate; ATP: adenosine triphosphate; TAN: total adenine nucleotides. Vaiues are meanHEM.
(n=8) were 0.3710.12, 3.56I0.47, 15.M.25, and 19.57I2.56 CIM/g
Those for Group Cp-Iso (n= 10) were OS4IO. 15, 3.9 lM.28.
2O.88+ 1.17 pM/g DW. respectively.
DW. respectively.
16.441 1.07. and
3.2.2 Post-Ischemic Functional Recovery Following Pm-Ischemic C~~trcliofumi~ir
Stimulation of Beating Myocardium
As described in Materials and Methods, hearts were randomly assigned to one of
two groups: 1) Group Pre-Iso/37 (n=l2): hearts stimulated with 25 nM Iso in the beating
state prior to arrest and 37°C ischemia for 60 min; 2) Group Pre-Iso/'>O (n= 10): hearts
stimulated with 25 nM Iso in the beating state prior to arrest and 20°C ischemia for 120
min. These two groups did not differ in their baseline values for systolic pressure.
diastolic pressure, and developed pressure (Table IV). A11 values are given as mem+SEM
and staûstical significance, as descnbed in Materials and Methods. is indicated as a
significant difference with ~ 4 . 0 5 using a two-tailed unpaired t-test.
3.2.2a IschemiaTemperature Effecton ChangeinSystolicPrerswe
Recovery of systolic function was evaluated as the absolute change (A) in systolic
pressure of each group, from baseline values just before arrest to systolic pressure values
Group Pre-Iso/37 ( 1 2) Pre-Is0/20 ( 1 O)
after 30 min reperfusion following ischemia (Figure 3.14). Hypothermia dunng ischemia
significantly protected systolic function of the Group Pre-Iso/2O hearts (p=0.003). These
hearts experienced only a 15.4fi.2 mmHg decrease in systolic pressure, compared to a
decrease of 3 1 .=.5 r n d g experienced by the Group Pre-Iso/37 hearts.
L
Table IV: Pre-ischemic baseline values. S P Systolic Pressure; DP: Diastolic Pressure: DevP: Developed Pressure. Al1 values are meaniSEM, rnmHg; numbers in brackets indicate siimple sizes.
D P 1 0.0+,0.4 9.6k0.4
SP 1 13.613.1 120.4I3.0
DevP 103.6k2.8 110.8+3.2
Group Pre-isd37 Gmup Pre-lsd2O
Figure 3.14. Effect of ischemia temperature on change ( A ) in systofic pressure. Beating hearts stimuiated with 25 n M Iso pnor to m s t and hypothermic ischemia (20°C. 120 min; Group Pre-lsoI20, n=lO) had significantly improved systolic function compared to hearts subjected to normothermic ischemia (37°C. 60 min; Group Pre-Iso/37. n= 12). Values are meanSEM. *~=û.003 vs. Group PR-Ison7; two-tailed, unpaired t-test.
3.2.2b Ischemia Temperature Effect on Change in Diastolic Presstcre
Recovery of diastolic function was evaluated as the absolute change (A) in diastolic
pressure of each group, from baseline values just before arrest to diastolic pressure values
after 30 min reperfusion foilowing ischemia (Figure 3.15). Hypothermia during ischemia
significantly protected diastolic function of the Group Pre-Iso/2O hearts (p<0.000 1 ).
These hearts experienced only a 8.8k1.6 mmHg increase in diastolic pressure. compared to
Group Pre-Isd37
Group Pre-Id20
Figure 3.15. Effect of ischernia temperature on change ( A ) in diastolic pressure. Beating hearts stimulated with 25 nM Iso prior to arrest and hypothermic ischemia (20°C. 120 min: Group Pre-Iso/20, n=10) had significantly improved diastolic relaxation. compared to hearts subjected to normothermic ischemia (37". 60 min). Values are meanSEM. *p<0.0001 vs. Group Pre-Iso/37; two-tailed, unpaired t-test.
an increase of 2 1.CE1.5 mmHg experienced by the Group Pre-Iso/37 hearts.
3.2 .2~ ischemia Temperature Ejfect on Change in Developecl Pressure
Recovery of developed pressure was evaluated as the absolute change ( A ) in
developed pressure of each group, from baseline values just before arrest to developed
pressure values after 30 min repemision following ischemia (Figure 3.16). Hypothermia
during ischemia significantly attenuated the decrease in developed pressure that occurred
under normothermic conditions (p<0.0001). Developed pressure of Group Pre-Iso137
hearts decreased by 52.6k4.0 mmHg whereas in Group Pre-Iso/20 hearts. the decrease
was by 2423 .4 rnmHg.
1 G r w p Preiso/37 * Group P r e - M O
Figure 3.16. Efecr of ischemia temperarure on change ( A ) in developed pressure. B eat i ng hearts stimuiated with 25 nM Iso pnor to arrest and hypothermic ischemia (20°C. 120 min; Group Pre-Iso/20, n=10) had significantly improved pressure development, compared to hearts subjected to normothermic ischemia (37", 60 min). Values rire meankSEM. *p<0.0001 vs. Group Pre-IsoL37; two-tailed, unpaired t-test.
3.2.M lschernia Temperature Effect on End-Reperjirsion Myoccrrclid
Adenine Nucleotide Concentrations
End-reperfusion ADP (p=0.0002), ATP (pc0.000 l ) , and total adenine nucleotide
(TAN) (p<0.000 1 ) concentrations in the myocardium were significan tl y prese rved in hearts
subjected to hypothermic ischemia foilowing catecholamine stimulation in the bea ting s tate
(Group Pre-Iso/2O, n=8), compared to hearts under normothermic conditions (Group Pre-
Iso/37, n=l 1) (Figure 3.17). AMP, ADP, ATP, and TAN values for Group Pre-Iso/20
were 0.3 lM.06, 3.66H.42, L5.62ki .49, and l9.58+1.93 prnoVg DW. respectively.
Those for Group Pre-Isol37 were 0.2 lM.06, L.8M. 12, 5.24k0.68. and 7.35M.76
p o V g DW. respec tively .
AlWP ADP
ATP TAN
Figure 3.17. Ischemia temperature effect on end-reperfrrsion ni~ocardiul udeninr nucleotide concentrations. Hearts treated with 25 nM Iso in the beating state p ior to 37°C arrest for 60 min (Group Re-Iso/37, n=l 1) were characterized by significantly lower ADP. ATP, and TAN tissue concentrations than hearts under hypothermic ischemia conditions (20°C. 120 min; Group Pre-Isof20, n=8). Vdues are rneanSEM. *p10.002 vs. Group Pre-Iso/37; two-tailed, unpaired t-test. AMP: adenosine monophosphate; ADP: adenosine diphosphate; ATP: adenosine triphosphate; TAN: totai adenine nucleotides.
3.2.3 Ischemia Temperature EIfect on Change in Diastolic Pressure in Control
and Cp-Iso Groups
Post-normothermic ischemia diastolic function in h e m exposed to catecholamines
during arrest (Group Cp-Iso) was the only sigrilficantly compromised parameter in the first
part of the ischemia study (i.e. Control vs. Cp-[so experirnents, Section 3.7.1). To funher
evaluate the protective effect of hypothermia, the change in diastolic pressure following
normothermic ischemia was compared to that foilowing hypothermic ischernia for both the
Control and Cp-Iso groups (Figure 3.18). Statistical analysis showed that hypothermia
during ischemia ~ i ~ c a n t l y attenuated the deletenous effects of normothermic conditions
during ischemia in both untreated (p=0.0001) and catecholamine-exposed hearts
(p<O.OOO 1).
e 3 m
20- V1
[p 1s *
= E io- s E a - 5 *
P d 0-
Control Cp-Iso
Nonnothermie lschemia
Hypothermie Isçhemia
Figure 3.18. Eflect of ischemia temperature on post-ischemic change ( A ) iri diusrolic pressure following catecholamine stimulation during arresr. Diastolic function of herirts subjected to 20°C hypothermic ischemia demonsuated improved recovery over herins subjected to 37OC normothermic ischemia. Normothermia n-values: ControI= I l . Grnup Cp-Iso= 12; hypothermia n-values: Control=8, Group Cplso= 1 O. Values are meanSEM. *p9.000 1 vs. corresponding group in nonnothennia; two-railed. unpaired t-test.
3.2.4 Summary of Ischemia Study Results
In this model, catecholarnine stimulation during arrest significantly compromised
post-nonnothermic ischemia diastolic functional recovery. The diastolic functional
compromise was drarnaticaiiy attenuated by lowering ischemic temperature and was not
linked to decreased levels of myocardial adenine nucleotides. in contrast, preserved levels
of myocardial adenine nucleotides accompanied the protective effect of hypothermic
ischemia Ui hearts stimulated with catecholamines in the beating state pnor to arrest.
DISCUSSION
Excessive catecholamine stimulation has been documented to be deleterious to the
non-ischemic [Rona et al., 1959; Rona et al., 1975; Mosinger et al.. 1977: Steen r r c d . .
1982; Caspi et al., 19931 and ischemic myocardium [Maroko et al.. 197 1 : Maroko et c d . .
1973; Karlsberg et al., 1979: Muntz et al., 1984; Yoshida and Iimura. 19891. Based on
such evidence, the effect of an elevated levei of catecholamines. such as that seen in blood-
based cardioplegia, on the globally ischemic rnyocardium, was questioned. Thus. it was
hypothesized that catecholarnines can affect basal metabolism of the heart. and that this
effect codd be iinked to a compromised pst-ischemic functional recovery.
The Langendorff system was used because of its widely accepted use in whole
heart studies. The modifiability, simplicity, and stability of this preparation dlow for the
studying of a large number of hearts quite efficiently . The system allows for ease of dnig
administration and removal and evaiuation of hem function wi thout the poten tial
complications arising from the body system in an in vivo preparation.
Myocardial oxygen consumption (MVO,) is largely represented by the eneqy
required for the contraction process of the beating state of the heart. However. the energy
required to maintain the integrity of the hem even in the arrested state is an important
determinant, although to a lesser degree, of MVO?. Basal energy requirements include
energy required for maintaining protein synthesis and compartmentalized differences in ion
concentrations against membrane leaks. Optimal cardioplegic arrest should maximize
myocardial protection from ischemia-induced injuries and minimize metabolic and
mechanical energy requirements while the rnyocardium is in its basal state. However. if an
elevated level of catecholamines is present within the cardioplegia. such as with blood-
based cardioplegias, the protective nature of the cardioplegia may be comprornised.
Studies in arrest metabolism have reported basal metabolism to range from about
12% in rats [Stembergh et al., 19891 to 46% in dogs [Gibbs et al., 19801 of the empty-
beating heart metabolism (see Table 1, p.15). Arrest MVOz values in this study ranged
from 15.88 (Figure 3.1, p.49) to 27.9% (Figure B.1, p.93). Comparably. a study by
Gibbs and Kotsanas [1986] on rabbit hearts demonstrated a 77% drop in the beating. non-
working MVO, upon arrest with 30 mM K+ solution. Considering the variations in
experimental conditions, the values presented in this study do roughly correspond with
MVO, values found by others and it appears that in this model. basal MVO, constitutes
about one-sixth ro over one-quater of empty-beating MVO,. However. one thing to
consider in the variability of the MVO* values is the fact that it is calculated as a unit per dry
weight. Dry weight of the hearts were, on average, 11% of wet weights (Appendix D.
pp.96-97). This indicates a 89% water content which may or may not have greatly
influenced the MVOZ results. If the majority of edema, due to the low oncotic property of
crystalloid perfisate. occurred in the beginning of the experiment. then the MVO, value
would be equally "erroneous" throughout ai i subsequent intervals. However, if the edema
occurred sometime throughout the perfusion intervals, then MVO, results could vary
largely from one perfusion interval to another.
4 . 1 Basal Metabolism Under Varying Arrest Conditions
4.1.1 Reasons for not Using Epinephhe as the Catecholamine
Although the protocol describes using isoproterenol as the catecholamine challenge.
initially. epinephrine had been chosen as the candidate catecholamine. Results using
epinephrine are given in Appendix A (pp.90-92). Pilot studies (unpublished data. LM.
Rebeyka) had shown that plasma epinephrine Ievels increased from less than 0.8 nM to
more than 2.8 nM after institution of the cardiopulmonary bypass apparatus. Thus. to
sirnulate the elevated catecholamine levels of the clinical situation, a dose of 2 nM Epi was
initially used. However, 2 n M Epi in the presence of potassium arrest (Figure A. 1.
Appendix A, p.90) did not significantly change MVO,. Although the focus of the study
was to determine the effect of the presence of catecholamines during arrest. the effect of 2
Discrissiorr
nM Epi on the beating heart was questioned. That is, the absence of any apparent effect of
2 nM Epi may have been due to a masking effect of the potassium arrest. or due to the lack
of any effect at dl. To address this issue, further experiments were done to confirm that
the dose of 2 nM Epi was experimentally measurable in this model of the beating. non-
working rabbit heart. The results are given in Figure A.2. Appendix A (p.91). No
sigmficant change in M V 4 was observed in the beating, non-working hem upon exposure
to 2 nM Epi.
Considering the nature of epinephrine and its known positively inotropic and
chronotropic effects [Kaufman et al., 1951; Lee and Yu. 1964: Suga et al.. 1983: Endoh
and Blinks, 19881, an increase in MVO, was expected in the beating heart model. The lack
of any effect of administe~g 2 nM Epi may be that the model is not sensitive enough to
detect the increase in MVOz caused by this particular dose of Epi. or that the human
sympathetic response is different from the rabbit's [Downing and Chen, 19851 and so. the
increased dose of 2 nM Epi found in humans rnay not have been transferable to the rabbit
rnodel. Furthemore, alpha,-receptor stimulation by epinephrine has k e n shown to have a
positive inotropic effect in the rabbit myocardium [Endoh et al.. 199 1 1. Generali y.
catecholamine cardiotoxicity has k e n considered to be through the beta-adrenergic receptor
pathway, as increased inotropy, chronotropy and energy demand exceed supply. thereby
creating "ischemic-like" injury. However, a study by Downing and Chen [1985]
investigating the effect of catecholamine cardiotoxicity in the rabbit rnyocardium reveded
that activation of the alpha-adrenergic pathway is the dominant rnechanism of injury in the
rabbit myocardium. Yet. in the rabbit, the maximal effects of beta-stimulation were
demonstrated to be greater than the maximal effects of alpha-stimulation [Endoh and
Blinks, 19881. The importance of the alpha,-response to positive inotropy in the rabbit
suggests that the alpha-mediated catecholamine effects seen in this expehental model rnay
not be as great as they could be.
Alpha, -receptor activation has been shown to increase intracellular Ca2+ throug h
CAMP-independent pathways, and thus, can result in inotropic stimulation of the
myocardium [Bruckner et al., 1984; Scholz et al., 1988: Endoh et al.. 199 11. If so. the
combined alpha and beta effects of epinephrine would be expected to increase MVO,. The
effects of 25 nM Epi and 50 nM Epi (Figures A.3 and A.4, Appendix A. pp.9 1-93 were
also tested in the empty-beating hem, and there was a tendency for MVOl to increase with
these higher doses but these changes were not statistically significant.
In contrast to the possible positive inotropic effects of alpha,-stimulation. Oleksa et
al. 119961 and Chen et al. [1996] have recently found an inhibitory effect of alpha,-
adrenergic stimulation on beta-adrenergic responses. Using norepinephrine. Oleksa et d.
[1996] found that the activation of the Cl- current through beta-adrenoceptor stimulation
was lirnited by the intrinsic ability of norepinephrine to also activate alpha,-adrenergic
receptors. (NormaUy, because & is about -50 mV, CAMP-dependent activation of the Cl-
channel has two roles in beta-stimulation: it enhances excitability by helping to depolarize
the cell, and it shortens AP duration to preserve diastole, and coronary blood flow. at faster
heart rates [Weiss, 19971). Sirnilarly, Chen et al. (19961 found that the L-type Ca2+ current
stimulated by beta-adrenergic agonists was inhibited by alpha, -adrenergic activation. W ith
the possibility that alpha, -adrenergic stimulation may be inhibitory to be ta-adrenerg ic
responses, and considering how beta-adrenergic stimulation increases MVOZ in the beating
heart, any alpha,-stimuiation rnay confound the MVO, effect of beta-stimulation in the
myocardium. In summary. the precise role the alpha-adrenergic response has in the
rnyocardium and how it interacts with the beta-adrenergic response is yet to be elucidated.
Thus, whether an alpha,-adrenergic antagonist could depress the possible inhibitory effect
of alpha,-stimulation on beta-stimulation or block the vasoconstrictive response to alpha, -
stimulation, an increase in MVOz would be expected upon isolating the beta-effects of
epinephrine.
Another possible reason for the lack of any expected effect from Epi may have
sternmed fiom a possible complication of its beta-adrenergic effects by its vasoac tive aipha-
adrenergic effects [Mohrman and Feigl. 1978: Feigl, 19871. That is. it may have been
difficult to detect the beta-effect of Epi since the parameter measured, MVO?. is directly
proportional to coronary flow, which is negatively affected by the vasocons tnctive alpha-
action of Epi Berne, 1958; Imai et al., 19751.
An additional study was therefore conducted with ephephrine to determine whether
its alpha-effects may indeed have substantiaily affected the MVO, results. In order to
determine whether the Iack of any change in MVO (in the beating hearts experiments) was
secondary to alpha,-stimulation. prazosin was used to block the alpha, -effects of
epinephrine while isolating its beta-effects. Prazosin (Prz) is a cornpetitive alpha, (post-
synaptic)-selective adrenergic antagonist which causes less tachycardia than other equi-
effective vasodilating dmgs [Cambridge et al., 19771. Thus, the effect of prazosin will
focus on its vasorelaxant properties with minimal interference with the other mec hanical
detemiinam of MVOz since the experiments with epinephrine and prazosin were done in
the beaùng heart model. The resuits of the effect of epinephrine in the presence of prazosin
in the beating, non-working heart are shown in Figure AS (Appendix A. p.92). After 30
min equilibration with KHB, the MVOz of hearis did not change with the addition of 25 nM
Epi (0.450M.04 1 ml 021rnin/g DW to 0.46 110.026 mL O,/min/g DW). Prazosin was then
added to the KHB+Epi perfusate and MVOz of hearts significantly decreased to
0.372M.030 mL O,/rnin/g DW (p<0.05, Figure A S , Appendix A, p.92).
Due to the conflicting reports on the action of alpha,-adrenergic receptors. it is
difficult to determine why such an effect was seen using prazosin. If alpha,-receptors do
indeed have a positive inotropic effect, then perhaps prazosin inhibited the positive
inotropic action of alpha,-stimulation complementary to beta-stimulation. However, since
epinephrine did not have any augmentative effect at ail on M V O in this model, the results
do not support this possibility. If, in contrast, alpha,-adrenergic receptor stimulation c m
suppress the upregulation of CaZ* and Cl- currents by beta-stimulation. then prazosin
should have potentiated the beta-effect of epinephrine. Yet. no effect was seen in these
experiments. Statistical analysis of the change in coronary flow (Table V(b). Appendix D.
p.97) from the Epi perhision interval to the Epi+Pn. perfusion interval (Intervals B to C .
Figure AS, Appendix A, p.92) indicates a significant increase in coronary flow in the
presence of prazosin (p4.02 , two-tailed, paired t-test). With the increased coronary flow.
the hearts decreased the amount of O2 extracted from the perfusate. and this decrease was
enough to significantly lower the MVOz value of the interval (Interval C). Hence. although
the dose of prazosin used was effective in this model. the dose of epinephrine used rnay
still not have been enough to demonstrate marked beta-adrenergic effects on MVO,.
The most plausible explanation for the lack of an Epi effect is that the dosage of Epi
may have k e n too low. The dosage study up to 50 nM Epi showed no significant effect.
indicating the possibility that even higher doses were required for a clear beta-effect to
occur. This possibility is supported by studies which have shown that low doses of Epi
have a greater alpha-effect with very little beta-effect, while higher doses of Epi
demonstrate a stronger beta-effect [Benfey and Varma. 1967: Endoh and Blinks. 19881. In
fact, most studies using Epi have used doses around the rnicromolar range [Century. 19%:
Lee and Yu, 1964; Challoner, 1968; Endoh and Blinks, 19881 compared to the nanomolar
value used in this study to simulate the clinical situation (see p.68). If the dose is too low
for a beta-effect, then any positive inotropy effected by the alpha-response would be
suppressed by prazosin, which may explain the decrease in mean MVO, as prazosin was
added to the KHB+Epi perfusate (Interval C, Figure AS, Appendix A. p.92).
The Epi experiments provided interesting insight into the complicated mechanisms
of catecholamine stimulation. Until M e r experirnents can be done with proper dosages.
no conclusions can be drawn except that upregulation of contractile function rnay aise from
the interaction of both alpha- and beta-stimulatory effects which regulate each other to
uitimately regdate myocardial function.
4.1.2 Catecholamine Stimulation of Arrested Myocardit<m
In consideration of the arnbiguous results with epinephrine. the catecholarnine of
this experimental mode1 was changed to a h g that was simpler in action for a more
unifactorial effect. Specifically, isoproterenol was considered because it has only a beta-
effect [Erlij and Mendez, 1964; Endoh and Blinks, 19881. Since the heart is predominantly
a beta,-receptor tissue, in both humans and rabbits Brodde et al.. 1986: Tenner et (11..
19891, the focus was on the beta-effect of catecholamines in the myocardium.
Isoproterenol could be used to do so and thereby, to eventually determine whether any
decreased post-ischemic functionai recovery due to pen-ischemic catecholamine stimulation
could possibly be attributed to the beta-effect of catecholamines and whether this beta-effect
affected basal MVO, despite the absence of mechanical activity.
Prier to starting with isoproterenol in the arrested state, its effect in the beating.
non-working heart was determined to c o d m its activity (Figure B. 1, Appendix B. p.93).
Unlike the results with epinephrine, 25 nM Iso demonstrated an unequivocal increÿse in
MVOz (pd.007 vs. initial KHB pefision interval). MV02 significantly increased by over
50% of its control period value (0.404W.067 vs. 0.622Hl.049 mL O2 Iminlg DW).
Furthemore, this dose of Iso did not appear to be cardiotoxic, as pre-Iso MVO, values
were recovered after 30 min of reperfusion with KHB following a 30 min perfusion
interval with Iso (Figure B.1, p.93). Thus, further experiments were continued using
isoproterenol.
The marked increase in MVOz observed in the isoproterenol study is not surprising
as isoproterenol, a synthetic catecholarnine, is a known potent beta-receptor agonist that is
stnicturally related to epinephrine [Kaufman et al., 195 11. Isoproterenol increases both the
heart rate (chronotropy) [Kaufman et al., 195 1 ; Kassebaum and Van, 1966: Hasenfuss et
al., 19891 and the contractility (inotropy) [Kaufman et al., 195 1 ; Hasenfuss et al.. 198%
Futaki et al.. 199 11 of the heart, both of which affect myocardial oxygen consumption of
the beating hem [Somenblick et al., 1965; Suga et al.. 1983: Hasenfuss et cd. . 1989:
Futaki et al., 199 11.
The fact that the myocardial response to Iso was a clear increase in basal MVOI.
compared to the response to Epi, could be attributed to both the dose and mechanism of
action. Whereas 25 nM Iso demonstrated a definitive increase in MVO, of beating hearts
(Figure B. 1, Appendix B, p.93). the same dosage for Epi did not (Figure A.3. Appendix
A, p.91). This is not surprising as Epi is 10-40 times less potent at betai-adrenoceptors
and 3-15 times less potent at betar-adrenoceptors than Iso [Biiezikian, 19871. In fact.
EUocke et al. [1965] showed that Iso could increase beating heart MVO, by 55% whereas
the same dose of Epi ody increased MVO, by 5%. As discussed in detail above. Epi haï
alpha-effects as well as beta-effects. In fact, Epi has a stronger alpha- than beta-action.
except at higher doses Penfey and Vanna, 1967: Endoh and Blinks, 19881 and thus. the
alpha-effects may be confounding any beta-effect.
Clearly, beta-adrenoceptor-mediated mechanicd stimulation increases the energy
expenditure of the heart, reflected in increased myocardiai oxygen consumption [Eckstein et
al., 1950; Fisher and Williamson, 196 1; Klocke et al., 1965; Sonnenblick et al.. 1965:
Gibbs et al., 1967; Coleman et al., 1971; Suga et al., 19831. However. whether
catecholarnines have any direct effect on myocardial metabolism. independent of their
cardiodynamic effects, is yet to be fully understood. The present study exarnining the
effect of isoproterenol on MV02 of the arrested myocardium addressed this question. By
administering catecholamines to a quiescent heart, any effect the beta-adrenergic agonist
would have on energy state would be reflected in a change in basal metabolism. As
hypothesized, isoproterenol did significantly affect basal MVO, (Figure 3.1. p.49).
increasing it by over 60% of the control arrest MVO, value.
Thus, results in both the beating (Figure B. 1, Appendix B, p.93) and arrest
experiments (Figure 3.1, p.49) with Iso dernonstrated a more than 50% increase in MVO,
from the control beating and control arrested States, respectively. Klocke et al. [1965],
Discussion
however, found that a dose of Iso which increased beating hem MVOz by 55% (0.30 pg)
ody increased basal MVO, by 5%. An interesting observation is that Klocke er cd. [1965)
found that their arrested heart MVOl averaged 78% of the beating heart MVO,. which is in
sharp contrast to the aforementioned 16-28% values found in the literature and these
studies. The unusually high basal MVOz could be attributed to the fact that the Klocke
group had also stimulated the hearts with Iso in the beating state pnor to arrest. As
Lochner et al. [1968] had previously shown, basal MVO, is influenced by the state of the
beating heart prîor to arrest and thus, the heans in Klocke et al.'s [1965] srudy had a high
basal MV02 Perhaps by raising basal MVO so much with high pre-arrest activity. any
effect of Iso was relatively smali, and therefore masked in the basal state.
Hanley and Loiselle [1992] have documented the great influence basal intracellular
Car+ concentration has on basal MV02. Thus, a possible explanation for the increase in
basal MVO, effected by Iso may be through an increase in Car+ mobilization within the
myocyte despite the absence of mechanicd activity. This increased Cd+ would not be to
the exrent seen in conjunction with the contractile state, but enough to affect the basal
metabolism of the heart as more energy must be used to maintain intracellular Ca2*
homeostasis, especially c o n s i d e ~ g that the SR Ca2+-ATPase may contribute 2 8 8 to the
basal MVO vdue [Ponce-Homos, 19901.
The active nature of the resting state of the myocardium with respect to intrücellular
Ca2+ concentration was demonstrated by Lappé and Lakatta [1980], who found that resting
force varied Linearly with Cal+ concentration in resting rat right ventricular papil lq muscle
exposed to stepwise increases and decreases in Gaz+ concentrations (0.44 mM) in the
bathing fluid. Further studies by this group supported their initial findings and sought to
determine the relationship between diastolic Ca2+ flux and subsequent twitch force in
isolated rat and cat papillary muscles Lakatta and Lappé, 198 11. Their results suggested
that an increase in the Ca2+ concentration during diastole could predict an increased twitch
force up to a certain concentration. This effect could possibly be explained by a "prirning"
effect of the Ca2+ concentration on the contractile proteins or. the uicreasing concentrations
could be inducing greater release of Ca2+ from the SR upon inward current activation. since
the elevated Ca2+ concentration already present in the cytosol would make it that rnuch
closer to the threshold of release. Thus, in diastole, or the resting state of the heart.
increased Cal+ activity rnay be occurring. enough to stimulate increased mitochondrial
respiration [Denton and McComack L980a; Denton et al., 1980: Denton and McCormack.
1980b; Denton et al., 1988; McCormack and Denton, 1979; Rutter and Denton. 1988:
Rutter et al., 19891 or the SR Ca2+-ATPase pumps. but not enough to cause a contraction.
This is what may have occurred in the Iso-treated arrested myocardium and may explain
how Iso increased basal MVO, in the arrested heart (Figure 3.1. p.49).
Further support of this possibility cornes from Solaro et al. [ 19741 who conducted
experiments in purified canine cardiac myofibrils to determine the amount of calcium
required for myofibrillar activation. For the rnost part. ATPase activity correlated almost
exactly with isornetric tension activation except for in the lower Ca?+ concentrations of IO1
to 106 M Cal+, where ATPase activity demonstrated elevated activity over tension
developrnent. Solaro et al. [1974] suggested that at these low Ca'+ concentrations, not
enough to cause a tme contraction, cross-bridge formation might be discontinuous dong
the length of the filament, where ATP rnay be gening hydrolyzed but overall. no tension is
generated at the fibre ends.
Although the results of the effect of catecholamine stimulation in the arrested
myocardium demonstrated an increase in basal MVO,, one cannot conclude whether the
catecholamine effect in the arrested heart in this mode1 was one that resulted from direct
stimulation of oxidative metabolism or whether the increase in basal energy expenditure
was secoadary to an increase in intracellular Gaz+. The augmented MVO, rnay even have
been a combination of both.
4.2 Peri-Ischemic Catecholamine Stimulation and Myocardial
Functional Recovery
If, as it was hypothesized, catecholamines can substantially affect basal MVOZ. then
this effect may be deleterious to the ischemic heart. Thus. catecholamines were
administered within the cardioplegia (Group Cp-Iso) to see if the basal effect of
catecholarnines seen in the pefision arrest experiments could be representative of the
ischemic situation. The control group (Control) had no catechoiamine stimulation.
Interestingly, hearts that were stimulated with 25 nM Iso in their arrested state (Le. Group
Cp-Iso) and then were subjected to normothermic ischemia had a significantly elevated
post-ischemic diastolic tone (Figure 3.7, p.57).
Many studies have shown that diastoiic relaxation is incornpiete and stiffness is
increased under conditions of ischemia [Mathey et al., 1974; McLaurin et cil.. 1973:
Paiacios et al., 1978; Serur et al., 1976; Weisfeidt et al., 1974; Diarnond and Forrester.
19721. Relaxation is an energy-dependent process, requiring the activity of the ATP-
dependent Ca2+ pumps of the SR. In contrat, activation is a passive process where Ca2-
can diffuse down its concentration gradient from the extracellular space and the
sarcoplasrnic reticulum, where the Caz+ concentration is in the rnillimolar range. into the
cytosol where the Ca" concentration is about 0.2 p M [ A h et al.. 1994: Cobbold and
Rink, 1987; Katz, 1992; Lee et al., 19871. The maximal rate at which Ca:+ c m be removed
from the cytosol to relax the heart is about an order of magnitude lower than the rate at
which influx occun to activate the heart [Katz, 19921. Thus, an energy-starved heart is
easily susceptible to Caf+ overload which would compromise the relaxation and tone of the
myocardium during diastole.
Although the relaxation property per se of the heart (Le. -dP/dtm,,) was not
measured, Mathey et al. [1974] demonstrated that an exponentiai relationship exists
between diastolic stiffness and relaxation ability. Stiffness is estirnated as dP/dV and since
the experimental mode1 used isovolumic conditions, an increase in diastolic pressure would
indicate increased stiffness. Katz [1992] indicates that stiffness may be considered an
index of the extent of Ca2+ cemoval from troponin. If so, the increased diastolic pressure
seen in hearts in Group Cp-Iso of normothermic ischemia and Group Pre-ho137 may be
indicative of increased levels of cytosolic Ca2+ that may have been induced by the excessive
catecholamine stimulation.
In Lakatta and Lappé's 1198 11 study on diastolic Cal+ concentration and twitch
force, a decline in twitch force was seen with supra-optimal Cal+ concentrations during the
resting state, indicating that if diastolic tone was too elevated with the presence of too much
Ca2+, mechanical function would be cornpromised. The higher levels of diastolic
myoplasmic calcium concentration could be a type of "diastolic Gaz+ overload" which
would contribute to a decline in mechanical function as diastolic tension would be greater
Kakatta and Lappé, 198 11. If the heart is too stiff, it cannot preload sufficiently and. in
relation to the force-length relationship curve of muscle. its ability to pnerate maximal
force is impaired at shorter lengths (Katz, 19921. This is reflected in decreased systolic
pressure deveiopment that accompanies increased diastolic stiffness. according to the
Frank-Stariing relatioaship [Katz, 19921. Eventuaily, if too much Cal+ enters the cell. such
as during the reperfusion process, the cell may undergo complete Ca2+ overload.
reminiscent of that resulting from catecholamine-induced myocardial injury. Appropriate
intracellular Ca?+ concentration, therefore, is crucial not only for proper relaxation but also
for proper contractile function.
It is interesthg to note that the changes in SP, DP, and DevP experienced by the
Group Fre-Iso/37 h e m (excessive catecholamine stimulation in beating state before arrest)
were even greater than those experienced by either the hypothermia Control or Group Cp-
Iso hearts. Furthemore, even though the change in diastolic pressure for Group Pre-
Iso/ZO was significantly attenuated by hypothermia (Figure 3.15, p. 63). this change was
in contrat to the lack of any change seen with the Control Group and Group Cp-Iso under
hypothermie ischemia conditions (Figure 3.1 1, p. 60). This suggests that excessive
catecholarnine stimulation exacerbates ischernic damage to a greater degree if the
myocardiurn is exposed to the catecholamines in the beating state just pnor to arrest (such
as with Groups PR-Isol2O and Pre-1~0137). rather than during arrest. The amount of CaL+
mobilized with each beat would be greater than the amount mobilized during mest (eg .
with Group Cp-Iso) and thus, the myocardiurn would be "pre-loaded with an increased
arnount of Ca2+ via the CAMP-dependent upregulation of CaL+ influx and SR Ca'+ uptake.
Because the level of basal meiabolism is influenced by the metabolic level of the preceding
beating state [Lochner et al., 19681, hearts stimulated with Iso in the beating state prior to
arrest would be predisposed to a higher basal metabolism during arrest. Thus. despite the
fact that coronary blood supply was elirninated during arrest, energy demand would be at
an elevated level.
With increased energy demand, ATP breakdown would occur. As ATP gets
catabolized to adenosine, then inosine, then hypoxanthine, energy levels would decrease
and Ca'+ homeostasis wouid be more difficult to maintain. The rise in intraceflular Ca2+
would activate a Ca"-dependent protease in the cytosol, whic h attac ks xanthine
dehydrogenase. Xanthine dehydrogenase normally reduces NAD+ to NADH while
oxidizing hypoxanthine to xanthine. However, when proteolytically rnodified. a new
enzyme activity emerges. xanthine oxidase, which reduces molecular oxygen to produce
the superoxide radical [McCord, 19841. Thus, re-introduction of oxygen upon reperfusion
will trigger the production of superoxide radicals which react with lipid molecules and lead
to the dismption of the cellular and intracellular membranes [McCord. 19841.
Consequently, further increase in intracellular Ca2+ concentration would occur through the
disrupted membranes, thereby exacerbating the damage.
Degradation of ATP past AMP (Le. to adenosine and its catabolites) results in the
loss of precursors for re-synthesis miedmeier et al., 1972; Snow et al., 1973; Berne and
Rubio, 19741. In rabbits, where the mitochondrial synthesis of adenine nucleotides is
primarily due to the salvage pathway which uses such precursors (in contrast to the de
novo pathway using ribose-5-phosphate Fossi, 19751). the loss of preformed precursors
could therefore bave a profound effect. Not only will loss of precursors limit ATP
resynthesis but aiso, the mitochondria rnay also be too damaged to synthesize ATP. Thus.
the decreased levei of ADP in Group Pre-IsoB7, could have limited ATP re-synthesis via
the creatine kinase-catalyzed reaction using phosphocreatine and ADP. Also. it may be
possible that the Ca'+ overload effect in Group Pre-[sol37 led to depletion of ATP and
production of superoxide radicals upon reperfusion, both of which contributed to disabling
mitochondrial energy metabolism by consequently overloading the mitochondna with Ca2+
as weil.
Decreased levels of adenine nucieotides, however. cannot account for the
comprornised diastolic function of Group Cp-Iso following normothermic ischemia. In
this case, the protection afforded by cardioplegia, despite the concomitant presence of
catecholamines, could have also rninunized the extent of ATP depletion. thereby allowing
for an easier recovery of ATP stores upon repemision. Engelman et al. [1979] studied the
time course of myocardial high energy phosphate degradation under conditions of both
normothennic and hypothermic (8-15°C) cardioplegic arrest. They found that ATP decay
was much more dramatic in the normothermic group. despite hyperkalernic cardioplegia.
compared to hypothermic potassium arrest, but both had better ATP preservation than
groups without cardioplegia at dl. In this study, because the myocardium was sampled for
adenine nucleotide concentrations at the end of the 30 min reperfusion period. the hearts in
Group Cp-Iso may have been able to restore their ATP levels. It may have been more
informative, therefore. to obtain biopsies immediately after the ischemic period. for a better
idea of the extent of ATP decay under each condition. Such data would also provide
information about the catecholamine effect on ATP during ischemia (eg. Group Cp-Iso
data). Due to the s m d size of the hearts used, though, this could not be feasible.
In considering the ATP data, a few factors should be taken into account. however.
ATP and its intermediates have been shown to be compartmentdized within the ce11 but the
method used to measure their concentrations only aüowed for determination of the average
tissue content. The ATP which determines tissue survival may be in relatively small
compartments whose contents may be masked by changes (or lack thereofl in other
compartments upon homogenization of the myocardial tissue [Miller and Horowitz. 19861 .
This theory is supported by reports that ischernic hearts can suddenly lose contractile
activity despite maintenance of relatively high ATP levels [Gudbjarnason et cil.. 19701.
Gudbjarnason et al. [1970] documented that contraction ceased when the ATP
concentration had dropped from only 5.7 to 4.5 pnoVg during ischemia. Thus. depletion
of ATP stored in a critical subcompartment of the cytosol may have occurred but could not
be detected upon andysis of the tissue homogenate of the normothermic ischemia Group
Cp-Iso hearts.
Although only Group Re-Iso/37 demonstrated any significant depletion of adenine
nucleotides, one cannot conclude that the control groups had fully preserved adenine
nucleotide concentrations since control levels were not recorded prior to the ischemic
penod. Vanoverschelde et al. [1994], however, found that isolated. Langendorff-perfused
rabbits hearts at 37°C had the following concentrations: AMP= 1 .OM.3: ADP=7.OM.7:
ATP=25.8+1.7; TAN=33.7+1.9 prnol/g DW, and Hearse et al. El9751 found similar ATP
concentrations in the Langendorff-perfûsed rat heart. Cornparison with the normothemic
ischemia Control Group values indicates a decrease of about 50% for al1 myocardial
adenine nucleotides.
In consideration of the protective effect of hypothermia and K+-cardioplegia. it is
not surprishg that only the hearts which experienced an excessive catecholarnine challenge
under normothermic conditions in addition to normothennic ischemia (i.e. norrnotherrnia
Group Cp-Iso and Re-Iso137) demonstrated significantly compromised diastolic function.
Even Group Pre-Iso/20. which was exposed to Iso under normothermic beating conditions
had an elevated DP compared to the hypothermie ischemia Control Group. The magnitude
of change in DP in Group Pre-Iso/20, however, was not as great as with Group Cp-Iso/37.
Discussion
Perhaps the elevated basal MVOl rnay have been suppressed enough by the hypothecmic
conditions of ischemia to minimize ischemic energy expendinire and allow for improved
post-isc hemic repemision recovery compared to Group C p-Iso/37.
The protection provided by hypothermia is also evident upon comparing the
diastolic hinctional recovery of Groups Control and Cp-Iso in normothermia with their
corresponding group in hypothennia (Figure 3.18). Hypothermie ischemia was found to
very significantly (pSO.0001) preserve diastolic function for both groups compared to the
corresponding groups under norrnothermic ischemia. Similady. the reason that no
significant increase in diastolic pressure was observed in hearts given Iso within the
cardioplegia and then subjected to hypothermïc ischernia may be due to the fact that cardiac
effects of beta-stimulation are reduced or even abolished in hypothennic conditions [Price
et al., 1967; Lauri, 19961. This may be partly attributed to the decreased fluidity of the
membrane proteins and phospholipids at lower temperatures [Darnell et al.. 19901. which
would affect the mobility of the stimulatory G-protein essential in beta-adrenoceptor signai
transmission. Fwthermore, the combined protective power of hyperkalemic cardioplegia
and hypothennia has been well documented to improve preservation of high energy
phosphates [Engelman et al., 1979; Hearse et al.. 1980: Ahn et al.. 19941. myocardial
integrity [Rosenfeldt et al., 19801, and myocardial function [Hearse et al.. 1975: Hearse er
al., 1980; Rosenfeldt et al., 19801.
4.3 Reversal of Catecholamine Effect on Basal MVO,
Esmolol was chosen as the candidate beta-adrenergic antagonisr for this study due
to its cardioselectivity and short-acting properties. Initiaily, no significant effect of esmolol
on basal MVO (without any excess catecholamine influence) was observed (Figure 3.2.
p.50). Since the heart was aLready in its basal state, any fixther depression of MVO, that
esmolol could have afforded on the heart may have k e n too small to measure. or perhaps
the dose was not high enough. or was indicative of minimal endogenous catecholamine
action. Thus, the effect of this dose (2 mg/L) on the beating, non-working heart was
determined (Appendix C, p.94). Because esmolol has been demonstrated to be a
cardioselective beta,-antagonist which has the ability to depress both resting heart rate
[Gorczynski et al., 1983; Iskandnan et al., 19851 and exercise- [Iskandrian et al.. 19851 or
catecholarnine- [Zaroslinski et al., 1982; Gorczynski et al.. 19831 induced tachycardia. a
decrease in MVOz was expected in concert with functional depression. However. a
significant inctease in beating MVO, was observed after 30 min perfusion with KHB + 2
mgR. Esm. Closer inspection of individual hearts revealed that both coronary fiow (Table
V(b), Appendix D, p.97) and artenovenous oxygen difference. both of which are directly
proportional to MV02, tended to increase with Esm administration. Munhy et rd. [ 1983 3
found that esmolol seemed to have an inexplicable hypotensive effect independent of its
beta-blocking activity. Furthemore, esmolol has been found to have some intrinsic
sympathornimetic activity at low concentrations of 109 to lD5 M [Gorczynski et cil.. 1983 3
and the dose of 2 mg/L Esm is less than 105 M, which may account for the increased
artenovenous oxygen difference. Indeed, higher doses of 10 and LOO mg/L Esm seerned to
have a more depressant effect on MVOz (Figure C.2 and C.3. Appendix C, p.95). With
100 mg/L Esm, MVO, decreased dramatically but the sarnple size was much too small to
evaluate statistical significance (Figure C.3).
An intermediate dose of 25 m g L Esrn (which is about IO--' M) was then
administered to hearts which were stimulated with 25 nM Iso while in the arrested state
(Figure 3.3, p.5 1 ). No signifcant change in MVO resulted, indicating that the subsequent
addition of a beta,-antagonist to a catecholamine-exposed arrested heart cannot reverse the
catecholamine-induced increase in basal MVO,. Evidently, the pre-administration of Iso
was enough to elicit an effect which a large dose of esmolol in the order of about five
magnitudes larger than the dose of Iso could not reverse. Thus, even though the antagonist
was able to "out-compete" the agonist for receptor sites, the action of the antagonist came
too late as the agonist had already stimulated the cell. Furthemore. because a beta,-
antagonist was used, the possibility of a be-rnediated effect exists as well.
BDM was another drug used to possibly reverse the deleterious effects of excessive
catecholamine stimulation in the arrested myocardium. Yet, whereas Esm was primarily
used to directly target the action of Iso at the site of the beta-adrenergic receptor. B DM was
used to intervene at the intracellular level. BDM has k e n shown to inhibit myofibrillar
ATPase activity [Higuchi and Takemori, 1989; Higuchi et al.. 1989: Ebus and Siienen.
19961, and decrease force production through increased detachment and decreased
attachent of actin-myosin cross-bridges [Ebus and Stienen, 19961. BDM energetically
stabilizes the unattached state of the myosin molecule [Higuchi and Takemori. 1989:
Higuchi et al., 19891. In addition, BDM has been implicated in the inhibition of C à +
influx via the slow inward channel [Bergey, 1978; Wiggins et al.. 1978: Wiggins et cil . .
1980; Coulombe et al., 1990: Gwathmey et al.. 19911. This could counteract the
upregulation of Ca'+ influx via beta-stimulation. BDM also appears to decrease
myofilament sensitivity to Ca2+ [Gwathmey et al., 199 1 ; Backx et (12.. 1994). Not only
does this contribute to a decrease in maximal force but also, more Ca'+ must bind to the
troponin-uopomyosin cornplex for cross-bridge attachent to occur in the presence of
BDM compared to in its absence. Thus, if intraceiiular Ca?+ increases with Iso stimulation
of the arrested myocardium, then BDM could interfere with the deletenous effects of
increased diastolic CaZ+ concentration.
The MVOt results demonstrating the effect of BDM on basal MVO, show that BDM
had no significmt effect on basal MVO, (Figure 3.4, p.53). indicating that basal
metabolism achieved with hyperkaiemic cardioplegia reduced MVOz low enough that any
effect BDM had was insignificant. Likewise, there was no significant effect of BDM on
the catecholamine-stimulated arrested myocardium (Figure 3 .S. p.54). S tatistically . B DM
did not change the elevated basai MVO, of the Iso-stimulated arrested heart, although the
error accompanying each interval mean value is fairly large and there appears to be a pattern
pattern of decrease in M V 4 induced by the addition of BDM. Further experiments would
have to be done to determine whether this effect is indeed a significant one. If. however.
BDM did significantly reverse the catecholamine-induced increase in basal MVO?. then this
would suggest that increased intracellular Gaz+ and ATP breakdown are the mechanisms by
which catecholarnines can stimulate basal MVO of the arrested myocardium.
5 CONCLUSIONS
Excessive catecholamines increase basai metabolism of the arrested myocardium.
This effect cannot be revened by the subsequent addition of a beta-adrenoceptor antagonist
or a Gaz+-modulator. Exposure of the arrested myocardium to excessive catecholamines is
deletenous to post-normothermic ischemia recovery of diastolic function. but hypothermia
during ischernia attenuates this effect and a decreased level of myocardial adenine
nucleotides is not a factor contrïbuting to the compromised hnction. Hypothennia during
ischemia also improves post-ischemic function of hearts stimulated with catecholamines in
the beating state prior to arrest. and this protective effect is associated with prese~ed
adenine nucleotide concentrations.
By gaining a better understanding of the mechanism of excessive catecholarnine
stimulation and its relevance to both non-ischemic and ischernic myocardiurn. measures for
myocardial protection during compromised states of disease or surgery c m be optirnized.
Various aspects of this study lirnited the fidi scientific value of the results:
1. Sample size: The sample size caicdation indicates that a sample of at l es t 9 hearts per
group would allow for detection of an intergroup difference of 10% with 95% confidence
and a power of 85%. However, most of the MVO, data, and some of the data in the
ischernia study, were represented by groups that were less than 9 in number. Although
statistical analysis was still performed for these smailer groups, the smaller sample size
tended to violate the basic d e s of pararnetric statistical analysis such as the requirement of
random sampling from a normally distributed population, and the requirement of having
enough data within each group to form a nomial distribution.
2. Drue administration: A more thorough dose-response study with al1 dmgs used should
have been done to minimize uncertainty about the effectivity of a dose.
3. Heart rate (HR): HR is a determinant of MVO, and thus, if the hearts were paced.
perhaps a more fair comparison could be made among the hearts.
4. Wet weieht vs. dry weight: As discussed in the Discussion. edema formation may have
greatly infiuenced MVO values if it occurred throughout the entire experïment.
6. Animal model: Although the rabbit hem is primarily a betai-receptor tissue [Tenner rr
al., 19891 and the maximal effects of beta-stimulation have been demonstrated to be greater
than the maximal effects of alpha-stimuiation [Endoh and Blinks, 19881, Downing and
Chen [1985] reported that the alpha-adrenergic pathway is the dominant mechanism of
injury in the rabbit myocardium. Thus, the rabbit myocardium rnay not provide cleÿr.
indisputable results with respect to the effect of betai-stimulation and its effects during
arrest and ischemia (see p.69).
FUTURE STUDIES
Further studies would need to be conducted to investigate the mechanism of the
catecholamine effect in arrested myocardium. If, by pre-treating hearts with a beta-
antagonist, the hearts remain unaffected upon catecholamine administration. then this
would indicate beta-adrenoceptor-mediation of catecholamine-induced increase in basal
MVOz of arrested myocardium. if Ca2+ is (also) a mediator of increased basai MVO,. then
hearts pre-treated with Ca2+-antagonists, or modulators, or substances which c m chelate
intracellular Ca2+, should maintain their basal MVOz upon exposure to catecholamines.
Increased intracellular Cal+ concentration and decreased ATP levels may be the
main factors of decreased post-ischemic functional recovery mediated by excess
catecholamine stimulation. Yet, these experiments do not reveal whether. or how rnuch.
these two are related. To clariQ their roles in compromising myocardial function. the
ischemia expenments could be done using Ca'+-free solution during reperfusion. If
diastolic tone is just as high as with the original pemisate (KHB), then the poor diastolic
functional recovery may be attributed to ATP depletion, rather than to an overload of Ca2-
in the cytosol. This may explain why Group Cp-Iso had compromised diastolic function
after normothermic ischemia, even though its levels of myocardial adenine nucleotides were
not different from the Control Group. However, in normocalcemic perfusion with KHB.
one cannot distinguish whether the increased diastolic tone was due to an already present
Cah overload, or one that was due to repemision injury.
Another way to distinguish between ischemic injury and reperhision injury could be
to use an adenosine dearriinase inhibitor and an adenine nucleoside transport blocker to
inhibit adenosine deamination and block nucleoside release, respectively. Use of these two
interventions would result in the site-specific entrapment of intrarnyocardid adenosine and
inosine generated during ischemia, thereby prevenhg degradation to free-radical substrates
during reperfusion. Thus. the effect of catecholamine stimulation during arrest, or pnor to
arrest, on myocardial functional recovery can be separated from reperfusion injury.
Subsequently, the extent of catecholamine-induced injury can be measured by pre-treatment
of the hearts with a beta-antagonist, Ca2+ antagonist, or Ca'+ modulator. Any improvement
demonstrated with these interventions would be indicative of the extent of the
catecholamine effect. Finaiiy, to differentiate between the damage incurred by ischemia and
that incurred by catecholamines, experiments could be repeated using hearts depleted of
catecholamine stores. Post-ischemic recovery can be assessed and the catecholamine
contribution to damage could be quantified.
8 APPENDICES
8.1 Appendix A: Effect of Epinephrine on MVO,
u - - ' - - - ' - O 30 60 90 1 20
Time (min)
Figure A.1. Ecffect of 2 n M epinephrine on basal MVO,. Basal MVO, did not significantly change ( p d . 7 and p d . 6 for each B-C sequence; paired t-test) upon the addition of 2 nM epinephrine to the arrest perfusate. The shaded areo shows the recovery of the beating state MVO:. Values are mean+SEM (n4). Perfusion intervals: A. KHB; B. K; C. K + 2 nM Epi.
0.50 - 0.50 -
0.40 - f 0.40 - S n , n
0" a" 0.30 - 0.30 - sq O 0.20 - 0.20- a w 0
0.10- 0.10-
: F I O 1 1 I 0 - I 1 1
O 30 60 90 O 30 60 90 Time (min) Timr (min)
Perfusion Interval A A Perfusion Iniervul A A
Figure A.2. Eg'cr of 2 nM epitiephrine on Figure A.3. Effecf u j 25 ~ I M epinephrine on bearing, tion-working MVO,. MVO, of the beuting, t iun-wrk ing MVO,, MVO, of the beating , non-working hcart did no1 signif'icuntly bcating , non-working henrt did not significantly change upon the addition of 2 nM epinephrhc to chungc upon the addition of 25 nM epinephrine to the çoronary pcrfusntc (p=0.6, ANOVA for the coronriry perfusute (p=0.1, ANOVA for repeaied mcnsurcs). Values are mcnnf SEM repested mcusures). Vulucs ure niennfSEM (n=4). Perfusion intcrvals: A, KHB; B, KHB + (n=6). Perfusion intervuls: A, KHB; B, KHB + 2 nM Epi. 25 nM Epi.
a % 'x ii. 8. \O 2 C
8.2 Appendix B: Effect of 25 n M Isoproterenol on Beating Heart MVOL
Time (min)
Perfusion Interval A I S ~ B I A I
Figure B.1. Effecr of 25 n M isoproterenol on bearing, non- working MVOp MVO: of the beating . non-working hean significantly increased upon the addition of 35 nM isoproterenol to the coronary perfusate. Values are mean+SEM (n=4). Perfusion intervals: A, KHB; B. KHB + 25 nM Iso. *p<0.01 vs. interval A; ANOVA for repeated measures.
Appendices
8.3 Appendix C: Effect of Esmolol on Beating Eeart MVO,
Appendices
Appendices
8.4 Appendix D: Coronary Flow and Heart Weight Data
Figure 3.1 (49)
3.2 (50)
3.3 (5 1)
3.4 (53)
3.5 (54)
A. 1 (90)
Coronary Flow (mL/min) Exat. n 1 B 1 1 A
1 so K+25mg/L 13 13.6f1.6 7.910.8 6.8I0.9 5.910.8 6,lf0.8 12.7I2. 1 Esm Kt 25 nM 1 1 32.7I2.0 32.2k2.9 19.8k2.4 20.7'2.1 17.6f2.3 31.0f2.5
'
Iso+ 25 rngL Esm K+30mM 6 28.7355 16.4f3.6 10.6k1.8 15.8 13.7 12.7k2.3 33.315.5 BDM K+ 25 nM 9 32.4r11.9 35.9f3.2 15.9f 1.5 24.13-2.3 16.4k2.0 37.1+3.1 Isoi 30 mM BDM K+2nMEpi 4 15.2I3.6 8.712.0 8.2320 7.6f 1.9 7.6I2.0 15.412.2
Table V(a). Coronary pow und heurt weight data for MVO, stttdy. The avcragc coronary llow of cach perfusion interval, indicaied by n letier (as markcd in ihc corrcsponding Figurc) is shown. The corrcsponding Figurc is givcn, with page rcference in brackeis. Values are meanfSEM. Expt. = expcrimcni. n = samplc six. WW = wei wcighi. DW = dry wcighi. Table V(b) is on following page.
Appendices
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