high levels of circulating epinephrine trigger apical cardiodepression...

DOI: 10.1161/CIRCULATIONAHA.112.111591

1

High Levels of Circulating Epinephrine Trigger Apical Cardiodepression in a

2-Adrenoceptor/Gi-Dependent Manner: A New Model of

Takotsubo Cardiomyopathy

Running title: Paur et al.; A physiological mechanism of Takotsubo Cardiomyopathy

Helen Paur, MSc1; Peter T. Wright, MSc1; Markus B. Sikkel, MD1; Matthew H. Tranter, BSc1; Catherine Mansfield, MSc1; Peter O’Gara, BSc1; Daniel J. Stuckey, PhD1; Viacheslav O.

Nikolaev, PhD2; Ivan Diakonov, PhD1; Laura Pannell, MSc1; Haibin Gong, PhD3; Hong Sun, PhD4, Nicholas S. Peters, MD1; Mario Petrou, PhD5; Zhaolun Zheng, PhD6; Julia Gorelik, PhD1;

Alexander R. Lyon, MD, PhD1,5*; Sian E. Harding, PhD1*

*These authors contributed equally.

1Myocardial Function Section, National Heart and Lung Inst, Imperial College London, London, United Kingdom; 2Emmy Noether Group of the DFG, Dept of Cardiology and Pneumology, Georg August Univ

Medical Ctr, Göttingen, Germany; 3Xuzhou Cardiovascular Disease Inst; 4Physiology Dept, Xuzhou Medical College, Xuzhou, China; 5Cardiovascular Biomedical Rsrch Unit, Royal Brompton Hosp,

London, United Kingdom; 6Cardiology Dept, UBC Hosp, Vancouver, Canada

Address for Correspondence:

Alexander Lyon

National Heart and Lung Institute

4th floor, Imperial Ctr for Translational and Experimental Medicine

Hammersmith Campus

Du Cane Road

London W12 0NN, United Kingdom

Tel: +44 (0) 207 594 3409

Fax: +44 (0) 207 886 6910

E-mail: [email protected]

Journal Subject Codes: [11] Other heart failure; [130] Animal models of human disease; [138] Cell signalling/signal transduction; [148] Heart failure - basic studies; [93] Receptor pharmacology

, , ; , ; g, ; ,Alexander R. Lyon, MD, PhD1,5*; Sian E. Harding, PhD1*

*These authors contributed equally.

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2

Abstract:

Background - Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by

myocardial hypocontractility from the mid left ventricle to apex. It is precipitated by extreme

stress and can be triggered by intravenous catecholamine administration, particularly

epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible

with generally good prognosis. We hypothesised that this represents switching of epinephrine

signalling through the pleiotropic 2-adrenoceptor ( 2AR) from canonical Gs-activated

cardiostimulant to Gi-activated cardiodepressant pathways.

Methods and Results - We describe an in vivo rat model in which a high intravenous

epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical

depression of myocardial contraction coupled with basal hypercontractility. The effect is

prevented via Gi inactivation by pertussis toxin pretreatment. 2AR number and functional

responses were greater in isolated apical cardiomyocytes compared to basal cardiomyocytes,

confirming higher apical sensitivity and response to circulating epinephrine. In vitro studies

demonstrated high dose epinephrine can induce direct cardiomyocyte cardiodepression and

cardioprotection in a 2AR-Gi dependent manner. Preventing epinephrine-Gi effects increased

mortality in the Takotsubo model, while -blockers which activate 2AR-Gi exacerbated the

epinephrine-dependent negative inotropic effects without further deaths. In contrast

levosimendan rescued the acute cardiac dysfunction without increased mortality.

Conclusions - We suggest that biased agonism of epinephrine for 2AR-Gs at low and Gi at high

concentrations underpins the acute apical cardiodepression observed in Takotsubo

cardiomyopathy, with an apical-basal gradient in 2ARs explaining the differential regional

responses. We suggest this epinephrine-specific 2AR-Gi signalling may have evolved as a

cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

Key words: acute heart failure; catecholamines; receptors, adrenergic, beta; Tako-tsubo syndrome

depression of myocardial contraction coupled with basal hypercontractility. The efffeectct iis

prevented via Gi inactivation by pertussis toxin pretreatment. 2AR number and d fffuncnn ttitionononalalal

esponses were greater in isolated apical cardiomyocytes compared to basal cardiomyocytes,

confirirmimingg hhigi heherr apa ical sensitivity and responsese tto o circulating epinepphrhrini e. In vitro studies

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Introduction

There has been a rapid increase in the recognition of a syndrome of acute and severe, but

reversible, heart failure called Takotsubo or Stress cardiomyopathy,1-3 also known as ‘Broken

Heart Syndrome’, which usually follows within hours of an identifiable emotional, psychological

or physical stress. Takotsubo cardiomyopathy mimics symptoms of acute myocardial infarction

(MI), but is distinguished by the lack of coronary occlusion and by characteristic regional wall

motion abnormalities, classically a virtual apical ballooning appearance due to a hypercontractile

base of the heart relative to hypo- or akinetic apical and mid left ventricular myocardium, the

latter extending beyond a single coronary artery territory.1, 2 Initial recognition in earthquake

survivors in Japan, plus the characteristic ventricular shape, led to the ‘Takotsubo’ (meaning

octopus-pot) label.3, 4 It has become apparent that ~1-2% of all presentations with suspected

acute coronary syndrome cases are finally diagnosed as Takotsubo cardiomyopathy.3

The pathophysiological mechanisms for this increasingly recognised syndrome are not

known. Evidence points to epinephrine as the precipitating factor. Physical or psychological

stress is a frequent precipitant, and serum catecholamine levels in Takotsubo patients 1-2 days

after presentation are higher than those in patients with myocardial infarction with pulmonary

oedema: epinephrine falls back to MI levels only after 7-9 days.1 Catecholamine storms, more

associated with epinephrine-secreting phaeochromocytomas than norepinephrine- and dopamine-

secreting phaeochromocytomas,5 can also precipitate Takotsubo cardiomyopathy.6 Particularly,

the reproduction of the signs of Takotsubo by accidental administration of epinephrine (including

single intramuscular 1mg doses from an ‘Epi-pen’) is most indicative of its central role.7

Although there is a significant mortality in the early period (1-1.5%) there is also a characteristic

rapid (days to weeks) recovery of patients surviving the acute period of profound depression in

urvivors in Japan, plus the characteristic ventricular shape, led to the ‘Takotsubbooo’ (((mmeeannaninining g g

octopus-pot) label.3, 4 It has become apparent that ~1-2% of all presentations with suspected

accutututeee ccocororoonanan rrry ssynynyndrd ome cases are finally diagnononoseseed as Takotsuboboo carardididiooomyopathy.3

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left ventricular contractile function,1 with excellent prognosis and absent, or minimal, residual

cardiac impairment. This striking difference from the normal prognosis of heart failure has led

us to propose previously that the cardiodepression has elements derived from a beneficial

physiological protective adaptation.8 Thus, the syndrome has interest for the cardiologist over

and above the design of optimal treatment for the individual Takotsubo patient.

We have previously proposed a mechanism based upon two overarching principles for

which there is prior evidence. Firstly the mammalian left ventricle contains apical-basal

gradients of ARs and sympathetic innervation, with the apex characterised by highest AR and

lowest sympathetic nerve density.8 Rat, feline, rabbit and dog left ventricles show increased

apical responses to global high dose isoproterenol challenges,9-12 with increased apical versus

basal AR levels measured directly in the dog ventricle.10 This pattern results in increased apical

responsiveness to circulating catecholamines, predominantly epinephrine from the adrenal

glands, as a compensatory mechanism for the sparse apical sympathetic innervation, to ensure

optimal ventricular ejection during times of stress. Conversely the sympathetic innervation is

highest in the basal myocardium, and lowest in the apex, and therefore cannot explain the

localised apical dysfunction. This is also true of human left ventricle,13 whereas presence of a

ventricular cardiomyocyte AR gradient in the human heart remains to be determined.

Secondly epinephrine, at high levels, can act as a negative inotrope via ligand mediated

trafficking of the 2AR from Gs to Gi subcellular signalling pathways. The 2AR is widely

reported as pleiotropic, having the potential to couple through Gs-AC-cAMP (like the 1AR) but

also though Gi , G and non-G-protein pathways.14, 15 2AR-Gi-mediated depression of

contraction was initially demonstrated using transgenic mice over expressing the 2AR (TG 2).16,

17 At high epinephrine concentrations, the 2AR switches its coupling from Gs protein to an

apical responses to global high dose isoproterenol challenges,9-12 with increased d aaapicici aalal vvererersususus s

basal AR levels measured directly in the dog ventricle.10 This pattern results in increased apical

eespspponononsisisiveveenenen sss ttooo ccicirculating catecholamines, prrreededooominantly epinnnepe hrhrininineee from the adrenal

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inhibitory Gi protein,16 a process described as ligand or stimulus directed-trafficking or biased

agonism. This switch would be favoured in conditions of high catecholamine stress, since it

depends upon 2AR phosphorylation by both protein kinase A (PKA)18 and G-protein receptor-

coupled kinases (GRKs).19 This is particularly relevant given the increased frequency of the

L41Q GRK5 polymorphism, known to increase cardiac GRK5 activity and AR

phosphorylation, in recent study genotyping Takotsubo cardiomyopathy patients.20 The negative

inotropic effect through Gi 21, 22 has contributions both from inhibition of Gs-cAMP production

and through other pathways such as p38 mitogen-activated protein kinase (MAPK) alteration of

myofilament sensitivity.23 No such role for 1ARs in this Gs-Gi trafficking switch has been

documented, and the phenomenon is epinephrine specific. Norepinephrine has 20-fold lower

affinity for the 2AR compared to the 1AR, and much weaker trafficking of 2AR stimulus

trafficking to the Gi pathway.16 While this negative inotropy is detrimental from a mechanical

perspective, the Gs to Gi switch is potentially both antiapoptotic and antiarrhythmic,24, 25 and

may represent a cardioprotective mechanism against 1AR-catecholamine cardiotoxicity. Both

p38 MAPK and PI3K/Akt pathways have been implicated in 2AR-Gi mediated antiapoptotic

effects in the adult cardiac myocytes,26, 27 and evidence for increased PI3K and Akt activation

has been reported in myocardial biopsies from Takotsubo cardiomyopathy patients during the

acute phase.28 Interestingly, direct negative inotropic effects of some -blockers in human

ventricular cardiomyocytes have been shown to depend on 2AR-Gi signalling,29 an observation

which may have implications for their use in Takotsubo Cardiomyopathy.

In this study we have developed an epinephrine-induced in vivo model of Takotsubo

cardiomyopathy which reproduces both the apically located negative inotropism and the

reversible nature of this cardiodepression. We have used this to explore the role of 2AR apical-

documented, and the phenomenon is epinephrine specific. Norepinephrine has 22000-fofofolddd lllowowowererer

affinity for the 2AR compared to the 1AR, and much weaker trafficking of 2AR stimulus

rrafafffififickckckiining gg tototo theee GGGi i pathway.16 While this negatititiveveve inotropy is detete rimemeentntntala from a mechanical

perssspep ctive, thehe GGss toto GGGiii swsws itititchcch iisss ppopotteennntialllylyl botthh aanttiiaapapopopptotootiticcc aandnd aantntiaiarrrrrhyhyhyththmimmicc,c,2424,24, 2555 aandndnd

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basal gradients; the involvement of Gi signalling and the cardioprotective nature of this

condition. It has been supplemented by an in vitro model of acute epinephrine exposure to

explore underlying cellular mechanisms. Potential pharmacological agents have been assessed in

terms of treatment of the established Takotsubo cardiomyopathy, with the intention to mitigate

the cardiodepression without disrupting any cardioprotective elements of the syndrome.

Methods

All studies complied with the United Kingdom Home Office Regulation Governing the Care and

use of Laboratory Animals and with the Guide for the Care and Use of Laboratory Animals

published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

In vivo Takotsubo cardiomyopathy model

Adult male Sprague-Dawley rats (250-350g) were anaesthetised and injected with 4.28x10-8

moles.100g-1 epinephrine or 1.43x10-7moles.100g-1 norepinephrine via the right jugular vein as a

bolus injection. Regional left ventricular responses were recorded using 2D echocardiography

(Visualsonics Vevo 770) in the parasternal long axis. Baseline scans were performed before

catecholamine administration. Preventative studies: a subgroup of animals were pretreated with

the Gi protein inhibitor pertussis toxin (PTX) (25 g.Kg-1), the p38MAP kinase antagonist

SB203580 (0.1-10mg.Kg-1) or the 2AR selective antagonist ICI 118,551 (1mg.Kg-1) followed

by intravenous epinephrine bolus. A separate cohort of cases had continuous aortic blood

pressure recording during the protocol using a 1.9F pressure-volume catheter (Scisense Inc,

Ontario, Canada). Rescue strategies: a subgroup of animals were treated with intravenous

propranolol (1.43x10-11 moles.100g-1), carvedilol (1.43x10-11 moles.100g-1), or levosimendan

infusion (4.7μg/kg/min) fifteen minutes post epinephrine injection.

published by the US National Institutes of Health (NIH Publication No. 85-23, rereevivisess dd d 191919969696).)).

n vivo Takotsubo cardiomyopathy model

AdAdululult t t mmamalelele SSSppragagguueue-Dawley rats (250-350g) weeererer aanaesthetised aaandn ininnjejejectc ed with 4.28x10-8

mmolleles.100g-1 eepipipineeephphhrirr nnene ooor r 1.1.1.4343x1x1x 000-7-77mmolesss.1100ggg-11 noorererepipinnnepphphririinee vviaiaia thhehe rrriiighgght t jujuuguguulallarr vveveinnin aaas a

boolululusss inininjejej ctctctioioionn. RRRegegegioioonanaal l lel ftftft vvenenentrtrtriciciculullararr rrresesespooonsnsn eees wweeereee rererecocoordrdrdeeded uuusisis nnng 22D DD ececechohoh cacacarrrdioioogrgrrapaphyhyhy

Visualsoniccss VeVeV vovovo 77707070)) innn tttheheh pppaaararaastss ererernananal l lololongngng aaaxixix s.s.s BBasasaselelelininneee scscscannns ss wewewererere pppererfofoformrmrmededed before



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Rat cardiomyocyte isolation and 2AR overexpression studies

Myocytes were isolated from adult male Sprague-Dawley rats (Harlan, Bicester,UK; weight 250-

350 grams) using the standard enzymatic technique as described previously.30 Isolated rat

cardiomyocytes were plated in culture medium at a field density of 10,000 cells/well and

infected with either Ad. 2AR.GFP, 2AR with mutations at the PKA phosphorylation sites 261,

262, 345, 346 S/A ( 2AR-PKA-KO) (Ad. 2AR-PKA-KO) or Ad.GFP (control) at a multiplicity

of infection (MOI) of 500 for 48 hours. For pertussis toxin (PTX) treatment, Ad. 2AR.GFP

infected rat ventricular myocytes were cultured in the presence or absence of PTX (1.5 μg/ml)

for 48 hours. Survival in culture was shown as a percentage of rod-shaped myocytes at the time

of plating: >100 cells per well were counted, with triplicates for each condition. 2AR-specific

contractile responses were measured on separately isolated apical and basal ventricular

cardiomyocytes using isoproterenol (100nM) plus the 1AR selective antagonist CGP20712A

(300nM) (see supplementary methods).21, 29

In vitro Takotsubo cardiomyopathy model

Freshly isolated rat ventricular cardiomyocytes were perfused with epinephrine (1μM) for

20mins followed by washout (10min). A subgroup of cells was preincubated with PTX for 3h at

35°C (1.5 μg.ml-1).

AR radioligand binding assay

Cell membranes, prepared from apical and basal-derived adult rat cardiomyocytes, were

incubated for 2 hours at RT in assay buffer (50mM Tris, 5mM MgCl2) (pH 7.4), with 0.1-10nM

of the non-selective -AR radioligand, [125I]-cyanopindolol ([125I]-CYP) (Amersham, Freiburg,

Germany), and increasing concentrations of the selective 2AR antagonist, ICI-118,551 (1x10-

11M to 1x10-2M). Non-specific binding was determined in the presence of 10μM of the non-

p g p y y

of plating: >100 cells per well were counted, with triplicates for each condition.n 22ARARAR s-s-spepepecicicififificc

contractile responses were measured on separately isolated apical and basal ventricular

cacaardrdrdiioiomym ococytytytes uusisingng iisosoprproto ererenenolol ((101 0n0nM)M) pplulusss tthhe 11ARAR sellecececttitiveve aantntaggononisist t CGCGP2P2070712A A

3330000nMn ) (seee suuppppplememementaarryy y mem ththododds)).21, 229

n vvittitroro TTTaakototsus bbobo ccarddidiomomyoyopapatht y momodedelll

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selective -AR antagonist, propranolol.

FRET-mediated cAMP assay

FRET studies in EPAC-cAMPS expressing apical and basal ventricular cardiomyocytes were

performed as previously described.31 Whole cell epinephrine-stimulated 2AR-mediated cAMP

transients were recorded. A subgroup of cells was preincubated with PTX for 3h at 35°C (1.5

μg.ml-1).

Human tissue samples and cardiomyocyte isolation

Left or right ventricular tissues were obtained from failing human hearts at the time of heart

transplantation; procedures for collecting human heart tissues conformed to the Ethics

Committee requirements of the Royal Brompton and Harefield Hospital, UK. Written informed

consent was provided by all patients. The investigation conforms to the principles outlined in the

Declaration of Helsinki. Single human ventricular myocytes were isolated from explanted failing

human hearts using a standard enzymatic technique as described before.32

Cardiomyocyte contractility studies

See online supplementary methods.

Statistical methods

Results are shown as mean ± SEM. Differences between cell responses for different treatments

were determined using paired or unpaired Student t-tests for two, or one-way ANOVA for more

than two, conditions. Concentration-response curves were compared using repeated measures

ANOVA (RMANOVA). Comparison between responses to bolus epinephrine and

norepinephrine, or ± pertussis toxin, beta-blockers or levosimendan, was performed using

RMANOVA with time as a second factor. For individual time points after bolus, significant

changes in fractional shortening compared to the pre-administration period are indicated: P

Committee requirements of the Royal Brompton and Harefield Hospital, UK. WWrirrittttenenen iinfnfnforormemmed d

consent was provided by all patients. The investigation conforms to the principles outlined in the

DeDeclclclarararatatiioionnn ooof HHHelelelsisinki. Single human ventriculllarra mmmyocytes wereee isololatatateeded from explanted failing

hhummaman hearts uusisiingg aa sstaaanddndararrd dd enenzyzyzymamamatiicc techchchniquuueee ass dddesescccriibbededd bbeffororee.3332

CaCardrdrdioioiomymymyocococytytee e cooontnttraractctc ili itty y y ststtudududiieies s

See online ssupupupplplplemememenenentatataryry mmmetete hohoodsdsd ..



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values less significant than P<0.01 are not shown because of the multiple comparisons.

Differences in mortality were analysed using the Chi-squared test, with Fisher’s exact test for

small numbers. P values of 0.05 or below are taken as significant, NS=non-significant.

Results

High dose epinephrine injection recapitulates Takotsubo Cardiomyopathy

High serum epinephrine levels are a common feature in Takotsubo Cardiomyopathy patients

suggesting a mechanistic link. We developed a model of Takotsubo Cardiomyopathy in which an

anaesthetised rat receives an intravenous (jugular) bolus of 4.3x10-8moles.100g-1 epinephrine

(equivalent to ~5mg in an adult human). Intravenous bolus delivery was selected to mimic the

human physiological response to sudden high stress. Initial dose-response curves determined the

highest catecholamine dose without excessive mortality (Figure S1). Epinephrine bolus

triggered a rapid hypertensive response with reflex bradycardia within seconds of administration,

which stabilised back to normotension after several minutes (Figure S2), and was associated

with an initial global increase in left ventricular contractility (Figure 1). However, this dropped

away to give a marked decrease in cardiac contraction, initiating at 15 min and reaching a nadir

between 20 and 25 min. Contraction normalised within an hour. One defining characteristic of

Takotsubo Cardiomyopathy is the apical and mid-ventricular localisation of dysfunction, and this

was clearly reproduced in our model (Figure 1), and was confirmed by cardiac MRI (Figure S3

and Supplementary movie).

Apical hypokinesia is epinephrine-specific

We and others have previously reported that epinephrine or isoproterenol at high concentrations

can switch the 2AR from positively inotropic Gs to negatively inotropic Gi coupling,17, 22, 33

while norepinephrine cannot.16 We found that equivalent high dose intravenous norepinephrine

equivalent to ~5mg in an adult human). Intravenous bolus delivery was selected totoo mmmimimmicicic ttthehehe

human physiological response to sudden high stress. Initial dose-response curves s dededettetermrmiininedded tthehhe

highg esest cacateechchollamamini e dose without excessive mmorortat lity (Figure S1). EEpipip nephrine bolus

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did not generate the negative effect observed after epinephrine bolus (Figure 1A-C), and

concentration-response curves (Figure S1) confirmed that no concentration of norepinephrine

was negatively inotropic. Changes in heart rate and systemic arterial blood pressure did not

differ between epinephrine and norepinephrine, indicating appropriate matching of effective

concentrations (Figure S2). Lack of negative effect of norepinephrine additionally eliminates

either myocardial 1AR, or 1AR-mediated vasoconstriction as the principal mediator of the

epinephrine-stimulated negative inotropic effect.

Epinephrine-induced apical hypokinesia is Gi dependent

We used pertussis toxin (PTX) to inhibit Gi by in vivo pre-treatment of the rats three days before

the intravenous epinephrine challenge. In vitro challenge of isolated cardiomyocytes from these

hearts with carbachol (after AR stimulation) was used to verify inhibition of Gi effects (not

shown). The negative effect of epinephrine was completely abolished by PTX (Figure 2A-C),

providing strong evidence for a Gi-dependent mechanism of action. Apical and mid-LV

switched completely to give an increase in contraction, and even basal hypercontractility was

significantly enhanced. PTX pretreatment did not alter baseline function, the systemic arterial

pressure response to epinephrine (Figure S3), or time-matched responses following control

saline bolus (Figure S4-5). PTX pretreatment reduced the vagally-mediated reflex bradycardia

during the first minutes after epinephrine injection (Figure S2E). However systemic vagal

blockade with atropine pretreatment failed to prevent epinephrine-induced hypokinesia as

observed with PTX pretreatment, and significantly increased mortality from cardiogenic shock

(Figure S6). This excluded systemic vagal inhibition as the explanation for the PTX-mediated

prevention of apical hypokinesia.

We also developed an in vitro model, in which isolated rat ventricular cardiomyocytes

he intravenous epinephrine challenge. In vitro challenge of isolated cardiomyocycycyteees frfrfromom tttheheh sese

hearts with carbachol (after AR stimulation) was used to verify inhibition of Gi effects (not

hhowowown)n)n). ThThTheee nnegagagattitivev effect of epinephrine was cooc mmmpletely aboliishshs edd bbbyy y PTX (Figure 2A-C),

provviding stroongng eevividedeencnccee fofofor r a a GiGiGi-d-d-depeppeenndentnt mecchhhaniiismsmsm ooof acacttiioonn. AAApiicaall l aanand d mmimid-d-d LVLVLV

wwitititchchhededed cccomomomplpleeetelelelyyy totto gggivive anana iiincncncrrereasasse e ininin ccconnntrtrt aacactitiiononn, , ananandd d evevvenenn bbbasasasalall hhyypypererrcocoontntntrararacctctililliiity y y wawaas

ignificantlyyy enenenhahahancncncededed.. PTPTTX X X prprp etetetrerer atatatmemementntnt dddididid nnnototot alala teteter bababaseseselililinenene fffununnctctctioioion,n,n, ttthhhe e sysysystststemememicicic arterial



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were treated for 20 min with epinephrine. These cells showed a decreased positive inotropic

response to a subsequent 2AR challenge (Figures 3A and S7). Maximum responses to high

calcium were unchanged, indicating that overall cellular and contractile function was not

compromised (Figure S8). The depression of 2AR response after epinephrine pretreatment

observed in this in vitro model was completely prevented (and the response became higher than

control) after PTX treatment (Figures 3A and S7). Notably, measurement of cAMP under the

same conditions showed much less marked changes: PTX treatment increased contraction 11-

fold without a significant increase in cAMP levels (Figure 3B). This implies a parallel negative

inotropic pathway activated through Gi. Since p38 MAPK has been shown to be both Gi-

dependent and negatively inotropic in rat ventricular myocytes we compared treatment with PTX

and a p38 MAPK inhibitor (Figure 3C). Both were able to increase 2AR responses to a similar

degree, and the effects of the two were not additive.

Apical ventricular cardiomyocytes have higher 2AR density and 2AR-mediated

contractile responses compared to basal cardiomyocytes

We have hypothesized that the increased apical sensitivity observed in Takotsubo

cardiomyopathy patients and our model is due to a greater proportion of 2ARs relative to 1ARs

in the apex,8 since the greater concentration of sympathetic innervation in the base of the heart34

is counterbalanced by increased apical AR functional responses to circulating catecholamines.9-

12 Using a radioligand binding-displacement assay to directly quantify the 2: 1AR ratio, we

found that apical cardiomyocytes demonstrated an increased 2: 1AR ratio (Figure 3D). The

functional consequences of a higher 2: 1AR ratio was studied and confirmed greater 2AR-

specific contractile responses in apical ventricular cardiomyocytes compared to paired basal

cardiomyocytes isolated from the same heart (Figure 3E). 2AR-dependent and maximal cAMP

dependent and negatively inotropic in rat ventricular myocytes we compared treaaeatmtmmenennt t wiwiwiththth PPTXTX

and a p38 MAPK inhibitor (Figure 3C). Both were able to increase 2AR responses to a similar

dedegrgrgreeeeee, , anndd d ththhe efefefffefects of the two were not additititivevev .

AApiicical ventricicuullaar ccaaarddidiomommyyoyocycytetetess hhahavve hhhigggherrr 2ARARAR ddeenensisitytyy aaandnd 2ARARAR-mmmedediaiai tteted d

cocontntntraraactctctililile e e rrerespspponnnsesesss cocoompmpaara ededed tttooo babaasasaalll cacac rdrdrdioioomymymyooocytytyteseses

We have hyypopopothththesesesizizizededed ttthahat t t thththe ininincrcrc eaeae seseed d d apapapicicicalalal sssenenensisisitit vivivitytyty ooobsbsbsererervevev dd d ininin TTTakakakototo sususubobobo



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responses demonstrated no difference between apical and basal cardiomyocytes (Figure S9), and

therefore could not explain the observed gradient and contractile response.

Epinephrine-induced 2AR-Gi signalling is cardioprotective

Since 2AR-Gi is widely reported to be antiapoptotic and cardioprotective,35-37 we hypothesized

that blocking 2AR-Gi signalling might increase the cardiotoxic effects of high epinephrine

levels via uninhibited 1AR-Gs and 2AR-Gs signalling. In the rat Takotsubo model in vivo,

epinephrine-induced mortality was significantly increased by prior selective 2AR-blockade with

ICI 118,551 (at concentrations insufficient to activate Gi) or p38MAPK inhibition with

SB203580 (Figure 4A). Death often occurred within 5-10 min, and was due to cardiogenic

shock and hypokinesia rather than primary ventricular fibrillation. In vitro, isoproterenol

increased cell death in cultured myocytes, an effect largely inhibited by 1AR blockade (Figure

4B) while overexpression of the 2AR (Figure 4B) or Gi (Figure 4C) protected against

catecholamine-induced cell death. 2AR switching from Gs to Gi coupling is thought to be

enhanced after strong AR-Gs activation, mediated by cAMP-dependent protein kinase (PKA).18

Overexpression of a 2AR construct in which PKA sites had been mutated to prevent

phosphorylation, not only failed to protect but produced 1AR-independent cell death (Figure

4B). This mutant was also unable to support 2AR-dependent negative inotropism, in contrast to

wild-type 2AR (Figure S10).

-blockers which activate 2AR-Gi do not rescue, and may worsen, established apical

hypokinesia.

In the previous section we note that pretreatment with a specific 2AR blocker before the

epinephrine bolus did not appear to be a therapeutically useful manoeuvre. We also predicted

that clinically used -blockers which activate 2AR-Gi might exacerbate the epinephrine-induced

SB203580 (Figure 4A). Death often occurred within 5 10 min, and was due to carrdiiogogeenic

hock and hypokinesia rather than primary ventricular fibrillation. In vitro, isoproroteteterereenononolll

ncreased cell death in cultured myocytes, an effect largely inhibited by 1AR blockade (Figure

4B4BB) ) whwhile ovovveere exexprprp esessisionon oof f ththe e 22ARAR ((FiFigugurere 44BBB)) or GGii (((FiF guurerere 44CC) ) prprp otececteted d agagaiainsnst t

caaateeechc olaminine-e innndduuceedd ccelll ddeeeathh. 22ARARAR swwwittchingngng ffrooom m m GsGG tto GGiGi coupppliingngg iss s tht ouuugghht t too be

enhahancnceded aaftf err stronong g g ARAR-GGs s aca titivationn,, memedidiatated bby y y cAcAMPMP-depeppenendedent ppprooteteinin kkinnasase (P(P( KAKA).).) 118

OO ii ff ARAR t t ii hhii hh PKPKAA iit hh dd bb t t dd t t



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negative inotropic effect. The Gi-dependent negative effect of -blockers is most readily seen in

myocytes from failing human hearts (where Gi is increased 29): we selected compounds that had

either strong (propranolol) or modest (carvedilol) effects on these cells (Figure 5A). Figures

5B-C show the effect of the two blockers added 15 min after epinephrine in the in vivo model,

when peak negative responses are developing. Propranolol, with higher 2AR-Gi agonism,

significantly enhanced and prolonged the negative effects of epinephrine at both apex and base

(Figure 5B), while carvedilol, with less pronounced 2AR-Gi agonism, had little effect on apex

but converted the base from positive to significant negative responses (Figure 5C). In contrast,

the 1AR-selective blocker bisoprolol reduced the positive effect of epinephrine at the base but

did not convert it to significant negative response: there was no effect on the apical epinephrine

response (Figure S11). These data support our hypothesis of synergistic effects of epinephrine

with propranolol (and to a minor extent carvedilol) upon 2AR-Gi signalling. While the

negative inotropic of epinephrine was enhanced, there was no increase in mortality with the

addition of propranolol or carvedilol (Figure 4A).

Levosimendan reverses epinephrine-induced apical dysfunction without increased

mortality

Levosimendan was selected for comparison as it is an inotrope with a cAMP-independent

mechanism of action, increasing myofilament calcium sensitivity.38 Global cardiac contraction in

untreated hearts was increased with infusion of this compound (not shown). In contrast to other

agents, application of levosimendan at the point where epinephrine negative effects were

beginning, was effective in preventing further decline in cardiac function (Figure 6). This

contractile benefit and rescue occurred with no deaths in the epinephrine-treated group (Figure

4A).

did not convert it to significant negative response: there was no effect on the apiciccalala epipipinenenephphphriririnenen

esponse (Figure S11). These data support our hypothesis of synergistic effectf s of epinephrine

wiwiiththth pproroprranannooololol ((anand to a minor extent carvedillooll) upon 22AR-GGGi i signgnalallil ngg. While the

nenen gagaative inotropiic of eeepiinepepphrhrrinine wawas enennhannnceed, tththeere wawawasss nooo innncrreasee innn mmmoorrtalilityyy wwwititth tht e

adaddidiitititiononon ooof f f prpropopprararanononolllololol ooorr r caarvrvvedededilililolol ((FiFiFigugugureree 444AAA).).)

Levosimendddananan rrreveve ererersesesess epepepinininepephrhrh inini e-e-ininindduducececedd d apappicicicaala dddyysysfufufuncncn titiononn wwwititthohohoutut iincnncrerer asasa eede



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Discussion

Takotsubo cardiomyopathy is an increasingly recognised acute cardiac syndrome in the modern

era of early access to diagnostic coronary angiography.1-3 As a cardiac response to extreme stress

levels it carries a relatively good prognosis, but has the intriguing feature of regional (apical)

hypokinesia, which is counterintuitive in relation to the systemic nature of the trigger and the

evolutionary drive for increased cardiac output during ‘flight or fight’ responses. We have

developed a rat model mimicking the clinical features with acute, reversible apical and mid-

ventricular myocardial hypokinesia, but preserved or enhanced basal contractility (Figure 1).

Rapid high dose intravenous epinephrine bolus, designed to mimic the serum catecholamine

response to acute stress compared with the traditional infusion protocols, recapitulated the

classical clinical findings, whereas the equivalent norepinephrine bolus did not (Figure 1). This

implied the mechanism is epinephrine-specific, and confirms the observation that dysfunction is

not typically observed in the region with the highest density of norepinephrine-releasing

sympathetic nerve terminals.13

We have further investigated this concept of apical-basal gradients of catecholamine

responsiveness to AR subtype, and demonstrate that apical ventricular cardiomyocytes have a

higher 2AR density and a greater 2AR-induced sensitivity compared with basal

cardiomyocytes isolated from the same heart (Figure 3D-E). The inability of norepinephrine at

equivalent (and higher) doses to initiate acute apical dysfunction excludes coronary vasospasm

or 1AR-mediated signalling as a primary effector (Figure 1). This agrees with clinical

observations that the apical dysfunction in Takotsubo cardiomyopathy extends beyond the

territory of a single coronary bed.1-3, 8 Supporting the predominance of a cardiomyocyte-based

explanation rather than a vascular one is also the ability of our in vitro cardiomyocyte model

esponse to acute stress compared with the traditional infusiond protocols, recapitutuulaaatetet dd d thththe ee

classical clinical findings, whereas the equivalent norepinephrine bolus did not (Figure 1). This

mmplplplieieieddd thththeee mmmechchhaananism is epinephrine-specific, anana ddd confirms the ooobsererrvavavatit on that dysfunction is

nnot tytypically obobbsserrrvededed innn thththe e e rereregigiononon wwiithhh theee hhhighhesest deeennsnsitityyy ooof f nonnorerepipiinnnepphphrrrininine-e-rerelelel aasasinining g

yympmpmpatatathehetitiicc c nenervrvve e tetermrmmiininala s.133

We hhavavaveee fufufurtrtrtheheer r r innvevevestss igggatatatededd thihihiss cococoncncncepepeptt t ofofof aapipicacacal-l-l babab sasasal l l grrradadadieieientntnts s s ofofo cccatatatececechohoholal mine dd



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used here to reproduce a number of the key in vivo observations (Figure 3), as well as the

matched responses of heart rate and blood pressure between epinephrine and norepinephrine

cohorts.

Norepinephrine also differs in that it does not couple 1ARs or 2ARs to Gi signalling,

while epinephrine at high concentrations produces a 2AR-Gs to Gi switch. 2AR-Gi coupling

has been reported in a number of experimental models including 2AR and Gi overexpression,

and importantly in chronic heart failure, where Gi levels are increased.29 2AR-Gi coupling

occurs via a process termed stimulus/ligand-directed trafficking or biased agonism. Other

agonists such as high dose isoproterenol also produce this switch, and we note a study in which

isoproterenol infusion over 2 weeks also produced a specific apical contraction defect.9 The key

role of Gi in the cardiodepression was shown by the ability of PTX to convert apical responses to

epinephrine from negative to positive (Figure 2). It should be noted that the response of basal

myocardium was also increased, implying that 2AR-Gi was operational even in this region

despite the 1AR predominance. Non-classical examples of Takotsubo Cardiomyopathy have

been observed where base or mid-LV is affected,39 and this may reflect individual patterns of

2AR expression. The in vivo observations were supported by those in isolated cells. In

untreated apical myocytes, positive inotropic responses to 2AR stimulation were enhanced by

PTX (Figures 3C and S7, and as previously reported40). In myocytes pretreated with

epinephrine, PTX was able to rescue and further enhance the depressed 2AR-mediated positive

responses (Figure 3A and S7). cAMP responses were decreased modestly in pretreated

myocytes (Figure 3B), though less affected than contraction. However, PTX was able to rescue

contractile responses with no significant effect on cAMP (Figure 3B), implying the existence of

a separate negatively inotropic Gi-dependent pathway. Inhibition of p38 MAPK produced similar

soproterenol infusion over 2 weeks also produced a specific apical contraction dddeefefeceectt.t.999 TTThehehe kkkeyey

ole of Gi in the cardiodepression was shown by the ability of PTX to convert apical responses to

epeppininineepephhrinne e e ffrfromm negegative to pop sitive (Figure 2).). Ittt should be nooteteted ththatat tthe respop nse of basal tt

mmym oococardium wasss aaalsoo innncreaaaseeed,d iimpmplyyyinnng thhhaaat 2ARARA -GGGiii wawawas opeereraaationnnall evevvennn iin ththhisiss reegegioion

dedespppitititeee thththe e e 11ARARR ppprereredododomimiminan ncncnce.e.e NoNNon-n-clclc asasassisis cacacal ll exexexamamamplplpleseses oooff f TaTaTakokokotststsubububooo CCaCardrddioioiomymymyoopopatatathyhyhy hhhavavaveee mmm

been observeveed dd whwhwhererereee bababasese ooorr r mimiid-d-d-LVLVLV iiisss afafaffefefectctctededed,,39399 aaandndn ttthihihiss mamamayy reeflflflececect tt ininindididivividududualalal pppatatatterns of



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and non-additive effects to PTX, consistent with the suggested role for this pathway as a Gi-

dependent negatively inotropic modulator.

The epinephrine-dependent 2AR-Gi mediated negative inotropism requires a preceding

high 1AR-Gs activation to initiate cAMP-dependent PKA- and GRK-phosphorylation of the

2AR.18, 41 This implies that, while norepinephrine does not directly couple receptors to Gi, the

rise in cAMP it produces will predispose the 2AR to traffic to Gi upon subsequent epinephrine

binding. Here we demonstrate that PKA-mediated 2AR phosphorylation is critical for Gi

coupling as deleting the phosphorylation sites prevented both negative inotropism and

cardioprotection attributable to 2AR-Gi coupling (Figures 4B and S10). This also explains the

reversibility of the Takotsubo cardiomyopathy syndrome. As the serum epinephrine levels fall,

2AR dephosphorylation, or internalisation and replacement with de novo unphosphorylated

2ARs, would reduced the 2AR-Gi stimulus trafficking and restore normal contractile function

in the surviving cardiomyocytes. Studies in model cell systems overexpressing fluorescently

labelled 2AR demonstrate the dependence of both PKA- and GRK-mediated 2AR

phosphorylation for 2AR internalisation from the surface membrane and recycling to different

surface microdomains.19 Interestingly they also demonstrate the epinephrine-specific dependence

of this trafficking.41 This is relevant to the Takotsubo cardiomyopathy patients as to date there

has been failure to identify any associated polymorphisms in the 1ARs, 1AR or 2ARs,42 but

one study, albeit with small patient numbers, found an increased prevalence of the GRK

polymorphism L41Q in the Takotsubo cardiomyopathy patient cohort compared to healthy

matched controls.20 This is a ‘gain-of-function’ mutation, previously referred to as genetic

betablockade,43 confers reduced responsiveness to AR agonists, and improved prognosis in the

population carrying this polymorphism,43 both conceivably consistent with enhanced myocardial

cardioprotection attributable to 2AR Gi coupling (Figures 4B and S10). This alsso exexplplainsns tthhe

eversibility of the Takotsubo cardiomyopathy syndrome. As the serum epinephhririinenen lllevevevelelelss s fafafallllll, ,,

2AR dephosphorylation, or internalisation and replacement with de novo unphosphorylated

222ARARARs,s wououldldld rededucuceded tthehe 2ARAR-G-Gi i ststimmululusus ttraraffffficcckingngg aandnd reestststororee nonormrmal cconontrtracactitilele ffununctctioion

nnn thhehe survivivingn ccaaardiomomomyooccycytttes. StStududdieees innn mmmodeeel cec lllll sssyysystetemms ovvverexxpxpreressssiinng g fluuouoreresscsceentlyy

abeelllleded 22ARAR ddemmononstraatete thehe ddepependencncee ofof bbototh PKPKA-A- aandnd GRKRK-m-mede iaiateted d 22ARAR

hphos hphor lyl tatiion ffor ARAR ii tnternalilisatition ffrom ththe su frface me bmbrane a dnd recycliling tto didifffferentt



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2AR-Gi coupling.

Although the final outcome for the Takotsubo patient is generally good, they have been

through an acute cardiac event requiring hospitalisation, and there is a significant incidence of

cardiogenic shock (~4%), malignant ventricular arrhythmias (1-2%) and death (1-1.5%). It

therefore seemed reasonable to try to block the depression of contraction with either a specific

2AR antagonist or a p38 MAPK inhibitor. However the marked increase in mortality produced

by this manoeuvre gave a clear indication that this was a counterproductive strategy (Figure

4A). The rapidity of the death, usually within 5-10 and always within 45 min, made it unlikely

that apoptosis was the underlying mechanism. The 2AR and/or Gi have been implicated in

suppression of arrhythmias,44 and 2AR variants with sudden cardiac death.45 2AR knockout

mice went into cardiogenic shock following doxorubicin, through a 1AR-related mechanism46

and 2AR/Gi mechanisms have also been implicated in post-ischemic stunning.47 All these are

potential mechanisms which could underlie the acute mortality. We suggest that the enhanced

2AR-Gi coupling initiated by high epinephrine levels is protective to dampen the effects of

toxic AR-Gs coupling, which is left unchecked would be fatal.

Few -blockers are pure neutral antagonists, with most having some other effect such as

partial agonism (intrinsic sympathomimetic activity), inverse agonism (reduction in activity of

constitutively active receptors) or biased agonism (ligand directed trafficking to other pathways).

It has been amply demonstrated that blockers of the 2AR can activate other signalling pathways,

both G-protein and non-G-protein-dependent.48 We were first alerted to the possibility that the

cardiodepressant effects in Takotsubo Cardiomyopathy were 2AR-Gi dependent by their

similarity to the 2AR-Gi-mediated effect of -blockers on myocytes from failing human heart.29

We therefore hypothesised that -blockers with strong 2AR-Gi agonism would synergise with

uppression of arrhythmias,44 and 2AR variants with sudden cardiac death.4522ARARAR knononockkckououout t

mice went into cardiogenic shock following doxorubicin, through a 1AR-related mechanism46

annd d d 22ARARAR/G/G/Gii meeechchchana isms have also been impliccatata eedd in post-ischeheemim cc stststuununning.mm 47 All these are

poteeentn ial mechhanannisssmsms wwwhihihichchch cccououldldd uuundnddeerrlie thhhe acccuutte momomortrtaaaliitty.y WWWe suugggggesestt t ththhatat tthehehe eennhnhaananceceedd d

22ARARAR-G-G-Giii cococouupupliliingngng iiinininittiatatatedede bybyby hhhigigighhh epepinininepepephhhrininne e e lelelevevevelslss is ss prprprotottecece titiivevee ttto o o dadaampmpm enenen ttthehehe eeefffffeeectstss ooofff

oxic AR-GGGs ss cococoupupuplililingngn ,, whwhwhicicich h isisis llefefeft t unununchchchecececkekeked dd wowowoululu d d bebebe fffatata alalal. .



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18

the negative inotropic effect of epinephrine. Propranolol, a particularly cardiodepressant agent,

markedly enhanced and prolonged the negative phase when given after the epinephrine bolus in

our in vivo model (Figure 5B). In support of the hypothesis of additive negative inotropic effects

of propranolol and epinephrine, we note a recent report that an acute dilated cardiomyopathy was

precipitated in a patient with phaeochromocytoma upon taking propranolol for migraine.49

Carvedilol had a more modest effect, reversing the basal hypercontractility whilst having a

neutral effect upon the apical hypokinesia (Figure 5C). Carvedilol (and propranolol) are also

able to produce biased agonism through G mechanisms,50 which would be PTX sensitive.

Although possibly exacerbating the syndrome, carvedilol could be useful in treatment in the

minority of Takotsubo cardiomyopathy patients with severe left ventricular outflow tract

obstruction secondary to the basal hypercontractility. It should be noted that neither blocker had

a deleterious effect on mortality in the Takotsubo Cardiomyopathy model. Bisoprolol, which is

predominantly 1AR selective, did not reproduce these effects to synergise with epinephrine.

We considered the implications for treating Takotsubo cardiomyopathy, and for heart

failure therapy more generally. For the Takotsubo patient, strategies to raise cAMP

(catecholaminergic inotropes or phosphodiesterase inhibitors) would clearly be contraindicated.

Indeed dobutamine administered for stress echocardiography testing has precipitated Takotsubo

Cardiomyopathy.7 A cAMP-independent inotrope, levosimendan, was effective in reversing the

negative inotropic effect of epinephrine and rescue occurred without increased (and a trend to

decreased) mortality (Figures 4A and 6). We suggest that this is likely to be a safe supporting

and bridging strategy for the sickest patients with cardiogenic shock until spontaneous recovery

occurs, and preliminary clinical reports support this view.51, 52 It should be noted that at the

higher doses levosimendan can inhibit phosphodiesterases,38 and increase cAMP, and thus we

minority of Takotsubo cardiomyopathy patients with severe left ventricular outffloloow ww trtracact tt

obstruction secondary to the basal hypercontractility. It should be noted that neither blocker had

a dedeeleleletetete iririouououss efe fefeectctct oon mortality in the Takotsubobobo CCCardiomyopathhhy yy momodededel.l Bisoprolol, which is

predddomo inantly y 1ARARAR ssselllececectititiveveve, didid dd nnonottt rreeproododuuce thhheseee eefefffeeecttsts ttooo ssynenerrrgiise e wiwiwithth eeepipipinenen phphp riririnenee. ffff

WeWee cccononsisiideeerereddd ththhe e immmplplplicicicatatatioioionsnss fffororor tttreeeaaatitit nngng TTaakakotototsususuboboo ccaarardididiomomomyoyoopapaaththt y,y,y, aaandndnd fooor hhheaearrrt

failure therapappy y y momomorerere gggenennerrralala lylyly. FoFoFor r ththt e e e TaTaTakokokotttsususuboboo pppatata ieentntnt,,, stststrararatetetegigg eseses ttto o o rararaisisise ee cAcAAMPMPMP



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19

only would recommend the lower (non-vasodilatory) doses. The value of non-selective -

blockers, which may also act as agonists at 2AR-Gi, is more difficult to predict, since they may

amplify both the negative inotropic and the protective effects of epinephrine. It could further be

suggested that the beneficial effects of -blockers in heart failure has taken serendipitous

advantage of cardioprotective 2AR-Gi biased agonism. If those two effects could be modulated

separately, this might point the way for an improved design of future -blockers by selecting for

cardioprotection through 2AR-Gi biased agonism.

Acknowledgements: We thank Orion Pharma for the gift of levosimendan, Drs Menick, Charleston and Wang, University of California, San Diego for the p38DN vector, and Professor Walter J. Koch, Center for Translational Medicine, Philadelphia for the 2AR-PKA-KO vector. We dedicate this paper to the memories of Sir James Black (Nobel laureate, 1924-2010) and Professor Philip Poole-Wilson (1943-2009). Funding Sources: This work was supported by grants from the Biotechnology and Biological Sciences Research Council (HP, SEH), Academy of Medical Sciences/Wellcome Trust Clinical Lecturer Start up grant (ARL), the British Heart Foundation (ARL (FS/11/67/28954), JG/SEH (NH/10/3/28574)) and the Wellcome Trust (SEH, JG (090594/Z/09/Z). ARL is supported by the National Institute for Health Research-funded Cardiovascular Biomedical Research Unit at the Royal Brompton Hospital. Conflict of Interest Disclosures: None

References: 1. Wittstein IS, Thiemann DR, Lima JAC, Baughman KL, Schulman SP, Gerstenblith G, Wu KC, Rade JJ, Bivalacqua TJ, Champion HC. Neurohumoral Features of Myocardial Stunning Due to Sudden Emotional Stress. N Engl J Med. 2005;352:539-548. 2. Prasad A. Apical Ballooning Syndrome: An Important Differential Diagnosis of Acute Myocardial Infarction. Circulation. 2007;115:56-59. 3. Gianni M, Dentali F, Grandi AM, Sumner G, Hiralal R, Lonn E. Apical ballooning syndrome or takotsubo cardiomyopathy: a systematic review. Eur Heart J. 2006;27:1523-1529. 4. Sato M, Fujita S, Saito A, Ikeda Y, Kitazawa H, Takahashi M, Ishiguro J, Okabe M, Nakamura Y, Nagai T, Watanabe H, Kodama M, Aizawa Y. Increased incidence of transient left ventricular apical ballooning (so-called 'Takotsubo' cardiomyopathy) after the mid-Niigata

g, y , g p ,Walter J. Koch, Center for Translational Medicine, Philadelphia for the 2AR-PPKAKAKA-K-KKOOO vevevectctctoroor. We dedicate this paper to the memories of Sir James Black (Nobel laureate, 19224-4-4-202020101010) ) ) ananand ddProfessor Philip Poole-Wilson (1943-2009).

Fundnddinining g g SoSoSouruu cecees::s: ThT is work was supported byy gggrararants from the Bioteechchchnnon logy and Biological ScScciieienncncese RReseseseearcrchh CoC ununcicil l (H(HP,P, SSEHE ),), AAcacadedemymy ooff Mededicical Sciciienene cees/s/WeW llcocomeme TTruustt CCliniicacalLLeLecctcturer Staartrt uuuppp gggrananantt (A(A(ARLRLRL),),), ttthehehe BBBririrititisssh h HeHeH aaart t FFoFouundadadatititiononon (((ARARARL L L (F(F(FSS/S/111/667/7/7/2828289595954)4)4 , , JGJGJG/S/S/SEHEHEH NNNHH/H/10/3/285744))) anddd tthhe WWWeelellcomme e TTrTruust ((S(SEH,,, JJG ((0909090505059944/ZZZ/009/Z)). ARARARL L is suuppppoporrrteedd byyy tththe

NaNaNatiiionono ala IInsnsstitititututee ffforor HHHeeaealtlth h ReReResesearararchchc -f-fuunundedeedd d CaCarrdrdioiovavvascscuullaarr BBioiomemedidiicaaal l RReReseses ararcchch Unnnitt t atat ttthee RoRoyayaall l BrBrBromomomptptononon HHHososospipipitatatal.ll

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Prefecture earthquake. Circ J. 2006;70:947-953.

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10. Mori H, Ishikawa S, Kojima S, Hayashi J, WWatanabe Y, Hoffman JJI,, Okino H. Increased eespspponononsisisiveveenenen sss oooff f lelel ft ventricular apical myocardrddiuiumm to adrenergigiiccc stimimmuululi. Cardiovasc Res.

1919993933;227:1992-2--1998.8..

1111. LLaL thers CMCM, LLLevinnn RRMRM,, SpSppivveyyy WWWHHH. Reegegiionaaal didiststtririribububuttiooon ooof f mmyoocaararddiialall bbetaaa-aaddrreennoceeptpttorsnn ttthehehe cccatat.. EuEuEurr J J PhPhhararmamamacocol. 119898986;6;6;13131 00::111111-1-1-11117.7.7

12. Mantravaadidid RR,, GaGaG brbrbrisi BBB,, LiLL uuu T,T,T CCChohohoi i BRBRBR, , deded GGGrorooata WWWC,C,C, NNNg g g GAGAGA,, , SaSaSalalaamamam GGG... AuAuAutotoonon mic NeNervrvee StStimimululatatioionn ReReveversrseses VVenentrtriciculularar RRepepololararizizatatioionn SeSeququenencece iinn RaRabbbbitit HHeaeartrtss CiCircrc RReses



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2444. ChChChesese leley y y A,AA, LLLununndbdbererg g g MSMSS,,, AsAsAsaiaiai TT, , XiXiXiaoaoao RRRP,P,P OhOhOhtat nnini SSS, , LaLaLakakak ttttta a EGEGE ,, CCrCrowoww MMMT.T.. TTTheee bbetete a(a((2)2))--adrerenenergicic rreccepeptoorr dedeliveersr aann anntitiapopptototiticc sisigngnal too cacardrdiaiac c mymyococytytese thrhrououghgh GG(ii)-)-deepependndennt coupling to phphphososo phphphatata idddylyly innnososositii ololol 333'--kikk nananasesee.. CiCiCircrcrc RRResese .. 220000000;0;0;878787:1:1117171 2-2-2-11111797979..



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33. Xiao RP, Zhang SJ, Chakir K, Avdonin P, Zhu W, Bond RA, Balke CW, Lakaatttttta a a EGEG, , , ChChenengH. Enhanced G(i) signaling selectively negates beta2-adrenergic receptor (AR)---bbubutt t noot tt bebebetatata1-1-AR-mediated positive inotropic effect in myocytes from failing rat hearts. Circululatattioioionn.2003;108:1633-1639.

344. . KaKaKawwawanonono HHH, OkOkOkada a R, Yano K. Histological stststududdy on the distrribibi utioioonn n ofo autonomic nerves in ddhhhee hhuhuman heheearrt.t. HeHeH arartt t VeVeVesssss elels.s.. 22200000 3;3;;1818:3:32-2-3939..

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3636 ToTongng HH BeBernrnststeieinn DD MMururphphyy EE SSteteenenbebergrgenen CC TThehe rrololee ofof bbetetaa a-adrdrenenerergigicc rerececeptptoror



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466. . BeBeBerrnrnstststeieieinn D,, FFFajajardo G, Zhao M, Urashima TTT,, PPPowers J, Berryryry G,, KoKoKobilka BK. Differential cacaardrddioioprottecctititiveve/c/ccarara didiotototoxoxxicici eefffffececectstst mmedediaiatteded bby y bebebetta-aadrdrdrenenenere gigiicc c rreececeptptptororo sububtytytypepepes.s. AmAmm JJJ PPPhyhyhysis oHHeaarart Circ Phyhysisis ooll. 22200000 5;5;5;2828289:9:9:H2H24444441-1-H2H244499.

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Figure Legends:

Figure 1. Takotsubo cardiomyopathy is epinephrine-specific. Effects of 4.28x10-8 moles.100g-1

epinephrine (dark red bars) and 1.43x10-7moles.100g-1 norepinephrine (dark blue bars) on apical

(A), mid left-ventricular (B) and basal myocardium contractility (C). Values are expressed as the

mean percentage change in LV fractional shortening (% FS) from baseline (untreated) levels ±

SEM at each 5 min time point following injection. N=6 (epinephrine), n=6 (norepinephrine)

(**p<0.01, ***p<0.001, ****p<0.0001 vs baseline FS = 0). Abbreviation: B (baseline). RM

ANOVA: epinephrine vs norepinephrine: p<0.001 (apex), p<0.001 (MLV), p=NS (base); Time:

P<0.001 (apex), P<0.001 (MLV), P<0.05 (base).

Figure 2. In vivo Takotsubo Cardiomyopathy model and prevention by Pertussis toxin

pretreatment. Contractile responses after an intravenous bolus injection of epinephrine (4.28x10-8

moles.100g-1 - dark red bars) on left ventricular apical (A), mid left-ventricular (B) and basal

myocardium (C). Values are expressed as the mean percentage change in LV fractional

shortening (% FS) from baseline (untreated) levels ± SEM at each 5 minute time point

following injection. Light blue bars show time-matched inotropic responses of the apical, mid-

left ventricular and basal myocardium in PTX (25 g.Kg-1) pre-treated animals after equivalent

i.v. epinephrine bolus, with loss of apical and MLV hypokinesis. N=6 (control epinephrine), n=5

(epinephrine+PTX) (**p<0.01, ***p<0.001 vs baseline FS = 0). Abbreviations: B (baseline).

RM ANOVA (epinephrine vs epinephrine+PTX): p<0.001 (apex), p<0.01 (MLV), p<0.05 (base);

Time: P<0.001 (apex), P<0.001 (MLV), P<0.001 (base).

P<0.001 (apex), P<0.001 (MLV), P<0.05 (base).

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myocardiumm (((CCC).).) VVValaa ueueues arararee exexexprprpresessseses d d d asass tthehehe mmmeaeaean nn pepep rcrccenenentatatagegege ccchahaangngngeee ininin LLLV V frfrfracacactitiionononal



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Figure 3. In vitro Takotsubo cardiomyopathy model induced by high dose epinephrine exposure.

Effect of 20 min pretreatment with epinephrine (Epi-pre, 1μM), followed by 10 min wash, on

subsequent 2AR contractile (A) and cAMP (B) responses with Gi (PTX-sensitive) component.

A, Contraction amplitude in isolated rat ventricular myocytes: peak fold increase over basal:

Control (n=15); Epi-pretreated alone (n=15); Epi-pretreated +PTX (n=7). B, Whole cell cyclic

AMP levels, measured using an EPAC2-FRET sensor. Control (n=40); Epi-pretreated alone

(n=10); Epi-pretreated +PTX (n=9). *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA

Kruskal-Wallis test. C, 2AR-mediated inotropic response to 100nM isoproterenol in the

presence of the 1AR blocker CGP20712A (300nM), peak fold increase over basal in control

(n=13) or PTX-treated rat ventricular myocytes (n=13), in the presence and absence of SB20380

2.5μM. **P<0.01, ***P<0.001 vs control, unpaired t-test. D and E, Apically-derived

cardiomyocytes demonstrate increased 2AR levels and responses. D, Proportion of 2ARs with

respect to total AR radioligand binding in ventricular myocytes from the apex and base of

normal rat heart. N=4 preparations, **P<0.01 vs base, paired t-test. E, Apical cardiomyocytes

(purple bars) show a larger increase in percentage cell shortening through the 2AR compared to

basal cardiomyocytes (green bars). Fold increase in shortening with 100nM isoproterenol +

300nM CGP20712A. (*p<0.05 apex vs base, paired t-test. Base: n=13 cells; Apex: n=13 cells,

n=13 animals).

Figure 4. Epinephrine-mediated 2AR-Gi signalling is cardioprotective. A, Mortality with in

vivo bolus epinephrine (4.28x10-8 moles.100g-1) in the absence (n=14) or presence of 0.1-

10mg.Kg-1 SB203580 (n=9), 1mg.Kg-1 ICI 118,551 (n=5), 1.43x10-11 moles.100g-1 propranolol

(n=9), 1.43x10-11 moles.100g-1 carvedilol (n=12), 4.7 μg/kg/min levosimendan (n=5). *P<0.05

n=13) or PTX-treated rat ventricular myocytes (n=13), in the presence and absenennceece ooof f SBSBSB20202038383 00

2.5μM. **P<0.01, ***P<0.001 vs control, unpaired t-test. D and Ed , Apically-derived

cacardrdrdioioiomym ococytytytes ddememonstrate increased 22AR levveeelsss and responseseess. DD, , PrPropo ortion of 2ARs with

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vs epinephrine alone, Fisher’s exact test. B, Survival of adult rat ventricular myocytes (%

remaining at 48h compared to time 0) after exposure to 1μM isoproterenol (ISO) in the presence

(light blue bars) and absence (red bars) of the 1AR blocker CGP20712A (300nM), compared to

untreated controls (white bars). Myocytes were transduced using adenoviral vectors with GFP

(control), the wild-type 2AR and 2AR with mutations at the PKA phosphorylation sites 261,

262, 345, 346 S/A ( 2AR-PKA-KO) to prevent switching to Gi. N=6, # P<0.05 vs con/GFP,

*p<0.05 vs GFP+ISO, One-way ANOVA. C, Effect of Gi expression upon ISO-induced

myocyte toxicity over 48hrs in culture. Myocytes were transduced using adenoviral vectors with

GFP (control), or Gi-GFP (Gi) at Day 0. N=6 preparations, *P<0.05, **P<0.01 vs respective

control, #P<0.05, ##P<0.01 vs ISO alone, one-way ANOVA.

Figure 5. Agonist-independent negative inotropic effect of betablockers and potentiation of

Takotsubo cardiomyopathy. A, Negative inotropic effect of AR blockers on contraction of

ventricular myocytes from failing human heart. Contraction amplitude relative to basal (open

bar) for ICI 118,551 (3μM, n=21), propranolol (Prop, 5μM, n=9) and carvedilol (Carv, 3μM,

n=24), *P<0.05, ***P<0.001 vs 100%, one-way ANOVA. B and C, The -blockers propranolol

(B) and carvedilol (C) (both 1.43x10-11 moles.100g-1 (i.v.)) either enhance or fail to prevent the

negative inotropic effects of epinephrine (4.28x10-8 moles.100g-1 (i.v.)) at the apex and also

reverse the positive effects of epinephrine at the base, in the in vivo rat model. Values are

expressed as the mean percentage change in LV FS from baseline (untreated) levels ± SEM at

each 5 min point following intravenous injection. N=6 (epi), n=6 (epi+propranolol), n=7

(epi+carvedilol). (**p<0.01, ***p<0.001, ****p<0.0001 vs baseline FS = 0). RM ANOVA epi

vs epi+Propranolol: apex, P=0.05; base P<0.01: time, apex P<0.001; base P<0.001. Epi vs

control, #P<0.05, ##P<0.01 vs ISO alone, one-way ANOVA.

FiFigugugurrere 5. AgAgAgoonisist-t-inindepep ndent negag tive inotropipiccc eeeffect of betablblloockekersrs aand potentiation of

TTaT kkokotsubo carddioioommmyooppaaathyy.. AAA, NeNegagatiivevee inoootrrropicc eeffecect t t ofofof ARARR bbblockkkerrs ononon contttraacctitionnn of f

veventntririricucuculalalarr r mymyocococytytyteseses fffrororomm m faailililininingg g hhuhumamaann n heheheararartt.t. CoCoContntntraraactctctioioion n n aamamplplplitititududude ee rererelalal titiveveve tttooo bbabasasaal ll (o(o(opepepennn

bar)) for ICI 111181818,5,5515151 (((3μ3μ3μM,M,M nnn=2=21)1)1), prprp opopprraranonoololooll (P(P(Prropopop, 5μ5μμM,M,M nnn=99=9)) ) ananandd d cacaarvrvr ededilillololol (((CaCaCarvrrv, 3μμM,



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27

epi+carvedilol: apex P=NS; base P<0.001. Time: apex P<0.001; base P<0.001.

Figure 6. Levosimendan rescues the Takotsubo cardiomyopathy model. Effects of 0.28mg/kg/h

(4.7 μg/kg/min) levosimendan infusion (i.v.) (black bars) on the inotropic responses of the apical

(A), mid left-ventricular (B) and basal myocardium contractility (C) after 4.26x10-8 moles.100g-1

epinephrine (i.v.), compared to epinephrine alone (grey bars). Values are expressed as the mean

percentage change in LV FS from baseline ± SEM at each 5 min time point following injection.

N=6 (epinephrine), n=5 (levosimendan + epinephrine) (**p<0.01, ***p<0.001, ****p<0.0001 vs

baseline FS = 0). RM ANOVA Epi vs epi+levosimendan: P<0.01 (apex), P<0.01 (MLV), P=NS

(base). Time: P<0.001 (apex); P<0.001 (MLV); P<0.001 (base). base). Time: P<0.001 (apex); P<0.001 (MLV); P<0.001 (base).



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Sian E. HardingHong Sun, Nicholas S. Peters, Mario Petrou, Zhaolun Zheng, Julia Gorelik, Alexander R. Lyon andO'Gara, Daniel J. Stuckey, Viacheslav O. Nikolaev, Ivan Diakonov, Laura Pannell, Haibin Gong, Helen Paur, Peter T. Wright, Markus B. Sikkel, Matthew H. Tranter, Catherine Mansfield, Peter

-Adrenoceptor/Gi-Dependent Manner: A New Model of Takotsubo Cardiomyopathy2βHigh Levels of Circulating Epinephrine Trigger Apical Cardiodepression in a

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2012 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation published online June 25, 2012;Circulation.

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High levels of circulating epinephrine trigger apical cardiodepression in a β2-adrenoceptor/Gi-

dependent manner: a new model of Takotsubo Cardiomyopathy

SUPPLEMENTAL MATERIAL

Cardiomyocyte contractility studies

Cardiomyocyte contraction experiments were performed using video edge tracking with the

IonOptix system. Ca2+ concentrations were adjusted to give contraction amplitude of 50% to 75% of

maximum to increase accuracy of contraction measurements (6-8mmol/L in human, 2mmol/L in rat).

Experiments were carried out at 32°C with field stimulation at 0.2 Hz in human, while rat

cardiomyocytes were paced at 0.5 Hz at 37°C. Cardiomyocyte contraction was expressed as the

percentage shortening of cell length upon field stimulation. Cardiomyocytes used to determine the

functional parameters were rod shaped without spontaneous contractions. 2AR-specific contractile

responses were measured in rat cardiomyocytes using isoproterenol (1 µmol/L) plus the 1AR

selective antagonist CGP20712A (300nmol/L).1, 2 Calcium studies were performed with 4mmol/L Ca

+/- epinephrine pretreatment. For β-blocker experiments, the stably contracting human failing

cardiomyocytes were subjected to blockers for 5-15 min, by which time a maximum decrease in

contraction was observed. Only cardiomyocytes showing full reversal of the negative inotropic effect

on washout of the -blockers were used.

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Catecholamine Dose (1.43 x 10 (-x) moles.100g-1)

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80Epinephrine

Norepinephrine

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A

B

C

%Δ

FS

%Δ

FS

%Δ

FS

Data Supplement Figure S1

Supplement Figure S1

Inotropic effect of incremental increases in concentration of epinephrine (i.v.) and norepinephrine

(i.v.) at the apex (a), MLV (b) and base (c). Values are expressed as the mean percentage change in

baseline (untreated) left ventricular fractional shortening (FS) ± SEM, measured for each

concentration at 5 minutes post-catecholamine injection. N=5 (epinephrine), n=4 (norepinephrine).


Haemodynamic arterial responses in in vivo Takotsubo cardiomyopathy model.

A. Representative example of aortic pressure trace recorded before and after intravenous

epinephrine bolus, demonstrating the rapid hypertensive response lasting several minutes, followed

by a secondary hypotension as left ventricular impairment develops. Horizontal broken lines mark

baseline systolic and diastolic blood pressure levels for reference. The tracing in the red box is

expanded in (B) to show the immediate changes in blood pressure and heart rate during the first 40

seconds post epinephrine bolus. Maximum systolic (C) and diastolic (D) blood pressure response in

cases treated with epinephrine (E), norepinephrine (NE), PTX-pretreated rats receiving epinephrine

(E+PTX) or saline control. E. Minimum heart rate during bradycardic reflex phase. *p<0.05 vs saline

controls, one-way ANOVA. N=5-6 per study arm.

20 40 60 80

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20Apex

BaseTime, min

% c

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ge f

rom

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#


Time-dependent changes in ejection fraction measured in cross-sectional area from cardiac MRI-

derived transverse apical (solid lines) and basal (broken lines) left ventricular slices after intravenous

epinephrine (4.28x10-8 moles.100g-1) # P<0.02, **P<0.01 compared to baseline, n=7.

Data Supplement Figure S4


Baseline heart rate and regional ventricular fractional shortening from the apex, mid left ventricle

(MLV) and basal left ventricle at baseline (pre-epinephrine) demonstrating no differences in animals

pretreated with PTX or ICI 118,551 in vivo.


Time matched saline injected controls demonstrating no significant changes in regional ventricular

fractional shortening from the apex, mid left ventricle (MLV) and basal left ventricle in cases

pretreated in vivo with ICI 118,551 (left, A-C) or PTX (right, D-F).


In vivo atropine pretreatment (10mg/kg, intravenous injection) reduced the reflex bradycardia

following subsequent epinephrine injection (above), but failed to prevent epinephrine-induced

apical hypokinesia (below). There was 100% mortality in the atropine-pretreated animals at 30 mins

post epinephrine injection. *p<0.05, unpaired t-test, n=5-6 per study arm.


Epinephrine pretreatment reduced β2AR-dependent isoproterenol responses in a PTX-sensitive

manner. Contraction amplitude (% shortening) of isolated rat ventricular myocytes in the presence

and absence of 1µM isoproterenol plus 300nM CGP20712A (iso); untreated (Con, n=15), pre-treated

for 20 min with 0.1µM epinephrine, 10 min wash (Epi-pre, n=15); preincubated with PTX (PTX, n=6);

pretreated with PTX then exposed to epinephrine (Epi-pre PTX, n=7). ISO studies: *p<0.05,

***p<0.001, paired t-test. Epi-pre vs Epi-pre +PTX +/- ISO ###p<0.0001, unpaired t-test.


Calcium-dependent inotropy is preserved after epinephrine pretreatment.

Contraction amplitude (% shortening) of isolated rat ventricular myocytes in the presence (black

bars) or absence (white bars) of 1µM isoproterenol plus 300nM CGP20712A (ISO); untreated (Con,

n=15), treated for 20 min with 0.1µM epinephrine, 10 min wash (Epi-pre, n=15); 4mM calcium (Ca,

n=5); 4mM calcium after epinephrine pretreatment (Epi-pre, Ca, n=4). ***p<0.001, paired t-test.


β2AR mediated cAMP response of apical and basal CMs (100nM Isoproterenol (ISO) + 100nM

CGP20712A) was not significantly different in terms of raw FRET (above) or %total cAMP production

(below) relative to NHK477 (a forskolin analog) (apical 46.7% ± 5.0 n=14 vs. basal 42.4% ± 4.5 n=16

p=NS: values are % maximal FRET response, P=NS unpaired t-test.)


Negative inotropic effect of the 2AR-specific blocker ICI 118,551 (1µM) is induced in rat ventricular

myocytes by overexpression of wild type 2AR but not 2AR with mutations at PKA phosphorylation

sites 261, 262, 345, 346 S/A (2AR-PKA-KO). Contraction amplitude in 2mM Ca2+ untreated (white

bars) or in the presence of 1µM ICI 118,551 (black bar, 10 min) N=5 preparations, 9 myocytes per

condition, ***P<0.001 vs respective control, paired t-test.


The -blocker bisoprolol (4.28x10-12 moles.100g-1 (i.v.)) did not alter epinephrine-mediated

contractility of the apex but it did reduce the increased contractility of the base, in the in vivo rat

model. Values are expressed as the mean percentage change in LV FS from baseline (untreated)

levels ± SEM at each 5 minute time point following intravenous injection of epinephrine. N=6 (epi),

n=6 (epi+bisoprolol). (**p<0.01, ***p<0.001, vs baseline FS = 0). RM ANOVA epi vs. epi+bisoprolol:

P=NS apex, P<0.05 base. Time: P<0.001 apex; P<0.05 base.

Supplementary Movie File S1

Representative in vivo cardiac cine-MRI of rat heart. Basal and apical short axis and 2 chamber long

axis views were acquired prior to and at multiple time points after adrenaline infusion. Note the

reduction in apical contractility at 15 and 30 mins.

1. Gong H, Sun H, Koch WJ, Rau T, Eschenhagen T, Ravens U, Heubach JF, Adamson DL, Harding SE.

The specific 2AR blocker, ICI 118,551, actively decreases contraction through a Gi-coupled

form of the 2AR in myocytes from failing human heart. Circulation. 2002;105:2497-503.

2. Gong H, Adamson DL, Ranu HK, Koch WJ, Heubach JF, Ravens U, Zolk O, Harding SE. The effect

of Gi-protein inactivation on basal, 1- and 2AR-stimulated contraction of myocytes from

trangenic mice overexpressing the 2-adrenoceptor. Br J Pharmacol. 2000;131:594-600.

high levels of circulating epinephrine trigger apical cardiodepression...

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