carvedilol induces greater control of β2- than β1-adrenoceptor-mediated inotropic and lusitropic...

ORIGINAL ARTICLE

Carvedilol induces greater control of β2-than β1-adrenoceptor-mediated inotropic and lusitropic effectsby PDE3, while PDE4 has no effect in human failing myocardium

PeterMolenaar & Torsten Christ & Emanuel Berk &Andreas Engel &Katherine T. Gillette &

Alejandro Galindo-Tovar & Ursula Ravens & Alberto J. Kaumann

Received: 7 November 2013 /Accepted: 9 March 2014 /Published online: 26 March 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The β-blockers carvedilol and metoprolol provideimportant therapeutic strategies for heart failure treatment.Therapy with metoprolol facilitates the control by phosphodi-esterase PDE3, but not PDE4, of inotropic effects of catechol-amines in human failing ventricle. However, it is not knownwhether carvedilol has the same effect. We investigatedwhether the PDE3-selective inhibitor cilostamide (0.3 μM)or PDE4-selective inhibitor rolipram (1 μM) modified thepositive inotropic and lusitropic effects of catecholamines inventricular myocardium of heart failure patients treated withcarvedilol. Right ventricular trabeculae from explanted heartsof nine carvedilol-treated patients with terminal heart failurewere paced to contract at 1 Hz. The effects of (-)-noradrena-line, mediated through β1-adrenoceptors (β2-adrenoceptors

blocked with ICI118551), and (-)-adrenaline, mediatedthrough β2-adrenoceptors (β1-adrenoceptors blocked withCGP20712A), were assessed in the absence and presence ofthe PDE inhibitors. The inotropic potency, estimated from –logEC50s, was unchanged for (-)-noradrenaline but decreased16-fold for (-)-adrenaline in carvedilol-treated compared tonon-β-blocker-treated patients, consistent with the previouslyreported β2-adrenoceptor-selectivity of carvedilol.Cilostamide caused 2- to 3-fold and 10- to 35-fold potentia-tions of the inotropic and lusitropic effects of (-)-noradrenalineand (-)-adrenaline, respectively, in trabeculae from carvedilol-treated patients. Rolipram did not affect the inotropic andlusitropic potencies of (-)-noradrenaline or (-)-adrenaline.Treatment of heart failure patients with carvedilol inducesPDE3 to selectively control the positive inotropic andlusitropic effects mediated through ventricular β2-adrenoceptors compared to β1-adrenoceptors. The β2-adrenoceptor-selectivity of carvedilol may provide protectionagainst β2-adrenoceptor-mediated ventricular overstimulationin PDE3 inhibitor-treated patients. PDE4 does not control β1-and β2-adrenoceptor-mediated inotropic and lusitropic effectsin carvedilol-treated patients.

Keywords Human heart failure .β1-andβ2-adrenoceptors .

Phosphodiesterases 3 and 4 . Noradrenaline and adrenaline .

Inotropism and lusitropism . Carvedilol

Introduction

Catecholamines increase human atrial and ventricular force,mediated through β1- and β2-adrenoceptors, via thecAMP/cAMP-dependent protein kinase (PKA) pathways(Kaumann et al. 1999, 1996;Molenaar et al. 2000, 2007). cAMPin human heart is mainly hydrolysed by phosphodiesterase

Peter Molenaar and Torsten Christ contributed equally to this work

Electronic supplementary material The online version of this article(doi:10.1007/s00210-014-0974-4) contains supplementary material,which is available to authorized users.

P. Molenaar (*) :K. T. GilletteFaculty of Health, QUT, Brisbane; School ofMedicine, University ofQueensland and Critical Care Research Group, The Prince CharlesHospital, Chermside, QLD 4032, Australiae-mail: [email protected]

T. Christ : E. Berk :A. Engel :U. RavensDepartment of Pharmacology and Toxicology, Dresden University ofTechnology, Dresden, Germany

A. Galindo-Tovar :A. J. Kaumann (*)Research Unit of the University Hospital Virgen de la Arrixaca andDepartment of Pharmacology, University of Murcia,30100 Murcia, Spaine-mail: [email protected]

Present Address:T. ChristDepartment of Experimental Pharmacology and Toxicology,UniversityMedical Center Hamburg Eppendorf, Hamburg, Germany

Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:629–640DOI 10.1007/s00210-014-0974-4

http://dx.doi.org/10.1007/s00210-014-0974-4

PDE3 (Benders and Beavo 2006; Movsesian et al. 1991),thereby controlling atrial (Christ et al. 2006; Kaumann et al.2007) and ventricular (Molenaar et al. 2013) β1- and β2-adrenoceptor-mediated positive inotropic and lusitropiceffects. In addition, ventricular PDE2 expression and func-tion was recently reported to be upregulated in human heartfailure and rat heart failure due to prolonged exposure toisoprenaline, thereby protecting against noradrenaline-inducedhypertrophy (Mehel et al. 2013).

In rodent hearts, however, PDE4 is the most abundantphosphodiesterase expressed (Richter et al. 2011), and theβ-adrenoceptor-mediated inotropic effects of catecholaminesare mainly controlled by PDE4 (reviewed in Kaumann 2011).PDE4 protects against β1-adrenoceptor-mediated arrhythmiasin rodent ventricle (Lehnhart et al. 2005; Galindo-Tovar andKaumann 2008) and human atrium (Molina et al. 2012).However, there is still controversy about the role of PDE4 inthe human heart (Eschenhagen 2013). PDE4 does not seem tolimit the positive inotropic effects of (-)-noradrenaline and (-)-adrenaline through β1- and β2-adrenoceptors, respectively, innon-failing human atrium obtained from patients treated withor without β-blockers (Christ et al. 2006; Kaumann et al.2007), and failing ventricle from patients treated with orwithout the slightly β1-adrenoceptor-selective metoprolol(Molenaar et al. 2013).

Chronic treatment of patients with terminal heart failurewith the β-blocker metoprolol appears to induce PDE3 activ-ity, thereby controlling positive inotropic and lusitropic effectsof the physiological catecholamines, mediated through β1-and β2-adrenoceptors (Molenaar et al. 2013). Metoprolol isnot the onlyβ-blocker currently indicated for the managementof heart failure (Bristow 2000). We, therefore, asked whetherthe facilitation of PDE3 activity is a general property of β-blockers or a specific characteristic property of metoprolol inheart failure patients. To answer this question, we investigatedthe interactions of PDE3 and PDE4 with carvedilol, anotherβ-blocker used in the therapy of heart failure (Poole-Wilsonet al. 2003).

The pharmacological profile of carvedilol is different fromthat of metoprolol. In contrast to quickly reversible blockadeby β1-adrenoceptor-selective metoprolol in heart failure pa-tients, blockade of β1- and β2-adrenoceptors by carvedilol islonger lasting (Kindermann et al. 2004; Molenaar et al. 2006).Furthermore, both the persistent blockade and affinity ofcarvedilol for β2-adrenoceptors are higher than for β1-adrenoceptors in the human heart (Molenaar et al. 2006) andrecombinant receptors (Baker 2005). We now investigatedwhether the inotropic and lusitropic effects of the catechol-amines (-)-noradrenaline and (-)-adrenaline, mediated throughβ1- and β2-adrenoceptors, respectively, are enhanced byPDE3 inhibition with cilostamide and/or PDE4 inhibitionwith rolipram in ventricular trabeculae from patients withterminal heart failure chronically treated with carvedilol.

Methods

Heart transplant patients

Written informed consent was obtained from all patients.Patients with terminal heart failure underwent heart transplantsurgery at The Prince Charles Hospital, Brisbane, ethics ap-proval EC9876, HREC10/QPCH/184 and Gustav Carus Hos-pital, Dresden Technological University ethics committee(Document EK 1140 82202). Clinical data for patients isshown in Table 1. The use of human myocardium conformswith the principles outlined in the Declaration of Helsinki(Rickham 1964).

Isolated right ventricular trabeculae from heart transplantpatients

Hearts from heart transplantation surgery at Brisbane wereplaced immediately into ice-cold pre-oxygenated modifiedKrebs solution containing the following (mM): Na+ 125, K+

5, Ca2+ 2.25, Mg2+ 0.5, Cl− 98.5, SO42− 0.5, HCO3

− 32,HPO4

2− 1 and EDTA 0.04, sealed and transported to thelaboratory where right ventricular trabeculae were dissectedunder continuous oxygenation with 95 % O2/5 % CO2. Dres-den experiments were carried out in Tyrode’s solution (mM):Na+ 149, K+ 5.4, Ca2+ 1.8, Mg2+ 1.05, Cl− 137.8, HCO3

− 22,HPO4

2− 0.42, EDTA 0.04, ascorbate 0.2 and glucose 5.5, andequilibratedwith 95%O2/5%CO2. Trabeculae were clampedto an electrode block and the other end attached to SwemaSG4-45 strain gauge transducers. Electrode blocks werehoused within 50-ml organ baths to accommodate two trabec-ulae in the oxygenated solution supplementedwith (mM): Na+

15, fumarate 5, pyruvate 5, L-glutamate 5 and glucose 10 at37 °C (Kaumann et al. 1999). Trabeculae were driven withsquare-wave pulses of 5-ms duration just above thresholdcurrent to contract at 1 Hz. Contractile force, time to peakforce (TPF) and time to half-relaxation (t0.5) were recordedthrough PowerLab amplifiers on a Chart forWindows, Version5.0 recording programme (ADInstruments Pty Ltd., CastleHill, Australia) or on Graphtec 8 and 12 channel recorders(Kaumann et al. 1999; Molenaar et al. 2006, 2013).

After determination of a length-tension curve, the length ofeach trabeculum was set to obtain a resting tension associatedwith maximum developed force. To irreversibly block tissueuptake of catecholamines and α-adrenoceptors, trabeculaewere incubated for 90 min with phenoxybenzamine (5 μM)followed by a washout (Kaumann et al. 1999; Molenaar et al.2006).

Specific activation of β1- and β2-adrenoceptors

To determine the effects of β1-adrenoceptor-selective activa-tion, concentration-effect curves for (-)-noradrenaline were

630 Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:629–640

obtained in the presence of ICI118551 (50 nM) to selectivelyblock β2-adrenoceptors (Kaumann et al. 1999; Molenaar et al.2006). To determine the effects of β2-adrenoceptor-selectiveactivation, concentration-effect curves for (-)-adrenaline weredetermined in the presence of CGP20712A (300 nM) toselectively block β1-adrenoceptors (Kaumann et al. 1999;Molenaar et al. 2006). To assess the influence of the PDE3-selective inhibitor cilostamide (300 nM) and the PDE4-specific inhibitor rolipram (1 μM) on the effects of the cate-cholamines, a single concentration-effect curve for a catechol-amine was obtained in the absence or presence of a PDEinhibitor. Trabeculae were incubated with PDE inhibitors for30–45 min prior to commencement of catecholamineconcentration-effect curves. At the completion ofconcentration-effect curves to catecholamines on right ven-tricular trabeculae, the effects of a maximal concentration of(-)-isoprenaline (200 μM) were determined. Since up to 20contracting trabeculae were obtained from the same heart, itwas often possible to compare the influence of the PDEinhibitors on responses mediated through both β1 and β2-adrenoceptors as shown in the representative experiment ofFig. 5.

Analysis and statistics

A single concentration-effect curve for (-)-noradrenaline or(-)-adrenaline was obtained in the absence or presence ofcilostamide (300 nM) or rolipram (1 μM). Responses to thecatecholamines were expressed as a percentage of the re-sponse to (-)-isoprenaline (200 μM). The catecholamine con-centrations producing a half maximum response, –LogEC50M(pEC50), were estimated from fitting a Hill function withvariable slopes to concentration-effect curves from individualexperiments. The data are expressed as mean±S.E.M. ofn=number of patients or trabeculae as indicated. Signifi-cance of differences between means were assessed withthe use of either Student’s t test or ANOVA followed byTukey-Kramer multiple comparisons ad hoc test atP<0.05 using InStat software (GraphPad Software Inc.,San Diego, CA).

Results

Decrease of inotropic and lusitropic potencies for (-)-adrenaline by chronic treatment with carvedilol

The potencies of (-)-adrenaline through activation of β2-adrenoceptors were reduced (inotropic 16- to 49-fold,P<0.0001; lusitropic 13- to 35-fold, P<0.0001) incarvedilol-treated patients compared to non-β-blocker-treatedpatients from previous studies (Kaumann et al. 1999;Molenaar et al. 2006, 2013) (inotropic, Fig. 1, Table 2;lusitropic Fig. 2, Table 3). In contrast, the inotropic andlusitropic potencies of (-)-noradrenaline for activation of β1-adrenoceptors in carvedilol-treated patients were only slightly(less than fourfold) or not reduced compared to non-β-blocker-treated patients (Kaumann et al. 1999; Molenaaret al. 2006, 2013) (Fig. 1, Table 2, Fig. 2, Table 3).

In trabeculae obtained from three carvedilol-treated pa-tients, the responses to (-)-adrenaline were greatly depressed,preventing the estimation of meaningful pEC50 values. Ino-tropic and lusitropic results from these three patients are,therefore, shown separately (Figs. 5, 6 and 7). There was nodifference with the daily dose of carvedilol administered to thetwo groups of patients (Fig. 1, 47.9±12.3 mg, n=6; Fig. 6,33.3±11.0 mg, n=3; P=0.5).

Cilostamide potentiates the effects of (-)-adrenaline morethan (-)-noradrenaline in trabeculae from carvedilol-treatedpatients

Cilostamide (300 nM) caused a small, not quite significant1.9-fold increase of the inotropic potency of (-)-noradrenaline(P=0.053; Fig. 3, Table 2) but significantly increased thepotency of (-)-noradrenaline to shorten TPF by 2.6-fold(P<0.01; Fig. 4, Table 3). Cilostamide did not significantlyalter (-)-noradrenaline-evoked shortening of t0.5 (P=0.12) intrabeculae from nine carvedilol-treated patients (Fig. 4,Table 3).

Cilostamide significantly potentiated the inotropic effectsof (-)-adrenaline, 10.2-fold in trabeculae from patients treatedwith carvedilol (P<0.05; Fig. 3, Table 2). The lusitropic

Table 1 Summary of patients chronically administered with carvedilol prior to heart transplantation

n Aetiology n Age (years) Sex n LVEF Dose (mg of carvedilol per day) Medication

9 IDCM 3 44±2.9 M 5 20±0.5 43±9 A7, B1, C8, D4, E4, F2, G6, H3, I2, J1

DCM 4 F 4

IHD 2

n number of patients, DCM dilated cardiomyopathy, IDCM idiopathic dilated cardiomyopathy, IHD ischaemic heart disease,M male, F female, LVEFleft ventricular ejection fraction, A angiotensin-converting enzyme inhibitor, B angiotensin-receptor blocker, C diuretic,D digoxin, Ewarfarin, F nitrate,G statin, H amiodarone, I thyroid hormone, J clopidogrel

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effects of (-)-adrenaline were potentiated by cilostamide (28-fold for TPF, P<0.03; 35-fold for t0.5, P<0.01; Fig. 4,Table 3).

The effects of cilostamide (300 nM) were also investigatedon additional trabeculae from the three carvedilol-treatedpatients that were poor responders to (-)-adrenaline (Figs. 5, 6and 7). In these patients, cilostamide partially restored the

inotropic responses to (-)-adrenaline, allowing estimates ofinotropic potentiation. The average log concentration ratios of(-)-adrenaline in the presence and absence of cilostamide at the10 and 20 % of maximum response levels to (-)-isoprenalinewere approximately 2.0 and 1.7 log units, i.e. equivalent to a100-fold and 50-fold potentiation, respectively. The responsesto (-)-noradrenaline of this group tended to be potentiated and

Fig. 1 Marked reductions in potency for inotropic effects of (-)-adrena-line (b) through β2-adrenoceptors, but not for (-)-noradrenaline (a)through β1-adrenoceptors, in right ventricular trabeculae from six ((-)-

adrenaline experiments) or nine carvedilol-treated patients ((-)-noradren-aline experiments), compared to five non-β-blocker-treated patients fromMolenaar et al. (2013)

Table 2 Effects of cilostamide (300 nM) and rolipram (1 μM) oninotropic potencies of (-)-noradrenaline and (-)-adrenaline, acting throughright ventricular β1- and β2-adrenoceptors, respectively, in trabeculaefrom patients chronically treated with carvedilol. Shown also is historical

data for inotropic potencies of (-)-noradrenaline and (-)-adrenaline in theabsence of PDE inhibitors, from right ventricular trabeculae of patientschronically treated with or without carvedilol

Carvedilol-treated patients (current study)

(-)-Noradrenaline (-)-Adrenaline

pEC50 (n) pEC50 (n)

Control 5.68±0.10 (24/9) 4.50±0.10 (18/6)

Cilostamide 5.89±0.14 (23/9) 5.51±0.25 (16/6)

ΔpEC50 0.285±0.1267 (9) 1.462±0.408 (6)

P1 0.0533 0.0491

P2 0.006

Rolipram 5.72±0.16 (13/7) 4.34±0.13 (10/5)

ΔpEC50 0.021±0.080 (7) 0.4495±0.25 (5)

P1 0.9374 0.3039

Historical data from carvedilol and non-β-blocker-treated patients


Non-β-blocker-treated Carvedilol-treated Non-β-blocker-treated Carvedilol-treated

pEC50 (n) pEC50 (n) pEC50 (n) pEC50 (n)

Historical dataa 5.93±0.13 (18/11) 5.68±0.16 (19/11)

Historical datab 6.21±0.11 (18/9) 5.62±0.12 (31/15) 6.19±0.13 (21/10) 4.63±0.12 (41/15)

Historical datac 5.65±0.15 (11/4) 5.70±0.27 (14/5)

ΔpEC50 is the difference in pEC50 values for (-)-noradrenaline or (-)-adrenaline obtained in the presence of phosphodiesterase inhibitor (cilostamide orrolipram) and without phosphodiesterase inhibitor (control) calculated frommean values for each patient. Values in parentheses are (trabeculae/patients).P1 are P values obtained from paired Student’s t test for comparisons between phosphodiesterase inhibitor (cilostamide or rolipram) and control (nophosphodiesterase inhibitor). P2 are comparisons of the effects of (-)-noradrenaline and (-)-adrenaline. P2 is the value obtained from Student’s t test forcomparisons ofΔpEC50 between (-)-noradrenaline and (-)-adrenaline. Values calculated for effects of cilostamide only.a Kaumann et al. 1999bMolenaar et al. 2006cMolenaar et al. 2013


were included in the group displayed in Fig. 3. Cilostamidealso potentiated the lusitropic responses of (-)-adrenaline by0.93±0.12 log units (TPF) and 1.47±0.64 log units (t50) inthese three patients (Fig. 7).

Rolipram does not modify inotropic and lusitropic potenciesof (-)-noradrenaline and (-)-adrenaline in trabeculaefrom patients treated with carvedilol

Rolipram (1 μM) did not significantly modify force, TPF ort50, in trabeculae from carvedilol-treated patients. The inotro-pic (Figs. 3, 5 and 6; Table 2) and lusitropic potencies (Fig. 4,Table 3) of (-)-noradrenaline and (-)-adrenaline were not sig-nificantly changed by rolipram (1 μM) in trabeculae fromcarvedilol-treated patients.

The effects of the combination of cilostamide (300 nM) androlipram (1 μM) on the inotropic and lusitropic potencies of(-)-noradrenaline and (-)-adrenaline were investigated in thethree carvedilol-treated patients that were poor responders toadrenaline (Figs. 5, 6 and 7). Cilostamide+rolipram potenti-ated the inotropic and lusitropic effects of both (-)-noradren-aline (inotropic and TPF, P<0.003, (t50, P=0.07)) and (-)-adrenaline (P<0.01), but the degree of potentiation did notsignificantly (P>0.07) differ from the potentiation caused bycilostamide alone (Figs. 5, 6 and 7).

Discussion

We confirm that chronic treatment of heart failure patientswith carvedilol reduces the inotropic and lusitropic effects andpotencies of (-)-adrenaline. Carvedilol therapy inducedmarked potentiation by cilostamide of the inotropic andlusitropic effects of (-)-adrenaline mediated through β2-adrenoceptors. In contrast, cilostamide had less ability toincrease the potency of (-)-noradrenaline for β1-adrenoceptor-mediated inotropic and lusitropic effects.Rolipram did not affect the inotropic and lusitropic responsesthrough β1- or β2-adrenoceptors.

Carvedilol treatment reduces responsesthrough β2-adrenoceptors but not β1-adrenoceptors

The β-adrenoceptor blocking effects of carvedilol partiallypersist after removal of tissues from the heart in isolated atrialand ventricular trabeculae (Kindermann et al. 2004; Molenaaret al. 2006). We have previously shown that persistent β2-adrenoceptor blockade is considerably greater than the resid-ual β1-adrenoceptor blockade after removal of tissue from thefailing hearts of carvedilol-treated patients (Molenaar et al.2006). Furthermore, we demonstrated with exogenously ad-ministered antagonist that carvedilol has a 13-fold higher

Fig. 2 Effects of chronic administration of carvedilol compared tonon-β-blocker on lusitropic effects (time to peak force and time to50 % relaxation (t50)) of (-)-noradrenaline through activation of β1-adrenoceptors (a, c) and (-)-adrenaline through activation of β2-adrenoceptors (b, d) in right ventricular trabeculae from failing hearts.Data from four ((-)-noradrenaline experiments) or five ((-)-adrenaline

experiments) patients with heart failure not treated with aβ-blocker (fromMolenaar et al. 2013) and nine ((-)-noradrenaline experiments) or six ((-)-adrenaline experiments) patients with heart failure treated with carvedilol.Carvedilol-treated patients showed marked reductions in potency forlusitropic effects of (-)-adrenaline (b, d). See Table 3 for more details


affinity for human atrial β2- than β1-adrenoceptors (Molenaaret al. 2006). The 16-fold reduction of the inotropic potency of(-)-adrenaline, mediated through β2-adrenoceptors, found inright ventricular trabeculae of carvedilol-treated patients com-pared to non-β-blocker-treated patients, is consistent with a

25-fold potency reduction reported previously (Molenaar et al.2006). In contrast, the inotropic and lusitropic potency of (-)-noradrenaline was not changed by the chronic treatment ofpatients with carvedilol, consistent with a mere 1.8-fold de-crease of (-)-noradrenaline inotropic potency (Molenaar et al.2006).

Therapy of heart failure patients with the β-blocker meto-prolol increases β1-adrenoceptor density and function(Heilbrunn et al. 1989; Gilbert et al. 1996; Sigmund et al.1996). The unchanged noradrenaline potency in trabeculaefrom carvedilol-treated patients could be the result of an actualupregulation of β1-adrenoceptor density combined with par-tial β1-adrenoceptor occupancy with carvedilol firmly boundto the receptors. Residual binding of carvedilol to β1-adrenoceptors would also explain the lack of increase of β1-adrenoceptor receptor density reported in heart failure patientstreated with carvedilol (Gilbert et al. 1996).

Carvedilol facilitates the control by PDE3 of β2- morethan β1-adrenoceptor-mediated effects

Cilostamide produced a small potentiation of the positiveinotropic and lusitropic effects of (-)-noradrenaline, mediatedthrough β1-adrenoceptors, but marked potentiation of theeffects of (-)-adrenaline mediated through β2-adrenoceptorsin ventricular trabeculae from carvedilol-treated patients. The-se data indicate that chronic administration of carvedilol topatients with severe heart failure differentially modifies thecontrol of PDE3 metabolism of cAMP accumulated by acti-vation of β1- and β2-adrenoceptors. The existence of spatialmicrodomains incorporating β1- or β2-adrenoceptors, G-proteins, cAMP, protein kinases, PDEs and AKAP scaffoldingproteins has been described in mouse and rat hearts (Nikolaevet al. 2006, 2010; Perera and Nikolaev 2013). In rat andmousenon-failing ventricular cardiomyocytes, β2-adrenoceptor sig-nalling was reported to be localized to deep transversetububles, whereas β1-adrenoceptor signalling was distributedacross the whole cell surface (Nikolaev et al. 2010). However,in a rat model of chronic heart failure induced by myocardialinfarction, β2-adrenoceptor signalling in ventricularcardiomyocytes became diffuse and was distributed acrossthe whole cell surface (Nikolaev et al 2010). The demonstra-tion of organized signalling structures and their ability, at leastfor β2-adrenoceptor signalling, to re-organize in rat failingheart may shed light on the current findings. It may be possi-ble that the chronic administration of carvedilol in patientswith human heart failure adjusts the spatial alignment ofPDE3 with β2- more than β1-adrenoceptor signalling so thatcAMP accumulated by activation ofβ2-adrenoceptors is moreefficiently metabolized by PDE3. This remains to be verifiedby high resolution imaging techniques (Nikolaev et al 2010),particularly in human myocardium.

Table 3 Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline,acting through right ventricular β1- and β2-adrenoceptors, respectively,from patients chronically treated with carvedilol. Effects of cilostamide(300 nM) and rolipram (1 μM). Historical data is given for non-β-blocked patients

(-)-Noradrenaline (-)-AdrenalinepEC50 (n) pEC50 (n)

TPF

Control 6.16±0.11 (24/9) 4.96±0.20 (18/6)

Cilostamide 6.49±0.12 (23/9) 6.40±0.17 (16/6)

ΔpEC50 0.423±0.126 (9) 1.27±0.18 (6)

P1 0.0086 0.0294

P2 0.0016

Rolipram 6.26±0.20 (12/7) 5.07±0.16 (10/5)

ΔpEC50 0.396±0.226 (7) 0.301±0.166 (5)

P1 0.1614 0.2051

t50Control 6.18±0.10 (24/9) 4.84±0.14 (16/6)

Cilostamide 6.52±0.17 (22/9) 6.38±0.18 (16/6)

ΔpEC50 0.418±0.24 (9) 1.54±0.277 (6)

P1 0.1181 0.0051

P2 0.0098

Rolipram 6.31±0.08 (12/7) 4.90±0.11 (10/5)

ΔpEC50 0.216±0.11 (7) 0.0879±0.304 (4)

P1 0.2518 0.7905

Historical data from non-β-blocker-treated patients


Non-β-blocker Non-β-blocker

pEC50 (n) pEC50 (n)

TPF

Historical Dataa 6.18±0.19 (18/11) 6.51±0.11 (19/11)

Historical Datab 6.45±0.20 (11/4) 6.33±0.28 (13/5)

t50Historical Dataa 6.43±0.17 (18/11) 6.24±0.15 (19/11)

Historical Datab 6.21±0.19 (11/4) 5.96±0.26 (13/5)

ΔpEC50 is the difference in pEC50 values for (-)-noradrenaline or (-)-adrenaline obtained in the presence of phosphodiesterase inhibitor(cilostamide or rolipram) and without phosphodiesterase inhibitor (con-trol) calculated from mean values for each patient. Values in parenthesesare (trabeculae/patients). P1 are P values obtained from paired Student’s ttest for comparisons between phosphodiesterase inhibitor (cilostamide orrolipram) and control (no phosphodiesterase inhibitor). P2 is the valueobtained from Student’s t test for comparisons of ΔpEC50 between (-)-noradrenaline and (-)-adrenaline. Values calculated for effects ofcilostamide onlya Kaumann et al. 1999bMolenaar et al. 2013


β2-Adrenoceptor-selective potentiation of the inotropicand lusitropic effects of (-)-adrenaline was also observed inventricular trabeculae from metoprolol-treated patients withheart failure (Molenaar et al. 2013) and in human atrial myo-cardium obtained from patients without heart failure, treatedwith or without a mixed group of β-blockers (metoprolol,bisoprolol, nebivolol, sotalol (∼70 % metoprolol)) which didnot include carvedilol (Christ et al. 2006). In this study, ourresults from carvedilol-treated patients with heart failure re-veal an even more pronounced control of PDE3 on β2-adrenoceptor-mediated effects. This may be due to a greaterability of carvedilol to localize PDE3 to cAMP accumulatedby activation of β2-adrenoceptors than metoprolol.

Although β2-adrenoceptors of non-failing human ventriclecouple tightly to Gs (Kaumann and Lemoine 1987), theyappear partially uncoupled from the Gs-cAMP pathway inchronic heart failure (Bristow et al. 1989). We speculate that

chronic treatment with carvedilol improves the coupling ofβ2-adrenoceptors to the Gs-cAMP pathway. Following β-adrenoceptor-mediated increases in cAMP levels and PKAactivity, PDE3 is phosphorylated (Gettys et al. 1987) and inturn hydrolyses cAMP, thereby reducing the positive inotropicand lusitropic effects of endogenous catecholamines. Thiseffect is likely to be more important for β2-adrenoceptors, atleast in the human heart, because these receptors are moreefficient than β1-adrenoceptors at activating Gs protein andstimulating atrial and ventricular adenylyl cyclase from non-failing human hearts (Gille et al. 1985; Kaumann andLemoine 1987), as later verified with recombinant receptors(Green et al. 1992; Levy et al. 1993).

The α subunits of Gi protein, Giα, are upregulated in heartfailure (Neumann et al. 1988), and β2-adrenoceptors cancouple to and activate Gi, in addition to Gs, when stimulatedby isoprenaline in human atrium (Kilts et al. 2000). A 3-month

Fig. 3 Cilostamide (300 nM)potentiates the inotropic effects of(-)-adrenaline (six patients) (b)more than (-)-noradrenaline (ninepatients) (a) in right ventriculartrabeculae from patients withheart failure chronicallyadministered with carvedilol. Nopotentiation by rolipram (1 μM)

Fig. 4 Cilostamide, but notrolipram, potentiates lusitropiceffects of (-)-adrenaline (TPFP<0.03; t50 P<0.01) and (-)-noradrenaline (TPF P<0.01, butnot t50 P=0.12) in rightventricular trabeculae from six((-)-adrenaline experiments) ornine ((-)-noradrenalineexperiments) patients with heartfailure chronically administeredwith carvedilol. Shown areconcentration-effect curves to (-)-noradrenaline (a, c) and (-)-adrenaline (b, d) in the absence orpresence of cilostamide (300 nM)or rolipram (1 μM). S.E.M. barsnot shown for rolipramexperiments to assist clarity. SeeTable 3 for more details


treatment of heart failure patients with metoprolol causes a74 % decrease in Giα (Sigmund et al. 1996). This reduction ofGiαwould allow a greater coupling ofβ2-adrenoceptors to Gs,thereby facilitating inotropic and lusitropic effects mediatedthrough these receptors. We speculate that a decrease of Gi

activity may also have occurred in chronic carvedilol-treatedpatients, as observed with metoprolol in heart failure patients(Sigmund et al. 1996), thereby facilitating coupling of β2-adrenoceptors to Gs. When the persistent β2-adrenoceptor

blockade by carvedilol is surmounted with (-)-adrenaline,improved coupling to Gs protein may become evident, andgreater PKA-catalyzed phosphorylation and activation ofPDE3 occurs.

As found with the inotropic effects, cilostamide potentiatedthe lusitropic effects mediated through β2- more than β1-adrenoceptors in trabeculae from carvedilol-treated patients(Figs. 4 and 7, Table 3). This was expected from similarincreases in PKA-catalyzed phosphorylation of the proteins

Fig. 5 Potentiation of inotropic effects of both (-)-noradrenaline and (-)-adrenaline in the presence of cilostamide (Cil, 300 nM) but not rolipram(Rol, 1 μM). Representative experiment carried out on right ventriculartrabeculae obtained from a 38-year-old male patient with DCM, leftventricular ejection fraction of 20 %, and chronically administered

carvedilol 37.5 mg daily. Shown are original traces for (-)-noradrenalineand (-)-adrenaline followed by (-)-isoprenaline (ISO, 200 μM). Thebottom panels show the corresponding graphical representation of theconcentration-effect curves to the catecholamines with non-linear fits

Fig. 6 Marked potentiation of theinotropic effects of (-)-adrenalineby cilostamide (b) in rightventricular trabeculae from threehearts chronically treated withcarvedilol and comparison with(-)-noradrenaline (a). The heartswere notable for poorresponsiveness to (-)-adrenalinein the absence of cilostamide.S.E.M. bars not shown forrolipram experiments in a to assistclarity


mediating myocardial relaxation, phospholamban (at Ser16),troponin I and cardiac myosin-binding protein C through β1-and β2-adrenoceptors in ventricular myocardium from failinghearts (Kaumann et al. 1999). Our lusitropic results are con-sistent with the localization of PDE3 in the vicinity of phos-pholamban (Movsesian et al. 1991; Lugnier et al. 1993).

PDE4 does not control the inotropic and lusitropic effectsof catecholamines in carvedilol-treated patients with heartfailure

Rolipram did not potentiate the positive inotropic andlusitropic effects mediated through β1- and β2-adrenoceptorsin trabeculae from carvedilol-treated patients with heart fail-ure. Furthermore, rolipram did not enhance the potentiationcaused by cilostamide (Fig. 6). A similar failure of rolipram topotentiate catecholamine effects was reported previously forhuman non-failing atrium (Christ et al. 2006; Kaumann et al.2007) and failing ventricle from metoprolol-treated patients(Molenaar et al. 2013). This evidence, taken together, allowsthe conclusion that under our conditions, PDE4 does notcontrol human atrial and ventricular inotropic and lusitropiceffects, mediated through β1- and β2-adrenoceptors. Howev-er, it has been reported that PDE4 reduces the incidence ofarrhythmias mediated through human atrial β1- and β2-adrenoceptors (Molina et al. 2012). Furthermore, althoughPDE4 protects against β1-adrenoceptor-mediated ventriculararrhythmias in mice (Lehnhart et al. 2005; Galindo-Tovar and

Kaumann 2008), it is unknownwhether this happens in failinghuman ventricle.

Plausible clinical relevance

By preventing cAMP hydrolysis, PDE3 inhibitors (e.g.milrinone and enoximone) enhance cardiac contractilitythrough activation of cAMP-dependent pathways. Short-lasting infusions or low-dose oral treatment with PDE3 inhib-itors have been shown to improve systolic function in chronicheart failure (Anderson 1991; Van Tassel et al. 2008). It isplausible that the potentiation of the effects of catecholaminescontributes to the positive inotropic effects of PDE3 inhibitorsin the clinic (Molenaar et al. 2013 and this paper). It could beargued that the consequences of the suppression bycilostamide of the PDE3-induced control of the inotropicand lusitropic responses mediated through β1- and β2-adrenoceptors are transient because they were only observedin a time frame of less than 2 h. Conceivably, at a later stage,compensatory PDEs could be activated through PKA-catalyzed phosphorylation by the cAMP accumulated duringPDE3 inhibition. Preliminary results reveal, however, thatcilostamide reduces the fade of the inotropic responses tonoradrenaline up to 21 h after cilostamide administration(Unpublished experiments of Galindo-Tovar and Kaumann),inconsistent with intervention of other PDEs.

High-dose chronic treatment with PDE3 inhibitors worsensheart failure and increases mortality, particularly through sud-den death (Packer et al. 1991; Amsallem et al. 2005),

Fig. 7 Marked potentiation oflusitropic effects of (-)-adrenalineby cilostamide in right ventriculartrabeculae from three heartschronically treatedwith carvediloland comparison with (-)-noradrenaline. The hearts werenotable for poor responsiveness to(-)-adrenaline in the absence ofcilostamide. Shown areconcentration-effect curves to (-)-noradrenaline and (-)-adrenalinein the absence or presence ofcilostamide (300 nM), or acombination of both cilostamideand rolipram (1 μM). Rolipramon its own had no effect on (-)-adrenaline- or (-)-noradrenaline-mediated lusitropic effects(not shown)


presumably due to ventricular fibrillation. Arrhythmias, elic-ited by endogenous catecholamines, may be facilitated byPDE3 inhibitors. (-)-Noradrenaline and (-)-adrenaline producea similar incidence of arrhythmic contractions, mediatedthrough β1- and β2-adrenoceptors, respectively, in humanatrial myocardium from patients without heart failure(Kaumann and Sanders 1993), despite the lower density ofβ2-adrenoceptors (Kaumann et al. 1995), probably related tothe tighter coupling of human β2-adrenoceptors to Gs protein.Elevations of endogenous plasma adrenaline levels duringstress (Wortsman 2002), stress cardiomyopathy (Wittsteinet al. 2005) and cardiopulmonary bypass surgery (Reveset al. 1982) are considerably higher than increases in nor-adrenaline. Human ventricle from failing hearts also showspro-arrhythmic β2-adrenoceptor-mediated after contractions,after transients and increases in Ca2+ transient amplitude, SRload and twitch [Ca2+] decline rate (De Santiago et al. 2008).

We have argued that the joint treatment of heart failurepatients with a PDE3 inhibitor and the slightly β1-adrenoceptor-selective blocker metoprolol could facilitate ad-verse stress-induced adrenaline effects such as arrhythmiasmediated through β2-adrenoceptors (Molenaar et al. 2013).In contrast, due to the selective blockage of β2-adrenoceptors,our quantitative assessment (Supplement) allows the predic-tion that carvedilol also protects the heart against adverseeffects of adrenaline in heart failure patients treated togetherwith a PDE3 inhibitor.

Carvedilol appears to produce more benefit than metopro-lol in heart failure as shown in the COMET trial (Poole-Wilson et al. 2003). The greater benefit could be partiallyrelated to the β2-adrenoceptor-selective (Molenaar et al.2006) blockage. Carvedilol, but not metoprolol, would pre-vent the appearance ofβ2-adrenoceptor-mediated arrhythmias(Remme et al. 2007; Di Lenarda et al. 1999) by endogenousadrenaline, which was even shown to decrease the incidenceof arrhythmias in heart failure patients carrying antibodieswith agonist activity through β2-adrenoceptors (Chiale et al.1995; Li et al. 2010). However, experimental evidence dem-onstrating β2-adrenoceptor-mediated arrhythmias, facilitatedby PDE3 inhibition, is still pending for human myocardium.

Conclusions

Chronic treatment of heart failure patients with carvedilolproduces a marked control by PDE3 of human ventricularinotropic and lusitropic effects of (-)-adrenaline mediatedthrough β2-adrenoceptors. Potentially adverse effects bystress-induced surges of (-)-adrenaline in the presence of aPDE3 inhibitor would be prevented by the selective blockageof β2-adrenoceptors with carvedilol. PDE4 does not controlthe inotropic and lusitropic effects mediated through β1- and

β2-adrenoceptors in a human heart from patients chronicallytreated with carvedilol.

Acknowledgments The authors thank the transplant heart surgeons andcoordinators of The Prince Charles Hospital, Chermside and Carl GustavCarus Hospital Dresden. This work was supported in part through thePrince Charles Hospital Foundation.

Conflict of interest None declared.

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carvedilol induces greater control of β2- than β1-adrenoceptor-mediated inotropic and lusitropic...

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