Br. J. Pharmacol. © Macmillan Press Ltd, 1993

Incomplete reversal of 3-adrenoceptor desensitization in humanand guinea-pig cardiomyocytes by cyclic nucleotidephosphodiesterase inhibitors

'Dylan G. Wynne, Philip A. Poole-Wilson & Sian E. Harding

Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY

1 The decreased response to P-adrenoceptor stimulation seen in heart failure may be related to a defectin cyclic AMP production. The inotropic effects of the selective phosphodiesterase (PDE) III inhibitors,SK&F 94120 and SK&F 94836, and the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine(IBMX), alone and when combined synergistically with isoprenaline, were studied in control andP-adrenoceptor-desensitized ventricular myocytes.2 Myocytes isolated from noradrenaline-treated guinea-pigs had a reduced maximum response toisoprenaline compared with control animals (60.0 ± 2.5%, n = 42 vs 79.5 ± 1.7% maximum calcium:n = 46, P <0.001). Together with an approximately 20 fold increase in the isoprenaline EC", this isindicative of ,-adrenoceptor desensitization as a result with chronic infusion with noradrenaline.3 The maximum inotropic response of IBMX was depressed following noradrenaline treatment, from74.9 ± 4.6% (n = 7) in control, to 61.7 ± 2.70% (n= 6), as a percentage of maximum calcium innoradrenaline-treated guinea-pig ventricular myocytes (P<0.02). The pD2 value for IBMX was alsoreduced (P <0.02). No significant differences in the inotropic effects of SK&F 94120 and SK&F 94836were seen between control and P-adrenoceptor desensitized myocytes.4 Threshold inotropic concentrations of SK&F 94120 and SK&F 94836 caused a five fold decrease inthe EC50 of control myocytes for isoprenaline, and an 11 fold decrease in the noradrenaline-treatedguinea-pig ventricular myocytes.5 The maximum response to isoprenaline in myocytes isolated from normal guinea-pigs was unaffectedby PDE inhibition; either at threshold or maximum inotropic concentrations, or by CPT cyclic AMP, ananalogue of cyclic AMP.6 A significant potentiation of the maximum isoprenaline response by threshold inotropic concentra-tions was observed with SK&F 94120 (P<0.05), but not with IBMX or SK&F 94836, in myocytesisolated from noradrenaline-treated guinea-pig hearts. This potentiation, however, did not completelyrestore the response to levels seen in control myocytes.7 The extent of potentiation of the maximum isoprenaline response by maximum inotropic concentra-tions of either IBMX or CPT cyclic AMP, was no greater than that by threshold concentrations ofIBMX, in myocytes isolated from noradrenaline-treated guinea-pig hearts.8 In cardiac myocytes isolated from the explanted hearts of 16 patients with heart failure, thresholdconcentrations of IBMX and SK&F 94120 decreased the isoprenaline EC50 by a factor of four and six,respectively, but potentiation of the maximum isoprenaline response occurred only with SK&F 94120.The attenuated isoprenaline response was increased from 60.3 ± 4.5% to 74.3 ± 4.2% as a % maximumcalcium (P<0.05, n = 6), but remained substantially lower than the 116 ± 7% (P<0.001, n = 6) seen inmyocytes isolated from non-failing hearts.9 We conclude that the reduced maximum contraction amplitude with isoprenaline in cardiac myocytesfrom either patients in end-stage failure, or noradrenaline-treated guinea-pigs, is partly but not solelydue to insufficient cyclic AMP levels, since inhibition of cyclic AMP degradation does not result incomplete reversal of the ,-adrenoceptor desensitization.

Keywords: Cardiac myocyte; phosphodiesterase inhibitor; heart failure; B-adrenoceptor desensitization; cyclic AMP

Introduction

Cyclic nucleotide phosphodiesterase (PDE) enzymes regulatemyocardial contraction. They terminate the action of adeno-sine 3':5'-cyclic monophosphate (cyclic AMP) by catalysingits hydrolysis to 5' adenosine monophosphate (Reeves et al.,1987). Inhibition of PDE results in an increase in cyclicAMP, similar to P-adrenoceptor agonist stimulation of ad-enylate cyclase. The resulting increased activation of cyclicAMP-dependent protein kinase elicits increased phosphoryla-tion of several regulatory proteins in the heart, with subse-quent increased contractility (Feldman et al., 1987; Karczewskiet al., 1990; Bristow et al., 1990). The initial aim of this studywas to characterize the inotropic effects of PDE inhibition inventricular myocytes isolated from both human and guinea-pig hearts. Previous work on PDE inhibition in cardiac

IAuthor for correspondence.

muscle has been carried out on whole muscle preparations(Feldman et al., 1987; Gristwood et al., 1987; Weishaar et al.,1987; Bohm et al., 1988a,d; Schmitz et al., 1989; Bethke etal., 1991; von der Leyen et al., 1991). The advantages ofmyocyte over multicellular preparations are numerous. Theyinclude the lack of scar tissue or fibrosis, and the avoidanceof external neural and humoral influences (Harding et al.,1991). We have previously shown that selected isolatedmyocytes retain the characteristics of whole heart and iso-lated papillary muscle in terms of response to isoprenalineand increased extracellular calcium (Harding et al., 1988).

Five isoenzymes of PDE have been defined; PDE I-V, andtheir inhibitors are classified according to their selectivity(Beavo & Reifsnyder, 1990). PDE III inhibition appears tobe necessary for eliciting a positive inotropic effect, irrespec-tive of whether the substance is a selective PDE III inhibitor

Br. J. Pharmacol. (1993), 109, 1071-1078 '." Macmillan Press Ltd, 1993

1072 D.G. WYNNE et al.

or inhibits cell isoenzymes with similar potencies (Schmitz etal., 1989). In this study, we therefore used the selective PDEIII inhibitors (4-acetamido)phenylpyrazin-2(1H)-one (SK&F94120) (Reeves et al., 1987; Gristwood et al., 1987) and2-cyano-1-methyl-3- [4-(4-methyl-6-oxo- 1,4,5,6-tetrahydropy-ridazin-3-yl)phenyl]guanidine (SK&F 94836) (Beavo, 1988).Also studied was 3-isobutyl-1-methylxanthine (IBMX), anon-selective PDE inhibitor (Schmitz et al., 1989; Nicholsonet al., 1991).

Attenuated contractile response to P-adrenoceptor stimula-tion, as a consequence of a decrease in both the receptornumber (Bristow et al., 1982) and cyclic AMP production(Feldman et al., 1987; Danielsen et al., 1989), is well docu-mented in heart failure (Bohm et al., 1988a,c; Bristow et al.,1990). This is believed to be a result of chronic exposure tothe observed high plasma noradrenaline levels of patientswith heart failure (Thomas & Marks, 1978). Therefore, oursecond aim in this study was to investigate the synergisticeffects of PDE inhibitors and 13-adrenoceptor stimulation byisoprenaline in myocytes isolated from both failing humanhearts and the hearts of noradrenaline-treated guinea-pigs.Such analysis will determine to what degree reversal of P-adrenoceptor desensitization is possible in these tissues.

Methods

Male Dunkin-Hartley guinea-pigs of weight 300-700 g wereanaesthetized with 2% Hypnorm (0.5 ml kg-') and a localinjection of lignocaine hydrochloride (2 ml kg-'). Osmoticminipumps (model 2001, Alzet, U.S.A.) containing (-)-noradrenaline hydrochloride dissolved in saline and 1 mMascorbate were incubated in saline for 4 h at 37°C andimplanted sub-cutaneously in the neck. The mean pumpingrate of the pumps was 0.97il h-' with a concentration ofnoradrenaline such that the guinea-pigs received, over aperiod of 7 days, 900 pg kg-' h-'.Human ventricular tissue was obtained from explanted

hearts at the time of transplant. All patients were in endstage heart failure; nine due to ischaemic cardiomyopathy,five due to dilated cardiomyopathy, one hypertrophic car-diomyopathy and one primary pulmonary hypertension. Twonon-failing hearts from normal donors were also obtained,the circumstances being patient incompatibility and excessivearteriosclerosis.

Isolation of the ventricular myocytes was as previouslydescribed (Harding et al., 1990), via enzymatic digestionprocedures, involving a Langendorff perfusion method forguinea-pig hearts and a modified chunk isolation for humanmyocardium. The isolated myocytes were chosen for contrac-tion experiments via strict selection criteria. These were; asarcomere length greater than 1.60 Am (human) and 1.701lm(guinea-pig), spontaneous contractions of a rate less than2 min-' when unstimulated, stable contraction amplitude anddiastolic length at a stimulation rate of 0.2 Hz (human) and0.5 Hz (guinea-pig), and a complete recovery of contractionamplitude after challenge with maximally stimulating concen-trations of calcium. Only those cells which contracted in linewith their long axis could be studied. Contractility experi-ments were carried out as previously described (Harding etal., 1988), and continuously monitored with a video camera/edge detection system.

Concentrations of inotropes were increased cumulativelyuntil no further increase in contraction amplitude was shownover one log unit, or until arrhythmic contractions wereobtained. The ECm, i.e. concentration at which half maximalresponse is obtained, values for isoprenaline and the isopren-aline/calcium ratio (the maximum response to isoprenalinerelative to that produced by maximally stimulating concen-trations of calcium) for ventricular myocytes have been deter-mined to be an indication of the severity of heart failure incells isolated from failing human hearts (Harding et al.,1992a), and an indication of the degree of desensitization in

myocytes from noradrenaline-treated animal hearts (Jones etal., 1990; Brown & Harding, 1992).The effectiveness of the PDE inhibitors was tested by their

ability to potentiate the positive inotropic effect of isoprena-line. Following initial maximum response to calcium andisoprenaline, a threshold inotropic concentration of PDEinhibitor was determined and the concentration-responsecurve to isoprenaline repeated in its presence. For concentra-tion-response curves, responses were normalised to their ownmaxima with basal and, when present, minimal PDE res-ponses, subtracted. In the graphs showing % cell shorteningand maximum contraction as a percentage of maximum cal-cium, unless otherwise indicated, no basal subtractions weremade. In experiments where the effect of maximum inotropicconcentrations of IBMX or 8-(4-chlorophenylthio)-adenosine3':5'-cyclic monophosphate (CPT cyclic AMP) were investi-gated, the compounds were added at the point of maximumresponse to isoprenaline.

Previous results have shown a decline in responsiveness ofguinea-pig ventricular myocytes to successive isoprenalineconcentration-response curves (a decrease of 9.3 ± 2.35%s.e.mean, n = 6, in the maximum contraction amplitude).Subtracting the preceding basal contraction away from eachmaximum isoprenaline response did not abolish the effect.Thus it was necessary to carry out second control calciumand isoprenaline concentration-response curves followingPDE inhibitor washout so that the stability of myocytescould be evaluated. The maximum inotropic effect of iso-prenaline in the presence of PDE inhibition was compared tothe mean of the two control isoprenaline maxima.A second control curve to isoprenaline was not routinely

carried out in human myocytes. Previous experiments haveindicated that there is little decrease in the maximum res-ponse to P-adrenoceptor stimulation with time in humancells. Three consecutive challenges with P-agonist were per-formed on the same cell, 1 h apart. There was no significantchange in maximum shortening (5.4 ± 0.5%, 5.5 ± 0.4% and5.3 ±0.5% respectively, n= 7) or pD2 (7.55 ±0.28, 7.32 ±0.22 and 7.22 ± 0.30, n = 5). Thus, comparisons were madeonly with the initial isoprenaline maximum.

Statistical significance was assessed by Student's t tests (forpaired data where possible) and P values quoted. Valuesquoted throughout are mean ± standard error and n numbersrepresent the numbers of animals or explanted hearts. Re-sults obtained from several cells for one heart preparationwere pooled. For EC50s, t tests were performed on the logtransformed variable (the pD2).

Materials

(-)-Noradrenaline hydrochloride, (-)-isoprenaline hydro-chloride and CPT cyclic AMP were obtained from Sigma.SK&F 94120 and SK&F 94836 were from Smithkline andBeecham, the kind gift of Brian Warrington, and werediluted from stock solutions of 10-2M dissolved in 0.1 MNaOH solution in dimethyl sulphoxide (DMSO), IBMX, wasobtained from Aldridge Chemical Group, Dorset, and dis-solved to l0'M stock solution in Krebs Henseleit solution.

Results

Guinea-pig ventricular myocytes

Characterization of myocytes isolated from noradrenaline-treated guinea-pigs The maximum percentage cell shorteningin the presence of high extracellular calcium was similar inmyocytes isolated from control and noradrenaline-treatedguinea-pigs. The responses were 10.8 ± 1.8% (n = 46) and10.8+ 0.6% (n = 42) respectively, with a maximal concentra-tion range of 8 to 20 mM Ca2+. Recovery from a challengewith maximum calcium was rapid for both groups. Thissuggests that those myocytes subjected to high concentrations

PHOSPHODIESTERASE INHIBITOR EFFECTS ON CARDIOMYOCYTES 1073

of noradrenaline for 7 days were structurally intact. Nomajor cellular defect of excitation-contraction coupling or inthe contractile apparatus was detected.The maximum isoprenaline/calcium ratio for the pooled

left ventricular myocytes isolated from the noradrenaline-treated guinea-pigs was 60.0 ± 2.5% (n = 42) which issignificantly less (P<0.001) than that of the myocytesisolated from the control guinea-pigs (79.5 ± 1.7%, n = 46).Such a depression is an indication of P-adrenoceptor desen-sitization as a result of the noradrenaline treatment. This isalso shown by the approximately 20 fold increase in the EC50of the isoprenaline concentration-response curves; pD2 valuesbeing 8.72 ± 0.07 JAM (n = 40) for the control myocytes and7.47 ± 0.09 JAM (n = 38) for the noradrenaline treated group(P< 0.001).The isoprenaline/calcium ratio and the pD2 value for

isoprenaline in left ventricular myocytes isolated from sham-operated guinea-pigs were not significantly different fromventricular myocytes isolated from normal/untreated guinea-pig hearts (data not shown). This is consistent with aprevious study where we used an animal model of,-agonistinduced desensitization (Jones et al., 1990). Thus data obtain-ed from myocytes isolated from sham-operated and untreatedguinea-pigs were pooled together and considered controldata.

Increase in contraction amplitude with PDE inhibitors Figure1 shows the concentration-response curves to IBMX in bothcontrol and noradrenaline-treated guinea-pig ventricular my-ocytes. There was a significant rightward shift in the nora-drenaline-treated guinea-pig ventricular myocytes comparedto control, pD2 values being 4.37 ± 0.20 JAM (n = 6) and5.36 ± 0.15 JAM (n = 7), respectively (P< 0.005). The max-imum contraction amplitudes compared to calcium weredepressed following noradrenaline treatment, from 74.9 +4.6% (n = 7) to 61.7 ± 2.7% (n = 6) (P<0.02).The necessary amount of solvent used to dissolve the

compounds SK&F 94120 and SK&F 94836 limited the max-imum concentration which could be used. In the case ofSK&F 94120, concentrations of 3 mM or greater had a det-rimental effect on the cells due to the negative inotropic effectof the DMSO concentration (a decline in contraction amp-litude of 12.7 ± 4.5%, n = 5, for dilutions of 3:1000, and19.8 ± 3.8 JAM, n = 4, for 1: 100 dilutions). For this reason itwas not always possible to obtain true EC,0 values forSK&F 94120 and SK&F 94836, particularly in the noradren-aline-treated animals.

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Those experiments where maximal inotropic concentra-tions of SK&F PDE inhibitors were possible, showed a slightshift to the right in the noradrenaline-treated guinea-pig ven-tricular myocyte-response curve, compared to control res-ponses. These shifts were smaller than those seen withIBMX, and were not statistically significant (pD2 shifts being5.01 ±0.2411M to 4.48±0.13.tLM, n=6, for SK&F94120and 4.60 ± 0.33 gM, n =8, to 4.36 0.30 gM, n = 6, forSK&F 94836). Noradrenaline treatment did not cause asignificant depression of the maximum obtainable responseby PDE inhibition with SK&F 94120 or SK&F 94836. How-ever, the changes seen are underestimated due to the neces-sary omission of cells from the most severely desensitizedanimals.

In Table 1, myocyte contraction amplitude was normalizedto the maximum calcium response. Mean response values toa common PDE inhibitor concentration: 10 JIM, were cal-culated from all the experiments on myocytes isolated fromcontrol and noradrenaline-treated guinea-pigs. A significantdepression of the response was seen for IBMX in noradren-aline-treated animals, but not for SK&F 94120 or SK&F94836.

Synergism of PDE inhibition and P-adrenoceptor stimula-tion Due to the varying degree of desensitization, we didnot use a standard concentration of PDE inhibition in theinvestigation of its synergistic effects with isoprenaline. In-stead a minimum inotropic effect was determined for eachcell. In the case of SK&F 94120, for the control myocytes themean threshold concentration used was 1.99 ± 1.08 JAM whichgave a mean increase in cell shortening over basal, as apercentage of maximum calcium, of 10.1 ± 1.9% (n = 9).These values are not significantly different from those of thenoradrenaline-treated guinea-pigs ventricular myocytes, wherea concentration of 7.33 ± 1.71 JiM was used, giving a responseof 8.43 ± 4.11% (n = 6). Similarly, the threshold inotropicresponse of IBMX was 20.6 ± 5.1% at a mean concentrationof 3.66 ± 0.99 JAM (n = 1 1) in control myocytes, not signifi-cantly different from 11.7 ± 5.6% at a mean concentration of5.67 ± 1.39 JAM (n = 9) in the noradrenaline-treated guinea-pig ventricular myocytes. Mean threshold concentrations ofSK&F 94836 were significantly different for the two groups:2.79 ± 1.10IAM for the control myocytes and a higher 15.5 +47. 1AM for the myocytes isolated from noradrenaline-treatedguinea-pigs (P <0.05). Despite this, the increase over basalwas significantly lower in the noradrenaline group as com-pared to controls: 3.25 ± 1.77% (n = 6) and 22.2 ± 4.6%(n = 8) (P< 0.01), respectively.

Table 2 gives the % maximum cell shortening data forisoprenaline concentration-response curves, both alone and incombination with the minimum inotropic concentration ofthe named PDE inhibitors. In the case of SK&F 94120, thesevalues are expressed graphically in Figure 2a,b; for myocytesisolated from control and noradrenaline-treated guinea-pigs.For every case of PDE inhibition, apart from IBMX incontrol myocytes, the isoprenaline concentration-responsecurve was significantly shifted to the left in the presence of a

Table 1 Contraction amplitude of control andnoradrenaline-treated guinea-pig ventricular myocytes with10iJM SK&F94120, SK&F94835 and 3-isobutyl-1-methyl-xanthine (IBMX)

-log [IBMXI M

Figure 1 Concentration-response curves to 3-isobutyl-1-methylxan-thine (IBMX) in ventricular myocytes isolated from control (0,n = 7) and noradrenaline-treated guinea-pig hearts (U, n = 6).Values are expressed as a percentage of the maximum isoprenalineresponse with basal contraction amplitudes subtracted, and aremean± s.e.mean. *P<0.001 statistically significantly different res-ponse at the corresponding concentration of isoprenaline.

SK&F 94120 SK&F 94836

Control

Noradrenaline-treated

25.7 ± 10.2(6)

11.0 ± 4.2(9)

22.8 ± 6.6(9)

11.7 ± 4.5(8)

IBMX

61.4± 9.1(7)

10.4 ± 4.9(6)*

Values expressed as % maximum calcium response,mean ± s.e.mean. Number of hearts in parentheses.*P<0.001 significantly different to corresponding control.

1074 D.G. WYNNE et al.

Table 2 Maximum contraction amplitudes and pD2 values of control and noradrenaline-treated guinea-pig ventricular myocytes toisoprenaline and isoprenaline in the presence of threshold inotropic concentrations of phosphodiesterase (PDE) inhibitors

SK&F 94120Control NA-treated

SK&F 94836Control NA-treated

IBMXControl NA-treated

IsoprenalineContraction amplitude(% maximum calcium response)pD2 (nM)

Isoprenaline & PDEIContraction amplitude(% maximum calcium response)pD2 (nM)

79.0 ± 3.8(7)

8.67 ± 0.08(6)

83.0 ± 6.8(6)

9.28 ± 0.10(7)tt

50.5 ± 7.3(7)*

7.08 ± 0.23(6)**

70.0 ± 2.0(6)t

8.11 ± 0.20(6)**tt

68.1 ±4.2(6)

8.81 ± 0.09(7)

65.4 ± 5.3(8)

9.48 ± 0.08(6)tt

45.1 ± 5.0(5)*

7.17 ± 0.15(6)**

57.3 ± 7.6(6)

8.23 ± 0.24(5)**tt

68.5 ± 3.7(9)

8.72 ± 0.23(6)

71.1 ±4.3(1 1)

9.06 ± 0.26(6)

50.3 ± 5.9(6)*

7.15 ± 0.15(7)**

61.9 ± 4.7(9)

8.26 ± 0.24(7)*tt

Mean values ± s.e.mean, number of hearts in parentheses. PDEI: PDE inhibition.*P <0.01, **P <0.001 significantly different from corresponding control.tP<0.05, ttP<0.01 significantly different from initial isoprenaline in corresponding group.

PDE inhibitor. There was a tendency for a greater shift inthe noradrenaline-treated guinea-pig ventricular myocytesthan in the control myocytes. Figure 2b shows an approxi-mate 12 fold shift in the isoprenaline EC_0 caused by SK&F94120 in the noradrenaline-treated guinea-pig ventricularmyocytes, comparable to only 4 fold in the control myocytes(Figure 2a).

Figure 3 represents the maximum contractile responses ofboth myocyte groups to mean control isoprenaline (pre andpost-SK&F 94120 treatment) and to isoprenaline in the pre-sence of the threshold inotropic concentration of SK&F94120. The values are expressed as a percentage of maximumcalcium response. Such analysis showed a significant poten-tiation of the inotropic response of isoprenaline by SK&F94120 only in the myocytes isolated from the noradrenaline-treated guinea-pigs. An increase in the % cell shorteningfrom 6.51 ± 0.97% (n = 5) to 9.45 ± 0.64% (n = 6) wasfound (P<0.05). In contrast, SK&F 94120 did not cause apotentiation of the mean isoprenaline response in controlmyocytes.IBMX and SK&F 94836 showed the same tendency to

increase the maximum contraction in cells from noradrena-line-treated, but not control, animals (Table 2) although theeffect escaped statistical significance. To examine the quan-titative nature of the effect of PDE inhibition more closely,the data for all PDE inhibitors were pooled (Figure 4). Apotentiation for the isoprenaline response was only noticed inthe noradrenaline-treated guinea-pig ventricular myocytes,with a significance level of P<0.01. However, the initialdepressed isoprenaline maximum was not completely reversedto control levels by PDE inhibition at minimal inotropicconcentrations.The effect of the minimum inotropic concentration of

IBMX on the initial isoprenaline maximum was compared tothat of maximum inotropic concentrations in both controlmyocytes (301tM IBMX) and in those from noradrenaline-treated guinea-pig hearts (100 iLM IBMX), as shown in Figure5. The synergistic response of isoprenaline plus PDE inhibi-tion is expressed as a percentage of the initial isoprenaline %cell shortening. There was no significant difference in maxi-mum isoprenaline responses in the presence of thresholdinotropic and maximally stimulating concentrations of IBMX,% maximum isoprenaline values being 108 ± 3% (n = 8) and102 ± 2% (n = 6), respectively. Similarly, synergistic responsesof noradrenaline-treated guinea-pig myocytes were compar-able for threshold (125 ± 6%, n = 6), and maximum concen-trations of IBMX (108 ± 3%, n = 6).The inotropic responses of PDE inhibition and P-adreno-

ceptor stimulation were further compared to that of max-imum concentrations of CPT cyclic AMP, an analogue ofcyclic AMP (Collis et al., 1989). Again there was nosignificant difference, from that of IBMX, in the extent of

potentiation over maximum isoprenaline in either group ofguinea-pig ventricular myocytes (Figure 5).

Human ventricular myocytes

Results were obtained on isolated ventricular myocytes from16 failing and two non-failing explanted human hearts. Themaximum isoprenaline/calcium ratio for the human failing

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Figure 2 Concentration-response curves to isoprenaline (0) andisoprenaline in the presence of threshold inotropic concentrations ofSK&F94120 (e) of guinea-pig ventricular myocytes isolated from(a) control (n = 9), and (b) noradrenaline-treated hearts (n = 6).Values are expressed as a percentage of the maximum isoprenalineresponse with basal contraction amplitudes subtracted, and aremean ± s.e.mean. *P<0.01; **P<0.001: statistically significantlydifferent response at the corresponding concentration of isoprenaline.

PHOSPHODIESTERASE INHIBITOR EFFECTS ON CARDIOMYOCYTES

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Figure 3 Maximum isoprenaline responses calculated from ventri-cular myocytes isolated from control and noradrenaline (NA)-treatedguinea-pig hearts. Open columns are pooled mean isoprenaline res-

ponses before and after PDE inhibition, and the cross hatchedcolumns are isoprenaline responses during exposure to a thresholdinotropic concentration of SK&F 94120. Values are expressed as apercentage of the maximum calcium response with no subtraction ofbasal amplitude, and are mean ± s.e.mean. Number of hearts indi-cated in boxes for each column.

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Figure 5 Potentiation of the maximum isoprenaline response bythreshold (min) and maximum inotropic (max) concentrations of3-isobutyl-l-methylxanthine (IBMX), and by maximum inotropicconcentrations of CPT cyclic AMP, of ventricular myocytes isolatedfrom control (open columns) and noradrenaline-treated (cross hat-ched columns) guinea-pig hearts. Concentrations were, respectively,3.6 ± 1.0IlM and 5.7 ± 1.4 ftM for threshold IBMX, 30 gM and100 gM for maximum IBMX, 100 gM and 1 mm for CPT cyclicAMP. Values are expressed as a percentage of the maximumisoprenaline response, and are mean ± s.e.mean. Number of heartsindicated in boxes for each column.

isoprenaline concentrations by IBMX. There was no

significant potentiation of the maximum effect of isoprenalineby IBMX in these 12 failing myocytes, see Figure 7. In thetwo non-failing myocytes used for this study, threshold ino-tropic concentrations of IBMX again potentiated the lowconcentrations of isoprenaline, but failed to potentiate themaximum. Figure 8 of a trace recording shows the actualresponses of one of these myocytes.SK&F 94120 at a mean threshold inotropic concentration

of 100 ± 45 tiM gave a response of 9.02 ± 4.13% in six failinghuman myocytes. Such concentrations caused an approx-

imate nine fold decrease in the isoprenaline EC50, pD2 valuesbeing 8.05 ± 0.49 for isoprenaline alone and 8.84 ± 0.23(n = 3), when in the presence of SK&F 94120. SK&F 94120was shown to cause a significant potentiation of the isopren-aline response in myocytes isolated from failing humanmyocardium (see Figure 7). The mean % cell shortening

Figure 4 Maximum isoprenaline responses calculated from ven-

tricular myocytes isolated from control and noradrenaline (NA)-treated guinea-pig hearts. Open columns are pooled mean isoprena-line responses before and after PDE inhibition, and the cross hatchedcolumns are pooled values for isoprenaline plus a threshold concent-ration of SK&F 94120, SK&F 94836 or 3-isobutyl-l-methylxanthine(IBMX). Values are expressed as a percentage of the maximumcalcium response with no subtraction of basal amplitude, and aremean ± s.e.mean. Number of hearts indicated in boxes for eachcolumn.

group was 56.4 ± 4.1%, n = 16. This is significantly less thanthe maximum response to isoprenaline previously shown inmyocytes from non-failing hearts which is close to, or higherthan the maximum calcium (116 ± 7%, n = 6, P<0.001)(Brown & Harding, 1992). The P-adrenoceptor desensitiza-tion was further shown in these failing myocytes by theirsignificantly lower isoprenaline pD2 values compared to non-

failing, 8.06 ± 0.19 (n= 16) and 8.89 ± 0.30 (n = 6) respec-tively, (P<0.05).

Twelve myocytes from 10 failing human hearts were chal-lenged with isoprenaline in the presence and absence ofIBMX. The mean concentration of IBMX was 40.3 ± 13.4ylM, which gave a minimal inotropic response of 25.0 ±6.19% (n = 10). Figure 6 shows the potentiation of low

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Figure 6 Contractile responses of failing human ventricular myo-cytes to four concentrations of isoprenaline, both alone (open col-umns) and in the presence of a threshold inotropic concentration of3-isobutyl-1-methylxanthine (cross hatched columns). Values are ex-pressed as a percentage of the maximum isoprenaline response withbasal contraction amplitudes subtracted, and are mean ± s.e.mean.Number of patients indicated in boxes for each column.

P< 0.01P < 0.02100

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1075

1076 D.G. WYNNE et al.

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Figure 7 Maximum contractile responses of failing human ven-tricular myocytes to isoprenaline (open columns) and isoprenaline inthe presence of threshold concentrations of 3-isobutyl-1-methylxan-thine (IBMX) or SK&F94120 (cross hatched columns). Values areexpressed as a percentage of the maximum calcium response, and aremean ± s.e.mean. Number of patients indicated in boxes for eachcolumn.

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t 1 t 3 t 10 i 30 +

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10

IBMX (KM)Figure 8 A typical trace showing the contraction amplitude of ahuman ventricular myocyte isolated from a non-failing donor heartresponding to 1 mM Ca2" and to cumulatively increasing concentra-tions of isoprenaline in the absence and presence of 10 jAM 3-isobutyl-l-methylxanthine (IBMX). An upward deflection represents cellshortening.

increased from 6.18 ± 0.59% (n = 6) to 7.54 ± 0.40% (n = 6)when in the presence of SK&F 94120. The maximum res-ponse was significantly (P < 0.05, n = 6), increased from60.3 ± 4.5% to 74.3 ± 4.2% maximum calcium. This is sub-stantially lower than the 116 ± 7% (P<0.001, n = 6) seen inmyocytes isolated from donor hearts.

Discussion

Cyclic nucleotide phosphodiesterase inhibition was shown toelicit a positive inotropic response in guinea-pig ventricularmyocytes. At concentrations of1O tLM, the non-specific PDEinhibitor IBMX was shown to be more effective than SK&F94120 and SK&F 94836 which are PDE III inhibitors. Themaximum mean response of IBMX was 60% of that causedby maximally stimulating concentrations of calcium, with anEC50 value of 4.4 ltM. An EC50 value of 70ylM has beenreported by Scholz & Meyer (1986), using guinea-pig ven-tricular papillary muscle preparations. Maximum responsevalues for the SK&F compounds were limited by solubilityrestrictions, however the mean threshold inotropic concentra-tion of 2tiM for SK&F 94120 is consistent with that ofprevious reports (Gristwood et al., 1987), which gave a valueof 1 i4M for guinea-pig papillary muscle preparations.PDE III is a major component of the pool of cardiac cyclic

nucleotide phosphodiesterases (Beavo & Reifsnyder, 1990)

and exerts a significant influence on myocardial contraction(Weishaar et al., 1987). Therefore, specific PDE III inhibitorswould be expected to be potent in the isolated myocytepreparation. The surprisingly large responses of IBMX maybe due to its additional effects, all of which may increasecontractility. These include its competitive reversible effect oninhibitory adenosine (Bohm et al., 1988b), its possible activa-tion of the sarcoplasmic reticulum calcium release channelsand sensitization of the myofilaments to calcium (Scholz &Meyer, 1986), reduction of the activity of inhibitory guaninenucleotide protein (Gi) function (Parsons et al., 1988) oradditional effects on other PDE isoenzymes, see Shahid &Nicholson (1990).We have previously demonstrated that in myocytes isolated

from failing human hearts, the positive inotropic effect viaP-adrenoceptor activation is diminished, the degree of whichcorrelates well with the severity of disease (Harding et al.,1992a). Such agonist subsensitivity was also shown in thisstudy where myocytes isolated from failing human ventriclehad a lower isoprenaline to calcium ratio and a higher EC50to isoprenaline compared to previous data from non-failingmyocytes. Groups working on whole muscle cardiac prepara-tions have also reported a depression in the isoprenalineresponse in human failing hearts compared to non-failinghearts (Brown et al., 1986; Bohm et al., 1988d; Nabauer etal., 1988; von der Leyen et al., 1991). In this study, myocytesisolated from noradrenaline treated guinea-pigs showed similardiminished isoprenaline responses compared with controlmyocytes. This is analogous to the P-adrenoceptor desensitiza-tion which is seen in patients with heart failure. Our presentresults are consistent with previous work on noradrenaline-induced desensitization in guinea-pig (Wynne et al., 1991;Brown & Harding, 1992) and rat (Jones et al., 1990) myocytes.The inotropic responses of noradrenaline treated guinea-

pig ventricular myocytes to the PDE inhibitor IBMX, weredepressed in comparison to control myocytes, the maximumresponse being significantly decreased. These results aresimilar to those of other groups who have reported adiminished response with PDE inhibition in isometricallycontracting trabeculae from failing human heart as comparedto those from non-failing hearts (Bohm et al., 1988d; von derLeyen et al., 1991; Steinfath et al., 1992). The same studiesshow no differences in the response to high calcium, or in thebasal activity of the PDE enzymes, between failing and non-failing human papillary muscle preparations. Similarly, inthis study the responsiveness of the noradrenaline treatedguinea-pig ventricular myocytes to calcium was unalteredcompared to control myocytes. Therefore, it is unlikely thatthe lack of efficiency of the PDE inhibitors in the myocytesisolated from noradrenaline-treated guinea-pig hearts wasdue to an impairment in the contractile apparatus, or due toan enhanced breakdown of cyclic AMP. The concentrationof IBMX giving half maximal inhibition of cardiac PDE isreported as 10 LM (Galvan & Schudt, 1990) in guinea-pigmyocardium. It has been shown that prenalterol-treated(Bobik & Little, 1984) and noradrenaline-treated (Reithmann& Werdan, 1988) chick embryo cells have depressed levels ofcyclic AMP under P-adrenoceptor stimulation. Depressedcyclic AMP levels have also been reported for human failingmyocardium compared to non-failing (Danielson et al.,1989). Therefore, the reduced inotropic responses of PDEinhibition seen in myocytes isolated from our animal modelof P-adrenoceptor desensitization may be attributable toimpaired cyclic AMP synthesis. The second part of this studywas aimed at investigating the synergistic effects of PDEinhibition and,-adrenoceptor stimulation. Such stimulationwould determine whether elevated cyclic AMP levels alonewould be sufficient to restore the diminished isoprenalineresponses of myocytes isolated from failing human heartsand noradrenaline-treated guinea-pig hearts, to non-failingand control levels, respectively.

In the presence of PDE inhibitors the concentration-response curves to isoprenaline were shifted to the left. This

_ - y y x

PHOSPHODIESTERASE INHIBITOR EFFECTS ON CARDIOMYOCYTES 1077

was the case for myocytes from human and guinea-pig, witha more pronounced shift seen in the P-adrenoceptor desen-sitized myocytes. This was also the case in the extent ofpotentiation of the maximum isoprenaline response. Nopotentiation of the maximum isoprenaline response by PDEinhibition was seen in control guinea-pig myocytes andmyocytes isolated from non-failing human hearts. Howeverin the P-adrenoceptor densensitized myocytes isolated fromhuman failing and noradrenaline-treated guinea-pig hearts,SK&F 94120 potentiated the maximum isoprenaline effect.No significant effect was seen with IBMX or SK&F 94836. Inthese respects the selective PDE III inhibitor was morepotent than the non-selective IBMX. This is consistent withresults of Gristwood et al. (1987) who, with concentrationswithin our range, showed potentiation of the maximumisoprenaline response by PDE III inhibition with SK&F94120 in failing human myocardium, but not in controlguinea-pig muscle.

In control, non-P-adrenoceptor desensitized myocytes, P-adrenoceptor agonist-stimulated cyclic AMP is likely to be atlevels adequate to generate a maximum inotropic response.Attempting to increase levels further through PDE inhibitionwould cause no additional increase in inotropic effect. How-ever, in myocytes isolated from failing human and nora-drenaline-treated guinea-pig hearts, it is possible for PDEinhibition to increase the reported depressed levels of cyclicAMP (Danielsen et al., 1989; von der Leyen et al., 1991) andhence potentiate the maximum inotropic effect of isopren-aline.Our results are in agreement with the hypothesis that

deficient 1-adrenoceptor-stimulated cyclic AMP synthesismay be an underlying defect in hearts of patients with end-stage heart failure, probably due to a defect in the receptor-adenylate cyclase coupling. This may be a result of P-adrenoceptor down regulation in addition to increased levelsof Gi in both noradrenaline-treated guinea-pig ventricularmyocytes (Reithmann et al., 1989) and in human failingmyocardium (Feldman et al., 1988; Neumann et al., 1988).

Pooling of all PDE inhibitor results showed incompletereversal of the noradrenaline-induced P-adrenoceptor desen-sitization in the myocytes isolated from the model. There stillremained a significant depression of the isoprenaline max-imum response, despite the fact that the effects of lowerconcentrations of isoprenaline were increased more than fourfold with these concentrations. Similar results were obtainedon human myocytes. The small potentiation observed in themaximum effect of isoprenaline in myocytes from failinghuman hearts did not restore responses to levels seen in cellsfrom normal hearts, where the isoprenaline response was116% of the maximum calcium. This is substantiated byadditional results which show that there was no significantdifference in the degree of potentiation of the maximumisoprenaline response with threshold IBMX concentrations ascompared to maximal inotropic concentrations (100 iM) ofIBMX. The latter concentration of IBMX was quoted byScholz & Meyer (1986) as inhibiting almost 100% of cardiacPDE III in guinea-pig papillary muscle preparations. Fur-thermore no increased potentiation was shown when PDEinhibition was substituted by maximum inotropic concentra-tions of the analogue of cyclic AMP, CPT cyclic AMP. The

results are in accord with our previous findings in myocytesfrom noradrenaline-treated rats (Jones et al., 1990) and fail-ing human heart (Harding et al., 1992b), where the effects ofN6,02 -dibutyryl-cyclic AMP (db-cyclic AMP, which pene-trates intracellularly to mimic cyclic AMP) were reducedcompared to control.

These results provide evidence for the existence of a reduc-tion in the effectiveness, as well as in the production, of cyclicAMP in failing human hearts and noradrenaline-treatedguinea-pig myocardium. Evidence from studies on intactmyocardium indicates that cyclic AMP-dependent phos-phorylation of regulatory proteins such as phospholamban inthe sarcoplasmic reticulum, troponin I in the myofibrils and a15 kDa protein in the sarcolemma, may be responsible for atleast some of the ,B-adrenergic effect on the mammalianmyocardium (Karczewski et al., 1990). Therefore, a possiblelesion at one or more of these pathways could explain theobserved post cyclic AMP abnormalities in these tissues.Our results differ from those of Bohm and co-workers

(1988d) who studied the inotropic-effects of P-adrenoceptoragonists, in combination with PDE inhibitors, on the con-tractility of whole muscle preparations. Bohm et al. (1988d)obtained a near maximal response with both P-adrenoceptoragonists and PDE inhibitors together (90% of that obtainedwith maximum concentrations of calcium). In the humanmyocytes treated with SK&F 94120, we obtained a maximumresponse to isoprenaline that was only 74% of maximumwith calcium. Various other authors have also reported com-plete restoration of the diminished positive inotropic res-ponse of isoprenaline in failing human myocardium by PDEinhibition (Bethke et al., 1991; von der Leyen et al., 1991;Bartel et al., 1992).However, all the above studies were carried out on whole

muscle preparations. Current studies are in progress in ourlaboratories to assess whether there is a true differencebetween myocytes and papillary muscle preparations fromthe same heart in the maximum attainable with cyclic AMP-raising agents. Our previous work has shown that there canbe some quantitative differences in the expression of ,B-adrenoceptor desensitization between intact muscle strips andisolated cells from the same failing human heart (Harding etal., 1991). Notably, in guinea-pig papillary muscle, unlikemyocytes, there is an accumulation of endogenous adenosinewhich mediates an anti-adrenergic effect upon P-adrenoceptorresponses (Hopwood et al., 1987). Alternatively, it may bethat patient populations were different between the presentstudy and those of Bohm & Scholz.To conclude, our findings indicate that reduced cyclic

AMP levels, both basal and P-adrenoceptor stimulated, are acontributing factor towards the reduced inotropic responsive-ness of P-agonists in myocytes isolated from both noradrena-line-treated guinea-pigs and failing human hearts. Incompleterestoration of the 0-adrenoceptor response with PDE inhibi-tion indicates that there exists an abnormality in the effect ofcyclic AMP distal to the adenylate cyclase system.

We are grateful for the technical assistance of Dr Federica DelMonte, Mr Peter O'Gara and Mr Graham Storey. This study wassupported by the British Heart Foundation.

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(Received December 10, 1992Revised March JO, 1993

Accepted March 23, 1993)