Can changes in sarcolemmal membranes account for the altered inotropic responsiveness in hypertrophied heart?

Biochimie, 69 (1987) 419- 425 © Soci6t~de Chimie biologique/Elsevier, Paris

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Can changes in sarcolemmal membranes account for the altered inotropic responsiveness in hypertrophied heart ? Eric MAYOUX, Lionel LELIEVRE and Dani61e CHARLEMAGNE

I N S E R M U127, H6pital Lariboisi~re, Universitd Paris 7, 41, boulevard de la Chapelle, 75010 Paris, France (Received 3-3-1987, accepted 11-3-1987)

Summary -

Hypertrophy is an adaptive mechanism of the heart subjected to pressure overload. Ultrastructural, electrophysiological and mechanical changes occur during this adaptation. A decrease in the inotropic responsiveness of the hypertrophied heart has often been observed as compared to the normal heart. Four sarcolemmal mechanisms that could account for this modification have been described. The mechanism of action of each system (calcium channel, =-and ~-adrenergic systems, (Na+,K+)-ATPase) of the hypertrophied heart has been compared to that of the normal heart. In spite of the paucity of results available relating to the calcium channel, the lengthening of the action potential in every case of compensatory hypertrophy could be explained by an altered functioning of the calcium channel. =- and fl-adrenergic systems in the hypertrophied heart could be modified at the receptor level itself, or at another level in the cascade of events under their control. For example, two different models of hypertrophy showed a decreased inotropic responsiveness correlated to a defect in the G s regulatory protein. The modification of the ouabainreceptor (Na+,K+)-ATPase mediates a decrease and a prolongation of the inotropic response. According to the modifications of each system, a direct relationship does not seem to exist between the stimulated membrane system and the inotropic responsiveness of the hypertrophied heart. hypertrophy / inotropic response / sarcolemma / Ca2+ channel

R~sum~ - Les modifications membranaires peuvent-elles rendre compte des particularit~s de ia r~ponse inotrope du cmur hypertrophi~ ? Lorsqu'un coeur est soumis ~ u n e surcharge de travail, il s'adapte en s'hypertrophiant. L "hypertrophie s'accompagne de changements ultrastructuraux, dlectrophysiologiques, et mdcaniques. La rdponse de ces c~eurs m~x agents qui produisent un effet inotrope positif, est !~ plus souvent diminude comparde ~ cello de coeurs ~ normaux >>.Nous nous sommes inMressds ~ quatre mdcanismes membranaires qui pouvaient intervenir clans cette modification. Les mdcanismes d'action de chacun de ces systbmes (canal calcique, systbmes =-et ~-adrenergiques, A TPase-(Na+,K+)) du coeur hypertrophid ant Otd compards ~ ceux du coeur normal. Bien que peu de rOsultats soient disponibles sur le canal calcique, l'allongement du potentiel d'action observd clans tousles cas d'hypertrophie compensde, pourrait Otre expliqu6 par un fonctionnement different du canal. Les syst~mes =- et fl-adrenergiques du coeur hypertrophid peuvent Etre modifids au niveau du rEcepteur lui-mOme ou de la cascade d'Ovdnements qu'ils contr61ent. Par exemple, clans deux modifies diffdrents d'hypertrophie, la rdponse inotrope diminuEe est sous le contr61e d'une ddfaillance de la protdine rdgulatrice Gs. La modification du rdcepteur de l'ouabaine (I'A TPase-(Na +,K +) entrMne une rdponse inotrope ldgbrement diminude et surtout prolongde. A la rue de l'ensemble des modifications q,, subit chacun des systbmes, il nepara~t pas exister obligatoirement une relation directe entre le systbme membranaire stimuld et la rdponse inotrope du coeur hypertrophiE.

hypertrophic / r6ponse inotrope / sarcolemme i canal ealcique

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Introduction This paper is a review of what is known about sarcolemmal changes in the hypertrophied heart. The different mechanisms responsible for an increase of free calcium at the sarcomere level, and leading to a positive inotropic response will be described with normal heart as a reference. In normal heart, contractile function is regulated by many important systems among which is excitationcontraction coupling. The transsarcolemmal influx of calcium is a key element of this coupling process (for a review, see [1]). It occurs through calcium channels, following depolarization mediated by a previous influx of sodium ions and is mainly responsible for the plateau phase of the cardiac action potential. The magnitude of the calcium influx depends upon both the density of activated channels and the period of time during which individual channels remain open. Activation of these channels is mediated by phosphorylation, which, in turn, is under the control of the adrenergic system [2, 3]. Calcium channels are inactivated by dephosphorylation and an elevated intraceUular calcium concentration [4]. Furthermore, they can be stimulated or inhibited by calcium channel agonist and antagonists [5]. This system is a major determinant of the inotropic state of the myocardium, but actually, three other sarcolemmal systems are involved in the inotropic responsiveness of the heart.

The [3-adrenergic system This is one of the more effective systems in the heart. It only involves ~l-receptors. This complex system consists of hormone-stimulated fl-receptors (R), nucleotide regulatory proteins (Gi,Gs), and a catalytic subunit (C). Stimulation of this system by coupling among R, G s and C ( R - G s - C ) increases the cellular cAMP level which specifically activates phosphorylation of the calcium channel [1, 6].

The ~-adrenergic system ~rReceptor stimulation produces an increase in the magnitude of the calcium influx, a deceleration of its normal decay [7] and an increased calcium sensitivity of the myofibrils [8]. However, the series of molecular events that mediate the cellular response of cardiac 0q-adrenergic receptors to norepinephrine are still undefined [9]. They involve an increased turnover of phosphatidyl inositol which results in the formation of inositol triphosphate and diacylglycerol [10, 11]. These second messengers in the heart could respectively con-

tribute to the release of calcium from sarcoplasmic reticulum (SR) [12], and to the activation of calcium channels [9]. The presence of ot2-receptors in the heart at the post-junctional level has not been demonstrated.

The cardiac glycoside system The mechanism of the inotropic effect of digitalis has not yet been clearly established [13, 14]. It is generally admitted that ouabain inhibits the (Na +, K+)-ATPase. As a consequence, there is an increase in intracellular sodium which could produce an increase in intracellular calcium via a Na+/ Ca 2+ exchange mechanism [15]. This increase of free calcium at the membrane level could also activate the slow inward current [16]. We shall consider if these four inotropic mechanisms maintain their characteristics in compensatory hypertrophy of the left ventricle. The heart responds to persistent long term stress by adaptive mechanisms which involve quantitative as well as qualitative changes (for a review see [17]). Cardiac hypertrophy is associated with a reduced basal myocardial contractile state. Whatever the positive inotropic agent, responsiveness of the hypertrophied heart is usually depressed. It must be pointed out that, at the sarcolemmal level, differences in the inotropic responsiveness between normal and hypertrophied hearts may be related to: 1) a direct change of receptor properties without any other modification of the subsequent cascade; 2) no difference in the receptor, but a defect in any one of the sequence of events between excitation and response; 3) the influence of the stimulation or inhibition of one of the abovementioned mechanisms while the others remain normal.

Calcium channel An almost universal finding in hearts subjected to pressure overload is an increase in action potential duration (for a review see [18]). The role of calcium influx in the determination of the duration of the plateau phase of the action potential suggests that calcium channels are involved. Calcium influx is determined by the density of channels, the number of functional channels and their opening and closing kinetics. The total number of inactivated calcium channels can be estimated in sarcolemmal preparations [19, 20]. However assessment of their dynamic, activated state requires electrophysiological studies.

lnotropic responsiveness of hypertrophied heart Inactivated channel In membrane preparations from spontaneously hypertensive rats (SHR), the density of inactivated channels, determined by nitrendipine binding, is similar to that observed in membranes from controls [21,22]. Our studies on highly enriched sarcolemmal preparations [23] from hypertrophied rat heart showed no changes in nitrendipine binding parameters as compared to similar parameters in sham-operated animals. These results are summarized in Table I.

Table I. Characteristics of [3H]nitrendipine binding to sarcolemmal preparations from normal and hypertrophied rat hearts. Normal heart

Hypertrophiedheart

(n=5)

(n=6)

Bmax

457 +_51(SEM)

486 _+48(SEM)

ro

0.47+0.06(SEM) 0.43 _+0.04(SEM)

421

p~Lanations have been proposed. Ten Eick ascribed this prolongation to slowed activation of an outward potassium current [18]. The mechanism of the prolonged duration of the action potential could be determined by patch-clamp recording in myocytes from hypertrophied heart. The action of the calcium channel agonists has not yet been tested in the hypertrophied myocytes or in the isolated hypertrophied heart. In the heart, the slow inward current is under the control of a wide variety of drugs, neurohormones and intraceUular metabolites. Thus a prolonged action potential could not only involve the calcium channel by itself but also its modulating systems. For example, in the normal heart, the fl-adrenergic system may modulate the slow inward current by modulating cytoplasmic adenosine 3',5'-cyclic monophosphate (c#~viP) levels [1]. fl-agonists increase the number of calcium channels available for voltage activation and prolong the proportion of time individual channels remain open [3].

(fmol/mg of protein) (nM)

In normal and hypertrophied heart sarcolemmal preparations, the (Na +,K +)-ATPase activity and the number of nitrendipine sites showed similar t..... r o n d s rt---,~. '~A1 ~r'k: t ^ a out the possibility of a bi-,,~. . . . ~u~u ased selection of a specific membrane domain during sarcolemmal preparation. Since the tubular(T)system developed very rapidly, so that the relationship between cellular membrane (sarcolemma and T- system) and the cell volume remained constant [25], we can assume that calcium channel density is unchanged in hypertrophied rat myocytes. This equality in the density of inactivated calcium channels does not reflect the number of functional channels, or their activation-inactivation process in the hypertrophied heart.

Activated channel Numerous electrophysiological studies have been conducted on left ventricular and papillary muscle from hypertrophied heart. An increased duration of the action potential has been observed in rat [26, 27] and in cat [28]. Aronson suggested that this prolonged action potential could be explained by a slower inactivation of the calcium inactivated inward current process. In right ventricular hypertrophy, where an increased duration of the action potential has also been observed [29- 31 ], other ex-

~-Adrenergic system Inotropic responsiveness to fl-adrenergic stimulation in the normally functioning myocardium far exceeds what may be achieved by maximal ouabam or histamine stimulation, or an increase in extracellular ionized calcium concentration. The importance of this accounts for the considerable amount of research in this area. Although reports in the literature are often contradictory, it is worth pointing out that the adrenergic system is modified in the hypertrophied heart and may play an important role in the establishment and reversal of hypertrophy [32, 33]. Since the inotropic response requires the coordinated stimulation of the E-receptor (R), coupling to the regulatory protein (Gs) and activation of the catalytic unit (C), we shall try to correlate differences in the inotropic response to modifications at each level of this casca,:;e me:-h~rdsm.

Inotropic responsivene~.s A reduction in the inotropic response to catecholamines has beer :iemonstrated by ,.nan~ investigators in a variety ni experimental u~.~aels. In the dog with pressure :werloa0, the increase in myocardial contractility after an injection of isoproterenol was less in t12 hypertrnphied heart than in the normal one [34]. The same type of reduced response is observed in hypertrophied rat heart, whatever the model. In SHR, as in the renal

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hypertensive rat (two-kidney, one clip hypertension, RHR) the reduction of the inotropic responsiveness is seen in vivo [35-37] and in viwo [37-38]. This reduction is correlated with the degree of left ventricular hypertrophy [37]. On the other hand, adequate responsiveness to catecholamines is retained in conscious dogs with severe left ventricular hypertrophy [39] or in the hypertensive dog [40]. No increased contractile response has been described,

Characteristics o f the fl-receptors fl-receptors have been characterized by studying the binding of [3Hldihydroalprenolol to crude or enriched myocardial membranes. Usually, the receptor affinity is not modified in hypertrophied heart [41-47] except in dog heart with pressure overload [39] or hypertensive hypertrophy [48], where a slight decrease in receptor affinity has been related. The number of fl-receptors is either decreased, increased or unchanged, according to the model studied; it seems to be regulated by ventricular catecholamines [6]. In RHR an increased number of fl-receptors [33, 47] could be correlated to a decreased myocardial catecholarnine concentration [49]. However, Ayobe and Tarazi [46] and Woodcock [43] have reposed the opposite result in a Goldblatt rat, 6 weeks after renal artery clipping. In dogs with pressure overload, the number of~-receptors is increasad: the plasma catecholamines are normal but myocardial norepinephrine is depressed [391. A comparative increase in the number is also observed in rat hearts induced to hypertrophy by abdominal aortic constriction [44]. When the myocardial catecholamine concentration is increased, as in SHR, the number of receptors might be decreased. In fact, in SHR a decreased [33, 41,47, 50, 51] or unchanged number has been observed [52].

Adenylate cyclase activity Changes in adenylate cyclase activity have been investigated less extensively than changes in the numbers of receptors. Results indicate unchanged [53, 54] or depressed [52] activity. Thus, the decreased or unchanged/3-adrenergic responsiveness seen in the different models of hypertrophy is due to biochemical changes in the different processes involved in the excitationcontraction coupling response. In SHR, the decreased inotropic responsiveness could be directly correlated to a down-regulation

of fl-adrenergic receptor density [41,47]. In RHR, Kumano [47] proposed a different biochemical pathway. It is possible that, independent of the number offl-receptors (which could even be increased) and the unchanged activity of the catalytic subunit, the major defect lies in the adenylate cyclase complex ( R - G s- C) and particularly in the nucleotide regulatory protein. This mechanism could also be operative in dog heart, since an increased number of fl-receptors is associated with a normal or depressed response to catecholamines [39]. This regulation of the inotropic response does not take into account the inhibitory role of muscarinic receptors [55]. These receptors have not been studied in compensatory hypertrophy. Their number is decreased in heart failure.

~-Adrenergic system To date, in the compensated hypertrophied heart, very few data on the 0c-adrenergic system have been published. In fact, the studies involve both 0c- and fl-adrenergic systems, because agonists for 0q-receptors usually also activate ~l-receptors. But under special experimental procedures, it is possible to elicit 0q-adrenergic effects alone [56, 57]. This may be effected by fl-blockade, low temperature, reserpine pretreatment, or specific rate of stimulation. Thus, as observed in SHR a decrease in the positive inotropic response to phenylephrine could suggest a subsensitivity in cardiac ~adJ[-{~J[]LU[I~f~CpLOrS [.~OJ. Lll I~I.~L, 111 LIII~;3q~.lll~;~IIIUU~I,

with specifically labeled agents, Y a m a d a found a decrease in the number of ~l-receptors without a change in affinity as compared to controls [51]. They ascribed this decreased number to a downregulation mediated by an increase of sympathetic stimulation. On the other hand, depletion of cardiac catechol~mines in the failing heart is associated with an increase in the number of ~q-receptors [5~1.

Responsiveness to cardiac glycosides Digitalis produces an inotropic response that is independent of activation of the adenylate cyclase system. Although ouabain is the oldest drug used to treat chronic heart failure, little information is available with respect to the response of hypertrophied hearts to it. In the hypertrophied dog heart, and also in the failing heart, the inotropic effect of digitalis does not appear to be reduced

lnotropic responsiveness of hypertrophied heart [60, 61]. In rats with cardiac hypertrophy due to renal hypertension or pressure overload, the increase of the contractile force by ouabain is slightly decreased or unchanged [45, 62] but, in the pressure overload model the responsiveness is prolonged [62]. Since ouabain is known to inhibit the (Na+,K+)-ATPase, alterations in the enzyme could be suspected.

Ouabain receptors The status of ouabain receptors has been evaluated by conducting [3H]ouabain binding studies on sarcolemmal membranes. In normal rat heart, two types of receptors (high- and low-ouabain affinity) have been described [63]. They are still present without any change ~n their affinities in hypertrophied rat hearts. In SHR, an increased number of receptors has been observed [64]. In rat hearts with pressure overload, the total number of receptors is not significantly d~fferent, but an increased number of high affinity sites is present [65]. In this model, it must be pointed out that the only important change is in the rates of drug release from the high- and low-affinity ouabain binding sites; these release ouabain at rates five times lower than the corresponding forms present in normal cardiac sarcolemmal vesicles [63, 65]° We demonstrated that these characteristics are those of the newborn heart enzyme [651.

(Na+,K +)-A TPase activity

modifications of the specific receptor system, or if they are derived from a common denominator. It clearly appears that a part of the pubh'shed data can be understood in terms of changes at the membrane level. This seems to be valid whatever the species (rat, dog), the type of hypertrophy (abrupt or progressive) and the inotropic agent considered. These changes at the receptor level might either be a modulation of the preexisting receptor system or reflect new systems absent or undetectable Ln normal hearts. The first possibility, Le., a modulation of preexisting sites, is illustrated by the responsiveness of hypertrophied hearts to fl-agonist compounds [35, 36]. There is a decreased number of fl-adrenergic receptors which, however, still exhibit the same apparent affinities as tho~e in norreal hearts [33, 41,47, 50, 511. The second type of change (i.e., alteration in the coupling process or expression of new receptor forms) seems to occur at the level of the fl-adrenergic sensitive adenylate cyclase system in RHR [33, 47]. An alteration of the G s protein is suspected and has also been considered in dog hearts with heart failure [67]. AI*.hough the other constituants of the system are normal, the response is modified. In rat cardiac hypertrophy, the two types of digitalis receptors which are expressed represent the neonatal cardiac forms undetectable in normal adult heart [65]. It faust be pointed out that two main limitations exist that require discussion. First, it has been assumed that a change in affinity for a ligand could represent the expression of a new receptor. The .

The results concerning (Na+,K +)-ATFase activity in hypertrophied rat heart are controversial. In SHR membranes, the enzyme activity was double that in membranes from control rats [64]; in RHR, it was decreased [66] and in pressure overload hypertrophy, this activity remained unchanged [65]. In all these models, results dealing with numbers of ouabain binding sites or (Na+,K+)-ATPase activity cannot be directly related to the inotropic effect of ouabain. On the other hand, in vivo and in vitro studies on ovedoaded hypertrophied rat heart suggest that the prolonged inotropic responsiveness to ouabain is due to lower rates of release, and thus, to modification of the digitalis receptors.

Conclusion From the data compiled above, the question arises as to whether changes in the inotropic responsiveness in hypertrophied hearts are related to

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a change in affinity can be induced by an alteration in the phospholipid composition of the membrane without any modification in the primary structure of the receptor under con~deration [68]. Conversely, we have shown that digitalis receptors may possess the same affinity for a drug, but differ widely in tiieir respective association and dissociation rate constants. ~"/l~isstrongly favors the existence of new forms of binding sites [65]. From a pharmacological point of view, all the mechanisms of action of the inotropic drugs analyzed here are dependent upon other cellular processes. For instance, as a consequence of the stimulation of the/3-adrenergic receptors, there is a phosphorylation of the calcium channel [1]. Similarly, the mechanism of action of digitalis in the heart involves both (Na+,K+)-ATPase activity and the Na+/Ca 2+ exchange; a stimulation of the slow calcium channels has also been observed [16]. There are many reported data that cannot be explained only in terms of changes at the receptor

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level. For example, there is a depressed inotropic response to fl-agonists that is seen in spite of an increased number of specific receptors [33, 39, 42, 47]. The number of digitalis receptors remains stable in normal and hypertrophied cardiac preparations, but the positive inotropic response is decreased in the latter group [63, 65]. So far the simplest interpretation is that there is (are) a difference(s) at a post-receptor level. In as much as calcium ions trigger contraction, they might be the c o m m o n denominator for all these pharmacological effects. However, if it is well known that in hypertrophy calcium movements are slowed [69] and the calcium sensitivity of the calcium binding proteins is increased, there is no information available about the behavior of these phenomena in response to inotropic agents in hypertrophied heart. The c o m m o n link between calcium ions, hypertrophy and inotropic agents could be the slow calcium channels. Indeed, at every contraction, the calcium channels collect and integrate the data coming from all the inotropic systems (negative or positive). Then, according to the resultant message, the calcium channels modulate the slow inward current which is a major determinant of the inotropic state of the myocardium. Thus it appears that the response of the heart to a specific inotropic agent (which stimulates one specific system) is in fact also dependent upon other non-specific systems. This context could explain the reason why in hypertrophied heart the modification of the response to one specific agent cannot always be directly correlated to changes in the corresponding system.

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Inotropic responsiveness o f hypertrophied heart

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