Regulation of Myocardial Contractility by a Downstream Mechanism

Correspondence to Masao Endoh, MD, Department of Pharmacology, Yamagata University School of Medicine, 2-2, 2-chome, Iida-nishi, Yamagata 990-9585, Japan.

Key Words: Ca²⁺ • myocardial contractility • myocardial cell • troponin C

Binding of intracellular Ca²⁺ ions to troponin C subsequent to membrane excitation triggers the interaction of actin with myosin molecules by displacing the inhibition induced by troponin I at diastolic levels of [Ca²⁺]_i. Therefore, in intact myocardial cells, the amplitude and rate of tension development and relaxation are primarily determined by the rate of Ca²⁺ mobilization and deprivation, by the crossbridge cycling rate, or by the contribution of both. From this perspective, there are 3 general types of mechanisms by which it should be possible to alter the contractile performance of cardiac muscle. Binding of Ca²⁺ to troponin C plays a key role and is considered to be the central mechanism of cardiac excitation-contraction coupling. The regulation of the Ca²⁺ mobilizing process is regarded as the upstream mechanism; the process subsequent to Ca²⁺ binding to troponin C (ie, an alteration of the response of the myofilaments to a given level of occupancy of Ca²⁺ binding sites on troponin C) is regarded as the downstream mechanism.¹

The mechanistic analysis of the role of Ca²⁺ ions in the cardiac contractile regulation in intact myocardial cells has progressed significantly since the introduction of methods to apply the Ca²⁺-sensitive photoprotein (aequorin) and fluorescent dyes (eg, fura-2, indo-1, and fluo-3) in intact myocardial cells.^{2 3 4} The majority of inotropic interventions alter the intracellular Ca²⁺ transient. The increase in frequency of contraction (force-frequency relationship) and cardiotonic agents, such as ß-adrenoceptor agonists, digitalis, and phosphodiesterase III inhibitors, act primarily through the upstream mechanism. By contrast, length-dependent regulation (Frank-Starling mechanism), activation of receptors coupled to the stimulation of phosphoinositide hydrolysis (-adrenoceptors, endothelin, and angiotensin AT₂ receptors), and actions of novel Ca²⁺ sensitizers (eg, EMD 57033 and Org 30029) are associated with a relatively small alteration or no alteration in Ca²⁺ transients. These are postulated to act through the central and/or downstream mechanism. The upstream mechanism can be easily distinguished from the other 2 mechanisms by examination of the relationship between the amplitude of the Ca²⁺ transient and the force developed in twitch contraction or by an analysis of the steady-state tension-pCa relationship during application of tetanic stimulation in the presence of a dysfunction of the cardiac sarcoplasmic reticular Ca²⁺ pump induced by ryanodine or thapsigargin in intact myocardial cells loaded with Ca²⁺ indicators.^{3 4} Application of these experimental procedures to analysis of the downstream mechanism, however, is difficult and limited. It is evident that the downstream mechanism includes 2 processes: (1) the interaction of the troponin-tropomyosin complex with actin and (2) crossbridge cycling itself. The combination of the in vitro motility assay, in which the movement of visualized thin filaments or actin molecules over a fixed layer of myosin can be directly investigated, the ⁴⁵Ca²⁺-binding assay, and the force-pCa relationship in cardiac skinned fibers differentiates the 2 processes. The first process shifts the force-pCa curve to the left in skinned fibers but does not increase the amount of ⁴⁵Ca²⁺ binding to troponin C and requires the troponin-tropomyosin complex for regulation in the in vitro motility assay, whereas the second process does not require the complex.^{3 4}

In this issue of Circulation Research, Palmer and Kentish⁵ have elegantly characterized the crossbridge cycling rate after isolation of the downstream mechanism from the central mechanism by application of caged compounds to skinned cardiac fibers isolated from rat and guinea pig ventricular trabeculae. The force-pCa relationships for both species were sigmoidal, fitted by the Hill equation, and superimposable, an indication that the myofibrillar Ca²⁺ sensitivity (Ca²⁺ binding affinity of troponin C) in the 2 species was the same. Since the Ca²⁺ binding sites of troponin C could be abruptly saturated by Ca²⁺ released from a caged Ca²⁺ compound, NP-EGTA, by photolysis with application of flash, the maximal rate of crossbridge cycling could be elicited in skinned fibers from the rat and guinea pig under the same experimental conditions. The former process of the downstream mechanism, ie, modulation at the level of interaction of the troponin-tropomyosin complex with actin could also be bypassed in the experiment of Palmer and Kentish⁵ because no interventions that might have potential action on this process were applied in their study. The rate constant of Ca²⁺ activation of force (k_act) at the maximum flash energy in rat trabeculae was 5-fold greater than that in guinea pig trabeculae, indicating that rat trabeculae were activated 5-fold faster than were trabeculae from guinea pigs. Interestingly and importantly, the rate constant for force redevelopment (k_tr) after forcible detachment of the crossbridges determined during maximal activation (pCa 4.5) in the rat was also 4.5-fold greater than that in the guinea pig, and these values were not significantly different from the maximum values of k_act measured in the respective species. This indicates that the different k_act values in the 2 species apparently reflect the relative rates of the force-generating processes. It is important to note that k_act was measured after sudden alteration of [Ca²⁺], whereas k_tr, which reflects the rate of crossbridge reattachment and transition to force-generating states, was determined under constant activation by Ca²⁺, thus bypassing any direct influence of [Ca²⁺] on the rate at which Ca²⁺ ions trigger conformational changes in the regulatory proteins of the thin filament. The consistency of these 2 values provides strong support for the correctness of the experimental procedure to produce an immediate elevation of [Ca²⁺]_i by means of photolysis of a caged Ca²⁺ compound in skinned cardiac fibers. Under such conditions, the authors have clearly demonstrated that the maximum rate of Ca²⁺-activated force development is limited by the rate at which detached or weakly bound crossbridges enter the force-generating state (downstream mechanism) rather than by the rate at which the thin filament is switched on by Ca²⁺ binding to troponin C (central mechanism). It has been known that the distribution of myosin isozymes involves a wide range of variation among mammalian species. The rat ventricle contains mostly V₁ myosin ( myosin heavy chains), whereas V₂ and V₃ myosins (ß and ßß heavy chains) predominate in the guinea pig. In various biochemical and mechanical assays, crossbridges containing V₁ myosin have a 2- to 6-fold higher cycling rate compared with those containing V₃ myosin, which may explain, in large part, the faster activation rates in the rat compared with the guinea pig. Since the human ventricle contains mostly V₃ myosins, analysis of the similarity and dissimilarity by comparison with these species by means of the methods of Palmer and Kentish⁵ may shed light on the regulation in human ventricular muscle. In this context, it is also noteworthy that the comparison of unitary displacements and forces between V₁ and V₃ myosin by means of optical trap techniques in the in vitro motility assay revealed that both the unitary displacements and forces of the 2 myosin isoforms are similar in amplitude but different in duration.⁶

The above consideration also accounts for the difference in the rate of relaxation between the rat and the guinea pig. The k_act values in the 2 species were not significantly different from the corresponding maximum rates of relaxation determined by a sudden drop of solution [Ca²⁺] by use of flash photolysis of diazo-2, a caged chelator of Ca²⁺. The rate of myocardial relaxation may be determined either by the rate at which Ca²⁺ dissociates from troponin C (k_off) and causes thin filament deactivation (central mechanism) or by the net rate at which myosin crossbridges detach from actin once Ca²⁺ ions are lost from troponin C (downstream mechanism). The similarity of the force-pCa relationships in the rat and guinea pig implies that k_off should be equivalent in both species. Thus, the 5-fold difference in relaxation rate between the 2 species suggests that the relaxation rate caused by photolysis of diazo-2 may reflect the detachment rate of crossbridges themselves but not k_off, which may be due to the difference between the myosin isozyme composition in the 2 species.⁵

Palmer and Kentish⁵ are the first to carry out a careful comparison of the rate of relaxation with k_act or k_tr under the same experimental conditions and in the same animal species. The rate of relaxation was similar to k_act or k_tr in a given species. These findings do not fit the crossbridge cycling model for skeletal muscle proposed previously⁷ : in the kinetic model, the rate of relaxation, which occurs at lower [Ca²⁺]_i than activation, should be lower than k_act or k_tr (see Palmer and Kentish⁵ for detailed discussion). The authors, therefore, postulated that during relaxation crossbridges may subsequently lose force by reversal of the force-generating transition (crossbridge working stroke) rather than by the slower forward detachment step.⁵ The fact that k_act is consistent with k_tr provides strong support for the conclusion that the k_act determined by the photolysis may reflect accurately the true rate of activation of crossbridges under the experimental conditions. In addition, the mode of dependence of k_act and k_tr on the extent of contractile activation corresponds with the activation dependence of the crossbridge cycling rate in the current concept of cardiac excitation-contraction coupling. On the other hand, the rate of relaxation solely determined by means of photolysis of the caged Ca²⁺-chelating compound diazo-2 may likely involve yet-unknown factors that contribute to the overestimation of the rate of relaxation in skinned cardiac fibers and also to the difference in relaxation kinetics between the rat (double exponential) and the guinea pig (single exponential).⁵ Simnett and coworkers^{8 9} have reported an even lower rate constant of activation (7.15 s^-1) than of relaxation (15.4 s^-1), determined by photolysis of nitr-5 and diazo-2, respectively, at 12°C. Further study focusing on the factors contributing to the rate of relaxation, including the development of this new experimental model in cardiac muscle as proposed by the authors,⁵ should be pursued.

Increasing interest has been focused on the elucidation of the basis of the downstream mechanism and its regulation by cardiotonic agents, because the downstream mechanism may bypass the risk of Ca²⁺ overload that leads to arrhythmias, myocardial cell injury, and energetic disadvantages that are associated with cardiotonic agents acting through the upstream mechanism. Furthermore, they may be effective in regulating force even under pathophysiological conditions such as acidosis and myocardial stunning. It has already been demonstrated that the actions of Ca²⁺ sensitizers acting primarily on the downstream mechanism reveal different characteristics among the compounds in respect to regulation of the rate of activation and detachment of crossbridges. EMD 57033 increased preferentially the rate of activation and accelerated slightly the rate of relaxation in skinned guinea pig cardiac trabeculae.^{8 9} In the rat cardiac skinned fibers loaded with diazo-2, CGP 48506 did not affect the rate of relaxation, whereas caffeine accelerated the relaxation rate.¹⁰ A potential and serious disadvantage accompanied by the action of Ca²⁺ sensitizers is an impairment of relaxation that may lead to diastolic dysfunction in the clinical setting. In contrast to EMD 57033, levosimendan did not impair the relaxation in intact myocardial cells.¹¹ In this respect, the experimental procedure to produce a rapid alteration of [Ca²⁺] by means of the photolysis of caged compounds in skinned cardiac fibers is important for elucidation of the mechanism of action of Ca²⁺ sensitizers, which may provide useful information for the clinical application of these agents as well as for analysis of basic molecular mechanisms of cardiac muscle contraction.

Footnotes

The opinions expressed in this article are not necessarily those of the editor or of the American Heart Association.

References

1. Blinks JR, Endoh M. Modification of myofibrillar responsiveness to Ca⁺⁺ as an inotropic mechanism. Circulation. 1986;73(Suppl III):III-85–III-98.

2. Blinks JR, Wier WG, Hess P, Prendergast FG. Measurement of Ca²⁺ concentrations in living cells. Prog Biophys Mol Biol.. 1982;40:1–114.[Medline] [Order article via Infotrieve]

3. Endoh M. Changes in intracellular Ca²⁺ mobilization and Ca²⁺ sensitization as mechanisms of action of physiological interventions and inotropic agents in intact myocardial cells. Jpn Heart J.. 1998;39:1–44.[Medline] [Order article via Infotrieve]

4. Kurihara S. Regulation of cardiac contraction by intracellular Ca²⁺. Jpn J Physiol.. 1994;44:591–611.[Medline] [Order article via Infotrieve]

5. Palmer S, Kentish JC. Roles of Ca²⁺ and crossbridge kinetics in determining the maximum rates of Ca²⁺ activation and relaxation in rat and guinea pig skinned trabeculae. Circ Res.. 1998;83:179–186.[Abstract/Free Full Text]

6. Sugiura S, Kobayakawa N, Fujita H, Yamashita H, Momomura S, Chaen S, Omata M, Sugi H. Comparison of unitary displacements and forces between 2 cardiac myosin isoforms by the optical trap technique: molecular basis for cardiac adaptation. Circ Res.. 1998;82:1029–1034.[Abstract/Free Full Text]

7. Brenner B. Effect of Ca²⁺ on crossbridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc Natl Acad Sci U S A.. 1988;85:3265–3269.[Abstract/Free Full Text]

8. Simnett SJ, Lipscomb S, Ashley CC, Mulligan IP. The effect of EMD 57033, a novel cardiotonic agent, on the relaxation of skinned cardiac and skeletal muscle produced by photolysis of diazo-2, a caged calcium chelator. Pflugers Arch.. 1993;425:175–177.[Medline] [Order article via Infotrieve]

9. Simnett SJ, Lipscomb S, Ashley CC, Potter JD, Mulligan IP. The thiadiazinone EMD 57033 speeds the activation of skinned cardiac muscle produced by photolysis of nitr-5. Pflugers Arch.. 1994;427:550–552.[Medline] [Order article via Infotrieve]

10. Palmer S, Kentish JC. Differential effects of Ca²⁺ sensitizers caffeine and CGP 48506 on the relaxation rate of rat skinned cardiac trabeculae. Circ Res.. 1997;80:682–687.[Abstract/Free Full Text]

11. Sato S, Talukder MAH, Sugawara H, Sawada H, Endoh M. Effects of levosimendan on myocardial contractility and Ca²⁺ transients in aequorin-loaded right ventricular papillary muscles and indo-1-loaded single ventricular cardiomyocytes of the rabbit. J Mol Cell Cardiol.. 1998;30:1115–1128.[Medline] [Order article via Infotrieve]

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