Sorcin inhibits calcium release and modulates excitation-contraction coupling in the heart

Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (ICa) gives rise to Ca2+-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. We reported previously (Lokuta, A. J., Meyers, M. B., Sander, P. R., Fishman, G. I., and Valdivia, H. H. (1997) J. Biol. Chem. 272, 25333-25338) that sorcin, a 22-kDa Ca2+-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Here we show that sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the ICa-triggered activity of RyRs that gives rise to [Ca2+]i transients. Because sorcin decreased the amplitude of the [Ca2+]i transient without affecting the amplitude or kinetics of ICa, the overall effect of sorcin was to reduce the "gain" of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = approximately 200 microM) appears to be reached only within the dyadic space. Rapid injection of 5 microM sorcin onto the cytosolic face of RyRs reconstituted in lipid bilayers resulted in complete inhibition of channel activity in < or = 20 ms. Thus, sorcin is a potent inhibitor of both spontaneous and ICa-triggered RyR activity and is kinetically capable of playing a role in terminating the positive feedback loop of CICR.     

Ontogeny of excitation-contraction coupling in the mammalian heart

The neonate mammalian heart is phenotypically different from the adult heart in many respects. Understanding these phenotypic differences are a fundamental component of understanding the mechanisms of congenital heart disease and its treatment. Differences in excitation-contraction (E-C) coupling of the neonatal heart from that of the adult include less reliance on intercellular sources of Ca(2+) such as that from sarcoplasmic reticulum (SR). Electron micrographs indicate that these immature cardiomyocytes lack transverse tubules and the SR is sparse. This paper focuses on the changes in the phenotype of E-C coupling during ontogeny in the mammalian heart and the molecular mechanisms underlying these changes.     

Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes

The phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 decreased steady-state contraction in neonatal rat ventricular myocytes (NRVM). To determine whether the effect on steady-state contraction could be due to decreased intracellular Ca(2+) content, Ca(2+) content was assessed with fluorescent plate reader analysis by using the caffeine-releasable Ca(2+) stores as an index of sarcoplasmic reticulum (SR) Ca(2+) content. Caffeine-releasable Ca(2+) content was diminished in a dose-dependent manner with LY-294002, suggesting that the decrease in steady-state contraction was due to diminished intracellular Ca(2+) content. Activation of the L-type Ca(2+) channel by BAY K 8644 was attenuated by LY-294002, suggesting the effect of LY-294002 is to reduce Ca(2+) influx at this channel. To investigate whether additional proteins involved in excitation-contraction (EC) coupling are likewise regulated by PI3K activity, the effects of compounds acting at sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), the ryanodine receptor, and the Na/Ca exchanger (NCX) were compared with LY-294002. Inhibition of SERCA2a by thapsigargin increased basal Ca(2+) levels in contrast to LY-294002, indicating that SERCA2a activity is sustained in the presence of LY-294002. Ryanodine decreased SR Ca(2+) content. The additive effect with coadministration of LY-294002 could be attributed to a decrease in Ca(2+) influx at the L-type Ca(2+) channel. The NCX inhibitor Ni(2+) was used to investigate whether the decrease in intracellular Ca(2+) content with LY-294002 could be due to inhibition of the NCX reverse-mode activity. The minimal effect of LY-294002 with Ni(2+) suggests that the primary effect of LY-294002 on EC coupling occurs through inhibition of PI3K-mediated L-type Ca(2+) channel activity.     

Modeling the cellular basis of altered excitation-contraction coupling in heart failure

Ca transients measured in failing human ventricular myocytes exhibit reduced amplitude and slowed relaxation [Beuckelmann, D.J., Nabauer, M., Erdmann, E., 1992. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85, 1046-1055; Gwathmey, J.K., Copelas, L., MacKinnon, R., Schoen, F.J., Feldman, M.D., Grossman, W., Morgan, J.P., 1987. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res. 61, 70-76; Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P.H., Kass, D.A., Marban, E., Tomaselli, G.F., 1996. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. 78(2); Li, H.G., Jones, D.L., Yee, R., Klein, G.J., 1992. Electrophysiologic substrate associated with pacing-induced hert failure in dogs: potential value of programmed stimulation in predicting sudden death. J. Am. Coll. Cardiol. 19(2), 444-449; Vermeulen, J.T., McGuire, M.A., Opthof, T., Colonel, R., Bakker, J.M.T.d., Klopping, C., Janse, M.J., 1994. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc. Res. 28, 1547-1554.] and blunted frequency dependence [Davies, C.H., Davia, K., Bennett, J.G., Pepper, J.R., Poole-Wilson, P.A., Harding, S.E., 1995. Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure. Circulation, 92, 2540-2549; Hasenfuss, G., Reinecke, H., Studer, R., Meyer, M., Pieske, B., Holtz, J., Holubarsch, C., Posival, H., Just, H., Drexler, H., 1994. Relation between myocardial function and expression of sarcoplasmic reticulum Ca-ATPase in failing and nonfailing human myocardium. Circ. Res. 75, 434-442; Hasenfuss, G., Reinecke, H., Studer, R., Pieske, B., Meyer, M., Drexler, H., Just, H., 1996. Calcium cycling proteins and force-frequency relationships in heart failure. Basic Res. Cardiol. 91, 17-22; Monte, F.D., O'Gara, P., Poole-Wilson, P.A., Yacoub, M., Harding, S.E., 1995. Cell geometry and contractile abnormalities of myocytes from failing human left ventricle. Cardiovasc. Res. 30, 281-290; Philips, P.J., Gwathmey, J.K., Feldman, M.D., Schoen, F.J., Grossman, W., Morgan, J.P., 1990. Post-extrasystolic potentiation and the force-frequency relationships: differential augmentation of myocardial contractility in working myocardium from patients with end-stage heart failure. J. Mol. Cell. Cardiol. 22, 99-110; Pieske, B., Hasenfuss, G., Holubarsch, C., Schwinger, R., Bohm, M., Just, H., 1992. Alterations of the force-frequency relationship in the failing human heart depend on the underlying cardiac disease. Basic Res. Cardiol. 87, 213-221.]. Analyses of protein levels in these failing hearts reveal that the SR Ca-ATPase is down-regulated on average by 50% and that the Na/Ca exchanger is up-regulated on average by a factor of two. In this paper, we test the hypothesis that this altered pattern of expression of Ca handling proteins is sufficient to account for changes in excitation-contraction coupling properties measured experimentally at the cellular level. To do this, we present an integrated model of excitation-contraction coupling in the guinea pig ventricular cell. The model is used to determine the effects of SR Ca-ATPase down-regulation and Na/Ca exchanger up-regulation on action potential duration, Ca transient shape and amplitude, and isometric force. Model analyses demonstrate that changes in Ca handling proteins play a direct and critical role in prolongation of action potential duration, and in reduction of contractile force in heart failure.     

Changes in the organization of excitation-contraction coupling structures in failing human heart

Background

The cardiac myocyte t-tubular system ensures rapid, uniform cell activation and several experimental lines of evidence suggest changes in the t-tubular system and associated excitation-contraction coupling proteins may occur in heart failure.

Methods and Results

The organization of t-tubules, L-type calcium channels (DHPRs), ryanodine receptors (RyRs) and contractile machinery were examined in fixed ventricular tissue samples from both normal and failing hearts (idiopathic (non-ischemic) dilated cardiomyopathy) using high resolution fluorescent imaging. Wheat germ agglutinin (WGA), Na-Ca exchanger, DHPR and caveolin-3 labels revealed a shift from a predominantly transverse orientation to oblique and axial directions in failing myocytes. In failure, dilation of peripheral t-tubules occurred and a change in the extent of protein glycosylation was evident. There was no change in the fractional area occupied by myofilaments (labeled with phalloidin) but there was a small reduction in the number of RyR clusters per unit area. The general relationship between DHPRs and RyR was not changed and RyR labeling overlapped with 51±3% of DHPR labeling in normal hearts. In longitudinal (but not transverse) sections there was an ∼30% reduction in the degree of colocalization between DHPRs and RyRs as measured by Pearson''s correlation coefficient in failing hearts.

Conclusions

The results show that extensive remodelling of the t-tubular network and associated excitation-contraction coupling proteins occurs in failing human heart. These changes may contribute to abnormal calcium handling in heart failure. The general organization of the t-system and changes observed in failure samples have subtle differences to some animal models although the general direction of changes are generally similar.     

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.     

Optical indications of electrical activity and excitation-contraction coupling in the early embryonic heart

Complete understanding of the ontogenesis and early development of electrical activity and its related contraction has been hampered by our inability to apply conventional electrophysiological techniques to the early embryonic heart. Direct intracellular measurement of electrical events in the early embryonic heart is impossible because the cells are too small and frail to be impaled with microelectrodes. Optical signals from voltage-sensitive dyes have provided a new and powerful tool for monitoring changes in membrane potential in a wide variety of living preparations. With this technique it is possible to make optical recordings from cells which are inaccessible to microelectrodes. An additional advantage of the optical method for recording membrane potential activity is that electrical activity can be monitored simultaneously from many sites in a preparation. Thus, applying a multiple-site optical recording method with a 100- or 144-element photodiode array and voltage-sensitive dyes, we have been able to monitor for the first time spontaneous electrical activity in pre-fused cardiac primordia in early chick embryos at the 6- and early 7-somite stages of development; we have been able to determine that the time of initiation of the heartbeat is the middle period of the 9-somite stage. In the rat embryonic heart, the onset of spontaneous electrical activity and contraction occurs at the 3-somite stage. This article describes ionic properties of the spontaneous action potential and genesis of excitation-contraction coupling in the early embryonic chick and rat hearts. In addition, an improved view of the ontogenetic sequence of spontaneous electrical activity and its implications for excitation-contraction coupling in the early embryonic heart are proposed and discussed.     

The effect of acidosis on excitation-contraction coupling in isolated ferret heart muscle

The contractile response to acidosis is the final product of a number of different changes in the excitation-contraction coupling pathway: (i) Ca_i increases and subsequently decreases during acidosis; (ii) the action potential becomes longer; (iii) the sensitivity of the contractile proteins to Ca²⁺ decreases. The increase of Ca_i and the lengthening of the action potential may help to maintain contractile function, although this advantage may be offset if spontaneous Ca^2– release from the s.r. occurs, secondary to the increase of Ca_i. The recovery of force shown in figure 1 occurs at a time when the calcium transient is decreasing, and therefore represents an increasing sensitivity of the contractile proteins to Ca_i, probably due to a recovery of intracellular pH(6), although it is also possible that a disappearance of spontaneous Ca²⁺ releases from the s.r. may be contributing [2].     

Overexpression of type 1 angiotensin II receptors impairs excitation-contraction coupling in the mouse heart

Transgenic mice that overexpress human type 1 angiotensin II receptor (AT(1)R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT(1)R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT(1)R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT(1)R overexpression on ventricular L-type Ca(2+) currents (I(CaL)), cell shortening, and Ca(2+) handling in 50-day and 6-mo-old male AT(1)R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT(1)R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, I(CaL), and Ca(v)1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT(1)R mice compared with controls. In addition, Ca(2+) transients and caffeine-induced Ca(2+) transients were reduced whereas the time to 90% Ca(2+) transient decay was prolonged in both age groups of AT(1)R mice. Western blot analysis revealed that sarcoplasmic reticulum Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger protein expression was significantly decreased in 50-day and 6-mo AT(1)R mice. Overall, the data show that cardiac contractility and the mechanisms that underlie excitation-contraction coupling are altered in AT(1)R mice. Furthermore, since the alterations in contractility occur before the development of cardiac hypertrophy, it is likely that these changes are attributable to the increased activity of the renin-angiotensin system brought about by AT(1)R overexpression. Thus it is possible that AT(1)R blockade may help maintain cardiac contractility in individuals with heart disease.     

Molecular properties of excitation-contraction coupling proteins in infant and adult human heart tissues

Excitation-contraction coupling (ECC) proteins in the human heart were characterized using human atrial tissues from different age groups. The samples were classified into one infant group (Group A: 0.2-7 years old) and three adult groups (Group B: 21-30; Group C: 41-49; Group D: 60-66). Whole homogenates (WH) of atrial tissues were assayed for ligand binding, 45Ca2+ uptake and content of ECC proteins by Western blotting. Equilibrium [3H]ryanodine binding to characterize the ryanodine receptor (RyR) of the sarcoplasmic reticulum (SR) showed that the maximal [3H]ryanodine binding (Bmax) to RyR was similar in all the age groups, but the dissociation constant (kd) of ryanodine was higher in the infant group than the adult groups. Oxalate-supported 45Ca2+ uptake into the SR, a function of the SR SERCA2a activity, was lower in the infant group than in the adult groups. Similarly, [3H]PN200-110 binding, an index of dihydropyridine receptor (DHPR) density, was lower in the infant group. Expression of calsequestrin and triadin assessed by Western blotting was similar in the infant and adult groups, but junctin expression was considerably higher in the adult groups. These differences in key ECC proteins could underlie the different Ca2+ handling properties and contractility of infant hearts.     

Nitric oxide modulates excitation-contraction coupling in the diaphragm

We investigated the enzymatic source, cellular production, and functional importance of nitric oxide (NO) in rat diaphragm. Neuronal and endothelial isoforms of constituitive nitric oxide synthase (nc-NOS, ec-NOS) were identified by immunostaining. NOS activity measured in diaphragm homogenates averaged 5.1 pmol/min/mg. Passive diaphragm fiber bundles produced NO derivatives (NOx) at the rate of 0.9 pmol/min/mg as measured by the cytochrome c reduction assay; NO production was confirmed by photolysis/ chemiluminescence measurements. Endogenous NO depressed diaphragm contractile function. The force of submaximal contraction was increased by NOS inhibitors, an effect that was stable for up to 60 min and was reversed by NO donors. We conclude that diaphragm muscle fibers express nc-NOS, ec-NOS, or both; passive myocytes produce NOx; and NO or NO-derivatives inhibit force production by modulating excitation-contraction coupling.     

Kinetic analysis of excitation-contraction coupling

Recent studies of isolated muscle membrane have enabled induction and monitoring of rapid Ca²⁺ release from sarcoplasmic reticulum (SR)⁵ in vitro by a variety of methods. On the other hand, various proteins that may be directly or indirectly involved in the Ca²⁺ release mechanism have begun to be unveiled. In this mini-review, we attempt to deduce the molecular mechanism by which Ca²⁺ release is induced, regulated, and performed, by combining the updated information of the Ca²⁺ release kinetics with the accumulated knowledge about the key molecular components.Abbreviations used: AMP-PCP, adenosine 5-(, -methylenetriphosphate); C_1/2, concentration a half-maximal activation or inhibition; Con-A, concanavalin A; DACM,N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide; DCCD, dicyclohexylcarbodiimide; SR, sarcoplasmic reticulum; DHP, dihydropyridine; E-C, excitation-contraction; EP, phosphorylated intermediate of the enzyme; IP₃, inositol 1,4,5-trisphosphate; JFM, junctional face membrane;M _r, molecular weight; T-tubule, transverse-tubular system.     

Cardiac excitation-contraction coupling in the portal hypertensive rat

Basal contractility and responses to beta-adrenoceptor activation are compromised in hearts from rats with chronic portal vein stenosis. Here we report the effect of partial ligation of the portal vein on myocardial G protein expression, beta-adrenoceptor-G protein coupling, and excitation-contraction coupling (ECC). Contractility (dT/dt) was reduced 30-50% in right and left ventricles, but the rate of relaxation (-dT/dt) was unaffected. Isoproterenol-induced positive inotropism was diminished, but there was no difference in ED(50). The concentration-dependent increase in -dT/dt was unaffected. G(s)alpha and G(i)alpha expression, cholera toxin- and pertussis toxin-induced ADP-ribosylation, and formation of the agonist-receptor-G(s) complex were unaffected by portal vein stenosis. Of the components of ECC examined, the caffeine-sensitive sarcoplasmic reticulum Ca(2+) pool was reduced 35%, although the Ca(2+) uptake and release processes were unchanged; the apparent density of L-type Ca(2+) channels decreased 60% with no change in affinity; the dihydropyridine Ca(2+) channel agonist BAY K 8644 produced relative changes in dT/dt that were similar in both groups, suggesting normal function in the remaining Ca(2+) channels; and Na(+)/Ca(2+) exchange was reduced 50% in the portal vein stenosis group. These data suggest that the effect of portal vein stenosis on the myocardium is the result of alterations to ECC.     

Force-frequency relations in hypertrophic heart muscle: a mathematical model for excitation-contraction coupling.

Static and dynamic chrono-inotropic responses were recorded from both normal and hypertrophic rat auricular myocardium. The slope of the static force-frequency relation for hypertrophic hearts was steeper than that for control hearts. Computer experiments were designed to study the cellular mechanisms underlying the changes in the force-frequency response associated with heart hypertrophy, with the aid of a mathematical model for excitation-contraction coupling in rat heart. A set of equations was derived which permitted to study the effects on the chronoinotropic relations of both the geometrical dimensions of cardiomyocytes and the sarcoplasmic reticulum, and of the variation in activity of mechanisms for Ca movements through the sarcolemma and the sarcoreticular membrane. A comparison of data obtained from simulated and real experiments suggested that the features characteristic of force-frequency relations for hypertrophic heart are a result of an enhanced volume of intracellular Ca-stores rather than of the total volume of the cardiomyocyte.     

Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.