The Regulation Of Excitation-contraction Coupling In Normal And Disease Heart

Part1KCNE2modulates cardiac L-type Ca2+channel, which is associated with a familial atrial fibrillation linked to KCNE2gain-of-function mutation (R27C)KCNE2plays an important role in maintaining cardiac electrical stability. Mutations in KCNE2have been linked to long-QT syndrome (LQT6) and atrial fibrillation/short QT syndrome. It has been suggested that KCNE2has the most promiscuity of function which can interact with multiple α-subunits of voltage-dependent cation channels and modulate their functions. However, whether KCNE2regulates voltage-dependent L-type Ca2+channel (LCC) remains unknown.Objective:This study investigated the possible role of KCNE2in regulating cardiac LCCs and the pathophysiological relevance of this regulation.Methods and Results:Adenoviral overexpression of KCNE2by2.5fold in rat cardiomyocytes decreased peak L-type Ca2+current (ICa,L) by18%, whereas79%KCNE2knockdown by RNA interference increased peak Ica,L by29%. Upregulation of KCNE2caused a positive shift of the voltage-dependent activation, a negative shift of the steady-state voltage-dependent inactivation, and slowed the recovery from inactivation of Ica,L, while downregulation of KCNE2had the contrary effects on the properties of Ica,L.Similar regulation of KCNE2on Ica,L had been observed in transfected HEK293cells. Coimmunoprecipitation and colocalization assays in both cardiomyocytes and the transfected cell line suggest that Cav1.2physically interacted with KCNE2. Furthermore, we found that the familial atrial fibrillation related KCNE2mutation R27C had a stronger suppression effect on Ica,L as compared to wild type KCNE2.Conclusion:These results indicate that KCNE2coassembles with Cav1.2and modulates ICa,L properties. This modulation is important in maintaining cardiac electrical stability, and the "gain of function" mutation in KCNE2(R27C) may induce familial atrial fibrillation partially through suppressing ICa,L and shortening action potential duration.Part2Polydatin modulates Ca2+handling, excitation-contraction coupling and β-adrenergic signaling in rat ventricular myocytesPolydatin (PD), a resveratrol glucoside, has recently been suggested to have cardioprotective effects against heart diseases, including ischemia-reperfusion injury and pressure-overload induced ventricular remodeling.However, the mechanisms are poorly understood. This study aims to investigate the direct effects of PD on cardiac Ca2+handling and excitation-contraction (EC) coupling to explore the potential role of which in PD-mediated cardioprotection. We found that micromolar PD decreased action potential-elicited Ca2+transient,but slightly increased cell shortening. The contradictory response could be attributed to PD increasing myofilament Ca2+sensitivity. Exploring the activities of the two types of Ca2+channels, L-type Ca2+channels (LCCs) and ryanodine receptors (RyRs), reveals that PD dose-dependently decreased LCC current (ICa),but increased frequency of spontaneous Ca2+sparks, the elementary Ca2+releasing events reflecting RyR activity in intact cells. PD dose-dependently increased the gain of EC coupling. In contrast, PD dose-dependently decreased SR Ca2+content. Furthermore, PD remarkably negated (3-adrenergic receptor (AR) stimulationinduced enhancement of ICa and Ca2+transients, but did not inhibit β-AR-mediated inotropic effect. Inhibition of nitric oxide synthase (NOS) with L-NAME abolished PD regulation of ICa and Ca2+spark rate, and significantly inhibited the alteration of Ca2+transient and myocyte contractility stimulated by PD. These results collectively indicate that PD modulated cardiac EC coupling mainly by inversely regulating LCC and RyR activity and increasing myofilament Ca2+sensitivity through increasing intracrine NO, resulting in suppression of Ca2+transient without compromising cardiac contractility. The unique regulation of PD on cardiac EC coupling and responsiveness to β-AR signaling implicates that PD has potential cardioprotective effects against Ca2+mishandling related heart diseases.Part3Cellular mechanism underlying burn serumygenerated bidirectional regulation of excitation-contraction coupling in isolated rat cardiomyocytesMyocardial depressant factors have long been recognized to be present in burn serum (BS) and contribute to burn-generated cardiac contractile dysfunction. However, much of the cellular and molecular mechanism for its role in the development of the cardiac deficiency remains unknown. In this study, we investigated the effect of BS on myocardial contractility and Ca2+handling in single rat cardiomyocytes. The results revealed that BS (5%by volume) bidirectionally regulated cardiac excitation-contraction (EC) coupling. The action potentialYelicited Ca2+transient and cell shortening were increased by28.0%±9.7%and34.7%±12.5%within20min after BS stimulation (the upregulation phase), but decreased by20.5%±6.8%and32.3%±5.1%at60min after BS stimulation (the downregulation phase). There was a32.0%±5.8%reduction in sarcoplasmic reticulum (SR) Ca2+content at the downregulation phase, whereas no alteration was detected at the upregulation phase. The incidences of spontaneous Ca2+sparks and Ca2+waves were significantly increased after BS stimulation, no matter at the upregulation or downregulation phase. The hyperactive Ca2+sparks and Ca2+waves could be completely abolished by antioxidative treatment (vitamin A,0.2mM; and vitamin E,1mM) and partially reversed by NOS inhibitor L-NAME (100uM), but not by blocking Ca2+influx with nifedipine (1uM). With the normalization of Ca2+sparks, BS-induced alterations of action potentialYelicited Ca2+transient and contractility were prevented by antioxidative therapy. Taken together, we propose that BS-associated bidirectional regulation of EC coupling is attributed largely to oxidative stressYinduced hyperactivity of ryanodine receptors, increasing EC coupling through enhancing intracellular Ca2release initially, but subsequently decreasing EC coupling by partially depleting SR Ca2+content through enhancement of Ca2+sparkYmediated SR leak.Part4Polydatin protects cardiac function against burn injury by inhibiting sarcoplasmic reticulum Ca2+leak through reducing oxidative modification of ryanodine receptorsOur recent studies demonstrate that burn trauma induces leaky sarcoplasmic reticulum (SR) in heart due to excessively active ryanodine receptor (RyR) function. SR Ca2+leak causes partial depletion of SR Ca2+content and disturbances of intracellular Ca2+homeostasis, resulting in the pathogenesis of burn-generated cardiac dysfunction. The present study investigated the role of polydatin, a resveratrol glucoside in preventing SR leak and the therapeutic effect against burn-generated cardiac dysfunction. We found that polydatin treatment improved cardiac function impaired by burn injury of30%of total body surface area. Parallel to the alteration of cardiac function, polydatin significantly increased the defective systolic Ca2+transient and contractility in burn-traumatized cardiomyocytes. Burn injury increased the occurrence of Ca2+sparks. The enhancement of Ca2+spark-mediated SR leak caused partial depletion of SR Ca2+content in burn-traumatized cardiomyocytes. Furthermore, we found that the content of free thiols (the number of reduced cysteines) in RyR2in cardiomyocytes determined by the monobromobimane (mBB) fluorescence of RyR2was decreased markedly in burn-traumatized hearts. Polydatin treatment decreased intracellular reactive oxygen species (ROS) levels and restored the amount of free thiols in RyR2in burns. Concomitantly, polydatin corrected Ca2+spark-mediated SR leak and restored SR Ca2+load. The systolic Ca2+transient and cellular contractility were significantly increased by polydatin treatment. Taken together, the present findings provide first evidence demonstrating that polydatin prevented enhanced Ca2+spark-mediated SR leak through reducing oxidative stress in RyR2in burn-traumatized heart, leading to protection of cardiac function against burn injury.