Partial and Controlled Depletion of SR Calcium by RyR Agonists Prevents Calcium-dependent Arrhythmias

摘要 交感神经刺激心室肌细胞激活蛋白激酶A，使L钙离子磷酸化 通道和磷蛋白等底物，以增加钙离子进入和肌浆网钙摄取。这个β- 肾上腺素能诱导的细胞内钙升高(钙超载)是一种自然而有效的机制 提高心脏功能，因为心脏收缩的幅度和力度在很大程度上取决于 提供给肌丝的钙离子的量。然而，钙超载也增加了心律失常的易感性 因为它使Ryanodine受体(RyR2)更接近自发钙释放(SCR)的阈值。可控硅 舒张期激活肌膜Na~+/Ca~(2+)交换器，产生去极化内向电流 (延迟后除极或DAD)，可能触发即兴动作电位。钙超载及其影响因素 随后的SCR确实是恶性心律失常发展的主要事件，正如在 儿茶酚胺能多形性室性心动过速(CPVT)，很可能存在于某些类型的心房 纤颤、QT延长和心力衰竭。为了防止这些钙依赖的心律失常，RyR2的一个紧急小组 阻滞剂旨在阻止SCR，尽管产生SCR的关键过程，即钙超载，可能 RyR2阻滞剂不会影响或在某些情况下甚至可能加重。我们发现， 一种高亲和力、膜通透性的RyRs选择性激动剂Imacalcin，矛盾地降低了 在慢性静脉曲张动物模型中，心律失常负荷和预防钙依赖的心律失常。我们假设 这种意想不到的显著抗心律失常作用是由于部分和可控的 肌浆网钙负荷耗竭，从而降低SCR事件的倾向，从而消散主要 CPVT的致心律失常底物。我们的研究计划将对此进行系统和严格的测试 在分子、细胞、整个心脏和完整动物水平使用已建立的(小鼠)和新的假说 (兔)CPVT模型。第一个具体目标将确定确定的结构和功能特征 这赋予了欧帕卡霉素和钙蛋白家族的其他成员他们渗透膜和 它们的抗心律失常特性。在第二个目标中，我们将在完整的 心肌细胞，兰登多夫灌流的工作心脏和完整的动物，为一种新颖的合理化设计 一组能够预防钙离子引发的心律失常的RyR2配体。这些研究使用的动物模型是 CPVT开发治疗钙超载引起的心律失常的新范例；结果 可应用于其他需要控制肌浆网钙离子卸载的心肌病。

ABSTRACT Sympathetic stimulation of ventricular myocytes activates PKA, which phosphorylates L-type Ca2+ channels and phospholamban, among other substrates, to increase Ca2+ entry and SR Ca2+ uptake. This β- adrenergic-induced increase of intracellular Ca2+ (Ca2+ overload) is a natural and efficient mechanism to increase cardiac performance, inasmuch as the magnitude and force of cardiac contractions greatly depend on the amount of Ca2+ supplied to the myofilaments. However, Ca2+ overload also increases arrhythmia vulnerability because it brings ryanodine receptors (RyR2) closer to threshold for spontaneous Ca2+ release (SCR). SCR during diastole activates the Na+/Ca2+ exchanger of the sarcolemma and generates a depolarizing inward current (delayed after-depolarization or DAD) that may trigger extemporaneous actions potentials. Ca2+ overload and its subsequent SCRs are indeed the primary events that evolve in malignant cardiac arrhythmias, as observed in Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) and quite possibly in some forms of atrial fibrillation, long QT, and heart failure. To prevent these Ca2+-dependent arrhythmias, an emergent group of RyR2 blockers aim to stop SCR, despite the fact that the key process that spawns SCR, namely, Ca2+ overload, may not be affected or in some cases may be even exacerbated by the RyR2 blockers. We have found that imperacalcin, a high-affinity, membrane-permeable selective agonist of RyRs, paradoxically decreases arrhythmia burden and prevents Ca2+-dependent arrhythmias in animal models of CPVT. We hypothesize that this unexpected and remarkable anti-arrhythmic effect of imperacalcin is due to a partial and controlled depletion of SR Ca2+ load, which decreases the propensity of SCR events and thus dissipates the main arrhythmogenic substrate in CPVT. Our research program will systematically and rigorously test this hypothesis at the molecular, cellular, whole heart and intact animal levels using established (mouse) and novel (rabbit) models of CPVT. The first specific aim will determine the defining structural and functional characteristics that confer to imperacalcin and other members of the calcin family their capacity to permeate membranes and their anti-arrhythmic properties. In the second aim, we will use native and modified calcins on intact cardiomyocytes, Langendorff-perfused working hearts and intact animals for a rationalized design of a novel group of RyR2 ligands capable of preventing Ca2+-triggered arrhythmias. These studies use animal models of CPVT to develop a novel paradigm for the treatment of Ca2+ overload-generated cardiac arrhythmias; results may be applied to other cardiomyopathies where controlled unloading of SR Ca2+ may be desirable.