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

摘要 心室肌细胞的交感神经刺激激活PKA，PKA磷酸化L型Ca 2 + 通道和受磷蛋白，以及其他底物，以增加Ca 2+进入和SR Ca 2+摄取。这个β- 肾上腺素能诱导的细胞内Ca 2+增加（Ca 2+超载）是一种自然而有效的机制， 增加心脏性能，因为心脏收缩的幅度和力量在很大程度上取决于 供应给肌丝的Ca 2+的量。然而，Ca 2+超载也增加心律失常的脆弱性 因为它使Ryanodine受体（RyR 2）更接近自发Ca 2+释放（SCR）的阈值。SCR 在此过程中，乙酰胆碱激活肌膜Na+/Ca 2+交换器，产生去极化内向电流 （延迟后去极化或DAD），可能触发临时动作电位。Ca 2+超载及其 随后的SCR确实是恶性心律失常中演变的主要事件， 儿茶酚胺能多态性室性心动过速（CPVT）和很可能在某些形式的心房 纤维性颤动QT间期延长和心力衰竭为了预防这些钙依赖性心律失常，一组新的RyR 2 阻滞剂旨在阻止SCR，尽管事实上产生SCR的关键过程，即Ca 2+过载，可能 不受影响，或在某些情况下，甚至可能加剧RyR 2阻滞剂。我们发现 Imperacalcin是RyR的高亲和力、膜渗透性选择性激动剂， 心律失常负荷和预防CPVT动物模型中的Ca 2+依赖性心律失常。我们假设 这种意想不到的和显著的抗糖尿病作用是由于部分的和受控的 SR Ca 2+负荷的耗尽，这降低了SCR事件的倾向，从而消耗了主要的 CPVT中的致瘤底物。我们的研究计划将系统和严格地测试这一点 在分子、细胞、整个心脏和完整动物水平上使用已建立的（小鼠）和新的 （兔）CPVT模型。第一个具体目标将确定定义结构和功能特征 其赋予imperacalcin和钙蛋白家族的其他成员渗透膜的能力， 它们的抗肿瘤特性。在第二个目标中，我们将使用天然和修饰的钙蛋白对完整的 心肌细胞，Langendorff灌注的工作心脏和完整的动物，以合理设计一种新的 一组RyR 2配体能够预防Ca 2+触发的心律失常。这些研究使用了 CPVT开发治疗Ca 2+超负荷引起的心律失常的新范例;结果 可以应用于其他心肌病，其中可能需要SR Ca 2+的受控卸载。

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.