Ryanodine receptor structure and function in heart failure

项目摘要 本PPG的总体目标是确定调节局部钙（Ca 2+）的分子机制 以前所未有的精确度在正常和衰竭的心脏中发出信号。这四个人有三个共同的目标 项目：1）探讨肾上腺素能信号在正常和衰竭心脏中调节钙的确切作用; 2)确定T-小管和肌浆网（SR）钙通道之间相互作用的新机制; 3)发展对涉及钙的遗传性CV疾病的遗传机制的新认识。 项目四通过研究结构-功能关系，为SR钙释放提供了必要的环节， 2型兰尼碱受体（RyR 2）/钙释放通道存在于肌浆和内质网 在心力衰竭（射血分数降低的慢性HF， HFrEF）。RyR 2通道是从细胞内储存的Ca 2+释放触发兴奋-收缩所必需的 (EC)心肌中的耦合。遗传性RyR 2突变可导致心律失常，包括运动诱发的心律失常。 猝死或儿茶酚胺能多形性室性心动过速（CPVT），以及应激诱导的后 RyR 2的翻译修饰有助于心力衰竭（HF）的进展。在这两种情况下，RyR 2通道 由于遗传性突变（CPVT）或获得性翻译后修饰（HF）而泄漏。没有答案 问题包括：1）RyR 2突变可以（如果是这样的话，如何）引起HF; 2）确切的机制是什么？ 哪种PKA磷酸化激活RyR 2并在HF中起作用？使用cryo-EM解决 人RyR 2在~2.4 nm处的结构表明，人CPVT突变RyR 2-R2474 S使通道进入一个 “引发状态”，从该状态可以容易地在低的、通常非活化的[Ca 2 +]细胞因子水平下病理性地活化。 这也解释了为什么这些通道在运动或压力下会发生泄漏并导致致命的室性心律失常。的 Rycal药物ARM 210与RyR 2结合并将突变通道恢复到稳定的闭合状态， 泄漏和心律失常。申请人假设RyR 2错义突变，其中14个在人中发现， 在来自巴基斯坦基因组资源（PGR）的患者中， 具有广泛表型数据和高近亲比率的个体的独特队列（核心A）可能是 功能增益（GoF）突变，也使通道进入启动状态，这对 与CPVT突变相比，因此，这些患者具有渗漏的RyR 2通道并发展为HF。 这是由于SR Ca 2+耗竭导致心肌收缩力受损。这些问题将使用 功能和结构分析，并使用一种新的药物，Rycal（ARM 210），固定RyR 2介导的SR Ca 2+通过明确的机制泄漏。提出了三个目标：1）评估功能的变化， 与心力衰竭（HFrEF）相关的RyR 2 GoF错义变体的结构; 2）评估Rycal对心力衰竭（HFrEF）的影响。 人RyR 2和疾病相关变体的结构/功能;以及3）通过以下方法检查分子机制： 肾上腺素能信号调节RyR 2在正常和衰竭心脏中的功能。

Project Summary The overarching goal of this PPG is to define the molecular mechanisms that regulate local calcium (Ca2+) signaling in normal and failing hearts with unprecedented precision. There are three goals shared by the four projects: 1) explore the precise role of adrenergic signaling in modulating calcium in normal and failing hearts; 2) define novel mechanisms of interactions between T-tubule and sarcoplasmic reticulum (SR) calcium channels; 3) develop new understandings of genetic based mechanisms of inherited forms of CV disease involving calcium. Project four provides the essential link to SR calcium release by studying the structure-function relationships of the type 2 ryanodine receptor (RyR2)/calcium release channel present on the sarcoplasmic and endoplasmic reticula (SR/ER) of many cell types in the context of heart failure (chronic HF with reduced ejection fraction, HFrEF). RyR2 channels are required for Ca2+ release from intracellular stores that triggers excitation-contraction (EC) coupling in the cardiac muscle. Inherited RyR2 mutations can cause arrhythmias including exercise-induced sudden death or catecholaminergic polymorphic ventricular tachycardia (CPVT), and stress-induced post- translational modifications of RyR2 contribute to heart failure (HF) progression. In both cases RyR2 channels are leaky either due to inherited mutations (CPVT) or acquired post-translational modifications (HF). Unanswered questions include: 1) can (and if so how) RyR2 mutations can cause HF; 2) what is the precise mechanism by which PKA phosphorylation activates RyR2 and plays a role in HF? Preliminary data using cryo-EM to solve the structure of human RyR2 at ~2.4 Å show that a human CPVT mutation RyR2-R2474S puts the channel into a “primed state” from which it can be readily and pathologically activated at low, normally non-activating [Ca2+]cyt explaining why these channels are leaky and cause fatal ventricular arrhythmias during exercise or stress. The Rycal drug ARM210 binds to RyR2 and restores the mutant channel back to a stable closed state preventing leak and arrhythmias. The applicant hypothesizes that RyR2 missense mutations, 14 of which are found in patients with a ClinVar prediction of cardiomyopathy in patients from the Pakistan Genome Resource (PGR), a unique cohort of individuals with extensive phenotype data and high rates of consanguinity, (Core A), may be gain of function (GoF) mutations that also put the channel into a primed state which is even more sensitive to activation compared to the CPVT mutations. Thus, these patients have leaky RyR2 channels and develop HF due to depletion of SR Ca2+ resulting in impaired cardiac contractility. These questions will be addressed using functional and structural assays and using a novel drug, Rycal (ARM210), that fixes the RyR2 mediated SR Ca2+ leak via a well-defined mechanism. Three aims are proposed: 1) Evaluate changes in the function and structure of RyR2 GoF missense variants linked to heart failure (HFrEF); 2) Evaluate the effect of Rycal on the structure/function of human RyR2 and disease related variants; and 3) Examine the molecular mechanism by which adrenergic signaling regulates RyR2 function in normal and failing hearts.