Pathophysiological Regulation of Cardiac Myocyte RyR Channel

描述（由申请人提供）：心脏ryanodine受体（RyR2）或肌浆网（SR）钙释放通道的突变和失调直接导致人类儿茶酚胺能多形性室性心动过速（CPVT）和心力衰竭（HF）。受影响的ryr比正常更活跃，在舒张期从SR漏钙，引起心律失常和功能障碍。FKBP12.6和钙调蛋白（CaM）是与RyR2紧密结合并稳定RyR2的蛋白。我们已经开发了新的定量方法来评估（在成人心肌细胞中）FKBP， CaM和其他肽如何结合并调节RyR2门控，并在结构上定位于心肌细胞RyR2。虽然FKBP12.6与RyR2具有高亲和力，但它不是一个关键的RyR2调节因子。在这里，我们研究了RyR2中CaM结合和改变的结构域-结构域相互作用如何参与疾病相关的RyR2功能障碍，使用荧光标记蛋白和成人心室肌细胞共聚焦成像中的新型靶向传感器来解决4个目标。我们将：1)测试hf相关的RyR改变是否会降低CaM结合并增加结构解压缩肽DPc10的可及性。2)检测已知的RyR抑制剂是否通过改变FKBP12.6、CaM或DPc10的结合作用于病理性RyR2。3)利用新型靶向Ca传感器测量裂隙[Ca]。4)通过绘制S100A1、CaMKII、IPTx和Sorcin位点，增强RyR2结构模型。这是一个高度合作的项目，两个团队在心衰和心律失常的心脏钙调节方面有着共同的兴趣，并在生理学、药理学、生物化学、分子生物学、荧光光谱和共聚焦成像方面具有互补的技术专长。这将极大地增强我们对健康和疾病中心肌细胞中RyR2结构和功能的理解，并为心脏中病理调节的RyR2的治疗方法的开发提供新的策略。

DESCRIPTION (provided by applicant): Mutation and dysregulation of the cardiac ryanodine receptor (RyR2) or sarcoplasmic reticulum (SR) calcium release channel contributes directly to catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure (HF) in humans. Affected RyRs are more active than normal, leaking Ca from the SR during diastole causing arrhythmias and dysfunction. FKBP12.6 and calmodulin (CaM) are proteins that bind tightly with and may stabilize RyR2. We have developed novel quantitative methods to assess (in adult cardiac myocytes) how FKBP, CaM and other peptides bind to and modulate RyR2 gating, and are structurally positioned on the myocyte RyR2. While FKBP12.6 binds RyR2 with high affinity, it is not a critical RyR2 regulator. Here we examine how CaM binding and altered domain-domain interaction in RyR2 are involved in disease-related RyR2 dysfunction, using fluorescent tagged proteins and novel targeted sensors in adult ventricular myocyte confocal imaging to address 4 aims. We will: 1) Test whether HF-related RyR alterations decrease CaM binding and increase the access of the structural unzipping peptide DPc10. 2) Test whether known RyR inhibitors work on pathological RyR2 by altering FKBP12.6, CaM or DPc10 binding. 3) Measure Cleft [Ca] using novel targeted Ca sensors. 4) Enhance RyR2 structural model by mapping sites of S100A1, CaMKII, IPTx and Sorcin. This is a highly collaborative project between two groups with shared interests in cardiac calcium regulation in HF and arrhythmias and with complementary technical expertise in physiology, pharmacology, biochemistry, molecular biology, fluorescence spectroscopy and confocal imaging. This will greatly enhance our understanding of RyR2 structure and function in cardiac myocytes in health and disease and provide novel strategies for the development of therapeutics for pathologically modulated RyR2 in the heart.