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Cardiac Calsequestrin (Casq2) function in excitation-contraction coupling and cardiac arrhythmias

心脏 Calsequestrin (Casq2) 在兴奋-收缩耦合和心律失常中的作用

基本信息

批准号：
10347169
负责人：
Matthew J Wleklinski
金额：
$ 5.18万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10347169
关键词：
Affect Affinity Arrhythmia Buffers Calcium Calcium Binding Calcium-Binding Proteins Calsequestrin Cardiac Cardiac Electrophysiologic Techniques Cardiac Myocytes Catecholaminergic Polymorphic Ventricular Tachycardia Coronary Arteriosclerosis Coupled Couples Coupling Data Dialysis procedure Electrocardiogram Electron Microscopy Electrophysiology (science)Equilibrium Exhibits Family Fluorescence Functional disorder Genetic Goals Heart Diseases Heart failure Human Hypertrophy Investigation Knock-in Mouse Lead Link Measures Methods Missense Mutation Modeling Molecular Sieve Chromatography Mus Mutation Patients Phenotype Physiological Play Polymers Process Proteins Recombinants Refractory Regulation Reporting Role Ryanodine Receptor Calcium Release Channel Sarcoplasmic Reticulum Spectrum Analysis Stress Sudden Death Syndrome Testing Therapeutic Variant Western Blotting absorption autosomal dominant mutation calcium indicator depolymerization genetic analysis genetic variant heart function human model human pluripotent stem cell improved in vivo induced pluripotent stem cell insight light scattering mechanical force mouse model mutant novel overexpression patch clamp polymerization protein protein interaction protein structure stem cell model sudden cardiac death targeted treatment treatment strategy triadin

项目摘要

PROJECT SUMMARY/ABSTRACT Calsequestrin-2 (Casq2) is a high capacity, low affinity calcium (Ca) binding protein located in the junctional sarcoplasmic reticulum (SR) of cardiac myocytes. As the major SR Ca buffer, Casq2 interacts with the ryanodine receptor (RyR2), a Ca release channel, to regulate the amount of Ca that is released during the excitation- contraction (EC) coupling cycle, a process that couples electrical activation to mechanical force (i.e. a heartbeat). Alterations in EC coupling can cause both contractile dysfunction and cardiac arrhythmias. Reduction or loss of Casq2 due to mutations causes a severe genetic arrhythmia syndrome known as catecholaminergic polymorphic ventricular tachycardia (CPVT). Genetic variants of Casq2 have also been associated with sudden cardiac death and heart failure in patients with coronary artery disease, while overexpression of Casq2 causes hypertrophy and heart failure in mice. Casq2-linked CPVT is usually autosomal-recessive, with mutations resulting in either a severe reduction or complete loss of Casq2 protein. As a result, SR Ca buffering is reduced, which leads to spontaneous Ca release and arrhythmias. In 2016, a genetic analysis conducted in a family that had an autosomal dominant inheritance of CPVT uncovered a novel missense mutation (K180R) within Casq2. This was the first autosomal dominant mutation found in Casq2. Initial studies in heterozygous K180R knock-in mice demonstrate that protein levels of Casq2 are normal but mice exhibit CPVT when stressed. This suggests that Casq2-K180R causes CPVT by a different mechanism than previously reported autosomal-recessive Casq2 mutations. I hypothesize that Casq2-K180R causes CPVT by disrupting its ability to regulate RyR2 Ca release channels, either directly or by altering SR Ca buffering, leading to spontaneous Ca release. To test this, I plan to use both mouse and human pluripotent stem cell models. Recent studies have shown that cardiomyocytes (CM) differentiated from human induced pluripotent stem cells (hiPSCs) can model CPVT and be used to screen potential therapeutics. Utilizing the K180R mouse and hiPSC models I already generated, the aims of this project are to determine how K180R affects SR calcium handling and to investigate how K180R affects Casq2 Ca binding, localization, and polymerization. I will investigate how CMs are effected at the physiological and cellular levels and determine how Casq2 is altered at the protein level. This project will provide new insight into the role of Casq2 during the EC coupling cycle, the functional interaction between Casq2 and RyR2, the termination of SR Ca release, and how Casq2 variants could lead to cardiac arrhythmias and/or heart failure. This improved understanding of Casq2 could lead to better treatment strategies for patients suffering from Casq2-dependent cardiac disorders.

项目摘要/摘要 Calequestrin-2(Casq2)是一种高容量、低亲和力的钙结合蛋白，位于结合部。心肌细胞的肌浆网(SR)。作为主要的肌质网钙缓冲液，Casq2与ryanodine相互作用受体(RyR2)，一种钙释放通道，调节在兴奋过程中释放的钙量。收缩(EC)耦合循环，将电激活与机械力(即心跳)相结合的过程。 EC偶联的改变可导致收缩功能障碍和心律失常。减少或损失 Casq2基因突变导致一种严重的遗传性心律失常综合征，称为儿茶酚胺能多态室性心动过速。Casq2的基因变异也与心源性猝死有关和冠心病患者的心力衰竭，而Casq2的过度表达导致肥厚以及小鼠的心力衰竭。Casq2连锁的CPVT通常是常染色体隐性遗传的，突变导致 Casq2蛋白的严重减少或完全丧失。因此，减少了SR Ca缓冲，从而导致自发性钙释放与心律失常。2016年，一项在一个家庭中进行的基因分析常染色体显性遗传的CPVT在Casq2中发现了一个新的错义突变(K180R)。这是在Casq2中发现的第一个常染色体显性突变。杂合子K180R敲除小鼠的初步研究证明Casq2的蛋白水平是正常的，但小鼠在应激时表现出CPVT。这表明 Casq2-K180R导致CPVT的机制与先前报道的常染色体隐性遗传Casq2不同突变。我推测Casq2-K180R通过破坏其调节RyR2钙通道的能力而导致CPVT 释放通道，直接或通过改变肌浆网钙缓冲，导致自发钙释放。至为了测试这一点，我计划同时使用小鼠和人类的多能干细胞模型。最近的研究表明，从人诱导多能干细胞(HiPSCs)分化的心肌细胞(CM)可以模拟CPVT和用于筛选潜在的治疗方法。利用我已经生成的K180R鼠标和HiPSC模型，该项目的目的是确定K180R如何影响SR钙处理，并调查K180R如何影响Casq2Ca的结合、定位和聚合。我将调查CMS是如何在并确定Casq2是如何在蛋白质水平上改变的。该项目将提供对Casq2在EC偶联循环中的作用、Casq2和Casq2之间的功能相互作用的新认识 RyR2，SR钙释放的终止，以及Casq2变异如何导致心律失常和/或心脏失败了。这种对Casq2的更好理解可能会为患者带来更好的治疗策略来自Casq2依赖型心脏疾病。