CMYA5 regulation of cardiac dyad structure and function

在心肌细胞中，二联体是由T-小管（质膜的管状内陷网络）和专门用于Ca 2+释放的内质网区域（称为连接肌浆网（jSR））并置形成的纳米级结构。二联体位于Z线附近，使得肌节Z线、jSR和T小管以规则的、横向的、线性模式共定位。二联体介导兴奋-收缩（E-C）偶联，其将快速传播的质膜电信号转化为整个心肌细胞中协调的Ca 2+瞬变，导致同步的、有力的肌节收缩。心肌细胞衰竭的一个标志是二分体的解体，这破坏了Ca 2+的处理，导致收缩减少和心律失常的风险增加。 二分体结构和定位的分子机制仍然是一个谜，尽管他们的心脏稳态和疾病的重要性。我们的初步数据建立了一个层次的二分体形成，其中一个小的研究蛋白质，CMYA 5，系jSR肌节Z线，T-小管与jSR形成二分体。我们进一步表明，CMYA 5是所需的正常二分体架构，保真度的E-C耦合，和RYR 2钙释放活性的调节。缺乏CMYA 5的小鼠患有扩张型心肌病，并且对压力超负荷敏感而发展为严重的心功能障碍。在衰竭的人类心脏中，T-小管和jSR组织的丧失与CMYA 5定位的扰动相关联。我们的研究将CMYA 5确立为研究二分体结构和邻近Z线定位的机制的新切入点，并暗示CMYA 5依赖性机制在人类心力衰竭二分体解体中的异常3 -5，这有助于心力衰竭发病机制。 基于这些新的发现，我们将进一步研究CMYA 5在调节CM Ca 2+释放和E-C偶联中的作用：（1）研究CMYA 5对RYR 2活性的调节。我们将检验CMYA 5与RYR 2的相互作用通过控制RYR 2通道活性和RYR 2通道聚集来调节RYR 2 Ca 2+释放的假设。(2)确定CMYA 5将RYR 2/jSR与Z线绑定的机制。我们将测试CMYA 5通过与目前未知的桥接蛋白相互作用将RYR 2/jSR锚定在Z线的假设。(3)评估CMYA 5错误定位对人类和实验性心脏病中二分体破坏的贡献。该提议将揭示负责二联体亚细胞组织的新机制，二联体是正常E-C耦合所必需的CM的标志性纳米结构。这些机制的阐明将提供对人类心力衰竭中干扰二联体、加剧收缩功能障碍和心律失常的机制的见解，并可能导致在遗传性和获得性心脏病中保护E-C偶联的途径。

In cardiomyocytes, dyads are nanoscale structures formed by the juxtaposition of T-tubules, a network of tubular invaginations of the plasma membrane, and regions of the endoplasmic reticulum specialized for Ca2+ release, known as the junctional sarcoplasmic reticulum (jSR). Dyads are positioned adjacent to Z-lines, such that sarcomere Z-lines, jSR, and T-tubules co-localize in a regular, transverse, linear pattern. Dyads mediate excitation-contraction (E-C) coupling, which converts rapidly propagating plasma membrane electrical signals into coordinated Ca2+ transients throughout the cardiomyocyte, resulting in synchronized, forceful sarcomere contraction. A hallmark of failing cardiomyocytes is disorganization of dyads, which disrupts Ca2+ handling and results in decreased contraction and increased risk of arrhythmia. The molecular mechanisms underlying dyad architecture and positioning have remained a mystery, despite their importance to heart homeostasis and disease. Our preliminary data establish a hierarchy for dyad formation in which a little studied protein, CMYA5, tethers jSR to sarcomere Z-lines, and T-tubules associate with jSR to form dyads. We further show that CMYA5 is required for normal dyad architecture, fidelity of E-C coupling, and regulation of RYR2 Ca2+ release activity. Mice lacking CMYA5 had dilated cardiomyopathy and were sensitized to develop severe cardiac dysfunction in response to pressure overload. In failing human hearts, loss of T-tubule and jSR organization were coupled to perturbed CMYA5 localization. Our studies establish CMYA5 as a novel entry point to study mechanisms responsible for dyad architecture and positioning adjacent to Z-lines, and implicate abnormalities of CMYA5-dependent mechanisms in the disorganization of dyads in human heart failure3–5, which contributes to heart failure pathogenesis. Building on these novel observations, we will pursue the following Specific Aims to gain further insights into the function of CMYA5 in regulating CM Ca2+ release and E-C coupling: (1) Investigate CMYA5 regulation of RYR2 activity. We will test the hypothesis that CMYA5 interaction with RYR2 regulates RYR2 Ca2+ release by controlling RYR2 channel activity and RYR2 channel clustering. (2) Identify mechanisms by which CMYA5 tethers RYR2/jSR to Z-lines. We will test the hypothesis that CMYA5 anchors RYR2/jSR at Z-lines through interaction with currently unknown bridging proteins. (3) Evaluate contribution of CMYA5 mislocalization to dyad disruption in human and experimental heart disease. This proposal will reveal novel mechanisms responsible for the subcellular organization of dyads, hallmark nanostructures of CMs that are essential for normal E-C coupling. Elucidation of these mechanisms will provide insights into the mechanisms that perturb dyads in human heart failure, exacerbating contractile dysfunction and arrhythmia, and may lead to avenues to protect E-C coupling in inherited and acquired forms of heart disease.