CMYA5 regulation of cardiac dyad structure and function

在心肌细胞中，二联体是由T管并列形成的纳米级结构，T管是质膜的管状凹陷网络，内质网专门用于释放钙离子的区域，称为连接肌浆网(JSR)。二联体位于Z线附近，因此肌节Z线、JSR和T管以规则的横向线性模式共同定位。DUADS介导兴奋-收缩(E-C)偶联，将快速传播的质膜电信号转化为遍及心肌细胞的协调钙瞬变，导致同步、有力的肌节收缩。心肌细胞衰竭的一个特征是二联体的紊乱，这会扰乱钙离子的处理，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，尽管它们对心脏动态平衡和疾病很重要。我们的初步数据建立了一个二聚体形成的层次结构，其中一个很少研究的蛋白质CMYA5将JSR与肌节Z线捆绑在一起，T-管与JSR结合形成二聚体。我们进一步证明，CMYA5是正常的二联体结构、E-C偶联的保真度和调节RYR2钙释放活性所必需的。缺乏CMYA5的小鼠有扩张性心肌病，并对压力超负荷反应敏感，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组织的丢失与CMYA5定位的干扰有关。我们的研究建立了CMYA5作为一个新的切入点来研究负责二联体结构和Z线附近定位的机制，并揭示了CMYA5依赖的机制异常在人类心力衰竭3-5的二联体解体中的作用，这有助于心力衰竭的发病。在这些新观察的基础上，我们将追求以下具体目标，以进一步了解CMYA5在调节CM钙释放和E-C偶联中的作用：(1)研究CMYA5对RYR2活性的调节。我们将验证CMYA5与RYR2相互作用通过控制RYR2通道活性和RYR2通道聚集来调节RYR2钙释放的假设。(2)确定CMYA5将RYR2/JSR系在Z线上的机制。我们将检验CMYA5通过与目前未知的桥接蛋白相互作用将RYR2/JSR锚定在Z线上的假设。(3)评价CMYA5错位在人类和实验性心脏病二联体断裂中的作用。这一提议将揭示负责二联体亚细胞组织的新机制，这是CMS的标志性纳米结构，对正常的E-C偶联至关重要。阐明这些机制将有助于深入了解扰乱人类心力衰竭中的二联体、加剧收缩功能障碍和心律失常的机制，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.