Novel Functions of the E-C Coupling Structural Protein Junctophilin-2 in the Heart

项目总结 连接蛋白2(JP2)是形成连接偶联所必需的结构蛋白(即， 心脏二联体)位于横形(T)管膜和肌浆网(SR)之间。JP2函数 因此，它是局部控制钙诱导的钙释放和有效收缩的基础。 心肌兴奋-收缩(E-C)偶联过程中的心肌细胞。JP2蛋白水平在衰竭时逐渐下降 人心脏和心力衰竭导致T小管重塑和E-C偶联丧失的动物模型 功能。JP2在E-C偶联位点的下调部分是由于钙激活的特异性切割所致 一种与多种心脏疾病有关的蛋白水解酶。在上一个资助期内，我们 证明了依赖于压力和钙蛋白的JP2裂解释放了一种新的，核转位，N- 末端片段(JP2NT)，抑制疾病心脏中的不适应转录重编程，因此 将E-C解偶联信息转化为独特的心脏保护性激发-转录(E-T)偶联 向原子核发出信号。然而，JP2介导的E-C和E-T耦合现象是如何被机械调节的 仍有待确定。我们的新的初步结果表明，在应激条件下，JP2被重复地磷酸化。 心脏靠近负责JP2裂解和JP2NT亚细胞定位的区域。在这场竞争中 续订申请，我们的目标是定义应力诱导的翻译后修饰如何调节结构， JP2/JP2NT的定位和功能。我们假设JP2NT介导的E-T偶联受到严格调控 通过心脏应激依赖的JP2磷酸化决定JP2对Calain和JP2NT的敏感性 核转位和转录活性。为了验证我们的假设，在目标1中，我们将使用突变分析 和细胞模型，以确定JP2磷酸化如何调节E-C偶联和切割诱导的JP2NT 发生、核转位和转录调控。在目标2中，我们将使用我们的新JP2 Calain 耐药小鼠与JP2NT过度表达相结合以确定这些靶向途径如何调节 体内心脏对应激的反应。我们将确定E-C如何将结构/功能和心脏基因偶联 这些小鼠在压力超负荷和心肌梗死时，转录会发生变化。我们预计 我们的研究将为调控JP2/JP2NT功能的调控机制提供重要的见解 他们对心脏病发病机制的有益贡献。

PROJECT SUMMARY Junctophilin 2 (JP2) is an essential structural protein required for the formation of junctional couplings (i.e., cardiac dyads) between the transverse (T)-tubule membrane and the sarcoplasmic reticulum (SR). JP2 function is therefore fundamental for the local control of Ca2+-induced Ca2+ release and efficient contraction in ventricular myocytes during cardiac excitation-contraction (E-C) coupling. JP2 protein levels progressively decline in failing human hearts and in animal models of heart failure leading to T-tubule remodeling and loss of E-C coupling function. The downregulation of JP2 at E-C coupling sites is in part due to specific cleavage by the Ca2+-activated protease calpain that is implicated in a variety of heart diseases. During the previous funding period, we demonstrated that stress- and calpain-dependent cleavage of JP2 liberates a novel, nuclear translocating, N- terminal fragment (JP2NT) that represses maladaptive transcriptional reprogramming in diseased hearts, thus transducing E-C uncoupling information into a unique cardio-protective excitation-transcription (E-T) coupling signal to the nucleus. However, how JP2-mediated E-C and E-T coupling phenomena are mechanistically regulated remains to be determined. Our new preliminary results show that JP2 is reproducibly phosphorylated in stressed hearts near regions responsible for JP2 cleavage and the subcellular localization of JP2NT. In this competitive renewal application, we aim to define how stress-induced post-translational modifications regulate the structure, localization, and function of JP2/JP2NT. We hypothesize that JP2NT-mediated E-T coupling is tightly regulated by cardiac stress-dependent phosphorylation of JP2 that determines JP2 sensitivity to calpain and JP2NT nuclear translocation and transcriptional activity. To test our hypothesis, in Aim 1, we will use mutation analysis and cell models to determine how JP2 phosphorylation regulates E-C coupling and cleavage-induced JP2NT generation, nuclear translocation and transcriptional regulation. In Aim 2, we will utilize our novel JP2 calpain resistant mice in combination with JP2NT overexpression to determine how these targeted approaches modulate cardiac responses to stress in vivo. We will determine how E-C coupling structure/function and cardiac gene transcription are altered in these mice in response to pressure overload and myocardial infarction. We expect our studies will provide significant insights into the regulatory mechanisms governing JP2/JP2NT function and their salutary contribution toward heart disease pathogenesis.