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

E-C 偶联结构蛋白 Junctophilin-2 在心脏中的新功能

• 批准号: 10058735
• 负责人: Long-Sheng Song
• 金额: $ 60.53万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058735
• 关键词: Animal Model; Attenuated; Biochemical; Calpain; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Culture Techniques; Cell Nucleus; Cell membrane; Cell model; Complex; Coupling; Data; Development; Disease; Down-Regulation; Functional disorder; Funding; Future; Generations; Genetic; Genetic Transcription; Goals; Heart; Heart Diseases; Heart failure; Homeostasis; Human; Knock-in Mouse; Link; Mediating; Mediator of activation protein; Membrane; Modification; Molecular; Mus; Muscle Cells; Mutate; Mutation; Mutation Analysis; Myocardial Infarction; N-terminal; Nuclear; Nuclear Import; Nuclear Translocation; Outcome; Pathogenesis; Pathologic; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Physiological; Physiology; Post-Translational Protein Processing; Prevalence; Process; Production; Proteins; Proteolysis; Regulation; Research; Resistance; Role; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Site; Stress; Structural Protein; Structure; Testing; Therapeutic; Transcript; Transcriptional Regulation; Translations; Treatment Failure; Tubular formation; Ventricular; base; biological adaptation to stress; cardioprotection; cardiovascular health; design; experimental study; heart function; heart preservation; improved; in vitro Assay; in vivo; insight; junctophilin; mimetics; multidisciplinary; mutant; novel; overexpression; preservation; pressure; prevent; response; tool

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.

项目概要 Junctophilin 2 (JP2) 是形成连接偶联所需的必需结构蛋白（即， 横管 (T) 膜和肌浆网 (SR) 之间的心脏二元体）。 JP2功能 因此，对于局部控制 Ca2+ 诱导的 Ca2+ 释放和心室有效收缩至关重要。 心脏兴奋-收缩（E-C）耦合过程中的肌细胞。 JP2蛋白水平在失败时逐渐下降 人类心脏和心力衰竭动物模型导致 T 管重塑和 E-C 耦合丧失 功能。 JP2 在 E-C 偶联位点的下调部分是由于 Ca2+ 激活的特异性裂解 蛋白酶钙蛋白酶与多种心脏病有关。在上一个资助期间，我们 证明 JP2 的应激和钙蛋白酶依赖性裂解释放出一种新型的核易位 N- 末端片段（JP2NT）抑制患病心脏中适应不良的转录重编程，因此 将 E-C 解偶联信息转换为独特的心脏保护性兴奋转录 (E-T) 偶联 向细胞核发出信号。然而，JP2介导的E-C和E-T耦合现象是如何机械调节的 仍有待确定。我们新的初步结果表明，JP2 在应激条件下可重复磷酸化 心脏靠近负责 JP2 裂解和 JP2NT 亚细胞定位的区域。在这场竞争激烈的 更新应用程序，我们的目标是定义压力诱导的翻译后修饰如何调节结构， JP2/JP2NT 的本地化和功能。我们假设 JP2NT 介导的 E-T 耦合受到严格调控 通过 JP2 的心脏应激依赖性磷酸化决定 JP2 对钙蛋白酶和 JP2NT 的敏感性 核易位和转录活性。为了检验我们的假设，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 偶联和裂解诱导的 JP2NT 产生、核易位和转录调控。在目标 2 中，我们将利用我们的新型 JP2 钙蛋白酶 耐药小鼠与 JP2NT 过度表达相结合，以确定这些靶向方法如何调节 心脏对体内应激的反应。我们将确定E-C耦合结构/功能和心脏基因如何 这些小鼠的转录因压力超负荷和心肌梗塞而发生改变。我们期望 我们的研究将为 JP2/JP2NT 功能的调控机制提供重要见解 它们对心脏病发病机制的有益贡献。