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: 10689197
• 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/10689197
• 关键词: Animal Model; Biochemical; Calpain; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Culture Techniques; Cell Nucleus; Cell membrane; Cell model; Complex; Coupling; Data; Development; Disease; Down-Regulation; Endowment; Functional disorder; Funding; Future; Generations; Genetic; Genetic Transcription; Goals; Heart; Heart Diseases; Heart failure; Homeostasis; Human; Knock-in Mouse; Link; Mediating; Mediator; Membrane; 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; Repression; Reproducibility; Research; Resistance; Role; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Site; Stress; Structural Protein; Structure; Testing; Therapeutic; Transcript; Transcriptional Regulation; Treatment Failure; Tubular formation; Ventricular; 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; transcriptional reprogramming

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.

项目摘要 嗜连接蛋白2（JP 2）是形成连接偶联（即， 心脏二分体）之间的横向（T）小管膜和肌浆网（SR）。JP 2函数 因此，对于局部控制Ca 2+诱导的Ca 2+释放和心室有效收缩是至关重要的。 在心脏兴奋-收缩（E-C）偶联期间，JP 2蛋白水平逐渐下降， 人心脏和心力衰竭动物模型中导致T-小管重塑和E-C偶联丧失 功能JP 2在E-C偶联位点的下调部分是由于Ca 2+激活的细胞因子的特异性切割。 蛋白酶钙蛋白酶，与多种心脏病有关。在上一个财政年度，我们 证明了JP 2的应激和钙蛋白酶依赖性裂解释放了一种新的核转位，N- 末端片段（JP 2NT），抑制患病心脏中的适应不良转录重编程，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 向原子核发出信号。然而，JP 2介导的E-C和E-T偶联现象如何被机械地调节， 还有待确定。我们新的初步结果表明，JP 2在应激条件下可重复磷酸化， 心脏靠近负责JP 2切割和JP 2NT亚细胞定位的区域。在这个竞争激烈的 更新申请，我们的目标是定义如何应激诱导的翻译后修饰调节结构， JP 2/JP 2NT的定位和功能。我们假设JP 2NT介导的E-T偶联受到严格调控， 通过JP 2的心脏应激依赖性磷酸化，其决定JP 2对钙蛋白酶和JP 2NT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JP 2磷酸化如何调节E-C偶联和裂解诱导的JP 2NT 生成、核转位和转录调控。在目标2中，我们将利用我们的新JP 2钙蛋白酶 联合JP 2NT过表达的耐药小鼠，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偶联结构/功能和心脏基因 在这些小鼠中，由于压力超负荷和心肌梗塞，转录发生了改变。我们预计 我们的研究将为JP 2/JP 2NT功能的调控机制提供重要的见解， 它们对心脏病发病机制的有益贡献。

项目成果

Calpain-2 specifically cleaves Junctophilin-2 at the same site as Calpain-1 but with less efficacy.

• DOI: 10.1042/bcj20210629
• 发表时间: 2021-10-15
• 期刊: The Biochemical journal
• 作者: Wang J;Ciampa G;Zheng D;Shi Q;Chen B;Abel ED;Peng T;Hall DD;Song LS
• 通讯作者: Song LS

Suppression of ryanodine receptor function prolongs Ca2+ release refractoriness and promotes cardiac alternans in intact hearts.

• DOI: 10.1042/bcj20160606
• 发表时间: 2016-11-01
• 期刊: The Biochemical journal
• 作者: Zhong X;Sun B;Vallmitjana A;Mi T;Guo W;Ni M;Wang R;Guo A;Duff HJ;Gillis AM;Song LS;Hove-Madsen L;Benitez R;Chen SR
• 通讯作者: Chen SR

Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity.

• DOI: 10.1007/s00204-019-02405-w
• 发表时间: 2019-04
• 期刊: Archives of toxicology
• 影响因子: 6.1

Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.

• DOI: 10.1161/circresaha.116.310283
• 发表时间: 2017-06-09
• 期刊: Circulation research
• 影响因子: 20.1
• 作者: Guo Y;VanDusen NJ;Zhang L;Gu W;Sethi I;Guatimosim S;Ma Q;Jardin BD;Ai Y;Zhang D;Chen B;Guo A;Yuan GC;Song LS;Pu WT
• 通讯作者: Pu WT

Targeting Calpain for Heart Failure Therapy: Implications From Multiple Murine Models.

靶向钙蛋白酶治疗心力衰竭

• DOI: 10.1016/j.jacbts.2018.05.004
• 发表时间: 2018-08
• 期刊: JACC. Basic to translational science
• 作者: Wang Y;Chen B;Huang CK;Guo A;Wu J;Zhang X;Chen R;Chen C;Kutschke W;Weiss RM;Boudreau RL;Margulies KB;Hong J;Song LS
• 通讯作者: Song LS