Novel functions of E-C coupling structural protein junctophilin-2 in the heart

 描述(由申请人提供)：JP2是一种结构蛋白，在横管(T)-小管膜和肌浆网(SR)之间形成连接偶联(即心脏二分体)所需的结构蛋白，是在心肌兴奋-收缩(E-C)偶联过程中局部控制钙离子诱导的钙释放和心室肌细胞有效收缩的基础。在心力衰竭动物模型中，JP2蛋白水平逐渐下降，导致T小管重塑和E-C偶联功能丧失。在人类衰竭的心脏中，JP2水平也显著降低。我们最近发表的初步数据表明，JP2是一种钙激活的蛋白酶，与多种心脏疾病有关，它是一种翻译后调控蛋白。最初的假设是，在衰竭的心脏中，Calain对JP2的切割是JP2下调的基础。在初步研究中，我们做了一个新颖和意想不到的观察到，与全长JP2不同，N端的JP2切割片段被输入到细胞核中，在心肌细胞的转录重编程中具有深刻的功能。然而，JP2NT核定位的(病理)生理后果和相关的分子机制完全未知。基于这些新的令人兴奋的初步发现，我们假设，在应激反应中，Calain介导的JP2裂解将JP2从结构蛋白转变为转录调节因子，重新编程受损心肌细胞的转录谱，抑制心肌肥厚的发展。这一分子事件可能是转录重塑的重要触发因素，在病理条件下作为一种未被认识的保护机制。我们将使用多学科方法来验证我们的假设，包括多篇小说转基因和基于CRISPR的小鼠模型、分子生物学、生物化学和我们实验室开发的一种新的原位共聚焦成像技术。在目标1中，我们将探索JP2NT在心肌细胞转录调控的分子机制，而目标2将研究JP2NT在心脏中的病理(生理)后果。我们预计，拟议的研究将为JP2NT的新功能提供重要的见解 在心脏应激背景下的膜到核信号转导，以及对心脏疾病中心肌细胞结构重构和转录重构之间的交叉交流的新见解。这些信息将作为未来开发新的心力衰竭疗法的平台。

 DESCRIPTION (provided by applicant): Junctophilin 2 (JP2) is a structural protein required for the formation of junctional couplings (i.e., cardiac dyads) between transverse (T)-tubule membrane and sarcoplasmic reticulum (SR) and is fundamental for local control of Ca2+-induced Ca2+ release and efficient contraction in ventricular myocytes during cardiac excitation- contraction (E-C) coupling. In animal models of heart failure, JP2 protein levels progressively decline, leading to T-tubule remodeling and loss of E-C coupling function. JP2 levels are also markedly reduced in human failing hearts. Our recently published and preliminary data demonstrate that JP2 is posttranslational regulated by calpain, a Ca2+-activated protease implicated in a variety of heart diseases. JP2 cleavage by calpain was originally hypothesized to underlie JP2 downregulation in failing hearts. In pilot studies, we made the novel and unexpected observation that an N-terminal JP2 cleavage fragment, termed "JP2NT," unlike full length JP2, is imported into nucleus, where it has a profound function in transcriptional reprogramming in cardiomyocytes. However, the (patho) physiological consequences of JP2NT nuclear localization and related molecular mechanisms are completely unknown. Based on these novel exciting preliminary findings, we hypothesize that, in response to stress, calpain-mediated cleavage of JP2 transforms JP2 from a structural protein into a transcriptional regulator that reprograms the transcriptional profile of damaged cardiomyocytes and represses the development of cardiac hypertrophy. This molecular event may be an important trigger for transcriptional remodeling, serving as an unappreciated protective mechanism under pathological conditions. We will test our hypothesis using a multidisciplinary approach, including multiple novels transgenic and CRISPR-based mouse models, molecular biology, biochemistry and a novel in situ confocal imaging technique developed by our lab. In Aim 1, we will explore the molecular mechanisms underlying JP2NT transcriptional regulation in cardiomyocytes, whereas Aim 2 will examine the patho(physiological) consequences of JP2NT in the heart. We anticipate the proposed studies will provide significant insights into the novel functions of JP2NT in membrane-to-nucleus signal transduction in the setting of cardiac stress and new insights into the cross- communication between cardiomyocyte structural remodeling and transcriptional remodeling in heart disease. This information will serve as a platform for the future development of novel heart failure therapeutics.