The role of Nexilin in cardiomyocyte and cardiomyopathy

项目摘要 Nexilin或NEXN（由NEXN编码）是最近发现的Z盘蛋白。NEXN是一种非常丰富的 心肌NEXN中的多个突变与心肌病相关，突出了其 心脏功能的重要性。NEXN中精氨酸279至半胱氨酸（R279 C）的单个氨基酸取代 被鉴定为引起家族性肥厚型心肌病（HCM），和一个3碱基对（bp）缺失（1948- 1950 del）导致650位甘氨酸缺失（G650 del），发现其与扩张型糖尿病高度相关。 心肌病（DCM）。斑马鱼中NEXN的缺失导致Z盘稳定性紊乱和心力衰竭。 有趣的是，心肌活检显示NEXN突变携带者表现出相同的心脏Z盘， 如在NEXN缺陷和突变斑马鱼中观察到的病理学。此外，小鼠体内NEXN的整体缺失， 据报道，可引起快速进展性心肌病，伴左心室扩张、室壁变薄， 心脏功能下降导致出生后不久死亡这些小鼠的过早死亡 NEXN在成人心脏功能中作用的预防性研究。此外，人们对以下方面的具体作用知之甚少： 心肌细胞中的NEXN，或小鼠中NEXN的整体丢失导致快速进展性心肌梗死的机制 心肌病此外，NEXN R279 C和G650 del突变导致细胞凋亡的机制也被证实。 心肌病进展仍有待解决。为了解决这些问题，我们成功地 产生了一个floxed NEXN小鼠系，并将使用它来产生NEXN心脏特异性敲除（KO）小鼠 无论是在发育阶段还是在成年阶段。此外，我们还生成了新型R274 C（相当于 人R279 C）和G645 del（等同于人G650 del）敲入小鼠模型以表征 人类NEXN R279 C和G650 del突变在心肌病中的作用。我们初步鉴定了 global NEXN KO小鼠也表明NEXN在调节心肌细胞周期中起重要作用 活动上述科学前提使我们假设NEXN在以下方面起着重要作用： 调节心肌细胞的细胞周期活性以及维持Z线的结构完整性， 肌肉收缩应激以及NEXN中R279 C和G650 del突变损害了 NEXN的功能分别导致HCM和DCM。我们的具体目标是：1。描述的角色 通过分析NEXN全局和心肌细胞特异性分析发育和成年心肌中的NEXN 在发育和成年阶段的心脏功能、心肌细胞细胞周期 活性、肌节完整性和心肌病的进展;以及2.阐明机制 通过分析NEXN的R274 C和G650 del突变导致的潜在心肌病，或 G645 del突变小鼠和含有NEXN R279 C或 G650 del突变。

PROJECT SUMMARY Nexilin or NEXN (encoded by NEXN) is a recently discovered Z-disc protein. NEXN is highly abundant in cardiac muscle. Multiple mutations in NEXN have been associated with cardiomyopathies, highlighting its importance for cardiac function. A single amino acid substitution of arginine 279 to cysteine (R279C) in NEXN was identified as causing familial hypertrophic cardiomyopathy (HCM), and a 3-base pair (bp) deletion (1948– 1950del) leading to loss of glycine at position 650 (G650del) was found to be highly associated with dilated cardiomyopathy (DCM). Loss of NEXN in zebrafish results in perturbed Z-disk stability and heart failure. Interestingly, myocardial biopsies showed that NEXN mutation carriers exhibit the same cardiac Z-disk pathology as observed in NEXN deficient and mutant zebrafish. Furthermore, global loss of NEXN in mice has been reported to cause rapidly progressive cardiomyopathy with left ventricular dilation, wall thinning, and decreased cardiac function, resulting in lethality shortly after birth. Premature lethality of these mice has prevented study of the role of NEXN in adult heart function. In addition, little is known as to the specific role of NEXN in cardiomyocytes, or mechanisms by which global loss of NEXN in mice results in rapidly progressive cardiomyopathy. Furthermore, mechanisms by which the NEXN R279C and G650del mutations lead to the progression of cardiomyopathy remain to be addressed. To address these questions, we have successfully generated a floxed NEXN mouse line and will use it to generate NEXN cardiac-specific knockout (KO) mice both during developmental and at adult stages. In addition, we have generated novel R274C (equivalent to human R279C) and G645del (equivalent to human G650del) knock-in mouse models to characterize the role of the human NEXN R279C and G650del mutations in cardiomyopathy. Our preliminary characterization of global NEXN KO mice also suggests that NEXN plays an important role in regulating cardiomyocyte cell cycle activity. The foregoing scientific premise leads us to the hypothesis that NEXN plays an essential role in regulating cardiomyocyte cell cycle activity as well as maintaining the structural integrity of the Z-line during the stress of muscle contraction, and that R279C and G650del mutations in NEXN impair specific aspects of NEXN function to lead to HCM and DCM, respectively. Our Specific Aims are: 1. To characterize the role of NEXN in the developing and adult myocardium by analyzing NEXN global and cardiomyocyte-specific knockout mice both during development and at adult stages for cardiac function, cardiomyocyte cell cycle activity, sarcomere integrity, and the progression of cardiomyopathy; and 2. To elucidate mechanisms underlying cardiomyopathy consequent to R279C and G650del mutations of NEXN by analysis of R274C or G645del mutant mice and human embryonic stem cell-derived cardiomyocytes containing NEXN R279C or G650del mutations.