The role of Nexilin in cardiomyocyte and cardiomyopathy

项目总结 Nexlin或NEXN(由NEXN编码)是最近发现的一种Z-Disc蛋白。NEXN的含量非常丰富 心肌。NEXN的多个突变与心肌病有关，突显了其 对心脏功能的重要性。NEXN中精氨酸279与半胱氨酸(R279C)的单一氨基酸取代 被确认为引起家族性肥厚型心肌病(HCM)，以及一个3碱基对(BP)缺失(1948- 1950del)导致650位甘氨酸丢失(G650del)被发现与扩张高度相关 心肌病(DCM)。斑马鱼中NEXN的丢失会导致Z-Disk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 在NEXN缺失和突变斑马鱼中观察到的病理学。此外，NEXN在小鼠体内的全球丢失 据报道可引起快速进展性心肌病，表现为左心室扩张、室壁变薄和 心脏功能下降，导致出生后不久死亡。这些小鼠的过早死亡已经 预防性研究NEXN在成人心脏功能中的作用。此外，人们对其具体作用知之甚少。 NEXN在心肌细胞中的表达，或NEXN在小鼠体内的整体缺失导致快速进展的机制 心肌病。此外，NEXN R279C和G650del突变导致 心肌病的进展仍有待解决。为了解决这些问题，我们成功地 产生了一种NEXN小鼠系，并将用它来产生NEXN心脏特异性基因敲除(KO)小鼠 无论是在发育阶段还是在成年阶段。此外，我们还生成了新颖的R274C(相当于 人R279C)和G645del(相当于人G650del)敲入小鼠模型中的作用 人类NEXN R279C和G650del突变在心肌病中的作用。我们的初步特征是 全球NEXN KO小鼠也提示NEXN在调节心肌细胞周期中发挥重要作用 活动。上述科学前提使我们得出这样的假设，即NEXN在 调节心肌细胞周期活动，并维持Z线的结构完整性 肌肉收缩应激，以及NEXN的R279C和G650del突变损害了 NEXN功能分别导致肥厚型心肌病和扩张型心肌病。我们的具体目标是：1.确定 NEXN在发育中和成年心肌中的表达 发育阶段和成体阶段的基因敲除小鼠的心功能、心肌细胞周期 活性、肌节完整性和心肌病的进展；以及2.阐明机制 NEXN基因R279C和G650del突变导致的潜在心肌病 G645del突变小鼠和人胚胎干细胞来源的心肌细胞含有NEXN R279C或 G650del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.