Role of ROCK isoform-mediated actin cytoskeleton modification in the pathogenesis of heart disease

项目摘要 心脏病的预防和治疗仍然具有挑战性。Rho激酶（也称为ROCK） 最近已成为各种心血管疾病的潜在治疗靶点。的长期目标 我们过去20年对ROCK病理生理学的研究是为了确定ROCK的作用和潜在机制， ROCK介导的信号通路在调节心脏重塑中的作用。摇滚家族的两个成员， ROCK 1和ROCK 2具有共同的和不同的细胞功能，并且可以在细胞内相互补偿。 许多单一同种型敲除条件。我们对细胞和分子的大部分知识 ROCK的功能来自于对ROCK调节肌动蛋白的增殖细胞类型的研究 通过促进肌动蛋白收缩和F-肌动蛋白稳定化来组织细胞骨架。心肌细胞 因为它们含有肌节和非肌节细胞骨架。 我们对ROCK如何调节肌节和非肌节F-肌动蛋白的知识存在空白， 心肌细胞以及这些过程如何促进整体心脏功能。近日，首次使用 通过诱导性方法删除心肌细胞中的两种ROCK亚型，我们发现，尽管 ROCK并不是维持成人心脏中肌节细胞骨架所必需的，它们确实参与了心肌细胞骨架的形成。 调节非肌节F-肌动蛋白组织，通过促进哺乳动物靶向 雷帕霉素（mTOR）活性，并有助于年龄相关的心脏纤维化。相反，非肌节F- 肌动蛋白动力学能够在心肌细胞中任一种亚型的存在下维持， 其他亚型已被删除;这可能是由于补偿过度激活的其余 在具有单个ROCK同种型缺失的心肌细胞中的ROCK同种型。这项研究旨在进一步 阐明ROCK介导的肌动蛋白细胞骨架的病理生理作用和下游通路 心肌细胞和成纤维细胞在病理应激情况下的变化。目标1将确定是否删除 ROCK 1和ROCK 2在心肌细胞中的表达限制了心力衰竭的病理进展， 心肌细胞肥大和心肌缺血损伤的机制可能是通过激活自噬和促进自噬流量来实现的。 抑制mTOR信号传导。目的2将确定是否从成人成纤维细胞中缺失ROCK 1和ROCK 2 通过抑制F-肌动蛋白调节的转录限制肌成纤维细胞的活化和纤维化反应 包括血清反应因子（SRF）和心肌素相关转录因子在内的因子激活 成纤维细胞中ROCKs/F-actin/MRTFs/SRF轴对心脏纤维化的直接作用从未被证实。 已经在体内得到证实，我们的初步结果表明，诱导的方法是必要的， 成纤维细胞中的双ROCK敲除。这项工作的生物医学意义在于提供了 理解ROCK在心力衰竭中的病理生理学作用的概念。最终的目标是开发 新的治疗干预，以改善受损的心脏功能。

PROJECT ABSTRACT The prevention and treatment of heart disease remain challenging. Rho kinase (also named ROCK) has recently emerged as a potential therapeutic target for various cardiovascular diseases. A long-term goal of our past twenty years of research on ROCK pathophysiology is to define the roles and underlying mechanisms of ROCK-mediated signal pathway in regulating cardiac remodeling. The two members of the ROCK family, ROCK1 and ROCK2, have both shared and distinct cellular functions and can compensate each other in numerous single isoform knockout conditions. The majority of our knowledge on the cellular and molecular function of ROCKs comes from research on proliferative cell types in which ROCKs modulate actin cytoskeleton organization through promoting actomyocin contraction and F-actin stabilization. Cardiomyocytes stand apart from other cell types because they contain both sarcomeric and non-sarcomeric cytoskeleton. There is a gap in our knowledge on how ROCKs regulate sarcomeric and non-sarcomeric F-actin in cardiomyocytes and how these processes contribute to overall heart function. Recently, for the first time to use inducible approach to delete both ROCK isoforms in cardiomyocytes, we have discovered that although ROCKs are not required for maintaining sarcomeric cytoskeleton in adult hearts, they do participate in the regulation of non-sarcomeric F-actin organization, inhibit autophagy by promoting mammalian target of rapamycin (mTOR) activity and contribute to age-related cardiac fibrosis. In contrast, the non-sarcomeric F- actin dynamics are able to be maintained with the presence of either isoform in the cardiomyocytes where the other isoform has been deleted; this might be attributed to compensatory over-activation of the remaining isoform in cardiomyocytes having single ROCK isoform deletion. The proposed research aims to further elucidate the pathophysiological roles and downstream pathways of ROCK-mediated actin cytoskeleton changes in cardiomyocytes and fibroblasts under pathological stress sceneries. Aim 1 will determine if deletion of both ROCK1 and ROCK2 from adult cardiomyocytes limits the progression of heart failure in pathological hypertrophy and myocardial ischemic injury through activating autophagy and facilitating autophagic flux by inhibiting mTOR signaling. Aim 2 will determine if deletion of both ROCK1 and ROCK2 from adult fibroblasts limits the activation of myofibroblasts and fibrotic response through inhibition of F-actin regulated transcription factor activation including the serum response factor (SRF) and myocardin-related transcription factors (MRTFs); the direct contribution of ROCKs/F-actin/MRTFs/SRF axis in fibroblasts to cardiac fibrosis has never been demonstrated in vivo, and our preliminary results indicate that the inducible approach is required for double ROCK knockout in fibroblasts. The biomedical significance of this work is to provide the cutting-edge concepts for understanding pathophysiological roles of ROCKs in heart failure. The ultimate goal is to develop new therapeutic intervention to ameliorate compromised cardiac function.