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(又称ROCK) 最近已成为各种心血管疾病的潜在治疗靶点。一个长期目标是 我们过去二十年来对岩石病理生理学的研究是为了确定其作用和潜在的机制 ROCK介导的信号通路在调节心脏重构中的作用。洛克家族的两名成员， ROCK1和ROCK2具有共同和不同的细胞功能，并可以在 许多单一的亚型敲除条件。我们关于细胞和分子的大部分知识 岩石的功能来自对增殖细胞类型的研究，在这种细胞类型中，岩石调节肌动蛋白 通过促进肌动蛋白收缩和F-肌动蛋白稳定来组织细胞骨架。心肌细胞 与其他类型的细胞区分开来，因为它们同时含有肌瘤和非肌瘤细胞骨架。 我们对岩石如何调节肌瘤和非肌瘤F-肌动蛋白的认识存在差距 心肌细胞以及这些过程如何对整体心脏功能做出贡献。最近，首次使用了 在心肌细胞中删除这两种ROCK异构体的诱导方法，我们发现尽管 岩石不是维持成人心脏肌节细胞骨架所必需的，它们确实参与了 调节非肌节F-肌动蛋白组织，通过促进哺乳动物靶向抑制自噬 雷帕霉素(MTOR)的活性，并促进年龄相关的心脏纤维化。相比之下，非肌瘤的F- 肌动蛋白的动力学能够在心肌细胞中存在任何一种异构体的情况下保持，其中 其他异构体已被删除；这可能归因于剩余的代偿性过度激活 心肌细胞中具有单一ROCK异构体缺失的异构体。拟议的研究旨在进一步 阐明岩石介导的肌动蛋白细胞骨架的病理生理作用及其下游途径 病理性应激状态下心肌细胞和成纤维细胞的变化。目标1将决定是否删除 成人心肌细胞中ROCK1和ROCK2的表达在病理上限制了心力衰竭的进展 激活自噬和促进自噬通量的肥大和心肌缺血性损伤 抑制mTOR信号转导。AIM 2将确定ROCK1和ROCK2在成人成纤维细胞中是否同时缺失 通过抑制F-肌动蛋白调节的转录来限制肌成纤维细胞的激活和纤维化反应 因子激活，包括血清反应因子(SRF)和肌钙蛋白相关转录因子 成纤维细胞中ROCKS/F-肌动蛋白/MRTFS/SRF轴在心脏纤维化中的直接作用 已经在体内得到证实，我们的初步结果表明，诱导方法对于 成纤维细胞中的双基因敲除。这项工作的生物医学意义在于为 了解岩石在心力衰竭中的病理生理作用的概念。最终目的是要发展 改善心功能受损的新治疗干预措施。

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.