Cell cycle activation for cardiac repair

描述(申请人提供)：尽管成年哺乳动物心肌再生能力有限，但固有的更新率不足以逆转心肌细胞的病理生理性丢失。因此，在受损心脏中重建丢失的心脏质量的能力可能具有相当大的治疗价值。实现这一目标的一种方法是在存活的心肌细胞中诱导细胞周期活动。初步数据表明，靶向表达细胞周期蛋白D2是调控复合体的关键成员，它驱动细胞周期通过G1/S细胞周期检查点，足以诱导成人心脏的心肌细胞周期活动。此外，细胞周期蛋白D2诱导的细胞周期活性可以逆转心肌损伤后的结构损伤，恢复功能。本申请中提出的实验将进一步阐明细胞周期蛋白D介导的心肌细胞周期调节的机制。具体目标1将检验这一假设，即D-型细胞周期蛋白的翻译后修饰调节其在心肌损伤后调节再生生长的能力。这些实验将利用新产生的表达D-型细胞周期蛋白的转基因小鼠，携带相关的模拟磷酸和非磷酸化氨基酸残基替代。Aim 1中的其他研究将检验这一假设，即特定的磷酸化事件介导D型细胞周期蛋白和p193/Cul7 E3泛素连接酶的相互作用，并且阻断这种相互作用可以增强受损心脏的细胞周期活性。在特定目的2中提出的实验将进一步检验细胞周期蛋白D2介导的心肌细胞周期激活可用于促进成人心脏心肌修复的假设。最初的实验将利用条件转基因小鼠模型来确定细胞周期蛋白D2是否能在成人心脏中诱导新生心肌细胞增殖。其他研究建议确定限制损伤后不良重塑的药物干预在多大程度上能够有利于长期的心肌细胞周期诱导的再生。总而言之，这些研究将有助于建立D型细胞周期蛋白调节心肌细胞周期进入的机制，以及细胞周期蛋白D2的靶向表达能够促进心肌再生的程度。总体目标是了解细胞周期调节通路如何被操纵以促进受伤心脏的修复。对这些分子靶点的识别最终可能导致开发促进疾病心脏再生生长的药理学药物。

DESCRIPTION (provided by applicant): Although the adult mammalian myocardium exhibits a limited ability to undergo regenerative growth, the intrinsic renewal rate is insufficient to reverse pathophysiologic cardiomyocyte loss. The ability to reconstitute lost cardiac mass in injured hearts could thus be of considerable therapeutic value. One approach to accomplish this entails inducing cell cycle activity in the surviving cardiomyocytes. Preliminary data indicate that targeted expression of cyclin D2, a key member of the regulatory complex which drives transit through the G1/S cell cycle check-point, is sufficient to induce cardiomyocyte cell cycle activity in adult hearts. Moreover, cyclin D2-induced cell cycle activity can reverse structural damage and restore function following myocardial injury. The experiments proposed in this application will further elucidate the mechanism of cyclin D-mediated cardiomyocyte cell cycle regulation. Specific Aim 1 will test the hypothesis that post-translational modification of the D-type cyclins regulates their ability to mediate regenerative growth following myocardial injury. These experiments will utilize newly generated transgenic mice expressing D-type cyclins carrying the relevant phospho-mimetic and non-phosphorylatable amino acid residue substitutions. Other studies in Aim 1 will test the hypothesis that specific phosphorylation events mediate the interaction of D-type cyclins and the p193/Cul7 E3 ubiquitin ligase, and that blocking this interaction enhances cell cycle activity in injured hearts. Experiments proposed in Specific Aim 2 will further test the hypothesis that cyclin D2-mediated cardiomyocyte cell cycle activation can be used to promote myocardial repair in the adult heart. Initial experiments will utilize a conditional transgenic mouse model to determine if cyclin D2 can induce de novo cardiomyocyte proliferation in adult hearts. Other studies are proposed to determine the degree to which pharmacologic interventions which limit adverse post-injury remodeling are able to benefit long-term, cardiomyocyte cell cycle-induced regeneration. Collectively, these studies will help establish the mechanism by which D-type cyclins regulate cardiomyocyte cell cycle entry, and the degree to which targeted expression of cyclin D2 is able to promote myocardial regeneration. The overall goal is to gain an understanding of how cell cycle regulatory pathways can be manipulation to promote the repair of injured hearts. Identification of such molecular targets may ultimately lead to the development of pharmacologic agents to promote regenerative growth in diseased hearts.