Cyclin Dependent Kinase 6 in Cardiac Development

这项建议的长期目标是促进我们对胚胎发生过程中涉及心脏细胞周期退出的调控级联的理解。这种戒断可能限制了心脏对病理性和老年性心肌细胞损失的反应能力。该建议的工作假设是，周期蛋白依赖性激酶cdk6在心肌细胞发育过程中通过心肌细胞特异性亚细胞区隔化，是连接心肌细胞周期退出和终末分化的关键节点。通过细胞分离、间接多标记免疫荧光和FACs分析，我们描述并量化了G1、S和G2/M调节蛋白在心脏发生过程中的时间和空间核表达模式。与已知存在的所有其他细胞周期调节蛋白相反，cdk6和细胞周期蛋白A水平的核减少与心肌细胞周期退出同步发生。进一步研究表明：1)核/细胞质cdk6的早期表达发生在心肌持续增殖期间；2)渐进式核下调有利于增加cdk6细胞质区隔化；3)体外tgf - β可影响细胞内cdk6的表达；4) cdk6与发育中的细胞质肌原纤维在体内共定位；5)成熟肌节特异性结合cdk6与肌球蛋白重链。考虑到cdk4敲除小鼠和其他细胞周期蛋白敲除小鼠中存在明显的器官特异性缺陷，我们的初步数据与cdk6是心肌细胞增殖和分化的组织特异性调节机制的假设一致。本提案将研究cdk6在哺乳动物发育过程中的作用，特别强调其在肌细胞周期退出中的作用。我们最近创造的新试剂，包括cdk6- GFP融合质粒、稳定过表达cdk6的CDC12细胞和转基因cdk6小鼠建立者，应该对验证我们的假设特别有用。成功完成我们的目标应该进一步我们对细胞周期调节的一般机制的理解，以及通过细胞周期调节重新激活心肌细胞增殖的潜力。

The long-term goal of this proposal is to advance our understanding of the regulatory cascade involved in cardiac cell cycle withdrawal during embryogenesis. This withdrawal presumably limits the heart's ability to respond to pathologic and senile myocyte loss. The working hypothesis of this proposal is that the cyclin-dependent kinase cdk6, via myocyte- specific subcellular compartmentalization during cardiomyocyte development, is a critical nodal point linking cardiomyocyte cell cycle withdrawal to terminal differentiation. Using cell fractionation, indirect multi-labeling immunofluorescence and FACs analysis, we have described and quantified the temporal and spatial nuclear expression patterns of G1, S, and G2/M regulatory proteins during cardiogenesis. In contrast to all other cell cycle regulatory proteins known to exist, the nuclear reduction in cdk6 and cyclin A protein levels occurs synchronously with cardiomyocyte cell cycle withdrawal. Further study has revealed: 1) Early nuclear/cytoplasmic cdk6 expression occurs during periods of persistent myocardial proliferation; 2) Progressive nuclear down-regulation in favor of increased cdk6 cytoplasmic compartmentalization; 3) Cytosolic egress of cdk6 can be effected in vitro by TGFbeta; 4) In vivo co- localization of cdk6 with developing cytoplasmic myofibrils ; and 5) Specific binding of cdk6 to myosin heavy chain with the maturing sarcomere. Given the striking and distinct organ organ-specific deficits in seen in cdk4 knockout mice and knockouts of other cyclins, our preliminary data are consistent with the hypothesis that cdk6 is a tissue- specific regulatory of cardiomyocyte proliferation and differentiation. This proposal will examine the role of cdk6 during mammalian development with special emphasis on its role in myocyte cell cycle withdrawal. Novel reagents we have recently created, including cdk6- GFP fusion plasmids, CDC12 cells stably over-expressing cdk6 and transgenic cdk6 mouse founders, should be especially useful in testing our hypothesis. Successful completion of our aims should further our understanding of the general mechanisms involved in cell cycle regulation and the potential to reactivate cardiomyocyte proliferation via cell cycle modulation.