Cyclin Dependent Kinase 6 in Cardiac Development

这一建议的长期目标是促进我们对胚胎发育过程中涉及心脏细胞周期退出的调节级联反应的理解。这种退缩可能限制了心脏对病理性和老年性心肌细胞丢失的反应能力。该提议的工作假设是，在心肌细胞发育过程中，细胞周期蛋白依赖的蛋白依赖性激酶CDK6通过肌细胞特异性的亚细胞区划，是连接心肌细胞周期退出和终末分化的关键节点。利用细胞分离、间接多标记免疫荧光和流式细胞仪分析，我们描述和定量了G1、S和G2/M调控蛋白在心脏发生过程中的时空核表达模式。与已知的所有其他细胞周期调节蛋白不同，CDK6和Cyclin A蛋白水平的核减少与心肌细胞周期退出同步发生。进一步的研究发现：1)早期的核/胞浆CDK6表达发生在持续性心肌增殖期；2)进行性的核下调有利于增加CDK6的胞浆分区；3)CDK6的胞浆外流可被TGFbeta影响；4)CDK6与发育中的肌原纤维在体内共定位；以及5)CDK6与肌球蛋白重链与成熟肌小室的特异性结合。考虑到CDK4基因敲除小鼠明显的器官特异性缺陷和其他周期蛋白的敲除，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的假设是一致的。这项建议将研究CDK6在哺乳动物发育过程中的作用，特别强调它在心肌细胞周期退出中的作用。我们最近开发的新试剂，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，应该对验证我们的假设特别有用。我们的目标的成功完成将进一步加深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可能性。

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.