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.
