The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclindependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono-and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono-to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21(Cip1) and p27(Kip1) but not p57(Kip2) showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21(Cip1) and p27(Kip1) bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21(Cip1) and p27(Kip1) knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21(Cip1) and p27(Kip) play important roles in the cell cycle exit of postnatal cardiomyocytes.

Background: How cell cycle exit is maintained in adult mammalian cardiomyocytes is largely unknown. Results: Cyclin D1 expression causes cell cycle reentry in Ͼ40% of adult mouse cardiomyocytes. Conclusion: Silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit. Significance: One of the mechanisms regulating cell cycle exit in mammalian cardiomyocytes has been uncovered.

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