Investigation of the Dynamics of Perinatal Cell Cycle Regulation in the Mouse Ventricular Myocardium

Objectives: Cardiac growth during embryonic development is primarily mediated by cardiomyocyte proliferation, which in mice drops continuously after birth, such that adult cardiomyocytes are almost unable to divide. If cell cycle arrest is induced by the extrauterine environment, the number of cardiomyocytes per heart might be reduced after preterm compared with term birth which can affect heart function later in life. Thus, deeper insights into regulation of cardiomyocyte proliferation upon birth would be beneficial for humans born preterm.

Methods: To determine proliferation in perinatal hearts, immunofluorescence staining for Ki67 (expressed at all cell cycle phases except for G0) and phosphohistone H3 (detecting mitotic cells) together with Caveolin3 as a cardiomyocyte marker was performed. Hearts were collected from mice at fetal stages E17.5 and E18.5, immediately after birth (NB18.5) and postnatal day 1 (P1). E18.5 and NB18.5 mice share the same gestational age but only NB18.5 mice were shortly exposed to the extrauterine environment.

Result: Immunofluorescence findings showed no difference in percentages of Ki67 positive cardiac cells and mitotic cardiomyocytes between fetal stages. E18.5 compared with NB18.5 ventricles showed a significant drop in cell cycle activity immediately after birth (mitosis rate: 4.7 vs. 1.8%, p < 0.05). Comparing NB18.5 with P1 mice revealed that although overall Ki67 positive cardiac cells did not vary, the rate of mitotic cardiomyocytes was further decreased at P1 (0.7%, p < 0.05 vs. NB18.5). Mitosis rates in nonmyocytes showed no changes between E18.5 and NB18.5 but dropped between NB18.5 and P1. Cardiac proliferation is influenced by cell-cycle regulators and growth promoting signaling pathways. Western blot analyses revealed no changes in Cyclin D1, D2, and D3 expression or activities of Akt, mTOR, and MAP-kinase (Erk1) pathways in ventricular myocardium of the two fetal stages. However, when comparing E18.5 and NB18.5 D-type cyclin expression, as well as phosphorylation of Akt, the mTORC1 target S6 and Erk1 dropped significantly directly after birth.

Conclusion: Our data show that within hours after birth, cardiomyocyte cell cycle activity decreases in the murine heart, paralleled by reduced activity of growth promoting signaling pathways and cell-cycle regulating gene expression. Therefore, our study could have implications for cardiomyocyte endowment in humans born preterm.

Publication History

Article published online:
21 February 2021