Introduction: Following cardiac injury, neonatal mouse hearts are able to completely regenerate. However, a few days after birth in mouse cardiomyocytes, DNA synthesis occurs without cytokinesis leading to most cardiomyocytes becoming binuclear instead of generating two daughter cells with one nucleus each. This results in cell cycle arrest of cardiomyocytes and the mouse heart is no longer able to regenerate. A long-standing unanswered question in the field is whether multinucleation of cardiomyocytes is a result of cytokinesis failure.

Methods and Results: To address this, we generated several cardiomyocyte-specific transgenic mouse models to determine whether forced induction of cardiomyocyte cytokinesis generates mononuclear cardiomyocytes and restores the endogenous regenerative properties of the myocardium. We focused on two complementary regulators of cytokinesis, namely Polo-like kinase 1 (Plk1) and epithelial cell-transformation sequence 2 (Ect2). Here we report that cardiomyocyte-specific overexpression of constitutively active Plk1(T210D) alone [αMHC-Plk1(T210D) mice] promotes mitosis and cytokinesis in adult hearts, while overexpression of Ect2 alone (αMHC-Ect2 mice) promotes cytokinesis. Intriguingly, cardiomyocyte-specific overexpression of both Plk1(T210D) and Ect2 concomitantly [αMHC-Plk1(T210D); αMHC-Ect2 mice] prevents binucleation of cardiomyocytes postnatally and results in widespread cardiomyocyte mitosis, cardiac enlargement, contractile failure, and death before two weeks of age.

To assess the effect of inducing Plk1(T210D) and Ect2 in the adult heart (a stage when the majority of cardiomyocytes in mice are already binucleated), we generated a transgenic mouse model [TRE-Plk1(T210D)-T2A-Ect2; αMHC-rtTA] of doxycycline inducible cardiomyocyte-specific overexpression of both Plk1(T210D) and Ect2 proteins. High-dose doxycycline inducible cardiomyocyte-specific overexpression of both Plk1(T210D) and Ect2 proteins in the adult heart results in reversible widespread cardiomyocyte mitosis, cardiac enlargement, and contractile failure, while low-dose transient induction also results in significant cardiomyocyte proliferation and lower ejection fraction that is reversible after doxycycline is removed. Finally, we show that transient low-dose induction of both Plk1(T210D) and Ect2 in adult cardiomyocytes improves left ventricular systolic function following myocardial infarction.

Conclusion: Collectively, these results demonstrate that cytokinesis failure mediates cardiomyocyte multinucleation and cell cycle exit of postnatal cardiomyocytes but may be a protective mechanism to preserve the contractile function of the myocardium. After myocardial infarction, transient overexpression of Plk1(T210D) and Ect2 improves heart function.