Alterations in membrane potential promote neonatal cardiomyocyte proliferation

Zebrafish and newt heart regeneration has been demonstrated to be the result of cardiomyocyte proliferation, which has suggested the feasibility of maintaining the proliferation capacity of native mammalian neonatal cardiomyocytes. Recent experiments demonstrating that stem cells are highly depolarized while terminally differentiated cells are hyperpolarized have indicated a potential role of membrane potential in maintaining a proliferative phenotype. In this study, we used titrated potassium gluconate and ouabain to depolarize primary neonatal rat cardiomyocytes and demonstrated the concomitant proliferative effect between postpartum day 3 (P3) and post partum day 7 (P7). After isolation, the cells were cultured for one day in normal media and before replacing the media with titrated depolarizing agents for four days. The effect of depolarization was validated by DiBAC, a slow-acting voltage-sensitive fluorescence dye. Total nuclei count and cardiomyocyte percentage were evaluated by microscopic cytometry. Cell cycle was analyzed by propidium iodide on flow cytometry. Both treatment groups had a dose dependent effect on cardiomyocyte percentage and induced proliferation at specific range of concentrations before eventually reaching toxic levels. A study of cell cycle showed increased populations of non-G0/G1 phase cardiomyocytes with treatment. Conclusion: We concluded that the control of membrane potential could modulate the proliferation of neonatal rat cardiomyocytes.