Abstract 262: Depolarization Stimulates Neonatal Cardiomyocyte Proliferation In Vitro

Abstract

Cardiac tissue engineering is a promising approach for treating children with congenital heart defects. However, as cardiomyocytes (CMs) undergo a rapid transition from hyperplastic to hypertrophic growth after birth, a major challenge to the development of engineered cardiac tissue is the limited proliferation of CMs. Mature CMs and other terminally differentiated cell types tend to have a highly negative resting membrane potential (Vmem) while stem cells and less mature cells tend to have Vmem closer to zero. Vmem has been shown to play an important role in cell differentiation and proliferation. We hypothesized that depolarization of cardiac cells would stimulate CM proliferation in vitro . To test our hypothesis, we isolated neonatal rat cardiac cells and cultured them for 24 hr under standard conditions. Cells were then subjected to depolarization treatment for 72 hr using potassium gluconate or ouabain at various concentrations. Samples were fixed and stained for cardiac α-actin (Fig 1A, red) and phospho-histone H3 (Fig 1A, green) to assess CM mitosis. We found that potassium gluconate had no significant effect while ouabain significantly increased CM mitosis, suggesting Vmem regulation via Na/K-ATPase. CM-specific proliferation was significantly higher with 10nM (p= 0.015) and 100nM (p=0.008) ouabain treatment compared to controls (n=3) (Fig 1B). Cell density was significantly higher with 100μM ouabain versus controls (2656 ± 50 vs. 2026 ± 117 cells/mm 2 ), indicating increased cardiac cell proliferation (Fig 1C). Our findings suggest that depolarization promotes CM proliferation and may be a novel approach to encourage growth of engineered cardiac tissue in vitro .