Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, and chronotropic response to isoproterenol.

Abstract

Proliferating nonmyocardial cells (NMC) complicate primary heart cultures and may influence myocardial cell (MC) differentiation. In cultures from the day-old rat ventricle, a method to arrest this proliferation was validated; cross-striated cells and the chronotropic response to isoproterenol were quantitated to assess MC differentiation. MC were cultured at single cell density using an improved method. By 4 days, all cells could be identified as MC or NMC. In cultures treated for 3 days with bromodeoxyuridine (BrdU), 0.1 mM, serial cell counts were unchanged through 11 days. Among 50,000 cells from 110 cultures, 75-80% were MC. In control cultures without BrdU, NMC density was 3- and 6-fold that in BrdU-treated cultures at days 5 and 8, respectively (P < 0.01), although the MC were not overgrown. The MC did not proliferate in either culture. The proportion of living MC with cross-striations was similar in treated and control cultures at day 5 (72.6% vs. 69.9%, P > 0.05), but was lower in controls at day 8 (86.3% vs. 50.4%, P < 0.01). A sensitive (ED50 10-100 pM), specific chronotropic response to L-isoproterenol was present in both treated and control cultures, but the maximum response was only 20-30% as great in controls on days 4 and 8 (P < 0.01). Baseline beating rates were the same. The MC had well-differentiated ultrastructure, including a T system. By autoradiography, a maximum 18.4% of MC had nuclear incorporation of 3H-BrdU at day 8. Media conditioned by NMC or by the control cultures did not change the cross-striations or isoproterenol response of BrdU-treated cultures. In a new culture preparation with few and stable NMC, morphological and functional MC characteristics differed from those of MC in cultures with proliferating NMC. Apparently, an MC-NMC interaction can alter MC properties, and this effect should be considered in studies of primary rat heart cultures. The pure, stable, well-differentiated MC in BrdU-treated cultures will be useful for studying MC growth, repair and other time-dependent phenomena.