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

Proliferating nonmyocardial cells (NMCs) complicate primary heart cultures and may influence myocardial cell (MC) differentiation.In cultures from the day-old rat ventricle, we validated a method to arrest this proliferation; and we quantitated cross-striated cells and the chronotropic response to isoproterenol to assess MC differentiation.MCs were cultured at single cell density using an improved method.By 4 days, all cells could be identified as MCs or NMCs.In cultures treated for 3 days with bromodeoxyuridine (BrdU), 0.1 miw, serial cell counts were unchanged through 11 days.Among 50,000 cells from 110 cultures, 75-80% were MCs.In control cultures without BrdU, NMC density was 3-and 6-fold that in BrdU-treated cultures at days 5 and 8, respectively (P 0.05) but was lower in controls at day 8 (86.3% vs. 50.4%,P < 0.01).A sensitive (ED 50 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 MCs had well-differentiated ultrastructure, including a T system.By autoradiography, a maximum 18.4% of MCs had nuclear incorporation of 3 H-BrdU at day 8. Media conditioned by NMCs or by the control cultures did not change the cross-striations or isoproterenol response of BrdU-treated cultures.Thus, in a new culture preparation with few and stable NMCs, morphological and functional MC characteristics were different from those of MCs in cultures with proliferating NMCs.We suggest that an MC-NMC interaction can alter MC properties and that this effect should be considered in studies of primary rat heart cultures.The pure, stable, well-differentiated MCs in BrdUtreated cultures will be useful for studying MC growth, repair, and other time-dependent phenomena.CircRes 50: 101102103104105106107108109110111112113114115116 1982 PRIMARY cultures of cells from the hearts of neonatal rats and embryonic chicks are used by many investigators.(See reviews by Lieberman and Sano, 1976;Sperelakis, 1978; Auclair and Freyss-Beguin, 1980).A major, unresolved problem with these cultures is contamination by a population of rapidly proliferating nonmyocardial cells (NMCs), the precise identity of which is unclear (Balk, 1980).The initial proportion of these contaminating cells can be reduced by the differential attachment technique (Hyde et al., 1969); but they eventually overgrow the myocardial cells (MCs), which are said to divide much more slowly (Kasten, 1972).This progressive increase in cell number is not charac-