Longevity of adult ventricular rat heart muscle cells in serum-free primary culture

Abstract

The study had two aims: first, to improve the longevity of isolated adult cardiomyocytes in serum-free culture, and, second, to investigate whether catecholamines which promote hypertrophy in vivo can prolong survival of isolated adult rat cardiomyocytes in serum-free culture. The basic cell culture medium consists of serum-free medium 199 with 10 −7 m insulin. In this medium 50% of the initially plated cardiomyocytes survive in elongated form for 2 days. Omission of glutamine and supplementation of the basic medium with 5 m m creatine, 2 m m carnitine and 5 m m taurine extends survival of elongated cells to 14 days. In supplemented medium, normal cell ATP content is maintained (27 nmol/mg protein after 15 days), but cells gradually atrophy and reduce their protein mass. The trophic effects of catecholamines (epinephrine, norepinephrine, phenylephrine; 10 μ m, added on day 3 of culture) were investigated. After addition of catecholamines the cells spread. Spreading can be prevented by prazosin (10 μ m) and phentolamin (10 μ m) but not by propranolol (10 μ m), indicating that spreading is stimulated via the α 1-adrenoreceptor. Cells also spread in the presence of the phorbol ester phorbolmyristate acetate (10 μ m). Catecholamines reduce the progressive cell atrophy and protein loss. With 10μ m phenyleprine, cellular ATP content remained constant at 27 nmol/mg protein until day 15. The results indicate that agents which stimulate protein kinase C ( α 1-agonists, phorbolesters) stimulate cell spreading, protein synthesis and long-term survival of cardiomyocytes in vitro.