Neonatal mouse cardiac myocytes exhibit cardioprotection induced by hypoxic and pharmacologic preconditioning and by transgenic overexpression of human Cu/Zn superoxide dismutase.

Although mouse models have been increasingly used for studies of cardiac pathophysiology, there is little information regarding cultured murine cardiac myocytes. Accordingly, we have developed a cell culture model of neonatal mouse cardiac myocytes by modifying a protocol used to prepare neonatal rat myocytes. The principal change is the substitution of cytosine arabinoside for bromodeoxyuridine to prevent fibroblast proliferation. Neonatal murine myocytes exhibited persistent spontaneous contraction and were viable for up to 14 days in culture. By flow cytometry 85% of the cells were cardiac myocytes. In sparse cultures (average cell density 259 cells/mm(2)), both hypoxic preconditioning (n=5) and phenylephrine pretreatment (n=8) produced significant protection of cardiac myocytes from cell death during a prolonged period of severe hypoxia (<0.5% O(2)for 18-20 h, both P<0.05). The phenylephrine effect was inhibited by the alpha(1)-adrenoceptor antagonist prazosin (n=4, P<0.05) and by an xi PKC peptide antagonist (xi V1-2) coupled to a TAT peptide (n=5, P<0. 05). Interestingly, the mixed alpha(1)- and beta -adrenoceptor agonist norepinephrine, which stimulates hypertrophy as measured by(14)[C]phenylalanine incorporation in neonatal rat cardiac myocytes, did not cause hypertrophy in mouse myocytes, suggesting that the signaling pathways for myocardial protection and hypertrophy are likely to be both divergent and species specific. In cardiac myocytes prepared from transgenic mice either homozygous or heterozygous for human Cu/Zn superoxide dismutase, there was protection from cell death (n=3) and restoration of(14)[C]phenyl- alanine uptake (n=4) during prolonged hypoxia (1% O(2)for 3 days, both P<0.05). We conclude that this cellular model, which is relatively simple to prepare, can be used for in-vitro examination of cardiac protection induced by preconditioning agents, various transgenes, and potentially by targeted gene deletions.