Isolation and Culture of Adult Mouse Cardiac Myocytes for Signaling Studies

Abstract

The Alliance for Cellular Signaling (AfCS) has chosen cultured adult mouse cardiac myocytes as a system for studying cellular signaling. We developed a reproducible protocol for the isolation and culture of large numbers of viable, rod-shaped myocytes. With this protocol, we routinely isolated 1.5to 1.7 million myocytes per heart, of which 65% to 74% were rod-shaped. After 24 hours in culture, myocytes were 80% rod-shaped, and 88% of the viable, rod-shaped myocytes originally plated were retained. Cultured myocytes demonstrated predictable ligand-induced changes in accumulation of cAMP, phosphorylation of signaling proteins, and excitation-contraction coupling (both calcium transients and contraction). By these criteria, the short-term cultured adult mouse cardiac myocytes are suitable for signaling studies. For future studies requiring the expression of exogenous proteins, protein mutants, or vector-based RNA interference (RNAi), we established a long-term culture system (72 hours) that relies on medium supplemented with 2, 3-butanedione monoxime as well as insulin, transferrin, and selenium. Myocytes cultured for 72 hours in this manner remained viable and were approximately 70% rod-shaped. Finally, using adenovirus-mediated gene transduction, we demonstrated expression of an exogenous β-galactosidase reporter gene for 72 hours in cultured myocytes.