Introduction

The Alliance for Cellular Signaling (AfCS) is a consortium of laboratories and scientists dedicated to understanding the complexities of signal transduction. To study signaling, the AfCS has chosen two model cell systems, B lymphocytes and cardiac myocytes from the mouse. The basis for the selection of these two cell types is described elsewhere(1,2), as is the description of the cell culture system for mouse B lymphocytes(3). This manuscript describes the cell culture system for adult mouse cardiac myocytes.

Cardiac myocytes respond to a variety of hormonal, neural, mechanical, and electrical stimuli by altering both their force and rate of contraction. Stimulation of calcium influx through sarcolemmal calcium channels, and subsequent activation of calcium release from the sarcoplasmic reticulum, initiate contraction (excitation-contraction coupling) (4). Activation of G protein-coupled receptors, such as b-adrenergic receptors (coupled to Gs) or muscarinic receptors (coupled to Gi), regulate contractile force by modulating production of cAMP and activation of the cAMP-dependent protein kinase (PKA)(5,6,7). In addition, PKA-mediated phosphorylation of troponin I regulates contractile force, and phosphorylation of phospholamban, a regulator of the sarcoplasmic reticulum calcium pump, controls reuptake of calcium and relaxation(4,8,9).

Cardiac myocytes also undergo hypertrophy as a compensatory response to either physiological stimuli, such as exercise, or pathological stimuli, such as hypertension(10). The signaling pathways regulating hypertrophy encompass a wide array of signaling molecules and pathways, including G protein-coupled receptors, cytokine receptors, protein kinase C, phosphotidylinositol 3-kinase (PI3-kinase), MAP kinases, and calcineurin(11,12,13).

Although adult mouse cardiac myocytes are an intriguing model for the AfCS, the isolation of large numbers of these cells and their subsequent culture under physiological conditions is not routine; however, recent reports in the literature suggest that it is feasible(14,15,16). The challenge for the AfCS was to develop a protocol for the rapid and consistent isolation of large numbers of myocytes (4 to 5 million) at one time. In addition, isolated myocytes had to survive short-term culture (24 hours) without a significant loss of viable, rod-shaped myocytes. Myocytes in short-term culture had to be suitable for both biochemical studies, such as measurement of protein phosphorylation, and studies of excitation-contraction coupling, such as calcium transients and contraction. A long-term culture system capable of high efficiency gene transduction was also needed in order to manipulate expression of individual signaling proteins, such as by vector-based RNA interference (RNAi)(17) or by dominant-negative signaling proteins. Finally, the protocols for the isolation and culture of myocytes had to be reproducible among different laboratories.

This report describes protocols for the isolation and culture of adult mouse cardiac myocytes and for their short- and long-term culture. To determine if the cultured myocytes were suitable for signaling studies, ligand-induced changes in the accumulation of cAMP, in the phosphorylation of signaling proteins, and in excitation-contraction coupling were measured. Finally, myocytes were infected with an adenovirus-b-galactosidase reporter gene to demonstrate successful gene transduction and expression for 72 hours. These experiments provide the basis for future signaling studies in cardiac myocytes and, ultimately, the examination of how different signaling modules interact.