Myocardial cells in culture to study effects of cytokines

This chapter describes the use of cultured adult primary ventricular cardiomyocytes as a model for cytokine-mediated septic shock. We describe the methods for preparation of adult cardiomyocytes from adult rat hearts for culture. We also describe techniques designed to assess the responses of single cells in culture to the proinflammatory cytokine, TNFα. We describe in detail the use of this cell-culture model to evaluate TNFα-induced voltage-dependent calcium fluxes as well as the expression of genes that mediate the TNFα response. Because cardiomyocyte cultures may be heterogeneous and may include a variety of other cell types (fibroblasts, endothelial cells, etc.), it is sometimes prudent to examine cells on a single-cell basis to ensure that the physiological or molecular process of interest is characteristic of the cardiomyocyte. The issue of heterogeneity is particularly problematic when attempting to examine the expression of low-abundant transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR). In our laboratory, we have employed single-cell RT-PCR as a tool for examining the expression of genes coding for TNFα receptors in isolated myocardial cells. The procedures can be applied to acutely isolated cells as well as those cultured and subjected to chronic treatments. By using these techniques, it is possible to implicate molecular mechanisms/pathways in the physiological responsiveness of the adult ventricular cardiomyocyte to cytokines.