Characterization of primary adult mouse cardiac fibroblast cultures

Theroleofcardiacfibroblasts (CFs) in disease states has been a focus of cardiovascular research over the past decade. Here, we briefly describe methods for isolation and characterization of CFs from adult mouse ventricles. Primary cultures were stained using antibodies for several marker proteins such as a-smooth muscle actin (aSMA), vimentin, and discoidin domain receptor 2 (DDR2) to confirm the identity of CFs or cardiac myofibroblasts (CMFs). Most cells in primary cultures consisted of CFs, with very low frequencies of endothelial cells, cardiomyocytes, and smooth muscle cells. We compared marker expression between cultures that were not passaged (P0) or passaged for few times (P1-3). When compared with P1-3 cultures, P0 cultures consistently displayed a lower percentage of cells positive for aSMA and DDR2, whereas vimentin expression was significantly higher in P0 cultures compared with P1-3 cultures. P0 cells were also smaller in area than P1-3 cells. Further, P1-3 mouse CFs were found to express both b1 andb2 adrenergic receptors (ARs) and b1ARs were more readily detected on the cell surface compared with b2ARs. In summary, mouse CF cultures underwent phenotype conversion into CMFs after passaging, consistent with what is seen with CF cultures from other species.