Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro

Abstract

Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro. Am J Physiol Cell Physiol 303: C1220 -C1228, 2012. First published August 29, 2012; doi:10.1152/ajpcell.00449.2011.-Long-term culture of primary neonatal rat cardiomyocytes is limited by the loss of spontaneous contractile phenotype within weeks in culture. This may be due to loss of contractile cardiomyocytes from the culture or overgrowth of the non-cardiomyocyte population. Using the mitochondria specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), we showed that neonatal rat cardiomyocytes enriched by fluorescence-activated cell sorting can be maintained as contractile cultures for long periods (24-wk culture vs. 2 wk for unsorted cardiomyocytes). Long-term culture of this purified cardiomyocyte (TMRM high) population retained the expression of cardiomyocyte markers, continued calcium cycling, and displayed cyclic electrical activity that could be regulated pharmacologically. These findings suggest that non-cardiomyocyte populations can negatively influence contractility of cardiomyocytes in culture and that by purifying cardiomyocytes, the cultures retain potential as an experimental model for longitudinal studies of cardiomyocyte biology in vitro.cardiomyocyte; tetramethylrhodamine methyl ester perchlorate; enrichment; contractility CARDIOVASCULAR DISEASE is a leading cause of morbidity and mortality in the world (21). The ongoing pandemic of cardiovascular disease has fueled much basic science research to achieve a better understanding of the pathophysiological function of cardiomyocytes (CMs) and for effective strategizing of potential therapies. Cell cultures of cardiomyocytes are commonly used in vitro model for investigation at a cellular level. Primary cardiomyocytes have been successfully isolated from hearts of a range of species [e.g., mouse (16), rat (4), guinea pig (15), rabbit (12), cat (31), and human (27)] using a simple enzymatic method and maintained as primary cultures for a short duration (13).Neonatal rat CM cultures have been widely employed as a cost-effective experimental model to study the contractility, electrophysiology, and morphology of CMs. CMs from postnatal hearts do not normally proliferate in culture (30), thus limiting the number of CMs available to the starting material. Long-term primary cultures of CMs are rarely reported in the literature; generally experimental time frames are limited to 2-3 wk. Previous studies have shown that neonatal CMs lose their contractile phenotype within weeks in culture and that this is associated with CM dedifferentiation and cytoskeleton remodeling (3, 26). One potential reason for this could be the cellular heterogeneity of the heart, containing both contractile CMs and noncontractile cells such as fibroblasts, endothelial cells, and smooth muscle cells. The noncontractile cells, especially fibroblasts, can proliferate rapidly in culture and typically overgrow CMs to a point where it is belie...

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