On Establishing Primary Cultures of Neonatal Rat Ventricular Myocytes for Analysis Over Long Periods

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Abstract
Primary cultures of neonatal rat ventricular myocytes include a population of rapidly dividing nonmyocardial cells that can alter the properties of myocytes and complicate experimental interpretations. Without any intervention, nonmyocyte proliferation restricts the utility of primary cultures in biochemical and electrophysiologic studies to 4-5 days. However, with the recent interest in regulation of cardiac gene expression and the effects of growth factors on cardiac function, long-term studies with stable heart cultures are warranted.In the present study an immunohistochemical staining strategy was developed that allowed for reliable quantitation of myocytes and nonmyocytes in cultures maintained for extended periods under different culture conditions. Density gradient purification of myocytes was found valuable in limiting nonmyocyte levels to < 20% at early times. Further treatment of cultures with a mitotic inhibitor, 0.1 mM bromodeoxyuridine, or 3500 rads of gamma-irradiation effectively blocked the proliferation of nonmyocardial cells, while it had no effect on cardiocyte levels. However, bromodeoxyuridine displayed side effects on the myocytes; the spontaneous beating rate and intracellular glycogen content were markedly depressed. In contrast, a systematic investigation of the properties of the irradiated myocytes, including spontaneous beating rates, dihydropyridine receptors, glycogen content, sarcoplasmic reticulum function, and phosphoinositide signaling, revealed that irradiation did not alter cardiac cell function. Although ionizing radiation can stimulate gene expression in some cell types, gamma-irradiation did not evoke c-fos expression or cause sarcomere formation, responses seen in cardiac cells to several trophic factors.This study establishes a system of stable, functional, primary cultured cardiac cells that can be used in long-term molecular and electrophysiologic studies of at least 2 weeks.