Magnesium Influx in Primary Cultured Ventricular Myocytes of Adult Rats

Abstract

We have characterized physiological pathway of Mg2+ influx in acutely isolated ventricular myocytes of rats [Biophys J 107:2049-2058, 2014]. In this study, we measured the rate of Mg2+ influx in primary cultured ventricular myocytes dissociated from adult rat hearts. Acutely isolated cells were plated onto laminin-coated glass bottom dishes, and maintained in DMEM supplemented with 10% FBS and 5% horse serum at 37°C and 5%CO2 for 72 hours. On the first day of culture, acutely isolated cells were in rod shape with sharp edges and clear cross-striations. The edges of cells became rounder gradually over time in culture; some cells were in round shape on the third day. We used the cells that kept rod shape and clear intracellular structure for experiments. Cytosolic free Mg2+ concentration ([Mg2+]i) was measured with a fluorescent indicator furaptra (mag-fura-2) introduced by AM-loading. The basal level of [Mg2+]i in primary cultured cell was 0.85±0.068 mM (n=7), which was not significantly different from that of acutely isolated cells (0.92±0.039 mM, n=10). [Mg2+]i decreased by incubation of the cells in a high-K+ (Ca2+- and Mg2+-free) solution for 20 min, and recovered by perfusion with Ca2+-free Tyrode's solution containing 1 mM Mg2+. The initial rate of Mg2+ influx was estimated from the time course of the [Mg2+]i recovery as described previously. The Mg2+ influx rate obtained from primary cultured cell was, on average, 0.28±0.11 μM/s with initial [Mg2+]i at 0.38±0.03 mM. This value was similar to that obtained from acutely isolated cells (0.27±0.043 μM/s at 0.35±0.016 mM [Mg2+]i). Since it is suggested that function of cellular Mg2+ regulation system is not altered by primary culture for 72 hours, the primary cultured ventricular myocytes may be used to study physiological roles of Mg2+ channel/transporter proteins.