Blebbistatin Protects Rodent Myocytes from Death in Primary Culture via Inhibiting Na/Ca Exchange

Abstract

Introduction: It has been long recognized that rodent myoyctes die during long-term primary culture, which limits the use of genetically altered myocytes for signaling studies. Blebbistatin (BLB), a myosin II ATPase inhibitor, has been used to protect rodent myocytes. However, the mechanisms underlying the protective effects of this drug are not clear and are the topics of this study. Materials & Methods: Adult rat ventricular myocytes (ARVM) were isolated and cultured with or without BLB (10μM) and BDM(10mM) for 72 hours. Myocyte death was evaluated by trypan blue staining. The effects of these two drugs on myocyte contraction, intracellular Ca transient ([Ca]i, Indo-1,410/480), SR Ca content, L-type calcium and Na/Ca exchanger currents were studied acutely. Results: 1, Both BDM (61.5± 6.4%) and BLB (74.0±3.2%) promoted myocyte survival in culture at 72 hours (control: 7.0±1.8%); 2. ARVM fractional shortening was reduced by BLB (1.7±0.4%) and BDM (0.5±0.1%, control: 6.5±0.7%); 3, Acutely, the amplitude of [Ca]i (Δ[Ca]i) was depressed by both BDM (0.038±0.005) and BLB (0.065±0.008) comparing to control (0.130±0.010). 4, Diastolic Ca was significantly increased by BLB (0.90±0.06) but not by BDM (0.73±0.06) comparing to control(0.70±0.05). 5. BLB and BDM significantly reduced the SR Ca content(Δ[Ca]i in BLB vs. BDM vs. control: 0.16±0.016, 0.09±0.01, 0.24±0.03). 5. The mechanisms of the protective effect of BDM and BLB are different in that BDM mainly reduced Ca influx through the L-type Ca channel (85% reduction) and Na/Ca exchanger (60% reduction) while BLB inhibited Na/Ca exchanger (100% inhibition) without altering the LTCC(<5% reduction). Conclusion: These results suggest both BDM and BLB protects rodent myocytes in culture by preventing cytosolic and SR Ca overload by both common and different mechanisms: both BDM and BLB inhibit NCX while BDM, but not BLB, reduces ICa-L.