Blebbistatin Protects Rodent Myocytes from Death in Primary Culture via Inhibiting the Sodium/ Calcium Exchanger and the L-type Calcium Channel

Abstract

Introduction: Cardiac disease is a leading cause of mortabity and morbidity in the developed countries. Cultured cardiac myocytes are widely used for exploring the underlying pathophysiology of cardiac disease. Rodents, especially mice with transgenes or gene ablation, have become popular animal models for heart disease research. However, 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. 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 (10 µM) for 72 hours in comparison with another protective chemical, BDM (10mM). Myocyte death was evaluated by morphology changes and trypan blue staining. The effects of these two drugs on myocyte contraction, intracellular Ca2+ transient ([Ca2+]i, indo-1,410/480), SR Ca2+ content, L-type calcium and Na+ /Ca2+exchanger currents were studied acutely. Neonatal rat ventricular myocytes (NRVM) were isolated from 1-3 days old neonatal rat hearts and cultured. The effect of BDM (10mM BDM) and BLB (10 µM) in the medium on NRVM growth and hypertrophy induced by norepinephrine (NE, 10µM) were determined. Results: 1. Both BDM and BLB promoted myocyte survival in culture at 72 hours but BLB protected more myocytes (Control: 7.0±1.8% vs. BDM: 61.5±6.4% vs. BLB: 74.0±3.2%); 2. ARVM fractional shortening was reduced by BLB to 1.7±0.4% and by BDM to 0.5±0.1% from the baseline of 6.5±0.7%; 3. Acutely, the amplitude of [Ca2+]i (∆ [Ca2+]i) evaluated with indo-1 AM (F410/F480) was depressed by both BDM (0.04±0.01) and BLB (0.07±0.01) compared to control (0.13±0.01). 4. Diastolic Ca2+ was significantly increased by BLB (0.90±0.06) but not by BDM (0.73±0.06) compared to pre-treat values (0.70±0.05); 5. BLB and BDM significantly reduced the SR Ca2+ content, as indicated by the reduced amplitudes of caffeine-induced Ca2+ transients in BLB- and BDM-treated ARVMs (∆[Ca2+]i in BLB vs. BDM vs. baseline: 0.20±0.03, 0.19±0.04, 0.30±0.03). 6. The mechanisms of the protective effects of BDM and BLB were similar but quantitatively different in that BDM reduced more Ca influx through the L-type Ca2+ channel (ICa-L) than BLB (the reduction in BDM-treated cells vs. BLB-treated cells: 70% vs. 40%) while BLB inhibited more Na+/Ca2+exchanger current (75% inhibition) than BDM (40% reduction); 7. Both BDM and BLB inhibited normal NRVM growth and NE-induced hypertrophy and NFAT translocation in NRVMs. Conclusion: These results suggest both BDM and BLB protect rodent myocytes in culture by preventing cytosolic and SR Ca2+ overload by similar mechanisms: inhibiting NCX and reducing the LTCC. The application of BLB to whole-heart studies and myocyte hypertrophy should be extremely cautioned because BLB does alter myocyte Ca2+ handling.