Abstract
Background: We have been reported that dantrolene (DAN) binds to N-terminal domain of the cardiac ryanodine receptor (RyR2) and corrects the aberrant Ca 2+ leak. We have been also reported that DAN enhances binding affinity of Calmodulin (CaM) to RyR2 in failing cardiomyocytes. In this study, we investigated whether CaM is essential for the anti-arrhythmogenic effect of DAN using pressure-overload heart failure induced by transverse aortic constriction (TAC). Methods and results: Pressure overload to 12 weeks old mice was induced by TAC using a 27-gauge needle. Cardiomyocytes isolated from mice 8 weeks after TAC operation were used in the present study. The survival rate of our TAC mice at 8 weeks after TAC operation was 32 %. LV end-diastolic dimension was significantly enlarged and LV ejection fraction was significantly decreased. Lethal arrhythmias were frequently observed after 2 mg epinephrine infusion in TAC mice, but not in Sham mice. All following experiments were done under 1 μM of KN93 to avoid the effect of CaMKII. The Ca 2+ spark frequency (SpF: s -1 ·100 μm -1 ; by fluo3) was much higher in TAC than in Sham cardiomyocytes {TAC (n=10): 7.6±4.7; Sham (n=5): 5.7±1.9; p<0.01}. Addition of DAN to saponin-permeabilized TAC cells normalize Ca 2+ spark frequency {(n=5): 5.6±1.4}. This effect of DAN disappeared after suramin, CaM remover from RyR2, treatment {(n=4): 7.2±3.2}. Spontaneous Ca 2+ transients (SCaT) were measured during sequential pacing of intact cardiomyocytes from 1 Hz to 5 Hz. SCaT was rarely observed in Sham cells even at 5 Hz pacing, whereas it was observed in 20 % of TAC cells. Pre-incubation with DAN to intact TAC cells normalize inceidence of SCaT (11 %). After suramin incorporation, effect of DAN was abolished (19 %). Conclusions: In conclusion, CaM is essential for the channel stabilization effect of DAN. DAN corrects the aberrant domain-domain interaction, and as the result, CaM binding affinity to the RyR2 improves. These are the possible mechanism underlying anti-arrhythmogenic effect of DAN.
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