Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca 2+ waves and elementary sarcoplasmic reticulum (SR) Ca 2+ release events, Ca 2+ sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca 2+ leak or SR Ca 2+ content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca 2+ leak, resulting in a significantly increased SR Ca 2+ content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca 2+ waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca 2+ fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca 2+ release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca 2+ waves resulting in triggered arrhythmias, such as CPVT.

In human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca 2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca 2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca 2+ -sensitizing agent EMD 57033 and prevented by myofilament Ca 2+ desensitization with blebbistatin. Ca 2+ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, myofilament Ca 2+ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca 2+ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.

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linescanning after 488nm excitation and recording emission at 505-530nm in intact Fluo-3-loaded cardiomyocytes (2uM) at 37 C and at [Ca2þ] 1.2mM and 5.0mM. These studies showed that spontaneous wave frequency was higher at 5.0mM than 1.2mM Ca2þ. Post-MI HF cardiomyocytes had~twice the wave frequency compared to sham-operated controls. Regular ExTr post-MI improved exercise capacity and induced reverse remodeling. ExTr also reduced the frequency of spontaneous waves at both Ca2þ 1.2mM and 5.0mM, although it did not completely normalize spontaneous Ca2þ waves. ExTr also increased the ratio between aborted and complete waves at Ca2þ 1.2mM, but not Ca2þ 5.0mM. No effects were found on spontaneous wave velocity. This suggests that ExTr partly improved the control of diastolic Ca2þ by reducing the frequency of spontaneous Ca2þ waves and by improving the ability of the cardiomyocyte to eliminate a spontaneous wave after its generation, but before its propagation. Finally, we repeated these studies in the presence of the nitric oxide synthase inhibitor L-NAME, to study the contribution of nitric oxide. This did not have any effects.