Abstract
Cardiac troponin T (cTnT) mutations cause Familial Hypertrophic Cardiomyopathy (FHC), which leads to sudden cardiac death. Ca2+ sensitivity in myofilaments was increased, however, the underlying mechanism by which the cTnT mutations lead to lethal arrhythmia and the therapeutic approach remains elusive. Here, we investigated the pathogenic role of phosphorylation-mediated aberrant Ca2+ release via the cardiac ryanodine receptor (RyR2) and the therapeutic effects of dantrolene, which was found to correct inter-domain interactions of RyR2 in failing hearts (i.e. unzipping to zipping), in transgenic mouse (TG) model with FHC-related cTnT mutation (delta160E). Methods and results: In 6thmonths of age, there was no appreciable difference in the structural or functional features of the hearts between non-TG and TG mice. In response to isoproterenol (ISO; 10 nmol/L), the Ca2+ spark frequency (SpF: s-1·100μm-1; by fluo4) was much higher in TG cardiomyocytes (ISO-TG: n=11: 7.7±0.7; p<0.01) than in non-TG cardiomyocytes (n=6: 3.5±0.5). It was largely reversed by CaMKII inhibitor {ISO-TG with KN-93(1μM), n=6: 5.2±0.4; p<0.05}}, but not by PKA inhibitor{ISO-TG with H-89(1μM), n=6: 7.3±0.4;n.s}}. ISO-TG (but not ISO-treated non-TG 0/14; n=14) showed spontaneous Ca2+ transient (sCaT) after 5 Hz pacing (12/18; n=13), whereas it was again attenuated by KN-93 (0/5; n=5), but not by H-89 (3/5; n=5)}. These abnormal events in ISO-TG were reproduced by adding EGTA-AM into ISO-treated non-TG, suggesting that increased Ca2+ buffering capacity, causing an increase in diastolic [Ca2+], predisposes to aberrant Ca2+ release events. Moreover, dantrolene (1μM) attenuated SpF (n=6: 5.2±0.3; p<0.05 vs ISO-TG), time from peak to 70% decline of Ca2+ transient and sCaT (3/10; n=10) in ISO-treated TG cardiomyocytes. Conclusions: In FHC-linked cTnT mutated hearts, aberrant local Ca2+ release through defective RyR2 was induced by beta-adrenergic stimulation, presumably due to mutation-linked, increased Ca2+ buffering capacity, and subsequent CaMKII activation. The inhibition of CaMKII-mediated aberrant Ca2+ release by stabilizing RyR2 could be a new therapeutic approach to prevent the development of arrhythmias in FHC.
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