RESUMO
OBJECTIVE: Enhanced late Na current (late INa) induces Na-dependent Ca overload as well as proarrhythmogenic events on the cellular level that include spatio-temporally uncoordinated diastolic Ca release from the sarcoplasmic reticulum (SR) and delayed afterdepolarizations (DADs). The Ca/calmodulin-dependent protein kinase II (CaMKII) gets activated upon increases in [Ca]i and mediates diastolic SR Ca leak as well as DADs. RATIONALE: We hypothesized that increased late INa (in disease-comparable ranges) exerts proarrhythmogenic events in isolated ventricular mouse myocytes in a manner depending on CaMKII-dependent SR Ca leak. We further tested whether inhibition of disease-related late INa may reduce proarrhythmogenic SR Ca leak in myocytes from failing human hearts. METHODS: Ventricular myocytes were isolated from healthy wildtype (WT), failing CaMKIIδC transgenic (TG) mouse, and failing human hearts. ATX-II (0.25-10 nmol/L) was used to enhance late INa. Spontaneous Ca loss from the SR during diastole (Ca sparks), DADs, non-triggered diastolic Ca transients in myocytes and premature beats of isometrically twitching papillary muscles were used as readouts for proarrhythmogenic events. CaMKII autophosphorylation was assessed by immunoblots. Late INa was inhibited using ranolazine (Ran, 10 µmol/L) or TTX (2 µmol/L), and CaMKII by KN-93 (1 µmol/L) or AIP (1 µmol/L). RESULTS: In WT myocytes, sub-nanomolar ATX-II exposure (0.5 nmol/L) enhanced late INa by ~60%, which resulted in increased diastolic SR Ca loss despite unaltered SR Ca content. In parallel, DADs and non-triggered diastolic Ca transients arose. Inhibition of enhanced late INa by RAN or TTX significantly attenuated diastolic SR Ca loss and suppressed DADs as well as mechanical alternans in mouse and diastolic SR Ca loss in failing human myocytes. ATX-II caused Ca-dependent CaMKII-activation without changes in protein expression, which was reversible by Ran or AIP. Conversely, CaMKII-inhibition decreased diastolic SR Ca loss, DADs and non-triggered diastolic Ca transients despite ATX-II-exposure. Finally, failing mouse myocytes with increased CaMKII activity (TG CaMKIIδC) showed an even aggravated diastolic SR Ca loss that was associated with an increased frequency of non-triggered diastolic Ca transients upon enhanced late INa. CONCLUSIONS: Increased late INa (in disease-comparable ranges) induces proarrhythmogenic events during diastole in healthy and failing mouse myocytes, which are mediated via CaMKII-dependent SR Ca loss. Inhibition of late INa not only attenuated these cellular arrhythmias in mouse myocytes but also in failing human myocytes indicating some antiarrhythmic potential for an inhibition of the elevated late INa/CaMKII signaling pathway in this setting.