Background—Timothy syndrome (TS) is a disease of excessive cellular Ca²⁺ entry and life-threatening arrhythmias caused by a mutation in the primary cardiac L-type Ca²⁺ channel (Ca_V1.2). The TS mutation causes loss of normal voltage-dependent inactivation of Ca_V1.2 current (I_Ca). During cellular Ca²⁺ overload, the calmodulin-dependent protein kinase II (CaMKII) causes arrhythmias. We hypothesized that CaMKII is a part of the proarrhythmic mechanism in TS.

Methods and Results—We developed an adult rat ventricular myocyte model of TS (G406R) by lentivirus-mediated transfer of wild-type and TS Ca_V1.2. The exogenous Ca_V1.2 contained a mutation (T1066Y) conferring dihydropyridine resistance, so we could silence endogenous Ca_V1.2 with nifedipine and maintain peak I_Ca at control levels in infected cells. TS Ca_V1.2–infected ventricular myocytes exhibited the signature voltage-dependent inactivation loss under Ca²⁺ buffering conditions, not permissive for CaMKII activation. In physiological Ca²⁺ solutions, TS Ca_V1.2–expressing ventricular myocytes exhibited increased CaMKII activity and a proarrhythmic phenotype that included action potential prolongation, increased I_Ca facilitation, and afterdepolarizations. Intracellular dialysis of a CaMKII inhibitory peptide, but not a control peptide, reversed increases in I_Ca facilitation, normalized the action potential, and prevented afterdepolarizations. We developed a revised mathematical model that accounts for CaMKII-dependent and CaMKII-independent effects of the TS mutation.

Conclusion—In TS, the loss of voltage-dependent inactivation is an upstream initiating event for arrhythmia phenotypes that are ultimately dependent on CaMKII activation.