Arrhythmogenic calmodulin mutations disrupt intracellular cardiomyocyte Ca²⁺ regulation by distinct mechanisms

Background: Calmodulin (CaM) mutations have been identified recently in subjects with congenital long QT syndrome (LQTS) or catecholaminergic polymorphic ventricular tachycardia (CPVT), but the mechanisms responsible for these divergent arrhythmia-susceptibility syndromes in this context are unknown. We tested the hypothesis that LQTS-associated CaM mutants disrupt Ca²⁺ homeostasis in developing cardiomyocytes possibly by affecting either late Na current or Ca²⁺-dependent inactivation of L-type Ca²⁺ current. Methods and Results: We coexpressed CaM mutants with the human cardiac Na channel (Na_V1.5) in tsA201 cells, and we used mammalian fetal ventricular cardiomyocytes to investigate LQTS- and CPVT-associated CaM mutations (LQTS- and CPVT-CaM). LQTS-CaM mutants do not consistently affect L-type Na current in heterologous cells or native cardiomyocytes, suggesting that the Na channel does not contribute to LQTS pathogenesis in the context of CaM mutations. LQTS-CaM mutants (D96V, D130G, F142L) impaired Ca²⁺-dependent inactivation, whereas the CPVT-CaM mutant N54I had no effect on Ca²⁺-dependent inactivation. LQTS-CaM mutants led to loss of Ca²⁺-transient entrainment with the rank order from greatest to least effect: CaM-D130G~CaM-D96V≫CaM-F142L. This rank order follows measured Ca²⁺-CaM affinities for wild-type and mutant CaM. Acute isoproterenol restored entrainment for CaM-130G and CaM-D96V but caused irreversible cytosolic Ca²⁺ overload for cells expressing a CPVT-CaM mutant. Conclusions: CaM mutations associated with LQTS may not affect L-type Na⁺ current but may evoke defective Ca²⁺-dependent inactivation of L-type Ca²⁺ current.