Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na + (Na V ) and L-type Ca 2+ channels (LTCC). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating Na V dysregulation within transverse (T)-tubules in D96V-CaM-associated arrhythmias. D96V-CaM induces pro-arrhythmic late Na + current (I Na ) by impairing inactivation of Na V 1.6, but not the predominant cardiac Na V isoform, Na V 1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Superresolution microscopy revealed close proximity of Na V 1.6 and RyR2 within T-tubules. Na V 1.6 density within these regions increased in cD96V relative to WT. Consistent with Na V 1.6 dysregulation by D96V-CaM in these regions, we observed increased late Na V activity in Ttubules. The resulting late I Na promoted aberrant Ca 2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia (VT) in vivo. Cardiac-specific Na V 1.6 knockout protected cD96V mice from increased T-tubular late Na V activity, and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promotes arrhythmias by dysregulating LTCC and Na V 1.6 within T-tubules and thereby, facilitating aberrant Ca 2+ release.

Dofetilide is a rapid delayed rectifier potassium current inhibitor widely used to prevent the recurrence of atrial fibrillation and flutter. The clinical use of this drug is associated with increases in QTc interval, which predispose patients to ventricular cardiac arrhythmias. The mechanisms involved in the disposition of dofetilide, including its movement in and out of cardiomyocytes, remain unknown. Using a xenobiotic transporter screen, we identified MATE1 (SLC47A1) as a transporter of dofetilide and found that genetic knockout or pharmacological inhibition of MATE1 in mice was associated with enhanced retention of dofetilide in cardiomyocytes and increased QTc prolongation. The urinary excretion of dofetilide was also dependent on the MATE1 genotype, and we found that this transport mechanism provides a mechanistic basis for previously recorded drug-drug interactions of dofetilide with various contraindicated drugs, including bictegravir, cimetidine, ketoconazole, and verapamil. The translational significance of these observations was examined with a physiologically-based pharmacokinetic model that adequately predicted the drug-drug interaction liabilities in humans. These findings support the thesis that MATE1 serves a conserved cardioprotective role by restricting excessive cellular accumulation and warrant caution against the concurrent administration of potent MATE1 inhibitors and cardiotoxic substrates with a narrow therapeutic window.