Introduction: Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a major cause of sudden cardiac death among young, athletic patients. Mutations in desmosomal proteins are frequently found in patients with ARVC and are suggested to predispose to arrhythmias and contractile dysfunction. Interestingly, it was shown that desmosomal function and integrity of the intercalated disc are mechanistically linked to the function of cardiac voltage-gated Na channels (Na_V1.5). It has been shown that a reduced expression of the desmosomal protein Plakophilin 2 (PKP2) leads to decreased sodium current in mouse cardiac myocytes. However, the exact cellular mechanisms of the arrhythmogenesis in ARVC patients remain unknown.

Objective: We investigated Na_V1.5 gating and action potential (AP) characteristic of induced-pluripotent stem cell(iPSC)-derived cardiomyocytes from a young female patient with ARVC, who survived sudden cardiac death. 

Methods and Results: Genetic testing of the index patient and her family revealed a loss-of-function mutation in the PKP2 gene in 12 of 19 tested family members including the index patient. After a three-month maturation period, iPSC cardiomyocytes were investigated from the index patient, an asymptomatic sibling with the same mutation and a healthy proband. Ruptured-patch whole-cell patch clamp technique was used to measure I_Na and APs. Interestingly, compared to control, peak I_Na was significantly reduced in iPSC cardiomyocytes from the index patient, as well as in comparison to the asymptomatic sibling harboring the same PKP2 mutation (Fig. 1). Surprisingly, late I_Na, which is known to be proarrhythmogenic, was found to be significantly reduced in the index patient but unaltered in the sibling. Late I_Na integrals (50-500 ms) were -106.2±10.3 (control) vs. -56.64±9.3 (index patient) vs. -95.55 ± 11.9 AmsF^-1 (sibling; n=16 vs. 8. vs. 15 cells, p<0.05 vs. control). Moreover, compared to control, resting membrane potential (RMP) was significantly less negative and AP duration was significantly prolonged in iPSC cardiomyocytes from the index patient but unaltered in cells from the sibling. RMP was -58.8±3.6 (index, n=11 cells) vs. 71±1.9 mV (control, n=21 cells, p<0.05) vs. -71.9±1.3 mV (sibling, n=21 cells). At 1 Hz stimulation frequency, APD90 was 370.3±23.9 (index, n=11 cells) vs. 247.8±21.1 ms (control, n=21 cells, p<0.05) vs. 287.6±23.1 ms (sibling, n=21 cells, p=n.s. vs control). Furthermore, western blotting and qPCR were used to measure expression of inward-rectifying potassium channel K_ir2.1. Interestingly, compared to control, K_ir2.1 expression was significantly reduced in iPSC cardiomyocytes from the index patient but only by trend for the sibling.

Conclusion: Our functional data show that peak I_Na and late I_Na are reduced in a symptomatic ARVC patient but not in an asymptomatic sibling harboring the same loss-of-function mutation of PKP2. Surprisingly, the changes observed in Na current did not correlate with the changes in RMP and APD. Instead, we found a less negative RMP and a prolonged APD. Interestingly, the cells from the asymptomatic sibling did not have altered electrophysiological properties in comparison to control cells. This indicates that further mechanisms may be relevant for arrhythmogenesis in ARVC.