Abstracts

Rationale: Mutations in SCN8A, encoding the voltage gated sodium channel Nav1.6,are associated with early-infantile epileptic encephalopathy, type 13 (EIEE13). Patients with EIEE13 are at an increased risk for Sudden Unexpected Death in Epilepsy (SUDEP). While the underlying mechanisms of SUDEP remain unknown, growing evidence suggests that cardiac arrhythmias may play an important role. In heart, Nav1.6 is preferentially localized in the T-tubules of ventricular myocytes where it plays a role in modulating the action potential duration. Our recent work in a mouse model of EIEE13 showed that ventricular myocyte calcium cycling is altered, and mice are at an increased risk of cardiac arrhythmias in vivo. Methods: To test if similar changes to cardiac excitability are observed in EIEE13 patients, we differentiated cardiac myocytes (CMs) from induced pluripotent stem cells (iPSCs). from one control and one patient. For action potential recordings, a subset of iPSC-CMs were infected with an adenovirus expressing Kir2.1-IRES-GFP and recorded 3-4 days after infection. Results: To detect differences in whole-cell sodium current (INa) between patients and controls, we performed patch clamp electrophysiology. As expected from our studies in a mouse model of SCN8A-linked EE, we observed no significant differences in peak INa (-124.9± 28.58 and -134.4± 14.89 for Control 3 and SCN8A pt. 2respectively; P=0.75; N=10 and 15). We next measured differences in cardiac action potentials. Initially, we tested spontaneously firing iPSC-CMs from one control and 1 patient iPSC-CM line. Results from these confluent iPSC-CMs suggest that iPSC-CMs from SCN8A patients display similar delayed afterdepolarization events, an arrhytmogenic substrate, as previously observed in the mouse model. However, these iPSC-CMs were immature, displaying spontaneous contraction and a depolarized resting membrane potential > - 60 mV. To accurately record mature action potential properties in iPSC-CMs, we used a virus to express Kir2.1. Expression of this viral construct allowed us to record iPSC-CMs with a hyperpolarized resting membrane potential of ≤ -60mV. Initial analysis of these cells suggests that the action potential duration is increased in EIEE13 patient iPSC-CMs, with a prolongation of phase 2 of the action potential. Conclusions: We propose that prolongation of action potential duration measured in EIEE13 iPSC-CMs is due to increased persistent INa resulting from mutant Nav1.6 expression at the cardiac T-tubules, in turn leading to reverse Na/Ca exchange and increased calcium transient duration. This mechanism may generate arrhythmogenic substrates within the intact heart. Our results suggest that EIEE13 patients may be at increased risk of cardiac arrhythmias, which may contribute to SUDEP. Using iPSC-CMs to model EIEE13 may provide important insights into the mechanism of SUDEP and lead to novel biomarkers and therapeutics to decrease SUDEP risk. Funding: Supported by a fellowship from NIH grant UL1TR000433 (CRF), and NIH grants R37-NS-076752(LLI) and U01-NS-090364(JP and LLI).