Research Investigator University of Michigan Ann Arbor, Michigan
Rationale: Sudden Unexpected Death in Epilepsy (SUDEP) is a leading cause of death in epilepsy patients. Cardiac arrhythmias are thought to contribute to the mechanisms of SUDEP in genetic epilepsies caused by variants of ion channel genes. Variants in DEPDC5, a gene encoding a member of the GATOR complex in the mTOR pathway, have recently been identified in epilepsy patients. SUDEP has been reported in 10% of epilepsy patients with DEPDC5 loss-of-function variants. This study aims to determine whether cardiac mechanisms contribute to SUDEP in epilepsy patients with variants in non-ion channel gene DEPDC5, and to identify SUDEP biomarkers. Methods: We used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to measure changes in cellular excitability that are known substrates for cardiac arrhythmias. CRISPR-derived isogenic DEPDC5 iPSC-CMs and DEPDC5 patient-derived iPSC-CMs, as well as their respective controls, were used in this study. Whole-cell patch-clamp was used to measure voltage-gated sodium current (INa) and calcium current (ICa) in single iPSC-CMs in voltage-clamp mode; and to measure action potentials (APs) in 3-dimentional iPSC-CM-derived micro-tissues in current-clamp mode. Confocal microscope imaging in line-scan mode was used to measure the intracellular calcium transients. Results: CRISPR generated heterozygous deletion of one base-pair in the first coding exon of the DEPDC5 gene, resulting in a premature stop codon, simulated the variants identified in DEPDC5 epilepsy patients. In CRISPR generated heterozygous DEPDC5 iPSC-CMs, whole-cell voltage-clamp recordings revealed that INa was increased and ICa was reduced compared with isogenic control iPSC-CMs. Whole-cell current-clamp recordings revealed that in CRISPR generated heterozygous DEPDC5 iPSC-CMs, AP duration at 80% and 90% of repolarization, APD80 and APD90, respectively, were prolonged; and AP amplitude was increased, compared with isogenic control iPSC-CMs. Intracellular calcium imaging revealed decreased calcium transient amplitude and increased time-to-peak in CRISPR generated heterozygous DEPDC5 iPSC-CMs. Similar measurements will be performed for iPSC-CMs derived from DEPDC5 patients. Conclusions: Epilepsy patients with non-ion channel gene variants in DEPDC5 have altered CM excitability and intracellular calcium dynamics, which may serve as a substrate for cardiac arrhythmias in DEPDC5 patients. Importantly, this work may help to identify biomarkers for future use of stratifying SUDEP risk in genetic epilepsy patients with DEPDC5 variants. Funding: Please list any funding that was received in support of this abstract.: This work is supported by MICHR’s CTSA grant UL1TR002240 (to Y.-T. Zhao), by R37 NS076752 (to L.L.I.), by U01 NS090364 (to L.L.I. and J.M.P), and by a grant from the Gerber Foundation (to L.L.I. and J.M.P).