SCN5A encodes the α subunit of the major cardiac sodium channel NaV1.5. Mutations in SCN5A are associated with conduction disease and ventricular fibrillation (VF); however, the mechanisms that link loss of sodium channel function to arrhythmic instability remain unresolved. Here, we generated a large-animal model of a human cardiac sodium channelopathy in pigs, which have cardiac structure and function similar to humans, to better define the arrhythmic substrate. We introduced a nonsense mutation originally identified in a child with Brugada syndrome into the orthologous position (E558X) in the pig SCN5A gene. SCN5AE558X/+ pigs exhibited conduction abnormalities in the absence of cardiac structural defects. Sudden cardiac death was not observed in young pigs; however, Langendorff-perfused SCN5AE558X/+ hearts had an increased propensity for pacing-induced or spontaneous VF initiated by short-coupled ventricular premature beats. Optical mapping during VF showed that activity often began as an organized focal source or broad wavefront on the right ventricular (RV) free wall. Together, the results from this study demonstrate that the SCN5AE558X/+ pig model accurately phenocopies many aspects of human cardiac sodium channelopathy, including conduction slowing and increased susceptibility to ventricular arrhythmias.
David S. Park, Marina Cerrone, Gregory Morley, Carolina Vasquez, Steven Fowler, Nian Liu, Scott A. Bernstein, Fang-Yu Liu, Jie Zhang, Christopher S. Rogers, Silvia G. Priori, Larry A. Chinitz, Glenn I. Fishman
(A) Precordial lead ECG from the 10-year-old child with SCN5AE555X/+ mutation, at baseline and during flecainide challenge. (B) The targeting vector contains a G-to-T point mutation (asterisk), resulting in replacement of a glutamic acid at amino acid 558 with a premature stop codon (GAG to TAG), as well as a floxed (triangles) neomycin resistance cassette (NeoR) driven by the phosphoglycerate kinase promoter in intron 11 for selection. (C and D) Properly targeted cells were identified by Southern blot (C) and direct sequencing (D). (E) Yucatan mini pigs harboring a single copy of the SCN5AE558X mutation (SCN5AE558X/+ pigs; E558X/+) were viable and appeared grossly normal. (F) WBs of membrane fractions detecting NaV1.5 and the sodium-potassium ATPase (Na/K ATPase) as loading control. Molecular weight markers (250 and 100 kDa) are indicated in lane 1. (G) NaV1.5 protein levels (normalized to Na/K ATPase), displayed relative to WT. *P < 0.05. (H) Patch-clamp analysis of atrial myocytes. Shown are representative whole-cell Na+ current recordings and average current/voltage relationship from adult WT (n = 23 cells) and SCN5AE558X/+ (n = 16 cells) left atrial myocytes. *P < 0.01.