Abstract 12752: Long QT Syndrome Type 3 Caused by a PY-motif Mutation Leading to Altered Ubiquitylation and Increased Expression of Nav1.5 in Knock-in Mice
Background: Congenital long QT syndrome type 3 (LQT3) is characterized by delayed repolarization leading to sudden cardiac death, and is caused by gain-of-function mutations in SCN5A, the gene encoding the sodium channel Nav1.5. This channel can be regulated upon its ubiquitylation by the ubiquitin ligase Nedd4–2, which binds to the PY motif of Nav1.5.
Aim: We have investigated the mechanisms underlying the phenotype of a LQT3 family with sudden cardiac death carrying a SCN5A mutation located in the PY motif (p.Y1981N) of Nav1.5.
Methods and results: First, HEK293 cells were transfected with Nav1.5 wild-type (WT) or p.Y1981N in the presence of Nedd4–2. Co-immunoprecipitation experiments revealed that the interaction of Nav1.5 with Nedd4–2 was abolished by the mutation. While the WT protein was ubiquitylated and the sodium current (INa) down-regulated upon Nedd4–2 co-expression, no such regulation was observed with the mutant channel. Second, the in vivo relevance of this regulatory mechanism was assessed by generating a knock-in (KI) mouse line bearing the p.Y1981N mutation. Ubiquitylation of the mutant channel expressed in KI hearts of homozygous mice was reduced by 28±4%, and the total level of Nav1.5 protein was increased by 35.4±5% compared to WT littermates. We found an increase of 19±5% of INa recorded in freshly isolated cardiomyocytes from KI hearts compared to WT. Finally, action potential duration at 90% repolarization of KI cardiomyocytes was increased by 38.5± 13.7%.
Conclusions: Together, these results demonstrate for the first time a central role for Nav1.5 ubiquitylation in the regulation of sodium channel density at the surface of cardiomyocytes. Alteration of this mechanism may lead to prolongation of action potential duration and LQT syndrome in patients harboring mutations that disrupt this pathway.
- © 2010 by American Heart Association, Inc.