The QT interval is an important diagnostic feature on surface electrocardiograms because it reflects the duration of the ventricular action potential. A previous genome-wide association study has reported a significant linkage between a single-nucleotide polymorphism 11.7 kb downstream of the gene encoding the RING finger ubiquitin ligase rififylin (RFFL) and variability in the QT interval. This, along with results in animal studies, suggests that RFFL may have effects on cardiac repolarization. Here, we sought to determine the role of RFFL in cardiac elec-trophysiology. Adult rabbit cardiomyocytes with adenovirus-expressed RFFL exhibited reduced rapid delayed rectifier current (IKr). Neonatal rabbit cardiomyocytes transduced with RFFL-expressing adenovirus exhibited reduced total expression of the potassium channel ether-a-go-go-related gene (rbERG). Using transfections of 293A cells and Western blotting experiments, we observed that RFFL and the core-glycosylated form of the human ether-a-go-go-related gene (hERG) potassium channel interact. Furthermore, RFFL overexpression led to increased polyubiquitination and proteasomal degradation of hERG protein and to an almost complete disappearance of IKr, which depended on the intact RING domain of RFFL. Blocking the ER-associated degradation (ERAD) pathway with a dominant-negative form of the ERAD core component, valosin-containing protein (VCP), in 293A cells partially abolished RFFL-mediated hERG degradation. We further substantiated the link between RFFL and ERAD by showing an interaction between RFFL and VCP in vitro. We conclude that RFFL is an important regulator of voltage-gated hERG potassium channel activity and therefore cardiac repolarization and that this ubiquitination-mediated regulation requires parts of the ERAD pathway.
Bibliographical noteFunding Information:
This work was supported by NHLBI, National Institutes of Health, Grants R01HL134706, R01HL110791, and R01HL046005 (to G. K.) and by the Rhode Island Foundation, Grant 20164378 (to K. S. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Acknowledgments—We are indebted to Drs. Wafik S. El-Deiry (Fox Chase Cancer Center, Department of Hematology/Oncology, Philadelphia, PA) for the RFFL plasmid, Ted Dawson (Johns Hopkins University, Institute for Cell Engineering, Baltimore, MD) for the ubiquitin plasmid, and Nico Dantuma (Karolinska Institutet, Department of Cell and Molecular Biology, Stockholm, Sweden) for the VCP expression plasmids. The data used for the analyses described in this article were obtained (on 10/2/2017) from the GTEx Portal (https:// www.gtexportal.org/home/).4 The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and the NCI, National Human Genome Research Institute (NHGRI), NHLBI, NIDA, NIMH, and NINDS.