Abstract
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 electrophysiology. 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.
Highlights
The QT interval is an important diagnostic feature on surface electrocardiograms because it reflects the duration of the ventricular action potential
Two genome-wide association studies [10, 11] of QT intervals identified two genetic variants downstream of the Ring finger and FYVE-like domain E3 ubiquitin protein ligase (RFFL)3 gene, viz. rs2074518 and rs1052536, which are associated with modest changes in QT interval duration
We noticed that RFFL overexpression significantly reduced rabbit ether-a-go-go-related gene (rbERG) levels (155-kDa band) by ϳ60% in neonatal rabbit cardiomyocytes (NRbCM) (Fig. 1, D and E)
Summary
Studies, RFFL has been linked to endocytic recycling, which requires an intact FYVE-like domain [15], p53 turnover through regulation of Mdm stability [17], tumor necrosis factor-induced NF-B activation [18], and destabilization of PRR5L, a suppressor of mTORC2, resulting in mTORC2-mediated protein kinase C␦ phosphorylation and cell migration downstream of G␣12 [19]. The aforementioned RFFL-proximal single nucleotide polymorphisms (SNPs) associated with modest changes in the QT interval duration in humans and the effect of elevated RFFL on the QT interval in a congenic rat strain prompted us to study the effect of RFFL on cardiac repolarization. We hypothesized that RFFL potentially acts through ubiquitination to alter ion channel synthesis, trafficking, and/or recycling or degradation through proteasomes or lysosomes [20]. We set out to identify RFFL-associated ion channels responsible for these effects. We present data that support a role for RFFL in the regulation of human ether-a-go-go-related gene (hERG) forward trafficking, thereby affecting cardiac repolarization
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
