A mechanism for NaV1.5 downregulation and sodium current decrease in heart failure

The pore-forming α-subunit of the cardiac voltage-gated sodium channel, NaV1.5, is responsible for the initial upstroke of the cardiac action potential. NaV1.5 cell surface expression and function are modulated by its interaction with regulatory proteins and by post-translational modifications, such as phosphorylation, arginine methylation or ubiquitination.1 Genetic mutations in the SCN5A gene, which encodes NaV1.5, have been associated with a variety of inherited cardiac arrhythmias, including long QT syndrome type 3, Brugada syndrome, atrial fibrillation and congenital sick sinus syndrome. In addition, abnormal NaV1.5 plasma membrane expression or sodium current (INa) density has also been observed in acquired cardiac disorders, such as heart failure (HF), although the molecular mechanisms that trigger these alterations are not well understood. In this issue of Acta Physiologica, Luo et al.2 describe a novel mechanism that may shed light into understanding why NaV1.5 function is altered in HF. They propose that downregulation of NaV1.5 in HF could be explained by increased intracellular Ca2+ ([Ca2+]i) and activation of the E3 ubiquitin ligase Nedd4-2 (neural precursor-cell expressed developmentally downregulated 4-2). First, Luo et al. provide evidence that increased [Ca2+]i by a Ca2+ ionophore downregulates NaV1.5 protein levels and INa in both neonatal rat cardiomyocytes and HEK293 cells and that these effects are restored by a Ca2+ chelator. Second, they show that reduction in INa and NaV1.5 protein levels is not observed in cells transfected with a Nedd4-2 catalytically inactive mutant or with the Y1977A-NaV1.5 mutant containing an impaired Nedd4-2 binding motif. They also detect that elevated [Ca2+]i increases Nedd4-2 expression levels, interaction between Nedd4-2 and NaV1.5, and NaV1.5 ubiquitination. Finally, they observe an upregulation of Nedd4-2 expression and downregulation of NaV1.5 protein levels in hearts from volume-overload HF rats. The same effects are observed in neonatal rat cardiomyocytes treated with isoproterenol or angiotensin II to induce hypertrophy and can be rescued with a Ca2+ chelator. It is well established that protein modification by polyubiquitin chains targets nuclear, cytosolic and membrane proteins of the endoplasmic reticulum for degradation by the 26S proteasome. On the contrary, a single ubiquitin moiety, that is, monoubiquitination, typically serves as an internalization signal of transmembrane proteins, such as signalling receptors, for their subsequent transport to the lysosome and degradation. The study by Luo et al. comes up with the conclusion that the ubiquitin ligase Nedd4-2 mediates NaV1.5 downregulation via the proteasome. It has been proposed that ion channels can be regulated by ubiquitination, including through the proteasome. This idea has already been addressed in various model systems. Indeed, NaV1.5 degradation via the ubiquitin-proteasome pathway has been shown in rat cardiomyocytes.3 In addition, reduced NaV1.5 expression in cardiomyocytes of dystrophin-deficient mice is dependent on proteasomal degradation; however, that work concluded that the proteasome is more likely implicated indirectly in regulating NaV1.5 levels and turnover.4 Thus, rather than directly degrading transmembrane proteins, the proteasome probably regulates stability of proteins of the endocytic machinery, which in turn affects the endocytosis rate of surface proteins and their trafficking to the lysosome. Alternatively, the components of the proteasome can themselves be regulated by the endocytic machinery. This may be exemplified in regulation of surface expression of the human ether-a-go-go-related gene (hERG), a potassium channel implicated in cardiomyocyte repolarization. In this regard, a model was proposed in which the small GTPase Rab4 increases Nedd4-2 levels by facilitating endosome recycling of Nedd4-2, consequently reducing its degradation. This in turn would cause a reduction in mature ion channels at the plasma membrane.5 Perhaps, as done in recent studies, genome editing combined with proteomic approaches could be used in the future to identify and distinguish between mono- and polyubiquitinated substrates headed to the proteasome instead of to the lysosome. The activity of Nedd4-2 ubiquitin ligase is modulated in response to different cellular stimuli. One of the well-established mechanisms is phosphorylation of Nedd4-2 by the serum- and glucocorticoid-inducible kinase SGK1, which inhibits Nedd4-2 activity, thus leading to increased INa. In addition, previous reports also showed that the C2 domain of Nedd4 E3 ubiquitin ligases functions as an autoinhibitory domain of their catalytic activity. Ca2+, by binding to and releasing the C2 domain-mediated autoinhibition, enhances E3 ligase activity of Nedd4 family members, including Nedd4-2.6 Luo et al. now propose another level of regulation of Nedd4-2 function induced by increased [Ca2+]i. Their experiments show that elevated [Ca2+]i leads to an increase in endogenous Nedd4-2 mRNA and protein levels. They also observe increased Nedd4-2 expression in left ventricle samples from a volume-overload HF rat model. Based on these results, they suggest that [Ca2+]i may also enhance transcription of the Nedd4l (Nedd4-2) gene, probably via an indirect mechanism. Ca2+ signalling has already been shown to regulate gene transcription in various cell types, including cardiomyocytes. In this process, molecules involved in excitation–contraction (EC) coupling activate Ca2+-dependent transcription factors (TFs) in a mechanism known as excitation–transcription coupling (ET coupling). In the adult heart, ET coupling is enhanced by hormonal/mechanical stress, which results in cardiac remodelling and hypertrophy. In particular, Ca2+/Calmodulin (CaM) has been shown to initiate a signalling cascade that leads to activation of the pro-hypertrophic TFs MEF2 and GATA4 via two different mechanisms: (i) phosphorylation of histone deacetylases 4 and 5 by CaM Kinase II (CaMKII), thereby promoting their translocation to the cytosol and the release and activation of MEF2; and (ii) dephosphorylation of the NFAT TF by calcineurin, which leads to NFAT translocation into the nucleus and activation of GATA4.7 Although the mechanisms that regulate transcription of Nedd4l still remain unclear, the results reported by Luo et al. suggest that Ca2+-activated TFs may be involved. Yet, future studies would be needed to further define the molecular mechanisms that lead to increased Nedd4l transcription by Ca2+. Interestingly, a recent study shows that miR-1, a highly conserved microRNA that plays a key role during cardiogenesis as well as post-natal cardiac function, downregulates Nedd4-2 expression. Given the electrophysiological abnormalities seen in miR-1 null mice, and the reported effect of murine Nedd4-2 in downregulation of cell surface ion channels in cardiomyocytes, the study proposes that the interaction of miR-1 with Nedd4-2 might influence heart excitability and connectivity in humans, including susceptibility to cardiac arrhythmias and sudden death.8 Human HF is characterized by action potential prolongation and arrhythmias. It has been suggested that a possible cause could be a decreased INa peak along with increased NaV1.5-generated late INa (INaL). The latter likely provides an important contribution to the reported increase in intracellular Na+ concentration in HF (Fig. 1). Indeed, long-term consequences of Na+ loading can arise from excessive Na+ entry during electrical activity. It is thought that excessive Na+ entry leads to Ca2+ loading through the Na+/ Ca2+ exchanger (NCX). As the Na+ gradient becomes insufficient to maintain the Ca2+ gradient, [Ca2+]i increases, and beyond a threshold, it triggers repetitive release of Ca2+ from the sarcoplasmic reticulum (SR). This process is a pathological version of the Ca2+-induced Ca2+ release process.9 On the other hand, a potential mechanism proposed for the increased INaL and Na+ influx is by CaMKII signalling.10 How such effects act on NaV1.5 are still under debate, similarly to regulation by protein kinase A, or by Ca2+-sensitive protein kinase C isoforms.1 This is illustrated by the many inconsistent results found in studies analysing the functional consequences of a reported direct interaction between CaM and the IQ motif of NaV1.5. An additional unsolved question in the field is whether these variations in [Ca2+]i are independent from the oscillations taking place during the contraction–relaxation cycles. In summary, the work by Luo and colleagues proposes yet one more potential mechanism for an effect on NaV1.5 due to [Ca2+]i increase, which could well be associated with HF, thereby opening a possible link between [Ca2+]i levels and altered INa in HF cardiomyocytes via regulation of NaV1.5 turnover. We have no conflict of interest