The epithelial Na ؉ channel (ENaC) functions as a pathway for epithelial Na ؉ transport, contributing to Na ؉ homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na ؉ transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na ؉ transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na ؉ transport.The transport of Na ϩ across epithelia in the distal nephron, colon, and airway is critical to maintain Na ϩ homeostasis and to control the composition of airway surface liquid. Defects in Na ϩ transport are responsible for genetic and acquired forms of hypertension and hypotension (1) and contribute to the pathogenesis of cystic fibrosis (2-4). Na ϩ transport is mediated by the epithelial Na ϩ channel (ENaC), 1 which forms a pathway for Na ϩ to cross the apical membrane (5, 6). At the basolateral membrane Na ϩ exits the cell via the Na ϩ -K ϩ -ATPase, resulting in transepithelial Na ϩ absorption. Nedd4-2, an E3 ubiquitin-protein ligase, is critical in the control of Na ϩ transport. Nedd4-2 inhibits Na ϩ transport by catalyzing ENaC ubiquitination and degradation, resulting in reduced ENaC expression at the cell surface (for review, see Refs. 7-9). This requires binding of multiple Nedd4-2 WW domains to PY motifs (PPPXYXXL) located in the C termini of each ENaC subunit (␣, ␤, and ␥). Disruption of this interaction by mutations that delete or alter the PY motifs increases ENaC surface expression and causes Liddle's syndrome, an inherited form of hypertension (10 -14).Recent work suggests that Nedd4-2 is negatively regulated by serum and glucocorticoid-inducible kinase (SGK), a Ser/Thr kinase. SGK binds to Nedd4-2 and phosphorylates it at three sites (see Fig. 3C), which reduces Nedd4-2 binding to ENaC (15, 16). Similar to Liddle's syndrome, decre...

Background Cardiovascular and arrhythmic events have been reported in hospitalized COVID-19 patients. However, arrhythmia manifestations and treatment strategies used in these patients have not been well-described. We sought to better understand the cardiac arrhythmic manifestations and treatment strategies in hospitalized COVID-19 patients through a worldwide crosssectional survey. Methods The Heart Rhythm Society (HRS) sent an online survey (via SurveyMonkey) to electrophysiology (EP) professionals (physicians, scientists, and allied professionals) across the globe. The survey was active from March 27 to April 13, 2020. Results A total of 1197 respondents completed the survey with 50% of respondents from outside the USA, representing 76 countries and 6 continents. Of respondents, 905 (76%) reported having COVID-19-positive patients in their hospital. Atrial fibrillation was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common bradyarrhythmias. Ventricular tachycardia/ventricular fibrillation arrest and pulseless electrical activity were reported by 4.8% and 5.6% of respondents, respectively. There were 140 of 631 (22.2%) respondents who reported using anticoagulation therapy in all COVID-19-positive patients who did not otherwise have an indication. One hundred fifty-five of 498 (31%) reported regular use of hydroxychloroquine/chloroquine (HCQ) + azithromycin (AZM); concomitant use of AZM was more common in the USA. Sixty of 489 respondents (12.3%) reported having to discontinue therapy with HCQ + AZM due to significant QTc prolongation and 20 (4.1%) reported cases of Torsade de Pointes in patients on HCQ/chloroquine and AZM. Amiodarone was the most common antiarrhythmic drug used for ventricular arrhythmia management. Conclusions In this global survey of > 1100 EP professionals regarding hospitalized COVID-19 patients, a variety of arrhythmic manifestations were observed, ranging from benign to potentially life-threatening. Observed adverse events related to use of HCQ + AZM included prolonged QTc requiring drug discontinuation as well as Torsade de Pointes. Large prospective studies to better define arrhythmic manifestations as well as the safety of treatment strategies in COVID-19 patients are warranted.

As a pathway for Na؉ reabsorption, the epithelial Na ؉ channel ENaC is critical for Na ؉ homeostasis and blood pressure control. Na ؉ transport is regulated by Nedd4-2, an E3 ubiquitin ligase that decreases ENaC expression at the cell surface. To investigate the underlying mechanisms, we proteolytically cleaved/activated ENaC at the cell surface and then quantitated the rate of disappearance of cleaved channels using electrophysiological and biochemical assays. We found that cleaved ENaC channels were rapidly removed from the cell surface. Deletion or mutation of the Nedd4-2 binding motifs in ␣, ␤, and ␥ENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXXØ endocytosis motif had no effect. ENaC endocytosis was also decreased by silencing of Nedd4-2 and by expression of a dominant negative Nedd4-2 construct. Conversely, Nedd4-2 overexpression increased ENaC endocytosis in human embryonic kidney 293 cells but had no effect in Fischer rat thyroid epithelia. In addition to its effect on endocytosis, Nedd4-2 also increased the rate of degradation of the cell surface pool of cleaved ␣ENaC. Together the data indicate that Nedd4-2 reduces ENaC surface expression by altering its trafficking at two distinct sites in the endocytic pathway, inducing endocytosis of cleaved channels and targeting them for degradation.The epithelial Na ϩ channel ENaC forms a pathway for Na ϩ reabsorption across epithelia, including the kidney, lung, and colon. Therefore, it plays a critical role in Na ϩ homeostasis and blood pressure control (reviewed in Refs. 1 and 2). Defects in ENaC function or regulation cause inherited forms of hypertension and hypotension (3) and may contribute to the pathogenesis of lung disease in cystic fibrosis (4).ENaC is regulated by Nedd4-2, a HECT domain E3 ubiquitin ligase that decreases ENaC expression at the cell surface (5-7). This regulation requires the binding of Nedd4-2 WW domains to PY motifs (PPPXYXXL) located in the C terminus of each of the three subunits that form the channel (␣, ␤, and ␥ENaC). Mutations in the PY motifs of ␤ or ␥ENaC disrupt binding, causing Liddle syndrome (8 -10). In this inherited form of hypertension, increased expression of ENaC at the cell surface results in excessive renal Na ϩ reabsorption (7,8,11). Binding is also modulated by aldosterone and vasopressin via serum and glucocorticoid-regulated kinase and protein kinase A, respectively; both kinases phosphorylate Nedd4-2, which reduces its binding to ENaC (6,12,13). However, the mechanism by which Nedd4-2 reduces ENaC surface expression is uncertain. It is possible that Nedd4-2 regulates ENaC trafficking in the biosynthetic pathway, targeting it for degradation in the proteasome. Consistent with this model, localization of Nedd4-2 at the cell surface is not required for Nedd4-2 to inhibit ENaC (14). Moreover, proteasome inhibitors decrease ENaC degradation (15-17) and increase ENaC surface expression.2 Alternatively, Nedd4-2 could regulate ENaC in the endocytic pathway, altering ENaC endocytosis and/or...

Cross-clamp time >60 min and concomitant mitral valve surgery were independent predictors of PPM implantation following TVR. Long-term PPM dependency is more prevalent after TVR than other types of valvular surgery.