The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15%) isoform and α2 comprising the bulk of the Na/K-ATPase pool. The acute and isoform-specific role of α2 in muscle performance and resistance to fatigue is well known, but the isoform-specific role of α1 has not been as thoroughly investigated. In vitro, we reported that α1 has a role in promoting cell growth that is not supported by α2. To assess whether α1 serves this isoform-specific trophic role in the skeletal muscle, we used Na/K-ATPase α1-haploinsufficient (α1) mice. A 30% decrease of Na/K-ATPase α1 protein expression without change in α2 induced a modest yet significant decrease of 10% weight in the oxidative soleus muscle. In contrast, the mixed plantaris and glycolytic extensor digitorum longus weights were not significantly affected, likely because of their very low expression level of α1 compared with the soleus. The soleus mass reduction occurred without change in total Na/K-ATPase activity or glycogen metabolism. Serum analytes including K, fat tissue mass, and exercise capacity were not altered in α1 mice. The impact of α1 content on soleus muscle mass is consistent with a Na/K-ATPase α1-specific role in skeletal muscle growth that cannot be fulfilled by α2. The preserved running capacity in α1 is in sharp contrast with previously reported consequences of genetic manipulation of α2. Taken together, these results lend further support to the concept of distinct isoform-specific functions of Na/K-ATPase α1 and α2 in skeletal muscle.

Aim: Highly prevalent diseases such as insulin resistance and heart failure are characterized by reduced metabolic flexibility and reserve. We tested whether Na/K-ATPase (NKA)-mediated regulation of Src kinase, which requires two NKA sequences specific to the α1 isoform, is a regulator of metabolic capacity that can be targeted pharmacologically. Methods: Metabolic capacity was challenged functionally by Seahorse metabolic flux analyses and glucose deprivation in LLC-PK1-derived cells expressing Src binding rat NKA α1, non-Src-binding rat NKA α2 (the most abundant NKA isoform in the skeletal muscle), and Src binding gain-of-function mutant rat NKA α2. Mice with skeletal muscle-specific ablation of NKA α1 (skα1−/−) were generated using a MyoD:Cre-Lox approach and were subjected to treadmill testing and Western diet. C57/Bl6 mice were subjected to Western diet with or without pharmacological inhibition of NKA α1/Src modulation by treatment with pNaKtide, a cell-permeable peptide designed by mapping one of the sites of NKA α1/Src interaction. Results: Metabolic studies in mutant cell lines revealed that the Src binding regions of NKA α1 are required to maintain metabolic reserve and flexibility. Skα1−/− mice had decreased exercise endurance and mitochondrial Complex I dysfunction. However, skα1−/− mice were resistant to Western diet-induced insulin resistance and glucose intolerance, a protection phenocopied by pharmacological inhibition of NKA α1-mediated Src regulation with pNaKtide. Conclusions: These results suggest that NKA α1/Src regulatory function may be targeted in metabolic diseases. Because Src regulatory capability by NKA α1 is exclusive to endotherms, it may link the aerobic scope hypothesis of endothermy evolution to metabolic dysfunction.

The N-terminal caveolin binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared to the parental wild type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated iSkm (induced Skm) cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells’ ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of β-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/β-catenin signaling.

Systemic levels of endogenous cardiotonic steroids (CTS) increase markedly during salt loading, volume expansion, and renal insufficiency, suggesting a physiological role in the regulation of renal Na+ handling. Rather than the classic CTS‐mediated inhibition of Na+/K+‐ATPase (NKA)‐mediated ion transport in the renal proximal tubule (RPT), in vitro pharmacological approaches have suggested that low concentrations of CTS (in the range of those reported in the blood) may initiate NKA/Src‐mediated signaling to reduce apical Na+/H+ Exchanger‐3 (NHE3) and transepithelial Na+ flux in the RPT. To obtain genetic evidence of this putative NKA/Src mechanism in the RPT and asses its physiological impact, we used a knockdown and rescue approach in pig renal epithelial cells (LLC‐PK1) and generated a PT‐specific NKA α1 knockout mouse (RPTα1−/−) by crossing SGLT2 (sodium glucose co‐transporter 2)‐Cre mice with Floxed Atp1a1 mice. A SGLT2‐Cre/Rosa 26 system was then used to re‐introduce expression of wild‐type NKA α1 (RPT α1WT) or a Src‐null mutant NKA α1Y260A (RPTα1Y260A) with intact ion‐pumping. In cells with 90% NKA α1 knockdown compared to the parent LLC‐PK1 cell line, we observed a 50% decrease in phosphorylated NHE3 (inactive form) without change in total NHE3, and a 50% increase in total Sodium‐Bicarbonate cotransporter‐1A (NBCe1A) expression. Comparable NHE3 activation with NBCe1A increase was observed when NKA α1 knockdown cells were rescued with a Src‐binding NKA α1 null‐mutant or non‐src binding NKA α2, but not with Src‐binding gain‐of‐function α2 mutant or the WT NKA α1, suggesting a role for NKA/Src receptor function in the tonic inhibition of NHE3. RPT‐specific KO and rescue confirmed by immunohistochemistry in kidney cross‐section from RPTα1−/−, RPTα1WT, and RPTα1Y260A mice did not alter kidney size, morphology or overall structure as assessed by periodic acid shift (PAS) and Masson’s trichrome staining. Western blot analyses of RPTα1−/− cortex indicated a decrease in phosphorylated NHE3 with no change in total NHE3 and an increase in NBCe1A expression comparable to those observed in vitro. Functionally, a 65% decrease in daily urine output and absolute Na+ excretion was observed in RPTα1−/− mice (n=6–10). Consistent with a NKA‐dependent tonic inhibition of NHE3, increased PT Na+ reabsorption was indicated by a 65% decrease in urinary lithium clearance in RPTα1−/−, with no change in glomerular filtration rate measured by FITC‐sinistrin clearance (n=10–12). The absolute Na+ excretion was rescued in the RPTα1WT but not in the RPTα1Y260A mouse, which supported the role of NKA α1/Src signaling in the PT Na+ reabsorption (n=5). These studies reveal a novel mechanism of tonic inhibition of NHE3 and NBCe1A by Atp1a1. In vitro results provide genetic evidence that NKA/Src receptor function is critical to this mechanism, which was corroborated in vivo. Animal studies further indicate a significant physiologically impact of this hitherto unrecognized regulation of Na+ reabsorption in the PT, which may be regulated by endog...

In the renal proximal tubule (PT), Na+/K+‐ATPase (NKA) is exclusively located in the basolateral domain. Through its classic ATP‐dependent ion‐pumping function, NKA generates the Na+ gradient that drives apical Na+ reabsorption, mostly through Na+/H+ exchanger (NHE3). Accordingly, activation of NKA‐mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical Na+ reabsorption (NHE3). In contrast, pharmacological evidence suggests that activation of the more recently discovered NKA signaling function triggers a cellular redistribution of NKA and NHE3 that decreases transcellular Na+ flux in cultured PT cells. To obtain genetic evidence of this NKA/Src mechanism in the PT and asses its physiological importance, we used a knockdown and rescue approach in pig renal epithelial cells (LLC‐PK1). Additionally, we genetically targeted NKA α1 in the mouse PT by crossing mice expressing a sodium glucose co‐transporter 2 promoter driven Cre transgene with Floxed NKA α1 mice (PTα1‐/‐). Knockdown of 90% of NKA α1 in PT LLC‐PK1 cells increased transepithelial 22Na flux by 2‐fold, activated NHE3 (50% decrease in inhibitory phosphorylation), and increased basolateral Na+/HCO3‐ cotransporter (NBCe1A) protein content. In the PTα1‐/‐ mouse (4‐month males and females in a 1:1 ratio), 70% decrease in PT NKA α1 expression decreased urine output (0.51±0.14 vs 1.57±0.21 mL/24h in PTα1+/+, p<0.001, n=16) and absolute Na+ excretion (0.14±0.05 vs 0.36±0.05 mmol/24h in PTα1+/+, p<0.05, n=8) by 65%, without histological or functional evidence of renal injury. Those changes were driven by increased PT Na+ reabsorption, as indicated by a 65% decrease in lithium clearance (4‐month males, 1344±220 vs 3932±697 mL/24h in PTα1+/+, p<0.001, n=12) with unchanged GFR. This hyper‐reabsorptive phenotype of PTα1‐/‐ mice was coupled to increased membrane abundance of NHE3 and NBCe1A, and rescued upon crossing with floxed NHE3 mice, consistent a NKA/NHE3‐dependent mechanism. A dismantlement of caveolar NKA/Src receptor complex and intracellular redistribution of pY418Src occurred in knockdown NKA α1 PT cells, and was also observed in the PTα1‐/‐ hypomorphic mouse. Rescue of PT cells with wild‐type but not Src signaling‐null NKA α1 restored NHE3 and NBCe1A to basal levels, indicative of a role for NKA/Src receptor function in the tonic inhibition of Na+ transporters in the PT. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, which is lifted upon NKA genetic suppression in cells and in vivo, tonically counteracts NKA's ATP‐driven function of basolateral Na+ reabsorption. Strikingly, NKA/Src signaling is not only physiologically relevant, it is functionally dominant over NKA ion‐pumping in the control of PT reabsorption. NKA signaling therefore provides a long sought‐after mechanism for the natriuretic action of endogenous NKA ligands such as cardiotonic steroids.