Isoform‐Specific Role of Na/K‐ATPase α1 in Skeletal Muscle Growth and Performance
Authors: Kutz Laura C , Mukherji Shreya , Marck Pauline , Cui Xiaoyu , Heiny Judith A , Blanco Gustavo , Pierre Sandrine V , Xie ZiJian 
Published: November 2023

The Na/K‐ATPase is vital to the maintenance of the membrane potential, especially in excitable tissues such as skeletal muscle. Additionally, recently published data from our lab revealed a role for the α1 but not α2 isoform in regulating cell growth in vitro. The importance of these distinct pumping and signaling roles in tissues expressing both α1 and α2 in vivo has not been established, in part due to a lack of appropriate models. This issue was investigated in the most abundant tissue with dual expression of α1 and α2 isoforms, the skeletal muscle. In this study, we tested whether deletion of Na/K‐ATPase α1, which only represent 13% of the total skeletal muscle Na/K‐ATPase pool, would impact muscle size and exercise performance. We used the MyoDiCre‐lox system to excise exons 15–18 of the ATPA1 gene exclusively in skeletal muscle. This resulted in viable and fertile mice. Deletion of ATP1A1 in skeletal muscle but not in heart, kidney, or brown adipose tissue of α1 KO mice was confirmed using PCR. Western blot analysis of α1 expression revealed that skeletal muscle expression in skα1 KO muscles was decreased to about 15% of that in control littermates (relative expression 1.00±0.11 in controls vs 0.15±0.04 in skα1 KO, p<0.0001, n=8–10), while α2 isoform expression was unaltered. Male and female skα1 KO mice exhibited normal growth as estimated by body weight at 16 weeks. The mass/tibia length ratio of the gastrocnemius muscle was decreased by 41% in 16 week old female skα1 KO mice (5.8±0.7 mg/g vs 3.4±0.3 mg/g, p=0.02, n=4). Twelve week old female skα1 KO mice and control littermates were subjected to a treadmill exercise testing protocol. Ability to tolerate high speeds was assessed by increasing speed by 2 m/min every 2 minutes up to 25 m/min. To measure endurance, speed was maintained at 25 m/min for up to 3 hours (4.8 km), and distance at fatigue was recorded for each animal. Running was encouraged with a shock grid on the platform, and the number of shocks administered to each animal was recorded each minute. Fatigue was defined as remaining on shock grid for 10 seconds. In spite of severely decreased muscle mass, all mice exhibited normal running at speeds of up to 25 m/s. In contrast, distance to fatigue decreased by 56% in skα1 KO mice (1.56±0.22 km vs 0.81±0.14 km, p<0.05, n=11–16). Because α1 signaling has been reported to increase skeletal muscle glycogen stores and glycogen storage is closely correlated with endurance capacity, we then measured total glycogen content in the gastrocnemius muscle of female mice. This revealed that skα1 KO muscles contained 50% less glycogen than controls (1.19±0.16 μg/mg vs 0.60±0.09 μg/mg, p=0.03, n=3). These data suggest unique roles for Na/K‐ATPase α1 isoform in skeletal muscle for which the α2 isoform does not compensate. The skeletal muscle specific α1 knockout mouse described here will be used in future studies to elucidate the distinct roles of the pumping and signaling functions of α1 and how they cause these physiological changes in muscle mass and performance.