Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples
Jannas-Vela, Sebastian1,2; Brownell, Stuart1; Petrick, Heather L.1; Heigenhauser, George J.F.3; Spriet, Lawrence L.1; Holloway, Graham P.1

Medicine & Science in Sports & Exercise: June 5, 2019 - Volume Publish Ahead of Print - Issue - p
doi: 10.1249/MSS.0000000000002063
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Introduction In skeletal muscle, the Na+/K+ ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism, however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle.

Methods Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca2+ ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize ‘background noise’.

Results We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as addition of 25 μM of blebbistatin (BLEB), a specific myosin ATPase inhibitor, considerably minimized ‘background noise’ (3-fold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. Specificity of the assay was demonstrated after addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, addition of BLEB did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively.

Conclusion The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

© 2019 American College of Sports Medicine