Digoxin and exercise effects on skeletal muscle Na⁺,K⁺‐ATPase isoform gene expression in healthy humans

In muscle, digoxin inhibits Na⁺,K⁺-ATPase (NKA) whereas acute exercise can increase NKA gene expression, consistent with training-induced increased NKA content. We investigated whether oral digoxin increased NKA isoform mRNA expression (qPCR) in muscle at rest, during and post-exercise in 10 healthy adults, who received digoxin (DIG, 0.25 mg per day) or placebo (CON) for 14 days, in a randomised, double-blind and cross-over design. Muscle was biopsied at rest, after cycling 20 min (10 min each at 33%, then 67% <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics> <mml:msub><mml:mover><mml:mi>V</mml:mi> <mml:mo>̇</mml:mo></mml:mover> <mml:mrow><mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> <mml:mi>peak</mml:mi></mml:mrow> </mml:msub> <mml:annotation>${{\dot{V}}_{{{{\mathrm{O}}}_2}{\mathrm{peak}}}}$</mml:annotation></mml:semantics> </mml:math> ), then to fatigue at 90% <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics> <mml:msub><mml:mover><mml:mi>V</mml:mi> <mml:mo>̇</mml:mo></mml:mover> <mml:mrow><mml:msub><mml:mi>O</mml:mi> <mml:mn>2</mml:mn></mml:msub> <mml:mi>peak</mml:mi></mml:mrow> </mml:msub> <mml:annotation>${{\dot{V}}_{{{{\mathrm{O}}}_2}{\mathrm{peak}}}}$</mml:annotation></mml:semantics> </mml:math> and 3 h post-exercise. No differences were found between DIG and CON for NKA α_1-3 or β_1-3 isoform mRNA. Both α₁ (354%, P = 0.001) and β₃ mRNA (P = 0.008) were increased 3 h post-exercise, with α₂ and β_1-2 mRNA unchanged, whilst α₃ mRNA declined at fatigue (-43%, P = 0.045). In resting muscle, total β mRNA (∑(β₁+β₂+β₃)) increased in DIG (60%, P = 0.025) and also when transcripts for each isoform were normalised to CON then either summed (P = 0.030) or pooled (n = 30, P = 0.034). In contrast, total α mRNA (∑(α₁+α₂+α₃), P = 0.348), normalised then summed (P = 0.332), or pooled transcripts (n = 30, P = 0.717) did not differ with DIG. At rest, NKA α_1-2 and β_1-2 protein abundances were unchanged by DIG. Post-exercise, α₁ and β_1-2 proteins were unchanged, but α₂ declined at 3 h (19%, P = 0.020). In conclusion, digoxin did not modify gene expression of individual NKA isoforms at rest or with exercise, indicating NKA gene expression was maintained consistent with protein abundances. However, elevated resting muscle total β mRNA with digoxin suggests a possible underlying β gene-stimulatory effect. HIGHLIGHTS: What is the central question of this study? Na⁺,K⁺-ATPase (NKA) in muscle is important for Na⁺/K⁺ homeostasis. We investigated whether the NKA-inhibitor digoxin stimulates increased NKA gene expression in muscle and exacerbates NKA gene responses to exercise in healthy adults. What is the main finding and its importance? Digoxin did not modify exercise effects on muscle NKA α_1-3 and β_1-3 gene transcripts, which comprised increased post-exercise α₁ and β₃ mRNA and reduced α₃ mRNA during exercise. However, in resting muscle, digoxin increased NKA total β isoform mRNA expression. Despite inhibitory-digoxin or acute exercise stressors, NKA gene regulation in muscle is consistent with the maintenance of NKA protein contents.