Digoxin and exercise effects on Na+,K+-ATPase isoform gene and protein expression in human skeletal muscle

This laboratory has shown that exercise in humans impairs skeletal muscle Na+,K+-ATPase maximal in vitro activity, whilst in isolated rat muscles, Na+,K+-ATPase inhibition with ouabain leads to early muscle fatigue. Hence, Na+,K+-ATPase function is likely to be important for skeletal muscle performance. Digoxin is a specific inhibitor of the Na+,K+- ATPase and is used to treat patients with severe heart failure. This thesis investigated whether in-vivo inhibition of Na+,K+-ATPase by digoxin adversely effected muscle performance and Na+,K+-ATPase isoform expression and protein abundance in skeletal muscle of healthy individuals. Ten active, but not well-trained healthy volunteers (9 M, 1 F) gave written informed consent. Subjects performed incremental cycle ergometer exercise to measure VO2peak and to determine 33, 67 and 90% VO2peak workrates. Exercise tests were performed after taking digoxin (DIG, 0.25 mg.d-1) or a placebo (CON) for 13 day (Cybex) or 14 day (cycling), in a randomised, counterbalanced, cross-over, double-blind design, with trials separated by at least 6 weeks. On day 13 subjects performed tests of quadriceps muscle strength and endurance of the dominant leg, on a Cybex isokinetic dynamometer ( Cybex Norm 770, Henley Healthcare, USA). On day 14 subjects completed 10 min cycling at each of 33% and 67% VO2peak, then to fatigue at 90% VO2peak on cycle ergometer (Lode Excalibur, Groningen, the Netherlands), with arterial blood sampling for plasma [K+] determinations. A muscle biopsy was taken at rest, after exercise at 67% and 90%VO2peak and at 3 hr recovery. Muscle was analysed for Na+, K+-pump isoform (alpha1-alpha3, beta1-beta3) mRNA expression (real-time RT-PCR, GeneAmp 7500 Sequence Detection System) and whole homogenate protein abundance (immunoblotting, Kodak Digital Science Image Station 400CF, Eastman Kodak Company, CT, USA). Serum digoxin was 0.7±0.1 nM at day 13 and 0.8±0.1 nM at day 14 (Mean±SEM) and was less than the lowest detection limit of 0.4 nM in control trials. There were no differences in VO2 or time to fatigue (DIG 262±156 vs CON 254 ±125 s) between DIG and CON during exercise. Arterial plasma [K+] increased above rest at 67% VO2peak and increased further at fatigue (P<0.05). No significant differences were found in [K+] between DIG and CON. Peak torque during dynamic isokinetic contractions was less at each increasing velocity (P<0.05). No differences were found in muscle strength between DIG and CON. Similarly, there were no differences in the leg extensor fatigue index between trials (DIG 0.54±0.03 vs CON 0.57±0.03). The mRNA expression of the alpha1, alpha2, alpha3, beta1, beta1 or beta3 isoforms was not significantly changed by DIG. However, DIG increased the total alpha mRNA expression (sum of alpha1, alpha2, alpha3) and also the total beta mRNA expression (sum of beta1, beta2 and beta3) at rest by 1.9- and 0.6-fold, respectively (P<0.05). An exercise effect was observed for alpha3 mRNA expression, which was 2.1-and 2.4-fold higher at 3 h post-exercise, than during exercise at 67% VO2peak and fatigue, respectively (P<0.05). Similarly, beta3 mRNA expression was increased at 3 h post-exercise by 1.8-, 1.4- and 1.6-fold, compared to rest, 67% VO2peak exercise and fatigue, respectively (P<0.05). No other significant changes with exercise or recovery were seen in mRNA expression. In resting muscle, protein relative abundance was not significantly changed by digoxin for alpha1 (1.14±0.19, P=0.50), alpha3 (1.12±0.18, P=0.52), beta1 (1.19±0.18, P=0.32), beta2 (1.86±0.57, P=0.17), beta3 (0.85±0.17, P=0.39) compared to control (1.00) although a tendency was observed for an increase in alpha2 with DIG (1.44±0.23, P=0.096). However, exercise affected both beta1 and beta3 isoform protein abundance. The beta1 protein abundance was increased at 3 h post-exercise by 2.2-and 1.5-fold compared to during exercise at 67% VO2peak and fatigue, respectively (P<0.05). Similarly, beta3 protein abundance was increased at 67% VO2peak and 3 h post-exercise compared to rest, by 1.5-and 1.6-fold, respectively (P