Intense exercise up‐regulates Na⁺,K⁺‐ATPase isoform mRNA, but not protein expression in human skeletal muscle

Abstract

Characterization of expression of, and consequently also the acute exercise effects on, Na + ,K + -ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at ∼40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na + ,K + -ATPase α 1 , α 2 , α 3 , β 1 , β 2 and β 3 mRNA and crude homogenate protein expression, using Real-Time RT-PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na + ,K + -ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 ± 69 s; mean ± S.E.M.) immediately increased α 3 and β 2 mRNA by 2.4-and 1.7-fold, respectively (P < 0.05), whilst α 1 and α 2 mRNA were increased by 2.5-and 3.5-fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for β 1 and β 3 mRNA, reflecting variable time-dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for α 1 , α 2 , α 3 , β 1 , β 2 and β 3 isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0-and 1.0-fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the α 1 -α 3 and β 1 -β 3 isoforms. Thus, human skeletal muscle expresses each of the Na + ,K + -ATPase α 1 , α 2 , α 3 , β 1 , β 2 and β 3 isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na + ,K + -ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na + ,K + -ATPase up-regulation.

Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

“…Likewise, an increase in PDK4 expression was apparent (35). GAPDH mRNA tended to increase after exercise, and when the changes observed at 0, 1, 3, and 5 h were averaged, a calculation termed average postexercise response in a previous study of mRNA expression (29), GAPDH mRNA expression was higher (P ϭ 0.005) after exercise. The analysis of the relative amount of Na ϩ -K ϩ -ATPase subunit mRNA in resting human skeletal muscle allows us to conclude that the changes in ␣ 2 and ␤ 1 mRNA are quantitatively the most important, even though the fold change for ␣ 1 was the highest.…”

“…The substantial, but typical, variation in mRNA expression data (29,30,33,37) underlines the need for caution when interpreting data where no differences are found. In the present study, we used duplicate RNA isolations from each biopsy specimen and normalization of target mRNA amount to the amount of RNA:cDNA hybrids in the sample used for real-time PCR.…”

“…In that study, expression of ␣ 1 and ␣ 2 protein increased, whereas ␤ 1 was unchanged after a period of training (32). One study has investigated ␣ 3 , ␤ 2 , and ␤ 3 mRNA after intense exercise in humans (29), and increases in mRNA for these subunits as well as for ␣ 1 , ␣ 2 , and ␤ 1 were found, whereas no change in protein expression was detected. Resistance training for 6 wk has been shown to increase ␣ 1 , ␣ 2 , and ␤ 1 protein expression (9), and 6 days of prolonged cycle training have resulted in a higher ␣ 2 and ␤ 1 but not ␣ 1 protein expression (15).…”

“…When investigating and interpreting the physiological importance of exercise-induced relative changes of the Na ϩ -K ϩ -ATPase subunit mRNA levels, it is important to know the basal subunit-specific abundance. Previously, only fold changes have been reported (29,33). Data on the relative abundance of Na ϩ -K ϩ -ATPase subunit mRNA in human skeletal muscle do not exist, but from rat muscle it is known that ␣ 2 and unspecified ␤ mRNA are the most abundant, followed by ␣ 1 mRNA (34).…”

“…Thus the effect of exercise on Na ϩ -K ϩ -ATPase subunit expression at the protein and mRNA level varies between studies, possibly dependent on the active muscle mass as well as the intensity and duration of the exercise investigated. In addition, methodological variation is seemingly larger in studies of mRNA changes (29,33,37) than the more traditional approach using [ 3 H]ouabain to quantify nonsubunit-specific changes in Na ϩ -K ϩ -ATPase protein expression (8). One mRNA transcript consistently reported to increase with exercise of various types is pyruvate dehydrogenase kinase 4 (PDK4) (35).…”