High-Intensity Training Represses FXYD5 and Glycosylates Na,K-ATPase in Type II Muscle Fibres, Which Are Linked with Improved Muscle K+ Handling and Performance

Abstract

Na⁺/K⁺ ATPase (NKA) comprises several subunits to provide isozyme heterogeneity in a tissue-specific manner. An abundance of NKA α, β, and FXYD1 subunits is well-described in human skeletal muscle, but not much is known about FXYD5 (dysadherin), a regulator of NKA and β1 subunit glycosylation, especially with regard to fibre-type specificity and influence of sex and exercise training. Here, we investigated muscle fibre-type specific adaptations in FXYD5 and glycosylated NKAβ1 to high-intensity interval training (HIIT), as well as sex differences in FXYD5 abundance. In nine young males (23.8 ± 2.5 years of age) (mean ± SD), 3 weekly sessions of HIIT for 6 weeks enhanced muscle endurance (220 ± 102 vs. 119 ± 99 s, p < 0.01) and lowered leg K⁺ release during intense knee-extensor exercise (0.5 ± 0.8 vs. 1.0 ± 0.8 mmol·min^-1, p < 0.01) while also increasing cumulated leg K⁺ reuptake 0-3 min into recovery (2.1 ± 1.5 vs. 0.3 ± 0.9 mmol, p < 0.01). In type IIa muscle fibres, HIIT lowered FXYD5 abundance (p < 0.01) and increased the relative distribution of glycosylated NKAβ1 (p < 0.05). FXYD5 abundance in type IIa muscle fibres correlated inversely with the maximal oxygen consumption (r = -0.53, p < 0.05). NKAα2 and β1 subunit abundances did not change with HIIT. In muscle fibres from 30 trained males and females, we observed no sex (p = 0.87) or fibre type differences (p = 0.44) in FXYD5 abundance. Thus, HIIT downregulates FXYD5 and increases the distribution of glycosylated NKAβ1 in type IIa muscle fibres, which is likely independent of a change in the number of NKA complexes. These adaptations may contribute to counter exercise-related K⁺ shifts and enhance muscle performance during intense exercise.

Keywords: ATPase; FXYD; NKA; Na-K; beta; dysadherin; isoform; phospholemman; pump; subunit.

Figure 1

Exercise-related K⁺ shifts during and 0–3 min into recovery from one-legged knee extensor exercise before (PRE) and after (POST) 6 weeks of high-intensity interval training (HIIT) 3 times weekly in young males (n = 9). (A,B) Femoral venous (A) and arterial (B) plasma K⁺ concentrations. (C) Leg K⁺ release and uptake. Data are presented as mean ± SD. * POST different from PRE (p < 0.05). ^## POST recovery values different from PRE (p < 0.01). ^$$ Cumulated leg K⁺ uptake after HIIT different from before (p < 0.01).

Figure 2

Abundance of Na, K-ATPase (NKA) subunits in human muscle type I and IIa fibre pools before (Pre) and after (Post) 6 weeks of high-intensity interval training (HIIT) 3 times weekly in young males (n = 8–9). (A) Frequency distribution of FXYD5 abundance in human type I and IIa muscle fibres before log-transformation. (B) Effect of HIIT on log-FXYD5 abundance. (C) Pearson’s correlation between log-FXYD5 abundance and maximal oxygen consumption (VO_2max). (D,E) Effect of HIIT on log-NKAα2 (D) and log-NKAβ1 abundance (E). Bars represent mean values.

Figure 3

Degree of glycosylation of the Na, K-ATPase (NKA) β1 subunit. (A) A control experiment indicating that the differences between the two upper signals migrating between 40 and 50 kDa are related to the degree of NKAβ1 glycosylation. Human skeletal muscle samples treated with 1 unit N-glycosidase per 40 µg protein (T) or controls without N-glycosidase (C) and a human skeletal muscle lysate (HS) were loaded on a gel, and afterwards, the membrane was incubated with an NKAβ1 antibody. (B) Representative Western blots for the pooled single fibres are shown. The NKAβ1 glycosylation ratio was determined as the ratio between the most glycosylated signal and the least glycosylated signal between 40 and 50 kDa. (C) NKAβ1 glycosylation ratios in human skeletal muscle type I and IIa fibres of healthy young men (n = 8–9) before (PRE) and after (POST) 6 weeks of high-intensity interval training (HIIT) 3 times weekly.

Figure 4

FXYD5 abundance in human skeletal muscle type I and IIa fibres of healthy young females (n = 15) and males (n = 15). (A) Frequency distribution of FXYD5 abundance before log-transformation. (B) Individual data points for both sexes and fibre types. Bars represent sample means.

Figure 5

Dot blotting and FXYD5 antibody validation (HPA010817 and SC-166782). (A) Shows dot blotting of 6 × 5 fibre segments incubated with either an MHC1 or MHC2a antibody. Each dot was evaluated as no signal (□), a weak signal (∆), or a clear signal (○). (B,C) HPA010817 (B) and SC-166782 (C) antibodies showed a clear multiple-band signal (19–40 kDa and 22–40 kDa, respectively) in FXYD5 overexpression samples (OE) compared to the control (C) and human muscle lysates. (D) In the first two lanes, one segment of type I (T1) and type IIa fibre (T2), respectively, was loaded. In the third lane, two pooled type IIa fibres were loaded, while pooled segments from three different fibres were loaded in lane four. In lane five, 3 µg human skeletal muscle lysate was loaded. The membrane was incubated with HPA010817. (E) The stain-free image is shown from loaded human skeletal muscle lysates either non-heated (NH) or heated for 3 min at 96 °C (96 °C) and FXYD5 OE and C samples. (F) Shows the membrane after incubation of the samples from (E) with an anti-actin antibody.