Increased FXYD1 and PGC-1α mRNA after blood flow-restricted running is related to fibre type-specific AMPK signalling and oxidative stress in human muscle

Abstract

Aim: This study explored the effects of blood flow restriction (BFR) on mRNA responses of PGC-1α (total, 1α1, and 1α4) and Na⁺ ,K⁺ -ATPase isoforms (NKA; α_1-3 , β_1-3 , and FXYD1) to an interval running session and determined whether these effects were related to increased oxidative stress, hypoxia, and fibre type-specific AMPK and CaMKII signalling, in human skeletal muscle.

Methods: In a randomized, crossover fashion, 8 healthy men (26 ± 5 year and 57.4 ± 6.3 mL kg^-1 min^-1 ) completed 3 exercise sessions: without (CON) or with blood flow restriction (BFR), or in systemic hypoxia (HYP, ~3250 m). A muscle sample was collected before (Pre) and after exercise (+0 hour, +3 hours) to quantify mRNA, indicators of oxidative stress (HSP27 protein in type I and II fibres, and catalase and HSP70 mRNA), metabolites, and α-AMPK Thr¹⁷² /α-AMPK, ACC Ser²²¹ /ACC, CaMKII Thr²⁸⁷ /CaMKII, and PLBSer¹⁶ /PLB ratios in type I and II fibres.

Results: Muscle hypoxia (assessed by near-infrared spectroscopy) was matched between BFR and HYP, which was higher than CON (~90% vs ~70%; P < .05). The mRNA levels of FXYD1 and PGC-1α isoforms (1α1 and 1α4) increased in BFR only (P < .05) and were associated with increases in indicators of oxidative stress and type I fibre ACC Ser²²¹ /ACC ratio, but dissociated from muscle hypoxia, lactate, and CaMKII signalling.

Conclusion: Blood flow restriction augmented exercise-induced increases in muscle FXYD1 and PGC-1α mRNA in men. This effect was related to increased oxidative stress and fibre type-dependent AMPK signalling, but unrelated to the severity of muscle hypoxia, lactate accumulation, and modulation of fibre type-specific CaMKII signalling.

Keywords: AMP-activated protein kinase; Na+-K+-ATPase; PGC-1α; blood flow restriction; oxidative stress; reactive oxygen species.

Figure 1

NKA‐α‐isoform mRNA responses to moderate‐intensity interval running performed without or with blood flow restriction or in systemic hypoxia. (A) α₁, (B) α₂, and (C) α₃, mRNA content. Individual changes from before (Pre) to 3 hours after exercise (+3 hours) are displayed on the left with each symbol representing one participant across trials and figures. On the right are bars representing mean (±SEM) changes relative to Pre for exercise alone (CON, white; n = 8), with blood flow restriction (BFR, blue; n = 6) or in systemic hypoxia (HYP, grey; n = 5). *P ≤ .05, different from Pre

Figure 2

NKA‐β‐isoform and FXYD1 mRNA responses to moderate‐intensity interval running performed without or with blood flow restriction or in systemic hypoxia. (A) β₁, (B) β₂, (C) β₃ and (D) FXYD1, mRNA content. Individual changes from before (Pre) to 3 hours after exercise (+3 hours) are displayed on the left with each symbol representing one participant across trials and figures. On the right are bars representing mean (±SEM) changes relative to Pre for exercise alone (CON, white; n = 8), with blood flow restriction (BFR, blue; n = 6) or in systemic hypoxia (HYP, grey; n = 5). *P < .05, different from Pre

Figure 3

PGC‐1α total and PGC‐1α‐isoform mRNA responses to moderate‐intensity interval running performed without or with blood flow restriction or in systemic hypoxia. (A) PGC‐1α total, (B) PGC‐1α1 and (C) PGC‐1α4, mRNA content. Individual changes from before (Pre) to 3 hours after exercise (+3 hours) are displayed on the left with each symbol representing one participant across trials and figures. On the right are bars representing mean (±SEM) changes relative to Pre for exercise alone (CON, white; n = 8), with blood flow restriction (BFR, blue; n = 6) or in systemic hypoxia (HYP, grey; n = 5).*P ≤ .05, different from Pre; †P ≤ .05, different from CON and HYP

Figure 4

Changes in muscle hypoxia and indicators of responses to oxidative stress in response to moderate‐intensity interval running performed without or with blood flow restriction or in systemic hypoxia. (A) Muscle hypoxia (ie deoxygenated haemoglobin, Muscle HHb) as assessed by near‐infrared spectroscopy during moderate‐intensity running without (CON, black symbols; n = 8) or with blood flow restriction (BFR, blue symbols; n = 8), or in systemic hypoxia (HYP, grey symbols; n = 8). Hashed bars represent exercise bouts. #P ≤ .05, BFR and HYP different from CON. *P ≤ .05, BFR different from CON. (B) Catalase, and (D) heat‐shock protein 70 (HSP70), mRNA expression. Individual changes from before (Pre) to 3 hours after exercise (+3 hours) are displayed on the left with each symbol representing one participant across trials and figures. On the right are bars representing mean (±SEM) changes relative to Pre for moderate‐intensity running without (CON, white) or with blood flow restriction (BFR, blue), or in systemic hypoxia (HYP, grey). *P ≤ .05, different from Pre. (C) Heat‐shock protein 27 protein content in type I (white bars) and type II fibres (grey bars) at rest before (Pre) and immediately after (+0 hour) exercise. Representative Western blots are indicated above the corresponding bars. *P ≤ .05, increase vs Pre within BFR. #P ≤ .05, BFR different from CON within fibre type. Data are means ± SEM

Figure 5

Changes in muscle ATP and lactate concentration in response to moderate‐intensity interval running performed without (CON) or with blood flow restriction (BFR) or in systemic hypoxia (HYP). (A) ATP, (B) lactate, (C) phosphocreatine (PCr), (D) creatine (Cr) and (E) PCr/Cr ratio before (Pre, white) and immediately after exercise (+0 hour, grey). n = 8 for all conditions. Data are means ± SEM. *P < .05, different from Pre. †P < .05, different from CON

Figure 6

Changes in venous blood lactate, pH and potassium ion (K⁺) concentration in response to moderate‐intensity interval running performed without or with blood flow restriction or in systemic hypoxia. (A) Lactate, (B) pH and (C) K⁺ concentration during moderate‐intensity interval running without (CON, black symbols) or with blood flow restriction (BFR, blue symbols), or in systemic hypoxia (HYP, grey symbols). Hashed bars represent running bouts. n = 8 for all conditions. Data are means ± SEM. *P < .05, different from rest; †P < .05, BFR different from CON; #P < .05, HYP different from CON

Figure 7

Representative blots for AMPKα, ACC, CaMKII and phospholamban (PLB) protein abundance and phosphorylation in type I and II human skeletal muscle fibres. Protein abundance and phosphorylation of (A) AMPK and ACC, and (B) CaMKII and PLB in human skeletal muscle in response to moderate‐intensity interval running without (CON) or with blood flow restriction (BFR), or in systemic hypoxia (HYP) before (Pre) and immediately after (+0 hour) exercise. Total protein was determined in each lane from the stain‐free gel images obtained after electrophoresis. CaMKII isoforms (β_M and σ/γ) are indicated in (B)

Figure 8

Changes in AMPKα and ACC protein abundance and phosphorylation in type I and II human skeletal muscle fibres in response to moderate‐intensity interval running performed without or with blood flow restriction or in systemic hypoxia. (A) AMPKα protein, (B) AMPKα phosphorylation at Thr¹⁷² normalized to AMPKα protein, (C) ACC protein and (D) ACC phosphorylation at Ser⁷⁹ normalized to ACC protein in type I (white bars) and type II (grey bars) fibres before (Pre) and immediately after (+0 hour) exercise. n = 8 for all conditions. Data are means ± SEM. *P < .05, different from rest within condition and fibre type; †P ≤ .05, BFR different from HYP in (A), and from CON in (D)

Figure 9

Changes in CaMKII and phospholamban (PLB) protein abundance and phosphorylation in type I and II human skeletal muscle fibres in response to moderate‐intensity interval running performed without or with blood flow restriction or in systemic hypoxia. (A) CaMKII protein, (B) CaMKII phosphorylation at Thr²⁸⁷ normalized to CaMKII protein, (C) PLB protein and (D) PLB phosphorylation at Ser¹⁶ normalized to PLB protein in type I (white bars) and type II (grey bars) fibres before (Pre) and immediately after (+0 hour) exercise. n = 8 for all conditions. Data are means ± SEM. *P < .05, different from rest within condition and fibre type

Figure 10

Summary of key findings. Effects of moderate‐intensity interval running without (CON) or with blood flow restriction (BFR), or in normobaric, systemic hypoxia (HYP) on the mRNA content of Na⁺,K⁺‐ATPase (NKAα_1‐3, NKAβ_1‐3, FXYD1) and PGC‐1α (total, 1α1, 1α4) isoforms, indicators of responses to oxidative stress (HSP27 protein content in type I and II muscle fibres, catalase and heat‐shock protein 70,HSP70, mRNA content), muscle hypoxia (ie deoxygenated haemoglobin as measured by near‐infrared spectroscopy), lactate concentration, and AMPK and CaMKII signalling in the skeletal muscle of men. “p‐” denotes phosphorylation; ACC, Acetyl‐CoA carboxylase; AMPK, 5′ AMP‐activated protein kinase; CaMKII, Ca²⁺/calmodulin‐dependent protein kinase II; PLB, phospholamban; LT, lactate threshold

Figure 11

Time‐aligned, schematic representation of the experimental design. The participants performed 3 exercise trials separated by 1 week consisting of running without (control) or with the muscle blood flow partially occluded (blood flow restriction, BFR), or in normobaric, systemic hypoxia (hypoxia). The exercise intensity was set according to the participants’ individual lactate threshold (~12 km h⁻¹). Muscle was sampled at rest before, immediately post (+0 hour) and after 3 hours (+3 hours) of recovery from each trial. Blood was sampled from an antecubital vein at the time points indicated. BFR was induced by inflation of a tourniquet (123 ± 12 to 226 ± 24 mm Hg during exercise and 320 mm Hg post‐exercise)