Exercise combined with corticoid/omega-3 therapy positively affected skeletal and cardiac muscles in middle aged mdx mice

Abstract

Exercise has an important impact on skeletal muscle quality, emerging as an adjuvant therapy to ameliorate muscle inflammation and fibrosis in Duchenne muscular dystrophy (DMD). The aim of the present study was to investigate the benefit of exercise alone or in association with corticoid and omega-3 therapy in the middle aged mdx mouse model of DMD, Mdx mice (12 months of age) performed treadmill exercise (12.4 m/min, for 15 min, twice a week) for 4 weeks. Exercised mdx received deflazacort (1.2 mg/kg; gavage) alone or combined with omega-3 (300 mg/kg; gavage). Sedentary mdx, C57BL/10 and exercised mdx received mineral oil and served as control. At the endpoint (14 months of age), muscle function, respiratory function, electrocardiography (ECG), blood markers of myonecrosis (creatine kinase, CK; alanine aminotransferase, ALT; and aspartate aminotransferase, AST), muscle biomarkers of inflammation and fibrosis (western blot), area of fibrosis and histopathology of tibialis anterior, biceps brachii and diaphragm muscles were evaluated. Exercise and exercise-associated therapies improved behavioural activity (open field), muscle function (grip strength, four limb hanging test, hanging wire test and rotarod), VO₂ consumption, VCO₂ production and ECG. Functional benefits correlated with reduced myonecrosis (decreased CK, ALT, AST) and fibrosis. The muscles studied showed a reduction in inflammation biomarkers (NF-κB, IL-6 and TNF-α) and fibrosis (TGF-β and fibronectin) and an increase in calsequestrin (calcium buffering protein) and PGC1-α (muscle integrity marker). In conclusion, exercise alone or associated with corticosteroid/omega-3 therapy benefited limb, diaphragm and cardiac dystrophic muscles in middle-aged mdx mice.

Keywords: PGC1‐α; TNF‐α; corticoid; dystrophy; exercise; fibronectin; rotarod.

FIGURE 1

Functional muscle test performance of the different groups over the 9 weeks of study. Rotarod latency (A) to fall and running times of wild‐type mice were significantly longer than those of the sedentary mdx mouse. Exercise associated with DFZ+O3 significantly increased the time to fall (B) of the mdx mouse. Grip strength (C) was significantly higher in wild‐type mice when compared to the sedentary dystrophic mouse. Exercise associated with DFZ greatly improved the grip strength of the mdx. Two (D) and four (E) limb hanging wire test performances were better for wild‐type than those of the sedentary mdx mouse. Exercise combined with DFZ or DFZ+O3 therapy significantly prolonged the four‐limb hanging time of the mdx compared with the sedentary mdx mouse. Open‐field horizontal (F) and vertical (G) activities were significantly better in wild‐type than in the sedentary mdx mouse. Exercise alone or associated with therapies (DFZ or DFZ+O3) improved mdx performance, but there were no statistical differences between the groups. Mdx horizontal activity in the dark (H) was increased by exercise alone or associated with therapies (DFZ or DFZ+O3). Mdx vertical activity in the dark (I) was improved only by the exercise associated with DFZ+O3 therapy. Data represent the mean and standard deviation per time point. ^aSignificantly different from wild‐type C57BL/10 Sed at Week 9. ^bSignificantly different from mdx sedentary at Week 9. ^cSignificantly different from exercised mdx at Week 9. p < .05.

FIGURE 2

Respiratory function performance was calculated at Week 5 (before exercise) and Week 9 (endpoint, after 4 weeks of exercise). Respiratory parameters were calculated in active mdx and C57BL/10 mice. (A) Oxygen consumption (VO₂), (B) the volume of carbon dioxide produced (VCO₂) and (C) total energy expenditure (EE) were significantly reduced in mdx (mdx Sed) compared with wild‐type mice (indicated by letter ‘a’; compared with C57BL/10 Sed). Exercise alone (mdx Exer) or combined with deflazacort (mdx Exer+DFZ) or with DFZ+omega‐3 (mdx Exer+DFZ‐O3) improved mdx (D) respiratory performance (indicated by letter ‘b’ in VO₂, VCO₂ and EE; compared with mdx Sed). Deflazacort therapies per se (DFZ alone or associated with O3) also improved mdx VO₂, VCO₂ and EE (indicated by letter ‘c’ at Week 5; given that DFZ and DFZ‐O3 therapies were performed from week 0 to the endpoint, at Week 5 the effects of DFZ therapies per se can be seen). Exercise alone or associated with DFZ therapies improved mdx respiratory exchange ratio (indicated by letter ‘b’ in RER; compared with mdx Sed). The wild‐type mouse (C57BL/10 Sed) had a longer distance (E) and time (F) to exhaustion than the dystrophic mouse (indicated by letter ‘a’; compared with C57BL/10 Sed). Exercise, only when associated with DFZ therapies (mdx Exer+DFZ and mdx Exer+DFZ‐O3), increased the distance and time to exhaustion of the dystrophic mouse (indicated by letters ‘b’ and ‘c’; compared with mdx Sed and mdx Exer, respectively). Data represent the mean and standard deviation per time point. ^a–cDenote significant differences, as indicated in the legend. ^γDenotes significant difference compared with Week 5 within the same group. p < .05.

FIGURE 3

Western blotting analysis of the inflammatory markers tumour necrosis factor alpha (TNF‐α), nuclear transcription factor kappa B (NF‐kB) and interleukin 6 (IL6); the fibrosis markers, transforming growth factor‐beta (TGF‐β) and fibronectin (fibrosis marker). Muscles used were tibialis anterior (TA), biceps brachii (BB), diaphragm (DIA) and heart from sedentary control mouse (C57BL/10/Sed), sedentary dystrophic mouse (mdx Sed), exercised dystrophic mouse (mdx Exer) and in the exercised dystrophic mouse under deflazacort (mdx Exer+DFZ) or under DFZ associated with omega‐3 (mdx Exer+DFZ‐O3) at 14 months of age. Graphs represent the levels of some proteins significantly different expressed in arbitrary units normalized to loading control levels in TA (A, B), BB (D, E), DIA (G, H) and Heart (J, K) muscles. Blots of proteins (top row) and of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; loading control; bottom row) in TA (C), BB (F), DIA (I) and Heart (L) muscles are shown. The molecular weight in kilodalton is indicated for each protein. Data represent the mean and standard deviation per time point. ^aSignificant difference from C57BL/10. ^bSignificant difference from sedentary mdx mice. ^cSignificant difference from mdx Exer. ^dSignificant difference from mdx Exer+DFZ. p < .05.

FIGURE 4

Western blotting of the peroxisome gamma coactivator 1‐alpha (PGC‐1α) and calsequestrin (CSQ). Muscles used were tibialis anterior (TA), biceps brachii (BB), diaphragm (DIA) and heart from sedentary control mouse (C57BL/10/Sed), sedentary dystrophic mouse (mdx Sed), exercised dystrophic mouse (mdx Exer) and in the exercised dystrophic mouse under deflazacort (mdx Exer+DFZ) or under DFZ associated with omega‐3 (mdx Exer+DFZ‐O3) at 14 months of age. Graphs represent the levels of proteins expressed in arbitrary units normalized to loading control levels in TA (A, B), BB (D, E), DIA (G, H) and Heart (J, K) muscles. The blots of proteins (top row) and of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; loading control; bottom row) in TA (C), BB (F), DIA (I) and Heart (L) muscles are shown. The molecular weight in kilodalton is indicated for each protein. Data represent the mean and standard deviation per time point. ^aSignificant difference from C57BL/10/Sed. ^bSignificant difference from sedentary mdx mice. ^cSignificant difference from mdx Exer. ^dSignificant difference from mdx Exer+DFZ. p < .05.