Episodic Binge-like Ethanol Reduces Skeletal Muscle Strength Associated with Atrophy, Fibrosis, and Inflammation in Young Rats

Abstract

Binge Drinking (BD) corresponds to episodes of ingestion of large amounts of ethanol in a short time, typically ≤2 h. BD occurs across all populations, but young and sports-related people are especially vulnerable. However, the short- and long-term effects of episodic BD on skeletal muscle function have been poorly explored. Young rats were randomized into two groups: control and episodic Binge-Like ethanol protocol (BEP) (ethanol 3 g/kg IP, 4 episodes of 2-days ON-2-days OFF paradigm). Muscle function was evaluated two weeks after the last BEP episode. We found that rats exposed to BEP presented decreased muscle strength and increased fatigability, compared with control animals. Furthermore, we observed that skeletal muscle from rats exposed to BEP presented muscle atrophy, evidenced by reduced fiber size and increased expression of atrophic genes. We also observed that BEP induced fibrotic and inflammation markers, accompanied by mislocalization of nNOSµ and high levels of protein nitration. Our findings suggest that episodic binge-like ethanol exposure alters contractile capacity and increases fatigue by mechanisms involving atrophy, fibrosis, and inflammation, which remain for at least two weeks after ethanol clearance. These pathological features are common to several neuromuscular diseases and might affect muscle performance and health in the long term.

Keywords: CCN2/CTGF; alcohol; alcoholic myopathy; atrophy; binge-drinking; ethanol; fibrosis; muscle fatigue; skeletal muscle.

Figure 1

Binge-like ethanol protocol decreased TA force and increased muscle fatigability. (A) Relative weight gain, calculated as a fraction of weight in day 1 (PND 25), and relativized to control animals. Control in black N = 8 and BEP in red N = 8. (B) Relative weight of TA from BEP-treated (N = 6) vs. control (N = 5) rats. Graphs represent mean ± SEM. (C) TA specific force values at various stimulation frequencies for each group: control (N = 7) and BEP-treated (N = 8) rats. (D) Fatigue protocol. For each time point, the maximal force from tetanic contraction was normalized to the initial maximal force. Control in black, N = 5; BEP in red, N = 6. (E) Profiles from tetanic contractions from the fatigue protocol in (D) (correspond to contractions N° 1, 22, 44 and 66). Each profile was escalated to 100%, corresponding to that specific contraction’s maximal force, and the group’s average was plotted. BEP-group shows a more pronounced force decline during tetanic contraction. (F) Comparison of maximal force for sampled contractions of the fatigue protocol. (G) Comparison of the average force at the last 50 ms of tetanus (plateau) in the sampled contractions of the fatigue protocol. (H) Ratio between the plateau and maximal force, as a measure of force decline in sampled contractions. (I) The velocity of the force fall was evaluated using the slope of the curve 50 ms after the peak force. For graphs (F–I), data were analyzed using two-way ANOVA with Fisher’s LSD multi-comparison test. Except for box and whiskers graphs, data represent mean ± SEM. p-values: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 2

Binge-like ethanol protocol induced skeletal muscle atrophy. (A,G) Representative images of WGA (green) stained muscle sections of TA (A) and DIAPH (G). Scale bar 100 µm. (B,H) Quantification of average minimum Feret’s diameter of skeletal muscle fibers of TA (B) and DIAPH (H). For TA, Control N = 5, BEP N = 6. For DIAPH, Control N = 2, BEP N = 4. (C,I) Histograms for relative frequencies of minimum Feret’s diameter in TA (C) and DIAPH (I). (D,J) Histograms for cumulative frequencies of minimum Feret’s diameter in TA (D) and DIAPH (J). (E,K) Relative mRNA expression of atrophy marker Atrogin-1 (E, TA; L, DIAPH). (F,L) Relative mRNA expression of atrophy marker Murf1 (F, TA; M, DIAPH). TA, Control N = 3, BEP N = 4. DIAPH, Control N = 3, BEP N = 3. p-values: * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.

Figure 3

Binge-like ethanol protocol induced ECM proteins accumulation. (A–D) Representative images of TA immunofluorescence using anti-fibronectin (A) and anti-collagen I (B) antibodies, and for Sirius Red staining in brightfield microscopy showing total collagen (C) and polarized light microscopy showing fibrillar collagen (D). Scale bar 100 µm. (E–H) Quantification of fibronectin (E), collagen I (F), total collagen (G), and fibrillar collagen (H) as a percentage of occupied area fraction. Control N = 5, BEP N = 6. (I) Immunoblot against fibronectin and GAPDH as a loading control, with the respective densitometric quantification. Control N = 4, BEP N = 4. p-values: * p ≤ 0.05; **** p ≤ 0.0001.

Figure 4

Binge-like ethanol protocol induced profibrotic factors TGF-β and CCN2/CTGF. TGF-β expression in TA muscle. TGF-β3 protein levels with the respective densitometric analysis (A,B). Control N = 6, BEP N = 4. Relative mRNA levels of TGF-β1 (C). Control N = 3, BEP N = 4. TGF-β expression in DIAPH muscle. TGF-β3 protein levels with the respective densitometric analysis (D,E). Control N = 4, BEP N = 3. Relative mRNA levels of TGF-β1 (F). Control N = 3, BEP N = 4. Relative mRNA levels of Ccn2/Ctgf in TA (G) and DIAPH (K). (H,L) Immunoblot against CCN2/CTGF and GAPDH as a loading control, with the respective densitometric analysis of the 37 and 50 kDa immunoreactive bands. WB performed from whole muscle extracts from TA (H–J) and diaphragm (L–N). Control N = 4, BEP N = 4. (O) Representative images of TA immunostaining using anti-CCN2/CTGF antibody. Scale bar 100 µm. (P) Quantification of CCN2/CTFG as a percentage of occupied area fraction. Control N = 3, BEP N = 4. p-values: * p ≤ 0.05; ** p ≤ 0.01.

Figure 5

Binge-like ethanol protocol increased skeletal muscle pathological markers. (A) Representative images of TA immunohistochemistry using anti-Rat IgG antibody. Scale bar 100 µm. (B) Quantification of IgG staining as a percentage of occupied area fraction. (C,D) Relative expression of Nf-kb mRNA in TA ((C), control N = 3, BEP N = 4) and DIAPH ((D), control N = 3, BEP N = 3). (E) Immunoblot against nNOSµ and GAPDH as a loading control on TA muscle. Densitometric analysis performed with control N = 4, BEP N = 4. (F) Representative images of TA immunofluorescence using an anti-nNOSµ antibody (red) and Hoescht (blue) to stain nuclei. Asterisks indicate fibers with nNOSµ lost from the sarcolemma. Scale Bar 100 µm (G). Immunoblot against nTyr, with GAPDH as a loading control on DIAPH muscle and the respective densitometric analysis of bands A, B, C, D. Control N = 3, BEP N = 4. (H) Representative images of TA immunofluorescence using anti-8-OHdG antibody. Scale bar 100 µm. Quantification of 8-OHdG as a percentage of occupied area fraction. Control N = 3, BEP N = 4. p-values: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.