Chronic Alcohol Dysregulates Skeletal Muscle Myogenic Gene Expression after Hind Limb Immobilization in Female Rats

Abstract

Alcohol use and aging are risk factors for falls requiring immobilization and leading to skeletal muscle atrophy. Skeletal muscle regeneration is integral to post-immobilization recovery. This study aimed to elucidate the effects of alcohol and ovarian hormone loss on the expression of genes implicated in muscle regeneration. Three-month-old female rats received an ovariectomy or a sham surgery, consumed an alcohol-containing or control diet for 10 weeks, were subjected to unilateral hind limb immobilization for seven days, and finally were allowed a three (3d)- or 14 (14d)-day recovery. Immobilization decreased the quadriceps weight at 3d and 14d, and alcohol decreased the quadriceps weight at 14d in the nonimmobilized hind limb (NI). At 3d, alcohol decreased gene expression of myoblast determination protein (MyoD) in the immobilized hind limb (IMM) and myocyte enhancer factor (Mef)2C and tumor necrosis factor (TNF)α in NI, and ovariectomy increased MyoD and decreased TNFα expression in NI. At 14d, alcohol increased the gene expression of Mef2C, MyoD, TNFα, and transforming growth factor (TFG)β in IMM and decreased monocyte chemoattractant protein (MCP)1 expression in NI; ovariectomy increased TNFα expression in NI, and alcohol and ovariectomy together increased Mef2C expression in NI. Despite increased TGFβ expression, there was no concomitant alcohol-mediated increase in collagen in IMM at 14d. Overall, these data indicate that alcohol dysregulated the post-immobilization alteration in the expression of genes implicated in regeneration. Whether alcohol-mediated molecular changes correspond with post-immobilization functional alterations remains to be determined.

Keywords: ethanol; immobilization; inflammation; ovarian hormone loss; ovariectomy; recovery; regeneration.

Figure 1

Schematic of study design. OVX: ovariectomy, SHAM: sham surgery, ALC: alcohol-containing diet, VEH: control diet.

Figure 2

Total body weight before immobilization (A) and after 3 (B) and 14 (C) days of post-immobilization recovery; quadriceps mass relative to tibia length after 3 (D) and 14 (E) days of post-immobilization recovery, and ratio of immobilized to nonimmobilized quadriceps mass after three (F) and 14 (G) days of post-immobilization recovery. Body weight shown at pre-immobilization includes all animals; all other figures depict the three- or 14-day post-immobilization recovery groups. Body weights and quadriceps mass ratios were analyzed using two-way ANOVA (alcohol × ovariectomy). A paired-samples t-test was used to detect differences due to immobilization for quadriceps weight relative to tibia length. Then, treatment effects were analyzed using two-way ANOVA (alcohol × ovariectomy) for the nonimmobilized and immobilized hind limbs separately. VEH: pair-fed, ALC: alcohol-fed, SHAM: nonovariectomized, OVX: ovariectomized, IMM: immobilized. “+” indicates that it includes that treatment; “−“indicates that it does not include that treatment. *p < 0.05, ***p < 0.001. Means ± SEM. N = 4‒5 per group.

Figure 3

mRNA expression of myogenic genes in vastus lateralis muscle: myogenic enhancer factor 2C (Mef2C) after three days of post-immobilization recovery in the nonimmobilized (A) and immobilized (B) hind limb, Mef2C after 14 days of post-immobilization recovery in the nonimmobilized (C) and immobilized (D) hind limb, myoblast determination protein 1 (MyoD) after three days of post-immobilization recovery in the nonimmobilized (E) and immobilized (F) hind limb, and MyoD after 14 days of post-immobilization recovery in the nonimmobilized (G) and immobilized (H) hind limb with or without alcohol (ALC) and ovariectomy (OVX). Data analyzed by two-way ANOVA (alcohol × ovariectomy), *p < 0.05, **p < 0.01, Mean ± SEM.

Figure 4

mRNA expression of inflammatory and fibrotic genes in the vastus lateralis muscle: tumor necrosis factor (TNF)α after three days of post-immobilization recovery in the nonimmobilized (A) and immobilized (B) hind limb; TNFα after 14 days of post-immobilization recovery in the nonimmobilized (C) and immobilized (D) hind limb; monocyte chemoattractant protein (MCP)1 after three days of post-immobilization recovery in the nonimmobilized (E) and immobilized (F) hind limb; MCP1 after 14 days of post-immobilization recovery in the nonimmobilized (G) and immobilized (H) hind limb; transforming growth factor (TGF)β after three days of post-immobilization recovery in the nonimmobilized (I) and immobilized (J) hind limb; and TGFβ after 14 days of post-immobilization recovery in the nonimmobilized (K) and immobilized (L) hind limb with or without alcohol (ALC) and ovariectomy (OVX). Data analyzed by two-way ANOVA (alcohol × ovariectomy), * p < 0.05, ** p < 0.01, Mean ± SEM.

Figure 5

Picrosirius red staining of the quadriceps muscle of the immobilized hind limb after 14 days of post-immobilization recovery, with or without alcohol (ALC) and ovariectomy (OVX). Data analyzed by two-way ANOVA, **p < 0.01, Mean ± SEM. Images obtained at 10x magnification.