Oxidant-induced atrogin-1 and transforming growth factor-beta1 precede alcohol-related myopathy in rats

Abstract

Alcohol-related chronic myopathy is characterized by severe biochemical and structural changes to skeletal muscle. Our goals were to: (1) identify early regulatory elements that precede the overt manifestation of plantaris atrophy; and (2) circumvent these derangements by supplementing alcohol-fed rats with the glutathione precursor, procysteine. After 6 weeks of daily ingestion, before the development of overt atrophy of the plantaris muscle, alcohol increased several markers of oxidative stress and increased gene expressions of atrogin-1 and transforming growth factor-beta1 (TGF-beta1) by approximately 60- and approximately 65-fold, respectively, which were attenuated by procysteine supplementation. Interestingly, after 28 weeks of alcohol ingestion, when overt plantaris atrophy had developed, atrogin-1 and TGF-beta1 gene expression had returned to baseline levels. Together, these findings suggest that alcohol-induced, redox-sensitive alterations drive pro-atrophy signaling pathways that precede muscle atrophy. Therefore, targeted anti-oxidant treatments such as procysteine supplementation may benefit individuals with chronic alcohol abuse, particularly if given prior to the development of clinically significant myopathy.

FIGURE 1

Total protein oxidation in rat plantaris muscles. The increase in total protein oxidation in rat plantaris muscles due to 6 weeks of chronic alcohol ingestion is attenuated by procysteine supplementation. Values are normalized to controls and expressed as mean + SEM. EtOH, alcohol-fed rats; PRO, procysteine-supplemented rats. Significant difference (P < 0.05) is shown compared with control group (*) or EtOH group (#).

FIGURE 2

GSH and GSSG levels in plantaris muscles. Chronic alcohol ingestion for 6 weeks decreased the available pool of GSH (A), but had no effect on GSSG levels (B). The GSSG/GSH ratio, a marker of the oxidative state of the GSH pool, was unchanged between the groups (C). Values are expressed as mean + SEM. GSH, glutathione; GSSG, glutathione disulfide; EtOH, alcohol-fed rats; PRO, procysteine-supplemented rats. Significant difference (P < 0.05) is shown compared with control group (*).

FIGURE 3

Cys and Cyss levels in plantaris muscles. Chronic alcohol ingestion for 6 weeks decreased Cys levels, which were restored to control levels following procysteine supplementation (A). Cyss levels were increased in plantaris muscles from EtOH + PRO animals (B). The Cyss/Cys ratio, a marker of the oxidative state of the Cys pool, was increased in alcohol-fed animals (C). Procysteine supplementation normalized this ratio to control levels. Values are expressed as mean + SEM. Cys, cysteine; Cyss, cystine; EtOH, alcohol-fed rats; PRO, procysteine-supplemented rats. Significant difference (P < 0.05) is shown compared with control group (*) or EtOH group (#).

FIGURE 4

Atrogin-1 and TGF-β1 gene expressions in plantaris muscles. Real-time polymerase chain reaction analyses showed increased atrogin-1 (A) and TGF-β1 (B) mRNA levels in plantaris muscles from rats chronically fed alcohol for 6 weeks. These expression levels were sensitive to procysteine supplementation. As a control, mRNA levels of skeletal muscle actin were quantified and showed no difference between groups. Data are presented as mean ± range of potential values based on the 2^−ΔΔCT method and expressed as fold changes relative to controls. EtOH, alcohol-fed rats; PRO, procysteine-supplemented rats. Significant difference (P < 0.05) is shown compared with control group (*) or EtOH group (#).

FIGURE 5

Plantaris fiber CSA, atrogin-1, and TGF-β1 gene expressions from rats chronically fed alcohol for 28 weeks. After 28 weeks of chronic alcohol ingestion, plantaris fiber CSA was significantly reduced compared with controls (A). However, no changes were observed in mRNA levels of atrogin-1 (B) or TGF-β1 (C). As a control, mRNA levels of skeletal muscle actin were quantified and showed no difference between groups. Data are presented as mean ± range of potential values based on the 2^−ΔΔCT method and expressed as fold changes relative to controls. EtOH, alcohol-fed rats; PRO, procysteine-supplemented rats. Significant difference (P < 0.05) is shown compared with control group (*).