Alcohol intoxication stimulates antioxidant gene expression in skeletal muscle in a time dependent manner

Abstract

Alcohol is a known myotoxin that disrupts skeletal muscle function and health. Mitochondria play an important role in maintaining proper skeletal muscle function as they contribute to the production of ATP. Mitochondrial oxidative metabolism as well as xenobiotics like alcohol, lead to the production of reactive oxygen species (ROS) which can cause cellular damage if the antioxidant response is exceeded. Chronic alcohol ingestion has been linked to enhanced oxidative stress in the muscle and liver, while the temporal progression of antioxidant gene programs in the skeletal muscle following acute alcohol intoxication remains unknown. Therefore, the goal of this work was to examine the effects of an acute alcohol binge on skeletal muscle oxidative stress and antioxidant pathways.MethodsC57BL/6Hsd female mice (15wks old) received an IP injection of either saline (CON; n=39) or ethanol (EtOH; n=39). Gastrocnemius muscles were collected from baseline (untreated; n=3), CON (n=3), and EtOH (n=3) mice every 4‐hours for 48‐hours to determine the time course of changes following intoxication. RNA was isolated, cDNA synthesized, and RT‐PCR was performed. Protein expression was measured via Western blotting.ResultsNuclear factor erythroid 2‐related factor 2 (Nrf2) is a transcription factor central to the control of cellular antioxidant responses along with its inhibitor, Kelch like ECH associated protein 1 (Keap1). Alcohol increased the mRNA expression of Nrf2 as well as Keap1 starting 12 hours after intoxication. Downstream target genes of this antioxidant pathway were also increased after acute alcohol starting at 12‐16 hours post intoxication including Cytochrome P450 oxidoreductase (Por),Heme oxygenase 1 (Hmox1),Peroxiredoxin 6 (Prdx6),Glutamate‐cysteine ligase catalytic subunit (Glcl),Glutamate‐cysteine ligase modifier subunit (Gclm),and Glutathione‐disulfide reductase (Gsr). EtOH also enhanced Superoxide dismutase 1 (Sod1) mRNA expression from 24‐48hr following intoxication and NADPH Oxidase 4 (Nox4) expression 24hrs after the binge. Meanwhile, mRNA expression of Sod2and Nox2 was suppressed over the 48hr period following alcohol intoxication.ConclusionThese results suggest that binge alcohol stimulates ROS production and thereby antioxidant systems, to assist in the breakdown of ROS in the skeletal muscle. Future work is needed to determine the contribution of a transient increase in these pathways to longer term skeletal muscle health, especially in the context of repeat binges, a common drinking behavior.