Abstract
Background: Cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) are enzymatic sources of hydrogen sulfide (H2S). Functions of H2S are mediated by several targets including ion channels and signaling proteins. Nuclear factor-erythriod 2-related factor 2 (NRF2) is responsible for the expression of antioxidant response element (ARE)-regulated genes and is known to be upregulated by H2S. We examined the levels of H2S producing enzymes, H2S, and NRF2 activation status in skeletal muscle obtained from CLI patients. Methods: Gastrocnemius tissues were attained post amputation from human CLI and aged-matched control patients. Tissue H2S levels were measured using gas chromatography methods coupled with sulfur chemiluminescence. RT-qPCR, immunoblot, and electrophoretic mobility shift assay (EMSA) were used to analyze respective gene expression, protein levels, and DNA binding activity, respectively. Results: We found mRNA and protein levels of CSE, CBS, and 3-MST were significantly decreased in skeletal muscle of CLI (~2 fold, p < 0.05) patients as compared to control. H2S and sulfane sulfur levels were significantly decreased in skeletal muscle of CLI patients. We also observed significant reductions in NRF2 activation (2 fold, p < 0.05) as well as antioxidant proteins, such as CuZn-superoxide dismutase (2 fold, p < 0.05), catalase (2 fold, p < 0.05), and glutathione peroxidase (2 fold, p < 0.05) in skeletal muscle of CLI patients. Biomarkers of oxidative stress, such as malondialdehyde and protein carbonyl formation were significantly increased (2 fold, p < 0.05) in skeletal muscle of CLI patients as compared to age-matched controls. Conclusions: The data demonstrate that H2S bioavailability and NRF2 activation are both attenuated in CLI tissues concomitant with significantly increased oxidative stress. Reductions in the activity of H2S producing enzymes may contribute to the pathogenesis of CLI.
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