Restoration of Contractile Protein Expression and Colonic Smooth Muscle Function by Hydrogen Sulfide in DMD Mice

Duchenne Muscular Dystrophy (DMD), characterized by the lack of dystrophin, results from a mutation in the Xp21 gene which encodes for the protein dystrophin that links the extracellular matrix to the actin cytoskeleton in skeletal, cardiac and smooth muscle. Slow colonic transit and chronic constipation are common in DMD patients due to the weakening of the abdominal wall muscles and gut smooth muscle. However, the cause of this hypocontractility in DMD patients and the expression of contractile proteins in smooth muscle are unknown. Expression of contractile proteins is regulated by the signaling pathways activated by excitatory (e.g., acetylcholine, ACh) and inhibitory transmitters (e.g., nitric oxide). Hydrogen sulfide (H2S) is well-known for its anti-oxidant effects; however, its utility to restore DMD-induced effects is unknown. Aim: To investigate the expression of contractile proteins and smooth muscle function in the colon of wild type mice and models of DMD (mdx and mdx/mTR mice) and the effect of H2S on these in mdx mice. Methods: Contraction of colonic segments was measured in the longitudinal orientation from 3-month old control and mdx mice, and 9-month old control and mdx/mTR mice, (mdx/mTR exhibit increased disease severity). The effect of SG1002, an orally active slow releasing H2S agent, was tested in mdx/mTR mice (40 mg/kg body weight in chow/every 3 days starting from 3 weeks to 9 months). Expression of contractile proteins was measured by qRT-PCR and western blot. Results: Expression of smoothelin, caldesmon, calponin and tropomyosin was decreased in colonic smooth muscle of mdx mice compared to control. This decrease was associated with a decrease in ACh-induced contraction in colonic segments (21 ± 3 mN/100 mg tissue in control and 3 ± 1 mN/100 mg tissue in mdx mice). To identify the specific involvement of smooth muscle dysfunction in the decrease in contraction, colonic muscle cells were isolated and contraction in response to ACh was measured by scanning micrometry and expressed as the percent decrease in cell length from control cell length. ACh-induced contraction was also inhibited in muscle cells isolated from mdx mice compared to control (43±5% in control and 27±3% in mdx mice). ACh-induced contraction was decreased in colonic segments from mdx/mTR mice (12 ± 2 mN/100 mg tissue in control versus 4 ± 1 mN/100 mg tissue in mdx/mTR) and the decrease was partly reversed by SG1002 treatment (9 ± 1 mN/100 mg tissue). mRNA expression of thin filament associated proteins was also decreased in colonic smooth muscle from mdx/mTR mice and the decrease was reversed by SG1002. These results suggest that H2S restores contractile protein mRNA expression and contraction in mdx/mTR mice. Conclusion: The data indicate that the lack of dystrophin in mdx mice adversely affects colonic smooth muscle contractility through the down regulation of contractile protein mRNA expression. Treatment of mdx/mTR mice with H2S restores contractile phenotype. Thus, H2S treatment would be an area of study in therapeutic control of DMD-induced gastrointestinal motility disorders