Resveratrol Decreases Oxidative Stress by Restoring Mitophagy and Improves the Pathophysiology of Dystrophin-Deficient mdx Mice

We previously showed that treatment with resveratrol (3,5,4′-trihydroxy-trans-stilbene), an activator of the NAD+-dependent deacetylase SIRT1 at 4 g/kg food for 32 weeks, significantly decreased the muscular reactive oxygen species (ROS) levels and ameliorated the pathology of mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Here, we treated mdx mice with various doses of resveratrol (0.04, 0.4, and 4 g/kg food) for 56 weeks and examined the effects on serum creatine kinase levels and physical activities. Because resveratrol promotes autophagy, we also investigated whether autophagy including mitochondrial autophagy (mitophagy) is involved in resveratrol’s effects. Autophagy/mitophagy-related genes and autophagic flux were downregulated in the muscle of mdx mice, and these phenomena were reversed by resveratrol with significant ROS reduction. Resveratrol at 4 g/kg food reduced the number of immature myofibers containing central nuclei and fine fibers < 400 μm2 and increased that of thicker myofibers in the quadriceps, suggesting that resveratrol decreased myofiber wasting and promoted muscular maturation. Accordingly, resveratrol at 0.4 g/kg food reduced the creatine kinase levels to one-third of those in untreated mdx mice and significantly increased the animals’ physical activities. In C2C12 myoblast cells, resveratrol promoted mitophagy and eliminated mitochondria containing high superoxide levels. The clearance of damaged mitochondria and ROS reduction by resveratrol was completely suppressed by an autophagy inhibitor (chloroquine) and by knocking down Atg5 or Pink1, essential genes for autophagy and mitophagy, respectively. Thus, resveratrol is a potential therapeutic agent for DMD, and the clearance of damaged mitochondria probably contributes to its action

SIRT1 deficiency interferes with membrane resealing after cell membrane injury.

Activation of SIRT1, an NAD+-dependent protein deacetylase, ameliorates muscular pathophysiology of δ-sarcoglycan-deficient TO-2 hamsters and dystrophin-deficient mdx mice. We found that SIRT1 was highly expressed beneath the cellular membranes of muscle cells. To elucidate functional roles of SIRT1 on muscles, skeletal muscle-specific SIRT1 knockout mice (SIRT1-MKO) were generated. SIRT1-MKO mice showed muscular pathology similar to mild muscular dystrophies with increased numbers of centrally nucleated small myofibers and decreased numbers of middle-sized (2000-3001 μm2) myofibers compared to those of wild-type (WT) mice. Accordingly, SIRT1-MKO mice showed significantly decreased exercise capacity in treadmill and inverted hanging tests with higher levels of serum creatine kinase activities compared with those in WT mice. Evans blue dye uptake after exercise was greater in the muscles of SIRT1-MKO than those of WT mice, suggesting membrane fragility in SIRT1-MKO mice. Because SIRT1 was dominantly localized beneath the membranes of muscular cells, SIRT1 may have a new role in the membranes. We found that levels of fluorescent FM1-43 dye intake after laser-induced membrane disruption in C2C12 cells were significantly increased by SIRT1 inhibitors or Sirt1-siRNA compared with those of control cells. Inhibition of SIRT1 or SIRT1-knockdown severely disturbed the dynamic aggregation of membrane vesicles under the injured site but did not affect expression levels of membrane repair proteins. These data suggested that SIRT1 had a critical role in the resealing of membrane-ruptured muscle cells, which could affect phenotypes of SIRT1-MKO mice. To our knowledge, this report is the first to demonstrate that SIRT1 affected plasma-membrane repair mechanisms