Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice

Abstract

Objective: Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by a mutation in DMD encoding dystrophin. Oxidative stress accounts for dystrophic muscle pathologies in DMD. We examined the effects of molecular hydrogen in mdx mice, a model animal for DMD.

Methods: The pregnant mother started to take supersaturated hydrogen water (>5 ppm) ad libitum from E15.5 up to weaning of the offspring. The mdx mice took supersaturated hydrogen water from weaning until age 10 or 24 weeks when they were sacrificed.

Results: Hydrogen water prevented abnormal body mass gain that is commonly observed in mdx mice. Hydrogen improved the spontaneous running distance that was estimated by a counter-equipped running-wheel, and extended the duration on the rota-rod. Plasma creatine kinase activities were decreased by hydrogen at ages 10 and 24 weeks. Hydrogen also decreased the number of central nuclei of muscle fibers at ages 10 and 24 weeks, and immunostaining for nitrotyrosine in gastrocnemius muscle at age 24 weeks. Additionally, hydrogen tended to increase protein expressions of antioxidant glutathione peroxidase 1, as well as anti-apoptotic Bcl-2, in skeletal muscle at age 10 weeks.

Discussion: Although molecular mechanisms of the diverse effects of hydrogen remain to be elucidated, hydrogen potentially improves muscular dystrophy in DMD patients.

Keywords: Duchenne muscular dystrophy; Molecular hydrogen; Muscle degeneration; Oxidative stress; mdx mouse.

Figure 1

Body mass (A) and motor functions (B–D) of wild-type BL/10 mice, untreated mdx mice (mdx), and hydrogen-treated mdx mice (mdx + H₂). The motor functions are estimated by counting the number of rotations on a running-wheel (B) and by the rota-rod tests with the fixed speed (‘fix’, C) and accelerated speed (‘accel’, D) protocols. The numbers of wild-type BL/10 mice, untreated mdx mice, and hydrogen-treated mdx mice were 6, 8, and 11, respectively. Mean and SEM are indicated. (A) Statistical differences with two-way repeated measures ANOVA are indicated in the symbol legend. (B–D) Statistical differences with two-way repeated measures ANOVA are indicated with the analyzed range.

Figure 2

CK activity in plasma. CK activities are measured at ages 10 (n = 6) and 24 (n = 4–6) weeks. CK activities of untreated mdx mice (mdx), and hydrogen-treated mdx mice (mdx + H₂) are normalized for that of wild-type BL/10 mice. Mean and SD are indicated. Statistical difference is observed between mdx and mdx + H₂ at age 24 weeks with Student t-test.

Figure 3

Histological analyses of gastrocnemius muscles at ages 10 (n = 6) and 24 (n = 4) weeks. (A–D) Hematoxylin-eosin staining of gastrocnemius muscles. Central nuclei are barely detectable in BL/10 mice, whereas they are frequently observed in untreated (mdx) and hydrogen-treated (mdx + H₂) mdx mice (A and C). The number of central nuclei is divided by the number of muscle fibers (B and D). (E–H) Nitrotyrosine immunostaining of gastrocnemius muscle. Representative images of BL/10 mice, mdx, and mdx + H₂ are indicated (E and G). As the secondary antibody is against mouse IgG, background signals may include incidental IgG in addition to nitrotyrosine. Morphometric analysis of signal intensities of nitrotyrosine (F and H). (I–L) TUNEL staining of gastrocnemius muscle. Representative images of BL/10 mice, mdx, and mdx + H₂ are indicated (I and K). The numbers of TUNEL-positive nuclei (arrowheads) are counted in each group of mice (J and L). Data are expressed by the mean and SEM (n = 36 visual fields at age 10 weeks and n = 40 visual fields at age 24 weeks). Statistical difference is calculated with Student's t-test.

Figure 4

mRNA expression levels in gastrocnemius muscle at ages 10 (n = 6) and 24 (n = 6–8) weeks. mRNA levels in untreated mdx mice (mdx) and hydrogen-treated mdx mice (mdx + H₂) are normalized for that in BL/10 mice in each gene. Mean and SD are indicated. Statistical difference is calculated with Student's t-test. n.s., not significant.

Figure 5

Protein levels in quadriceps muscle at ages 10 (n = 6) and 24 (n = 6) weeks. Representative immunoblots of BL/10 mice, untreated mdx mice (mdx), and hydrogen-treated mdx mice (mdx + H₂) are shown (A and C). Densitometric analysis of immunoblots (B and D). Signal intensities are normalized for that in BL/10 for each protein. Mean and SD are indicated. No statistical difference is observed between mdx and mdx + H₂ with Student t-test.