Ana Paula Tiemi Taniguti

Prevention of muscle fibrosis and myonecrosis in mdx mice by suramin, a TGF-β1 blocker.

Fibrosis is a pathological feature observed in patients with Duchenne muscular dystrophy (DMD) and in mdx mice, the experimental model of DMD. We evaluated the effect of suramin, a transforming growth factor‐beta 1 (TGF‐β1) blocker, on fibrosis in mdx mice. mdx mice (6 months old) received suramin for 7 weeks. Suramin‐ and saline‐treated (control) mdx mice performed exercise on a treadmill to worsen disease progression. Immunoblotting showed an increase of TGF‐β1 in mdx diaphragm, limb, and cardiac muscles. Suramin decreased creatine kinase in mdx mice and attenuated fibrosis in all muscles studied, except for cardiac muscle. Suramin protected limb muscles against damage and reduced the exercise‐induced loss of strength over time. These findings support a role for TGF‐β1 in fibrinogenesis and myonecrosis during the later stages of disease in mdx mice. Suramin might be a useful therapeutic alternative for the treatment of dystrophinopathies. Muscle Nerve, 2011

Eicosapentaenoic acid decreases TNF-α and protects dystrophic muscles of mdx mice from degeneration

In dystrophin-deficient fibers of mdx mice and in Duchenne muscular dystrophy, inflammation and increased production of tumor necrosis factor alpha (TNF-α) contribute to myonecrosis. We examined the effects of eicosapentaenoic acid (EPA) on dystrophic muscle degeneration. Mdx mice (14 days old) received EPA for 16 days. The sternomastoid, diaphragm and biceps brachii muscles were removed. Control mdx mice received vehicle. EPA decreased creatine kinase and myonecrosis and reduced the levels of TNF-α. These results suggest that EPA plays a protective role in dystrophic muscle degeneration, possibly by reducing TNF-α, and support further investigations of EPA as a potential therapy for dystrophinopathies.

Stretch-activated calcium channel protein TRPC1 is correlated with the different degrees of the dystrophic phenotype in mdx mice

In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin is related to enhanced calcium influx and muscle degeneration. Stretch-activated channels (SACs) might be directly involved in the pathology of DMD, and transient receptor potential cation channels have been proposed as likely candidates of SACs. We investigated the levels of transient receptor potential canonical channel 1 (TRPC1) and the effects of streptomycin, a SAC blocker, in muscles showing different degrees of the dystrophic phenotype. Mdx mice (18 days old, n = 16) received daily intraperitoneal injections of streptomycin (182 mg/kg body wt) for 18 days, followed by removal of the diaphragm, sternomastoid (STN), biceps brachii, and tibialis anterior muscles. Control mdx mice (n = 37) were injected with saline. Western blot analysis showed higher levels of TRPC1 in diaphragm muscle compared with STN and limb muscles. Streptomycin reduced creatine kinase and prevented exercise-induced increases of total calcium and Evans blue dye uptake in diaphragm and in STN muscles. It is suggested that different levels of the stretch-activated calcium channel protein TRPC1 may contribute to the different degrees of the dystrophic phenotype seen in mdx mice. Early treatment designed to regulate the activity of these channels may ameliorate the progression of dystrophy in the most affected muscle, the diaphragm.

Nerve terminal contributes to acetylcholine receptor organization at the dystrophic neuromuscular junction of mdx mice.

Changes in the distribution of acetylcholine receptors have been reported to occur at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration rather than the absence of dystrophin. In the present study, we examined whether the nerve terminal determines the fate of acetylcholine receptor distribution in the dystrophic muscle fibers of mdx mice. The left sternomastoid muscle of young (1‐month‐old) and adult (6‐month‐old) mdx mice was injected with 60 μl lidocaine hydrochloride to induce muscle degeneration‐regeneration. Some mice had their sternomastoid muscle denervated at the time of lidocaine injection. After 10 days of muscle denervation, nerve terminals and acetylcholine receptors were labeled with 4‐Di‐2‐ASP and rhodamine‐α‐bungarotoxin, respectively, for confocal microscopy. In young mdx mice, 75% (n = 137 endplates) of the receptors were distributed in islands. The same was observed in 100% (n = 114 endplates) of the adult junctions. In denervated‐regenerated fibers of young mice, the receptors were distributed as branches in 89% of the endplates (n = 90). In denervated‐regenerated fibers of adult mice, the receptors were distributed in islands in 100% of the endplates (n = 100). These findings show that nerve‐dependent mechanisms are also involved in the changes in receptor distribution in young dystrophic muscles. In older dystrophic muscles, other factors may play a role in receptor distribution. Anat Rec 290:181–187, 2007.

Suramin affects metalloproteinase-9 activity and increases beta-dystroglycan levels in the diaphragm of the dystrophin-deficient mdx MOUSE

Introduction: In Duchenne muscular dystrophy and in the mdx mouse, muscle fiber degeneration and subsequent fibrosis lead to cardiorespiratory failure. Previously, we demonstrated that the anti‐fibrotic agent suramin was effective in decreasing fibrosis in mdx muscles. In this study, we were interested to see whether suramin could affect metalloproteinases (MMP) and improve the functional activity of the mdx diaphragm muscle. Methods: Zymography was performed to evaluate MMP‐2 and MMP‐9 activity. Western blotting was used to analyze the levels of beta‐dystroglycan. Muscle function was assessed in hemidiaphragm in vitro preparations. Results: We found that suramin affects metalloproteinase‐9 activity and increases beta‐dystroglycan. Furthermore, suramin also protects against diaphragm muscle fatigue over time. Conclusions: These results show the potential benefits of suramin in maintaining the structure of the dystrophin‐glycoprotein complex. Muscle Nerve, 46:810–813, 2012

Doxycycline ameliorates the dystrophic phenotype of skeletal and cardiac muscles in mdx mice

Introduction: We examined whether doxycycline, an antibiotic member of the tetracycline family, improves the histopathology and muscle function in mdx mice, the experimental model of DMD. Methods: Doxycycline was administered for 36 days (starting on postnatal day 0) and for 9 months (starting at 8 months of age) in drinking water. Histopathological, biochemical (creatine kinase), and functional (forelimb muscle grip strength) parameters were evaluated in limb, diaphragm, and cardiac muscle. Results: Doxycycline significantly minimized the dystrophic phenotype of skeletal and cardiac muscles and improved forelimb muscle strength. The drug protected muscle fibers against myonecrosis and reduced inflammation. Furthermore, it slowed the progression of myocardial fibrosis. Conclusions: This study provides evidence that doxycycline may be a potential therapeutic agent for DMD. Muscle Nerve 46: 400–406, 2012

Suramin attenuates dystrophin-deficient cardiomyopathy in the mdx mouse model of duchenne muscular dystrophy.

Introduction: The purpose of this study was to determine the effects of suramin, an antifibrotic agent, on cardiac function and remodeling in mdx mice. Methods: mdx mice (8 months old) received intraperitoneal injections of suramin twice a week for 3 months. Control mdx mice (8 months old) were injected with saline. Results: Suramin improved the electrocardiography profile with the main corrections seen in S‐ to R‐wave ratio, PR interval, and Q amplitude, and a significant decrease in the cardiomyopathy index. Suramin decreased myocardial fibrosis, inflammation, and myonecrosis. Conclusions: These findings suggest that suramin may be a new adjunctive therapy to help improve cardiomyopathy in DMD. Muscle Nerve 48: 911–919, 2013

Myocardial fibrosis is unaltered by long-term administration of L-arginine in dystrophin deficient mdx mice: a histomorphometric analysis.

Cardiac failure secondary to myocardial fibrosis (MF) significantly contributes to death in Duchenne muscular dystrophy (DMD), a fatal form of muscle disease. In aging, the mdx mice, an animal model of DMD, MF is similar to that observed in humans. Nitric oxide-based therapy has been proposed to retard MF in DMD and a candidate is L-arginine (L-arg). In this study we evaluated the effects of long-term therapy with L-arg in the MF of mdx mice. mdx mice (6 months old) were treated with L-arg in drinking water. Control mdx mice received water only. After 15 months of treatment, hearts were stained with Massons trichrome for analysis of MF and with hematoxilyn and eosin for analysis of inflammation and cardiomyocyte damage. We observed that MF was not affected (29.5 +/- 2.5% of MF area for control vs 31.4 +/- 2% for L-arginine-treated animals; P > 0.05). The density of inflammatory cells was reduced (169 +/- 12 cells/mm 2 in control vs 102 +/- 9 cells/mm 2 in L-arg-treated; P < 0.05). The present study shows that long-term administration of L-arg is not effective in retarding MF in mdx dystrophinopathy.