Maria Julia Marques

Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells

Duchenne muscular dystrophy is one of the most devastating myopathies. Muscle fibers undergo necrosis and lose their ability to regenerate, and this may be related to increased interstitial fibrosis or the exhaustion of satellite cells. In this study, we used mdx mice, an animal model of Duchenne muscular dystrophy, to assess whether muscle fibers lose their ability to regenerate after repeated cycles of degeneration-regeneration and to establish the role of interstitial fibrosis or exhaustion of satellite cells in this process. Repeated degenerative-regenerative cycles were induced by the injection of bupivacaine (33 mg/kg), a myotoxic agent. Bupivacaine was injected weekly into the right tibialis anterior muscle of male, 8-week-old mdx (N = 20) and C57Bl/10 (control, N = 10) mice for 20 and 50 weeks. Three weeks after the last injection, the mice were killed and the proportion of regenerated fibers was counted and reported as a fibrosis index. Twenty weekly bupivacaine injections did not change the ability of mdx muscle to regenerate. However, after 50 weekly bupivacaine injections, there was a significant decrease in the regenerative response. There was no correlation between the inability to regenerate and the increase in interstitial fibrosis. These results show that after prolonged repeated cycles of degeneration-regeneration, mdx muscle loses its ability to regenerate because of the exhaustion of satellite cells, rather than because of an increase in interstitial fibrosis. This finding may be relevant to cell and gene therapy in the treatment of Duchenne muscular dystrophy.

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

Intrinsic laryngeal muscles are spared from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy.

Intrinsic laryngeal muscles share many anatomical and physiological properties with extraocular muscles, which are unaffected in both Duchenne muscular dystrophy and mdx mice. We hypothesized that intrinsic laryngeal muscles are spared from myonecrosis in mdx mice and may serve as an additional tool to understand the mechanisms of muscle sparing in dystrophinopathy. Intrinsic laryngeal muscles and tibialis anterior (TA) muscle of adult and aged mdx and control C57Bl/10 mice were investigated. The percentage of central nucleated fibers, as a sign of muscle fibers that had undergone injury and regeneration, and myofiber labeling with Evans blue dye, as a marker of myofiber damage, were studied. Except for the cricothyroid muscle, none of the intrinsic laryngeal muscles from adult and old mdx mice showed signs of myofiber damage or Evans blue dye labeling, and all appeared to be normal. Central nucleation was readily visible in the TA of the same mdx mice. A significant increase in the percentage of central nucleated fibers was observed in adult cricothyroid muscle compared to the other intrinsic laryngeal muscles, which worsened with age. Thus, we have shown that the intrinsic laryngeal muscles are spared from the lack of dystrophin and may serve as a useful model to study the mechanisms of muscle sparing in dystrophinopathy. Muscle Nerve, 2006

On the incidence of the biceps brachii third head in Brazilian whites and blacks

The incidence of a third head of the biceps brachii muscle has been reported in several articles, and there is a general idea that it is a race-dependent variation. The aim of this investigation was to study the biceps brachii muscle with regard to the incidence of its third head in a mixed white and black Brazilian population. A total of 200 upper arms from adult white and black cadavers (100 whites and 100 blacks) fixed in a 10% formol solution were examined and compared. It was observed that for white subjects the incidence of the third head was 20% against 9% for the black subjects: a statistically significant difference. We suggest that other factors, in addition to racial ones, might play a part in determining the incidence of the biceps brachii third head in a population.

Muscle regeneration in dystrophic mdx mice is enhanced by isosorbide dinitrate

Activation of muscle satellite cells, a fundamental step in the success of muscle regeneration is mediated by nitric oxide (NO). In this study, we investigated whether isosorbide dinitrate (ISD), an NO donor, could improve muscle regeneration in dystrophic mdx mice. The right tibialis anterior muscle of mdx and C57Bl/10 mice was injected with bupivacaine (0.3 ml, 33 mg/kg), a myotoxic agent, to induce muscle fiber regeneration. After bupivacaine injection, mice were treated with ISD (30 mg/kg; i.p.), verapamil (a non-NO donor vasodilator, 15 mg/kg, i.p.) or saline solution (vehicle, 0.3 ml, i.p.) for 20 days. Some bupivacaine-injected mice received no pharmacological treatment (control group). Muscle regeneration was evaluated by counting the total number of muscle fibers and measuring myofiber cross-sectional area. ISD significantly improved bupivacaine-induced muscle regeneration in mdx by increasing by 20% the total number of muscle fibers compared to the other groups. Spontaneous muscle regeneration, evaluated in the contralateral non-injected muscle, was not affected. ISD treatment did not affect myofiber cross-sectional area. Verapamil and saline had no effect on muscle regeneration. These results suggested that NO derived from ISD stimulated and/or recruited satellite cells. Pharmacological treatment with ISD could be clinically useful for improving muscle regeneration in Duchenne muscular dystrophy.

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.

N-acetylcysteine treatment reduces TNF-α levels and myonecrosis in diaphragm muscle of mdx mice.

BACKGROUND & AIMS Duchenne muscular dystrophy (DMD) is a genetic muscle disease caused by the absence of dystrophin. An established animal model of DMD is the mdx mouse, which is unable to express dystrophin. Inflammation, particularly the proinflammatory cytokine tumor necrosis factor alpha (TNF-α), strongly contributes to necrosis in the dystrophin-deficient fibers of the mdx mice and in DMD. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) decreases TNF-α levels and protects the diaphragm muscle of mdx mice against necrosis. METHODS Mdx mice (14 days old) received daily intraperitoneal injections of NAC for 14 days, followed by removal of the diaphragm muscle. Control mdx mice were injected with saline. RESULTS NAC reduced TNF-α and 4-HNE-protein adducts levels, inflammation, creatine kinase levels, and myonecrosis in diaphragm muscle. CONCLUSIONS NAC may be used as a complementary treatment for dystrophinopathies. However, clinical trials are needed to determine the appropriate dose for patients with Duchenne muscular dystrophy.

Sarcoplasmic-endoplasmic-reticulum Ca2+-ATPase and calsequestrin are overexpressed in spared intrinsic laryngeal muscles of dystrophin-deficient mdx mice.

In the mdx mouse model of Duchenne muscular dystrophy, the lack of dystrophin is associated with increased calcium levels and skeletal muscle myonecrosis. The intrinsic laryngeal muscles (ILM) are protected and do not undergo myonecrosis. We investigated whether this protection is related to an increased expression of calcium‐binding proteins, which may protect against the elevated calcium levels seen in dystrophic fibers. The expression of sarcoplasmic–endoplasmic–reticulum Ca2+‐ATPase and calsequestrin was examined in ILM and in nonspared limb muscles of control and mdx mice using immunofluorescence and immunoblotting. Dystrophic ILM presented a significant increase in the proteins studied when compared to controls. The increase of Ca2+‐handling proteins in dystrophic ILM may permit better maintenance of calcium homeostasis, with the consequent absence of myonecrosis. The results further support the concept that abnormal Ca2+‐handling is involved in dystrophinopathies. Muscle Nerve, 2009

Acetylcholine receptors and neuronal nitric oxide synthase distribution at the neuromuscular junction of regenerated muscle fibers

We investigated whether the changes in acetylcholine receptor (AChR) distribution and neuronal nitric oxide synthase (nNOS) expression reported for the skeletal muscle of mdx mice were a consequence of muscle fiber regeneration rather than of the absence of dystrophin. Degenerative‐regenerative changes in muscle fibers of the sternomastoid muscle of normal mice were induced by injecting lidocaine hydrochloride. Twenty‐one days later, AChRs were labeled with alpha‐bungarotoxin and nNOS with anti‐nNOS antibody, and observed under a confocal microscope. AChRs were distributed in continuous branches in normal fibers. Regenerated fibers showed disruption of AChRs distribution similar to that seen in muscle of mdx mice. This suggests that changes in AChRs distribution seen in mdx mice were probably a consequence of muscle fiber degeneration and regeneration, rather than a symptom of dystrophin deficiency. Conversely, there were no changes in nNOS distribution and expression in normal regenerated fibers, suggesting that the decrease in nNOS expression reported for mdx mice might be attributed to the absence of dystrophin.

Diltiazem and verapamil protect dystrophin-deficient muscle fibers of MDX mice from degeneration: A potential role in calcium buffering and sarcolemmal stability

The lack of dystrophin in mdx mice and in Duchenne muscular dystrophy causes sarcolemmal breakdown and increased calcium influx followed by myonecrosis. We examined whether the calcium channel blockers diltiazem and verapamil protect dystrophic muscles from degeneration. Mdx mice received daily intraperitoneal injections of diltiazem or verapamil for 18 days, followed by removal of the sternomastoid, diaphragm, tibialis anterior, and cardiac muscles. Control mdx mice were injected with saline. Both drugs significantly decreased blood creatine kinase levels. Total calcium content was significantly higher in mdx muscles than in control C57Bl/10. Verapamil and diltiazem reduced total calcium content only in diaphragm and cardiac muscle. Histological analysis showed that diltiazem significantly attenuated myonecrosis in diaphragm. Immunoblots showed a significant increase of calsequestrin and β‐dystroglycan levels in some diltiazem‐ and verapamil‐treated muscles. Possible interactions of these drugs with the sarcoplasmic reticulum and sarcolemma may also contribute to the improvement of the dystrophic phenotype. Muscle Nerve 39: 167–176, 2009