USA – neste congresso americano de pesquisas experimentais serão apresentadas mais de 30 trabalhos relacionados com distrofia muscular, sendo que as 10 abaixo são as que tem maior relação com o tratamento da doença. Há pesquisas sobre o uso de suplementos como resveratrol e quercetina, pesquisas com o uso de vetor viral diretamente no diafragma de camundongos, com o uso de antioxidantes como a acetilcisteína, com o uso de anti-inflamatórios doadores de óxido nítrico como o flurbiprofen e com o uso de inibidores da fosfodiesterase (drogas habitualmente usadas na disfunção erétil).

Os resumos dos trabalhos em inglês pode ser lido abaixo:

1) Acute phosphodiesterase inhibition improves functional muscle ischemia in patients with Becker muscular dystrophy

Elizabeth Anne Martin1, Ashley E Walker1, Bryan L Scott1, Teresa C Malott1, Nirmal Singh1, Swaminatha V Gurudevan1, Jimmy Johannes1, Robert M Elashoff2, Gail D Thomas1 and Ronald G Victor1

1 The Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA
2 Biomathematics, UCLA, Los Angeles, CA

Loss of sarcolemmal nitric oxide synthase (nNOS) engenders ischemia of exercising dystrophin-deficient muscles of mdx mice and boys with Duchenne muscular dystrophy. We tested if muscle ischemia also occurs in Becker muscular dystrophy (BMD), a milder disease often caused by dystrophin mutations involving the nNOS binding site, and is improved by tadalafil, a phosphodiesterase (PDE5A) inhibitor that enhances cGMP/NO signaling. We measured reflex vasoconstriction (decreased forearm muscle oxygenation [Hb02, near infrared spectroscopy] during lower body negative pressure [LBNP]) at rest and during rhythmic handgrip (HG) in 5 male controls (Ctrls) and 10 men with BMD who underwent a placebo-controlled cross-over trial of single-dose (20 mg) tadalafil. At baseline, HG greatly attenuated vasoconstriction in Ctrls (Hb02:–393±89 vs. –91±40 units, p<.01; rest vs. HG) but caused no attenuation in BMD (–381±45 vs. – 374±46). Tadalafil markedly improved ischemia in BMD (Hb02:– 439±70 vs. –230±54, rest vs. HG; p=0.014) whereas placebo had no effect. These data provide the first evidence in man that PDE5A inhibition can improve blood flow regulation in dystrophin-deficient skeletal muscle. Funded by MDA 201149.

2) Early Anatomical Identification Markers for Duchenne Muscular Dystrophy in a Subadult Subject

Jasmine H. Harris1, Ellen Godwin2 and Samuel Marquez3

1 College of Medicine, SUNY Downstate Medical Center, Brooklyn, NY
2 Department of Orthopaedic Surgery & Rehabilitation Medicine, SUNY Downstate Medical Center, Brooklyn, NY
3 Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY

Duchenne Muscular Dystrophy (DMD) readily affects gait and posture in subadult populations afflicted with the disease. This study used instrumented motion analysis (IMA) to identify how gait and posture changes respond to DMD disease. 3-D motion analysis was performed with the Vicon Motion Capture System on a 5-year-old boy suspected with DMD. Thirty-nine reflective markers were placed on specific anatomical landmarks according to the Plug-in-Gait Model used with the Vicon system. The child walked across the 25 feet gait analysis laboratory for 10 trials. A single representative trial was selected for analysis. Kinematic parameters of gait were compared to those of a typically developing child. IMA identified specific changes in joint kinematics in the child with DMD as compared to the typically developing child. Changes include: increase in anterior pelvic tilt and hip flexion in swing, genu recurvatum in stance, plantar flexion on initial contact with ground, and lack of dorsiflexion in swing. These gait deviations are commonly found in boys with DMD. The results show the ability to identify these changes translating in the early diagnosis of DMD as they represent a specific pattern of walking that is related to the progression of weakness observed. Identification of anatomical gait deviations can assist in the development of treatment interventions to assist the child to be ambulant as long as possible.

3) Resveratrol decreases inflammation and oxidative stress in the mdx mouse model of duchenne muscular dystrophy

Bradley Scott Gordon, Patti Weed, Emily Learner, Drew Schoenling and Matthew C Kostek

Exercise Science, University of South Carolina, Columbia, SC

Duchenne Muscular Dystrophy (DMD) is a genetic disease characterized by muscle damage, oxidative stress, chronic inflammation, and fibrosis. Resveratrol (RES) is an antioxidant and anti-inflammatory. We have shown that RES improves muscle function in the mdx mouse model of DMD, and others have shown resveratrol decreases fibrosis and oxidative stress in older mdx mice. However, its effect on pathology in young mdx mice is unknown. The purpose of this study was to investigate the effect of resveratrol on muscle pathology in young mdx mice. RES (100 mg/kg) or vehicle was administered to 4–5 week old mdx mice everyday for 10 days or every other day for 8 weeks. Muscle fiber integrity, inflammation, and oxidative stress were assessed by H&E staining and 4-HNE content. Total inflammation was reduced 21 ± 6% (p < 0.05) after 10 days of treatment with no change in oxidative stress. After 8 weeks of RES treatment, centrally located nuclei were reduced 12 ± 4% (p < 0.05), oxidative stress measured through 4-HNE content decreased 2 ± 0.13 fold (p < 0.05), and total inflammation and fibrosis did not change. We conclude that RES enhances muscle membrane integrity by reducing inflammation during the peak pathological period and long term oxidative stress. Therefore, resveratrol could be a treatment for boys with DMD. This project was funded by The Center for Alternative Medicine at The University of South Carolina School of Medicine.

4) Mdx mice have a defect in autophagy that is restored by rapamycin-loaded nanoparticle treatment

Allison Jinquan Li1, Kristin P. Bibee1, Jon N. Marsh1, Conrad C. Weihl2 and Samuel A. Wickline1

1 Department of Medicine, Division of Cardiology, Washington University in St. Louis, St. Louis, MO
2 Department of Neurology, Washington University in St. Louis, St. Louis, MO

Duchenne Muscular Dystrophy (DMD) is genetic disorder caused by mutations in dystrophin, a cytoskeletal protein in muscles, leading to progressive muscle wasting and ultimately death in the second or third decade of life. The current standard of care for DMD patients is corticosteroid therapy which slows down the natural progression of the disease but causes unwanted side effects. Our lab’s previous studies of therapeutics in an in vivo DMD model has demonstrated that mdx mice treated with rapamycin-loaded nanoparticles showed an increase in strength that was not observed with oral rapamycin treatment. Because rapamycin is known to induce autophagy, we assayed for autophagy in mdx mice treated with rapamycin-loaded nanoparticles. Western blot analysis of LC3B-II, the processed form of a protein used in autophagy, suggests that there is a previously unknown defect in autophagy in mdx mice, as shown by a lack of LC3 3B-II accumulation after blockade of autophagic flux by colchicine (Fig. 1A). Rapamycin nanoparticle treatment rescues autophagy to levels comparable to the control (Fig. 1B), suggesting that defective autophagy may contribute to the physical manifestations of muscular dystrophy in mdx mice and that restoration to normal levels may lead to the observed strength increase. Supported by NIH grant (R01 AR056223 to S.A.W.)

5) Acute phosphodiesterase inhibition ameliorates functional muscle ischemia in dystrophin-deficient mdx mice

Liang Li, Ronald G Victor and Gail D Thomas

The Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA

We previously have shown that the loss of sarcolemmal nitric oxide synthase (nNOS) in the dystrophin-deficient muscles of mdx mice and boys with Duchenne muscular dystrophy (DMD) renders the diseased muscles susceptible to ischemia during exercise. We now are extending this finding to men with Becker muscular dystrophy (BMD). We therefore hypothesized that treatment with a phosphodiesterase (PDE) inhibitor to reduce cGMP breakdown and enhance the NO signal from residual nNOS would prevent functional muscle ischemia. To test this, we compared norepinephrine (NE)-mediated vasoconstriction in resting and contracting hindlimbs of mdx mice after acute treatment with vehicle or a PDE inhibitor (tadalafil, 8 mg/kg; zaprinast, 4 mg/kg). In vehicle-treated mice, the usual effect of muscle contraction to attenuate NE-mediated vasoconstriction was impaired as shown by similar decreases in femoral vascular conductance in contracting vs resting hindlimbs (attenuation ratio = 0.87 ± 0.11; n = 10). NE-induced ischemia in the contracting hindlimbs was partially reversed in mice treated with the selective PDE5A inhibitor tadalafil (0.61 ± 0.06; n = 6; P < 0.05 vs vehicle) or the nonselective PDE inhibitor zaprinast (0.46 ± 0.10; n = 7; P < 0.05 vs vehicle). The effect of PDE inhibition to ameliorate functional muscle ischemia in mdx mice suggests a novel potential use for the treatment of DMD/BMD patients. Supported by MDA, 201149.

6) Dietary quercetin supplementation alleviates disease related muscle injury in dystrophic muscle

Katrin Hollinger1, Elizabeth Snella1, R. Andrew Shanely2 and Joshua T. Selsby1

1 Animal Science, Iowa State University, Ames, IA
2 Human Performance Laboratory; North Carolina Research Campus, Appalachian State University, Kannapolis, NC

Duchenne muscular dystrophy is the most common, fatal, X-linked muscle disease and is modeled by the mdx mouse. Dystrophic muscle shows signs of progressive necrosis and fibrosis leading to a loss of muscle function. Peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) up-regulation has been shown to alleviate some aspects of dystrophic pathology. Quercetin (QCN), a natural polyphenolic compound derived from foods such as red apples and red onions, is a potent sirtuin 1 (SIRT1) activator capable of entering muscle cells via oral delivery. SIRT1, in turn, activates PGC-1α by deacetylation. To determine the extent to which a diet containing QCN could alter the progression of disease related muscle injury 3 mo old mdx mice were fed a diet containing 0% or 0.2% QCN for 6 mo, sacrificed, and diaphragms removed. Control and treated mice ate similar amounts of food and grew at a similar rate during the study period. Dietary QCN reduced the number of extracellular nuclei/mm2 by 37% (p<0.05). The number of muscle cells/mm2 was increased by 20% (p<0.05) and muscle cells with centralized nuclei were reduced by 33% (p<0.05) in diaphragms from treated animals compared to control. Fibrosis was similar between groups. These data suggest that dietary QCN is beneficial to dystrophic muscle and warrants greater exploration as a potential therapeutic agent. Partially supported by the Martin Fund.

7) PCG-1 alpha over-expression rescues dystrophic muscle by modifying gene expression

Katrin Hollinger, Drance Rice, Elizabeth Snella and Joshua T Selsby

Animal Science, Iowa State University, Ames, IA

Duchenne muscular dystrophy is caused by the inability to produce a functional dystrophin protein. Typically, diagnosis is in the preschool years due to locomotor deficits, indicating muscles have already been damaged by the disease. Thus, it is critical that treatments be able to rescue muscle from further deterioration. We have shown that Peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) gene transfer rescues dystrophic muscle from disease related decline. To better understand the mechanism underlying the benefits of PGC-1α over expression, 3 wk old mdx mice were injected in one hind limb with null AAV6 (empty capsid) and in the other with an AAV6 driving expression of PGC-1α. At six weeks of age solei were collected. Utrophin protein expression was measured by immunohistochemistry and was increased nearly 3-fold (p<0.05) in PGC-1α over-expressing limbs compared to control limbs. PCR arrays were performed to identify genes regulated by PGC-1α over-expression. In the PGC-1α treated soleus expression of genes associated with the dystrophinglycoprotein complex (DGC) were increased by 40–92% (p<0.05), oxidative metabolism by 35–87% (p<0.05), muscle repair by 56–92% (p<0.05), and structural components by 20–300% (p<0.05). These data indicate that PGC-1α-mediated rescue of dystrophic muscle is accomplished through numerous contributing mechanisms. Partially supported by CIAG.

8) The effect of N-acetylcysteine on contractile function and protein-thiol oxidation in skeletal muscles of mdx mice

Gavin Jon Pinniger1, Evanna Binti Assan1, Jessica Terrill2 and Peter Arthur3

1 Physiology, University of Western Australia, Crawley, Australia
2 Anatomy and Human Biology, University of Western Australia, Crawley, Australia
3 Biochemistry, University of Western Australia, Crawley, Australia

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked recessive disease characterized by severe muscle weakness. We hypothesized that oxidation of skeletal muscle proteins such as myosin contributes to dystrophic muscle weakness seen in DMD boys and dystrophic mdx mice and that this muscle weakness will be attenuated by treatment with the antioxidant N-acetylcysteine (NAC). Six week old mdx mice and non-dystrophic, C57 mice were treated with 2% NAC in drinking water for six weeks and compared to untreated mdx and C57 mice. Grip strength and body weight were measured weekly during the treatment period. After six weeks of treatment, the 12 week old mice were anaesthetized (sodium pentobarbitone; 40 mg/kg; IP) and the extensor digitorum longus (EDL) muscles were excised for analysis of contractile function and protein thiol-oxidation. n mdx mice, NAC treatment significantly increased normalized grip strength and maximum specific force in isolated EDL muscles (NAC = 13.1 ± 1.2 N/cm2; Untreated = 9.8 ± 0.8 N/cm2, p<0.05), and significantly reduced myosin protein-thiol oxidation (NAC = 10.6 ± 0.8 %; Untreated = 13.7 ± 0.8 %, p<0.05). In non-dystrophic C57 mice, NAC treatment significantly increased normalized grip strength by 36%, but had no significant effect on maximum specific force or myosin protein-thiol oxidation in EDL muscles.

9) Treatment with a nitric oxide-donating NSAID counteracts functional muscle ischemia in dystrophin-deficient mdx mice

Gail D Thomas1, Angela Monopoli2, Claudio De Nardi2, Ennio Ongini2 and Ronald G Victor1

1 The Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA
2 NicOx Research Institute, Bresso, Italy

he dystrophin-deficient muscles of boys with Duchenne muscular dystrophy (DMD) and mdx mice, a model of DMD, are susceptible to ischemia during exercise due to loss of neuronal nitric oxide synthase (nNOS) from the sarcolemma. We hypothesized that treatment with a NO-donating drug would compensate for nNOS deficiency and counteract functional muscle ischemia. We fed mdx mice a standard diet containing 1% soybean oil (vehicle) or a low (15 mg/kg) or high (45 mg/kg) dose of a NO-releasing derivative of the NSAID flurbiprofen (n = 12/group). After 1 month of treatment, we compared vasoconstrictor responses to intra-arterial norepinephrine (NE) in resting and contracting hindlimbs. In vehicle-treated mice, the usual effect of muscle contraction to attenuate NE-mediated vasoconstriction was impaired as shown by similar decreases in femoral vascular conductance in contracting vs resting hindlimbs (attenuation ratio = 0.88 ± 0.03). NE-induced ischemia was also seen in the contracting hindlimbs of mice treated with low dose drug (0.92 ± 0.04; P > 0.05 vs vehicle), but was markedly attenuated in mice treated with high dose drug (0.22 ± 0.03; P < 0.05 vs vehicle or low dose). The beneficial effect of the high dose was maintained with treatment up to 3 months. These data demonstrate a robust anti-ischemic effect of a NO-donating drug in mdx mice and suggest a potential use in the treatment of DMD patients. Supported by NicOx, 801130.

10) Administration of recombinant adeno-associated virus vector to the diaphragm through direct intramuscular injection

Ashley J Smuder1, Darin J Falk2, W Bradley Nelson1 and Scott K Powers1

1 Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL
2 Department of Pediatrics, University of Florida, Gainesville, FL

Ventilatory insufficiency due to impaired diaphragm function is the leading cause of morbidity and mortality in many conditions (e.g. muscular dystrophy). Currently, pharmacological inhibitors and genetically modified animals are used to study many diseases affecting the diaphragm. However, these methodologies are problematic due to the occurrence of off-target effects and possible consequences of life-long genetic alterations. Further, conventional approaches to gene transfection (i.e., plasmid injection and electroporation) are not possible due to the size and location of the diaphragm and thus alternative methods are needed to alter gene expression. Therefore, we have developed a method for the delivery of recombinant adeno-associated virus vectors (rAAV) to the rat diaphragm via direct intramuscular injection. We hypothesized that by directly injecting rAAV we could selectively target diaphragm muscle fibers and establish a novel animal model for studying signaling pathways and also provide a strategy for effectively using gene therapy to rescue the diaphragm in disease states. Our results demonstrate that the morphology of the rat diaphragm is sufficient to allow direct injection and provide support for the use of rAAV as an intervention to study the diaphragm during conditions that promote diaphragm dysfunction.

Fonte: http://distrofiamuscular.net/noticias.htm