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1.
Biochem Biophys Res Commun ; 505(1): 51-59, 2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30236982

RESUMO

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45-55 produces an internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45-55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.


Assuntos
Distrofina/metabolismo , Proteínas Musculares/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Proteolipídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Distrofina/genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Fenótipo , Proteolipídeos/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Transgenes/genética
2.
Am J Pathol ; 187(5): 1147-1161, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28315675

RESUMO

Inflammatory events occurring in dystrophic muscles contribute to the progression of Duchenne muscular dystrophy (DMD). Low-intensity training (LIT) attenuates the phenotype of mdx mice, an animal model for DMD. Therefore, we postulated that LIT could have anti-inflammatory properties. We assessed levels of inflammatory cytokines and infiltrated immune cells in gastrocnemius muscle of mdx mice after LIT. We detected high levels of complement component C5a, chemokine ligand (CCL) 2, CD68+ monocytes/macrophages, and proinflammatory M1 macrophages in muscles of mdx mice. LIT decreased CCL2 levels, increased CD68+ cell numbers, and shifted the macrophage population to the regenerative M2 type. We investigated whether inhibition of C5a or CCL2 with L-aptamers could mimic the effects of LIT. Although no effect of CCL2 inhibition was detected, treatment with the C5a inhibitor, NOX-D21, rescued the phenotype of nonexercised mdx mice, but not of exercised ones. In both cases, the level of CD68+ cells increased and macrophage populations leaned toward the inflammatory M1 type. In muscles of nonexercised treated mice, the level of IL-1 receptor antagonist increased, damage decreased, and fibers were switched toward the glycolytic fast type; in muscles of exercised mice, fibers were switched to the oxidative slow type. These results reveal the effects of LIT on the inflammatory status of mdx mice and suggest that NOX-D21 could be an anti-inflammatory drug for DMD.


Assuntos
Complemento C5a/antagonistas & inibidores , Distrofia Muscular Animal/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Quimiocina CCL2/antagonistas & inibidores , Citocinas/metabolismo , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Membro Anterior , Macrófagos/fisiologia , Masculino , Camundongos Endogâmicos mdx , Força Muscular/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/fisiopatologia , Miosite/fisiopatologia , Miosite/prevenção & controle , Fenótipo , Natação/fisiologia
3.
Int J Mol Sci ; 19(9)2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30231482

RESUMO

Intracellular signaling pathways, including the mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK) pathway, are activated by exercise, and promote skeletal muscle hypertrophy. However, the mechanisms by which these pathways are activated by physiological stimulation are not fully understood. Here we show that extracellular ATP activates these pathways by increasing intracellular Ca2+ levels ([Ca2+]i), and promotes muscle hypertrophy. [Ca2+]i in skeletal muscle was transiently increased after exercise. Treatment with ATP induced the increase in [Ca2+]i through the P2Y2 receptor/inositol 1,4,5-trisphosphate receptor pathway, and subsequent activation of mTOR in vitro. In addition, the ATP-induced increase in [Ca2+]i coordinately activated Erk1/2, p38 MAPK and mTOR that upregulated translation of JunB and interleukin-6. ATP also induced an increase in [Ca2+]i in isolated soleus muscle fibers, but not in extensor digitorum longus muscle fibers. Furthermore, administration of ATP led to muscle hypertrophy in an mTOR- and Ca2+-dependent manner in soleus, but not in plantaris muscle, suggesting that ATP specifically regulated [Ca2+]i in slow muscles. These findings suggest that ATP and [Ca2+]i are important mediators that convert mechanical stimulation into the activation of intracellular signaling pathways, and point to the P2Y receptor as a therapeutic target for treating muscle atrophy.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Músculo Esquelético/patologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Hipertrofia/metabolismo , Hipertrofia/patologia , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia
4.
Chimia (Aarau) ; 72(4): 238-240, 2018 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-29720316

RESUMO

Drug discovery is a long, expensive and risky process. Evaluating drugs that have already been proved safe for use in humans and testing them for a new indication greatly reduces the time and monetary costs involved in finding treatments for life-threatening conditions. Here tamoxifen, a drug that is used for the treatment of breast cancer, is investigated in a mouse model of Duchenne muscular dystrophy. Tamoxifen was efficacious in countering the symptoms of the disease without affecting the underlying genetic cause. Based on these results, tamoxifen has been tested in other forms of muscle disease with success. Drug repurposing may not only be a cost-effective manner for treating a variety of diseases, it may also help us uncover common mechanisms between conditions that were previously thought to be unrelated.


Assuntos
Distrofia Muscular de Duchenne/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Tamoxifeno/uso terapêutico , Animais , Feminino , Humanos , Masculino , Camundongos
5.
Am J Pathol ; 182(2): 485-504, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23332367

RESUMO

Duchenne muscular dystrophy (DMD) is a severe disorder characterized by progressive muscle wasting,respiratory and cardiac impairments, and premature death. No treatment exists so far, and the identification of active substances to fight DMD is urgently needed. We found that tamoxifen, a drug used to treat estrogen-dependent breast cancer, caused remarkable improvements of muscle force and of diaphragm and cardiac structure in the mdx(5Cv) mouse model of DMD. Oral tamoxifen treatment from 3 weeks of age for 15 months at a dose of 10 mg/kg/day stabilized myofiber membranes, normalized whole body force, and increased force production and resistance to repeated contractions of the triceps muscle above normal values. Tamoxifen improved the structure of leg muscles and diminished cardiac fibrosis by~ 50%. Tamoxifen also reduced fibrosis in the diaphragm, while increasing its thickness,myofiber count, and myofiber diameter, thereby augmenting by 72% the amount of contractile tissue available for respiratory function. Tamoxifen conferred a markedly slower phenotype to the muscles.Tamoxifen and its metabolites were present in nanomolar concentrations in plasma and muscles,suggesting signaling through high-affinity targets. Interestingly, the estrogen receptors ERa and ERb were several times more abundant in dystrophic than in normal muscles, and tamoxifen normalized the relative abundance of ERb isoforms. Our findings suggest that tamoxifen might be a useful therapy for DMD.


Assuntos
Antineoplásicos/uso terapêutico , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/patologia , Tamoxifeno/uso terapêutico , Animais , Antineoplásicos/farmacologia , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Fenômenos Biomecânicos/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Creatina Quinase/sangue , Diafragma/patologia , Diafragma/fisiopatologia , Modelos Animais de Doenças , Comportamento Alimentar/efeitos dos fármacos , Fibrose , Camundongos , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/sangue , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/fisiopatologia , Miocárdio/patologia , Tamanho do Órgão/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Tamoxifeno/sangue , Tamoxifeno/farmacologia
6.
J Biol Chem ; 287(18): 14524-34, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22399301

RESUMO

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.


Assuntos
Diferenciação Celular/fisiologia , Mioblastos Esqueléticos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPC/metabolismo , Animais , Cálcio/metabolismo , Ativação Enzimática/fisiologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteína MyoD/biossíntese , Proteína MyoD/genética , Mioblastos Esqueléticos/citologia , Fator Regulador Miogênico 5/biossíntese , Fator Regulador Miogênico 5/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Canais de Cátion TRPC/genética
7.
Curr Opin Neurol ; 26(5): 577-84, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23995279

RESUMO

PURPOSE OF REVIEW: The most encouraging recent advances regarding pharmacological agents for treating Duchenne muscular dystrophy (DMD) are summarized. Emphasis is given to compounds acting downstream of dystrophin, the protein lacking in DMD, on cellular pathways leading to pathological consequences. The author highlights the progress that may have the greatest potential for clinical use in DMD. RECENT FINDINGS: Modifying the transcripts of the mutated gene by exon skipping has led to expression of shortened dystrophins in DMD patients. Currently, the most promising potential drugs are the exon-skipping agents eteplirsen and drisapersen. Biglycan and SMTC1100 upregulate utrophin. The steroid receptor modulating compounds VBP15 and tamoxifen, and specific antioxidants appear promising agents for symptomatic therapy. SUMMARY: The past 18 months have seen a strong increase in the number of exciting reports on novel therapeutic agents for DMD. Exon-skipping agents have been fine-tuned to improve tissue delivery and stability. Impressive discoveries regarding pathogenic events in cellular signalling have revealed targets that were unknown in the context of DMD, thus enabling approaches that limit inflammation, fibrosis and necrosis. The targets are nuclear hormone receptors, NADPH-oxidases and Ca channels. Inhibition of NF-KB, transforming growth factor-alpha (TGF-α) and transforming growth factor-beta (TGF-ß)/myostatin production or action are also promising routes in counteracting the complex pathogenesis of DMD.


Assuntos
Distrofina/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Animais , Distrofina/genética , Humanos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Mutação/genética , Transdução de Sinais/fisiologia , Utrofina/genética , Utrofina/metabolismo
8.
Am J Pathol ; 180(2): 749-62, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22192627

RESUMO

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.


Assuntos
Acetilcisteína/análogos & derivados , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eritromicina/análogos & derivados , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular Animal/tratamento farmacológico , Urocortinas/farmacologia , Acetilcisteína/antagonistas & inibidores , Acetilcisteína/metabolismo , Animais , Brefeldina A/farmacologia , Cálcio/metabolismo , Canais de Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Eritromicina/antagonistas & inibidores , Eritromicina/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Injeções Intradérmicas , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Força Muscular/fisiologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Molécula 1 de Interação Estromal
9.
Histochem Cell Biol ; 137(6): 811-27, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22331205

RESUMO

In two separate previous studies, we reported that subcutaneous (sc) or oral administration of (-)-epigallocatechin-3-gallate (EGCG) limited the development of muscle degeneration of mdx mice, a mild phenotype model for Duchenne muscular dystrophy (DMD). However, it was not possible to conclude which was the more efficient route of EGCG administration because different strains of mdx mice, periods of treatment and methods of assessment were used. In this study, we investigated which administration routes and dosages of EGCG are the most effective for limiting the onset of dystrophic lesions in the same strain of mdx mice and applying the same methods of assessment. Three-week-old mdx mice were injected sc for 5 weeks with either saline or a daily average of 3 or 6 mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effects of EGCG were assessed quantitatively by determining the activities of serum muscle-derived creatine kinase, isometric contractions of triceps surae muscles, integrated spontaneous locomotor activities, and oxidative stress and fibrosis in selected muscles. Oral administration of 180 mg/kg/day EGCG in the diet was found the most effective for significantly improving several parameters associated with muscular dystrophy. However, the improvements were slightly less than those observed previously for sc injection started immediately after birth. The efficacy of EGCG for limiting the development of dystrophic muscle lesions in mice suggests that EGCG may be of benefit for DMD patients.


Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Chá/química , Animais , Peso Corporal , Catequina/farmacologia , Modelos Animais de Doenças , Ingestão de Alimentos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Estresse Oxidativo , Polifenóis/farmacologia
10.
J Pineal Res ; 51(2): 163-71, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21486366

RESUMO

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. In addition to abnormal calcium handling, numerous studies point to a crucial role of oxidative stress in the pathogenesis of the disease. Considering the impressive results provided by antioxidants on dystrophic muscle structure and function, we investigated whether melatonin can protect the mdx(5Cv) mouse, an animal model for DMD. Male mdx(5Cv) mouse pups were treated with melatonin by daily intraperitoneal (i.p.) injection (30 mg/kg body weight) or by subcutaneous (s.c.) implant(s) (18 or 54 mg melatonin as Melovine® implants) from 17/18 to 28/29 days of age. Isometric force of the triceps surae was recorded at the end of the treatment. The i.p. treatment increased the phasic twitch tension of mdx(5Cv) mice. The maximal tetanic tension was ameliorated by 18 mg s.c. and 30 mg/kg i.p. treatments. Melatonin caused the dystrophic muscle to contract and relax faster. The force-frequency relationship of melatonin-treated dystrophic mice was shifted to the right. In accordance with improved muscle function, melatonin decreased plasma creatine kinase activity, a marker for muscle injury. Melatonin treatment increased total glutathione content and lowered the oxidized/reduced glutathione ratio, indicating a better redox status of the muscle. In light of the present investigation, the therapeutic potential of melatonin should be further considered for patients with DMD.


Assuntos
Antioxidantes/farmacologia , Contração Isométrica/efeitos dos fármacos , Melatonina/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculo Esquelético , Distrofia Muscular de Duchenne , Animais , Creatinina/sangue , Modelos Animais de Doenças , Glutationa/sangue , Humanos , Contração Isométrica/genética , Masculino , Camundongos , Camundongos Endogâmicos mdx , Relaxamento Muscular/genética , Força Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Oxirredução/efeitos dos fármacos
11.
Biochem Biophys Res Commun ; 391(1): 401-6, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19914213

RESUMO

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca(2+) entry through sarcolemmal cationic channels. Using the Mn(2+) quench method, we show here that Mn(2+) entry triggered by Ca(2+) store depletion but not basal Mn(2+) entry relies on Ca(2+)-independent PLA(2) (iPLA(2)) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx(5cv) mouse. iPLA(2) was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA(2) lipid product lysophosphatidylcholine was found to trigger Ca(2+) entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA(2) and lysophospholipid production may be of interest to reduce Ca(2+) entry and subsequent degeneration of dystrophic muscle.


Assuntos
Cálcio/metabolismo , Lisofosfatidilcolinas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Fosfolipases A2 Independentes de Cálcio/metabolismo , Animais , Ácido Araquidônico/farmacologia , Lisofosfatidilcolinas/farmacologia , Manganês/metabolismo , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular de Duchenne/patologia , Fosfolipases A2 Independentes de Cálcio/antagonistas & inibidores , Sarcolema/enzimologia
12.
Mol Ther ; 17(1): 19-25, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19002166

RESUMO

Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution.


Assuntos
Distrofina/genética , Terapia Genética/métodos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia , Animais , Dependovirus/genética , Elasticidade , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Microscopia de Força Atômica , Distrofia Muscular Animal/fisiopatologia
13.
Dev Cell ; 4(5): 673-85, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12737803

RESUMO

The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. We show here that extracellular matrix (ECM) receptors of the beta1 integrin family regulate myoblast fusion. beta1-deficient myoblasts adhere to each other, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of beta1-deficient myoblasts, suggesting that beta1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, beta1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of beta1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers.


Assuntos
Fusão Celular , Integrina beta1/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Sarcômeros/metabolismo , Animais , Morte Celular , Divisão Celular , Movimento Celular , Células Cultivadas , Citoesqueleto/metabolismo , Integrina beta1/genética , Camundongos , Microscopia Eletrônica , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Músculo Esquelético/ultraestrutura , Mioblastos/ultraestrutura
14.
J Pineal Res ; 47(3): 238-52, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19664004

RESUMO

Oxidative stress-induced mitochondrial dysfunction plays a crucial role in the pathogenesis of a wide range of diseases including muscle disorders. In this study, we demonstrate that melatonin readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptosis of primary muscle cultures prepared from C57BL/6J mice. In particular, melatonin (10(-4)-10(-6) m) fully prevented myotube death induced by tert-butylhydroperoxide (t-BHP; 10 microm-24 hr) as assessed by acid phosphatase, caspase-3 activities and cellular morphological changes. Using fluorescence imaging, we showed that the mitochondrial protection provided by melatonin was associated with an inhibition of t-BHP-induced reactive oxygen species generation. In line with this observation, melatonin prevented t-BHP-induced mitochondrial depolarization and mitochondrial permeability transition pore (PTP) opening. This was associated with a highly reduced environment as reflected by an increased glutathione content and an increased ability to maintain mitochondrial pyridine nucleotides and glutathione in a reduced state. Using isolated mitochondria, in a similar manner as cyclosporin A, melatonin (10(-8)-10(-6) m) desensitized the PTP to Ca(2+) and prevented t-BHP-induced mitochondrial swelling, pyridine nucleotide and glutathione oxidation. In conclusion, our findings suggest that inhibition of the PTP essentially contributes to the protective effect of melatonin against oxidative stress in myotubes.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Melatonina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Células Musculares , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Espécies Reativas de Oxigênio , terc-Butil Hidroperóxido/farmacologia
15.
Biofactors ; 35(3): 279-94, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19322813

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by the absence of the protein dystrophin. Because oxidative stress contributes to the pathogenesis of DMD, we investigated if a green tea polyphenol blend (GTP) and its major polyphenol (-)-epigallocatechin gallate (EGCg), could protect muscle cell primary cultures from oxidative damage induced by hydrogen peroxide (H(2)O(2)) in the widely used mdx mouse model. On-line fluorimetric measurements using an H(2)O(2)-sensitive probe indicated that GTP and EGCg scavenged peroxide in a concentration-dependent manner. A 48 h exposure to EGCg increased glutathione content but did not alter the expression of proteins involved in membrane stabilization and repair. Pretreatment of dystrophic cultures with GTP or EGCg 48 h before exposure to H(2)O(2) improved cell survival. Normal cultures were protected by GTP but not by EGCg. 67LR, a receptor for EGCg, was seven times more abundant in dystrophic compared with normal cultures. Altogether our results demonstrate that GTP and EGCg protect muscle cells by scavenging H(2)O(2) and by improving the glutathione balance. In addition, the higher levels of 67LR in dystrophic muscle cells compared with normal ones likely contribute to EGCg-mediated survival.


Assuntos
Flavonoides/farmacologia , Glutationa/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Esquelético/citologia , Fenóis/farmacologia , Receptores de Laminina/metabolismo , Chá/química , Animais , Animais Recém-Nascidos , Catequina/análogos & derivados , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Mutantes , Estresse Oxidativo/efeitos dos fármacos , Polifenóis , Inibidores de Proteases/farmacologia
16.
Biochem J ; 395(2): 267-76, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16393138

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal disease caused by the lack of the cytoskeletal protein dystrophin. Altered calcium homoeostasis and increased calcium concentrations in dystrophic fibres may be responsible for the degeneration of muscle occurring in DMD. In the present study, we used subsarcolemmal- and mitochondrial-targeted aequorin to study the effect of the antiapoptotic Bcl-2 protein overexpression on carbachol-induced near-plasma membrane and mitochondrial calcium responses in myotubes derived from control C57 and dystrophic (mdx) mice. We show that Bcl-2 overexpression decreases subsarcolemmal and mitochondrial calcium overload that occurs during activation of nicotinic acetylcholine receptors in dystrophic myotubes. Moreover, our results suggest that overexpressed Bcl-2 protein may prevent near-plasma membrane and mitochondrial calcium overload by inhibiting IP3Rs (inositol 1,4,5-trisphosphate receptors), which we have shown previously to be involved in abnormal calcium homoeostasis in dystrophic myotubes. Most likely as a consequence, the inhibition of IP3R function by Bcl-2 also inhibits calcium-dependent apoptosis in these cells.


Assuntos
Apoptose , Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Canais de Cálcio , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Expressão Gênica , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Estaurosporina/farmacologia
17.
Cell Calcium ; 40(4): 359-71, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16806462

RESUMO

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.


Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Equorina/genética , Equorina/metabolismo , Animais , Aorta/citologia , Soluções Tampão , Canais de Cálcio/metabolismo , Células Cultivadas , Citosol/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipertensão/metabolismo , Miócitos de Músculo Liso/citologia , Purinas/metabolismo , Ratos , Receptores Purinérgicos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sódio/metabolismo
18.
FASEB J ; 17(1): 28-37, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12522109

RESUMO

We investigated the role of mitochondria (MT) in calcium signaling in a culture of rat aortic smooth muscle cells. We used targeted aequorin to selectively measure [Ca2+] in this organelle. Our results reveal that smooth muscle cell stimulation with agonists causes a large, transient increase in mitochondrial [Ca2+] ([Ca2+]m). This large transient can be blocked with inhibitors of the sarco-endoplasmic reticulum Ca2+-ATPase, suggesting a close relationship between the sarcoplasmic reticulum (SR) and the mitochondria. FCCP completely abolished the response to agonists, and targeted mitochondrial GFP revealed a vast tubular network of MT in these cells. When added before stimulation with ATP, IP3 inhibitors partially blocked the ATP-induced rise in mitochondrial Ca2+ release. The role of the Na+/Ca2+ exchanger (NCX) was examined by removing extracellular Na+. This procedure prevented the decrease in the [Ca2+]m transient normally seen on removal of extracellular Ca2+. We propose a functional linkage of MT and SR dependent on a narrow junctional space between the two organelles in which Ca2+ diffusion is restricted. Approximately half of the mitochondria appear to be associated with the superficial SR, which communicates with the extracellular space via NCX.


Assuntos
Sinalização do Cálcio , Mitocôndrias/metabolismo , Músculo Liso Vascular/metabolismo , Trifosfato de Adenosina/farmacologia , Equorina/genética , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular , Células Cultivadas , Receptores de Inositol 1,4,5-Trifosfato , Modelos Biológicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/ultraestrutura , Ratos , Receptores Citoplasmáticos e Nucleares/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Trocador de Sódio e Cálcio/fisiologia , Desacopladores/farmacologia , Vasopressinas/farmacologia
19.
Free Radic Res ; 39(9): 913-9, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16087472

RESUMO

Cyclosporin A (CsA) generates superoxide in smooth muscle cells. Our earlier studies have demonstrated that the increase in the vasopressin type 1 receptor induced in vascular smooth muscle cells in the presence of CsA is probably due to superoxide (Krauskopf et al., J Biol Chem 278, 41685-41690, 2003). This increase in vasopressin receptor is likely at the base of increased vascular responsiveness to vasoconstrictor hormones and hypertension induced by CsA. Here, we demonstrate that CsA produces superoxide. In addition, our data show that superoxide generation does not originate from the major cellular superoxide generating systems NAD(P)H oxidase or xanthine oxidase. Our results suggest that the side effects of CsA could be diminished with the help of SOD mimetic drugs.


Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Superóxidos/metabolismo , Animais , Aorta/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Etídio/análogos & derivados , Fluoresceínas , Sequestradores de Radicais Livres/farmacologia , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética
20.
J Neuromuscul Dis ; 2(4): 325-342, 2015 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-27858750

RESUMO

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene coding for dystrophin and leads to muscle degeneration, wheelchair dependence and death by cardiac or respiratory failure. Physical exercise has been proposed as a palliative therapy for DMD to maintain muscle strength and prevent contractures for as long as possible. However, its practice remains controversial because the benefits of training may be counteracted by muscle overuse and damage.The effects of physical exercise have been investigated in muscles of dystrophin-deficient mdx mice and in patients with DMD. However, a lack of uniformity among protocols limits comparability between studies and translatability of results from animals to humans. In the present review, we summarize and discuss published protocols used to investigate the effects of physical exercise on mdx mice and DMD patients, with the objectives of improving comparability between studies and identifying future research directions.

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