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1.
Bio Protoc ; 13(13): e4759, 2023 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-37456334

RESUMO

In vitro models are essential for investigating the molecular, biochemical, and cell-biological aspects of skeletal muscle. Still, models that utilize cell lines or embryonic cells do not fully recapitulate mature muscle fibers in vivo. Protein function is best studied in mature differentiated tissue, where biological context is maintained, but this is often difficult when reliable detection reagents, such as antibodies, are not commercially available. Exogenous expression of tagged proteins in vivo solves some of these problems, but this approach can be technically challenging because either a mouse must be engineered for each protein of interest or viral vectors are required for adequate levels of expression. While viral vectors can infect target cells following local administration, they carry the risk of genome integration that may interfere with downstream analyses. Plasmids are another accessible expression system, but they require ancillary means of cell penetration; electroporation is a simple physical method for this purpose that requires minimal training or specialized equipment. Here, we describe a method for in vivo plasmid expression in a foot muscle following electroporation.

2.
J Cell Biol ; 220(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33496727

RESUMO

Mutations in ANO5 (TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.


Assuntos
Anexinas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Anoctaminas/química , Anoctaminas/deficiência , Anoctaminas/genética , Anoctaminas/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Cinética , Camundongos Knockout , Mutação/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Domínios Proteicos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Hum Mol Genet ; 26(10): 1952-1965, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28334834

RESUMO

Limb Girdle Muscular Dystrophies type 2I (LGMD2I), a recessive autosomal muscular dystrophy, is caused by mutations in the Fukutin Related Protein (FKRP) gene. It has been proposed that FKRP, a ribitol-5-phosphate transferase, is a participant in α-dystroglycan (αDG) glycosylation, which is important to ensure the cell/matrix anchor of muscle fibers. A LGMD2I knock-in mouse model was generated to express the most frequent mutation (L276I) encountered in patients. The expression of FKRP was not altered neither at transcriptional nor at translational levels, but its function was impacted since abnormal glycosylation of αDG was observed. Skeletal muscles were functionally impaired from 2 months of age and a moderate dystrophic pattern was evident starting from 6 months of age. Gene transfer with a rAAV2/9 vector expressing Fkrp restored biochemical defects, corrected the histological abnormalities and improved the resistance to eccentric stress in the mouse model. However, injection of high doses of the vector induced a decrease of αDG glycosylation and laminin binding, even in WT animals. Finally, intravenous injection of the rAAV-Fkrp vector into a dystroglycanopathy mouse model due to Fukutin (Fktn) knock-out indicated a dose-dependent toxicity. These data suggest requirement for a control of FKRP expression in muscles.


Assuntos
Distrofia Muscular do Cíngulo dos Membros/terapia , Proteínas/genética , Proteínas/uso terapêutico , Animais , Modelos Animais de Doenças , Distroglicanas/metabolismo , Expressão Gênica , Regulação da Expressão Gênica/genética , Terapia Genética/métodos , Glicosilação , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Distrofias Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação , Pentosefosfatos/metabolismo , Pentosiltransferases , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Transferases
4.
Skelet Muscle ; 6: 20, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27257474

RESUMO

BACKGROUND: Secondary dystroglycanopathies are a subset of muscular dystrophy caused by abnormal glycosylation of α-dystroglycan (αDG). Loss of αDG functional glycosylation prevents it from binding to laminin and other extracellular matrix receptors, causing muscular dystrophy. Mutations in a number of genes, including FKTN (fukutin), disrupt αDG glycosylation. METHODS: We analyzed conditional Fktn knockout (Fktn KO) muscle for levels of mTOR signaling pathway proteins by Western blot. Two cohorts of Myf5-cre/Fktn KO mice were treated with the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) for 4 weeks and evaluated for changes in functional and histopathological features. RESULTS: Muscle from 17- to 25-week-old fukutin-deficient mice has activated mTOR signaling. However, in tamoxifen-inducible Fktn KO mice, factors related to Akt/mTOR signaling were unchanged before the onset of dystrophic pathology, suggesting that Akt/mTOR signaling pathway abnormalities occur after the onset of disease pathology and are not causative in early dystroglycanopathy development. To determine any pharmacological benefit of targeting mTOR signaling, we administered RAPA daily for 4 weeks to Myf5/Fktn KO mice to inhibit mTORC1. RAPA treatment reduced fibrosis, inflammation, activity-induced damage, and central nucleation, and increased muscle fiber size in Myf5/Fktn KO mice compared to controls. RAPA-treated KO mice also produced significantly higher torque at the conclusion of dosing. CONCLUSIONS: These findings validate a misregulation of mTOR signaling in dystrophic dystroglycanopathy skeletal muscle and suggest that such signaling molecules may be relevant targets to delay and/or reduce disease burden in dystrophic patients.


Assuntos
Distroglicanas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular Animal/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Regulação para Baixo , Estimulação Elétrica , Feminino , Predisposição Genética para Doença , Glicosilação , Masculino , Camundongos Knockout , Contração Muscular , Força Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/fisiopatologia , Fator Regulador Miogênico 5/deficiência , Fator Regulador Miogênico 5/genética , Fenótipo , Processamento de Proteína Pós-Traducional , Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Torque , Transferases
5.
Am J Physiol Cell Physiol ; 311(2): C190-200, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27281480

RESUMO

The primary objective of this study was to determine whether alterations in mitochondria affect recovery of skeletal muscle strength and mitochondrial enzyme activity following myotoxic injury. 3-Methyladenine (3-MA) was administered daily (15 mg/kg) to blunt autophagy, and the creatine analog guanidionpropionic acid (ß-GPA) was administered daily (1% in chow) to enhance oxidative capacity. Male C57BL/6 mice were randomly assigned to nontreatment (Con, n = 6), 3-MA-treated (n = 6), and ß-GPA-treated (n = 8) groups for 10 wk. Mice were euthanized at 14 days after myotoxic injury for assessment of mitochondrial remodeling during regeneration and its association with the recovery of muscle strength. Expression of several autophagy-related proteins, e.g., phosphorylated Ulk1 (∼2- to 4-fold, P < 0.049) was greater in injured than uninjured muscles, indicating a relationship between muscle regeneration/remodeling and autophagy. By 14 days postinjury, recovery of muscle strength (18% less, P = 0.03) and mitochondrial enzyme (e.g., citrate synthase) activity (22% less, P = 0.049) were significantly lower in 3-MA-treated than Con mice, suggesting that the autophagy process plays an important role during muscle regeneration. In contrast, muscle regeneration was nearly complete in ß-GPA-treated mice, i.e., muscle strength recovered to 93% of baseline vs. 78% for Con mice. Remarkably, 14 days allowed sufficient time for a near-complete recovery of mitochondrial function in ß-GPA-treated mice (e.g., no difference in citrate synthase activity between injured and uninjured, P = 0.49), indicating a robust mitochondrial remodeling process during muscle regeneration. In conclusion, autophagy is likely activated following muscle injury and appears to play an important role in functional muscle regeneration.


Assuntos
Autofagia/fisiologia , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/fisiologia , Recuperação de Função Fisiológica/fisiologia , Regeneração/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Força Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , Doenças Musculares/tratamento farmacológico , Doenças Musculares/fisiopatologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
6.
PLoS One ; 11(1): e0147049, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26751696

RESUMO

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury.


Assuntos
Distroglicanas/genética , Fibras Musculares Esqueléticas/fisiologia , Distrofia Muscular Animal/genética , Proteínas/genética , Regeneração , Animais , Cardiotoxinas/química , Modelos Animais de Doenças , Distroglicanas/metabolismo , Éxons , Deleção de Genes , Genótipo , Glicosilação , Laminina/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/metabolismo , Necrose , Transdução de Sinais , Transferases
7.
J Clin Invest ; 122(9): 3330-42, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22922256

RESUMO

Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. Disruption of the extensive carbohydrate structure normally present on α-dystroglycan causes an array of congenital and limb girdle muscular dystrophies known as dystroglycanopathies. The essential role of dystroglycan in development has hampered elucidation of the mechanisms underlying dystroglycanopathies. Here, we developed a dystroglycanopathy mouse model using inducible or muscle-specific promoters to conditionally disrupt fukutin (Fktn), a gene required for dystroglycan processing. In conditional Fktn-KO mice, we observed a near absence of functionally glycosylated dystroglycan within 18 days of gene deletion. Twenty-week-old KO mice showed clear dystrophic histopathology and a defect in glycosylation near the dystroglycan O-mannose phosphate, whether onset of Fktn excision driven by muscle-specific promoters occurred at E8 or E17. However, the earlier gene deletion resulted in more severe phenotypes, with a faster onset of damage and weakness, reduced weight and viability, and regenerating fibers of smaller size. The dependence of phenotype severity on the developmental timing of muscle Fktn deletion supports a role for dystroglycan in muscle development or differentiation. Moreover, given that this conditional Fktn-KO mouse allows the generation of tissue- and timing-specific defects in dystroglycan glycosylation, avoids embryonic lethality, and produces a phenotype resembling patient pathology, it is a promising new model for the study of secondary dystroglycanopathy.


Assuntos
Modelos Animais de Doenças , Distroglicanas/metabolismo , Distrofias Musculares/genética , Proteínas/genética , Animais , Feminino , Deleção de Genes , Glicosilação , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofias Musculares/metabolismo , Fenótipo , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/metabolismo , Transferases
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