RESUMO
Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn(-/-) mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn(-/-) mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn(-/-) mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn(-/-) mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.
Assuntos
Metabolismo Energético , Contração Muscular , Músculo Esquelético/metabolismo , Miostatina/metabolismo , Resistência Física , Animais , Genótipo , Glicólise , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Fadiga Muscular , Miostatina/deficiência , Miostatina/genética , Consumo de Oxigênio , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fenótipo , Fosfopiruvato Hidratase/metabolismo , Corrida , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.
Assuntos
Processamento Alternativo , Núcleo Celular/metabolismo , Éxons , Multimerização Proteica , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Núcleo Celular/patologia , Células HeLa , Humanos , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Expansão das Repetições de TrinucleotídeosRESUMO
Myotonic dystrophy type 1 (DM1) is an RNA-dominant disease whose pathogenesis stems from the functional loss of muscleblind-like RNA-binding proteins (RBPs), which causes the formation of alternative-splicing defects. The loss of functional muscleblind-like protein 1 (MBNL1) results from its nuclear sequestration by mutant transcripts containing pathogenic expanded CUG repeats (CUGexp). Here we show that an RBP engineered to act as a decoy for CUGexp reverses the toxicity of the mutant transcripts. In vitro, the binding of the RBP decoy to CUGexp in immortalized muscle cells derived from a patient with DM1 released sequestered endogenous MBNL1 from nuclear RNA foci, restored MBNL1 activity, and corrected the transcriptomic signature of DM1. In mice with DM1, the local or systemic delivery of the RBP decoy via an adeno-associated virus into the animals' skeletal muscle led to the long-lasting correction of the splicing defects and to ameliorated disease pathology. Our findings support the development of decoy RBPs with high binding affinities for expanded RNA repeats as a therapeutic strategy for myotonic dystrophies.
Assuntos
Distrofia Miotônica , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Humanos , Camundongos , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/terapia , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Mutant DMPK transcripts containing expanded CUG repeats (CUGexp) are retained within the nucleus of myotonic dystrophy type 1 (DM1) cells as discrete foci. Nuclear CUGexp-RNA foci that sequester MBNL1 splicing factor represent a hallmark of this RNA dominant disease caused by the expression of expanded microsatellite repeats. Here we described fluorescent in situ hybridization (FISH) techniques to detect either RNA containing CUG expansion or DMPK transcripts in human DM1 or WT cells. In addition, we propose a combined FISH/immunofluorescence protocol to visualize the colocalization of MBNL1 with CUGexp-RNA foci in DM1 cells.
Assuntos
Hibridização in Situ Fluorescente/métodos , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Proteínas de Ligação a RNA/genética , Núcleo Celular/genética , Células Cultivadas , Imunofluorescência , Humanos , Mutação , Imagem Individual de Molécula , Expansão das Repetições de TrinucleotídeosRESUMO
The adeno-associated virus (AAV) vector is an efficient tool for gene delivery in skeletal muscle. AAV-based therapies show promising results for treatment of various genetic disorders, including muscular dystrophy. These dystrophies represent a heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency through direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different recombinant AAV (rAAV) serotypes is therefore needed. In this study, we investigated the transduction efficiency of rAAV serotypes 1-10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of ß-galactosidase activity on muscle cryosections demonstrated that rAAV serotypes 1, 7, 8, 9, and 10 were more efficient than the others, with rAAV9 being the most efficient in mice. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle, rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that the human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.
Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Músculo Esquelético/metabolismo , Transdução Genética , Animais , Dependovirus/classificação , Feminino , Expressão Gênica , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Injeções Intramusculares , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Sorogrupo , TransgenesRESUMO
Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.
Assuntos
Peptídeos Penetradores de Células/farmacologia , Músculo Esquelético/metabolismo , Distrofia Miotônica , Miotonina Proteína Quinase , Oligodesoxirribonucleotídeos Antissenso , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Músculo Esquelético/patologia , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Miotonina Proteína Quinase/genética , Miotonina Proteína Quinase/metabolismo , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
α-Sarcoglycanopathy (limb-girdle muscular dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by deficiency in α-sarcoglycan, a transmembrane protein part of the dystrophin-associated complex. To date, no treatment exists for this disease. We constructed recombinant pseudotype-1 adeno-associated virus (rAAV) vectors expressing the human α-sarcoglycan cDNA from a ubiquitous or a muscle-specific promoter. Evidence of specific immune response leading to disappearance of the vector was observed with the ubiquitous promoter. In contrast, efficient and sustained transgene expression with correct sarcolemmal localization and without evident toxicity was obtained with the muscle-specific promoter after intra-arterial injection into the limbs of an LGMD2D murine model. Transgene expression resulted in restoration of the sarcoglycan complex, histological improvement, membrane stabilization, and correction of pseudohypertrophy. More importantly, α-sarcoglycan transfer produced full rescue of the contractile force deficits and stretch sensibility and led to an increase of the global activity of the animals when both posterior limbs are injected. Our results establish the feasibility for AAV-mediated α-sarcoglycan gene transfer as a therapeutic approach.
RESUMO
alpha-Sarcoglycanopathy (limb-girdle muscular dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by deficiency in alpha-sarcoglycan, a transmembrane protein part of the dystrophin-associated complex. To date, no treatment exists for this disease. We constructed recombinant pseudotype-1 adeno-associated virus (rAAV) vectors expressing the human alpha-sarcoglycan cDNA from a ubiquitous or a muscle-specific promoter. Evidence of specific immune response leading to disappearance of the vector was observed with the ubiquitous promoter. In contrast, efficient and sustained transgene expression with correct sarcolemmal localization and without evident toxicity was obtained with the muscle-specific promoter after intra-arterial injection into the limbs of an LGMD2D murine model. Transgene expression resulted in restoration of the sarcoglycan complex, histological improvement, membrane stabilization, and correction of pseudohypertrophy. More importantly, alpha-sarcoglycan transfer produced full rescue of the contractile force deficits and stretch sensibility and led to an increase of the global activity of the animals when both posterior limbs are injected. Our results establish the feasibility for AAV-mediated alpha-sarcoglycan gene transfer as a therapeutic approach.
Assuntos
Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Músculos/metabolismo , Sarcoglicanas/deficiência , Sarcoglicanas/metabolismo , Animais , Permeabilidade da Membrana Celular , Dependovirus/classificação , Distrofina/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Hipertrofia/genética , Hipertrofia/metabolismo , Hipertrofia/patologia , Injeções Intra-Arteriais , Cinética , Camundongos , Especificidade de Órgãos , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Sarcoglicanas/genéticaRESUMO
We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT) transcripts. We developed 5'-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD) mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV) delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.
RESUMO
Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Músculo Esquelético/patologia , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/patologia , Adulto , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Linhagem Celular Transformada , Criança , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteína MyoD/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , RNA/metabolismoRESUMO
Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.
Assuntos
Processamento Alternativo/genética , Arritmias Cardíacas/complicações , Arritmias Cardíacas/genética , Sistema de Condução Cardíaco/fisiopatologia , Distrofia Miotônica/complicações , Distrofia Miotônica/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adulto , Idoso , Animais , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Fenômenos Eletrofisiológicos , Éxons/genética , Feminino , Células HEK293 , Sistema de Condução Cardíaco/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Motivos de Nucleotídeos/genética , Proteínas de Ligação a RNA/metabolismo , Canais de Sódio/metabolismo , XenopusRESUMO
Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1.
Assuntos
Distrofina/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Distrofia Miotônica/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Peixe-Zebra/genética , Animais , Cromatografia Líquida , Distrofina/metabolismo , Éxons , Homeostase , Humanos , Imuno-Histoquímica , Imunoprecipitação , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Eletrônica , Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/metabolismo , Distrofia Miotônica/patologia , Reação em Cadeia da Polimerase em Tempo Real , Retículo Sarcoplasmático/ultraestrutura , Espectrometria de Massas em Tandem , Proteínas de Peixe-Zebra/metabolismoRESUMO
Dystrophin contributes to force transmission and has a protein-scaffolding role for a variety of signaling complexes in skeletal muscle. In the present study, we tested the hypothesis that the muscle adaptive response following mechanical overloading (ML) would be decreased in MDX dystrophic muscle lacking dystrophin. We found that the gains in muscle maximal force production and fatigue resistance in response to ML were both reduced in MDX mice as compared to healthy mice. MDX muscle also exhibited decreased cellular and molecular muscle remodeling (hypertrophy and promotion of slower/oxidative fiber type) in response to ML, and altered intracellular signalings involved in muscle growth and maintenance (mTOR, myostatin, follistatin, AMPKα1, REDD1, atrogin-1, Bnip3). Moreover, dystrophin rescue via exon skipping restored the adaptive response to ML. Therefore our results demonstrate that the adaptive response in response to ML is impaired in dystrophic MDX muscle, most likely because of the dystrophin crucial role.
Assuntos
Adaptação Fisiológica , Músculo Esquelético/fisiologia , Animais , Fenômenos Biomecânicos , Camundongos , Fadiga Muscular , Distrofia Muscular de Duchenne/fisiopatologia , Transdução de SinaisRESUMO
Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with metastatic melanoma (B16-F10) or carcinoma (LLC1) cells including conversion to the myogenic lineage in vitro and in vivo, as well as inhibition of melanin production in melanoma cells coupled with cytotoxic and cytostatic effects. No effect is seen with non-tumorigenic cells. Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene. Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers.
Assuntos
Músculo Esquelético/citologia , Mioblastos/citologia , Neoplasias Experimentais/patologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Citotoxicidade Imunológica/imunologia , Desmina/genética , Desmina/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Melaninas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Músculo Esquelético/imunologia , Músculo Esquelético/metabolismo , Mioblastos/imunologia , Mioblastos/metabolismo , Metástase Neoplásica , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
With the aim of simplifying recombinant-adeno-associated virus (rAAV) delivery in muscle, a new femoral intra-arterial technique was designed and tested in rodents (rats and mice). Two serotypes, several promoters and transgenes (reporter or therapeutic) were tested using this administration route. The new route is both easy to perform and efficient. Its usefulness as a tool to assess gene delivery constructs in the muscle was established in the context of recombinant AAV serotypes 1 and 2, and with the ubiquitous CMV and two muscle-specific (C5-12 and CK6) promoters. Both serum monitoring of a secreted protein (murine alkaline phosphatase: muSEAP) and slide staining were used to compare the different constructs. Significantly different patterns of expression in kinetics of expression (muSEAP) and homogeneity of fiber transduction (staining) were evidenced with the different promoters tested, and compared with intra-muscular expression patterns. Detailed studies of differential transduction in leg and thigh muscles showed equivalent efficacy, except in rectus femoris, and to a lesser extent in soleus. In light of these results and prior data, intra-arterially mediated gene transfer mechanism is discussed.