RESUMO
Null mutation of titin-cap (TCAP) causes limb-girdle muscular dystrophy type 2G (LGMD2G). LGMD2G patients develop muscle atrophy, and lose the ability to walk by their third decade. Previous findings suggest that TCAP regulates myostatin, a key regulator of muscle growth. We tested the hypothesis that TCAP knockdown with RNA interference will lead to differential expression of genes involved in muscle proliferation and differentiation, impairing muscle cell growth. mRNA from cultured cells treated with TCAP siRNA duplex constructs was analyzed using Northern blots and real-time RT-PCR. siRNA treatment decreased TCAP mRNA expression in differentiating muscle cells. Significant (p<0.05) decreases in mRNA were observed for myogenic regulatory factors. siRNA treatment also prevented development of the normal phenotype of muscle cells. Our findings suggest that TCAP knockdown with RNA interference alters normal muscle cell differentiation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas Musculares/fisiologia , Mioblastos Esqueléticos/metabolismo , Interferência de RNA , Animais , Sequência de Bases , Northern Blotting , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células , Conectina , Imunofluorescência , Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Proteína MyoD/genética , Mioblastos Esqueléticos/citologia , Miogenina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Intact dystrophin-deficient canine muscles were previously shown to incur greater-than-normal stretch-induced force deficits. Here we tested the hypothesis that maximally activated detergent-treated (skinned) single fibers from normal and dystrophin-deficient dogs would incur comparable force deficits after stretch. Skinned cranial sartorius (CS) fibers from dystrophin-deficient and normal dogs were calcium-activated (pCa 4.5) and rapidly stretched. A single 30% stretch induced force deficits of 27.07 +/- 3.9% and 29.7 +/- 4.8% in dystrophin-deficient (n = 22) and normal (n = 18) fibers, respectively. Our data support the hypothesis that maximally activated skinned single fibers from normal and dystrophin-deficient dogs incur comparable force deficits after stretch. Our findings suggest that knowledge of the extent of stretch-induced force deficits following repeated stretch-activations in the GRMD dog may be useful to assess future therapeutic interventions aimed at replacing dystrophin in the sarcolemmal membrane.