RESUMO
In myotonic ADR mice that are homozygous for a defect in the muscular chloride channel gene adr/Clc-1, the hyperexcitability of fast muscles is associated with secondary changes in gene expression and fibre type composition. cDNA clones derived from a set of genes down regulated in fast muscles of the myotonic ADR mouse were isolated by a subtractive cloning procedure. A total of 1200 clones were analysed for high expression in fast muscle of wild type and low expression in mutant mouse. Differential transcript levels were verified by northern blot hybridizations. The identities of the corresponding transcripts were determined by sequencing as myosin heavy chain IIB, alpha-tropomyosin, troponin C, a Ca2+ ATPase and parvalbumin mRNAs. Of these, mRNAs for parvalbumin and myosin heavy chain IIB were drastically downregulated in myotonic muscle (to < 10% of control). A full length cDNA clone for skeletal muscle alpha-tropomyosin was homologous to the mouse fibroblast tropomyosin isoform 2, except for the portion encoding the alpha-tropomyosin specific amino acids 258-284. A cDNA derived from the 1100 nucleotide parvalbumin transcript was cloned and the sequence for the as yet unknown 3' extended trailer, generated by alternative polyadenylation, was determined.
Assuntos
Clonagem Molecular , DNA Complementar/biossíntese , Doenças Musculares/genética , Miotonia/genética , Animais , Sequência de Bases , Northern Blotting , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Regulação para Baixo/fisiologia , Biblioteca Gênica , Camundongos , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Doenças Musculares/metabolismo , Miotonia/metabolismo , Hibridização de Ácido Nucleico , RNA/biossínteseRESUMO
Skeletal muscle fibers from a patient with Schwartz-Jampel syndrome were studied in vitro. The fibers had normal resting membrane potentials, but their resting [Ca2+]i was elevated. The resting potentials were unstable and spontaneous depolarizations caused twitching in all fibers. Stimulated contractions were characterized by markedly slowed relaxation which was due to electrical after-activity. Neither curare (0.7 microM), tocainide (50 microM), nor phenytoin (80 microM) had an effect on the myotonic activity. In contrast, procainamide (200 microM) suppressed the hyperexcitability without affecting the twitch amplitude. The steady-state current-voltage relation was normal in 5 fibers, but altered in 3 others. These latter fibers had an increased specific membrane resistance owing to a decreased Cl- conductance. The Na+ channels were investigated in the cell-attached patch clamp mode. In all patches on either type of fiber, depolarizing pulses elicited delayed, synchronized openings of Na+ channels. These abnormal openings occurred even after the surface membrane repolarized. We hypothesize that these altered membrane conductances are responsible for the hyperexcitability and the associated slowed relaxation.