Two dihydropyridine receptor mRNA isoforms (cardiac and skeletal) are expressed in rat skeletal muscle cells in primary culture. The progressive changes in excitation-contraction coupling mode from dual mode (‘skeletal’ and ‘cardiac’) to predominant ‘skeletal’ one during in vitro myogenesis are thought to be linked to the developmental changes in the relative expression of the two types of molecular entity previously observed in this preparation. In order to test this hypothesis, myotube cultures (5- to 7-day-old) were treated with antisense phosphorothioated oligodeoxynucleotides against cardiac or skeletal alpha1 subunit of L-type calcium channel. The oligodeoxynucleotide uptake by cells was checked by means of imaging of fluorescent oligodeoxynucleotide derivatives within the cells. Optimum concentration used (10 microM in the extracellular medium) and incubation time (70 hours) were empirically determined. Antisense directed against the cardiac type led to a 54% decrease in the averaged L-type calcium current peak density at −10 mV. The same type of experiment was performed with antisense against the skeletal isoform and led to a same order of inhibition (46%). This result clearly shows that the two isoforms can work as a calcium channel. Conversely, analysis of the shape of T-V (relative contractile amplitude versus membrane potential) curves shows that the treatment with ‘skeletal’ antisense depressed the contractile response in the medium membrane potential range whereas treatment with ‘cardiac’ antisense had no effect. This and other results taken together suggest that the skeletal isoform of dihydropyridine receptor is involved in both ‘cardiac’ and ‘skeletal’ types of EC coupling mechanisms at work in early stages of myotubes in vitro development. The type of coupling probably depends on the proximity of the skeletal dihydropyridine receptor and the ryanodine receptor.
Accumulation and nuclear import of HIF1 alpha during high and low oxygen concentration in skeletal muscle cells in primary culture
