Regulation of dihydropyridine receptor and ryanodine receptor gene expression in regenerating skeletal muscle

Abstract

One of the the major properties of mature skeletal muscle is its ability to regenerate after injury. The purpose of the present study was to determine whether the expression of genes encoding the dihydropyridine receptor calcium channel (DHPR) and the ryanodine receptor (RyR), which play a critical role in excitation-contraction coupling, is regulated by skeletal muscle regeneration. The process of regeneration was induced by bupivacaine injection in surgically exposed rat extensor digitorum longus (EDL) muscle. After total RNA isolation from the injected and the contralateral control EDL muscles performed 3, 7, 15 and 30 days following injection, Northern blot and RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac muscle isoforms of both the DH-PR α1-subunit and the RyR. After 3 days, an initial precipitous decrease in the expression of the genes encoding the skeletal muscle isoforms of the DHPR and RyR was observed, followed by an increase. Moreover, regenerating skeletal muscle transiently expressed mRNA for the DHPR cardiac isoform, mainly at the beginning of regeneration. No expression of mRNA for the cardiac RyR was observed. Contraction experiments, performed using EDL muscle at the same times after bupivacaine injection, showed that twitch amplitude was markedly decreased in the absence of external calcium, but only during the early stages of regeneration. Similar findings in relation to expression of skeletal and cardiac muscle DH-PR message were previously reported from experiments conducted during early developmental stages using fetal skeletal muscle and muscle cell cultures [Chaudhari N, Beam KG (1993) Dev Biol 155:507-515]. These results suggest that expression of the DHPR cardiac isoform in skeletal muscle could explain certain cardiac-like aspects of excitation-contraction coupling of regenerating skeletal muscle and developing skeletal muscle as well.& k w d :

Differential expression of KvLQT1 isoforms across the human ventricular wall

“…Antisense RNA probes were produced using appropriate polymerase (T3, T7, or SP6, Ambion) on linearized KvLQT1 isoform 1, KvLQT1 isoform 2, IsK, and cyclophilin templates in the presence of [␣-32 P]rUTP (DuPont NEN). RNase protection assays (RPAs) were carried out as previously described (17) using the RPA II Kit (Ambion). For each tissue source, 15 µg of total RNA were hybridized to 0.5-1 ϫ 10 5 counts/min of the RNA probe.…”

Sodium withdrawal contractures in developing and regenerating rat extensor digitorum longus muscles

“…20 The sodium withdrawal contractures observed in the present study seem to resemble contractures seen in cardiac muscle which are considered to reflect a Na/Ca exchange. 5,6 Taken together, these results confirmed that excitation-contraction coupling of developing and regenerating EDL muscles is rather different from that observed in adult EDL muscles.…”

Dihydropyridine receptor isoform expression in adult rat skeletal muscle

“…The RNase protection assay (RPA) was used in the present study to assess mRNA expression levels, as it provides a greater sensitivity than Northern blots. RPAs were carried out as previously described [28]. Antisense RNA probes were produced using T7 polymerase (Stratagene, La Jolla, Calif., USA) on linearized 1fb, C32, GAPDH or actin templates in the presence of [α-32 P]rUTP (DuPont NEN, Boston, Mass., USA).…”