Effects of chronic electrical stimulation on myosin heavy chain expression in satellite cell cultures derived from rat muscles of different fiber-type composition.

Myotube cultures were established from satellite cells of three rat muscles of different fiber-type composition, slow-twitch soleus, diaphragm, and fast-twitch tibialis anterior (TA). Effects of chronic electrical stimulation were studied by exposing these cultures for up to 13 days to a stimulus pattern consisting of 250 ms impulse trains of 40 Hz, repeated every 4 s. Changes in myosin expression were assessed at the mRNA level by Northern blotting and in situ hybridization. Expression of slow myosin at the protein level was analysed by immunoblotting and immunohistochemistry with two antibodies, one specific to adult slow myosin, the other reacting with developmental and adult slow myosin heavy chain (MHCI) isoforms. In all three myotube cultures stimulation enhanced the mRNA and protein expression of a developmental isoform of slow myosin (MHCI). However, the three myotube cultures differed in the extent of the increase in MHCI. It was greatest in soleus-derived myotubes, least in TA-derived myotubes, and intermediate in diaphragm-derived myotubes. In addition to the increase in slow myosin, long-term stimulation led to an isoform switch, as indicated by an increase in myotubes reacting with the antibody specific for the adult MHCI. Our results suggest that enhanced contractile activity promotes the expression of the slow phenotype predetermined in satellite cells of slow-twitch, type I fibers. The different extents of increased slow myosin expression may thus be explained as reflecting different percentages of type I fibers and consequently of slow-type satellite cells in the corresponding donor muscles.

Fast myosin heavy chain diversity in skeletal muscles of the rabbit: heavy chain IId, not IIb predominates.

The myosin heavy chain (HC) composition of various rabbit muscles was analysed at both the mRNA and the protein level. S1-nuclease mapping was performed with a cDNA probe specific for myosin HCIIa, yielding a fully protected sequence for HCIIa, a partially protected sequence for HCIIb, and an additional signal putatively assigned to HCIId. At the protein level, three fast myosin HC isoforms, HCIIa, HCIIb and HCIId, were separated by gradient PAGE. The results obtained at the protein level were in agreement with the findings at the mRNA level. The expression of appreciable amounts of myosin HCIIb, the predominating isoform of fast-twitch muscles in rat and mouse, was restricted in the rabbit to only a few muscles, i.e. adductor magnus, gastrocnemius, latissimus dorsi and vastus lateralis. Typical fast-twitch muscles such as extensor digitorum longus, tibialis anterior and psoas contained only minute amounts of HCIIb. The HCIId isoform, demonstrated in the present study for the first time in rabbit, is the predominating fast myosin HC isoform in this species. Electrophoretic analyses of myosin HC in histochemically defined single fibers confirmed the lack of fibers expressing only HCIIb in tibialis anterior, whereas such fibers were found in the adductor magnus. In addition to fiber types IIB, IID, and IIA expressing HCIIb, HCIId, and HCIIa, respectively, an appreciable amount of hybrid fibers coexpressing two HC isoforms at various ratios were found: HCIIb > HCIId; HCIId > HCIIb; HCIId > HCIIa; HCIIa > HCIId; HCIIa > HCI; HCI > HCIIa. This fiber-type spectrum indicates possible fiber-type transitions in the order IIB<==> IIB<==>IIDB<==>IID<==>IIDA<==>IIAD<==>IIA<==>IIC<==>IC <==>I.