Subcellular localization of newly incorporated myosin in rabbit fast skeletal muscle undergoing stimulation-induced type transformation

Abstract

Immunogold labelling was used to study the distribution of newly synthesized slow muscle myosin (SM) at the ultrastructural level as it replaced fast muscle myosin (FM) in rabbit muscles undergoing stimulation-induced type transformation. Control fast muscle was labelled strongly with antibody to FM and control slow muscle with antibody to SM; label was confined to the A-band. Well-defined differences in the distribution of label within the A-band suggested that the monoclonal antibodies used corresponded to epitopes on different parts of the myosin molecule; this was confirmed by Western blots of subfragments prepared from FM and SM. After 4 weeks of continuous stimulation at 10 Hz, fibres of the tibialis anterior muscle reacted with antibodies to both isoforms; after 6 weeks, labelling was obtained only with antibody to SM. After a 7-week period of stimulation and 3 further weeks of recovery, fibres again reacted with both antibodies. In all positively-labelled sections, the distribution of gold particles was characteristic of the antibody and independent of the origin or history of the fibres. This observation supports the conclusion that newly synthesized myosin is capable of being incorporated throughout the length and cross-section of the A-band.

Atrophy, but not necrosis, in rabbit skeletal muscle denervated for periods up to one year

“…Expression of developmental MHCs was demonstrated immunohistochemically with a mouse anti-rat developmental (embryonic) myosin (NCL-MHCd; Novocastra Laboratories, Newcastle-upon-Tyne, UK) and the antibody WB-MHC neo, specific to neonatal MHC. The latter and antibodies WB-MHCf and WB-MHCs were first raised in this research group by Dr. W. E. Brown (16,17). For all the antibodies, binding was detected with biotinylated horse anti-mouse IgG (Vector, Peterborough, UK) as the secondary antibody, amplified by the avidin-biotin horseradish peroxidase technique (ABC Elite, Vector), and visualized by incubation with 3,3Ј-diaminobenzidine tetrazolium hydrochloride.…”

Myosin heavy-chain mRNA is present in both myofibrillar and subsarcolemmal regions of muscle fibres

“…Such a mechanism could be of particular importance in skeletal muscle, where the synthesis and turnover of myofibrillar proteins presents extreme logistical problems in synthesizing large quantities of actin and myosin which are required in precise locations within a highly organized framework (Millward, 1980). Although the turnover of myofibrillar proteins is asynchronous (Zak et al, 1979), the replacement of these components appears to take place without loss of sarcomere integrity and, at least in the case of myosin, the protein is incorporated throughout the length and cross-section of the A-band (Franchi et al, 1990). If myofibrillar proteins are synthesized close to their site of assembly, then one would expect the mRNAs for proteins such as myosin to be directed to the intermyofibrillar cytoplasm and to be translated in the polyribosomes that various data suggest are present in these regions ofthe myofibres, around the myosin-containing A-bands (Clavert et al, 1949;Larson et al, 1971;Home & Hesketh, 1990a).…”

The dose-related response of rabbit fast muscle to long-term low-frequency stimulation

“…The fiber type transformation induced by stimulation at 10 Hz proceeds over a period of many weeks, during which new myosin mRNA species are expressed, 6,7,22 the corresponding heavy and light chains are synthesized, 5 and the new isoforms are interpolated progressively into the thick filaments in place of the preexisting myosin molecules. 13 When the muscle is stimulated at 2.5 Hz the metabolic adaptation is more gradual than for stimulation at 10 Hz. 16,27 Thus, the question arises: do changes in myosin also continue to take place, but at a reduced rate, under these conditions?…”