T-tubule Formation Research Articles

Co-localization of 1,4-dihydropyridine receptor alpha 2/delta subunit and N-CAM during early myogenesis in vitro.

The surface distribution of the alpha 2/delta subunit of the 1,4-dihydropyridine receptor and its topographical relationship with the neural cell adhesion molecule (N-CAM) were investigated during early myogenesis in vitro, by double immunocytochemical labeling with the monoclonal antibody 3007 and an anti-N-CAM polyclonal antiserum. The monoclonal antibody 3007 has been previously shown to immunoprecipitate dihydropyridine receptor from skeletal muscle T-tubules. In further immunoprecipitation experiments on such preparations and muscle cell cultures, it was demonstrated here that the monoclonal antibody 3007 exclusively recognizes the alpha 2/delta subunit of the 1,4-dihydropyridine receptor. In rabbit muscle cell cultures, the labeling for both alpha 2/delta and N-CAM was first detected on myoblasts, in the form of spots on the membrane and perinuclear patches. Spots of various sizes organized in aggregates were then found on the membrane of myotubes. At fusion (T0), aggregates of N-CAM spots alone were found at the junction between fusing cells. At T6 and later stages, all alpha 2/delta aggregates present on myotubes co-localized with N-CAM, while less than 3% of N-CAM aggregates did not co-localize with alpha 2/delta. A uniform N-CAM staining also made its appearance. At T12, when myotubes showed prominent contractility, alpha 2/delta-N-CAM aggregates diminished in size. Dispersed alpha 2/delta spots of a small regular size spread over the whole surface of the myotubes and alignments of these spots became visible. Corresponding N-CAM spots were now occasionally seen, and uniform N-CAM staining was prominent. These results show that alpha 2/delta and N-CAM are co-localized and that their distributions undergo concomitant changes during early myogenesis until the T-tubule network starts to be organized. This suggest that these two proteins might jointly participate in morphogenetic events preceding the formation of T-tubules.

Behavior of sarcotubular system formation in experimentally induced regeneration of muscle fibers

To examine the behavior of transverse (T)-tubule formation in experimentally-induced regenerating fibers, a local anesthetic, bupivacaine hydrochloride, was injected directly into the rat soleus muscle to cause myonecrosis. The regenerating fibers following necrosis were then examined by electron microscopy using lanthanum nitrate which clearly demonstrated the sarcotubular system. In the early stage of regeneration within 7 days after muscle necrosis, the T-tubules seemed to be composed of markedly proliferated subsarcolemmal caveolae with occasional honeycomb structure formation. Around 10 days, the T-tubules in regenerating fibers were tortuously and irregularly arranged with focal dilatation in diameter, and extended longitudinally along the axis of well organized myofibrils. As the regenerating fibers matured, the sarcotubular system, irregular in course and in shape, gradually became organized into a regular transverse position against the myofibrils, along with a marked decrease in longitudinally arranged tubular components. These morphological findings of the early T-tubule formation seen in the present study were similar to those found in early myogenesis, and in biopsied muscles from cases of polymyositis and progressive muscular dystrophy.

Satellite cells and muscle regeneration in the developing dystrophic chicken

An electron microscopic study was carried out on the pectoralis superficialis muscle in 18 dystrophic (line 413) and 18 control (line 412) chickens at different stages of development (5 to 40 days after hatching). Three chickens of each group were killed at 7-day intervals. The numbers of satellite cell nuclei and true muscle nuclei per 100 cross-sectioned muscle fibers were calculated from the electron microscopic observation of approximately 200 fibers in each of the total number of chickens. The most striking featue was the large number of satellite cell nuclei in dystrophic chickens compared with control chickens, a difference which became significant beginning at 19 days of age ( P < 0.05). The increased number of satellite cell nuclei and true muscle nuclei suggested that the incorporation of the daughters of satellite cells into the fibers was promoted. In dystrophic chickens, regenerative activity was represented by the activated satellite cells with proliferation of vesicles and early T-tubule formation, and of myoblasts and myotubes beneath the same basement membrane of the degenerating fibers as well as at some distance from the foci of degeneration. Poorly aligned myofibrils were demonstrated in the myotube, possibly in the early stage of fusion with the parent fiber. This observation may shed light on the genesis of the aberrant myofibrils encountered in dystrophic muscles.

Postnatal differentiation of ventricular myocardial cells of the opossum (Didelphis virginiana Kerr) and T-tubule formation.

Cardiac muscle cells in the right ventricle of the postnatal opposum were studied ultrastructurally, with particular attention to the developmental stage of T-tubule formation. Animals ranging from 5.5 cm (day 29 of postnatal life) to 41 cm (young adult) in body length were used. Typical T-tubules were first recognized in a few myocardial cells of the 7.5 cm (day 43) opossum. T-tubules increased in number as cardiac muscle cells continued to differentiate until 22 cm in body length (about 105 days after birth). At this stage of development most of the T-tubules were completely established. The general mode of differentiation and development of cardiac muscle cells appeared essentially the same as in other mammals.

Myosin synthesis by fusion-arrested chick embryo myoblasts in cell culture

The synthesis and accumulation of myosin was studied in subcultures of fusion-blocked, postmitotic embryonic chicken myogenic cells. Electron micrographs and fluorescent microscopy with antimyosin revealed that most, if not all, of these cells contain myosin. It was also found that these cells are capable of accumulating myosin at rates comparable to fused cells. Incipient T-tubule formation was also present in some of the blocked cells. It is concluded that cell fusion is not a prerequisite for myosin synthesis and accumulation or T-tubule formation during myogenesis in vitro.