dy2J Mice Research Articles

Histochemical and structural analyses of the phenotypic expression of the dystrophic gene in the [formula omitted] and the [formula omitted] mice

Although both slow- and fast-twitch fibers of dystrophic genotype are affected by the disease hereditary muscular dystrophy, our histochemical evidence indicates a differential temporal involvement of different fiber types in the two strains of dystrophic mice investigated. Whereas in the 129 ReJ dy dy mice the disease is expressed early in white, fast-twitch glycolytic fibers, in the C57BL 6J dy 2J dy 2J mice the primary expression of the disease is noted in red, slow-twitch oxidative fibers. In the former, wasting progresses rapidly in leg muscles characterized by a large proportion of fast-twitch fibers; in the latter, there is a prolonged maintenance of the integrity of regions rich in these fibers. Of equal importance with the C57BL 6J dy 2J dy 2J mouse is the detection of small foci of destruction in regions rich in slow-twitch fibers as early as 2 to 3 weeks postnatally; wasting of these fibers ensues rapidly. Thus, in both strains, the fibers which are affected primarily show early alterations with a subsequent rapid progression of disease characteristics and the fibers which seem secondarily affected remain intact for prolonged periods. Our observations suggest an important role of the dy 2J dy 2J mouse as a “transitional” model to investigate a relationship between genotypically dystrophic muscle in which the phenotypic expression of the disease is present and genotypically dystrophic muscle spared phenotypic signs of the disease. The latter, characterized by multiple innervation, is never challenged to alter its metabolic or physiologic behavior during ontogeny.

DISEASED MUSCLE CELLS IN CULTURE

Summary(1) Cultures of differentiated muscle cells have been grown from diseased human, mouse and chick skeletal muscle, and from cardiac muscle of the myopathic hamster.(2) Methods of culture established for normal embryonic and adult skeletal muscle cells have proved suitable for cultures of diseased muscle cells.(3) Myoblasts obtained from dy2J mouse muscle crushed in vivo before explanting fuse in culture and form morphologically normal myotubes. Studies of the effects of innervation by dy2J spinal cord neurones on the differentiation of normal, dy2J and dy myotubes have been inconclusive but it is probable that innervation does not play a part in the pathogenesis of this disorder.(4) Myoblasts prepared by trypsinization of embryonic dy muscle behave normally in culture and fuse to form myotubes that appear normal. It is not clear if myoblasts that migrate from explants of adult muscle in vitro fuse. Aggregates of non‐fusing cells have been described, but under other culture conditions normal and abnormal forms of myotube have been observed. dy muscle fibres fail to regenerate even when cultured with normal spinal cord explants and dy nerves are without effect on regenerating normal muscle fibres. These tissue‐culture studies suggest that the dy mouse mutation is a myopathic disorder.(5) Embryonic mdg myoblasts have a normal cell cycle in vitro and fuse to form well‐differentiated myotubes with cross‐striations. mdg myotubes have normal electro‐physiological properties but do not contract spontaneously or on depolarization. The defect in the muscle of the mdg mutant appears to be a failure of excitation‐contraction coupling.(6) Cells migrate earlier from explants of adult dystrophic chick muscle than from normal muscle but dystrophic chick myotubes appear morphologically normal. Myotubes prepared from embryonic dystrophic chick muscle become vacuolated and degenerate, changes that can be prevented by anti‐proteases such as antipain. Lactic dehydrogenase isozyme subunit M4 is absent from dystrophic muscle in vivo but reappears in cultured myotubes. Dystrophic myotubes innervated in culture by either normal or dystrophic neurones exhibit bi‐directional lcoupling and multiple innervation. These results suggest that there are changes in dystrophic myotubes and that chick muscular dystrophy is a myopathy.(7) Cardiac muscle cells from the cardiomyopathic hamster synthesize less actin and myosin than normal cells, and Z lines in dystrophic cells are irregularly arranged. The beat frequency of myopathic cardiac cells is lower than that of normal cells and declines more rapidly. Tissue‐culture studies have not been made of hamster skeletal muscle.(8) Human dystrophic myotubes do not show degenerative changes in culture and have normal histochemical reactions. RNA synthesis appears normal in dystrophic myotubes but there may be changes in adenyl‐cyclase activity and protein synthesis in dystrophic cells. Morphological and biochemical changes have been found in muscle cells cultured from a case of acid‐maltase deficiency but phosphorylase activity re‐appeared in myotubes cultured from biopsies of phosphorylase‐deficient muscle. Innervation by normal mouse nerves does not induce degenerative changes in dystrophic myotubes.(9) Studies on the origins of myoblasts in explants of muscle fibres in culture suggest that in these conditions myoblasts are derived only from satellite cells and that this process may be the same in normal and diseased muscle.

A study of functional denervation in fast and slow muscles of dystrophic mice of various ages

It has been claimed that some fibers in dystrophic mouse muscle are denervated and, furthermore, that there is no increase in motor unit size which would be expected to occur if reinnervation took place. If these observations are correct, it ought to be possible to demonstrate a mechanical correlate of the denervation phenomenon. The functional innervation ratio (ratio of maximum twitch tension in response to nerve stimulation × 100 to maximum twitch tension in response to direct muscle stimulation) has been determined for the extensor digitorum longus and soleus muscles of normal ( C57B1 6J ) and dystrophic ( dy 2J dy 2J ) mice of various ages. It is shown that although a fall in the functional innervation ratio is seen in both muscles, it occurs some time after the symptoms of the disease are apparent. This suggests that denervation may not be the primary event in murine muscular dystrophy. The fall in functional innervation ratio is apparently not due to an enhanced susceptibility of the dystrophic neuromuscular junction to anoxia, because dystrophic muscles showed the same response to a raised bath temperature as did normal muscles.