Freeze etching electron microscopic study of muscle plasma membranes and myofilaments of human dystrophic muscles.

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Our previous freeze fracture studies showed that one of the characteristics of muscle plasma membrane of Duchenne muscular dystrophy (DMD) was the marked decrease of orthogonal arrays. The defect of orthogonal arrays is also seen in the plasma membrane of immature myofibers. Therefore the criticism raised that the marked scarcity of orthogonal arrays is due to the presence of regenerating fibers in DMD muscles. To investigate this problem more precisely, we intended to observe the muscle plasma membrane and myofilaments simultaneously by quick freeze, deep etch, rotary shadow (QDR) replica method, since the cytoplasmic structure such as myofibrillar arrangement hints the degree of maturation of myofibers. Histochemically normal 6 human quadriceps femoris muscles and the quadriceps femoris muscles from 3 patients with DMD were immediately cut into small pieces and rapidly frozen at helium temperature by metal contact method with Eiko RF 23 rapid freeze device. The frozen muscle samples … More were transferred to the Eiko FD 5A freeze fracture apparatus and fractured at -120ﾟC and a vacuum of 1-2 X 10^<-7> Torr, and deep etched at -90ﾟC for 30 minutes. Then the specimen were rotary shadowed at shadow angle of 30ﾟ by electron beam gunned platinum and carbon. The muscle tissues were digested by bleaching solution and the detached replicas were washed 3 times in distilled water. Electron microscopy of the replicas showed clearly the fine structure of myofiber cytoskeletal elements such as myosin and actin filaments, but failed to reveal the orthogonal arrays in normal muscle plasma membrane. The results of this study demonstrated that we could identify the regenerating myofibers by the appearance of cytoplasmic structure but the ultrastructure of normal muscle plasma membrane seen in the replicas made by QDR method was quite different from that of replicas made by conventional freeze fracture method using chemically fixed glycerinated normal human muscles. Although we made a lot of replicas of normal human skeletal myofibers and examined them extensively by electron microscope, we could not find any orthogonal arrays at their plasma membranes. Therefore we could not solve the above mentioned criticism in this study. However, it became clear from this study that the QDR replica method was most suitable for the observation of myofiber cytoskeletal elements such as dystrophin. Less