Proteolysis Pathways via FoxO1 Reduce the Diameter of Myotube under Hypoxia Exposure / 中国生物化学与分子生物学报

The high altitude/hypoxic environment induced skeletal muscle atrophy is considered to be the interaction of multi-system and multi-organ, but the direct mechanism of hypoxia on muscle cells in this process is not clear. This study intended to investigate the effects of hypoxia exposure on proteins in ubiquitin and autophagy pathways, and explored the possible mechanism of hypoxia induced change of myotube diameter. The expression of myosin, hypoxia inducible factor-1 α (HIF-1α), forkhead box protein O1 (FoxO1), and ubiquitin protease pathway (MuRF1 and Atrogin1) and autophagy lysosomal pathway (p62, Beclin1, LC3) related proteins were detected by Western blot; The integrated optical density (IOD) of Myosin and LC3 was detected by IF. The results showed that the diameters of myotube at 6 h and 12 h were significantly reduced, and the expression of myosin was significantly reduced at 6 h after hypoxia exposure (P<0. 05); the protein levels of HIF-1α and FoxO1 were significantly increased at 6 h (P<0. 05); The expression of MuRF1 in each time points of hypoxia was significantly higher than 0 h (P<0. 05), but no difference of Atrogin1 expression was detected; Compared with 0 h, the expression of p62 was reduced significantly in response to hypoxia. The protein expression of Beclin1 and the IOD of LC3 was increased significantly at 6 h, and the LC3Ⅱ/Ⅰ ratio was significantly higher at 6 h, but significantly lower at 12 h and 24 h (P<0. 05).The results above indicated that the reduction of the myotube diameter of L6 skeletal muscle cells was induced by hypoxia exposure (1% O

Culture and identification of human embryo-derived myoblasts / 中国组织工程研究

BACKGROUND:There are myoblasts in human embryonic skeletal muscle. It remains poorly understand whether myoblasts in vitro can form myotube and what are the corresponding markers for identifying myoblasts and myotubes. OBJECTIVE:To investigate whether in vitro cultured myoblasts from human embryonic skeletal muscle can form myotube and whether they can express neural markers. METHODS:Human embryonic muscle-derived myoblasts were cultured in serum-containing medium. When the primary culture was established, cultured cel s were identified with immunocytochemistry for neural markers, such asβ-tubulin markers (desmin, myogenin, smooth muscle actin and myosin). RESULTS AND CONCLUSION:A population of myoblasts could migrate from human embryonic muscle tissues. They could express the markers for skeletal muscle such as desmin and myogenin, and they could express neuron specific enolase, nestin and neurofilament 200. They could form myotubes in vitro, and myotubes expressedβⅢ-tubulin, neurofilament 200 and glial fibril ary acidic protein. The data support the hypothesis that myoblasts from human embryonic muscle express neural markers and muscle markers, and cultured myoblasts and myotubes expressed neuron specific enolase,β-tubulin Ⅲ, nestin, neurofilament 200 and glial fibrillary acidic protein. This indicates that these markers could not be used for cel identification of trans-differentiation study from muscle origin to nervous system.

MORPHOLOGICAL OBSERVATION ON THE DEVELOPMENT OF NEUROMUSCULAR JUNCTIONS IN FETAL MOUSE SPINAL CORD AND MUSCLE CO-CULTURE / 解剖学报

Neuromuscular junctions were formed in vitro between spatially separated explants of the fetal mouse spinal cord and the skeletal muscle. The interdependence between nerve and muscle and the changes of pre- and postsynaptic structures were observed during the formation of junctions in living and stained specimens. On the 7th day in vitro, the myotube was innervated by the neurite to form a small bulblike ending, and the cholinesterase activity was not observed at the contact region between nerve and myotube. On the 14th day in vitro, the nerve fiber sent out its branches, and the myofibers were innervated by these branches. Except some bulblike endings, some simple arborized nerve endings were also observed on the myofibers. At this stage, the cholinesterase activity began to appear at the contact region between the ending and the myofiber, and the multiple innervation of myofiber also occurred. Sometimes there were more than one nerve-muscle contact on a single myofiber, and sometimes a myofiber was innervated by several axons to form a "mixed-contact" at same place. The cholinesterase activity can only be examined in one of the multiple contacts but it is absent in the "mixed-contact". On the 21st day, both the number and the cholinesterase activity of the endplate were increased markedly. After 30 days in culture, there were intense cholinesterase activities on some endplates which were similar to young endplates in vivo, these endplates were innervated separately by single axons. When the spinal cord and muscle were co-cultured, the differentiation of muscle fiber depended on the innervation of motor nerve, and not on sensory nerve. On the other hand, the growth and maintenance of the neuron is closely related to the presence of the muscle fiber.