Dystrophic Myoblasts Research Articles

Dystrophin expression in myotubes formed by the fusion of normal and dystrophic myoblasts

Mdx mouse dystrophy is characterized by the absence in the muscle cytoplasmic membrane of a high molecular weight protein called dystrophin. A possible avenue for treatment of muscular dystrophies is to inject normal myoblasts in a dystrophic muscle to form hybrid muscle fibers. Hybrid myotubes were formed in vitro by the fusion of normal rat and dystrophic mouse (mdx) myoblasts. Staining with Hoechst dye 33258 permitted the clear distinction of mouse and rat nuclei. Immunostaining demonstrated that dystrophin was present over the entire membrane of all hybrid myotubes even when nuclei ratio normal/dystrophic was low.

Ornithine and S-adenosylmethionine decarboxylase activities and polyamine contents in developing muscle tissues and primary cultures of normal and polymyopathic hamsters

Polyamine (putrescine, spermidine, and spermine) contents and ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylase activities have been assessed in an age-dependent manner, in normal and polymyopathic (dystrophic) hamster skeletal muscle, heart, and tongue extract and in primary tongue myoblast and skin fibroblast cultures. At 2 weeks of age, polyamine contents were significantly elevated in all of the dystrophic hamster tissues studied when compared with their age-matched controls. The degree of this elevation decreased with the age of the animals, generally, to a level where no significant difference in polyamine contents could be noted between normal and dystrophic hamster tissues. ODC and SAMDC activities in whole tissue extracts were consistently highest in 2-week-old muscle extracts and also declined with age. However, no significant changes in ODC or SAMDC activities were evident in any of the dystrophic muscle tissues studied when compared with their age-matched controls. Polyamine contents in dystrophic hamster myoblast and fibroblast primary cultures were also during proliferation (1 and 2 days after the initial seeding) compared with cultures prepared from normal hamsters. ODC and SAMDC activities in primary myoblast and fibroblast cultures clearly reflected the rate of cell proliferation, with highest activities found in subconfluent cell cultures. However, in general, no significant dystrophic-related abnormality in ODC or SAMDC activity was evident in proliferating myoblast or fibroblast cultures. These results suggest that the elevated polyamine contents of dystrophic hamster tissues and primary cultures may be due to a deficiency in polyamine catabolism or transport.

Calmodulin levels in developing muscle tissues and primary cultures of normal and dystrophic (UM-X7.1) hamsters.

Calmodulin levels have been assessed in whole muscle and primary culture extracts in order to examine the relationship between calmodulin and the accumulation of calcium in dystrophic hamster muscle tissues. Significant decreases in both normal and dystrophic skeletal muscle, tongue, and heart calmodulin levels were observed between 2 and 12 weeks of age. Dystrophic values, however, tended to be somewhat higher than normal, especially in 12-week-old skeletal muscle total and soluble extracts (normal 29.7 and 0.6 and dystrophic 117.0 and 3.1 micrograms/g wet weight, respectively). No significant differences were observed in dystrophic myoblast (total 2.22-2.78, soluble 2.85-3.26 micrograms/mg protein) or fibroblast (total 2.64-2.94, soluble 2.54-3.60 micrograms/mg protein) calmodulin levels, except for a significant decrease in dystrophic fibroblast levels (total 1.97, soluble 2.18 micrograms/mg protein) at 7 days in culture. Elevated calmodulin levels in dystrophic muscle are discussed in terms of increases in intracellular Ca2+ concentrations and immature regenerating fibers.

Normal and dystrophic hamster myoblast and fibroblast growth in culture.

Normal and dystrophic hamster myoblasts and fibroblasts were compared for characteristic indicators of growth and differentiation. Clonal analysis of myoblast cultures indicated that 80% of colonies judged to be fusion-competent had differentiated. Dystrophic myoblasts were identical to normal in terms of their morphology, fusion potential (81.4%), and myokinase activity (59.6-49.1 mU/mg at 2-7 days), but displayed a significantly higher plating efficiency (normal: 52.6%; dystrophic: 82.1%), a longer doubling time (normal: 21.7 hours; dystrophic: 33.3 hours), and a lower day-7 creatine kinase activity (normal: 60.7 mU/mg; dystrophic: 41.3 mU/mg). Dystrophic fibroblasts were indistinguishable from normal ones in terms of their morphology, plating efficiency (90.4%), and doubling time (32.5 hours), but displayed a significantly lower day-2 creatine kinase activity (normal 58.3 mU/mg; dystrophic: 35.8 mU/mg) and day-7 myokinase activity (normal: 52.7 mU/mg; dystrophic: 39.9 mU/mg). The results are suggestive of an early and differential expression of the primary defect in dystrophic hamster myoblasts and fibroblasts in culture.

Phospholipid biosynthesis during normal and dystrophic avian muscle cell differentiation in culture

The biosynthesis of phospholipids during differentiation of normal and dystrophic muscle cells was studied using [32Pi] added to primary culture media. Labeled phosphatides were separated by two-dimensional thin-layer chromatography, exposing the plates to HCl vapors between solvents to cleave the alk-1-enyl bonds. Phosphatidalethanolamine labeling did not vary during myotube formation. Although lower labeling of phosphatidalcholine was observed in dystrophic myoblasts at the single cell stage, no differences were observed between normal and dystrophic cultures after cell contact and myotube formation. A sharp decrease in phosphoinositide content was observed as myoblasts first achieved cell contact and continued to decline slowly as myotubes developed. During myogenesis, phosphatidylcholine content gradually increased, from 18.4% of the total labeled phospholipid in dividing myoblasts to 48.6% of the labeled phospholipids in fully differentiated myotubes.

Myoblast senescence in muscular dystrophy

The limited proliferative capacity of normal diploid cells predicts that the utilization of cell divisions in vivo should reduce the lifespan of cells in culture. Because of the continuing demands for muscle regeneration in muscular dystrophy, myoblasts isolated from affected muscles should thus show a decrease in the number of cell divisions they are capable of expressing in culture. This hypothesis was tested by examining the proliferative capacity of myoblasts from different muscles for normal line 412 and dystrophic line 413 chickens of various ages. Prior to approx. 2 months of age, dystrophic myoblasts exhibited a relatively normal proliferative lifespan. By 5 months of age, myoblasts from the severely affected pectoralis major showed a 40% reduction in their proliferative potential, while myoblasts from the less affected posterior latissimus dorsi muscle showed a 25 % decrease in their cultured lifespan. The time course of the appearance of a decreased proliferative capacity only after the disease has been clinically manifested strongly supports it representing a secondary response rather than it being an intrinsic property of dystrophic myoblasts. A hypothesis for manipulating the pattern of stem cell division in order to increase the mass of muscle produced from a constant number of cell divisions is presented. If myoblast senescence and the consequent failure of muscle regeneration is a contributing factor in the progressive deterioration of muscle function in the disease, then this hypothesis might provide an important therapeutic strategy for ameliorating the course of muscular dystrophy.

A myogenic malfunction in adult dystrophic mice studied in vitro

Using Bar Harbor 129/ReJ dystrophic mice an in vitro study has been carried out in order to assess the capability of regenerating adult dystrophic muscle to proceed through myogenesis. An inability of dystrophic myoblasts to complete fusion was observed light-microscopically although the myoblasts became aggregated into muscle structures (called pseudostraps) bearing a superficial resemblance to normal multinucleate muscle straps. When investigated ultrastructurally the myoblasts constituting the pseudostraps were preserved in an immobile state by electron-dense attachment plaques. The location and structure of the dystrophic attachment plaques is described and compared with the plaques known to occur in normal myogenesis. Several additional myogenic features are described which occur consistently in pseudostrap myoblasts, including the formation of a basement membrane and elongation of the mitochondria. Well-developed myofibrillar protein synthesis could not be observed although swathes of filaments with primitive Z-bodies were present. A gross abnormal dilatation of the cisternae of the rough endoplasmic reticulum is described which is seen in approximately one-third of the dystrophic cultures. The results reported here are compared with those in which genetic variants of the Bar Harbor 129/ReJ dystrophic mouse have been used in coupled spinal cord and muscle cultures.