Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice

Embryonic stem (ES) cells have great therapeutic potential because of their capacity to proliferate extensively and to form any fully differentiated cell of the body, including skeletal muscle cells. Successful generation of skeletal muscle in vivo, however, requires selective induction of the skeletal muscle lineage in cultures of ES cells and following transplantation, integration of appropriately differentiated skeletal muscle cells with recipient muscle. Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disease due to a mutation in the dystrophin gene and the mdx mouse, an animal model for DMD, are characterized by the absence of the muscle membrane associated protein, dystrophin. Here, we show that co-culturing mouse ES cells with a preparation from mouse muscle enriched for myogenic stem and precursor cells, followed by injection into mdx mice, results occasionally in the formation of normal, vascularized skeletal muscle derived from the transplanted ES cells. Study of this phenomenon should provide valuable insights into skeletal muscle development in vivo from transplanted ES cells.     

Accelerated protein turnover in the skeletal muscle of dystrophic mice

The excretion of 3-methylhistidine increased in the urine of dystrophic mice C57BL/6J. The content of 3-methylhistidine residue decreased in the muscle proteins of dystrophic mice, but not in other organs. Methylated proteins in the skeletal muscle, actin and myosin, were partially purified from the dystrophic and control muscles. The amount of 3-methylhistidine residue in unit weight of the actin and myosin preparations was normal in dystrophic muscle. These three facts indicate that the turnover rates of actin and myosin are increased in the muscle of the dystrophic mice.     

Properties of creatine kinase from skeletal muscle mitochondria

Creatine kinase from pigeon breast muscle was obtained in a homogeneous (as evidenced from polyacrylamide gel SDS electrophoresis) state. The molecular mass of the enzyme monomer is 43,000. Ultracentrifugation in a sucrose density gradient and gel filtration revealed that the enzyme is present in solution as a mixture of two major forms, i.e., octamer and dimer, which differ in their activity. The decrease of ionic strength from 0.25 to 0.02 results in reversible dissociation of the octameric form. A temperature rise from 5 degrees to 20 degrees C or the nature of monovalent anions (e.g., Cl-, CH3COO-, NO3-) and cations (K+, Na+) present in the medium do not influence the distribution of oligomeric forms. At pH 6.0 the major form is represented by the octamer; its dissociation is caused by an increase of pH. The octamer dissociation occurs in a mixture of substrates of the creatine kinase reaction in the presence of Mg2+; no such dissociation is observed in the absence of Mg2+ and in the presence of each of the reaction substrates. The non-interacting pair of substrates--ADP and creatine--causes the dissociation of the octamer in the presence of nitrate ions but not acetate. It is concluded that the dissociating effect of substrates is due to the conformational changes of subunits during catalysis. At physiological concentrations of nucleotide substrates the degree of octamer dissociation depends on the ratio of creatine phosphate and creatine concentrations, as well as on the presence of chlorine and phosphate ions. A qualitative estimation of the rate of pH- and substrate-dependent dissociation of creatine kinase octamer revealed that under the given experimental conditions the pH-dependent dissociation is completed within hours, whereas the substrate-dependent one--within seconds or minutes. According to its properties, mitochondrial creatine kinase from pigeon breast muscle is close to its bovine heart counterpart; the observed differences were found to be quantitative.     

Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca²⁺/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg²⁺-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD⁺ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD⁺ in the case of NAD⁺-linked substrates.     

Contribution of human muscle-derived cells to skeletal muscle regeneration in dystrophic host mice

Background

Stem cell transplantation is a promising potential therapy for muscular dystrophies, but for this purpose, the cells need to be systemically-deliverable, give rise to many muscle fibres and functionally reconstitute the satellite cell niche in the majority of the patient''s skeletal muscles. Human skeletal muscle-derived pericytes have been shown to form muscle fibres after intra-arterial transplantation in dystrophin-deficient host mice. Our aim was to replicate and extend these promising findings.

Methodology/Principal Findings

Isolation and maintenance of human muscle derived cells (mdcs) was performed as published for human pericytes. Mdscs were characterized by immunostaining, flow cytometry and RT-PCR; also, their ability to differentiate into myotubes in vitro and into muscle fibres in vivo was assayed. Despite minor differences between human mdcs and pericytes, mdscs contributed to muscle regeneration after intra-muscular injection in mdx nu/nu mice, the CD56+ sub-population being especially myogenic. However, in contrast to human pericytes delivered intra-arterially in mdx SCID hosts, mdscs did not contribute to muscle regeneration after systemic delivery in mdx nu/nu hosts.

Conclusions/Significance

Our data complement and extend previous findings on human skeletal muscle-derived stem cells, and clearly indicate that further work is necessary to prepare pure cell populations from skeletal muscle that maintain their phenotype in culture and make a robust contribution to skeletal muscle regeneration after systemic delivery in dystrophic mouse models. Small differences in protocols, animal models or outcome measurements may be the reason for differences between our findings and previous data, but nonetheless underline the need for more detailed studies on muscle-derived stem cells and independent replication of results before use of such cells in clinical trials.     

Soluble cholinesterase of muscle from dystrophic and normal mice

The percentage of cholinesterase extractable by isotonic sucrose from dystrophic mouse muscle was greater than that found in normal muscle. Of the total cholinesterase found in normal and dystrophic muscle about 60% was specific AChE and 40% was non-specific cholinesterase. The extract from dystrophic muscle showed, on sucrose sedimentation, one major peak of AChE activity with a sedimentation constant of approximately 4.3 S. This was much higher than that from normal muscle.     

Sarcotubular development in dystrophic skeletal muscle cells

Summary Dilations of the sarcotubular system and misaligned myofilaments have been reported as early indicators of muscular dystrophy in skeletal muscle. Since the developing tubular component is believed instrumental in initial myofilament alignment during myogenesis, tubular development is evaluated using normal and dystrophic chick embryo skeletal muscle and cultures of normal and dystrophic embryonic pectoral muscle incubated in the presence of horse spleen ferritin. Comparisons of the findings show that periodic tubules are absent from dystrophic somitic muscle and that invaginating tubules from the sarcolemma are found in fewer, randomly located areas of dystrophic pectoral muscle cells. The results indicate that the tubular component is not involved in the bizarre vesiculations seen in mature dystrophic muscle, however, the malalignment of dystrophic myofilaments is probably the result of the poorer development of the T system in this muscle.     

A defect of purine nucleotide cycle in the skeletal muscle of hereditary dystrophic mice

The purine nucleotide cycle in the hind leg skeletal muscle of hereditary dystrophic mice ($\text{C57BL}\text{6J}\text{-}\text{dy}\text{dy}$) was investigated. The amount of adenine nucleotide produced from adenylosuccinate by the muscle extract in the dystrophic group was less than 3 % of that in the control group, while adenine nucleotide plus adenylosuccinate converted from IMP in the dystrophic group was about 70 % of that of the control group. Moreover, the activity of AMP deaminase of the dystrophic group was about 50 % of that of the control group. These results indicate that the purine nucleotide cycle is defective in the dystrophic muscle. This abnormality was suggested to be caused by the considerably low activity of adenylosuccinase.