Simulation of the purine nucleotide cycle as an anaplerotic process in skeletal muscle

A computer model of purine nucleotide and citric acid cycles joined through fumarate is given. Steady-state equations corresponding to metabolic enzymes are written based on the information from the literature about their kinetic behavior. Numerical integration of this set of equations is performed and in order to maintain an overall stabilization between the two cycles, enzymatic activities, in the form of V, have been calculated. Sensitivity coefficients for enzymes indicate that the control is exerted, depending upon the intermediate concentrations, and furthermore, it is demonstrated that AMP concentration in muscle should be very low. From stabilization, simulation of exercise conditions has been performed by diminishing [ATP] and increasing accordingly [ADP] and [AMP]. In such conditions the operation of purine nucleotide cycle leads to a considerable increase in the level of citric acid cycle intermediates. Disruption of purine nucleotide cycle by altering some of the three enzymatic steps leads to a lesser increase of these intermediates. The set of results presented seems to confirm the hypothesis that purine nucleotide cycle acts as an anaplerotic process in muscle, as the experimental results of Aragon and Lowenstein (Aragon, J.J., and Lowenstein, J.M. (1980) Eur. J. Biochem. 110, 371-377) suggest.     

Purine nucleotide cycle enzymes in dystrophic and normal mouse muscle

A comparative study of the operation of the purine nucleotide cycle and of the activity of adenylosuccinase in extracts of muscle from the two strains of dystrophic mouse shows that the cycle is defective in both cases in the conversion of adenylosuccinate to AMP. However, adenylosuccinase activity is only slightly reduced in the standard conditions for its direct assay.     

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.     

Protein and RNA synthesis in the skeletal muscle of hereditary dystrophic mouse.

Protein and RNA contents in muscle of normal and hereditary dystrophic mice C57BL/6J-dy/dy were reexamined on the basis of DNA. It was observed that protein and RNA contents in dystrophic muscle decreased at the early stage of the disease, in disagreement with the reported results on a wet weight basis, in which RNA content in dystrophic muscle had been found to increase. Rates of protein and RNA systhesis in the early stage of the disease were also determined with a concomitant check of the specific activities of free amino acids and free nucleotides. The rates of both protein and RNA synthesis (i.e., specific activities of protein and RNA) were higher in the dystrophic muscle, but when they were expressed on a DNA basis, the total protein synthesis per cell was the same as that of normal muscle and the total RNA synthesis per cell showed a smaller increase in dystrophic muscle. These apparent increases of protein and RNA synthesis were discussed in connection with the decreased protein and RNA contents in the cells of dystrophic muscle. The synthesized RNAs seemed to contain mRNA on the basis of sedimentation character and Millipore filter binding ability. However, no particular RNA was mainly synthesized in dystrophic muscle.     

Control of the purine nucleotide cycle in extracts of rat skeletal muscle: effects of energy state and concentrations of cycle intermediates

The enzymes of the purine nucleotide cycle-AMP deaminase, adenylosuccinate synthetase, and adenylosuccinate lyase-were examined as a functional unit in an in vitro system which simulates the purine nucleotide composition of sarcoplasm. Activity of each cycle enzyme in extracts of rat skeletal muscle was observed to increase as ATP/ADP, reflecting the energy state of the system, was lowered from approximately 50 to 1. The increase in AMP deaminase activity could be attributed to effects of energy state and factors such as AMP concentration, which are obligatorily coupled to energy state. The increases in synthetase and lyase activities were accounted for by increases in the concentration of IMP and adenylosuccinate, respectively. The inhibitory influence of IMP concentration on synthetase activity reported in other systems was not observed in this system; synthetase activity progressively increased as IMP concentration was raised to approximately 4 mM, and apparent saturation occurred at concentrations above 4 mM. Also, adenylosuccinate was found to be an activator of AMP deaminase. The results of this study document that the activities of the enzymes of the purine nucleotide cycle increase in parallel at low energy states, and the components of the cycle function as a coordinated unit with individual enzyme activities linked via concentrations of cycle intermediates.     

Studies on mitochondria from dystrophic skeletal muscle of mice

Mitochondrial respiration and oxidative phosphorylation were compared in normal and dystrophic mouse skeletal muscles. To obtain the maximum respiration control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio from isolated muscle mitochondria, it is found that there is an advantage in having a low concentration of proteinase and EGTA present in the medium during preparation of mitochondria by centrifugation fractionation. Using pyruvate, acetylcarnitine, and palmitylcarnitine as substrates for oxidation, a highly significant reduction (40-60%) is shown in oxygen uptake by dystrophic muscle mitochondria as compared to normal muscle mitochondria. Studies of the integrity of the oxidative phosphorylation apparatus in these samples showed that there is a reduction of the RCR and ADP/O ratio in dystrophic muscle mitochondria as compared to normal muscle mitochondria.     

Thermodynamics of the purine nucleotide cycle

Since the standard Gibbs energies of formation are known for all the species in the purine nucleotide cycle at 298.15 K, the functions of pH and ionic strength that yield the standard transformed Gibbs energies of formation of the ten reactants can be calculated. This makes it possible to calculate the standard transformed Gibbs energies of reaction, apparent equilibrium constants, and changes in the binding of hydrogen ions for the three reactions at desired pHs and ionic strengths. These calculations are also made for the net reaction and a reaction that is related to it. The equilibrium concentrations for the cycle are calculated when all the reactants are initially present or only some are present initially. Since the concentrations of GTP, GDP, and P(i) may be in steady states, the equilibrium concentrations are also calculated for the system at specified steady-state concentrations.     

Metabolic interaction between purine nucleotide cycle and oxypurine cycle during skeletal muscle contraction of different intensities: a biochemical reappraisal

Background

A substrate cycle is a metabolic transformation in which a substrate A is phosphorylated to A?P at the expense of ATP (or another “high energy” compound), and A?P is converted back to A by a nucleotidase or a phosphatase. Many biochemists resisted the idea of such an ATP waste. Why a non-phosphorylated metabolite should be converted into a phosphorylated form, and converted back to its non-phosphorylated form through a “futile cycle”?

Aim of review

In this Review we aim at presenting our present knowledge on the biochemical features underlying the interrelation between the muscle purine nucleotide cycle and the oxypurine cycle, and on the metabolic responses of the two cycles to increasing intensities of muscle contraction.

Key scientific concepts of review

Nowadays it is widely accepted that the substrate cycles regulate many vital functions depending on the expense of large amounts of ATP, including skeletal muscle contraction, so that the expense of some extra ATP and “high energy” compounds, such as GTP and PRPP via substrate cycles, is not surprising. The Review emphasizes the strict metabolic interrelationship between the purine nucleotide cycle and the oxipurine cycle.

     

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.     

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.     

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.