Nerve fiber formation and catecholamine content in adult rat adrenal medullary transplants after treatment with NGF, NT-3, NT-4/5, bFGF, CNTF, and GDNF

Forward (-->ATP) and reverse (-->CrP) fluxes through the creatine kinase reaction were determined in isolated rat and bovine heart mitochondria and with soluble MM-CK from rabbit skeletal muscle, using 31P-saturation transfer NMR. With soluble MM-CK forward and reverse fluxes were identical in the absence and presence of BSA or rat liver mitochondria. Addition of liver mitochondria decreased fluxes with increasing mitochondria concentration. The fluxf/Vmax(f) ratio was 0.006 with 10 mg BSA and 0.04 with 10 mg rat liver mitochondria, respectively. With heart mitochondria, fluxr was considerably higher than fluxf and the fluxf/Vmax(f) ratio was 1.7 for rat heart and 0.22 for bovine heart. It is concluded that in the presence of isolated mitochondria, the flux through the creatine kinase is driven by the mitochondrial ATP-ADP turnover. Therefore the fluxf/Vmax(f) ratio is highest for rat heart mitochondria with a high ATP-ADP turnover, intermediate for bovine heart mitochondria and low for MM-CK in the presence of liver mitochondria. It is lowest with MM-CK alone, where the creatine kinase reaction is at equilibrium and external ATP-ADP turnover is absent. The higher reverse than forward fluxes of mitochondrial creatine kinase determined at steady state by saturation transfer NMR, are caused mainly by a high ATP<-->Pi exchange in heart mitochondria preparations, having a high ATPase activity, compared to liver mitochondria.     

Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).     

The universality of bioenergetic disease and amelioration with redox therapy

Overt mitochondrial diseases associated with mitochondrial DNA mutations are characterized by a decline in mitochondrial respiratory function. Similarly, a progressive decline in mitochondrial respiratory function associated with mitochondrial DNA mutations is clearly evidenced in aged human subjects. This communication is concerned with the development of a rat model for the study of bioenergy decline associated with the ageing process and overt mitochondrial diseases. The model involves the treatment of young rats with AZT to induce skeletal and cardiac myopathies. It has shown that there is a decline in soleus muscle function in vivo and that this decline is mirrored in the capacity of heart sub-mitochondrial particles to maintain bioenergy function. Coenzyme Q10 and several analogs were administered with AZT as potential therapeutics for the re-energization of affected tissues. Coenzyme Q10 and especially decyl Q were found to be therapeutically beneficial by both in vivo improvement in soleus muscle function and in vitro cardiac mitochondrial membrane potential capacity. Sub-mitochondrial particles were also prepared from heart mitochondria of young and aged rats. The particles prepared from the aged rats were found to have a decreased ability to maintain membrane potential as compared to those derived from the young rats.     

Maintained coupling of oxidative phosphorylation to creatine kinase activity in sarcomeric mitochondrial creatine kinase-deficient mice

The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponin-skinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K(m) of mitochondrial respiration for ADP in ventricle and soleus (232 +/- 36 and 381 +/- 63 microM, respectively) was significantly reduced in the presence of creatine (52 +/- 8 and 45 +/- 12 microM, respectively). There was no change in the K(m) in oxidative fibres from mutant mice (258 +/- 27 and 399 +/- 66 microM, respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144 +/- 8 and 150 +/- 27 microM, respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.     

Muscle creatine kinase-deficient mice. II. Cardiac and skeletal muscles exhibit tissue-specific adaptation of the mitochondrial function

Functional properties of in situ mitochondria and of mitochondrial creatine kinase were studied in saponin-skinned fibers taken from normal and M-creatine kinase-deficient mice. In control animals, apparent Km values of mitochondrial respiration for ADP in cardiac (ventricular) and slow-twitch (soleus) muscles (137 +/- 16 microM and 209 +/- 10 microM, respectively) were manyfold higher than that in fast-twitch (gastrocnemius) muscle (7.5 +/- 0.5 microM). Creatine substantially decreased the Km values only in cardiac and slow-twitch muscles (73 +/- 11 microM and 131 +/- 21 microM, respectively). As compared to control, in situ mitochondria in transgenic ventricular and slow-twitch muscles showed two times lower Km values for ADP, and the presence of creatine only slightly decreased the Km values. In mutant fast-twitch muscle, a decrease rather than increase in mitochondrial sensitivity to ADP occurred, but creatine still had no effect. Furthermore, in these muscles, relatively low oxidative capacity was considerably elevated. It is suggested that in the mutant mice, impairment of energy transport function in ventricular and slow-twitch muscles is compensated by a facilitation of adenine nucleotide transportation between mitochondria and cellular ATPases; in fast-twitch muscle, mainly energy buffering function is depressed, and that is overcome by an increase in energy-producing potential.     

An assay method for nitric oxide-related compounds in whole blood

The tibialis anterior muscle and soleus muscle of apolipoprotein-E-deficient mice were examined by light and electron microscopy. By light microscopy, sarcoplasmic inclusions were seen in tibialis anterior muscle and 40% of type 2 myofibers were affected in all animals over 8 months of age. These inclusions reacted for nonspecific esterase, cytochrome oxidase, and myoadenylate deaminase and were also periodic acid Schiff positive and stained basophilic with hematoxylin. Moreover, they reacted immunocytochemically with an antibody specific to fragment 17 to 24 of the published sequence of Alzheimer's cerebrovascular amyloid peptide. Immunoreactivity was lost when the antibody was adsorbed with the appropriate synthetic peptide. Ultrastructurally, the inclusions consisted of tubular arrays and were similar to those observed in human muscle in several pathological conditions. In type 1 myofibers of both tibialis anterior and soleus muscle, however, mitochondrial abnormalities including an increase in their number and size were detected, but tubular aggregates were not seen. These large mitochondria possessed an electron-dense inner chamber with an increased number of tightly packed cristae. The results obtained suggest that in these mice there is a disturbed lipid metabolism in skeletal muscle fibers that manifests itself with an accumulation of phospholipid in the form of sarcoplasmic reticulum tubules in the type 2 fibers and enlarged mitochondria with tightly packed cristae in the type 1 fibers. In addition, beta-amyloid protein was closely associated with the accumulated tubules and vesicles of sarcoplasmic reticulum and may represent dysregulation of amyloid precursor protein metabolism.     

Improvement of muscular oxidative capacity by training is associated with slight acidosis and ATP depletion in exercising muscles

Metabolic and mechanical properties of female rat skeletal muscles, submitted to endurance training on a treadmill, were studied by a 60-min in vivo multistep fatigue test. 31P-NMR was used to follow energy metabolism and pH. Mechanical performance was greatly improved in trained muscles. The oxidative capacity of the skeletal muscles was evaluated from the relationship between ADP calculated from the creatine kinase equilibrium and work and from the measure of the rate of phosphocreatine (PCr) resynthesis following exercise. In trained muscles, ADP production was lower per unit of mechanical performance, showing an improvement of oxidative metabolism. However, the PCr resynthesis rate was not modified. Slight acidosis and ATP depletion were observed from the beginning of the fatigue test. These modifications suggest changes of the creatine kinase equilibrium favoring mitochondrial ATP production. Our results indicate that muscle status improvement could be accompanied by ATP depletion and minimal acidosis during contraction; this would be of particular importance for objective evaluation of muscle regeneration processes and of gene therapy in muscle diseases.     

Muscle unloading induces slow to fast transitions in myofibrillar but not mitochondrial properties. Relevance to skeletal muscle abnormalities in heart failure

Muscle deconditioning is a common observation in patients with congestive heart failure (CHF), chronic obstructive pulmonary disease, neuromuscular diseases or prolonged bed rest. To gain further insight into metabolic and mechanical properties of deconditioned slow-twitch (soleus) or fast-twitch (EDL) skeletal muscles, we induced experimental muscle deconditioning by hindlimb suspension (HS) in rats for 3 weeks. Cardiac muscle was also studied. Besides profound muscle atrophy, increased proportion of fast type II fibers as well as fast myosin isoenzymes, we found decreased calcium sensitivity of Triton X-100 skinned fiber bundles of soleus muscle directed towards the fast muscle phenotype. Glycolytic enzymes such as hexokinase and pyruvate kinase were increased, and the LDH isoenzyme pattern was clearly shifted from an oxidative to an anaerobic profile. Creatine kinase (CK) and myokinase activities were increased in HS soleus towards EDL values. Moreover, the M-CK mRNA level was greatly increased in soleus, with no change in EDL. However, oxygen consumption rate assessed in situ in saponin skinned fibers (12.5 +/- 0.8 in C and 15.1 +/- 0.9 micromol O2/min/g dw in HS soleus compared to 7.3 +/- 1.3 micromol O2/min/g dw in control EDL), as well as mitochondrial CK (mi-CK) and citrate synthase activities, were preserved in HS soleus. Following deconditioning no change in Km for ADP of mitochondrial respiration, either in the absence (511 +/- 92 in C and 511 +/- 111 microM in HS soleus compared to 9 +/- 4 microM in control EDL) or presence of creatine (88 +/- 10 in C and 95 +/- 16 microM in HS soleus compared to 32 +/- 9 microM in control EDL), was found. The results show that muscle deconditioning induces a biochemical and functional slow to fast phenotype transition in myofibrillar and cytosolic compartments of postural muscle, but not in the mitochondrial compartment, suggesting that these compartments are differently regulated under conditions of decreased activity.     

The development of creatine kinase in rat skeletal muscle. Changes in isoenzyme ratio, protein, RNA and DNA during development

The activity of cytosolic creatine kinase in rat skeletal muscle rises stepwise during development. The increases occur simultnaeously with transient increases in DNA content. The second increase is accompanied by a rise in total protein, soluble sarcoplasmic protein and RNA/DNA ratio. Such changes are not observed at 20 days after birth, when creatine kinase finally accumulates to the adult level. Transient higher amounts of the MB and BB isoenzymes are observed after the first and second stepwise increase. The increase in creatine kinase activity observed after birth is predominantly due to an activation of the M gene. The BB isoenzyme is still present in adult skeletal muscle, but contributes little to the total activity.     

t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria

Rat liver mitochondria have a specific Ca2+ release pathway which operates when NAD+ is hydrolysed to nicotinamide and ADPribose. NAD+ hydrolysis is Ca(2+)-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca2+ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD+ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD+ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca2+ release pathway activated by both tbh and GT. Ca2+ release increases with the mitochondrial Ca2+ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca2+ ('Ca2+ cycling') is prevented. These data support the notion that both tbh- and GT-induced Ca2+ release are not the consequence of an unspecific increase of the inner membrane permeability ('pore' formation). Tbh induces Ca2+ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca2+ loads and high tbh concentrations), Ca2+ release to about the same extent and rate as the Na+/Ca2+ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca2+ cycling and damage to mitochondria.     

Development of high-performance liquid chromatography-tandem mass spectrometric methods for the determination of a new oxytocin receptor antagonist (L-368,899) extracted from human plasma and urine: a case of lack of specificity due to the presence of metabolites

P-31 nuclear magnetic resonance (NMR) is uniquely suited to measure the kinetics of the phosphoryl-exchange reaction catalyzed by creatine kinase in intact mammalian tissue, especially striated muscle. Recently developed transgenic mouse models of the creatine kinase iso-enzyme system open novel opportunities to assess the functional importance of the individual iso-enzymes and their relative contribution to the total in situ flux through the CK reaction. This chapter reviews the most recent findings from NMR flux measurements on such genetic models of CK function. Findings in intact mouse skeletal and cardiac muscle in vivo are compared to data from purified mitochondrial and cytosolic creatine kinase in vitro. The relevance of findings in transgenic animals for the function of CK in wild-type tissue is described and the perspectives of transgenic techniques in future quantitative studies on the creatine kinase iso-enzyme system are indicated.     

Muscular ultrasound in idiopathic inflammatory myopathies of adults

To evaluate the value of myosonography in inflammatory myopathies ultrasound of skeletal muscles was performed in 70 patients, aged 21-82 years, suffering from histologically proven polymyositis (n = 30), dermatomyositis (n = 18), granulomatous myositis (n = 9), inclusion body myositis (n = 13), and in 102 control persons. The sensitivity of muscle ultrasound in detecting histopathologically proven disease (82.9%) was not significantly different from electromyography (92.4%) or serum creatine kinase activity (68.7%). The positive predictive value of ultrasound was 95.1%, the negative predictive value 89.2%, and the accuracy 91.3%. The different types of inflammatory myopathies presented with typical, but not specific ultrasound features. Polymyositis showed atrophy and increased echointensity predominantly of lower extremity muscles, whereas in dermatomyositis clear muscle atrophy was rare and echointensities were highest in forearm muscles. Echointensities were lower in dermatomyositis compared to poly- and granulomatous myositis. Granulomatous myositis was characterized by the highest echointensities and a tendency towards muscle hypertrophy. Severe muscle atrophy was the most impressive feature in the majority of patients with inclusion body myositis. Comparison of ultrasound and histopathological findings indicates that muscle lipomatosis has a much greater impact on muscular echointensity than does muscle fibrosis. Ultrasound of myositis improved clinical assessment of patients by supplying differential diagnostic clues based on precise muscle size measurements and identification of mesenchymal abnormalities, particularly muscle lipomatosis.     

Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) <==> ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output. Single ScCKmit deficiency has no noticeable effects but in combination the mutations cause slowing of the relaxation rate. Importantly, our studies revealed that there is metabolic and cytoarchitectural adaptation to CK defects in energy metabolism. The effects involve mutation type-dependent alterations in the levels of AMP, IMP, glycogen and phosphomonoesters, changes in activity of metabolic enzymes like AMP-deaminase, alterations in mitochondrial volume and contractile protein (MHC isoform) profiles, and a hyperproliferation of the terminal cysternae of the SR (in tubular aggregates). This suggests that there is a compensatory resiliency of loss-of-function and redirection of flux distributions in the metabolic network for cellular energy in our mutants.     

Quantitative studies of enzyme-substrate compartmentation, functional coupling and metabolic channelling in muscle cells

Some historical aspects of development of the concepts of functional coupling, metabolic channelling, compartmentation and energy transfer networks are reviewed. Different quantitative approaches, including kinetic and mathematical modeling of energy metabolism, intracellular energy transfer and metabolic regulation of energy production and fluxes in the cells in vivo are analyzed. As an example of the system with metabolic channelling, thermodynamic aspects of the functioning the mitochondrial creatine kinase functionally coupled to the oxidative phosphorylation are considered. The internal thermodynamics of the mitochondrial creatine kinase reaction is similar to that for other isoenzymes of creatine kinase, and the oxidative phosphorylation process specifically influences steps of association and dissociation of MgATP with the enzyme due to channelling of ATP from adenine nucleotide translocase. A new paradigm of muscle bioenergetics-the paradigm of energy transfer and feedback signaling networks based on analysis of compartmentation phenomena and structural and functional interactions in the cell is described. Analysis of the results of mathematical modeling of the compartmentalized energy transfer leads to conclusion that both calcium and ADP, which concentration changes synchronously in contraction cycle, may simultaneously activate oxidative phosphorylation in the muscle cells in vivo. The importance of the phosphocreatine circuit among other pathways of intracellular energy transfer network is discussed on the basis of the recent data published in the literature, with some experimental demonstration. The results of studies of perfused rat hearts with completely inhibited creatine kinase show significantly decreased work capacity and respectively, energy fluxes, in these hearts in spite of significant activation of adenylate kinase system (Dzeja et al. this volume). These results, combined with those of mathematical analysis of the energy metabolism of hearts of transgenic mice with switched off creatine kinase isoenzymes confirm the importance of phosphocreatine pathway for energy transfer for cell function and energetics in mature heart and many other types of cells, as one of major parts of intracellular energy transfer network and metabolic regulation.     

Ontogenetic transformations in the energy metabolism system of the skeletal muscles under different conditions of animal development

Age-related changes in the general activity of creatin kinase (CrK), mitochondrial fraction of this enzyme (CrKmit), general activity of lactate dehydrogenase (LDH) and its anaerobic fractions (LDHan), content and ratio of cytochromes of the mitochondrial respiration chain, respiration rate of isolated mitochondria on various substrates in the 3rd state have been studied in the skeletal muscle of Wistar rats developing under the conditions of normo- (NK) and hypokinesia (HK). General patterns of changes in these indices have been shown, that do not depend on the conditions of animal development: the minimal CrK and CrKmit activity and increased saturation of the respiration chain with cytochrome b562 during the playing period, days 30 to 40 of postnatal period. The highest level of cytochrome aa3 was observed in the peak of sexual maturation (Day 45). However the conditions of development affect the timing of extreme indices and their level in adult animals. For example, in the animals developing under the NK conditions the highest saturation of the respiration chain with cytochrome b562 was observed on Day 30, while in those developing under the HK conditions by five days later. In 30-day old rats from the NK group the cytochrome aa3 content of skeletal muscle was almost maximal, while in the HK group the maximum was observed only at the peak of sexual maturation. In the adult animals of the HK group the activity of CrKmit, LDH and LDHan was higher, while the content of cytochrome aa3 and saturation of the respiration chain with cytochrome b562 was lower. A lesser b562/aa3 ratio is related to the predominance of NADH2-dependent electron transport pathways of oxidation in the mitochondria from the rat skeletal muscle. Thus, the conditions of development in the early postnatal period, especially during the playing period, determine the state of energy metabolism in the adult skeletal muscle.     

Mitochondrial abnormalities and peripheral neuropathy in inflammatory myopathy, especially inclusion body myositis

Computer retrieval in a database, comprising 7,225 muscle cases, revealed that mitochondrial myopathies do not occur more frequently in inflammatory myopathies (3.74%) than in the whole series (3.69%). A more detailed study of inclusion body myositis (IBM), however, showed that severe mitochondrial alterations were apparent in about twice as many IBM cases as expected. This confirms recent studies of others although a causal relationship has thus far not been established. Identification of mitochondrial deletions by Southern blotting corresponded to the presence of severe structural abnormalities of mitochondria. Peripheral neuropathy of variable severity was noted in all cases of IBM and mitochondrial myopathy. By contrast, the association of severe mitochondrial abnormalities with polymyositis, systemic scleroderma, and vasculitis observed in some cases of the present series may be incidental or age dependent.     

Differential myogenicity of satellite cells isolated from extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro

Following muscle damage, fast- and slow-contracting fibers regenerate, owing to the activation of their satellite cells. In rats, crush-induced regeneration of extensor digitorum longus (EDL, a fast muscle) and soleus (a slow muscle) present different characteristics, suggesting that intrinsic differences exist among their satellite cells. An in vitro comparative study of the proliferation and differentiation capacities of satellite cells isolated from these muscles is presented there. We observed several differences between soleus and EDL satellite cell cultures plated at high density on gelatin-coated dishes. Soleus satellite cells proliferated more actively and fused into myotubes less efficiently than EDL cells. The rate of muscular creatine kinase enzyme appeared slightly lower in soleus than in EDL cultures at day 11 after plating, when many myotubes were formed, although the levels of muscular creatine kinase mRNA were similar in both cultures. In addition, soleus cultures expressed higher levels of MyoD and myogenin mRNA and of MyoD protein than EDL satellite cell cultures at day 12. A clonal analysis was also carried out on both cell populations in order to determine if distinct lineage features could be detected among satellite cells derived from EDL and soleus muscles. When plated on gelatin at clonal density, cells from both muscles yielded clones within 2 weeks, which stemmed from 3-15 mitotic cycles and were classified into three classes according to their sizes. Myotubes resulting from spontaneous fusion of cells from the progeny of one single cell were seen regardless of the clone size in the standard culture medium we used. The proportion of clones showing myotubes in each class depended on the muscle origin of the cells and was greater in EDL- than in soleus-cell cultures. In addition, soleus cells were shown to improve their differentiation capacity upon changes in the culture condition. Indeed, the proportions of clones showing myotubes, or of cells fusing into myotubes in clones, were increased by treatments with a myotube-conditioned medium, with phorbol ester, and by growth on extra-cellular matrix components (Matrigel). These results, showing differences among satellite cells from fast and slow muscles, might be of importance to muscle repair after trauma and in pathological situations.     

Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P < 0.05) over a 13-d period. Total protein synthesis rates could be determined accurately in the bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions necessary for space flight studies were validated as part of the studies. For example, the anabolic autocrine/paracrine skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.     

Dual projection from the medial septum to the supramammillary nucleus in the rat

Hereditary inclusion body myopathies comprise autosomal recessive and autosomal dominant muscle disorders that have a variable clinical phenotype but share similar morphological features. These include rimmed vacuoles within muscle fibres and collections of intrasarcoplasmic and intranuclear tubulofilamentous inclusions, 16-18 nm in external diameter. The resemblances and the differences between the sporadic and the hereditary inclusion body myopathies are discussed. Recent advances in the identification of various proteins involved in these diseases are mentioned because they have provided better insight into their underlying pathophysiological mechanisms. Linkage studies have allowed the localization of the genetic defect of some hereditary inclusion body myopathies and related disorders, contributing to their individualization.     

Non-insulin and non-exercise related increase of glucose utilization in rats and mice

The effects of high-energy phosphate contents in muscles on glucose tolerance and glucose uptake into tissues were studied in rats and mice. Enhanced glucose tolerance associated with depleted high-energy phosphates and elevated glycogen content in muscles and liver was observed in animals fed creatine analogue beta-guanidinopropionic acid (beta-GPA). Distribution of infused 2-[1-14C]deoxy-D-glucose in tissues especially in the soleus muscle, kidney, and brain was greater in mice fed beta-GPA than controls. The glucose uptake was decreased when the contents of ATP and glycogen were normalized following creatine supplementation. Plasma insulin in animals at rest was lower and its concentration after intraperitoneal glucose infusion tended to be less in animals fed beta-GPA than controls (p > 0.05), although the pattern of insulin response to glucose loading was similar to the control. The daily voluntary activity in beta-GPA fed mice was also less than controls. These results suggest that improved glucose tolerance is not related to elevated insulin concentration and/or decreased glycogen following exercise. Such improvement may be due to an increased mitochondrial energy metabolism caused by depletion of high-energy phosphates.