Associations between muscle strength, spirometric pulmonary function and mobility in healthy older adults.

Pathological obstruction in lungs leads to severe decreases in muscle strength and mobility in patients suffering from chronic obstructive pulmonary disease. The purpose of this study was to investigate the interdependency between muscle strength, spirometric pulmonary functions and mobility outcomes in healthy older men and women, where skeletal muscle and pulmonary function decline without interference of overt disease. A total of 135 69- to 81-year-old participants were recruited into the cross-sectional study, which was performed as a part of European study MyoAge. Full, partial and no mediation models were constructed to assess the interdependency between muscle strength (handgrip strength, knee extension torque, lower extremity muscle power), spirometric pulmonary function (FVC, FEV1 and FEF50) and mobility (6-min walk and Timed Up and Go tests). The models were adjusted for age, sex, total fat mass, body height and site of enrolment. Partial mediation models, indicating both direct and pulmonary function mediated associations between muscle strength and mobility, fitted best to the data. Greater handgrip strength was significantly associated with higher FVC, FEV1 and FEF50 (p < 0.05). Greater muscle power was significantly associated with better performance in mobility tests. Results suggest that decline in mobility with aging may be caused by decreases in both muscle strength and power but also mediated through decreases in spirometric pulmonary function. Future longitudinal studies are warranted to better understand how loss of function and mass of the respiratory muscles will affect pulmonary function among older people and how these changes are linked to mobility decline.

Androgen replacement therapy improves function in male rat muscles independently of hypertrophy and activation of the Akt/mTOR pathway.

AIM: We analysed the effect of physiological doses of androgens following orchidectomy on skeletal muscle and bone of male rats, as well as the relationships between muscle performance, hypertrophy and the Akt/mammalian target of rapamycin (mTOR) signalling pathway involved in the control of anabolic and catabolic muscle metabolism. METHODS: We studied the soleus muscle and tibia from intact rats (SHAM), orchidectomized rats treated for 3 months with vehicle (ORX), nandrolone decanoate (NAN) or dihydrotestosterone (DHT). RESULTS: Orchidectomy had very little effect on the soleus muscle. However, maximal force production by soleus muscle (+69%) and fatigue resistance (+35%) in NAN rats were both increased when compared with ORX rats. In contrast, DHT treatment did not improve muscle function. The relative number of muscle fibres expressing slow myosin heavy chain and citrate synthase activity were not different in NAN and ORX rats. Moreover, NAN and DHT treatments did not modify muscle weights and cross-sectional area of muscle fibres. Furthermore, phosphorylation levels of downstream targets of the Akt/mTOR signalling pathway, Akt, ribosomal protein S6 and eukaryotic initiation factor 4E-binding protein 1 were similar in muscles of NAN, DHT and ORX rats. In addition, trabecular tibia from NAN and DHT rats displayed higher bone mineral density and bone volume when compared with ORX rats. Only in NAN rats was this associated with increased bone resistance to fracture. CONCLUSION: Physiological doses of androgens are beneficial to muscle performance in orchidectomized rats without relationship to muscle and fibre hypertrophy and activation of the Akt/mTOR signalling pathway. Taken together our data clearly indicate that the activity of androgens on muscle and bone could participate in the global improvement of musculoskeletal status in the context of androgen deprivation induced by ageing.

Slow myosin heavy chain expression in the absence of muscle activity.

Innervation has been generally accepted to be a major factor involved in both triggering and maintaining the expression of slow myosin heavy chain (MHC-1) in skeletal muscle. However, previous findings from our laboratory have suggested that, in the mouse, this is not always the case (30). Based on these results, we hypothesized that neurotomy would not markedly reduced the expression of MHC-1 protein in the mouse soleus muscles. In addition, other cellular, biochemical, and functional parameters were also studied in these denervated soleus muscles to complete our study. Our results show that denervation reduced neither the relative amount of MHC-1 protein, nor the percentage of muscle fibers expressing MHC-1 protein (P > 0.05). The fact that MHC-1 protein did not respond to muscle inactivity was confirmed in three different mouse strains (129/SV, C57BL/6, and CD1). In contrast, all of the other histological, biochemical, and functional muscle parameters were markedly altered by denervation. Cross-sectional area (CSA) of muscle fibers, maximal tetanic isometric force, maximal velocity of shortening, maximal power, and citrate synthase activity were all reduced in denervated muscles compared with innervated muscles (P < 0.05). Contraction and one-half relaxation times of the twitch were also increased by denervation (P < 0.05). Addition of tenotomy to denervation had no further effect on the relative expression of MHC-1 protein (P > 0.05), despite a greater reduction in CSA and citrate synthase activity (P < 0.05). In conclusion, a deficit in neural input leads to marked atrophy and reduction in performance in mouse soleus muscles. However, the maintenance of the relative expression of slow MHC protein is independent of neuromuscular activity in mice.

Genetic inactivation of acetylcholinesterase causes functional and structural impairment of mouse soleus muscles.

Acetylcholinesterase (AChE) plays an essential role in neuromuscular transmission. Not surprisingly, neuromuscular transmission during repetitive nerve stimulation is severely depressed in the AChE knockout mouse (KO). However, whether this deficit in AChE leads to skeletal muscle changes is not known. We have studied the in vitro contractile properties of the postural and locomotor soleus muscles of adult KO and normal (wildtype, WT) mice, and this was completed by histological and biochemical analyses. Our results show that muscle weight, cross-sectional area of muscle fibres and absolute maximal isometric force are all reduced in KO mice compared with WT mice. Of interest, the relative amount of slow myosin heavy chain (MHC-1) in muscle homogenates and the percentage of muscle fibres expressing MHC-1 are decreased in the KO mice. Surprisingly, AChE ablation does not modify twitch kinetics, absolute maximal power, fatigue resistance or citrate synthase activity, despite the reduced number of slow muscle fibres. Thus, a deficit in AChE leads to alterations in the structure and function of muscles but these changes are not simply related to the reduced body weight of KO mice. Our results also suggest that this murine model of congenital myasthenic syndrome with endplate AChE deficiency combines alterations in both neurotransmission and intrinsic muscle properties.

Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

Impact on oxidative phosphorylation of immortalization with the telomerase gene.

Skin fibroblasts are essential tools for biochemical, genetic and physiopathological investigations of mitochondrial diseases. Their immortalization has been previously performed to overcome the limited number of divisions of these primary cells but it has never been systematically evaluated with respect to efficacy and impact on the oxidative phosphorylation (OXPHOS) characteristics of the cells. We successfully immortalized with the human telomerase gene 15 human fibroblasts populations, 4 derived from controls and 11 from patients with diverse respiratory chain defects. Immortalization induced significant but mild modification of the OXPHOS characteristics of the cells with lower rates of oxygen consumption and ATP synthesis associated with their loose coupling. However, it never significantly altered the type and severity of any genetic OXPHOS defect present prior to immortalization. Furthermore, it did not significantly modify the cells' dependence on glucose and sensitivity to galactose thus showing that immortalized cells could be screened by their nutritional requirement. Immortalized skin fibroblasts with significant OXPHOS defect provide reliable tools for the diagnosis and research of the genetic cause of mitochondrial defects. They also represent precious material to investigate the cellular responses to these defects, even though these should afterwards be verified in unmodified primary cells.

Myogenic stem cells: regeneration and cell therapy in human skeletal muscle.

Human skeletal muscle has been considered as an ideal target for cell-mediated therapy. However, the positive results obtained in dystrophic animal models using the resident precursor satellite cell population have been followed by discouraging evidences obtained in the clinical trials involving Duchenne muscular dystrophy patients. This text reviews the recent advances that many groups have achieved to identify from the stem cell compartment putative candidates for cell therapy. We focused our attention on stem cells with myogenic potential which might be able to improve transplantation efficiency and therefore could be used as a therapeutic tool for neuromuscular diseases.

The mitotic clock in skeletal muscle regeneration, disease and cell mediated gene therapy.

The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo-Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease.

Myoblast transfer therapy: is there any light at the end of the tunnel?

Myoblast transfer therapy (MTT) was proposed in the 70's as a potential treatment for muscular dystrophies, based upon the early results obtained in mdx mice: dystrophin expression was restored in this model by intramuscular injections of normal myoblasts. These results were quickly followed by clinical trials for patients suffering from Duchenne Muscular Dystrophy (DMD) in the early 90's, based mainly upon intramuscular injections of allogenic myoblasts. The clinical benefits obtained from these trials were minimal, if any, and research programs concentrated then on the various pitfalls that hampered these clinical trials, leading to numerous failures. Several causes for these failures were identified in mouse models, including a massive cell death of myoblasts following their injection, adverse events involving the immune system and requiring immunosuppression and the adverse events linked to it, as well as a poor dispersion of the injected cells following their injection. It should be noted that these studies were conducted in mouse models, not taking into account the fundamental differences between mice and men. One of these differences concerns the regulation of proliferation, which is strictly limited by proliferative senescence in humans. Although this list is certainly not exhaustive, new therapeutic venues were then explored, such as the use of stem cells with myogenic potential, which have been described in various populations, including bone marrow, circulating blood or muscle itself. These stem cells presented the main advantage to be available and not exhausted by the numerous cycles of degeneration/regeneration which characterize muscle dystrophies. However, the different stem candidates have shown their limits in terms of efficiency to participate to the regeneration of the host. Another issue was raised by clinical trials involving the injection of autologous myoblasts in infacted hearts, which showed that limited targets could be aimed with autologous myoblasts, as long as enough spared muscle was available. This resulted in a clinical trial for the pharyngeal muscles of patients suffering from Oculo-Pharyngeal Muscular Dystrophy (OPMD). The results of this trial will not be available before 2 years, and a similar procedure is being studied for Fascio-Scapulo-Humeral muscular Dystrophy (FSHD). Concerning muscular dystrophies which leave very few muscles spared, such as DMD, other solutions must be found, which could include exon-skipping for the eligible patients, or even cell therapy using stem cells if some cell candidates with enough efficiency can be found. Recent results concerning mesoangioblasts or circulating AC133+ cells raise some reasonable hope, but still need further confirmations, since we have learned from the past to be cautious concerning a transfer of results from mice to humans.

Human muscle precursor cell regeneration in the mouse host is enhanced by growth factors.

The aim of this study was to optimize human muscle formation in vivo from implanted human muscle precursor cells. We transplanted donor muscle precursor cells (MPCs) prepared from postnatal or fetal human muscle into immunodeficient host mice and showed that irradiation of host muscle significantly enhanced muscle formation by donor cells. The amount of donor muscle formed in cryodamaged host muscle was increased by exposure of donor cells to growth factors before their implantation into injured host muscle. Insulin-like growth factor type I (IGF-I) significantly increased the amount of muscle formed by postnatal human muscle cells, but not by fetal human MPCs. However, treatment of fetal muscle cells with IGF-I, in combination with basic fibroblast growth factor and plasmin, significantly increased the amount of donor muscle formed. In vivo, human MPCs formed mosaic human-mouse muscle fibers, in which each human myonucleus was associated with a zone of human sarcolemmal protein spectrin.

IGF-1 induces human myotube hypertrophy by increasing cell recruitment.

Insulin-like growth factor-1 (IGF-1) has been shown in rodents (i) in vivo to induce muscle fiber hypertrophy and to prevent muscle mass decline with age and (ii) in vitro to enhance the proliferative life span of myoblasts and to induce myotube hypertrophy. In this study, performed on human primary cultures, we have shown that IGF-1 has very little effect on the proliferative life span of human myoblasts but does delay replicative senescence. IGF-1 also induces hypertrophy of human myotubes in vitro, as characterized by an increase in the mean number of nuclei per myotube, an increase in the fusion index, and an increase in myosin heavy chain (MyHC) content. In addition, muscle hypertrophy can be triggered in the absence of proliferation by recruiting more mononucleated cells. We propose that IGF-1-induced hypertrophy can involve the recruitment of reserve cells in human skeletal muscle.

Extended amplification in vitro and replicative senescence: key factors implicated in the success of human myoblast transplantation.

The limited success of human myoblast transplantation has been related to immune rejection, poor survival, and limited spread of injected myoblasts after transplantation. An important issue that has received little attention, but is nevertheless of fundamental importance in myoblast transplantation protocols, is the proliferative capacity of human satellite cells. Previous studies from our laboratory have demonstrated that the maximum number of divisions that a population of satellite cells can make decreases with age during the first two decades of life then stabilizes in adulthood. These observations indicate that when satellite cells are used as vectors in myoblast transplantation protocols it is important to consider donor age and the number of divisions that the cells have made prior to transplantation as limiting factors in obtaining an optimal number of donor derived muscle fibers. In this study, myoblasts derived from donors of different ages (newborn, 17 years old, and 71 years old) were isolated and amplified in culture. Their potential to participate in in vivo muscle regeneration in RAG2(-/-)/gamma(c)/C5 triple immunodeficient hosts after implantation was evaluated at 4 and 8 weeks postimplantation. Our results demonstrate that prolonged amplification in culture and the approach to replicative senescence are both important factors that may condition the success of myoblast transplantation protocols.

Satellite cells and training in the elderly.

In the present review, we describe the effects of ageing on human muscle fibres, underlining that each human muscle is unique, meaning that the phenotype becomes specifically changed upon ageing in different muscles, and that the satellite cells are key cells in the regeneration and growth of muscle fibres. Satellite cells are closely associated with muscle fibres, located outside the muscle fibre sarcolemma but beneath the basement lamina. They are quiescent cells, which become activated by stimulation, like muscle fibre injury or increased muscle tension, start replicating and are responsible for the repair of injured muscle fibres and the growth of muscle fibres. The degree of replication is governed by the telomeric clock, which is affected upon excessive bouts of degeneration and regeneration as in muscular dystrophies. The telomeric clock, as in dystrophies, does not seem to be a limiting factor in ageing of human muscle. The number of satellite cells, although reduced in number in aged human muscles, has enough number of cell divisions left to ensure repair throughout the human life span. We propose that an active life, with sufficient general muscular activity, should be recommended to reduce the impairment of skeletal muscle function upon ageing.

[The persistence of ontogenic characteristics in the adult masseter muscle]. / Persistance de caractères ontogéniques dans le muscle masséter adulte.

During embryonic and foetal development, the masseter is formed from two successive generations of muscle fibers in a manner which is very similar to that which has been previously described for other skeletal muscles. This phenotype is characterised by the persistence of ontogenic myosin isoforms (embryonic and foetal myosin heavy chains, embryonic light chain) and by the presence of two distinct populations of fibers: small diameter fibers which coexpress the embryonic, foetal and fast isoforms of the myosin heavy chains but never express the slow isoform; large diameter fibers which express the slow myosin heavy chain either exclusively or in variable associations with the other isoforms. These characteristics of the human masseter muscle probably correspond not only to its embryological origin and its special innervation, but also to the functional constraints to which it is submitted after birth.

Defective satellite cells in congenital myotonic dystrophy.

In this study we have developed an in vitro cell culture system which displays the majority of the defects previously described for congenital myotonic dystrophy (CDM) muscle in vivo. Human satellite cells were isolated from the quadriceps muscles of three CDM fetuses with different clinical severity. By Southern blot analysis all three cultures were found to have approximately 2300 CTG repeats. This CTG expansion was found to progressively increase in size during the proliferative life span, confirming an instability of this triplet in skeletal muscle cells. The CDM myoblasts and myotubes also showed abnormal retention of mutant RNA in nuclear foci, as well as modifications in their myogenic program. The proliferative capacity of the CDM myoblasts was reduced and a delay in fusion, differentiation and maturation was observed in the CDM cultures compared with unaffected myoblast cultures. The clinical severity and delayed maturation observed in the CDM fetuses were closely reflected by the phenotypic modifications observed in vitro. Since the culture conditions were the same, this suggests that the defects we have described are intrinsic to the program expressed by the myoblasts in the absence of any trophic factors. Altogether, our results demonstrate that satellite cells are defective in CDM and are probably implicated in the delay in maturation and muscle atrophy that has been described previously in CDM fetuses.

[Metabolic differentiation of the human longus colli muscle]. / Différenciation métabolique du muscle longus colli chez l'Homme.

The cervical muscles have a dual postural and dynamic function, in order to ensure both the stability and the motility of the cervical spine. The functional duality together with the complexity of the cervico-cephalic system render the study of the cervical muscles difficult, and their physiology is not fully understood in humans. This study has been carried out on ten samples from the m. longus colli, taken during a surgical procedure in patients aged between 36 to 62 years. The histological study combined enzyme histochemical (ATPases) and immunohistochemical techniques (using antibodies specific for the slow and the fast isoforms of the myosin heavy chains). Our results indicate that, in all cases, the m. longus colli is composed of muscle fibers with peripheral nuclei and with a relative dispersion in size. Histochemically, the type 1 and type 2 fibers express exclusively either the slow or the fast myosin heavy chain. From a quantitative point of view, the proportion of the slow fibers varies between extreme values of 30 and 73%; in addition, the dispersion in fiber size predominates on the fast type 2 fibers which are smaller than the slow type 1 fibers. Thus, most of the muscles that we have studied have histologically a slow predominance. This predominant expression of a slow phenotype in the m. longus colli corresponds to its important postural function, in addition to its phasic role during the flexion of the cervical spine.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Influence of early postnatal cold exposure on myofiber maturation in pig skeletal muscle.

Early after birth, piglets rely almost exclusively on muscular shivering thermogenesis to produce heat in the cold and this can possibly modulate skeletal muscle development. An experiment involving 10 individually housed piglets was conducted to determine the influence of cold (24-15 degrees C, D5C group) vs. thermoneutrality (34-30 degrees C, D5TN group) between birth and 5 days on myosin heavy chain (MyHC) polymorphism and metabolic characteristics of longissimus lumborum (LL) and rhomboideus (RH) muscles. Five additional piglets were sacrificed at birth. Piglets exposed to cold received 43% more artificial milk on a liveweight basis in order to achieve similar growth rates. D5C piglets produced 93% more heat and exhibited intense shivering during the whole experiment. Contractile and metabolic characteristics of muscles were determined by immunocytochemistry, electrophoresis and enzyme activities. At least eight MyHC isoforms were detected, including atypical expressions of the alpha-cardiac and extraocular isoforms. Dramatic changes in MyHC composition, myofiber cross-sectional area (CSA) and energy metabolism occurred between birth and 5 days. Cold exposure did not affect either the total number of fibers or the CSA, but it did influence muscle maturation. In particular, it increased the expression of alpha-cardiac and type I MyHC, and decreased that of fetal MyHC, confirming an acceleration in the rate of postnatal maturation. An increase in oxidative enzyme activities was observed in both muscles in the cold, whereas the activity of a glycolytic enzyme, lactate dehydrogenase, remained unchanged. Cold exposure also induced an increase in T3 plasma levels. The extent to which these changes are the result of sustained shivering or are due to the action of hormonal factors, such as thyroid hormones, are discussed.

Skeletal muscle regeneration and the mitotic clock.

Regeneration of muscle fibers following damage requires activation of quiescent satellite cells, their proliferation and finally their differentiation and fusion into multinucleated myotubes, which after maturation will replace the damaged fiber. The regenerative potential of human skeletal muscle will be determined, at least partly, by the proliferative capacity of the satellite cells. In this study, we have measured the proliferative life span of human satellite cells until they reach senescence. These analyses were performed on cell populations isolated from old and young donors as well as from one child suffering from Duchenne muscular dystrophy, where extensive regeneration had occurred. In order to see if there are any age-related changes in the myogenic program we have also compared the program of myogenic differentiation expressed by satellite cells from these subjects at different stages of their proliferative lifespan.

Shorter telomeres in dystrophic muscle consistent with extensive regeneration in young children.

Muscular dystrophies are characterised by continuous cycles of degeneration and regeneration resulting in an eventual diminution of the muscle mass and extensive fibrosis. In somatic cells chromosomal telomeres shorten with each round of cell division and telomere length is considered to be a biomarker of the replicative history of the cell. We have previously shown that human myoblasts have a limited proliferative capacity, and that normal skeletal muscle has a very low level of nuclear turnover. However, in patients suffering from muscular dystrophy the satellite cells will be forced to make repeated rounds of cell division, driving the cells towards senescence. In this study we have used the telomere length to quantify the intensity of the muscle cell turnover in biopsies from dystrophic patients of different ages. Our results show that as soon as the first clinical symptoms become apparent the muscle has already undergone extensive regeneration and the rate of telomere loss is 14 times greater than that observed in controls. This confirms that the decline in regenerative capacity is due to the premature senescence of the satellite cells induced by their excessive proliferation during muscle repair.