The data on satellite cell frequency obtained in young and old human muscles can serve as a standard when comparing satellite cell frequency in pathological, disused or differently trained muscles. Number of M-cadherin stained satellite cells in 1cm3 of human vastus lateralis muscles: 2.36 �8.97 x 105 (in the young), 1.85 �3.55 x 105 (in the old muscle) (non-significant difference). Frequency of M-cadherin stained satellite cells in human vastus lateralis muscle: 1.85 ± 0.23 (SEM) per mm fibre length in the young, 1.19 ± 0.06 in the old muscle (p<0.05); - Percentage of M-cadherin stained SC nuclei from total muscle nuclei 2.02 ± 0.33 in the young, 1.09± 0.12 in the old muscle (p<0.05); - Per fibre number: 2.4 ±0.4 in the young, 1.6 ±0.1 in the old muscle (p<0.05). These results might also be interesting for comparison among different species.
Characterisation of membrane electrical properties and of calcium homeostasis during the differentiation process of human myoblasts from donors of different ages
To understand the possible effect of ageing on functional properties of human satellite cells we performed electrophysiological and videoimaging experiments on human satellite cells isolated from biopsies taken from donors of different ages. More in detail, we used different configurations of the patch clamp technique to study the biophysical properties of acetylcholine receptor and voltage-dependent sodium channels. We observed only minor changes in the kinetics of sodium channels, while acetylcholine receptor channels had similar properties, showing only some delay in the appearance in older cells. Fluorescence videomicroscopy, using Fura-2 as calcium dye, showed a delayed maturation of excitation-contraction coupling mechanism in developing myotubes from older donors. This latter phenomenon can be mimicked in vitro by artificially ageing of satellite cells. Our results help to understand the cellular basis of muscle ageing.
Exhaustion of the human muscle organotypic stem cell with ageing; prognostic tool for human cell therapy
We investigated whether the lifelong consumption of the organotypic muscle stem cells, the satellite cells, might lead to their exhaustion in aged people. Human myogenic cells derived from donors aged 2-82 years and from biopsies of 3 children suffering from Duchenne Muscular Dystrophy (DMD) in which satellite cells are thought to suffer premature ageing, were implanted into immunincompetent mice. The progeny of the grafted cells was quantified by structural features and by the amount of human DNA extractable from these muscles. When considering highly pure myogenic cell preparations, we observed an inverse relation between the donor's age and the proliferation capacity in vitro as well as in vivo with about 2 divisions of each satellite cell per 10 years of life. Myogenic cells from the oldest donors still grew human muscle tissue, indicating a lack of exhaustion even in aged donors in spite of a continual loss of growth capacity of satellite cells in the living muscle. On the other hand, cell preparations with initially low or decreasing myogenicity during expansion in vitro showed completely different growth characteristics, e.g. increased proliferation capacity, longer telomeres and lower myogenicity of the tissue formed upon implantation. We suggest a "Desmin Factor" (DF) to predict myogenicity of the human tissue formed in vivo from the growth characteristics during expansion in vitro. This may serve as a prognostic tool in human trials.
The specificity and suitability of antibodies, muscle specimens and membrane preparations was evaluated. This included the testing of various monoclonal and polyclonal antibodies to muscle proteins involved in fibre contraction, the regulation of ion homeostasis and excitation-contraction coupling. Various tissues from animal models and humans biopsies were analysed and the suitability of subcellular fractionation procedures evaluated. Optimum visualization of immuno-decorated proteins was shown to occur with the enhanced chemiluminescence technique. This knowledge will be used for the future analysis of normal, diseased and aged muscle samples. It is planed to perform a comprehensive comparison of the protein expression patterns between normal and aged fibres over the next three years.
Different staining techniques for identification of satellite cells in human muscles have been evaluated. Several methods give an estimation of frequency of satellite cells however no golden standard has been established, as different markers for satellite cells do not give the same numbers. This suggests that either there are different populations of satellite cells or they show different degrees of activation. One efficient staining method using antibodies against N-CAM to visualize satellite cells has been used to quantify the number of satellite cells in persons at different ages, from newborns, children aged 2-7 years, young adults and aged persons (over 70 years). In children the number of satellite cells are 2,5-3 folds higher than in young adults that in turn are 2-3 folds higher than in aged people.
The regenerative capacity of human muscle relies on a population of cells called satellites cells, and is known to decrease with age. The amount of muscle these cells will be able to regenerate depends on the quantity of cells available at the time of degeneration, and their capacity to proliferate before forming new fibres. We measured in vitro the number of divisions that satellite cells isolated from adults of different ages can achieve in vitro before reaching senescence, and this measurement was correlated with telomere length, which act as a mitotic clock by shortening at each divisions. We found that adult human satellite cells have a proliferation capacity that does not change significantly with age in the adult, and this is confirmed by telomere length. However, we also have also shown that this capacity is significantly reduced in patients suffering from degenerative diseases where repeated cycles of degeneration-regeneration decrease prematurely the proliferation capacity of human satellite cells. Although these parameters are not the only ones involved in muscle regeneration, telomere length as well as remaining proliferation capacity as measured in vitro are indicative of the regenerative capacity in vivo. Potential applications include therapeutic trials for cell-mediated gene therapy or cellular therapy. It should be noted that pre-clinical studies emanating from these results are currently being achieved for a clinical trial on patients suffering from Oculo-Pharyngeal Muscular Dystrophy (OPMD).
We have established protocols for the detection of satellite cells in thick muscle sections and establish sampling design of very rare structures as satellite cells are, starting from muscles of individual persons and ending with completely registered stacks of optical images captured by a confocal microscope. Here, satellite cells were defined from their association with M-Cadherin and their location beneath the basal lamina. To avoid the biased counting of satellite cell profiles or myonuclear profiles used so far, which can lead to erroneous results, because the same satellite cells or the same myonuclei can be seen in several successive sections, we have introduced an unbiased counting method, the Disector.
Biophysical properties of sodium and potassium voltage-dependent channels during myogenesis of satellite cells from donors of different ages
To understand if the electrophysiological features of the cells such as the resting membrane potential and the properties of the voltage-gated potassium and sodium currents depend on age we performed electrophysiological experiments on cells cultured from differently aged donors. Recordings of Na-currents showed the presence of both TTX-resistant and TTX-sensitive components. Current density and kinetics properties were only slightly and not significantly different in the old and middle-aged cells. Outward K-currents with delayed rectifier or non-inactivating properties and inward K-currents have been recorded. The percentage of cells expressing outward and inward potassium currents were lower in cells derived from the oldest donors. Cells from the older donors showed, on the average a more negative activation potential of outward currents. Potassium current properties seem to be related to the donor age.
During the grant period, a blot overlay assay procedure was developed and used to analyse aged muscle fibres. The purified probe was characterized by mass spectrometry, peptide sequencing and immunoblotting. Using a calsequestrin-peroxidase conjugate, it was shown that the major 63-kDa Ca2+-binding protein of the sarcoplasmic reticulum forms complexes with itself, as well as junctin of 26-kDa, triadin of 94-kDa and the ryanodine receptor of 560-kDa. Variations in the relative abundance of the four central elements of excitation-contraction coupling in different fibre types, during myogenesis, ageing and fibre type shifting were reflected by distinct alterations in the calsequestrin overlay binding patterns. In future application, we will use the overlay procedure for an intraproteomic approach to determine protein-protein interactions within the mature and aged sarcoplasmic reticulum.
In order to extend the animal studies to human skeletal muscle, we have determined whether potential changes in the relative expression of the voltage sensor occur in senescent human fibres. Besides small inter-individual variations in expression levels, the microsomal immunoblot analysis of vastus lateralis autopsy specimens from male humans aged 18 to 82 years of age showed no major changes in the relative abundance of the dihydropyridine receptor, fast calsequestrin and the slow/fast myosin heavy chains. The oligomeric status of the a1S-dihydropyridine receptor was unaltered in aged human fibres. One potential way forward is the use of proteomics research tools such as the global comparative survey of mature versus aged muscle protein complement using 2D gel electrophoresis in combination with mass spectrometry. Depending on the availability of proteomics equipment, such an analysis is planned for the next 3 years. This should lead to the identification of novel candidate proteins involved in the molecular pathogenesis of sarcopenia of old age and such data can then be complemented with detailed biomedical studies into the mechanism of age-related muscle weakness.
To determine age-induced changes in animal muscles, specimens from ageing male New Zealand white rabbits (3 weeks to 2.4 years of age) and male WI/HicksCar rats (4 and 28 months of age) were examined. Immunoblotting of the microsomal fraction from aged rabbit and rat muscle revealed a drastic decline in the voltage-sensing alpha-1-subunit of this transverse-tubular receptor, and a shift to slower fibre type characteristics was indicated by an age-related increase in the slow calsequestrin isoform. Based on our analysis of aged animal muscle fibres, we plan to further study the potential involvement of abnormal calcium handling in sarcopenia of animals. This will include the identification and characterization of the microsomal subproteome by 2D electrophoresis over the next 3 years.
Frequency of satellite cells in young and old rat extensor digitorum longus muscle and in cross-aged grafts
The data on satellite cell frequency obtained in young and rat extensor digitorum longus muscles can serve as a standard when comparing satellite cell frequency in pathological, disused or experimental rat muscles. These results might also be interesting for comparison among different species.
The regenerative capacity of human muscle relies on a population of cells called satellites cells. Although many parameters may have an influence of the regenerative capacity, some of them can be monitored in vitro. However, models in vitro can only be indicative. We have set up along with partners 1 and 3 (University of Bonn, MRC) a model to evaluate the regenerative capacity of human satellite cells in vivo. This model is based upon the injection of human myoblasts expanded in vitro into regenerating muscles of "humanized" mice (i.e. immunodeficient strains which do not reject human cells). This model has been used to monitor expression of genes that are not expressed in vitro, but only in matured fibres. Using this model, we have confirmed the results obtained in vitro (see IP, former result), which is that the proliferation capacity is one of the parameters involved in the regenerative capacity of human myoblasts. However, we also established that long period of expansion in vitro are also detrimental to the regenerative capacity. This result applies to cell-mediated gene therapy and cellular therapy in general, where the expansion of cells is usually needed to obtain a sufficient number of cells of relevant therapeutic potential. The model can also be applied to searching myogenic potential among embryonic or adult stem cells.
The data on the frequency of myonuclei obtained in young and old human muscles can serve as a standard when comparing the distribution and frequency of myonuclei in pathological, disused or differently trained muscles. Absolute numbers of myonuclei within unit tissue volumes are important references for in vitro studies: - Number of muscle nuclei per mm fibre length: 99.88 ± 5.97 in the young, 123.83 ± 14.38 in the old muscle (non significant difference); - Number of myonuclei in 1cm3 of human vastus lateralis muscles:2.73 ± 0.13 (SEM) x 107 in the young, 3.00 ± 0.11 (SEM) x 107(non significant difference); - Nuclear domains: 37.96 ± 2.56 mm3x 103 in the young, 35.54 ± 2.01 mm3x 103 in the old muscle. These results might also be interesting for comparison among different species.
Highly prolific myogenic cells are found in skeletal muscle and are commonly said to be derived from the satellite cells that are tightly associated with the muscle fibre. To study the myogenic potential of satellite cells we have studied isolated muscle fibres in tissue culture and found that only a minority of satellite cells are highly proliferative and myogenic. We have also grafted isolated muscle fibres and found that a minority of fibres contain a satellite cell that is highly myogenic in that it can repair a large volume of muscle around the graft site and replace the satellite cell population on these fibres: properties that define them as stem cells. We calculate that such cells constitute about 1-2% of the total number and that they differ in behaviour but not in frequency between different muscles. Further characterization of this category of cell would provide a basis for improving our control over muscle repair and for myoblast transplantation strategies.
To understand the role of satellite cells and myonuclei in adaptation of muscle fibre size, analyses have been performed using muscle biopsies from elite strength trained athletes, some of which had also used anabolic steroids as supplement to the training. An important implication of that study is that satellite cells are incorporated in pre-existing muscle fibres to match the increase in muscle fibre area allowing a permissible nuclear domain size. Satellite cells were also found to be the basis for new fibres suggesting that, if the number of new fibres made is in excess over those destroyed, hyperplasia can occur. Muscle biopsies have also been obtained from persons, before and after they have been in bed rest for 10 weeks. These biopsies allow us to estimate which processes are related to inactivity of the muscles. A significant decrease in mean area was observed without committing decrease in myonuclear number or satellite cells. This suggests that the capacity to recover after a relative long period of inactivity is still possible. This finding has also an impact on elderly people indicating that activity is of great value keeping the muscle fit during ageing.
Muscle regeneration is commonly held to be accomplished by endogenous myogenic cells but recent evidence has implicated circulating stem cells as a source of muscle precursors for regeneration. We have used single fibres isolated from the Myf5 nlacZ knock-in mouse to measure the amount of myogenic material produced by proliferation of satellite cells derived from a known quantity of skeletal muscle, to show that the satellite cells present in a young muscle are fully able to reconstitute that muscle over a 3-4 day period. This corresponds closely to the in vivo time course for a cycle of muscle regeneration following acute injury and that there is no requirement for input from circulating sources. When we graft muscle derived myogenic cells into a damaged muscle, they affect a much better repair when the graft site has been pre-irradiated. Using a muscle cell line, we have shown that this effect is largely achieved by driving proliferation of the grafted cells and have begun to try to identify the changes in gene expression in the recipient muscle that underlie this effect with the idea of exploiting such knowledge to enhance muscle repair and regeneration.