SLOWING DOWN AGE-RELATED MUSCLE LOSS AND SARCOPENIA
P. NOIREZ 12 AND G. BUTLER-BROWNE 1
3. INTRINSIC FACTORS
74 NOIREZ AND BUTLER-BROWNE
by the discovery that their proliferation is evoked not only by acute muscle injury but also by muscle overuse and increased muscle tension. A number of factors are involved in this regulation of satellite cell activation (Hawke and Garry,2001).
SLOWING DOWN AGE-RELATED MUSCLE LOSS AND SARCOPENIA 75 acetylcholine, by the nerve cell. The neurotransmitter then binds to its receptor located on the muscle cell membrane and induces the formation of an electric current across the membrane. Excitation-contraction coupling is defined as the biological phenomenon that transforms an order arriving in the form of an electrical signal into a mechanical event: contraction of the muscle cell. This phenomenon is made possible by the presence in certain parts of the cell of calcium reservoirs termed sarcoplasmic reticulum (SR). The SR is bound by its own membrane, which is linked to the cell membrane by binding molecules (one located on the cell membrane, the dihydropyridine receptor (DHPR), and the other on the reservoir membrane, the Ryanodine receptors (RyR). These two binding molecules constitute channels through which the calcium passes and whose opening is controlled by the electric current. Thus, SR discharges its calcium inside the muscle cell when the channels open under the effect of the current (Ryan and Ohlendieck,2004).
In humans, the speed of contraction and the force developed by the muscles both deteriorate with age. Similar results have been obtained in mice. This loss of force could be explained by excitation-contraction decoupling. In effect, it has been shown that the number of calcium – channels diminishes with age (Delbono,2003).
It was therefore assumed that if for the same electric current fewer channels opened, this should limit the amount of calcium entering the cell and thus lead to a weaker contraction. However, experiments carried out on isolated human muscle cells moderate this theory. The experimental results obtained in vitro on muscle fibres from different subjects in which the reservoirs had been rendered inactive show a drop in developed force in the fibres of elderly subjects compared to that of young subjects (Frontera et al.,2000). Moreover DHPR expression seem to be preserved during the aging process of human skeletal muscle fibres (Ryan et al.,2003). This would indicate that excitalion-contraction decoupling is not the limiting factor in the loss of developed force with age. The number and the force of the actin-myosin crossbridges appear to be the preponderant factors.
However, the question of alterations in the neural control of the expression of muscle genes such as myosin or actin, which also depends on the quantity of calcium discharged by the reservoirs, remains unanswered.
In conclusion, excitation-contraction decoupling due to the reduction in the number of calcium channels in the calcium reservoir membranes is not the direct cause of the loss of muscular force observed in elderly people. Nevertheless, it cannot be excluded that this reduction may modify the expression of the genes encoding myosin, for example, which would lead to a modification in the actin- myosin cross-bridges. The cause-and- effect relationship should be explored in more detail in the forth coming years. It has however been shown that the myosin molecule is susceptible to post-translational modification such as glycation.
In addition it has been shown that glutathione can reverse these modifications (Ramamurthy et al.,2003). It could therefore be imagined that physical activity can maintain the number of functional receptors and thus maintain sufficient expression of the muscle genes, thereby making it possible to maintain a high level of force production.
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3.2 Mitochondria, oxidative stress and aging
Mitochondria are cell structures that produce energy that is vital to the cells;
moreover, they participate in the cascade of cell signalling events. The number of mitochondria varies according to muscle fibre type. Type I fibres have the greatest number, followed by Type lla, and finally Type llx fibres. In addition to this heterogeneous number of mitochondria in muscle cells, it is interesting to note that regular physical activity increases the number of mitochondria in the cells. As previously discussed, the main effects of age on skeletal muscle are sarcopenia and cell death. These two events could be linked to dysfunction of the mitochondria.
In effect, these structures responsible for cell respiration can, in certain cases, form reactive oxygen species (ROS) that are toxic for the cells. ROS production increases drastically during aging (Fulle et al.,2004). Free radicals cause severe damage if they are not promptly eliminated by the action of anti-oxidant agents. However, some of these toxic molecules may escape and bind to the mitochondrial DNA causing punctual mutations of the DNA molecule. These mutations could trigger a cascade of events leading to cell death by apoptosis: formation of chemically unstable molecules, induction of mutations on the DNA, formation of mutated enzymes, alteration of the respiratory activity of the mitochondria, which triggers either the accumulation of other unstable molecules (and thus other mutations) or cell death by apoptosis (Kujoth et al.,2005). This is a lengthy process.
Although this is an interesting theory, it is nevertheless controversial. Many questions still remain unanswered. It is undeniable that cells accumulate mutations with age, but not all these mutations induce modifications in mitochondrial activity.
Moreover, the induced modifications are not always bad for the cells. We can add to this argument by saying that the mutations that trigger cell death disappear and that the muscle cells reformed by satellite cells no longer present these mutations.
This leads us to discuss the advantages of regular exercise in respect of changes in mitochondria in the skeletal muscles of elderly people. The first experiments carried out on patients suffering from mitochondrial myopathies type pathologies are encouraging (Chabi et al.,2005). It is already well know that physical activity improves endurance capacities in healthy subjects, but the same also appears to be true for myopathic patients. The working hypothesis currently put forward by researchers is that, by allowing satellite cells to renew the mitochondria or to strengthen the existing muscle fibres, exercise diminishes the chances of mitochon- drial DNA mutations to accumulate.
3.3 Satellite cells and Telomeres
When a muscular lesion occurs, the satellite cells are rapidly activated, proliferate and then fuse either with the damaged fibres in order to repair them, or among themselves in order to form new fibres. One part of the activated satellite cells does not differentiate and renews the stock of quiescent satellite cells. The satellite cells are involved in maintaining the fibre size/muscle nuclei ratio.
SLOWING DOWN AGE-RELATED MUSCLE LOSS AND SARCOPENIA 77 The reduction in the number of satellite cells with age could therefore be one of the factors that could explain the loss of muscle mass linked to aging and alterations in the regenerative capacity. Modification, with age, in the capacity of satellite cells to proliferate or fuse could be another factor limiting the action of repairing these cells and of maintaining muscle mass during the aging process.
How the pool of satellite cells evolves during normal aging in human skeletal muscle is still controversial. Using EM, human satellite cells represent 15% of all the myonuclei at birth, 6–10% at two years of age, and 4% in the adult (Tome and Fardeau,1986;Schmalbruch and Hellhammer,1976). For older subjects, this value varies between 0.6 and 3.4% in different studies (Thornell et al.,2003).
In our own studies, we have observed values around 5% for the young biceps brachii and masseter, a value which is in close agreement with previous studies which were carried out on the trapezius muscle of young female subjects (Kadi and Thornell, 2000). The proportion of satellite cells we found in corresponding muscles in aged persons (mean age: 74±425 years) were relatively low; 1.44%
in the biceps brachii and 1,77% in the masseter (Renault et al.,2002). We have also examined in the same way the number of satellite cells in the vastus lateralis of four subjects with a mean age of 88 years. Values obtained were 1.49%, 1.33%, 1.07% and 1.67% giving a mean value of 1.39% (unpublished data). This suggests that there is a significant decrease in the satellite cell number between young and old adults for three different muscles. Further analysis is needed to find out if there is a progressive decrease in satellite cells number during adulthood or whether at some critical time there is a sudden decrease due to altered trophic enviroment in the aged muscle. To obtain this knowledge it will be necessary to carry out a transversal analysis.
It has previously been described in birds and rodents that the satellite cell popula- tions isolated in vitro from fast or slow muscle fibres expressed myosin heavy chain isoforms that reflected the phenotype of the muscle from which they were isolated (Dusterhoft and Pette,1993;Feldman and Stockdale,1991;Rosenblatt et al.,1996).
In our laboratory, we have shown both by clonal (Edom et al.,1994) and by single fibre (Bonavaud et al.,2001) analyses that all of the myogenic satellite cells when differentiated in culture co-express both fast and slow myosin heavy chains. This suggests that human satellite cells are not lineage restricted, and that the regulation of the program they can express is open and will depend on external factors such as innervation (Edom et al.,1994). One should keep in mind that although human muscle contains in general mixed fibres, the ratio of which is specific for each muscle, there are no specific fast and slow satellite cell lineages in human skeletal muscle. Since human satellite cells upon differentiation are not oriented towards a precise fibre type programme this will allow them to participate in the growth and repair of any fibre in their vicinity regardless of its programme of differentiation (Mouly et al.,2005).
In order to provide sufficient nuclei to repair damaged muscle fibres following activation the satellite cells undergo successive cycles of cell division; Proliferation is therefore one of the key steps involved in muscle regeneration. However it has
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been well established that human diploid cells are limited in their proliferation capacity. During their life span human cells will gradually replicate more slowly until they reach a non replicative state called replicative senescence. We have studied the number of divisions that human satellite cells can make when they are isolated from donors of different ages. Previous studies on skin fibroblasts have shown that there is a gradual decline in proliferative capacity with increasing donor age. When we carried out a similar study on human satellite cells isolated from donors of increasing age, we did not observe a regular loss of proliferative capacity with donor age. Instead, we have found that there was a rapid loss of proliferative capacity during the first two decades of life (from about 55–60 divisions at birth to about 20 divisions at 20 years of age. Satellite cells isolated from adult muscle independent of age were always able to make between 15–20 divisions (Decary et al.,1997;Renault et al.,2000). The fact that the proliferative potential does not change in adult skeletal muscle would suggest that during normal healthy aging the ability to regenerate skeletal muscle is maintained throughout life even into old age. We can however predict that the situation will be different if proliferation of the satellite cells were to be highly solicited as has been observed in muscular dystrophies (Decary et al.,2000).
One mechanism, which has been suggested to control this limited proliferation, or mitotic clock, is the shortening of the telomeric sequences. Telomeres are specialized DNA fragments located at the end of all eucaryotic chromosomes. In mammals, they consist of short repeated non coding DNA sequences, (TTAGGG)n, which in human are 5–20 kb in length (Harley et al.,1990). During DNA replication, DNA polymerase is unable to copy the 3 ’92 terminal segment of each DNA strand. This results in chromosome shortening at each round of cell division (Olovnikov,1973).
In somatic cells, telomere length decreases regularly with cell division. In vivo, a decrease in the length of telomeric DNA with aging has been demonstrated in many human mitotic tissues (Klapper et al.,2001). In a series of studies carried out on three different human muscles, quadriceps (Decary et al.,1997), masseter and biceps (Renault et al.,2002) we found that there is only a very small decrease in the length of the telomeric DNA in skeletal muscle with increasing donor age. However a dramatic decrease in telomeric DNA length was observed in the muscles of children with muscular dystrophy (Decary et al.,2000). Our results would confirm previous observations that skeletal muscle is a very stable tissue and that during the lifetime there is a low turnover of the myonuclei. The results that we have obtained so far seem to point to the fact that number and quality of satellite cells and hence regenerative capacity are not a limiting factor during healthy aging. Limitations would only arise if these factors were to be oversolicited during the lifetime of an individual by sore chronic disease or if the quality of the satellite cell would become modified by a decrease in trophic factors which accompanies aging (Mouly et al.,2005).
SLOWING DOWN AGE-RELATED MUSCLE LOSS AND SARCOPENIA 79 Consequently, alternative hypothesis have been proposed based on a defect in the activation of the satellite cells due to changes in their environment caused by age-related changes in the body, such as modification of the hormone status, reduction in certain local factors, or changes in neuromuscular activity.
4. EXTRINSIC FACTORS