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This short review discusses changes in the fibre type distribution, myosin heavy chain isoform composition and histological appearance of the very elderly human skeletal muscle. Point of origin of the discussion comes from data that we have obtained from muscle biopsies from the vastus lateralis muscle of a group of frail very elderly subjects (age: 88 6 3 years, range 8597). Myosin heavy chain composition of muscle homogenates and single fibres, fibre type distribution, fibre size and capillary density were examined and compared with muscle biopsies from the young vastus lateralis muscle. Histological preparations of the muscle biopsies from our elderly subjects showed extended grouping (Nygaard & Sanchez, Anat Rec 1992: 202: 451459) of the fibre types as well as significant changes in the appearance and size of the individual muscle fibres.
On average, the fibre type composition of our very elderly subjects do not seem to be different to what is observed in a corresponding young group when examined with ATPase histochemistry. Likewise, the MHC composition of the muscle homogenates is comparable to what is observed in young subjects. Nevertheless, a detailed examination of the MHC composition of single fibres from the old subjects revealed that the most prominent phenotype was fibres coexpressing MHC I and MHC IIA. This is very different from what is observed in the young muscle. Detailed investigation of longitudinally cut fibres indicated that some fibres in the very old muscle, in contrast to the young muscle, switch fibre type along the length of the fibre or contain areas or nuclear domains in which the MHC expression is different from the remaining part of the fibre.
Discussion The proportions of the human skeletal muscle change, as we get older. Pronounced muscle atrophy, muscle weakening and an apparent slowing are inevitable followers of increasing age. These changes have significant implications on the individual and his or hers ability to overcome everyday tasks (Porter, Vandervoort, Lexell, 1995). The present review aim at giving an overview of the morphological and biochemical fibre type related modifications that occurs with increased ageing as they were manifested in a small population of very elderly subjects that we have investigated. We analysed needle biopsies obtained from the vastus lateralis muscle of a group (n 12) of frail very elderly men and women (age: 88 + 3 years, range 8597) (Andersen, Terzis, Kryger, 1999). The muscle biopsies were very thoroughly examined in order to establish fibre type distribution, fibre size and myosin heavy chain (MHC) composition in muscle homogenates and single fibres, as well as density of capillaries. Furthermore, the biopsies were, in a qualitative manner, examined for morphological appearance and abnormalities as they appeared in comparison with the young vastus lateralis muscle.
Muscle morphology
In a number of studies examining either whole muscle preparations or muscle biopsies of elderly subjects an apparent grouping of the muscle fibre types have been observed (Nygaard & Sanchez, 1982; Lexell, Downham, Sjostrom, 1986; Lexell & Downham, 1991). Grouping describes the phenomenon that certain fibre types seem to be distributed in clusters rather than in the random fashion commonly observed in younger muscle. The grouping phenomenon was quite pronounced in our group of frail elderly (fig. 1). Almost all individuals showed some sign of fibre type grouping, although the range was considerable, ranging from biopsies showing a normal random appearance of the fibre types, to biopsies with very pronounced clustering of the fibre types (fig. 1). The mechanisms for the obvious grouping of the fibre types in the ageing human muscle still need to be fully understood. Presently, the most evident explanation seems to be that the fibre type grouping arises from a continuous process of denervation and partial reinnervation that is claimed to accelerate with advancing age (for review see Lexell, 1995). Apart from the grouping phenomenon other morphological features separate the old from the young muscle.
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Fibre size
An obvious consequence of ageing is a marked atrophy of the skeletal muscles. This atrophy is also evident in the individual muscle fibres (Larsson, Sjodin, Karlsson, 1978; Larsson, Grimby, Karlsson, 1979; Scelsi, Marchetti, Poggi, 1980; Larsson, 1982; Lexell, Taylor, Sjostrom, 1988; Klitgaard, Mantoni, Schiaffino et al., 1990a; Lexell, 1995; Borges & Essen-Gustavsson, 1989; Essen-Gustavsson & Borges, 1986). Nevertheless, the age-related fibre atrophy is very inhomogeneous when looking at the fibre pool as a whole. In muscle biopsies from very elderly subjects it
Young muscle
Old muscle
Fig. 1. Grouping of muscle fibre types in the ageing muscle. ATPase staining of a young muscle (age: 22 years) and old (87 years) muscle. Note the random distribution of the fibre types in the young muscle vs. the pronounced grouping of the fibre types in the old muscle. Dark fibres, type I fibres; White fibres, type IIA fibres; Grey fibres, type IIX (or I/IIA) fibres; Bar 100 mm. ATPase staining (pH 4.6).
Young muscle
Old muscle
Fig. 2. Fibre shapes are often different in young and very old human skeletal muscle. Muscle fibres in the young muscle most often appear angular with four to six angels or corners, whereas many fibres in the elderly muscle appear as if they have been flattened or crushed. This flattening of the fibres is much more pronounced among the type II fibres than the type I fibres. Dark fibres, type I fibres; White fibres, type IIA fibres; Grey fibres, type IIX (or I/IIA) fibres; Bar 50 mm. ATPase staining (pH 4.6).
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can often be observed that in some areas the fibres have almost normal size, and right next to these are areas in which the fibres are small, or even extremely small (fig. 3). Furthermore, the atrophy also affects the different fibre types differently. Numerous studies have demonstrated that ageing-related fibre atrophy is less hard on type I fibres than on type II fibres (for review see Porter et al., 1995). When we compared our 88 year old subjects with a matching group of 25 year old subjects, we found that the type I fibres were reduced in size, but only to 75% of the young type I fibres (2891 + 853 mm2 vs. 3822 + 755 mm2). In contrast, the type II fibres were reduced to 43% of the young fibre size (1704 + 936 mm2 vs. 3974 + 873 mm2) (fig. 4). taken into account the difference between the young and the old muscle is erased. Thus, the old muscle will contain equal amounts of capillaries per square millimetre (Coggan, Spina, King et al., 1992a, b). Using the same protocol (Qu, Andersen, Zhou, 1997) we analyzed capillary density of both a young group of subjects (Qu et al., 1997) and of our group of very elderly subjects. When calculated as capillaries/fibre we found that the young subjects had more than twice the number of capillaries per fibre compared to the elderly group, but since the younger group as mentioned earlier have larger fibres this may not be the correct way to compare the two groups. Thus, we also looked at the number of capillaries per square millimetre in the two groups. If calculated in this manner it seems that the very old subjects per area have the same, or even more, capillaries than the young group (536 vs. 474 capillaries/mm2) (Qu et al., 1997; Andersen & Kryger, unpublished).
Capillary density
Most studies have indicated no severe age-related influence on skeletal muscle capillary density (Cartee, 1994). If measured as capillaries/fibre the ageing muscle will show a decrease in capillary density (Frontera, Meredith, O'Reilly, Evans, 1990; Cartee, 1994), but if the reduced fibre size of the elderly muscle fibres are
Fibre types
An often-debated issue is whether or not fibre type composition of the skeletal muscle change with ageing.
Young muscle
Old muscle
Fig. 3. Muscle fibre sections from a normal young muscle containing fibres of normal size and a section from a very old subject (> 85 years) in which areas with extremely atrophied fibres occurred. Bar 50 mm. ATPase staining (pH 4.6).
Type I fibre size 6000 5000 4000 m2 3000 2000 1000 0 0 20 40 60 Age (years) 80 100 6000 5000 4000 m2 3000 2000 1000 0 0 20
40 60 Age (years)
80
100
Fig. 4. Size of type I and type II fibres in a group of young (avg. age 25 years) and old subjects (avg. age 88 years). Notice the apparent lager difference between the young and the old group in size of the type II fibres in comparison to the type I fibres (Andersen & Kryger, unpublished).
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(a)
40 35 30 25 Percent 20 15 10 5 0 MHC I MHC I/IIA MHC IIA MHC IIA/IIX MHC IIX MHC I/IIX MHC I/IIA/IIX
(b)
80 70 60 Percent
(c)
80 70 60 Percent 50 40 30 20 10 0
MHC I/IIA (dominant I) MHC I/IIA MHC I/IIA (no dominance) (dominant IIA)
50 40 30 20 10 0
MHC IIA/IIX MHC IIA/IIX MHC IIA/IIX (dominant IIA) (no dominance) (dominant IIX)
Fig. 5. (a) MHC composition (%) of single muscle fibres from the vastus lateralis muscle of a group of very old subjects (avg. age 88 years) (Andersen et al., 1999); (b) Single fibres co-expressing MHC I and MHC IIA, subdivided into groups according to the dominant MHC isoform; (c) Single fibres co-expressing MHC IIA and MHC IIX, subdivided into groups according to the dominant MHC isoform.
histochemical terminology) than expected from the single fiber data. This apparent divergence in results, generated from the different methods of analysis, provided a problem. One obvious difference between the two methods is that with ATPase histochemistry only very narrow part of the fibre (10 mm thick) is used in the analysis, whereas the single fibres used for the gelelectrophoretic determination of the MHC isoforms are often several millimetre long, thus the amount of muscle fibre examined is often several hundred fold longer. Traditionally, mixed or hybrid fibres are often looked upon as having a fairly even distribution of the two MHC isoforms expressed throughout the entire length of the fibre, which can to some extent be verified by the fact that a type I/IIA (IIC) fibres can be followed in serial sections of good quality biopsies (Andersen, unpublished observations). Animal experiments using chronic electrical stimulation, have shown that at least under certain conditions the MHC isoform distribution might not be so uniform along the length of the fibre (Staron & Pette, 1987). Skeletal muscle fibres are multinucleated and a large number of nuclei are distributed along the length of the individual fibres. At least for the contractile proteins, the individual nuclei have some sort of autonomy of the surrounding area, so that transcribed mRNA is translated into protein and incorporated locally in the domain. However, under normal conditions and certainly in the young muscle there seems to be a strict coordination of the expression of mRNA for the various MHC isoforms, so that all the nuclei behave alike. A condition for this type of organization is the before
mentioned strict coordination of the individual nuclei within the fibre. Consequently, the individual nucleus within the fibre transcribes the same mRNA of the MHC isoforms or the same two MHC mRNAs in the same ratio. This mechanism will ensure that a type I fibre stays a type I fibre and a type II fibre stays a type II fibre. Furthermore, when a certain stimuli are induced this mechanism ensures that a fibre type shift happens throughout the entire fibre. What happens if for some reason this coordination is lost or is reeling, if the individual nuclei looses the coordination and steps out, either alone or in clusters? If this would happen, nuclear domains or parts of fibres would probably transcribe mRNA and additional locally incorporate a MHC isoform that could be different from the rest of the fibre or different from areas close to. It is not difficult to imagine that at very high age this coordination could be disturbed and some or many nuclei could start misbehaving either for inbuilt intrinsic or other reasons. This scenario could in part explain why we found only a minor part of the fibres to be type I/IIA fibres with ATPase staining, using only a 10-mm crosssection of the fibres representing only one or two nuclei domains. In contrast, the several millimetre long fibre pieces used in the single fibre analysis representing several hundred nuclear domains. Although we have not systematically cut in the length direction of the fibres, cross-sectional cutting often have part of the biopsy cut longitudinally. Thus, we re-evaluated our ATPase stainings in an attempt to find evidence of fibres with different MHC expression along the length of the fibres. Interestingly, we found several examples
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Fig. 6. Longitudinally cut muscle fibre originating from the muscle of a very old subject (> 85 years). The fibre apparently shifts phenotype along the length-axis of the fibre. Below is shown a theoretical sketch of how different transcription of mRNA for the MHC isoforms on each side of the shifting-zone might provide a fibre type shift along the length-axis of the fibre. For further information see text. ATPase staining (pH 4.3).
Fig. 7. Longitudinally cut muscle fibre originating from the muscle of a very old subject (> 85 years). Notice how the staining pattern changes in a small area within the fibre. Below is shown a theoretical sketch of how different transcription of mRNA for the MHC isoforms in a small area corresponding to the domain of a single nucleus could provide a very local area expressing a different MHC isoform than the remaining part of the fibre. For further information see text. ATPase staining (pH 4.3).
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Perspectives Ageing causes muscle fibre loss and fibre atrophy, progressively leading towards a state at which an elderly person will be unable to conduct most every-day tasks. Thus, it is of the outmost importance that studies examining the most effective ways of strengthening the elderly muscle as well as preventing fibre atrophy or even introducing hypertrophy are initiated. I am convinced that the most effective and simple advice to give to the elderly population, in an attempt to counteract agerelated muscle degeneration, is to prescribe regular resistance training. Our finding of extremely high relative amount of fibres co-expressing MHC I and MHC IIA in the very elderly muscle are currently difficult to link to any clinical significance at the whole muscle level. Nevertheless, it will be interesting to pursue our hypothesis of local shift in MHC expression along the length of the individual muscle fibres, in an attempt to establish if this apparently age related phenomenon can be liked to changes in the expression of various factors controlling fibre type specificity. Key words: ageing; capillary density; co-expression; fibre size; MHC isoforms; myosin; nuclear domain. Acknowledgement
The author would like to acknowledge the skilful work on the single fibre project done by Greasimos Terzis. Likewise, Ann Kryger worked hard on obtaining the muscle biopsies from this very unique group of subjects. The Danish National Research Foundation supported this work, grant number 50414.
References
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