Acta Physiol Scand 1990, 140, 55-62
Ageing alters the myosin heavy chain composition of single fibres from human skeletal muscle H . KLITGAARD, M. ZHOU, S. S C H I A F F I N O " , R.BETTO", G. S A L V I A T I " and B. S A L T I N August Krogh Institute, University of Copenhagen, Denmark, and Centro de Studio per la Biologia e la Fisiopatologia Muscolare del CNR, c/o Institute of General Pathology, University of Padova, Italy
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KLITGAARD, H., ZHOU,M., SCHIAFFINO, S., BETTO,R., SALVIATI, G. & SALTIN,B. 1990. Ageing alters the myosin heavy chain composition of single fibres from human skeletal muscle. Acta Ph,yszol Scand 140, 55-62. Received 1 1 August 1989, accepted 27 March 1990. ISSN 0001-6772. August Krogh Institute, University of Copenhagen, Denmark, and Centro di Studio per la Biologia e la Fisiopatologia Muscolare del CNR, c/o Institute of General Pathology, University of Padova, Italy. The myosin heavy chain composition of single fibres (n = 1088) was analysed with an electrophoretic technique in biopsy material from m. vastus lateralis (n = 5) and m. biceps brachii (n = 4) of young (23-31 years old) and elderly men (68-70 years old). In m. vastus lateralis, elderly subjects had a higher proportion of fibres showing a coexistence of myosin heavy chain types I and IIa (20 & 3 yo us 8 f 1 yo,P < 0.05) and of myosin heavy chain types IIa and IIb (33f2Y0 us 12+4%, P < 0.05). In contrast, the young subjects had a higher proportion of fibres containing only myosin heavy chain type I (50+5% us 33fy0,P < 0.05) and type IIa (26+_3y0 us 12+2%, P < 0.05). A similar pattern of myosin heavy chain expression was found in single fibres from m. biceps brachii, with the exception that the elderly subjects had a lower proportion of fibres with coexistence of types IIa and IIb (23f 1 Yo us 34+2y0, P < 0.05) and a higher proportion of fibres containing only myosin heavy chain type IIa (25 _+ 5 % us 12 2Y0, P < 0.05). Three fibres from m. biceps brachii contained all three isoforms. These results indicate that coexistence of myosin heavy chain isoforms in single fibres is present in skeletal muscles of young adults, and that there is an increased occurrence of this phenomenon with ageing. Whether this reflects an ongoing transition process or a 'dynamic equilibrium' between the fibre populations is presently unsettled.
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Key words : ageing, coexistence of myosin isoforms, human skeletal muscle, myofibrillar ATPase histochemistry, myosin heavy chains, single fibres.
(Aniansson et al. 1980, Grimby et al. 1982, Lexell et al. 1983, Essen-Gustavsson & Borges 1986). Electrophysiological studies of ageing human skeletal muscle demonstrate a selective loss of the large and fast-conducting motor neurons which innervate type I1 fibres (Campbell et al. 1973). A preferential loss of type I1 fibres is therefore likely with ageing. T h e increased Correspondence : Henrik Klitgaard, August Krogh incidence of type grouping and enclosed fibres, Institute, Universitetsparken 13, 2100 Copenhagen 0, mainly of type I fibres (Grimby et al. 1982), and Denmark. the larger size of the motor units observed in
A common approach in the study of the ageing human skeletal muscle has been to stain for actomyosin myofibrillar ATPase, based on the p H stability following acid and alkaline preincubations (Brooke & Kaiser 1970). An agerelated decrease in the relative proportion of type I1 fibres was found by Larsson et al. (1978). T h i s was not, however, confirmed in later reports
55
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N. Khtgaard et
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ageing human skeletal muscle (Stalberg 8i Fai\cett 1982) are indications that some of thc dencrvated type I1 fibres become reinnen ated. Thus, it is remarkable that the histochemicall! determined relative occurrence of the u r i o u s fibre types remains unaltered with ageing. One possibility is that the samples obtained \vith the needle h i o p y technique are too small :o he representath e of the muscles' total fibre population. However, in a histochemical study of the fibre type cornposition in cross-sections of a.hole m. vastus latcralis, I~,exell~ ' fill. (1983) were not able to demonstrate any change in the proportion of type I fibres with ageing. T h e histochemical actomyosin m!-ofibrillar ATPase reaction of a muscle fibre is determined b - its myosin heavy chain (hlHC) composition (Danieli-Retto et (11. 1986). .I recent elcctrophoretic study examined the SIHC composition of single tibres from skeletal muscies of young subjects (Biral rt al. 1988). A relatively high number of fibres evinced coexistence of two \IHC isoforms, especiallJ- of type IIa and type 111) lII-IC. Similar studies on single fibres from cndurance-trained muscles revealed a large coesisicncc of ST€IC type I and type IIa (Klitgaard t t lii. 1990a). These data suggest that a d!-namic equilibrium may exist betv-een the main fibre t!j>es, i.c. expression of onc form of l l l I ( : and films containing more than one form of UH(:. 'rhis appcars to be the case for the myosin light chain (l1I.C:) composition of single fibres from sbeleti!l muscles of young and elderly subjects (S:iliiiti t'i I , / . IUX.?). 3 decrease in fibres cc?ntnirlin;; i m l ! f a s t isoforins of \ I I L \%as found i:i cldcrl! \uhjccts concomitant with an
SI.4TERI.4LS A N D METHODS
subjrcls. Using suction, needle biopsies were taken from the middle portion of m. vastus lateralis from a group of young (27 years old, range 23-31, n = 5 ) and elderl!- men (69 )-ears old, range 68-70, n = S), and from m. biceps brachii of a group of young (28 years old, range 26-30, n = 4) and elderly men (69 years old, range h8--70, n = 4). The muscle specimens were dirided into two parts. One part was frozen immediately in liquid nitrogen and used for single-fibre analysis of LIHC composition, the rest being trimmed, mounted and frozen in isopentane cooled with liquid nitrogen for histochemical analyses. None of the subjects suffered from any cardiovascular and/or neuromuscular disease. The study was approved by the Copenhagen Ethical Committee. Single-jibre electrophoresis. Single fibres were prepared and analysed for MHC composition as previously described by Biral et al. (1988). Electrophoresis of the fibres was performed in 6qb SDS-PAGE gels with 37.5O, glycerol (w/v) in the separating gel (Klitgaard et al. 1990 b). This electrophoretic system has demonstrated a clear resolution of MHC types I, IIa and IIb as verified by monoclonal antibodies specific to human MHC types I, IIa and IIb (Fig. 1a). In each gel, a sample known from immunoblotting to contain MHC types I, IIa and IIb was used as an internal standard (Fig. 2). On average, 5&80 fibres were analysed in each biopsy. A total of 344 fibres were analysed from the young vastus lateralis muscles, 288 fihres from the elderly vastus lateralis muscles, 224 fibres from the young biceps brachii muscles and 232 fibres from the elderly biceps brachii muscles. Immiinoblotting. Purified myosin from both muscles studied a a s separated in 60:) SDS-PAGE gels with hieh glycerol content as described above (Klitgaard r f ( i i . 1990 h). Procedures for electrophoretic transfer to nitrocellulose, incubation with monoclonal antibodies (mrtbs) and detection of bound antibody by peroxidase-conjugated second antibody were as previously described (Schiaffino et al. 1989). Monoclonal antibodies specific to human MHC type I (mAh RA-FS), human 3lHC tl-pe IIa (mAb SC-71) and human typing nith myofibrillar JIHC tqpe IIb (mAb BF-34) were used. Immunoblotting with these three mAbs has previously demonstrated that the antibodies react selectively with vastus Iatcralis of young and elderly men. In rat skeletal muscles known to contain predominantly addition, tissue \\as obtained from m. biceps npes I, IIa and IIb fibres as determined by the brachii. T h e reason for the inclusion of arm histochemical reaction for myofibrillar ATPase (Schiafino et u1. 1989). muscle i i w ~ ri + thar, in contrast to the leg Hislochrrnistry. Serial transverse sections (10 pm) intains its fibre size with were cut in a cryotome at - 20 "C. The sections were 1. 1982). This finding has mounted on coverslips and stained for myofibrillar reliitcd to a more forceful .4TPase at pH 0.4 after both alkaline (pII 10.3) and ,I:* cornpiired with the I m e r acid (pH 4.3 and 4.6) preincubations (Brooke & pcople (Cirimby 8i Saltin Kaiser 1970). Fibres were characterized as I, IIa, IIb and intermediate (IM) fibres. The latter fibre type has 1983I
Myosin isoforms in ageing human musclejbres
57
also been referred to as IIc and/or Ib (Schantz & Dhott 1987). On average, 459 (range 322-646) fibres were classified in each sample. Fibre areas were analysed from photographs of the ATPase stain after acid (pH 4.6) preincubation. The areas of a minimum of 50 type I, 30 type IIa and 20 type IIb fibres were measured in each sample with a digitizer. Statistics. The non-parametric Mann-Whitney test was applied for statistical comparisons between the groups. The 0.05 confidence level was chosen for statistical significance.
RESULTS Myosin purified from both muscles analysed was resolved into three distinct bands in 6% SDS-PAGE gels with high glycerol content (Fig. l a , lane i). Using immunoblotting these bands were shown to correspond to M H C types I, IIa and I I b (Fig. l a , lanes ii and iv). Single fibres containing only M H C type I, type IIa or type I I b were identified and are depicted in Fig. 1(b), lanes (ii-iv) (see also Fig. 2). Coexpression of two MHC isoforms in the same fibre was also observed (Fig. lc, lanes ii-iv) (see also Fig. 2). The electrophoretic analysis of single fibres from m. vastus lateralis demonstrated that out of 288 fibres more than half of the fibres showed coexistence in the elderly subjects (Fig. 4). Comparing the young and the elderly subjects, M H C type I and type IIa were observed in 8f 1 % vs 2 0 + 3 % (P< 0.05) of the fibres and M H C type IIa and type I I b in 12f4y0 as 3 3 + 2 % (P< 0.05) of the fibres. In contrast, the young subjects manifested more fibres containing only M H C type I (50*5% vs 33 f3 yo,P < 0.05) and M H C type IIa (26 f3 yo vs 12 f2 %, P < 0.05). The histochemical fibre typing of the vastus lateralis muscle from young and elderly subjects revealed a relative distribution of 64f4Yo vs 48+3y0 in type I (P< 0.05), 29 & 3 yous 37 f4% in type IIa and 7 2 3 % vs 15f2y0 in type I I b fibres. These values are within the range that has been reported from biopsy material in other studies (Aniansson et al. 1980, Essin-Gustavsson & Borges 1986). Hardly any I M fibres were found in the muscles studied. The electrophoretic analysis demonstrated a similar pattern of M H C expression in muscle fibres from m. biceps brachii and m. vastus lateralis, with the exception that elderly subjects had a lower proportion of fibres with coexistence of IIa and I I b (23+ 1% vs 34+2y0, P < 0.05)
Fig. 1. Identification of human myosin heavy chain isoforms by SDS-PAGE in 6% gels with high glycerol content. Only the MHC region is shown. (a) Identification of MHC types I, IIa and IIb from myosin purified from human m. vastus lateralis by immunoblotting analysis. Myosin was separated by SDS-PAGE in 6% gels and either stained with Coomassie blue (i) or transferred to nitrocellulose filters and reacted with mAb BA-F8, specific to human MHC type I (ii), mAb SC-71 specific to human MHC type IIa (iii) or mAb BF-34 specific to human MHC type IIb (iv). (b) Lane (i), purified myosin from human m. vastus lateralis after SDSPAGE in 6% gels and silver staining; (c) Lane (i), purified myosin from human m. vastus lateralis after SDS-PAGE in 6% gels and silver staining; lanes (ii-iv), single fibres from m. vastus lateralis of elderly subjects demonstrating coexistence of MHC type IIa and type IIb (ii) and coexistence of MHC type I and type IIa (iii) and (iv), after SDS-PAGE in 6Sb gels and silver staining.
and a higher proportion of fibres containing only M H C type IIa (25 f5 yo vs 12 k 2Y0,P < 0.05). Fibre typing from myofibrillar ATPase histochemistry in m. biceps brachii of young and elderly subjects revealed a similar distribution for type I (48+2y0 as 44+3%), type IIa (25+6y0 us 27f5%) and type I I b fibres (27+6% vs 29f5y0). No fibres exhibiting coexistence of M H C type I and type I I b were found in any of the muscles analysed. However in m. biceps brachii one of the fibres from the young group and two of the fibres from the elderly group contained all three isoforms of M H C (Fig. 3). These fibres were characterized by trace amounts of M H C types I
5$
H . Klitgaard et al.
Fig. 2. 6”,, SDS-P.4GE gels with high glycerol content. ‘ AfHC’ indicates the M H C region of the gel. T h e lane next to ‘XIHC’ is an internal standard known to contain MHC types I, IIa and I I b from immunoblotting. The other lanes contain single fihres. In gel (A), one fibre showed coexistence of 11HC types I and IIa (u), and in (B) two fibres showed coexistence of MHC types IIa and IIb (ss). The other fibres in both gels contained either M H C type I or IIa.
M ,.HC
iLsl .-MHC .--MHC
I1 b IIa
I
vastus lateralis, and a reduction in fibre size occurs with increasing age. However, this hypotrophy is less pronounced in the upper than in the lower extremities (Table 1). DISCUSSION
Fig. 3. Demonstration of three m!osin hear!- chain isoforms in a single fibre from human m. biceps brachii. Lane (i), a single fibre from m. biceps brachii of a !oung subject containing both M H C types IIa and IIh, lane (ii), a single fibre containing M H C types 1 , IIa and IIb, after SDS-PAGE in 6O,, gels and Yil\ er staining.
and IIb, with the dominant fraction being M H C typc I I a i n analysis of the sizes of the major fibre types re\caled that the fibres in m. biceps brachii mere larger than the corresponding fibres in m.
The results of the two methods used in the present study to identify fibres give the impression that major differences exist between the ti+o muscle and age groups studied (Figs. 5 and 6). This may not be the case, taking into account that in most fibres showing coexistence of M H C isoforms one of them usually dominates. Since the dominating fraction of M H C ‘sets’ the histochemically determined fibre type, the two methods seem to agree. Thus, the 33 y o of fibres H ithin the elderly m. vastus lateralis containing only M H C type I would be histochemical type I fibres (Fig. 5 ) . Within the 2Oyo of fibres showing
Myosin isoforms in ageing human muscleJibres
59
Table 1. Fibre areas of m. vastus lateralis and m. biceps brachii in young and elderly subjects
M. vastus lateralis (,urnz) Young Old
Type 1
Type IIa
Type IIb
5.053 k438 4.526+395
5.460f479, 3.957k364
4.870+479, 3.362+341
5.519f533 4.915_+624
7.286f241 6.329+588
6.21 1 294 5.414+621
M. biceps brachii (,am2) Young 01d
The values are meansfSEM * Statistically significant (P < 0.05) difference between the groups.
coexistence of M H C types I and Ha, 11yo of the fibres had a clear dominance of M H C type 1. These fibres would also react as histochemical type I fibres, giving a total of 44%. This is similar to the 48% of type I fibres found with LO myofibrillar ATPase histochemistry (Fig. 5 ) . However, 9% of the latter had a majority of 30 M H C type IIa (Fig. 1c, lane iii), whereas nearly s none of these fibres were observed within the ; 20 0 young group. This suggests an increase in the .c proportion of histochemical type IIa fibres which 2 10 .contain M H C type I within the elderly m. vastus * lateralis. In contrast to the young group, the .TI major part of the histochemically determined a, a types IIa and I I b fibres within the elderly m. x 601 M.BICEPS BRACHII c vastus lateralis contained M H C types IIa and I I b in different relative amounts, as seen in Fig. 5 from the large group (33%) of fibres showing coexistence of M H C types IIa and IIb. These results may then indicate that most of the 30 histochemical type I I b fibres in the young m. vastus lateralis contain only M H C type IIb, 20 whereas nearly all histochemical type IIb fibres in the elderly m. vastus lateralis contain both 10 M H C types IIa and IIb. In m. biceps brachii of the elderly subjects, MHC I M H C l l l l a MHCIla MHC llalllb M H C Ilb the lower proportion of fibres containing only Fig. 4. Comparison of the fibre type composition in M H C type I (36f 2% uus 26f 5%, P < 0.05) m. vastus lateralis of a group of young (0) (27 years and the higher proportion of fibres showing old, range 23-31, n = 5) and elderly (m) men (69 coexistence of M H C types I and IIa (6 & 2 yo us years old, range 68-70, n = 5) and in m. biceps 2 0 k 2%, P < 0.05) (Fig. 4) are indicative of an brachii of a group of young (28 years old, range 2&30, increase in the relative proportion of histon = 4) and elderly men (69 years old, range 68-70, chemical type I fibres which contain minor n = 4). Values are means k SEM. Ir Indicates statistical difference (P < 0.05) between the groups. amounts of MHC type IIa within the ageing Single fibres were classified according to their content human m. biceps brachii (Fig. 6). Furthermore, of myosin heavy chain isoforms after SDS-PAGE in a lower proportion of fibres containing only 6% gels and silver staining. Between 224 and 344 M H C type I I b (12i=1y0 us 6f2y0, P < 0.05) fibres were analysed in each group. and a lower proportion with a coexistence of 60
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Fig. 5. Comparison of the fibre type composition, determined bx single-fibre analysis of MHC composition (XIHC) and myofibrillar ATPase histochemistr! (histochemistrl-), in m. vastus lateralk of young (27 years old, range 23-31, n = 5 ) and elderllmen (69 years old, range 68-70, n = 5 ) . On average, 434 (range 240-646) fibres were t\-ped in each sample from m!-ofibrillar ATPase histochemistry. A total of 344 fibres was analysed for myosin heavy composition in the !oung group and 288 fibres in the elderll-.
M H C types IIa and I I b ( 3 4 i 2 w 23F l o o , P < 0 05) together with a higher proportion of fibres containing only M H C type IIa ( 1 2 * 2 O , z's 23 k soO, P < 0.05) were found uithin the elderly group (Fig. 4). This indicates that a relativel! higher proportion of the histochemical type IIb fibres and a relatively lower proportion of the histochemical type IIa fibres contain both MHC types IIa and I I b in the ageing human m. biceps brachii (Fig. 6). In addition, of the 20 F 2 O o of fibres showing coexistence of M H C types I and IIa in the elderly subjects, 504 contained major amounts of M H C type IIa and only minor amounts of M H C type I. Few of
Fig. 6. Comparison of the fibre type composition, determined by single-fibre analysis of MHC composition (MHC) and myofibrillar ATPase histochemistrl- (histochemistry), in m. biceps brachii of young (28 !-ears old, range 26-30, n = 4) and elderly men (69 years old, range 68-70, n = 4). On average, 416 (range 322-640) fibres were typed in each sample from myofibrillar ATPase histochemistry. A total of 224 fibres was analysed for myosin heavy chain composition in the young group and 232 fibres in the elderll- group.
these fibres were found within the young m. biceps brachii. As this type of fibre was also found in the ageing m. vastus lateralis, this seems to indicate a special population of histochemical type IIa fibres containing major amounts of M H C type IIa and only small amounts of M H C type I. Recent studies of rat skeletal muscles (DanieliBetto et a/. 1986) and human m. vastus lateralis (Biral et af. 1988, Klitgaard et al. 1990a) have demonstrated a specific pattern in the expression of M H C isoforms within single fibres. Thus, a coexistence was observed for M H C types I and IIa and M H C types IIa and IIb, but not for M H C types I and IIb. This agrees with the
~
Myosin isoforms in ageing human muscle j h e s hypothesis that the expression of M H C isoforms within a fibre follows the sequence: I + I I a + I I b (Biral et al. 1988). The present study is the first to report the coexistence of three M H C isoforms within the same fibre. The trace amounts of M H C types I and I I b and the major amount of M H C type IIa within these fibres indicate that they have a high expression of M H C type IIa, with either a down-regulation of M H C type I expression accompanied by an up-regulation of M H C type I I b expression or vice versa. It appears that these fibres are close to manifesting coexistence of only M H C types I and IIa or M H C types IIa and IIb. The human m. biceps brachii contains a relatively larger proportion of histochemical type I1 fibres, as determined from myofibrillar ATPase histochemistry, than m. vastus lateralis (for ref. see Saltin & Gollnick 1983). This study demonstrates that the histochemical type I1 fibre population of the two muscles differs not only in its relative occurrence but also in its M H C composition. Thus, only 30% of the histochemical type I1 fibres of m. vastus lateralis manifested a coexistence of M H C types IIa and IIb, whereas 60% of the histochemical type I1 fibres in m. biceps brachii demonstrated a coexistence of these two M H C isoforms. Using myofibrillar ATPase histochemistry, there are several reports of an unchanged fibre type composition of the human skeletal muscle with ageing (Aniansson et al. 1980, Grimby et al. 1982, Lexell et al. 1983, Esstn-Gustavsson & Borges 1986). However, a selective atrophy of the type I1 fibres has been reported in m. vastus lateralis but not in m. biceps brachii (Grimby et al. 1982). It has been proposed that the morphology of m. biceps brachii is better maintained owing to differences in the physical activity pattern ;faster and more forceful contractions are thought to be performed in the upper than in the lower extremities. In the light of this explanation, it is noteworthy that the alteration in M H C composition of single fibres from m. biceps brachii is similar to that observed in m. vastus lateralis. Thus an increased coexistence of M H C as well as M L C isoforms may be a general phenomenon of ageing human skeletal muscle fibres. Electrical stimulation experiments on skeletal muscles from animals indicate that coexistence of myosin isoforms within a fibre is related to a transformation process (Pette & Vrbova 1985).
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Several recent reports have demonstrated an increased proportion of fibres showing coexistence of M H C isoforms in the human skeletal muscle after endurance training (Schantz & Dhott 1987, Klitgaard et al. 1990a). The crosssectional design of the present study makes it impossible to be conclusive, but the higher proportion of fibres showing coexistence of M H C types I and IIa within both muscles of the elderly subjects strongly suggests that a changed activity pattern with ageing might induce a transition process within the fibres of the ageing human skeletal muscle. However, this phenomenon may also be caused by other factors. A selective denervation of large fibres (Grimby & Saltin 1983) containing only M H C type IIa or M H C type IIb, and a reinnervation of these by smaller motor neurons, could explain the increased proportion of fibres possessing a coexistence of M H C types I and IIa in both muscles studied and of M H C types IIa and I I b in m. vastus lateralis. Another possibility is agerelated changes in the neuromuscular junction (Gutmann & Hanzlikova 1972), which could alter the traffic of neurotrophic factors and/or impulses reaching the fibres, thereby changing their M H C expression. These changes could be related to a disuse phenomenon since regular usage seems to maintain morphology, fibre size and M H C expression of the ageing human skeletal muscle (Klitgaard et al. 1990b). In conclusion, analysis of M H C composition in single fibres from human skeletal muscles of young and elderly subjects demonstrated a higher proportion of fibres possessing a coexistence of M H C types I and IIa in both muscles studied and also a higher proportion of fibres with coexistence of M H C types IIa and I I b in m. vastus lateralis of the elderly subjects. This indicates an increased coexistence of M H C isoforms in histochemically determined fibre types with ageing. The explanation for this phenomenon is at present unknown. This study was supported by grants from the Danish Research Academy, the Danish Research Council for the Natural Sciences, the Danish Sports Research Council, the Obel Family Foundation, the Knud H~jgaardFoundation, the John Meyer Foundation, the former Director Leo Nielsen and wife Karen Margrethe Nielsen grant for the medical sciences and, in part, by Institutional Funds from the Italian National Research Council and grants from MPI and MDA. We are grateful to Mrs S. Ceoldo, Mrs Lise
62
H. Klitgaard et al.
Kiis-Jacobsen and Mrs Marianne Hemmingsen for their skillful technical assistance and to Mrs Lis H. Christensen for tj-ping of the manuscript.
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Ageing alters the myosin heavy chain composition of single fibres from human skeletal muscle H . KLITGAARD, M. ZHOU, S. S C H I A F F I N O " , R.BETTO", G. S A L V I A T I " and B. S A L T I N August Krogh Institute, University of Copenhagen, Denmark, and Centro de Studio per la Biologia e la Fisiopatologia Muscolare del CNR, c/o Institute of General Pathology, University of Padova, Italy
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KLITGAARD, H., ZHOU,M., SCHIAFFINO, S., BETTO,R., SALVIATI, G. & SALTIN,B. 1990. Ageing alters the myosin heavy chain composition of single fibres from human skeletal muscle. Acta Ph,yszol Scand 140, 55-62. Received 1 1 August 1989, accepted 27 March 1990. ISSN 0001-6772. August Krogh Institute, University of Copenhagen, Denmark, and Centro di Studio per la Biologia e la Fisiopatologia Muscolare del CNR, c/o Institute of General Pathology, University of Padova, Italy. The myosin heavy chain composition of single fibres (n = 1088) was analysed with an electrophoretic technique in biopsy material from m. vastus lateralis (n = 5) and m. biceps brachii (n = 4) of young (23-31 years old) and elderly men (68-70 years old). In m. vastus lateralis, elderly subjects had a higher proportion of fibres showing a coexistence of myosin heavy chain types I and IIa (20 & 3 yo us 8 f 1 yo,P < 0.05) and of myosin heavy chain types IIa and IIb (33f2Y0 us 12+4%, P < 0.05). In contrast, the young subjects had a higher proportion of fibres containing only myosin heavy chain type I (50+5% us 33fy0,P < 0.05) and type IIa (26+_3y0 us 12+2%, P < 0.05). A similar pattern of myosin heavy chain expression was found in single fibres from m. biceps brachii, with the exception that the elderly subjects had a lower proportion of fibres with coexistence of types IIa and IIb (23f 1 Yo us 34+2y0, P < 0.05) and a higher proportion of fibres containing only myosin heavy chain type IIa (25 _+ 5 % us 12 2Y0, P < 0.05). Three fibres from m. biceps brachii contained all three isoforms. These results indicate that coexistence of myosin heavy chain isoforms in single fibres is present in skeletal muscles of young adults, and that there is an increased occurrence of this phenomenon with ageing. Whether this reflects an ongoing transition process or a 'dynamic equilibrium' between the fibre populations is presently unsettled.
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Key words : ageing, coexistence of myosin isoforms, human skeletal muscle, myofibrillar ATPase histochemistry, myosin heavy chains, single fibres.
(Aniansson et al. 1980, Grimby et al. 1982, Lexell et al. 1983, Essen-Gustavsson & Borges 1986). Electrophysiological studies of ageing human skeletal muscle demonstrate a selective loss of the large and fast-conducting motor neurons which innervate type I1 fibres (Campbell et al. 1973). A preferential loss of type I1 fibres is therefore likely with ageing. T h e increased Correspondence : Henrik Klitgaard, August Krogh incidence of type grouping and enclosed fibres, Institute, Universitetsparken 13, 2100 Copenhagen 0, mainly of type I fibres (Grimby et al. 1982), and Denmark. the larger size of the motor units observed in
A common approach in the study of the ageing human skeletal muscle has been to stain for actomyosin myofibrillar ATPase, based on the p H stability following acid and alkaline preincubations (Brooke & Kaiser 1970). An agerelated decrease in the relative proportion of type I1 fibres was found by Larsson et al. (1978). T h i s was not, however, confirmed in later reports
55
56
N. Khtgaard et
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ageing human skeletal muscle (Stalberg 8i Fai\cett 1982) are indications that some of thc dencrvated type I1 fibres become reinnen ated. Thus, it is remarkable that the histochemicall! determined relative occurrence of the u r i o u s fibre types remains unaltered with ageing. One possibility is that the samples obtained \vith the needle h i o p y technique are too small :o he representath e of the muscles' total fibre population. However, in a histochemical study of the fibre type cornposition in cross-sections of a.hole m. vastus latcralis, I~,exell~ ' fill. (1983) were not able to demonstrate any change in the proportion of type I fibres with ageing. T h e histochemical actomyosin m!-ofibrillar ATPase reaction of a muscle fibre is determined b - its myosin heavy chain (hlHC) composition (Danieli-Retto et (11. 1986). .I recent elcctrophoretic study examined the SIHC composition of single tibres from skeletal muscies of young subjects (Biral rt al. 1988). A relatively high number of fibres evinced coexistence of two \IHC isoforms, especiallJ- of type IIa and type 111) lII-IC. Similar studies on single fibres from cndurance-trained muscles revealed a large coesisicncc of ST€IC type I and type IIa (Klitgaard t t lii. 1990a). These data suggest that a d!-namic equilibrium may exist betv-een the main fibre t!j>es, i.c. expression of onc form of l l l I ( : and films containing more than one form of UH(:. 'rhis appcars to be the case for the myosin light chain (l1I.C:) composition of single fibres from sbeleti!l muscles of young and elderly subjects (S:iliiiti t'i I , / . IUX.?). 3 decrease in fibres cc?ntnirlin;; i m l ! f a s t isoforins of \ I I L \%as found i:i cldcrl! \uhjccts concomitant with an
SI.4TERI.4LS A N D METHODS
subjrcls. Using suction, needle biopsies were taken from the middle portion of m. vastus lateralis from a group of young (27 years old, range 23-31, n = 5 ) and elderl!- men (69 )-ears old, range 68-70, n = S), and from m. biceps brachii of a group of young (28 years old, range 26-30, n = 4) and elderly men (69 years old, range h8--70, n = 4). The muscle specimens were dirided into two parts. One part was frozen immediately in liquid nitrogen and used for single-fibre analysis of LIHC composition, the rest being trimmed, mounted and frozen in isopentane cooled with liquid nitrogen for histochemical analyses. None of the subjects suffered from any cardiovascular and/or neuromuscular disease. The study was approved by the Copenhagen Ethical Committee. Single-jibre electrophoresis. Single fibres were prepared and analysed for MHC composition as previously described by Biral et al. (1988). Electrophoresis of the fibres was performed in 6qb SDS-PAGE gels with 37.5O, glycerol (w/v) in the separating gel (Klitgaard et al. 1990 b). This electrophoretic system has demonstrated a clear resolution of MHC types I, IIa and IIb as verified by monoclonal antibodies specific to human MHC types I, IIa and IIb (Fig. 1a). In each gel, a sample known from immunoblotting to contain MHC types I, IIa and IIb was used as an internal standard (Fig. 2). On average, 5&80 fibres were analysed in each biopsy. A total of 344 fibres were analysed from the young vastus lateralis muscles, 288 fihres from the elderly vastus lateralis muscles, 224 fibres from the young biceps brachii muscles and 232 fibres from the elderly biceps brachii muscles. Immiinoblotting. Purified myosin from both muscles studied a a s separated in 60:) SDS-PAGE gels with hieh glycerol content as described above (Klitgaard r f ( i i . 1990 h). Procedures for electrophoretic transfer to nitrocellulose, incubation with monoclonal antibodies (mrtbs) and detection of bound antibody by peroxidase-conjugated second antibody were as previously described (Schiaffino et al. 1989). Monoclonal antibodies specific to human MHC type I (mAh RA-FS), human 3lHC tl-pe IIa (mAb SC-71) and human typing nith myofibrillar JIHC tqpe IIb (mAb BF-34) were used. Immunoblotting with these three mAbs has previously demonstrated that the antibodies react selectively with vastus Iatcralis of young and elderly men. In rat skeletal muscles known to contain predominantly addition, tissue \\as obtained from m. biceps npes I, IIa and IIb fibres as determined by the brachii. T h e reason for the inclusion of arm histochemical reaction for myofibrillar ATPase (Schiafino et u1. 1989). muscle i i w ~ ri + thar, in contrast to the leg Hislochrrnistry. Serial transverse sections (10 pm) intains its fibre size with were cut in a cryotome at - 20 "C. The sections were 1. 1982). This finding has mounted on coverslips and stained for myofibrillar reliitcd to a more forceful .4TPase at pH 0.4 after both alkaline (pII 10.3) and ,I:* cornpiired with the I m e r acid (pH 4.3 and 4.6) preincubations (Brooke & pcople (Cirimby 8i Saltin Kaiser 1970). Fibres were characterized as I, IIa, IIb and intermediate (IM) fibres. The latter fibre type has 1983I
Myosin isoforms in ageing human musclejbres
57
also been referred to as IIc and/or Ib (Schantz & Dhott 1987). On average, 459 (range 322-646) fibres were classified in each sample. Fibre areas were analysed from photographs of the ATPase stain after acid (pH 4.6) preincubation. The areas of a minimum of 50 type I, 30 type IIa and 20 type IIb fibres were measured in each sample with a digitizer. Statistics. The non-parametric Mann-Whitney test was applied for statistical comparisons between the groups. The 0.05 confidence level was chosen for statistical significance.
RESULTS Myosin purified from both muscles analysed was resolved into three distinct bands in 6% SDS-PAGE gels with high glycerol content (Fig. l a , lane i). Using immunoblotting these bands were shown to correspond to M H C types I, IIa and I I b (Fig. l a , lanes ii and iv). Single fibres containing only M H C type I, type IIa or type I I b were identified and are depicted in Fig. 1(b), lanes (ii-iv) (see also Fig. 2). Coexpression of two MHC isoforms in the same fibre was also observed (Fig. lc, lanes ii-iv) (see also Fig. 2). The electrophoretic analysis of single fibres from m. vastus lateralis demonstrated that out of 288 fibres more than half of the fibres showed coexistence in the elderly subjects (Fig. 4). Comparing the young and the elderly subjects, M H C type I and type IIa were observed in 8f 1 % vs 2 0 + 3 % (P< 0.05) of the fibres and M H C type IIa and type I I b in 12f4y0 as 3 3 + 2 % (P< 0.05) of the fibres. In contrast, the young subjects manifested more fibres containing only M H C type I (50*5% vs 33 f3 yo,P < 0.05) and M H C type IIa (26 f3 yo vs 12 f2 %, P < 0.05). The histochemical fibre typing of the vastus lateralis muscle from young and elderly subjects revealed a relative distribution of 64f4Yo vs 48+3y0 in type I (P< 0.05), 29 & 3 yous 37 f4% in type IIa and 7 2 3 % vs 15f2y0 in type I I b fibres. These values are within the range that has been reported from biopsy material in other studies (Aniansson et al. 1980, Essin-Gustavsson & Borges 1986). Hardly any I M fibres were found in the muscles studied. The electrophoretic analysis demonstrated a similar pattern of M H C expression in muscle fibres from m. biceps brachii and m. vastus lateralis, with the exception that elderly subjects had a lower proportion of fibres with coexistence of IIa and I I b (23+ 1% vs 34+2y0, P < 0.05)
Fig. 1. Identification of human myosin heavy chain isoforms by SDS-PAGE in 6% gels with high glycerol content. Only the MHC region is shown. (a) Identification of MHC types I, IIa and IIb from myosin purified from human m. vastus lateralis by immunoblotting analysis. Myosin was separated by SDS-PAGE in 6% gels and either stained with Coomassie blue (i) or transferred to nitrocellulose filters and reacted with mAb BA-F8, specific to human MHC type I (ii), mAb SC-71 specific to human MHC type IIa (iii) or mAb BF-34 specific to human MHC type IIb (iv). (b) Lane (i), purified myosin from human m. vastus lateralis after SDSPAGE in 6% gels and silver staining; (c) Lane (i), purified myosin from human m. vastus lateralis after SDS-PAGE in 6% gels and silver staining; lanes (ii-iv), single fibres from m. vastus lateralis of elderly subjects demonstrating coexistence of MHC type IIa and type IIb (ii) and coexistence of MHC type I and type IIa (iii) and (iv), after SDS-PAGE in 6Sb gels and silver staining.
and a higher proportion of fibres containing only M H C type IIa (25 f5 yo vs 12 k 2Y0,P < 0.05). Fibre typing from myofibrillar ATPase histochemistry in m. biceps brachii of young and elderly subjects revealed a similar distribution for type I (48+2y0 as 44+3%), type IIa (25+6y0 us 27f5%) and type I I b fibres (27+6% vs 29f5y0). No fibres exhibiting coexistence of M H C type I and type I I b were found in any of the muscles analysed. However in m. biceps brachii one of the fibres from the young group and two of the fibres from the elderly group contained all three isoforms of M H C (Fig. 3). These fibres were characterized by trace amounts of M H C types I
5$
H . Klitgaard et al.
Fig. 2. 6”,, SDS-P.4GE gels with high glycerol content. ‘ AfHC’ indicates the M H C region of the gel. T h e lane next to ‘XIHC’ is an internal standard known to contain MHC types I, IIa and I I b from immunoblotting. The other lanes contain single fihres. In gel (A), one fibre showed coexistence of 11HC types I and IIa (u), and in (B) two fibres showed coexistence of MHC types IIa and IIb (ss). The other fibres in both gels contained either M H C type I or IIa.
M ,.HC
iLsl .-MHC .--MHC
I1 b IIa
I
vastus lateralis, and a reduction in fibre size occurs with increasing age. However, this hypotrophy is less pronounced in the upper than in the lower extremities (Table 1). DISCUSSION
Fig. 3. Demonstration of three m!osin hear!- chain isoforms in a single fibre from human m. biceps brachii. Lane (i), a single fibre from m. biceps brachii of a !oung subject containing both M H C types IIa and IIh, lane (ii), a single fibre containing M H C types 1 , IIa and IIb, after SDS-PAGE in 6O,, gels and Yil\ er staining.
and IIb, with the dominant fraction being M H C typc I I a i n analysis of the sizes of the major fibre types re\caled that the fibres in m. biceps brachii mere larger than the corresponding fibres in m.
The results of the two methods used in the present study to identify fibres give the impression that major differences exist between the ti+o muscle and age groups studied (Figs. 5 and 6). This may not be the case, taking into account that in most fibres showing coexistence of M H C isoforms one of them usually dominates. Since the dominating fraction of M H C ‘sets’ the histochemically determined fibre type, the two methods seem to agree. Thus, the 33 y o of fibres H ithin the elderly m. vastus lateralis containing only M H C type I would be histochemical type I fibres (Fig. 5 ) . Within the 2Oyo of fibres showing
Myosin isoforms in ageing human muscleJibres
59
Table 1. Fibre areas of m. vastus lateralis and m. biceps brachii in young and elderly subjects
M. vastus lateralis (,urnz) Young Old
Type 1
Type IIa
Type IIb
5.053 k438 4.526+395
5.460f479, 3.957k364
4.870+479, 3.362+341
5.519f533 4.915_+624
7.286f241 6.329+588
6.21 1 294 5.414+621
M. biceps brachii (,am2) Young 01d
The values are meansfSEM * Statistically significant (P < 0.05) difference between the groups.
coexistence of M H C types I and Ha, 11yo of the fibres had a clear dominance of M H C type 1. These fibres would also react as histochemical type I fibres, giving a total of 44%. This is similar to the 48% of type I fibres found with LO myofibrillar ATPase histochemistry (Fig. 5 ) . However, 9% of the latter had a majority of 30 M H C type IIa (Fig. 1c, lane iii), whereas nearly s none of these fibres were observed within the ; 20 0 young group. This suggests an increase in the .c proportion of histochemical type IIa fibres which 2 10 .contain M H C type I within the elderly m. vastus * lateralis. In contrast to the young group, the .TI major part of the histochemically determined a, a types IIa and I I b fibres within the elderly m. x 601 M.BICEPS BRACHII c vastus lateralis contained M H C types IIa and I I b in different relative amounts, as seen in Fig. 5 from the large group (33%) of fibres showing coexistence of M H C types IIa and IIb. These results may then indicate that most of the 30 histochemical type I I b fibres in the young m. vastus lateralis contain only M H C type IIb, 20 whereas nearly all histochemical type IIb fibres in the elderly m. vastus lateralis contain both 10 M H C types IIa and IIb. In m. biceps brachii of the elderly subjects, MHC I M H C l l l l a MHCIla MHC llalllb M H C Ilb the lower proportion of fibres containing only Fig. 4. Comparison of the fibre type composition in M H C type I (36f 2% uus 26f 5%, P < 0.05) m. vastus lateralis of a group of young (0) (27 years and the higher proportion of fibres showing old, range 23-31, n = 5) and elderly (m) men (69 coexistence of M H C types I and IIa (6 & 2 yo us years old, range 68-70, n = 5) and in m. biceps 2 0 k 2%, P < 0.05) (Fig. 4) are indicative of an brachii of a group of young (28 years old, range 2&30, increase in the relative proportion of histon = 4) and elderly men (69 years old, range 68-70, chemical type I fibres which contain minor n = 4). Values are means k SEM. Ir Indicates statistical difference (P < 0.05) between the groups. amounts of MHC type IIa within the ageing Single fibres were classified according to their content human m. biceps brachii (Fig. 6). Furthermore, of myosin heavy chain isoforms after SDS-PAGE in a lower proportion of fibres containing only 6% gels and silver staining. Between 224 and 344 M H C type I I b (12i=1y0 us 6f2y0, P < 0.05) fibres were analysed in each group. and a lower proportion with a coexistence of 60
M . VASTUS LATERALIS
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Fig. 5. Comparison of the fibre type composition, determined bx single-fibre analysis of MHC composition (XIHC) and myofibrillar ATPase histochemistr! (histochemistrl-), in m. vastus lateralk of young (27 years old, range 23-31, n = 5 ) and elderllmen (69 years old, range 68-70, n = 5 ) . On average, 434 (range 240-646) fibres were t\-ped in each sample from m!-ofibrillar ATPase histochemistry. A total of 344 fibres was analysed for myosin heavy composition in the !oung group and 288 fibres in the elderll-.
M H C types IIa and I I b ( 3 4 i 2 w 23F l o o , P < 0 05) together with a higher proportion of fibres containing only M H C type IIa ( 1 2 * 2 O , z's 23 k soO, P < 0.05) were found uithin the elderly group (Fig. 4). This indicates that a relativel! higher proportion of the histochemical type IIb fibres and a relatively lower proportion of the histochemical type IIa fibres contain both MHC types IIa and I I b in the ageing human m. biceps brachii (Fig. 6). In addition, of the 20 F 2 O o of fibres showing coexistence of M H C types I and IIa in the elderly subjects, 504 contained major amounts of M H C type IIa and only minor amounts of M H C type I. Few of
Fig. 6. Comparison of the fibre type composition, determined by single-fibre analysis of MHC composition (MHC) and myofibrillar ATPase histochemistrl- (histochemistry), in m. biceps brachii of young (28 !-ears old, range 26-30, n = 4) and elderly men (69 years old, range 68-70, n = 4). On average, 416 (range 322-640) fibres were typed in each sample from myofibrillar ATPase histochemistry. A total of 224 fibres was analysed for myosin heavy chain composition in the young group and 232 fibres in the elderll- group.
these fibres were found within the young m. biceps brachii. As this type of fibre was also found in the ageing m. vastus lateralis, this seems to indicate a special population of histochemical type IIa fibres containing major amounts of M H C type IIa and only small amounts of M H C type I. Recent studies of rat skeletal muscles (DanieliBetto et a/. 1986) and human m. vastus lateralis (Biral et af. 1988, Klitgaard et al. 1990a) have demonstrated a specific pattern in the expression of M H C isoforms within single fibres. Thus, a coexistence was observed for M H C types I and IIa and M H C types IIa and IIb, but not for M H C types I and IIb. This agrees with the
~
Myosin isoforms in ageing human muscle j h e s hypothesis that the expression of M H C isoforms within a fibre follows the sequence: I + I I a + I I b (Biral et al. 1988). The present study is the first to report the coexistence of three M H C isoforms within the same fibre. The trace amounts of M H C types I and I I b and the major amount of M H C type IIa within these fibres indicate that they have a high expression of M H C type IIa, with either a down-regulation of M H C type I expression accompanied by an up-regulation of M H C type I I b expression or vice versa. It appears that these fibres are close to manifesting coexistence of only M H C types I and IIa or M H C types IIa and IIb. The human m. biceps brachii contains a relatively larger proportion of histochemical type I1 fibres, as determined from myofibrillar ATPase histochemistry, than m. vastus lateralis (for ref. see Saltin & Gollnick 1983). This study demonstrates that the histochemical type I1 fibre population of the two muscles differs not only in its relative occurrence but also in its M H C composition. Thus, only 30% of the histochemical type I1 fibres of m. vastus lateralis manifested a coexistence of M H C types IIa and IIb, whereas 60% of the histochemical type I1 fibres in m. biceps brachii demonstrated a coexistence of these two M H C isoforms. Using myofibrillar ATPase histochemistry, there are several reports of an unchanged fibre type composition of the human skeletal muscle with ageing (Aniansson et al. 1980, Grimby et al. 1982, Lexell et al. 1983, Esstn-Gustavsson & Borges 1986). However, a selective atrophy of the type I1 fibres has been reported in m. vastus lateralis but not in m. biceps brachii (Grimby et al. 1982). It has been proposed that the morphology of m. biceps brachii is better maintained owing to differences in the physical activity pattern ;faster and more forceful contractions are thought to be performed in the upper than in the lower extremities. In the light of this explanation, it is noteworthy that the alteration in M H C composition of single fibres from m. biceps brachii is similar to that observed in m. vastus lateralis. Thus an increased coexistence of M H C as well as M L C isoforms may be a general phenomenon of ageing human skeletal muscle fibres. Electrical stimulation experiments on skeletal muscles from animals indicate that coexistence of myosin isoforms within a fibre is related to a transformation process (Pette & Vrbova 1985).
-
N
61
Several recent reports have demonstrated an increased proportion of fibres showing coexistence of M H C isoforms in the human skeletal muscle after endurance training (Schantz & Dhott 1987, Klitgaard et al. 1990a). The crosssectional design of the present study makes it impossible to be conclusive, but the higher proportion of fibres showing coexistence of M H C types I and IIa within both muscles of the elderly subjects strongly suggests that a changed activity pattern with ageing might induce a transition process within the fibres of the ageing human skeletal muscle. However, this phenomenon may also be caused by other factors. A selective denervation of large fibres (Grimby & Saltin 1983) containing only M H C type IIa or M H C type IIb, and a reinnervation of these by smaller motor neurons, could explain the increased proportion of fibres possessing a coexistence of M H C types I and IIa in both muscles studied and of M H C types IIa and I I b in m. vastus lateralis. Another possibility is agerelated changes in the neuromuscular junction (Gutmann & Hanzlikova 1972), which could alter the traffic of neurotrophic factors and/or impulses reaching the fibres, thereby changing their M H C expression. These changes could be related to a disuse phenomenon since regular usage seems to maintain morphology, fibre size and M H C expression of the ageing human skeletal muscle (Klitgaard et al. 1990b). In conclusion, analysis of M H C composition in single fibres from human skeletal muscles of young and elderly subjects demonstrated a higher proportion of fibres possessing a coexistence of M H C types I and IIa in both muscles studied and also a higher proportion of fibres with coexistence of M H C types IIa and I I b in m. vastus lateralis of the elderly subjects. This indicates an increased coexistence of M H C isoforms in histochemically determined fibre types with ageing. The explanation for this phenomenon is at present unknown. This study was supported by grants from the Danish Research Academy, the Danish Research Council for the Natural Sciences, the Danish Sports Research Council, the Obel Family Foundation, the Knud H~jgaardFoundation, the John Meyer Foundation, the former Director Leo Nielsen and wife Karen Margrethe Nielsen grant for the medical sciences and, in part, by Institutional Funds from the Italian National Research Council and grants from MPI and MDA. We are grateful to Mrs S. Ceoldo, Mrs Lise
62
H. Klitgaard et al.
Kiis-Jacobsen and Mrs Marianne Hemmingsen for their skillful technical assistance and to Mrs Lis H. Christensen for tj-ping of the manuscript.
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