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Am J Physiol Cell Physiol 283: C1228-C1241, 2002. First published June 26, 2002; doi:10.1152/ajpcell.00206.2002
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Vol. 283, Issue 4, C1228-C1241, October 2002

Aging-related satellite cell differentiation defect occurs prematurely after Ski-induced muscle hypertrophy

Sophie B. P. Chargé, Andrew S. Brack, and Simon M. Hughes

Medical Research Council (MRC) Muscle and Cell Motility Unit and MRC Centre for Developmental Neurobiology, Guy's Campus, King's College London, London SE1 1UL, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the cause of skeletal muscle weakening during aging we examined the sequence of cellular changes in murine muscles. Satellite cells isolated from single muscle fibers terminally differentiate progressively less well with increasing age of donor. This change is detected before decline in satellite cell numbers and all histological changes examined here. In MSVski transgenic mice, which show type IIb fiber hypertrophy, initial muscle weakness is followed by muscle degeneration in the first year of life. This degeneration is accompanied by a spectrum of changes typical of normal muscle aging and a more marked decline in satellite cell differentiation efficiency. On a myoD-null genetic background, in which satellite cell differentiation is defective, the MSVski muscle phenotype is aggravated. This suggests that, on a wild-type genetic background, satellite cells are capable of repairing MSVski fibers and preserving muscle integrity in early life. We propose that decline in myogenic cell differentiation efficiency is an early event in aging-related loss of muscle function, both in normal aging and in some late-onset muscle degenerative conditions.

muscle regeneration; MyoD; myonuclear domain size


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AGING HUMAN SKELETAL MUSCLE becomes weaker and undergoes atrophy, changes that frequently lead to physical dependence in the elderly. Muscle aging atrophy results from the combined effects of loss of muscle fibers and atrophy of the remaining fibers (2, 4, 28, 31, 40, 44, 51). Decline in fiber number is paralleled by the loss of motor neurons occurring throughout life (39). Consequently, because each motor neuron is thought to have a limited innervation capacity, useful reversal of fiber number decline may be difficult. In contrast, various interventions can reverse muscle atrophy in aging humans or rodents by enhancing fiber size. Exercise is effective at preserving human muscle mass and function in the elderly (24, 25). In rodents, overexpression of IGF-I in muscle has also been shown to enhance fiber size, mitigating muscle atrophy and weakness (4, 16). Nevertheless, muscle mass declines with age even in individuals who exercise, consistent with the view that although factors such as neural or hormonal changes contribute to muscle aging (11, 37, 39), changes in muscle tissue itself, such as reduced force, enhanced susceptibility to injury, and poor repair capacity, are important (3, 8, 10, 21, 27, 35, 41, 55, 66, 67). In particular, in pathological situations, such as Duchenne muscular dystrophy, in which muscle function is gradually lost, enhanced cellular turnover has been implicated in exacerbation of the deficit and control of disease progression (20, 73). However, the causal links between the various cellular changes leading to muscle weakness and atrophy in aging or disease are unclear.

The cellular mechanisms underlying fiber atrophy are poorly understood. Fiber size consists of two elements, the number of nuclei inside each multinucleated muscle fiber and the volume of cytoplasm supported by each nucleus, hereafter referred to as the nuclear domain size (35). Both elements vary among fibers of different type, during fiber development and in response to changes in innervation (23, 65). During development, ablation of the transforming growth factor (TGF)-beta superfamily member growth and differentiation factor (GDF)-8 leads to increased nuclear number and fiber size (48, 76). Conversely, ablation of IGF-I leads to decreased muscle mass (56). Whether these pathways only control nuclear number or also affect domain size is unclear. To understand the long-term effects of increase in domain size in the absence of change in nuclear number on muscle integrity and function and their consequences for aging, we examined transgenic mice overexpressing a truncated form of the Ski protooncogene, a histone deacetylase complex component that has been implicated in various signaling pathways, including that activated by TGF-beta superfamily members (1, 46, 52, 71). MSVski transgenic mouse muscle hypertrophies without increase in fiber or myonuclear number (69). Expression levels of the transgene are highest in fast muscles, and hypertrophy specifically occurs in fast type IIb fibers (43). A similar, but poorly characterized, hypertrophic muscle phenotype occurs in MSVski transgenic cattle, leading to death within 10-15 wks of birth accompanied by muscle weakness and elevated serum creatine phosphokinase, a marker of muscle damage (9). Other studies have demonstrated muscle damage after hypertrophy (13), and these findings, together with the observations made in the transgenic cattle, suggest that muscle nuclear domain enlargement may be detrimental to muscle integrity.

We report that MSVski-induced nuclear domain enlargement is accompanied by loss of force but no other detectable changes in young mice. However, over time, muscle degeneration becomes apparent with defective regeneration and other signs typical of aggravated muscle aging. The premature aginglike changes in MSVski muscle are preceded by decreased satellite cell differentiation potential, a change similar to that observed in aging wild-type mice. The data raise the possibility that long-term muscle nuclear domain enlargement may have deleterious effects in later life and that decreased satellite cell differentiation efficiency may contribute to the changes occurring during muscle aging.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of transgenic mice. Fertilized mouse eggs were injected with the FB29 MSVski transgene encoding a truncated version of chicken c-ski protooncogene driven by the murine sarcoma virus LTR as previously described (69). Three of four founder mice carrying the MSVski transgene identified by Southern blot analysis transmitted the transgene to F1 progeny, termed lines 6, 16, and 33. Transgenic mice were backcrossed onto a CBA/J background for at least four generations before further analysis or crossing. Northern blot analysis of skeletal muscle total RNA from each line at postnatal day 60 (P60) showed that line 33, the highest expresser, accumulated ~3.3-fold more MSVski mRNA than line 6, the lowest expresser, and that MSVski mRNA was undetectable in soleus and high in extensor digitorum longus (EDL), as described previously (43, 69). MSVski line 16 heterozygous mice were bred to homozygous myoDm1 (hereafter referred to as myoD-/-) mice (59). F1 progeny heterozygous for MSVski and myoD-/- were backcrossed with homozygous myoD-/- mice, and progeny were genotyped for MSVski by Southern blot analysis and for myoD by PCR on tail DNA. Mice were kept in plastic cages with wire mesh lids in a 12:12-h light-dark cycle and fed ad libitum.

Histology, immunocytochemistry, and morphometry. For morphometric analysis, full-length EDL muscle was isolated, clamped at rest length, and frozen in isopentane cooled in liquid nitrogen. For total fiber number and fiber type proportion, whole lower hind legs were frozen as above and fiber counts were made on the entire transverse sections of the midbelly EDL. Ten-micrometer serial cryosections were stained with myosin heavy chain (MyHC) antibodies specific for MyHC IIb (BF-F3), MyHC IIa (A4.74), embryonic fast MyHC (F1.652), and slow beta -cardiac MyHC (A4.951) (47, 64) as described previously (33). Total nuclei counts were made on BF-F3-stained EDL sections after DNA staining with 4',6- diamidino-2-phenylindole (DAPI) on areas containing ~50 fibers. Cross-sectional areas (CSAs) of at least 200 IIb and non-IIb fibers contained within the same area were determined by outlining fibers manually in NIH Image captured with a Pulnix TM-765E camera attached to a Zeiss Axiophot. To ensure transverse sectioning, images with fiber profiles showing no significant major axis orientation were analyzed.

Northern blot analysis. Total RNA was extracted from skeletal muscles of a single upper leg with a single-step method (15). Approximately 30 µg of total RNA were separated on 1% agarose gels containing 0.41 M formaldehyde in 1× MOPS buffer, transferred to a nylon membrane, and probed with a 32P random-primed MSVski cDNA and human beta -actin cDNA (which readily cross-reacts to mouse alpha -actin) as a loading control.

Nuclear turnover and creatine kinase assay. Mice were injected intraperitoneally with 30 mg/kg body wt of 5-bromo-2'-deoxyuridine (BrdU) and 100 mg/kg Evans blue dye in saline. After 17 h, lower hindlimbs were collected and cryosectioned as above. BrdU staining of acid-treated sections followed (72). Terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) staining on fixed cryosections used the Apoptag kit (Invitrogen). Serum was prepared by coagulation and centrifugation of blood samples, stored frozen, and assayed with a Boehringer Mannheim creatine kinase (CK) NAC-activated kit using UV detection.

Satellite cell cultures derived from single muscle fibers. Single fibers dissociated by collagenase treatment of EDL muscle of MSVski or wild-type mice of various ages were plated, and outgrowing mononucleate cells were analyzed as described previously (57). Fibers derived from at least two mice of each genotype and age group were compared. Single EDL fibers were individually plated on Matrigel (Becton Dickinson)-coated wells of Permanox chamber slides incubated in a humid environment at 37°C and 5% CO2. After plating, fibers were incubated for 3 days in plating medium consisting of 10% (vol/vol) horse serum (GIBCO) and 0.5% chick embryo extract (CEE, Imperial) in DMEM containing 2% L-glutamine and 1% penicillin and streptomycin (GIBCO). At day 3, fibers were removed and medium was replaced with a rich medium containing 20% FCS (GIBCO), 10% horse serum, and 2% CEE in DMEM to promote cell growth. After a further 2 days, cultures were differentiated for 2 days in a medium consisting of 2% FBS in DMEM. Cells were rinsed in PBS, fixed in cold methanol, blocked in 5% horse serum in PBS, and incubated with MAb against desmin (1/500, clone no. DE-U-10, IgG1; Sigma) and MyHC (1/10, A4.1025, IgG2a; Ref. 19). Primary antibodies were successively detected with rat anti-mouse IgG1 (1:1,000; Serotec), FITC-conjugated goat anti-mouse IgG2a (1:100; Serotec), and Cy3-conjugated donkey anti-rat IgG (1:100; Jackson). In the last antibody incubation, the DNA dye DAPI was added. Cells were fixed in cold methanol before being mounted with antifading agent. At day 3 after plating and after 2 days in growth medium, the total number of cells was analyzed for each single-fiber culture. After two additional days in differentiation medium, a representative number of cells were analyzed by randomly analyzing 10 fields of view equivalent to a total area of 5.45 mm2. In this study, differentiation efficiency is the ratio of nuclei associated with MyHC-desmin over the total number of nuclei associated with desmin.

Single permeabilized fiber isometric force measurement. The protocol has been described by Sabido-David et al. (61). Briefly, single-fiber 2- to 3-mm segments were dissected from permeabilized EDL of P60 MSVski heterozygote or wild-type littermates and aluminum T clips were crimped to their ends and attached to a fixed hook and to a force transducer (AE801; Aksjelskapet). Sarcomere lengths were adjusted to 2.4 µm, and fiber CSAs were calculated by assuming a circular circumference. Peak tension was recorded in maximally Ca-activated and Mg-rigor conditions, as was the time to reach 90% of maximum calcium activation (t90) and relaxation to 50% (t50).

Muscle DNA and protein contents. Each weighed EDL muscle was homogenized with 1 ml of lysis buffer (0.1 M KPO4 pH 7.8, 0.2% Triton X-100, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin). Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay (Pierce). DNA concentrations were determined with a modification of the Hoechst 33258 assay (26). Briefly, a 50-µl sample was diluted in 450 µl of DNA assay buffer (0.15 M NaCl, 15 mM Na citrate) before incubation with 250 µl of Hoechst 33258 (0.8 mg/l in distilled H2O) for 10 min at room temperature in the dark. Fluorescence was read with a fluorometer (Spex; FluoroMax Instruments) at an excitation wavelength of 360 nm and an emission wavelength of 450 nm, and DNA concentration was determined from a standard curve generated with salmon sperm DNA.

Statistical analysis. Fiber CSA data were analyzed with a multiple ANOVA test. Data on fiber type proportion, total fiber number, and nuclear number and data from single permeabilized fiber force experiments were analyzed with an unpaired t-test. Data from single-fiber cultures were analyzed with the Kolmogorov-Smirnov two-sample (1-tailed) test and the Spearman rank-order correlation coefficient rs.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MSVski IIb fiber size increase is not paralleled by increased force generation. To examine the effect of nuclear domain enlargement, we made MSVski transgenic mice. Each of three independent lines of such mice showed increases in CSA of postnatal fast muscle fibers compared with control littermates. At P17, wild-type and MSVski transgenic EDL IIb muscle fibers were similar (Fig. 1, A-C, J). By P60, IIb fibers in transgenic animals had increased in size over twofold, from 1,325 ± 52 µm2 (n = 6) in controls to 3,042 ± 360 µm2 (n = 5) in MSVski (Fig. 1, D-F, J). The distribution of IIb fiber sizes in MSVski mice is skewed toward larger size, with some very large fibers present (>6,000 µm2; Fig. 1F). In MSVski lines 16 and 33 the type IIb fiber size increase was paralleled by a doubling of EDL wet mass and extractable protein but without a significant increase in muscle DNA content or nuclear number (Fig. 2). Thus MSVski causes approximately twofold nuclear domain enlargement, in all respects confirming published results (43, 69). In our lines, in contrast to previous reports, MSVski-induced hypertrophy occurred without bone malformation or significant increase in body mass, so hypertrophy is unlikely to be a response to increased load (38). We also analyzed the ability of MSVski fibers to generate force. Isometric force measurements on single permeabilized fibers demonstrated that hypertrophied MSVski fibers from P60 mice generate less force per unit CSA than control fibers, in both maximally activated and Mg-rigor conditions (Table 1 and data not shown). However, the kinetics of activation (t90) and relaxation (t50) between the MSVski and control fibers remained similar (Table 1 and data not shown). The reduction in specific force without change in kinetics of MSVski EDL fibers suggests either that each active myosin molecule produces less force or that fewer myosins are functioning. Immunohistochemical fiber typing using MAbs to MyHC isoforms revealed no differences between young adult control and MSVski limbs (see below). Evans blue dye perfusion failed to detect a change in fiber integrity: all fibers excluded the dye in EDL muscles of P90 control and MSVski mice (data not shown). Similarly, histological comparisons of P60 MSVski and control muscles showed no signs of pathology (Figs. 1 and 3). However, occasional isolated MSVski fibers had misalignment of sarcomeres in adjacent myofibrils. Thus, although MSVski fibers are larger than controls, they do not generate greater force.


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  		Fig. 1.   MSVski-induced IIb fiber hypertrophy declines with age. Transverse cryosections of extensor digitorum longus (EDL) from wild-type (A, D, G) and MSVski (B, E, H) mice at postnatal day (P)17 (A, B), P60 (D, E), and aging (P >550; G, H) stained with MAb specific to type IIb myosin heavy chain (MyHC). In MSVski mice IIb fibers are hypertrophied (arrows) from P60, but with aging there is additional appearance of small IIb fiber profiles (arrowhead). Bar, 50 µm. Distribution of mean ± SE IIb fiber cross-sectional (CSA) at P17 (C), P60 (F), and aging (I) in wild-type and MSVski mouse EDL is shown (n = 3, 6, and 3 for wild type and 4, 5, and 5 for MSVski at P17, P60, and P >550, respectively). With aging, there is a decrease in very large and an increase in very small IIb fibers in MSVski mice. J: average ± SE IIb fiber CSA with age in wild-type and MSVski mouse EDL. Data are from C, F, and I (**P < 0.01). On aging, the mean IIb fiber size is comparable between wild-type and MSVski mice. K: Northern analysis of total RNA from skeletal muscle shows a 2.5-kb chicken ski transcript from the MSVski transgene detectable from P17, reaching high levels at P60 that are maintained in aging (P550) mice. Actin loading control is shown at bottom.



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  		Fig. 2.   Increase in nuclear domain size of adult MSVski mice. A: muscle mass is doubled (left), extractable DNA concentration is halved (center), and extractable protein concentration is unaltered (right) in P60 MSVski EDL compared with wild type. Values are means ± SE (n = 4, 9 for P17 and n = 5, 11 for P60 wild type and MSVski, respectively). B: total nuclear numbers per fiber [calculated by dividing number of 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei by number of fibers in an area of >50 fibers without visible interstitial tissue] are little changed in P60 MSVski EDL. Values are means ± SE (n = 1, 3 for line 33 and n = 4, 5 for line 16 wild type and MSVski, respectively). C: both IIb fiber CSA per nucleus (left) and perimeter per nucleus (right) are significantly increased in P60 MSVski EDL (P = 0.01 and 0.0004, respectively). Fiber area and perimeter of IIb fibers were measured in each of several cryosections from EDL midbelly of 3 MSVski (265, 22, and 24 total fibers) and 2 wild-type littermate (89 and 27 total fibers) mice with NIH Image. Values are means ± SE (n = 116 and 311, wild type and MSVski, respectively).


                              
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  		Table 1.   Reduced force per CSA in P60 MSVski mice



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  		Fig. 3.   Nonperipheral myonuclei are abundant in aging MSVski mice. A: hematoxylin and eosin (H & E) staining on EDL transverse sections of wild-type (A, C) or MSVski (B, D) mice at P60 (A, B) or aging (C, D) shows an increase in nonperipheral myonuclei (arrowheads) in aging MSVski mice. Bar, 50 µm.

Aging MSVski mice undergo muscle damage and regeneration. Histological examination of P60 MSVski mice showed no signs of damaged fibers or nonperipheral myonuclei (Fig. 3). Control aging mice (defined as animals over 550 days of age) showed relatively few nonperipheral nuclei, although slightly more than at P60 (Fig. 3C). However, aging MSVski muscle revealed that all kinds of fibers (large and small, IIb and non-IIb) have abundant nonperipheral nuclei (>70% of fibers analyzed in some sections; Fig. 3D), suggesting that regeneration had occurred within the muscle. Infiltration by cells of the immune system was not observed, nor were serum CK levels raised in mice of either genotype or age group [CK levels were 632 ± 208, 352 ± 93, 477 ± 94, and 409 ± 101 U/l in P42-P106 control (n = 6), MSVski expressing (n = 6) and aging control (n = 2), and MSVski expressing (n = 4), respectively]. Therefore, MSVski mouse muscle has a contractile deficit but apparently healthy histology in early life but shows marked histological damage in aging animals.

Exacerbated aging-related changes in MSVski muscle. Accompanying the damage in aging MSVski mice were a series of changes typical of normal muscle aging but more severe. Depending on genetic strain, mice begin to show aging changes late in their second year of life and are often defined as "aged" over 2 yr of age. Average IIb fiber CSA begins to decline in aging CBA/J mice, and this change was enhanced in aging MSVski: although highly hypertrophied fibers (>6,000 µm2) were still present, many smaller fiber profiles were also observed (0-1,199 µm2) (Fig. 1, G-I). The mean MSVski IIb fiber size was greatly reduced to a value similar to the wild-type level, despite continued expression of the transgene (Fig. 1, J and K). These data suggest that a long-term effect of MSVski is to promote the appearance of small IIb fibers typical of muscle aging (2, 44).

Exacerbation of other changes typical of normal muscle aging was also apparent in MSVski muscle (Fig. 4). Type IIb fibers become less frequent compared with other fiber types as rodent muscle ages (2, 4, 40). In our control cohort this trend was not apparent, perhaps because of strain differences in the onset of aging changes. In control mice, ~65% of EDL fibers were of type IIb at all ages [61% (n = 3), 66% (n = 4) and 69% (n = 3) at P17, P60, and >P550, respectively]. In P17 and P60 MSVski mice, the EDL had a proportion of IIb fibers comparable with that of control littermates [67% (n = 3) and 60% (n = 5), respectively]. However, in aging MSVski, IIb fibers were less frequent than in controls (Fig. 4, E-G), dropping to 48% (n = 3) from 69% in controls (P < 0.05). In place of IIb fibers, significantly increased numbers of IIx and slow fibers were observed (20 to 38% for IIx and 0.4 to 2.2% for slow in aging control and MSVski, respectively), with little change in IIa fiber frequency (10.9% in controls and 11.3% in MSVski) (Fig. 4, A-D and G). These non-IIb fibers were increased in size but were significantly smaller than normal IIb fibers in wild-type animals (Figs. 4H and 1J). At P17 and P60, when the non-IIb fiber type proportion was unchanged from control, the non-IIb fiber size was also unaffected by the MSVski transgene (Fig. 4H). One interpretation of this spectrum of changes is that reduced IIb fiber number combined with increased non-IIb fiber size and frequency reflects a shift of some hypertrophied IIb fibers to a non-IIb phenotype. Thus aging MSVski mice show an enhancement of changes in fiber type proportion that occur during normal aging.


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  		Fig. 4.   MSVski expression leads to increase in non-IIb fiber type proportion and size in aging mice. Serial EDL transverse sections from aging wild-type (A, C, E) and MSVski (B, D, F) mice stained with MAb specific to slow MyHC (A, B), type IIa MyHC (C, D), and type IIb MyHC (E, F). Type I, IIa, and IIb are specifically stained by these antibodies, whereas type IIx fibers are not stained by any of these antibodies. Arrowheads show examples of the 3 most abundant fiber types present in the EDL. Arrows highlight a branched fiber. Bar, 100 µm. G: quantification of fiber type frequency was performed by scoring all slow (dark gray bars), IIa (light gray bars), IIx (open bars), and IIb (filled bars) MyHC-containing fibers present at the muscle midbelly. Data are means ± SE (n = 3). H: analysis of non-IIb fiber size in wild-type and MSVski mice EDL with age. Wild-type non-IIb fiber CSA increases during growth and plateaus between P60 and aging. MSVski non-IIb fibers are indistinguishable from those in wild-type littermates until P60 but increase in size in aging mice. Size increase in aging MSVski IIa fibers compared with control IIa fibers is similar to that in all non-IIb fibers, suggesting that IIa and IIx fibers increase in size to a similar extent in aging MSVski mice. Data are means ± SE (n = 3, 4 and 3 for wild type and 3, 5, and 5 for MSVski at P17, P60, and P550, respectively). wt, Wild type. **P < 0.01.

Counts of the total number of fiber profiles present at the EDL midbelly at P60 revealed no significant difference between MSVski and control littermates [1,391 ± 62 (n = 8) and 1,268 ± 47 (n = 6), respectively]. Like humans, rodents lose a significant number of fibers during aging (31, 39, 44). This appeared to occur in our control cohort, with total fiber profile number dropping to 995 ± 56 (n = 3) in aging animals (P = 0.01). In aging MSVski mice, in contrast, total fiber profile numbers were unchanged compared with P60 [1,268 ± 32 (n = 3) in aging MSVski, P = 0.27], showing that IIb fiber loss alone does not account for the change in fiber proportions. On the contrary, the increased number of fiber profiles in aging MSVski compared with aging controls suggests that the transgene protects fibers from death, triggers generation of additional fibers, or causes fiber branching. Analysis of serial sections demonstrated the presence of some branched IIb fibers in aging MSVski muscle (Fig. 4B, D, and F). To test the hypothesis that enhanced apparent fiber number was due to fiber branching, aging EDL muscle was dissociated into single fibers and large IIb fibers were analyzed optically for branching after plating in culture (Fig. 5A). Increased fiber branching was a characteristic of aging mice (Fig. 5B). Single fibers from young (P75-P150) MSVski mice were indistinguishable from wild-type littermates with respect to branching (Fig. 5B). However, as MSVski mice aged, fibers showed earlier onset and quantitative increase in branching (Fig. 5, A and B). By P720-P785 many individual fibers had more than one branch (data not shown). Thus formation of these apparently branched fibers is likely to account for the apparent maintenance of fiber number and to contribute to the apparent decline to wild-type mean IIb fiber size in aging MSVski mice.


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  		Fig. 5.   Exacerbation of aging-related fiber branching in aging MSVski mice. A: isolated single EDL fibers show that branching (arrows) is observed more commonly in fibers from P240 MSVski mice than from age-matched wild-type mice. Mononucleate cells migrate away from single fibers (arrowheads). B: analysis of isolated EDL fiber branching in wild-type and MSVski mice at P75-P150, P220-P240, and P720-P785. Fiber branching increases slightly with normal aging but markedly in MSVski mice from P220 onwards. Data are means ± SE [n = 2 animals for all controls (110, 75, and 102 total fibers) and 3, 3, and 2 animals for MSVski (87, 136 and 164 fibers) at P75-P150, P220-P240, and P720-P785, respectively].

Rapid muscle damage in myoD-/- mice induced by MSVski hypertrophy. The spectrum of changes present in aging MSVski mice, involving nonperipheral myonucleation, increase in slower fiber type proportion, loss of IIb fibers, decreased size of the remaining IIb fibers, and enhanced fiber branching, is reminiscent of the changes observed in aging muscle. Moreover, the poor contractile properties of fibers from young MSVski mice suggested that an early defect may precipitate the later degenerative pathology. Human satellite cells isolated from aging people show decreased replicative capacity, suggesting that defective repair may contribute to the changes observed in aging muscle (55). A similar phenomenon has been suggested to contribute to impaired muscle regeneration in aging rodents (66) and in the later stages of Duchenne muscular dystrophy (73). These considerations raised the possibility that the damage and regeneration in aging MSVski mice might result from prolonged covert muscle damage due to the overexpression of MSVski or the resultant hypertrophy. To determine whether the MSVski transgene leads to covert muscle damage in young animals, MSVski mice were bred onto a myoD-/- background, which has impaired regeneration because of a reduced satellite cell differentiation capacity (49, 62).

We first demonstrated that the absence of myoD did not prevent the MSVski-induced hypertrophy. The MSVski transgene is expressed at similar levels in the absence or presence of myoD (data not shown). Hypertrophy of IIb fibers is seen in all MSVski genotypes, with or without myoD (Fig. 6A). Moreover, quantitative fiber size analysis showed that the removal of myoD does not prevent IIb hypertrophy or significantly alter non-IIb fiber size. However, MSVski-induced hypertrophy may be slightly reduced in the absence of myoD (Figs. 6B and 1F). In particular, there appear to be fewer IIb fibers of the largest sizes (>5,600 µm2) and somewhat more IIb fibers of very small size (<1,600 µm2). Thus a functional myoD gene is not required for MSVski-induced hypertrophy, although MSVski.myoD-/- muscles are slightly different from those of MSVski mice.


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  		Fig. 6.   Muscle hypertrophy and damage in young MSVski.myoD-/- mice. A: EDL transverse sections from P60 wild-type and MSVski mice in a myoD+/- or myoD-/- background stained for IIb MyHC. B: analysis of P60 EDL IIb fiber size distribution as in Fig. 1 (n = 6 for each genotype). The absence of MyoD does not prevent the Ski-induced hypertrophy (hatched bars). C: spaced transverse sections of a P60 MSVski.myoD-/- EDL stained with MAb to embryonic (Emb), IIb MyHC, and H & E. Distances between sections are: Emb-IIb and IIb-H & E = 48 µm, H & E-IIb = 80 µm. For orientation, a IIb fiber () and a IIa fiber (*) are marked. Nonperipheral myonuclei are clearly visible after H & E stain (arrowheads), and characteristic fibers that appear split are apparent (highlighted on the drawings, right). D: quantitation of TUNEL and BrdU labeling in MSVski and control mice. At least 5 sections/leg were analyzed. To avoid ambiguity, no attempt was made to distinguish myogenic from nonmyogenic nuclei. BrdU-labeled nuclei were scored in whole EDL cross sections of MSVski mice or wild-type littermates. TUNEL-labeled nuclei were less numerous and were consequently scored in entire lower hindlimb sections from sibling myoD+/- or myoD-/- mice with or without the MSVski transgene. Values are means ± SE; from left to right, n = 6, 3, 4, 4, 2, and 2 mice. No differences were significant, chiefly owing to high interanimal variation that appeared to be independent of genotype. Scale bars, 100 µm.

MSVski.myoD-/- EDL muscle displayed two signs of degeneration. First, analysis of serial sections showed apparent fiber branching in young MSVski.myoD-/- mice not observed in myoD-/- or MSVski.myoD+/- mice (Fig. 6C). The small branched fibers expressed IIb MHC and did not show signs of acute regeneration, such as the expression of embryonic MHC (Fig. 6C). Second, nonperipheral nuclei were more numerous in the MSVski.myoD-/- mice (Fig. 6C) than in wild-type mice or mice carrying either myoD-/- or MSVski alone. Up to 50 nonperipheral nuclei were observed in a single EDL section of one MSVski.myoD-/- mouse and at least 24 ± 6 (n = 6) nonperipheral nuclei/EDL section on average. This compared with an average of 2.2 ± 0.5 (n = 6), 1.5 ± 0.3 (n = 6) and 4.5 ± 1.4 (n = 8) nonperipheral nuclei/EDL section in myoD+/-, myoD-/-, and MSVski.myoD+/-, respectively. Although myoD-/- mice have fewer total fibers, the EDL IIb fiber proportion is unchanged (34, 60). The MSVski transgene does not alter either property (data not shown). However, the number of nuclei/fiber profile is increased from 2.7 ± 0.1 (n = 4) in myoD-/- (similar to wild type, see Fig. 2B) to 4.2 ± 0.4 (n = 5) in MSVski.myoD-/- (P < 0.01), consistent with the view that hyperplasia is triggered by the MSVski transgene in young myoD-/- animals. Presumably, many of these additional nuclei are mononucleate cells (see below). So, by several criteria, young MSVski.myoD-/- mice appear to undergo significant muscle degeneration and show other changes reminiscent of older MSVski mice containing functional MyoD.

The slight increase in nonperipheral nuclei in MSVski.myoD+/- compared with myoD+/- animals suggested that increased nuclear turnover might be occurring in young MSVski mice. We assessed this possibility in a cohort of MSVski and wild-type littermates by analyzing BrdU incorporation into nuclei in S phase and TUNEL staining for apoptotic nuclei within lower hindlimb muscle. No gross turnover of nuclei was detected, although a slight increase in nuclear turnover in MSVski animals could not be excluded (Fig. 6D). Both BrdU-labeled and TUNEL-labeled nuclei were rare in muscle tissue of either genotype (averaging around 1 BrdU-labeled nucleus per EDL cryosection and many fewer TUNEL-labeled nuclei). Thus, although functioning satellite cells appear to be required to maintain MSVski muscle integrity, enhanced nuclear turnover is not detected.

Decreased satellite cells with aging and increased desmin- cells in MSVski EDL. The phenotypes of young MSVski.myoD-/- mice, older MSVski mice, and aged wild-type mice share many similarities. It is possible, therefore, that a defect in satellite cells contributes to the failure of muscle regeneration in aging MSVski and/or wild-type mice, as it appears to do in MSVski.myoD-/- mice. Skeletal muscle satellite cell number decreases with increasing age (8, 27, 67), and this may contribute to the poor regeneration capacity observed in aging muscle. Individual culture of single intact fibers and their associated mononucleate cells induces activation, proliferation, and, subsequently, differentiation of satellite cells and permits assessment of fiber-associated cell properties without the cell selection inherent in bulk culture methods (7, 8, 57). In single-fiber cultures from young and adult wild-type mice, most fibers yielded numerous cells (Table 2, Fig. 7), generally over 10 cells/fiber, most of which contained desmin (Table 2, Fig. 7), an intermediate filament protein expressed in myoblasts and differentiated muscle fibers (36). In cultures from aging wild-type mice, most mononucleate cells still contain desmin (Fig. 7), but desmin+ cell numbers are significantly reduced (P < 0.001; Table 2, Fig. 7). Consistent with this, almost 50% of fibers from aging wild-type mice yielded no mononucleate cells, whereas almost all fibers from younger mice yield myogenic cells (Table 2). Thus normal aging leads to a decline in myogenic cells recoverable in single-fiber cultures, confirming the finding of Bockold et al. (8) on C57Bl/10 mice.

                              
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  		Table 2.   Cultured single fibers yield fewer desmin+ cells with normal aging and MSVski



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  		Fig. 7.   Changes in mononucleate cell yield with aging from wild-type and MSVski single EDL fiber. Data from individual EDL fiber cultures, ranked according to the number of desmin+ cells present after 3 days in culture are displayed, following Bockold et al. (8), by representing the group of cells derived from a single fiber as a point on the y-axis. The number of desmin+ cells and the total number of cells derived from each fiber are read from the x-axis. To permit visual comparison between conditions from which distinct numbers of fibers were analyzed, the data are normalized for sample size by expressing individual rank as a percentage of the total number of fibers analyzed for each condition. In wild-type cultures (left), desmin+ cell numbers were a high proportion of total cell numbers from each fiber at all ages, but total yield declined with age (P < 0.01). In MSVski cultures (right), the number of desmin+ cells shows no significant decline with age, although cultures are more heterogeneous than wild type. Also, total cell yield is more variable, with many individual MSVski fibers at P220-P240 and P720-P785 yielding more desmin- cells than wild-type fibers (P < 0.05 and P < 0.01, respectively). Number of nonhypercontracted fibers analyzed (from n animals) was 27 (2) and 25 (3) at P75-P150, 16 (2) and 13 (3) at P220-P240, and 32 (2) and 32 (2) at P720-P785 for control and MSVski, respectively. All trends discussed were also observed from hypercontracted fibers (data not shown; such fibers constituted approximately one-half those cultured) and from single fibers analyzed for desmin expression from these and 2 further mice after between 2 and 5 days in culture.

Young MSVski mice appear to have normal mononucleate cells. The numbers of both desmin+ and desmin- cells are comparable to those in young wild-type mice. On average, each cultured wild-type fiber yielded 30 ± 6 (n = 27) desmin+ cells (84% ± 4 of total cells; n = 27), whereas MSVski fibers yielded 32 ± 5 (n = 25) desmin+ cells (81% ± 7 of total cells; n = 23). However, as MSVski mice mature, changes in mononucleate cells become apparent. By 8 mos of age, two changes are observed. First, the number of desmin- cells obtained from MSVski fibers increases to 45 ± 24 (n = 13) from 2 ± 1 in wild type (P < 0.01). Second, although desmin+ cell numbers are unchanged overall, their distribution between fibers is less uniform: some fibers yielded more desmin+ cells, whereas more fibers yielded no desmin+ cells. These changes were also observed in aged MSVski mice. Aged MSVski fibers yielded more desmin+ and also more desmin- cells than age-matched controls (P < 0.001; Fig. 7, Table 2). The normal aging-related decline in desmin+ cells is not so marked in MSVski mice, which are undergoing continual regeneration.

Decreased myoblast differentiation efficiency with normal aging and with MSVski. The increased number of desmin+ cells in aged MSVski mice compared with age-matched controls is clearly not sufficient to repair the Ski-induced damage efficiently and prevent the exacerbated aging-related changes in these mice. This is surprising in view of the efficient repair of damage that occurs in young MSVski mice and raises the possibility that decrease in muscle regenerative capacity may be the result of a decrease in myoblast differentiative capacity. We therefore examined whether the properties of myogenic cells were altered in aging wild-type and MSVski mice.

Analysis of proliferative capacity of desmin+ or desmin- cells from all three ages of both wild-type and MSVski mice revealed no significant differences in the rate of cell doubling, which occurred in ~13 h in all cases (data not shown). No evidence of apoptosis was observed in any cultures. Thus changes in intrinsic myoblast proliferation capacity are unlikely to account for either the decline in desmin+ cell yield with age or the enhanced numbers of desmin+ cells in aged MSVski mice.

In contrast to proliferative capacity, myogenic differentiative capacity showed marked changes with age, and these were exacerbated in MSVski mice. After challenge with low-growth factor conditions for 2 days, aging wild-type fiber cultures yielded fewer differentiated myocytes than cultures from younger animals (Fig. 8). In cultures from P75-P150 mice, nearly all desmin+ cells differentiated, as detected by MyHC staining, forming long multinucleated myotubes (Fig. 8A). Quantitation of the proportion of nuclei in desmin+ and MyHC+ cytoplasm revealed that differentiation efficiency gradually decreased in wild-type cultures with increasing age of the mice (P < 0.01; Fig. 8B). Desmin+ cell number itself was significantly decreased at P720-P785 (P < 0.01), with 99 ± 9 (n = 18) desmin+ cells compared with 430 ± 56 (n = 20) at P75-P150 and 554 ± 69 (n = 19) at P220-P240, probably because of the lower numbers of desmin+ cells yielded by aged fibers. This observation raised the possibility that the decline in differentiation efficiency could be due to the reduced density of desmin+ cells in the well. However, several observations argue against this possibility. First, the number of desmin+ cells per culture was similar at P75-P150 and P220-P240, yet the differentiation efficiency declined (P < 0.01, Fig. 9A, right). Second, all but one aged fiber culture contained between 51 and 200 desmin+ cells, for which the mean differentiation efficiency was 46.5% ± 6.7 (n = 17) compared with 88.6% ± 7.5 (n = 4) for young fiber cultures that yielded similarly low numbers of desmin+ cells (Fig. 9A, right). Third, analysis of individual fiber cultures showed no significant correlation between desmin+ cell number and differentiation efficiency at any age (Fig. 9B). The number of desmin- cells was low in wild-type cultures at all ages and did not affect differentiation efficiency of desmin+ cells (Fig. 9A, left). Thus, in wild-type cultures, the differentiation efficiency of myocytes declines in parallel with increasing age, and this decline appears to be intrinsic to the myocytes.


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  		Fig. 8.   Decreased satellite cell differentiation with aging and MSVski. A: fluorescence micrographs of single-fiber cultures stained for the expression of the myogenic cell marker desmin (Cy3, red), all MHC (FITC, green) and DNA (DAPI, blue). Cultures were established from EDL fibers of wild-type and MSVski mice aged P75-P90 and P720. After 3 days in plating medium and 2 days in growth medium, cells were induced to differentiate in a medium low in growth factors for a further 2 days. Differentiated desmin+ cells (arrowheads) were numerous in the cultures from young muscles, whereas in the older cultures, there were more nondifferentiated desmin+ cells (arrows). B: proportion of desmin+ cells expressing MHC in cultures from wild type and MSVski EDL fibers of P75-P150, P220-P240, and P720-P785 mice. Values are means ± SE (n = 20, 19, 18 for wild type and 22, 22, 29 for MSVski at P75-P150, P220-P240, and P720-P785, respectively). Significant differences: *P < 0.05, **P < 0.01.



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  		Fig. 9.   Satellite cell differentiation defect is independent of both myogenic and nonmyogenic cell density. A: independence of differentiation efficiency from desmin+ (right) and desmin- (left) cell numbers at each age. Values are means ± SE with n as indicated in bars. B: comparison of differentiation efficiency of wild-type and aged MSVski fiber cultures yielding similar numbers of desmin+ cells.

In MSVski cultures, a more marked trend of decreased differentiation efficiency was observed with aging (Fig. 8). Young MSVski cultures did not differ significantly from wild-type cultures in any respect, with 85% ± 2 (n = 22) of desmin+ cells containing MHC (Figs. 8, 9). However, cultures from P220-P240 and P720-P785 MSVski fibers showed progressively worse differentiation compared with age-matched wild-type fiber cultures (P < 0.05 and P < 0.01, respectively). Eight-month-old MSVski myoblasts differentiated as poorly [57% ± 5 (n = 22) desmin+ cells containing MyHC] as those of aged wild-type cultures, and aged MSVski myogenic cells differentiated half as well as [23% ± 4 (n = 29) desmin+ cells containing MyHC]. Thus the decline in differentiative efficiency occurs earlier and is more dramatic as MSVski mice age. As in fiber cultures assessed 3 days after explant, older MSVski fibers yielded significantly more desmin- cells after the differentiation period (Fig. 9A, left; P < 0.05 at P220-P240 and P < 0.01 at P720-P785). However, correlation analysis showed that differentiation efficiency declined with age, independent of desmin- cell number. No significant correlation was apparent in any age group (Fig. 9A, left). Similarly, the decline in differentiation efficiency of MSVski fiber cultures with age was not accounted for by decreasing desmin+ cell numbers (Fig. 9A, right). When young and aged MSVski fibers yielding similarly low numbers of desmin- cells (1-400) were compared, aged cells differentiated poorly (Fig. 9A, right). Moreover, the aged MSVski cultures had more desmin+ cells than age-matched wild-type cultures yet differentiated less well independent of desmin+ cell number (Fig. 9). Together, the data show that MSVski desmin+ cells undergo a more marked and earlier loss of differentiation efficiency than wild-type myogenic cells, paralleling the earlier onset of a spectrum of aging-related changes in MSVski muscles.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Four major findings arise from this study of aging and the long-term effects of MSVski-induced muscle hypertrophy. First, fiber-associated myogenic cells lose differentiation efficiency with age. Second, although MSVski mice have larger fibers, these fibers are relatively weak. Third, the MSVski transgene leads to muscle changes suggestive of premature muscle aging, possibly triggered by "covert" muscle damage. Fourth, the premature aging and onset of obvious degeneration in MSVski hypertrophied muscle is preceded by decline in differentiation efficiency of fiber-associated mononucleate myogenic cells.

Defective differentiation of myoblasts from aging mice. Aging mammalian muscle undergoes a characteristic set of changes including loss of force, slower contraction, and poor regenerative ability (44, 63). The primary causes of and the sequence of events leading to such changes are unclear (29). Our finding that myogenic cells from aging muscle have a decreased intrinsic efficiency of differentiation focuses attention on whether this change contributes to the deficits of aging muscle.

Despite extensive analysis in the past of myoblasts derived from aging muscle, poor differentiation has not been described hitherto. Yet the decrease in satellite cell numbers, which we also observed, is well known (8, 66, 67). We believe the key to our analysis is the single-fiber culture method, which allows a high proportion of satellite cells associated with individual fibers to be assessed in a unique culture environment. Satellite cell abundance in mouse EDL muscle is ~1-2% of myonuclei, i.e., five per fiber, and this is consistent with the number of distinct proliferative foci observed during the early days of single-fiber cultures (5, 68). Whole muscle dissociation methods usually have estimated yields of ~0.01% of satellite cells and, therefore, may select for subsets of myogenic cells (57). Such cells frequently show reduced replication capacity but satisfactory differentiation when derived from older tissue (73). We observed neither altered morphology nor altered proliferation in the generality of myogenic cells derived from aged mouse muscle fibers, yet we found poor differentiation. Together with recent demonstrations of the heterogeneity of the myogenic cell compartment in postnatal muscle tissue (5, 6, 30, 42), these findings raise the possibility that distinct myogenic precursor cell pools may show different changes during aging. In situations in which aged satellite cells are forced to undergo myogenesis in a young environment, they perform well (11, 12, 37, 39), so perhaps environmental factors can revive the differentiation capacity of aged satellite cells in a way similar to the reported effect of short-term IGF-I exposure on replicative capacity of myoblasts isolated from aging rats (14). Nevertheless, during the slow aging of muscle the lack of such factors may account for the gradual loss of satellite cell performance and thus contribute to other aging-related changes.

Previously, the large numbers of nonmyogenic fibroblastic cells in whole muscle dissociates hampered analysis of differentiation because fibroblastic cells are more abundant in aging muscle and can inhibit differentiation (53). Our analysis provides two arguments against an inhibitory effect of nonmyogenic cells accounting for reduced differentiation. First, very few desmin- nonmyogenic cells are present in single-fiber cultures from wild-type mice at any age that we examined, and the few there are do not significantly increase with age, either in absolute numbers or as a proportion of desmin+ cells. Second, there is no correlation between differentiation efficiency of desmin+ cells and number or proportion of desmin- cells in cultures from individual fibers of the same mouse at any age examined. Neither does the known cooperativity of myoblast differentiation, together with the reduced numbers of desmin+ cells from aging muscle, account for the poor differentiation: single-fiber cultures yielding similar total desmin+ cell numbers still showed reduced differentiation if derived from aging mice compared with younger animals. Moreover, there was no correlation between desmin+ cell number and differentiation efficiency in single-fiber cultures from young mice. Thus, in contrast to data from selected populations of highly proliferative myogenic cells isolated from aging muscle, which show defects in proliferation (66), our analysis of the behavior of the general population of fiber-associated satellite cells reveals little alteration in satellite cell proliferation. However, deficits in myoblast differentiation efficiency become more marked with age.

Does decreased myoblast differentiation efficiency result from or contribute toward other changes in aging muscle? Our data provide evidence that it is an early marker of muscle aging. CBA/J mice show very limited signs of aging by P550. Yet already by P240, there was a significant reduction in myoblast differentiation efficiency, which preceded any detectable drop in myogenic cell numbers. When other markers of aging such as fiber branching and decreased numbers of satellite cells become apparent, myogenic cell differentiation efficiency is even poorer. As discussed in more detail below, MSVski mice show premature changes similar to those occurring during aging, and these are preceded by premature decline in myoblast differentiation capacity. Thus it appears that decline in myoblast differentiation efficiency is a very early marker of aging-related changes and progressively worsens with age. This suggests that decrease in differentiation efficiency is not a consequence of other known changes. On the contrary, it has the potential to cause or contribute to the later changes.

MSVski, MyoD, and muscle hypertrophy. Our data confirm that the MSVski transgene leads to specific IIb fiber hypertrophy (43, 69). This hypertrophy results from a postnatal increase in type IIb fiber cytoplasmic volume, accompanied by proportionate increase in muscle protein, without significant changes in total fiber number, fiber type, number of nuclei per fiber, or extractable DNA content, suggesting strongly that the over twofold increase in cytoplasmic volume of IIb fibers occurred without a change in the number of myofiber nuclei. Consistent with the view that extra myoblast differentiation is not required for the hypertrophy, we find similar hypertrophy in MSVski.myoD-/- mice that have defective myogenic cell differentiation (49, 62). Moreover, our findings show that MyoD is not required either for the larger size of type IIb fibers (which normally express more MyoD; Ref. 34) or for the differential expression of MSVski between fast and slow muscle (18, 70) or for MSVski-driven muscle hypertrophy. In contrast, manipulation of myogenin, myocyte enhancer factor-2 (MEF-2), and Ras proteins has implicated each in fiber size regulation in rodents (32, 50, 75).

MSVski transgene causes late-onset muscle degeneration. Muscle from young MSVski transgenic mice appears healthy, despite marked hypertrophy. However, we report that subtle early defects in muscle function are followed by a profound late-onset degeneration. During both maximal activation and Mg-rigor conditions, young MSVski fibers generated less force per CSA than control fibers. The reason for this apparent force deficit is unclear, although the lack of change in activation and relaxation kinetics in permeabilized fibers argues against changes in regulatory machinery and suggests a decrease in force generation by myosin heads. Decrease in rigor force, assuming that all myosin heads are attached to actin in normal rigor, also suggests either a decrease in strongly attached myosin heads or a decrease in the tension generated per myosin head (17, 45). Disorganization of intracellular structure, as suggested by the misaligned myofibrils, may prevent force generation or effective transmission of force along the fiber.

In MSVski mice, altered muscle function is followed by onset of muscle degeneration in later life. We have been unable to detect any increase in classic features of fiber damage in young MSVski mice. In particular, BrdU labeling experiments in young MSVski mice failed to demonstrate a significant activation of satellite cell proliferation. Only around one nucleus per whole muscle cross section was labeled in these experiments, and it is unclear whether in control animals these nuclei were satellite cells or nonmuscle cells, so it is possible that a significant increase on satellite cell activation has been overlooked. Whatever the case, the data suggest that muscle degeneration/regeneration in young MSVski muscle does not involve extensive satellite cell activation comparable to that caused by muscle crush or toxin injection. Nevertheless, our observation that genetic ablation of the myoD gene causes premature onset of muscle damage in MSVski mice suggests that satellite cells can mitigate the effects of the transgene in early life. The major described function of myoD in postnatal muscle is to promote satellite cell differentiation during muscle regeneration, so inefficient satellite cell-mediated repair of covert damage could explain the MSVski.myoD-/- phenotype, just as described for mdx.myoD-/- mice (49). Although we cannot rule out some more complex genetic interaction of MSVski and myoD, the simplest view of these findings suggests that covert muscle degeneration occurs in young MSVski mice, which is normally efficiently repaired. As MSVski mice age, abundant central nucleation, fiber branching, and very small fibers appear. These probable degenerative signs are accompanied by a spectrum of changes characteristic of normal murine muscle aging, including fiber atrophy, a shift to slower fiber types, and altered myogenic cell behavior (see below). Because aging-related changes, such as fiber type shifts, may in part be attributed to changes of innervation, the MSVski mice raise the possibilities either that MSVski affects the motor neurons directly or that changes in innervation with age may be "myogenic" in origin.

Whether the observed decline in specific force is pathological and might trigger later changes in MSVski muscle is unclear. Muscle hypertrophy resulting from either overload or compensatory hypertrophy or dystrophy is also accompanied by a decline in specific force (22, 58), so it is uncertain whether the high expression of c-ski is directly harmful to fibers or whether the hypertrophy induced by c-ski expression is the cause. However, the literature is replete with examples of degenerative changes associated with muscle fiber hypertrophy. Muscle overload hypertrophy in rodents is associated with muscle fiber damage (74). In addition, muscle hypertrophy after weight lifting in humans, which involves increase in myonuclear number as well as fiber volume, is associated with elevated serum and urinary markers for muscle damage (54). Hypertrophy-associated damage is often attributed to the training regimes or forces applied to the muscle that elicit hypertrophy. This interpretation may be partially correct. However, our data together with the elevated CK and muscle weakness in MSVski transgenic cattle (9) suggest that muscle with nuclear domain enlargement caused by genetic manipulation may be more fragile and, without a change in stress, may become damaged. No comparative studies of muscle aging in bodybuilders or weight lifters, proper analysis of the relative contribution of nuclear domain enlargement and increase in myonuclear number, or long-term follow-ups of the effects of strength training to promote muscle function in the elderly have been reported. So it is important to determine whether, in susceptible individuals, long-term muscle hypertrophy elicited through training regimes may also contribute, through long-term covert damage and resultant alteration in regenerative capacity of satellite cells, to an ultimately worsened aging prognosis.

Myoblast changes precede most late-onset degenerative changes. Regardless of the exact relationship between early force deficit and the late-onset muscle degeneration in MSVski mice, what triggers muscle degenerative changes when they appear? Several lines of argument point to a contribution of changes in the mononucleate cell population of MSVski mice. First, single-fiber cultures of MSVski muscle show premature aging-related decline in differentiative capacity of myogenic cells. In the context of an enhanced need for muscle repair caused by the transgene, such a decline could account for the appearance of degeneration. Second, the fibers in MSVski mice are highly branched even at P240, before most degenerative changes are apparent, suggesting that the differentiation deficit observed in cell culture is also occurring in vivo. We never observed embryonic myosin in MSVski muscle, so de novo muscle formation is unlikely. Instead, the branched fibers are consistent with inefficient repair of damaged fiber regions. Third, no differences in mononucleate cells between young MSVski and wild-type mice were observed, suggesting that MSVski does not act directly in mononucleate cells but rather triggers the changes indirectly. However, significant changes were present by P240, and these worsened in older MSVski mice. Fourth, nonmyogenic desmin- cell numbers are enhanced in P240 MSVski compared with wild-type mice. Thus mononucleate cell changes arise early, whereas most fiber changes occur later.

We examined the myogenic desmin+ and nonmyogenic desmin- cells for their contribution to the MSVski phenotype. Myogenic cells from P240 MSVski are defective in differentiation compared with both age-matched controls and MSVski myoblasts from younger mice. This defect is not accounted for by decline in myogenic cell number because such decline does not occur in MSVski mice, nor it is caused by increase in nonmyogenic cells because some single-fiber cultures yield no nonmyogenic cells yet still show poor differentiation of myogenic cells. It is possible that the marked increase in nonmyogenic cells is important. However, the clear changes in myogenic cells make it unnecessary to evoke effects of nonmyogenic cells. Instead, premature changes in myogenic cells akin to those occurring in normal aging are sufficient to account for both the exacerbated aging-related changes and the late onset of muscle degeneration in MSVski mice. In future, it will be essential to test this hypothesis and to examine whether human aging and late-onset muscle degenerations (such as muscular dystrophies or spinal muscular atrophies) share elements in common with these murine models.


    ACKNOWLEDGEMENTS

We thank Charlotte Peterson, Steve Hughes, Bernd Krippl, Michael Rudnicki, Kim Wells, Terry Partridge, and Louise Heslop for help and reagents.


    FOOTNOTES

This work was supported by the MRC and European Commission BMH4-CT96-0174 and QLK6-2000-530 to S. M. Hughes. S. B. P. Chargé and A. S. Brack held MRC PhD studentships.

Address for reprint requests and other correspondence: S. M. Hughes, MRC Centre for Developmental Neurobiology, 4th floor south, New Hunt's House, Guy's Campus, King's College London, London SE1 1UL, UK (E-mail: simon.hughes{at}kcl.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 26, 2002;10.1152/ajpcell.00206.2002

Received 3 May 2002; accepted in final form 19 June 2002.


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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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