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
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ABSTRACT |
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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
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INTRODUCTION |
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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)- 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-
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.
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MATERIALS AND METHODS |
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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 -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 -actin cDNA (which readily cross-reacts to mouse
-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.
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RESULTS |
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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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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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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).
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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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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
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DISCUSSION |
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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 desminMSVski, 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.myoDMyoblast 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.
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ACKNOWLEDGEMENTS |
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We thank Charlotte Peterson, Steve Hughes, Bernd Krippl, Michael Rudnicki, Kim Wells, Terry Partridge, and Louise Heslop for help and reagents.
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FOOTNOTES |
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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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