ARTICLE |
Correspondence to: Jason D. White, U. of Western Australia, Dept. of Anatomy and Human Biology, Nedlands, WA, Australia 6907. E-mail: jwhite@anhb.uwa.edu.au
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Summary |
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We compared the time course of myogenic events in vivo in regenerating whole muscle grafts in MyoD(-/-) and control BALB/c adult mice using immunohistochemistry and electron microscopy. Immunohistochemistry with antibodies to desmin and myosin revealed a striking delay by about 3 days in the formation of myotubes in MyoD(-/-) autografts compared with BALB/c mice. However, myotube formation was not prevented, and autografts in both strains appeared similar by 8 days. Electron microscopy confirmed myotube formation in 8- but not 5-day MyoD(-/-) grafts. This pattern was not influenced by cross-transplantation experiments between strains examined at 5 days. Antibodies to proliferating cell nuclear antigen demonstrated an elevated level of replication by MyoD(-/-) myoblasts in autografts, and replication was sustained for about 3 days compared with controls. These data indicate that the delay in the onset of differentiation and hence fusion is related to extended proliferation of the MyoD(-/-) myoblasts. Overall, although muscle regeneration was delayed it was not impaired in MyoD(-/-) mice in this model. (J Histochem Cytochem 48:15311543, 2000)
Key Words: MyoD, skeletal muscle, regeneration, whole muscle graft, fusion, proliferation
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Introduction |
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The myogenic regulatory factors (MRFs) are a group of transcription factors whose expression is exclusive to skeletal muscle. To date, the MRFs consist of MyoD (
Mice that lack a functional MyoD gene develop normally, with no overt morphological or physiological skeletal muscle abnormalities, and remain viable and fertile (
Skeletal muscle has an exceptional ability to regenerate after mechanical or chemical injury or pathological insult, and regeneration follows a series of clearly defined events, consisting of infiltration of inflammatory cells, satellite cell (myoblast) activation, and myoblast proliferation and fusion (
Because expression of MyoD ( (
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Materials and Methods |
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Animals
Whole muscle grafts were performed on a total of 38 MyoD(-/-) and 24 BALB/c mice aged 8 weeks (see Table 1). A colony of MyoD(-/-) mice (originally provided by Michael Rudnicki) was established in the Animal Resource Centre at Murdoch University in Perth, Australia. Experiments were conducted in strict accordance with guidelines of the University of Western Australia Animal Ethics Committee and the National Health and Medical Research Council, Canberra, Australia. All animals were housed in individual cages under a 12-hr day/night cycle and allowed access to food and water ad libitum. The null mutation (exon 1 deletion) in MyoD(-/-) mice was confirmed using PCR with primers kindly provided by Dr. Marcia Ontell (Pittsburgh, PA). The BALB/c strain was used as the wild-type control in these studies for two reasons: (a) MyoD(-/-) mice are on a BALB/c-enriched genetic background, and (b) BALB/c muscle exhibits the least efficient regenerative capacity among the different strains investigated to date (
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Surgical Procedure for Muscle Grafts
The transplantation procedure for whole muscle grafts has been described in detail previously (
To examine the relative influence of the (exogenous) host environment compared with (intrinsic) factors within the graft itself, muscles were cross-transplanted between strains. MyoD(-/-) muscles were implanted into both BALB/c and SJL/J hosts (and vice versa) because SJL/L mice show superior regeneration compared with BALB/c mice (
Tissue Collection and Processing
Mice were sacrificed by cervical dislocation at various times (214 days) after grafting. In most cases, hindlegs were fixed immediately in 4% (w/v) paraformaldehyde in PBS for 60 min before transfer to 70% ethanol, in which muscles were dissected out and processed for paraffin sections. Fixed muscles were placed in a Shandon automatic tissue processor, immersed in 70% ethanol, and dehydrated through a series of graded ethanols before being infiltrated and embedded in paraffin. Other grafts were dissected out immediately and snap-frozen in isopentane quenched in liquid nitrogen for immunohistochemistry on frozen sections. In a few cases, muscles were fixed in 2.5% glutaraldehyde in cacodylate buffer for electron microscopy. The numbers of autografted mice sampled at various times after transplantation and processing of grafts for paraffins (P), frozens (F), or electron microscopy (G) are summarized in Table 1.
Immunohistochemistry
Antibodies.
The primary antibodies used were a polyclonal rabbit anti-desmin (Biogenex; San Roman, CA), a mouse monoclonal anti-myosin fast heavy chain (Sigma, St Louis, MO; cat. M-4276), a mouse monoclonal anti-PCNA (NovaCastra Laboratories; Newcastle upon Tyne, UK). Biotinylated secondary antibodies used were a donkey anti-mouse IgG (Jackson Immunoresearch Laboratories; West Chester, PA) and donkey anti-rabbit IgG (Jackson Immunoresearch). The biotin conjugates were detected with horseradish peroxidase-conjugated avidin D (Vector Laboratories; Burlingame, CA).
Immunohistochemical Staining. Desmin and myosin were used to identify myoblasts and myotubes in the muscle grafts.
Desmin immunohistochemistry on paraffin sections required high-temperature antigen retrieval in citrate buffer, pH 6.0 (
Myosin immunostaining was also carried out on paraffin sections using the antigen retrieval conditions described above. The Histomouse Kit (Zymed Laboratories; So. San Francisco, CA) was used to stain tissue sections for myosin on paraffin sections and for tropomyosin on tissue-cultured cells, because these monoclonals were mouse-derived and specific blocking procedures were required to reduce the background staining due to endogenous mouse immunoglobulins. The staining protocol used was per the manufacturer's instructions, based on the biotinavidin amplification system with the secondaries conjugated to peroxidase.
Replicating cells in grafts were stained on paraffin sections using the LSAB kit (DAKO; Carpinteria, CA) for PCNA immunohistochemistry per the manufacturer's instructions.
Analysis of Tissues
Histological analysis and cell counts were performed using Image Pro Plus 4.0. The pattern of regeneration in whole muscle grafts is similar throughout its length. Therefore, all counts were based on representative sections from the mid-region of each graft. The area of the central necrotic zone, the width of the peripheral regenerating zone, and the area occupied by myofibers before (Day 3) and after (Day 14) were calculated to assess the rate of regeneration in all grafts. In myosin-stained sections, the number of positive myotubes in an entire transverse section were counted and tagged to avoid double counting. PCNA-positive cells were counted in five sequential fields at x400 magnification along the edge of each graft immediately adjacent to the underlying TA.
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Results |
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Whole Muscle grafts (Autografts)
Histology.
The typical histological appearance of a transverse section through a whole EDL muscle autograft is shown in Fig 1, and the sequence of histological events during graft regeneration is well described elsewhere (
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Desmin Immunostaining of Paraffin Sections
Desmin is an excellent marker for identifying activated myoblasts and myotubes in vivo (
The pattern of desmin immunostaining was markedly different in MyoD(-/-) autografts. In most grafts up to 7 days, the desmin staining was consistently weak and patchy and the cell boundaries of desmin-positive cells in these grafts were hard to define (Fig 2A and Fig 2C) compared to BALB/c grafts (Fig 2B and Fig 2D). At 7 days, three of the five grafts contained many desmin-positive myotubes and these grafts resembled the Day 5 BALB/c grafts. By Day 8, the number of myotubes in MyoD(-/-) grafts increased to a level similar to BALB/c grafts of the same age.
Myotube Formation in Whole Muscle Autografts
Myosin was used as a marker for the initial stages of myoblast differentiation and subsequent fusion to form myotubes (Fig 2E and Fig 2F). The formation of myosin-positive myotubes was delayed in MyoD(-/-) compared to BALB/c grafts, and the numbers of myosin positive myotubes in typical autografts are summarized in Fig 3. Intensely stained myotubes were present in all four BALB/c grafts at Day 5 (278 myotubes in one typical graft; Fig 2F). In contrast, only very few or no myosin-positive myotubes were seen in all four MyoD(-/-) grafts at Day 5. Similarly, two of the three MyoD(-/-) grafts at Day 6 contained few myotubes (0 and 9 myotubes), although a single MyoD(-/-) graft had significant numbers of myosin-positive myotubes at this time (177 myotubes). At 7 days after grafting, significant numbers of myosin-positive myotubes were seen in most of the five BALB/c grafts. Only two of the five MyoD grafts (Fig 2E) contained many myotubes (229 and 289 myotubes), with the remaining three having 1, 38, and 63 myotubes. At Day 7, the myotubes in both MyoD(-/-) and BALB/c grafts were present mainly in the peripheral regenerating zone, whereas two BALB/c grafts had myotubes in the central regions of the graft. In all grafts examined on Day 8 and later, the distribution of myosin-positive myotubes appeared similar in MyoD(-/-) and BALB/c grafts, although a quantitative analysis was not undertaken.
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EM Studies
Electron microscopy was carried out to observe myoblasts and myotube formation in vivo in MyoD(-/-) and BALB/c Day 5 autografts (Fig 4A and Fig 4B), partially because of difficulties in distinguishing between myoblasts and young myotubes in transverse sections by desmin immunostaining in the MyoD(-/-) grafts. In transverse sections, large numbers of apparently mononucleated "cuffing" cells were seen lying within the contour of the persisting basement membrane of myofibers in MyoD(-/-) autografts (data not shown). Examination of longitudinal sections (LS) clearly defined these as unfused mononucleated cells in the 5-day grafts (Fig 4A). Extensive observations at high power revealed no evidence of sarcomeric organization (indicative of differentiation) in these mononucleated cells but, on the basis of their location and appearance, it appeared likely that they were indeed myoblasts. Large numbers of cells with mitotic figures were noted in these MyoD(-/-) autografts compared with the BALB/c grafts (data not shown). In contrast, in LS sections of BALB autografts at Day 5, mutinucleated myotubes were conspicuous adjacent to surviving myofibers (Fig 4B), in areas where no necrotic sarcoplasm remained, and in areas where many inflammatory cells were present (not shown).
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Additional EM was carried out on 8- and 21-day autografts from both strains to specifically observe the myotubes and numbers of potential satellite cells. The myotubes in Day 8 MyoD(-/-) grafts had many closely packed nuclei that were a little "disorganized" (Fig 4C), and these myotubes resembled the (newly formed) myotubes in Day 5 BALB/c grafts (Fig 4B). In the 8-day MyoD(-/-) grafts, although many unfused cells (presumably myoblasts) were present, none was observed closely apposed to the surface of new myotubes or in a classical satellite cell position (beneath the basement membrane) in either MyoD(-/-) or BALB/c autografts. It was considered that such satellite cells might not be evident until the newly formed myofibers had matured further, and therefore grafts from both strains were also sampled at 21 days for EM analysis. Myotubes in 21-day grafts from both strains had highly organized contractile apparatus (Fig 4D and Fig 4E) and appeared of similar size in terms of both diameter and length. In the four MyoD(-/-) grafts examined, mononuclear cells in the classical satellite cell position under the basement membrane (Fig 4F) appeared more frequent compared with the two BALB/c grafts examined. A comprehensive quantitation was not undertaken, but counting of all satellite cells in 10 grid squares (one grid square is 55 x 55 nm) in single sections (11 nm thick) revealed five and 11 satellite cells in the two MyoD(-/-) grafts (from different animals) and two and two satellite cells in the two BALB/c grafts (from the same animal). There was no marked difference between the two strains in the number of unfused mononuclear cells lying outside the basement membrane. All grafts had extensive interstitial connective tissue and many endothelial cells in intimate contact with myotubes. Nerve endplates were obvious in both MyoD(-/-) and BALB/c grafts, indicating innervation of the grafts.
Cell Proliferation in Grafts
The mononuclear cells labeled by PCNA immunohistochemistry fell into two broad groups: (a) those that were within or closely associated with persisting or new myofibers, which were considered to be myoblasts (Fig 5A); and (b) other cells that included macrophages, fibroblasts, endothelial cells, and unidentified cells, some of which may have been myogenic (Fig 5A). It is acknowledged that some of the cells located in and closely apposed to the basement membrane of necrotic myofibers may also have been macrophages, but for the purposes of this study this was not considered to be significant. When PCNA-stained sections were examined in parallel zones of both strains of autografts (as described earlier), marked differences in the distribution of PCNA-positive cells were noted, and this was especially apparent in Day 5 grafts. In regions of MyoD(-/-) grafts where little necrotic muscle remained, there were few or no myotubes and many PCNA-positive cells (Fig 5B). In contrast, in BALB/c grafts where little necrotic tissue remained, myotube formation was conspicuous and few PCNA-positive cells were present (Fig 5C).
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The number of replicating myoblasts (identified as described above) in grafts at 314 days after transplantation was quantitated and expressed as a percentage of the total number of proliferating cells using PCNA immunohistochemistry (Fig 6). The peak in myogenic cell replication (50% of total replicating cells) in BALB/c grafts occurred by 6 days, after which time replication declined markedly. In MyoD(-/-) grafts, extensive myogenic cell replication was seen from 3 days to 8 days. The decrease in replication observed in BALB/c grafts after 6 days was not seen until 9 days in the MyoD(-/-) grafts. After 8 days in the MyoD(-/-) grafts, the level of replication decreased rapidly to that observed in BALB/c grafts (Fig 6).
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Cross-transplantation Studies
In cross-transplantation studies (Table 1), BALB/c and SJL/J EDL muscles were allografted into MyoD(-/-) hosts to see whether the rate and extent of myotube formation in "normal" grafts was affected by the MyoD-deficient environment. Five days after grafting these were directly compared to autografts of the same species (e.g., SJL/J cross-transplants were compared with SJL/J autografts of the same age). Histological comparison revealed no marked differences between these cross-transplants and the equivalent autograft controls in terms of the infiltration of mononuclear cells and the number and distribution of myotubes (data not shown). Where MyoD(-/-) grafts were allografted into BALB/c or SJL/J hosts to see whether the normal host environment would modify regeneration in the null graft, the time of myotube formation within the MyoD(-/-) graft was not affected (data not shown), and these grafts were indistinguishable from MyoD(-/-) autografts at 5 days, when no or few myotubes were present.
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Discussion |
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Myotube Formation Is Delayed During Regeneration of Whole MyoD(-/-) Muscle Grafts
The whole muscle graft is a highly reproducible model of regeneration, and this study demonstrates a striking delay in the timing, but not an inhibition, of myoblast fusion to form myotubes in vivo in skeletal muscle deficient in MyoD. Long-term grafts of MyoD(-/-) and control BALB/c muscle appeared very similar at the light and EM levels. The delayed formation of hypaxial muscle during embryogenesis also indicates that a similar delay in fusion may occur in developing MyoD(-/-) muscles (
Is Myotube Formation Impaired in MyoD(-/-) Muscles?
Previous studies in MyoD(-/-) muscle regenerating after crush injury (
The question arises of whether myotube formation by MyoD(-/-) myoblasts (apart from being delayed) is also impaired in vivo. This was the conclusion of the crush injury study by
The severe pathology resulting from MyoD deficiency in dystrophic mdx mice (
Sustained Replication of MyoD(-/-) Myoblasts
Comparison of cell replication in MyoD(-/-) and control BALB/c whole muscle autografts showed a high level of PCNA immunostaining in MyoD(-/-) grafts in areas that, in normal grafts at this time, are associated with myotube formation. The delay in fusion of MyoD(-/-) myoblasts shown here and in other studies (
Tissue culture studies in our laboratory confirm an extended phase of myoblast proliferation and delayed myotube formation in primary cultures of MyoD(-/-) muscle (data not shown). This agrees with the detailed tissue culture studies from other laboratories showing that MyoD(-/-) myoblasts continue to proliferate even after the addition of fusion media, and it is suggested that the inefficient withdrawal from the cell cycle of MyoD(-/-) myoblasts is the basis of the delay in differentiation and fusion (
Although it might be considered that enhanced myoblast proliferation would result in "superior" new muscle formation, this does not generally seem to be the case. It was not seen in the present study nor in two other studies of muscle regeneration. The local injection of hepatocyte growth factor (HGF) after freeze injury resulted in increased numbers of myoblasts, but overall myotube formation and regeneration were inhibited in a time- and dose-dependent manner (
Other Factors that May Contribute to the Delayed Myoblast Fusion
Differences in the ability of myoblasts to proliferate, differentiate, and fuse in vivo exist among strains (
There are other instances in which myoblasts can fuse in the absence of MyoD. Fusion is well documented in L6, L8, and BC3H1 myogenic cell lines, which express little or no MyoD (Braun et al. 1989). Most notably, during early myotome development two sub-lineages of myogenic precursors exist, one expressing MyoD and the other Myf5 (
In normal muscle, MyoD+ cells rapidly exit the cell cycle and differentiate (
The Appearance of Desmin-positive Myoblasts Is Altered in MyoD(-/-) Grafts
In MyoD(-/-) grafts, the appearance of desmin-positive myoblasts differed strikingly from BALB/c controls. The desmin staining of MyoD(-/-) myoblasts in vivo was weak and generally ill-defined within the cytoplasm, suggesting that MyoD may play some role in the production or organization of desmin. Decreased or variable desmin immunostaining has been reported previously for MyoD myoblasts in primary cultures (
In desmin(-/-) mice, distinct morphological abnormalities, such as non-aligned fibers with sparse filaments as well as abnormal sarcomeric structure, have been observed in embryonic (
In conclusion, these data demonstrate that myoblasts deficient in MyoD in regenerating adult muscle have sustained proliferation leading to delayed fusion, but that this problem is overcome with time and normal myotubes eventuate. Once myoblasts initiate the differentiation cascade, the rate of myotube formation appears similar to that of wild-type cells. The alternative mechanisms independent of MyoD expression that facilitate fusion remain to be elucidated. These observations suggest that sustained proliferation of myoblasts increases the pool of myoblasts that settle into the satellite cell position. The significance of the observation of unfused "leftover" mononuclear cells and whether these MyoD(-/-) satellite cells might have stem cell properties remain to be clarified.
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Acknowledgments |
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Supported by a grant from the National Health and Medical Research Council, Canberra, Australia.
We would like to acknowledge the considerable technical contribution of Mr Jim Hopley in the processing and sectioning of tissues for autoradiography, immunohistochemistry, and histology. We also thank Mr Mike Archer for all of the electron microscopy and analysis.
Received for publication February 2, 2000; accepted June 7, 2000.
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