Studies of the dynamics of skeletal muscle regeneration - Canadian ...

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the myosatellite cell, a quiescent precursor cell located between the mature muscle fiber and its sheath of external lamina. To form new fibers in a muscle ...
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REVIEW / SYNTHÈSE

Murray L. Barr Award Lecture 1997 / La conférence Murray L. Barr 1997

Studies of the dynamics of skeletal muscle regeneration: the mouse came back! Judy E. Anderson

Abstract: Regeneration of skeletal muscle tissue includes sequential processes of muscle cell proliferation and commitment, cell fusion, muscle fiber differentiation, and communication between cells of various tissues of origin. Central to the process is the myosatellite cell, a quiescent precursor cell located between the mature muscle fiber and its sheath of external lamina. To form new fibers in a muscle damaged by disease or direct injury, satellite cells must be activated, proliferate, and subsequently fuse into an elongated multinucleated cell. Current investigations in the field concern modulation of the effectiveness of skeletal muscle regeneration, the regeneration-specific role of myogenic regulatory gene expression distinct from expression during development, the impact of growth and scatter factors and their respective receptors in amplifying precursor numbers, and promoting fusion and maturation of new fibers and the ultimate clinical therapeutic applications of such information to alleviate disease. One approach to muscle regeneration integrates observations of muscle gene expression, proliferation, myoblast fusion, and fiber growth in vivo with parallel studies of cell cycling behaviour, endocrine perturbation, and potential biochemical markers of steps in the disease-repair process detected by magnetic resonance spectroscopy techniques. Experiments on muscles from limb, diaphragm, and heart of the mdx dystrophic mouse, made to parallel clinical trials on human Duchenne muscular dystrophy, help to elucidate mechanisms underlying the positive treatment effects of the glucocorticoid drug deflazacort. This review illustrates an effective combination of in vivo and in vitro experiments to integrate the distinctive complexities of post-natal myogenesis in regeneration of skeletal muscle tissue. Key words: satellite cell, cell cycling, HGF/SF, c-met receptor, MyoD, myogenin, magnetic resonance spectroscopy, mdx dystrophic mouse, deflazacort. Résumé : La régénération du muscle squelettique fait intervenir une séquence de prolifération, d’engagement et de fusion des cellules musculaires, de différenciation des fibres musculaires et de communication entre cellules de différents tissus. La cellule myosatellite, une cellule précurseur quiescente localisée entre la fibre musculaire mature et sa lamina externe, est essentielle dans ce processus. Pour former de nouvelles fibres dans un muscle endommagé au cours d’une maladie ou lors d’une blessure, les cellules satellites doivent être activées, proliférer et par la suite fusionner en une cellule multinucléée allongée. Les recherches actuelles portent sur la modulation de l’efficacité de la régénération du muscle squelettique, sur le rôle de l’expression de gènes de régulation de la myogenèse, spécifique de la régénération et distincte de celle notée au cours du développement, sur le rôle de facteurs de croissance, de facteurs de dispersion et de leurs récepteurs spécifiques dans l’amplification du nombre de cellules précurseurs et dans la promotion de la fusion et de la maturation de nouvelles fibres et sur les applications thérapeutiques de ces informations pour atténuer les maladies. Une approche de la régénération musculaire intègre les résultats d’études in vivo de l’expression de gènes musculaires, de la prolifération et la fusion des myoblastes et de la croissance des fibres et d’études parallèles du cycle cellulaire, des changements endocriniens et des marqueurs biochimiques des étapes de la réparation suivie par spectroscopie de résonance magnétique au cours d’une maladie. Des expériences effectuées avec des muscles de patte, de diaphragme et de coeur de souris dystrophiques mdx, faites en parallèle à des essais de thérapie de la dystrophie musculaire de Duchenne, ont aidé à élucider les mécanismes sous-jacents aux effets thérapeutiques positifs du déflazacort, un glucocorticoïde. Cette revue présente une série d’expériences in vivo et en culture permettant d’intégrer la complexité distinctive de la myogenèse postnatale dans la régénération du muscle squelettique.

Received December 5, 1997. Revised February 3, 1998. Accepted February 5, 1998. Abbreviations: HGF/SF, hepatocyte growth factor – scatter factor; TA, tibialis anterior; T3, thyroid hormone. J.E. Anderson. Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, MB R3E 0W3, Canada (e-mail: [email protected]). Biochem. Cell Biol. 76: 13–26 (1998)

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Biochem. Cell Biol. Vol. 76, 1998 Mots clés : cellule satellite, cycle cellulaire, HGF/SF, récepteur c-met, MyoD, myogénine, spectroscopie de résonance magnétique, souris dystrophique mdx, déflazacort. [Traduit par la rédaction]

Introduction The general sequence of morphological steps in the process of muscle regeneration is well characterized by recent reviews (Grounds 1991; Grounds and Yablonka-Reuveni 1993; Bischoff 1994). Differentiated mature fibers transduce a mechanical injury to the sarcolemma, and while debris is removed by phagocytosis, quiescent myosatellite precursor cells are activated to cycle. Satellite cell progeny must then fuse into new fibers called myotubes that subsequently grow and mature. The broad literature on muscle regeneration using autografts, denervation and devascularization, minced muscle grafts, and isografts shows that this sequence more or less repeats itself in each instance of recovery, with variations in timing and outcome that are dependent on strain, age of host, innervation or denervation, graft size, vascular supply, activity, and other factors. Always constant in studies of skeletal muscle tissue regeneration, however, is the huge task of approaching the questions using tools of classical cell and tissue morphology. Studies of cell ultrastructure during postnatal myogenesis, similar to those for developmental muscle formation (Ontell and Kozeka 1984a, 1984b; Ontell et al. 1988a, 1988b), have tracked the details of events during accumulation of fiber debris and membranous vesicles, the sealing of damaged fiber ends, and the fusion of myogenic precursor cells to those remnant fiber stumps (e.g., Robertson et al. 1993). While morphological studies are large in their burden of preparation, sampling, and systematic analysis (e.g., Grounds and McGeachie 1989a; McIntosh et al. 1994; Garrett and Anderson 1995; Pernitsky et al. 1996), the hard-won conclusions from those comprehensive experiments are valuable although somewhat unfortunately find only narrow application. Such narrow application of results is ironically due to the impressively beautiful and unique complexity of skeletal muscle regeneration (e.g., Fig. 1) and the relationship between tissue structure and the functional capacity of muscle. A review, for example of fiber typing, may glean numerous reports that concern the same anatomical muscle but that seldom repeat the experimental approach, the hypothesis, or the species of interest. Quixotically, the accumulating literature on the subject of regeneration seems to focus our attention on increasingly narrow questions to be addressed by powerful tests using single cell systems and bench-top methodologies. Therefore, frequent reminders of context using in vivo experiments are needed to verify (or refute) the results of experiments using in vitro models: those reminders account for the phrase, “the mouse came back.” This paper addresses some key phases of regeneration in vivo and demonstrates a role for our interdisciplinary approach to the dynamics of that process.

DNA synthesis in muscle regeneration Experiments using in vivo injections of tritiated-thymidine for autoradiography have characterized the precursor potential in muscle that is unique to satellite cells (Mauro et al. 1961; Moss

and Leblond 1971). The size and distribution of the satellite cell pool varies among the different types of muscle and under different conditions (Carlson and Faulkner 1996; Carlson 1995; Schmalbruch 1991). The sequence of satellite cell proliferation can be studied by labelling DNA synthesis at specific times postinjury and observing the later dilution of grain density over time (e.g., Roberts et al. 1989). Concrete data on the possible asymmetry of daughter cell fates and the number of generations between activation and fusion can be derived from those experiments. However, for the many investigators and students involved, the frustration of elaborate sampling methodologies, potential or inadequate stains for satellite cells, onerous sectioning and counting processes, and the complex architectural anatomy of muscles have combined to constrain examination of critical events in new muscle formation that rely on satellite cell populations for their success, and which are arguably best examined in vivo. For example, our studies and those of other investigators of disease- or injury-induced muscle regeneration have necessarily relied on important caveats about interpreting cell behaviour on the basis of assumptions of representative, presumptive, or putative precursor populations (Anderson et al. 1987; Zacharias and Anderson 1991; McIntosh and Anderson 1995; Pernitsky et al. 1996). Notwithstanding such qualifications, the formation of myotubes in regeneration can be examined to determine the extent of myoblast proliferation and fusion as an index of new muscle formation (e.g., Anderson et al. 1987; Grounds and McGeachie 1989a, 1989b). Experiments that control variables by age-matching and use of a particular crush-injury model show that regeneration can vary between strains (e.g., BALB/c versus SJL/J: Mitchell et al. 1992; Anderson et al. 1995; C57BL/10 versus mdx: Zacharias and Anderson 1991; McIntosh et al. 1994), with SJL/J and mdx strains demonstrating better regeneration than their respective comparison strains. Interference with new myotube formation results from any divergence of thyroid hormone levels from the euthyroid state, since treatments that induce hyper- and hypo-thyroidism reduce the level of effective regeneration in vivo (Anderson et al. 1994; McIntosh et al. 1994; McIntosh and Anderson 1995; Pernitsky et al. 1996). Other experiments have shown similar effects of increased anabolic steroid levels (Krahn and Anderson 1994). Detailed study of those effects complements, and benefits from, ongoing explorations by others using in vitro culture studies to approach myogenic cell activities in particular. However, the identification of quiescent satellite cells is now possible using their expression of the c-met receptor protein (Cornelison and Wold 1997; Tatsumi et al. 1998) as shown in Fig. 1. While accurate counts of the quiescent and activated precursors will remain onerous, owing to the configuration of thin satellite cell cytoplasm at the extreme periphery of the fiber profile seen by light microscopy, that data will be valuable in future investigation of many disease and regeneration processes in skeletal muscle, including the sequelae of myoblast transplantation experiments. Cell proliferation in a population can also be measured © 1998 NRC Canada

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Review / Synthèse

Fig. 1. In situ hybridization, autoradiography, and immunocytochemistry views showing features of skeletal muscle regeneration. (A) Myoblasts containing myf5 mRNA transcripts (arrows) are present just inside the external lamina sheath of a degenerating myofiber in mdx diaphragm muscle. (B) In this field of muscle regenerating 4 days after a crush injury, cell proliferation is labelled by silver grains over nuclei that have incorporated tritiated-thymidine 24 h prior to tissue collection. A short necrotic segment (open arrow) remains among many mononuclear cells, the identity of which cannot be determined with routine staining. (C) In this autoradiograph of an in situ hybridization experiment, many myf5+ mononuclear cells (arrows) are present between two fiber segments persisting in a region adjacent to the site of injury. The upper arrow indicates a cell that is myf5+ and replicating. (D) The cytoplasm of two myosatellite cells are indicated by immunofluorescence staining for c-met receptor protein, while nuclei and the parent fibers are unstained. (E) A low magnification view of a field of regenerating muscle that contains scattered persistent fiber remnants. Myf5+ mononuclear cells are arranged radially around the segments. (K.L. Garrett and J.E. Anderson, unpublished data; R.E. Allen and J.E. Anderson, unpublished data.) Scale bars = 10 µm (A and D), 20 µm (B and C), and 50 µm (E).

through scintillation counts of isotope or by bromodeoxyuridine uptake in DNA synthesis. More detailed study in vitro of proliferation events can be made using flow cytometry based on DNA content, as is more commonly applied in immunological studies. My colleagues and I recently reported studies of proliferation by myoblasts and fibroblasts that were initially separated using Percoll density gradient centrifugation and light scatter sorting. The differential effects of thyroid hormone (T3) on cycling normal myoblasts were demon-

strated in comparison to myoblasts of the mdx strain, which is resilient to fiber damage and to precocious differentiation induced by T3 treatment (Pernitsky and Anderson 1996; Moor et al.1). Normal control myoblasts show less active proliferation than cycling mdx myoblasts. The mdx dystrophic mouse 1

A.N. Moor, E.S. Rector, and J.E. Anderson. The transition into S-phase is increased by T3 in primary muscle cell cultures of normal but not mdx dystrophic mice. Submitted for publication. © 1998 NRC Canada

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regenerates limb muscle very effectively after the onset of dystrophin-deficient myopathy (reviewed in Partridge 1993). We also found interesting distinctions between cycling of myoblasts alone and cycling by myoblasts recombined with fibroblasts at a known (1:1) ratio, owing to nonadditive differences in cycling between the two cell types (Pernitsky and Anderson 1996). Those observations and others (Anderson et al. 1998) suggest that it is too simplistic to model regeneration in vitro without considering communication from nonmuscle cells and signalling from ligands in the environment of myoblasts. The co-expression of the mitogen basic fibroblast growth factor (bFGF or FGF2) and muscle regulatory genes stands as another example. The bHLH gene myogenin, which is expressed during late myoblast differentiation in embryological development, and bFGF are co-expressed in myoblasts in adult muscle regenerating in vivo (Garrett and Anderson 1995). FGF is also present in muscle in proportion to the effective capacity for regeneration (Anderson et al. 1993, 1994, 1995). By comparison, in vitro studies find a more mutually exclusive expression of the two proteins in cell lines. This and other examples show clearly that results should be interpreted in context of the test or model system and the magnitude of resolution that each system can detect. Such complexity helps to explain the discrepancies between reports of in vivo and in vitro experiments and can give exciting insights into problematic contradictions in the literature. The application of bromodeoxyuridine pulse-labelling of newly synthesized DNA, and its detection with flow cytometry and 2-colour fluorescence, helped to reveal more information about the dynamic process of myoblast cycling. It now appears that there can be phase-specific influences on myoblasts depending on their degree of differentiation or commitment to the myogenic lineage. T3 treatment of normal primary muscle cell cultures slowed the proliferation of differentiating MyoDpositive myoblasts while increasing proliferation by myoblasts prior to MyoD expression (Moor et al., see footnote 1). By comparison, T3 did not affect the transitions into or out of S-phase by cycling mdx myoblasts, providing further evidence of the uniqueness of that dystrophic strain. Such tissue culture studies strongly corroborate the observations from in vivo experiments that excess serum levels of T3 impair regeneration by normal but not mdx mouse muscles (Pernitsky et al. 1996). The data on isolated cell cycling behaviours complemented in vivo studies that earlier had demonstrated the net outcome of cycling changes during regeneration.

Protein localization in muscle regeneration Studies of proteins expressed during muscle development have demonstrated that contractile proteins like myosin heavy chain isoforms, troponins and tropomyosins, and cytoskeletal proteins such as dystrophin are similarly made during fiber differentiation, matrix deposition, and the maturation of innervation, hormonal status, and activity patterns (e.g., Bischoff and Heintz 1994; Maley et al. 1995; Ontell et al. 1995; Walker and Luff 1995). As determined using Western blot assays and tissue culture studies, the sequence of proteins expressed during regeneration was originally presumed to reiterate their expression during development of skeletal muscle (e.g., Miranda et al. 1988) following the rationale that the similar outcomes

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of initial formation and later reformation are due to similar signal processing. That notion is less acceptable since recent studies of muscle regeneration in vivo have reexamined the process (Miller et al. 1993; Rantanen et al. 1995; Bhagwati et al. 1995). For example, our recent studies on a variety of markers previously used to identify differentiated muscle showed that contractile and cytoskeletal proteins such as troponin T (data not shown), developmental myosin heavy chain, and dystrophin can all be localized in mononuclear muscle precursor cells before fusion, in addition to their presence in newly formed differentiating myotubes in regenerating muscle (Fig. 2). The identification of growth factors such as PDGF, FGF-2, TGF-β, and IGF-I (Kardami et al. 1985; Jennische 1989; Yamada et al. 1989; Grounds and Yablonka-Reuveni 1993; Olwin et al. 1994; Husmann et al. 1996; Yablonka-Reuveni and Rivera 1997; Bischoff 1997) and molecules like neural cell adhesion molecule (N-CAM) and m-cadherins (Dickson et al. 1990; Beauchamp et al. 1992; Dubois et al. 1994; Eng et al. 1997; Kuch et al. 1997) has also been used to examine events prior to fusion. For example, Fig. 3 shows that T3-treated normal regenerating muscles have a change in the overall distribution of bFGF, N-CAM, or both epitopes on mononuclear cells, after immunolocalization studies, compared with regenerating muscles in untreated normal control mice and mdx mice with or without T3 treatment. Such data were initial findings from in vivo studies, now known to be consistent with subsequent in vivo and in vitro experiments (Pernitsky et al. 1996; Pernitsky and Anderson 1996; Anderson et al. 1998). Recently, the localization of hepatocyte growth factor– scatter factor (HGF/SF) and c-met receptor protein was examined in vivo in tissue sections (Tatsumi et al. 1998). HGF/SF was previously shown to activate quiescent satellite cells in culture (Allen et al. 1995). We have now shown that HGF/SF is the activating and mitogenic component of crushed muscle extract and is present on intact myofibers in normal muscle (Tatsumi et al. 1998). By comparison, in undamaged muscle c-met is confined to quiescent satellite cells (e.g., Fig. 1D). However, c-met and HGF/SF are intensely colocalized in activated satellite cells. Figures 4A and 4B show a small focal region of damaged muscle above an intact fiber (indicated by the curved arrow, and presented at lower magnification in Fig. 4C). HGF/SF (Fig. 4A) is localized over the periphery of the intact fiber and intensely stains many mononuclear cells in the damaged region. Activated satellite cells (indicated by the straight arrow in Figs. 4A, 4B, and 4C) and later generations of muscle precursors contain both HGF/SF and c-met. The intensity of immunostaining for c-met and HGF/SF gradually declines as myoblasts fuse into myotubes (Tatsumi et al. 1998). This can be observed in the lower row of Fig. 4. Figures 4D and 4E show immunofluorescence for HGF/SF and c-met, respectively, and Fig. 4F shows a partial phase contrast image of the same field. An intact myofiber (m) is shown to the left of the field in each panel, and myoblasts are aligned and fusing into a branched myotube (outlined by broken lines in which two nuclei (n) are also indicated by broken lines). The intensity of staining for both HGF/SF and c-met on the fusing myoblasts and in the myotube itself are very low relative to that observed in the newly damaged muscle shown in Figs. 4A–4C, run in the same experiment. While not strictly quantitative, immunolocalization studies of the satellite cell © 1998 NRC Canada

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Review / Synthèse Fig. 2. Fluorescence micrographs of regenerating muscle showing new myotubes (indicated along their length by the black arrows) and mononuclear cells aligned longitudinally in the muscle (indicated by the white arrows). (A) Developmental myosin heavy chain and (B) dystrophin are present in mononuclear cells. Note that in A the cytoplasm of both small and large diameter new myotubes is filled with devMHC+ fluorescence, which demonstrates early myofibril and sarcomeric striation patterns, whereas in B the small diameter myotube is filled with dystrophin+ fluorescence while the larger fiber (right side) is stained only at the fiber periphery. Scale bar = 10 µm.

and precursor expression of the two epitopes under different conditions can now be viewed in direct relation to the speed or success of muscle regeneration. For example, co-localization in regenerating muscles of bFGF and N-CAM in mononuclear cells (Fig. 3, J.E. Anderson, unpublished data) or HGF/SF and c-met in myoblasts (Fig. 4, R.E. Allen and J.E. Anderson, unpublished data) suggests that the levels of co-expression are correlated with the success and timing of regeneration in vivo. Another example suggests that important data are missing from the literature. Shortly after dystrophin deficiency was identified as the result of mutations causing human Duchenne and mdx mouse muscular dystrophies, the appearance of somatic revertant fibers in dystrophic muscles that express segmental dystrophin (Fig. 5) was described (Hoffman et al. 1990). It is interesting that the push to find treatments to increase dystrophin expression using cell or gene therapy has not found the relationship between the proportion of segmental expression and functional muscle strength. Only very recent experiments connected dystrophin deficiency and physiological alterations with membrane recycling and the electrical integrity of muscle cells (McNeil and Steinhardt 1997). Also not known are the effects of myoblast transfer-induced expression of dystrophin on the density or activation of satellite cells in the dystrophin-positive segments. That information may be important in determining the number of myoblasts

required during treatments, since partial segmental dystrophin expression could further target the mechanical contractile strain on fiber membranes at the muscle–tendon junction to produce new fiber injury. For example, we have noted that c-met-positive satellite cells in stable regenerated mdx muscle are larger and more prominent than in quiescent normal mouse muscle (R.E. Allen and J.E. Anderson, unpublished data). Perhaps clinically important studies can now occur in the new context of HGF/SF, c-met receptor, and dystrophin expression using in vivo experiments and intact fiber studies, where fiber segments with central and peripheral nuclei can be visualized (e.g., Z. Yablonka-Reuveni and J.E. Anderson, manuscript in preparation, Fig. 6). Intact fiber studies may also provide a means to investigate cell diversity in the myogenic lineage and possible multiple pools or subsets of satellite cell precursors (Cossu and Molinaro 1987; Rantanen et al. 1995; Rosenblatt et al. 1995, 1996; Schultz 1996; Pin and Merrifield 1993, 1997). Protein expression can also be used to track the influence of segmental differences in gene expression and reexamine earlier ideas. Very early reports of muscle-like cells in the thymus prompted the hypothesis that the gland held a potential reserve of muscle precursor cells. Examination of thymic myoid cells that express troponins, acetylcholine receptors (Schluep et al. 1987; Meinl et al. 1991), and muscle regulatory genes like © 1998 NRC Canada

18 Fig. 3. Histograms of staining for bFGF and N-CAM in regenerating normal control (CON) and mdx (MDX) mice (n = 4–7), either untreated or treated with thyroid hormone (T3). The proportions (mean % ± SD) of mononuclear cells immunostained for bFGF (open bars), for N-CAM (shaded bars), and for both epitopes (cross-hatched bars) were determined from two-colour photographs of regenerating fields adjacent to the crush injury of TA muscles (McIntosh et al. 1994; Pernitsky et al. 1996). The proportion of N-CAM+ mononuclear cells (i.e., differentiated myoblasts) in this distribution study is increased in regenerating control T3-treated muscles (χ2 statistics). The distribution of the single- or double-stained cells is unchanged in either T3-treated or untreated mdx muscles. The results are consistent with T3 effects that reduce control but not mdx muscle regeneration in vivo (Pernitsky et al. 1996) and decrease only control myoblast proliferation in cycling cultures in vitro (Pernitsky and Anderson 1996). (A.N. Moor (Pernitsky), L.M. McIntosh, and J.E. Anderson, unpublished data.)

MyoD (Grounds et al. 1992a) showed that myoid cells also contain dystrophin and can divide (Wong et al.2). As well, the density of myoid cells is reduced during responses to acute damage of limb muscle injury and lower than normal during dystrophy-induced chronic damage (Wong et al.2). All of these studies show that the expression of particular proteins can be used as valuable adjuncts to studies of muscle regeneration.

Regulatory gene expression in muscle regeneration Suggestions that simpler studies in vitro would be the best test 2

A. Wong, K.L. Garrett, and J.E. Anderson. Myoid cell density in the thymus is lower during mdx dystrophy and is reduced after muscle crush injury. Submitted for publication.

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of new ideas are frequently given to counter proposals for whole animal experiments. However, techniques combining in situ hybridization with protein immunolocalization can bring more specific details to the mechanistic analysis of muscle regeneration. Studies of muscle regulatory gene expression in vivo during muscle regeneration using in situ techniques with radiolabelled or digoxigenin-labelled probes began with the work of Grounds et al. (1992b). Grounds and colleagues showed the sequential appearance of transcripts for the four bHLH genes in activated precursor cells following crush injury-induced regeneration. The identification of committed myogenic cells and the rich background of their detailed studies of regeneration allowed the specific reporting of bHLH gene expression in the advancing front of myogenic precursor proliferation and new myotube formation. In combination with immunostaining for the product of second genes like developmental myosins and growth factors, deductions of colocalization can now be used to test for the possible interplay of gene products in vivo. Unfortunately, despite the clear importance in clinical medicine of basic science observations of in vivo muscle tissue repair, such basic molecular and cell biology applications can appear too specific (i.e., only relevant to muscle) or too descriptive (as interpretation depends on detailed histopathology). Now, expression markers like β-galactosidase production from lac-Z gene insertion at the promotor region of particular genes or mutations can show the sequence and intensity of gene expression and can be applied to regeneration studies (e.g., Li et al. 1997). Such expression studies offer the exciting potential to reveal in vivo processes at the resolution of single cell nuclei. Since satellite cells are definitively myogenic in adult skeletal muscle, the expression of MyoD, myogenin, and other regulatory genes upon activation (Grounds et al. 1992b) suggests that some reiteration of their myogenic nature. Alternatively, a synchronized expression of gene families during myogenic differentiation may be required to optimize the subsequent regeneration of muscle. The application of molecular tools for tracing gene expression with β-galactosidase and blue and green fluorescent proteins will demonstrate exciting new details of the molecular regulation of regeneration. Studies on the MyoD knockout transgenic mouse showed that normal muscle formation occurs during development in that mutant, subsequent to the upregulation of myf5 expression (reviewed in Megeney and Rudnicki 1995; Rudnicki and Jaenisch 1995). Those studies and many others in vivo and in vitro confirmed the functional redundancy of two pairs of muscle regulatory genes, MyoD and myf5, and myogenin and myf6 (Weintraub et al. 1991; Weintraub 1993), overlain on the sequential expression pattern (reviewed in Yun and Wold 1996; Buonanno and Rosenthal 1996; Ludolph and Konieczny 1995). In sharp contrast, studies on muscle regeneration clearly showed that muscles lacking MyoD expression are much less effective than normal muscle in meeting the challenge to regenerate (Megeney et al. 1996). The absence of MyoD expression interfered with the proliferation of committed myoblasts in adult regenerating muscles, and myf5 expression was not able to compensate for that deficiency. Those findings indicated that genes with functional redundance in prenatal muscle development can each have a unique role in postnatal myogenesis. The observation of regenerationspecific gene functions adds to the growing body of evidence that regeneration is not a reiteration of developmental myogenesis © 1998 NRC Canada

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Fig. 4. Fluorescence (A, B, D, E) and phase contrast (C, F) micrographs of mdx dystrophic muscle showing the localization of hepatocyte growth factor/scatter factor (HGF/SF) (A, D) and c-met receptor (B, E). Soon after fiber damage, activated satellite cells (one is shown by the short arrow) contain both HGF/SF (A) and c-met receptor (B), while an undamaged fiber nearby demonstrates peripheral HGF/SF staining (long arrow in A). The same activated satellite cell and intact fiber are indicated in the small focal region of damaged muscle in C, at a lower magnification for orientation. At a later stage of myogenic repair, new multinucleated myotubes (indicated by the broken outlines in D–F) are forming. At this stage, staining for HGF/SF (D) and c-met receptor (E) is less than was present in mononuclear cells immediately after activation. Nuclei in myotubes (n) are unstained for both proteins and appear centrally within the elongated region of staining. (R.E. Allen and J.E. Anderson, unpublished data.) Scale bar = 10 µm.

(e.g., Miller et al. 1993; Bhagwati et al. 1996). Recent experiments further demonstrated that proliferation of committed muscle precursor cells (identified by their expression of muscle regulatory factor genes and the uptake of tritiated-thymidine) is targeted by the lack of MyoD expression and somehow

modulated by background strain. While the population of myogenic precursors early in commitment (Myf5-positive) does not cycle in regenerating MyoD(–/–) muscle, there is some proliferation of fully differentiated precursors that are myogeninpositive in that mutant. By comparison, during the effective © 1998 NRC Canada

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Fig. 5. Light micrographs of normal control (A, B) and mdx dystrophic (C, D) muscle showing anti-dystrophin immunostaining (A, C) and nuclear localization with bisbenzimide staining of DNA (B, D). (A) In normal muscle, each fiber cross section is outlined by a peripheral rim of dystrophin staining, and nuclei are found at the periphery of fibers (B). In one small focus of mdx muscle (C), three dystrophin+ fiber segments are shown (numbered 1, 2, and 3) in a field of dystrophin-negative fibers (data not shown). Of the three dystrophin+ segments in C, most exhibit only nuclei located at the fiber periphery (D). However, one of the dystrophin+ segments (segment 3 in C) contains a central nucleus (arrow in C and D), which indicates that fiber damage and regeneration has occurred where a dystrophin+ (somatic revertant) myosatellite cell can respond to the injury. (E. Kardami and J.E. Anderson, unpublished data.) Scale bar = 25 µm.

regeneration of limb muscles in the mdx mouse, substantial proportions of precursors cycle during expression of myf5 (18%) and myogenin (30%) genes (McIntosh et al.3). Therefore, the net amplification of myoblasts is correlated with the outcome of new muscle formation in the two strains. As yet unexplained is the mechanism that underlies an intermediate level of proliferation by myogenin-positive precursors in regenerating muscles of the double mutant MyoD(–/–):mdx strain. However, the relation between proliferation of differentiated precursors and the success of muscle regeneration in an animal strain is strongly supported. Other studies of myogenic regulatory gene expression were undertaken to test whether proliferation of precursors could account for the difference in the phenotype of dystrophy between the mdx mouse limb and diaphragm muscles. Since mdx 3

L.M. McIntosh, K.L. Garrett, M.A. Rudnicki, and J.E. Anderson. Regeneration and myogenic cell proliferation correlate with taurine levels in dystrophin and MyoD-deficient muscles. Submitted for publication.

diaphragm muscle is more severely affected by the dystrophin deficiency than the limb muscles (Stedman et al. 1991; Petrof et al. 1993; Dupont-Versteegden and McCarter 1992; DupontVersteegden 1996), we were interested to know whether some reduction in precursor proliferation was involved in the severe phenotypic expression in the mdx diaphragm. We found that while precursors can proliferate during expression of all four muscle regulatory genes in mdx muscle, only the level of proliferation by myf5-positive myoblasts differed between the tibialis anterior (TA) and diaphragm in that mutant strain. The difference also showed that in a severely affected tissue, the level of proliferation in the myogenic precursor populations could not account for the differences between diaphragm and TA muscle phenotypes. Thus, while proliferation of committed precursors might be modified in compensation for a severe expression of dystrophy (e.g., myf5+ cell proliferation is higher in diaphragm, Anderson et al. 1998), proliferation of precursors prior to commitment (pre-MyoD cells, YablonkaReuveni and Rivera 1997; Anderson et al. 1997) and activities of nonmyogenic cells early in regeneration are also important. © 1998 NRC Canada

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Fig. 6. Comparative light micrographs of myogenic tissue culture (A) and an intact fiber preparation (B). (A) Typical branched myotubes are present, formed by the fusion of normal myoblasts in tissue culture. (B) The intact flexor digitorum brevis fiber is cultured on a dish coated with Vitrogen 100 (Collagen Corporation, Freemont, Calif.). B shows a fiber with nuclei stained by DAPI fluorescence. The fiber was isolated from an mdx mouse (10 weeks of age). In the upper part of the panel (above short arrow) a fiber segment is intact and shows many peripheral nuclei above, through, and below the plane of focus. Below the short arrow the fiber exhibits a row of closely placed central nuclei that are all in focus (bent arrow) and satellite cell nuclei at the periphery (e.g., at curved arrow). (Z. Yablonka-Reuveni and J.E. Anderson, manuscript in preparation.) The clear visualization of the two different segments by this preparation suggests that the possibility of differential gene expression and satellite cell activation can be examined between the two pools of satellite cells and myonuclei. Scale bar = 20 µm.

Nuclear magnetic resonance spectroscopy studies of dystrophy and regeneration There is a clinical need for new means to detect muscle tissue status in a noninvasive fashion, since many patients with neuromuscular diseases, for example, are too young to participate in manual assessments of muscle strength. Magnetic resonance (MR) techniques are routinely used to image structure and disease at larger medical centres. Since young patients are most likely to benefit from disease treatments that could prevent further muscle damage, we investigated the use of proton NMR spectroscopy to find potential biochemical signatures of disease progression and treatment. Earlier studies showed that the onset of dystrophy rapidly accelerated at 3 weeks of age and that fiber damage was later reduced as regenerated fibers grew in the mdx limb muscles. Detailed biochemical studies of alpha tropomyosin showed that levels of phosphorylation increased very early in mdx muscles in concert with the onset of regeneration and later reached a low-level plateau slightly above normal levels, since damage was ongoing (D.H. Heeley and J.E. Anderson, unpublished data, Fig. 7). That difference and other changes in other proteins could have some signature

in the resonance of proton bonds and could potentially be used in tracking tissue status. That notion was tested on biopsy samples of limb and diaphragm muscles from normal and mdx mice to determine if proton MR spectroscopy could distinguish patterns of aging or growth in normal muscle, distinguish normal from dystrophic muscle, and follow the progression of muscular dystrophy and muscle regeneration. Tissue samples were collected from animals that were young (< 3 weeks), adolescent (3–6 weeks), and older and examined by spectroscopy using peak measurements and linear discriminant analysis techniques. The peak values were also tested for correlation with a centronucleation index, the histological marker of accumulated regeneration, from the same samples (McIntosh et al. 1998). The normal samples of TA and diaphragm were always distinguished from respective dystrophic muscles and from each other within both strains. Progressive changes associated with aging and growth of normal muscle and dystrophy and regeneration were also distinguished by MR spectroscopy. Interestingly, the distinctions between young normal and young dystrophic limb muscle prior to the onset of fiber damage indicated that proton MR spectroscopy might potentially be © 1998 NRC Canada

22 Fig. 7. Developmental changes in the phosphorylation of alpha-tropomyosin (α-TM) in tibialis anterior muscles of normal C57BL/10 control mice and mdx mutant mice. Phosphorylation is expressed as moles of phosphate per mole of protein 3 100 and was determined by densitometry of two-dimensional electropherograms when the two forms of α-TM had been sufficiently well resolved. Solid circles are mdx muscles. Open circles are control muscles. Each data point corresponds to a separate sample scanned 2 or 3 times. Muscles in dystrophic mice have higher than normal muscle levels of α-TM phosphorylation when muscle regeneration is very active during disease onset and progression, and the level decreases as regeneration is reduced in older animals. However, the levels of α-TM phosphorylation in muscle from older mdx mice are still significantly higher than in normal muscles because there is a persistent low level of ongoing dystrophic damage and regeneration. (D.H. Heeley and J.E. Anderson, unpublished data.)

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and appears to have fewer side effects than prednisone, although both drugs are immunosuppressive compounds (e.g., Reitter 1995; Markham and Bryson 1995; discussed in Anderson et al. 1996a). In a double blind study, deflazacort treatment of young mdx mice for 4 weeks enhanced the formation of new muscle during regeneration from injury and dystrophy and also improved the growth of fibers in both limb and diaphragm muscles. By comparison, prednisone treatment promoted growth but not the formation of new myotubes when compared with the placebo treatment (Anderson et al. 1996b; McIntosh et al., see footnote 3). The spectroscopy data from the deflazacort study corroborated earlier MR results and showed again that the broad taurine peak intensity increased in the mdx muscles after treatment-induced increases in new muscle formation (McIntosh et al. 1997). An infrared spectroscopy study of the same set of mdx muscles from the deflazacort trial showed that the absorbance peaks of the muscle tissues also reproducibly separated groups on the basis of treatment with deflazacort (high and low dose), prednisone, or placebo (Shaw et al. 1996). While more work is needed to account for the results of muscle spectroscopy experiments, studies on the mdx, MyoD-deficient, and double mutant mice (Anderson et al. 1996b; McIntosh et al., see footnote 3) support the ideas that taurine levels are greater in muscles with a larger ability to regenerate, and that the levels decrease in concert with lower regeneration and less proliferation by myogenic precursors. The large potential for noninvasive monitoring of disease progression and treatment effects in skeletal muscle is exciting, even though the precise explanation for a relation between proliferation, myogenic commitment, and taurine levels is not understood.

Why study regeneration?

applied in early diagnosis of some disease states where fiber pathology may not be explained by a known genetic mutation. Finally, there was a strong, significant correlation between the centronucleation index of accumulated regeneration and various spectral peaks measured in the same sample. Peaks attributed to taurine plus carnitine were particularly identified to increase with age and growth in normal mice. The same peaks were larger in normal than mdx mice at the youngest age, and then increased in mdx muscles during active dystrophy and reached a level higher than normal in the oldest mdx group. The regions of the muscle spectrum were also highly correlated to centronucleation that is accumulated from prior regeneration in the muscles, even though the peaks in ex vivo spectra were very broad and were identified only by the major apparent contributions to their intensity. Since taurine stabilizes membranes and promotes proliferation in addition to playing a cardioprotectant role, the findings are under further investigation in our laboratory. In a final test, the effects of deflazacort treatment on mdx mouse dystrophy were investigated by MR spectroscopy in parallel with the histopathological studies of the treated groups. Deflazacort is a glucocorticoid derived from prednisone and is currently in use in clinical trials and by permission to treat boys with Duchenne muscular dystrophy. Deflazacort slows or reduces progression of muscle weakness

These illustrations of in vivo tissue studies emphasize that the complexity of muscle regeneration can be useful in formulating and testing clinically relevant hypotheses. Studies of muscle regulatory and other gene expression by satellite cells on intact fibers (e.g., Yablonka-Reuveni and Rivera 1994, 1997; Bischoff 1990, 1997) as one model of in vivo muscle tissue, and the study of individual nuclei in transgenic models with coloured or fluorescent protein markers of gene activity will undoubtedly permit investigators to make huge strides in compiling a more complete profile of muscle regeneration. We know that development can proceed with apparent normalcy in the face of mutations in putatively critical regulatory genes. However, the outward function of muscle in the adult depends on the ability of muscles to meet the ultimate challenge: to regenerate new muscle after injury or during disease-induced damage. The achievement of regeneration through satellite cell activation and myogenic precursor proliferation, differentiation, and fusion into myofibers involves multiple regulatory influences on gene expression by myogenic and nonmyogenic cells, each of which may place its own limitations on the outcome. The linkage of observations from wide-ranging perspectives of cell and molecular biology to NMR and infrared spectroscopy emphasizes the complexity of muscle regeneration processes. Ongoing studies of neuromuscular diseases, myogenic development, and satellite cell biology will each gain from the increasing integration of data that is demanded by in © 1998 NRC Canada

Review / Synthèse

vivo analysis of the sequential processes that underlie functional muscle regeneration.

Acknowledgments The author acknowledges the fruitful collaborations with Drs. M.D. Grounds, Z. Yablonka-Reuveni, E. Kardami, M.A. Rudnicki, R.E. Allen, and D.H. Heeley, and the work by students (M. Krahn, L.M. McIntosh, A.N. Moor (Pernitsky), A. Wong, R. Poettcker, K.-E. Granberg) and a postdoctoral fellow (Dr. K.L. Garrett). Support for the research program was through grants from the Manitoba Health Research Council, the Paul H.T. Thorlakson Foundation, the Children’s Hospital Research Foundation, the Muscular Dystrophy Association of Canada, the Muscular Dystrophy Association (U.S.A.), and the Canadian Heart and Stroke Foundation in addition to numerous studentships and fellowships. The technical expertise of R. Simpson and C. Vargas is gratefully recognized.

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24 Kardami, E., Spector, D., and Strohman, R.C. 1985. Myogenic growth factor present in skeletal muscle is purified by heparinaffinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 82: 8044–8047. Krahn, M.J., and Anderson, J.E. 1994. The effects of anabolic steroid treatment on muscular dystrophy in the mdx mouse. J. Neurol. Sci. 125: 138–146. Kuch, C., Winnekendonk, D., Butyz, S., Unvericht, U., Kemler, R., and Starzinski-Powitz, A. 1997. M-cadherin-mediated cell adhesion and complex formation with the catenins in myogenic mouse cells. Exp. Cell Res. 232: 331–338. Li, Z., Mericskay, M., Agbulut, O., Butler-Browne, G., Carlsson, L., Thornell, L.-E., Babinet, C., and Paulin, D. 1997. Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. J. Cell Biol. 139: 129–144. Ludolph, D.C., and Konieczny, S.F. 1995. Transcription factor families: muscling in on the myogenic program. FASEB J. 9: 1595–1604. Maley, M.A.L., Davies, M.J., and Grounds, M.D. 1995. Extracellular matrix, growth factors, genetics: their influence on cell proliferation and myotube formation in primary cultures of adult mouse skeletal muscle. Exp. Cell Res. 219: 169–179. Markham, A., and Bryson, H.M. 1995. Deflazacort. A review of its pharmacological properties and therapeutic efficacy. Drugs, 50: 317–333. Mauro, A. 1961. Satellite cells of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9: 493–495. McIntosh, L.M., and Anderson, J.E. 1995. Hypothyroidism prolongs and increases mdx muscle precursor proliferation and delays myotube formation in normal and dystrophic limb muscle. Biochem. Cell Biol. 73: 181–190. McIntosh, L., Pernitsky, A.N., and Anderson, J.E. 1994. The effects of altered metabolism (hypothyroidism) on muscle repair in the mdx dystrophic mouse. Muscle Nerve, 17: 444–453. McIntosh, L.M., Granberg, K.-E., Brière, K.M., and Anderson, J.E. 1998. An 1H-NMR spectroscopy study of muscle growth, mdx dystrophy, and glucocorticoid treatment: correlation with repair. NMR Biomed. In press. McNeil, P., and Steinhardt, R.A. 1997. Loss, restoration, and maintenance of plasma membrane integrity. J. Cell Biol. 137: 1–4. Megeney, L., and Rudnicki, M.A. 1995. Determination versus differentiation and the MyoD family of transcription factors. Biochem. Cell Biol. 73: 723–732. Megeney, L.A., Kablar, B., Garrett, K.L., Anderson, J.E., and Rudnicki, M.A. 1996. MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev. 10: 1173–1183. Meinl, E., Klinkert, W.E.F., and Wekerle, H. 1991. The thymus in myasthenia gravis: changes typical for the human disease are absent in experimental autoimmune myasthenia gravis of the Lewis rat. Am. J. Pathol. 139: 995–1008. Miller, J.B., Everitt, E.A., Smith, T.H., Block, N.E., and Dominov, J.A. 1993. Cellular and molecular diversity in skeletal muscle development: news from in vitro and in vivo. BioEssays, 15: 191–196. Miranda, A.F., Bonilla, E., Martucci, G., Moraes, C.T., and DiMauro, S. 1988. Immunocytochemical study of dystrophin in muscle cultures from patients with Duchenne muscular dystrophy and unaffected control patients. Am. J. Pathol. 132: 410–416. Mitchell, C.A., McGeachie, J.K., and Grounds, M.D. 1992. Cellular differences in the regeneration of skeletal muscle: a quantitative histological study in SJL/J and BALB/C mice. Cell Tissue Res. 269: 159–166. Moss, F.P., and Leblond, C.P. 1971. Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170: 421–436. Olwin, B.B., Hannon, K., and Kudla, A.J. 1994. Are fibroblast growth factors regulators of myogenesis in vivo? Prog. Growth Factor Res. 5: 145–158.

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Notes about the author

Notes sur l’auteure

This article is based on Dr. Judy E. Anderson’s Murray L. Barr Award Lecture of the Canadian Society of Anatomy, Neurobiology and Cell Biology that was presented at the 41st Annual Meeting of the Canadian Federation of Biological Societies in Quebec City, June 1997. Dr. Anderson was born in Vancouver, B.C., and obtained a B.Sc. (Zoology) from the University of British Columbia. She completed a Ph.D. from the University of Manitoba on “Morphometric, Histochemical and Hormonal Studies of the Testis in Experimental Diabetes Mellitus in the Rat” under the supervision of Dr. J. Thliveris. Dr. Anderson did postdoctoral work on muscle regeneration and the mdx dystrophic mouse with Drs. B.H. Bressler and W.K. Ovalle at the University of British Columbia. In 1988, she joined the Department of Anatomy at the University of Manitoba, Winnipeg, and now holds the position of Associate Professor of Human Anatomy and Cell Science at the University of Manitoba. She has just completed a term as the President of the Canadian Federation of Biological Societies.

Cet article est basé sur la conférence d’acceptation du prix Murray L. Barr de l’Association canadienne d’anatomie, neurobiologie et biologie cellulaire qui a été décerné au Dr Judy E. Anderson lors du 41e congrès annuel de la Fédération canadienne des sociétés de biologie, tenu à Québec en juin 1997. Dr Anderson est née à Vancouver, B.C. Elle a obtenu un B.Sc. en zoologie de l’Université de la Colombie-Britannique. Puis, sous la direction du Dr J. Thliveris, elle a effectué une étude morphométrique, histochimique et hormonale des testicules de rats rendus diabétiques au cours de son programme de doctorat à l’Université du Manitoba. Après avoir obtenu un Ph.D., Dr Anderson a fait un stage postdoctoral avec les Dr B.H. Bressler et W.K. Ovalle de l’Université de la ColombieBritannique pour étudier la régénération des muscles chez la souris dystrophique mdx. Depuis 1988, elle est membre du Département d’anatomie de l’Université du Manitoba. Elle est maintenant professeure adjointe au Département de biologie cellulaire et d’anatomie humaine de l’Université du Manitoba. Elle vient de compléter un mandat de présidente de la Fédération canadienne des sociétés de biologie.

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