In limb muscles of adult rodents, four distinct fiber types have been recognized (slow type I and fast types IIa, IIx and IIb) that express myosin heavy chains MyHCI, MyHCIIA, MyHCIIX, and MyHCIIB, respectively. In larger muscles such as tibialis anterior and gastrocnemius, there is a heterogeneous distribution of fiber types such that the superficial regions are composed of largely or exclusively types IIb and IIx (≈ 80:20%, respectively, in mouse), with types I and IIa limited to deeper regions. In addition to these four “pure” fiber types, various “hybrid” fiber types exist in which more than one of the MyHCs is expressed. The existence of such hybrid fibers may be taken as evidence in favor of conversion of one fiber type to another and reflects the plasticity of MyHC expression. Perhaps the most striking demonstration of plasticity is that which follows cross-reinnervation of fast-twitch limb muscles by a nerve that previously innervated a slow-twitch muscle such as soleus (SOL) or vice versa. Both the contractile characteristics and the fiber type distribution of the cross-reinnervated muscle are shifted toward those of the muscle originally innervated by the reinnervating nerve. The role of the pattern of activity (number and frequency of action potentials) in determining these characteristics has been clearly demonstrated by the application of different patterns of stimuli either to the muscle directly or to the nerve distal to the site of blockade of motor neuronal action potentials by tetrodotoxin. Indeed, it appears that there are subtly different neuronal activity patterns underlying the expression of each of the four known skeletal muscle–specific MyHCs. Other situations in which such conversion occurs include exercise training, altered thyroid hormone status, and several pathological conditions. The pattern of coexpression of the various MyHCs has led to general acceptance of the following sequence of changes in fiber type. EQUATION
A phasic pattern of activity or increased thyroid hormone level will shift MyHC expression toward the IIB isoform, whereas tonic activity or reduced thyroid hormone levels have the opposite effect.
LIMITED ADAPTIVE RANGE
However, there is some suggestion that the extent of transformation of MyHC expression may be restricted in any given fiber, leading to the concept of a “limited adaptive range.” For example, the slow-twitch SOL of adult rodents is normally composed of type I and IIa fibers, and regardless of the nature of stimulus applied, it has proven difficult or impossible to induce expression of MyHCIIB protein in these fibers, provided that degeneration and subsequent regeneration from satellite cells do not occur (see later). However, a marked increase in MyHCIIX expression, after the stimulation of denervated rat SOL with a pattern of activity characteristic of that normally observed in type IIb fibers, has been reported (2). In addition, stimulation of the denervated fast-twitch extensor digitorum longus muscle (EDL), with a pattern of activity similar to that of the normal SOL, resulted in an increase in expression of MyHCIIA and MyHCIIX but failed to induce MyHCI expression. In rats that were rendered hypothyroid from embryonic day 10 for periods up to 6 months, none of the fibers in the superficial region of the tibialis anterior muscle (TAS), an area that normally contains almost exclusively type IIb fibers, expressed type I MyHC, although in the deeper region of TA, which has a more mixed fiber type distribution, there was a marked increase in the number of fibers expressing the slow isoform (5). It was suggested that these latter fibers were those that would have normally become type IIa fibers (see later) but were driven by the hypothyroid stimulus to maintain MyHCI expression. Furthermore, when the TAS of mice was randomly reinnervated by sectioning the lateral popliteal nerve and rotating the proximal stump through 180 degrees so as to minimize or prevent rematching of the cut ends of the axons, no type I or IIa fibers were detected in the TAS (8), suggesting that these fibers could not be ”respecified“ to express type I or IIA MyHC, thus supporting the concept of a limited range of MyHC expression.
One possible explanation for the putative limited range of adaptation may lie in the existence of discrete populations of myoblasts, each giving rise to a specific subset of myonuclei with distinct patterns of gene expression, as initially proposed for avian muscle (12). It is known that two temporally distinct populations of myoblasts play a role in the development of muscle in utero. In the mouse EDL, for example, one population of myoblasts (primary or embryonic) begins to proliferate around day 11 or 12 of gestation (E11–12), subsequently fusing to give rise to primary myotubes. Secondary (also known as fetal) myoblast proliferation, and subsequent fusion to form secondary myotubes, does not commence until around E16. In SOL (and probably in other muscles of the posterior crural group), both phases are delayed ≈ 48 h (7).
In the rat, the first myotubes to form in vivo initially express MyHCemb and subsequently coexpress MyHCI, whereas secondary myotubes, first seen at E18 lying beneath the basal lamina of preexisting primary myotubes, coexpress MyHCemb and MyHCneo (3). Most of the primary myotubes, however, progressively increase their expression of MyHCneo so that by the late fetal stage, the level of expression of this isoform is greater than that of MyHCI in all muscles except SOL, in which the slow MyHC continues to be the predominant isoform. The population of primary myotubes that continue to express only the slow isoform tends to be localized to the deeper portions of larger muscles. Because these are the areas that, in the adult, are the site of type I fibers, this led to the suggestion that primary myotubes eventually develop into adult slow fibers, whereas secondary myotubes develop into fast fibers. It is now clear, however, that this is an oversimplification: some primary myofibers must subsequently change their MyHC expression to a fast type, because adult EDL and TA muscles contain few, if any, fibers that express MyHCI. Although secondary myotubes in future fast-twitch muscles, maintain their expression of MyHCneo,, it has been suggested that in SOL some secondary myotubes coexpress MyHCI and MyHCneo. However, because these myotubes were reported to be larger than the remaining secondary myotubes and were not associated with primary myotubes, it is possible that these were actually late developing primary myotubes. Indeed, this raises the question of whether primary and secondary myoblasts are defined simply by the time at which they begin to proliferate and/or fuse or whether there are distinct differences between the two populations.
DO PRIMARY AND SECONDARY MYOBLASTS REPRESENT DISTINCT POPULATIONS?
A number of reports have indicated that primary and secondary myoblasts have distinct characteristics. For example, their expression of β-enolase and α integrin differ, and both have been suggested as markers to distinguish between primary and secondary myoblast lineages. In addition, the two populations respond quite differently to transforming growth factor-β under culture conditions. It has also been suggested that myotubes derived from primary and secondary myoblasts exhibit different patterns of MyHC expression. For example, cultures of somitic or early embryonic (E9–12) limb bud myoblasts differentiated almost exclusively (> 93%) into myocytes expressing MyHCemb and MyHCI, with only a very small proportion (< 5%) expressing another developmental isoform, MyHCneo (referred to in the literature as either the neonatal or perinatal isoform) in place of MyHCI. In cultures of myoblasts taken from E13 mice, MyHCneo was detected in about one third of differentiated myocytes and myotubes, whereas as many as 95% of myotubes seen in a 10-d culture of E18 limb muscle myoblasts expressed the neonatal isoform (11). Similar findings have been observed in cultures of rat embryonic and fetal myoblasts, although the different phases of myogenesis occur somewhat later in gestation. These findings are schematically represented in Figure 1.
There is, however, no a priori reason to assume that primary and secondary myoblast populations should fuse only within their own cohort. Although there are no markers of these myonuclear populations, there is some evidence that suggests both primary and secondary myoblasts can actually contribute myonuclei to a single muscle fiber. For example, bromodeoxyuridine labeling indicates that in the mouse at E17, new nuclei (presumably from the secondary myoblast population predominantly present at that stage of development) continue to be incorporated into preexisting (presumably primary) myotubes. Furthermore, both primary and secondary myotubes were labeled in E15–17 mouse hind limb muscles after the injection of a retroviral construct carrying the LacZ gene as a reporter. This suggests that a common pool of cells is able to contribute to both primary and secondary myotubes. Thus, the vast majority of myotubes will likely be “hybrids” resulting from the fusion of variable proportions of primary and secondary myoblasts. Although it has not been possible to date to identify myonuclei derived from primary or secondary myoblasts, the observation that some primary myotubes seem to progressively up-regulate MyHCneo expression during late fetal development at the expense of MyHCI would be entirely consistent with the progressive incorporation of secondary myoblast nuclei into these myotubes.
A HYPOTHETICAL SCHEME TO EXPLAIN FIBER TYPE DIFFERENCES IN MyHC EXPRESSION AND LIMITED PLASTICITY
The results reported here can be incorporated into an hypothetical scheme that, I believe, can explain not only the MyHC expression of any given muscle fiber but also the degree of plasticity exhibited by that fiber. It is assumed that in an adult muscle fiber, those myonuclei derived from primary myoblasts are capable of expressing a limited range of MyHC, specifically types I and IIA, whereas myonuclei of secondary myoblast origin are capable of expression of MyHCIIB and MyHCIIX only. Given the fact that most muscle fibers will contain myonuclei of both origins, the MyHC expression of any fiber will reflect the proportions of primary and secondary myonuclei, which in turn reflects the availability of myogenic precursor cells from each of these populations at the time and location of myotube formation (Figure 2). The effects of neural (e.g., activity pattern) and endocrine (e.g., thyroid hormone) influences on MyHC expression in the adult animal would then be superimposed on a developmental background that limits the extent of plasticity.
The limited adaptive range of both SOL and TAS may be explained by the relative paucity of myonuclei of secondary origin in the former and of primary origin in the latter (Figure 3). As mentioned here, SOL primary myotubes, unlike those in fast-twitch muscles, fail to express MyHCneo; this suggests that most primary myotubes may have formed before the secondary myoblasts begin to proliferate. Furthermore, many secondary myotubes in SOL eventually coexpress MyHCI. This may reflect both the relatively high proportion of primary myoblasts in this muscle as well as the fact that their proliferative phase, being delayed, may overlap substantially with that of the secondary myoblasts. This would result in late-forming myotubes with relatively high proportions of myonuclei of primary origin. As a consequence, cross-reinnervation by a fast nerve or even application of a stimulation pattern typical of type IIb fibers would drive these myonuclei to express the fastest MyHC isoform within their repertoire (i.e., MyHCIIA). The relatively small population of secondary myonuclei should be driven by this stimulus to express either MyHCIIX or MyHCIIB. In fact, at least initially, only IIX protein is detected (2), although with prolonged stimulation, it has been reported that MyHCIIB can be induced. By contrast with SOL, almost all fibers in TAS are derived from secondary myoblasts, because this population forms the majority of myoblasts available at the time and location of the formation of the superficial region of this muscle (5). This would explain the absence of types I and IIa fibers in this region, even as a result of interventions that should drive MyHC expression toward the “slow” end of the spectrum, such as hypothyroidism (5) or reinnervation by axons originally supplying fibers in the deep region of the muscle (8).
Fibers containing both myonuclear cohorts might be expected to express MyHCs characteristic of both populations. However, the vast majority of fibers express only a single MyHC, at least at the protein level, and coexpression of MyHCI and MyHCIIB is never seen (at least not without the simultaneous expression of the IIA and IIX isoforms). This raises the question, “What is the mechanism by which the MyHC expression of a fiber containing two populations of myonuclei is determined?” Is MyHC expression within any given fiber determined by a “simple majority” of myonuclei of one population; do fibers with more myonuclei of primary origin express MyHCI/IIA/IIX, whereas those with a preponderance of secondary myonuclei express MyHCIIB? Alternatively, the MyHC expression of any given myotube may be determined by the characteristics of the first myoblast to differentiate, which subsequently entrains the same MyHC expression in all the other myonuclei within the myotube—the “founder-cell” hypothesis that has been proposed for myogenesis in Drosophila. In either case, what is the mechanism of repression of the MyHC genes within the other population of myonuclei? The progressive shift in MyHC expression from type I to neonatal during the later stages of fetal development would certainly be inconsistent with the founder-cell hypothesis. Further evidence against this hypothesis has been adduced from experiments in which clonal populations of mouse myoblasts have been implanted into chick embryos (9). These clones, when grown in culture, expressed MyHCemb but not MyHCI and were therefore deemed to be “fast.” When they were implanted into regions of the chick limb containing a high proportion of slow fibers, the mouse myoblasts fused to form myotubes expressing MyHCI. This suggests that secondary myoblasts can be induced to express MyHCI and was taken as evidence for an influence of the local environment on MyHC expression of the implanted myoblasts. It is worth noting, however, that no evidence was presented to show that myotubes obtained from these cloned myoblasts could actually express fast (or neonatal) MyHC; the absence of MyHCI was used as an indicator of the “fast” profile of these myoblasts. As pointed out earlier, however, most primary myotubes initially express only MyHCemb, with MyHCI appearing after a delay of 24 to 48 h (3).
Studies with heterokaryons have permitted the conclusion that the expression of some genes by one of the nuclear populations may be repressed. In the case of skeletal muscle, nuclear domains with localized MyHC expression have been shown to exist. The absence of expression of a given gene (e.g., MyHCIIB) in one myonuclear population within any fiber might result from a subthreshold concentration of a specific transcription factor or from the production, by the “dominant” myonuclei, of a substance that represses expression of the “minority” gene. It was recently demonstrated that an increase in intracellular calcium in myotubes results in translocation of the transcription factor NF-ATc (nuclear factor of activated T cells) to myonuclei. In some myotubes, all myonuclei appeared to be labeled with the antibody to NF-ATc, whereas in other myotubes, NF-ATc was detected only in a subset of myonuclei (1). Although the possibility was considered that the labeled myonuclei may be involved in synapse-specific gene expression, it is also noteworthy that the calcium/calcineurin/NF-AT pathway has been suggested to play a role in the regulation of several slow fiber–specific genes (6), raising the possibility that under these conditions, NF-ATc is translocated specifically to myonuclei derived from primary myoblasts (Figure 4).
The myogenic regulatory factor MyoD has also been implicated in specific fiber type specification. MyoD expression is down-regulated by tonic activation of muscle, a pattern characteristic of type I fibers. MyoD has been shown to be localized rather specifically to myonuclei of fibers in areas of muscles that are enriched in either type IIb or IIx but is expressed at very low levels in muscles such as SOL (4). Because the MyHCIIB promoter region contains an E-box with which MyoD interacts, independently of its role in myogenesis, it is tempting to speculate that up-regulation and localization of MyoD in secondary myonuclei might play a pivotal role in the regulation of MyHCIIB expression as a result of interventions known to induce fiber type conversion (Figure 4).
ARE THERE DISCRETE POPULATIONS OF SATELLITE CELLS?
So far, we have considered only the potential role of myoblast populations that are present during fetal development. However, it is also necessary to take into account the possible contribution of satellite cells to the MyHC expression of adult muscle fibers. Satellite cells are myogenic precursors that lie beneath the basal lamina but outside the sarcolemma of muscle fibers in the adult. They are normally in a suspended Go phase of the cell cycle but can be triggered to divide during normal growth, compensatory hypertrophy, or regeneration or after denervation of muscle. Thus, under any of these circumstances, these cells might be expected to add their genetic information to the fiber, thereby potentially altering the gene expression. It is therefore of interest to determine whether distinct lineages of satellite cells exist as proposed earlier for the embryonic and fetal myoblasts.
To answer this, we introduced a method to culture satellite cells derived from single muscle fibers, the MyHC expression of which was subsequently determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Immunocytochemistry of the myotubes arising from these satellite cells indicated that there is a preferential (but in most cases, not absolute) association of a particular satellite cell population with a specific fiber type (10). Thus, in cultures obtained from type I fibers from SOL, a mean of 82% of the myonuclei were located in myotubes expressing MyHCI. When the culture was derived from satellite cells from type IIa fibers, a mean of 64% of the myonuclei were in MyHCI-expressing myotubes, whereas in the case of satellite cells from type IIb fibers from EDL, this number was only 31% (Figure 5). If the distribution of satellite cells on any given fiber reflects the distribution of primary and secondary myonuclei, these results could be interpreted to mean that ≈ 18% of the myonuclei in a SOL type I fiber, ≈ 36% in a type IIa fiber, and 69% in an EDL IIb fiber arise from secondary myoblasts. Interestingly, these numbers are rather similar to those reported for the percentage of β-galactosidase– positive myonuclei in myotubes derived from satellite cells from transgenic mice in which β-galactosidase expression is under the control of the fast MyLC3f promoter (13). Given that MyLC3f preferentially associates with MyHCIIB, it is conceivable that myonuclei expressing the MyLC3f gene may be derived from secondary myoblasts (i.e., those proposed to express MyHCIIB).
In summary, until markers are available to allow distinction between myonuclei or satellite cell nuclei derived from primary and secondary myoblasts, the myogenic lineage hypothesis remains a plausible but as yet unproven basis for the limited adaptive range of MyHC expression.
This work was supported by a grant from the Medical Research Council of Canada.
1. Abbott, K.L. Friday, B.B. Thaloor, D. Murphy, T.J. Pavlath G.K.. Adaptation and cellular localization of the cyclosporine A-sensitive transcription factor NF-AT in skeletal muscle
cells. Mol. Biol. Cell. 9: 2905–2916, 1998.
2. Ausoni, S. Gorza, L. Schiaffino, S. Gundersen, K. Lomo T.. Expression of myosin heavy chain isoforms in stimulated fast and slow rat muscles. J. Neurosci. 10: 153–160, 1990.
3. Condon, K. Silberstein, L. Blau, H.M. Thompson W.J.. Development of muscle fiber types in the prenatal rat hind limb. Dev. Biol. 138: 256–274, 1990.
4. Hughes, S.M. Taylor, J.M. Tapscott, S.J. Gurley, C.M. Carter, W.J. Peterson C.A.. Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle
is controlled by innervation and hormones. Development 118: 1137–1147, 1993.
5. Narusawa, M. Fitzsimons, R.B. Izumo, S. Nadal-Ginard, B. Rubinstein, N.A. Kelly A.M.. Slow myosin in developing rat skeletal muscle
. J. Cell. Biol. 104: 447–459, 1987.
6. Olson, E.N, Williams R.S.. Remodelling muscles with calcineurin. Bioessays 22: 510–519, 2000.
7. Ontell, M. Kozecka K.. The organogenesis of murine striated muscle: a cytoarchitectural study. Am. J. Anat. 171: 133–148, 1984.
8. Parry, D.J. Wilkinson R.S.. The effect of reinnervation on the distribution of muscle fiber types in the tibialis anterior muscle of the mouse. Can. J. Physiol. Pharmacol. 68: 596–602, 1990.
9. Robson, L.G.. Hughes S.M.. Local signals in the chick limb bud can override myoblast lineage commitment: induction of slow myosin heavy chain in fast myoblasts. Mech. Dev. 85: 59–71, 1999.
10. Rosenblatt, J.D. Parry, D.J. Partridge T.A.. Phenotype of mouse muscle myoblasts reflects their fiber type of origin. Differentiation 60: 39–45, 1996.
11. Smith, T.H.. Miller J.B.. Distinct myogenic programs of embryonic and fetal mouse muscle cells: expression of the perinatal myosin heavy chain isoform in vitro. Dev. Biol. 149: 16–26, 1992.
12. Stockdale, F.E. Myogenic cell lineages. Dev. Biol. 154: 284–298, 1992.
13. Yang, J. Kelly, R. Daood, M. Ontell, M. Watchko, J. Ontell M.. Alteration in myosatellite cell commitment with muscle maturation. Dev. Dyn. 211: 141–152, 1998.