myosin isoforms in mammalian skeletal muscle · fibers showing an immunoreactivity profile...

Myosin isoforms in mammalian skeletal muscle

STEFANO SCHIAFFINO AND CARLO REGGIANI Department of Biomedical Sciences and Consiglio Nazionale delle Ricerche Unit for Muscle Biology and Physiopathology, University of Padova, 35121 Padua; and Department of Human Physiology, University of Pavia, 27100 Pavia, Italy

Schiaffino, Stefano, and Carlo Reggiani. Myosin isoforms in mammalian skeletal muscle. J. Appl. Physiol. 77(Z): 493-501, 1994.-Skeletal muscles of different mammalian species contain four major myosin heavy- chain (MHC) isoforms: the “slow” or ,&MHC and the three “fast” IIa-, 11x-, and IIb-MHCs; and three major myosin light-chain (MLC) isoforms, the “slow” MLCls and the two “fast” MLClf and MLC3f. The differential distribution of the MHCs defines four major fiber types containing a single MHC isoform and a number of intermediate hybrid fiber populations containing both ,Wslow- and IIa-MHC, IIa- and 11x-MHC, or 11x- and IIb- MHC. The IIa-, 11x-, and IIb-MHCs were first detected in neonatal muscles, and their expression in developing and adult muscle is regulated by neural, hormonal, and mechanical factors. The transcriptional mechanisms responsible for the fiber type-specific regulation of MHC and MLC gene expression are not known and are presently being explored by in vivo transfection experiments. The functional role of MHC isoforms has been in part clarified by correlated biochemical-physiological studies on single skinned fibers: these studies, in agreement with results from in vitro motility assays, indicate that both MHC and MLC isoforms determine the maximum velocity of shortening of skeletal muscle fibers.

myosin heavy chains; myosin light chains; muscle fiber types; muscle fiber differentiation; velocity of muscle shortening

THE STRUCTURAL and functional diversity of skeletal muscles reflects a variety of myosin isoforms. The myosin molecule is composed of two myosin heavy chains (MHCs), the carboxy-terminal portions of which associate to form an a-helical coiled-coil rod (myosin tail), whereas the amino-terminal portions are separated and form two elongated globular domains (myosin heads). At the head-tail junction each MHC associates with two myosin light chains (MLCs), one belonging to the alkali or MLCl family and one to the regulatory or MLCZ family. Both MHCs (mol mass -220 kDa) and MLCs (mol mass 17-23 kDa) exist in multiple isoforms that are differentially distributed in the various fiber types. Our un- derstanding of the complexity of myosin diversity and of the functional significance of MHC and MLC isoforms has rapidly progressed during the last few years. Major advances in the study of mammalian skeletal muscle myosins have been the recognition of a new “fast-type” MHC isoform that is widely distributed in most skeletal muscles, the characterization of the developmental MHC transitions that lead to the emergence of the adult myosin isoform profile, and the demonstration that both MHCs and MLCs influence the velocity of muscle shortening.

In this brief review we focus on three major aspects: 1)

the fiber type distribution of MHC isoforms in mammalian skeletal muscles and their developmental changes, with particular reference to the “fast” or type II MHCs; 2) the myosin gene families and the regulation of their expression; and 3) the functional role of MHC and MLC variants. Our aim is not to present a comprehensive review of myosin diversity but rather to illustrate recent important advances, discuss open issues, and show the direction of ongoing research.

SARCOMERIC MHC AND MLC ISOFORMS ARE

THE PRODUCT OF MULTIGENE FAMILIES

The catalog of myosin isoforms so far identified is still incomplete: most studies have focused on a few muscles of selected mammalian species, and additional myosin variants are probably present in fibers so far considered to be homogeneous in terms of myosin composition (e.g., the slow fibers; see below). On the basis of available information, it is useful to distinguish between major isoforms, which are widely distributed in most muscles and most animal species, and minor isoforms, which have a restricted distribution in specialized muscles. A list of the major MHC and MLC isoforms identified in rat skeletal muscle is shown in Table 1. Similar forms exist in other

0161-7567/94 $3.00 Copyright 0 1994 the American Physiological Society 493

494 BRIEF REVIEW

TABLE 1. Major MHC and MLC isoforms in rat skeletal muscle

Isoforms Expressed Mainly in

Gene Family

MHC

Developing muscles

Emb-MHC Neo-MHC

Fast muscles

(type II fibers)

IIa-MHC IIb-MHC 11x-MHC

Slow muscles

(type I fibers)

/!USlow-MHC

MLC1 (alkali)

MLC2 (regulatory)

MLClemb MLClf MLCls MLClf MLC3f

MLC2f MLC2f MLC2s

MHC, myosin heavy chain; MLC, myosin light chain; emb, embryonic; neo, neonatal.

mammalian species, such as mouse, guinea pig, and rabbit. The structure of each MHC or MLC isotype is generally conserved between species, whereas greater sequence divergence may be found between different iso- types within the same organism.

Sarcomeric MHCs and MLCs are the product of three multigene families, each presumably derived from a single ancestor gene. The MHC gene family comprises several genes, each coding a distinct isoform, that are located in two clusters. The ,@lslow-MHC gene is closely linked to the cardiac cu-MHC gene on chromosome 14 in both human and mouse (42, 58, 80). The other skeletal muscle genes, including the embryonic and neonatal (or perinatal) isoforms and the adult fast MHCs, are clustered on human chromosome 17 and mouse chromosome 11 (36, 80). At least six MHC genes, some of which are still unidentified, are present in the human chromosome 17 cluster (69,85). At variance with the MHC genes, the MLC genes are dispersed in different chromosomes. Each sarcomeric MLC is coded by a distinct gene, with the exception of the MLClf and MLC3f isoforms, which originate from a single gene by alternative promoters and alternative splicing of the first exons (for review see Ref. 5).

The distribution of the various MHC and MLC isoforms is not always strictly confined to specific muscle fibers or specific stages of development. For example, the embryonic and neonatal MHCs persist in adult extraocular muscles (62, 83) and intrafusal nuclear chain fibers (56), as well as in the adult masseter muscle (10, 18), which also contains the embryonic MLCl (68). Embry- onic and neonatal MHCs and embryonic MLCl are also reexpressed in regenerating (13, 61) and denervated muscle (65). Furthermore, the MLCs typical of fast muscles can be detected in a number of type I (slow) fibers, and, conversely, MLCs typical of slow muscles are found in some type II (fast) fibers. MHC and MLC isoforms can in fact associate in various combinations, generating a large number of myosin molecules (see Ref. 48).

In addition to the major isoforms indicated in Table 1, other MHC and MLC variants have been identified in certain muscles and in certain species. A specific MHC and specific MLCl and MLC2 isoforms have been detected in the fast-contracting jaw-closing muscles and in

the tensor timpani muscle of some carnivores and pri- mates (44, 57). The cardiac cu-MHC protein and the corresponding mRNA are expressed in the rabbit masseter muscle (see Ref. 17). A unique MHC form, coded by a specific gene, is present in most type II fibers of extraocular muscles (62,83). A number of type I fibers in extraocular muscles, as well as the bag fibers of muscle spindles, contain an MHC form antigenically different from the ,&/slow-MHC present in normal slow-twitch fibers and similar to that present in the “slow-tonic” fibers of am- phibians and birds (50). However, this slow-tonic MHC has not yet been characterized, and it is not known whether it derives from a distinct gene. Other plslow- MHC isoforms are probably present in mammalian skeletal muscle. According to a recent report, three antigeni- tally different slow MHC isoforms are sequentially expressed in developing mammalian muscle (28). Two types of MHC transcripts, differing in the 3’ nontrans- lated region, have been recognized by in situ hybridization in two populations of type I fibers present in human skeletal muscle (V. Smerdu, I. Karsch-Mizrachi, M. Campione, L. Leinwand, and S. Schiaffino, unpublished data). A second form of MLCls, called MLClsa while the major isoform is also referred to as MLClsb, was initially identified in rabbit slow skeletal muscles (78). The corresponding gene has recently been identified and found to be expressed in both muscle and nonmuscle tissues (26).

TYPE IIA-, IIB-, AND 11X-MHCS DEFINE DISTINCT

FIBER POPULATIONS IN MAMMALIAN SKELETAL

MUSCLE

Three major populations of type II fibers, including a novel type 11x subset distinct from the IIa and IIb fibers, have been identified in rat skeletal muscle by immunohistochemical staining with different anti-MHC monoclonal antibodies (23, 66, 67). The 11x fibers are not easily distinguished from IIa and IIb fibers by the usual adeno- sinetriphosphatase (ATPase) histochemical reactions and in previous studies were generally confused with the IIb fibers. The 11x fibers are numerous in most leg muscles and are especially abundant in the diaphragm (66), are rich in oxidative enzymes (67), and belong to motor units relatively resistant to fatigue (34). Immunoblotting analyses showed that muscles rich in 11x fibers contain a 11x-MHC isoform distinct from IIa- and IIb-MHCs (66). Three type II MHC isoforms were independently identified by gel electrophoresis (3). Single-fiber studies showed that the band of intermediate mobility between the IIa- and IIb-MHCs, which is called IId-MHC, is distributed in a specific type II fiber population (72). Be- cause 11x-MHC was also separated and identified by immunoblotting as a band of intermediate electrophoretic mobility (3l), it appears that 11x- and IId-MHCs corre- spond to the same isoform (Fig. 1).

Definitive evidence that the novel MHC is a distinct protein and not the product of posttranslational modifi- cation of other MHCs was recently obtained with the isolation of cDNAs corresponding to the specific IIx- MHC mRNA (19). Type IIa-, IIb-, and 11x-MHC cDNAs display different restriction endonuclease maps, thus must derive from different genes, and have unique 3’

BRIEF REVIEW 495

C

DIA TA DIA TA DIA TA FIG. 1. Identification of 4 myosin heavy-chain (MHC) isoforms in rat skeletal muscle by electrophoresis and immu-

noblotting. MHCs present in diaphragm (DIA) and tibialis anterior (TA) muscles of adult rat were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either silver stained (A) or transferred to nitrocellulose and stained with monoclonal antibody BF-32, which reacts with /Glow- and IIa-MHC (B). This same nitrocellulose sheet was subsequently incubated with another monoclonal antibody, RT-D9, which reacts with 11x- and IIb-MHC (C). [From La Framboise et al. (31).]

untranslated sequences that can be used to generate specific probes. In situ hybridization studies using these probes show that IIa-, IIb-, and 11x-MHC mRNAs are differentially distributed in the corresponding fiber types identified by staining of serial sections with specific anti- MHC antibodies (Fig. 2). In agreement with the results of immunocytochemical (66) and electrophoretic studies (72), in situ hybridization also shows that, in addition to fibers containing a single type of MHC mRNA, there are

fibers coexpressing different MHC genes, i.e., fibers containing both p/slow- and IIa-MHC, IIa- and 11x-MHC, or 11x- and IIb-MHC. These hybrid fibers are very numerous in rat muscles; for example, the extensor digitorum longus muscle contains -26% pure 11x fibers, 10% IIa/ 11x hybrid fibers, and 20% IIx/IIb hybrid fibers (19).

Fibers showing an immunoreactivity profile characteristic of type 11x-MHC are numerous in mouse and guinea pig muscles (23), and fibers containing the type IId-MHC

FIG. 2. Distribution of type IIb- (B), 11x- (X), and IIa-MHC (A) mRNAs, determined by /* /* n . . ̂ . . in situ hybridization,

matcnes tnat or corresponcimg protem isotorms, determmed by immunoperoxidase staining. Serial cryosections of rat extensor digitorum longus muscle were incubated with %labeled cRNA probes specific for IIa- (a), 11x- (b), and IIb-MHC mRNA (c): reaction was revealed by autoradiography and visualized by dark field microscopy. Serial cryosections were incubated with monoclonal antibodies reactive with IIa-MHC (d), all MHCs but 11x-MHC (e), and IIb-MHC (f). Bound antibodies were revealed by peroxidase-conjugated secondary antibodies. Hybrid fiber coexpressing 11x- and IIb-MHC mRNA is marked by an open circle.

496 BRIEF REVIEW

Fetal

Neonatal

Postnatal

PRIMARY GENERATION

SECONDARY GENERATION

embryonic (slow, neonatal)

embryonic slow

embryonic neonatal

embryonic neonatal

emb/neo emb/neo emb/neo emb/neo slow c---, 2A M 2x f----, 2B

1 1 1 1

slow 2A

represent a major population in rabbit skeletal muscles (1). A type 11x-MHC is probably expressed also in human skeletal muscle, as suggested by the finding that a human MHC cDNA (59) shows very high homology with the rat 11x-MHC gene in the distinctive 3’ untranslated region and has an identical amino acid sequence at the carboxy- terminal end (19). In situ hybridization shows that this 11x-like MHC transcript is distributed in a specific type II fiber subset in human skeletal muscles (Smerdu et al., unpublished data).

MYOSIN ISOFORM SWITCHING AND FIBER TYPE

DIVERSIFICATION DURING DEVELOPMENT

The development of fast skeletal muscles is accompanied by the progressive substitution of the embryonic. and neonatal MHC isoforms with the adult type II MHC isoforms (82). As discussed above, the appearance of the definitive ,Wslow-MHC is also apparently preceded by two antigenically distinct “slow” isoforms; however, these MHCs have not yet been characterized biochemi- cally (28). A simplified scheme of muscle fiber diversification in rat muscles, based on major and well-docu- mented MHC isoform transitions, is shown in Fig. 3. The first fetal stage is characterized by the differentiation of fibers strongly expressing ,B/slow-MHC, derived from primary generation fibers, and fibers strongly expressing neonatal MHC, derived from both primary and secondary generation fibers (15,40,46). The appearance of the three type II MHC mRNAs, which are first detected a few days after birth in rat hindlimb muscles, defines a second neonatal phase in muscle fiber differentiation (19). Interestingly, the various mRNAs display since the earliest appearance a specific fiber distribution; for example, many fibers of soleus muscle contain IIa- but not IIx- or IIb-MHC transcripts, and many fibers of tibialis anterior contain IIb- but not IIa- or 11x-MHC transcripts, whereas other fibers express 11x-MHC mRNA exclusively. The distribution of the various transcripts prefi- gures the pattern found in adult muscles. During postnatal development the embryonic and neonatal MHC isoforms are progressively eliminated at various time

< l 2x

FIG. 3. Scheme highlights sequential phases of fiber type differentiation in developing rat skeletal muscle based on MHC isoform transitions. First fetal phase is characterized by differential expression of ,Olslow- and neonatal MHC among primary generation fibers and by appearance of secondary generation fibers also expressing high levels of neonatal MHCs. Emergence of IIa-, 11x-, and IIb-MHC in neonatal muscle marks 2nd major phase of fiber type differentiation. Further changes in MHC expression, including disappearance of embryonic (emb) and neonatal (neo) MHCs, take place during postnatal development. ++, Existence of hybrid fibers with mixed MHC composition, i.e., coexpressing slow/IIa-, IIa/IIx-, or IIx/IIb- MHCs. [From DeNardi et al. (19), by copyright permission of the Rockefeller University Press.]

periods according to muscle type and fiber type (65), and further MHC transitions occur with age in different muscles. For example, a switch from IIa-MHC to ,Blslow- MHC expression is found in the rat soleus muscle during early postnatal development (ll), and a progressive disappearance of ,Blslow-MHC is seen in mouse fast leg muscles (81). In old rat muscles there is a relative de- crease of IIb-MHC and a corresponding increase in IIx- MHC (33,70). Accordingly, type 11x motor units become the predominant motor unit type in the tibialis anterior muscle of old rats, apparently as a result of an age-related transition from IIb to 11x motor units (32).

Muscle fiber diversification is also accompanied by changes in MLC composition during fetal and postnatal development. MLClf and MLClemb are the major transcripts in both developing fast and slow fetal muscles, whereas MLCls transcripts are first detected at relatively later fetal stages and are exclusively expressed in prospective type I fibers, namely, the same fibers showing greater accumulation of P/slow-MHC (40, 46). Post- natal development is characterized by the disappearance of MLClemb, which is accompanied by a progressive increase in MLC3f in fast muscles and a switch from the mixed MLClf/MLCls profile to the pure MLCls profile in slow muscles (43). Electrophoretic analyses on single fibers indicate that MLClf and MLC3f generally coexist within the same type II fibers. In rat skeletal muscles, the relative proportion of the two isoforms is variable but MLC3f is significantly more abundant in IIb than in 11x or IIa fibers, with the lowest concentrations being found in IIa fibers (6, 77).

REGULATION OF FIBER TYPE-SPECIFIC MYOSIN

GENE EXPRESSION

Multiple mechanisms, including myoblast predeter- mination, neuronal influences, and thyroid hormone, regulate muscle fiber diversification and myosin gene expression during development (for review see Ref. 25). The expression of myosin genes can be further modu- lated in the adult stages by a variety of factors, such as

BRIEF REVIEW 497

6

5

4

75 23

8

2

1

0

. . . 2A

0.0 0.5 1.0 MLC3fI MLC2f

FIG. 4. Relationship between unloaded shortening velocity (V,) and relative content of MLC3f, expressed with reference to amount of regulatory light chain MLC2, in 3 sets of fast fibers from rat hindlimb skeletal muscles. Each set was formed of fibers containing a single MHC isoform (either IIa, 11x, or IIb) with various proportions of MLC3f. Linear regression analysis (regression lines are shown) showed that V, and MLC3f relative content were significantly correlated in all 3 sets. Significant differences between values of slope (between IIb and 11x) and intercept (between IIa and IIb) were present. [From Bottinelli et al. (6).]

innervation (see Ref. 49), thyroid hormone (see Ref. 30), electrical stimulation (see Refs. 2 and 73), and mechanical factors (see Ref. 37). For example, 11x-MHC can be induced in the rat soleus muscle, in which this isoform is normally absent, by either thyroid hormone treatment, electrical stimulation at high frequency, or mechanical unloading after hindlimb suspension (2, 12, 19). The MHC transitions appear to reflect an obligatory pathway of MHC gene expression in the order I +-+ IIa ++ 11x - IIb (64). Changes in the pattern of activity or in thyroid hormone levels can push the transformation of the fiber phe- notype to the right or to the left along this pathway. This interpretation helps to explain the apparently paradoxi- cal finding (30) that the IIa-MHC gene is upregulated by thyroid hormone in the soleus muscle, which contains a majority of type I fibers, but is downregulated in the extensor digitorum longus muscle, which is composed al- most exclusively of type II fibers. It remains to be established whether the specific patterns of MHC transitions reflect specific rules in the transcriptional regulation of MHC genes and whether MHC gene regulation is af- fected by the chromosomal organization of the MHC gene clusters.

The regulatory DNA sequences and the transcription factors responsible for fiber type-specific myosin gene

expression are not. known. It has been suggested (29,76) that the transcription factors MyoD and myogenin, which are known to play a role in muscle cell differentiation during development (for review see Ref. 79), may be implicated in differential muscle gene expression. MyoD mRNAs are more abundant in type II muscle fibers, whereas myogenin mRNAs are more abundant in type I fibers, and the transformation of fiber types induced by thyroid hormone or by cross-reinnervation is accompanied by a corresponding alteration in the MyoD and myogenin mRNA levels. However, at present there is no direct evidence for a specific role of MyoD and myogenin in the differential regulation of myosin genes in skeletal muscle fibers.

In vitro transfection assays, which are generally used to study muscle gene regulation, are not appropriate to identify fiber type-specific sequences because cultured muscle cells do not undergo fiber type differentiation. On the other hand, in transgenic animals the pattern of transgene expression may differ from that of the corresponding endogenous gene. For example, a transgene made of regulatory sequences of the MLClf/3f gene linked to a bacterial reporter gene is expressed in fast but not in slow muscle fibers like the corresponding endogenous gene. However, at variance with the endogenous gene, the level of expression of the transgene varies according to the type II fiber subtype, in the order IIb > 11x > IIa fibers (20). Similar variations in level of expression between type II fiber subtypes have been observed with a quail fast troponin I transgene (27). It has been suggested that the IIb > 11x > IIa expression hierarchy may represent a fundamental pattern, also possibly involved in the regulation of MHC genes, whereas additional, as yet unidentified, elements would be responsible for en- hancing the expression in 11x and IIa fibers, leading to the homogeneous expression of the endogenous gene in the three type II fiber populations (27). An alternative interpretation is that the abnormal pattern of expression of these transgenes results from differences in genomic imprinting, possibly due to methylation processes, between transgenes and their endogenous counterparts: genomic imprinting has been implicated in the generation of the aberrant rostrocaudal gradient of MLClf/3f transgene expression (21). It will be of interest to determine whether similar variations in gene expression are seen using alternative procedures of in vivo transfection, such as intramuscular injection of plasmid DNA into skeletal muscles of adult animals (84). The efficiency of gene transfer is very low in normal rat muscles, but the level of expression of the transgene can be increased by two orders of magnitude by DNA injection into regenerating muscles (75). We are presently using this experi- mental model to identify the regulatory sequences responsible for the fiber type-specific expression of muscle genes. In a recent study plasmids containing fragments of the Y-flanking region of the mouse MLCls gene linked to bacterial reporter genes showed a much higher level of expression in slow than in fast muscles, like the corresponding endogenous gene, and a specific regulatory ele- ment has been identified by deletion analysis in the MLCls gene promoter (74).

498 BRIEF REVIEW

.25

~/slow 2A 2X 2B 0 .2 .4 .6 MHC MLC3f / MLC2f

.6

FIG. 5. Influence of MHC and MLC isoform composition on myofibrillar ATPase activity was determined in iso- lated fibers from NADH oxidation enzymatically coupled to ATP regeneration, as described by Potma et al. (51). A: differences in ATP hydrolysis rate during isometric contractions between 4 sets of fibers, each containing single MHC isoform. Differences between IIb and 11x and between IIb and IIa fibers were statistically significant, whereas difference between IIa and 11x fibers was not significant. B: V, and ATPase activity plotted vs. relative content of MLC3f in set of 18 fibers containing IIb-MHC. Regression analysis (regression lines are shown) showed that V, (solid line) was significantly related to MLC3f content, whereas ATPase (dashed line) was not.

BOTH MHC AND MLC ISOFORMS DETERMINE

MUSCLE CONTRACTILE PROPERTIES

Since the early studies of Close (14) and Barany (4), the maximum velocity of shortening of fast and slow skeletal muscles has been correlated with the presence of myosin isozymes having high and low ATPase activity, respectively. The higher efficiency in chemomechanical conversion of the slow myosin isozymes has been considered responsible for the greater economy in tension maintainance of slow muscles compared with fast muscles (see Ref. 16). Subsequent studies have shown that shortening velocity may also differ between muscles containing different fast-type myosins. For example, the rat extensor digitorum longus muscle, containing predominantly IIb-MHC, has a higher maximum shortening velocity than the soleus muscle chronically stimulated at high frequency, which contains predominantly 11x-MHC (63). However, interpretation of studies at the whole muscle level is complicated by the heterogeneous composition of most skeletal muscles. Single-fiber studies have shown that the presence of slow MHCs and MLCs is correlated with a lower shortening velocity, a lower power output, and a higher curvature of the force-velocity relationship than fast myosin isoforms (9,24,54,71). The relative role of the MHC and MLC isoforms in determining these differences has not been clearly established. MHC isoforms have been considered as major de- terminants of the different speed of fast and slow muscle fibers (54); however, MLCs may be important as well, as

suggested by the finding that human type IIa fibers that contain exclusively MLCZf have a higher shortening velocity than hybrid IIa fibers containing both MLCZf and MLC2s (35).

The functional role of fast-type MHC and MLC isoforms has been investigated in a number of correlated biochemical-physiological studies on mammalian single fibers. Fibers containing IIa-MHC were found to display a lower maximum velocity of shortening than fibers containing IIb-MHC (9, 22, 35, 55, 71). Fibers containing 11x-MHC are more similar to IIa fibers with respect to shortening velocity, whereas they are similar to IIb fibers with respect to power output and curvature of the force- velocity curve (9). On the other hand, a role of the alkali MLCs was suggested by the finding that the maximum velocity of shortening is higher in fibers that contain higher amounts of MLC3f (22, 24, 71). Interpretation of these results is complicated by the preferential associa- tion of MLClf with IIa-MHC and MLC3f with IIb-MHC (6,41,60,77). Therefore it is not clear whether IIb fibers are faster because they contain IIb-MHC or because they contain more MLC3f. The relative role of MHC and MLC isoforms can only be established by comparing the contractile properties of fibers containing the same MHC isoform but differing in MLC3f relative content or in fibers with similar MLC3f content but different MHC isoform composition. This has been done in two recent studies on single human (35) and rat muscle fibers (6). By using rat muscle fibers containing a single type of MHC, and thus avoiding the further complication of the

BRIEF REVIEW 499

coexistence of multiple MHCs (7), Bottinelli et al. (6) found that the maximum shortening velocity is closely related to the relative content of MLC3f. This finding is in agreement with previous results in skinned fibers in which MLClf was partially substituted with MLC3f (45). However, as shown in Fig. 4, the impact of MLC3f is more pronounced in IIb fibers, where a 10% variation in MLC3f content gives a 16% change in maximum shortening velocity, than in IIa and 11x fibers, where the same 10% variation induces a 4 and 8% change, respectively, of the mechanical parameters. Thus it appears that in rat muscle fibers both MHCs and alkali MLCs determine the maximum shortening velocity: at a given value of MLC3f relative content, velocity depends on MHC composition, and vice versa. On the other hand, Larsson and Moss (35) were not able to find any correlation between maximum shortening velocity and MLC3f relative content in human muscle fibers. However, as suggested by the same authors, interpretation of these results is complicated by the frequent coexistence of fast and slow MLC2 isoforms in a large proportion of type II human fibers and the aforementioned possible influence of MLC2 isoforms in determining the maximum shortening velocity.

The alkali MLC isoform composition does not seem to play a correspondingly important role in the ATP consumption and economy of tension development of isomet- rically contracting fast fibers. As shown in Fig. 5A, during isometric contraction the ATPase activity is significantly higher in rat type II than in type I fibers and, among type II fibers, in fibers containing IIb-MHC than in fibers containing IIa- or 11x-MHC (8). However, no significant relationship was observed between ATPase activity and the relative proportion of MLC3f (Fig. 5B). This conclusion is in agreement with the results of studies on avian fast myosins: the actin-activated ATPase activity of myosin from chicken pectoralis muscle is only little decreased by removal of alkali MLCs (38), and no significant difference in ATPase activity is found between two myosin fractions containing either MLClf or MLC3f (47). Lowey et al. (38,39) have also explored the functional role of MLCs using an in vitro motility assay, i.e., a reconstituted actomyosin system made of fluores- cent actin filaments sliding over chicken pectoralis myosin adhered to nitrocellulose-coated coverslips. The velocity of actin filaments, which can be considered an ana- logue of unloaded velocity of shortening of muscle, is markedly decreased by removal of MLCs (38) and is significantly higher in myosin containing MLC3f than in myosin containing MLClf (39). Using the same ap- proach it was found that myosins from embryonic and early posthatch chicken pectoralis, which contain similar MLC complement but different MHCs, translocate actin at different velocities, suggesting that the heavy chain is the principal determinant of the lower velocity of embryonic compared with neonatal and of neonatal compared with adult muscle myosin (39).

In conclusion, it appears that both MHC and MLC isoforms are responsible for determining the maximum velocity of shortening, whereas only MHCs determine the ATP consumption and the isometric tension cost. This conclusion is consistent with current molecular

models of muscle contraction based on a variety of biochemical and biophysical approaches and confirmed by the recent crystallographic reconstruction of the myosin Sl subfragment (52, 53). The structural model clearly shows that the ATPase site and the actin binding region are located on opposite sides of the distal globular part of the myosin head, which represents the motor domain. This domain undergoes small conformational changes in relation to the hydrolytic cycle; these changes are ampli- fied and transmitted to the thick filament through a 85- A-long a-helix, around which are wrapped the regulatory and alkali MLCs (light-chain binding domain). Within the framework of the new structural information, one can now ask specific questions concerning the properties of the various myosin isoforms. I) Which sequences of the MHC are critical for the molecular events that determine the speed of movement or the ATPase hydrolysis rate? 2) Is the lower shortening velocity associated with MLClf due to changes in the physical properties of the 85-A MHC a-helix (possibly caused by the long amino- terminal extension of MLClf) or to a direct interaction between MLClf and actin that could place a mechanical load on the cycling cross bridges? Several converging lines of research, including molecular biology studies providing the complete sequence of various MHCs, crystallographic studies, and in vitro motility assays with re- combinant normal and mutated MHCs and MLCs, should contribute to answering these and similar questions and to further progress in defining the functional role of the different myosin isoforms present in mammalian skeletal muscle.

Our research was supported by grants from Minister0 dell’univer- sita e della Ricerca Scientifica e Tecnologica of Italy (to S. Schiaffino and C. Reggiani), Agenzia Spaziale Italiana (to S. Schiaffino), and Te- lethon-Italia Grant 343 (to C. Reggiani).

Address for reprint requests: S. Schiaffino, Dept. of Biomedical Sciences, Via Trieste 75, 35121 Padova, Italy.

REFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

Aigner, S., B. Gohlsch, N. Hamalainen, R. S. Staron, A. Uber, U. Wehrle, and D. Pette. Fast myosin heavy chain diversity in skeletal muscles of the rabbit: heavy chain IId, not IIb, pre- dominates. Eur. J. Biochem. 211: 367-372, 1993. Ausoni, S., L. Gorza, S. Schiaffino, K. Gundersen, and T. Lomo. Expression of myosin heavy chain isoforms in stimulated fast and slow rat muscles. J. Neurosci. 10: 153-160, 1990. Bar, A., and D. Pette. Three fast myosin heavy chain in adult rat skeletal muscle. FEBS Lett. 235: 153-155, 1988. Barany, M. ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50: 197-218, 1967. Barton, P. J. R., and M. E. Buckingham. The myosin alkali light chain proteins and their genes. Biochem. J. 231: 249-261, 1985. Bottinelli, R., R. Betto, S. Schiaffino, and C. Reggiani. Both myosin heavy chain and alkali light chain isoforms determine unloaded shortening velocity in rat muscle fibers. J. Physiol. Lond. In press. Bottinelli, R., R. Betto, S. Schiaffino, and C. Reggiani. Maxi- mum shortening velocity and coexistence of myosin heavy chain isoforms in single skinned fast fibers of rat skeletal muscle. J. Mus- cle Res. Cell Motil. In press. Bottinelli, R., M. Canepari, C. Reggiani, and G. J. M. Stienen. Myofibrillar ATPase activity during isometric contraction and isomyosin composition of rat single skinned muscle fibres J. Physiol. Lond. In press.

9. Bottinelli, R., S. Schiaffino, and C. Reggiani. Force-velocity

500 BRIEF REVIEW

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

relation and myosin heavy chain isoform composition in skinned fibres of rat skeletal muscle. J. Physiol. Lond. 437: 655-672, 1991. Butler-Browne, G. S., P. 0. Eriksson, C. Laurent, and L. E. Thornell. Adult human masseter muscle fibres express myosin isozymes characteristic of development. Muscle Nerve 11: 610-620, 1988. Butler-Browne, G. S., and R. G. Whalen. Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Deu. Biol. 102: 324-334, 1984. Campione, M., S. Ausoni, C. Y. Guezennec, and S. Schiaf- fino. Myosin and troponin changes in rat soleus muscle after hindlimb suspension. J. Appl. Physiol. 74: 1156-1160, 1993. Carraro, U., L. Dalla Libera, and C. Catani. Myosin light and heavy chains in muscle regenerating in the absence of the nerve: transient appearance of the embryonic light chain. Exp. Neurol. 79: 106-117, 1983. Close, R. The relation between intrinsic speed of shortening and duration of the active state of muscle. J. Physiol. Lond. 180: 542- 559, 1965. Condon, K., L. Silberstein, H. M. Blau, and W. J. Thompson. Development of fiber types in the prenatal rat hindlimb. Dev. Biol. 138: 256-274, 1990. Crow, M. T., and M. J. Kushmerick. Chemical energetics of slow- and fast-twitch muscles of the mouse. J. Gen. Physiol. 79: 147-166, 1982. D’Albis, A., M. Anger, and A.-M. Lompre. Rabbit masseter expresses the cardiac CY myosin heavy chain gene. FEBS Lett. 324: 178-180, 1993. D’Albis, A., C. Janmot, and J.-J. Bechet. Comparison of myosins from the masseter muscle of adult rat, mouse and guinea pig. Persistence of neonatal-type isoforms in the murine muscle. Eur. J. Biochem. 156: 291-296, 1986. DeNardi, C., S. Ausoni, P. Moretti, L. Gorza, M. Velleca, M. Buckingham, and S. Schiaffino. Type 2X myosin heavy chain is coded by a muscle fiber type-specific and developmentally regulated gene. J. Cell Biol. 123: 823-835, 1993. Donoghue, M. J., J. D. Alvarez, J. P. Merlie, and J. R. Sanes. Fiber type- and position-dependent expression of a myosin light chain-CAT transgene detected with a novel histochemical stain for CAT. J. Cell Biol. 115: 423-434, 1991. Donoghue, M. J., B. L. Patton, J. R. Sanes, and J. P. Merlie. An axial gradient of transgene methylation in murine skeletal muscle: genomic imprinting of rostrocaudal position. Development 116: 1101-1112, 1992. Eddinger, T. J., and R. L. Moss. Mechanical properties of skinned single fibers of identified types from rat diaphragm. Am. J. Physiol. 253 (Cell Physiol. 22): C210-C218, 1987. Gorza, L. Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J. Histochem. Cy- tochem. 38: 257-265, 1990. Greaser, M. L., R. L. Moss, and P. J. Reiser. Variations in contractile properties of rabbit single muscle fibres in relation to troponin T isoforms and myosin light chains. J. Physiol. Lond. 406: 85-98, 1988. Gunning, P., and E. Hardernan. Multiple mechanisms regulate muscle fiber diversity. FASEB J. 5: 3064-3070, 1991. Hailstones, D. L., and P. W. Gunning. Characterization of human myosin light chains Isa and 3nm: implications for isoform evolution and function. Mol. Cell. Biol. 10: 1095-1104, 1990. Hallauer, P. L., H. L. Bradshaw, and K. E. M. Hastings. Complex fiber -type-specific expression of fast skeletal muscle troponin I gene constructs in transgenic mice. Development 119: 691- 701,1993. Hughes, S. M., M. Cho, I. Karsch-Mizrachi, M. Travis, L. Silberstein, L. A. Leinwand, and H. Blau. Three slow myosin heavy chains sequentially expressed in developing mammalian muscle. Dev. Biol. 158: 183-199, 1993. Hughes, S. M., J. M. Taylor, S. J. Tapscott, C. M. Gurley, W. J. Carter, and C. A. Peterson. Selective accumulation of MyoD and myogenin in fast and slow adult skeletal muscle is con- trolled by innervation and hormones. Development 118: 1137-1147, 1993. Izumo, S., B. Nadal-Ginard, and V. Mahdavi. All members of

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science Wash. DC 231: 597-600, 1986. LaFramboise, W. A., M. J. Daood, R. D. Guthrie, P. Moretti, S. Schiaffino, and M. Ontell. Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle. Biochim. Biophys. Acta 1035: 109-112, 1990. Larsson, L., T. Ansved, L. Edstrom, L. Gorza, and S. Schiaf- fino. Effects of age on physiological, immunohistochemical and biochemical properties of fast-twitch single motor units in the rat. J. Physiol. Lond. 443: 257-275, 1991. Larsson, L., D. Biral, M. Campione, and S. Schiaffino. An age related type IIB to 11X myosin heavy chain switching in rat skeletal muscle. Acta Physiol. Stand. 147: 227-234, 1993. Larsson, L., L. Edstrom, B. Lindergren, L. Gorza, and S. Schiaffino. Myosin heavy chain composition and enzyme-histochemical and physiological properties of a novel fast-twitch motor unit type. Am. J. Physiol. 261 (Cell Physiol. 30): C93-C101, 1991. Larsson, L., and R. L. Moss. Maximum velocity of shortening in relation to myosin isoform composition in single fibres from human skeletal muscles. J. Physiol. Lond. 472: 595-614, 1993. Leinwand, L. A., R. E. K. Fournier, B. Nadal-Ginard, and T. B. Shows. Multigene family for sarcomeric myosin heavy chain in mouse and human DNA: localization on a single chromosome. Science Wash. DC 221: 766-769, 1983. Loughna, P. T., S. Izumo, G. Goldspink, and B. Nadal-Gin- ard. Disuse and passive stretch cause rapid alterations in expression of developmental and adult contractile protein genes in skeletal muscle. Development 109: 217-223, 1990. Lowey, S., G. S. Waller, and K. M. Trybus. Skeletal muscle myosin light chains are essential for physiological speeds of shortening. Nature Lond. 365: 454-456, 1993. Lowey, S., G. S. Waller, and K. M. Trybus. Function of skeletal muscle myosin heavy and light chains isoforms by an in vitro motility assay. J. Biol. Chem. 268: 20414-20418, 1993. Lyons, G., M. Ontell, R. Cox, D. Sassoon, and M. Buck- ingham. The expression of myosin genes in developing skeletal muscle in the mouse embryo. J. Cell Biol. 111: 1465-1476, 1990. Mabuchi, K., D. Szvetko, K. Pinter, and F. Sreter. Type IIb to IIa fiber transformation in intermittently stimulated rabbit muscles. Am. J. Physiol. 242 (Cell Physiol. 11): C373-C381, 1982. Mahdavi, V., A. P. Chambers, and B. Nadal-Ginard. Cardiac cy- and ,&myosin heavy chain genes are organized in tandem. Proc. Natl. Acad. Sci. USA 81: 2626-2630, 1984. Margreth, A. Changes in myosin during development. In: Recent Advances in Myology, edited by W. G. Bradley, D. Gardner-Med- win, and J. N. Walton. Amsterdam: Excerpta Med., 1975, p. 307- 309. Mascarello, F., A. Veggetti, E. Carpene, and A. Rowlerson. The tensor tympani muscle of cat and dog contains IIM and slow- tonic fibres: an unusual combination of fibre types. J. Muscle Res. Cell Motil. 3: 363-374, 1982. Moss, R. L., P. J. Reiser, M. L. Greaser, and T. J. Eddinger. Varied expression of myosin alkali light chains is associated with altered speed of contraction in rabbit fast twitch skeletal muscles. In: The Dynamic State of Muscle Fibers, edited by D. Pette. Berlin: de Gruyter, 1990, p. 355-368. Ontell, M. P., M. M. Sopper, G. Lyons, M. Buckingham, and M. Ontell. Modulation of contractile protein gene expression in fetal murine crural muscles: emergence of muscle diversity. Deu. Dyn. 198: 203-213, 1993. Pastra-Landis, S. C., T. Huiatt, and S. Lowey. Assembly and kinetic properties of myosin light chain isozymes from fast skeletal muscle. J. Mol. Biol. 170: 403-409, 1983. Pette, D., and R. S. Staron. Cellular and molecular diversities of mammalian skeletal muscle fibers. Rev. Physiol. Biochem. Pharma- col. 116: l-76, 1990. Pette, D., and G. Vrbova. Neural control of phenotypic expression in mammalian muscle fibers. Muscle Nerve 8: 676-689, 1985. Pierobon, B. S., S. Sartore, M. Vitadello, and S. Schiaffino. “Slow” myosins in vertebrate skeletal muscle. An immunofluores- cence study. J. Cell Biol. 85: 672-681, 1980. Potma, E. J., G. J. M. Stienen, J. P. F. Barends, and G. El- zinga. Myofibrillar ATPase activity and mechanical performance

BRIEF REVIEW 501

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

of skinned fibers from rabbit psoas. J. Physiol. Lond. 474: 303-317, 1994. Rayment, I., H. M. Holden, M. Whittaker, C. B. Yohn, M. Lorenz, K. C. Holmes, and R. A. Milligan. Structure of the actin-myosin complex and its implications for muscle contraction. Science Wash. DC 261: 58-65, 1993. Rayment, I., W. R. Rypniewski, K. Schmidt-Base, R. Smith, D. R. Tomchick, M. M. Benning, D. A. Winkelman, G. We- senberg, and H. M. Holden. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science Wash. DC 261: 50-58, 1993. Reiser, P. J., R. L. Moss, G. G. Giulian, and M. L. Greaser. Shortening velocity in single fibres from adult rabbit soleus muscles is correlated with myosin heavy chain composition. J. Biol. Chem. 260: 9077-9080, 1985. Rome, L. C., A. A. Sosnicki, and D. 0. Goble. Maximum velocity of shortening of three fibre types from horse soleus muscle: implications for scaling with body size. J. Physiol. Lond. 431: 173- 185, 1990. Rowlerson, A., L. Gorza, and S. Schiaffino. Immunohisto- chemical identification of spindle fibre types in mammalian muscle using type-specific antibodies to isoforms of myosin. In: The Mus- cle Spindle, edited by I. A. Boyd and M. W. Gladden. London: Mac- Millan, 1985, p. 29-34. Rowlerson, A., P. Pope, J. Murray, R. B. Whalen, and A. G. Weeds. A novel myosin present in cat jaw-closing muscles. J. Mus- cle Res. Cell Motil. 2: 415-438, 1981. Saez, L. J., K. M. Gianola, E. McNally, R. Feghali, R. Eddy, T. Shows, and L. Leinwand. Human cardiac myosin heavy chain genes and their linkage in the genome. Nucleic Acids Res. 15: 5443-5459, 1987. Saez, L., and L. A. Leinwand. Characterization of diverse forms of myosin heavy chain expressed in adult human skeletal muscle. Nucleic Acids Res. 14: 2951-2969, 1986. Salviati, G., R. Betto, and D. Danieli-Betto. Polymorphism of myofibrillar proteins of rabbit skeletal-muscle fibres. An electrophoretic study of single fibres. Biochem. J. 207: 261-272, 1982. Sartore, S., L. Gorza, and S. Schiaffino. Fetal myosin heavy chains in regenerating muscle. Nature Lond. 298: 294-296, 1982. Sartore, S., F. Mascarello, A. Rowlerson, L. Gorza, S. Au- soni, M. Vianello, and S. Schiaffino. Fibre types in extraocular muscles: a new myosin isoform in the fast fibers. J. Muscle Res. Cell Motil. 8: 161-172, 1987. Schiaffino, S., S. Ausoni, L. Gorza, L. Saggin, K. Gunder- sen, and T. Lsmo. Myosin heavy chain isoforms and velocity of shortening of type 2 skeletal muscle fibres. Acta Physiol. Stand. 134: 565-566, 1988. Schiaffino, S., L. Gorza, S. Ausoni, R. Bottinelli, C. Reg- giani, L. Larsson, L. Edstrom, K. Gundersen, and T. Lsmo. Muscle fiber types expressing different myosin heavy chain isoforms. Their functional properties and adaptive capacity. In: The Dynamic State of Muscle Fibers, edited by D. Pette. Berlin: de Gruyter, 1990, p. 329-341. Schiaffino, S., L. Gorza, G. Pitton, L. Saggin, S. Ausoni, S. Sartore, and T. Lsmo. Embryonic and neonatal myosin heavy chain in denervated and paralyzed rat skeletal muscle. Deu. Biol. 127: 1-11, 1988. Schiaffino, S., L. Gorza, S. Sartore, L. Saggin, S. Ausoni, M. Vianello, K. Gundersen, and T. Lsmo. Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J. Muscle Res. Cell Motil. 10: 197-205, 1989. Schiaffino, S., L. Saggin, A. Viel, S. Ausoni, S. Sartore, and L. Gorza. Muscle fiber types identified by monoclonal antibodies to myosin heavy chains. In: Biochemical Aspects of Physical Exer- cise, edited by G. Benzi, L. Packer, and N. Siliprandi. Amsterdam: Elsevier, 1986, p. 27-34.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

Soussi-Yanicostas, N., J. P. Barbet, C. Laurent-Winter, P. Barton, and G. S. Butler-Browne. Transition of myosin isozymes during development of human masseter muscle. Persistence of developmental isoforms during postnatal stages. Development 108: 239-249, 1990. Soussi-Yanicostas, N., R. G. Whalen, and C. Petit. Five myosin heavy chain genes are organized as a multigene complex in the human genome. Hum. Mol. Genet. 2: 563-569, 1993. Sugiura, Y., H. Matoba, H. Mihata, Y. Kawai, and N. Mura- kami. Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat. Acta Physiol. Stand. 144: 419-423, 1992. Sweeney, H. L., M. J. Kushmerick, K. Mabuchi, F. A. Sreter, and J. Gergely. Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of iso- lated skeletal muscle fibres. J. Biol. Chem. 263: 9034-9039, 1988. Termin, A., R. S. Staron, and D. Pette. Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle. Histo- chemistry 92: 453-457, 1989. Termin, A., R. S. Staron, and D. Pette. Changes in myosin heavy chain isoforms during chronic low-frequency stimulation of rat fast hindlimb muscles. A single fiber study. Eur. J. Biochem. 186: 749-754, 1989. Vitadello, M., R. Jerkovic, R. Di Lisi, R. Kelly, M. Buck- ingham, and S. Schiaffino. Muscle fiber type-specific gene regulatory elements identified by in vivo transfection (Abstract). J. Cell. Biochem. 18D: 532, 1994. Vitadello, M., M. V. Schiaffino, A. Picard, M. Scarpa, and S. Schiaffino. Gene transfer in regenerating muscle. Hum. Gene Ther. 5: 11-18, 1994. Voytik, S. L., M. Przyborski, S. F. Badylak, and S. Kon- ieczny. Differential expression of muscle regulatory factor genes in normal and denervated adult rat hindlimb muscles. Deu. Dy- namics 98: 214-224, 1993. Wada, M., and D. Pette. Relationship between alkali light-chain complement and myosin heavy-chain isoforms in single fast-twitch fibers of rat and rabbit. Eur. J. Biochem. 214: 157-161, 1993. Weeds, A. G. Light chains from slow-twitch muscle myosin. Eur. J. Biochem. 66: 157-173, 1976. Weintraub, H., R. Davis, S. Tapscott, M. Thayer, M. Krause, R. Benezra, T. K. Blackwell, D. Turner, R. Rupp, S. Hollen- berg, Y. Zhuang, and A. Lassar. The myoD gene family: nodal points during specification of the muscle cell lineage. Science Wash. DC 251: 761-766, 1991. Weydert, A., P. Daubas, I. Lazaridis, P. Barton, I. Garner, D. Leader, F. Bonhomme, J. Catalan, D. Simon, J. Guenet, F. Gros, and M. Buckingham. Genes for skeletal muscle myosin heavy chains are clustered and are not located on the same mouse chromosome as a cardiac myosin heavy chain gene. Proc. Natl. Acad. Sci. USA 82: 7183-7187, 1985. Whalen, R. G., D. Johnstone, P. S. Bryers, G. S. Butler- Browne, M. S. Ecob, and E. Jaros. A developmentally regulated disappearance of slow myosin in fast-type muscles of the mouse. FEBS Lett. 177: 51-56, 1984. Whalen, R. G., S. M. Sell, G. S. Butler-Browne, K. Schwartz, P. Bouveret, and I. Pinset-Harstrom. Three myosin heavy chain isozymes appear sequentially in rat muscle development. Nature Lond. 292: 805-809, 1981. Wieczoreck, D. F., M. Periasamy, G. S. Butler-Browne, R. G. Whalen, and B. Nadal-Ginard. Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature. J. Cell Biol. 101: 618-629, 1985. Wolff, J. A., R. W. Malone, P. Williams, W. Chong, G. Ac- sadi, A. Jani, and P. L. Felgner. Direct gene transfer into mouse muscle in vivo. Science Wash. DC 247: 1465-1468, 1990. Yoon, S.-J., S. H. Seiler, R. Kucherlapati, and L. Leinwand. Organization of the human skeletal myosin heavy chain gene cluster. Proc. Natl. Acad. Sci. USA 89: 12078-12082, 1992.

myosin isoforms in mammalian skeletal muscle · fibers showing an immunoreactivity profile...

Documents