Contractile protein isoforms in muscle development

DEVELOPMENTAL

BIOLOGY

154,273-283

(1992)

Contractile

Protein lsoforms in Muscle Development EVERETT BANDMAN

Department

of Food

Science

and Technology, Accepted

University August

of California,

Davis,

Califomzia

95616

7, 1992

The contractile proteins of skeletal muscle are often represented by families of very similar isoforms. Protein isoforms can result from the differential expression of multigene families or from multiple transcripts from a single gene via alternative splicing. In many cases the regulatory mechanisms that determine the accumulation of specific isoforms via alternative splicing or differential gene expression are being unraveled. However, the functional significance of expressing different proteins during muscle development remains a key issue that has not been resolved. It is widely believed that distinct isoforms within a family are uniquely adapted to muscles with different physiological properties, since separate isoform families are often coordinately regulated within functionally distinct muscle fiber types. It is also possible that different isoforms are functionally indistinguishable and represent an inherent genetic redundancy among critically important muscle proteins. The goal of this review is to assess the evidence that muscle proteins which exist as different isoforms in developing and mature skeletal and cardiac muscles are functionally unique. Since regulation of both transcription and alternative splicing within multigene families may also be an important factor determining the accumulation of specific protein isoforms, evidence that genetic regulation rather than protein coding information provides the functional basis of isoform diversity is also examined. 0 1992 Academic Press, h.

wand et al., 1982; Wydro et al., 1983; Robbins et al., 1986; Radice and Malacinski, i989) and distinct patterns of developmental regulation (Crow and Stockdale, 1986; Bandman and Bennett, 1988; Bandman et al, 1982) which suggests that the generation of myosin heavy chain isoform diversity is a recurrent theme in evolution. This is underscored by recent studies in Drosophila melanogaster, where though only a single sarcomeric myosin heavy chain gene has been identified (Bernstein et al., 1983; Rozek and Davidson, 1983), myosin heavy chain isoform diversity has been maintained by alternative splicing (Bernstein e2 aZ., 1986; Rozek and Davidson, 1986; Wassenberg et aZ., 1987; George et ah, 1989; Collier et ak, 1990). Studies on the differential regulation of multigene families have focused on characterizing the various gene products and the genomic sequences which may modulate expression. Similarly, studies focusing on genes that yield spliced transcripts have attempted to correlate the appearance of a particular mRNA transcript with a developing or mature muscle fiber type or have focused on the cis- and trans-acting factors and the mechanisms governing the splicing process. The goal of this paper is not to chronicle the extent and regulation of contractile protein isoform diversity since numerous in-depth reviews exist (Obinata et al., 1981; Bandman, 1985; Swynghedauw, 1986; Syrovy, 1987). Instead, the intent is to examine the unique functionality of contractile protein isoforms and the genes that encode them. This article focuses predominantly upon the proteins of vertebrate muscle. Studies of the unique proteins of in-

INTRODUCTION Many proteins are uniquely expressed in skeletal muscle. Among them are the functional and structural proteins of the myofibril and the sarcoplasmic reticulum, as well as essential enzymes involved in muscle metabolism. A common property of many of these proteins is that they are members of isoform families. Distinct isoforms may be found in different adult muscle fibers and in some cases novel isoforms are expressed in developing muscle cells. Virtually all of the proteins that constitute the myofibril--a-actin, C-protein, myomesin, and the subunits of myosin, tropomyosin, and troponin-are represented by protein isoform families. The generation of isoform diversity has been accomplished predominantly by two mechanisms: the differential expression of multigene families and the production of multiple proteins from a single gene via alternative splicing. An example of the former is the heavy chain subunit of myosin which is encoded by a multigene family in all vertebrates that have been studied (Nguyen et aZ., 1982; Buckingham, 1985; Robbins et al., 1986). In contrast, the isoform family of troponin T subunits results from the generation of as many as 64 different transcripts from a single gene (Breitbart et al., 1985). Whichever mechanism of maintaining isoform diversity is employed, the importance of maintaining an assortment of very similar proteins is clear from studies of myosin heavy chain multigene families. Birds, amphibians, and mammals have disparate numbers of myosin heavy chain genes (Nguyen et al., 1982; Lein273

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DEVELOPMENTALBIOLOGY

vertebrate muscle are detailed in recent reviews describing the genetic dissection of contractile protein function in Caenorhubditis elegans (Epstein, 1990) and in D. melanogaster (Fyrberg et ah, 1991). MYOSIN

HEAVY

CHAIN

ISOFORMS

The initial observations that different myosin heavy chain (MYHC) isoforms existed were based on studies of myosins from rabbit skeletal muscle (Huszar, 1972, 1975). Subsequent studies have shown that sarcomeric MYHCs are represented by protein isoform families in species as diverse as nematode and human. With the single exception of D. melanogaster (Bernstein et al., 1983; Rozek and Davidson, 1983), all MYHC isoforms are encoded by unique genes which are differentially regulated in muscle cells (Mahdavi et aZ., 1987; Buckingham, 1985; Buckingham et aZ., 1986). Different MYHCs are expressed in embryonic, neonatal, and adult muscle fibers (Rushbrook and Stracher, 1979; Whalen et aL, 1981; Bader et ak, 1982; Bandman et al., 1982; Winkelmann et aL, 1983). Also, MYHCs differ in fast or slow muscle fiber types (Masaki, 1974; Arndt and Pepe, 1975; Gauthier and Lowey, 1977, 1979). Considerable effort has gone into characterizing the complexity of MYHC multigene families and their regulation. In-depth reviews of this area have been published (Bandman, 1985; Buckingham, 1985). The myosin molecule has two main functions: an enzyme activity that provides the driving force for muscle contraction, and a structural role in forming the thick filament of the myofibril. MYHC specialization is evident for both of these functions. The ATPase activity of myosin was correlated with the speed of muscle shortening and has been proposed as the basis for the distinction between fast- and slow-twitch muscles (Barany, 1967). These observations were extended to the singlefiber level (Reiser et ak, 1985, 1988). That fast MYHCs with a high rate of intrinsic ATPase activity can generate more movement in an in vitro motility assay than slow MYHCs with a lower rate of ATPase activity further supports this hypothesis (Sheetz et aL, 1984). Two forms of cardiac MYHCs, (Y and /3, have been characterized (Hoh, 1979; Mahdavi et al., 1984). These isoforms likely have physiological importance since the CX-MYHC has a Ca2+-ATPase activity three times that of P-MYHC (Pope et al., 1980) and their expression varies according to species, development, hormonal state, hemodynamic and localization (for review see characteristics, Schwartz et al., 1992). Observations from myofibrils of the nematode C. eleguns provide the best evidence that MYHC isoforms have unique roles in fibrillogenesis. The thick filaments of this nematode are composed of two distinct MYHC

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isoforms, myosin A in the center of the filament and myosin B at the periphery (Miller et ah, 1983; Epstein et ah, 1986). Since myosins in the center of the bipolar filament form an antiparallel orientation, whereas myosins at the ends are oriented parallel with respect to each other, it appears that each isoform may be specialized for a different role in the myosin assembly process. Nematode mutants lacking either isoform have abnormal sarcomere morphology, indicating that neither myosin can completely replace the other (Waterston et aL, 1980; Epstein et aL, 1986, 1987; Waterston, 1989). Although observations in vertebrates have indicated that there may be differential localization of isoforms within thick filaments (Taylor and Bandman, 1989) and myofibrils (Gauthier, 1990), no corroborating evidence supports this as an inherent function of a particular isoform. Studies of the alternatively spliced MYHC gene of D. melanogaster also provide insights into the functional specialization of myosin isoforms. There are alternative versions of four exons within the myosin head, one exon within the myosin hinge, and two versions of the carboxy terminus at the end of the myosin tail (George et aL, 1989; Collier et aL, 1990). Only one version of each set of exons is included in a mature transcript. The two copies of exon 15 in the myosin hinge are correlated with differences in contraction speed and force generation, thus implicating alternative versions of the hinge region in regulating these processes (Collier et aL, 1990). The functional significance of the alternative exons in the myosin head may contribute directly to differences in ATPase activity, actin binding, or myosin light chain binding, whereas the alternative carboxy termini could play unique roles in filament formation. The differential expression of MYHC multigene families has been extensively studied and in a few cases correlations with physiological properties of muscles have been demonstrated. However, other MYHC isoforms appear to be functionally equivalent and their existence may evolve from the need to maintain adequate MYHC levels, rather than appropriate MYHC types, in a dynamic tissue like muscle that may need to respond to different signals by altering expression of many contractile proteins. The molecular mechanisms that govern the accumulation of MYHCs at the mRNA level are being unraveled. Differential regulation of MYHC genes appears to result from interaction of &-acting sequences and trans-acting factors. The regulation of the cardiac (YMYHC gene by thyroid hormone has been extensively studied. A consensus thyroid-responsive element (TRE), positive and negative transcriptional regulatory elements, and the nuclear thyroid hormone receptors which interact with the TRE of specific genes have been identified (Gustafson et al., 1987; Izumo and Mahdavi,

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1988; Mahdavi et al., 1989; Nadal-Ginard and Mahdavi, 1989). Thyroid hormone has also been found to modulate the expression of developmentally regulated rat and mouse skeletal MYHC genes (Gambke et aZ., 1983; Izumo et aZ., 1986). However, efforts to locate similar regulatory elements have been limited by the fact that relatively few MYHC genes have been sufficiently characterized. Sequences that play a positive or negative role in regulating transcription of chicken MYHC genes and that interact with tram-acting protein factors present in muscle nuclei have been demonstrated (Subramaniam et ab, 1990). Unlike other muscle contractile protein genes which are dispersed in the genome (Robert et ab, 1985), a characteristic of sarcomeric MYHC genes is that they appear to be clustered. The two cardiac MYHC genes are 4 kb apart in both mouse and human (Mahdavi et al., 1984). Fast skeletal MYHC genes in mouse and human are also found on a single chromosome within a few hundred kilobase pairs (Saez et al., 1987; Weydert et al., 1985). Similar observations have also been made on the chicken embryonic and neonatal fast MYHC genes (Gulick et ak, 1987). While this may be a random consequence resulting from gene duplication, it may also have functional significance for the sequential expression of the genes. However, recent studies indicate that the clustered fast MYHC genes have independent transcriptional regulation (Cox et aZ., 1990). Gene conversion-like processes also appear to be playing a role in the evolution of MYHC multigene families (Moore et al, 1992). Extensive regions of nucleotide identity between different MYHC genes are most readily explained if constraints upon the protein sequence combined with gene clustering are sufficient to maintain the requisite nucleotide sequence homology that result in hot spots for gene conversion (Hibner et al., 1991). MYOSIN

LIGHT

CHAIN

ISOFORMS

Each MYHC is associated with two light chains. There are two classes of light chains in sarcomeric myosins, the alkali light chains and the phosphorylatable or regulatory light chains. In mammals the alkali light chains are encoded by three genes. In fast skeletal muscle, two different isoforms (MLCl, and MLC3,) result from different transcription initiation sites and alternative splicing of the 5’ end of a single gene (Nabeshima et al., 1984; Robert et al., 1984; Strehler et al., 1985). Accumulation of these two isoforms is also regulated developmentally, with the appearance of MLCl, preceding that of MLCS, (Sreter et al, 1975; Barton and Buckingham, 1985). A second gene encodes an alkali light chain isoform that is expressed in cardiac ventricle and

slow skeletal muscle (Barton et ab, 1985), while the third gene encodes an isoform that is expressed in cardiac atria and embryonic skeletal muscle (Barton and Buckingham, 1985; Barton et ab, 1985). Similar to the alkali light chains, the regulatory light chains are encoded by three unique genes: one expressed in fast skeletal muscle (MLCZ,), one expressed in cardiac ventricle and slow skeletal muscle (MLCB, or MLCZ,), and a third expressed in cardiac atria (Arnold and Siddiqui, 1979; Whalen et al, 1982; Kumar et ah, 1986). Both essential and regulatory myosin light chain genes contain enhancer sequences shown to be involved in muscle-specific gene transcription (Braun et al., 1989; Wentworth et ak, 1991; Ernst et al., 1991). A set of light chain isoforms (e.g., fast or slow) need not exclusively combine with a specific MYHC (Billeter et ah, 1981; Staron and Pette, 1987). Thus given the complexity of the heavy chain and light chain isoforms, a multitude of molecular species of myosin can be generated. Although the removal of regulatory light chains results in the reversible loss of Ca2+ control of tension in molluscan muscles (Simmons and Szent-Gyorgyi, 1978), there is little evidence that either the alkali or regulatory light chains modulate myosin ATPase activity in vertebrate striated muscle (Wagner and Giniger, 1981), making it difficult to envision their functional significance. Nevertheless, given the extensive conservation of both essential and regulatory sarcomeric light chain sequences throughout evolution (Collins, 1991) and the fact that functional hybrid myosins can be generated with scallop heavy chains and vertebrate light chains (Sellars et ah, 1980), it is likely that the light chain subunits have critical sequences which interact with the myosin head and possibly the actin filament as well. Perhaps more sensitive assays, such as in vitro motility measurements, may clarify the functional role of vertebrate light chains in force generation. a-ACTIN

ISOFORMS

Two a-actin isoforms are expressed in striated muscles: skeletal cY-actin and cardiac a-a&in. Skeletal a-actin is the predominant isoform found in adult striated muscles. Cardiac a-a&in, which is the main a-a&in isoform in the adult heart, is the predominant isoform expressed in early developing muscle and downregulated after birth (Minty et al., 1982; Mayer et al., 1984; Schwartz et al, 1986; Sassoon et aZ., 1988). Unlike MYHC sequences, the a-actin sequences vary very little. Skeletal and cardiac cu-actin differ by only four amino acids over 375 residues (Elzinga et al., 1973; Vandekerckhove and Weber, 1979). While the relative abundance of the two forms varies with species, muscle type, and developmental stage, their ubiquitous expression in developing

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skeletal and cardiac muscles, combined with their extensive homology, makes it unlikely that the two genes encode functionally unique proteins. The lack of pathology in a mutant mouse that expresses high levels of skeletal a-actin in adult cardiac tissue provides further evidence that these two isoforms are redundant (Garner et ah, 1986). The functional significance of these two genes is presumably related to their differential regulation in cardiac and skeletal muscles. While the promoter regions of the human cardiac and skeletal Lw-actin genes have been extensively analyzed, the sequences and the factors that interact with these sequences to confer muscle specificity and positive and/or negative transcriptional regulation are poorly understood (Gustafson and Kedes, 1989). Future studies identifying factors specifying cardiac differentiation, analogous to the muscle regulatory proteins and their DNA binding sites (Davis et ah, 1987; Tapscott et aZ., 1988; Edmondson and Olson, 1989; Lassar et al., 1989; Wright et al., 1989), may provide the key to unraveling this question. Thyroid hormone administration has been shown to upregulate the accumulation of skeletal Lu-actin transcripts (Winegrad et al., 1990). It is unclear whether this is a direct effect of thyroid hormone on the gene or a secondary effect of the hormone. The lack of a thyroid responsive element in the skeletal cy-actin gene similar to that observed in the a-cardiac MYHC gene suggests the indirect mechanism is more likely. TROPONIN ISOFORMS Troponin is composed of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT) (Ebashi and Endo, 1968), each of which exists as multiple isoforms. These isoforms are differentially expressed in fast, slow, and cardiac muscle and are also developmentally regulated (Perry, 1985). TnC isoforms are encoded by two genes, one expressed in fast skeletal muscle and the other expressed in both slow skeletal and cardiac tissue (Chen et al, 1986; Gahlmann et aa, 1988; Reinach and Karlsson, 1988; Toyota et ah, 1989; Parmacek and Leiden, 1989). TnI isoforms are encoded by three genes: a fast skeletal, a slow skeletal, and a cardiac form (Nikovits et al., 1986; Koppe et al., 1989; Vallins et al., 1990; Murphy et ctL, 1991; Ausoni et ah, 1991). TnT isoforms are also encoded by fast skeletal, slow skeletal, and cardiac genes (Cooper and Ordahl, 1985; Breitbart and NadalGinard, 1986; Gahlmann et aZ., 1987; Smillie et al., 1988). Each of the TnC and TnI genes appears to code for a single protein, while each of the TnT genes produces multiple variants as a result of alternative splicing (Medford et aZ., 1984; Breitbart et al., 1985). The troponin C subunit binds Ca2+ ions and confers Ca2+ sensitivity to the myofibrils (Schaub and Perry,

1969; Greaser and Gergely, 1973). Based upon common structural features, it has been proposed that TnC and other Ca2’ binding proteins such as the calmodulins, the essential and regulatory light chains of molluscan muscles, as well as the regulatory myosin light chains of vertebrate smooth muscles have evolved from a common ancestor (Collins, 1991). TnC sequences are highly conserved, with only a single amino acid difference between human and rabbit fast skeletal isoforms. A single amino residue difference is also found in a comparison of turkey and chicken fast TnC. All mammalian slow/cardiac TnC sequences differ by only one or two residues. The fast TnC isoform can bind four Ca2+ ions per mole, while the slow/cardiac isoform can only bind three (Van Eerd and Takahashi, 1976). This difference in Ca2+ binding activity appears to have resulted from a natural mutation in one of the binding sites in vertebrate cardiac/ slow TnC (Putkey et aZ., 1989). Troponin I is the subunit of the troponin complex that inhibits actomyosin ATPase activity. TnI is phosphorylated by a CAMP-dependent protein kinase which results in a loss of myofibrillar Ca2+ sensitivity (England, 1976; Winegrad et aZ., 1983). Cardiac TnI contains a much longer amino-terminal domain compared to the skeletal TnIs (Leszyk et ah, 1988). It is in this region where phosphorylation of a serine occurs when hearts are perfused with catecholamines that results in altered Ca2+ sensitivity (Solar0 et al, 1976; Mope et ah, 1980). Skeletal TnI isoforms that lack this region do not display phosphorylation-mediated regulation by /3-adrenergic agents. The cardiac TnI isoform is developmentally regulated, being expressed at low levels in the fetal myocardium and markedly increasing in the perinatal period (Saggin et al., 1989; Sabry and Dhoot, 1989). In the fetal rat and human heart the slow skeletal isoform represents the predominant transcripts (Ausoni et al, 1991; Bhavsar et al., 1991). As noted above since the slow skeletal form lacks the extended N-terminal sequence, the fetal heart would be expected to exhibit less sensitivity to adrenergic stimulation. Fetal skeletal muscle also appears to contain isoforms distinct from the adult fast and slow TnIs (Nikovits et aZ., 1986; Saggin et cd., 1990) although it is unclear whether these are encoded by distinct genes. Numerous fast skeletal TnT isoforms have been observed in both adult and developing muscles. These variants appear to result from alternative splicing of the TnT, gene which may generate up to 64 different mRNAs (Breitbart et ab, 1985; Breitbart and NadalGinard, 1986). The splicing of alternative and constitutive exons is controlled by muscle-specific tram-acting factors that are also developmentally regulated (Breitbart and Nadal-Ginard, 1987). In late fetal and early neonatal chicken muscle as many as 40 fast TnT vari-

REVIEWS

ants have been identified (Abe et al., 1986; Imai et al., 1986; Dhoot, 1988; Smillie et aZ., 1988). Significantly less diversity at the protein level has been observed in developing mammalian fast muscles (Briggs et aZ., 1984,1987, 1990; Hartner et aL, 1989) and even fewer slow TnT variants have been identified (Moore et al., 1987; Schmitt and Pette, 1988). The cardiac TnT gene has been shown to generate isoforms that are developmentally regulated and expressed in a tissue-specific manner (Cooper and Ordahl, 1985; Anderson et al., 1988). Fast TnT isoform heterogeneity results predominantly from variation of the amino-terminal region as a consequence of alternative splicing of several 5’ exons in the fast TnT gene (Breitbart et ak, 1985; Briggs and Schachat, 1989). Significantly, variants of bovine heart TnT confer different Ca2+ sensitivities to the thin filament-activated Mg2+-ATPase activity of cardiac myosin Sl in vitro (Tobacman and Lee, 1987). The amino-terminal sequences of three different fast TnTs have now been determined (Briggs and Schachat, 1989). Recent studies have shown that the Ca2+ affinity of reconstituted troponin using a TnT variant lacking the first 38 amino acids is reduced (Tobacman, 1988). These data in conjunction with observations on permeabilized rabbit and avian skeletal muscle fibers showing a correlation between TnT isoform composition and tension generated in response to Ca2+provide convincing evidence that fast TnT variants are functionally different proteins (Schachat et al., 1987; Reiser et al., 1992). TROPOMYOSIN

ISOFORMS

277

1982; Heeley et aZ., 1985; Pernelle et al, 1986), suggesting that phosphorylation or other post-translational modifications may also generate distinctive isoforms. The LYand ,f3subunits are developmentally regulated. In the chicken pectoralis muscle, the /3-tropomyosin gene is repressed at hatching (Meinnel et al, 1989) which accounts for the presence of only the a-isoform in adult muscle (Roy et ah, 1979; Montarras et ak, 1982; Matsuda et aZ.,1983). In mammals the ratio of a/a and CY//~dimers is modulated during neonatal development (Briggs et al, 1990) and appears to be regulated by the accumulation of the two transcripts. The observation that the ratio of (Y/(Y, a/p, p//? dimers differs between mature muscle fiber types (Bronson and Schachat, 1982) and can be altered by denervation or muscle pathology (Takeda and Nonomura, 1980; Matsuda et ah, 1984) suggests that muscle activity plays a role in regulation. The tight correlation between troponin and tropomyosin isoform expression has led to the suggestion that these genes are coregulated although no direct evidence has been presented. Studies on permeabilized rabbit muscle fibers have demonstrated that the presence of different troponin-tropomyosin combinations in isolated and permeabilized rabbit muscle fibers alters the slope of the pCa/tension relations (Schachat et al., 1987). Since this parameter reflects the concerted transition from the relaxed to the active state in the presence of Ca2+by all the troponintropomyosin complexes of the thin filament (Brandt et al., 1984), these data provide a justification for coregulation. Additionally, the variation in the broad constellation of thin filament regulatory proteins has been used as a model to explain the evolution of functional muscle fiber diversity (Schachat et ah, 1990).

Tropomyosin is a threadlike protein composed of two subunits which form an a-helical coiled coil, which along with the troponin complex regulate calcium activation of the actomyosin complex (Cummins and Perry, OTHER MYOFIBRILLAR PROTEINS 1973; Smillie, 1979). Muscle cells express two isoforms, C-protein is a myosin binding protein with a molecua-tropomyosin and fi-tropomyosin (Bronson and Schachat, 1982; Kardami et al., 1983). Tropomyosin may be a lar weight of 140,000 located in the central region of coiled-coil homodimer or heterodimer. The a-p heterovertebrate sarcomeres (Offer et ah, 1973; Moos et aZ., dimer appears to be the preferential form when both 1975). At the ultrastructural level, C-protein is responsiisoforms are expressed within a single cell (Brown and ble for a characteristic seven- to nine-transverse-stripe Schachat, 1985). Thus, the ratio of a/(~, cu/fi, p//3 forms is pattern in the A-band region of the myofibril (Pepe and predominantly determined by the accumulation of the Drucker, 1975; Dennis et al., 1984). Recent studies on different transcripts that arise from two genes. The (Y- isolated and skinned single rat muscle fibers suggest and /3-tropomyosin genes have been characterized in rat that partial extraction of C-protein leads to a reversible and chicken and are alternatively spliced to give rise to increase in the Ca2+-dependent tension (Hoffman et al., skeletal muscle, smooth muscle, and nonmuscle tropo1991), suggesting that contractile function is affected by myosin isoforms (Ruiz-Opazo and Nadal-Ginard, 1987; C-protein. Fast skeletal, slow skeletal, and cardiac isoWieczorek et al, 1988; Libri et al., 1989,199O; Lemonnier forms of C-protein have been distinguished in striated et al., 1991). In addition there appear to be unique fast muscles of mammals and birds (Callaway and Bechtel, 1981; Reinach et al., 1982; Yammamoto and Moos, 1983; and slow (Yand p subunits (Kardami et al., 1983). Twodimensional electrophoretic separations have demon- Dhoot et al., 1985; Kawashima et al, 1986; Takano-Ohstrated additional complexity (Romero-Herrera et ab, muro et ab, 1989). Sequential changes in C-protein iso-

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form expression appear to be correlated with sequential changes in MYHC isoforms (Obinata et al., 1984; Kawashima et aL, 1986; Takano-Ohmuro et aZ., 1989), leading to suggestions that specific interactions between myosin binding proteins and different myosin isoforms may facilitate transitions within the myofibril (Kerwin and Bandman, 1991). The chicken fast skeletal C-protein cDNA has been cloned and sequenced and shown to be an intracellular member of the immunoglobulin superfamily (Einheber and Fischman, 1990). However, until the gene encoding this isoform is characterized, and additional mRNA transcripts identified and sequenced, it cannot be determined whether the different C-protein isoforms identified immunologically, by two-dimensional gel electrophoresis, and by peptide mapping are the products of separate genes, result from alternative splicing, or are a consequence of post-translational modifications. Different M-line patterns have been observed ultrastructurally (Wallimann and Eppenberger, 1985; Thornell et aZ., 1987,199O). The M-line protein myomesin has been shown to exist as distinct isoforms in fast and slow chicken skeletal muscles (Grove et ah, 1989). Previous single-fiber studies have identified different M-protein isoforms electrophoretically (Salviati et al., 1982). However, our knowledge of the role of the M-line in the myofibril is limited and thus the significance of any diversity in M-line protein isoforms would be speculative. Evidence for size diversity on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of the filamentous proteins titin (Maruyama et ak, 1977) and nebulin (Wang and Williamson, 1980) has been demonstrated. Measurements of resting tension as a function of sarcomere length in muscles expressing different ratios of the three size classes of titin have shown that there is a correlation between the expressed titin isoform, the sarcomere length at the onset of exponential resting tension, and the elastic limit of the muscle (Wang, 1984; Wang et aZ., 1991). If muscle cells modulate stiffness and elasticity by selective expression of titin and nebulin isoforms, the molecular analysis of these genes will undoubtedly be difficult due to their extremely large size. There is presently no evidence for isoform diversity among the minor components of the myofibril including H-protein (Starr and Offer, 1983) and X-protein (Starr et ak, 1980). SUMMARY AND CONCLUSIONS

The flected found fibers.

functional diversity of skeletal muscle cells is reby the wide array of protein isoforms which are in differentiating, developing, and mature muscle As documented within this review, differential

expression of specific members of multigene families and alternate RNA processing from a single gene are both involved in generating protein isoform diversity. While in a few cases the unique function of a particular isoform has been documented, in many cases there is no apparent explanation for the observed changes in isoform expression. Considerable efforts are being made to try to understand the mechanisms that induce or repress a particular gene and to correlate the appearance of specific isoforms with unique physiological or functional demands on the muscle fiber. Furthermore, since many isoform families exhibit coordinate expression, the regulatory mechanisms that control the transcription or processing of these genes are likely to share common factors and/or pathways. These studies ultimately may clarify how gene regulation itself can contribute to the functional significance of isoform diversity. Studies of isolated proteins have been useful in identifying protein function but may be less helpful in distinguishing properties of closely related isoforms. Furthermore, the complexity of the muscle cell and the difficulty in reconstructing the interactions of the components of the myofibril in vitro are additional hurdles to overcome. However, even when it has been possible to study the interaction between contractile proteins within experimental systems, it is unclear whether differences observed are experimentally significant. Perhaps, current studies taking advantage of recent advances in molecular genetic approaches to gene inactivation and gene replacement may provide new insights into the importance of protein isoforms in who. As a consequence of the wide array of contractile protein isoforms expressed in muscle, virtually no two muscle cells contain identical components. There have been many attempts to classify muscle cells into discrete “fiber types.” While such classifications have been helpful to biologists defining functional, metabolic, or enzymatic properties, fiber type grouping is not only misleading in that it often overlooks considerable molecular heterogeneity within a group, but also illustrates how difficult it is to uncover the functional significance of isoform divergence. However, recent proposals for defining protein diversity of multicomponent systems within a molecular continuum (Schachat et ak, 1990) may provide a new perspective for understanding the advantage of maintaining isoform diversity in systems such as muscle which must respond rapidly to changes in physiological demands. Clearly there is no single explanation for isoform diversity in muscle development. The mechanisms by which multigene families are expanded and new isoforms fixed within the genome must be considered as well, since genetic redundancy of critically important

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genes may also be important. While it is easy to envision the selective advantage of protein specialization, specific programs of muscle gene expression have also been maintained. Little is known regarding the &-acting sequences and the tram-acting factors which govern developmental regulation of multigene families such as MYHC. Future studies on the genomic organization and the structure of individual genes may provide key insights into the role that regulatory regions play in generating and maintaining protein isoform diversity. REFERENCES Abe, H., Komiya, T., and Obinata, T. (1986). Expression of multiple troponin T variants in neonatal chicken breast muscle. Dev. BioL 11842-51. Anderson, P. A. W., Moore, G. E., and Nassar, R. N. (1988). Developmental changes in the expression of rabbit left ventricular troponin T. Circ. Res. 63,742-747. Arndt, I. and Pepe, F. (1975). Antigenic specificity of red and white muscle myosin. J. X.&&em. C@oohem 23,159-168. Arnold, H. H., and Siddiqui, M. (1979). Control of embryonic development: Isolation and purification of chick heart myosin light chain mRNA and quantitation with a cDNA probe. Biochemistry l&647654. Ausoni, S., De Nardi, C., Moretti, P., Gorza, L., and Schiaffino, S. (1991). Developmental expression of rat cardiac troponin I mRNA. Development 112,1041-1051. Bader, D., Masaki, T., and Fischman, D. A. (1982). Immunochemical analysis of myosin heavy chain during avian myogenesis in viva and in vitro.

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Bandman, E. (1985). Myosin isozyme transitions during muscle development, maturation, and disease. Int. Rev. Cytol. 97,97-131. Bandman, E. (1992). The avian myosin heavy chains. In “Gene Expression in Neuromuscular Development” (H. Blau and A. M. Kelly, Eds.). Raven Press, New York. Bandman, E., and Bennett, T. (1988) Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens. Dev. BioL 130,220-231. Bandman, E., Matsuda, R., and Strohman, R. C. (1982). Developmental appearance of myosin heavy and light chain isoforms in viva and in vitro in chicken skeletal muscle. Dev. BioL 93,508-518. Barany, M. (196’7). ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. PhysioL 50,197-218. Barton, P., and Buckingham, M. E. (1985). The myosin alkali light chain proteins and their genes. Biochem. J. 231,249-261. Barton, P. J. R., Robert, B., Fiszman, M. Y., Leader, D. P., and Buckingham, M. E. (1985). The same myosin alkali light chain is expressed in adult cardiac atria and in fetal skeletal muscle. J. Muscle Res. Cell MotiL

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