Changes in tropomyosin subunits and myosin light chains during development of chicken and rabbit straited muscles

DEVELOPMENTAL

BIOLOGY

69, 15-30 (197%

Changes in Tropomyosin Subunits and Myosin Light Chains during Development of Chicken and Rabbit Striated Muscles

Department

RAMAN K. ROY, F.A. SRETER,* ANDSATYAPRIYASARKAR of Muscle Research, Boston Biomedical Research Institute, and Departments Harvard

Medical

School and *Massachusetts General Hospital, Boston, Massachusetts 02114

20 Staniford

of Neurology, Street,

Received May 15, 1978; accepted in revised form September 28, 1978 We have selected tropomyosin subunits and myosin light chains as representative markers of the myofibrillar proteins of the thin and thick filaments and have studied changes in the type of proteins present during development in chicken and rabbit striated muscles. The /3 subunit of tropomyosin is the major species found in all embryonic skeletal muscles studied. During development the proportion of the (Ysubunit of tropomyosin gradually increases so that in adult skeletal muscles the (Ysubunit is either the only or the major species present. In contrast, cardiac muscles of both chicken and rabbit contain only the o( subunit which remains invariant with development, Two subspecies of the (Y subunit of tropomyosin which differ in charge only were found in adult and embryonic chicken skeletal muscles. Only one of these subspecies seems to be common to chicken cardiac tropomyosin. With respect to myosin light chains, embryonic skeletal fast muscle myosin of both species resembles the adult fast muscle myosin except that the LC3 light chain characteristic of the adult skeletal fast muscle is present in smaller amounts. The significance of these isozymic changes in the two myofibrillar proteins is discussed in terms of a model of differential gene expression during development of chicken and rabbit skeletal muscles, INTRODUCTION

Avian and mammalian skeletal muscles have been classified as either fast or slow twitch muscles by a variety of physiological, histochemical, and biochemical criteria (Peachey, 1968; Hess, 1970; Close, 1972; Barany et al., 1967). It is now well known that several contractile proteins, e.g., myosin (Sarkar et aZ., 1971; Lowey and Risby, 1971; Jean et al., 1975; Hoh et al., 1976), tropomyosin (Cummins and Perry, 1973, 1974; Hayashi et al., 1977; Izant and Lazarides, 1977; Leger et al., 1976), and troponin (Syska et al., 1974; Wilkinson, 1978), exist in multiple forms in vertebrate striated muscles. On the biochemical level the distinction between fast and slow muscles seems to be reflected in the presence and relative amounts of some of these isozymes (for a review, see Perry, 1974). Cardiac muscles resemble slow muscles with respect to some physiological and biochemical param-

eters (Perry, 1974; Sarkar et al., 1971). Changes in the subunit pattern of myofibrillar proteins have been observed during muscle development (Sreter et al., 1972; Pelloni-Muller et al., 1976a,b; Sreter et al., 1975; Roy et al,, 1976; Amphlett et al., 1976; Rubinstein et al., 1977; Syrovy and Gutman, 1977; Dabrowska et al., 1977). Embryonic skeletal muscles, as judged by physiological parameters such as rate of contraction and force-velocity measurements, show similarities to adult slow muscles (Buller et al., 1960 a,b). Similarly, reports on biochemical and immunological properties of embryonic skeletal myosin (Perry, 1970; Holland and Perry, 1969; Trayer et al., 1968; Trayer and Perry, 1966) have stressed the similarities between myosins of embryonic skeletal and adult slow muscles. Reports from other laboratories, however, have indicated that myosins isolated from embryonic skeletal and adult fast muscles are similar (Sreter et al., 1972, 0012-1606/79/030015-16$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved

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DEVELOPMENTAL BIOLOGY

1975; Takahashi and Tonomura, 1975; Pelloni-Muller et al., 1976 a,b; Rubinstein et al., 1977). According to recent immunocytochemical studies (Gauthier et al., 1978), in the early stages of development all fibers react with antibody against slow myosin as well as antibody against fast light-chain subunits. In this work we have studied in parallel the developmental changes in isozymic forms of tropomyosin subunits and myosin light chains selected as two representative myofibrillar proteins in order to correlate these changes with the functional type of muscle. The significance of the changes in the subunit pattern of these proteins is discussed in terms of a model of differential gene expression in the fiber.

VOLUME 69, 1979

and isoelectric precipitation at pH 4.6 (Greaser and Gergely, 1971; Roy et al., 1976). Rabbit skeletal pure (Y-and /?-tropomyosin subunits separated by ion-exchange chromatography (Betcher-Lange and Lehrer, 1978) were kind gifts from Dr. Sherwin S. Lehrer of this department. Myosin was isolated from skeletal muscles and purified by chromatography on a column of DEAE-Sephadex A-50 by previously described procedures (Sarkar and Cooke, 1970; Sarkar et al., 1971; Sreter et al., 1972). Purified myosin preparations were tested for their Ca’+-activated ATPase activities by published procedures (Sreter et al., 1972). The typical values of ATPase activity for myosins isolated from rabbit adult fast and slow muscles and embryonic EXPERIMENTAL PROCEDURES skeletal (3- to 4-week-old) muscles were Preparation of tropomyosin and myosin. 0.85, 0.22, and 0.61 pmole/mg/min, respecBoth tropomyosin and myosin were iso- tively. The corresponding values for lated from chicken and rabbit skeletal and chicken adult fast, slow, and embryonic cardiac muscles at various stages of devel- breast (18-day-old) muscles were 0.68,0.17, opment (for details, see figure legends). In and 0.65 pmole/mg/min, respectively. the case of embryos and newborn rabbits, Preparation of actin. Actin was prepared where the amount of muscles available is from rabbit skeletal muscles by the method rather limited, the superficial muscles of of Spudich and Watt (1971). the hindlegs and backs were pooled as a Assay of tropomyosin activity. The biosource of skeletal muscles. In other cases, logical activity of tropomyosin was meae.g., rabbit fast (extensor digitorum longus) sured by determining the EGTA inhibition and slow (soleus) muscles, chicken fast of Mg2+ ATPase of reconstituted rabbit twitch (m. pectoralis major and latissimus actomyosin in the presence of rabbit trodorsi posterior) and slow tonic (m. latissi- ponin complex. Reconstituted rabbit actomus dorsi anterior) as well as leg muscles, myosin was freshly made by combining rabindividual muscles were isolated separately. bit myosin and actin at a 4:l ratio by weight Tropomyosin was isolated and purified and assayed as described previously (Roy from extracts of ether-dried muscle powder et al., 1976). by two methods. In the case of adult musGet electrophoresis. Protein samples for cles, a combination of (NHJ2S04 fractionpolyacrylamide gel electrophoresis were alation and isoelectric precipitation at pH 4.6 kylated with a-iodoacetamide as previously as described previously (Greaser and described (Bag and Sarkar, 1975). The alGergely, 1971) was used. In the case of kylated proteins were then dialyzed either embryonic muscles, because of its high con- against 50 mM sodium phosphate buffer, tent of nucelic acids, the tropomyosinpH 7.0, containing 1% NaDodSOl and 1% troponin complex was first isolated by our P-mercaptoethanol for analysis by Narecently reported chromatographic method DodS04-polyacrylamide gel electrophoreon columns of DEAE-cellulose (Roy et al., sis or against 5 M urea containing 5.4% 1976). Tropomyosin was then purified from acetic acid and 1% /3-mercaptoethanol for this complex by (NH&S04 fractionation analysis by urea-polyacrylamide gel elec-

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Myofibrillar

trophoresis. The urea solutions used were freshly prepared from ultrapure-grade urea (Schwarz/Mann) in order to prevent cyanate ion-dependent carbamylation of proteins (Stark et al., 1960). Electrophoresis under acidic conditions (pH 3.4) also minimizes the cyanate ion formation (SchmidtUllrich and Wallach, 1977). NaDodS04-polyacrylamide gel electrophoresis in 10% gels was carried out according to the procedure of Weber and Osborn (1969). Densitometric scans of the Coomassie blue-stained gels were done using a From Joyce-Loebel microdensitometer. the scans the molar stoichiometry and relative ratios of the myofibrillar proteins were calculated as described previously (Sarker, 1972; Potter, 1974; Roy et aZ., 1976). Urea-polyacrylamide gel electrophoresis was carried out using gels containing 7.5% polyacrylamide at pH 3.4. The gels were first prerun for 1 hr at 1 mA/tube. After

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application of the samples the final electrophoresis was performed at 3 mA/tube. RESULTS

Biological activity of adult and embryonic skeletal muscle tropomyosins. Samples of purified preparations of tropomyosins isolated from rabbit and chicken skeleta1 muscles were routinely tested for their biological activity (for details, see also Experimental Procedures). Figure 1 shows the degree of inhibition of Mg”+-stimulated ATPase activity of reconstituted rabbit, actomyosin obtained with tropomyosins isolated from adult and embryonic (15-dayold) chicken breast (pectoralis) muscles in the presence of rabbit troponin complex. Both preparations were found to have comparable biological activity over a wide concentration range. Similar biological activities were also observed among other adult and embryonic skeletal muscles of both

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FIG. 1. Effect of tropomyosins isolated from chicken embryonic (Xi-day-old) and adult breast muscles on the MC ATPase activity of reconstituted rabbit actomyosin in the presence of rabbit skeletal troponin complex. Assays were carried out at 25°C in a 2-ml incubation mixture containing 25 mJ4 Tris-HCl, pH 7.5,25 mM KCl, 1 mi%f MgC12, 1 m&f ATP, 1 mM EGTA, and 600 pg actomyosin and troponin in amounts equal to the added tropomyosin. The activities are expressed as percentages of that containing all additions except tropomyosin.

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DEVELOPMENTAL BIOLOGY

chicken and rabbit (results not shown here). Tropomyosin subunits of adult rabbit and chicken skeletal muscles. The tropomyosin subunit patterns of adult rabbit and chicken skeletal muscles are shown in Fig. 2. Both fast (gel a, Fig. 2A) and slow (gel b, Fig. 2A) muscles of rabbit contain the (Yand p subunits of tropomyosin, although the ratio varies with the muscle type. Thus in rabbit fast muscle the 01to /? ratio is 80:20, while in slow muscles the ratio is 5545. These results are in agreement with previously published reports (Cummins and Perry, 1974) on the subunit distribution of tropomyosin in rabbit skeletal muscles. Chicken leg muscle (gel a, Fig. 2B), which consists of a mixture of both slow and fast fibers, contains the two subunits of tropomyosin in approximately equal amounts (a$ = 55:45; see also scan c, Fig. 5B). On the other hand, chicken breast muscle (which is considered a fast muscle; gel b, Fig. 2B) contains only the o subunit of tropomyosin (see also scan c, Fig. 5A). This was further confirmed by coelectrophoresis of chicken breast muscle tropomyosin with marker rabbit pure (Ytropomyosin subunit (gel e, Fig. 2B) which shows that the two protein samples comigrated. It has been postulated that the speed of contraction of vertebrate skeletal muscles is correlated with the subunit composition of tropomyosin, the skeletal slow muscles containing a relatively higher proportion of the p subunit of tropomyosin compared to fast muscles (Cummins and Perry, 1974). However, tropomyosins isolated from anterior latissimus dorsi (ALD)’ (gel c, Fig. 2B) and posterior latissimus dorsi (PLD) (gel d, Fig. 2B) muscles of chicken, which are considered as slow and fast muscles, respectively, surprisingly gave identical subunit pictures (a:/? = 55:45). Tropomyosin subunits of embryonic ’ Abbreviations used: ALD, anterior latissimus dorsi; PLD, posterior latissimus dorsi; LG, LG, and LG, light chains 1,2, and 3, respectively; NaDodSOs, sodium dodecyl sulfate.

VOLUME 69, 1979

skeletal muscles. In contrast to adult skeletal muscle tropomyosin, embryonic tropomyosin of both chicken and rabbit shows a different subunit picture (Fig. 3). Chicken embryonic tropomyosin of both leg (gel a, Fig. 3A) and breast (gel c, Fig. 3A) muscles contains predominantly the fi subunit, which amounts to about 65-70% of the total tropomyosin in either muscle of 12-day-old embryos. Furthermore, the subunits present in the embryonic and adult stages of these two muscles have identical mobilities, as shown by coelectrophoresis experiments (gels b and d, Fig. 3A). Rabbit embryonic skeletal muscle is also found to contain predominantly the /3 subunit of tropomyosin (Fig. 3B), the relative proportion of which changed with development. Thus, the /? subunit, which amounts to 70% of the total tropomyosin in 20-day-old embryonic skeletal muscles (gel a, Fig. 3B), decreases to about 50% in 26-day-old embryonic muscles (gel b, Fig. 3B), and further to only 45% in l-day-old rabbit skeletal muscles (gel c, Fig. 3B). Similar changes in the distribution of tropomyosin subunits have been reported in longissimus dorsi muscle of rabbit during development (Amphlett et al., 1976). Our results, while confirming this report, further indicate that in both avian and mammalian skeletal muscles it is the /3 subunit of tropomyosin which is present as the major species in the embryonic stage and the c@ ratio changes significantly during development. Time course of developmental changes in tropomyosin subunitpatterns in chicken leg and breast muscles. The striking difference in the subunit pattern of tropomyosins in chicken embryonic and adult skeletal muscles led us to study the time course of this developmental change in both leg and breast muscles (Fig. 4). During the early stages of development the two muscles show similar rates of change in the subunit pattern, which ranges from 30-35% of a tropomyosin subunit in 12-day-old embryos (see also scans a, Figs. 5A and B) to about 55-60% in 2-day-old chickens. In leg mus-

ROY, SRETER, AND SARKAR

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FIG. 2. NaDodS04-polyacrylamide gel electrophoresis of adult rabbit and chicken skeletal muscle tropomyosins. Amount of protein loaded, 6-12 pg. (A) Tropomyosins of rabbit skeletal muscles: gel a, extensor digitorum longus; gel b, soleus; gel c, skeletal pure u-tropomyosin; gel d, mixed sample of soleus muscle tropomyosin and skeletal muscle a-tropomyosin. (B) Tropomyosins of chicken skeletal muscles: gel a, leg muscle; gel b, breast muscle; gel c, anterior latissimus dorsi muscle; gel d, posterior latissimus dorsi muscle; gel e, mixed sample of breast muscle tropomyosin and rabbit skeletal muscle oc-tropomyosin.

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FIG. 3. NaDodSOa-polyacrylamide gel electrophoresis of embryonic chick and rabbit skeletal nuscle tropomyosins. Amount of protein loaded, 6-12 ag. (A) Tropomyosins isolated from chick skeletal muscles: gel a, 12day-old embryonic leg muscles; gel b, mixed sample from 12-day-old embryonic and adult leg muscles; gel c, 12day-old embryonic breast muscles; gel d, mixed sample from 12-day-old embryonic and adult breast muscles. (13) Gels a, b, and c, tropomgosins isolated from 20-day-old embryonic, 26-day-old embryonic, and l-day-old rabbit skeletal muscles, respectively.

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DEVELOPMENTAL BIOLOGY

VOLUME 69, 1979

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FIG. 4. Time course of developmental changes in the subunit patterns of chick leg and breast muscle tropomyosins. Results are expressed in terms of the o( subunit of tropomyosin as percentages of the total tropomyosin subunit (a and j3) content of muscles at each stage of development studied. The values represent the means of four separate gel runs with each of two different preparations of tropomyosin at each age group studied. The variation in the estimation of different experiments was f 8%.

cles the subunit pattern of tropomyosin of Z-day-old chickens is identical to that observed in adult muscles (see also scan c, Fig. 5B). In contrast, in breast muscles the subunit ratio of tropomyosin continues to change and the single a-subunit pattern characteristic of the adult muscles does not appear before Day 8 ex ouo (see also scan c, Fig. 5A). Cardiac tropomyosins. Tropomyosin isolated from cardiac muscles of both chicken and rabbit gave a simple subunit picture in NaDodS04-polyacrylamide gel electrophoresis (Fig. 6). In both species, a single protein band was obtained with tropomyosins isolated from both adult (gels a and e, Fig. 6), and embryonic (gels b and f, Fig. 6) cardiac muscles. Coelectrophoresis of adult and embryonic cardiac tropomyosins of both chicken (gel c, Fig. 6) and rabbit (gel g, Fig. 6) did not show any detectable difference in their electrophoretic mobilities. Moreover, when rabbit skeletal pure (Ysubunit of tropomyosin was coelectrophoresed with mixed samples of either adult and embryonic chicken cardiac tropomyosins (gel d, Fig. 6) or adult and embryonic rabbit cardiac tropomyosins (gel h, Fig. 6), the single subunits of each of these cardiac

tropomyosins had mobilities identical to that of rabbit skeletal ar subunit of tropomyosin. Our observations, while confirming some earlier reports on cardiac tropomyosin (Cummins and Perry, 1973, 1974), further indicate that the single a-tropomyosin subunit pattern of both chicken and rabbit cardiac muscles remains invariant during development, in contrast to the changes observed in skeletal muscles. Urea-polyacrylamide gel electrophoresis of skeletal tropomyosins. Multiple forms of tropomyosin subunits in skeletal muscles have been reported on the basis of differences in electrophoretic mobilities (Cummins and Perry, 1973) and immunological properties (Hayashi et al., 1977). We have examined the tropomyosin subunit patterns of rabbit and chicken skeletal muscles by urea-polyacrylamide gel electrophoresis (Fig. 7) in order to look for multiple forms of these subunits which may differ in charge only and for possible changes in their distribution during development. Urea gel electrophoresis at alkaline pH (pH 8.4) did not resolve the tropomyosin subunits very well. However, electrophoresis in 5 M urea at pH 3.4 gave well-resolved protein bands, as can be seen with the single band obtained

ROY, SRETER, AND SARKAR

Myofibrillar

with rabbit skeletal pure (Y (gel c, Fig. 7A) and p (gel b, Fig. 7B) subunits of tropomyosin. Tropomyosin isolated from adult chicken breast muscles, which consists of only the (Ysubunit as shown by NaDodS04 gel electrophoresis (see also Fig. 2), is resolved into two bands of equal intensity (gel a, Fig. 7A) in acidic urea gel electrophoresis. Neither of these two bands comigrates with rabbit skeletal pure (Y (gel d, Fig. 7A) or ,8 (gel e, Fig: 7A) subunit of tropomyosin. Adult chicken leg muscle tropomyosin, which consists of both (Y and /3 subunits (see also Fig. 2), is resolved into three bands on urea gel electrophoresis (gel a, Fig. 7B). Among these three bands, the fastest- and the slowest-moving bands have mobilities similar to those of the two corresponding components of adult chicken breast muscle (Y subunit of tropomyosin (compare gel a, Fig. 7A, and gel a, Fig. 7B), suggesting that the middle band is due to the p subunit. This was further confirmed in coelectrophoresis experiments which show that only the middle band of chicken leg tropomyosin comigrates with rabbit skeletal pure /I subunit of tropomyosin (gel d, Fig. 7B). When a mixed sample of rabbit skeletal (Y subunit and chicken leg muscle tropomyosin was analyzed, the resulting gel pattern gave the expected four well-resolved protein bands with mobilities corresponding to those of the bands obtained in separate gel runs (compare gel c, Fig. 7A, and gel a, Fig. 7B) of the two protein samples (results not shown here). Tropomyosin isolated from embryonic chicken leg muscles also gave three bands in acidic urea gel electrophoresis (gel b, Fig. 7B). Coelectrophoresis of tropomyosins isolated from adult and embryonic chicken leg muscles clearly shows that the corresponding bands of the two protein samples comigrated (gel c, Fig. 7B) indicating the strong similarities of their subunits. The three-band gel pattern was also obtained with embryonic chick breast muscle tropomyosin (results not shown here), which according to NaDodS04 gel electrophoresis, consists of

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A

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FIG. 5. Densitometric scans of electrophoretograms of tyopomyosins isolated from chicken leg and breast muscles at various stages of development. (A) Scans a and b, 12- and la-day-old embryonic chicken breast muscles, respectively; scan c, adult chicken breast muscle. (B) Scans a and b, 12s and 18-day-old embryonic chicken leg muscles, respectively; scan c, adult chicken leg muscle.

both LYand ,8 subunits (see also Fig. 3). It appears from these results that while chicken skeletal muscle ,8 subunit of tropomyosin is very similar to the corresponding subunit of rabbit skeletal muscle, with respect to both molecular weight and charge, the two components of chicken skeletal (Y subunit of tropomyosin are clearly distinct from the corresponding subunit of rabbit skeletal muscle tropomyosin. Furthermore, the multiple forms of the chicken skeletal muscle (Y subunit of tropomyosin seen in adult muscles are also present at the embryonic stage. Urea-polyacrylamide gel electrophoresis of cardiac tropomyosins. When cardiac

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DEVELOPMENTAL BIOLOGY VOLUME 69, 1979

abcdefQh FIG. 6. NaDodSOd-polyacrylamide gel electrophoresis of tropomyosins isolated from adult and embryonic chicken and rabbit cardiac muscles. Amount of protein loaded, 6-13 pg. Gels a and b, adult and 12-day-old embryonic chicken cardiac tropomyosin, respectively; gel c, mixed sample of adult and embryonic chicken cardiac tropomyosin; gel d, mixed samples of adult and embryonic chicken cardiac and rabbit skeletal (Ysubunit of tropomyosin; gels e and f, adult and embryonic (3- to 4-week) rabbit cardiac tropomyosin, respectively; gel g, mixed sample of adult and embryonic rabbit cardiac tropomyosin; gel h, mixed samples of adult and embryonic rabbit cardiac and rabbit skeletal oi subunit of tropomyosin.

tropomyosins of chicken and rabbit were analyzed by acidic urea gel electrophoresis (Fig. 8), the electrophoretograms revealed complexities not apparent in NaDodS04 gel runs. Cardiac tropomyosins of both rabbit (gel a, Fig. 8) and chicken (gel b, Fig. 8) migrate as well-resolved single bands, the rabbit cardiac tropomyosin moving much faster than that of the chicken (gel c, Fig. 8). The large difference in mobilities in urea gel runs indicates that the cardiac tropomyosins of rabbit and chicken are significantly different from each other. Coelectrophoresis of mixed samples shows that only rabbit cardiac tropomyosin comigrates with rabbit skeletal cu subunit of tropomyosin (gel d, Fig. 8), indicating the similarity between the two proteins. Of the two bands of chicken breast muscle tropomyosin described in the previous section, only the slower-moving component comigrates with

the chicken cardiac tropomyosin (gel e, Fig. 8), indicating that the polypeptide chain corresponding to this band may be the only common tropomyosin subunit present in both skeletal and cardiac muscles in chicken. Myosin light chains of adult and embryonic skeletal muscles of rabbit and chicken. In addition to tropomyosin we have also used the myosin light chains of rabbit and chicken skeletal muscles in order to study in parallel the developmental changes in the isozymic distribution of these proteins. Figures 9 and 10 show the densitometric scans of NaDodS04-polyacrylamide gel runs of purified myosin preparations of rabbit and chicken skeletal muscles, respectively. As judged by the electrophoretic mobilities of the three light chains, LC, LG, and LG, myosins isolated from both embryonic rabbit (scan A, Fig. 9)

ROY, SRETER, AND SARKAR

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FIG. 7. Urea-polyacrylamide

gel electrophoresis of chicken and rabbit skeletal muscle tropomyosins. (A) Amount of protein loaded, 8-15 pg. Gel a, adult chicken breast muscle tropomyosin; gel b, rabbit skeletal p subunit of tropomyosin; gel c, rabbit skeletal (Y subunit of tropomyosin; gels d and e, mixed samples of adult chicken breast muscle tropomyosin and rabbit skeletal a and /3 subunits of tropomyosin, respectively. (B) Amount of protein loaded, 5-12 pg. Gels a, b. and c. adult chicken leg, 15-day-old embryonic chicken leg, and mixed sample of adult and 15-day-old embryonic chicken leg muscle tropomyosin, respectively; gel d. mixed sample of adult chicken leg muscle tropomyosin and rabbit skeletal j3 subunit of tropomyosin.

Fro. 8. Urea-polyacrylamide gel electrophoresis of tropomyosins of chicken and rabbit cardiac muscles. Amount of protein loaded, 6-12 pg. Gels a and b, rabbit and chicken cardiac tropomyosin, respectively; gels c and d, mixed samples of rabbit and chicken cardiac, and rabbit cardiac and rabbit skeletal (1 subunit of tropomyosin, respectively; gel e, mixed sample of chicken cardiac and breast muscle tropomyosin.

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VOLUME 69, 1979

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FIG. 9. Densitometric scans of the light-chain regions of myosins isolated from adult and embryonic (3- to 4-week-old) rabbit skeletal muscles. For details, see text. (A) Adult rabbit extensor digitorum longus msucle (-) and embryonic rabbit skeletal muscle (- -); (B) adult rabbit soleus muscle; (C) mixed sample of adult rabbit extensor digitorum longus and embryonic rabbit skeletal muscle; (D) mixed sample of adult rabbit soleus and embryonic rabbit skeletal muscle.

and embryonic chicken (scan C, Fig. 10) skeletal muscles seem to resemble the myosin isolated from adult fast muscles of the corresponding animals. This similarity in light-chain patterns of embryonic skeletal and adult fast muscles is further confirmed by coelectrophoresis of the two protein samples purified from both rabbit (scan C, Fig. 9) and chicken (scan C, Fig. 10) muscles. The only difference between embryonic skeletal and adult fast muscle myosin light chains of the above two species, which is apparent from the densitometric scans of the myosin light chains, lies in the content of LC3 light chain. In both embryonic rabbit (scan A, Fig. 9) and chicken (scan C, Fig. 10) skeletal muscles, the LG light chain of myosin is found to be present in reduced quantities compared to that present in adult fast muscles, This point is further documented in Fig. 11, which shows that

while the LC2 light-chain content remains unchanged during development (about 2 moles/mole of myosin) of chicken breast muscle, the molar ratio of LCi:LCa changes significantly between Day 1 before birth and Day 8 ex ovo. Similar changes in the relative contents of LCI and LC3 light chains have also been observed in developing rabbit skeletal muscles (Takahashi and Tonomura, 1975). Studies with slow adult muscle myosins of both rabbit (scan B, Fig. 9) and chicken (scan A, Fig. 10) indicate that they are different from embryonic fast muscle myosin with respect to two criteria: (i) Slow muscle myosin of neither of the above two species contains any LG myosin light chain, and (ii) both LC1 and LC2 myosin light chains of slow muscles have mobilities different from those of the corresponding myosin light chains of embryonic skeletal

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since sequence information for some of these proteins, particularly the embryonic forms, is not currently available, The developmental patterns of tropomyosin subunits indicate several interesting features. The p subunit, which is predominant in embryonic skeletal muscles of both chicken and rabbit, is gradually replaced during development by the (Y subunit in skeletal fibers. Depending on the species and the muscle type, these changes subseDISCUSSION quently lead to either (a) a single or preThe tropomyosin subunits and myosin dominantly a-subunit pattern or (b) a patlight chains have been characterized in this tern consisting of almost equal amounts of (Y and p subunits (Fig. 2). In contrast to work by comparison of the electrophoretic skeletal muscles, only the (Ysubunit of tromobilities of the subunits of the purified proteins according to both size (Figs. 2, 3, pomyosin is present in chicken and rabbit 6, 9, and 10) and charge (Figs. 7 and 8) and cardiac muscles at all stages of developassay for their biological activity. However, ment. Changes in the tropomyosin subunits the similarities between the protein sub- associated with the development of various units present in embryonic and adult stages striated muscles of the rabbit and the should be interpreted with due reservation, chicken as described here are schematically

muscles in both rabbit (scan D, Fig. 9) and chicken (scan B, Fig. 10). Comparison of the time courses of developmental changes in myosin light chains (Fig. 11) and tropomyosin subunits (Fig. 4) of chicken skeletal muscles indicates that the changes in the two myofibrillar protein subunits do not take place in a strictly parallel way, the tropomyosin subunit pattern characteristic of adult muscles being reached earlier.

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FIG. 10. Densitometric scans of the light-chain regions of myosins isolated from adult and 20-day-old embryonic chicken skeletal muscles. For details, see text. (A) Adult chicken ALD (--) and PLD (- -) muscle; (B) mixed sample of adult chicken ALD and embryonic chicken twitch muscle; (C) mixed samples of adult chicken PLD and embryonic chicken twitch muscle (---) and embryonic chicken twitch muscle (- 4; (D) mixed sample of adult chicken ALD and PLD muscle.

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AGE (DAYS) FIG. 11. Time course of developmental changes in the myosin light-chain patterns of chicken breast muscle. For details, see also Experimental Procedures. Results are expressed in terms of moles of light chain per mole of myosin. Each point represents the mean of four separate gel runs with each of two different preparations of myosin. The variation in the estimation of different experiments was f 10%.

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L FIG. 12. Schematic representation of the relative various striated muscles. q , (Ysubunit of tropomyosin;

summarized in Fig. 12. Urea gel electrophoresis of tropomyosin indicates that only the cysubunit is present in multiple forms in the same muscle among different species, and also in different muscles of the same species (Figs. 6 and 7). Of

proportions of the a and p subunits q , fi subunit of tropomyosin.

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the two electrophoretically distinct forms of the cr subunit present in adult chicken skeletal muscles, only one seems to be common to both skeletal and cardiac muscles. Furthermore, both subspecies are also present in embryonic chicken skeletal muscles.

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With respect to myosin light chains, our results indicate that both avian and mammalian embryonic fast muscle myosins resemble the adult fast type (Figs. 9 and 10). This observation is also supported by recent histochemical studies (Rubenstein and Kelly, 1978) which indicate that embryonic rat skeletal muscles contain predominantly type II fibers characteristic of adult fast muscle. The difference between embryonic and adult fast muscle myosin light chains is reflected only in the LCI:LC$ ratio, which shows a gradual change, like the tropomyosin subunits, during development (Fig. 11). Previous reports on myosins isolated from embryonic PLD muscles (Rubenstein et al., 1977) indicated the absence of the LC3 light chain. However, the changes in the LC:LCZ ratio during development shown in Fig. 11 indicate that the LC3 light chain is indeed present in embryonic myosin. In contrast to the presence of only fast myosin light chains in developing fast muscles (Figs. 9 and lo), both fast and slow myosin light chains have been reported to be present during early development of slow muscles (Pelloni-Muller et al., 1976a; Rubinstein et al., 1977). Gauthier et al. (197&J),in a recent report, have observed that all embryonic fibers from rat diaphragm, a fast twitch muscle, react with anti-slow myosin antibody. This seems to be somewhat puzzling, since Rubinstein et al. (1977) did not find any cross-reaction between anti-slow myosin antibody and embryonic fast muscle myosin. Considered together with other reports in the literature (Sreter et al., 1973, 1975; Pelloni-Muller et al., 1976a,b: Rubinstein et al., 1977; Rubinstein and Kelly, 1978), our results rule out the previously held view that all embryonic myosins are of the adult slow type (Holland and Perry, 1969; Perry, 1970; Trayer et al., 1968; Trayer and Perry, 1966) and that definitive fast fibers are produced from a pool of embryonic slow fibers (for a review, see Perry, 1974). The present studies also suggest that the distribution of the types of tropomyosin

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and myosin present in skeletal muscles is altered during development. In terms of the currently accepted isozyme concept of the two-headed myosin molecule (Holt and Lowey, 1977)) our results indicate that homodimers containing two LC, light chains per mole of myosin are the major species present in embryonic skeletal muscle. Subsequently, with the appearance of L&-containing homodimers during development, changes in the isozyme populations of myosin must take place in the fiber until the pattern characteristic of adult muscle is reached (Sarkar, 1972; Holt and Lowey, 1977; Matsuda et al., 1977). Similarly, the high P-subunit content of embryonic skeletal muscle tropomyosin indicates that the /3,B dimer, which is absent in adult skeletal muscles (Eisenberg and Kielley, 1974; Lehrer, 1975; Ookubo, 1977; Yamaguchi et al., 1974), must be present as the major species in embryonic stages. With respect to the postulated correlation between the speed of contraction and the tropomyosin subunit ratio of skeletal muscle (Cummins and Perry, 1974), our results indicate that embryonic muscles with a higher proportion of the p subunit should be considered as slow. However, in terms of myosin light chains, it appears that embryonic skeletal muscle resembles adult fast muscle. Thus it is inappropriate to term an embryonic skeletal muscle as fast or slow solely on the basis of the type distribution of any single myofibrillar protein. The developmental changes of myofibrillar proteins reported here could be the result of either a differential growth of different fiber populations or, alternatively, a switch in the expression of one set of genes to another within the same fiber. Recent experiments, involving one of us (Rubinstein et al., 1978), using antimyosin antibodies specific to either fast or slow muscle myosin have shown that during the transformation of fast muscle to the slow type by chronic electrical stimulation, the individual fibers are reprogrammed to switch from the synthesis of fast to the synthesis

28

DEVELOPMENTAL BIOLOGY

of slow myosin. Since the developmental changes reported here take place in terminally differentiated fibers, it is most likely that the isozymic changes are due to a switch in the expression of genes in the same fiber and not due to a differential growth of fiber populations. This view is further supported by recent studies of Kugelberg (1976) which showed that in developing rat soleus muscle there is a smooth and continuous transition from type II to type I fibers due to progressive differentiation of one and the same motoneuron. The characteristic tropomyosin and myosin subunit pattern seen in embryonic skeletal muscles could be attributed to an intrinsic developmental program in all embryonic skeletal muscles regardless of their maturation into fast or slow type. Subsequently, under the influence of exogenous factors or stimuli, such as innervation, activity pattern, etc., other genes coding for different isozymic forms of the myofibrillar proteins are turned on, and this leads to the differentiation of individual muscle fibers into specific types. The appearance of the LC3 light chain in fast muscles, the switch from the synthesis of both fast and slow types of myosin to that of only slow myosin in slow muscles (Rubinstein et al., 1977), and the change in the a$ ratio of tropomyosin subunits during muscle development may be interpreted as being due to the response of the fibers to such factors. The authors thank Drs. John Gergely and John Seidel for critical reading of the manuscript and Ms. Cynthia Mis for her expert assistance in photography. This work was supported by grants from the National Institutes of Health (AM 13238 and AG 00262), the National Science Foundation (7704289PCM), and the Muscular Dystrophy Associations of America, Inc.; it was carried out during the tenure of a research fellowship from the American Heart Association, Massachusetts Affiliate, awarded to Raman K. Roy. REFERENCES AMPHLETT,G. W., SYSKA,H., and PERRY,S. V. (1976). The polymorphic forms of tropomyosin and troponin in developing rabbit skeletal muscle. FEBS Lett. 63,22-25. BAG, J., and SARKAR,S. (1975). Cytoplasmic nonpol-

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ysomal messenger ribonucleoprotein containing actin messenger RNA in chick embryonic muscles. Biochemistry 14,3800-3807. BARANY, M. (1967). ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50, 197-218. BETCHER-LANGE, S. L., and LEHRER, S. S. (1978). Pyrene excimer fluorescence in rabbit skeletal (YOI tropomyosin labeled with N-(1-pyrene)maleimide: A probe of sulfhydryl proximity and local chain separation. J. Biol. Chem. 253, 3757-3760. BULLER, A. J., ECCLES,J., and ECCLES,R. (1960a). Differentiation of fast and slow muscles in the cat hind limb. J. Physiol. 160,399-416. BULLER, A. J., ECCLES, J. C., and ECCLES, R. M. (1960b). Interactions between motoneurones and muscles in respect to the characteristic speeds of their responses. J. Physiol. 150,417-439. CLOSE,R. (1964). Dynamic properties of fast and slow skeletal muscles of the rat during development. J. Physiol. 173,74-95. CLOSE,R. (1972). Dynamic properties of mammalian skeletal muscles. Physiol. Rev. 52,129-197. CUMMINS, P., and PERRY, S. V. (1973). The subunits and biological activity of polymorphic forms of tropomyosin. Biochem. J. 133.765-777. CUMMINS,P., and PERRY, S. V. (1974). Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle. Biochem. J. 141, 43-49. DABROWSKA,R., SOSINSKI,J., and DRABIKOWSKI,W. (1977). Changes in the composition of the myofibrillar fraction during development of rabbit. FEBS Lett. 79, 295-300. EISENBERG,E., and KIELLEY, W. Y. (1974). Troponintropomyosin complex: Column chromatographic separation and activity of the three active troponin components with and without tropomyosin present. J. Biol. Chem. 249,4724-4748. GAUTHIER,G. F., LOWEY,S., and HOBBS,A. W. (1978). Fast and slow myosin in developing muscle fibers. Nature (London) 274,25-29. GREASER,M. L., and GERGELY,J. (1971). Reconstitution of troponin activity from three protein components. J. Biol. Chem. 246,4226-4233. HAYASHI, J., ISHIMODA, T., and HIRABAYASHI, T. (1977). On the heterogeneity and organ specificity of chicken tropomyosins. J. Biochem. 81,1487-1495. HESS, A. (1970). Vertebrate slow muscle fibers. Physiol. Rev. 50, 40-62. HOH, J. F. Y., MCGRATH, P. A., and WHITE, R. I. (1976). Electrophoretic analysis of multiple forms of myosin in fast-twitch and slow-twitch muscles of the chick. Biochem. J. 157,87-95. HOLLAND, D. I., and PERRY, S. V. (1969). The adenosine triphosphatase and calcium ion transporting activity of the sarcoplasmic reticulum of developing muscles. Biochem. J. 114, 161-170. HOLT, J. C., and LOWEY, S. (1977). Distribution of

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