Emergence of the mature myosin phenotype in the rat diaphragm muscle

DEVELOPMENTAL

BIOLOGY

144, 1-15 (1991)

Emergence of the Mature Myosin Phenotype in the Rat Diaphragm Muscle W. A.LAFRAMBOISE,*,~ M.J. DAooD,t R.D. GuTHRIE,t S. SCHIAFFINO,$P. MORETTI,$ B. BROZANSKI,t M.P.ONTELL,* G.S.BUTLER-BROWNE,§ R.G.WHALEN,IIAND M. ONTELL* *Department of Neurobiology, Anatomy and Cell Science and tDepartment of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; *Institute of General Patholagy and CNR Unit for Muscle Biology and Physiopathology, University of Padova, Italy; QINSERM U 262, Clinique Universitaire Baudeloque, 123 bd de Port Royal, 75014 Paris, France; and “Departement de Biologie Moleculaire, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, France Accepted November

20, 1990

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHCax and MHC, appeared perinatally, while MHC, did not appear until 30 days postnatal. By Day 60 this isoform was present in -27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day > 115, ~4% of the myofibers expressed MHC,). Fibers which contained MHC&,,, during the late fetal and early neonatal period coexpressed MHC,,. A marked increase in the frequency of fibers containing MHCO,.i, occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHC,, and MHC, during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was 3115 days old. o 1991 Academic press, IN.

myosin heavy chain (MHC,,,), and adult isoforms coexist in single myofibers during development (Whalen et The contractile protein myosin exists in multiple, tis- ab, 1981; Butler-Browne and Whalen, 1984). sue-specific and developmentally regulated isoforms In the present study, it is demonstrated that an affinencoded by a family of genes (cf. Whalen et al., 1981; ity-purified polyclonal antibody, previously designated Buckingham, 1985; Mahdavi et al, 1987; Barton et ah, anti-fast MHC (Butler-Browne and Whalen, 1984), im1989). Seven myosin heavy chain (MHC) genes, each munoblots the band containing MHC,n and the band coding for a different protein, have been identified in containing MHC,, but fails to react with bands containthe rat (Wieczorek et aZ., 1985). Of these, five are ex- ing MHC,, , MHC,,,, and MHC, on transfers of MHCs pressed in typical rat muscle, while two are expressed in which have been separated on 5% SDS-PAGE. Morehighly specialized rat head muscles. Recent evidence in- over, it is also demonstrated that this antibody reacts dicates the existence of an additional MHC that is found with myofibers containing MHC, and with myofibers in typical rat muscles and that is particularly promi- containing MHC, but not with myofibers containing nent in the adult diaphragm. This isoform had been des- MHCZA. Therefore, we have renamed this antibody antiignated as MHC,x (Schiaffino et al., 1986, 1989) or MH%B+zx. By reacting serial sections of developing MHC,, (Bar and Pette, 1988; Termin et a& 1989). While muscle with this antibody and with a monoclonal antithe gene coding for this isoform has not been identified, body specific for MHC, (BF-F3; Schiaffino et ah, 1986), evidence for the existence of MHC,x in rodents has been it is possible to identify by subtraction myofibers conobtained in separate studies using either immunocytotaining MHC, (i.e., fibers containing MHCvr will react chemistry or SDS-PAGE. Schiaffino et al. (1986) have with anti-MHC,,,,, and will be unstained with the used a monoclonal antibody (BF-35) which reacts with monoclonal antibody specific for MHC&. This permits all known striated muscle MHC isoforms (including de- determination of the time of appearance of MHC,, even velopmental isoforms) except MHC& to define, by ex- when MHC,,, and/or MHC,,, are the predominant clusion, those fibers which contain only the MHC, iso- myosin isoforms in the perinatal myofibers. form. However, this antibody cannot be used to deterIn the present study, serial cryosections of late fetal, mine the time of onset of the expression of MHC, since early neonatal, and adult rat diaphragm muscles have embryonic myosin heavy chain (MHC,,,), neonatal been reacted with anti-MHC,,,, and with a battery of INTRODUCTION

1

00121606/91 $3.00 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form resewed.

2

DEVELOPMENTALBIOLOGYVOLUME144,1991

monoclonal and polyclonal antibodies which allow the identification of all of the known MHC isoforms found in typical developing and adult rat muscles. This study provides the first immunohistochemical evaluation of the time course of the appearance of all known MHC isoforms and facilitates consideration of the sequential changes, on an individual myofiber level, which occur as the adult phenotype gradually emerges. Parallel studies with SDS-PAGE and two-dimensional gel electrophoresis of myosin light chains further elucidate the pattern of emergence of the myosin isoforms expressed in the adult diaphragm. METHODS

Sample Origin Diaphragm muscles were removed from fetal rats (Sprague-Dawley) at 18 and 21 days in utero (E18, E21) and from male rats at 4,21,30,40,60,90, and >115 days postnatal [D4, D21, D30, D40, D60, D90, and D115-D156 (adult)]. Midcostal sections originating from the 8th through the 10th rib were excised and prepared for either immunohistochemical or electrophoretic analysis. Additionally, diaphragm muscles of D8, Dll, and D14 rats were removed for analysis of myosin light chain (MLC) composition by electrophoresis, and the tibialis anterior and soleus muscles of adult rats were removed for analysis of MHCs by electrophoresis. Each sample was obtained from rats of different litters, except for the fetal tissue where diaphragm muscles from littermates of three litters (at each age) were used. Immunohistochemistry Muscle samples were embedded in liver, frozen in isopentane cooled in liquid nitrogen and sectioned in a cryostat at -20°C. Serial sections (5-8 pm thick) were reacted with either monoclonal or polyclonal antibodies to MHC isoforms. The specificity of three monoclonal antibodies (mouse anti-bovine) to MHC,A (SC-71), MHCzB (BF-F3), and to all MHC isoforms except MHC& (BF-35) had been determined with rat muscle, by immunotransfer analysis, and by comparison of histochemically and immunochemically reacted serial cryostat sections (Schiaffino et ah, 1986, 1989). In the present study these antibodies were referred to as anti-MHC&, and anti-MHC,+,x, respectively (Table anti-MH&, 1A). Schiaffino et al. (1989) showed that anti-MHC,, and anti-MHC, reacted with epitopes which were exclusive to MHCzA and MHC=, respectively. Anti-MHC,,,-, reacted with an epitope common to all known striated muscle MHCs but did not react with MHC,,. Therefore, anti-MHC,,,-, can only be used to identify myofibers in which MHC,, is the sole isoform of MHC present (Schiaffino et ah, 1989) (Table 1B). The specificity of

affinity-purified, polyclonal antibodies (rabbit anti-rat) against embryonic (MHC,,,), neonatal (MHC,,,), and slow (MHC B,slOw) myosin heavy chains used in the present study had been previously established, both by immunotransfer analysis and indirect immunofluorescence of cryostat sections (Butler-Browne and Whalen, 1984). An affinity-purified polyclonal antibody, antifast MHC, had been previously shown to react with myosin prepared from fast rat muscle, but not to myosin from slow, neonatal, or embryonic muscle (ButlerBrowne and Whalen, 1984). In the present study we sought to determine which of the three fast MHCs known to be present in the rat limb muscle and diaphragm muscle (i.e., MHCZA, MHC&, and MHC,x) would react with this antibody. Accordingly, serial sections of the adult diaphragm were reacted with this antibody and with the specific monoclonal antibodies: antiMHCZA, anti-MHC,, and anti-MHC,,-,. Since it was determined that the polyclonal anti-fast MHC (ButlerBrowne and Whalen, 1984) reacted with MHCzB and MHCw but not with MHC,A (Fig. 1) (an observation confirmed by Western blot analysis, Fig. 9), we changed the name of the antibody to anti-MHC,,,,, to reflect its specificity (Table 1A). In the present study, antiMHCm+zx was used to determine the presence of MHCvr in myofibers which had less than 100% of their total MHC composition as the MHCvr isoform. Fibers which in serial sections are positive with anti-MHC,,,, but are negative with the monoclonal antibody, antiMHC,, contain some MHC,,. A description of the antibodies used in the immunohistochemical analysis and an immunohistochemical method for demonstrating the presence of MHC, when it is the exclusive MHC isoform or when it is one of two or more isoforms in a single myofiber are found in Table 1. Primary antibodies, diluted in phosphate-buffered saline (PBS) containing 0.5% (w/v) bovine serum albumin, were applied to sections for overnight incubation in a humid chamber at 4°C. Slides were washed in PBS (3 x 15 min) and reacted with a rhodamine-labeled secondary antibody [goat anti-rabbit polyvalent Ig for polyclonal antibodies, goat anti-mouse IgG for all monoclonal antibodies except anti-MHC,,, and goat anti-mouse IgM for anti-MHC, (Fisher Biotech, NY)]. Slides were incubated (45 min at 37”C), washed (3 X 15 min in PBS), mounted in Mowiol, and viewed with a microscope equipped for epifluorescence. Sections receiving only the second antibody served as a control for background activity. Serial sections of adult diaphragm were included as a control in all immunohistochemical studies. For all muscle samples, photographic montages (magnified 250X) were constructed of each of the serial sections reacted with the different MHC antibodies. A minimum of 300 myofibers for each E21 muscle sample (n = 3) and at least 500 but typically 2500 fibers for each older sam-

MHC Isoform

LAFRAMBOISE ET AL.

TABLE A. Specificity

1

of Antibodies for MHCs Myosin heavy chains

Antibody Original

name

3

Expressim

New name

Emb

TYP@

Neo

Slow

2A

2B

2x

-

-

-

-

-

f + +

+ -

+ -

+ -

f

+

Immunohistochemistry” Anti-MHC,,t,c Anti-MHC,,” Anti-MHC,,,,,” Anti-fast MHCd SC-71e BF-F3* BF-35”

Anti-MHC,,, Anti-MHC, Anti-MHC, Anti-MHC,,,.,

+ -

P P P P M M M

+ -

+

-

+ -

+

+

+

+ -

-

-

+ -

+ -

+ -

Immunoblottin$ RT-D9” BF-32” Anti-fast

MHCd

Anti-MHC,,,, B. Identijcatimz

-

M M P of Myojibers

with MHC,,

with Immunohistochemistry

1. Fibers containing MHC, exclusively: a. Fail to react with anti-MHC,,.,. b. React with anti-MHC,,, and fail to react with MHC,. 2. Fibers containing MHC, which is eoexpressed with any other MHC except MHC,. a. React with anti-MHC,,,, but fail to react with MHC,,. 3. Fibers containing MHC, and MHC, It is not possible to determine with the available antibodies whether fibers containing MHC, coexpress MHC,, as these fibers would exhibit the same staining as fibers which contain MHC,, exclusively (i.e., they would stain with anti-MHC,+,x and they would stain with anti-MHC,,). a The names of all monoclonal antibodies against MHCs used for immunohistochemistry were changed to reflect the MHC isoform(s) with which they react. b The original names of the antibodies used for immunoblotting were retained. ‘The specificity of these antibodies were demonstrated in Butler-Browne and Whalen (1984). d This antibody, reportedly specific for fast-MHCs (Butler-Browne and Whalen, 1934), has been shown in the present study to be specific for MHC, and MHC,, but not for MHC,. In the present study it has been demonstrated that this antibody does not react with MHC,,,+,or MHC,,. The new name reflects this specificity. e The specificity of these antibodies had been demonstrated in Schiaffino et al. (1989). *The specificity of this antibody had been demonstrated in Schiaffino et al. (1986). UP, polyclonal; M, monoclonal.

ple (D4, n = 6; D21, n = 5; D30, n = 2; D60, n = 5; adult, n = 7) were followed in serial sections and evaluated according to their reactivity with each antibody. Additional immunohistochemical studies were performed at certain ages to evaluate the presence of MHC,,, and MHC,, (D30 and D90, n = 4). Electrophoresis

Minced muscle samples were extracted on ice for 40 min in 4 vol of buffer (300 mMNaC1, 100 mMNaH,PO,, 50 mM Na,HPO,, 1 mM MgCl,, 10 mM Na,P,07, 10 mM EDTA) at pH 6.5 as previously described (ButlerBrowne and Whalen, 1984). Extracts were centrifuged at 4°C [13,OOOgfor 30 min in a Model 235C microfuge (Fisher Scientific)] and the supernatants recovered for

gel electrophoresis. A minimum of five samples from every age group was analyzed by each electrophoretic technique. (1) 5% SDS-PAGE of myosin heavy chains. Extracted myosin was diluted in 9 vol of 1 mM EDTA and 0.1% 2-mercaptoethanol (v/v), stored overnight at 4°C to allow precipitation of myosin filaments, and centrifuged (13,000~ for 30 min) to form a pellet. The pellet was dissolved in buffer (0.5 M NaCl, 10 m&f NaH,PO,) and diluted 1:200 in SDS buffer [62.5 mMTris/HCl, 2% (w/v) SDS, 10% (v/v) glycerol, and 0.001% (w/v) bromophenol blue] at pH 6.8 (Laemmli, 1970). Samples were boiled for 2 min and stored at-80°C. Electrophoresis was carried out using a modification (LaFramboise et aZ.,1990) of previously described protocols (Laemmli, 1970; Carraro and Catani, 1983; Danieli-

DEVELOPMENTALBIOLOGY

vOLUME144,1991

FIG. 1. Serial frozen sections of an adult (D150) diaphragm muscle reacted with (A) anti-MHC,,, (B) anti-MHCs,,,,,, (C) anti-MHC,, (D) anti-MHC,, (E) anti-MHC,,l-zx, and (F) anti-MHC,,,. Numbers indicate the same fiber in serial sections. Fiber 5 can be identified as containing MHCzx exclusively, because it fails to react with anti-MHC,,,-, (E). Anti-MHC,s+zx (F) reacts with all myofibers which react with anti-MHC, (D), and with all myofibers which fail to react with anti-MHC,,,-, (E), but it fails to react with any myofibers which contain MHCm., W or MHC, (C). Therefore, it is possible to identify fibers containing MHC, (e.g., fiber 5) (1) by their failure to react with anti-MHC,,,-, or (2) by their reactivity with MHC,s+zx and their failure to react with anti-MHC,s. This region of the diaphragm was chosen because it had an unusually high frequency of fibers containing MHC,, and it was suitable, therefore, for this demonstration. Size bar = 20 pm.

Betto et al., 1986). Five percent separating and 3% stacking gels were prepared from a stock solution of 30% (w/v) acrylamide: 1.5% (w/v) bis(N,N-bis-methylene acrylamide) and 28.5% (w/v) acrylamide. Glycerol [25-33% (v/v)] was added to the separating gel (Carraro and Catani, 1983; Danieli-Betto et ah, 1986; Biral et ab, 1988). Myosin extracts (l-3 ~1) containing 500 to 1500 ng of protein were loaded onto the gels. Electrophoresis (120 V for 22-24 hr) was performed using a vertical slab gel unit (SESOO,Hoefer Sci. Instr.), with Tris/glycine running buffer (Laemmli, 1970) maintained at 15°C (Neslab, Endocal RBC-3). Separating gels were stained with silver nitrate (Oakley et aZ.,1980). Immunoblot analysis was performed after the electrophoretic transfer of MHCs from unstained 5% gels to nitrocellulose sheets (Towbin et aL, 1979). Two protocols for immunoblot analysis were followed. With the first protocol, the transfers were incubated, initially, with a

monoclonal antibody BF-32 specific for immunoblotting the band containing MHC,,, and the band containing MHC,, (Schiaffino et al., 1989; LaFramboise et aL, 1990). The primary antibody was visualized with a peroxidaselabeled second antibody [goat anti-mouse Ig (Dakopatts)] followed by development with diaminobenzidine in the presence of imidazole (Trojanowski et al., 1983). The immunoblot was then incubated with a monoclonal antibody RT-D9 [previously determined to stain the band containing MHC, and the band containing MHC, on Western blots of adult muscle (Schiaffino et aL, 1989; LaFramboise et ak, 1990) and which also reacted with MHC,,, in developing muscle]. It was visualized as described above. With the second protocol, the transfers were incubated with the polyclonal anti-fast MHC (Butler-Browne and Whalen, 1984), which we had renamed anti-MHC,,+,x. The primary antibody was visualized with an Amersham RPN.23 blotting detection kit,

LAFRAMBOISEETAL. MHC Isofm

Expression

5

All of the myofibers in E21 and in D4 diaphragm muscles contained some MHC,,, (Figs. 2A and 2B) and/or MHC,,, plus MHC,,, (Figs. 3A and 4A). Therefore, all of these fibers reacted with anti-MHC,,,-, (Fig. 3E), confirming that it would not be possible to use this antibody to demonstrate the presence of fibers which coexpressed MHC,, if such fibers were present (see Table 1). After immunostaining with anti-MHC,,,,,, -20% of the myofibers in the diaphragm muscle at E21 (Figs. 3F and 5) reacted weakly and -80% of the myofibers on D4 (2) Two-dimensicmal gel electrophoresis of light chains. Extracted myosin was diluted in 9 vol of buffer contain- (Figs. 4E and 5) reacted with greater avidity. That so ing 1 mM EDTA and 0.1% (v/v) Z-mercaptoethanol and many of the fibers with MHC,,, and/or MHC,,, plus stored overnight at 4°C to allow formation of myosin MHC,,, did not react with anti-MHCzs+,x suggested filaments. The solution was centrifuged (13,000g for 30 that this antibody did not react with any known developmin) and the supernatant removed. Myosin pellets were mental isoforms of MHC. (See also Butler-Browne and resuspended in lysis buffer [9.5 M urea, 2% (v/v) Noni- Whalen, 1984.) This was confirmed by the failure of this antibody to react with bands containing either MHC,,, det-P40, 2% ampholines, and 5% (v/v) 2-mercaptoethanol], and lo-20 pg of myosin was loaded into tubes con- or MHC,,, on immunoblots of MHCs transferred from taining the isoelectric focusing gel (13 cm X 2.5 mm) for 5% SDS-PAGE of neonatal muscle (see below and Fig. electrophoresis according to Whalen et al. (1978). Am- 9). To determine whether the weak reaction with antipholines (Pharmacia) in the pH range of 4.5-5.4 (1.6%) MHC,B+ZX at E21 (Fig. 3F) might reflect cross-reactivity and 3-10 (0.4%) were utilized, and isoelectric focusing with an, as yet undiscovered, embryonic isoform present gels were run in the second dimension on SDS-PAGE at prior to E21, sections of El8 muscles were reacted with 20 mA through a 5% stacking gel and 25 mA through a this antibody. None of the fibers in the El8 diaphragm 12.5% separating gel (Laemmli, 1970). Gels were stained reacted with anti-MHC,,,, (not shown). with 0.1% Coomassie blue in 25% isopropanol and 10% Given the failure of anti-MHC,,,,, to react with any acetic acid. When necessary, silver stain was used to known developmental MHC, its failure to react with developmental isoforms present in the diaphragm prior to improve the detection of trace quantities of myosin light chains (Oakley et al., 1980). E21, and its failure to react with MHCs,,i, or MHCzA, this antibody could be used alone to determine the presRESULTS ence of MHC,, at all stages prior to the appearance of MHCZB in the rat diaphragm (i.e., in the absence of I. Immunohistochemistry MHC,, , all myofibers staining with anti-MHC,,,,, IdentQication of myojibers containing MHC,,. The would contain MHC,,). After MHC, begins to be expolyclonal antibody previously designated as anti-fast pressed in the diaphragm (this isoform is not expressed MHC (Butler-Browne and Whalen, 1984) was renamed until D30, when ~1% of the fibers react with anti anti-MHc,+, because it reacted with all of the myo- MHC,,; Fig. 6), a positive reaction with anti-MHC,,, fibers of the adult (D115-D156) diaphragm which and a failure to react with anti-MHC, would be evistained with the monoclonal antibody specific for dence of MHC, expression, even if this isoform was beMHCzs, stained all of the myofibers which failed to ing coexpressed with one or more of the following isostain with the anti-MHC,,,-,x and because it failed to forms of MHC: MHC,,,, MHC,,,, MHC@,,,,,,,and MHC, react with fibers which contained MHC,, (fiber 5, Fig. 1; (e.g., fiber 2, Fig. 3; fiber 3, Figs. 4A-4E). However, it Table 1). Confirmation of the specificity of this antibody would not be possible to identify myofibers which confor MHC,s and for MHC,, was demonstrated on West- tain MHC, if they were coexpressing MHC,, as these ern blots of transfers of denatured MHCs which had fibers would react similarly to fibers containing only been separated by 5% SDS-PAGE (see below and Fig. MHC,o (i.e., they would react positively with anti9). Thus, the new name reflected the fact that the anti- MHCall-2X, positively with anti-MHC,,,, and with antibody reacted with two of the three fast MHC isoforms MHC,,; fiber 4, Figs. 4N and 40). Table 1B summarizes found in adult muscle. It was possible, therefore, to the methods for assessing the presence of MHC, with identify fibers which contained MHC, exclusively ei- immunofluorescence. ther by (1) their failure to react with anti-MHC,,,-, Developmental analysis of MHC isofim transitions. (Schiaffino et aL, 1989) or (2) their staining with anti- All myofibers from E21 muscles (n = 3) contained MHCU~+~ and their failure to stain with the monoclonal MHC,,,, and -90% of the myofibers also contained antibody specific for MHCzB (fiber 5, Figs. lD-1F; Ta- MHC,,, (Figs. 2A, 3A, and 5). Fibers failing to stain with ble 1B). anti-MHC,,, were the largest in diameter and reacted which is based on biotinylated second antibody (antirabbit) detected with a streptavidin-alkaline phosphatase conjugate. A description of the antibodies used for immunoblotting is found in Table 1A. For clarity, the antibodies used for immunohistochemical analyses will be referred to by the MHCs for which they are specific. We will refer to the monoclonal antibodies used for immunoblotting by the original name given in Schiaffino et aZ.(1989) (see Table 1A).

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DEVELOPMENTALBIOLOGY

VOLUME144,1991

FIG. 2. Frozen sections of diaphragm muscle (A) E21, (B) D4, and (C) D21 reacted with the polyclonal antibody against MHCemb.Myofibers contain MHC,,, through D4, although there is a reduction in the intensity of staining between E21 and D4. MHC,, is not present at D21. Bar = 20 pm.

intensely with anti-MHCg,,,,, (compare Figs. 3A and 3B). Approximately 20% of the fibers containing MHGrn, and MHC,,, also stained, albeit lightly, with anti-MHC,,,, . Since no fibers at this age stained with the monoclonal antibody specific for MHC, (Fig. 3D), this population of fibers contained a small amount of MH&x. None of the E21 samples showed reactivity with anti-hfHczA (Fig. 3C). At D4 (n = 6), there was a diminution in the intensity of fluorescence of fibers which reacted with antihowever, all fibers did show fluorescence MHG,,,; above background level (Fig. 2B). The percentage of fibers containing MHC,,, (Fig. 4A) was unchanged from that observed on E21 (Figs. 5 and 6). Those fibers which contained MHC&, but not MHC,,, stained intensely (fiber 1, Figs. 4A and 4B). These with the anti-MHC,,,,,,, fibers accounted for -10% of the total fiber population, unchanged from the frequency found at E21. None of the fibers containing MHCB,,,,, coexpressed MHC,,, (Figs. 4A, 4B, and 5). At this stage most fibers stained with the (Fig. 4E) but failed to stain with the anti-MHC,B+zx monoclonal antibody specific for MHC,, (Fig. 4D), indicating that a substantial number of myofibers contained MHC,. All myofibers stained with the anti-MHGall-2x (not shown). Thus, no fibers contained MHCvr exclusively. A substantial but variable percentage (X + SD = 43 + 24%) of D4 fibers contained MHC,, (Figs. 4C and 6). Whenever MHC,, was found in a fiber, it coexisted with MHC,,,, MHC,,, and MHC, (Figs. 4A4E and 5). Only 7 + 4% of the myofibers on D4 did not contain “adult” isoforms of MHC. By D21, none of the diaphragm myofibers (n = 5 muscle samples) contained MHC,,,, (Fig. 2C). Concomitant with the disappearance of MHC,,,, a population of myofibers containing a single MHC, MHCa,,,,, was observed (fiber 1, Figs. 4F-4J). Fibers containing only comprised 13 + 3% of the D21 population MHCmow

(Fig. 5), suggesting that they may be the same fibers which at earlier developmental stages contained both and MHC$,,i,, (i.e., the -10% of the fibers at MJKm, D4 which coexpressed these isoforms). During the 17day interval from D4 to D21, the percentage of fibers containing MH&,, was increased by ~100% (Fig. 6). At D21 many of the fibers which expressed MHCs,,,,, coexpressed MHC,,, (fiber 3, Figs. 4F-4J) or MHC,,, and MHCzA (fiber 7, Figs. 4F-4J). At D21, a second population of myofibers expressed a single MHC isoform. Approximately 7% of the myofibers stained with antiand did not stain with the monoclonal antiMHCm+ax body specific for MHC,,. These fibers failed to stain with the anti-MHC,,,-,x (not shown). Thus, by both staining criteria (Table lB), a population of fibers containing MHC,x, exclusively, was present by D21 (fiber 2, Figs. 4F-4J). The frequency of finding myofibers containing MHC,x decreased between D4 and D21 (Figs. 5 and 6), indicating that this isoform was only transiently expressed in some fibers. Of the remaining myofibers present at D21, -65% contained a single adult MHC isoform coexisting with MHC,,,, while all of the remaining myofibers in the D21 diaphragm contained three MHCs (Figs. 4F-4J and 5). As at earlier stages, there was no reaction with the monoclonal antibody specific for MHC,a (Fig. 41). Four D30 muscles were analyzed in serial sections. More fibers contained exclusively MH&,i,, (21 & 4%) or exclusively MHCvr (24 f 1%) at this age than at D21; however, all myofibers containing MHC= continued to coexpress other isoforms. At D30, a small number of fibers (~1%) stained faintly with the antibody specific for MHC,,, indicating that a limited amount of this isoform was present in two of the four muscles analyzed. By D60, none of the extrafusal myofibers of the diaphragm contained MHC,,, (Fig. 4K). At this age 31+ 4% of the fibers contained MHC,,,,,, 26 + 4% contained

LAFRAMBOISEETAL.

MHC Isofbrm Expression

7

FIG. 3. Serial cryosections of E21 diaphragm muscle reacted with (A) anti-MHC,,, (B) anti-MHCa,,,,,, (C) anti-MHC,, (D) anti-MHC,, (E) None of the fibers contain MHC,* (C) or MHC,a (D). All myofibers of E21 diaphragm muscle react with anti-MHC,,-,, and (F) anti-MHC,,,. anti-MHC,,, (see Fig. 2A), and all fibers react with anti-MHC,,,-, (E) (as all of them contain developmental isoforms). A few fibers react and are unstained with anti-MHC,,, indicating that they contain MHCzx in addition to the developmental isoforms. weakly with anti-MHC,,, Three groups of fibers can be identified: those which contain MHC,,, + MHC,, (fiber l), those which express MHC,,, + MHC,, + MHCU( (fiber 2), and fibers which contain MHC,,, + MHCBlslow(fiber 3). Bar = 20 pm.

MHCzA, and 15 + 6% were found to contain MHC, exclusively [as defined by criteria 1 and 2, Table 1B (Figs. 4K-40 and 5)]. Numerous fibers at D60 (27 + 8%) stained with the monoclonal antibody specific for MHC, (Fig. 4N). These fibers were generally among the largest in diameter and exhibited varying levels of fluorescence. As previously mentioned, with the antibodies currently available, it was not possible to determine whether some or all of these fibers also contained MHC, (Table 1B). However, electrophoresis suggested that MHC,n and MHC,, were coexpressed in many of these fibers (Fig. 7). At D115 the adult MHC phenotype, as determined with immunohistochemistry, was established. The extrafusal fibers of the adult diaphragms (D115-D156) were unreactive with anti-MHC,,, (not shown) and anti-MHC,,, (Fig. 1A). Approximately one-third (35 + 10%) of the fibers contained only MHC,,,,,, and onethird (31 f 3%) contained only MHC,, (Figs. 1 and 5). Occasionally, slow fibers exhibited some reactivity with

the antibody to MHC,,, but this occurred in less than 0.5% of fibers (fiber 3, Fig. 1). Five of the seven adult diaphragm muscles had a few large fibers (4 + 9% of the total fiber population) which reacted with the monoclonal antibody specific for MHC,n (Fig. 1D). The two remaining muscles had no fibers containing MHC,,, despite exposure of over 5000 fibers in each muscle to the antibody. Approximately 30% (30 + 7%) of the fibers of the adult diaphragm contained MHC,x exclusively (Figs. 1, 5, and 6). II. Electrophoresis Electrophoresis of myosin from diaphragm muscles and from tibialis anterior muscles of adult (>115D) rats, using 5% SDS-PAGE, produced a total of four resolvable bands (Fig. 7; see also LaFramboise et d., 1990), one more band than is usually reported with conventional 6% SDS-PAGE (Danieli-Betto et al, 1986; Schiaffino et ah, 1989). The identity of the MHCs in each of the four

8

DEVELOPMENTALBIOLOGY

D4

anti NE0

anti 0 /SLOW

anti 2A

anti 2B

anti 2B+ 2X

VOLUME144,1991

D21

D60

LAFRAMBOISE ET AL.

bands had been previously determined by Western blotting [with the same monoclonal antibodies (BF-32 and RT-D9) used for immunoblotting in the present study] to be in order of mobility MH&,i,, > MHC,s > MHCvr > MHCZA (LaFramboise, 1990; see also Fig. 8). To confirm the specificity of the polyclonal anti-fast MHC (Butler-Browne and Whalen, 1984), which had been renamed anti-MHCZB+,x in the present study, Western blot analysis with this antibody was also performed. Transfers of MHCs from 5% SDS-PAGE of myosin from the tibialis anterior muscle and myosin from the diaphragm muscle of adult (>115D) rats were reacted with anti-MHC,s+,x. This antibody stained both the band containing MHC, and the band containing MHC,, and failed to stain any other MHC (Fig. 9). Myosin obtained from El8 diaphragm muscles contained three bands, including one band which comigrated with the MHC&,,,,, band found in the adult diaphragm. The slower migrating of the two remaining bands diminished in intensity with age, while the other band became increasingly prominent through D4 (Fig. 7). Previous studies (Schiaffino et ab, 1988; Bar and Pette, 1988) have established that these bands were MHC,,, and MHC,,, with the neonatal species migrating in advance of the embryonic isoform. A band with similar mobility to MHC,, in adult muscle was present on D4 and at subsequent ages (Fig. 7). Immunoblotting with BF-32 confirmed that this band contained MHCZA (Fig. 8B). D4 samples also contained a band with an electrophoretic mobility comparable to either MHC, or MHC,,,,, which typically comigrated in 50% SDS-PAGE (Fig. 7A), although on occasional gels MHGm, appeared to have a slightly greater mobility than MHC, (Fig. ‘7B). Western blot analysis with RTD9 stained the band containing MHC,,, in D4 samples, but it failed to stain the band with the electrophoretic mobility of MHC,x (Fig. 8), despite the fact that immunohistochemical data indicated the presence of MHC, on D4. Immunoblot analysis with the polyclonal antifailed to stain any band in the D4 sample (Fig. MHCZB+ZX 9). The failure to stain the bands on the D4 sample which contained MHC,,, and MHC,,, confirmed that the immunohistochemical reaction seen with antiat D4 was not a result of cross-reactivity with MHC,,+, any known developmental isoforms. These data suggested that the polyclonal anti-MHC,,,,, used in the

9

MHC Isofbrm Expressim?,

immunohistochemical studies had a very high affinity for MHC&, detecting it in many myofibers on D4, although this isoform did not represent enough of the total myosin (-5%) to be detected by SDS-PAGE at this age. A band corresponding to MHC, was not present until D60 (Fig. 8), the stage when 27 + 8% of the myofibers could be shown to contain this isoform by immunohistochemistry. This isoform was not present or was present in trace amounts in the diaphragm muscles of rats > D115. Thus, the adult myosin phenotype is not established until the rat is 2115 days old. Analysis of light chain composition indicated that an embryonic isoform (ML&,,) was present in El8 (Fig. lo), E21, D4, and D8 samples. This isoform was not present at Dll or at any subsequent ages (not shown). All of the light chains identified in the adult diaphragm (MLCsl, MLC,,, MLC,, , MLCFz, ML(&) were present in samples obtained at El8 and at all subsequent ages. DISCUSSION

The present study provides the first immunohistochemical evaluation of the alterations in the MHC composition of perinatal muscle as it matures to an adult phenotype, with antibodies capable of identifying all of the known MHCs found in typical developing and adult rat skeletal muscles. The transition of stage-specific MHC isoforms is more complex than has been previously described in mammals in that the emergence of the final adult phenotype does not simply involve an embryonic to neonatal to adult transition. As many as four different isoforms may be concurrently expressed in a single maturing myofiber. The elimination of the developmental isoforms and the production of an adult MHC does not necessarily result in the myofiber immediately achieving its final adult phenotype. Activation of genes for specific adult isoforms may be delayed until puberty, and once activated the isoform may be transiently or permanently expressed in a given myofiber as part of its normal maturation process. Given that de nova myotube formation is completed at the time of birth (Ontell and Kozeka, 1984; Ontell et al, 1988) and that we have quantitatively evaluated the pattern of myosin heavy chain expression at multiple time periods with antibodies which recognize all known developmen-

FIG. 4. Serial sections of diaphragm muscle of D4, D21, and D60 rats reacted with antibodies indicated. All D4 fibers contain MHC,,,, but no fibers at D21 contain this isoform (see Fig. 2). At D4 no fibers contain MHC,a (D). At D4, four groups of fibers are present which contain MHCe,b + MHCBlalow (fiber 1); MHC,,,, + MHC,, + MHCU( (fiber 2); MHC,,, + MHC,, + MHCzx + MHC, (fiber 3); or MHC,,, + MHC,, (fiber 4). At D21 no fibers contain MHCzB (I). At D21, seven fiber groups are present, containing MHC,,, (fiber 1); MHC, (fiber 2); MHC,,,,, + MHC,, (fiber 3); MHCzx + MHC,, (fiber 4); MHC, + MHC,,, (fiber 5); MHC, + MHC,* + MHC,, (fiber 6); or MHCBlsloP, + MHC, + MHC,, (fiber 7). At D60 no myofibers express MHC, (K), and fibers containing MHC,, are abundant (N). There are four fiber groups present at D60 which contain MHC B,slopr(fiber 1); MHC, (fiber 2); MHC, (fiber 3); or MHCzB (fiber 4). With the antibodies used, it is not possible to determine whether fibers containing MHCas also contain MHC,. Bar = 20 pm.

10

DEVELOPMENTAL

BIOLOGY

144,1991

antibody to fast MHC (Butler-Browne and Whalen, 1984), establishing that the antibody recognizes both MHC,, and MHCzx but not MHCzA and renaming it anti-MHC,,,,, . By reacting serial sections with this antibody and the monoclonal antibody specific for MHC, and determining which fibers stain with anti MHC,s+, but fail to stain with anti-MHC,, it has been possible to determine the presence of MHCzx even when it is a minor component of the total MHC present within a myofiber. With this protocol, it has been possible to find

D4

7-T

VOLUME

D21

D60

80 70

>D115

601

S E

N E

N E 2x

N E 2x 2A

s N

2x N

2A N

2A 2X N

s N 2A

s

2x

28

2A

i: 2x

MYOSIN HEAVY CHAIN COMPOSITON FIG. 5. Percentage of diaphragm myofibers containing various MHC isoforms as determined with immunohistochemistry, expressed as X zk SD. E, MHC,,,; N, MHC,,; S, MHC,,,,; 2X, MHC,; 2A, MHC,; 2B, MHCaa. Multiple MHC isoforms are found in individual myofibers at E21, D4, and D21. The first fibers with a single MHC isoform appear by D21 and they contain either MHC,,,,,, or MHC&. By D60 MHC,,, is not present in any myofibers. Fibers which contain MHC, represent a substantial percentage of the total myofiber population at D60, but the frequency of these fibers is markedly reduced in the adult. It was not possible, with the antibodies used in this study, to determine whether these fibers also contain MHCax.

AGE (DAYS)

tal and adult myosin heavy chain isoforms, it is possible to follow the emergence of each adult isoform, commenting on the isoform switching that may be involved in the emergence of the final adult phenotype. The specificity of all of the antibodies used in the present study, both for immunohistochemistry and Western blotting, has been previously established (ButlerBrowne and Whalen, 1984; Schiaffino et al., 1989). In the present study we further defined the specificity of the

6. Age-related changes in MHC expression in diaphragm myofibers based on immunohistochemical assay with antibodies which permit the identification of all known MHC isoforms. N > 8 at D4, D21, D30, D60, and >D115. N = 3 at E21. To further clarify the time course of loss of MHC,,, D40 diaphragm muscles (N = 2) have been reacted with anti-MHC,,. At D60, 16 f 6% of the myofibers contain only MHC, (dotted line). However, the antibodies cannot exclude the possibility that some or all of the fibers which reacted with antiMHCzm+sxalso contain MHCvr (solid line), particularly since gel electrophoresis indicates no decline in this isoform at D60 (Fig. ‘7;see also Termin et al, 1383). FIG.

LAFRAMBOISEETAL.

MHC Isoform

Expression

11

FIG. 7. Separation of MHC isoforms by 5% SDS-PAGE. (A) The first seven lanes contain diaphragm muscle samples obtained at specified ages. Adult muscles were obtained on D135. TA, tibialis anterior muscle; SOL, soleus; DIA, diaphragm muscle. MHC,, and MHC,, are abundant in fetal muscles (El8 and E21). The ratio of MHC,,, to MHC,,, increases at E21 and D4 as compared with E18. A MHC,, band is is identifiable in the diaphragm at every age. MHC,, the fast MHC with the lowest barely detectable at D21 and is not present at D30. MHC,,,,, mobility, is present at D4 (see Fig. 8). In this gel, as in most 5% SDS-PAGE, MHC, and MHC,,, have similar mobility. Only on an occasional gel is it possible to obtain two discrete bands for these isoforms (see Fig. 7B). MHC,, is abundant in the TA, but is consistently present in the diaphragm only at D60. (B) Lane 1 contains a sample of myosin obtained from the diaphragm muscle of an E21 rat, lane 2 from a D4 rat, and lane 3 from a D21 rat. On this gel, it was possible to separate the band containing MHC,,, from that containing MHC,. Using these two gels, the migration of MHCs in order of electrophoretic mobility is MHCBlslow > MHC,n > MHC,,, > MHC,,, > MHCU( > MHC,.

a few (-20%) myofibers in El8 muscle which react, albeit weakly, with the anti-MHC,,,,. Approximately 80% of the fibers present at D4 exhibit staining with this antibody. It is surprising, therefore, not to find a discrete band with similar mobility to MHC, on 5% gel electrophoresis. However, these two techniques provide fundamentally different information. Immunohistochemistry can show the presence of a MHC isoform found in a single myofiber even when that isoform may be a small fraction of the total MHC in that myofiber. In contrast, SDS-PAGE will detect individual MHC isoforms only if they constitute >5% of the total amount of myosin in the sample (unpublished data). The failure to find a discrete band suggests that the percentage of total MHC, in the D4 diaphragm is below the resolution of the 5% SDS-PAGE. The ability of anti-MHC,,,,, to react with myofibers in which MHC,,, and MHC,,, are clearly the dominant isoform indicates that this antibody has a very strong affinity for MHC,. The following observations are consistent with this hypothesis: (1) anti-MHC,ncu( does not react with any known developmental MHC or any yet to be discovered embryonic

MHC present prior to E21 and (2) anti-MHC,B+2x does not react with any known adult MHC isoform except MHC, and MHCvr. Consistent with the hypothesis that MHC,, is expressed very early in development is the observation that the first fibers with fast MHC to express a single isoform are those fibers which express MHC, exclusively. At D21 these fibers can be identified by their failure to stain with anti-MHC,,,-,, leaving no doubt as to their MHC content. It would appear unlikely, given the absence of de novo myotube formation after birth (Ontell and Kozeka, 1984; Ontell et aZ.,1988), that one would find fibers containing only MHC,, at this stage if this isoform was turned on late in development. Myofibers containing MHC, initially arise from the population of fibers which contain both MHC,,, and MHGeo. As the fibers containing MHC,, mature, they have three options: (1) they continue to produce MHC,; (2) they coexpress MHC,A and they cease to produce MHC,; or (3) they transiently or permanently produce MHC,. We cannot determine unequivocally with immunohistochemistry whether this latter group of myofibers contains MHC,, exclusively or whether MHC,, is

12

DEVELOPMENTALBIOLOGY

VOLUME144,199l

FIG. 8. Western blotting of a nitrocellulose transfer sheet containing MHCs which have been separated by 5% SDS-PAGE. (A) A silverstained gel that contains samples of EN, D4, and adult (D148) diaphragm muscle (DIA) and adult (D148) tibialis anterior muscle (TA). The identity of the bands of the adult muscle has been previously demonstrated (LaFramboise et al., 1990). Bands representing MHCemt,,MHC,,, MHC,, MHC,, and MHC, are seen. MHCs in an adjacent segment of the gel to that seen in A, containing four lanes loaded with the M%v,,, same proteins, have been transferred to nitrocellulose and reacted first with antibody BF-32 (specific for MHCs,sl,,wand for MHC& (B). Bands containing MHCBIsLoar are present in D4 diaphragm muscles but are not detected in the TA or El8 diaphragm muscle of this gel due to insufficient amounts of this protein, but see Fig. ‘7.MHC, is present in D4, but is not detected in the El8 sample. The nitrocellulose transfer was then blotted with RT-D9 (C), which had been previously demonstrated to react with bands containing MHC, or MHC, in adult muscle (LaFramboise et al, 1990). MHC,, is not present in the DIA samples. MHC, is present in both the DIA and TA of D148 rats, but cannot be demonstrated in El8 and D4 samples. RT-D9 also stains the band containing MHC,, in El8 and D4 diaphragm muscle samples.

also expressed in these fibers. However, the results which have been obtained with 5% SDS-PAGE support the conclusion that MHCvr expression is not diminished during the transient period when many myofibers expressed MHC,. Further, Termin et al. (1989) have determined, by electrophoretic analysis of MHCs from single fibers, that individual myofibers of the “adult” rat diaphragm can produce two MHC bands, one corresponding to MHC, and the other which has been designated MHC,, (Bar and Pette, 1988). The MHC,, band appears to migrate in the same position as the band

which has been identified by immunoblotting as MHCz in the present study. What is clear from the present study is that most of the myofibers in the adult diaphragm which contain MHCti or MHCzB express MHCvr sometime during their development. Fibers containing MHC,,,,, are present in the diaphragm prior to birth, and the percentage of these slow fibers does not change between E21 and D4. At these early ages, fibers containing MHCB,s,o, coexpress MHGn, 9 as in the developing soleus muscle (ButlerBrowne and Whalen, 1984). It is not until D21 that a

A

ADULT TA DIA

D4

ADULT TA DiA

D4

FIG. 9. Western blotting of a nitrocellulose transfer sheet containing MHCs which have been separated by 5% SDS-PAGE. (A) A silverstained gel that contains samples of adult (D150) tibialis anterior muscle (TA), adult (D150) diaphragm muscle (DIA), and D4 diaphragm muscle. The identity of the bands of the adult muscle has been previously demonstrated (LaFramboise et al, 1990). MHCs in an adjacent segment of the gel to that seen in A, containing three lanes loaded with the same proteins, have been transferred to nitrocellulose and reacted with the polyclonal anti-fast MHC (Butler-Browne and Whalen, 1984) which has been renamed anti-MHC 2B+2x(B). This antibody reacts with the band containing MHC, and with the band containing MHC, in the adult samples. (Compare with Fig. 7.) This antibody fails to react with is specific for MHC= and MHCvr and shows no cross-reacany of the bands present in the D4 sample. This demonstrates that anti-MHC,,, tivity with any other adult MHC isoforms or with MHC,, and MHC,,. Thus, although MHCzx can be shown immunohistochemically to be present in myofibers of the D4 diaphragm (Fig. 4), the amount of this isoform is insufficient (i.e., ~5% of the total MHC) to produce a discrete band on 5% SDS-PAGE.

LAFRAMBOISEETAL.

*

M LCst,

M=gml-

MLC , emb

c

El8

M LCF3-

FIG. 10. Two-dimensional gel electrophoresis of diaphragm myosin at El% MLC,, and MLC,,, slow light chains; MLC,,, ML&,, and ML&,, fast light chains; ML&,,, embryonic light chain. All myosin light chains found in the adult diaphragm are present in El8 samples, being coexpressed with ML&,,.

substantial increase in the percentage of fibers containing MHCa/,~,, occurs, and these additional slow myofibers coexpress MHC,,. The timing of the increase in the number of slow myofibers coincides with the elimination of polyneuronal innervation in the rat diaphragm (Redfern, 1970). Thus the removal of polyneuronal innervation may be a factor in the production of MH’&,I,, in fibers initially containing MHC,,,. If both future “fast” and future “slow” motoneurons innervate a single developing myofiber, and fast motoneurons withdraw their axon terminals, slow motoneurons could gain influence over a population of fibers during this time. An alternate hypothesis to explain the timing of the increase in slow myofibers is that slow motoneurons require a postnatal maturation period before they can exert sufficient influence to direct a myofiber producing MHC,,, to express MHCs,,,,. Alternately, changes in circulating levels of hormones may provide a critical influence in directing the MHC composition of muscle fibers either through an effect on the motoneuron or by a direct influence on developing myofibers. For example, previous studies have established that changing thyroid status affected both motoneuron maturation (Kawa and Obata, 1982) and MHC expression, particularly MHC,, expression (Russell et ah, 1988; Rubinstein et al., 1988). However, a relationship between circulating hormones and slow fiber development has not been established. An unexpected observation was that MHC,, was expressed abundantly, albeit for a transient period. MHC,, was first seen in diaphragm muscle at D30, when ~1% of the myofibers express this isoform. This is considerably later than the time of appearance of this isoform in rat hindlimb muscle (Russell et aZ, 1988). At

MHC Isoj’m

Expression

13

D60, 27 + 8% of the myofibers contained MHC&, and this fell to 4 + 9% by D90. The factors involved in the initiation and in the subsequent “down-regulation” of the gene coding for this isoform in the diaphragm have not been determined. Thyroid hormone is known to play a role in the production of MHC,, in developing hindlimb muscles (Gambke et al., 1983; Butler-Browne et ab, 1984; Narusawa et ah, 1987; Russell et ab, 1988). Male rats are undergoing puberty during the period when this isoform appears in the diaphragm, and elevated testosterone levels (Aguilar et aZ.,1988) may play a role in its expression. Testosterone has been determined to be responsible for sexually dimorphic variations in MHC expression in certain guinea pig muscles [e.g., the temporalis muscle (Lyons et al., 1986)]. Clearly, future studies are required to provide insight into the factors controlling the unique pattern of MHCzs expression observed in the diaphragm. The diaphragm was selected for this study of the developmental expression of MHC, because it has been previously determined to contain more fibers with MHC,, than other muscles of the adult rat. Nevertheless, most adult rat muscles do have significant, albeit small, populations of fibers containing MHC, (Bar and Pette, 1988; Schiaffino et ah, 1989). It has not been determined whether MHC, is transiently expressed in developing hindlimb myofibers destined to become type IIA or type IIB fibers in the adult. This is an important consideration since we cannot exclude the possibility that MHC,, may be a developmental isoform, transiently expressed in many muscles but retained with high frequency in the diaphragm. The persistence of developmental isoforms (MHC,,) in the adult rat and human masseter muscle (D’Albis et ab, 1986; Soussi-Yanicostas et ah, 1990) and the rat extraocular muscle (Wieczorek et al., 1985) has been previously reported. It has been demonstrated with histochemical analysis that the pH sensitivity of the myosin ATPase in fibers containing MHC,, is nearly indistinguishable from fibers containing MHC,, according to standard histochemical procedures (Termin et al., 1989; Gorza, 1990). However, fibers containing MHC,, are more oxidative than those containing MHCzs (Schiaffino et al., 1988; Termin et al., 1989). Previous studies have characterized the adult rat diaphragm as a mixed muscle comprised of nearly equal populations of type I, IIA, and IIB fibers based on histochemical myosin ATPase assays (Metzger et aZ.,1985; Smith et al., 1988). The inability of previous investigators to discriminate between fibers containing MHCzn and MHC,, may explain the higher oxidative enzyme capacity reported for type IIB fibers of the rat diaphragm compared with hindlimb type IIB myofibers (Smith et al., 1988). That the diaphragm contains a large number of motor units comprised of type 11X fibers may

14

DEVELOPMENTALBIOLOGY

be related to its high fatigue resistance compared to many hindlimb muscles (Schiaffino et cd., 1990; Sieck and Fournier, 1990). This represents an important feature of the diaphragm since this muscle, unlike appendicular muscles, must contract repetitively to sustain viability. In summary, our results establish that MHC,x is present in the fetal rat diaphragm prior to birth. Moreover, fibers containing MHCvr appear to be the source of most future type IIA and IIB fibers, suggesting that MHC, may be regarded as a transitional as well as an adult isoform in the diaphragm muscle. MHCzB is transiently expressed beginning at D30, is present in a substantial percentage of D60 myofibers, but is only minimally produced in the adult. Fibers containing MHC,,,,, immediately prior to birth coexpress MHC,,, but the majority (~75%) of slow fibers in the adult diaphragm arise from a population of myofibers which express MHC, during their development. The presence of large numbers of fibers containing MHC, in the neonatal and adult diaphragm may account for the unique physiological properties ascribed to this muscle. The competent secretarial assistance of Ms. J. Lieberman is gratefully acknowledged. Supported by NIH Grants AR36294 and HD25630 to Dr. M. Ontell.

VOLUME144.1991

sins 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. DANIELI-BETTO, D., ZERBATO, E., and Bmo, R. (1986). Type I, 2A, and 2B myosin heavy chain electrophoretic analysis of rat muscle fibers. Biochem. Biophys. Res. Commun. 138,981-987. GAMBKE,B., LYONS,G. E., HASELGROVE,J., KELLY, A. M., and RUBINSTEIN,N. A. (1983). Thyroidal and neural control of myosin transitions during development of rat fast and slow muscles. FEBS Lett. 156,335-339.

GORZA,L. (1990). Identification of a novel type 2 population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J. Histochem. Cytothem. 38,257-265. KAWA, K., and OBATA, K. (1982). Altered developmental changes of neuromuscular junction in hypo- and hyperthyroid rats. J PhysioL (London) 329,143-161. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lvmkm) 227,680685.

LAFRAMBOISE, W. A., DAOOD, M. J., GUTHRIE, R. D., MORETTI, P., SCHIAFFINO,S., and ONTELL, M. (1996). Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle. Biochim. Biophys. Acta 1035,109-112. LYONS, G. E., HASELGROVE,J., KELLY, A. M., and RUBINSTEIN,N. A. (1983). Myosin in developing fast and slow muscles of the rat hindlimb. Bferentiaticm 25,168-175. LYONS, G. E., KELLY, A. M., and RUBINSTEIN,N. A. (1986). Testosterone-induced changes in contractile protein isoforms in the sexually dimorphic temporalis muscle of the guinea pig. J. BioL Chem. 261, 13,278-13,284.

REFERENCES AGUILAR, R., BELLIDO, C., SANCHEZ-CRIADO,J. E., and AGUILAR, E. (1988). Mechanisms of precocious puberty induced in male rats by pituitary grafts. J. Reprod. Fertil. 83,879~883. BAR, A., and PETITE, D. (1988). Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett. 235,153-155. BARTON, P., ALONSO,S., BIBEN, C., BILLEN, P., COHEN, A., Cox, R., DAUBAS, P., GARNER, I., ROBERT,B., SASSOON,D., VANDEKERCKHOVE, J., and BUCKINGHAM,M. (1989). Aetin and myosin gene expression during mouse myogenesis. In “Cellular and Molecular Biology of Muscle Development.” (F. Stockdale and L. Kedes, Eds.) pp. 701-711. Alan R. Liss Inc., New York. BIRAL, D., BET~O, R., DANIELI-BETTO, D., and SALVIATI, G. (1988). Myosin heavy chain composition of single fibers from normal human muscle. B&hem. J. 250,30’7-308. BUCKINGHAM, M. E. (1985). Actin and myosin multigene families: Their expression during the formation of skeletal muscle. Essays B&hem. 20,77-109. BUTLER-BROWNE,G. S., HERLICOVIEZ,D., and WHALEN, R. G. (1984). Effects of hypothyroidism on myosin isozyme transitions in developing rat muscle. FEBS L&t. 166,71-75. BUTLER-BROWNE,G. S., and WHALEN, R. G. (1984). Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Dev. Biol. 102, 324-334. CARRARO,U., and CATANI, C. (1983). A sensitive SDS-PAGE method separating myosin heavy chain isoforms of rat skeletal muscles reveals the heterogeneous nature of the embryonic myosin. Biochem. Biophys. Res. Commun. 116,793-802. D’ALBIS, A., JANMOT,C., and BECHET,J. (1986). Comparison of myo-

MAHDAVI, V., IZUMO,S., and NADAL-GINARD, B. (1987). Developmental and hormonal regulation of sarcomeric myosin heavy chain gene family. Circ. Res. 60,804-814. MALLOY, J. M., RIEKER, J. P., and RIZZO,C. F. (1984). Quantitation of proteins on Coomassie blue-stained polyacrylamide gels based on spectrophotometrie determination of electroeluted dye. Anal. Bie them. 141,503-509. METZGER,J. M., SCHEIDT,K. B., and F~rrs, R. H. (1985). Histochemical and physiological characteristics of the rat diaphragm. J. AppL PhysioL 58,1085-1091. NARUSAWA,M., FITZSIMONS,R. B., IZUMO,S., NADAL-GINARD, B., RuBINSTEIN,N. A., and KELLY, A. M. (1987). Slow myosin in developing rat skeletal muscle. J. Cell BioL 104,447-459. OAKLEY, B. R., KIRSCH, D. R., and MORRIS,N. R. (1980). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. B&hem. 105,361-363. O’FARRELL,P. H. (1975). High resolution two-dimensional gel electrophoresis of proteins. J. Biol. Chem. 250,4007-4021. ONTELL, M., and KOZEKA, K. (1984). Organogenesis of the mouse extensor digitorum longus muscle: A quantitative study. Amer. J. Anat. 171,149-162. ONTELL,M., HUGHES,D., and BOURKE,D. (1988). Morphometric analysis of the developing mouse soleus muscle. Amer. J. Anat. 181,279288.

REDFERN,P. A. (1970). Neuromuscular transmission in newborn rats. J. Physiol. (London) 209.701-709. RUBINSTEIN,N. A., LYONS,G. E., and KELLY, A. M. (1988). Hormonal control of myosin heavy chain genes during development of skeletal muscles. In “Plasticity of the Neuromuscular System,” Vol. 138, pp. 35-51, Wiley, Chichester (Ciba Foundation Symposium). RUSSELL,S. D., CAMBON,N., NADAL-GINARD, B., and WHALEN, R. G. (1988). Thyroid hormone induces a nerve-indenendent nrecocious

LAFRAMBOISEET AL. expression of fast myosin heavy chain mRNA in rat hindlimb skeletal muscle. J. Biol. Chem. 263,6370-6374. SCHIAFFINO,S., SAGGIN, L., VIEL, A., AUSONI, S., SARTORE,S., and GORZA,L. (1986). Muscle fibre types identified by monoclonal antibodies to myosin heavy chains. In “Biochemical Aspects of Physical Exercise” (G. Benzi, L. Packer, and N. Siliprandi, Eds.), pp. 27-34. Elsevier, Amsterdam. SCHIAFFINO,S., GORZA,L., SARTORE,S., SAGGIN,L., AUSONI, S., VIANELLO, M., GUNDERSEN,K., and LOMO, T. (1989). Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J. Muscle Res. Cell Motil. 10, 197205. SCHIAFFINO,S., GORZA,L., and AUSONI, S. (1990). Muscle fiber types expressing different myosin heavy chain isoforms: Their functional properties and adaptive capacity. In “The Dynamic State of Muscle Fibers” (D. Pette, Ed.). pp. 329-341 De Gruyter, Berlin. SIECK, G. C., and FOURMER,M. (1990). Developmental aspects of diaphragm muscle cells: Structural and functional organization. In “Developmental Neurobiology of Breathing” (G. Haddad and J. Farber, Eds.). Saunders, Philadelphia, in press. SMITH, D., GREEN,H., THOMSON,J., and SHARRATT,M. (1988). Oxidative potential in developing rat diaphragm, EDL, and soleus muscle fibers. Amer. J. Physiol 254, C661-C668. SOUSSI-YANICOSTAS, N., BARBET,J. P., LAURENT-WINTER,C., BARTON, P., and BUTLER-BROWNE,G. S. (1990). Transition of myosin isozymes during development of human masseter muscle: Persistence

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15

of developmental isoforms during postnatal stage. Development 108, 239-249. TERMIN, A., STARON,R. S., and PEXTE,D. (1989). Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle. Hi&p chemistry

92.453-457.

TOWBIN,H., STAEHELIN, T., and GORDON,J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad Sci. USA 76,4350-4354.

TROJANOWSKI,J. Q., OBROCKA,M. A., and LEE, V. M. Y. (1983). A comparison of eight different chromogen protocols for the demonstration of immunoreactive neurofilaments in rat cerebellum using the peroxidase-antiperoxidase method and monoclonal antibodies. J. Histochem. Cytochem. 31,1217-1223. WHALEN, R. G., BUTLER-BRO~NE,G. S., and GROS,F. (1978). Identification of a novel form of myosin light chain present in embryonic muscle tissue and cultured muscle cells. J. Mol. Biol. 126,415-431. WHALEN, R. G., SELL, S. M., BUTLER-BROWNE,G. S., SCHWARTZ,K., BOUVERET, P., and PINSET-HARSTROM, I. (1981). Three myosin heavy-chain isozymes appear sequentially in rat muscle development. Nature @ondo+ 292,805-809. WIECZOREK,D. F., PERIASAMY,M., BUTLER-BROWNE,G. S., WHALEN, R. G., and NADAL-GINARD, B. (1985). Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature. J. Cell Bid 101,618-629.