DEVELOPMENTAL DYNAMICS 210:106–116 (1997) Transitory Expression of Alpha Cardiac Myosin Heavy Chain in a Subpopulation of Secondary Generation Muscle Fibers in the Pig L. LEFAUCHEUR,1,2* R. HOFFMAN,1 C. OKAMURA,3 D. GERRARD,4 J.J. LÉGER,5 N. RUBINSTEIN,6 AND A. KELLY1 1School of Veterinary Medicine, Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania 2Station de Recherches Porcines, INRA, St-Gilles, France 3Department of Food Science, University of Missouri, Columbia, Missouri 4Department of Animal Science, Purdue University, West Lafayette, Indiana 5INSERM U300, Faculté de Pharmacie, Montpellier, France 6School of Medicine, Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania ABSTRACT Unlike the random distribution of fiber types seen in skeletal muscles of most mammals, pig muscle exhibits a rosette pattern consisting of islets of slow fibers surrounded by concentric circles of type IIA and IIB fibers. Within each islet of slow fibers, one of the central fibers is a primary myofiber, whereas all others are secondary fibers. The present study demonstrates that a subpopulation of the slow secondary fibers transiently expresses a-myosin heavy chain (MHC). Two cDNA libraries were made from longissimus dorsi skeletal muscle of 14-dayold piglet and adult pig atrium; the latter muscle is mainly composed of a-MHC. Screening of the libraries with a human anti-a-MHC mAb (F8812F8) demonstrated the presence of positive MHC clones in both libraries; the nucleotide sequence of the 38-untranslated region (38-UTR) was identical in both libraries. As this MHC 38-UTR had 75% homology with the human a-MHC, it was identified as pig a-MHC. Using specific cRNA probes and mAbs against pig a-cardiac and b/slow/type I MHC, we studied the expression of these MHCs in developing pig semitendinosus muscle by combining in situ hybridization and immunocytochemistry on serial sections at 90 days of gestation, and at 1, 6, 35 days and 6 months of age. The results showed that a subpopulation of secondary fibers that directly abut primary fibers, transiently produced a-MHC, both at the levels of the protein and its transcript. Subsequently, these fibres expressed b-MHC. At 1 day, immunocytochemistry showed that 16% of the secondary fibers expressed a-MHC, among which 20% did not yet express b-MHC. At 6 days, a- and b-MHCs were mostly present in the same fibers, i.e., 23% of the secondary fibers. Thereafter, the proportion of secondary fibers reacting with a-MHC mAb decreased to 10% at 5 weeks and 0% at 6 months, whereas b-MHC was still accumulating in about 38% of the secondary fibers. During the period studied, the distribution of a- and b-MHC tranr 1997 WILEY-LISS, INC. scripts closely matched that of the corresponding proteins. Expression of a-MHC was not detected in primary type I muscle fibers and slow type I secondary fibers at the periphery of the rosettes of slow fibers. This study is the first unequivocal demonstration of a transitory expression of a-MHC in a subpopulation of secondary fibers in a limb skeletal muscle during mammalian development. Dev. Dyn. 1997;210:106–116. r 1997 Wiley-Liss, Inc. Key words: alpha-cardiac myosin heavy chain; skeletal muscle; immunocytochemistry; in situ hybridization; pig INTRODUCTION In vertebrate skeletal muscles, myosin heavy chains (MHC) are encoded by a highly conserved multigene family and are subjected to developmental stage- and muscle type-specific regulation (for review, see Pette and Staron, 1990). Thus far, four major MHC isoforms have been identified in adult mammalian limb muscles (I, IIA, IIB, and IIX) (Schiaffino et al., 1989), and two isoforms (embryonic and fetal/neonatal/perinatal) are developmentally expressed (Whalen et al., 1981). All these isoforms are coded by single genes that are differentially regulated in muscle cells (Mahdavi et al., 1987; Buckingham et al., 1986). Genes coding for types IIA, IIB, IIX, and developmental isoforms are clustered on the same chromosome in human (Leinwand et al., 1983; Rappold and Vosberg, 1983; Yoon et al., 1992; Soussi-Yanicostas et al., 1993) and mouse (Weydert et al., 1985), whereas genes coding for b- and a-MHC are tandemly linked on another chromosome in both human and mouse (Mahdavi et al., 1984; Saez et al., 1987; Grant sponsor: Muscular Dystrophy Association of America. *Correspondence to: Louis Lefaucheur, Station de Recherches Porcines, INRA, 35590, St. Gilles, France. E-mail: firstname.lastname@example.org Received 7 February 1997; Accepted 11 June 1997 MYOFIBER DIVERSITY IN PIG DEVELOPMENT Emerson and Bernstein, 1987; Matsuoka et al., 1991; Gulick et al., 1991). It has been suggested that a- and b-MHC genes originated from an evolutionarily recent duplication of a common ancestral gene (Mahdavi et al., 1984). Unlike the b-cardiac MHC, which is identical to the slow/type I MHC expressed in slow-twitch skeletal muscle fibers, a-MHC was initially thought to be exclusively expressed in the heart (Lompré et al., 1984; Mahdavi et al., 1987). However, recent data have demonstrated that this isoform is also present in other adult skeletal muscles in human and rabbit, such as the masseter, retractor mandibulae, temporalis, and extraocular muscles (Bredman et al., 1991, 1992a,b; D’Albis et al., 1991, 1993; Pedrosa-Domellöf et al., 1992; Hämäläinen and Pette, 1994). Furthermore, it is also present in some intrafusal fibers of human muscle spindles (Pedrosa et al., 1990; Pedrosa-Domellöf et al., 1992; Kucera et al., 1992; Pedrosa-Domellöf and Thornell, 1994). A transitory expression has also been reported in the diaphragm of the rabbit at 4–5 weeks of age (D’Albis et al., 1991). More recently, in pig semitendinosus (ST) muscle, a subpopulation of secondary fibers in the direct vicinity of primary myotubes has been shown to be transiently labeled with a monoclonal antibody (mAb) raised against human a-MHC (Lefaucheur et al., 1995). However, possible cross-reaction with an as yet uncharacterized MHC isoform could not be ruled out. This possibility is consistent with data suggesting the existence of new developmental isoforms such as three slow MHC isoforms in developing human and rat skeletal muscle (Hughes et al., 1993). The present study was undertaken to identify unequivocally the MHC isoform labeled with the human a-cardiac mAb during development in pig ST muscle. Because of the high degree of polymorphism in the 38-untranslated regions (38-UTR) of different MHC mRNAs in laboratory animals and humans (Saez and Leinwand, 1986), we used the 38-UTRs to generate specific probes. Our objectives were (1) to clone and sequence the 38-UTRs of the mRNAs coding for the isoforms recognized by the anti-a and anti-b-MHC monoclonal antibodies in the pig and (2) to combine immunocytochemistry and in situ hybridization techniques on serial transverse sections of the pig ST muscle during development. MATERIALS AND METHODS Production of the cDNA Libraries Total RNA was extracted from frozen samples of 14-day-old longissimus dorsi (LD) muscle and adult atrium of Yorkshire pigs by the acid-guanidinium method (Chomczynski and Sacchi, 1987) and quantified by measuring optical density at 260 nm. Fourteen-dayold LD muscle corresponds to a stage of intense maturation where a subpopulation of secondary fibers is transiently labeled with the anti-a-MHC mAb F8812F8 in pig skeletal muscle, whereas adult pig atrium has been shown to be mainly composed of a-MHC (Lefaucheur et al., 1995). Poly(A)1 mRNA was isolated by two passages 107 over an oligo(dT)-cellulose column. First strand cDNA synthesis was primed with an oligodT/XhoI primer/ adapter and carried out for 60 min at 42°C with 5 µg of poly(A)1 using 250 U RNase H2 Moloney murine leukemia virus reverse transcriptase (Stratagene, La Jolla, CA) and 5-methyl dCTP. By construction, the poly(A)1 tail was near the added Xho I site, which allowed the production of a directional cDNA library. After second strand synthesis, cDNAs were made blunt by using 5 U cloned Pfu DNA polymerase (Stratagene) and EcoR I adaptors were added. After digestion with Xho I, cDNAs were size fractionated through a Sephacryl S-400 spin column. Fractions over 0.5 kb were cloned into the EcoR I/Xho I sites of the expression Uni-Zap XR vector (Stratagene). The plating of the packaged reaction was done by infecting XL1-blue MRF8 Escherichia coli bacteria (Stratagene). From 100 ng cDNA and 1 µg vector, 5.7 and 5.2 3 106 independent recombinants were obtained from the 14-day-old LD and adult atrium, respectively. Screening of the cDNA Libraries Amplified libraries (1.35 and 1.56 1010 plaque forming units [pfu]/ml) were screened using two mAbs on duplicate filters from the same plates, NOQ7-5-4D, which is specific for b-MHC (Narusawa et al., 1987), and F8812F8, which has been shown to be specific for a-MHC in rat, cat, and man (Dechesne et al., 1985, 1987; Léger et al., 1985; Pons et al., 1986; PedrosaDomellöf et al., 1992). The anti-a-MHC mAb (F8812F8) has also been shown to label a very strong band on transblots of MHC from adult pig atrium with no reaction in 75-day gestation and adult pig ST muscle (Lefaucheur et al., 1995). Several clones reactive with the antibodies were recovered from the agar plates and purified twice to homogeneity by rescreening with the same antibodies. Excision and recircularization of the Bluescript phagemids containing the cloned inserts was achieved in vivo by simultaneously infecting another strain of E. coli (SOLR) with both the Uni-ZAP XR vector and a f1 bacteriophage (ExAssisty) (Stratagene). Plasmid DNA was prepared using the QIAprepspin plasmid miniprep protocol (QIAGEN Inc., Chatsworth, CA). The size of the inserts and their restriction map were analyzed to select the clones to be further studied. Sequencing and Subcloning of the Selected Clones Double-stranded plasmid DNA was sequenced using the Sequenase DNA sequencing kit (U.S. Biochemical Corp., Cleveland, OH), [35S]dATP (Amersham, Arlington Heights, IL), and the M13 primer to start the sequencing of the insert from the poly(A) tail. Only the 38-UTR and the beginning of the coding region were sequenced. The homology with known sequences of MHC in human (Jaenicke et al., 1990; Liew et al., 1990; Yamauchi-Takihara et al., 1989; Matsuoka et al., 1991; Eller et al., 1989) and rat (DeNardi et al., 1993) was 108 LEFAUCHEUR ET AL. analyzed using the NMATPUS program from the PC Gene software package (PC Gene, Intelligenetics, Inc.). The RESTRI program (PC Gene, Intelligenetics, Inc.) was used to analyze the sequence for restriction enzyme cutting sites. The 38 ends (38 end of the coding region 1 38-UTR) of the cDNA inserts from selected clones were subcloned by PCR into Bluescript (Stratagene, Mountain View, CA) to get rid of the poly(A) tail before making the corresponding riboprobes. Riboprobe Synthesis Vectors containing subcloned cDNAs specific to band a-MHC mRNAs were linearized with StyI and MaeIII, respectively. The complementary RNA probes were transcribed using T3 RNA polymerase (Promega, Madison, WI) and 35S-UTP (New England Nuclear, Boston, MA) as the labeled nucleotide. The b-MHC probe length was 202 nucleotides, of which 19 nucleotides correspond to 38 end of the coding region, 115 nucleotides to the 58 end of the 38-UTR, and 68 nucleotides to vector sequence. The a-MHC probe length was 125 nucleotides, of which 57 nucleotides correspond to the 58 end of the 38-UTR and 68 nucleotides to vector sequence. Histological Analysis In situ hybridization with riboprobes, immunocytochemistry with anti-MHC mAbs, and conventional histochemistry were performed on the deep portion of semitendinosus (ST) muscle from 90-day-old fetus, from 1-, 6-, and 35-day-old piglets and from 6-monthold pig of the Yorkshire breed. Muscle samples were frozen in isopentane cooled with liquid nitrogen and stored at 280°C until further analysis. Transverse serial sections of 10 µm were cut on a cryostat (Microm 505M, Walldorf, Germany) at 220°C, dried at room temperature, and stored at 280°C. Cryosections were fixed for 15 min with 10% formalin in PBS and subsequently processed for in situ hybridization as described by Lyons et al. (1990), except that sections were treated for 7.5 min with 5 µg/ml instead of 20 µg/ml proteinase K. Sections were hybridized overnight at 50°C in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM NaH2PO4, 10% (w/v) dextran sulfate, 1 3 Denhardt’s, 0.5 mg/ml yeast tRNA. Riboprobes were used at a final concentration of 20,000 cpm/µl. Slides were washed at RT for 30 min in 4 3 SSC containing 10 mM DTT; followed by washings at RT for 10 min in 4 3 SSC and two times at 37°C for 10 min in 1 3 Washing Solution (WS) (10 mM Tris-HCl, pH 7.5, 400 mM NaCl, 5 mM EDTA). After treatment with 20 µg/ml RNAse A at 37°C for 30 min in 1 3 WS, slides were rinsed at 37°C for 5 min in 1 3 WS, followed by washes for 15 min at 37°C in 2 3 SSC and for 45 min at 43°C in 0.1 3 SSC. Slides were air dried and dipped in Kodak NTB-2 nuclear track emulsion and exposed for 1 week in light-tight boxes with desiccant at 4°C. After development in Kodak D-19, slides were analyzed with bright-field, phase-contrast, and dark-field optics of a Nikon microscope (Nikon microphot-FXA). In addition to in situ hybridization, unfixed serial cryosections were processed for immunocytochemistry using the immunofluorescence technique described by Pons et al. (1986). The reaction was carried out with the same antibodies used for the screening of the libraries: NOQ7-5-4D, reactive with b-MHC (Narusawa et al., 1987) and F8812F8, reactive with a-MHC. Monoclonal Ab NOQ7-5-4D was a generous gift from Dr. R.B. Fitzsimons (Med. Res. Council Lab. Mol. Biol., Cambridge, UK) and F8812F8 was obtained from Dr. J. Léger (INSERM, U300, Montpellier, France). Finally, two serial sections were stained for Ca21 actomyosin ATPase activity after preincubation at pH 4.6 and 10.4 (Brooke and Kaiser, 1970; Guth and Samaha, 1970). At birth, primary generation fibers can be identified by a combination of characteristics: larger size, central location within the islets, high expression of slow MHC, and absence of fetal MHC (Lefaucheur et al., 1995). At 6 days postpartum, primary myotubes are still identifiable by these criteria; later, however, primary myotubes are not distinguishable from secondary slow fibers any longer. RESULTS Isolation of b- and a-MHC Clones For both libraries, double screening with NOQ7-5-4D (b-MHC) and F8812F8 (a-MHC) mAbs showed that reactive clones were mutually exclusive between the two mAbs. In both libraries, several positive clones were identified with each antibody. However, the ratios between the number of positive clones with a- and b-MHC mAbs on serial filters were 12:88 and 93:7 for the 14-day-old LD and adult atrium cDNA libraries, respectively. Partial sequencing from the 38 end was used to confirm the nature of the clones as MHC cDNAs, based on the presence of the poly(A) tail and the high degree of homology of the coding region with that of the human embryonic MHC (Eller et al., 1989). Two groups of clones were identified in each library on the basis of restriction endonuclease maps and 38 end sequences (Figs. 1, 2, and 3). The two groups will be referred to as a- and b-MHC cDNAs, based on the mAbs used to identify them, i.e., F8812F8 and NOQ7-5-4D, respectively. The size of the a- and b-MHC clones ranged from 3.0 to 5.8 kb and 3.2 to 5.2 kb, respectively. The 38 end sequences of the a and b clones are closely related to the human a- (Yamauchi-Takihara et al., 1989) and human b- (Jaenicke et al., 1990) MHC cDNA sequences, respectively (Table 1). The first 48–50 carboxy-terminal amino acids are identical between pig and human for both isoforms with a high divergence between the a- and b-MHC isoforms for the last 9 (a-MHC)/7 (b-MHC) amino acids from the carboxy-terminus. The 38-UTRs of the pig a- and b-MHC mRNAs show 75.0 and 79.6% similarity with the human a- and b-MHC mRNAs, respectively (Table 1). MYOFIBER DIVERSITY IN PIG DEVELOPMENT 109 TABLE 1. 38 End Sequence Homology of a- and b-MHC cDNAs Between Pig (P), Human (H), and Rat (R) (Calculated From Figure 2)a Coding region (%) AA N a-MHC P vs H P vs R H vs R b-MHC P vs H P vs R H vs R 38-UTR (N, %) 100 100 100 94.7 93.3 93.3 75.0 73.1 70.0 100 97.9 97.9 94.7 91.7 94.4 79.6 63.7 66.4 Fig. 1. Restriction maps of pig a- and b-MHC cDNA clones. Black area, 38 end of the coding region; hatched area, 38-untranslated region; open area, poly-(A) tail. Restriction enzyme abbreviations: B, Bst NI; D, Dde I; Ha, Hae III; Hd, Hind III; M, Mae III; N, Nla III; S, Sty I. aAA, amino acids; N, nucleotides; 38-UTR, 38-untranslated region. Expression of b- and a-MHC in the Deep Medial Portion of Pig Semitendinosus Muscle at 6 Days of Age Serial sections in the deep medial portion of ST muscle at 6 days of age were processed for acto-myosin ATPase, reacted with NOQ7-5-4D and F8812F8 mAbs, and hybridized with RNA probes complementary to 38-UTRs of a- and b-MHC transcripts (Fig. 4). The b-MHC cRNA probe specifically hybridized with the islets of type I fibers as evidenced by ATPase staining and reaction with NOQ7-5-4D (Fig. 4a,c,e). Primary myotubes (labelled P on Fig. 4) were strongly reactive with NOQ7-5-4D mAb (Fig. 4c) and unreactive with F8812F8 (Fig. 4d), whereas a subpopulation of secondary fibers, in the direct vicinity of primary myotubes, were reactive with both mAbs (Fig. 4c,d). Fast-twitch secondary fibers surrounding the islets of slow fibers did not react with any of these mAbs. Figure 4e and f shows that the distribution of b- and a-MHC transcripts closely matches that of the corresponding proteins (Fig. 4c,d). In particular, primary myotubes, labelled P on Figure 4e and f, were labeled with the b-MHC probe (Fig. 4e) and unlabeled with the a-MHC probe (Fig. 4f). Developmental Pattern of a-MHC Expression Alpha-MHC transcripts could be detected using PCR in RNA from fetal muscle at 70 days of gestation, but this technique cannot discriminate between expression from intrafusal (bag) fibers and extrafusal muscle fibers (data not shown). We detected a-MHC in intrafusal fibers by immunocytochemistry as early as 90 days of gestation, but not in extrafusal fibers. At this stage, b-MHC transcript and protein were only expressed in primary myofibers (Fig. 5a,b), not in secondary fibers. At 103 days of gestation, the first a-MHC was detected in a few extrafusal fibers spread out in small areas of the muscle (data not shown). Thereafter, the number of fibers staining positively with F8812F8 mAb increased through late gestation and birth (113 days gestation). At 1 day postpartum, the primary fibers displayed the same reactivity as at 90 days gestation whereas some secondary fibers, in the direct vicinity of the primary fibers, began to express a- and sometimes b-MHC transcripts and proteins (Fig. 5e–h). At 1 day, immunocytochemistry showed that 16% of the secondary fibers expressed a-MHC, among which 20% did not yet express b-MHC. The proportion of secondary fibers reacting with anti-a-MHC mAb reached a maximum value of 23% at 6 days (Fig. 5k), plateaued until 11 days (not shown), and thereafter decreased to 10% at 5 weeks (Fig. 5o) and 0% at 6 months (Fig. 5s). By contrast, the proportion of secondary fibers reacting with anti-b-MHC mAb increased to reach a plateau of 37% from 5 weeks onwards. Therefore, only 60% (i.e., 23/37%) of the secondary fibers maturing to type I fibers, those located in the direct vicinity of primary myotubes, transiently expressed a-MHC. Alpha-MHC was never expressed in primary myotubes nor in fast secondary fibers at the periphery of the rosettes. During development, the distribution of b- (Fig. 5b, f, j, n,r) and a- (Fig. 5d,h,l,p,t) MHC transcripts closely matched that of their corresponding MHC proteins (Fig. 5a,e,i,m,q and Fig. 5c,g,k,o,s, respectively). DISCUSSION a-MHC Is Expressed in Pig Semitendinosus During Development This study is the first to demonstrate that a MHC isoform that is identical to a-MHC is transiently expressed during development in a mammalian hind limb muscle. The clones screened with F8812F8 mAB (aMHC) in the 14-day-old LD cDNA library are identical to those screened in the library made from adult atrium, a muscle mainly composed of a-MHC (Lefaucheur et al., 1995). The sequence of the a-MHC 38-UTR is very different from that of the b- (Fig. 3a,b), embryonic, perinatal, fast IIA, IIB, and IIX mRNAs (Lefaucheur et al., unpublished data). The amino acid and nucleotide sequences of the pig a-MHC show remarkable homology with that of human a-isoform (YamauchiTakihara et al., 1989; Matsuoka et al., 1991), which confirmed that this isoform does correspond to the pig a-MHC. As in human, the extreme carboxyl terminus amino acid sequence is highly divergent between a- and b-MHC isoforms (Fig. 2). 110 LEFAUCHEUR ET AL. Fig. 2. Nucleotide and amino acid sequences of the 38 ends of a- and b-MHC coding regions. Stop codons are underlined. A: a-MHC. B: b-MHC. Bottom lines correspond to published nucleotide sequences for human (h) (Yamauchi-Takihara et al., 1989; Matsuoka et al., 1991; Jaenicke et al., 1990) and rat (r) (Mahdavi et al., 1982, 1984) MHC. Dots and asterisks indicate identical nucleotides and amino acids with pig, respectively. MYOFIBER DIVERSITY IN PIG DEVELOPMENT 111 Fig. 3. Nucleotide sequences of the 38 untranslated regions of a- and b-MHC clones. A: a-MHC 38-UTR. B: b-MHC 38-UTR. Bottom lines correspond to published nucleotide sequences for human (h) (YamauchiTakihara et al., 1989; Matsuoka et al., 1991; Jaenicke et al., 1990) and rat (r) (Mahdavi et al., 1982, 1984) MHC. Dots indicate identical nucleotides with pig, blanks indicate missing nucleotides. Polyadenylation signals are underlined. Alpha-MHC Is Under Spatial and Temporal Regulation ATPase activity, which is, in turn, strongly correlated with MHC composition (Barany, 1967; Schwartz et al., 1981; Reiser et al., 1985). Thus, it seems likely that muscle fibers containing a-MHC would exhibit an intermediate speed of contraction between those containing b- and adult fast type II MHCs. A similar transitory expression of a-MHC has also been reported to occur in the pig ventricle during the first 3 weeks after birth (Lompré et al., 1981) suggesting common regulatory factors. During normal skeletal and cardiac development, it has been well established that although some of the myosin heavy chain transitions are nerve dependent (Jolesz and Sreter, 1981), others are regulated by the level of thyroid hormones (Ianuzzo et al., 1977; Gambke et al., 1983; Izumo et al., 1986; Butler-Browne et al., 1987; D’Albis et al., 1987). In particular, the regulation of the a-MHC gene by thyroid hormone has been extensively studied. Thyroid responsive elements have been characterized in the a-MHC promoter (Izumo and Mahdavi, 1988; Tsika et al., 1990; Flink and Morkin, 1990; Subramaniam et al., 1991; Rindt et al., 1995) and thyroid hormones increased the expression of a-MHC both at the levels of mRNA and protein in the ventricular myocardium of rats and mice (Butler-Browne et al., 1987; D’Albis et al., 1987; Lompré et al., 1984). Since a significant increase in thyroxin (T4) has been reported to occur in the pig during the last days of gestation (Berthon et al., 1993), and very high levels of triiodothyronine (T3) and T4 have been measured during the first week following birth in the pig (Slebodzinski et al., 1981), thyroid hormones could therefore be the factor that triggers the transitory expression of a-MHC in both the heart (Lompré et al., 1981) and skeletal muscle (present study). Why thyroid hormones might act selectively on a subpopulation of secondary fibers, close to the primary The combined use of antibodies and riboprobes on serial sections confirmed the occurrence of a-MHC in developing pig ST muscle both at the levels of the transcript and corresponding protein. The red portion of mature porcine ST muscle is characterized by a typical arrangement of fibers in rosettes, with islets of type I fibers occupying the central core position surrounded by a ring of type II fibers (Fig. 4a). This ordered pattern of fiber distribution is unique to the pig and contrasts with the checkerboard pattern of fiber types seen in skeletal muscles of most other mammals. The expression of a- and b-MHC within a rosette of fibers from 90-day gestation to 6 months of age is schematized in Figure 6. It is noteworthy that the most important expression of a-MHC occurs during the first postnatal weeks, a period when the greatest changes occur in the maturation of pig muscle fibers (Suzuki and Cassens, 1980; Lefaucheur and Vigneron, 1986). Our results show that the expressions of a- and b-MHC isoforms are differentially regulated in a spatial and temporal manner within the pig ST muscle. The close correlation observed between the presence of the a- and b-MHC mRNAs and corresponding isozymes during development suggests that transcriptional mechanisms are involved in the regulation of MHC phenotype, as suggested by Medford et al. (1983), Lompr[41d] ae et al. (1984), Cox and Buckingham (1992), Boheler et al. (1992). The biological significance of the transitory expression of a-MHC in pig ST muscle is unknown but one could speculate that it influences the contractile performance of skeletal muscle. Indeed, the a-MHC has been shown to have an ATPase activity three times that of b-MHC (Pope et al., 1980), and the velocity of contraction of a particular fiber is directly proportional to its 112 LEFAUCHEUR ET AL. Fig. 4. Serial sections of the deep medial portion of semitendinosus muscle at 6 days of age. Serial sections were processed for histochemical demonstration of Ca21 activated acto-myosin ATPase after preincubation at pH 4.6 (a) and 10.4 (b), reacted with NOQ7-5-4D (b-MHC) (c) and F8812F8 (a-MHC) (d) mAbs, and processed for in situ hybridization with b (e) and a- (f) cRNA probes. Phase contrast pictures corresponding to (e) and (f) were used to identify the fibers. Three primary fibers are labelled P on each serial section. Scale bar 5 50 µm. myotube, is an interesting question. Fiber-specific differences in nuclear T3 receptors, local growth factors and hormones, and/or neuronally imposed mechanical work could be involved. Recent results reported by Peuker and Pette (1995) demonstrate that low-frequency stimulation strongly induced a-MHC mRNA in rabbit tibialis anterior muscle, which suggests that neural input and its associated mechanical work are likely to be involved in the spatial distribution of a-MHC expression. Interestingly, the expression of a-MHC we observed seemed to precede that of b-MHC in the same fibers, as suggested by Peuker and Pette (1995) in their stimulation study. The significance of the a-MHC in the maturation of muscle fibers and the mechanisms involved Fig. 5. Expression of b- and a-MHCs on serial sections of the deep medial portion of the semitendinosus muscle at 90 days of gestation (a–d), and 1 day (e–h), 6 days (i–l), 5 weeks (m–p), and 6 months (q–t) of age. The sections were reacted with NOQ7-5-4D (b-MHC) (a,e,i,m,q) and F8812F8 (a-MHC) (c,g,k,o,s) mAbs and processed for in situ hybridization with b (b,f,j,n,r) and a (d,h,l,p,t) cRNA probes. White arrowheads denote corresponding myofibers on serial sections at a given age. Scale bars 5 50 µm. 114 LEFAUCHEUR ET AL. Fig. 6. Schematic representation of a- and b-MHC expression within a rosette of fibers in the red portion of pig semitendinosus muscle from 90-day gestation (dg) to 6 month of age (6 mo). The central fiber of each rosette is a primary fiber (thick circle) and the islet of slow fibers is formed by the gradual conversion of some surrounding secondary generation fibers to type I during the first weeks after birth. During the period of maturation that we have analyzed, the expression of a-MHC was limited to a subpopulation of secondary muscle fibers which later mature to become slow fibers. Within each rosette, a-MHC was only expressed in those secondary fibers which were in the direct vicinity of the primary fibers and which were also the first of the secondary generation muscle fibers to express slow MHC. At 6d, a- and b-MHCs were mostly present in the same fibers, i.e., in 23% of the secondary fibers. During the subsequent maturation, the a-MHC was gradually eliminated, while b-MHC continued to accumulate. These fibers progressively matured to type I fibers, giving rise to the typical islets of type I fibers surrounded by type II fibers encountered in the adult pig. It should be noted that the primary generation type I muscle fibers never expressed a-MHC nor did the more peripheral type I fibers of each slow islet and the fast secondary fibers. This demonstrates that even though all the fibers within a slow islet are type I in mature muscle, they got there by different pathways. Scale bars 5 50 µm. remain to be established. On the other hand, it is well documented that expression of b-MHC would be mostly neurally maintained by maturation of the type I motor units and their associated contractile activity and excitation-contraction coupling (Butler-Browne et al., 1982). Since the piglet can immediately walk and run after birth, the dramatic increase in physical activity at birth, i.e., the increased contractile activity, could be part of the mechanism leading to myosin transitions during the perinatal period in the pig. by sequencing and in situ hybridization. Therefore, a-MHC can be assigned as an additional element in the succession of MHC isoforms in skeletal muscle during development in the pig. Up to now, no transitory expression of a-MHC in a limb skeletal muscle has been reported in other mammals, but one could speculate that one of the ‘‘slow’’ isoforms recognized by antibodies in human and rat developing muscle in Hughes et al.’s paper (1993) was actually a-MHC. However, they did not identify the corresponding cDNAs, and further studies are needed to know if the pig is unique in that respect. It is likely that interplay between hormonal and neuronal factors is involved in the regulation of the spatial and temporal expression of a-MHC in pig skeletal muscle, and more research is still needed to answer the question. Finally, the present study shows that a-MHC can be considered as a developmental isoform in pig semitendinosus muscle, joining a grow- CONCLUSION This study in the pig is the first unequivocal demonstration of a transitory expression of a-MHC in a subpopulation of secondary fibers in a limb skeletal muscle during development in mammals. The presence of a-MHC has been demonstrated both at the levels of the protein by immunocytochemistry and its transcript MYOFIBER DIVERSITY IN PIG DEVELOPMENT ing amount of other studies that show that expression of a-MHC is far from being exclusive to the myocardium. ACKNOWLEDGMENTS We are grateful to Dr. R.B. Fitzsimons for her generous gift of the NOQ7-5-4D monoclonal antibody. 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