SUMMARY

Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp (Cyprinus carpio L.), MYHemb1,MYHemb2 and MYHemb3. MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10°C (MYHS10)and 30°C (MYHS30). Phylogenetic analysis demonstrated that MYHemb1 and MYHemb2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYHemb3 was similar to the adult slow muscle isoforms, MYHS10 and MYHS30. MYHemb1 and MYHemb2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYHemb1 continued to be expressed at low levels in 7-month-old juveniles when MYHemb2 was not detectable. MYHemb3transcripts appeared at almost the same stage as MYHemb1transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYHemb1 and MYHemb2 were expressed in the inner part of myotome, whereas MYHemb3 was expressed in the superficial compartment. MYHS10 and MYHS30 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYHS10 mRNA was expressed at an acclimation temperature of 10 and 20°C, but not at 30°C. In contrast, MYHS30 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10°C-acclimated adult fish (MYHcard). MYHcard mRNA was first detected at 61 h.p.f., but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered.

Introduction

Skeletal muscle is composed of different muscle fiber types. Differential expression of muscle structural proteins, including the class II myosin heavy chain (MYH) is a major contributing factor to the diversity of skeletal muscle fibers (for a review, see Schiaffino and Reggiani, 1996). The fiber type composition of individual muscles has been shown to vary dramatically during ontogeny. In the mouse a transition of MYH isoforms has been reported during embryogenesis(Lyons et al., 1990; Whalen et al., 1981) and in response to unloading and exercise(Linderman et al., 1996; Sugiura et al., 1993; Talmadge et al., 1996).β-Cardiac and embryonic skeletal MYHs were first detected between 9 and 10 days post fertilization (d.p.f.) in the myotomes of the most rostral somites. Perinatal MYH transcripts started to accumulate at 10.5 d.p.f. By 12.5 d.p.f., both embryonic and perinatal MYH mRNAs were found to an equal extent, whereas by 15.5 d.p.f., perinatal MYH mRNA was predominantly expressed and mRNA levels of embryonic MYH had decreased. The mRNAs of adult fast and slow MYHs become segregated according to fiber types at later stages.β-Cardiac MYH transcripts are always present as a minor component in trunk muscle of embryonic stages (Lyons et al., 1990). At least three forms of slow twitch MYH accumulate sequentially during mouse fetal development. Slow MYH maturation occurs in slow fibers before expression of the adult fast MYHs in fast fibers(Maggs et al., 2000). Allen and Leinwand (2001) analyzed the expression of seven sarcomeric MYHs in the hindlimb muscles of wild-type mice at several time points from 1 day of postnatal life (d.p.n.) to 20 d.p.n. In early postnatal life, the developmental isoforms (embryonic and perinatal)constitute >90% of the total MYH expression, whereas three adult fast isoforms (IIa, IIb, and IId) constitute <1% of the total MYH protein. However, between 5 and 20 d.p.n. their expression increases to be >90% of the total MYH. In developing cardiac muscle, there is a small but significant decrease in the level of MYH alpha in the developing human ventricle between 7 and 12 weeks of gestation (Reiser et al.,2001).

Three distinct-type skeletal muscle fibers, slow, fast and intermediate are formed during ontogeny in the zebrafish Danio rerio(van Raamsdonk et al., 1978; Devoto et al., 1996; Weinberg et al., 1996). The embryonic slow and fast muscle fibers express tissue-specific MYH isoforms(Devoto et al., 1996). Blagden et al. (1997) suggested that Sonic hedgehog (Shh) acts to induce myoblasts committed to slow muscle differentiation from uncommitted presomitic mesoderm. Mutant fish lacking Shh expression failed to form slow muscle but did form fast muscle. Ectopic expression of Shh, either in wild-type or mutant embryos, lead to ectopic slow muscle at the expense of fast muscle. In rainbow trout Oncorhynchus mykiss, there is a developmental reduction in the number of MYH isoforms present in red, white and ventricular muscle between smaller parr and older juveniles (smolts; Weaver et al.,2001).

Many teleosts can modify their swimming performance following several weeks acclimation to a new temperature regime(Fry and Hart, 1948). The mechanisms underlying the plasticity of locomotory performance are complex and vary between species (Johnston and Temple,2002). In the goldfish Carassius auratus(Johnston et al., 1975) and common carp Cyprinus carpio L.(Heap et al., 1985) changes in muscle myofibrillar ATPase activity constitute an important element of the acclimation response. The common carp has become a model species for the study of temperature acclimation in fish and the subject of a large number of investigations (Watabe, 2002). Studies with fully activated skinned fibers found that maximum tension and shortening speed increased at low temperatures in both fast and slow muscles following a period of cold acclimation(Johnston et al., 1985). We have shown that changes in the expression of MYH isoforms play a key role in the plasticity of myofibrillar ATPase activity and contractile properties with temperature acclimation (Hwang et al.,1990; Watabe et al., 1992, 1995; Guo et al., 1994). Three distinct MYH DNAs were cloned from the fast myotomal muscle of carp acclimated to either 10°C or 30°C for a minimum of 6 weeks(Imai et al., 1997; Hirayama and Watabe, 1997). The nomenclature used for these clones was based on the acclimation temperature at which the corresponding mRNAs were most strongly expressed. Thus, the 10°C-type MYH (MYHF10) and the 30°C-type MYH(MYHF30) cDNAs were the predominant transcripts in 10°C- and 30°C-acclimated fish, respectively. The relative proportions of each isoform varied with acclimation temperature(Imai et al., 1997). A third cDNA, which was named the intermediate-type because of its intermediate DNA nucleotide and deduced amino acid sequences between those of the 10°C- and 30°C-type MYH isoforms, was also isolated from fast muscle, which was expressed over a relatively broad temperature range (MYHFint). Studies with chimeric myosins constructed with Dictyostelium discoideum myosin heavy chain backbone indicate that amino acid substitutions in the surface loops 1 and 2, involved in ATP-binding and actin-binding, respectively, are responsible for the different temperature-dependent properties of myosin ATPase(Hirayama et al., 2000; Watabe, 2002).

Adult common carp can overwinter in frozen ponds and survive 35°C in the summer, however, embryonic and early larval stages are not viable below 15°C (Rothbard and Yarron,1995). In fast muscle, the ability to change myofibrillar ATPase activity is acquired gradually during ontogeny, concomitant with the development of cold tolerance (Cole and Johnston, 2001). Peptide mapping studies have shown that the MYH composition characteristic of 10°C-acclimated carp appears in juveniles after several months at around 37 mm total length(Wakeling et al., 2000). Studies of expressed MYH have shown a complex developmental-stage-specific expression of isoforms in different tissue types that continues throughout much of the life cycle (Huriaux et al.,1991; Martinez et al.,1991; Mascarello et al.,1995; Johnston et al.,1998). In common carp, two developmentally regulated MYH isoform genes (Eggs22 and Eggs24) have been described(Ennion et al., 1999). These genes were expressed from 22 h.p.f. until 2 weeks after the larvae hatched. It is therefore likely that further developmental stage-specific MYH genes remain to be discovered. Furthermore, nothing is known about changes in slow muscle MYH gene expression with temperature acclimation.

The aim of the present study was to comprehensively clone MYHs from embryonic muscle, adult slow muscle and adult cardiac muscle in the common carp and to characterize their expression patterns in relation to developmental stage.

Materials and methods

Fish

Common carp Cyprinus carpio L. were reared at the Yoshida Research and Training Station, Tokyo University of Fisheries, Japan. Embryos and larvae were cultured in freshwater aquaria at approximately 20°C and experimental samples were collected during ontogeny as described in our previous paper(Kobiyama et al., 1998). Somite formation was observed with a light microscope to assess the developmental stages of embryos. The whole embryos (3-somite, 15-somite, first heart-beat and eyed-stage) and the whole larvae at hatching were frozen in liquid N2 and stored at -80°C until use. Larvae aged 2 days were transported to the laboratory of the University of Tokyo and further cultured by feeding commercial pellets twice daily for 7 months under a 14 h:10 h L:D photoperiod at approximately 20°C. The whole trunk muscle was isolated from juveniles aged 1 and 7 months, frozen in liquid N2,and stored at -80°C until use. Fast and slow skeletal muscles and myocardium were taken from adult carp aged 2 years acclimated to 10°C,20°C and 30°C for a minimum of 6 weeks.

Reverse-transcription PCR

Total RNAs were prepared by the method of Chomczynski and Sacchi(1987) and first strand cDNAs were synthesized using the 3′RACE system (rapid amplification of cDNA ends; Invitrogen, Carlsbad, CA, USA). RNA was extracted from embryos at hatching and from the slow and cardiac muscle of adult fish acclimated to either 10°C or 30°C for a minimum of 6 weeks. 3′RACE was performed using gene-specific primers for embryonic MYH(5′-ACCTGCAGCACCGTCTGGAT-3′) or for cardiac/slow MYH(5′-CCATGATGGCTGAGGAGCTG-3′) with abridged universal amplification primer (AUAP; 5′-GGCCACGCGTCGACTAGTAC-3′). PCR amplifications were carried out for 3 min at 94°C, followed by 30 cycles of denaturation for 30 s at 94°C, 1 min of annealing at 58°C and 1 min of extension at 72°C with the final extension step for 5 min, using a model 2400 DNA thermal cycler (Applied Biosystems, Foster City, CA, USA). Forward and reverse primers (25 pmol) and 20 ng of the first strand cDNA template were added to 8μl of a solution containing 2 mmol l-1 dNTP mixture and 10 μl 10× PCR buffer in 100 mmol l-1 Tris-HCl (pH 8.3), 500 mmol l-1 KCl and 15 mmol l-1 MgCl2. The final volume was adjusted to 99 μl with sterilized water, and a 1 μl aliquot containing 1 U Taq DNA polymerase was added to start the reaction. Amplified DNA fragments were ligated into pBluescript SKII-(Stratagene, La Jolla, CA, USA) for sequence analysis. DNA sequencing was performed using a Dye-terminator Cycle Sequencing Ready Reaction Kit with model 373A and 310 DNA sequencers (Applied Biosystems).

Fig. 1.

Comparison of the partial deduced amino acid sequences of carp embryonic(emb), slow skeletal and cardiac types of myosin heavy chain (MYH) with those of corresponding regions of carp fast skeletal MYH isoforms. The data on adult fast muscle MYH sequences were taken from Imai et al.(1997). Slow 10, slow 30 and 10°C fast, 30°C fast refer to the isoforms predominantly expressed at 10°C and 30°C, respectively. I fast is an intermediate type, which is expressed over a broad range of temperature. Amino acid residues identical to those of carp MYHemb1 are indicated by dots. Numbers in the right margin represent amino acid residues from the N terminus. Primer binding regions of MYHemb1, MYHemb2 and MYHemb3 are boxed.

Fig. 1.

Comparison of the partial deduced amino acid sequences of carp embryonic(emb), slow skeletal and cardiac types of myosin heavy chain (MYH) with those of corresponding regions of carp fast skeletal MYH isoforms. The data on adult fast muscle MYH sequences were taken from Imai et al.(1997). Slow 10, slow 30 and 10°C fast, 30°C fast refer to the isoforms predominantly expressed at 10°C and 30°C, respectively. I fast is an intermediate type, which is expressed over a broad range of temperature. Amino acid residues identical to those of carp MYHemb1 are indicated by dots. Numbers in the right margin represent amino acid residues from the N terminus. Primer binding regions of MYHemb1, MYHemb2 and MYHemb3 are boxed.

Phylogenetic tree construction

The deduced amino acid sequences obtained in this study contained part of a myosin rod region corresponding to amino acid residues 1792-1924 from the N terminus of MYHF10 (BAA22067; Hirayama and Watabe, 1997). These sequences together with those for the corresponding regions in the fast and cardiac/slow type MYHs reported from human Homo sapiens,zebrafish Danio rerio and torafugu pufferfish Takifugu rubripes were used for phylogenetic analysis following paired alignment of the sequences with CLUSTAL W (Thompson et al., 1994). The neighbor-joining (NJ) and maximum parsimony(MP) methods were employed to obtain phylogenetic trees using the software Mega3 (Kumar et al., 2004). Bootstrap resampling analysis from 1000 replicates was used to evaluate internal branches.

Northern blot analysis

Samples for northern blot analysis were: the whole carp embryos, the whole larvae at hatching, the whole trunk muscle from juveniles aged 1 and 7 months(Kobiyama et al., 1998), and pure samples of slow and cardiac muscle isolated from adult carp acclimated to 10°C, 20°C and 30°C. Total RNAs were fractionated in 1% (w/v)agarose gels containing 18% (v/v) formamide and transferred onto the nylon membrane Hybond N+ (Amersham Bioscience, Buckinghamshire, UK). The membranes were prehybridized at 65°C for 1 h in a solution containing 0.5 mol l-1 Church buffer (Church and Gilbert, 1984), 1 mmol l-1 EDTA and 7% SDS, and then hybridized at 65°C for 20 h in the same solution used for prehybridization but containing 32P-labelled probe. The membranes were washed sequentially with 2× SSC (1× SSC is 0.15 mol l-1 sodium chloride, 0.015 mol l-1 sodium citrate) plus 0.1% SDS at room temperature for 20 min, 1× SSC plus 0.1% SDS at 65°C for 30 min and 0.1× SSC plus 0.1% SDS at 65°C for 10 min,and then exposed to X-ray films.

The DNA fragments used as probes encoding MYHemb1,MYHemb2, MYHemb3, MYHS10, MYHS30and MYHcard isoforms (refer to Results) were amplified by PCR and labeled with [α-32P]dCTP using a Random Primer DNA labeling kit Ver.2 (Takara, Otsu, Japan). The probes correspond to nucleotides (nt)397-494 for MYHemb1, nt 414-505 for MYHemb2, nt 411-506 for MYHemb3, nt 417-539 for MYHS10, nt 417-539 for MYHS30 and nt 184-581 for MYHcard cDNAs, and these have been registered in the DDBJ/EMBL/GenBank databases with the accession numbers of AB104622, AB104623, AB104624, AB104625, AB104626 and AB104627,respectively. Alternatively, a probe was amplified from the DNA sequence encoding residues 534-717 from the N terminus for MYHF10 of carp fast skeletal muscle, which is thought to react with all known skeletal muscle-type MYH mRNAs of carp (consensus)(Hirayama and Watabe, 1997). The oligonucleotide probes specific to MYHF10 and MYHF30mRNA were 5′-TCCTTTCTTTCCAGCGTCCTCTGCT-3′ and 5′-GCCCTCAGCTTCAGCTCCATGAGTGG3′, respectively, which were labeled with [γ-32P]dATP using a Megalabel DNA end labeling kit(Takara) (Hirayama and Watabe,1997).

Fig. 2.

Comparison of the partial deduced amino acid sequence of carp cardiac myosin heavy chain (MYH) with the corresponding regions of zebrafish ventricular, rat α and β cardiac, and chicken atrial MYHs. Comparative data are from the zebrafish(Yelon et al., 1999), ratα (McNally et al.,1989), rat β (Kraft et al., 1989) and chicken (Yutzey et al., 1994). Amino acid residues identical to those of carp are indicated by dots. Numbers in the right margin represent amino acid residues from the N terminus.

Fig. 2.

Comparison of the partial deduced amino acid sequence of carp cardiac myosin heavy chain (MYH) with the corresponding regions of zebrafish ventricular, rat α and β cardiac, and chicken atrial MYHs. Comparative data are from the zebrafish(Yelon et al., 1999), ratα (McNally et al.,1989), rat β (Kraft et al., 1989) and chicken (Yutzey et al., 1994). Amino acid residues identical to those of carp are indicated by dots. Numbers in the right margin represent amino acid residues from the N terminus.

The specificity of probes used for northern blot analysis was examined by dot blot analysis. Plasmid DNAs encoding partial sequences of eight MYH isoforms were spotted onto a nylon membrane and allowed to react with the same probes as those used in northern blot analysis(Hirayama and Watabe, 1997),which were derived from MYHemb1, MYHemb2,MYHemb3, MYHS10, MYHS30 and MYHcard cDNAs.

Whole-mount in situ hybridization

Whole-mount in situ hybridization (WISH) was performed according to the method of Westerfield(1993). RNA probes derived from nt 357-527 for MYHemb1, nt 357-512 for MYHemb2 and nt 1-512 for MYHemb3 were synthesized from plasmids incorporating MYHemb1, MYHemb2 and MYHemb3. Sample tissues were reacted with the probes and cut at a thickness of 18 μm using a cryostat.

Results

cDNA cloning of embryonic MYH isoforms

We employed 3′ RACE for identification of embryonic-type MYH cDNAs including the untranslated region. PCR amplification of RNA from larvae at hatching using universal primer for fast skeletal MYHs with AUAP yielded a single band with high staining intensities in agarose gel electrophoresis at approximately 600 bp (data not shown). The DNA fragments in this band were subcloned into pBluescript

SK II-, and the resulting clones were sequenced. As a result,three different clones encoding carp embryonic MYHs were isolated and named MYHemb1 (AB104622), MYHemb2 (AB104623) and MYHemb3 (AB104624). The corresponding mRNAs of these three genes were predominantly expressed at early developmental stages of carp as revealed by northern blot analysis (see below) and, therefore, we refer to them as embryonic. Two independent clones were sequenced for each gene.

Fig. 3.

The neighbor-joining (A, NJ) and maximum parsimony (B, MP) tree based on deduced amino acid sequences in partial rod domains of MYHs. Carp MYHs identified in this study (in bold) were compared with H. sapiens: Hs MYH3 (embryonic), NM_002470; Hs MYH2 (adult fast IIa), AF111784; Hs MYH1(adult fast IId/x), AF11785; Hs MYH4 (adult fast IIb), AF11783; Hs MYH8(perinatal), NM_002474; Hs MYH13 (extraocular), AF11782; Hs MYH6(alpha-cardiac), NM_002471; Hs MYH7 (beta-cardiac), NM_000257; C. carpio: Cc F10 (adult 10°C-acclimated fast), D89990; Cc Fint (adult 20°C-acclimated fast), D89991; Cc F30 (adult 30°C-acclimated fast),D89992; D. rerio: Dr myhz1 (embryonic fast), AF180893; Dr myhz2(embryonic fast), NM_152982; Dr myhc4 (embryonic fast), AY921650; Dr artial(atrial), AY138982; Dr ventricular (ventricular), AF114427; Dr smyhc1(embryonic slow), AY921649. MYH sequences of T. rubripes (Tr) were constructed by using Fugu genomic sequence assembly data version 3.0(Ikeda et al., 2004).

Fig. 3.

The neighbor-joining (A, NJ) and maximum parsimony (B, MP) tree based on deduced amino acid sequences in partial rod domains of MYHs. Carp MYHs identified in this study (in bold) were compared with H. sapiens: Hs MYH3 (embryonic), NM_002470; Hs MYH2 (adult fast IIa), AF111784; Hs MYH1(adult fast IId/x), AF11785; Hs MYH4 (adult fast IIb), AF11783; Hs MYH8(perinatal), NM_002474; Hs MYH13 (extraocular), AF11782; Hs MYH6(alpha-cardiac), NM_002471; Hs MYH7 (beta-cardiac), NM_000257; C. carpio: Cc F10 (adult 10°C-acclimated fast), D89990; Cc Fint (adult 20°C-acclimated fast), D89991; Cc F30 (adult 30°C-acclimated fast),D89992; D. rerio: Dr myhz1 (embryonic fast), AF180893; Dr myhz2(embryonic fast), NM_152982; Dr myhc4 (embryonic fast), AY921650; Dr artial(atrial), AY138982; Dr ventricular (ventricular), AF114427; Dr smyhc1(embryonic slow), AY921649. MYH sequences of T. rubripes (Tr) were constructed by using Fugu genomic sequence assembly data version 3.0(Ikeda et al., 2004).

The nucleotide sequences of MYHemb1 and MYHemb2 cDNAs showed 95% and 98% identity to those of Eggs22 and Eggs24 from carp embryos previously characterized by Ennion et al.(1999). However,MYHemb3 cDNA showed a low sequence identity compared to MYH isoforms reported previously. All embryonic cDNA clones encoded a part of the carboxy-terminal region of l-meromyosin(Fig. 1) and contained polyadenylation signals (AATAAA) and poly(A) tails (data not shown). The deduced amino acid sequences of MYHemb1 and MYHemb2showed about 90% identity with those of the MYHF10,MYHFint and MYHF30 isoforms from adult carp fast skeletal muscle (Imai et al.,1997) (Table 1). By contrast, the amino acid sequence of MYHemb3 had only 75% identity with those of the three carp fast skeletal MYH isoforms(Table 1).

Table 1.

Percentage comparison of amino acid sequences of myosin heavy chains from carp

emb210°C fastI fast30°C fastemb3slow10slow30cardiac
embl 95 88 88 89 78 79 78 79 
emb2  90 90 90 78 78 79 78 
10°C fast   97 92 75 74 75 75 
I fast    92 75 75 76 75 
30°C fast     76 75 77 75 
emb3      90 96 88 
slow10       91 87 
slow30        88 
emb210°C fastI fast30°C fastemb3slow10slow30cardiac
embl 95 88 88 89 78 79 78 79 
emb2  90 90 90 78 78 79 78 
10°C fast   97 92 75 74 75 75 
I fast    92 75 75 76 75 
30°C fast     76 75 77 75 
emb3      90 96 88 
slow10       91 87 
slow30        88 

cDNA cloning of slow MYH isoforms

Starting with RNA extracted from adult slow muscle, PCR using universal primer for cardiac/slow MYHs with AUAP, amplified a single band of about 700 bp from first strand cDNA. The PCR products were cloned into a plasmid vector,randomly selected and sequenced. Sequencing demonstrated the presence of two types of slow muscle MYH, MYHS10 (AB104625) and MYHS30(AB104626), which were predominantly expressed in carp acclimated to 10°C and 30°C, respectively. We sequenced three independent clones for each gene. The slow muscle from adult carp acclimated to 20°C contained both MYH isoforms. MYHS10 and MYHS30 cDNAs had polyadenylation signals and poly(A) tails, and their deduced amino acid sequences encoded part of l-meromyosin(Fig. 1), showing 74-96%identity with the MYH isoforms found in the present study and those reported previously (Imai et al., 1997)(Table 1). It was noted that the MYHemb3 isoform was more similar to MYHS10 and MYHS30 (90-96% identity) than to other carp embryonic and adult fast skeletal MYH isoforms (74-79% identity).

Fig. 4.

Specificity of probes for northern blot hybridization from eight types of cDNA clones encoding MYH isoforms of carp. Nylon membranes incorporating MYHF10, MYHF30, MYHS10, MYHS30,MYHemb1, MYHemb2, MYHemb3 and MYHcard cDNA clones were hybridized with probes specific to MYHS10, MYHS30, MYHemb1,MYHemb2, MYHemb3 and MYHcardused for northern blot analysis. S10, S30, slow cDNA, emb1-3 embryonic 1-3 cDNA, cardiac, cardiac cDNA (all at 1, 10 or 100 ng).

Fig. 4.

Specificity of probes for northern blot hybridization from eight types of cDNA clones encoding MYH isoforms of carp. Nylon membranes incorporating MYHF10, MYHF30, MYHS10, MYHS30,MYHemb1, MYHemb2, MYHemb3 and MYHcard cDNA clones were hybridized with probes specific to MYHS10, MYHS30, MYHemb1,MYHemb2, MYHemb3 and MYHcardused for northern blot analysis. S10, S30, slow cDNA, emb1-3 embryonic 1-3 cDNA, cardiac, cardiac cDNA (all at 1, 10 or 100 ng).

cDNA cloning of cardiac MYH

We synthesized first strand cDNA from total RNAs of the myocardium from carp acclimated to 10°C and 30°C. PCR using universal primer for cardiac/slow MYHs with AUAP gave an intense single band of about 700 bp for carp acclimated to both 10°C and 30°C. Sequencing of PCR products revealed that the clone isolated was that of the cardiac type,MYHcard (AB104627), since its deduced amino acid sequence was different from any fast skeletal MYHs from carp (75-79% identity; Imai et al., 1997), but resembled carp slow skeletal MYHs in the present study (87-88% identity) and cardiac types from other vertebrates (72-96% identity; Yelon et al., 1999; McNally et al., 1989; Kraft et al., 1989; Yutzey et al., 1994; Figs 1 and 2). We sequenced two independent clones for this gene.

Phylogenetic analysis

The topology in NJ and MP trees were almost the same, implying that the trees obtained were highly reliable (Fig. 3). MYHS30 and MYHS10 as well as MYHemb3 were located in the same clade with zebrafish slow type MYH encoded by smyhc1(Bryson-Richardson et al.,2005). However, MYHcard formed monophyly with zebrafish MYH expressed in ventricular muscle, but was different from zebrafish MYH expressed in atrial muscle (Berdougo et al., 2003).

MYHemb1 and MYHemb2 were monophyletic with zebrafish MYHs encoded by myhz1, myhz2 and myhc4 expressed in embryos(Bryson-Richardson et al.,2005; Peng et al.,2002; Xu et al.,2000). This group also contained torafugu MYHM743-2 and MYHM10392 (Ikeda et al.,2004). MYHF10, MYHFint and MYHF30isolated from carp adult fast muscle (Imai et al., 1997; Hirayama et al.,2000; Watabe,2002) were located in the same clade with torafugu MYHM86-2 and different from other four torafugu fast skeletal MYHs(Ikeda et al., 2004)(Fig. 3). It was noted that human slow/cardiac MYHs and fast skeletal MYHs formed separate groups from fish counterparts, respectively.

Northern blot analysis

Although the specificities of probes for MYHF10 and MYHF30 had been previously confirmed(Hirayama and Watabe, 1997),it was not clear whether or not the DNA fragments used as probes for MYHemb1, MYHemb2, MYHemb3,MYHS10, MYHS30 and MYHcard were specific to their respective genes. Dot blot analysis revealed that all probes except MYHcard were highly specific to each target mRNA(Fig. 4). Only MYHcard gave faint positive signals with MYHemb3, MYHS10 and MYHS30(Fig. 4).

We then performed northern blot analysis to determine the accumulated mRNA levels of MYH DNAs for carp at various developmental stages as well as for adult carp acclimated to 10°C, 20°C and 30°C(Fig. 5). The expression pattern of MYHemb1 was similar to that of MYHemb2, and both species of mRNA were first detected in embryos 61 h.p.f. at the heartbeat stage. The peak expression of these mRNAs occurred in juveniles 1 month post-hatching. Faint expression of MYHemb1, but not MYHemb2 was found 7 months post-hatching. Signals for MYHemb1 mRNA were also detected in fast skeletal muscle from adult carp acclimated to 10°C and 20°C. MYHemb3 was also first detected in embryos 61 h.p.f., and faintly detected in fast and slow skeletal muscles of adult carp acclimated to 10°C and 20°C as well as in fast skeletal muscle of adult carp acclimated to 30°C. MYHS10 and MYHS30 mRNAs were faintly expressed at hatching and the transcripts increased in abundance between 1 month and 7 months post-hatching. The transcripts of MYHS10 and MYHS30 were detected in slow skeletal muscles of adult carp acclimated to 10°C and 20°C,whereas 30°C-acclimated fish only expressed MYHS30. The transcripts of MYHcard were first detected in embryos 61 h.p.f. and were found in juvenile and in fast and slow skeletal muscles from adult fish.

Fig. 5.

Changes in the accumulated mRNA levels of carp MYH isoforms during ontogeny. Total RNAs (20 μg) were electrophoresed in a 1% agarose gel and transferred on to the nylon membranes, which were then hybridized with 32P-labeled probe. Lanes contain total RNAs from the whole embryos at 30 h, 42 h, 61 h, and 77 h.p.f., from the whole larvae at hatching (96 h),from the muscle of juveniles aged 1 and 7 months post-hatching and from the fast and slow skeletal muscles of thermally acclimated adult fish. An ethidium bromide-stained gel shows 28S and 18S rRNA. Consensus indicates the membrane hybridized with the probe, which is supposed to react with all skeletal MYH mRNA of carp. Each lane contains 5 μg of total RNA.

Fig. 5.

Changes in the accumulated mRNA levels of carp MYH isoforms during ontogeny. Total RNAs (20 μg) were electrophoresed in a 1% agarose gel and transferred on to the nylon membranes, which were then hybridized with 32P-labeled probe. Lanes contain total RNAs from the whole embryos at 30 h, 42 h, 61 h, and 77 h.p.f., from the whole larvae at hatching (96 h),from the muscle of juveniles aged 1 and 7 months post-hatching and from the fast and slow skeletal muscles of thermally acclimated adult fish. An ethidium bromide-stained gel shows 28S and 18S rRNA. Consensus indicates the membrane hybridized with the probe, which is supposed to react with all skeletal MYH mRNA of carp. Each lane contains 5 μg of total RNA.

The transcripts of MYHemb1 and MYHemb2 were not found, even at very low levels, in 7-month-old juveniles, by which time the MYHF10 gene was showing strong expression. In adult stages, the fast skeletal muscle of fish acclimated to 10°C expressed the MYHF10,MYHemb1, MYHemb3, MYHS10,MYHS30 and MYHcard mRNAs, while 30°C-acclimated fish expressed MYHF30, MYHS10,MYHS30, MYHemb3 and MYHcardmRNAs. The transcripts of MYHS10 and MYHS30 were found in embryos at hatching and juveniles of 1-month and 7-months old. It was noted that fast skeletal muscle from adult carp acclimated to 20°C expressed MYHS10,MYHS30 and MYHcard mRNA. The slow skeletal muscle of adult carp acclimated to 10°C and 20°C contained both MYHS10 and MYHS30. By contrast,30°C-acclimated fish predominantly expressed MYHS30.

As a next step in the analysis a universal probe was adopted for northern blot analysis. This DNA probe encoded residues 534-717 from the 3′region of the MYHF10 cDNA, which is thought to react with all known skeletal MYH isoforms of carp (Hirayama and Watabe, 1997). The signals to this probe were first detected in embryos 61 h.p.f., and then in fast and slow skeletal muscles from adult fish (Fig. 5).

Whole-mount in situ hybridization

Whole-mount in situ hybridization (WISH) was carried out for MYHemb1, MYHemb2 and MYHemb3 to localize their transcripts in 77 h.p.f.(eyed-stage) and 95 h.p.f. (hatching stage) fish. Probes used for northern blot analysis consisting of about 100 nt gave no signals (data not shown),therefore, the size of probes was enlarged to about 150 nt as described in the Materials and methods. The transcripts of MYHemb1(Fig. 6G,I) and MYHemb2 (Fig. 6J) were expressed in the inner part of the myotome, whereas that of MYHemb3 was expressed in the superficial compartment(Fig. 6H,K). The superficial compartment of the myotome largely comprises slow muscle fibers(Devoto et al., 1996), thus MYHemb3 is probably predominantly expressed in slow embryonic muscle. Whereas the transcripts of MYHemb1 were detected in almost the whole trunk muscle(Fig. 6A,B), those of MYHemb2 were observed in the region of anterior to middle trunk muscle (Fig. 6C,D) at 77 and 95 h.p.f. Observation of this region revealed that the transcripts of MYHemb2 were localized only in four corners of trunk(Fig. 6J, arrows).

Fig. 6.

Localization of the transcripts of MYHemb1,MYHemb2 and MYHemb3 in carp embryos. Carp embryos were hybridized with probes specific to MYHemb1(A,B), MYHemb2 (C,D) and MYHemb3 (E,F)at 77 (A,C,E) and 95 (B,D,F) h.p.f. G and H are higher magnifications of the caudal region shown in B and F, respectively. (I,J,K) Transverse sections of B,D,F, respectively, in the regions indicated by arrowheads. Arrows in J indicate the transcripts of MYHemb2 in the four `corners'of the trunk.

Fig. 6.

Localization of the transcripts of MYHemb1,MYHemb2 and MYHemb3 in carp embryos. Carp embryos were hybridized with probes specific to MYHemb1(A,B), MYHemb2 (C,D) and MYHemb3 (E,F)at 77 (A,C,E) and 95 (B,D,F) h.p.f. G and H are higher magnifications of the caudal region shown in B and F, respectively. (I,J,K) Transverse sections of B,D,F, respectively, in the regions indicated by arrowheads. Arrows in J indicate the transcripts of MYHemb2 in the four `corners'of the trunk.

Discussion

In the present study, we isolated six types of MYH DNA from common carp,the deduced amino acid sequences of which were classified into two groups(Table 1 and Fig. 3). One was fast skeletal muscle type, which contained MYHemb1, MYHemb2,MYHF10, MYHFint, MYHF30 and the other,slow/cardiac muscle type, which consisted of MYHemb3,MYHS10, MYHS30 and MYHcard. The slow/cardiac muscle group of MYHs was further divided into slow and cardiac (MYHcard) types(Fig. 3). Carp MYHs belonging to the fast skeletal muscle clade were divided into two types, one expressed in adult fast muscle (MYHF10, MYHFintand MYHF30) and the other in embryonic stages(MYHemb1 and MYHemb2). The two carp embryonic type MYHs formed the other group with zebrafish myhz1,myhz2 and myhc4 expressed in embryos(Xu et al., 2000; Peng et al., 2002; Bryson-Richardson et al., 2005)and also with torafugu MYHM743-2 and MYHM10392 (Ikeda et al., 2004) (Fig. 3).

The expression patterns of various MYHs of carp identified in relation to tissue type and developmental stage are summarized in Fig. 7. MYHemb1 was not exclusively expressed in the embryonic and larval stages, since transcripts were also detected in the fast skeletal muscle of adult fish acclimated to 20°C. This may reflect its expression in newly formed myotubes. Small amounts of embryonic and neonatal MYHmRNAs are known to be expressed in adult rat plantaris and soleus muscles during hypertrophic growth(Periasamy et al., 1989). In addition, Ennion et al. (1995)isolated a MYH clone (FG2) from carp that hybridized exclusively to small diameter fibers in warm-acclimated fish. Ennion et al.(1999) isolated two developmentally regulated MYHs, Eggs22 and Eggs24,from the carp. These transcripts were first detected in embryos 22 h.p.f. and continued to be expressed over 2 weeks post-hatching, but were not detected in juveniles 21 and 28 d post-hatching or in adult stages. In contrast, the mRNA levels of MYHemb1 and MYHemb2,homologues of Eggs22 and Eggs24, respectively, were still high in 1-month-old juveniles. MYHemb1 transcripts were also detected at low levels in 7-month-old juveniles, and in the fast skeletal muscle of adult carp acclimated to 10°C and 20°C (see Fig. 4). Although both MYHemb1 and MYHemb2 had nucleotide sequences very similar to each other, their expression patterns were markedly different. WISH analysis demonstrated no obvious differences in expression patterns between MYHemb1 and MYHemb2(Fig. 6). Wakeling et al.(2000) demonstrated, by peptide mapping, that the expression of carp fast type MYHs was only altered with temperature acclimation in fish greater than 37 mm total length. Furthermore, larval MYH isoforms were replaced by juvenile isoforms when larvae reached 20 mm. In the present study, only weak signals for MYHemb1 were detected at 7 months post-hatching(Fig. 7), which suggests that an as yet uncharacterized MYH isoform(s) is expressed in juvenile fish.

MYHemb3 had DNA nucleotide and deduced amino acid sequences very similar to those of MYHS10 and MYHS30 isolated from slow muscle in adult fish and these three isoforms were located on the same clade in the phylogenetic tree(Fig. 2). The transcripts of MYHemb3 were first detected in 61 h.p.f. embryos by northern blot analysis and in the superficial layer of myotomes by WISH (Figs 5 and 6). It has been demonstrated,using immunohistochemistry, that expression of the slow type MYH gene is followed by expression of the fast type in zebrafish(van Raamsdonk et al., 1978; Devoto et al., 1996; Weinberg et al., 1996). The initial slow muscle fibers in zebrafish are formed from an array of adaxial cells in response to hedgehog signaling from the notochord(Blagden et al., 1997). The adaxial cells elongate to span the somite width and migrate through the somite to form a superficial layer of slow muscle fibers(Devoto et al., 1996). However, these adaxial-derived slow muscle fibers account for about half the number of slow fibers present at the time the embryo hatches. In the late embryo, additional slow muscle fibers are added from discrete germinal zones by stratified hyperplasia (Barresi et al.,2001). Northern blot analysis in the present study revealed that in common carp MYHS10 and MYHS30transcripts were not expressed until hatching.

Fig. 7.

Schematic representation of the mRNA expression patterns of the various MYHs identified in relation to muscle type, developmental stage and acclimation temperature. Since northern blot analysis was only performed on pure muscle types in adults, the tissue-specific expressions in embryonic and juvenile fish were based on inference (see text for discussion), and therefore remain a hypothesis. Putative MYH possibly expressed in juveniles are indicated by question marks.

Fig. 7.

Schematic representation of the mRNA expression patterns of the various MYHs identified in relation to muscle type, developmental stage and acclimation temperature. Since northern blot analysis was only performed on pure muscle types in adults, the tissue-specific expressions in embryonic and juvenile fish were based on inference (see text for discussion), and therefore remain a hypothesis. Putative MYH possibly expressed in juveniles are indicated by question marks.

We previously cloned Myf-5, MyoD, myogenin, MEF2A and MEF2C from carp and showed that these genes were expressed in carp embryos, larvae and juveniles(Kobiyama et al., 1998). It was noted that no differences in the timing of expression of Myf-5, MyoD and myogenin mRNAs were seen between different temperature groups of carp embryo(Cole et al., 2004). The signals of carp MEF2C and MEF2A mRNA were first detected in carp embryos at 30 and 42 h.p.f., respectively (Kobiyama et al., 1998). The MEF2 family is a key regulator of cardiac muscle lineage (Gossett et al., 1989;Olson, 1995). In zebrafish, the transcripts of MEF2C were detected in primordial cardiac cells from embryos at 16 h.p.f., whereas those of MEF2A andα-tropomyosin were first observed in embryos at 10 h.p.f.(Ticho et al., 1996). Yelon et al. (1999) showed that ventricular MYH was first expressed in zebrafish embryos at 30 h.p.f., and that of cardiac myosin light chain 2 mRNA appeared at the same stage and was expressed only in the heart tube. In the present study MYHcard was first expressed in embryos at 61 h.p.f. and it continued to be expressed during subsequent embryonic stages. The transcripts of MYHcard were detected not only in myocardium of adult carp, but also in adult slow and fast skeletal muscles. Since the present probe for MYHcard showed weak signals against MYHemb3, MYHS10 and MYHS30,a new specific probe for MYHcard is required to distinguish between the expression patterns of MYHcard,MYHemb3MYHS10 and MYHS30. However, Allen and Leinwand(2001) showed thatα MYH, which is expressed almost exclusively in the heart, is expressed in scattered fibers in all the hindlimb muscles of the mouse during postnatal development. Single skinned fibers from slow and fast muscle of adult rats contained MYHI/β and MYHIIa, although the frequency of this co-expression was very low (Bottinelli et al., 1994). Bisaha and Bader(1991) showed that a type of cardiac MYH, VMHC 1, expressed exclusively in the chicken heart during embryogenesis, was also transiently expressed in all embryonic skeletal muscles.

In summary, we isolated six MYH DNAs from carp, three embryonic, two slow skeletal and one cardiac. According to the deduced amino acid sequence, each MYH isoform was classified into either fast or slow type using phylogenetic analysis. Our initial hypothesis that there would be multiple slow muscle MYH genes that are expressed at different acclimation temperatures was accepted although we do not rule out the possibility that some of these genes might correspond to the alternatively spliced transcripts from the same gene. However, we have revealed a complex pattern of expression of MYHs in relation to developmental stage, muscle type and acclimation temperature. For example, MYHs predominantly associated with slow muscle or early developmental stages were expressed in the fast muscle of adult fish at some acclimation temperatures but not others. Since none of the nine skeletal muscle MYHs, including the three previously reported, were strongly expressed in 7-month-old juveniles it is likely that further members of the MYH family remain to be identified.

Acknowledgements

This study was supported partially by Grant-in-Aids for Scientific Research from the Ministry of Education, Science, Technology, Sports and Culture of Japan, and from the Asahi Glass Foundation, Japan. We thank Dr K. Sakai and Dr G. Yoshizaki, Tokyo University of Fisheries, for assistance with the sampling of carp embryos. Yoshiaki Nihei was supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. Ian Johnston is grateful to the Natural Environment Research Council, UK for support.

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