ARTICLE |
Correspondence to: Karin Eizema, Utrecht University, Faculty of Veterinary Medicine, Div. of Anatomy, PO Box 80.157, NL-3508 TD Utrecht, The Netherlands. E-mail: c.g.h.eizema@vet.uu.nl
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Summary |
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The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei. (J Histochem Cytochem 51:12071216, 2003)
Key Words: horse, fiber type, in situ hybridization, immunohistochemistry, myosin heavy-chain
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Introduction |
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SKELETAL MUSCLE is composed of different types of myofibers, each expressing a distinct set of structural proteins and metabolic enzymes (], each with its own ATPase activity and each encoded by a separate gene (
The MyHC proteins are believed to be primarily transcriptionally controlled. The time course and threshold stimulus needed to trigger changes at the mRNA level are therefore important aspects of gene regulation. (
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Materials and Methods |
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All chemicals were obtained from Merck (Amsterdam, The Netherlands) unless otherwise indicated.
Muscle Biopsies
Percutaneous muscle biopsies from the gluteus medius were taken according to the protocol of
Oligonucleotides for 3' RACE PCR
The oligonucleotides RoRi-dT17 and Ro were synthesized according to
First-strand cDNA Synthesis
Total RNA was isolated using acid guanidinium thiocyanate-phenol-chloroform according to
RACE-PCR
Double-stranded cDNA was amplified from the first-strand cDNA reaction in a PCR using either FG2Exn40 or RnExn40 primer. Reactions were performed in a 100-µl volume containing reaction buffer (final concentrations 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 0.2 mM each of dNTPs (Roche Molecular Biochemicals; East Sussex, UK), 25 µM each Ro and exon 40 primers, and 10 µl first-strand cDNA. Using a "hot start" 2.5 U of Taq polymerase (Roche Molecular Biochemicals) was added. Amplification was then carried out by 32 cycles of denaturation at 94C for 1 min followed by annealing at 55C for 1 min, followed by elongation at 72C for 2 min. A final elongation step at 72C for 10 min was included to ensure that all PCR products had 3' A overhangs. PCR products were analyzed by gel electrophoresis on 1% agarose gels. PCR products were excised and purified using Wizard PCR purification kit (Promega) and subcloned into the pGEM-T vector (Promega) or the TpCRII vector (Invitrogen; Breda, The Netherlands) using the T-A cloning method. DNA sequencing was performed by the chain termination method (
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In Situ Hybridization
Transverse serial sections (10 µm) were made with a cryostat at -20C, placed on Superfrost Plus slides (Menzel; Merck, Amsterdam, The Netherlands), dried for 1 hr, fixed for 20 min with 4% paraformaldehyde in 1 x PBS, pH 7.4 (
The cDNAs included MyHC 1 (276 bp) in pGEM-T, 2a (278 bp) in pGEM-T, and 2d/x (282 bp) in pCRII-TOPO. Riboprobes were synthesized with digoxigenin-labeled UTP in antisense direction by in vitro transcription from the linearized cDNA templates, according to the manufacturer's guidelines (Roche Molecular Biochemicals; Almere, The Netherlands) and purified by a Qiagen RNeasy kit (Westburg; Leusden, The Netherlands). The riboprobes (500 ng/ml final concentration) were suspended in 40% (deionized) formamide, 1 x SSC, 10% dextran sulfate, 1 x Denhardt solution [0.02% Ficoll, 0.02% polyvynilpyrrolidine, 0.02% bovine serum albumin (BSA)], 0.67 M NaCl, 0.1 µg/µl yeast tRNA, and 0.1 µg/µl herring sperm DNA, heated at 80C for 5 min. Prehybridization (45 µl) was performed for 30 min. Approximately 30 µl of probe was used per slide, overlaid with a coverslip. Hybridization was performed overnight at 45C in a humidified In Slide Out incubator (Boekel Scientific; Merck, Amsterdam, The Netherlands). Coverslips were removed by rinsing in 6 x SSC at 60C, followed by two high-stringency steps at 60C for 20 min in 0.5 x SSC and 20% formamide and two rinses in 2 x SSC at room temperature. Unhybridized probe was digested with 2 µg/ml RNase A in 0.5 M NaCl, 10 mM Tris-HCl, pH 8.0, at 37C for 30 min, followed by five washes in 2 x SSC at room temperature and another high-stringency wash for 10 min. The sections were rinsed twice with 2 x SSC and maleic buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5). Methods for the detection of hybridized probes were adapted from the manufacturer's protocols (Roche Molecular Biochemicals). Tissue sections were blocked twice for 15 min and once for 1 hr with 5% inactivated BSA in maleic buffer at RT. Blocking was replaced with sheep anti-digoxigenin Fabalkaline phosphatase conjugate (1:2000 in 1% BSA in maleic buffer) overnight at RT. After several rinses with the same buffer, sections were washed twice with 0.1 M NaCl, 0.1 M Tris-HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20 at RT. Alkaline phosphatase activity was visualized by incubation with 0.18 mg/ml BCIP, 0.34 mg/ml NBT, in the buffer described above. The staining was allowed to develop for approximately 16 hr at RT, rinsed with distilled water, and embedded in Aquamount. Control sense probes were negative (results not shown).
Immunohistochemical Staining
The monoclonal antibodies (MAbs) used were previously shown to crossreact with horse myosins (
Analyses
A group of 296 contiguous fibers was used for fiber typing and calculation of fiber type composition. The muscle fibers were classified into type 1, type 2a, or type 2d/x, or type 2ad on the basis of their reactions with the different in situ probes and the MAbs.
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Results |
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Isolation of Equine MyHC Isoforms
Sequence analysis of the isolated cDNA clones revealed that three different isoforms had been isolated. Identification of the isoforms was done by performing a BLAST search against all MyHC genes known in combination with the comparisons shown in this article. From these analyses, we could conclude that the isolated clones were the slow MyHC (type 1) and the two fast MyHC [type 2a and type 2d/x (called in the Fig 2d)] known in horse. We did not identify cDNAs encoding the 2b gene. The complete sequences of the different isoforms were aligned as depicted in Fig 1. The two fast isoforms isolated showed higher homology relative to each other (79%) than to type 1 (type 1 vs type 2a 61% homology; type 1 vs 2d/x 62% homology). Fig 2 depicts the alignment of the nucleotide and deduced amino acid sequence of the last 15 amino acids of the coding regions of the isolated horse clones. A comparison was performed with known sequences of human, mouse, rabbit, rat, bovine, and pig. From this comparison, it can be concluded that the type 2 isoforms show more homology to other type 2 isoforms than to the type 1 isoform. Of the 15 amino acids presented, six showed consistent differences between the type 1 and type 2 isoforms. These are the first A/V, the fifth D/E, the sixth I/V, the seventh G/H, the eleventh L/I, the twelfth N/S, resulting in a slight (2) charge difference.
Fig 3 shows the nucleotide sequence alignment of the horse MyHC genes 3' untranslated region (UTR) with sequences of the same species as in Fig 2. Interestingly, it can be seen that between species the sequence identity of the 3'UTR is much higher for the type 2 MyHC isoforms, suggesting that this region, as previously suggested, is either under stringent evolutionary constraints or that they have diverged much later than the type 1 isoform. There were no clear distinctions possible between a 5' and 3' part as previously suggested from the pig sequence (
Identification of Myofiber Types by ISH and IHC
The complete equine MyHC sequences depicted in Fig 1 were used to generate isoform specific non-radioactive RNA probes. Because most ISH studies used radioactive probes and the equine muscle was to our knowledge not investigated by this type of analysis, we had to optimize the non-radioactive protocol for our experiments based on protocols for embryonic mouse and adult human muscle (
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Using serial sections we compared the MyHC mRNA content of the fibers (Fig 5, left) with their MyHC protein composition, determined by IHC (Fig 5, right). Fibers 1, 2, and 3 exhibit specific staining for type 1, 2a, and 2d/x, MyHC protein, respectively. Clearly, the staining patterns of the ISH and IHC experiments show a high correlation. We analyzed the same 296 fibers used for the mRNA fiber typing again for the protein fiber typing. On the protein level more (21.4%) hybrid fibers were detected, and they all expressed the type 2a and type 2d/x isoform (type 2ad; Table 1). Of all fibers analyzed, 76.5% had the same RNA and protein expression; the rest showed a mismatch (Table 1). Most of these mismatches concerned fibers expressing type 2ad protein. An example is fiber no. 5 expressing type 2a RNA but type 2ad protein. Of the type 2ad fibers at the protein level, 19.7% had only type 2a RNA, 1.4% had only type 2d/x RNA, and 0.3% (one fiber) had type 1 RNA (Table 1). On the other hand, fiber no. 4 in Fig 5 expressed type 2ad at the RNA level but only type 2d/x protein. This is a typical result because type 2ad RNA expression was never matched by type 2ad protein expression (Table 1).
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Discussion |
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We have isolated and characterized for the first time part of the genes encoding the equine MyHC type 1, 2a, and 2d/x. Isolation was performed based on conserved sequences in exon 40 of the fast and slow isoforms. We did not isolate any cDNA fragment encoding the 2b gene, making it unlikely that this gene is expressed in the gluteus medius of the horse. This finding is in line with previous publications on the expression of MyHC protein in equine skeletal muscle (
The isolated MyHC genes presented in this article cluster together with their expected counterparts when a relationship analysis is performed (Fig 2). Comparing the last 15 amino acids of the coding region reveals some characteristic differences between the type 1 and the type 2 isoform (Fig 2). In the type 2 isoforms, such differences do not appear to exist, although not many sequences are known, especially not of the 2b gene.
This article presents for the first time the application of a non-radioactive ISH procedure on skeletal muscle of a large animal. The technique allows comparison of the expression of MyHC isoform transcripts and protein on a fiber-to-fiber basis, using serial sections. Analyses of a large number of fibers (around 300) per biopsy are possible, an important advantage over single-fiber analysis.
The majority (76.5%) of the fibers analyzed expressed the same MyHC isoform on the RNA and protein levels. Mismatches were due to the occurrence of hybrid fibers. At the transcriptional level, hardly any hybrid fibers were detected, whereas at the translational level a substantial number of hybrid fibers were seen. Recently, the same type of result was presented in the skeletal muscle of the pig (
Fibers that contained two MyHCs at the protein level, but only one transcript, could be fibers that are converting to the type corresponding to the expressed mRNA (
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In conclusion, we have isolated and characterized part of the genes encoding the equine MyHC type 1, 2a, and 2d/x isoforms. The type 2b isoform was not identified. The isolated genes were used to generate isoform-specific probes for non-radioactive ISH experiments using a biopsy from the equine gluteus medius muscle. We compared the expression on the RNA level with the expression on the protein level on a fiber-to-fiber basis. Discrepancies were found that were not due to differences in expression between adjacent myonuclei. Co-expression of MyHCs was more common at the protein level than at the mRNA level and was mostly observed for 2a and 2d/x MyHCs, suggesting a fine tuning of these two genes and a strong influence of their expression on myofiber plasticity. The present study also shows that only the combination of IHC with ISH allows a clear understanding of the dynamic process involved in fiber type transitions by giving a potential clue to the direction of change in MyHC gene expression. Finally, because MyHC is the most abundant protein in muscle and because of the influence of its polymorphism on contractile, metabolic, and size properties of myofibers, further research is now possible to establish and understand the importance of MyHC transcriptional and translational polymorphism in growing and exercising horses.
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Acknowledgments |
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We thank Dr A.F.M. Moorman and J.A.M. Korfage (University of Amsterdam) for the generous gift of the monoclonal antibodies. We are grateful for the skillful technical assistance of Miriam van der Belt and Ellen van der Wiel. Dr M. Horton (University of Pittsburgh) was invaluable for the development of the in situ hybridization protocol.
Received for publication January 6, 2003; accepted April 2, 2003.
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