Journal of Histochemistry and Cytochemistry, Vol. 50, 353-364, March 2002, Copyright © 2002, The Histochemical Society, Inc.


ARTICLE

Quantifying the Temporospatial Expression of Postnatal Porcine Skeletal Myosin Heavy Chain Genes

Nuno da Costaa, Ross Blackleya, Hadi Alzuherria, and Kin-Chow Changa
a Veterinary Molecular Medicine Laboratory, Department of Veterinary Pathology, University of Glasgow, Glasgow, Scotland

Correspondence to: Kin-Chow Chang, Veterinary Molecular Medicine Laboratory, Dept. of Veterinary Pathology, Univ. of Glasgow, Bearsden Road, G61 1QH Glasgow, Scotland. E-mail: k.chang@vet.gla.ac.uk


  Summary
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Materials and Methods
Results
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Postnatal skeletal muscle fiber type is commonly defined by one of four major myosin heavy chain (MyHC) gene isoforms (slow/I, 2a, 2x, and 2b) that are expressed. We report on the novel use of combined TaqMan quantitative real-time RT-PCR and image analysis of serial porcine muscle sections, subjected to in situ hybridization (ISH) and immunocytochemistry (IHC), to quantify the mRNA expression of each MyHC isoform within its corresponding fiber type, termed relative fiber type-restricted expression. This versatile approach will allow quantitative temporospatial comparisons of each MyHC isoform among muscles from the same or different individuals. Using this approach on porcine skeletal muscles, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a given muscle and between muscles. Marked differences were also observed among pig breeds. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber type-restricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that although 2a fibers are a minor fiber type, they may be disproportionately more important as a determinant of overall muscle function than was previously believed. (J Histochem Cytochem 50:353–364, 2002)

Key Words: quantitative real-time PCR, myosin heavy chain, fiber type, temporospatial expression, porcine


  Introduction
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Summary
Introduction
Materials and Methods
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Literature Cited

SKELETAL MUSCLE is composed of different types of myofibers, which are the results of co-ordinate expression of distinct sets of structural proteins and metabolic enzymes (Schiaffino and Reggiani 1996 ). Fiber type is often defined by the isoform of myosin heavy chain (MyHC) that is present. MyHCs are the major structural proteins of myofibrillar thick filaments that are able to convert chemical energy to mechanical energy for muscle contraction. As for other structural muscle proteins, MyHCs are encoded by a highly conserved multigene family, of which eight isoforms are known in mammals (2a, 2x, 2b, embryonic, perinatal, slow/I/ß, extraocular, and {alpha}), each with its own ATPase activity and each encoded by a separate gene (Weiss and Leinwand 1996 ). In postnatal mammalian muscles (rodents and pigs), there are four major fiber types characterized by the expression of the slow/I/ß, 2a, 2x, and 2b MyHC gene isoforms. We can readily identify the four postnatal fiber types (slow/ß, 2a, 2x, and 2b) in porcine skeletal muscle by in situ hybridization (ISH), with isoform-specific cRNA probes from the 5'-untranslated cDNA regions of the respective MyHC isoforms (Chang and Fernandes 1997 ).

In addition to temporal regulation (Lefaucheur et al. 1995 ), the expression of each member of the MyHC gene family is subjected to specific spatial control, such that the number and distribution of fibers expressing each isoform often vary among anatomic muscles, e.g. soleus and longissimus dorsi (Schiaffino and Reggiani 1996 ). Variation in fiber type composition among muscles is a reflection of functional differences in contractile activity. The same MyHC isoform in different muscles can also show differential response to the same stimulus, e.g., thyroid hormone administration (Haddad et al. 1998 ). To study temporospatial changes in MyHC expression or the effects of exogenous manipulations on MyHC expression, reliable mRNA quantification of each MyHC gene isoform is necessary (Schiaffino and Reggiani 1994 ). Previous work has shown that the protein level of a MyHC isoform in muscle largely correlates with its corresponding normal steady-state mRNA level (Stedman and Sarkar 1988 ; McKoy et al. 1998 ). Conventional methods of measuring gene expression, i.e., Northern analysis and RNase protection assays, do perform satisfactorily but are time-consuming and are limited in precise quantification. Such problems have been largely overcome with the recent introduction of quantitative real-time reverse-transcription polymerase chain reaction (TaqMan real-time RT-PCR), which measures an accumulating PCR product in real time by using an internal fluorogenic TaqMan oligonucleotide probe that is activated by the endogenous 5'->3' nuclease activity of Taq polymerase (Freeman et al. 1999 ). However, the use of real-time RT-PCR alone, as applied to skeletal muscle, could not distinguish differences in fiber type composition.

We report here on the development of a novel and versatile approach to accurately quantify the mRNA expression of each of the four major postnatal MyHC isoforms (slow/ß, 2a, 2x, and 2b) within its fiber type, termed relative fiber type-restricted expression. This was achieved by the combined use of TaqMan quantitative real-time RT-PCR and fiber type image analysis. One main advantage of this approach is the ability to make quantitative comparisons of each MyHC isoform among muscles from the same or different individuals, which could significantly advance our knowledge of the temporospatial regulation of MyHCs. This approach can also be readily extended to the quantification of other muscle genes. Experimentally, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a muscle and among muscles. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber type-restricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that 2a fibers, although a minor fiber type, may be disproportionately more important in muscle function than was previously believed.


  Materials and Methods
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Materials and Methods
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Literature Cited

Pigs
Detailed analysis was carried out on muscles from a 6-week-old Large White-based pig, three 22-week-old Duroc-based pigs (pigs 4, 5, and 6), and three 22-week-old Berkshire pigs (pigs 1, 2, and 3). All Durocs and Berkshires were intact males reared under identical husbandry and feeding conditions. Pigs were individually fed (20% protein, 14 MJ DE/kg with 1.14% lysine) twice a day at 90% of the predicted ad-lib feed intake based on body weight. Muscles analyzed were selected for their varying mixtures of adult skeletal MyHC isoforms. They were the supraspinatous (supra), the longissimus dorsi (LD), and the psoas. Muscles were sampled from the same anatomic sites of each animal within 30 min of slaughter, frozen in pre-chilled isopentane, and stored at -70C until further processing. Porcine fetal back muscles (47- and 104-day-old, based on known mating dates) were collected from a commercial abattoir and frozen in isopentane on site.

Quantitative Real-time RT-PCR
Quantitative real-time RT-PCR was performed on nine porcine genes: MyHC embryonic, MyHC perinatal, MyHC slow/I/ß, MyHC 2a, MyHC 2x, MyHC 2b, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), skeletal {alpha}-actin, and ß-actin. Each cDNA template for real-time PCR was prepared from mRNA directly extracted from 50 mg of skeletal muscle tissues (Dynabeads mRNA direct kit), by first-strand reverse transcription using 0.25 µg of random hexadeoxynucleotides (First-strand cDNA synthesis kit; Pharmacia, Piscataway, NJ). Owing to the high sequence homology among MyHC gene isoforms, all primers and probes were targeted towards the 5'-untranslated regions of their respective cDNAs, which were shown to be isoform-specific (Chang and Fernandes 1997 ). Random hexamers gave consistently higher yield of 5'-end-untranslated MyHC cDNAs than oligo-d(T) primers. TaqMan probe and primer sequences (Table 1) were designed using Primer Express software (version 1; PE Applied Biosystems, Tucson, AZ). For each gene, at least one oligonucleotide, either probe or primer, was designed to span two exons to avoid genomic DNA amplification. TaqMan PCR assays for each target gene were performed in triplicate on cDNA templates in 96-well optical plates in an ABI Prism 7700 Sequence Detection System according to the manufacturer's recommendations (PE Applied Biosystems). For each sample, an amplification plot was generated, displaying an increase in the reporter dye fluorescence ({Delta}Rn) with each PCR cycle. From it, a threshold cycle (Ct) value was calculated, which was the PCR cycle number at which fluorescence was detected above threshold, based on the variability of baseline data in the first 15 cycles. Each Ct value was determined from the mean of four separate real-time PCR experiments. Relative standard curves for each gene were plotted showing Ct (y-axis) vs log (initial cDNA) diluted 10-fold sequentially (x-axis). The random hexamer-derived cDNA used to generate the relative standard curves was from the LD of a sow. The slope (m) of the standard curve describes the efficiency of PCR, and is defined by the equation


 
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Table 1. Oligonucleotides and TaqMan fluorogenic probesa

Ct = m (logQ) + c,

where Ct is the threshold cycle, Q is the initial amount, and c is the intercept on the y-axis. When PCR amplification is maximally efficient, resulting in a doubling of product in every cycle, the slope will be -3.3 (Medhurst et al. 2000 ).

Quantitative Dot-blot Analysis
Total RNA was extracted from the LD of a sow, and from the psoas, supraspinatous, and LD of a 6-week-old Large White pig (Chomcznski and Sacchi 1987 ). Total muscle RNA from each muscle was spotted onto a nylon membrane (Hybond-N+; Amersham, Arlington Heights, IL) with a dot-blot apparatus as a dilution series (10.0, 5.0, 2.5, 1.25, and 0.16 µg), after which it was hybridized with 32P-labeled total cDNA, made by reverse transcription with oligo-d(T) primers derived from the LD of a sow. The filter was scanned by phosphor imaging (Molecular Image System Fx; Biorad, Richmond, CA), stripped, re-probed with 32P-labeled skeletal {alpha}-actin, and re-scanned. The 300-bp {alpha}-actin probe, derived from the 3'-end of its cDNA, shows high sequence homology with ß-actin and will therefore hybridize to both actin isoforms. In this way, the amount of actin mRNA relative to total mRNA in each muscle can be calculated.

In Vivo Expression
Specific 35S-labeled cRNA probes for porcine slow/I/ß, 2a, 2x, and 2b MyHCs, and porcine skeletal {alpha}-actin, were generated for ISH as previously described (Chang et al. 1995 ). A rabbit fast MyHC monoclonal (M-32) (Sigma; St Louis, MO) and a human slow MyHC monoclonal antibody (NOQ7.5.4D) (Sigma) were used in IHC on serial muscle sections. M32 shows specificity for all three porcine fast MyHC fibers, but not slow fibers, and NOQ7.5.4D shows specificity only for slow fibers (unpublished data). A biotinylated rabbit anti-mouse antibody (E 0354; Dako, Carpinteria, CA) was used together with the avidin–biotin peroxidase complex (ABC) immunocytochemical procedure for the localization of primary antibody binding, following the manufacturer's instructions (K0377; Dako). Diaminobenzidine tetrahydrochloride (DAB kit D493; Sigma) was used as a chromogen to localize peroxidase on bound secondary antibodies.

Relative Fiber Type-restricted Expression of MyHC Isoforms
Image analysis on serial muscle sections, subjected to ISH and IHC was performed using the KS 300 V.3 software package (Image Associates/Zeiss; Oberkochen, Germany), to determine the relative cross-sectional fiber area of each of the four fiber types in a given muscle. MyHC slow fibers, based on IHC or ISH, and MyHC 2a fibers, based on ISH, were individually identified and measured for cross-sectional area. Owing to their greater abundance, MyHC 2x- and 2b-positive fibers, as determined by ISH, were measured collectively in each field as total cross-sectional area by using an in house-written computer macro for KS 300. Between 400 and 800 fibers were measured in each muscle sample. Relative fiber type-restricted expression of each isoform, defined as the relative MyHC mRNA level per unit cross-sectional fiber area, was calculated by dividing the relative amount of normalized (to ß-actin) MyHC mRNA with the corresponding relative cross-sectional area of expression.

Protein Extraction and Western Analysis
Whole muscle extracts were prepared from frozen samples by homogenization in extraction buffer (1 M HEPES, pH 7.8, 5 M NaCl, 0.5 M NaF, 0.5 M EDTA, and 25% glycerol) with a cocktail of protease inhibitors (0.1 M PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 M DTT) (White et al. 1995 ). Whole muscle extracts were subjected to 7.8% SDS-PAGE gels under denaturing conditions and blotted onto Hybond-P membrane (Amersham). Membranes were blocked in BLOTTO (4% milk, 0.1% Tween-20 in TBS, pH 8.0) for 30 min and incubated overnight with primary antibody at 4C (Laemmli 1970 ). Immunoblotting was performed with the rabbit fast MyHC monoclonal (M-32; Sigma), the human slow MyHC monoclonal antibody (NOQ7.5.4D; Sigma), and a mouse ß-actin monoclonal antibody (AC-15; Sigma). Bound antibodies were developed using the ECL Plus kit and exposed to Hyperfilm ECL-Plus film (Amersham). Autoradiographs were analyzed using the Phoretics 1D Plus (Non Linear Dynamics; Newcastle upon Tyne, United Kingdom) software package by measuring the integrated intensities of each band (volume) and normalized against the corresponding ß-actin signal to give a semi-quantitative assessment of each sample.


  Results
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Materials and Methods
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Quantifying Muscle Gene Expression by TaqMan Quantitative Real-time RT-PCR
The relative standard curve method was used to quantify the relative gene expression of MyHC slow, 2a, 2x, 2b, GAPDH, and of skeletal {alpha}-actin and ß-actin (Johnson et al. 2000 ) (Fig 1). No amplification of porcine genomic DNA was ever detected with any of the primer and TaqMan probe set (data not shown). The two-base difference between the TaqMan probes of {alpha}- and ß-actin (Table 1) was sufficient to confer specificity, in that no skeletal {alpha}-actin amplification product was detected in non-skeletal muscle cDNAs, such as kidney and liver (data not shown). The standard curve gradients of all four MyHC genes were sufficiently similar (mean value 3.65 ± 0.13) to suggest comparable PCR amplification efficiency, which would facilitate quantitative comparisons among isoforms (Fig 1). To correct for variations in the quality and quantity of cDNA templates, all MyHC isoform expression results presented were normalized to corresponding endogenous ß-actin levels (Table 2). GAPDH has been recently shown to be unsuitable for normalization in skeletal muscle (Lowe et al. 2000 ; and our data not shown). One notable finding was that normalized mRNA expression level of each MyHC gene isoform showed wide variation among muscles within an animal and among the same muscles of different individuals (Table 2). For example, MyHC slow/I/ß level in the LD of Berkshire (pig 3) was over 80-fold greater than in the LD of Duroc (pig 4).



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Figure 1. Relative standard curves of seven porcine genes: MyHC slow/I, MyHC 2a, MyHC 2x, MyHC 2b, ß-actin, {alpha}-actin, and GAPDH. Note that their gradients suggest highly similar amplification efficiencies.


 
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Table 2. Results of TaqMan quantitative real-time RT-PCR, relative fiber type area, and relative fiber type-specific expression of a 6-week-old Large White, three 22-week-old Berkshires, and three 22-week-old Durocsa

In a separate TaqMan experiment on porcine fetal muscles to quantify the relative mRNA expression of the embryonic, perinatal, and slow MyHC isoforms, similarly wide variations in temporal expression between the 47-day and 104-day fetal stages were found for each isoform (Fig 2). However, skeletal muscle is a heterogeneous tissue, with widely different fiber type composition among functional muscles. A more informative way of quantifying the expression level of each MyHC isoform in muscle is to take into account the proportion of the corresponding fiber type, calculated as normalized relative expression per unit relative cross-sectional area, termed relative fiber type-restricted expression (see below). This approach is particularly suited to postnatal muscles, which have undergone phenotypic maturation.



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Figure 2. TaqMan real-time RT-PCR to quantify MyHC embryonic, perinatal, and slow/I mRNA isoforms in two porcine fetal back muscles. (A) Relative standard curves show comparable amplification efficiencies of all three genes. (B,C) Like the postnatal muscles (Table 2), each isoform normalized to ß-actin showed considerable temporal variation.

Total mRNA Levels Among Fiber Types Within a Muscle Are Similar
A recent report suggests that total RNA could vary widely among fiber types within rat muscles (Habets et al. 1999 ). This finding could complicate the normalization procedure necessary for quantitative real-time PCR and the subsequent determination of relative fiber type-specific expression. To ascertain differences in total mRNA levels among fiber types, ISH using a 35S-labeled porcine skeletal {alpha}-actin cRNA probe, which hybridized to both {alpha}- and ß-actin mRNA in each fiber, was performed on a range of porcine skeletal muscles (Fig 3). All results showed evenly diffuse expression throughout each muscle, indicating that there was little variation in total actin levels among fiber types. In addition, the ratio of total actin mRNA to total mRNA of several muscles, determined by dot-blot analysis, showed little variation between phenotypically and developmentally distinct muscles (Fig 4). Taken together, unlike the rat muscle report (Habets et al. 1999 ), our results suggest that there is little variation in total mRNA among fiber types within a porcine muscle.



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Figure 3. Typical ISH shows evenly diffuse expression of actin throughout the psoas muscle of a 22-week-old Duroc (A) and semitendinosus of a 100-day pig fetus (B), indicating little variation in total actin levels between fiber types.



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Figure 4. Ratio of total actin to total mRNA, determined by dot-blot hybridization, showed little change among muscles. Together with actin ISH results, it suggests that total mRNA varies little among fiber types of different porcine muscles. Note that the specific radioactivity of individual cDNA species in the labeled total cDNA probe was considerably lower than that in the {alpha}-actin probe, which would account for a weaker hybridization signal from total cDNA probe. The gradual rise in the total actin to total mRNA ratio for each dilution series of total RNA indicates that the phosphor imaging scale was not strictly linear. LD, longissimus dorsi.

In Vivo MyHC Expression to Determine the Relative Cross-sectional Area of each Fiber Type
The four major MyHC fiber types in postnatal porcine skeletal muscles were identified, on the same muscle samples used for TaqMan quantitative PCR, by the combined use of ISH with MyHC isoform-specific probes and IHC on serial muscle sections. Slow/I and fast 2a fibers were readily resolved (Fig 5 Fig 6 Fig 7). However, the abundance of 2x and 2b fibers, as identified by 35S-labeled ISH, made them difficult to localize as individual fibers. Fiber type composition was therefore expressed as relative cross-sectional area (Table 2; Fig 8). All data were analyzed using a mixed model ANOVA on untransformed data for significant differences both between muscles (LD and psoas) and breed (Berkshire and Duroc). In brief, within a breed, fiber type composition between the LD and psoas was significantly different (p<0.05), consistent with their functional differences. Further analysis on a larger set of animals (nine Berkshires and nine Durocs) gave similar results (data not shown). Based on the combined cross-sectional area of the four fiber types, co-expression of more than one MyHC isoform in a fiber appeared more widespread in muscles of the 6-week-old Large White than in the older Berkshire and Duroc pigs (Fig 8). However, contrary to expectation, there was no significant difference (ANOVA) in mean cross-sectional fiber size among fiber types within each muscle (data not shown).



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Figure 5. Serial sections of psoas muscle from a 6-week-old pig, probed by ISH for MyHc 2a (A), MyHC 2x (B), MyHC 2b (C), and immunostained for MyHC slow/I/ß (D).

Figure 6. Serial sections of psoas muscle from a 22-week-old Berkshire pig, probed by ISH for MyHc 2a (A), MyHC 2x (B), MyHC 2b (C), and immunostained for MyHC slow/I/ß (D).



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Figure 7. Serial sections of longissimus dorsi muscle from a 22-week-old Berkshire pig, probed by ISH for MyHc 2a (A), MyHC 2x (B), MyHC 2b (C), and immunostained for MyHC slow/I/ß (D).



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Figure 8. Relative cross-sectional areas of MyHC slow/I, 2a, 2x, and 2b fibers in psoas and longissimus dorsi (LD) muscles of individual pigs, determined by ISH and immunostaining. The greater total cross-sectional areas of the supraspinatous (Supra), psoas, and LD muscles in the 6-week-old pig than in the older pigs suggest greater MyHC co-expression in the younger animal.

Relative Fiber Type-restricted Expression of MyHC Isoforms
Relative fiber type-restricted expression of an MyHC gene is defined as normalized relative mRNA expression per unit relative cross-sectional area of the corresponding fiber isoform. It is independent of gross muscle size and individual fiber length and is mathematically equivalent to normalized relative mRNA expression per unit volume of the corresponding fiber type (Lieber and Friden 2000 ). Importantly, because the efficiency of PCR amplification for each of the four postnatal MyHC genes was similar (Fig 1), comparisons of relative fiber type-restricted expression could be made among the four gene isoforms within an animal or among animals.

Within-animal Comparisons: MyHC 2a Gene Showed the Highest Relative Fiber Type-restricted Expression
An elevated relative fiber type-restricted expression level implies a raised level of steady-state MyHC mRNA in the corresponding fiber type. Relative fiber type-restricted expression of each MyHC isoform showed wide temporal variation in the same muscle as well as spatial variation among muscles of the same animal, suggesting that the same MyHC gene was differentially regulated among muscles in the same animal (Fig 9).



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Figure 9. Relative fiber type-restricted expression of the four major postnatal MyHC isoforms in (A) a 6-week-old Large White, (B) three 22-week-old Berkshires, and (C) in three 22-week-old Durocs. MyHC 2a had the highest level of expression within each muscle, with the exception of the LD in Duroc pig 4. Expression level for each MyHC isoform in the Berkshires ranged from several-fold to at least an order of magnitude greater than in the Durocs. Standard deviations shown. Muscle abbreviations as in Fig 8.

Although MyHC 2a fibers are a minor fiber type in porcine skeletal muscles (Fig 8), their relative fiber type-restricted expression, with the exception of one muscle (LD of Duroc pig 4), was consistently the highest among the four isoforms within each muscle (Fig 9). By contrast, MyHC 2x fibers, one of the most common fiber types, had the lowest relative fiber type-restricted expression. Data were analyzed using a mixed model ANOVA using the mixed program in the SAS statistical package. The fixed effect of muscle fiber type was highly significant (p<0.001). Relative fiber type-restricted expression of each MyHC isoform showed a highly statistically significant difference (p<0.001) among muscles within the same animal (Fig 9).

Among-animal Comparisons: Possible Breed Differences in Relative Fiber Type-restricted Expression
The relative fiber type-restricted expression of each MyHC isoform in the 6-week-old Large White was higher than in the 22-week-old Duroc pigs, with the exception of one muscle (LD of Duroc pig 6) (Fig 9). Surprisingly, the relative fiber type-restricted expression of each MyHC isoform in the Berkshires ranged from several-fold to at least an order of magnitude higher than in the Durocs or 6-week-old Large Whites (Fig 9). Such breed differences were also reflected in normalized {alpha}-actin mRNA levels (Fig 10). This difference could be a genetic effect. Indeed, this fixed effect of breed was significantly different (p<0.05), as was the effect of animal nested within breed (p<0.05). All data were skewed and log-transformed before analysis.



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Figure 10. Total {alpha}-actin mRNA, normalized to endogenous ß-actin, in the longissimus dorsi (LD) and psoas muscles of the Duroc and Berkshire pigs. The levels of total {alpha}-actin in the Berkshires were at least several-fold higher than in the Durocs. Standard deviations shown.

One obvious question arising from the apparent difference in MyHC mRNA expression between the two breeds of pig (Fig 9) was whether a similar difference was present at the protein level. Immunohistochemistry was performed on identically prepared serial muscle sections of both breeds to detect fast MyHCs (2a, 2x, and 2b) and slow MyHC. All Berkshire muscles examined showed generally higher signal intensities than their Duroc counterparts (Fig 11A). Western analysis also showed generally higher levels of fast and slow MyHC proteins, normalized to the corresponding endogenous ß-actin, in the Berkshires than in the Durocs, but not apparently to the same degree as the corresponding mRNA differences (Fig 11B).



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Figure 11. Apparent breed difference in MyHC proteins expression. (A) Typical set of IHC results on the presence of fast MyHCs (2a, 2x, and 2b) and slow MyHC in serial longissimus dorsi (LD) and psoas muscle sections of a Berkshire and a Duroc pig. All muscle sections were 10 µm thick and immunostained at the same time with each antibody as a batch to ensure uniformity of treatment between slides. Visually, both fast and slow MyHC proteins were consistently higher in the Berkshire than in the Duroc. (B) Typical set of Western results on the relative amounts of fast MyHCs (2a, 2x, and 2b) and slow MyHC in the LD and psoas muscles of two Berkshire and two Duroc pigs. Each lane of the fast MyHC immunoblot was loaded with 2 µg of total proteins. The blot was subsequently stripped and immunostained for slow MyHC. Each lane of the ß-actin immunoblot was loaded with 10 µg of total proteins. Both fast and slow MyHC proteins were normalized to the corresponding endogenous ß-actin protein levels. Because the exposure times of the fast and slow MyHC blots were not the same, quantitative comparisons could be made only between individual samples in the same row. MyHC expression was generally higher in the Berkshires than in the Durocs. Western analysis was performed in triplicate.


  Discussion
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Previous quantitative PCR techniques used in the study of skeletal muscle gene expression tend to suffer from a number of drawbacks (Jankala et al. 1997 ; Wright et al. 1997 ; Tanabe et al. 1999 ; Harjola et al. 2000 ), such as variation in PCR amplification efficiency among primer sets, inability to measure accumulation of PCR products accurately in real time, lack of an internal control to normalize template quality and quantity, and handling loss. With the advent of quantitative real-time RT-PCR, such as the TaqMan PCR assay used here, such deficiencies have been largely overcome, making it the most accurate and sensitive method available for mRNA quantification. In this study we have enhanced the quantification of individual MyHCs by combining the results of fiber type composition and quantitative real-time RT-PCR, termed relative fiber type-restricted expression. Contrary to expectations, we found that each MyHC isoform showed wide variation of mRNA expression among muscles, even within the same animal. This differential expression is likely to reflect differences in the physiological state of individual muscles, and indicates that the regulation of muscle genes is subjected to major local modulations. Surprisingly, MyHC 2a was consistently the most highly expressed fiber type-restricted isoform, regardless of muscle, age, and breed. An elevated level of an MyHC mRNA isoform within a fiber could be the result of either raised transcription or decreased mRNA turnover. At present it is unclear which of the two mechanisms is at work leading to differential levels of MyHC mRNAs. We have not shown whether elevated MyHC 2a mRNA level was translated into structural protein, but our data appear to indicate that raised total fast MyHC or MyHC slow/I mRNAs correlated with slightly increased MyHC protein levels (Fig 11). Previous work has shown that the porcine MyHC 2a gene possesses a secondary promoter, located in the second intron of the gene, which suggests that this member of the MyHC family could be under more complex regulation than its counterparts (Chang 2000 ). In this context, even though MyHC 2a fibers contribute only to a relatively small percentage of the total fibers (Chang et al. 1995 ), they could be a major determinant of overall muscle function and fiber type switching under modulating conditions.

We further found possible breed differences at both the mRNA and protein MyHC levels between the Berkshire and Duroc pigs. However, this difference may have no bearing on total protein content within muscle fibers of the two breeds. It should be pointed out that although Durocs and Berkshires are ostensibly two distinct breeds, there is in fact enormous genetic variation within each breed, which could account for the variation in relative fiber-type restricted expression among individuals of the same breed. Clearly, more work is needed to better characterize this possible breed difference in muscle gene expression. Finally, the novel approach described here could be readily extended to quantify the mRNA expression of a whole host of fiber type-specific and non-fiber type-specific muscle genes in a range of research areas, e.g., on muscles undergoing aging, regeneration, and gene therapy (Balagopal et al. 1997 ; Frontera et al. 2000 ).


  Acknowledgments

Supported by the Biotechnology and Biological Sciences Research Council, the Pig Improvement Company, and the Rare Breeds Survival Trust.

Received for publication May 30, 2001; accepted October 3, 2001.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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