ARTICLE |
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
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Summary |
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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 50:353364, 2002)
Key Words: quantitative real-time PCR, myosin heavy chain, fiber type, temporospatial expression, porcine
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Introduction |
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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 (), each with its own ATPase activity and each encoded by a separate gene (
In addition to temporal regulation (3' nuclease activity of Taq polymerase (
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.
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Materials and Methods |
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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 -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 (
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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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 (
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 (-actin, and re-scanned. The 300-bp
-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 -actin, were generated for ISH as previously described (
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) (
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Results |
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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 -actin and ß-actin (
- and ß-actin (Table 1) was sufficient to confer specificity, in that no skeletal
-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 (
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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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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 (-actin cRNA probe, which hybridized to both
- 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 (
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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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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 (
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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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 -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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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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Discussion |
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Previous quantitative PCR techniques used in the study of skeletal muscle gene expression tend to suffer from a number of drawbacks (
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 (
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Acknowledgments |
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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.
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