Skeletal muscle development in normal and double-muscled cattle
Article first published online: 5 NOV 2004
Copyright © 2004 Wiley-Liss, Inc.
The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology
Volume 281A, Issue 2, pages 1363–1371, December 2004
How to Cite
Martyn, J. K., Bass, J. J. and Oldham, J. M. (2004), Skeletal muscle development in normal and double-muscled cattle. Anat. Rec., 281A: 1363–1371. doi: 10.1002/ar.a.20140
- Issue published online: 22 NOV 2004
- Article first published online: 5 NOV 2004
- Manuscript Accepted: 22 MAR 2004
- Manuscript Received: 18 SEP 2003
- New Zealand Foundation for Research, Science and Technology
- fiber type;
- fiber size;
- myosin heavy chain;
This study examined the effect of genotype on prenatal muscle development in both normal-muscled (NM) animals and in double-muscled (DM) animals harboring a mutation in the gene for myostatin that results in the production of a functionally inactive protein. The following muscle development parameters were analyzed at four gestational ages: muscle weight, fiber type, by both enzyme histochemistry and myosin heavy-chain (MHC) immunocytochemistry, and average fiber area. The weights of both M. vastus lateralis and M. vastus medialis were greater throughout prenatal development in the DM animals compared to NM. The percentage of type 1 muscle fibers initially declined with gestational age and subsequently increased in both NM and DM. The percentage of type 1 fibers was consistently lower in DM than in NM. A pattern of MHC isoform localization was shown in DM muscle that is indicative of a delay in muscle development relative to NM. Muscle fiber size was differentially regulated in NM and DM, depending on fiber type. Type 1 fibers were smaller in DM than NM in late gestation, while type 2 fibers were smaller throughout gestation. This study suggests that the inactivating myostatin mutation in DM animals may be associated with changes in both skeletal muscle fiber type and fiber size during bovine muscle development. © 2004 Wiley-Liss, Inc.
The major determinants of skeletal muscle mass are muscle fiber number and muscle fiber size. During development, these factors are controlled by a series of events, including myoblast proliferation, myotube formation, and myofiber maturation. A number of regulatory factors can influence each of these stages of muscle development, including the genetic background and breed of an animal.
Within double-muscled (DM) cattle breeds, there is an overall increase in muscle mass (Boccard, 1981). The genetic basis of the DM condition in Belgian Blue animals arises from an 11 bp deletion mutation in the gene for myostatin in these animals, resulting in a severely truncated myostatin protein (Grobet et al., 1997; Kambadur et al., 1997; McPherron and Lee, 1997). Myostatin is a negative regulator of muscle mass (McPherron et al., 1997), which has been shown to inhibit both myoblast proliferation (Thomas et al., 2000; Joulia et al., 2003) and differentiation (Langley et al., 2002; Rios et al., 2003). Each of these effects is mediated through differential regulation of a key component of cell cycle progression, the cyclin-dependent kinase inhibitor, p21. In addition to regulation of cell cycle progression, myostatin also has a direct effect on myogenesis through its action in regulating levels of the myogenic regulatory factors (Langley et al., 2002; Joulia et al., 2003). This dysregulation of myogenic proliferation and differentiation leads to muscle hyperplasia and hypertrophy as seen in myostatin-null animals (Langley et al., 2002).
The extent of the muscular hypertrophy exhibited by DM animals has been reported to vary among different muscles in a number of DM breeds (Butterfield, 1966; Rollins et al., 1969; Boccard, 1981). Muscles with a large surface area tend to be the most enlarged, while deeper muscles tend to be reduced in size relative to NM. As all of these muscles have increased muscle fiber numbers, it follows that the reduced muscles must have a smaller fiber size (Ouhayon and Beaumont, 1968). No studies have been carried out to determine if these differences in muscle mass and fiber size are present in prenatal animals, so we examined the development of the M. vastus lateralis, which is 15% heavier in adult DM than NM, and M. vastus medialis, which is 38% lighter in mature animals (Boccard, 1981).
Double-muscled cattle have a different composition of histochemical fiber types compared with NM, both during prenatal development (Ashmore et al., 1974) and in the postnatal animal (Holmes and Ashmore, 1972), where there are increased proportions of type 2 muscle fibers and fewer type 1 fibers. In addition to histochemical staining, myosin heavy-chain (MHC) immunohistochemistry has been widely used in the investigation of muscle development in DM animals. The sequence of MHC isoform transitions which myotubes undergo as they mature is an important determinant of skeletal muscle fiber type. The relationship between the patterns of MHC isoforms expressed by primary myotubes and the fiber type composition of mature muscles is not yet fully understood (Whalen et al., 1984; Dhoot, 1986; Zhang and McLennan, 1998). Immunohistochemistry using antibodies against developmental MHC isoforms has enabled changes in MHC isoform expression to be examined during normal bovine development (Robelin et al., 1993; Picard et al., 1994, 1995b) and in DM animals (Picard et al., 1995a). This latter study reported developmental differences in MHC expression between NM and DM, with DM tending to express more immature isoforms at the same gestational age during the first two-thirds of fetal life (Picard et al., 1995a).
In normal bovine muscle, fiber size varies according to fiber type. Type 2B fibers have the largest cross-sectional area, type 2A fibers are intermediate, and type 1 fibers are the smallest. In DM cattle, muscle fiber size is altered relative to NM, with both increases and decreases in fiber size being reported. These variations in fiber size are related to differences in fiber type composition and animal age (Holmes and Ashmore, 1972; Ashmore, 1974). In postnatal animals, there is an increase in the size and frequency of type 2B fibers in DM animals (Holmes and Ashmore, 1972) and this contributes to the overall increase in the whiteness of meat from these animals. In muscles from adult DM animals that are decreased in size relative to normal animals, average muscle fiber size is smaller (Ouhayon and Beaumont, 1968).
The aims of this study were to test the hypothesis that the relative difference in size between M. vastus lateralis and M. vastus medialis that occurs in adult NM and DM animals was present during prenatal development, and that differences in fiber type composition and fiber size during prenatal development contribute to overall differences in skeletal muscle mass between NM and DM fetuses.
MATERIALS AND METHODS
Normal-muscled (NM) and double-muscled calves were generated as previously described (Oldham et al., 2001). Fetuses at 120, 160, 210, and 260 days of gestation were collected after slaughter of the recipient cows. The M. vastus lateralis and M. vastus medialis were dissected out of one hind limb from each animal and weighed. A 5 mm slice was taken through the mid belly of the muscle, at right angles to the direction of the muscle fibers. These samples were stored at −80°C for fiber typing and immunohistochemistry. There were five fetuses in each of the age groups for the normal animals and in the 120- and 160-day groups for the DM and three in the 210- and 260-day groups for the DM. These gestational ages were selected to cover the late stages of primary myotube formation, which is nearing completion by 120 days (Stickland, 1978; Robelin et al., 1991), a period of active secondary fiber formation at around 160 days (Stickland, 1978) and a period when all fibers were undergoing hypertrophic growth (210–260 days). This study was carried out with the approval of the Animal Ethics Committee of Ruakura Research Center.
Muscle fiber typing was carried out on the M. vastus lateralis according to a modification of the myosin ATPase method of Guth and Samaha (1969). After fixation, slides were incubated in 0.1 M potassium acetate buffer at pH 5.0 for 10 min. The remainder of the procedure was identical to that of Guth and Samaha (1969). This procedure results in a staining pattern identical to that of fixed sections preincubated at pH 9.4, with type 1 fibers showing lighter staining and type 2 fibers darker, but with improved histology.
Immunohistochemistry was carried out on serial cryostat sections of M. vastus lateralis. Slides were fixed in neutral buffered formaldehyde, blocked in dilute normal serum, and incubated using primary antibodies specific for fast MHC (MY32), slow MHC (NOQ.4.D; Sigma, St. Louis, MO), and embryonic MHC (2B6; gift of Dr. D. A. chman). Detection of the primary antibody was carried out using a biotinylated secondary antibody followed by streptavidin-biotin complex and diaminobenzidine substrate.
Three sections stained for mATPase activity were analyzed from each group for fiber type proportions and average area for each fiber type. All fibers within each of five fascicles were analyzed for each animal, giving a total of 200–300 fibers per muscle. The rationale behind sampling entire fascicles to enable a more accurate assessment of fiber type proportions was based on reports suggesting that the fiber type composition of fascicles recapitulates that of entire muscles (Maier et al., 1992). Quantitative image analysis was carried out using the NIH image system for the Macintosh. Digital images were captured using the ScionCorp CMS-700 image analysis system.
Statistical analysis of muscle weight, fiber type, and fiber size was carried out using analysis of variance with age and breed as the main effects. Data were log-transformed before analysis as required and back-transformed for presentation of results. Values are presented as means; errors are pooled or individual standard errors of the means (SEMs). Covariate analysis was carried out within groups using sex ratio and body weight and established that these factors did not contribute to the breed effects.
Both M. vastus lateralis and M. vastus medialis weights showed a highly significant increase with increasing gestational age (P ≤ 0.001) and both muscles were significantly larger in the DM animals relative to NM (54% for VL, 30% for VM; P ≤ 0.001; Table 1).
|Days||Vastus lateralis||Vastus medialis|
|NM||DM||NM||DM||Sex ratio (M:F)|
|wt (g)||SEM||wt (g)||SEM||wt (g)||SEM||wt (g)||SEM||NM||DM|
Fiber Typing and Morphological Analysis
Muscle fiber type.
Qualitative analysis of sections stained using mATPase histochemistry showed similar fiber morphology between NM and DM at all gestational ages. In both breeds, however, there was a noticeable difference between 120 and 160 days gestation in the morphology of the presumptive primary myotubes, from being large and vacuolated to more closely resembling mature myofibers. The smaller size of the type 1 fibers in the DM muscles was readily seen at 260-day gestation (Fig. 1H).
The percentage of type 1 fibers in both DM and NM showed a biphasic pattern of change throughout development (Fig. 2), with numbers decreasing between 120 and 160 days and then increasing again between 210 and 260 days (P ≤ 0.001). There were consistently fewer type 1 muscle fibers in DM than in NM (P ≤ 0.001; Fig. 2).
This study has shown a similar pattern of MHC expression in both DM and NM at 120-day gestation, with both presumptive primary and presumptive secondary fibers staining positively for embryonic MHC (Fig. 3). All primary fibers were also positive for slow MHC and all secondary fibers were also positive for fast MHC. At 160-day gestation, the pattern was similar, but there were a number of fibers in NM that were negative for embryonic MHC (Fig. 3G). At 210 days, a number of presumptive secondary fibers in the DM were negative for embryonic MHC and others were positive for slow MHC (Fig. 4). Some presumptive secondary fibers in NM and DM were positive for all MHC isoforms (Fig. 4A–F). At 260 days, all fibers were negative for embryonic MHC in NM (Fig. 4G), while immunostaining remained quite strong in smaller presumptive secondary fibers in DM (Fig. 4J).
Muscle fiber size.
The average area of type 1 fibers in both DM and NM decreased from 120 to 160 days of gestation (Fig. 5a), as their morphology changed from that of primary myotubes with a central region devoid of myofibrils to more mature muscle fibers surrounded by developing secondary fibers. By 210 days, type 1 fibers of NM muscles markedly increased in size as they continued to mature, while DM fibers had only very small increases in size, failing to regain the size they had previously been at 120 days. By main-effect analysis, type 1 fibers were significantly smaller overall in DM than in NM (P ≤ 0.001) due to the reduced size at 210 and 260 days (Fig. 5a). The average area of type 2 muscle fibers increased with age (P ≤ 0.001) and was less in DM than in NM (P ≤ 0.05). This effect was consistent across age groups from 160 to 260 days (Fig. 5b).
One of the most sensitive measures of overall fiber type composition is total % area of a specific fiber type, which is the product of average fiber area and average numerical percentage of each fiber type. The total % area of type 1 fibers declined between 120 and 160 days of gestation in both NM and DM, then remained relatively constant throughout the remainder of the period studied. The net effect of the changes in size and proportions of type 1 fibers in the DM animals was that the muscle overall had a significantly lower proportion of total area given over to type 1 fibers at all gestational ages (P ≤ 0.001; Fig. 6).
This study describes the quantitative and qualitative analysis of bovine muscle development in normal animals and in animals harboring a mutation in the gene for myostatin. Skeletal muscle mass was increased in DM fetuses relative to NM in both the M. vastus medialis and the M. vastus lateralis. This contrasts with observations from postnatal DM animals in which the M. vastus medialis is 38% smaller in size relative to NM animals, while the M. vastus lateralis is 15% larger (Boccard, 1981). This suggests that the difference in muscle mass between DM and NM is not due to a direct effect of the myostatin mutation on muscle development. The atrophy may occur as a result of an effect that is not expressed until the postnatal period, or it may also be a consequence of postnatal environmental effects. In humans, injury to or diseases of the knee joint may be associated with atrophy of the M. vastus medialis and hypertrophy of the M. vastus lateralis (Speakman and Weisberg, 1977). Animals exhibiting extreme muscular hypertrophy have some abnormalities in stance and in the anatomy of the front limbs and hocks (Kieffer et al., 1972). This postural abnormality may therefore result in atrophy of the M. vastus medialis in DM animals during postnatal life.
Quantitative analysis of histochemical fiber type showed the percentage of type 1 muscle fibers initially declines with gestational age and then increases again. In developing human muscle, a proportion of primary myotubes degenerate between 16 and 20 weeks of gestation (Fidzianska and Goebel, 1991), a time period that coincides with the decrease in the percentage of type 1 fibers seen in the current study. This would have the net effect of decreasing the percentage of type 1 fibers, as seen in this study. An alternative mechanism for the reduction in the percentage of type 1 fibers may be that a proportion of fibers underwent transformation from type 1 to type 2 fibers (Whalen et al., 1984). This possibility was not directly tested in this study, as individual fibers could not be followed throughout gestation.
During late gestation, the percentage of type 1 fibers increases in both NM and DM. A similar observation to this has been previously made in developing ovine muscle (Maier et al., 1992). Maier et al. (1992) suggested that the majority of the fibers transforming to slow MHC initially expressed an adult fast MHC isoform. This was not, however, supported by other investigators, who suggested that in predominantly fast twitch muscles, all the slow fibers originated from primary generation myotubes (Picard et al., 1994). Although the current study did not investigate temporal changes in MHC isoforms in individual fibers, this remains an area for future investigation in order to determine whether those fibers that express slow MHC isoforms in late gestation are indeed the original population of primary myofibers.
The pattern of change in fiber type proportions described in this study was similar in both NM and DM, although in the DM the percentage of type 1 fibers was consistently lower than in the NM. This result had been previously reported both during prenatal development (Ashmore et al., 1974) and in adult animals (Holmes and Ashmore, 1972). On that basis, it appears that the initial decrease in type 1 fibers and the subsequent increase again are unrelated to the DM condition. The overall fiber type composition is, however, affected by the mutation. Previous studies have generally shown a positive association between myostatin expression and fast MHC isoforms. Myostatin mRNA was undetectable in the slow MHC expressing soleus muscle in mice (Carlson et al., 1999), and in vitro myostatin was associated with myotubes expressing the MHC type II isoform (Artaza et al., 2002). In rats, in which muscle atrophy was induced following dexamethazone treatment, myostatin mRNA was elevated, and there was an increase in type 2 muscle fibers (Ma et al., 2003). The mechanism through which myostatin mediates effects on MHC expression remains unknown.
In the earliest periods of gestation covered by this study, differences in MHC isoform expression between NM and DM were restricted to a population of fibers in NM that were negative for embryonic MHC at 160 days. As this is a developmental isoform, expression of which is lost as fibers mature, this suggests a relatively more advanced stage of development in the NM at this gestational age. Ninety and 130 days of gestation have been identified as the ages during which developmental differences were most marked between NM and DM (Picard et al., 1995a). The current study extended the period of gestation a further 50 days beyond that of Picard et al. (1995a) and has demonstrated that at 260 days of gestation NM no longer expresses embryonic MHC, although it is still relatively abundant in DM. These results suggest that later muscle development in DM fetuses is delayed relative to NM with respect to the expression of MHC isoforms.
An alternative explanation for the prolonged period of expression of immature MHC isoforms is that the period of secondary fiber formation is extended in DM. This is consistent with the observation of an overall increase in muscle fiber number in DM animals. An elevation in satellite cell numbers in muscle fibers from mstn−/− mice also suggests that myostatin deficiency may be a mechanism through which muscle fiber number may be increased in DM animals (McCroskery et al., 2003). A number of in vitro studies have identified a role for myostatin in blocking cell cycle progression and differentiation (Langley et al., 2002; Rios et al., 2003), with inhibition of myostatin synthesis leading to enhanced cell cycle withdrawal and the stimulation of myoblast differentiation (Joulia et al., 2003). One proposed mechanism for the mediation of cell cycle progression may be a heightened response to MyoD in DM animals (Oldham et al., 2001; Spiller et al., 2002). A C313Y mutation in the myostatin gene results in hyperplasia but not hypertrophy in both cattle (Berry et al., 2002) and mice (Nishi et al., 2002), suggesting that different dominant negative mutations in the myostatin gene can have different effects on muscle fiber number and fiber size.
The average size of primary myotubes initially decreased from 120 to 160 days of gestation as they developed into mature myofibers. A similar result has been previously reported in bovine muscle (Stickland, 1978). After the initial decrease in fiber size that occurred in both breeds, type 1 fibers in muscles from NM fetuses began to enlarge, but fibers from DM fetuses did not. This result suggests that during late gestation, some hypertrophic stimulus induces growth in type 1 fibers of NM only, while in DM, either this stimulus is not present or the muscle fibers are unable to respond. The time period during which a difference develops in the average area of type 1 fibers between NM and DM is from 160 to 210 days of gestation.
Type 2 fibers grew at a relatively constant rate throughout the time period studied and were consistently smaller in DM than NM. The smaller fiber size in DM may be explained by the apparent developmental delay in these animals with myofiber hypertrophy lagging behind that of NM, in the same way as expression of more mature MHC isoforms was delayed. In postnatal animals, type 2 fibers have been shown to be larger in myostatin-deficient DM animals than in NM (Holmes and Ashmore, 1972). This is consistent with studies in humans, in which chronic disuse atrophy of the vastus muscles, resulting in a reduction in the area of type 2A and 2B muscle fibers, was associated with an increase in myostatin expression (Reardon et al., 2001). It is well established that fiber type composition between DM and NM changes from prenatal to postnatal life (Holmes and Ashmore, 1972; Ashmore et al., 1974). Fiber size differences between DM and NM also appear to vary with developmental age.
The total area of muscle classified as type 1 or type 2 fibers is a more accurate measure of overall fiber type composition than measurement of either fiber type percentage or average fiber area in isolation (Holmes and Ashmore, 1972; West, 1974). The total area of type 1 muscle fibers in NM and DM declines between 120 and 160 days, largely because of the remodeling of the vacuolated primary myotubes, then remains essentially level. The increase in the percentage of type 1 fibers in the DM at 210 and 260 days of gestation is able to compensate fully for the smaller average fiber size, with no overall decrease in the total area occupied by type 1 fibers. Although myosin ATPase activity in developing muscle is not necessarily a reliable indicator of contraction speed or metabolic activity (Guth and Samaha, 1972), this decline may represent a change in the metabolic pathway being employed by the muscles at this stage of development. Previous researchers proposed that a transformation toward more glycolytic fiber types in DM animals may reflect an inability of the cardiovascular system to supply the excess musculature, resulting in a compensatory shift toward more anaerobic metabolism (Ashmore, 1974).
In summary, in contrast to postnatal animals, the M. vastus medialis in DM animals is not reduced in mass relative to NM during prenatal growth. In M. vastus lateralis, type 1 muscle fibers in both NM and DM exhibited a biphasic change in proportions with gestational age, and proportions of type 1 fibers were consistently lower in DM, suggesting that differences in fiber type composition are associated with the myostatin mutation. There are patterns of MHC isoform localization in DM muscle that are indicative of a delay in development relative to NM. There is an increase in the proportion of type 2 muscle fibers that may contribute to the increase in muscle mass seen in DM animals during postnatal growth. Finally, type 1 fibers are smaller in DM than NM in late gestation only and type 2 fibers are smaller throughout gestation. We provide evidence that in this myostatin-deficient model of muscular hypertrophy, the increase in muscle mass in prenatal animals is not associated with muscle fiber hypertrophy and may therefore be largely accounted for by the increase in muscle fiber number seen in these animals.
The authors thank N. Cox for assistance with statistical analysis.
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