Genetic determinants of weight of fast- and slow-twitch skeletal muscle in 500-day-old mice of the C57BL/6J and DBA/2J lineage
A. Lionikas1,2,
D. A. Blizard1,
G. S. Gerhard4,
D. J. Vandenbergh1,3,
J. T. Stout1,
G. P. Vogler1,3,
G. E. McClearn1,3 and
L. Larsson1,2
1 Center for Developmental and Health Genetics
3 Department of Biobehavioral Health, The Pennsylvania State University, University Park
4 Geisinger Medical Center, Weis Center for Research, Danville, Pennsylvania
2 Department of Clinical Neurophysiology, Uppsala University, Uppsala, Sweden
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ABSTRACT
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C57BL/6J (B6) and DBA/2J (D2) strains and two derivative populations, BXD recombinant inbred strains (BXD RIs) and B6D2F2, were used to explore genetic basis for variation in muscle weight at 500 days of age. In parallel with findings in 200-day-old mice (Lionikas A, Blizard DA, Vandenbergh DJ, Glover MG, Stout JT, Vogler GP, McClearn GE, and Larsson L. Physiol Genomics 16: 141152, 2003), weight of slow-twitch soleus, mixed gastrocnemius, and fast-twitch tibialis anterior (TA) and extensor digitorum longus (EDL) muscles was 1322% greater (P < 0.001) in B6 than in D2. Distribution of BXD RI strain means indicated that genetic influence on muscle weight (strain effect P < 0.001, all muscles) was of polygenic origin, and effect of genetic factors differed between males and females (strain-by-sex interaction: P < 0.01 for soleus, EDL; P < 0.05 for TA, gastrocnemius). Linkage analyses in B6D2F2 population identified QTL affecting muscle weight on Chr 1, 2, 6, and 9. Pleiotropic influences were observed for QTL on Chr 1 (soleus, TA), 2 (TA, EDL, gastrocnemius), and 9 (soleus, TA, EDL) and were not related to muscle type (fast/slow-twitch) or function (flexor/extensor). Effect of QTL on Chr 9 on soleus muscle was male specific. QTL on Chr 2 and 6 were previously observed at 200 days of age, whereas QTL on Chr 1 and 9 are novel muscle weight QTL. In summary, muscle weight in B6/D2 lineage is affected by a polygenic system that has variable influences at different ages, between males and females, and across muscles in a manner independent of muscle type.
muscle size; quantitative trait loci
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INTRODUCTION
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ASIDE FROM ITS primary power-generating function, skeletal muscle serves as a source of energy and amino acids during critical illness and starvation, contributes to thermoregulation, and also protects bones and viscera. Muscle size is of major importance for all of these functions. Skeletal muscle is characterized by an extraordinary capacity to adapt to different external influences. However, there is also a substantial genetic component determining its size. It has been estimated that in humans, heritability of muscle size ranged between 0.53 and 0.9 (9, 13, 17). The quest for specific genes causing alterations in muscle mass has identified several candidates. Myostatin has been recognized as a powerful suppressor of muscle mass growth in cattle and mouse (22, 23, 52) and recently in humans (41). Insulin-like growth factors-1 and -2 (IGF-1 and IGF-2) are also well-known modulators of muscle weight in rodents (1, 10, 34). Recent findings indicate that mutations of the intronic region of the IGF-2 gene induced a substantial increase in pig muscle size via imprinting mechanisms (25, 49). A more complex influence on muscle weight, involving a single nucleotide polymorphism (SNP) in a long-range control element and interactions of reciprocally imprinted genes, was observed in sheep muscles (21, 42). However, studies in agricultural animals, especially Sus scrofa (the domestic pig), and rodents indicate that genetic factors influencing muscle size are likely to include multiple genes (6, 29, 32, 36, 53). In addition, muscle-specific quantitative trait loci (QTL) in pig (32, 36, 53) and mouse (29) as well as findings in sheep (14, 15, 21) revealed that the genetic architecture is not uniform for different muscles.
The present study is a continuation of a comprehensive analysis of age-related changes in a large number of behavioral, physiological, morphological, and pathological phenotypes in C57BL/6J (B6) x DBA/2J (D2) lineages. A QTL analysis of muscle phenotypes at 200 days of age demonstrated the existence of a polygenic system with muscle- and sex-specific influence on variation in muscle weight (29). The aim of the present analysis was to explore genetic architecture of muscle weight at 500 days and compare it with the data from younger animals. The weights of slow-twitch soleus, fast-twitch tibialis anterior (TA) and extensor digitorum longus (EDL), and mixed gastrocnemius muscles in B6 and D2 strains, BXD recombinant inbred (BXD RI) strains, and a B6D2F2 intercross were measured at 500 days of age. These muscles perform plantarflexions (soleus and gastrocnemius) or dorsiflexions (TA and EDL) in the ankle joint. The results support previous findings of polygenic and muscle- but not muscle type-specific genetic effects and reveal that the genetic architecture of muscle weight varies with age.
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MATERIALS AND METHODS
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Animals.
This study was a part of a broad project investigating genetic architecture of various traits in two populations derived from C57BL/6J (B6) and DBA/2J (D2) mice: a B6D2F2 intercross and 22 BXD RI strains, at the age of 450500 days. All animals were bred in the barrier colony developed at the Center for Developmental and Health Genetics, The Pennsylvania State University (29). The B6D2F2 intercross was generated in four birth cohorts of
112120 mice at 1-mo intervals. Each birth cohort of B6D2F2 mice consisted of an equal number of four possible mating types of F1 males and females, i.e., B6D2XB6D2, B6D2XD2B6, D2B6XB6D2, and D2B6XD2B6 [D2B6 is the F1 offspring of D2 female (female is the first-named member of a mating pair) and B6 male, B6D2 is the offspring of B6 female and D2 male, and so on]. There were 464 B6D2F2 mice weaned for the study from both primiparous and multiparous females. A total of 187 females and 190 males of these survived until dissection. Mice of the 22 BXD RI strains (BXD-2, -6, -8, -11, -12, -14, -16, -19, -22, -24, -27, -28, -29, -30, -31, -32, -33, -34, -38, -39, -40, -42) were weaned from both primiparous and multiparous females. The BXD RI study design called for 12 males and 12 females within each strain. Death before euthanasia reduced the number of animals to 810 in some strains. A total of 205 females and 191 males from BXD RIs survived until dissection. Twenty-three B6 and seventeen D2 mice were also euthanized. All animals were housed at room temperature (7072°F) on a 12:12-h light-dark cycle and fed ad libitum with sterilizable Purina 5010 (designed to be equivalent to Purina 5001 after sterilization). At the age of
500 days, B6 (518 ± 2 days, range 516521 days), D2 (518 ± 2, range 515520 days), B6D2F2 (511 ± 9 days, range 490527 days), and BXD (515 ± 4 days, range 494521 days) mice were killed by cervical dislocation between 8 AM and 12 noon, i.e., in the first one-half of the light cycle. Procedures were approved by the The Pennsylvania State University Animal Care and Use Committee.
Phenotypic measures.
Animals were weighed on an electronic balance (Ohaus Scout) to 0.1 g accuracy before death, and nose-to-anus distance (with an accuracy of ±1 mm) was recorded immediately after death. The gastrocnemius, soleus, TA, and EDL muscles were gently dissected from the right hindlimb and weighed on a Mettler AE 50 balance to 0.1 mg accuracy. The muscles were frozen and stored for further analyses.
QTL analyses.
The B6D2F2 mice (n = 396) were genotyped at 97 microsatellite markers spaced at
15- to 20-cM intervals throughout the genome. The previously described set of markers (50) was employed, with the exception that D12Mit153 was added to chromosome (Chr) 12 (15.0 cM). DNA for genotyping was extracted from tail tips obtained at weaning. There were 763 microsatellite markers selected for the 22 BXD RIs (54).
Interval mapping (IM) analysis in the B6D2F2 intercross was carried out using the R/qtl package (8). This program package permits single-QTL genome scans and the search for epistatic interactions between two loci in the presence of covariates. In a search for confounding variables, using General Linear Model (SPSS 11.0 statistical software package), sex was found to account for 27.3, 59.4, 36.9, and 52.9% of phenotypic variance of soleus, TA, EDL, and gastrocnemius, respectively. In addition, the effect of litter size accounted for 4.9, 3.1, 2.7, and 2.6%, respectively. These two variables were included in the IM model as additive covariates. To assist identification of the sex-specific effects of QTL, sex was also included as the interacting covariate (43). Linkages to the X Chr were analyzed separately from autosomes. The IM procedure has been carried out in males with litter size included as a covariate. To account for the random inactivation of X Chr in females, it was divided into four intervals, each flanked by two adjacent markers, and only females homozygous for the same allele in flanking markers were selected for the one-way ANOVA with an interval as a factor (29). Interval mapping in the BXD RI strains was carried out using the QTL Cartographer package (2).
Suggestive, significant, and highly significant thresholds of statistical significance (values corresponding to 37th, 95th and 99.9th percentiles, respectively) were determined for each muscle from 10,000 permutations (12). Permutations were carried out, including the same covariates into the model as for the IM, and resulted in the estimates of thresholds of 3.1 LOD (suggestive), 4.9 LOD (significant), and 6.8 LOD (highly significant). Significant epistatic interaction was determined by requiring a joint LOD >11 and a significance level of the interaction component itself of P < 0.001 (3).
To understand how QTL and interactions contribute simultaneously to the traits, all identified QTL (suggestive or better), epistatic interactions and sex (as interacting covariate) and sex and litter size (as additive covariates), were included in a multiple-regression analysis of individual muscle. Individual terms were dropped by backward elimination until all terms of the model were significant at P < 0.05.
Statistical analysis.
There were 370 and 394 solei, 372 and 393 TA, 363 and 391 EDL, and 361 and 392 gastrocnemius muscles available for analyses of B6D2F2 and BXD RI populations, respectively.
The weight of soleus, TA and EDL, and gastrocnemius muscles approximated normality in the B6D2F2 population. Within sex, strain means were calculated for each muscle in BXD RIs. The distribution of the strain means did not deviate from normality.
Heritability of muscle weight in the BXD RIs (hRI2) was estimated from a one-way ANOVA by strain as SSbetween strains/SStotal (19). Heritability was predicted for the B6D2F2 population (hF22) as proposed by Belknap (4): hF22 = 1/2hRI2/(1 1/2hRI2).
The statistical significance of effects is presented by P value at three different levels: <0.05, 0.01, or 0.001.
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RESULTS
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Analyses of muscle weight in parental and BXD RI strains, and B6D2F2 intercross.
In parental strains, muscle weight was 1322% greater (P < 0.001) in the B6 than the D2 strain (Table 1). There was a significant effect of strain (P < 0.001) and sex (P < 0.001) on weight of soleus, TA, EDL, and gastrocnemius muscles among the BXD RIs (for details, see Supplemental Table S1; available at the Physiological Genomics web site).1
Strain-by-sex interactions in the BXD RIs were statistically significant for soleus, EDL (P < 0.01) and TA, and gastrocnemius (P < 0.05) muscles. Genetic correlations between muscles, calculated from the BXD strain means (5), were positive and statistically significant (P < 0.01) among all muscles in females and males. The relationship of muscle weight to body weight and body length was weaker, and a statistically significant (P < 0.05) correlation was only found between body weight and the gastrocnemius muscle in males (Table 2), indicating that at 500 days of age, genetic mechanisms that affect muscle weight and body size (weight and length) only partially overlap. The distribution of BXD RI strain means suggests a polygenic effect rather than a major gene affecting muscle weight (Fig. 1). The estimates of heritability derived for the B6D2F2 population (hF22; for details see MATERIALS AND METHODS) were 0.37 and 0.43 for soleus, 0.43 and 0.41 for TA, 0.40 and 0.49 for EDL, and 0.38 and 0.39 for gastrocnemius in females and males, respectively. This indicates that the narrow sense heritability of muscle weight is similar for slow-twitch (soleus), fast-twitch (TA, EDL), and mixed muscles (gastrocnemius) and between males and females.
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Table 1. Weight of 4 muscles, body weight, and length in C57BL/6J and DBA2/J strains and B6D2F2 intercross at 500 days of age
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Table 2. Genetic correlation between body weight, body length, and muscle weight in females (above diagonal) and males (below diagonal) of 22 BXD RI strains at 500 days of age
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Fig. 1. Distribution of strain means of weight of different muscles in the 500-day-old BXD recombinant inbred strains (BXD RIs) and progenitor strains C57BL/6J (B2) and DBA/2J (D2) sorted in order of increasing weight in males. TA, tibialis anterior; EDL, extensor digitorum longus; , females; , males. Vertical arrows indicate the parental strains. Error bars represent SD.
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QTL analyses of muscle weight in B6D2F2 intercross.
Linkage analysis was employed to locate the genetic factors responsible for variation in muscle weight. The suggestive, significant, and highly significant QTL influencing different muscles were identified on Chr 1, 2, 6, and 9. Soleus muscle was affected by the QTL on Chr 1 and 9; TA by the QTL on Chr 1, 2, and 9; EDL by the QTL on Chr 2 and 9; and gastrocnemius by the QTL on Chr 2 and 6 (Fig. 2). On Chr 6 and 9, the B6 allele was associated with increased muscle weight (B6, dominant), whereas the allelic relationship with muscle weight for Chr 1 QTL was reversed (D2, dominant). The B6 allele was also associated with increased muscle weight for the Chr 2 QTL, but the mode of action varied by muscle (TA, dominant; gastrocnemius, additive; EDL, approximated an additive model; Fig. 3).

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Fig. 2. LOD plots of interval mapping in the 500-day-old B6D2F2 intercross. Black line, sex, litter size as the covariates; gray line, sex, litter size and body length as the covariates. Horizontal dotted lines represent threshold of significance determined by 10,000 permutations. The suggestive, significant, and highly significant thresholds were 3.2, 4.8, and 6.5 LOD, respectively, for soleus; 3.1, 4.8, and 6.8 LOD, respectively, for TA; 3.2, 4.8, and 6.8 LOD, respectively, for EDL; and 3.2, 4.9, and 7.1 LOD, respectively, for gastrocnemius (Gastroc).
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Fig. 3. Allelic effects of the markers closest to the quantitative train loci (QTL) peak in the B6D2F2 intercross. Values are means ± SE across sex. B6, homozygous for C57BL/6J; H, heterozygous; D2, homozygous for DBA/2J.
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The search for the epistasis detected several locus-by-locus interactions affecting the weight of the TA, EDL, and gastrocnemius (Table 3).
The QTL and epistatic interactions that influenced individual muscles as well as sex and litter size were included in the multiple-regression models for each trait. None of the identified epistatic two-locus interactions was retained in the multiple-regression model, whereas the effects of the QTL on Chr 1, 2, and 9 were retained in the regression model at the P < 0.01 level of statistical significance, and the QTL on Chr 6 influencing gastrocnemius muscle was significant at the P < 0.05 level. A statistically significant (P < 0.05) sex-by-QTL interaction on Chr 9 was also retained in the model for soleus muscle. Whereas the QTL peaking at the same centimorgan position were affecting one or more muscles on Chr 1, 6, and 9, there were three different peaks that affected one muscle each on the Chr 2 with overlapping 1.5-LOD drop-off support intervals (Table 4). The QTL retained in the regression models were named Skmw611, and their characteristics are summarized in Table 5. Phenotypic variance accounted for by the model varied between 38.4 and 69.1% and was mainly contributed by sex, whereas the effects of individual QTL contributed 14% (Table 4). The X Chr was analyzed separately from autosomes within sex with litter size as a covariate (for details, see MATERIALS AND METHODS). The analysis, however, did not identify any significant linkages with the X Chr in females or males.
To test specificity of the genetic effects on muscle tissue, a separate IM analysis was carried out in the B6D2F2 population with body length included as a covariate (in addition to sex and litter size). The QTL on Chr 1, 2, 6, and 9 remained statistically significant. This observation is consistent with the absence of significant genetic correlations between muscle weight and body length in the BXD RIs (Table 2). The analysis with body length as a covariate also revealed novel suggestive QTL on Chr 7 (23 cM), 10 (65 cM), and 15 (62 cM) that affected TA (Chr 7) and gastrocnemius (Chr 10, 15) muscles (Fig. 2). All the QTL that were identified with body length as a covariate were subsequently subjected to multiple-regression analysis as described in MATERIALS AND METHODS. The effects of the QTL on Chr 1, 2, 6, and 9 were retained in the multiple-regression model, whereas the novel QTL were not.
QTL verification and linkage analysis in BXD RI strains.
The QTL nominations for all phenotypes were submitted to the RI panel for confirmation in the following manner. The 1.5-LOD drop-off intervals were calculated for each QTL nominated in the B6D2F2 population. Then, the markers in the BXD RIs that fell within the 1.5-LOD support intervals were identified. ANOVA on a single marker was conducted for strain means of each nominated muscle, and statistically significant relationships within each interval were corrected for multiple comparisons (i.e., the number of markers within the interval in the BXD RI database) by the method of Bonferroni. In each case, directional prediction of the B6D2F2 data regarding the allele associated with increased muscle weight pointed to the appropriateness of a one-tailed test (D. A. Blizard, personal communication). It has to be noted that this is a conservative protection for multiple comparisons because, due to the linkage of microsatellites, comparisons are not completely independent. None of the effects tested retained a statistical significance after the adjustment for multiple comparisons.
Search for the QTL affecting strain means of muscle weight was done using interval mapping in males and females separately. This analysis identified several suggestive QTL on Chr 1, 2, 14, 17, 18, and 19. Characteristics of the QTL may be viewed in Table 6. More than one muscle was affected by the QTL on Chr 1 (proximal) and 19. The B6 allele was associated with a greater muscle weight for QTL on Chr 2, 17, and 18 and the D2 allele on Chr 1, 14, and 19.
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DISCUSSION
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QTL analyses of mouse quadriceps weight at 6 wk of age (6) and cross-sectional area of forearm muscles at 7 wk of age (33) revealed a polygenic genetic architecture consisting of both additive and epistatic effects. Our own studies of four different hindlimb muscles in 200-day-old mice confirmed the polygenic nature of the genetic architecture but added the important information that QTL may be muscle and/or sex specific (29). Muscle mass changes throughout life in response to a variety of internal and external stimuli and may reflect the influence of different genes at various ages. To explore age-related changes in genetic architecture, we studied muscle weight in B6D2F2 and BXD RI populations at 500 days of age.
A number of autosomal QTL were identified [Chr 1, 2, 3, 4, 5 (two), 6, 7, 8, 9, 11, and 16], each affecting only one muscle but not influencing others in the analysis of 200-day-old B6D2F2 mice (29). In the present study, four suggestive, significant, and highly significant autosomal QTL were found on Chr 1, 2, 6, and 9 (Fig. 2). Although the B6 strain had larger muscles than the D2, the results of linkage analysis in the B6D2F2 intercross indicated that both B6 and D2 alleles were associated with the increased muscle weight in different QTL (Table 5). The QTL found in the present study were not detected in previous analyses of muscle weight in other mouse crosses (6, 33, 51). The total phenotypic variance accounted for by the regression model including QTL and covariates accounted for 38.4, 69.1, 44.8, and 58.2% of phenotypic variance of soleus, TA, EDL, and gastrocnemius muscles, respectively (Table 4). Most of it was attributed to the effect of sex, whereas the influence of the QTL accounted for a fraction of what was predicted by the estimates of heritability. These results are consistent with the hypothesis that many genes and perhaps epistatic interactions of a small effect size are responsible for the residual variation.
In contrast with the 200-day results, where all identified QTL appeared muscle specific, in the present study there was also the evidence of pleiotropy, e.g., the Skmw11 locus exerted a dominant effect on three of four muscles studied, whereas the Skmw6 locus affected two muscles in a dominant manner as well (Fig. 3). Interestingly, pleiotropy was observed across fast- and slow-twitch muscles, providing evidence that the genetic control of muscle mass may be unrelated to the fiber type, even though previous analyses of soleus muscle suggested a different genetic architecture for fast and slow fibers (48). The Chr 2 harbored three loci (Skmw7, Skmw8, Skmw9) influencing different muscles. Although the support intervals of the QTL were overlapping (Table 4), the following features of the loci suggest that the proximal part of the Chr 2 might be harboring several linked genes with an influence on muscle weight: 1) the effect on each of the muscles peaked at the different chromosomal locations (Skmw7, EDL, 20 cM; Skmw8, TA, 45 cM; gastrocnemius, Skmw9, 62 cM), and 2) the mode of action of the QTL differed among the muscles (Fig. 3). Muscle specificity with regard to the effects of a QTL has also been reported in other species. For instance, QTL affected weight of back muscles (loin) but not hindlimb (ham) and vice versa in the domestic pig (32, 36, 53). Similarly, the effect of the callipyge locus was more pronounced in the hindquarters of sheep than in other muscles (21). An illustration of the variable effect of a single gene on different muscles was given by the effect of constitutively active calcineurin, a cell-signaling molecule influencing gene expression in skeletal muscle. Transgenic mice with constitutively active calcineurin exhibited an increase in mass of soleus but a decrease in that of TA muscles compared with wild-type animals (47). Possible mechanisms of muscle-specific influence may involve substantially distinct durations of contractile activity; e.g., in freely moving rats during a 24-h period, the soleus was shown to be contracting for 2235% of time, whereas EDL was active for only 0.045.0% (24). If a hypothetical polymorphic gene was involved in a pathway that is sensitive to the amount of activity, it would likely have a different impact on active vs. less-active muscles. Another plausible explanation of the muscle-specific effects proceeds from the complex control of muscle-specific transcription factors, e.g., expression of Myf5, muscle tissue-specific transcription factor, is controlled by discrete enhancers specific for a particular population of muscle precursors in the embryo (23, 46).
Sex specificity was not addressed in the MRL/MPJ x SJL/J intercross, as only females were studied (33). No sex-specific QTL for quadriceps weight were reported in the DU6i x DBA/2 intercross (6). Sex specificity of QTL, however, was a feature of the genetic architecture in the B6D2F2 intercross. A sex-specific QTL (Chr 3, affecting gastrocnemius weight in females only) was found in B6D2F2 at 200 days of age (29). Statistically significant strain-by-sex interactions for all four muscles in the BXD RIs at 500 days of age were consistent with the hypothesis that some of the genes exert sex-specific effects in the B6/D2 lineage. The hypothesis was further supported by the sex-specific influence of the Skmw11 on soleus muscle in the B6D2F2 population. As illustrated by the within-sex IM, the QTL was predominantly influencing males (Fig. 4). The mechanisms underlying sex-specific effects are unknown but may arise from sex hormone regulation of the polymorphic genes or interactions between mitochondrial or sex chromosome-linked genes. Analyses of the possible influence of the Y chromosome are currently in progress. In support of the possibility of altered hormonal regulation at different ages, the level of circulating estrogen in 500-day-old females is expected to be lower because of lengthening of the estrus cycle at
8 mo (240 days) (38) followed by a cessation of cycling
1215 mo (360450 days) of age (20). In passing, it should be noted that detection of sex-specific QTL is hampered by halving the sample and a consequent reduction in power.

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Fig. 4. LOD plot of male-specific QTL for soleus muscle on chromosome (Chr) 9 of 500-day-old B6D2F2 intercross. Top: black line, females; gray line, males; litter size was included as covariate. Bottom: allelic effect of the marker closest to the QTL peak in females (F) and males (M).
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None of the QTL nominated in the B6D2F2 population was validated in the BXD RIs at the age of 500 days. As previously discussed (29), considerations such as low statistical power and nonsyntenic association (54) may limit the ability to verify QTL. For example, with sufficient power to identify one or a few multiple segregating QTL, successive studies aimed at replication may detect different QTL (7). In addition, the inbred background of the RIs may impact the measured phenotypes in ways different than the heterogeneity present in the F2s. Although age specificity of the genetic architecture is supported by our results (see below) and the observations of others (11, 40), further studies are needed to exclude the possibility that we have identified a nonoverlapping subset of QTL at each age from a larger group of QTL operating at both ages.
Exploring influences of age.
Comparison of the results of analyses at 200 and 500 days of age in the B6/D2 lineage permits evaluation of the age specificity of the genetic architecture. The QTL analyses in the B6D2F2 population at the two ages identified both age-independent and age-specific linkages. The loci Skmw8 and Skmw9 and the support interval of the Skmw7 were within the support interval of the QTL on Chr 2 that affected EDL muscle at the age of 200 days (29). The position of the Skmw10 almost coincided with the peak of the QTL on Chr 6 that influenced gastrocnemius weight at the age of 200 days. The allele (B6) associated with the increase in muscle weight was consistent between the two ages for both QTL. The mode of action, however, was predominantly additive for both QTL (Chr 2 and 6) at 200 days, as well as that of the loci Skmw7 and Skmw9, whereas the loci Skmw8 and Skmw10 favored the recessive (2 increasing alleles were required for the increase in weight) and dominant (1 increasing allele was sufficient for the increase) modes of action, respectively. In contrast, the influence of the loci Skmw6 and Skmw11 was not observed at the age of 200 days. Complex traits like body weight demonstrate age-specific patterns of QTL in the early stages of life in the mouse (11, 40) and are believed to be an outcome of temporal patterns of expression of polymorphic genes in various tissues. The present findings of a difference in genetic architecture determining muscle weight in the B6D2F2 intercross at 200 and 500 days of age are novel with regard to both the tissue and the periods of life span studied. Mechanisms behind the age-specific genetic architecture remain speculative, but the changes in the hormonal status discussed above may be partially responsible.
Candidate genes.
Muscle weight is determined by many factors: the number and size of muscle fibers, the amount of constituent proteins, and retained fluid. Therefore, genes involved in control of number and size of fibers, cell signaling, transcription, translation, and degradation of proteins as well as ion transport may contribute to the difference in muscle weight. Studies in mice and domestic animals indicated that myostatin (22), IGF-1, and IGF-2 (30) are powerful modifiers of muscle mass. However, there are likely many more protein-coding genes that could influence muscle weight. The search for candidate genes contributing to QTL effects (http://www.ensembl.org and http://www.ncbi.nlm.nih.gov) was restricted to those whose function could influence muscle weight, e.g., via expression in muscle tissue and/or systemic effect, and which were within the 1.5-LOD support interval of the QTL identified in the 500-day-old B6D2F2 population. The search resulted in a set of seven candidate genes. Further studies are necessary to determine the possible influence of those genes on muscle weight. Among the candidates on Chr 1, the 2310002A08Rik gene is involved in translation and codes for ribosomal proteins (26, 27), the Usp21 gene codes for an ubiquitin-specific protease-21 (18) that may be involved in myofibrillar protein degradation (16, 37), and the Casq1 gene (39) is involved in regulation of contractility. Candidate genes on Chr 2 and 6, respectively, are Capn3 (large subunit of calpain), coding for proteins involved in protein degradation, and Clcn1 (chloride channel), both genes implicated in human myopathies (28, 44). Two genes on Chr 9 are Mras, a small GTPase (31, 32a, 35) involved in cell signaling, and Mylc, coding for contractility modifying protein.
In conclusion, the present results with 500-day-old mice support and extend our previous observations of 200-day-old mice that the influence of genes varies among muscles; i.e., some of the QTL appeared to be muscle specific, whereas others acted pleiotropically. The genetic architecture in the B6D2F2 intercross appears to be independent of muscle type (fast or slow twitch) and may vary between sexes. Several linkages that were absent at 200 days of age emerged at 500 days of age, i.e., QTL on Chr 1 and 9, whereas others were present at both ages, i.e., QTL on Chr 2 and 6. This pattern indicates that only some of the factors influence muscle weight at both 200 and 500 days of age, whereas others are switched on or off at different ages.
One of the major advantages of the QTL approach is its ability to define the genetic architecture underlying a trait. The fact that >30 muscle QTL have been discovered in mice emphasizes the importance of considering muscle phenotypes in the context of a polygenic system. Adoption of this polygenic concept will be necessary for understanding the diversity of clinical manifestations of hereditary muscular disorders in humans. The difference in severity of the symptoms caused by mutation in a single gene might be modified according to contribution of the other genes that may be polymorphic, even among the closest relatives. Exploration of the mechanisms behind age-, muscle-, and/or sex-specific patterns of the QTL in mice may provide some explanations for differential time of the onset of disease and muscles affected in humans. In addition, it may offer new possibilities for therapies that target the pathways of modifier genes. This bespeaks the need for further studies leading to identification of specific genes involved in genetic architecture of muscle weight.
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GRANTS
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The study was supported by grants from the National Institutes of Health (AG-14731, AG-00276, AR-45627, AR-47318), the European Commission (QLK6-CT-2000-0530), and the Swedish Medical Research Council (8651).
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ACKNOWLEDGMENTS
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We are grateful to Drs. F. Yu and N. Sharkey, C. Vandenberg, A. Lionikiene, M. Teklu, and J. Heckman, and M. G. Glover for excellent help with the tissue harvesting. We also thank Dr. Karl W. Broman for advice with R/qtl analyses and Dr. Shengchu Wang for advice with QTL Cartographer analyses.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: A. Lionikas, Center for Developmental and Health Genetics, Penn State Univ., 101 Amy Gardner House, Univ. Park, PA 16802 (E-mail: aul104{at}psu.edu).
10.1152/physiolgenomics.00209.2004.
1 The Supplemental Material for this article (Supplemental Table S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00209.2004/DC1. 
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