Fine-mapping of genes determining extrafusal fiber properties in murine soleus muscle.

Muscle fiber cross-sectional area (CSA) and proportion of different fiber types are important determinants of muscle function and overall metabolism. Genetic variation plays a substantial role in phenotypic variation of these traits; however, the underlying genes remain poorly understood. This study aimed to map quantitative trait loci (QTL) affecting differences in soleus muscle fiber traits between the LG/J and SM/J mouse strains. Fiber number, CSA, and proportion of oxidative type I fibers were assessed in the soleus of 334 genotyped female and male mice of the F34 generation of advanced intercross lines (AIL) derived from the LG/J and SM/J strains. To increase the QTL detection power, these data were combined with 94 soleus samples from the F2 intercross of the same strains. Transcriptome of the soleus muscle of LG/J and SM/J females was analyzed by microarray. Genome-wide association analysis mapped four QTL (genome-wide P < 0.05) affecting the properties of muscle fibers to chromosome 2, 3, 4, and 11. A 1.5-LOD QTL support interval ranged between 2.36 and 4.67 Mb. On the basis of the genomic sequence information and functional and transcriptome data, we identified candidate genes for each of these QTL. The combination of analyses in F2 and F34 AIL populations with transcriptome and genomic sequence data in the parental strains is an effective strategy for refining QTL and nomination of the candidate genes.

Myostatin dysfunction is associated with reduction in overload induced hypertrophy of soleus muscle in mice.

The aim of the study was to investigate if myostatin dysfunction would promote the gain in muscle mass and peak isometric force (P0 ) of soleus muscle (SOL) in response to functional overloading (FO) after ablation of the gastrocnemius muscle. Fifteen male Berlin high (BEH) mice homozygous for the compact mutation causing dysfunction of myostatin and 17 mice with the corresponding wild-type allele (BEH+/+) were subjected to FO of SOL for 28 days at the age of 14 weeks. Compared with BEH+/+ mice, SOL of BEH was heavier (mean ± SD, 13.5 ± 1.5 vs 21.4 ± 1.8 mg, respectively, P < 0.001). After FO, SOL mass increased relatively more in BEH+/+ than BEH strain (34.9 ± 11.5 vs 17.7 ± 11.9%, respectively, P < 0.01). P0 fell (P < 0.01) only in BEH strain, which also showed an increase (P < 0.01) in optimal muscle length. Specific P0 became even more depressed in BEH compared with BEH+/+ strain (8.4 ± 1.4 vs 10.8 ± 1.3 N/g, respectively, P < 0.001). Phosphorylation p70 S6 kinase did not differ between the strains. In summary, myostatin dysfunction impairs adaptation of SOL muscle to high functional demands.

Discovery and refinement of muscle weight QTLs in B6 × D2 advanced intercross mice.

The genes underlying variation in skeletal muscle mass are poorly understood. Although many quantitative trait loci (QTLs) have been mapped in crosses of mouse strains, the limited resolution inherent in these conventional studies has made it difficult to reliably pinpoint the causal genetic variants. The accumulated recombination events in an advanced intercross line (AIL), in which mice from two inbred strains are mated at random for several generations, can improve mapping resolution. We demonstrate these advancements in mapping QTLs for hindlimb muscle weights in an AIL (n = 832) of the C57BL/6J (B6) and DBA/2J (D2) strains, generations F8-F13. We mapped muscle weight QTLs using the high-density MegaMUGA SNP panel. The QTLs highlight the shared genetic architecture of four hindlimb muscles and suggest that the genetic contributions to muscle variation are substantially different in males and females, at least in the B6D2 lineage. Out of the 15 muscle weight QTLs identified in the AIL, nine overlapped the genomic regions discovered in an earlier B6D2 F2 intercross. Mapping resolution, however, was substantially improved in our study to a median QTL interval of 12.5 Mb. Subsequent sequence analysis of the QTL regions revealed 20 genes with nonsense or potentially damaging missense mutations. Further refinement of the muscle weight QTLs using additional functional information, such as gene expression differences between alleles, will be important for discerning the causal genes.

Divergent physiological characteristics and responses to endurance training among inbred mouse strains.

Both baseline values and adaptive changes in mice can vary depending on the genetic background. We aimed to assess variation in a battery of variables and their adaptations to endurance training in six inbred mouse strains. Males, n = 184, from A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, and PWD/PhJ strains were assigned to a control or an endurance group (5 weeks swimming exercise). Enzyme activity, histology of soleus (SOL) muscle, swimming endurance, cardiac ventricular and hind limb muscle weight, and femur length were examined. Endurance capacity, morphological and histological variables, and enzyme activity substantially differed among strains. For example, SOL weight was twofold higher and cross-sectional area (CSA) of fibers was ≈ 30% greater in C57BL/6J than in PWD/PhJ strain. The CSA of type 1 fibers were larger than type 2A in PWD/PhJ (P < 0.01); however, the reverse was true in DBA/2J and BALB/cByJ strains (P < 0.05). Swimming endurance in DBA/2J strain was ≈ 9 times better than in BALB/cByJ. Endurance training increased the activity of citrate synthase in gastrocnemius across strains (P < 0.01), however, changes in endurance were strain-specific; the C57BL/6J and DBA/2J strains improved substantially, whereas A/J and BALB/cByJ strains did not. In conclusion, genetic background is a potent determinant of the physiological characteristics and adaptations to training in mice.

Fine-mapping of muscle weight QTL in LG/J and SM/J intercrosses.

Genetic variation plays a substantial role in variation in strength, but the underlying mechanisms remain poorly understood. The objective of the present study was to examine the mechanisms underlying variation in muscle mass, a predictor of strength, between LG/J and SM/J strains, which are the inbred progeny of mice selected, respectively, for high and low body weight. We measured weight of five hindlimb muscles in LG/J and SM/J males and females, in F(1) and F(2) intercrosses, and in an advanced intercross (AI), F(34), between the two. F(2) mice were genotyped using 162 SNPs throughout the genome; F(34) mice were genotyped at 3,015 SNPs. A twofold difference in muscle mass between the LG/J and SM/J mouse strains was observed. Integrated genome-wide association analysis in the combined population of F(2) and AI identified 22 quantitative trait loci (QTL; genome-wide P < 0.05) affecting muscle weight on Chr 2 (2 QTL), 4, 5, 6 (7 QTL), 7 (4 QTL), 8 (4 QTL), and 11 (3 QTL). The LG/J allele conferred greater muscle weight in all cases. The 1.5-LOD QTL support intervals ranged between 0.3 and 13.4 Mb (median 3.7 Mb) restricting the list of candidates to between 5 and 97 genes. Selection for body weight segregated the alleles affecting skeletal muscle, the most abundant tissue in the body. Combination of analyses in an F(2) and AI was an effective strategy to detect and refine the QTL in a genome-wide manner. The achieved resolution facilitates further elucidation of the underlying genetic mechanisms affecting muscle mass.

Genetic architecture for hole-board behaviors across substantial time intervals in young, middle-aged and old mice.

A quantitative trait locus (QTL) analysis of behaviors across the life span was conducted in F(2) mice from a C57BL/6J x DBA/2J cross and 22 BXD recombinant inbred (RI) strains. Mice of three age groups were tested in a hole-board apparatus for 3 min on three occasions approximately 1 month apart (average age at test 150, 450 and 750 days, approximately 400 mice per group, divided equally by sex). Quantitative trait loci with small effect size were found on 11 chromosomes for hole-board activity (Hbact) and hole-board rearing (Hbrear). Analysis of 22 RI strains tested at 150 and 450 days of age found only suggestive linkage, with four QTL for Hbact overlapping with those from the F(2) analysis. There was a significant phenotypic correlation between Hbact and Hbrear (approximately 0.55-0.69) and substantial commonality among QTL for the two behaviors. QTL analyses of head-pokes (HP) and fecal boli (FB) only identified QTL at the suggestive level of significance. Age accounted for approximately 15% of the phenotypic variance (sex approximately 3%), and there were genotype by age interactions at approximately 25% of the Hbact and Hbrear QTL. Quantitative trait loci for Hbrear were relatively stable across the three measurement occasions (those for Hbact somewhat less so), although mean levels of each index declined markedly comparing the first to subsequent trials. Considered as a whole, the polygenic system influencing exploratory behaviors accounts for approximately the same amount of phenotypic variance as age (within the range studied), is stable across substantial periods of time, and acts, for the most part, independently of age and sex.

Muscle cell and motor protein function in patients with a IIa myosin missense mutation (Glu-706 to Lys).

The pathogenic events leading to the progressive muscle weakness in patients with a E706K mutation in the head of the myosin heavy chain (MyHC) IIa were analyzed at the muscle cell and motor protein levels. Contractile properties were measured in single muscle fiber segments using the skinned fiber preparation and a single muscle fiber in vitro motility assay. A dramatic impairment in the function of the IIa MyHC isoform was observed at the motor protein level. At the single muscle fiber level, on the other hand, a general decrease was observed in the number of preparations where the specific criteria for acceptance were fulfilled irrespective of MyHC isoform expression. Our results provide evidence that the pathogenesis of the MyHC IIa E706K myopathy involves defective function of the mutated myosin as well as alterations in the structural integrity of all muscle cells irrespective of MyHC isoform expression.

Human skeletal muscle myosin function at physiological and non-physiological temperatures.

AIM: The aim of the study was to assess the function of human skeletal muscle myosin across a wide range of temperatures, including physiological. METHODS: We used a single fibre in vitro motility assay. The in vitro motility speed of actin filaments propelled by myosin extracted from fibres expressing type I myosin heavy chain (MyHC; n = 9), IIa MyHC (n = 6), IIax MyHC (n = 4) and I/IIa MyHC (n = 1) was measured at 15, 20, 25, 30 and 35 degrees C. RESULTS: The motility speed between groups of fibres expressing different MyHC differed significantly (P << 0.001). The increase in motility speed with an increase in temperature was statistically significant (P << 0.001) between all temperatures. The relative difference in motility speed between the slow type I and the fast IIax MyHC fibres decreased with increasing temperature, i.e. a 7.5-fold difference at 15 degrees C was reduced to twofold at 35 degrees C. Furthermore, the twofold difference in motility speed between type IIa and IIax MyHC at 15 degrees C disappeared completely at 35 degrees C. The activation energy, E(A), and temperature coefficient, Q(10), over the 15-35 degrees C temperature range was higher for type I MyHC, 54.47 +/- 4.37 kJ mol(-1) and 2.09 +/- 0.12, respectively, than for type IIa MyHC, 45.41 +/- 3.12 kJ mol(-1) (P < 0.001) and 1.85 +/- 0.08 (P < 0.001), or IIax MyHC, 34.71 +/- 1.75 kJ mol(-1) (P << 0.001) and 1.60 +/- 0.04 (P << 0.001). CONCLUSION: The present results suggest a significantly reduced difference in shortening velocity between different human muscle fibre types at physiological temperature than previously reported at lower temperatures (12 or 15 degrees C) with implications for human in vivo muscle function.

Genetic determinants of weight of fast- and slow-twitch skeletal muscle in 500-day-old mice of the C57BL/6J and DBA/2J lineage.

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: 141-152, 2003), weight of slow-twitch soleus, mixed gastrocnemius, and fast-twitch tibialis anterior (TA) and extensor digitorum longus (EDL) muscles was 13-22% 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.

Genetic architecture of fast- and slow-twitch skeletal muscle weight in 200-day-old mice of the C57BL/6J and DBA/2J lineage.

The aim of the study was to explore the genetic architecture influencing weight of fast- and slow-twitch skeletal muscles. The weights of the slow-twitch soleus, the mixed gastrocnemius, the fast-twitch tibialis anterior (TA), and extensor digitorum longus (EDL) muscles were 11-34% greater (P < 0.001) in 200-day-old C57BL/6J (B6) than in DBA/2J (D2) mice. Male muscles were 13-28% larger than female (P < 1 x 10(-5), no strain by sex interaction). The sex-related difference in muscle weight, however, varied significantly among the 23 derivative BXD recombinant inbred (RI) strains (strain by sex interaction for soleus, P < 0.01; TA, P < 1 x 10(-4); EDL, not significant; and gastrocnemius, P < 0.001). Quantitative trait loci (QTL) affecting muscle weight were mapped in an F2 intercross of B6 and D2 mice (B6D2F2) and BXD RIs. A total of 10 autosomal, muscle-specific, but not muscle-type-specific, QTL, explaining a total of 5.4, 7.7, 22.9, and 8.6% of phenotypic variance for soleus, TA, EDL, and gastrocnemius muscles, respectively, were found across chromosomes 1 (Chr 1), 2, 3 (female-specific), 5 (two), 6, 7, 8, and 9 in B6D2F2 mice. The QTL on Chr 8 for EDL and the female-specific QTL on Chr 3 for gastrocnemius muscles were statistically significant, but the remaining QTL were at the suggestive level of statistical significance. Ten QTL on Chr 1, 2, 4, 5, 7, 8, 14, 17 (two), and 19 were identified in BXD RIs. Half of the QTL in BXD RIs had pleiotropic effects and were at the suggestive level of significance (except for the significant QTL for gastrocnemius muscle on Chr 17). The B6D2F2 nominated QTL on Chr 8 for EDL weight was validated in BXD RIs (P < 0.03). Support intervals for the QTL on Chr 1 and 5 overlapped between B6D2F2 and BXD RIs. An epistatic interaction between markers on Chr 1 and 17 affected gastrocnemius weight in BXD RIs. The interaction was not, however, validated in the B6D2F2 population. Our results indicate that the differences in muscle weight in the B6 and D2 segregating populations were the outcome of a polygenic system, with each factor contributing a small amount to the phenotypic variance and the genetic architecture affecting muscle weight was muscle specific, but not muscle-type specific, and in some instances sex specific.

Effects of concentric and eccentric contractions on phosphorylation of MAPK(erk1/2) and MAPK(p38) in isolated rat skeletal muscle.

1. Exercise and contractions of isolated skeletal muscle induce phosphorylation of mitogen-activated protein kinases (MAPKs) by undefined mechanisms. The aim of the present study was to determine exercise-related triggering factors for the increased phosphorylation of MAPKs in isolated rat extensor digitorum longus (EDL) muscle. 2. Concentric or eccentric contractions, or mild or severe passive stretches were used to discriminate between effects of metabolic/ionic and mechanical alterations on phosphorylation of two MAPKs: extracellular signal-regulated kinase 1 and 2 (MAPK(erk1/2)) and stress-activated protein kinase p38 (MAPK(p38)). 3. Concentric contractions induced a 5-fold increase in MAPK(erk1/2) phosphorylation. Application of the antioxidants N-acetylcysteine (20 mM) or dithiothreitol (5 mM) suppressed concentric contraction-induced increase in MAPK(erk1/2) phosphorylation. Mild passive stretches of the muscle increased MAPK(erk1/2) phosphorylation by 1.8-fold, whereas the combination of acidosis and passive stretches resulted in a 2.8-fold increase. Neither concentric contractions, nor mild stretches nor acidosis significantly affected phosphorylation of MAPK(p38). 4. High force applied upon muscle by means of either eccentric contractions or severe passive stretches resulted in 5.7- and 9.5-fold increases of phosphorylated MAPK(erk1/2), respectively, whereas phosphorylation of MAPK(p38) increased by 7.6- and 1.9-fold (not significant), respectively. 5. We conclude that in isolated rat skeletal muscle an increase in phosphorylation of both MAPK(erk1/2) and MAPK(p38) is induced by mechanical alterations, whereas contraction-related metabolic/ionic changes (reactive oxygen species and acidosis) cause increased phosphorylation of MAPK(erk1/2) only. Thus, contraction-induced phosphorylation can be explained by the combined action of increased production of reactive oxygen species, acidification and mechanical perturbations for MAPK(erk1/2) and by high mechanical stress for MAPK(p38).

Differential activation of mitogen-activated protein kinase signalling pathways by isometric contractions in isolated slow- and fast-twitch rat skeletal muscle.

Activation of mitogen-activated protein (MAP) kinases has been implicated in the signal transduction pathways linking exercise to adaptive changes of muscle protein expression. In the present study, we investigated whether contractions of isolated muscles induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 MAPK in a fibre-type dependent manner. Slow-twitch (soleus) and fast-twitch (epitrochlearis, extensor digitorum longus) rat skeletal muscles were exposed to intermittent tetanic stimulation. Compared with the contralateral non-stimulated muscle, contractions increased ERK1/2 phosphorylation to the same extent in fast- and slow-twitch muscles. Significant increase in phosphorylation of p38 MAPK was observed in the fast-twitch muscles only. The total amount of ERK1/2 and p38 MAPK proteins was higher in the slow-twitch soleus muscle. In conclusion, MAP kinase signalling pathways are differentially activated and expressed in slow- and fast-twitch muscles. In addition, this activation is owing to muscle contraction per se and do not demand additional external influence.

Influence of exercise intensity on ERK/MAP kinase signalling in human skeletal muscle.

The mitogen-activated protein (MAP) kinase pathways have been highlighted as a possible link between exercise and adaptive changes in skeletal muscle. In this study, the effect of exercise intensity on the activation of the ERK/MAP kinase pathway was investigated in human skeletal muscle. One-leg exercise at low (40% maximal oxygen consumption, VO2max for 30 min) and high (75% VO2max for 30 min) intensity resulted in 11.5+8. I-fold and 39.7+/-6.3-fold (mean +/-SEM) increases in ERK1/2 phosphorylation (P<0.001), respectively. The phosphorylation of MEK1/2, the upstream kinase of ERK1/2, increased with exercise intensity (P<0.05) to 2.5+/-0.9 and 4.8+/-1.1 times the basal level at the low and high intensity, respectively. The statistical analysis revealed a systematic difference between basal, low and high intensity exercise levels for both kinases. There was no change in the phosphorylation of either kinase in the non-exercised leg. The phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB), a possible downstream target of the ERK/MAP kinase signalling pathway, was unaffected by exercise. The phosphorylation of ERK1/2 was significantly higher in purified freeze-dried compared to crude wet muscle after exercise, whereas the opposite pattern was observed for CREB. In conclusion, phosphorylation of ERK1/2 and MEK1/2 increases in an exercise intensity-dependent manner in human skeletal muscle and this seems to originate in the muscle fibres themselves.