Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability.

Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle "increasing" allele C (n = 15) or "decreasing" allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/- and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the "increasing" compared with the "decreasing" CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/- mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.

Stanniocalcin-2 inhibits skeletal muscle growth and is upregulated in functional overload-induced hypertrophy.

AIMS: Stanniocalcin-2 (STC2) has recently been implicated in human muscle mass variability by genetic analysis. Biochemically, STC2 inhibits the proteolytic activity of the metalloproteinase PAPP-A, which promotes muscle growth by upregulating the insulin-like growth factor (IGF) axis. The aim was to examine if STC2 affects skeletal muscle mass and to assess how the IGF axis mediates muscle hypertrophy induced by functional overload. METHODS: We compared muscle mass and muscle fiber morphology between Stc2-/- (n = 21) and wild-type (n = 15) mice. We then quantified IGF1, IGF2, IGF binding proteins -4 and -5 (IGFBP-4, IGFBP-5), PAPP-A and STC2 in plantaris muscles of wild-type mice subjected to 4-week unilateral overload (n = 14). RESULTS: Stc2-/- mice showed up to 10% larger muscle mass compared with wild-type mice. This increase was mediated by greater cross-sectional area of muscle fibers. Overload increased plantaris mass and components of the IGF axis, including quantities of IGF1 (by 2.41-fold, p = 0.0117), IGF2 (1.70-fold, p = 0.0461), IGFBP-4 (1.48-fold, p = 0.0268), PAPP-A (1.30-fold, p = 0.0154) and STC2 (1.28-fold, p = 0.019). CONCLUSION: Here we provide evidence that STC2 is an inhibitor of muscle growth upregulated, along with other components of the IGF axis, during overload-induced muscle hypertrophy.

Analysis of independent cohorts of outbred CFW mice reveals novel loci for behavioral and physiological traits and identifies factors determining reproducibility.

Combining samples for genetic association is standard practice in human genetic analysis of complex traits, but is rarely undertaken in rodent genetics. Here, using 23 phenotypes and genotypes from two independent laboratories, we obtained a sample size of 3076 commercially available outbred mice and identified 70 loci, more than double the number of loci identified in the component studies. Fine-mapping in the combined sample reduced the number of likely causal variants, with a median reduction in set size of 51%, and indicated novel gene associations, including Pnpo, Ttll6, and GM11545 with bone mineral density, and Psmb9 with weight. However, replication at a nominal threshold of 0.05 between the two component studies was low, with less than one-third of loci identified in one study replicated in the second. In addition to overestimates in the effect size in the discovery sample (Winner's Curse), we also found that heterogeneity between studies explained the poor replication, but the contribution of these two factors varied among traits. Leveraging these observations, we integrated information about replication rates, study-specific heterogeneity, and Winner's Curse corrected estimates of power to assign variants to one of four confidence levels. Our approach addresses concerns about reproducibility and demonstrates how to obtain robust results from mapping complex traits in any genome-wide association study.

Genome-wide Associations Reveal Human-Mouse Genetic Convergence and Modifiers of Myogenesis, CPNE1 and STC2.

Muscle bulk in adult healthy humans is highly variable even after height, age, and sex are accounted for. Low muscle mass, due to fewer and/or smaller constituent muscle fibers, would exacerbate the impact of muscle loss occurring in aging or disease. Genetic variability substantially influences muscle mass differences, but causative genes remain largely unknown. In a genome-wide association study (GWAS) on appendicular lean mass (ALM) in a population of 85,750 middle-aged (aged 38-49 years) individuals from the UK Biobank (UKB), we found 182 loci associated with ALM (p < 5 × 10-8). We replicated associations for 78% of these loci (p < 5 × 10-8) with ALM in a population of 181,862 elderly (aged 60-74 years) individuals from UKB. We also conducted a GWAS on hindlimb skeletal muscle mass of 1,867 mice from an advanced intercross between two inbred strains (LG/J and SM/J); this GWAS identified 23 quantitative trait loci. Thirty-eight positional candidates distributed across five loci overlapped between the two species. In vitro studies of positional candidates confirmed CPNE1 and STC2 as modifiers of myogenesis. Collectively, these findings shed light on the genetics of muscle mass variability in humans and identify targets for the development of interventions for treatment of muscle loss. The overlapping results between humans and the mouse model GWAS point to shared genetic mechanisms across species.

Myostatin dysfunction is associated with lower physical activity and reduced improvements in glucose tolerance in response to caloric restriction in Berlin high mice.

Myostatin is an inhibitor of skeletal muscle growth and might be involved in adaptations to caloric restriction (CR). We compared responses to 12-week 30% CR in male mice of Berlin high strain with myostatin dysfunction (BEH) and wild-type myostatin (BEH+/+). BEH mice were heavier than BEH+/+ mice (58.8 ±â€¯2.0 versus 53.1 ±â€¯2.7 g, p < 0.001), had 1.8-fold greater hind limb muscle mass and were less (p < 0.05) physically active when fed ad libitum. After CR, BEH and BEH+/+ strains experienced similar weight loss (24.7 ±â€¯5.7 versus 20.6 ±â€¯6.5%, p > 0.05, respectively) and decreases (p < 0.001) in plasma IGF-1 and total cholesterol, but loss of hind limb muscle mass was greater (p < 0.001) in BEH mice than BEH+/+ mice. BEH mice had better (p < 0.001) glucose tolerance and showed smaller (p < 0.05) improvements of it than BEH+/+ mice after CR (1038.2 ±â€¯174.7 versus 744.4 ±â€¯95.8 glucose mM× 120 min, p < 0.01 for BEH; 1365.8 ±â€¯218.5 versus 831.5 ±â€¯134.4 glucose mM ×120 min, p < 0.001, for BEH+/+, respectively). In summary, myostatin dysfunction is associated with muscle hypertrophy and high glucose tolerance, but greater muscle wasting and smaller improvements in glucose tolerance in response to CR.

Myostatin dysfunction does not protect from fasting-induced loss of muscle mass in mice.

OBJECTIVES: The aim of the study was to investigate if myostatin dysfunction can ameliorate fasting-induced muscle wasting. METHODS: 18-week old males from Berlin high (BEH) strain with myostatin dysfunction and wild type myostatin (BEH+/+) strain were subjected to 48-h food deprivation (FD). Changes in body composition as well as contractile properties of soleus (SOL) and extensor digitorum longus (EDL) muscles were studied. RESULTS: BEH mice were heavier than BEH+/+ mice (56.0±2.5 vs. 49.9±2.8 g, P<0.001, respectively). FD induced similar loss of body mass in BEH and BEH+/+ mice (16.6±2.4 vs. 17.4±2.2%, P>0.05), but only BEH mice experienced wasting of the gastrocnemius, tibialis anterior and plantaris muscles. FD induced a marked decrease in specific muscle force of SOL. EDL of BEH mice tended to be protected from this decline. CONCLUSION: Myostatin dysfunction does not protect from loss of muscle mass during fasting.

Corrigendum to "Low Citrate Synthase Activity Is Associated with Glucose Intolerance and Lipotoxicity".

[This corrects the article DOI: 10.1155/2019/8594825.].

Low Citrate Synthase Activity Is Associated with Glucose Intolerance and Lipotoxicity.

Citrate synthase (CS) is a key mitochondrial enzyme. The aim of this study was to test the hypothesis that low CS activity impairs the metabolic health of mice fed a high fat diet (HFD) and promotes palmitate-induced lipotoxicity in muscle cells. C57BL/6J (B6) mice and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A), a strain which carries the A/J allele of CS on the B6 strain background, were fed HFD (45% kcal from fat) for 12 weeks. C2C12 mouse muscle cells were used to investigate effects of CS knockdown on cell viability and signalling after incubation in 0.8 mM palmitate. CS activity, but not that of ß-hydroxyacyl-coenzyme-A dehydrogenase was lower in the gastrocnemius muscle and heart of B6.A mice compared to B6 mice (P < 0.001). During HFD feeding, glucose tolerance of mice decreased progressively and to a greater extent in B6.A females compared to B6 females, with males showing a similar trend. Body weight and fat gain did not differ between B6.A and B6 mice. After an 18 h incubation in 0.8 mM palmitate C2C12 muscle cells with ∼50% shRNA mediated reduction in CS activity showed lower (P < 0.001) viability and increased (P < 0.001) levels of cleaved caspase-3 compared to the scramble shRNA treated C2C12 cells. A/J strain variant of CS is associated with low enzyme activity and impaired metabolic health. This could be due to impaired lipid metabolism in muscle cells.

Genome wide association analysis in a mouse advanced intercross line.

The LG/J x SM/J advanced intercross line of mice (LG x SM AIL) is a multigenerational outbred population. High minor allele frequencies, a simple genetic background, and the fully sequenced LG and SM genomes make it a powerful population for genome-wide association studies. Here we use 1,063 AIL mice to identify 126 significant associations for 50 traits relevant to human health and disease. We also identify thousands of cis- and trans-eQTLs in the hippocampus, striatum, and prefrontal cortex of ~200 mice. We replicate an association between locomotor activity and Csmd1, which we identified in an earlier generation of this AIL, and show that Csmd1 mutant mice recapitulate the locomotor phenotype. Our results demonstrate the utility of the LG x SM AIL as a mapping population, identify numerous novel associations, and shed light on the genetic architecture of mammalian behavior.

Replication and discovery of musculoskeletal QTLs in LG/J and SM/J advanced intercross lines.

The genetics underlying variation in health-related musculoskeletal phenotypes can be investigated in a mouse model. Quantitative trait loci (QTLs) affecting musculoskeletal traits in the LG/J and SM/J strain lineage remain to be refined and corroborated. The aim of this study was to map muscle and bone traits in males (n = 506) of the 50th filial generation of advanced intercross lines (LG/SM AIL) derived from the two strains. Genetic contribution to variation in all musculoskeletal traits was confirmed; the SNP heritability of muscle mass ranged between 0.46 and 0.56; and the SNP heritability of tibia length was 0.40. We used two analytical software, GEMMA and QTLRel, to map the underlying QTLs. GEMMA required substantially less computation and recovered all the QTLs identified by QTLRel. Seven significant QTLs were identified for muscle weight (Chr 1, 7, 11, 12, 13, 15, and 16), and two for tibia length, (Chr 1 and 13). Each QTL explained 4-5% of phenotypic variation. One muscle and both bone loci replicated previous findings; the remaining six were novel. Positional candidates for the replicated QTLs were prioritized based on in silico analyses and gene expression in muscle tissue. In summary, we replicated existing QTLs and identified novel QTLs affecting muscle weight, and replicated bone length QTLs in LG/SM AIL males. Heritability estimates substantially exceed the cumulative effect of the QTLs, hence a richer genetic architecture contributing to muscle and bone variability could be uncovered with a larger sample size.

H55N polymorphism is associated with low citrate synthase activity which regulates lipid metabolism in mouse muscle cells.

The H55N polymorphism in the Cs gene of A/J mice has been linked to low activity of the enzyme in skeletal muscles. The aim of the study was to test this hypothesis and examine effects of low citrate synthase (CS) activity on palmitate metabolism in muscle cells. Results of the study showed that carriers of the wild type (WT) Cs (C57BL/6J and Balb/cByJ mouse strains) had higher CS activity (p < 0.01) than carriers of the A/J variant (B6.A-(rs3676616-D10Utsw1)/KjnB6 and A/J mouse strains) in the heart, liver and gastrocnemius muscle. Furthermore, the recombinant CS protein of WT showed higher CS activity than the A/J variant. In C2C12 muscle cells the shRNA mediated 47% knockdown of CS activity reduced the rate of fatty acid oxidation compared to the control cells. In summary, our results are consistent with the hypothesis that H55N substitution causes a reduction in CS activity. Furthermore, low CS activity interferes with metabolic flexibility of muscle cells.

Forced Running Endurance Is Influenced by Gene(s) on Mouse Chromosome 10.

Phenotypic diversity between laboratory mouse strains provides a model for studying the underlying genetic mechanisms. The A/J strain performs poorly in various endurance exercise models. The aim of the study was to test if endurance capacity and contractility of the fast- and slow-twitch muscles are affected by the genes on mouse chromosome 10. The C57BL/6J (B6) strain and C57BL/6J-Chr 10A/J/NaJ (B6.A10) consomic strain which carries the A/J chromosome 10 on a B6 strain background were compared. The B6.A10 mice compared to B6 were larger in body weight (p < 0.02): 27.2 ± 1.9 vs. 23.8 ± 2.7 and 23.4 ± 1.9 vs. 22.9 ± 2.3 g, for males and females, respectively, and in male soleus weight (p < 0.02): 9.7 ± 0.4 vs. 8.6 ± 0.9 mg. In the forced running test the B6.A10 mice completed only 64% of the B6 covered distance (p < 0.0001). However, there was no difference in voluntary wheel running (p = 0.6) or in fatigability of isolated soleus (p = 0.24) or extensor digitorum longus (EDL, p = 0.7) muscles. We conclude that chromosome 10 of the A/J strain contributes to reduced endurance performance. We also discuss physiological mechanisms and methodological aspects relevant to interpretation of these findings.

Baseline Muscle Mass Is a Poor Predictor of Functional Overload-Induced Gain in the Mouse Model.

Genetic background contributes substantially to individual variability in muscle mass. Muscle hypertrophy in response to resistance training can also vary extensively. However, it is less clear if muscle mass at baseline is predictive of the hypertrophic response. The aim of this study was to examine the effect of genetic background on variability in muscle mass at baseline and in the adaptive response of the mouse fast- and slow-twitch muscles to overload. Males of eight laboratory mouse strains: C57BL/6J (B6, n = 17), BALB/cByJ (n = 7), DBA/2J (D2, n = 12), B6.A-(rs3676616-D10Utsw1)/Kjn (B6.A, n = 9), C57BL/6J-Chr10A/J/NaJ (B6.A10, n = 8), BEH+/+ (n = 11), BEH (n = 12), and DUHi (n = 12), were studied. Compensatory growth of soleus and plantaris muscles was triggered by a 4-week overload induced by synergist unilateral ablation. Muscle weight in the control leg (baseline) varied from 5.2 ± 07 mg soleus and 11.4 ± 1.3 mg plantaris in D2 mice to 18.0 ± 1.7 mg soleus in DUHi and 43.7 ± 2.6 mg plantaris in BEH (p < 0.001 for both muscles). In addition, soleus in the B6.A10 strain was ~40% larger (p < 0.001) compared to the B6. Functional overload increased muscle weight, however, the extent of gain was strain-dependent for both soleus (p < 0.01) and plantaris (p < 0.02) even after accounting for the baseline differences. For the soleus muscle, the BEH strain emerged as the least responsive, with a 1.3-fold increase, compared to a 1.7-fold gain in the most responsive D2 strain, and there was no difference in the gain between the B6.A10 and B6 strains. The BEH strain appeared the least responsive in the gain of plantaris as well, 1.3-fold, compared to ~1.5-fold gain in the remaining strains. We conclude that variation in muscle mass at baseline is not a reliable predictor of that in the overload-induced gain. This suggests that a different set of genes influence variability in muscle mass acquired in the process of normal development, growth, and maintenance, and in the process of adaptive growth of the muscle challenged by overload.

Genome-wide association of multiple complex traits in outbred mice by ultra-low-coverage sequencing.

Two bottlenecks impeding the genetic analysis of complex traits in rodents are access to mapping populations able to deliver gene-level mapping resolution and the need for population-specific genotyping arrays and haplotype reference panels. Here we combine low-coverage (0.15×) sequencing with a new method to impute the ancestral haplotype space in 1,887 commercially available outbred mice. We mapped 156 unique quantitative trait loci for 92 phenotypes at a 5% false discovery rate. Gene-level mapping resolution was achieved at about one-fifth of the loci, implicating Unc13c and Pgc1a at loci for the quality of sleep, Adarb2 for home cage activity, Rtkn2 for intensity of reaction to startle, Bmp2 for wound healing, Il15 and Id2 for several T cell measures and Prkca for bone mineral content. These findings have implications for diverse areas of mammalian biology and demonstrate how genome-wide association studies can be extended via low-coverage sequencing to species with highly recombinant outbred populations.

Genome-wide association study of behavioral, physiological and gene expression traits in outbred CFW mice.

Although mice are the most widely used mammalian model organism, genetic studies have suffered from limited mapping resolution due to extensive linkage disequilibrium (LD) that is characteristic of crosses among inbred strains. Carworth Farms White (CFW) mice are a commercially available outbred mouse population that exhibit rapid LD decay in comparison to other available mouse populations. We performed a genome-wide association study (GWAS) of behavioral, physiological and gene expression phenotypes using 1,200 male CFW mice. We used genotyping by sequencing (GBS) to obtain genotypes at 92,734 SNPs. We also measured gene expression using RNA sequencing in three brain regions. Our study identified numerous behavioral, physiological and expression quantitative trait loci (QTLs). We integrated the behavioral QTL and eQTL results to implicate specific genes, including Azi2 in sensitivity to methamphetamine and Zmynd11 in anxiety-like behavior. The combination of CFW mice, GBS and RNA sequencing constitutes a powerful approach to GWAS in mice.

Deficiency of the zinc finger protein ZFP106 causes motor and sensory neurodegeneration.

Zinc finger motifs are distributed amongst many eukaryotic protein families, directing nucleic acid-protein and protein-protein interactions. Zinc finger protein 106 (ZFP106) has previously been associated with roles in immune response, muscle differentiation, testes development and DNA damage, although little is known about its specific function. To further investigate the function of ZFP106, we performed an in-depth characterization of Zfp106 deficient mice (Zfp106(-/-)), and we report a novel role for ZFP106 in motor and sensory neuronal maintenance and survival. Zfp106(-/-) mice develop severe motor abnormalities, major deficits in muscle strength and histopathological changes in muscle. Intriguingly, despite being highly expressed throughout the central nervous system, Zfp106(-/-) mice undergo selective motor and sensory neuronal and axonal degeneration specific to the spinal cord and peripheral nervous system. Neurodegeneration does not occur during development of Zfp106(-/-) mice, suggesting that ZFP106 is likely required for the maintenance of mature peripheral motor and sensory neurons. Analysis of embryonic Zfp106(-/-) motor neurons revealed deficits in mitochondrial function, with an inhibition of Complex I within the mitochondrial electron transport chain. Our results highlight a vital role for ZFP106 in sensory and motor neuron maintenance and reveal a novel player in mitochondrial dysfunction and neurodegeneration.

Myostatin dysfunction impairs force generation in extensor digitorum longus muscle and increases exercise-induced protein efflux from extensor digitorum longus and soleus muscles.

Myostatin dysfunction promotes muscle hypertrophy, which can complicate assessment of muscle properties. We examined force generating capacity and creatine kinase (CK) efflux from skeletal muscles of young mice before they reach adult body and muscle size. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of Berlin high (BEH) mice with dysfunctional myostatin, i.e., homozygous for inactivating myostatin mutation, and with a wild-type myostatin (BEH+/+) were studied. The muscles of BEH mice showed faster (P < 0.01) twitch and tetanus contraction times compared with BEH+/+ mice, but only EDL displayed lower (P < 0.05) specific force. SOL and EDL of age-matched but not younger BEH mice showed greater exercise-induced CK efflux compared with BEH+/+ mice. In summary, myostatin dysfunction leads to impairment in muscle force generating capacity in EDL and increases susceptibility of SOL and EDL to protein loss after exercise.

Regenerated soleus muscle shows reduced creatine kinase efflux after contractile activity in vitro.

Regenerated skeletal muscles show less muscle damage after strenuous muscle exercise. The aim of the studies was to investigate if the regeneration is associated with reduced muscle creatine kinase (CK) efflux immediately after the exercise. Cryolesion was applied to the soleus muscle of 3-month-old C57BL/6J male mice. Then total CK efflux was assessed in vitro in the regenerated muscles without exercise or after 100 eccentric contractions. The same measurements were performed in the control muscles, which were not exposed to cryolesion. Regenerated muscles generated weaker (P < 0.05) twitches, but stronger (P < 0.05) 150-Hz and 300-Hz tetani with prolonged (P < 0.01) contraction times compared with the control muscles. There was no difference between regenerated and control muscles in the total CK efflux without exercise, but only control muscles showed an increase (P < 0.001) in the CK efflux after the exercise. Our results suggest that muscle regeneration is associated with modulation of contractile properties and improvement in muscle resistance to damage after eccentric exercise.

Novel mutations in human and mouse SCN4A implicate AMPK in myotonia and periodic paralysis.

Mutations in the skeletal muscle channel (SCN4A), encoding the Nav1.4 voltage-gated sodium channel, are causative of a variety of muscle channelopathies, including non-dystrophic myotonias and periodic paralysis. The effects of many of these mutations on channel function have been characterized both in vitro and in vivo. However, little is known about the consequences of SCN4A mutations downstream from their impact on the electrophysiology of the Nav1.4 channel. Here we report the discovery of a novel SCN4A mutation (c.1762A>G; p.I588V) in a patient with myotonia and periodic paralysis, located within the S1 segment of the second domain of the Nav1.4 channel. Using N-ethyl-N-nitrosourea mutagenesis, we generated and characterized a mouse model (named draggen), carrying the equivalent point mutation (c.1744A>G; p.I582V) to that found in the patient with periodic paralysis and myotonia. Draggen mice have myotonia and suffer from intermittent hind-limb immobility attacks. In-depth characterization of draggen mice uncovered novel systemic metabolic abnormalities in Scn4a mouse models and provided novel insights into disease mechanisms. We discovered metabolic alterations leading to lean mice, as well as abnormal AMP-activated protein kinase activation, which were associated with the immobility attacks and may provide a novel potential therapeutic target.