Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.

The TGFß family member myostatin (growth/differentiation factor-8) is a negative regulator of skeletal muscle growth. The hypermuscular Compact mice carry the 12-bp Mstn(Cmpt-dl1Abc) deletion in the sequence encoding the propeptide region of the precursor promyostatin, and additional modifier genes of the Compact genetic background contribute to determine the full expression of the phenotype. In this study, by using mice strains carrying mutant or wild-type myostatin alleles with the Compact genetic background and nonmutant myostatin with the wild-type background, we studied separately the effect of the Mstn(Cmpt-dl1Abc) mutation or the Compact genetic background on morphology, metabolism, and signaling. We show that both the Compact myostatin mutation and Compact genetic background account for determination of skeletal muscle size. Despite the increased musculature of Compacts, the absolute size of heart and kidney is not influenced by myostatin mutation; however, the Compact genetic background increases them. Both Compact myostatin and genetic background exhibit systemic metabolic effects. The Compact mutation decreases adiposity and improves whole body glucose uptake, insulin sensitivity, and 18FDG uptake of skeletal muscle and white adipose tissue, whereas the Compact genetic background has the opposite effect. Importantly, the mutation does not prevent the formation of mature myostatin; however, a decrease in myostatin level was observed, leading to altered activation of Smad2, Smad1/5/8, and Akt, and an increased level of p-AS160, a Rab-GTPase-activating protein responsible for GLUT4 translocation. Based on our analysis, the Compact genetic background strengthens the effect of myostatin mutation on muscle mass, but those can compensate for each other when systemic metabolic effects are compared.

Anxious and nonanxious mice show similar hippocampal sensory evoked oscillations under urethane anesthesia: difference in the effect of buspirone.

Hippocampal oscillations recorded under urethane anesthesia are proposed to be modulated by anxiolytics. All classes of clinically effective anxiolytics were reported to decrease the frequency of urethane theta; however, recent findings raise concerns about the direct correlation of anxiolysis and the frequency of hippocampal theta. Here, we took advantage of our two inbred mouse strains displaying extremes of anxiety (anxious (AX) and nonanxious (nAX)) to compare the properties of hippocampal activity and to test the effect of an anxiolytic drugs. No difference was observed in the peak frequency or in the peak power between AX and nAX strains. Buspirone (Bus) applied in 2.5 mg/kg decreased anxiety of AX but did not have any effect on nAX as was tested by elevated plus maze and open field. Interestingly, Bus treatment increased hippocampal oscillatory frequency in the AX but left it unaltered in nAX mice. Saline injection did not have any effect on the oscillation. Paired-pulse facilitation was enhanced by Bus in the nAX, but not in the AX strain. Collectively, these results do not support the hypothesis that hippocampal activity under urethane may serve as a marker for potential anxiolytic drugs. Moreover, we could not confirm the decrease of frequency after anxiolytic treatment.

Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling.

Myostatin, a member of the transforming growth factor ß family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation-contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (Mstn(Cmpt-dl1Abc) or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa-force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca(2+) concentration ([Ca(2+)]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nM, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nM, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 ΔF/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mM s(-1)) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mM s(-1)) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.

The compact mutation of myostatin causes a glycolytic shift in the phenotype of fast skeletal muscles.

Myostatin is an important negative regulator of skeletal muscle growth. The hypermuscular Compact (Cmpt) mice carry a 12-bp natural mutation in the myostatin propeptide, with additional modifier genes being responsible for the phenotype. Muscle cellularity of the fast-type tibialis anterior (TA) and extensor digitorum longus (EDL) as well as the mixed-type soleus (SOL) muscles of Cmpt and wild-type mice was examined by immunohistochemical staining of the myosin heavy chain (MHC) proteins. In addition, transcript levels of MHC isoforms were quantified by qPCR. Based on our results, all investigated muscles of Cmpt mice were significantly larger compared with that of wild-type mice, as characterized by fiber hyperplasia of different grades. Fiber hypertrophy was not present in TA; however, EDL muscles showed specific IIB fiber hypertrophy while the (I and IIA) fibers of SOL muscles were generally hypertrophied. Both the fast TA and EDL muscles of Cmpt mice contained significantly more glycolytic IIB fibers accompanied by a decreased number of IIX and IIA fibers; however, this was not the case for SOL muscles. In summary, despite the variances found in muscle cellularity between the different myostatin mutant mice, similar glycolytic shifts were observed in Cmpt fast muscles as in muscles from myostatin knockout mice.

The anxiolytic buspirone shifts coping strategy in novel environmental context of mice with different anxious phenotype.

Patients suffering from anxiety disorders show increased fear when encounter a novel environment. Rodents, placed in new environmental context may respond either with increased novelty seeking (active), or enhanced anxiety (passive coping style), which may depend on the trait anxiety of the animal. Here, the connection between the initial level of anxiety and the behavioral responses in a novel environment was investigated. Two inbred mouse strains having either high- or low-anxiety related behavior (AX and nAX) were exposed to elevated plus maze (EPM), a standard test for assessing anxiety level, for 8 consecutive days. The initial anxiety level was modulated by chronic treatment with buspirone (bus) treatment, a clinically effective anxiolytic, using 2.5mg/kg and 5.0mg/kg doses. Both strains showed a gradual decrease of open-arm exploration, which was not prevented by bus treatment. Another cohort of animals was exposed to EPM for 2 days, and then we changed to blue light illumination and used a different cleaning substance with citrus odor (context change, CC). It was found that upon CC AX mice exhibited increased, while nAX mice showed decreased anxiety. Bus in 2.5mg/kg changed the coping strategy from passive to active exploration after CC in the AX mice; however, the same treatment rendered nAX mice passive upon CC. Bus in 5.0mg/kg failed to alter the overall coping style in the novel environment of both strains. These results suggest that these mouse lines use different coping strategy in novel context, which can be changed with bus treatment.

Functional changes in transcriptomes of the prefrontal cortex and hippocampus in a mouse model of anxiety.

Anxiety is a multi-etiology disorder influenced by both genetic background and environment. To study the impact of a genetic predisposition, we developed a novel mouse model of anxiety using a combination of crossbreeding and behavioral selection. Comparison of the transcriptomes from the prefrontal cortex and hippocampus of anxious and control mice revealed that the numbers of significantly up- and down-regulated genes were modest, comprising approximately 2% of the tested genes. Functional analysis of the significantly altered gene sets showed that functional groups such as nervous system development, behavior, glial cell differentiation and synaptic transmission were significantly enriched among the up-regulated genes, whereas functional groups such as potassium ion transport, Wnt signaling and neuropeptidergic signaling were significantly enriched among the down-regulated genes. Many of the identified genes and functional groups have been previously linked to the molecular biology of anxiety, while several others, such as transthyretin, vasoactive intestinal polypeptide and various potassium ion channels, are novel or not as well described in this context. Supporting the gene expression data, we also found increased excitability in the hippocampi of anxious mice, which can be a phenotypic result of decreased potassium channel density. Our transcriptome screen showed that the initiation and/or effect of anxiety involve multiple pathways and cellular processes. The identified novel genes and pathways could be involved in the molecular pathogenesis of anxiety and provide potential targets for further drug development.

Characterisation of the variation of mouse brain proteome by two-dimensional electrophoresis.

Neuroproteomics is aimed to study the molecular organisation of the nervous system at the protein level. Two-dimensional electrophoresis is the most frequently used technique in quantitative proteomics. The aim of this study was to assess the experimental and biological variations on this proteomic platform using mouse brain tissue. Mice are the most generally used lab animals for modelling human disease or investigating the effect of a drug-candidate or a treatment. Experimental design plays a crucial role in quantitative proteomics, hence understanding and minimizing the variables is essential. Our results indicate that the technical variance dominantly contributes to the total variance in mouse brain and the genetic background has a negligible effect on the total variation. The results also characterise the anticipated variation using mouse brain for proteomic study hence they should be useful for future experimental design in other proteomics laboratories.

A mouse model of anxiety molecularly characterized by altered protein networks in the brain proteome.

Recently, several attempts have been made to describe changes related to certain anxiety states in the proteome of experimental animal models. However, these studies are restricted by limitations regarding the number and correct identification of separated proteins. Moreover, the application of a systems biology approach to discover the molecular mechanisms of anxiety requires genetically homogenous inbred animal models. Therefore, we developed a novel mouse model of anxiety using a combination of crossbreeding (inbred for 35 generations) and behavioral selection. We found significant changes in 82 proteins in the total brain proteome compared to the control proteome. Thirty-four of these proteins had been previously identified in other anxiety, depression or repeated psychosocial stress studies. The identified proteins are associated with different cellular functions, including synaptic transmission, metabolism, proteolysis, protein biosynthesis and folding, cytoskeletal proteins, brain development and neurogenesis, oxidative stress, signal transduction. Our proteomics data suggest that alterations in serotonin receptor-associated proteins, in the carbohydrate metabolism, in the cellular redox system and in synaptic docking are all involved in anxiety.

Mapping a syntenic modifier on mouse chromosome 1 influencing the expressivity of the compact phenotype in the myostatin mutant (MstnCmpt-dl1Abc) compact mouse.

A novel method for mapping a modifier gene that is syntenic to its major gene was used to map a male-sex-limited modifier of the expressivity of the Compact phenotype in the myostatin mutant (Mstn(Cmpt-dl1Abc)) Compact mouse. The modifier was mapped to the general region of D1Mit262, 40 cM distal to Mstn on chromosome 1. Myogenin, a postulated downstream target of myostatin, maps to the same region.

Mapping modifiers affecting muscularity of the myostatin mutant (Mstn(Cmpt-dl1Abc)) compact mouse.

The hypermuscular Compact phenotype was first noted in a line of mice selected for high body weight and protein content. A new line, based on mice showing the Compact phenotype, was formed and selected for maximum expression of the Compact phenotype. Previously we mapped and identified a 12-bp deletion in the myostatin gene, denoted Mstn(Cmpt-dl1Abc), which can be considered as a major gene responsible for the hypermuscular phenotype. Genetic analysis revealed that full expression of the hypermuscular phenotype requires the action of modifier loci in addition to Mstn(Cmpt-dl1Abc). To map these modifier loci, an interspecific F(2) population was generated between Comp9, an inbred line homozygous for Mstn(Cmpt-dl1Abc), and CAST/Ei, an inbred line generated from Mus musculus castaneus. Selective DNA pooling and genotyping, separately by gender, was carried out within a subpopulation of the F(2) consisting of individuals homozygous for Mstn(Cmpt-dl1Abc). Significant association with hypermuscularity at a false discovery rate (FDR) of 0.05 was found for markers on chromosomes 3, 5, 7, 11, 16, and X. In all cases, the marker allele derived from the Comp9 parent showed a higher frequency in the hypermuscular group and the CAST/Ei allele in the normal group. The modifier loci apparently exerted their effects on muscularity only in the presence of Mstn(Cmpt-dl1Abc).