Myostatin-deficient Mice Research Articles

Myostatin-deficient mice exhibit reduced insulin resistance through activating the AMP-activated protein kinase signalling pathway

Myostatin-null mice (Mstn(-/-)) have reduced body fat and increased tolerance to glucose. To date the molecular mechanisms through which myostatin regulates body fat content and insulin sensitivity are not known. Therefore, the aim of the current study was to identify signalling pathways through which myostatin regulates insulin sensitivity. Wild-type (WT) mice and Mstn(-/-) mice were fed either a control chow diet or a high fat diet (HFD) for 12 weeks. Glucose tolerance testing and insulin stimulated glucose uptake by M. extensor digitorum longus (EDL) were used as variables to determine insulin sensitivity. Quantitative PCR, Western blotting and enzyme assays were used to monitor AMP-activated protein kinase (AMPK) levels and activity. Mstn(-/-) mice exhibited reduced fat accumulation and peripheral insulin resistance when compared with WT mice, even when they were fed an HFD. Furthermore, treatment with a myostatin antagonist also increased insulin sensitivity during HFD. Consistent with increased insulin sensitivity, we also detected elevated levels of GLUT4, AKT, p-AKT and insulin receptor substrate-1 in Mstn(-/-) muscles. Molecular analysis showed that there is increased expression and activity of AMPK in Mstn(-/-) muscles. Furthermore, we also observed an increase in the AMPK downstream target genes, Sirt1 and Pgc-1α (also known as Ppargc1a), in skeletal muscle of Mstn(-/-) mice. We conclude that myostatin inactivation leads to increased AMPK levels and activity resulting in increased insulin sensitivity of skeletal muscle. We propose that, by regulating AMPK in skeletal muscle and adipose tissues, myostatin plays a major role in regulating insulin signalling.

Myonuclear Domain Size and 3d Myonuclear Organization in Single Muscle Fibers from Myostatin Deficient Or IGF1 Overexpressing Mice

Myostatin deprived or IGF-1 over-expressing mice are characterized by a 2-3 fold increase in muscle size compared to controls. Despite the hypertrophy these mice show significant difference in force generating capacity, i.e., maximum force normalized to muscle fiber cross-sectional area or specific force. That is, specific tension in IGF1 overexpressing transgenic mice is similar to controls while significantly lower in the myostatin knock out mice. The mechanism underlying this compromised muscle function is unknown. In an attempt to explore this mechanism we have investigated the size of cytoplasmic volume (myonuclear domain MND) supported by individual myonuclei in single muscle fiber segments from myostatin deficient, IGF1 over-expressing and control mice, using a novel algorithm to measure the MND in 3D. Single skinned muscle fiber segments were mounted at fixed sarcomere length corresponding to optimum filament overlap for force generation and stained with DAPI (myonuclei) and rhodamine (actin). Our image analysis algorithm was highly effective in determining the spatial organization of myonuclei and distribution of MNDs along the length of the fiber. Early results point towards an inverse relationship between MND and specific force. This implies that hypertrophy is primarily due to expansion of existing MNDs in myostatin knock-outs, and addition of more myonuclei in IGF1 over-expressing mice. This is suggested to have significant effects on transcriptional control of protein synthesis/degradation, turnover rates and/or posttranslational modifications of contractile proteins. We conclude that a maintained MND size is a prerequisite for force generation capacity in hypertrophied muscle fibers.

Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice

There is no consensus about whether making muscles abnormally large by reducing myostatin activity affects force-generating capacity or the ability to perform activities requiring muscular endurance. We therefore examined grip force, contractile properties of extensor digitorum longus (EDL) muscles, and voluntary wheel running in mice in which myostatin was depleted after normal muscle development. Cre recombinase activity was induced to knock out exon 3 of the myostatin gene in 4-mo-old mice in which this exon was flanked by loxP sequences (Mstn[f/f]). Control mice with normal myostatin genes (Mstn[w/w]) received the same Cre-activating treatment. Myostatin depletion increased the mass of all muscles that were examined (gastrocnemius, quadriceps, tibialis anterior, EDL, soleus, triceps) by approximately 20-40%. Grip force, measured multiple times 2-22 wk after myostatin knockout, was not consistently greater in the myostatin-deficient mice. EDL contractile properties were determined 7-13 mo after myostatin knockout. Twitch force tended to be greater in myostatin-deficient muscles (+24%; P=0.09), whereas tetanic force was not consistently elevated (mean +11%; P=0.36), even though EDL mass was greater than normal in all myostatin-deficient mice (mean +36%; P<0.001). The force deficit induced by eccentric contractions was approximately twofold greater in myostatin-deficient than in normal EDL muscles (31% vs. 16% after five eccentric contractions; P=0.02). Myostatin-deficient mice ran 19% less distance (P<0.01) than control mice during the 12 wk following myostatin depletion, primarily because of fewer running bouts per night rather than diminished running speed or bout duration. Reduced specific tension (ratio of force to mass) and reduced running have been observed after muscle hypertrophy was induced by other means, suggesting that they are characteristics generally associated with abnormally large muscles rather than unique effects of myostatin deficiency.

Myostatin knockout mice increase oxidative muscle phenotype as an adaptive response to exercise

Myostatin-deficient mice (MSTN (-/-)) display excessive muscle mass and this is associated with a profound loss of oxidative metabolic properties. In this study we analysed the effect of two endurance-based exercise regimes, either a forced high-impact swim training or moderate intensity voluntary wheel running on the adaptive properties of the tibialis anterior and plantaris muscle from MSTN (-/-) mice. MSTN (-/-) and wild type (MSTN (+/+)) animals had comparable performances in the wheel running regime in terms of distance, average speed and time, but MSTN (-/-) mice showed a reduced ability to sustain a high-impact activity via swimming. Swim training elicited muscle specific adaptations on fibre type distribution in MSTN (-/-); the tibialis anterior displaying a partial transformation in contrast to the plantaris which showed no change. Conversely, wheel running induced similar changes in fibre type composition of both muscles, favouring transitions from IIB-to-IIA. Succinate dehydrogenase activity, an indicator of mitochondrial oxidative potential was increased in response to either exercise regime, with wheel running eliciting more robust changes in the MSTN (-/-) muscles. Examination of the cross sectional area of individual fibre types showed genotype-specific responses with MSTN (-/-) mice exhibiting an incapability of fibre enlargement following the wheel running regime, as opposed to MSTN (+/+) mice and a greater susceptibility to muscle fibre area loss following swimming. In conclusion, the muscle fibre hypertrophy, oxidative capacity and glycolytic phenotype of myostatin deficient muscle can be altered with endurance exercise regimes.

The relationship between bone mechanical properties and ground reaction forces in normal and hypermuscular mice.

Understanding the relationship between external load and bone morphology is critical for understanding adaptations to load in extant animals and inferring behavior in extinct forms. Yet, the relationship between bony anatomy and load is poorly understood, with empirical studies often producing conflicting results. It is widely assumed in many ecological and paleontological studies that bone size and strength reflect the forces experienced by the bone in vivo. This study examines that assumption by providing preliminary data on gait mechanics in a hypermuscular myostatin-deficient mouse model with highly mineralized and hypertrophied long bones. A small sample of hypermuscular and wild-type mice was video recorded while walking freely across a force platform. Temporal gait parameters, peak vertical and transverse (mediolateral) ground reaction forces (GRFs), vertical impulse, and loading rates were measured. The only gait parameters that differed between the two groups were the speeds at which the animals traveled and the transverse forces on the hind limb. The myostatin-deficient mice move relatively slowly and experienced the same magnitude of vertical forces on all limbs and transverse forces on the forelimb as the wild-type mice; though the myostatin-deficient mice did experience lower mediolateral forces on their hindlimbs compared with the wild-type mice. These preliminary results call into question the hypothesis that skeletal hypertrophy observed in hypermuscular mice is a result of larger GRFs experienced by the animals' limbs during locomotion. This calls for further analysis and a cautious approach to inferences about locomotor behavior derived from bony morphology in extant and fossil species.

Mechanisms involved in the enhancement of mammalian target of rapamycin signalling and hypertrophy in skeletal muscle of myostatin-deficient mice

Myostatin deficiency leads to both an increased rate of protein synthesis and skeletal muscle hypertrophy. However, the mechanisms involved in mediating these effects are not yet fully understood. Here, we demonstrate that genetic loss of myostatin leads to enhanced muscle expression of both protein kinase B and mammalian target of rapamycin/S6K signalling components, consistent with their elevated activity. This is associated with a reduction in the expression of PGC1α and COX IV, proteins which play important roles in maintaining mitochondrial function. Furthermore, we show that these changes in signalling and protein expression are largely independent of alterations in intramuscular amino acid content. Our findings, therefore, reveal potential new mechanisms and further contribute to our understanding of myostatin-regulated skeletal muscle growth and function.

Role of myostatin (GDF‐8) signaling in the human anterior cruciate ligament

Myostatin, also referred to as growth and differentiation factor-8 (GDF-8), is expressed in muscle tissue where it functions to suppress myoblast proliferation and myofiber hypertrophy. Recently, myostatin and its receptor, the type IIB activin receptor (ActRIIB), were detected in the leg tendons of mice, and recombinant myostatin was shown to increase cellular proliferation and the expression of type 1 collagen in primary fibroblasts from mouse tendons. We sought to determine whether myostatin and its receptor were present in human anterior cruciate ligament (ACL) tissue, and if myostatin treatment had any effect on primary ACL fibroblasts. ACL tissue samples were obtained from material discarded during ACL reconstruction surgery. Real-time PCR and immunohistochemistry demonstrate that both myostatin and its receptor are abundant in the human ACL. Primary fibroblasts isolated from human ACL specimens were treated with recombinant myostatin, and myostatin treatment increased fibroblast proliferation as well as the expression of tenascin C (TNC), type 1 collagen, and transforming growth factor-beta1. Real-time PCR analysis of TNC and type 1 collagen expression in ACL specimens from normal mice and mice lacking myostatin supported these findings by showing that both TNC and type 1 collagen were downregulated in ACL tissue from myostatin-deficient mice. Together, these data suggest that myostatin is a pro-fibrogenic factor that enhances cellular proliferation and extracellular matrix synthesis by ACL fibroblasts. Recombinant myostatin may therefore have therapeutic applications in the area of tendon and ligament engineering and regeneration.

Age‐Related Changes in Craniofacial Morphology in GDF‐8 (Myostatin)‐Deficient Mice

It is well recognized that masticatory muscle function helps determine morphology, although the extent of function on final form is still debated. GDF-8 (myostatin), a transcription factor is a negative regulator of skeletal muscle growth. A recent study has shown that mice homozygous for the myostatin mutation had increased muscle mass and craniofacial dysmorphology in adulthood. However, it is unclear whether such dysmorphology is present at birth. This study examines the onset and relationship between hypermuscularity and craniofacial morphology in neonatal and adult mice with GDF-8 deficiency. Fifteen (8 wild-type and 7 GDF-8 -/-), 1-day-old and 16 (9 wt and 7 GDF-8 -/-), 180-day-old male CD-1 mice were used. Standardized radiographs were taken of each head, scanned, traced, and cephalometric landmarks identified. Significant mean differences were assessed using a group x age, two-way ANOVA. Myostatin-deficient mice had significantly (P < 0.01) smaller body and masseter muscle weights and craniofacial skeletons at 1 day of age and significantly greater body and masseter muscle weights at 180 days of age compared to controls. Myostatin-deficient mice showed significantly (P < 0.001) longer and "rocker-shaped" mandibles and shorter and wider crania compared to controls at 180 days. Significant correlations were noted between masseter muscle weight and all cephalometric measurements in 180-day-old Myostatin-deficient mice. Results suggest that in this mouse model, there may be both early systemic skeletal growth deficiencies and later compensatory changes from hypermuscularity. These findings reiterate the role that masticatory muscle function plays on the ontogeny of the cranial vault, base, and most notably the mandible.

EM.P.1.07 Effects of muscle hypertrophy on individual myonuclear domain sizes in single muscle fibers from myostatin deficient or IGF-1 over-expressing mice

EM.P.1.07 Effects of muscle hypertrophy on individual myonuclear domain sizes in single muscle fibers from myostatin deficient or IGF-1 over-expressing mice

Myostatin in tendon maintenance and repair

Myostatin, a negative regulator of muscle growth, has recently been found to be expressed in tendons. Myostatin-deficient mice have weak and brittle tendons, which suggest that myostatin could be important for tendon maintenance. Follistatin expression in the callus tissue after tendon transection is influenced by loading. We found that follistatin antagonises myostatin, but not GDF-5 or OP-1 in vitro. To study if myostatin might play a physiological role in soft tissue, we transected 64 rat Achilles tendons and studied the gene expression for myostatin and its receptors at four different time-points during healing. Intact tendons were also studied. All samples were studied with or without mechanical loading. Unloading was achieved with botulinum toxin injections in the calf muscles. The expression of the myostatin gene was more than 40 times higher in intact tendons than in the callus tissue during tendon healing. The expression of myostatin was also influenced by loading status in both intact and healing tendons. Thereafter, we measured the mechanical properties of healing tendons after local myostatin administration. This treatment increased the volume and the contraction of the callus after 8 days, but did not improve its strength. Our results indicate that myostatin plays a positive role in tendon maintenance and that exogenous protein administration stimulates proliferation and growth of early repair tissue. However, no effect on further development towards connective tissue formation was found.

Myostatin (GDF-8) deficiency increases fracture callus size, Sox-5 expression, and callus bone volume

Myostatin (GDF-8) is a negative regulator of skeletal muscle growth and mice lacking myostatin show increased muscle mass. We have previously shown that myostatin deficiency increases bone strength and biomineralization throughout the skeleton, and others have demonstrated that myostatin is expressed during the earliest phase of fracture repair. In order to determine the role of myostatin in fracture callus morphogenesis, we studied fracture healing in mice lacking myostatin. Adult wild-type mice (+/+), mice heterozygous for the myostatin mutation (+/−), and mice homozygous for the disrupted myostatin sequence (−/−) were included for study at two and four weeks following osteotomy of the fibula. Expression of Sox-5 and BMP-2 were significantly upregulated in the fracture callus of myostatin-deficient (−/−) mice compared to wild-type (+/+) mice at two weeks following osteotomy. Fracture callus size was significantly increased in mice lacking myostatin at both two and four weeks following osteotomy, and total osseous tissue area and callus strength in three-point bending were significantly greater in myostatin −/− mice compared to myostatin +/+ mice at four weeks post-osteotomy. Our data suggest that myostatin functions to regulate fracture callus size by inhibiting the recruitment and proliferation of progenitor cells in the fracture blastema. Myostatin deficiency increases blastema size during the early inflammatory phase of fracture repair, ultimately producing an ossified callus having greater bone volume and greater callus strength. While myostatin is most well known for its effects on muscle development, it is also clear that myostatin plays a significant, direct role in bone formation and regeneration.

G.P.8.07 Increased oxidative metabolism in mdx muscle treated by a combination of exon skipping and myostatin blockade

Myostatin blockade stimulates growth of skeletal muscle and has been suggested as a potential therapy for muscular dystrophies. Previously we observed a fibre type conversion towards glycolytic fibres in myostatin deficient mice, which was associated with a decreased number of mitochondria and reduction of oxidative enzymes SDH and COX, implicating a reduced oxidative metabolism of skeletal muscle as a possible adverse effect following lack of myostatin. Here we investigated the effect on muscle metabolism when myostatin blockade was used as a supportive therapy combined with skipping of dystrophin exon 23 in mdx mouse, the animal model for Duchenne muscular dystrophy. We used a AAV construct harboring a double vector which blocks myostatin signalling by RNA interference of the activin receptor IIb (sh-AcvRIIb-3) and which rescues dystrophin expression by using a modified exon skipping strategy (U7 snRNA targeting exon 23). Remarkably, here we demonstrate that combined exon skipping and myostatin blockade increased oxidative properties of treated mdx muscle. Intramuscular injection of U7/sh-AcvRIIb-inv vector into tibialis anterior muscles of mdx mice increased the activity of COX by 78% and of respiratory chain complexes II by 104% compared to the treatment with a control vector harboring scramble sh-RNA. Furthermore, we found an increase in citrate synthase activity of 13% compared to the controls. From these results we hypothesize that myostatin blockade has a different effect on muscle metabolism and subsequently on muscle function when used in combination with dystrophin rescue than myostatin blockade on its own. Therefore, side effects resulting from myostatin blockade may be reduced if applied as a supportive therapy. However, above demonstrated data do not permit to differentiate whether observed effects resulted from myostatin blockade or from exon skipping. Experiments to compare the effect of each strategy are on course.

Building a better organismal model: The role of the mouse--Introduction to the symposium

‘‘The literature is filled with statements about the mechanical function of this or that trait, or about the ‘‘superiority’’ of this or that design without a single experiment to back up the claim’’ (Lauder 1996, p. 86). One of the obstacles preventing a more in-depth understanding of the determinants of complex musculoskeletal traits has been a relative paucity of suitable organismal-level models for investigating their genesis. A complex trait, in this sense, is defined as having multiple contributing factors including behavioral, developmental, and environmental inputs, as well as interactions between any of these (Hofmann 2003). This symposium was organized around a central theme—demonstrating the unique capabilities of mouse models in investigating the manifestation of complex traits of the mammalian musculoskeletal system. In comparison to choosing other animals for organismal-level models, selecting a house mouse has several distinct advantages. The small body size and relatively rapid maturation of mice encourages large samples and more cost-effective experimentation relative to most other animal models. A growing number of inbred strains exist (Vogel 2003; Wergedal et al. 2005), each of which essentially eliminates genetic variability from the list of factors contributing to the morphology or behavior in question. This provides an invaluable opportunity to investigate musculoskeletal responses in experimental designs (e.g., differences related to performance criteria) without introducing confounding genetic variability. Perhaps currently undervalued in importance, mice are behaviorally plastic (Crawley et al. 1997), which is a boon for functional morphologists studying masticatory and locomotor adaptations in the musculoskeletal system, or for conducting selection experiments to study evolutionary outcomes (Garland 2003). Continual advances in the field of genomics, particularly in the technology sector, have resulted in a more comprehensive understanding of the mouse genome, including the creation of new laboratory strains (Schughart and Churchill 2007). Relatedly, experimental manipulation of the mouse genome (e.g., knock-outs or transgenics) is an expanding area of research using mouse models (Austin et al. 2004). Thorough documentation of the mouse genome creates the possibility for tracing phenotypic responses (and differences therein) to precise genes through comparisons among inbred strains. This provides a powerful tool for studying evolutionary morphology (Cheverud et al. 1998). Having briefly described why mouse models would be beneficial for functional morphologists, an eclectic series of investigations were assembled into a symposium in order to demonstrate how mouse models are beneficial in practice. The goal was to craft a program that featured a range of musculoskeletal adaptations, including both cranial and postcranial studies. Three of the contributions use a mouse model to address specific form–function relationships between the cranium/mandible and feeding adaptations. Byron et al. (2008) use a myostatin-deficient mouse model to investigate the role of presumed compressive forces (e.g., those generated during mastication) on the morphology of the temporal bone and squamosal suture. While they anticipated that more heavily muscled myostatin-deficient mice should have exhibited expanded temporal bones and more extensive overlap of adjoining cranial bones at the squamosal suture relative to wild-type controls, the opposite was observed. Myostatin-deficient mice exhibit less overlap of adjoining cranial bones at the squamosal suture relative to wild-type mice. Byron et al. suggest implications for interpreting cranial morphology of fossil hominins. Ravosa et al. (2008) also use a

Using "Mighty Mouse" to understand masticatory plasticity: myostatin-deficient mice and musculoskeletal function

Knockout mice lacking myostatin (Mstn), a negative regulator of the growth of skeletal muscle, develop significant increases in the relative mass of masticatory muscles as well as the ability to generate higher maximal muscle forces. Wild-type and Mstn-deficient mice were compared to investigate the postnatal influence of elevated masticatory loads due to increased jaw-adductor and bite forces on the biomineralization of mandibular articular and cortical bone, the internal structure of the jaw joints, and the composition of temporomandibular joint (TMJ) articular cartilage. To provide an interspecific perspective on the long-term responses of mammalian jaw joints to altered loading conditions, the findings on mice were compared to similar data for growing rabbits subjected to long-term dietary manipulation. Statistically significant differences in joint proportions and bone mineral density between normal and Mstn-deficient mice, which are similar to those observed between rabbit loading cohorts, underscore the need for a comprehensive analysis of masticatory tissue plasticity vis-à-vis altered mechanical loads, one in which variation in external and internal structure are considered. Differences in the expression of proteoglycans and type-II collagen in TMJ articular cartilage between the mouse and rabbit comparisons suggest that the duration and magnitude of the loading stimulus will significantly affect patterns of adaptive and degradative responses. These data on mammals subjected to long-term loading conditions offer novel insights regarding variation in ontogeny, life history, and the ecomorphology of the feeding apparatus.

Bigger Muscles and Smaller Tendons ‐ Tendons of Myostatin Deficient Mice are Small, Brittle, and Hypocellular

Tendons play a significant role in the modulation of forces transmitted between bones and skeletal muscles and consequently protect muscle fibers from contraction‐induced, or high strain, injuries. Myostatin (MSTN) is a negative regulator of muscle mass. Inhibition of myostatin not only increases the mass and maximum isometric force of muscles, but also increases the susceptibility of muscle fibers to contraction‐induced injury. Due to the protective role of tendons in preventing contraction‐induced injury, and the greater susceptibility of muscles from MSTN−/− mice to contraction‐induced injury, we hypothesized that myostatin would regulate the morphology and mechanical properties of tendons. The expression of myostatin and the myostatin receptors, ACVR2B and ACVRB, were detectable in tendons. Surprisingly, compared with MSTN+/+ mice, the tendons of myostatin‐deficient mice MSTN−/− were smaller, had a decrease in fibroblast density and a decrease in the expression of type I collagen. Tendons of MSTN−/− mice also had a decrease in the expression of two genes that promote tendon fibroblast proliferation, scleraxis and tenomodulin. Treatment of tendon fibroblasts with myostatin activated the p38 MAPK and Smad2/3 signaling cascades, increased cell proliferation and increased the expression of type I collagen, scleraxis and tenomodulin. Compared with the tendons of MSTN+/+ mice, the mechanical properties of tibialis anterior tendons from MSTN−/− mice had a greater peak stress, lower peak strain and a increased stiffness. We conclude that in addition to the regulation of muscle mass and force, myostatin regulates the structure and function of tendon tissues.

Tendons of myostatin-deficient mice are small, brittle, and hypocellular

Tendons play a significant role in the modulation of forces transmitted between bones and skeletal muscles and consequently protect muscle fibers from contraction-induced, or high-strain, injuries. Myostatin (GDF-8) is a negative regulator of muscle mass. Inhibition of myostatin not only increases the mass and maximum isometric force of muscles, but also increases the susceptibility of muscle fibers to contraction-induced injury. We hypothesized that myostatin would regulate the morphology and mechanical properties of tendons. The expression of myostatin and the myostatin receptors ACVR2B and ACVRB was detectable in tendons. Surprisingly, compared with wild type (MSTN(+/+)) mice, the tendons of myostatin-null mice (MSTN(-/-)) were smaller and had a decrease in fibroblast density and a decrease in the expression of type I collagen. Tendons of MSTN(-/-) mice also had a decrease in the expression of two genes that promote tendon fibroblast proliferation: scleraxis and tenomodulin. Treatment of tendon fibroblasts with myostatin activated the p38 MAPK and Smad2/3 signaling cascades, increased cell proliferation, and increased the expression of type I collagen, scleraxis, and tenomodulin. Compared with the tendons of MSTN(+/+) mice, the mechanical properties of tibialis anterior tendons from MSTN(-/-) mice had a greater peak stress, a lower peak strain, and increased stiffness. We conclude that, in addition to the regulation of muscle mass and force, myostatin regulates the structure and function of tendon tissues.

Craniofacial Morphology in Myostatin-deficient Mice

GDF-8 (myostatin) is a negative growth regulator of skeletal muscle, and myostatin-deficient mice are hypermuscular. Muscle size and force production are thought to influence growth of the craniofacial skeleton. To test this relationship, we compared masticatory muscle size and craniofacial dimensions in myostatin-deficient and wild-type CD-1 control mice. Myostatin-deficient mice had significantly (p < 0.01) greater body (by 18%) and masseter muscle weight (by 83%), compared with wild-type controls. Significant differences (p < 0.05) were noted for cranial vault length, maxillary length, mandibular body length, and mandibular shape index. Significant correlations were noted between masseter muscle weight and mandibular body length (r = 0.68; p < 0.01), cranial vault length (r = -0.57; p < 0.05), and the mandibular shape index (r = -0.56; p < 0.05). Masticatory hypermuscularity resulted in significantly altered craniofacial morphology, probably through altered biomechanical stress. These findings emphasize the important role that masticatory muscle function plays in the ontogeny of the cranial vault, the maxilla, and, most notably, the mandible.

Loss of myostatin (GDF8) function increases osteogenic differentiation of bone marrow-derived mesenchymal stem cells but the osteogenic effect is ablated with unloading

Myostatin (GDF8) is a negative regulator of skeletal muscle growth and mice lacking myostatin show a significant increase in muscle mass and bone density compared to normal mice. In order to further define the role of myostatin in regulating bone mass we sought to determine if loss of myostatin function significantly altered the potential for osteogenic differentiation in bone marrow-derived mesenchymal stem cells in vitro and in vivo. We first examined expression of the myostatin receptor, the type IIB activin receptor (AcvrIIB), in bone marrow-derived mesenchymal stem cells (BMSCs) isolated from mouse long bones. This receptor was found to be expressed at high levels in BMSCs, and we were also able to detect AcvrIIB protein in BMSCs in situ using immunofluorescence. BMSCs isolated from myostatin-deficient mice showed increased osteogenic differentiation compared to wild-type mice; however, treatment of BMSCs from myostatin-deficient mice with recombinant myostatin did not attenuate the osteogenic differentiation of these cells. Loading of BMSCs in vitro increased the expression of osteogenic factors such as BMP-2 and IGF-1, but treatment of BMSCs with recombinant myostatin was found to decrease the expression of these factors. We investigated the effects of myostatin loss-of-function on the differentiation of BMSCs in vivo using hindlimb unloading (7-day tail suspension). Unloading caused a greater increase in marrow adipocyte number, and a greater decrease in osteoblast number, in myostatin-deficient mice than in normal mice. These data suggest that the increased osteogenic differentiation of BMSCs from mice lacking myostatin is load-dependent, and that myostatin may alter the mechanosensitivity of BMSCs by suppressing the expression of osteogenic factors during mechanical stimulation. Furthermore, although myostatin deficiency increases muscle mass and bone strength, it does not prevent muscle and bone catabolism with unloading.

Plasticity of mandibular biomineralization in myostatin‐deficient mice

Compared with the normal or wild-type condition, knockout mice lacking myostatin (Mstn), a negative regulator of skeletal muscle growth, develop significant increases in relative masticatory muscle mass as well as the ability to generate higher maximal muscle forces. Wild-type and myostatin-deficient mice were compared to assess the postweaning influence of elevated masticatory loads because of increased jaw-adductor muscle and bite forces on the biomineralization of mandibular cortical bone and dental tissues. Microcomputed tomography (microCT) was used to quantify bone density at a series of equidistant external and internal sites in coronal sections for two symphysis and two corpus locations. Discriminant function analyses and nonparametric ANOVAs were used to characterize variation in biomineralization within and between loading cohorts. Multivariate analyses indicated that 95% of the myostatin-deficient mice and 95% of the normal mice could be distinguished based on biomineralization values at both symphysis and corpus sections. At the corpus, ANOVAs suggest that between-group differences are due to the tendency for cortical bone mineralization to be higher in myostatin-deficient mice, coupled with higher levels of dental biomineralization in normal mice. At the symphysis, ANOVAs indicate that between-group differences are related to significantly elevated bone-density levels along the articular surface and external cortical bone in the knockout mice. Both patterns, especially those for the symphysis, appear because of the postweaning effects of increased masticatory stresses in the knockout mice versus normal mice. The greater number of symphyseal differences suggest that bone along this jaw joint may be characterized by elevated plasticity. Significant differences in bone-density levels between normal and myostatin-deficient mice, coupled with the multivariate differences in patterns of plasticity between the corpus and symphysis, underscore the need for a comprehensive analysis of the plasticity of masticatory tissues vis-à-vis altered mechanical loads.

Lack of myostatin results in excessive muscle growth but impaired force generation.

The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null (Mstn(-/-)) and compact (Berlin High Line, BEH(c/c)). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn(-/-) muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.