Effect of storage conditions on the quality of equine and canine mesenchymal stem cell derived nanoparticles including extracellular vesicles for research and therapy.

Nanoparticles including extracellular vesicles derived from mesenchymal stem cells are of increasing interest for research and clinical use in regenerative medicine. Extracellular vesicles (EVs), including also previously named exosomes, provide a promising cell-free tool for therapeutic applications, which is probably a safer approach to achieve sufficient healing. Storage of EVs may be necessary for clinical applications as well as for further experiments, as the preparation is sometimes laborious and larger quantities tend to be gained. For this purpose, nanoparticles were obtained from mesenchymal stem cells from adipose tissue (AdMSC) of horses and dogs. The EVs were then stored for 7 days under different conditions (- 20 °C, 4 °C, 37 °C) and with the addition of various additives (5 mM EDTA, 25-250 µM trehalose). Afterwards, the size and number of EVs was determined using the nano tracking analyzing method. With our investigations, we were able to show that storage of EVs for up to 7 days at 4 °C does not require the addition of supplements. For the other storage conditions, in particular freezing and storage at room temperature, the addition of EDTA was found to be suitable for preventing aggregation of the particles. Contrary to previous publications, trehalose seems not to be a suitable cryoprotectant for AdMSC-derived EVs. The data are useful for processing and storage of isolated EVs for further experiments or clinical approaches in veterinary medicine.

Optimizing the Adipogenic Induction Protocol Using Rosiglitazone Improves the Physiological Parameters and Differentiation Capacity of Adipose Tissue-Derived Mesenchymal Stem Cells for Horses, Sheep, Dogs, Murines, and Humans.

The investigation of adipose tissue-derived mesenchymal stem cells (ASCs) has received considerable interest in regenerative medicine. A nontoxic adipogenic induction protocol valid for cells of different mammalian species has not been described. This study aims to establish an adipogenic differentiation protocol suitable for horses, sheep, dogs, murines, and human cells. An optimized rosiglitazone protocol, consisting of 5% fetal calf serum in Dulbecco's Modified Eagle's Medium, 10 µg/mL insulin, 0.55 µg/mL transferrin, 6.8 ng sodium selenite, 1 µM dexamethasone, and 1-5 µM of rosiglitazone, is compared to the 3-isobutyl-1-methylxantine (IBMX) protocol, where rosiglitazone was replaced with 0.5 mM IBMX and 0.2 mM indomethacin. Cell viability, cytotoxicity, a morphometric analysis of the lipid, and the expression of adipogenic markers for 14 days were assessed. The data revealed that using 5 µM of rosiglitazone promotes the adipogenic differentiation capacity in horse, sheep, and dog cells compared to IBMX induction. Meanwhile, marked reductions in the cell viability and cell number with the IBMX protocol were detected, and rosiglitazone increased the cell number and lipid droplet size, prevented apoptosis, and upregulated FABP-4 and Leptin expression in the cells of most of the species. Our data revealed that the rosiglitazone protocol improves the adipogenesis of ASCs, together with having less toxicity, and should be considered for cell reproducibility and clinical applications targeting obesity.

The effect of hypoxia on myogenic differentiation and multipotency of the skeletal muscle-derived stem cells in mice.

BACKGROUND: Skeletal muscle-derived stem cells (SC) have become a promising approach for investigating myogenic differentiation and optimizing tissue regeneration. Muscle regeneration is performed by SC, a self-renewal cell population underlying the basal lamina of muscle fibers. Here, we examined the impact of hypoxia condition on the regenerative capacity of SC either in their native microenvironment or via isolation in a monolayer culture using ectopic differentiation inductions. Furthermore, the effect of low oxygen tension on myogenic differentiation protocols of the myoblasts cell line C2C12 was examined. METHODS: Hind limb muscles of wild type mice were processed for both SC/fiber isolation and myoblast extraction using magnetic beads. SC were induced for myogenic, adipogenic and osteogenic commitments under normoxic (21% O2) and hypoxic (3% O2) conditions. SC proliferation and differentiation were evaluated using histological staining, immunohistochemistry, morphometric analysis and RT-qPCR. The data were statistically analyzed using ANOVA. RESULTS: The data revealed enhanced SC proliferation and motility following differentiation induction after 48 h under hypoxia. Following myogenic induction, the number of undifferentiated cells positive for Pax7 were increased at 72 h under hypoxia. Hypoxia upregulated MyoD and downregulated Myogenin expression at day-7 post-myogenic induction. Hypoxia promoted both SC adipogenesis and osteogenesis under respective induction as shown by using Oil Red O and Alizarin Red S staining. The expression of adipogenic markers; peroxisome proliferator activated receptor gamma (PPARγ) and fatty acid-binding protein 4 (FABP4) were upregulated under hypoxia up to day 14 compared to normoxic condition. Enhanced osteogenic differentiation was detected under hypoxic condition via upregulation of osteocalcin and osteopontin expression up to day 14 as well as, increased calcium deposition at day 21. Hypoxia exposure increases the number of adipocytes and the size of fat vacuoles per adipocyte compared to normoxic culture. Combining the differentiation medium with dexamethasone under hypoxia improves the efficiency of the myogenic differentiation protocol of C2C12 by increasing the length of the myotubes. CONCLUSIONS: Hypoxia exposure increases cell resources for clinical applications and promotes SC multipotency and thus beneficial for tissue regeneration.

Characterisation of stemness and multipotency of ovine muscle-derived stem cells from various muscle sources.

Muscle stem cells (MSCs) are a promising tool for cell-based therapy and tissue regeneration in veterinary medicine. Evaluation of MSCs from muscles of different origins improves our understanding of their regenerative potential. The present study compared the stemness, cell proliferation, migration potential, myogenic differentiation (MD), and multipotency of MSCs for four developmentally different muscles of ovine origin. MSCs were isolated from the hind limb (HL), diaphragm (DI), extraocular (EO), and masseter (MS) muscles. Cell proliferation, migration, and stemness were examined using sulforhodamine B, and colony formation assays. Evaluation of multipotency was examined using histological and morphometric analyses, alkaline phosphatase (ALP) activity, and the expression of myogenic, adipogenic, and osteogenic markers using RT-qPCR. Data were statistically analysed using analysis of variance. The results revealed that all experimental groups expressed stem cell markers paired box transcription factor Pax7, α7-integrin, CD90, and platelet-derived growth factor receptor alpha. DI and HL muscle cells displayed higher proliferation, migration, and colony formation capacities compared to the EO and MS muscle cells. HL and DI muscle cells showed increased MD, as indicated by myotube formation and relative expression of MyoD at day 7 and Myogenin at day 14. Although MS and EO muscle cells displayed impaired MD, these cells were more prone to adipogenic differentiation, as indicated by Oil Red O staining and upregulated fatty acid-binding protein 4 and peroxisome proliferator-activated receptor gamma expression. DI muscle cells demonstrated a higher osteogenic differentiation capability, as shown by the upregulation of osteopontin expression and an elevated ALP activity. Our data indicate that ovine HL and DI MSCs have a higher regenerative and multipotent potential than the EO and MS muscle cells. These results could be valuable for regional muscle biopsies and cell-based therapies.

Combined macromolecule biomaterials together with fluid shear stress promote the osteogenic differentiation capacity of equine adipose-derived mesenchymal stem cells.

BACKGROUND: Combination of mesenchymal stem cells (MSCs) and biomaterials is a rapidly growing approach in regenerative medicine particularly for chronic degenerative disorders including osteoarthritis and osteoporosis. The present study examined the effect of biomaterial scaffolds on equine adipose-derived MSC morphology, viability, adherence, migration, and osteogenic differentiation. METHODS: MSCs were cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and combined B30 with strontium (B30Str) biomaterials in osteogenic differentiation medium either under static or mechanical fluid shear stress (FSS) culture conditions. The data were generated by histological means, live cell imaging, cell viability, adherence and migration assays, semi-quantification of alkaline phosphatase (ALP) activity, and quantification of the osteogenic markers runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression. RESULTS: The data revealed that combined mechanical FSS with MC but not B30 enhanced MSC viability and promoted their migration. Combined osteogenic medium with MC, B30, and B30Str increased ALP activity compared to cultivation in basal medium. Osteogenic induction with MC, B30, and B30Str resulted in diffused matrix mineralization. The combined osteogenic induction with biomaterials under mechanical FSS increased Runx2 protein expression either in comparison to those cells cultivated in BM or those cells induced under static culture. Runx2 and ALP expression was upregulated following combined osteogenic differentiation together with B30 and B30Str regardless of static or FSS culture. CONCLUSIONS: Taken together, the data revealed that FSS in conjunction with biomaterials promoted osteogenic differentiation of MSCs. This combination may be considered as a marked improvement for clinical applications to cure bone defects.

Canine Adipose-Derived Mesenchymal Stem Cells (cAdMSCs) as a "Trojan Horse" in Vaccinia Virus Mediated Oncolytic Therapy against Canine Soft Tissue Sarcomas.

Several oncolytic viruses (OVs) including various human and canine adenoviruses, canine distemper virus, herpes-simplex virus, reovirus, and members of the poxvirus family, such as vaccinia virus and myxoma virus, have been successfully tested for canine cancer therapy in preclinical and clinical settings. The success of the cancer virotherapy is dependent on the ability of oncolytic viruses to overcome the attacks of the host immune system, to preferentially infect and lyse cancer cells, and to initiate tumor-specific immunity. To date, several different strategies have been developed to overcome the antiviral host defense barriers. In our study, we used canine adipose-derived mesenchymal stem cells (cAdMSCs) as a "Trojan horse" for the delivery of oncolytic vaccinia virus Copenhagen strain to achieve maximum oncolysis against canine soft tissue sarcoma (CSTS) tumors. A single systemic administration of vaccinia virus-loaded cAdMSCs was found to be safe and led to the significant reduction and substantial inhibition of tumor growth in a CSTS xenograft mouse model. This is the first example that vaccinia virus-loaded cAdMSCs could serve as a therapeutic agent against CSTS tumors.

Effects of testosterone and 17ß-estradiol on osteogenic and adipogenic differentiation capacity of human bone-derived mesenchymal stromal cells of postmenopausal women.

Progressive bone loss is a predominant symptom of aging and osteoporosis. Therefore, the effects of sex steroids (i.e. testosterone and 17ß-estradiol) on the differentiation capacity of human bone-derived mesenchymal stromal cells (hMSCs), as progenitors of osteoblasts and adipocytes, are of particular interest. The objectives of the present study were, thus, to elucidate whether bone-derived hMSCs of postmenopausal women produce aromatase (CYP19A1) and, whether they modulate their differentiation behaviour in response to testosterone and 17ß-estradiol (E2), in relation to their steroid receptor expression. Supplementation of testosterone resulted in a considerable formation of E2 under osteogenic and adipogenic culture conditions, whereas E2 synthesis remained minimal in the cells cultured in basal medium. Concomitant with high aromatase expression and 17ß-estradiol formation of the cells cultured in osteogenic medium supplemented with testosterone, a distinct promotion of late-stage osteogenesis was found, as shown by significant matrix mineralization and a notable increase in osteogenic markers. These effects were abrogated by the aromatase inhibitor anastrozole. Under adipogenic conditions, testosterone reduced the occurrence of lipid droplets and led to a decrease in PPARγ and AR expression, independent of anastrozole. Regardless of the culture conditions, ERα was detectable whilst ERß was not. In conclusion, aromatase activity is limited to differentiated hMSCs and the resulting 17ß-estradiol enhances late osteogenic differentiation stages via ERα. Adipogenic differentiation, on the other hand, is reduced by both sex steroids: testosterone via AR and 17ß-estradiol.

Investigation of stemness and multipotency of equine adipose-derived mesenchymal stem cells (ASCs) from different fat sources in comparison with lipoma.

BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ASCs) offer a promising cell source for therapeutic applications in musculoskeletal disorders. The appropriate selection of ASCs from various fat depots for cell-based therapy is challenging. The present study aims to compare stemness and multipotency of ASCs derived from retroperitoneal (RP), subcutaneous (SC), and lipoma (LP) fat to assess their usefulness for clinical application. METHODS: Equine ASCs from the three fat tissue sources were isolated and characterized. The cell viability, proliferation, and self-renewal were evaluated using MTT, sulforhodamine B, and colony forming unit (CFU) assays. Stem cell relative marker CD44, CD90, and CD105 and tumor marker CA9 and osteopontin (OPN) expression were quantified using RT-qPCR. Multipotency of ASCs for adipogenic, osteogenic, and chondrogenic differentiation was examined by quantifying Oil Red O and Alizarin Red S staining, alkaline phosphatase activity (ALP), and expression of differentiation relative markers. All data were statistically analyzed using ANOVA. RESULTS: RP fat-derived ASCs showed a higher cell proliferation rate compared to SC and LP derived cells. In contrast, ASCs from lipoma displayed a lower proliferation rate and impaired CFU capacities. The expression of CD44, CD90, and CD105 was upregulated in RP and SC derived cells but not in LP cells. RP fat-derived cells displayed a higher adipogenic potential compared to SC and LP cells. Although ASCs from all fat sources showed enhanced ALP activity following osteogenic differentiation, SC fat-derived cells revealed upregulated ALP and bone morphogenetic protein-2 expression together with a higher calcium deposition. We found an enhanced chondrogenic potency of RP and SC fat-derived cells as shown by Alcian blue staining and upregulation of aggrecan (Aggre), cartilage oligomeric matrix protein precursor (COMP), and collagen 2a1 (Col2a1) expression compared to LP. The expression of OPN and CA9 was exclusively upregulated in the ASCs of LP. CONCLUSIONS: The results provide evidence of variation in ASC performance not only between normal fat depots but also compared to LP cells which suggest a different molecular regulation controlling the cell fate. These data provided are useful when considering a source for cell replacement therapy in equine veterinary medicine.

The effect of high-fat diet on the morphological properties of the forelimb musculature in hypertrophic myostatin null mice.

Obesity is a worldwide nutritional disorder affecting body performance, including skeletal muscle. Inhibition of myostatin not only increases the muscle mass but also it reduces body fat accumulation. We examined the effect of high-fat diet on the phenotypic properties of forelimb muscles from myostatin null mice. Male wild-type and myostatin null mice were fed on either a normal diet or a high-fat diet (45% fat) for 10 weeks. Musculus triceps brachii Caput longum; M. triceps brachii Caput laterale; M. triceps brachii Caput mediale; M. extensor carpi ulnaris and M. flexor carpi ulnaris were processed for fiber type composition using immunohistochemistry and morphometric analysis. Although the muscle mass revealed no change under a high-fat diet, there were morphometric alterations in the absence of myostatin. We show that high-fat diet reduces the cross-sectional area of the fast (IIB and IIX) fibers in M. triceps brachii Caput longum and M. triceps brachii Caput laterale of both genotypes. In contrast, increases of fast fiber areas were observed in both M. extensor carpi ulnaris of wild-type and M. flexor carpi ulnaris of myostatin null mice. Meanwhile, a high-fat diet increased the area of the fast IIA fibers in wild-type mice; myostatin null mice display a muscle-dependent alteration in the area of the same fiber type. The combined high-fat diet and myostatin deletion shows no effect on the area of slow type I fibers. Although a high-fat diet causes a reduction in the area of the peripheral IIB fibers in both genotypes, only myostatin null mice show an increase in the area of the central IIB fibers. We provide evidence that a high-fat diet induces a muscle-dependent fast to slow myofiber shift in the absence of myostatin. The data suggest that the morphological alterations of muscle fibers under a combined high-fat diet and myostatin deletion reflect a functional adaptation of the muscle to utilize the high energy intake.

Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells.

Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes osteogenic differentiation of equine adipose-derived stem cells (ASCs). ASCs were induced into osteogenic fate using osteogenic differentiation medium (ODM) protocol or additional supplementation of 5 mM CaCl2 and 7.5 mM CaCl2 protocol compared to cells cultivated in basal medium (BM) up to 21 day. The ASCs proliferation pattern was evaluated using the sulforhodamine B (SRB) protein assay. Osteogenic differentiation examined via semi-quantification of alizarin red staining (ARS) and alkaline phosphatase activity (ALP) as well as, via quantification of osteocalcin (OC), alkaline phosphatase (ALP), osteopontin (OP), and collagen type-1 (COL1) gene expression using RT-qPCR. We show that mechanical FSS increased the proliferation pattern of ASCs compared to the static conditions. Mechanical FSS together with 5 mM CaCl2 and 7.5 mM CaCl2 promoted osteogenic nodule formation and increased ARS intensity compared to the standard osteogenic protocols. We observed that combined mechanical FSS with ODM protocol increase ALP activity compared to static culture conditions. We report that ALP and OC osteogenic markers expression were upregulated under mechanical FSS culture condition particularly with the ODM protocol. Taken together, it can be assumed that mechanical stress using FSS promotes the efficiency of the osteogenic differentiation protocols of ASCs through independent mechanisms.

Exosomes isolation and identification from equine mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characterization and usage in functional assays. RESULTS: It was possible to obtain exosomes secreted from equine adipose derived mesenchymal stem cells with three common techniques: a stepwise ultracentrifugation at 100.000 g, an ultrafiltration with 3 kDa exclusion membranes and a charge-based precipitation method. The mean sizes and amounts of exosomes isolated with the different techniques were measured by the nanoparticle tracking analysis. The diameter ranged between 116.2 nm (ultracentrifugation), 453.1 nm (precipitation) and 178.7 nm (ultrafiltration), the counts of particles / ml ranged between 9.6 × 108 (ultracentrifugation), 2.02 × 109 (precipitation) and 52.5 × 109 (ultrafiltration). Relevant marker for exosomes, tetraspanins CD9, CD63 and CD81 were detectable by immunofluorescence staining of the investigated exosomes secreting mesenchymal stem cells. In addition, transmission electron microscopy and immunogold labeling with CD9 and CD90 was performed to display the morphological shape of exosomes and existence of marker relevant for exosomes (CD9) and mesenchymal stem cells (CD90). Western blot analysis of CD9 and CD90 of exosomes ensured the specificity of the rare available respectively cross reacting antibodies against equine antigens. CONCLUSION: Exosomes generated by equine mesenchymal stem cells can be obtained by ultrafiltration and ultracentrifugation in an equal quality for in vitro experiments. Especially for later therapeutic usage we recommend ultrafiltration due to a higher concentration without aggregation of extracellular vesicles in comparison to exosomes obtained by ultracentrifugation.

Osteogenic differentiation of equine adipose tissue derived mesenchymal stem cells using CaCl2.

Adipose tissue derived mesenchymal stem cells (ASCs) may be used to cure bone defects after osteogenic differentiation. In this study we tried to optimize osteogenic differentiation for equine ASCs using various concentrations of CaCl2 in comparison to the standard osteogenic protocol. ASCs were isolated from subcutaneous adipose tissue from mixed breed horses. The osteogenic induction protocols were (1) the standard osteogenic medium (OM) composed of dexamethasone, ascorbic acid and ß-glycerol phosphate; (2) CaCl2 based protocol composed of 3, 5 and 7.5mM CaCl2. Differentiation and proliferation were evaluated at 7, 10, 14 and 21days post-differentiation induction using the alizarin red staining (ARS) detecting matrix calcification. Semi-quantification of cell protein content, ARS and alkaline phosphatase activity (ALP) were performed using an ELISA reader. Quantification of the transcription level for the common osteogenic markers alkaline phosphatase (ALP) and Osteopontin (OP) was performed using RT-qPCR. In the presence of CaCl2, a concentration dependent effect on the osteogenic differentiation capacity was evident by the ARS evaluation and OP gene expression. We provide evidence that 5 and 7mM CaCl2 enhance the osteogenic differentiation compared to the OM protocol. Although, there was a clear commitment of ASCs to the osteogenic fate in the presence of 5 and 7mM CaCl2, cell proliferation was increased compared to OM. We report that an optimized CaCl2 protocol reliably influences ASCs osteogenesis while conserving the proliferation capacity. Thus, using these protocols provide a platform for using ASCs as a cell source in bone tissue engineering.

Multipotency of skeletal muscle stem cells on their native substrate and the expression of Connexin 43 during adoption of adipogenic and osteogenic fate.

Muscle regeneration is performed by resident muscle stem cells called satellite cells (SC). However they are multipotent, being able to adopt adipogenic and osteogenic fate under the correct stimuli. Since SC behavior can be regulated by the extra-cellular matrix, we examined the robustness of the myogenic programme of SC on their native substrate-the surface of a myofiber. We show that the native substrate supports myogenic differentiation judged by the expression of members of the Myogenic Determination Factor (MRF) family. However SC even on their native substrate can be induced into adopting adipogenic or osteogenic fate. Furthermore conditions that support adipose or bone formation inhibit the proliferation of SC progeny as well as their migration. We show that Connexin43 (Cx43), a gap junction complex protein, is only expressed by activated and not quiescent SC. Furthermore, it is not expressed by SC that are in the process of changing their fate. Lastly we show that intact adult mouse muscle contains numerous cells expressing Cx43 and that the density of these cells seems to be related to capillary density. We suggest the Cx43 expression is localized to angioblasts and is more prominent in oxidative slow muscle compared to glycolytic fast muscle.

The effect of caloric restriction on the forelimb skeletal muscle fibers of the hypertrophic myostatin null mice.

Skeletal muscle mass loss has a broad impact on body performance and physical activity. Muscle wasting occurs due to genetic mutation as in muscular dystrophy, age-related muscle loss (sarcopenia) as well as in chronic wasting disorders as in cancer cachexia. Food restriction reduces muscle mass underpinned by increased muscle protein break down. However the influence of dietary restriction on the morphometry and phenotype of forelimb muscles in a genetically modified myostatin null mice are not fully characterized. The effect of a five week dietary limitation on five anatomically and structurally different forelimb muscles was examined. C57/BL6 wild type (Mstn+/+) and myostatin null (Mstn-/-) mice were either given a standard rodent normal daily diet ad libitum (ND) or 60% food restriction (FR) for a 5 week period. M. triceps brachii Caput laterale (T.lateral), M. triceps brachii Caput longum (T.long), M. triceps brachii Caput mediale (T.medial), M. extensor carpi ulnaris (ECU) and M. flexor carpi ulnaris (FCU) were dissected, weighted and processed for immunohistochemistry. Muscle mass, fibers cross sectional areas (CSA) and myosin heavy chain types IIB, IIX, IIA and type I were analyzed. We provide evidence that caloric restriction results in muscle specific weight reduction with the fast myofibers being more prone to atrophy. We show that slow fibers are less liable to dietary restriction induced muscle atrophy. The effect of dietary restriction was more pronounced in Mstn-/- muscles to implicate the oxidative fibers compared to Mstn+/+. Furthermore, peripherally located myofibers are more susceptible to dietary induced reduction compared to deep fibers. We additionally report that dietary restriction alters the glycolytic phenotype of the Mstn-/- into the oxidative form in a muscle dependent manner. In summary our study shows that calorie restriction alters muscle fiber profile of forelimb muscles of Myostatin null mice.

Characterisation of connective tissue from the hypertrophic skeletal muscle of myostatin null mice.

Myostatin is a potent inhibitor of muscle development. Genetic deletion of myostatin in mice results in muscle mass increase, with muscles often weighing three times their normal values. Contracting muscle transfers tension to skeletal elements through an elaborate connective tissue network. Therefore, the connective tissue of skeletal muscle is an integral component of the contractile apparatus. Here we examine the connective tissue architecture in myostatin null muscle. We show that the hypertrophic muscle has decreased connective tissue content compared with wild-type muscle. Secondly, we show that the hypertrophic muscle fails to show the normal increase in muscle connective tissue content during ageing. Therefore, genetic deletion of myostatin results in an increase in contractile elements but a decrease in connective tissue content. We propose a model based on the contractile profile of muscle fibres that reconciles this apparent incompatible tissue composition phenotype.

Exercise training attenuates the hypermuscular phenotype and restores skeletal muscle function in the myostatin null mouse.

Myostatin regulates both muscle mass and muscle metabolism. The myostatin null (MSTN(-/-)) mouse has a hypermuscular phenotype owing to both hypertrophy and hyperplasia of the myofibres. The enlarged muscles display a reliance on glycolysis for energy production; however, enlarged muscles that develop in the absence of myostatin have compromised force-generating capacity. Recent evidence has suggested that endurance exercise training increases the oxidative properties of muscle. Here, we aimed to identify key changes in the muscle phenotype of MSTN(-/-) mice that can be induced by training. To this end, we subjected MSTN(-/-) mice to two different forms of training, namely voluntary wheel running and swimming, and compared the response at the morphological, myocellular and molecular levels. We found that both regimes normalized changes of myostatin deficiency and restored muscle function. We showed that both exercise training regimes increased muscle capillary density and the expression of Ucp3, Cpt1α, Pdk4 and Errγ, key markers for oxidative metabolism. Cross-sectional area of hypertrophic myofibres from MSTN(-/-) mice decreased towards wild-type values in response to exercise and, in this context, Bnip3, a key autophagy-related gene, was upregulated. This reduction in myofibre size caused an increase of the nuclear-to-cytoplasmic ratio towards wild-type values. Importantly, both training regimes increased muscle force in MSTN(-/-) mice. We conclude that impaired skeletal muscle function in myostatin-deficient mice can be improved through endurance exercise-mediated remodelling of muscle fibre size and metabolic profile.

Axon and muscle spindle hyperplasia in the myostatin null mouse.

Germline deletion of the myostatin gene results in hyperplasia and hypertrophy of the tension-generating (extrafusal) fibres in skeletal muscle. As this gene is expressed predominantly in myogenic tissues it offers an excellent model with which to investigate the quantitative relationship between muscle and axonal development. Here we show that skeletal muscle hyperplasia in myostatin null mouse is accompanied by an increase in nerve fibres in major nerves of both the fore- and hindlimbs. We show that axons within these nerves undergo hypertrophy. Furthermore, we provide evidence that the age-related neural atrophic process is delayed in the absence of myostatin. Finally, we show that skeletal muscle hyperplasia in the myostatin null mouse is accompanied by an increase in the number of muscle spindles (also called stretch receptors or proprioceptors). However, our work demonstrates that the mechanisms regulating intrafusal fibre hyperplasia and hypertrophy differ from those that control the aetiology of extrafusal fibres.

Altered primary and secondary myogenesis in the myostatin-null mouse.

Skeletal muscle fiber generation occurs principally in two myogenic phases: (1) Primary (embryonic) myogenesis when myoblasts proliferate and fuse to form primary myotubes and (2) secondary (fetal) myogenesis when successive waves of myoblasts fuse along the surface of the primary myotubes, giving rise to a population of smaller and more numerous secondary myotubes. This sequence of events determines fiber number and is completed at or soon after birth in most muscles of the mouse. The adult myostatin null mouse (MSTN(-/-)) displays both an increase in fiber number and size relative to wild type (MSTN(+/+)), suggesting a developmental origin for the hypermuscular phenotype. The focus of the present study was to determine at which point during myogenesis do MSTN(-/-) animals diverge from MSTN(+/+). To achieve this, we focused on the extensor digitorum longus (EDL) muscle and evaluated primary myotube number at embryonic day (E) 13.0 and E14.5 and secondary to primary myotube ratios at E18.5. We show that primary myotube number and size were significantly increased in the MSTN(-/-) mice by E14.5 and the secondary to primary myotube ratio increased at E18.5. This increase in the rate of fiber formation resulted in MSTN(-/-) mice harboring 87% of their final adult fiber number at E18.5, compared to only 73% in MSTN(+/+). An accelerated myogenic program in the MSTN(-/-) mice was further confirmed by our finding of an initial expansion in the myogenic stem cell (identified through Pax7 expression) and myoblast (identified through myogenin expression) cell pools at E14.5 in the EDL muscle of these animals that was, however, followed by a reduction of both populations of cells at E18.5 relative to MSTN(+/+). Overall these data suggest that the genetic loss of myostatin accelerates the developmental myogenic program of primary and secondary skeletal myogenesis.

Morphology and myofiber composition of skeletal musculature of the forelimb in young and aged wild type and myostatin null mice.

Most current research into therapeutic approaches to muscle diseases involves the use of the mouse as an experimental model. Furthermore, a major strategy to alleviate myopathic symptoms through enhancing muscle growth and regeneration is to inhibit the action of myostatin (Mstn), a transforming growth factor-beta (TGF-beta) family member that inhibits muscle growth. Presently, however, no study has expanded the morphological analysis of mouse skeletal muscle beyond a few individual muscles of the distal hindlimb, through which broad conclusions have been based. Therefore, we have initially undertaken an expansive analysis of the skeletal musculature of the mouse forelimb and highlighted the species-specific differences between equivalent muscles of the rat, another prominently used experimental model. Subsequently, we examined the musculature of the forelimb in both young and old adult wild-type (mstn(+/+)) and myostatin null (mstn(-/-)) mice and assessed the potential beneficial and detrimental effects of myostatin deletion on muscle morphology and composition during the aging process. We showed that: (1) the forelimb muscles of the mouse display a more glycolytic phenotype than those of the rat; (2) in the absence of myostatin, the induced myofiber hyperplasia, hypertrophy, and glycolytic conversion all occur in a muscle-specific manner; and, importantly, (3) the loss of myostatin significantly alters the dynamics of postnatal muscle growth and impairs age-related oxidative myofiber conversion.