Myostatin Deficiency Protects C2C12 Cells from Oxidative Stress by Inhibiting Intrinsic Activation of Apoptosis.

Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn-/- cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn-/- cells and could serve as basis for further research.

Deficiency of myostatin protects skeletal muscle cells from ischemia reperfusion injury.

Ischemia reperfusion (IR) injury plays a pivotal role in many diseases and leads to collateral damage during surgical interventions. While most studies focus on alleviating its severity in the context of brain, liver, kidney, and cardiac tissue, research as regards to skeletal muscle has not been conducted to the same extent. In the past, myostatin (MSTN), primarily known for supressing muscle growth, has been implicated in inflammatory circuits, and research provided promising results for cardiac IR injury mitigation by inhibiting MSTN cell surface receptor ACVR2B. This generated the question if interrupting MSTN signaling could temper IR injury in skeletal muscle. Examining human specimens from free myocutaneous flap transfer demonstrated increased MSTN signaling and tissue damage in terms of apoptotic activity, cell death, tissue edema, and lipid peroxidation. In subsequent in vivo MstnLn/Ln IR injury models, we identified potential mechanisms linking MSTN deficiency to protective effects, among others, inhibition of p38 MAPK signaling and SERCA2a modulation. Furthermore, transcriptional profiling revealed a putative involvement of NK cells. Collectively, this work establishes a protective role of MSTN deficiency in skeletal muscle IR injury.

Myostatin deficiency not only prevents muscle wasting but also improves survival in septic mice.

Sepsis remains a leading cause of mortality in critically ill patients. Muscle wasting is a major complication of sepsis and negatively affects clinical outcomes. Despite intense investigation for many years, the molecular mechanisms underlying sepsis-related muscle wasting are not fully understood. In addition, a potential role of muscle wasting in disease development of sepsis has not been studied. Myostatin is a myokine that downregulates skeletal muscle mass. We studied the effects of myostatin deficiency on muscle wasting and other clinically relevant outcomes, including mortality and bacterial clearance, in mice. Myostatin deficiency prevented muscle atrophy along with inhibition of increases in muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1 expression and phosphorylation of signal transducer and activator of transcription protein 3 (STAT3; major players of muscle wasting) in septic mice. Moreover, myostatin deficiency improved survival and bacterial clearance of septic mice. Sepsis-induced liver dysfunction, acute kidney injury, and neutrophil infiltration into the liver and kidney were consistently mitigated by myostatin deficiency, as indicated by plasma concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase activity in the organs. Myostatin deficiency also inhibited sepsis-induced increases in plasma high-mobility group protein B1 (HMGB1) and macrophage inhibitory cytokine (MIC)-1/growth differentiation factor (GDF)-15 concentrations. These results indicate that myostatin plays an important role not only in muscle wasting but also in other clinically relevant outcomes in septic mice. Furthermore, our data raise the possibility that muscle wasting may not be simply a complication, but myostatin-mediated muscle cachexia and related changes in muscle may actually drive the development of sepsis as well.NEW & NOTEWORTHY Muscle wasting is a major complication of sepsis, but its role in the disease development is not known. Myostatin deficiency improved bacterial clearance and survival and mitigated damage in the liver and kidney in septic mice, which paralleled prevention of muscle wasting. These results raise the possibility that muscle wasting may not simply be a complication of sepsis, but myostatin-mediated cachexic changes may have a role in impaired bacterial clearance and mortality in septic mice.

Muscle differentiation induced by p53 signaling pathway-related genes in myostatin-knockout quail myoblasts.

The myostatin (MSTN) gene is of interest in the livestock industry because mutations in this gene are closely related to growth performance and muscle differentiation. Thus, in this study, we established MSTN knockout (KO) quail myoblasts (QM7) and investigated the regulatory pathway of the myogenic differentiation process. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate MSTN KO QM7 cells and subsequently isolated a single cell-derived MSTN KO QM7 subline with 10- and 16-nucleotide deletions that induced translational frameshift mutations. The differentiation capacity and proliferation rate of MSTN KO QM7 cells were enhanced. We conducted next-generation-sequencing (NGS) analysis to compare the global gene expression profiles of wild-type (WT) QM7 and MSTN KO QM7 cells. Intriguingly, NGS expression profiles showed different expression patterns of p21 and p53 in MSTN KO QM7 cells. Moreover, we identified downregulated expression patterns of leukemia inhibitory factor and DNA Damage Inducible Transcript 4, which are genes in the p53 signaling pathway. Using quantitative RT-PCR (qRT-PCR) analysis and western blotting, we concluded that p53-related genes promote the cell cycle by upregulating p21 and enhancing muscle differentiation in MSTN KO QM7 cells. These results could be applied to improve economic traits in commercial poultry by regulating MSTN-related networks.

Enhanced skeletal muscle growth in myostatin-deficient transgenic pigs had improved glucose uptake in stretozotocin-induced diabetes.

The size of skeletal muscle mass plays a significant role in glucose uptake in healthy and diabetic human subjects. Previously, we have generated myostatin-deficient (MSTN-/-) transgenic pigs via animal cloning technology. MSTN-/- pigs had dramatic phenotype with individual muscle mass increase by 100% over their wild-type controls, which provides a unique large animal model to investigate how enhanced skeletal muscles are beneficial to glucose update in diabetes. We employed intravenous administration of stretozotocin (STZ) to male MSTN-/- and wild-type pigs (100 mg/kg body weight). One month later, blood glucose and insulin concentrations and pancreas histology were examined, STZ-induced diabetes occurred in both MSTN transgenic and wild-type pigs. Histology of pancreas, analysis of pAKT and Glut4 transporter proteins by Western blotting, and real-time qPCR for MSTN gene expression were used in the study. The STZ-treated pigs had increased levels of fasting plasma glucose and insulin levels in comparison with animals receiving sodium citrate buffer, their pancreas also had reduced beta cells and slight increases in lymphocyte. There are significant lower concentrations of fasting plasma glucose and insulin in MSTN-/- pigs than that of wild-type pigs after STZ administration. Detections of pAKT and Glut4 transporter proteins by Western blotting in muscle tissue indicates significant elevations of both proteins in MSTN-/- pigs compared with the wild-type pigs. The results from this pig model suggest that enhanced skeletal muscle by manipulation of myostatin function can improve glucose uptake even in the status of diabetes.

Mstn knockdown decreases the trans-differentiation from myocytes to adipocytes by reducing Jmjd3 expression via the SMAD2/SMAD3 complex.

Myostatin (Mstn) is an important growth/differentiation factor, and knockdown of Mstn reduces fat content. Here, we knocked down Mstn expression in C2C12 myoblasts and then induced adipogenic trans-differentiation in the cells. The effects of Mstn knockdown on lipid droplet contents and H3K27me3 marker expression on adipocyte-specific genes were detected. The results showed that Mstn knockdown reduced the formation of lipid droplets, downregulated the expression of adipocyte-specific genes, and increased H3K27me3 marker expression on adipocyte-specific genes. Chromatin immunoprecipitation analysis showed that the SMAD2/SMAD3 complex could combine with the Jumonji D3 (Jmjd3) promoter and that Mstn regulated Jmjd3 expression through this process. Jmjd3 overexpression removed the H3K27me3 marker and increased the expression of adipocyte-specific genes. Overall, our results showed that Mstn regulated Jmjd3 expression through SMAD2/SMAD3, thus affecting the H3K27me3 marker on adipocyte-specific genes and the trans-differentiation from myocytes to adipocytes.

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.

Regulation of the dystrophin-associated glycoprotein complex composition by the metabolic properties of muscle fibres.

The dystrophin-glycoprotein complex (DGC) links the muscle cytoskeleton to the extracellular matrix and is responsible for force transduction and protects the muscle fibres from contraction induced damage. Mutations in components of the DGC are responsible for muscular dystrophies and congenital myopathies. Expression of DGC components have been shown to be altered in many myopathies. In contrast we have very little evidence of whether adaptive changes in muscle impact on DGC expression. In this study we investigated connection between muscle fibre phenotype and the DGC. Our study reveals that the levels of DGC proteins at the sarcolemma differ in highly glycolytic muscle compared to wild-type and that these changes can be normalised by the super-imposition of an oxidative metabolic programme. Importantly we show that the metabolic properties of the muscle do not impact on the total amount of DGC components at the protein level. Our work shows that the metabolic property of a muscle fibre is a key factor in regulating the expression of DGC proteins at the sarcolemma.

The Possible Role of Complete Loss of Myostatin in Limiting Excessive Proliferation of Muscle Cells (C2C12) via Activation of MicroRNAs.

Myostatin (MSTN) is a member of the TGF-ß superfamily that negatively regulates skeletal muscle growth and differentiation. However, the mechanism by which complete MSTN deletion limits excessive proliferation of muscle cells remains unclear. In this study, we knocked out MSTN in mouse myoblast lines using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system and sequenced the mRNA and miRNA transcriptomes. The results show that complete loss of MSTN upregulates seven miRNAs targeting an interaction network composed of 28 downregulated genes, including TGFB1, FOS and RB1. These genes are closely associated with tumorigenesis and cell proliferation. Our study suggests that complete loss of MSTN may limit excessive cell proliferation via activation of miRNAs. These data will contribute to the treatment of rhabdomyosarcoma (RMS).

Effects of myostatin on the mechanical properties of muscles during repeated active lengthening in the mouse.

The aim of the present study was to investigate how myostatin dysfunction affects fast and slow muscle stiffness and viscosity during severe repeated loading. Isolated extensor digitorum longus (EDL) and soleus muscles of young adult female mice of the BEH (dysfunctional myostatin) and BEH+/+ (functional myostatin) strains were subjected to 100 contraction-stretching loading cycles during which contractile and mechanical properties were assessed. BEH mice exhibited greater exercise-induced muscle damage, although the effect was muscle- and age-dependent and limited to the early phases of simulated exercise. The relative reduction of the EDL muscle isometric force recorded during the initial 10-30 loading cycles was greater in BEH mice than in BEH+/+ mice and exceeded that of the soleus muscle of either strain. The induced damage was associated with lower muscle stiffness. The effects of myostatin on the mechanical properties of muscles depend on muscle type and maturity.

Efficient genome editing in cultured cells and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system.

Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutation efficiency in fibroblasts was as high as 87.5% in pig and 78.9% in buffalo. We then obtained single-cell clones with mutations at the specific sites of the GDF8 gene by screening with G418 in fibroblasts of pig and buffalo. In addition, the frequencies of Cas9/gRNA-mediated mutations were at 36 and 25% in the intracytoplasmic sperm injection embryos of pig and in vitro fertilization embryos of buffalo, respectively. Our work demonstrates that the Cas9/gRNA system is a highly efficient and fast tool for genome editing in cultured cells and embryos of Debao pig and swamp buffalo. These results can be helpful for the establishment of a new animal strain that can generate more meat.

Absence of Myostatin Improves Cardiac Function Following Myocardial Infarction.

BACKGROUND: Myostatin inhibits the development of skeletal muscle and regulates the proliferation of skeletal muscle fibroblasts. However, the role of myostatin in regulating cardiac muscle or myofibroblasts, specifically in acute myocardial infarction (MI), is less clear. This study sought to determine whether absence of myostatin altered left ventricular function post-MI. METHODS: Myostatin-null mice (Mstn-/-) and wild-type (WT) mice underwent ligation of the left anterior descending artery to induce MI. Left ventricular function was measured at baseline, days 1 and 28 post-MI. Immunohistochemistry and immunofluorescence were obtained at day 28 for cellular proliferation, collagen deposition, and myofibroblastic activity. RESULTS: Whilst left ventricular function at baseline and size of infarct were similar, significant differences in favour of Mstn-/- compared to WT mice post-MI include a greater recovery of ejection fraction (61.8±1.1% vs 57.1±2.3%, p<0.01), less collagen deposition (41.9±2.8% vs 54.7±3.4%, p<0.05), and lower mortality (0 vs. 20%, p<0.05). There was no difference in the number of BrdU positive cells, percentage of apoptotic cardiomyocytes, or size of cardiomyocytes post-MI between WT and Mstn-/- mice. CONCLUSIONS: Absence of myostatin potentially protects the function of the heart post-MI with improved survival, possibly by limiting extent of fibrosis.

Myostatin deficiency is associated with lipidomic abnormalities in skeletal muscles.

Myostatin (Mstn) deficiency leads to skeletal muscle overgrowth and Mstn inhibition is considered as a promising treatment for muscle-wasting disorders. Mstn gene deletion in mice also causes metabolic changes with decreased mitochondria content, disturbance in mitochondrial respiratory function and increased muscle fatigability. However the impact of MSTN deficiency on these metabolic changes is not fully elucidated. Here, we hypothesized that lack of MSTN will alter skeletal muscle membrane lipid composition in relation with pronounced alterations in muscle function and metabolism. Indeed, phospholipids and in particular cardiolipin mostly present in the inner mitochondrial membrane, play a crucial role in mitochondria function and oxidative phosphorylation process. We observed that Mstn KO muscle had reduced fat membrane transporter levels (FAT/CD36, FABP3, FATP1 and FATP4) associated with decreased lipid oxidative pathway (citrate synthase and ß-HAD activities) and impaired lipogenesis (decreased triglyceride and free fatty acid content), indicating a role of mstn in muscle lipid metabolism. We further analyzed phospholipid classes and fatty acid composition by chromatographic methods in muscle and mitochondrial membranes. Mstn KO mice showed increased levels of saturated and polyunsaturated fatty acids at the expense of monounsaturated fatty acids. We also demonstrated, in this phenotype, a reduction in cardiolipin proportion in mitochondrial membrane versus the proportion of others phospholipids, in relation with a decrease in the expression of phosphatidylglycerolphosphate synthase and cardiolipin synthase, enzymes involved in cardiolipin synthesis. These data illustrate the importance of lipids as a link by which MSTN deficiency can impact mitochondrial bioenergetics in skeletal muscle.

Differential effects of myostatin deficiency on motor and sensory axons.

INTRODUCTION: Deletion of myostatin in mice (MSTN-/- ) alters structural properties of peripheral axons. However, properties like axon diameter and myelin thickness were analyzed in mixed nerves, so it is unclear whether loss of myostatin affects motor, sensory, or both types of axons. METHODS: Using the MSTN-/- mouse model, we analyzed the effects of increasing the number of muscle fibers on axon diameter, myelin thickness, and internode length in motor and sensory axons. RESULTS: Axon diameter and myelin thickness were increased in motor axons of MSTN-/- mice without affecting internode length or axon number. The number of sensory axons was increased without affecting their structural properties. DISCUSSION: These results suggest that motor and sensory axons establish structural properties by independent mechanisms. Moreover, in motor axons, instructive cues from the neuromuscular junction may play a role in co-regulating axon diameter and myelin thickness, whereas internode length is established independently. Muscle Nerve 56: E100-E107, 2017.

Effect of constitutive inactivation of the myostatin gene on the gain in muscle strength during postnatal growth in two murine models.

INTRODUCTION: The effect of constitutive inactivation of the gene encoding myostatin on the gain in muscle performance during postnatal growth has not been well characterized. METHODS: We analyzed 2 murine myostatin knockout (KO) models, (i) the Lee model (KOLee ) and (ii) the Grobet model (KOGrobet ), and measured the contraction of tibialis anterior muscle in situ. RESULTS: Absolute maximal isometric force was increased in 6-month-old KOLee and KOGrobet mice, as compared to wild-type mice. Similarly, absolute maximal power was increased in 6-month-old KOLee mice. In contrast, specific maximal force (relative maximal force per unit of muscle mass was decreased in all 6-month-old male and female KO mice, except in 6-month-old female KOGrobet mice, whereas specific maximal power was reduced only in male KOLee mice. CONCLUSIONS: Genetic inactivation of myostatin increases maximal force and power, but in return it reduces muscle quality, particularly in male mice. Muscle Nerve 55: 254-261, 2017.

Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle.

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes (Dlk1, Gtl2, Rtl1/Rtl1as, and Rian) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge) locus in the skeletal muscle.

Decreasing maternal myostatin programs adult offspring bone strength in a mouse model of osteogenesis imperfecta.

During fetal development, the uterine environment can have effects on offspring bone architecture and integrity that persist into adulthood; however, the biochemical and molecular mechanisms remain unknown. Myostatin is a negative regulator of muscle mass. Parental myostatin deficiency (Mstntm1Sjl/+) increases muscle mass in wild-type offspring, suggesting an intrauterine programming effect. Here, we hypothesized that Mstntm1Sjl/+ dams would also confer increased bone strength. In wild-type offspring, maternal myostatin deficiency altered fetal growth and calvarial collagen content of newborn mice and conferred a lasting impact on bone geometry and biomechanical integrity of offspring at 4 mo of age, the age of peak bone mass. Second, we sought to apply maternal myostatin deficiency to a mouse model with osteogenesis imperfecta (Col1a2oim), a heritable connective tissue disorder caused by abnormalities in the structure and/or synthesis of type I collagen. Femora of male Col1a2oim/+ offspring from natural mating of Mstntm1Sjl/+ dams to Col1a2oim/+sires had a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure compared with age, sex, and genotype-matched offspring from natural mating of Col1a2oim/+ dams to Col1a2oim/+ sires. Finally, increased bone biomechanical strength of Col1a2oim/+ offspring that had been transferred into Mstntm1Sjl/+ dams as blastocysts demonstrated that the effects of maternal myostatin deficiency were conferred by the postimplantation environment. Thus, targeting the gestational environment, and specifically prenatal myostatin pathways, provides a potential therapeutic window and an approach for treating osteogenesis imperfecta.

Myostatin inhibition therapy for insulin-deficient type 1 diabetes.

While Type 1 Diabetes Mellitus (T1DM) is characterized by hypoinsulinemia and hyperglycemia, persons with T1DM also develop insulin resistance. Recent studies have demonstrated that insulin resistance in T1DM is a primary mediator of the micro and macrovascular complications that invariably develop in this chronic disease. Myostatin acts to attenuate muscle growth and has been demonstrated to be elevated in streptozotocin-induced diabetic models. We hypothesized that a reduction in mRNA expression of myostatin within a genetic T1DM mouse model would improve skeletal muscle health, resulting in a larger, more insulin sensitive muscle mass. To that end, Akita diabetic mice were crossed with Myostatin(Ln/Ln) mice to ultimately generate a novel mouse line. Our data support the hypothesis that decreased skeletal muscle expression of myostatin mRNA prevented the loss of muscle mass observed in T1DM. Furthermore, reductions in myostatin mRNA increased Glut1 and Glut4 protein expression and glucose uptake in response to an insulin tolerance test (ITT). These positive changes lead to significant reductions in resting blood glucose levels as well as pronounced reductions in associated diabetic symptoms, even in the absence of exogenous insulin. Taken together, this study provides a foundation for considering myostatin inhibition as an adjuvant therapy in T1DM as a means to improve insulin sensitivity and blood glucose management.

Enhanced exercise and regenerative capacity in a mouse model that violates size constraints of oxidative muscle fibres.

A central tenet of skeletal muscle biology is the existence of an inverse relationship between the oxidative fibre capacity and its size. However, robustness of this relationship is unknown. We show that superimposition of Estrogen-related receptor gamma (Errγ) on the myostatin (Mtn) mouse null background (Mtn(-/-)/Errγ(Tg/+)) results in hypertrophic muscle with a high oxidative capacity thus violating the inverse relationship between fibre size and oxidative capacity. We also examined the canonical view that oxidative muscle phenotype positively correlate with Satellite cell number, the resident stem cells of skeletal muscle. Surprisingly, hypertrophic fibres from Mtn(-/-)/Errγ(Tg/+) mouse showed satellite cell deficit which unexpectedly did not affect muscle regeneration. These observations 1) challenge the concept of a constraint between fibre size and oxidative capacity and 2) indicate the important role of the microcirculation in the regenerative capacity of a muscle even when satellite cell numbers are reduced.

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.