The effect of modification of DNA interference on myostatin gene expression in mice.

Myostatin is a known negative regulator of muscle tissue growth. Thus, an inhibitor of myostatin may be therapeutically useful as an anabolic agent for the muscle tissue. A promising gene-silencing approach for gene therapy is DNA interference (DNAi), a sequence that is complementary to the promoter region of a target gene. To confer resistance to nuclease digestion, several modifications such as methylphosphonate or phosphorothioate have been proposed, wherein a nonbridging oxygen atom in the oligonucleotide phosphate backbone is replaced by sulphur. The aim of the present study was to assess the effectiveness of the DNAi molecule with phosphorothioate (PS) and without phosphorothioate (WPS) modification for inhibition of myostatin gene expression in mice. Eighteen four-week-old male BALB/c mice were randomly divided into three groups: DNAi-PS (n = 6), DNAi-WPS (n = 6) and control (n = 6). Intraperitoneal injections of DNAi (10 mg/kg) were given once a week, and mice body weights were measured weekly and sacrificed after three weeks. The expression of myostatin was assessed using real-time quantitative polymerace chain reaction. For histological evaluation, the skeletal muscle tissue was dissected from the biceps. The results were analysed by a t-test. Results demonstrated that administration of DNAi intraperitoneally with modification could suppress myostatin expression by up to 70%. Leg weight and histological analysis proved that chemically modified DNAi significantly suppressed the myostatin gene in mice. Overall, the results on DNA-induced gene silencing by antisense DNA oligonucleotides in animals can provide insight into the treatment of inherited diseases.

Unveiling the Potential of Nelumbo nucifera-Derived Liensinine to Target The Myostatin Protein and to Counteract Muscle Atrophy.

Muscle atrophy refers to a decline in muscle mass and function, which has become a global concern due to the aging population. Various clinical trials have investigated the inhibitors of myostatin (MSTN). They have shown promising improvements in muscle function and quality of life. However, there are no drugs specifically targeting MSTN that have been approved for clinical use. In this study, we virtually screened liensinine (LIE), a food (Nelumbo nucifera)-derived compound, with low toxicity, from over 1.1 million compounds. We subsequently identified it as a potential candidate that targets MSTN by a cellular thermal shift assay (CETSA) and drug affinity response target stability (DARTS) assay. Further validation through cellular and in vivo studies demonstrated its promising potential in combating muscle atrophy. The mechanism of action may involve hindering the interaction between MSTN and the activin receptor type IIB (ActRIIB) and downregulating the expression of downstream proteins, including the muscle RING-finger protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx)/Atrogin-1, ultimately promoting muscle regeneration. These results provide a strong foundation for future studies to explore the therapeutic potential of LIE in clinical settings.

Skeletal muscle myostatin mRNA expression is upregulated in aged human adults with excess adiposity but is not associated with insulin resistance and ageing.

Myostatin negatively regulates skeletal muscle growth and appears upregulated in human obesity and associated with insulin resistance. However, observations are confounded by ageing, and the mechanisms responsible are unknown. The aim of this study was to delineate between the effects of excess adiposity, insulin resistance and ageing on myostatin mRNA expression in human skeletal muscle and to investigate causative factors using in vitro models. An in vivo cross-sectional analysis of human skeletal muscle was undertaken to isolate effects of excess adiposity and ageing per se on myostatin expression. In vitro studies employed human primary myotubes to investigate the potential involvement of cross-talk between subcutaneous adipose tissue (SAT) and skeletal muscle, and lipid-induced insulin resistance. Skeletal muscle myostatin mRNA expression was greater in aged adults with excess adiposity than age-matched adults with normal adiposity (2.0-fold higher; P < 0.05) and occurred concurrently with altered expression of genes involved in the maintenance of muscle mass but did not differ between younger and aged adults with normal adiposity. Neither chronic exposure to obese SAT secretome nor acute elevation of fatty acid availability (which induced insulin resistance) replicated the obesity-mediated upregulation of myostatin mRNA expression in vitro. In conclusion, skeletal muscle myostatin mRNA expression is uniquely upregulated in aged adults with excess adiposity and insulin resistance but not by ageing alone. This does not appear to be mediated by the SAT secretome or by lipid-induced insulin resistance. Thus, factors intrinsic to skeletal muscle may be responsible for the obesity-mediated upregulation of myostatin, and future work to establish causality is required.

Genetic variants in myostatin and its receptors promote elite athlete status.

BACKGROUND: While product of the myostatin gene (MSTN) is an important factor influencing muscle growth, which is well confirmed in nonhuman species, it has not been clearly confirmed whether MSTN expression influences interindividual differences in skeletal muscle mass, affects posttraining changes, or plays a role in the age-related loss of muscle mass and function in humans. Although the inconclusive results are usually explained by ethnic differences and the low frequency of some alleles, it is possible that the role of receptors (ACVR2A and ACVR2B) that affect the biological activity of myostatin is crucial. Therefore, we investigated the sequences of the MSTN, ACVR2A, and ACVR2B genes and determined the interaction between allelic variants and athletic performance and competition level in the Caucasian population. One hundred-two athletes were recruited for the sequencing study, and whole-genome sequencing (WGS) was performed. Second, 330 athletes and 365 controls were included, and real-time PCR was performed. RESULTS: The sequence analysis revealed two polymorphisms relatively common in the athlete cohort, and the alternate allele showed overrepresentation in athletes: MSTN rs11333758 and ACVR2A rs3764955. Regarding the polymorphic site MSTN rs11333758, there was a significant overrepresentation of the -/- genotype in all high-elite and mixed-sport high-elite athletes. Carriers of the ACVR2A rs3764955 CC and GG genotypes were more likely to be elite and high-elite athletes. In addition, carriers of the CC genotype were more likely to be in the mixed-sport subelite group. The gene‒gene interaction analysis revealed that mixed-sport high elite athletes showed significant underrepresentation of the ACVR2A rs3764955 GC - MSTN rs11333758 AA genotype combination. In the same group, we observed a significant overrepresentation of the ACVR2A rs3764955 GC - MSTN rs11333758 -/- and the ACVR2A rs3764955 CC - MSTN rs11333758 -/- genotype combinations. CONCLUSIONS: We showed that the specific genotypes of the MSTN rs11333758 and ACVR2A rs3764955, either individually or in gene‒gene combination, are significantly associated with athletes' competition level in the Polish population, especially in the mixed-sports athlete group. Thus, although further research is required, these polymorphisms, alone or in combination with other polymorphisms, are among the numerous candidates that could explain individual variations in muscle phenotypes.

Myostatin and the Heart.

Myostatin (growth differentiation factor 8) is a member of the transforming growth factor-ß superfamily. It is secreted mostly by skeletal muscles, although small amounts of myostatin are produced by the myocardium and the adipose tissue as well. Myostatin binds to activin IIB membrane receptors to activate the downstream intracellular canonical Smad2/Smad3 pathway, and additionally acts on non-Smad (non-canonical) pathways. Studies on transgenic animals have shown that overexpression of myostatin reduces the heart mass, whereas removal of myostatin has an opposite effect. In this review, we summarize the potential diagnostic and prognostic value of this protein in heart-related conditions. First, in myostatin-null mice the left ventricular internal diameters along with the diastolic and systolic volumes are larger than the respective values in wild-type mice. Myostatin is potentially secreted as part of a negative feedback loop that reduces the effects of the release of growth-promoting factors and energy reprogramming in response to hypertrophic stimuli. On the other hand, both human and animal data indicate that myostatin is involved in the development of the cardiac cachexia and heart fibrosis in the course of chronic heart failure. The understanding of the role of myostatin in such conditions might initiate a development of targeted therapies based on myostatin signaling inhibition.

Associations between polymorphisms in the myostatin gene with calving difficulty and carcass merit in cattle.

A fully functional myostatin gene inhibits muscle fiber growth. The objective of the present study was to quantify the association between 21 known myostatin mutations with both calving and carcass traits in 12 cattle breeds. The myostatin genotypes of 32,770 dam-progeny combinations were used in the association analysis of calving dystocia, with the genotypes of 129,803 animals used in the mixed model association analyses of carcass weight, conformation, and fat score. The mixed model included additive genetic, maternal, and permanent environmental effects where appropriate. The mutant genotypes of nt821, Q204X, and F94L were all associated (P < 0.01) with more calving difficulty when present in either the dam or the progeny. The nt821 deletion had the greatest association with calving difficulty when the homozygous deletion was present in either the calf (0.37 points greater calving difficulty score relative to calves carrying no copies of the deletion based on a one to four scale) or the dam (1.30 points greater calving difficulty score relative to dams carrying no copies of the deletion), although the association between the calf's nt821 genotype and calving difficulty differed depending on the nt821 genotype of the dam. With the exception of nt748_78, nt414, and nt374_51, all other seven segregating myostatin variants were associated (range of allele substitution effect size relative to animals with no copies of the mutant allele) with carcass weight (2.36 kg lighter to 15.56 kg heavier), all 10 segregating variants with conformation (0.15 units less conformed to 2.24 units more conformed assessed on a scale of 1 to 15), and all segregating variants other than E226X with carcass fat (0.23 units less carcass fat cover to 3.85 units more carcass fat cover assessed on a scale of 1 to 15). Of these, the F94L, Q204X, and nt821 mutations generally had the greatest association with all three carcass traits, giving rise to heavier and more conformed carcasses. Despite the antagonistic genetic relationship between calving difficulty and carcass traits, the nt374_51, F94L, and E226X mutations were all associated with improved carcass merit while having minimal expected consequences on calving difficulty. Thus, animals carrying these mutation(s) may have favorable genetic merit for calving difficulty and carcass merit. Furthermore, depending on the dam genotype, a bull with two copies of the nt821 mutation can produce progeny with improved carcass merit while minimizing calving problems.

The objective of the present study was to quantify the association between 21 known myostatin mutations with calving difficulty, carcass fat, carcass conformation, and carcass weight in 12 prominent cattle breeds. Out of all segregating myostatin variants, the nt821 deletion had the greatest observed association with calving difficulty when the homozygous deletion was carried by either the calf or the dam. However, the association between the calf's nt821 genotype and calving difficulty varied depending on the nt821 genotype of the dam. The F94L, Q204X, and nt821 mutations generally had the greatest association with all three carcass traits, resulting in heavier and more conformed carcasses. The nt374_51, F94L, and E226X mutations were all associated with improved carcass merit while having minimal expected consequences on calving difficulty; therefore, animals carrying these mutations could potentially have desirable genetic merit for both calving difficulty and carcass merit. Similarly, depending on the genotype of the dam, a bull carrying two copies of the nt821 deletion could give rise to progeny that have improved carcass merit while minimizing the associated risk of calving difficulty.

GDF8 inhibition enhances musculoskeletal recovery and mitigates posttraumatic osteoarthritis following joint injury.

Musculoskeletal disorders contribute substantially to worldwide disability. Anterior cruciate ligament (ACL) tears result in unresolved muscle weakness and posttraumatic osteoarthritis (PTOA). Growth differentiation factor 8 (GDF8) has been implicated in the pathogenesis of musculoskeletal degeneration following ACL injury. We investigated GDF8 levels in ACL-injured human skeletal muscle and serum and tested a humanized monoclonal GDF8 antibody against a placebo in a mouse model of PTOA (surgically induced ACL tear). In patients, muscle GDF8 was predictive of atrophy, weakness, and periarticular bone loss 6 months following surgical ACL reconstruction. In mice, GDF8 antibody administration substantially mitigated muscle atrophy, weakness, and fibrosis. GDF8 antibody treatment rescued the skeletal muscle and articular cartilage transcriptomic response to ACL injury and attenuated PTOA severity and deficits in periarticular bone microarchitecture. Furthermore, GDF8 genetic deletion neutralized musculoskeletal deficits in response to ACL injury. Our findings support an opportunity for rapid targeting of GDF8 to enhance functional musculoskeletal recovery and mitigate the severity of PTOA after injury.

Novel Polymorphisms in Caprine Myostatin Gene and its Relationship with Growth Traits in Saburai Does (Capra hircus).

<b>Background and Objective:</b> Saburai goat (<i>Capra hircus</i>) is a crossbred goat from Boer buck (75%) and Ettawa doe (25%) for meat production purposes. This study was carried out to detect the mutation points or Single Nucleotide Polymorphisms (SNPs) in the myostatin (<i>MSTN</i>) gene of Saburai with the forward sequencing method. <b>Materials and Methods:</b> A total of twenty-one blood samples of Saburai does (75% Boer, 25% Ettawa) were collected from the Villager Breeder Center (VBC) at Tanggamus Regency of Lampung Province, Indonesia. The DNA analysis consists of DNA isolation, PCR analysis and sequencing analysis. The record data were used for association study with the mathematical model: Y_ij = µ+G_i+Є_ij. <b>Results:</b> Research showed that one common SNP of g.217_218.indel.TTTTA (5'UTR) and three novel SNPs of c.386G>C (exon 1), g.641_642.indel.T (intron 2) and c.4957G>C (exon 3) were detected in the present study. In this study, a novel SNP on exon 1 and intron 2 of Saburai <i>MSTN</i> gene has a moderate PIC value (>0.30). In addition, a novel SNP on exon 1 and exon 3 of the Saburai <i>MSTN</i> gene was detected as a missense mutation of A55P and A43P, respectively. Goats with the heterozygous genotype have higher growth traits compared to goats with the homozygous genotype. <b>Conclusion:</b> The goats with heterozygous genotypes can be further developed to increase the productivity of Saburai goats.

Pharmacological Inhibition of Myostatin in a Mouse Model of Typical Nemaline Myopathy Increases Muscle Size and Force.

Nemaline myopathy is one of the most common non-dystrophic congenital myopathies. Individuals affected by this condition experience muscle weakness and muscle smallness, often requiring supportive measures like wheelchairs or respiratory support. A significant proportion of patients, approximately one-third, exhibit compound heterozygous nebulin mutations, which usually give rise to the typical form of the disease. Currently, there are no approved treatments available for nemaline myopathy. Our research explored the modulation of myostatin, a negative regulator of muscle mass, in combating the muscle smallness associated with the disease. To investigate the effect of myostatin inhibition, we employed a mouse model with compound heterozygous nebulin mutations that mimic the typical form of the disease. The mice were treated with mRK35, a myostatin antibody, through weekly intraperitoneal injections of 10 mg/kg mRK35, commencing at two weeks of age and continuing until the mice reached four months of age. The treatment resulted in an increase in body weight and an approximate 20% muscle weight gain across most skeletal muscles, without affecting the heart. The minimum Feret diameter of type IIA and IIB fibers exhibited an increase in compound heterozygous mice, while only type IIB fibers demonstrated an increase in wild-type mice. In vitro mechanical experiments conducted on intact extensor digitorum longus muscle revealed that mRK35 augmented the physiological cross-sectional area of muscle fibers and enhanced absolute tetanic force in both wild-type and compound heterozygous mice. Furthermore, mRK35 administration improved grip strength in treated mice. Collectively, these findings indicate that inhibiting myostatin can mitigate the muscle deficits in nebulin-based typical nemaline myopathy, potentially serving as a much-needed therapeutic option.

From correlation to causation using directed topological overlap matrix: Applications in genomics.

Most causal discovery tools assume the local causal Markov condition. However, the theoretical assumptions that underlie the local causal Markov condition are often not met in practice. This is especially marked in genomics, where the unwanted presence of measurement errors, averaging effects, and feedback loops significantly undermine the legitimacy of the local causal Markov condition. Furthermore, these causal discovery algorithms require very large samples, orders above what is often available. In this paper, relaxing the local causal Markov condition and using Reichenbach's common cause principle instead, we present a more flexible approach to causal discovery, the directed topological overlap matrix (DTOM). DTOM is robust w.r.t. the presence of measurement errors, averaging effects, feedback loops, and is significantly more sample efficient. We study the utility of DTOM for discovering causal relations in biological data using three real gene expression data-sets. We first examine if DTOM can help distinguish the Myostatin mutation in the Piedmontese cattle by contrasting the muscle transcriptomes of the Piedmontese and Wagyu crosses: the Myostatin mutation is the cause of the double-muscling the Piedmontese cattle are famous for. We then consider a large-scale gene deletion study in yeast. We show that DTOM allows us to distinguish the deleted gene in a sample knowing only the set of differentially expressed genes in that sample. We then examine the progression of Alzheimer's disease (AD) under the lens of DTOM. The genes implicated as having a causal role in the progression of AD by our DTOM analysis were significantly enriched in cellular components that had been repeatedly implicated in the progression of AD.

Transcriptional Knock-down of mstn Encoding Myostatin Improves Muscle Quality of Nile Tilapia (Oreochromis niloticus).

Myostatin (encoded by mstn) negatively regulates skeletal muscle mass and affects lipid metabolism. To explore the regulatory effects of mstn on muscle development and lipid metabolism in Nile tilapia (Oreochromis niloticus), we used antisense RNA to transcriptionally knock-down mstn. At 180 days, the body weight and body length were significantly higher in the mstn-knock-down group than in the control group (p < 0.05). Additionally, fish with mstn-knock-down exhibited myofiber hyperplasia but not hypertrophy. Oil red O staining revealed a remarkable increase in the area of lipid droplets in muscle in the mstn-knockdown group (p < 0.05). Nutrient composition analyses of muscle tissue showed that the crude fat content was significantly increased in the mstn-knock-down group (p < 0.05). The contents of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids were all significantly increased in the mstn-knock-down group (p < 0.05). Comparative transcriptome analyses revealed 2420 significant differentially expressed genes between the mstn-knock-down group and the control group. KEGG analysis indicates that disruptions to fatty acid degradation, glycerolipid metabolism, and the PPAR signaling pathway affect muscle development and lipid metabolism in mstn-knock-down Nile tilapia: acaa2, eci1, and lepr were remarkably up-regulated, and acadvl, lpl, foxo3, myod1, myog, and myf5 were significantly down-regulated (p < 0.05). These results show that knock-down of mstn results in abnormal lipid metabolism, acceleration of skeletal muscle development, and increased adipogenesis and weight gain in Nile tilapia.

Effects of the F94L myostatin gene mutation in beef × dairy crossed cattle on muscle fiber type, live performance, carcass characteristics, and boxed beef and retail cut yields.

Producer live performance data and carcasses from steers (n = 116) resulting from the mating of four Limousin/Angus sires heterozygous for the F94L myostatin mutation to Jersey/Holstein dams were utilized to evaluate the effects of one copy of the F94L allele on live performance, carcass traits and USDA grades, and boxed beef and retail yields. Slaughter data were collected at time of harvest and carcass data were collected 48 hours postmortem. One side from each of the 58 carcasses was fabricated into boxed beef and retail cuts by experienced lab personnel 5-8 d postmortem. One copy of the F94L allele did not affect gestation length, birth weight, percentage of unassisted births, feedlot average daily gain, live weight at harvest, hot carcass weight, or dressing percentage (P > 0.05). Muscle fiber analysis indicated that the increase in muscularity by the F94L allele in the semitendinosus and longissimus was likely due to hyperplasia as there was a 19% increase in the quantity of myosin heavy chain type IIA and IIX fibers in the semitendinosus (P < 0.05) with no effect on muscle fiber size (P > 0.05). Carcasses from steers with one F94L allele had larger ribeye areas (99.2 vs. 92.3 sq.cm.), greater ribeye width:length ratios (0.498 vs. 0.479), lower USDA yield grades (2.21 vs. 2.66), and lower marbling scores (438 vs. 480) (P < 0.05). Additionally, for boxed beef yields, one F94L allele, vs. zero F94L alleles, increased (P < 0.05) 85/15 trimmings (+0.59%), top round (+0.28%), strip loin (+0.12%), eye round (+0.11%), tenderloin (+0.07%), boneless foreshank (+0.07%), cap/wedge (+0.06%), and tri-tip (+0.04%). Overall, carcasses from steers with one F94L allele had a greater boxed beef yield (+1.06%), boxed beef plus 85/15 trimmings yield (+1.65%), and total retail cuts plus ground beef 85/15 yield (+1.78%) than carcasses from steers with zero F94L alleles (P < 0.05). One copy of the F94L allele utilized in beef-on-dairy breeding system had no significant impact on live performance traits but resulted in lower marbling scores and increased muscularity as evidenced through larger, more beef-shaped ribeyes, lower USDA yield grades, and greater carcass cutout yields (both boxed beef and retail yields).

In a beef-on-dairy system, one copy of the F94L myostatin allele caused increased muscling, resulting in larger, more beef-shaped ribeyes, more desirable yield grades, and greater boxed beef and retail yields, all of which address inherent deficiencies in dairy and dairy-cross carcasses. These improvements were realized with no negative effects on calving ease or live performance. The F94L did cause a significant and meaningful reduction in marbling score; therefore, marbling ability should be paramount in sire selection if F94L sires are utilized. Using a beef sire homozygous for F94L myostatin in a beef-on-dairy system would ensure that all resulting progenies have exactly one copy of the F94L allele, meaning that this genetic tool could be rapidly implemented in the beef-on-dairy industry segment. When selecting sires for beef-on-dairy programs, accurate EPDs should remain the primary evaluation tool as the F94L effects are reflected in accurate EPDs; however, using a sire homozygous for F94L (2 or 0 copies) should result in more consistent progeny.

Growth differentiation factor myostatin regulates epithelial-mesenchymal transition genes and enhances invasion by increasing serine protease inhibitors E1 and E2 in human trophoblast cells.

Placental insufficiency disorders, including preeclampsia and intrauterine growth restriction, are major obstetric complications that can have devastating effects on both the mother and the fetus. These syndromes have underlying poor placental trophoblast cell invasion into uterine tissues. Placental invasion is controlled by many hormones and growth factors. Myostatin (MSTN) is a transforming growth factor-ß superfamily member recognized for its important role in muscle growth control. MSTN has also been shown to be secreted and functioning in the placenta, and its serum and/or placental levels were found to be upregulated in preeclampsia and intrauterine growth restriction. Considering that the mechanistic role of MSTN in placentation remains poorly understood, we hypothesized that MSTN uses ALK4/5-SMAD2/3/4 signaling to increase human trophoblast invasion through a group of epithelial-mesenchymal transition genes including SERPINE2, PAI-1, and SOX4. mRNA sequencing of control and MSTN-treated primary human trophoblast cells (n = 5) yielded a total of 610 differentially expressed genes (false discovery rate <0.05) of which 380 genes were upregulated and 230 were downregulated. These differentially expressed genes were highly enriched in epithelial-mesenchymal transition genes, and a subset including SERPINE2, PAI-1, and SOX4 was investigated for its role in MSTN-induced trophoblast cell invasion. We found that MSTN induced upregulation of SERPINE2 via ALK4/5-SMAD2/3/4 signaling; however, SMAD2 was not involved in MSTN-induced PAI-1 upregulation. SOX4 was involved in MSTN-induced upregulation of SERPINE2, but not PAI-1. Collectively, this study discovers novel molecular mechanisms of MSTN-induced human trophoblast cell invasion and provides insight into the functional consequences of its dysregulation in placental insufficiency disorders.

Association of myostatin gene polymorphism with echocardiographic and muscular ultrasonographic measurements in Hungarian thoroughbreds horses.

The g.66493737C/T polymorphism of the myostatin gene (MSTN) majorly influences muscle fiber composition and best race distance of Thoroughbreds. Thus, a better understanding of this process may lead to superior genetic exploitation for maximizing Thoroughbred athletic potential. Our objective is to investigate whether myostatin genotypes are associated with muscular development and cardiac variables of Thoroughbreds. Echocardiography and muscular ultrasonography were performed on three groups having C/C, C/T, and T/T genotypes, respectively. Each group consisted of 22 animals. Homogeneity of variance between the groups was checked by Levene's test. Multivariate analysis of variance was applied to determine differences in measured variables vs. MSTN genotypes. Fascicle length of anconeus and thickness of triceps brachii muscles showed significant differences between C/C and T/T genotypes (pFascicle-length-of-anconeus = 0.004, pthickness-of-triceps-brachii < 0.001). According to the primary outcome, there are associations between myostatin genotypes and cardiac variables. Aortic diameter at the sinus of Valsalva (end-diastole and end-systole) and aortic diameter at the valve (end-systole) indicated significant differences between C/C and T/T genotypes (paortic-diameter-at-the-sinus-of-Valsalva-end-diastole = 0.015, paortic-diameter-at-the-sinus-of-Valsalva-end-systole = 0.011, paortic-diameter-at-the-valve-end-systole = 0.014). Pearson correlation effect sizes were rFascicle-length-of-anconeus = 0.460, rthickness-of-triceps-brachii = 0.590, raortic-diameter-at-the-sinus-of-Valsalva-end-diastole = 0.423, raortic-diameter-at-the-sinus-of-Valsalva-end-systole = 0.450, and raortic-diameter-at-the-valve-end-systole = 0.462. C/C genotypes gave 22.1, 12.2, 6.3, 6.0, and 6.7% higher values compared to T/T genotypes, respectively. Differences regarding aortic diameter between genotype groups support the hypothesis that C/C animals have consequently increased cardiac output and aerobic capacity.

Dual single guide RNAs-mediating deletion of mature myostatin peptide results in concomitant muscle fibre hyperplasia and adipocyte hypotrophy in pigs.

Myostatin (MSTN) is a major gene target for skeletal muscle overgrowth in animals. We hypothesized that deletion of the entire mature peptide encoded by MSTN in pigs would knock out its bioactive form and accordingly stimulate skeletal muscle overgrowth. Thus, we engineered two pairs of single-guide RNAs (sgRNAs) to target exons 1 and 3 of MSTN in primary fetal fibroblasts of Taoyuan black pigs. We found that sgRNAs targeting exon 3, which encodes the mature peptide, had higher biallelic null mutation efficiency than those targeting exon 1. Somatic cell nuclear transfer was conducted using the exon 3 mutation cells as donor cells to generate five cloned MSTN null piglets (MSTN-/-). Growth testing revealed that both the growth rate and average daily weight gain of MST-/- pigs were greater than those of wild-type (MSTN+/+) pigs. Slaughter data demonstrated that the lean ratio of MSTN-/- pigs was 11.3% higher (P < 0.01) while the back-fat thickness was 17.33% lower (P < 0.01) than those of MSTN+/+ pigs. Haematoxylin-eosin staining indicated that the increased leanness of MSTN-/- pigs resulted from muscle fibre hyperplasia rather than hypertrophy.HE staining showed markedly decreased adipocyte size in MSTN-/- pigs. We also critically examined the off-target and random integration by resequencing, which showed that the founder MSTN-/- pigs contained no non-target mutations or exogenous plasmid elements. This study is the first to report the successful knock out of the mature MSTN peptide using dual sgRNA-mediated deletion, leading to the most prominent alteration of meat production traits in pigs published thus far. This new strategy is expected to have a wide impact on genetic improvements in food animals.

Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR.

Hu sheep, an indigenous breed in China known for its high fecundity, are being studied to improve their growth and carcass traits. MSTN is a negative regulator of muscle development, and its inactivation results in muscularity. The C-CRISPR system, utilizing multiple neighboring sgRNAs targeting a key exon, has been successfully used to generate genes for complete knockout (KO) monkeys and mice in one step. In this study, the C-CRISPR system was used to generate MSTN-edited Hu sheep; 70 embryos injected with Cas9 mRNA and four sgRNAs targeting exon 3 of sheep MSTN were transferred to 13 recipients. Out of 10 lambs born from five recipients after full-term pregnancies, nine had complete MSTN KO with various mutations. No off-target effects were found. These MSTN-KO Hu sheep showed a double-muscled (DM) phenotype, characterized by a higher body weight at 3 and 4 months old, prominent muscular protrusion, clearly visible intermuscular groves, and muscle hypertrophy. The molecular analysis indicated enhanced AKT and suppressed ERK1/2 signaling in the gluteus muscle of the edited Hu sheep. In conclusion, MSTN complete KO Hu sheep with a DM phenotype were efficiently and specifically generated using C-CRISPR, and the C-CRISPR method is a promising tool for farm animal breeding.

Effects of elastase-induced emphysema on muscle and bone in mice.

Chronic obstructive pulmonary disease (COPD) causes sarcopenia and osteoporosis. However, the mechanisms underlying muscle and bone loss as well as the interactions between muscle and bone in the COPD state remain unclear. Therefore, we herein investigated the effects of the COPD state on muscle and bone in mice intratracheally administered porcine pancreatic elastase (PPE). The intratracheal administration of PPE to mice significantly reduced trabecular bone mineral density (BMD), trabecular bone volume, trabecular number, cortical BMD and cortical area. It also significantly decreased grip strength, but did not affect muscle mass or the expression of myogenic differentiation-, protein degradation- or autophagy-related genes in the soleus and gastrocnemius muscles. Among the myokines examined, myostatin mRNA levels in the soleus muscles were significantly elevated in mice treated with PPE, and negatively related to grip strength, but not bone parameters, in mice treated with or without 2 U PPE in simple regression analyses. Grip strength positively related to bone parameters in mice treated with or without PPE. In conclusion, we showed that a PPE model of COPD in mice exerts dominant effects on bone rather than skeletal muscles. Increased myostatin expression in the soleus muscles of mice in the COPD state may negatively relate to a reduction in grip strength, but not bone loss.

Long-read sequencing assays designed to detect potential gene editing events in the myostatin gene revealed distinct haplotype signatures in the Thoroughbred horse population.

We present here the use of targeted, long-read sequencing of the myostatin (MSTN) gene as a model to detect potential gene editing events in Thoroughbred horses. MSTN is a negative regulator of muscle development, making the gene a prime candidate target for gene doping. By sequencing the complete gene in one PCR product, we can catalogue all mutations without the need to produce short-fragment libraries. A panel of reference material fragments with defined mutations was constructed and successfully sequenced by both Oxford Nanopore and Illumina-based methods, showing that gene doping editing events can be detected using this technology. To ascertain the normal variation within the population, we sequenced the MSTN gene in 119 UK Thoroughbred horses. Variants from the reference genome were assigned to haplotypes and eight distinct patterns, designated Hap1 (reference genome) to Hap8, were determined with haplotypes Hap2 and Hap3 (which includes the 'speed gene' variant) being far the most prevalent. Hap3 was most abundant in flat-racing horses, whereas Hap2 was most abundant in jump-racing. Within this data set, results for 105 racehorses from out-of-competition sampling were compared between matrices of extracted DNA and direct PCR of whole blood from lithium heparin gel tubes, and strong agreement was found between the two methods. The direct-blood PCR was achieved without compromising the sample prior to plasma separation for analytical chemistry, and could thus be used as part of a routine screening workflow for gene editing detection.

Characterisation of Single Nucleotide Polymorphisms and Haplotypes of MSTN Associated with Growth Traits in European Sea Bass (Dicentrarchus labrax).

The myostatin (MSTN) gene, known as growth differentiation factor-8 (GDF-8), is a member of the transforming growth factor-ß (TGF-ß) superfamily and plays a specific inhibitory role during the critical phases of skeletal muscle mass development in vertebrates. This study was conducted to investigate MSTN polymorphisms in harvest size European sea bass reared in Turkey. Nine single nucleotide polymorphisms (SNPs) and two indels were identified in exons 1-3 of MSTN in the European sea bass population The associations between the g.16612A indel located in intron 1 and standard length were significant. The MSTN g.15252 T > A locus in intron 2 was significantly related to the total weight, fillet weight and standard length (P < 0.05). The relationship between the g.14873C > T locus in exon 3 of MSTN and standard height, head length, body length, pre-anal length, abdominal length, post-anal length and head width was significant (P < 0.05). According to the results of the haplotype analysis, two haplogroup and eight haplotype combinations were detected in the population. The haplogroup 2 had significant associations with all measured growth traits (P < 0.05). Thus, SNPs and haplotypes identified in this study could be useful for European sea bass breeding and marker-assisted selection.

Nutritional status affects Igf1 regulation of skeletal muscle myogenesis, myostatin, and myofibrillar protein degradation pathways in gopher rockfish (Sebastes carnatus).

Insulin-like growth factor-1 (Igf1) regulates skeletal muscle growth in fishes by increasing protein synthesis and promoting muscle hypertrophy. When fish experience periods of insufficient food intake, they undergo slower muscle growth or even muscle wasting, and those changes emerge in part from nutritional modulation of Igf1 signaling. Here, we examined how food deprivation (fasting) affects Igf1 regulation of liver and skeletal muscle gene expression in gopher rockfish (Sebastes carnatus), a nearshore rockfish of importance for commercial and recreational fisheries in the northeastern Pacific Ocean, to understand how food limitation impacts Igf regulation of muscle growth pathways. Rockfish were either fed or fasted for 14 d, after which a subset of fish from each group was treated with recombinant Igf1 from sea bream (Sparus aurata). Fish that were fasted lost body mass and had lower body condition, reduced hepatosomatic index, and lower plasma Igf1 concentrations, as well as a decreased abundance of igf1 gene transcripts in the liver, increased hepatic mRNAs for Igf binding proteins igfbp1a, igfbp1b, and igfbp3a, and decreased mRNA abundances for igfbp2b and a putative Igf acid labile subunit (igfals) gene. In skeletal muscle, fasted fish showed a reduced abundance of intramuscular igf1 mRNAs but elevated gene transcripts encoding Igf1 receptors A (igf1ra) and B (igf1rb), which also showed downregulation by Igf1. Fasting increased skeletal muscle mRNAs for myogenin and myostatin1, as well as ubiquitin ligase F-box only protein 32 (fbxo32) and muscle RING-finger protein-1 (murf1) genes involved in muscle atrophy, while concurrently downregulating mRNAs for myoblast determination protein 2 (myod2), myostatin2, and myogenic factors 5 (myf5) and 6 (myf6 encoding Mrf4). Treatment with Igf1 downregulated muscle myostatin1 and fbxo32 under both feeding conditions, but showed feeding-dependent effects on murf1, myf5, and myf6/Mrf4 gene expression indicating that Igf1 effects on muscle growth and atrophy pathways is contingent on recent food consumption experience.