Age-induced gene expression in Thoroughbred horse skeletal muscle highlights genes that enhance muscle architecture and function.

Cattle breeds are optimized either for milk or meat production and secrete consumed nutrients in the form of milk or accrete nutrients as skeletal muscle tissue, respectively. Surplus energy is usually stored in the form of fat in adipose tissues. To gain more insight into the physiological and genetic background of nutrient accretion as either protein or fat, an experimental F2 population was generated crossing Charolais (CH) bulls and German Holstein (GH) cows. Mutations in two genes with known, profound effects on growth were segregating in this population: the I442M mutation in the non-SMC condensin I complex, subunit G (NCAPG) gene, and the Q204X mutation in the myostatin (MSTN) gene. The major aim of this study was to close the gap between the described effects of the NCAPG/LCORL region and MSTN SNPs on carcass and meat quality traits, as well as on the structure and composition of the underlying tissues. Whole carcass data, meat quality traits, composition of major cuts and their dominating muscles, including muscle and fat cell structure, were analyzed as well as chemical and fatty acid composition. Mutant alleles of both loci were associated with higher weights, increased muscularity, and reduced fatness, e.g., each explaining about 15% of the observed variance. However, both loci apparently affect traits in a specific manner, influencing either dimensional traits or mass accretion.

Crosstalk between myostatin and callipyge in CRISPR/Cas9-edited goat fibroblast cells.

Can myostatin editing together with gut microbiota modulation produce more and tastier meat?

SpCas9-mediated gene editing in bovine embryo via single adeno-associated virus infection using a novel micro-sized promoter.

A mismatch refractory (Mr.) LAMP-lateral flow assay biosensor for highly specific and ultraportable detection of the MSTN g.66493737T/C variant.

Osteoporosis is highly prevalent in postmenopausal women, causing chronic pain, fractures, and limited mobility that burden individuals and society. While resistance exercise benefits bone health, its role in osteoporotic bone injury healing and underlying mechanisms remain unclear. This study aimed to explore the effects of 10-week weight-bearing ladder climbing exercise on ovariectomy (OVX)-induced osteoporosis and subsequent bone injury healing, and to investigate whether these effects are associated with the myostatin (MSTN) and Wnt/β-catenin pathways. Fifty-four 12-week-old female SD rats were randomized into Sham, OVX, and OVX + EX groups. Rats in the OVX and OVX + EX groups underwent ovariectomy to induce postmenopausal osteoporosis, and those in the OVX + EX group received 10-week weight-bearing ladder climbing. After the exercise intervention, 6 rats in each group were sacrificed; the remaining rats underwent femoral midshaft drilling to establish bone injury. The improvement in osteoporosis was evaluated via Micro-CT, biomechanical tests, RT-qPCR for mRNA detection, and Western blot for measuring protein levels of MSTN and Wnt/β-catenin pathway-related molecules at post-exercise and 21 days post-injury. Bone healing was reflected by the bone volume fraction at the bone injury site detected via Micro-CT at 10 and 21 days post-injury. This exercise significantly enhanced muscle strength and improved femoral bone mineral density (BMD), trabecular microstructure, and biomechanical properties in OVX rats. Meanwhile, the level of MSTN in the OVX + EX group was decreased, the expression of its downstream signaling pathways was inhibited, and the mRNA and protein expressions of Wnt/β-catenin were upregulated. Moreover, 21 days after exercise intervention, the biomechanical properties and bone microstructure of the OVX + EX group were still significantly superior to those of the OVX group, and the aforementioned molecular regulatory effect remained. In addition, pre-conducted exercise was able to promote increases in bone volume fraction at the bone injury site 10 and 21 days after drilling, which was conducive to bone injury healing. Ten-week weight-bearing ladder climbing ameliorates OVX-induced bone loss and promotes osteoporotic bone repair via regulating the MSTN/ActRIIB/Smad3 and Wnt/β-catenin pathways, providing evidence for exercise as a safe non-pharmacological intervention.

Small interfering RNAs (siRNAs) hold promise for treating cardiac and muscular diseases, but robust and scalable delivery remains a hurdle. While biologic–siRNA conjugates (e.g. antibodies) are in clinical development, their manufacturing is complex. Lipophilic siRNAs are readily chemically synthesized at scale and support effective heart and muscle delivery. Here, we refine siRNA chemical design for enhanced potency and durability to support clinically relevant silencing. Targeting myostatin (MSTN), a key gene in muscle-wasting, a single subcutaneous dose in mice achieved potent silencing (80% inhibition up to 6 weeks, 30% up to 14 weeks). Biweekly dosing led to over 95% MSTN reduction for half-a-year with no observed toxicity. This resulted in muscle growth, increased lean mass, and improved grip strength. Phenotypical benefits extended beyond direct target silencing, suggesting prolonged effects. The siRNA scaffold was effective across multiple muscle groups, with its modularity confirmed by three additional targets. Optimized dosing extended durability to 20 weeks without compromising phenotypic outcomes. As a proof of concept, MSTN inhibition with siRNAs successfully combated muscle wasting in an inflammatory myopathy model (cardiotoxin). These findings pave the way for long-lasting gene modulation in heart and muscle, offering new therapeutic strategies for muscular diseases.

The fast growth of livestock production worldwide demands new approaches to boost the growth performance and feed ratio as well as sustainability and animal welfare. Traditional breeding methods have been applied to genetic advancement although they have been slow and relied on natural genetic variations that restrict their application in addressing the present-day farming needs. This paper examines how gene editing using CRISPR/Cas9 can be used to enhance sheep growth and feed efficiency by targeting a negative muscle development regulator, the myostatin (MSTN) gene. The microinjection and electroporation of sheep zygotes were done with subsequent embryo culture, transfer and phenotypic analysis of the resulting progeny. There was high editing efficiency, whereby the mass of muscles, average daily gain, and a 20-30 percent increase on feed conversion ratio of edited lambs was significantly higher than those of controls. Off target analysis showed that there were low numbers of unwanted mutations that validate the specificity and safety of CRISPR/Cas9 system. These results indicate that a fine-tuned genome editing of MSTN can be an effective method to increase growth characteristics and feed efficiency in sheep, which provides a promising instrument to achieve a fast-based genetic enhancement and provide livestock production with sustainability. This study forms the basis of the multi-gene editing approaches in the future to maximize economically valuable characteristics in livestock.

CRISPR-Cas9 genome editing offers significant opportunities to improve livestock traits; however, its application in buffalo has been very limited, with no prior reports of live gene-edited animals. Here, we report the successful birth of a buffalo edited in the myostatin (MSTN) gene. To achieve this, five single-guide RNAs (sgRNAs) targeting the buffalo MSTN gene were designed and tested in skin-derived fibroblasts. Among these, sgRNA5 exhibited the highest editing efficiency, approaching ∼50%, as confirmed by T7 Endonuclease I assay, Tracking of Indels by Decomposition, and Inference of CRISPR Edits analyses. Single-cell cloning identified six edited fibroblast clonal populations, including one with a bi-allelic frameshift mutation predicted to severely truncate the MSTN protein. These bi-allelic clonal cells were subsequently used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos, which were transferred into recipient buffaloes (n = 15). This effort established three pregnancies and resulted in the birth of one live MSTN knockout buffalo calf. Phenotypically, the calf displayed accelerated growth and increased muscle fiber number and size while maintaining normal meat composition. In conclusion, this study reports the world's first gene-edited buffalo generated through CRISPR-Cas9-mediated genome editing combined with SCNT. These findings provide a proof-of-concept for genome editing in buffalo and demonstrate that MSTN disruption can effectively enhance muscle growth and meat production traits.

The problem of improving the breeding process in order to breed highly productive animals remains relevant and requires current approaches to its solution. Preserving local breeds of sheep is a key task for animal breeders and specialists in animal genetics research. One of the well-established breeds with high productivity and adaptability is Soviet Merino of Gashunsky type, which was bred in the Rostov region. The results of research the polymorphism of the genes CAST and MSTN and its influence on the meat productivity of sheep of Soviet merino breed have been presented in the article. Allele polymorphism was studied by PCR-RFLP assay. The gene CAST is considered as one of the promising markers affecting the growth intensity and meat quality. The gene MSTN plays an important role in the increase of muscle tissue. The results obtained confi rm the presence of the diversity of allelic variants of the genes calpastatin and myostatin in the sheep of Soviet merino breed. The use of genetic technologies in combination with traditional breeding methods will allow us to improve the productive characteristics of sheep in a relatively short time. The results of polymorphism of the genes CAST and MSTN in the sheep of Soviet merino breed have been obtained. Signifi cant associations between the gene CAST and MSTN genotypes have been revealed. The influence of genes CAST and MSTN polymorphism on meat indicators has been established, and the desirable genotypes CAST_MN and MSTN_AG have been determined. These studies indicate the potential for using genes CAST and MSTN as markers for the meat productivity of sheep. It is possible by combining genetic technologies with traditional breeding practices to improve the productivity of sheep Soviet merino breed of the type Gashunsky in a relatively short period of time.

The MSTN gene plays a critical role in muscle development, and its knockout has been shown to significantly enhance growth performance in animals. In this study, we utilized MSTN knockout Chinese Merino sheep (Xinjiang type), which were previously generated in our laboratory, to compare their growth performance, hematological, and biochemical parameters with those of a control group. The results revealed that the MSTN knockout sheep exhibited significantly improved traits, including body weight, body length, and body height, without adverse effects on blood or metabolic parameters. Transcriptomic analysis identified 121 differentially expressed genes (DEGs) involved in key pathways such as amino acid metabolism, muscle contraction, and immune response. A protein-protein interaction (PPI) network was constructed, highlighting 10 core genes that may play pivotal roles in the biological processes regulated by MSTN. RT-qPCR validation of several key genes confirmed the results of the transcriptomic analysis. This study provides both theoretical foundations and practical insights for gene-edited breeding in meat sheep.

Follistatin is a potent regulator of various TGF-β superfamily members, including myostatin (MSTN) and activin A. Previous studies have shown that follistatin is crucial in enhancing myogenesis during acute muscle injury. The mechanism by which fibro-adipogenic progenitors (FAPs)-specific follistatin influences muscle homeostasis in obese mice remains unknown. Therefore, we investigated the physiological role of follistatin in PDGFRα-positive FAPs in the regulation of muscle homeostasis and exercise in obese mice. A PDGFRα-specific follistatin knockout (follistatin KO) mouse model was generated using PDGFRα-GFP-CreERT2 (PDGFRα-GCE) and follistatinflox/flox mice. These mice were fed a 60% high-fat diet (HFD) for 20weeks, followed by a series of analyses, including exercise tolerance test, grip strength test, glucose and insulin tolerance assays, gene expression analysis, histology, western blotting, and immunohistochemistry. We showed that follistatin KO mice had reduced expression of Fst in skeletal muscle and white adipose tissue. We also showed that follistatin KO mice exhibited decreased exercise performance and altered skeletal homeostasis during obesity. Deletion of follistatin in FAPs activated the MSTN: Activin A/SMADs signaling pathways, which negatively impacted muscle homeostasis. Furthermore, follistatin KO mice showed reduced muscle mass, increased muscle degradation, and atrophic myofibers. Mitochondrial biogenesis, oxidative phosphorylation, and fatty acid oxidation were also altered in the skeletal muscles of follistatin KO mice. Follistatin plays a protective role in mice by maintaining the metabolic health of skeletal muscles; it restores muscle function during HFD challenge, thereby reducing diet-induced obesity-related complications.

Myocyte Enhancer Factor 2 (Mef2) is a transcription factor that exert crucial functions in muscle cells, neurons and other cell types. These Mef2 family members modulate the expression of target genes to participate in a broad spectrum of biological processes, such as, but not restricted to, muscle differentiation and heart development. Myostatin (mstn), a protein from the Transforming Growth Factor-β (TGF-β) superfamily, is of great significance in muscle growth and development. Its principal function lies in suppressing the proliferation and differentiation of skeletal muscle cells, thus controlling muscle mass. In this study, we obtained the genomic sequence of mstn1 from Acanthopagrus latus, which is 2,198bp in length and encodes 384 amino acids. It consists of three distinct domains: a TGF-β domain, a TGF-β propeptide domain and a signal peptide. Through phylogenetic analysis, it was found that mstn1 and mstn2 from A. latus are closely grouped with those from Sparus aurata, which implies a high level of similarity between the two species. Additionally, mstn1 was mainly expressed in the brain, white muscle, and skin. To further investigate the regulatory mechanisms underlying mstn1 in muscle growth, we analyzed the transcriptional levels of mstn1 in white muscle under conditions of starvation and refeeding. The results indicated that during the 56-day experimental period, the expression of the mstn1 gene decreased notably as the starvation period extended. Truncation experiments revealed that the region from - 645 to + 112bp constitutes the core promoter region responsive to Mef2a and Mef2b. The point mutation analysis verified that the transcriptional activity of mstn1 is contingent upon the mutation of binding site 3 (M3) regulated by Mef2a and Mef2b. Moreover, siRNA-mediated knockdown experiments demonstrated that downregulation of mef2a or mef2b significantly decreased the transcription of mstn1. These findings provide novel insights into how Mef2 transcription factors regulatemstn1expression, enhancing our understanding of the molecular mechanisms underlying muscle development in teleost fish.

Data on the interplay between muscle, bone, and adipose tissue metabolism in normal-weight children with Prader–Willi syndrome (PWS) undergoing growth hormone (GH) therapy and dietary interventions are limited. This study aimed to assess the myokine profile and explore the associations between myokines, bone markers, adipokines, and body composition in these patients. The study included 26 children with PWS and 26 age-matched healthy controls. Serum levels of irisin, myostatin (MSTN), fibroblast growth factor-2, insulin-like growth factor-I (IGF-I), IGF-binding protein-2, bone alkaline phosphatase (BALP), osteocalcin (OC), carboxylated OC (Gla-OC), periostin, soluble receptor activator of nuclear factor kappa-B ligand, tartrate-resistant acid phosphatase 5b, leptin/soluble leptin receptor, adiponectin, and proinsulin were measured using immunoenzymatic assays. Children with PWS had significantly lower lean mass (p = 0.047) and a higher fat mass/lean mass ratio (p < 0.001) than controls. Irisin levels were lower in the PWS group (p = 0.031), while MSTN levels were similar between the groups. In patients, irisin positively correlated with BALP (p = 0.025) and negatively correlated with Gla-OC (p = 0.041) and periostin (p = 0.005). MSTN was positively associated with proinsulin (p = 0.001) and negatively associated with lean mass (p = 0.015). OC concentration was lower in the PWS group and correlated positively with lean mass (p = 0.052). Children with PWS exhibit altered myokine, osteokine, and adipokine profiles, as well as differences in body composition. Reduced irisin and osteocalcin levels, along with the negative association between MSTN and lean mass, may impair muscle development and bone metabolism. These imbalances could also contribute to future metabolic disorders in patients with PWS.

MSTN gene editing does not alter reproductive capacity but modifies gestational metabolism in Mongolian cattle

Disclosure: K. Stefanakis: None. P. Veeragandham: None. V. Gutierrez de Pineres: None. C.S. Mantzoros: Novo Nordisk.The recent FDA approval of generic Liraglutide necessitates a better understanding of its downstream molecular mechanisms to develop obesity treatments that reduce adiposity while preserving lean body mass, key factors in mitigating sarcopenia associated with GLP-1 agonists. In a 5-week crossover randomized, double-blind, placebo-controlled trial, 20 participants with obesity received Liraglutide (3.0 mg/day) or placebo in random sequence, separated by a minimum 3-week washout period. The SomaScan v4.1 platform was used to quantify approximately 7,000 proteins across serum and plasma samples with robust quality controls. Longitudinal proteomic changes were assessed using FDR-corrected pre-to-post comparisons and adjusted mixed models, whereas enrichment analyses were performed using gene and disease ontology, and data from deCODE GWAS. Liraglutide treatment resulted in a significant weight reduction (-2.99 kg placebo-subtracted) and improved glucose and lipid profiles. Out of 6,249 common proteins analyzed, 338 proteins had a significant Time*Treatment interaction, of which Liraglutide significantly upregulated 84 and downregulated 104 proteins, whereas placebo had minimal effects. Specifically, Liraglutide increased exocrine pancreatic proteins (e.g., PNLIPRP1, PRSS2, CTRB2) and fatty acid–binding proteins (e.g., FABP4), and decreased proteins related to fibrosis, inflammation, collagen deposition, and neural development - potentially related to known effects of Liraglutide on appetite regulation and hypothalamic centers. Most proteomic changes were independent of weight and glucose; however, myostatin (MSTN), a negative regulator of muscle growth, was downregulated in a weight-dependent manner, and its inhibitor WFIKKN2, alongside TGF-β receptor BMPR1a were upregulated, suggesting a compensatory mechanism to preserve lean mass during early weight loss. ELISA validation confirmed the proteomic measurements for MSTN (r=0.78, p<10-14). Pathway enrichment analyses indicated reductions in fibrosis and inflammatory processes, and in pathways associated with cardiovascular, renal, and liver diseases, alongside differential regulation of apoptosis, and enhancements in digestion, lipid metabolism, and humoral immunity. Secondary analyses identified distinct proteomic signatures in seven participants who lost more than 3% of their baseline weight, involving neurotransmitter and insulin-regulating proteins. Unlike longer-term weight loss studies, these results capture early molecular adaptations, with the myostatin axis emerging as a target for combination therapies to minimize lean tissue loss. These insights are critical to better understand the mechanisms of weight loss and to optimize the development of novel compounds aimed towards myostatin antagonism to concomitantly lose fat and preserve lean body mass and metabolic health.Presentation: Saturday, July 12, 2025

Maintaining skeletal muscle mass is crucial for health, as muscle atrophy caused by drugs, cancer, or aging poses serious risks. However, there are few effective pharmacological interventions targeting muscle atrophy, highlighting the need for new therapeutic strategies. In this study, in vivo self‐assembled siRNA is designed to silence myostatin (MSTN), a key regulator of muscle growth and atrophy, aiming to prevent muscle atrophy. Using synthetic constructs and the host liver as a scaffold, the assembly of MSTN‐siRNA is guided into muscle‐specific peptide MSP‐tagged small extracellular vesicles (sEVs). These MSP‐tagged sEVs selectively deliver MSTN‐siRNA to muscle tissue. Treatment significantly reduces MSTN protein levels in skeletal muscle, promotes muscle mass gain in healthy mice, and protectes skeletal muscles from atrophy in cancer‐ and dexamethasone‐induced muscle atrophy models. Notably, the sEV‐encapsulated MSTN‐siRNA is produced in a nontoxic, nonimmunogenic, and biocompatible manner. This study offers a promising therapeutic approach for muscle atrophy, addressing a key gap in current treatment options and potentially improving outcomes for patients with muscle‐wasting conditions.

Characterization of myostatin genes in Black Sea trout, Salmo labrax, and their differential responses to high temperature and starvation stressors.

Diabetic skeletal muscle atrophy is one of the most serious complications among diabetes-related complications. LIPUS enhances muscle regeneration and repair in skeletal muscle injuries. However, whether LIPUS can improve skeletal muscle atrophy in mice with T1DM has not been studied. This study involves forty male C57BL/6 mice randomly divided into four groups: normal control group (NC), streptozocin (STZ)-induced T1DM mice (T1D), T1DM mice treated with LIPUS (DL), and T1DM mice treated with insulin (DI). The DL group was treated on the quadriceps of mice with LIPUS (1 MHz, 80 mW/cm2, 20 min/day) for 6 weeks. The results demonstrated that LIPUS significantly improved muscle function by increasing the cross-sectional area, mass, and strength of skeletal muscles. In addition, LIPUS significantly effectively lowered the blood glucose levels of T1DM mice. The knockout of myostatin (MSTN) (MSTN-/-) and knockin of MSTN (MSTN+/+) mice were employed to verify the underlying mechanism. The results indicated that LIPUS reduces blood glucose levels in T1DM mice by improving their muscle atrophy. This study demonstrated that LIPUS will become a novel therapy for the treatment of skeletal muscle atrophy caused by T1DM.