Advances in sarcopenia: mechanisms, therapeutic targets, and intervention strategies.

Sarcopenia is a multifactorial condition characterized by loss of muscle mass. It poses significant health risks in older adults worldwide. Both pharmacological and non-pharmacological approaches are reported to address this disease. Certain dietary patterns, such as adequate energy intake and essential amino acids, have shown positive outcomes in preserving muscle function. Various medications, including myostatin inhibitors, growth hormones, and activin type II receptor inhibitors, have been evaluated for their effectiveness in managing sarcopenia. However, it is important to consider the variable efficacy and potential side effects associated with these treatments. There are currently no drugs approved by the Food and Drug Administration for sarcopenia. The ongoing research aims to develop more effective strategies in the future. Our review of research on disease mechanisms and drug development will be a valuable contribution to future research endeavors.

Licochalcone A and B enhance muscle proliferation and differentiation by regulating Myostatin.

BACKGROUND: Myostatin (MSTN) inhibition has demonstrated promise for the treatment of diseases associated with muscle loss. In a previous study, we discovered that Glycyrrhiza uralensis (G. uralensis) crude water extract (CWE) inhibits MSTN expression while promoting myogenesis. Furthermore, three specific compounds of G. uralensis, namely liquiritigenin, tetrahydroxymethoxychalcone, and Licochalcone B (Lic B), were found to promote myoblast proliferation and differentiation, as well as accelerate the regeneration of injured muscle tissue. PURPOSE: The purpose of this study was to build on our previous findings on G. uralensis and demonstrate the potential of its two components, Licochalcone A (Lic A) and Lic B, in muscle mass regulation (by inhibiting MSTN), aging and muscle formation. METHODS: G. uralensis, Lic A, and Lic B were evaluated thoroughly using in silico, in vitro and in vivo approaches. In silico analyses included molecular docking, and dynamics simulations of these compounds with MSTN. Protein-protein docking was carried out for MSTN, as well as for the docked complex of MSTN-Lic with its receptor, activin type IIB receptor (ACVRIIB). Subsequent in vitro studies used C2C12 cell lines and primary mouse muscle stem cells to acess the cell proliferation and differentiation of normal and aged cells, levels of MSTN, Atrogin 1, and MuRF1, and plasma MSTN concentrations, employing techniques such as western blotting, immunohistochemistry, immunocytochemistry, cell proliferation and differentiation assays, and real-time RT-PCR. Furthermore, in vivo experiments using mouse models focused on measuring muscle fiber diameters. RESULTS: CWE of G. uralensis and two of its components, namely Lic A and B, promote myoblast proliferation and differentiation by inhibiting MSTN and reducing Atrogin1 and MuRF1 expressions and MSTN protein concentration in serum. In silico interaction analysis revealed that Lic A (binding energy -6.9 Kcal/mol) and B (binding energy -5.9 Kcal/mol) bind to MSTN and reduce binding between it and ACVRIIB, thereby inhibiting downstream signaling. The experimental analysis, which involved both in vitro and in vivo studies, demonstrated that the levels of MSTN, Atrogin 1, and MuRF1 were decreased when G. uralensis CWE, Lic A, or Lic B were administered into mice or treated in the mouse primary muscle satellite cells (MSCs) and C2C12 myoblasts. The diameters of muscle fibers increased in orally treated mice, and the differentiation and proliferation of C2C12 cells were enhanced. G. uralensis CWE, Lic A, and Lic B also promoted cell proliferation in aged cells, suggesting that they may have anti-muslce aging properties. They also reduced the expression and phosphorylation of SMAD2 and SMAD3 (MSTN downstream effectors), adding to the evidence that MSTN is inhibited. CONCLUSION: These findings suggest that CWE and its active constituents Lic A and Lic B have anti-mauscle aging potential. They also have the potential to be used as natural inhibitors of MSTN and as therapeutic options for disorders associated with muscle atrophy.

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.

Network pharmacology based pharmacokinetic assessment and evaluation of the therapeutic potential of catechin derivatives as a potential myostatin inhibitor: A special view on Sarcopenic Obesity.

Sarcopenic obesity has become a significant age-related metabolic problem. Catechins are flavanol, derivatives which poses a strong antioxidant activity. The major components of catechin derivatives. were identified through our physicochemical and pharmacokinetic parameters estimation. Therefore, in this study, network pharmacology was used to explore the multiple targets related to Sarcopenia, Metabolic syndrome, and obesity. The targets were identified from network analysis. The catechin derivatives were screened using Lipinski's rule of five, Veber scale, Egan scale, and Muegge scale. From this drugglikness property catechin and Epicatechin was selected which were docked towards the myostatin inhibition PDB ID: 3HH2. Furthermore, the computational docking method on Catechin and Epicatechin with the stronger interaction towards myostatin inhibition receptor with the binding energy of -6.90 kcal/mol. and -7.0 kcal/mol from autodock software, respectively, for catechin and Epicatechin. Higher binding energy confirms the pharmacotherapeutic activity of Catechin and Epicatechin toward the myostatin inhibitor target.

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.

Myostatin and CXCL11 promote nervous tissue macrophages to maintain osteoarthritis pain.

Pain is the most debilitating symptom of knee osteoarthritis (OA) that can even persist after total knee replacement. The severity and duration of pain do not correlate well with joint tissue alterations, suggesting other mechanisms may drive pain persistence in OA. Previous work identified that macrophages accumulate in the dorsal root ganglia (DRG) containing the somas of sensory neurons innervating the injured knee joint in a mouse OA model and acquire a M1-like phenotype to maintain pain. Here we aimed to unravel the mechanisms that govern DRG macrophage accumulation and programming. The accumulation of F4/80+iNOS+ (M1-like) DRG macrophages was detectable at day 3 after mono-iodoacetate (MIA)-induced OA in the mouse. Depletion of macrophages prior to induction of OA resolved pain-like behaviors by day 7 without affecting the initial development of pain-like behaviors. Analysis of DRG transcript identified CXCL11 and myostatin. CXCL11 and myostatin were increased at 3 weeks post OA induction, with CXCL11 expression partially localized in satellite glial cells and myostatin in sensory neurons. Blocking CXCL11 or myostatin prevented the persistence of OA pain, without affecting the initiation of pain. CXCL11 neutralization reduced the number of total and F4/80+iNOS+ DRG macrophages, whilst myostatin inhibition diminished the programming of F4/80+iNOS+ DRG macrophages. Intrathecal injection of recombinant CXCL11 did not induce pain-associated behaviors. In contrast, intrathecal myostatin increased the number of F4/80+iNOS+ DRG macrophages concurrent with the development of mechanical hypersensitivity that was prevented by macrophages depletion or CXCL11 blockade. Finally, myostatin inhibition during established OA, resolved pain and F4/80+iNOS+ macrophage accumulation in the DRG. In conclusion, DRG macrophages maintain OA pain, but are not required for the induction of OA pain. Myostatin is a key ligand in neuro-immune communication that drives the persistence of pain in OA through nervous tissue macrophages and represent a novel therapeutic target for the treatment of OA pain.

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.

Current Nutritional and Pharmacological Approaches for Attenuating Sarcopenia.

Sarcopenia is characterized by a gradual slowing of movement due to loss of muscle mass and quality, decreased power and strength, increased risk of injury from falls, and often weakness. This review will focus on recent research trends in nutritional and pharmacological approaches to controlling sarcopenia. Because nutritional studies in humans are fairly limited, this paper includes many results from nutritional studies in mammals. The combination of resistance training with supplements containing amino acids is the gold standard for preventing sarcopenia. Amino acid (HMB) supplementation alone has no significant effect on muscle strength or muscle mass in sarcopenia, but the combination of HMB and exercise (whole body vibration stimulation) is likely to be effective. Tea catechins, soy isoflavones, and ursolic acid are interesting candidates for reducing sarcopenia, but both more detailed basic research on this treatment and clinical studies in humans are needed. Vitamin D supplementation has been shown not to improve sarcopenia in elderly individuals who are not vitamin D-deficient. Myostatin inhibitory drugs have been tried in many neuromuscular diseases, but increases in muscle mass and strength are less likely to be expected. Validation of myostatin inhibitory antibodies in patients with sarcopenia has been positive, but excessive expectations are not warranted.

Resistance exercise alleviates dexamethasone-induced muscle atrophy via Sestrin2/MSTN pathway in C57BL/6J mice.

AIM: It has long been recognized that resistance exercise can substantially increase skeletal muscle mass and strength, but whether it can protect against glucocorticoid-induced muscle atrophy and its potential mechanism is yet to be determined. This study aimed to investigate the protective effects of resistance exercise in dexamethasone-induced muscle atrophy and elucidate the possible function of exercise-induced protein Sestrin2 in this process. METHODS: Eight-week-old male C57BL/6J mice carried out the incremental mouse ladder exercise for 11 weeks. Two weeks before the end of the intervention, mice were daily intraperitoneally injected with dexamethasone. Body composition, muscle mass, and exercise performance were examined to evaluate muscle atrophy. In vitro, C2C12 cells were used for RT-qPCR, Western Blot, and immunofluorescence experiments to elucidate the potential mechanism. RESULTS: Our results showed that long-term resistance exercise is an effective intervention for dexamethasone-induced muscle atrophy. We also found that Sestrin2 plays a vital role in dexamethasone-induced muscle atrophy. In both animal (P = .0006) and cell models (P = .0266), dexamethasone intervention significantly reduced the protein expression of Sestrin2, which was increased (P = .0112) by resistance exercise. Inversely, overexpression of Sestrin2 improved (P < .0001) dexamethasone-induced myotube cell atrophy by reducing the activation of the ubiquitin-proteasome pathway via inhibiting Forkhead box O3 (FoxO3a) and myostatin (MSTN)/small mother against decapentaplegic (Smad) signaling pathways. CONCLUSION: Taken together, our results indicated that Sestrin2 may serve as an effective molecule that mimics the protective effect of resistance exercise on dexamethasone-induced muscle atrophy.

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.

Senescent adipocytes as potential effectors of muscle cells dysfunction: An in vitro model.

Recently, there has been a growing body of evidence showing a negative effect of the white adipose tissue (WAT) dysfunction on the skeletal muscle function and quality. However, little is known about the effects of senescent adipocytes on muscle cells. Therefore, to explore potential mechanisms involved in age-related loss of muscle mass and function, we performed an in vitro experiment using conditioned medium obtained from cultures of mature and aged 3 T3-L1 adipocytes, as well as from cultures of dysfunctional adipocytes exposed to oxidative stress or high insulin doses, to treat C2C12 myocytes. The results from morphological measures indicated a significant decrease in diameter and fusion index of myotubes after treatment with medium of aged or stressed adipocytes. Aged and stressed adipocytes presented different morphological characteristics as well as a different gene expression profile of proinflammatory cytokines and ROS production. In myocytes treated with different adipocytes' conditioned media, we demonstrated a significant reduction of gene expression of myogenic differentiation markers as well as a significant increase of genes involved in atrophy. Finally, a significant reduction in protein synthesis as well as a significant increase of myostatin was found in muscle cells treated with medium of aged or stressed adipocytes compared to controls. In conclusion, these preliminary results suggest that aged adipocytes could influence negatively trophism, function and regenerative capacity of myocytes by a paracrine network of signaling.

Increased Ca2+ signaling through CaV 1.2 induces tendon hypertrophy with increased collagen fibrillogenesis and biomechanical properties.

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone, and cartilage development and homeostasis, knowledge about Ca2+ signaling and the source of Ca2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca2+ signaling through CaV 1.2 voltage-gated Ca2+ channel in tendon formation. Using a reporter mouse, we found that CaV 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;CaV 1.2TS mice that express a gain-of-function mutant CaV 1.2 in tendon. We found that mutant tendons were hypertrophic, with more tendon fibroblasts but decreased cell density. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendons. Biomechanical testing revealed that the hypertrophic tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomic analysis showed no significant difference in the abundance of type I and III collagens, but mutant tendons had about two-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2, and cathepsin K. Taken together, these data highlight roles for increased Ca2+ signaling through CaV 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.

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.

Myostatin: a potential therapeutic target for metabolic syndrome.

Metabolic syndrome is a complex metabolic disorder, its main clinical manifestations are obesity, hyperglycemia, hypertension and hyperlipidemia. Although metabolic syndrome has been the focus of research in recent decades, it has been proposed that the occurrence and development of metabolic syndrome is related to pathophysiological processes such as insulin resistance, adipose tissue dysfunction and chronic inflammation, but there is still a lack of favorable clinical prevention and treatment measures for metabolic syndrome. Multiple studies have shown that myostatin (MSTN), a member of the TGF-ß family, is involved in the development and development of obesity, hyperlipidemia, diabetes, and hypertension (clinical manifestations of metabolic syndrome), and thus may be a potential therapeutic target for metabolic syndrome. In this review, we describe the transcriptional regulation and receptor binding pathway of MSTN, then introduce the role of MSTN in regulating mitochondrial function and autophagy, review the research progress of MSTN in metabolic syndrome. Finally summarize some MSTN inhibitors under clinical trial and proposed the use of MSTN inhibitor as a potential target for the treatment of metabolic syndrome.

Snail mediates GDF-8-stimulated human extravillous trophoblast cell invasion by upregulating MMP2 expression.

BACKGROUND: Extravillous trophoblast (EVT) cell invasion is a tightly regulated process that requires for a normal pregnancy. The epithelial-mesenchymal transition (EMT) has been implicated in EVT cell invasion. Growth differentiation factor-8 (GDF-8), a member of the transforming growth factor-beta (TGF-ß) superfamily, is expressed in the human placenta and promotes EVT cell invasion by upregulating the expression of matrix metalloproteinase 2 (MMP2). However, the underlying molecular mechanism of GDF-8-induced MMP2 expression remains undetermined. Therefore, the present study aims to examine the role of Snail and Slug, the EMT-related transcriptional regulators, in GDF-8-stimulated MMP2 expression and cell invasion in HTR-8/SVneo human EVT cell line and primary cultures of human EVT cells. METHODS: HTR-8/SVneo and primary cultures of human EVT cells were used to examine the effect of GDF-8 on MMP2 expression and explore the underlying mechanism. For gene silencing and overexpression, the HTR-8/SVneo cell line was used to make the experiments more technically feasible. The cell invasiveness was measured by Matrigel-coated transwell invasion assay. RESULTS: GDF-8 stimulated MMP2 expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effect of GDF-8 on MMP2 expression was blocked by the inhibitor of TGF-ß type-I receptors, SB431542. Treatment with GDF-8 upregulated Snail and Slug expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effects of GDF-8 on Snail and Slug expression were blocked by pretreatment of SB431542 and siRNA-mediated knockdown of SMAD4. Interestingly, using the siRNA knockdown approach, our results showed that Snail but not Slug was required for the GDF-8-induced MMP2 expression and cell invasion in HTR-8/SVneo cells. The reduction of MMP2 expression in the placentas with preeclampsia (PE) was also observed. CONCLUSIONS: These findings discover the physiological function of GDF-8 in the human placenta and provide important insights into the regulation of MMP2 expression in human EVT cells. Video Abstract.

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.

Combined Transcriptome and Metabolome Analysis of Smooth Muscle of Myostatin Knockout Cattle.

Myostatin (MSTN), a growth and differentiation factor, plays an important role in regulating skeletal muscle growth and development. MSTN knockout (MSTN-KO) leads to skeletal muscle hypertrophy and regulates metabolic homeostasis. Moreover, MSTN is also detected in smooth muscle. However, the effect of MSTN-KO on smooth muscle has not yet been reported. In this study, combined metabolome and transcriptome analyses were performed to investigate the metabolic and transcriptional profiling in esophageal smooth muscles of MSTN-KO Chinese Luxi Yellow cattle (n = 5, 24 months, average body weight 608.5 ± 17.62 kg) and wild-type (WT) Chinese Luxi Yellow cattle (n = 5, 24 months, average body weight 528.25 ± 11.03 kg). The transcriptome was sequenced using the Illumina Novaseq™ 6000 sequence platform. In total, 337 significantly up- and 129 significantly down-regulated genes were detected in the MSTN-KO cattle compared with the WT Chinese Luxi Yellow cattle. Functional enrichment analysis indicated that the DEGs were mainly enriched in 67 signaling pathways, including cell adhesion molecules, tight junction, and the cGMP-PKG signaling pathway. Metabolomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 130 differential metabolites between the groups, with 56 up-regulated and 74 down-regulated in MSTN knockout cattle compared with WT cattle. Differential metabolites were significantly enriched in 31 pathways, including glycerophospholipid metabolism, histidine metabolism, glutathione metabolism, and purine metabolism. Transcriptome and metabolome were combined to analyze the significant enrichment pathways, and there were three metabolically related pathways, including histidine metabolism, purine metabolism, and arginine and proline metabolism. These results provide important references for in-depth research on the effect of MSTN knockout on smooth muscle.

Fecal transplant from myostatin deletion pigs positively impacts the gut-muscle axis.

The host genome may influence the composition of the intestinal microbiota, and the intestinal microbiota has a significant effect on muscle growth and development. In this study, we found that the deletion of the myostatin (MSTN) gene positively regulates the expression of the intestinal tight junction-related genes TJP1 and OCLN through the myosin light-chain kinase/myosin light chain pathway. The intestinal structure of MSTN-/- pigs differed from wild-type, including by the presence of a thicker muscularis and longer plicae. Together, these changes affect the structure of intestinal microbiota. Mice transplanted with the intestinal microbiota of MSTN-/- pigs had myofibers with larger cross-sectional areas and higher fast-twitch glycolytic muscle mass. Microbes responsible for the production of short-chain fatty acids (SCFAs) were enriched in both the MSTN-/- pigs and recipient mice, and SCFAs levels were elevated in the colon contents. We also demonstrated that valeric acid stimulates type IIb myofiber growth by activating the Akt/mTOR pathway via G protein-coupled receptor 43 and ameliorates dexamethasone-induced muscle atrophy. This is the first study to identify the MSTN gene-gut microbiota-SCFA axis and its regulatory role in fast-twitch glycolytic muscle growth.

Red blood cell extracellular vesicles deliver therapeutic siRNAs to skeletal muscles for treatment of cancer cachexia.

Cancer cachexia is a multifactorial syndrome characterized by a significant loss of skeletal muscle, which negatively affects the quality of life. Inhibition of myostatin (Mstn), a negative regulator of skeletal muscle growth and differentiation, has been proven to preserve muscle mass in muscle atrophy diseases, including cachexia. However, myostatin inhibitors have repeatedly failed clinical trials because of modest therapeutic effects and side effects due to the poor efficiency and toxicity of existing delivery methods. Here, we describe a novel method for delivering Mstn siRNA to skeletal muscles using red blood cell-derived extracellular vesicles (RBCEVs) in a cancer cachectic mouse model. Our data show that RBCEVs are taken up by myofibers via intramuscular administration. Repeated intramuscular administrations with RBCEVs allowed the delivery of siRNAs, thereby inhibiting Mstn, increasing muscle growth, and preventing cachexia in cancer-bearing mice. We observed the same therapeutic effects when delivering siRNAs against malonyl-CoA decarboxylase, an enzyme driving dysfunctional fatty acid metabolism in skeletal muscles during cancer cachexia. We demonstrate that intramuscular siRNA delivery by RBCEVs is safe and non-inflammatory. Hence, this method is useful to reduce the therapeutic dose of siRNAs, to avoid toxicity and off-target effects caused by systemic administration of naked siRNAs at high doses.

Global Long Noncoding RNA Expression Profiling of MSTN and FGF5 Double-Knockout Sheep Reveals the Key Gatekeepers of Skeletal Muscle Development.

Improving livestock and poultry growth rates and increasing meat production are urgently needed worldwide. Previously, we produced a myostatin (MSTN) and fibroblast growth factor 5 (FGF5) double-knockout (MF-/-) sheep by CRISPR Cas9 system to improve meat production, and also wool production. Both MF-/- sheep and the F1 generation (MF+/-) sheep showed an obvious "double-muscle" phenotype. In this study, we identified the expression profiles of long noncoding RNAs (lncRNAs) in wild-type and MF+/- sheep, then screened out the key candidate lncRNAs that can regulate myogenic differentiation and skeletal muscle development. These key candidate lncRNAs can serve as critical gatekeepers for muscle contraction, calcium ion transport and skeletal muscle cell differentiation, apoptosis, autophagy, and skeletal muscle inflammation, further revealing that lncRNAs play crucial roles in regulating muscle phenotype in MF+/- sheep. In conclusion, our newly identified lncRNAs may emerge as novel molecules for muscle development or muscle disease and provide a new reference for MSTN-mediated regulation of skeletal muscle development.