Expression Of Myogenic Markers Research Articles

Derivation and long-term maintenance of porcine skeletal muscle progenitor cells

Culture of muscle cells from livestock species has typically involved laborious enzyme-based approaches that yield heterogeneous populations with limited proliferative and myogenic differentiation capacity, thus limiting their use in physiologically-meaningful studies. This study reports the use of a simple explant culture technique to derive progenitor cell populations from porcine muscle that could be maintained and differentiated long-term in culture. Fragments of semitendinosus muscle from 4 to 8 week-old piglets (n = 4) were seeded on matrigel coated culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Cell outgrowths appeared within a few days and were serially passaged and characterised using RT-qPCR, immunostaining and flow cytometry. MDPCs had an initial mean doubling time of 1.4 days which increased to 2.5 days by passage 14. MDPC populations displayed steady levels of the lineage-specific markers, PAX7 and MYOD, up until at least passage 2 (positive immunostaining in about 40% cells for each gene), after which the expression of myogenic markers decreased gradually. Remarkably, MDPCs were able to readily generate myotubes in culture up until passage 8. Moreover, a decrease in myogenic capacity during serial passaging was concomitant with a gradual increase in the expression of the pre-adipocyte markers, CD105 and PDGFRA, and an increase in the ability of MDPCs to differentiate into adipocytes. In conclusion, explant culture provided a simple and efficient method to harvest enriched myogenic progenitors from pig skeletal muscle which could be maintained long-term and differentiated in vitro, thus providing a suitable system for studies on porcine muscle biology and applications in the expanding field of cultured meat.

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

Acetyl genistin modulates myotube differentiation and attenuates dexamethasone-induced muscle atrophy through the FoxO1/3 signaling pathway in C2C12 myotubes

Muscle atrophy, a debilitating condition characterized by loss of muscle mass and strength, is a major concern in various clinical settings. Acetyl genistin (AG), a bioactive compound, was evaluated for its role in muscle cell differentiation and its potential protective effects against dexamethasone (dexa)-induced muscle atrophy. Our study demonstrated that AG significantly promoted C2C12 myotube differentiation, as evidenced by enhanced myotube width and increased fusion index. Notably, AG treatment upregulated the expression of myogenic markers, including MHC, MyoD, and MyoG. Moreover, AG displayed protective properties by attenuating dexa-induced muscle atrophy, mainly by suppressing the expression of the atrophy-related genes MAFbx and MuRF1. AG's protective effects are mechanistically attributed to its regulation of the AMPK/FoxO-dependent signaling pathway. Our results highlighted the dual benefits of AG in fostering muscle differentiation and safeguarding against muscle atrophy, positioning it as a promising agent for muscle health and therapeutic applications.

Exosomes Secreted During Myogenic Differentiation of Human Fetal Cartilage-Derived Progenitor Cells Promote Skeletal Muscle Regeneration through miR-145-5p.

Currently, there is no apparent treatment for sarcopenia, which is characterized by diminished myoblast function. We aimed to manufacture exosomes that retain the myogenic differentiation capacity of human fetal cartilage-derived progenitor cells (hFCPCs) and investigate their muscle regenerative efficacy in myoblasts and a sarcopenia rat model. The muscle regeneration potential of exosomes (F-Exo) secreted during myogenic differentiation of hFCPCs was compared to human bone marrow mesenchymal stem cells-derived (hBMSCs) exosomes (B-Exo) in myoblasts and sarcopenia rat model. The effect of F-Exo was analyzed through known microRNAs (miRNAs) analysis. The mechanism of action of F-Exo was confirmed by measuring the expression of proteins involved in the Wnt signaling pathway. F-Exo and B-Exo showed similar exosome characteristics. However, F-Exo induced the expression of muscle markers (MyoD, MyoG, and MyHC) and myotube formation in myoblasts more effectively than B-Exo. Moreover, F-Exo induced greater increases in muscle fiber cross-sectional area and muscle mass compared to B-Exo in a sarcopenia rat. The miR-145-5p, relevant to muscle regeneration, was found in high concentrations in the F-Exo, and RNase pretreatment reduced the efficacy of exosomes. The effects of F-Exo on the expression of myogenic markers in myoblasts were paralleled by the miR-145-5p mimics, while the inhibitor partially negated this effect. F-Exo was involved in the Wnt signaling pathway by enhancing the expression of Wnt5a and β-catenin. F-Exo improved muscle regeneration by activating the Wnt signaling pathway via abundant miR-145-5p, mimickingthe remarkable myogenic differentiation potential of hFCPCs.

Directing cellular responses in a nanocomposite 3D matrix for tissue regeneration with nanoparticle-mediated drug delivery

Hydrogels play an important role in tissue engineering due to their native extracellular matrix-like characteristics, but they are insufficient in providing the necessary stimuli to support tissue formation. Efforts to integrate bioactive cues directly into hydrogels are hindered by incompatibility with hydrophobic drugs, issues of burst/uncontrolled release, and rapid degradation of the bioactive molecules. Skeletal muscle tissue repair requires internal stimuli and communication between cells for regeneration, and nanocomposite systems offer to improve the therapeutic effects in tissue regeneration. Here, the versatility of mesoporous silica nanoparticles (MSN) was leveraged to formulate a nanoparticle-hydrogel composite and to combine the benefits of controlled delivery of bioactive cues and cellular support. The tunable surface characteristics of MSNs were exploited to optimize homogeneity and intracellular drug delivery in a 3D matrix. Nanocomposite hydrogels formulated with acetylated or succinylated MSNs achieved high homogeneity in 3D distribution, with succinylated MSNs being rapidly internalized and acetylated MSNs exhibiting slower cellular uptake. MSN-hydrogel nanocomposites simultaneously allowed efficient local intracellular delivery of a hydrophobic model drug. To further study the efficiency of directing cell response, a Notch signaling inhibitor (DAPT) was incorporated into succinylated MSNs and incorporated into the hydrogel. MSN-hydrogel nanocomposites effectively downregulated the Notch signaling target genes, and accelerated and maintained the expression of myogenic markers. The current findings demonstrate a proof-of-concept in effective surface engineering strategies for MSN-based nanocomposites, suited for hydrophobic drug delivery in tissue regeneration with guided cues.

HMGB1 Promotes In Vitro and In Vivo Skeletal Muscle Atrophy through an IL-18-Dependent Mechanism

Skeletal muscle atrophy occurs due to muscle wasting or reductions in protein associated with aging, injury, and inflammatory processes. High-mobility group box-1 (HMGB1) protein is passively released from necrotic cells and actively secreted by inflammatory cells, and is implicated in the pathogenesis of various inflammatory and immune diseases. HMGB1 is upregulated in muscle inflammation, and circulating levels of the proinflammatory cytokine interleukin-18 (IL-18) are upregulated in patients with sarcopenia, a muscle-wasting disease. We examined whether an association exists between HMGB1 and IL-18 signaling in skeletal muscle atrophy. HMGB1-induced increases of IL-18 levels enhanced the expression of muscle atrophy markers and inhibited myogenic marker expression in C2C12 and G7 myoblast cell lines. HMGB1-induced increases of IL-18 production in C2C12 cells involved the RAGE/p85/Akt/mTOR/c-Jun signaling pathway. HMGB1 short hairpin RNA (shRNA) treatment rescued the expression of muscle-specific differentiation markers in murine C2C12 myotubes and in mice with glycerol-induced muscle atrophy. HMGB1 and IL-18 signaling was suppressed in the mice after HMGB1 shRNA treatment. These findings suggest that the HMGB1/IL-18 axis is worth targeting for the treatment of skeletal muscle atrophy.

Inhibition of GSK3β Promotes Proliferation and Suppresses Apoptosis of Porcine Muscle Satellite Cells

As the global population increases, interest in cultured meat (a new research field) is gradually increasing. The main raw material for the production of cultured meat is muscle stem cells called satellite cells isolated from livestock. However, how to mass proliferate and maintain satellite cells in vitro without genetic manipulation remains unclear. In the present study, we isolated and purified porcine muscle satellite cells (PMSCs) from the femur of a 1-day-old piglet and cultured PMSCs by treating them with an inhibitor (XAV939, Tankyrase (TNKS) inhibitor) or an activator (CHIR99021, glycogen synthase kinase 3 beta (GSK3β) inhibitor) of Wnt signaling. The CHIR group treated with 3 μM CHIR99021 showed a significantly increased proliferation rate of PMSCs compared to the SC group (control), whereas the XAV group treated with 1 μM XAV939 showed a significantly decreased proliferation rate of PMSCs. CHIR99021 also inhibited the differentiation of PMSCs by reducing the expression of MyoD while maintaining the expression of Pax7 and suppressed apoptosis by regulating the expression of apoptosis-related proteins and genes. RNA sequencing was performed to obtain gene expression profiles following inhibition or activation of the Wnt signaling pathway and various signaling mechanisms related to the maintenance of satellite cells were identified. Our results suggest that inhibition of GSK3β could dramatically improve the maintenance and mass proliferation ability of PMSCs in vitro by regulating the expression of myogenic markers and the cell cycle.

Molecular subtypes of leiomyosarcoma: Moving toward a consensus

Molecular subtypes of leiomyosarcoma: Moving toward a consensus

Mesenchymal stromal cell spheroids in sulfated alginate enhance muscle regeneration

The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is critically linked to the potency of the complex mixture of growth factors, cytokines, exosomes, and other biological cues that they secrete. The duration of cell-based approaches is limited by rapid loss of cells upon implantation, motivating the need to prolong cell viability and extend the therapeutic influence of the secretome. We and others demonstrated that the secretome is upregulated when MSCs are formed into spheroids. Although the efficacy of the MSC secretome has been characterized in the literature, no studies have reported the therapeutic benefit of in situ sequestration of the secretome within a wound site using engineered biomaterials. We previously demonstrated the capacity of sulfated alginate hydrogels to sequester components of the MSC secretome for prolonged presentation in vitro, yet the efficacy of this platform has not been evaluated in vivo. In this study, we used sulfated alginate hydrogels loaded with MSC spheroids to aid in the regeneration of a rat muscle crush injury. We hypothesized that the use of sulfated alginate to bind therapeutically relevant growth factors from the MSC spheroid secretome would enhance muscle regeneration by recruiting host cells into the tissue site. The combination of sulfated alginate and MSC spheroids resulted in decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions 2 weeks after injury. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreased fibrosis and increased functional regeneration of muscle. Statement of significanceThe therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is attributed to the complex diversity of the secretome. Cell-based approaches are limited by rapid cell death, motivating the need to extend the availability of the secretome. We previously demonstrated that sulfated alginate hydrogels sequester components of the MSC secretome for prolonged presentation in vitro, yet no studies have reported the in situ sequestration of the secretome. Herein, we transplanted MSC spheroids in sulfated alginate hydrogels to promote muscle regeneration. MSC spheroids in sulfated alginate decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreasing fibrosis and increasing functional muscle regeneration.

The Effect of Heat Shock on Myogenic Differentiation of Human Skeletal-Muscle-Derived Mesenchymal Stem/Stromal Cells.

Muscle injuries, degenerative diseases and other lesions negatively affect functioning of human skeletomuscular system and thus quality of life. Therefore, the investigation of molecular mechanisms, stimulating myogenic differentiation of primary skeletal-muscle-derived mesenchymal stem/stromal cells (SM-MSCs), is actual and needed. The aim of the present study was to investigate the myogenic differentiation of CD56 (neural cell adhesion molecule, NCAM)-positive and -negative SM-MSCs and their response to the non-cytotoxic heat stimulus. The SM-MSCs were isolated from the post operation muscle tissue, sorted by flow cytometer according to the CD56 biomarker and morphology, surface profile, proliferation and myogenic differentiation has been investigated. Data show that CD56(+) cells were smaller in size, better proliferated and had significantly higher levels of CD146 (MCAM) and CD318 (CDCP1) compared with the CD56(−) cells. At control level, CD56(+) cells significantly more expressed myogenic differentiation markers MYOD1 and myogenin (MYOG) and better differentiated to the myogenic direction. The non-cytotoxic heat stimulus significantly stronger stimulated expression of myogenic markers in CD56(+) than in CD56(−) cells that correlated with the multinucleated cell formation. Data show that regenerative properties of CD56(+) SM-MSCs can be stimulated by an extracellular stimulus and be used as a promising skeletal muscle regenerating tool in vivo.

Cultured Myoblasts Derived from Rat Soleus Muscle Show Altered Regulation of Proliferation and Myogenesis during the Course of Mechanical Unloading.

The structure and function of soleus muscle fibers undergo substantial remodeling under real or simulated microgravity conditions. However, unloading-induced changes in the functional activity of skeletal muscle primary myoblasts remain poorly studied. The purpose of our study was to investigate how short-term and long-term mechanical unloading would affect cultured myoblasts derived from rat soleus muscle. Mechanical unloading was simulated by rat hindlimb suspension model (HS). Myoblasts were purified from rat soleus at basal conditions and after 1, 3, 7, and 14 days of HS. Myoblasts were expanded in vitro, and the myogenic nature was confirmed by their ability to differentiate as well as by immunostaining/mRNA expression of myogenic markers. The proliferation activity at different time points after HS was analyzed, and transcriptome analysis was performed. We have shown that soleus-derived myoblasts differently respond to an early and later stage of HS. At the early stage of HS, the proliferative activity of myoblasts was slightly decreased, and processes related to myogenesis activation were downregulated. At the later stage of HS, we observed a decrease in myoblast proliferative potential and spontaneous upregulation of the pro-myogenic program.

Irisin/FNDC5 influences myogenic markers on skeletal muscle following high and moderate-intensity exercise training in STZ-diabetic rats.

In the present study, we investigated the effects of high-intensity interval training (HIIT) versus moderate-intensity continuous training (MICT) on irisin and expression of myogenic markers (paired box 7 (Pax7), myogenic differentiation 1 (MyoD), myogenin) in skeletal muscle of diabetic rats. Eighty-four male Wistar rats (6weeks of age) were randomly divided into seven groups (n = 12): basic control (Co Basic), 8weeks control (Co 8w), diabetes mellitus (DM), HIIT, DM + HIIT, MICT, and DM + MICT groups. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). The protocol was characterized by running on a rodent treadmill with moderate intensity (60-70% ), 60min per session, 5days/week, for 6weeks. HIIT consisted of six 3-min runs at a high intensity (80-90% ) alternated with 2-min running at low intensity (50% ), 30min per session, 5days/week, for 6weeks. Results showed that DM decreased myoblast markers compared to Co Basic and Co 8w groups. Fibronectin type III domain-containing protein 5 (FNDC5) mRNA decrease was correlated with myoblast markers (Pax7 r = 0.632, p = 0.027; MyoD r = 0.999, p = 0.001; myogenin r = 1.000, p = 0.001) in DM group. DM + MICT significantly increased gene expression of MyoD, myogenin, and FNDC5 compared to DM and DM + HIIT. The results also showed that the intensity and duration of exercise on the treadmill were effective in stimulating irisin and myogenic markers after DM.

Emulsion-templated microparticles with tunable stiffness and topology: Applications as edible microcarriers for cultured meat

Cultured meat has potential to diversify methods for protein production, but innovations in production efficiency will be required to make cultured meat a feasible protein alternative. Microcarriers provide a strategy to culture sufficient volumes of adherent cells in a bioreactor that are required for meat products. However, cell culture on inedible microcarriers involves extra downstream processing to dissociate cells prior to consumption. Here, we present edible microcarriers that can support the expansion and differentiation of myogenic cells in a single bioreactor system. To fabricate edible microcarriers with a scalable process, we used water-in-oil emulsions as templates for gelatin microparticles. We also developed a novel embossing technique to imprint edible microcarriers with grooved topology in order to test if microcarriers with striated surface texture can promote myoblast proliferation and differentiation in suspension culture. In this proof-of-concept demonstration, we showed that edible microcarriers with both smooth and grooved surface topologies supported the proliferation and differentiation of mouse myogenic C2C12 cells in a suspension culture. The grooved edible microcarriers showed a modest increase in the proliferation and alignment of myogenic cells compared to cells cultured on smooth, spherical microcarriers. During the expansion phase, we also observed the formation of cell-microcarrier aggregates or ‘microtissues’ for cells cultured on both smooth and grooved microcarriers. Myogenic microtissues cultured with smooth and grooved microcarriers showed similar characteristics in terms of myotube length, myotube volume fraction, and expression of myogenic markers. To establish feasibility of edible microcarriers for cultured meat, we showed that edible microcarriers supported the production of myogenic microtissue from C2C12 or bovine satellite muscle cells, which we harvested by centrifugation into a cookable meat patty that maintained its shape and exhibited browning during cooking. These findings demonstrate the potential of edible microcarriers for the scalable production of cultured meat in a single bioreactor.

Schwann Cells Promote Myogenic Differentiation of Myoblasts and Adipogenic Mesenchymal Stromal Cells on Poly-ɛ-Caprolactone-Collagen I-Nanofibers.

For the purpose of skeletal muscle tissue engineering, different cell types have been investigated regarding their myogenic differentiation potential, including co-cultured myoblasts and adipogenic mesenchymal stromal cells (Mb/ADSC). As neural cells enhance synaptic junction formation, the aim of this study was to co-culture Schwann cells (SCs) with Mb/ADSC on biocompatible electrospun aligned poly-ε-polycaprolacton (PCL)-collagen I-nanofibers. It was hypothesized that SCs, as part of the peripheral nervous system, promote the myogenic differentiation of Mb/ADSC co-cultures. Mb/ADSC were compared to Mb/ADSC/SC regarding their capacity for myogenic differentiation via immunofluorescent staining and gene expression of myogenic markers. Mb/ADSC/SC showed more myotubes after 28 days of differentiation (p ≤ 0.05). After 28 days of differentiation on electrospun aligned PCL-collagen I-nanofibers, gene expression of myosin heavy chains (MYH2) and myogenin (MYOG) was upregulated in Mb/ADSC/SC compared to Mb/ADSC (p ≤ 0.01 and p ≤ 0.05, respectively). Immunofluorescent staining for MHC showed highly aligned multinucleated cells as possible myotube formation in Mb/ADSC/SC. In conclusion, SCs promote myogenic differentiation of Mb/ADSC. The co-culture of primary Mb/ADSC/SC on PCL-collagen I-nanofibers serves as a physiological model for skeletal muscle tissue engineering, applicable to future clinical applications.

Myogenic Precursor Cells Show Faster Activation and Enhanced Differentiation in a Male Mouse Model Selected for Advanced Endurance Exercise Performance.

Satellite cells (SATC), the most abundant skeletal muscle stem cells, play a main role in muscle plasticity, including the adaptive response following physical activity. Thus, we investigated how long-term phenotype selection of male mice for high running performance (Dummerstorf high Treadmill Performance; DUhTP) affects abundance, creatine kinase activity, myogenic marker expression (Pax7, MyoD), and functionality (growth kinetics, differentiation) of SATC and their progeny. SATC were isolated from sedentary male DUhTP and control (Dummerstorf Control; DUC) mice at days 12, 43, and 73 of life and after voluntary wheel running for three weeks (day 73). Marked line differences occur at days 43 and 73 (after activity). At both ages, analysis of SATC growth via xCELLigence system revealed faster activation accompanied by a higher proliferation rate and lower proportion of Pax7+ cells in DUhTP mice, indicating reduced reserve cell formation and faster transition into differentiation. Cultures from sedentary DUhTP mice contain an elevated proportion of actively proliferating Pax7+/MyoD+ cells and have a higher fusion index leading to the formation of more large and very large myotubes at day 43. This robust hypertrophic response occurs without any functional load in the donor mice. Thus, our selection model seems to recruit myogenic precursor cells/SATC with a lower activation threshold that respond more rapidly to external stimuli and are more primed for differentiation at the expense of more primitive cells.

Tongue Muscle for the Analysis of Head Muscle Regeneration Dynamics

Tongue muscle damage impairs speaking and eating, thereby degrading overall health and quality of life. Skeletal muscles of the body are diverse in embryonic origin, anatomic location, and gene expression profiles. Responses to disease, atrophy, aging, or drugs vary among different muscles. Currently, most muscle studies are focused on limb muscles and the tongue is neglected. The regenerative ability of tongue muscle remains unknown, and thus there is need for tongue muscle research models. Here, we present a comprehensive characterization of the spatiotemporal dynamics in a mouse model of tongue muscle regeneration and establish a method for the isolation of primary tongue-derived satellite cells. We compare and contrast our observations with the tibialis anterior (TA) limb muscle. Acute injury was induced by intramuscular injection of cardiotoxin, a cytolytic agent, and examined at multiple timepoints. Initially, necrotic myofibers with fragmented sarcoplasm became infiltrated with inflammatory cells. Concomitantly, satellite cells expanded rapidly. Seven days postinjury, regenerated myofibers with centralized nuclei appeared. Full regeneration, as well as an absence of fibrosis, was evident 21 d postinjury. Primary tongue-derived satellite cells were isolated by enzymatic separation of tongue epithelium from mesenchyme followed by magnetic-activated cell sorting. We observed that tongue displays an efficient regenerative response similar to TA but with slightly faster kinetics. In vitro, tongue-derived satellite cells differentiated robustly into mature myotubes with spontaneous contractile behavior and myogenic marker expression. Comparison of gene expression signatures between tongue and TA-derived satellite cells revealed differences in the expression of positional-identity genes, including the HOX family. In conclusion, we have established a model for tongue regeneration useful for investigations of orofacial muscle biology. Furthermore, we showed that tongue is a viable source of satellite cells with unique properties and inherited positional memory.

Dickkopf-1 Inhibition Reactivates Wnt/β-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo.

The Wnt/β-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/β-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to β-catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression.

Deletion of intestinal SIRT1 exacerbated muscle wasting in cirrhotic mice by decreasing the intestinal concentration of short-chain fatty acids and inflammation

Systemic sirtuin 1 (SIRT1) activation alleviates muscle wasting and improves muscle function by downregulation of myotropic and proteolytic markers. In this study, we evaluated the effects of the intestinal Sirt1 deletion on the dysregulated gutmuscle axis in cirrhotic mice. Cirrhosis-related muscle wasting was induced by common bile duct ligated (BDL) in either wild-type (WT) or intestine-specific Sirt1-deleted (Sirt1IEC-KO) mice, including WT-BDL, WT-sham, Sirt1IEC-KO-BDL and Sirt1IEC-KO-sham mice. Compared with WT-BDL mice, Sirt1IEC-KO-BDL mice showed worsened low lean mass, exacerbated muscle wasting, increased expression of myotropic markers, increased muscular protein degradation, and decreased expression of myogenic markers through aggravation of intestinal inflammation (as evidenced by increased fecal calprotectin/lipocalin-2 levels, increased intestinal macrophage infiltration, and increased intestinal TNFα/IL-6 levels), decrease in abundance of short-chain fatty acid (SCFA)-producing bacteria, decrease in levels of intestinal SCFAs (with anti-inflammatory effects), and downregulation of SCFA receptor GPR43. In biliary cirrhotic mice, a decrease in the abundance of SCFA-producing bacteria and an increase in the levels of intestinal/muscular inflammatory markers are involved in the pathogenesis of dysregulated gut-muscle axis-related muscle wasting, and intestinal deletion of Sirt1 exacerbated these changes.

Role of differentially expressed LBX1 in Adolescent Idiopathic Scoliosis (AIS) paraspinal muscle phenotypes and muscle-bone crosstalk through modulating myoblasts.

AIS is three-dimensional spinal deformity with unclear etiopathogenesis. LBX1 is so far the only multi-centers validated AIS predisposing gene. The imbalance of posterior paraspinal muscles is an important factor in AIS etiopathogenesis. It is poorly understood how LBX1 contributes to the abnormal paraspinal muscles and onset/progression of AIS. We aimed to evaluate the expression of LBX1 in paraspinal muscles at the concave and convex side in AIS, and whether alternation of LBX1 expression could affect myoblastsactivities and potentially influence muscle-bone interaction via myokines expression. Paraspinal muscles from AIS and age- and curvature-matched congenital scoliosis (CS) patients were collected for fiber types analysis. Biopsies were also subjected to qPCR to validate expression of myogenic markers, selected myokines and LBX1. Human skeletal muscle myoblast (HSMM) was used for LBX1 loss-of-function study in vitro. Muscle fiber types analysis showed type I and type IIX/IIAX fibers proportion were significantly different between AIS concave and convex but not in two sides of CS. LBX1, myogenic markers and one myokine were significantly imbalanced in AIS but not in CS. Loss-of-function study showed knockdown of LBX1 could inhibit myogenic markers expression and myokines as well. This study provides new insight into the association between imbalanced paraspinal muscle and potential muscle-bone crosstalk in AIS patients and the biological function of predisposing gene LBX1. Further investigation with appropriate animal models is warranted to explore if asymmetric expression of LBX1 could result in distinct muscle phenotypes and bone qualities thus affect the progression of spine curvature in AIS.

MiR-21-5p Regulates the Proliferation and Differentiation of Skeletal Muscle Satellite Cells by Targeting KLF3 in Chicken.

The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) play an important role in the development of skeletal muscle. Our previous sequencing data showed that miR-21-5p is one of the most abundant miRNAs in chicken skeletal muscle. Therefore, in this study, the spatiotemporal expression of miR-21-5p and its effects on skeletal muscle development of chickens were explored using in vitro cultured SMSCs as a model. The results in this study showed that miR-21-5p was highly expressed in the skeletal muscle of chickens. The overexpression of miR-21-5p promoted the proliferation of SMSCs as evidenced by increased cell viability, increased cell number in the proliferative phase, and increased mRNA and protein expression of proliferation markers including PCNA, CDK2, and CCND1. Moreover, it was revealed that miR-21-5p promotes the formation of myotubes by modulating the expression of myogenic markers including MyoG, MyoD, and MyHC, whereas knockdown of miR-21-5p showed the opposite result. Gene prediction and dual fluorescence analysis confirmed that KLF3 was one of the direct target genes of miR-21-5p. We confirmed that, contrary to the function of miR-21-5p, KLF3 plays a negative role in the proliferation and differentiation of SMSCs. Si-KLF3 promotes cell number and proliferation activity, as well as the cell differentiation processes. Our results demonstrated that miR-21-5p promotes the proliferation and differentiation of SMSCs by targeting KLF3. Collectively, the results obtained in this study laid a foundation for exploring the mechanism through which miR-21-5p regulates SMSCs.