Myogenic Precursor Research Articles

CPEB2-activated Prdm16 translation promotes brown adipocyte function and prevents obesity

ObjectiveBrown adipose tissue (BAT) plays an important role in mammalian thermogenesis through the expression of uncoupling protein 1 (UCP1). Our previous study identified cytoplasmic polyadenylation element binding protein 2 (CPEB2) as a key regulator that activates the translation of Ucp1 with a long 3′-untranslated region (Ucp1L) in response to adrenergic signaling. Mice lacking CPEB2 or Ucp1L exhibited reduced UCP1 expression and impaired thermogenesis; however, only CPEB2-null mice displayed obesogenic phenotypes. Hence, this study aims to investigate how CPEB2-controlled translation impacts body weight. MethodsBody weight measurements were conducted on mice with global knockout (KO) of CPEB2, UCP1 or Ucp1L, as well as those with conditional knockout of CPEB2 in neurons or adipose tissues. RNA sequencing coupled with bioinformatics analysis was used to identify dysregulated gene expression in CPEB2-deficient BAT. The role of CPEB2 in regulating PRD1-BF1-RIZ1 homologous-domain containing 16 (PRDM16) expression was subsequently confirmed by RT-qPCR, Western blotting, polysomal profiling and luciferase reporter assays. Adeno-associated viruses (AAV) expressing CPEB2 or PRDM16 were delivered into BAT to assess their efficacy in mitigating weight gain in CPEB2-KO mice. ResultsWe validated that defective BAT function contributed to the increased weight gain in CPEB2-KO mice. Transcriptomic profiling revealed upregulated expression of genes associated with muscle development in CPEB2-KO BAT. Given that both brown adipocytes and myocytes stem from myogenic factor 5-expressing precursors, with their cell-fate differentiation regulated by PRDM16, we identified that Prdm16 was translationally upregulated by CPEB2. Ectopic expression of PRDM16 in CPEB2-deprived BAT restored gene expression profiles and decreased weight gain in CPEB2-KO mice. ConclusionsIn addition to Ucp1L, activation of Prdm16 translation by CPEB2 is critical for sustaining brown adipocyte function. These findings unveil a new layer of post-transcriptional regulation governed by CPEB2, fine-tuning thermogenic and metabolic activities of brown adipocytes to control body weight.

138 Regulation of post protein translational modification in adipogenesis and fat accumulation in pigs

Abstract In this study, we explored the role of protein transcriptional modification (PTM) in the adipogenesis and fat accumulation of intramuscular adipocytes. Intramuscular fat (IMF) is mainly stored in adipocytes interspersed in the perimysial space or within fascicles, and its content is a key factor attributing to meat quality in farm animals, especially in terms of tenderness, flavor and juiciness. The early stage from the fetus to the neonate is crucial for skeletal muscle development involving myogenesis, adipogenesis, and fibrogenesis, and is susceptible to environmental factors. Previous studies have been subjected to explore the molecular mechanism involved in IMF development. However, it was obscured by muscular background of IMF adipocytes. Therefore, we investigated PTM dynamics from three aspects: 1) first analyzed PTM levels of lysine acetylation (Kac), 2-hydroxyisobutyrylation (Khib), crotonylation (Kcr) and succinylation (Ksu) in three distinct differentiation potenctial primary stem cells, including myogenic precursor cells (MPCs), fibro-adipogenic progenitors (FAPs) and MSCs isolated from neonatal longissimus dorsi muscle of pigs within 3-d of age; 2) Comparing the Khib levels of proliferative and differentiated FAPs; and 3) Exploring the role of Khib modification in the adipose tissue of obese- and lean-types of pigs. We found that only Khib level in FAPs was significantly greater than MSCs. During adipogenic differentiation of FAPs, the modification level of Khib was significantly increased quadratically in parallel with protein levels of acylation regulatory enzymes KAT5 (writer) and HDAC2 (Eraser). In comparison of Khib levels in FAPs in proliferation and in differentiation and Khib levels in backfat tissues from obese pigs (Laiwu pigs) and lean pigs (Duroc pigs), we also demonstrated that Khib modification level was positive correlated with adipogenic differentiation and fat accumulation. Accordingly, the expression patterns of KAT5 and HDAC2 were parallel to the degree of lipid accumulation in the backfat adipose tissue of finishing pigs. Furthermore, we showed that KAT5 knockdown in FAPs inhibited adipogenic differentiation, while HDAC2 knockdown enhanced adipogenic differentiation of FAPs. By employing C3H10T1/2 cells, we identified EON1 involved in Khib modification in regulation of adipo gesis and fat accumulation with analysis of Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the Tandem Mass Tag (TMT Through conditional loss- and gain-of-function mutations, we further demonstrated that khib modification of ENO1 is necessary for preadipocyte differentiation and subsequent fat accumulation by mediating cellular glycolysis. In conclusion, we are the first to reveal the role of Khib modification in adipogenesis and fat deposition in pigs, and provided new clues for the improvement of fat accumulation and distribution via genetic selection and nutritional strategy in pigs.

Single cell transcriptomic profiling identifies tumor-acquired and therapy-resistant cell states in pediatric rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric tumor that resembles undifferentiated muscle cells; yet the extent to which cell state heterogeneity is shared with human development has not been described. Using single-cell/nucleus RNA sequencing from patient tumors, patient-derived xenografts, primary in vitro cultures, and cell lines, we identify four dominant muscle-lineage cell states: progenitor, proliferative, differentiated, and ground cells. We stratify these RMS cells/nuclei along the continuum of human muscle development and show that they share expression patterns with fetal/embryonal myogenic precursors rather than postnatal satellite cells. Fusion-negative RMS (FN-RMS) have a discrete stem cell hierarchy that recapitulates fetal muscle development and contain therapy-resistant FN-RMS progenitors that share transcriptomic similarity with bipotent skeletal mesenchymal cells. Fusion-positive RMS have tumor-acquired cells states, including a neuronal cell state, that are not found in myogenic development. This work identifies previously underappreciated cell state heterogeneity including unique treatment-resistant and tumor-acquired cell states that differ across RMS subtypes.

Lysine 2-hydroxyisobutyrylation levels determined adipogenesis and fat accumulation in adipose tissue in pigs

BackgroundExcessive backfat deposition lowering carcass grade is a major concern in the pig industry, especially in most breeds of obese type pigs. The mechanisms involved in adipogenesis and fat accumulation in pigs remain unclear. Lysine 2-hydroxyisobutyrylation (Khib), is a novel protein post-translational modification (PTM), which play an important role in transcription, energy metabolism and metastasis of cancer cells, but its role in adipogenesis and fat accumulation has not been shown.ResultsIn this study, we first analyzed the modification levels of acetylation (Kac), Khib, crotonylation (Kcr) and succinylation (Ksu) of fibro-adipogenic progenitors (FAPs), myogenic precursors (Myo) and mesenchymal stem cells (MSCs) with varied differentiation potential, and found that only Khib modification in FAPs was significantly higher than that in MSCs. Consistently, in parallel with its regulatory enzymes lysine acetyltransferase 5 (KAT5) and histone deacetylase 2 (HDAC2) protein levels, the Khib levels increased quadratically (P < 0.01) during adipogenic differentiation of FAPs. KAT5 knockdown in FAPs inhibited adipogenic differentiation, while HDAC2 knockdown enhanced adipogenic differentiation. We also demonstrated that Khib modification favored to adipogenic differentiation and fat accumulation by comparing Khib levels in FAPs and backfat tissues both derived from obese-type pigs (Laiwu pigs) and lean-type pigs (Duroc pigs), respectively. Accordingly, the expression patterns of KAT5 and HDAC2 matched well to the degree of backfat accumulation in obese- and lean-type pigs.ConclusionsFrom the perspective of protein translational modification, we are the first to reveal the role of Khib in adipogenesis and fat deposition in pigs, and provided new clues for the improvement of fat accumulation and distribution as expected via genetic selection and nutritional strategy in obese-type pigs.

Muscle Fiber Heterogeneity of Insulin Sensitivity in Syntaxin-4 Enriched Skeletal Muscle

Skeletal muscle insulin resistance (IR) is a primary cause for prediabetes, an intermediary condition of higher-than-normal blood glucose levels that left untreated progresses into type 2 diabetes (T2D). A number of contributing factors associated with skeletal muscle IR include aberrant mitochondrial function, elevated reactive oxygen species, inflammation, and reduction in oxidative slow-twitch type 1 skeletal muscle fibers. One leading candidate for reversing skeletal muscle IR is Syntaxin 4 (STX4), traditionally a cell surface transmembrane SNARE protein for GLUT4 vesicle exocytosis that our lab has recently discovered is also localized in the skeletal muscle outer mitochondrial membrane. Importantly, we previously demonstrated that doxycycline-induced skeletal muscle specific STX4 overexpression in obese, prediabetes male transgenic mice (skmSTX4-iOE) reversed IR and mitochondrial dysfunction associated with diabetes muscle. However, the precise mechanisms of how STX4 restores skeletal muscle insulin sensitivity remains to be determined. To test glucose uptake and insulin sensitivity we used the “gold standard” method of hyper-insulinemic-euglycemic clamp in chow-fed skmSTX4-iOE STX4 enriched mice against non-induced controls, we identified improved glucose clearance was only associated with increased insulin sensitivity in type 1 fiber rich soleus muscle. On the other hand, insulin tolerance test studies in doxycycline-induced skeletal muscle STX4 knockout (skmSTX4-iKO) male mice revealed the opposite phenotype, suggesting that STX4 activity in slow-twitch skeletal muscle may be a contributing factor for the reversal of IR. To determine the heterogeneity of STX4 activity between different skeletal muscle fibers, myogenic precursors and intercalating non-muscular tissue in skeletal muscle STX4 enriched skmSTX4-iOE mice, we aim to use our established single nuclear RNA-Sequencing platform (snRNA-Seq) to identify pathways in distinct fiber types associated with STX4 enrichment. Overall, our work aims to determine the functional requirements of STX4 in slow-and-fast twitch skeletal muscle fiber types and identify novel molecular pathways that may serve as useful targets for the reversal/protection against skeletal muscle IR in prediabetes and T2D. This study was supported by grants from the National Institutes of Health (DK067912, DK1129712 and DK102233) and City of Hope Shared Resources Pilot Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lineage tracing reveals a novel PDGFRβ+ satellite cell subset that contributes to myo-regeneration of chronically injured rotator cuff muscle

Massive rotator cuff (RC) tendon tears are associated with progressive fibro-adipogenesis and muscle atrophy that altogether cause shoulder muscle wasting. Platelet derived growth factor β (PDGFRβ) lineage cells, that co-express PDGFRα have previously been shown to directly contribute to scar formation and fat accumulation in a mouse model of irreversible tendon and nerve transection (TTDN). Conversely, PDGFRβ+ lineage cells have also been shown to be myogenic in cultures and in other models of skeletal muscle injury. We therefore hypothesized that PDGFRβ demarcates two distinct RC residing subpopulations, fibro-adipogenic and myogenic, and aimed to elucidate the identity of the PDGFRβ myogenic precursors and evaluate their contribution, if any, to RC myo-regeneration. Lineage tracing revealed increasing contribution of PDGFRβ+ myo-progenitors to the formation of GFP+ myofibers, which were the most abundant myofiber type in regenerated muscle at 2 weeks post-TTDN. Muscle regeneration preceded muscle atrophy and both advanced from the lateral site of tendon transection to the farthest medial region. GFP+/PDGFRβ+Sca-1−lin−CXCR4+Integrin-β1+ marked a novel subset of satellite cells with confirmed myogenic properties. Further studies are warranted to identify the existence of PDGFRβ+ satellite cells in human and other mouse muscles and to define their myo-regenerative potential following acute and chronic muscle injury.

Chromatin profiling reveals TFAP4 as a critical transcriptional regulator of bovine satellite cell differentiation

BackgroundSatellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle regeneration, maintenance, and growth. Like embryonic myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study, we aimed to identify additional transcription factors that control gene expression during bovine satellite cell proliferation and differentiation.ResultsUsing chromatin immunoprecipitation followed by sequencing, we identified 56,973 and 54,470 genomic regions marked with both the histone modifications H3K4me1 and H3K27ac, which were considered active enhancers, and 50,956 and 59,174 genomic regions marked with H3K27me3, which were considered repressed enhancers, in proliferating and differentiating bovine satellite cells, respectively. In addition, we identified 1,216 and 1,171 super-enhancers in proliferating and differentiating bovine satellite cells, respectively. Analyzing these enhancers showed that in proliferating bovine satellite cells, active enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation whereas repressed enhancers were associated with genes essential for myoblast differentiation, and that in differentiating satellite cells, active enhancers were associated with genes essential for myoblast differentiation or muscle contraction whereas repressed enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation. Active enhancers in proliferating bovine satellite cells were enriched with binding sites for many transcription factors such as MYF5 and the AP-1 family transcription factors; active enhancers in differentiating bovine satellite cells were enriched with binding sites for many transcription factors such as MYOG and TFAP4; and repressed enhancers in both proliferating and differentiating bovine satellite cells were enriched with binding sites for NF-kB, ZEB-1, and several other transcription factors. The role of TFAP4 in satellite cell or myoblast differentiation was previously unknown, and through gene knockdown and overexpression, we experimentally validated a critical role for TFAP4 in the differentiation and fusion of bovine satellite cells into myofibers.ConclusionsSatellite cell proliferation and differentiation are controlled by many transcription factors such as AP-1, TFAP4, NF-kB, and ZEB-1 whose roles in these processes were previously unknown in addition to those transcription factors such as MYF5 and MYOG whose roles in these processes are widely known.

Skeletal muscle regeneration after extensive cryoinjury of caudal myomeres in adult zebrafish

Skeletal muscles can regenerate after minor injuries, but severe structural damage often leads to fibrosis in mammals. Whether adult zebrafish possess the capacity to reproduce profoundly destroyed musculature remains unknown. Here, a new cryoinjury model revealed that several myomeres efficiently regenerated within one month after wounding the zebrafish caudal peduncle. Wound clearance involved accumulation of the selective autophagy receptor p62, an immune response and Collagen XII deposition. New muscle formation was associated with proliferation of Pax7 expressing muscle stem cells, which gave rise to MyoD1 positive myogenic precursors, followed by myofiber differentiation. Monitoring of slow and fast muscles revealed their coordinated replacement in the superficial and profound compartments of the myomere. However, the final boundary between the muscular components was imperfectly recapitulated, allowing myofibers of different identities to intermingle. The replacement of connective with sarcomeric tissues required TOR signaling, as rapamycin treatment impaired new muscle formation, leading to persistent fibrosis. The model of zebrafish myomere restoration may provide new medical perspectives for treatment of traumatic injuries.

A Pharmacological Investigation of the TMEM16A Currents in Murine Skeletal Myogenic Precursor Cells.

TMEM16A is a Ca2+-activated Cl- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

ADVANCING MUSCULAR DYSTROPHY THERAPY EVALUATION THROUGH MUSCLE-ON-CHIP DEVICES

Duchenne muscular dystrophy (DMD) is a prevalent childhood neuromuscular disease characterized by progressive skeletal and cardiac muscle degeneration due to dystrophin protein deficiency. Despite ongoing drug development efforts, no cure exists, with limited success in preclinical studies. To expedite DMD drug development, we introduce an innovative organ-on-a-chip (OOC) platform. This microfluidic device sustains up to six 3D patient-derived skeletal muscle tissues, enabling real-time evaluation of anti-DMD treatments. Our in vitro model recreates myotube integrity loss, a hallmark of DMD, by encapsulating myogenic precursors in a fibrin-composite matrix using a PDMS casting mold. Continuous contractile regimes mimic sarcolemmal instability, monitored through tissue contractibility and Creatine Kinase (CK) levels—an established marker of muscle damage. We further enhance our platform with a nanoplasmonic CK biosensor, enabling rapid, label-free, and real-time sarcolemmal damage assessment. Combining these elements, our work demonstrates the potential of OOCs in accelerating drug development for DMD and similar neuromuscular disorders.

Indole Family and Neomycin Sulfate: Inductors of Differentiation in C2C12 and RD cell Lines

Rhabdomyosarcoma (RMS) is a highly aggressive tumor primarily affecting the pediatric population, that generally originates from a failure in the embryonic differentiation of myogenic precursor cells. Standard treatment involves surgery, chemotherapy and radiation therapy, with a poor prognosis, especially when it spreads to other parts of the body, highlighting the need for new treatment approaches. In this study, cellular differentiation was investigated as a potential therapeutic approach for an embryonal subtype of RMS (eRMS) employing a human eRMS cell line, RD; and comparing it with a normal murine myoblast cell line, C2C12. We observed that not only a serum free or a low serum concentration in the cultured medium induce morphological changes towards differentiations, but also staurosporine, bisindolylmaleimide, and neomycin sulfate (direct or indirect protein kinase inhibitors) induces differentiation in RD and C2C12 cell lines, suggesting a crucial role of PKC, PLC and its signaling pathways in a proliferation/differentiation switch. These findings underscore the importance of exploring cellular differentiation as a therapeutic strategy against RMS and highlight the need for further research into the underlying mechanisms of kinase inhibitor-induced cellular differentiation as a potential cancer treatment.

Optimization of Xenografting Methods for Generating Human Skeletal Muscle in Mice.

Xenografts of human skeletal muscle generated in mice can be used to study muscle pathology and to test drugs designed to treat myopathies and muscular dystrophies for their efficacy and specificity in human tissue. We previously developed methods to generate mature human skeletal muscles in immunocompromised mice starting with human myogenic precursor cells (hMPCs) from healthy individuals and individuals with facioscapulohumeral muscular dystrophy (FSHD). Here, we examine a series of alternative treatments at each stage in order to optimize engraftment. We show that (i) X-irradiation at 25Gy is optimal in preventing regeneration of murine muscle while supporting robust engraftment and the formation of human fibers without significant murine contamination; (ii) hMPC lines differ in their capacity to engraft; (iii) some hMPC lines yield grafts that respond better to intermittent neuromuscular electrical stimulation (iNMES) than others; (iv) some lines engraft better in male than in female mice; (v) coinjection of hMPCs with laminin, gelatin, Matrigel, or Growdex does not improve engraftment; (vi) BaCl2 is an acceptable replacement for cardiotoxin, but other snake venom preparations and toxins, including the major component of cardiotoxin, cytotoxin 5, are not; and (vii) generating grafts in both hindlimbs followed by iNMES of each limb yields more robust grafts than housing mice in cages with running wheels. Our results suggest that replacing cardiotoxin with BaCl2 and engrafting both tibialis anterior muscles generates robust grafts of adult human muscle tissue in mice.

Foxk1 stimulates adipogenic differentiation via a peroxisome proliferator-activated receptor gamma 2-dependent mechanism.

Adipogenesis is a tightly regulated process, and its dysfunction has been linked to metabolic disorders such as obesity. Forkhead box k1 (Foxk1) is known to play a role in the differentiation of myogenic precursor cells and tumorigenesis of different types of cancers; however, it is not clear whether and how it influences adipocyte differentiation. Here, we found that Foxk1 was induced in mouse primary bone marrow stromal cells (BMSCs) and established mesenchymal progenitor/stromal cell lines C3H/10T1/2 and ST2 after adipogenic treatment. In addition, obese db/db mice have higher Foxk1 expression in inguinal white adipose tissue than nonobese db/m mice. Foxk1 overexpression promoted adipogenic differentiation of C3H/10T1/2, ST2 cells and BMSCs, along with the enhanced expression of CCAAT/enhancer binding protein-α, peroxisome proliferator-activated receptor γ (Pparγ), and fatty acid binding protein 4. Moreover, Foxk1 overexpression enhanced the expression levels of lipogenic factors during adipogenic differentiation in both C3H/10T1/2 cells and BMSCs. Conversely, Foxk1 silencing impaired these cells from fully differentiating. Furthermore, adipogenic stimulation induced the nuclear translocation of Foxk1, which depended on the mTOR and PI3-kinase signaling pathways. Subsequently, Foxk1 is directly bound to the Pparγ2 promoter, stimulating its transcriptional activity and promoting adipocyte differentiation. Collectively, our study provides the first evidence that Foxk1 promotes adipocyte differentiation from progenitor cells by promoting nuclear translocation and upregulating the transcriptional activity of the Pparγ2 promoter during adipogenic differentiation.

Nystagmus Associated With the Absence of MYOD Expression Across the Lifespan in Extraocular and Limb Muscles.

The extraocular muscles (EOMs) undergo significant levels of continuous myonuclear turnover and myofiber remodeling throughout life, in contrast to limb skeletal muscles. Activation of the myogenic pathway in muscle precursor cells is controlled by myogenic transcription factors, such as MYOD. Limb muscles from MyoD-/- mice develop normally but have a regeneration defect, and these mice develop nystagmus. We examined MyoD-/- mice to determine if they have an aging phenotype. Eye movements of aging MyoD-/- mice and littermate controls (wild type) were examined using optokinetic nystagmus (OKN). We assessed limb muscle function, changes to myofiber number, mean cross-sectional area, and abundance of the PAX7 and PITX2 populations of myogenic precursor cells. Aging did not significantly affect limb muscle function despite decreased mean cross-sectional areas at 18+months. Aging wild type mice had normal OKN responses; all aging MyoD-/- mice had nystagmus. With OKN stimulus present, the MyoD-/- mice at all ages had shorter slow phase durations compared to wild type age matched controls. In the dark, the MyoD-/- mice had a shorter slow phase duration with age. This correlated with significantly decreased fiber numbers and cross-sectional areas. The EOM in MyoD-/- mice had increased numbers of PAX7-positive satellite cells and significantly decreased PITX2-positive myonuclei. The absence of MYOD expression in aging mice causes a decrease in on-going myofiber remodeling, EOM fiber size, and number, and is associated with the development of spontaneous nystagmus. These results suggest that muscle-specific mutations can result in nystagmus, with increasing aging-related changes in the MyoD-/- EOM.

Newt A1 cell-derived extracellular vesicles promote mammalian nerve growth

Newts have the extraordinary ability to fully regenerate lost or damaged cardiac, neural and retinal tissues, and even amputated limbs. In contrast, mammals lack these broad regenerative capabilities. While the molecular basis of newts’ regenerative ability is the subject of active study, the underlying paracrine signaling factors involved remain largely uncharacterized. Extracellular vesicles (EVs) play an important role in cell-to-cell communication via EV cargo-mediated regulation of gene expression patterns within the recipient cells. Here, we report that newt myogenic precursor (A1) cells secrete EVs (A1EVs) that contain messenger RNAs associated with early embryonic development, neuronal differentiation, and cell survival. Exposure of rat primary superior cervical ganglion (SCG) neurons to A1EVs increased neurite outgrowth, facilitated by increases in mitochondrial respiration. Canonical pathway analysis pinpointed activation of NGF/ERK5 signaling in SCG neurons exposed to A1EV, which was validated experimentally. Thus, newt EVs drive neurite growth and complexity in mammalian primary neurons.

Myofiber directs macrophages IL-10-Vav1-Rac1 efferocytosis pathway in inflamed muscle following CTX myoinjury by activating the intrinsic TGF-β signaling

BackgroundTo explore the role of skeletal muscle specific TGF-β signaling on macrophages efferocytosis in inflamed muscle caused by Cardiotoxin (CTX) injection.MethodsCTX myoinjury was manipulated in TGF-βr2flox/flox (control) mice or transgenic mice with TGF-β receptor 2 (TGF-βr2) being specifically deleted in skeletal muscle (SM TGF-βr2−/−). Gene levels of TGF-β signal molecules, special inflammatory mediators in damaged muscle or in cultured and differentiated myogenic precursor cells (MPC-myotubes) were monitored by transcriptome microarray or qRT-PCR. TGF-β pathway molecules, myokines and embryonic myosin heavy chain in regenerating myofibers, the phenotype and efferocytosis of macrophages were evaluated by immunofluorescence, immunoblotting, Luminex, or FACS analysis. In vitro apoptotic cells were prepared by UV-irradiation.ResultsIn control mice, TGF-β-Smad2/3 signaling were significantly up-regulated in regenerating centronuclear myofibers after CTX-myoinjury. More severe muscle inflammation was caused by the deficiency of muscle TGF-β signaling, with the increased number of M1, but the decreased number of M2 macrophages. Notably, the deficiency of TGF-β signaling in myofibers dramatically affected on the ability of macrophages to conduct efferocytosis, marked by the decreased number of Annexin-V−F4/80+Tunel+ macrophages in inflamed muscle, and the impaired uptake of macrophages to PKH67+ apoptotic cells transferred into damaged muscle. Further, our study suggested that, the intrinsic TGF-β signaling directed IL-10-Vav1-Rac1 efferocytosis signaling in muscle macrophages.ConclusionsOur data demonstrate that muscle inflammation can be suppressed potentially by activating the intrinsic TGF-β signaling in myofibers to promote IL-10 dependent-macrophages efferocytosis.2-7di1wFqhyvGwQ3tfntcnVideo

Potential Application of Muscle Precursor Cells from Male Specific-Pathogen-Free (SPF) Chicken Embryos in In Vitro Agriculture.

This study examined the potential benefits of male specific-pathogen-free (SPF) White Leghorn embryos in cellular agriculture for sustainable and ethical poultry meat production-addressing traditional farming challenges, including disease outbreaks of Salmonella and Avian influenza. We isolated myogenic precursor cells (MPCs) from the thigh muscles (Musculus femoris) of 12.5-day-old embryos from 10 SPF White Leghorns that tested negative for Salmonella. We randomly selected MPCs from three males and three females, isolated them using a modified pre-plating (pp) method, and compared their in vitro development. After 1 h (pp1) and 2 h (pp2) of incubation, they were transferred to a new dish to remove fast-adhering cells and cultured (pp3). Isolated MPCs had a 69% positive reaction to Pax7. During proliferation, no differences were observed in PAX7, MYF5, or MYOD expression between the male and female MPCs. However, after five days of differentiation, the expression of late myogenic factors-MYOG and MYF6-significantly increased in all MPCs. Notably, MYOG expression was 1.9 times higher in female than in male MPCs. This impacted MYMK's expression pattern. Despite this, the myotube fusion index did not differ between the sexes. Muscle cells from male SPF-laying chicken embryos are promising for developing clean animal-cell-derived protein sources via resource recycling.

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

IRE1α arm of unfolded protein response in muscle-specific TGF-β signaling-mediated regulation of muscle cell immunological properties

Endoplasmic reticulum stress (ERS) and the unfolded protein response (UPR) are involved in various muscle pathological states. The IRE1α arm of UPR can affect immunological properties of myofiber through restraining p38 mitogen-activated protein kinases (MAPK) activation under inflammatory milieu. However, the relevant pathway molecules regulating the initiation of the IRE1α arm in myofiber remain unclear. In this work, expression of transforming growth factor-beta (TGF-β) and TGF-β receptor II (TGF-βr2), and UPR pathway activation were examined in cardiotoxin (CTX)-damaged mouse muscle, which revealed the activation of TGF-β signaling and UPR in CTX-damaged muscle and in regenerating myofibers. Using control or transgenic mice with TGF-βr2 deleted in skeletal muscle (SM TGF-βr2−/−) and the derived primary differentiating myogenic precursor cells (MPCs) treated with/without ERS activator or inhibitor, IRE1α pathway inhibitor, or TGF-β signaling activator, this study further revealed an essential role of intrinsic TGF-β signaling in regulating muscle cell to express inflammation-related molecules including H-2Kb, H2-Eα, TLR3, and special myokines. TGF-β signaling prompted UPR IRE1α arm and restrained p38 MAPK activation in myofiber under inflammatory milieu. This study uncovers a previously unrecognized function of TGF-β signaling acting as an upstream factor controlling myofiber immune capacities in the inflamed state through the UPR–IRE1α–p38 MAPK pathway.

Denervation Atrophy of Skeletal Muscle is Not Influenced by Numb Levels in Mice.

Skeletal muscle undergoes rapid and extensive atrophy following nerve transection though the underlying mechanisms remain incompletely understood. We previously showed transiently elevated Notch 1 signaling in denervated skeletal muscle that was abrogated by administration of nandrolone (an anabolic steroid) combined with replacement doses of testosterone. Numb is an adaptor molecule present in myogenic precursors and skeletal muscle fibers that is vital for normal tissue repair after muscle injury and for skeletal muscle contractile function. It is unclear whether the increase in Notch signaling observed in denervated muscle contributes to denervation and whether expression of Numb in myofibers slows denervation atrophy. To address these questions, the degree of denervation atrophy, Notch signaling, and Numb expression was studied over time after denervation in C57B6J mice treated with nandrolone, nandrolone plus testosterone or vehicle. Nandrolone increased Numb expression and reduced Notch signaling. Neither nandrolone alone nor nandrolone plus testosterone changed the rate of denervation atrophy. We next compared rates of denervation atrophy between mice with conditional, tamoxifen-inducible knockout of Numb in myofibers and genetically identical mice treated with vehicle. Numb cKO had no effect on denervation atrophy in this model. Taken together, the data indicate that loss of Numb in myofibers does not alter the course of denervation atrophy and that upregulation of Numb and blunting of the denervation-atrophy induced activation of Notch do not change the course of denervation atrophy.