Process Of Myogenesis Research Articles

Early-age heat exposure improved growth performance and heat tolerance in broilers

A total of 440 one-day-old healthy male Arbor Acres broilers were equally assigned to a control group (CTL) and an early-age high-temperature exposure (EHT) group (4 replicates per group, 55 chickens per replicate). At d 3, the broilers in CTL group were reared in the normal temperature 33 ± 1°C, while the broilers in EHT group were exposed to 36 ± 1°C for 24 h. At d 43, all broilers were treated with an acute high temperature 35 ± 1°C for 5 h. The results showed that average daily gain in EHT group was decreased at d 3, but average daily gain in EHT group was increased at d 36 to 42 (P < 0.05). Plasma GLU level in EHT group was lower in broilers at d 7 or facing subsequently high temperature for 5 h (P < 0.05). The relative expression of myogenic differentiation (MyoD) gene in pectoralis major and myogenic factor 5 (Myf5) gene in biceps femoris were significantly improved at d 42 after early-age heat exposure (P < 0.05). Broilers in EHT group have a higher temperature tolerance with a lower mortality than control broilers (P < 0.05). Broilers in EHT group have a lower rectal temperature and a higher comb and ear temperature when facing subsequently acute high temperature than control broilers (P < 0.05). In addition, our study demonstrated that early-age heat exposure significantly decreased the mortality and increased the heat tolerance of broilers when facing an acute short-term heat exposures. Early-age heat exposure increased the process of myogenesis via up-regulating the MyoD and Myf5 gene expression in skeletal muscle, which accelerated average daily gain.

Mechanism of Stimulation of Myogenesis under the Action of Succinic Acid through the Succinate Receptor SUCNR1.

Effect of succinic acid on the processes of myogenesis was investigated in the study with the cells of C2C12 line. In the concentration range 10-1000µM, succinic acid stimulated the process of myogenic differentiation, increasing the levels of myogenesis factors MyoD (at all stages of myogenesis) and myogenin (at the stage of terminal differentiation). Presence of the succinate receptors SUCNR1 was revealed in the C2C12 cells using Western blotting, level of which decreased during myogenesis. When succinic acid was added to the cells, the level of intracellular succinate did not change significantly and decreased during myogenic differentiation. Using a specific Gai protein inhibitor, pertussis toxin, it was found that stimulation of myogenesis in the C2C12 cells under the action of succinic acid is realized through SUCNR1-Gai interaction.

CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.

RNA Sequencing Reveals the Inhibitory Effect of High Levels of Arachidonic Acid and Linoleic Acid on C2C12 Differentiation and Myogenic Biomarkers.

Over the past three decades, studies have shown that consuming polyunsaturated fatty acids (PUFAs) can enhance animal and human health and welfare through biological, biochemical, pathological, and pharmacological impacts. Furthermore, omega-6 plays key roles in the cardiopulmonary system, including promoting airway relaxation and inhibiting atherosclerosis and hypertension. However, findings from investigations of the effects of omega-6 fatty acids on molecular and cellular activity and discussions on their influence on biomarkers are still unclear. Therefore, the present study aimed to evaluate omega-6 fatty acids, the arachidonic acid (AA), and linoleic acid (LA) effects on C2C12 proliferation, myogenesis morphology, and relative myogenic biomarker expression through the Wnt pathway. C2C12 cells were cultured with and without 25, 50, 100, and 150 µM of LA and AA and then subjected to CCK8, Giemsa staining, RT qPCR, Western blotting, and RNA Sequencing. The CCK8 Assay results showed that 25, 50, 100, and 150 µM LA significantly decreased the viability after 72 h for 25, 50, 100, and 150 µM concentrations. Also, AA supplementation decreased cell viability after 24 h for 150 µM, 48 h for 150 µM, and 72 h for 50, 100, and 150 µM concentrations. Moreover, the LA and AA inhibitory effects noticed through Gimesa staining were morphological changes during myoblast differentiation. Both LA and AA showed inhibiting IGF1, Cola1, Col6a2, Col6a1, Itga10, Itga11, SFRP2, DAAM2, and NKD2 effects; however, the depressing effect was higher for AA compared to LA. The previous results were confirmed through Western blotting, which showed that 50 µM LA and AA significantly reduced DAAM2 and SFRP2 protein levels compared to the control. Regarding RNA sequencing results, LA and AA increased the number of differentially expressed (DE) Mt-rRNA and snoRNA; however, the numbers of lncRNA detected decreased compared to the control. Our findings demonstrate that high and moderate LA and AA concentrations reduce primary myoblast proliferation and differentiation. Also, they highlight novel biomarkers and regulatory factors to improve our understanding of how the nutrition of fatty acids can control and modulate the myogenesis and differentiation process through different biomarker families.

Neonatal resveratrol administration promotes skeletal muscle growth and insulin sensitivity in intrauterine growth-retarded suckling piglets associated with activation of FGF21-AMPKα pathway.

Skeletal muscle is a major insulin-sensitive tissue with a pivotal role in modulating glucose homeostasis. This study aimed to investigate the effect of resveratrol (RES) intervention during the suckling period on skeletal muscle growth and insulin sensitivity of neonates with intrauterine growth retardation (IUGR) in a pig model. Twelve pairs of normal birth weight (NBW) and IUGR neonatal male piglets were selected. The NBW and IUGR piglets were fed basal formula milk diet or identical diet supplemented with 0.1% RES from 7 to 21 days of age. Myofiber growth and differentiation, inflammation and insulin sensitivity in skeletal muscle were assessed. Early RES intervention promoted myofiber growth and maturity in IUGR piglets by ameliorating the myogenesis process and increasing thyroid hormone level. Administering RES also reduced triglyceride concentration in skeletal muscle of IUGR piglets, along with decreased inflammatory response, increased plasma fibroblast growth factor 21 (FGF21) concentration and improved insulin signaling. Meanwhile, the improvement of insulin sensitivity by RES may be partly regulated by activation of the FGF21/AMP-activated protein kinase α/sirtuin 1/peroxisome proliferator activated receptor-γ coactivator-1α pathway. Our results suggest that RES has beneficial effects in promoting myofiber growth and maturity and increasing skeletal muscle insulin sensitivity in IUGR piglets, which open a novel field of application of RES in IUGR infants for improving postnatal metabolic adaptation. © 2023 Society of Chemical Industry.

Systematic Myostatin Expression Screening Platform for Identification and Evaluation of Myogenesis-Related Phytogenic in Pigs.

Skeletal muscle growth in livestock impacts meat quantity and quality. Concerns arise because certain feed additives, like beta-agonists, may affect food safety. Skeletal muscle is a specialized tissue consisting of nondividing and multinucleated muscle fibers. Myostatin (MSTN), a protein specific to skeletal muscle, is secreted and functions as a negative regulator of muscle mass by inhibiting the proliferation and differentiation of myoblasts. To enhance livestock muscle growth, phytogenic feed additives could be an alternative as they inhibit MSTN activity. The objective of this study was to establish a systematic screening platform using MSTN activity to evaluate phytogenics, providing scientific evidence of their assessment and potency. In this study, we established a screening platform to monitor myostatin promoter activity in rat L8 myoblasts. Extract of Glycyrrhiza uralensis (GUE), an oriental herbal medicine, was identified through this screening platform, and the active fractions of GUE were identified using a process-scale liquid column chromatography system. For in vivo study, GUE as a feed additive was investigated in growth-finishing pigs. The results showed that GUE significantly increased body weight, carcass weight, and lean content in pigs. Microbiota analysis indicated that GUE did not affect the composition of gut microbiota in pigs. In summary, this established rodent myoblast screening platform was used to identify a myogenesis-related phytogenic, GUE, and further demonstrated that the active fractions and compounds inhibited MSTN expression. These findings suggest a novel application for GUE in growth performance enhancement through modulation of MSTN expression. Moreover, this well-established screening platform holds significant potential for identifying and assessing a diverse range of phytogenics that contribute to the process of myogenesis.

Regulation mechanism and functional verification of key functional genes regulating muscle development in black Tibetan sheep

Black Tibetan sheep is a branch of Tibetan sheep on the Qinghai-Tibet Plateau (QTP). It is mainly distributed in Guinan County, Qinghai Province. In order to accurately identify the core regulatory genes in the process of muscle development of black Tibetan sheep, further explore the physiological processes of growth, development and myogenesis of black Tibetan sheep, and carry out molecular breeding of black Tibetan sheep, this experiment took the unique black Tibetan sheep on the Qinghai-Tibet Plateau as the experimental object, and selected three stages of 4-month-old embryo (embryonic stage, MF group), 10-month-old(breeding stage, ML group) and 36-month-old (adult, MA group). The longissimus dorsi tissues of 3 sheep were taken at each stage to quantify the expression of genes during muscle development at different developmental stages. Meanwhile, overexpression and interference techniques were used to detect the role of core genes in the proliferation of primary muscle cells of black Tibetan sheep. In the process from embryonic stage to mature stage and adulthood, more than 1000 genes were up-regulated and more than 4000 down-regulated in black Tibetan sheep, while from breeding to adulthood, there were only 51 up-regulated genes and 83 down-regulated genes. About 998 genes were newly identified in each group. During muscle development from embryonic stage to mature stage to adulthood, two significant differential trend gene sets of Profile1 and Profile 6 were screened and identified, in which there were 121 and 31 core regulatory genes identified, respectively. In the trend of first decreasing and then stable expression in the whole development stage, 121 genes are core regulatory transcripts, which are mainly related to axonal guidance, cell cycle and other functions. 31 genes are core regulatory transcripts in the first rising and then stable expression, which are mainly related to biological metabolic pathway, oxidative phosphorylation and other processes. In the MF-ML stage, 75 genes were selected as the core regulatory gene set, the core genes were PTEN, AKT3, etc., and there were 134 differentially expressed genes in the ML-MA stage, and the core regulatory genes were IL6, ABCA1 and so on. In the MF-ML stage, the core gene set widely plays a role in cell components, cell matrix and other biological processes, while in the ML-MA stage, the core gene set widely plays a role in cell migration, cell differentiation, tissue development and so on. Adenovirus vector overexpressed and interfered with the core gene PTEN in primary muscle satellite cells of black Tibetan sheep shown that, interference and overexpression of PTEN would correspondingly increase and decrease the expression of other core genes, like AKT3, CKD2, CCNB1, ERBB3, HDAC2, but the specific interaction mechanism of each gene still needs to be further explored.

Vitamin D3 decreases myoblast fusion during the growth and increases myogenic gene expression during the differentiation phase in muscle satellite cells from Korean native beef cattle.

The process of myogenesis, which involves the growth and differentiation of muscle cells, is a crucial determinant of meat yield and quality in beef cattle. Essential nutrients, such as vitamins D and A, play vital roles in the development and maintenance of various tissues, including muscle. However, limited knowledge exists regarding the specific effects of vitamins A and D in bovine muscle. Therefore, the aim of this study was to investigate the impact of vitamins A and D treatment on myogenic fusion and differentiation in bovine satellite cells (BSC). BSC were isolated from Korean native beef cattle, specifically from four female cows approximately 30 mo old. These individual cows were used as biological replicates (n = 3 or 4), and we examined the effects of varying concentrations of vitamins A (All-trans retinoic acid; 100 nM) and D (1,25-dihydroxy-vitamin D3; 1 nM, 10 nM, and 100 nM), both individually and in combination, on myoblast fusion and myogenic differentiation during the growth phase (48 h) or differentiation phase (6 d). The results were statistically analyzed using GLM procedure of SAS with Tukey's test and t-tests or one-way ANOVA where appropriate. The findings revealed that vitamin A enhanced the myoblast fusion index, while vitamin D treatment decreased the myoblast fusion index during the growth phase. Furthermore, vitamin A treatment during the differentiation phase promoted terminal differentiation by regulating the expression of myogenic regulatory factors (Myf5, MyoD, MyoG, and Myf6) and inducing myotube hypertrophy compared to the control satellite cells (P < 0.01). In contrast, vitamin D treatment during the differentiation phase enhanced myogenic differentiation by increasing the mRNA expression of MyoG and Myf6 (P < 0.01). Moreover, the combined treatment of vitamins A and D during the growth phase increased myoblast fusion and further promoted myogenic differentiation and hypertrophy of myotubes during the differentiation phase (P < 0.01). These results suggest that vitamin A and D supplementation may have differential effects on muscle development in Korean native beef cattle during the feeding process.

A Novel Rat Model for Muscle Regeneration and Fibrosis Studies in Surgical Lip Repair.

Lip muscle undergoes suboptimal regeneration after surgical repair, but the mechanism underlying this observation remains obscure. This study provided a rat model to investigate lip muscle regeneration after surgical intervention. This work provided a detailed description of the rat orbicularis oris muscle anatomy, and a surgically injured model was established based on the muscle anatomy. Morphological and histological features of the rat orbicularis oris muscle were characterized. The processes of myogenesis and fibrogenesis were examined between the untreated and surgically injured groups. Rat orbicularis oris muscle is encapsulated by the vermilion and oral mucosa. Although it remains a thin layer of flat muscle with tight myocutaneous and myomucosal junctions, if accessed properly, the rat orbicularis oris muscle could be isolated as a cylindrical muscle bundle with considerable size, facilitating further surgical manipulations of the muscle fibers. Muscles in steady state and after surgical intervention demonstrated distinct molecular features in the myogenesis and fibrogenesis processes, which were quantifiable in tissue section analysis. The orbicularis oris muscle dissection procedures and injury model provided in this work clarify the rat lip muscle anatomy. The injury model offered a platform to analyze the effects of surgical interventions commonly used in lip repair on orbicularis oris muscle regeneration.

Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells to Model Myogenesis and Muscle Regeneration.

In vitro organoids derived from human pluripotent stem cells (hPSCs) have been developed as essential tools to study the underlying mechanisms of human development and diseases owing to their structural and physiological similarity to corresponding organs. Despite recent advances, there are a few methodologies for three-dimensional (3D) skeletal muscle differentiation, which focus on the terminal differentiation into myofibers and investigate the potential of modeling neuromuscular disorders and muscular dystrophies. However, these methodologies cannot recapitulate the developmental processes and lack regenerative capacity. In this study, we developed a new method to differentiate hPSCs into a 3D human skeletal muscle organoid (hSkMO). This organoid model could recapitulate the myogenesis process and possesses regenerative capacities of sustainable satellite cells (SCs), which are adult muscle stem/progenitor cells capable of self-renewal and myogenic differentiation. Our 3D model demonstrated myogenesis through the sequential occurrence of multiple myogenic cell types from SCs to myocytes. Notably, we detected quiescent, non-dividing SCs throughout the hSkMO differentiation in long-term culture. They were activated and differentiated to reconstitute muscle tissue upon damage. Thus, hSkMOs can recapitulate human skeletal muscle development and regeneration and may provide a new model for studying human skeletal muscles and related diseases.

The expression, function, and coding potential of circular RNA circEDC3 in chicken skeletal muscle development

As an emerging class of non-coding transcripts, circular RNAs (circRNAs) are proved to participate in the complex process of myogenesis in diverse species. A previous study has identified circular RNA EDC3 (circEDC3) as a typical covalently closed circular RNA abundant in chicken skeletal muscle. This study found that circEDC3 is a conservative circular RNA and performed functional analysis to investigate the role of circEDC3 in chicken muscle growth. The results indicated that circEDC3 could inhibit (P<0.05) chicken skeletal muscle satellite cells (SMSCs) proliferation and differentiation but had no significant influence on SMSCs apoptosis. Additionally, bioinformatics analysis showed that circEDC3 had promising coding potential. The open reading frames (ORF) were found in circEDC3 in this study. Furthermore, this study predicted that circEDC3 had internal ribosome entry sites (IRES) and N6-methyladenosine (m6A) motifs in different species, implying that circEDC3 might be translatable. This study revealed that circEDC3 might be a negative regulator in chicken muscle development and suggested it has protein-coding potential in different species.

Stretch-Induced Healing of Injured Muscles Is Associated With Myogenesis and Decreased Fibrosis

Background: Alghouth therapeutic stretching exercise has been applied to accelerate the healing of injured skeletal muscles, mechanisms behind the mechanical stretch-induced muscle recovery remain unclear. Purpose: To examine stretch-associated antifibrotic and myogenic responses in injured muscles and to evaluate the feasibility of the ultrasonic Nakagami parametric index (NPI) in assessing muscle morphology during recovery. Study Design: Controlled laboratory study. Methods: Skeletal muscle fibrosis was induced in the right hind legs of 48 rats by making a posterior transverse incision in the gastrocnemius muscle; the left hind legs remained intact as a comparative normal reference. After surgery, the 48 rats were randomly divided into the stretch (S) and control (C) groups. The S group received stretching interventions on the injured hind leg from week 3 to week 7 after surgery, while the C group did not receive stretching throughout the study period. The muscle fibrosis percentage and the ultrasonic NPI were examined sequentially after surgery. Relative expressions of myogenesis-related proteins, including myoblast determination protein 1 (MyoD), myogenin, and embryonic myosin heavy chain (MHCemb), were also evaluated during the follow-up. Results: Mean fibrosis percentages in the injured hind leg were approximately 25% at week 3 in both groups, but they were significantly decreased by approximately 20% from week 4 to the end of the follow-up in the S group only (all, P < .05). Upon injury, the NPI values of injured hind legs in both groups dramatically dropped. Within the S group, stretching increased the NPI values of injured hind legs, which approached those of control hind legs at weeks 6 and 7. The highest MyoD, myogenin, and MHCemb levels were observed at week 6 in both groups. The NPI values corresponded to the MyoD expression in the S group during the follow-up. Conclusion: Stretching induced a decrease in muscle fibrosis and an increase in myogenesis in injured muscles. The NPI values correspond to the myogenesis process. Clinical Relevance: The NPI may be capable of continuously monitoring the injured skeletal muscle morphology during the healing process in clinical settings.

Piezoelectric nanocomposite bioink and ultrasound stimulation modulate early skeletal myogenesis.

Despite the significant progress in bioprinting for skeletal muscle tissue engineering, new stimuli-responsive bioinks to boost the myogenesis process are highly desirable. In this work, we developed a printable alginate/Pluronic-based bioink including piezoelectric barium titanate nanoparticles (nominal diameter: ∼60 nm) for the 3D bioprinting of muscle cell-laden hydrogels. The aim was to investigate the effects of the combination of piezoelectric nanoparticles with ultrasound stimulation on early myogenic differentiation of the printed structures. After the characterization of nanoparticles and bioinks, viability tests were carried out to investigate three nanoparticle concentrations (100, 250, and 500 μg mL-1) within the printed structures. An excellent cytocompatibility was confirmed for nanoparticle concentrations up to 250 μg mL-1. TEM imaging demonstrated the internalization of BTNPs in intracellular vesicles. The combination of piezoelectric nanoparticles and ultrasound stimulation upregulated the expression of MYOD1, MYOG, and MYH2 and enhanced cell aggregation, which is a crucial step for myoblast fusion, and the presence of MYOG in the nuclei. These results suggest that the direct piezoelectric effect induced by ultrasound on the internalized piezoelectric nanoparticles boosts myogenesis in its early phases.

Head muscle fibro-adipogenic progenitors account for the tilted regeneration towards fibrosis

Branchiomeric head muscle is ontogenetically and phylogenetically distinct from somitic limb muscle, and they exhibit different regenerative capacity. Unique satellite cell property of head muscle could explain the impaired myofiber formation, but the underlying mechanism for fibrosis is still elusive. In this work, we first established a freezing-induced skeletal muscle regeneration model and made comparisons between the regeneration characteristics in tibialis anterior (TA) muscle and masseter (MAS) muscle. The process of myogenesis and fibrogenesis were investigated by histological, immunohistochemical and cellular analysis, to characterize the role of muscle satellite cell (MuSCs) and fibro-adipogenic progenitors (FAPs) in TA and MAS muscle regeneration. Our results revealed that FAPs infiltrated the fibrotic area during MAS muscle regeneration. In contrast to the rapid rise and fall of FAPs number at the early regeneration stages in TA muscle, the number of MAS FAPs increased to a plateau without descending till 14 days after injury. It is the first time that the pivotal role of FAPs in head muscle regeneration was characterized. The persistence of FAPs without timely clearance in the first two weeks of regeneration could be accountable for the head muscle fibrosis.

Inhibition of Postn Rescues Myogenesis Defects in Myotonic Dystrophy Type 1 Myoblast Model.

Myotonic dystrophy type 1 (DM1) is an inherited neuromuscular disease caused by expanded CTG repeats in the 3′ untranslated region (3′UTR) of the DMPK gene. The myogenesis process is defective in DM1, which is closely associated with progressive muscle weakness and wasting. Despite many proposed explanations for the myogenesis defects in DM1, the underlying mechanism and the involvement of the extracellular microenvironment remained unknown. Here, we constructed a DM1 myoblast cell model and reproduced the myogenesis defects. By RNA sequencing (RNA-seq), we discovered that periostin (Postn) was the most significantly upregulated gene in DM1 myogenesis compared with normal controls. This difference in Postn was confirmed by real-time quantitative PCR (RT-qPCR) and western blotting. Moreover, Postn was found to be significantly upregulated in skeletal muscle and myoblasts of DM1 patients. Next, we knocked down Postn using a short hairpin RNA (shRNA) in DM1 myoblast cells and found that the myogenesis defects in the DM1 group were successfully rescued, as evidenced by increases in the myotube area, the fusion index, and the expression of myogenesis regulatory genes. Similarly, Postn knockdown in normal myoblast cells enhanced myogenesis. As POSTN is a secreted protein, we treated the DM1 myoblast cells with a POSTN-neutralizing antibody and found that DM1 myogenesis defects were successfully rescued by POSTN neutralization. We also tested the myogenic ability of myoblasts in the skeletal muscle injury mouse model and found that Postn knockdown improved the myogenic ability of DM1 myoblasts. The activity of the TGF-β/Smad3 pathway was upregulated during DM1 myogenesis but repressed when inhibiting Postn with a Postn shRNA or a POSTN-neutralizing antibody, which suggested that the TGF-β/Smad3 pathway might mediate the function of Postn in DM1 myogenesis. These results suggest that Postn is a potential therapeutical target for the treatment of myogenesis defects in DM1.

The role of IL-1 in adipose browning and muscle wasting in CKD-associated cachexia

Cytokines such as IL-6, TNF-α and IL-1β trigger inflammatory cascades which may play a role in the pathogenesis of chronic kidney disease (CKD)-associated cachexia. CKD was induced by 5/6 nephrectomy in mice. We studied energy homeostasis in Il1β−/−/CKD, Il6−/−/CKD and Tnfα−/−/CKD mice and compared with wild type (WT)/CKD controls. Parameters of cachexia phenotype were completely normalized in Il1β−/−/CKD mice but were only partially rescued in Il6−/−/CKD and Tnfα−/−/CKD mice. We tested the effects of anakinra, an IL-1 receptor antagonist, on CKD-associated cachexia. WT/CKD mice were treated with anakinra (2.5 mg/kg/day, IP) or saline for 6 weeks and compared with WT/Sham controls. Anakinra normalized food intake and weight gain, fat and lean mass content, metabolic rate and muscle function, and also attenuated molecular perturbations of energy homeostasis in adipose tissue and muscle in WT/CKD mice. Anakinra decreased serum and muscle expression of IL-6, TNF-α and IL-1β in WT/CKD mice. Anakinra attenuated browning of white adipose tissue in WT/CKD mice. Moreover, anakinra normalized gastrocnemius weight and fiber size as well as attenuated muscle fat infiltration in WT/CKD mice. This was accompanied by correcting the increased muscle wasting signaling pathways while promoting the decreased myogenesis process in gastrocnemius of WT/CKD mice. We performed qPCR analysis for the top 20 differentially expressed muscle genes previously identified via RNAseq analysis in WT/CKD mice versus controls. Importantly, 17 differentially expressed muscle genes were attenuated in anakinra treated WT/CKD mice. In conclusion, IL-1 receptor antagonism may represent a novel targeted treatment for adipose tissue browning and muscle wasting in CKD.

Targeting interleukin-1 for reversing fat browning and muscle wasting in infantile nephropathic cystinosis.

Background Ctns −/− mice, a mouse model of infantile nephropathic cystinosis, exhibit hypermetabolism with adipose tissue browning and profound muscle wasting. Inflammatory cytokines such as interleukin (IL)‐1 trigger inflammatory cascades and may be an important cause for cachexia. We employed genetic and pharmacological approaches to investigate the effects of IL‐1 blockade in Ctns −/− mice.MethodsWe generated Ctns −/− Il1β −/− mice, and we treated Ctns −/− and wild‐type control mice with IL‐1 receptor antagonist, anakinra (2.5 mg/kg/day, IP) or saline as vehicle for 6 weeks. In each of these mouse lines, we characterized the cachexia phenotype consisting of anorexia, loss of weight, fat mass and lean mass, elevation of metabolic rate, and reduced in vivo muscle function (rotarod activity and grip strength). We quantitated energy homeostasis by measuring the protein content of uncoupling proteins (UCPs) and adenosine triphosphate in adipose tissue and skeletal muscle. We measured skeletal muscle fiber area and intramuscular fatty infiltration. We also studied expression of molecules regulating adipose tissue browning and muscle mass metabolism. Finally, we evaluated the impact of anakinra on the muscle transcriptome in Ctns −/− mice.ResultsSkeletal muscle expression of IL‐1β was significantly elevated in Ctns −/− mice relative to wild‐type control mice. Cachexia was completely normalized in Ctns −/− Il1β −/− mice relative to Ctns −/− mice. We showed that anakinra attenuated the cachexia phenotype in Ctns −/− mice. Anakinra normalized UCPs and adenosine triphosphate content of adipose tissue and muscle in Ctns −/− mice. Anakinra attenuated aberrant expression of beige adipose cell biomarkers (UCP‐1, CD137, Tmem26, and Tbx1) and molecules implicated in adipocyte tissue browning (Cox2/Pgf2α, Tlr2, Myd88, and Traf6) in inguinal white adipose tissue in Ctns −/− mice. Moreover, anakinra normalized gastrocnemius weight and fiber size and attenuated muscle fat infiltration in Ctns −/− mice. This was accompanied by correction of the increased muscle wasting signalling pathways (increased protein content of ERK1/2, JNK, p38 MAPK, and nuclear factor‐κB p65 and mRNA expression of Atrogin‐1 and Myostatin) and the decreased myogenesis process (decreased mRNA expression of MyoD and Myogenin) in the gastrocnemius muscle of Ctns −/− mice. Previously, we identified the top 20 differentially expressed skeletal muscle genes in Ctns −/− mice by RNAseq. Aberrant expression of these 20 genes have been implicated in muscle wasting, increased energy expenditure, and lipolysis. We showed that anakinra attenuated 12 of those top 20 differentially expressed muscle genes in Ctns −/− mice.ConclusionsAnakinra may provide a targeted novel therapy for patients with infantile nephropathic cystinosis.

The LIM domain protein nTRIP6 modulates the dynamics of myogenic differentiation

The process of myogenesis which operates during skeletal muscle regeneration involves the activation of muscle stem cells, the so-called satellite cells. These then give rise to proliferating progenitors, the myoblasts which subsequently exit the cell cycle and differentiate into committed precursors, the myocytes. Ultimately, the fusion of myocytes leads to myofiber formation. Here we reveal a role for the transcriptional co-regulator nTRIP6, the nuclear isoform of the LIM-domain protein TRIP6, in the temporal control of myogenesis. In an in vitro model of myogenesis, the expression of nTRIP6 is transiently up-regulated at the transition between proliferation and differentiation, whereas that of the cytosolic isoform TRIP6 is not altered. Selectively blocking nTRIP6 function results in accelerated early differentiation followed by deregulated late differentiation and fusion. Thus, the transient increase in nTRIP6 expression appears to prevent premature differentiation. Accordingly, knocking out the Trip6 gene in satellite cells leads to deregulated skeletal muscle regeneration dynamics in the mouse. Thus, dynamic changes in nTRIP6 expression contributes to the temporal control of myogenesis.

Temporomandibular Disorders Slow Down the Regeneration Process of Masticatory Muscles: Transcriptomic Analysis.

Background and Objectives: Musculoskeletal injuries represent a pathological condition due to limited joint motility and morphological and functional alterations of the muscles. Temporomandibular disorders (TMDs) are pathological conditions due to alterations in the musculoskeletal system. TMDs mainly cause temporomandibular joint and masticatory muscle dysfunctions following trauma, along with various pathologies and inflammatory processes. TMD affects approximately 15% of the population and causes malocclusion problems and common symptoms such as myofascial pain and migraine. The aim of this work was to provide a transcriptomic profile of masticatory muscles obtained from TMD migraine patients compared to control. Materials and Methods: We used Next Generation Sequencing (NGS) technology to evaluate transcriptomes in masseter and temporalis muscle samples. Results: The transcriptomic analysis showed a prevalent downregulation of the genes involved in the myogenesis process. Conclusions: In conclusion, our findings suggest that the muscle regeneration process in TMD migraine patients may be slowed, therefore therapeutic interventions are needed to restore temporomandibular joint function and promote healing processes.

The Effects of Muscle Cell Aging on Myogenesis.

The process of myogenesis gradually deteriorates as the skeletal muscle ages, contributing to muscle mass loss. The aim of this study is to investigate the effect of senescence/aging on skeletal myogenesis, in vitro. A model of multiple cell divisions of C2C12 myoblasts was used to replicate cell senescence. Control and aged myoblasts were investigated during myogenesis, i.e., at days 0, 2, and 6of differentiation. SA-β-gal activity and comet assay were used as markers of aging and DNA damage. Flow cytometry was performed to characterize potential differences in cell cycle between control and aged cells. Alterations in the mRNA and/or protein expression of myogenic regulatory factors (MRFs), IGF-1 isoforms, apoptotic, atrophy, inflammatory, metabolic and aging-related factors were evaluated. Compared with the control cells, aged myoblasts exhibited G0/G1 cell cycle arrest, DNA damage, increased SA-β-gal activity, and increased expression of aging-related factors p16 and p21 during differentiation. Moreover, aged myoblasts showed a reduction in the expression of MRFs and metabolic/anabolic factors, along with an increased expression of apoptotic, atrophy and inflammatory factors. A diminished differentiation capacity characterized the aged myoblasts which, in combination with the induction of apoptotic and atrophy factors, indicated a disrupted myogenic lineage in the senescent muscle cells.