Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro

Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications.

Skeletal muscle phenotyping of Hippo gene-mutated mice reveals that Lats1 deletion increases the percentage of type I muscle fibers

The Hippo signal transduction network regulates transcription through Yap/Taz-Tead1-4 in many tissues including skeletal muscle. Whilst transgenic mice have been generated for many Hippo genes, the resultant skeletal muscle phenotypes were not always characterized. Here, we aimed to phenotype the hindlimb muscles of Hippo gene-mutated Lats1−/−, Mst2−/−, Vgll3−/−, and Vgll4+/− mice. This analysis revealed that Lats1−/− mice have 11% more slow type I fibers than age and sex-matched wild-type controls. Moreover, the mRNA expression of slow Myh7 increased by 50%, and the concentration of type I myosin heavy chain is 80% higher in Lats1−/− mice than in age and sex-matched wild-type controls. Second, to find out whether exercise-related stimuli affect Lats1, we stimulated C2C12 myotubes with the hypertrophy agent clenbuterol or the energy stress agent AICAR. We found that both stimulated Lats1 expression by 1.2 and 1.3 fold respectively. Third, we re-analyzed published datasets and found that Lats1 mRNA in muscle is 63% higher in muscular dystrophy, increases by 17–77% after cardiotoxin-induced muscle injury, by 41–71% in muscles during overload-induced hypertrophy, and by 19–21% after endurance exercise when compared to respective controls. To conclude, Lats1 contributes to the regulation of muscle fiber type proportions, and its expression is regulated by physiological and pathological situations in skeletal muscle.

Decreased miR-497-5p Suppresses IL-6 Induced Atrophy in Muscle Cells

Interleukin-6 (IL-6) is a pro-inflammatory cytokine associated with skeletal muscle wasting in cancer cachexia. The control of gene expression by microRNAs (miRNAs) in muscle wasting involves the regulation of thousands of target transcripts. However, the miRNA-target networks associated with IL6-induced muscle atrophy remain to be characterized. Here, we show that IL-6 promotes the atrophy of C2C12 myotubes and changes the expression of 20 miRNAs (5 up-regulated and 15 down-regulated). Gene Ontology analysis of predicted miRNAs targets revealed post-transcriptional regulation of genes involved in cell differentiation, apoptosis, migration, and catabolic processes. Next, we performed a meta-analysis of miRNA-published data that identified miR-497-5p, a down-regulated miRNAs induced by IL-6, also down-regulated in other muscle-wasting conditions. We used miR-497-5p mimics and inhibitors to explore the function of miR-497-5p in C2C12 myoblasts and myotubes. We found that miR-497-5p can regulate the expression of the cell cycle genes CcnD2 and CcnE1 without affecting the rate of myoblast cellular proliferation. Notably, miR-497-5p mimics induced myotube atrophy and reduced Insr expression. Treatment with miR-497-5p inhibitors did not change the diameter of the myotubes but increased the expression of its target genes Insr and Igf1r. These genes are known to regulate skeletal muscle regeneration and hypertrophy via insulin-like growth factor pathway and were up-regulated in cachectic muscle samples. Our miRNA-regulated network analysis revealed a potential role for miR-497-5p during IL6-induced muscle cell atrophy and suggests that miR-497-5p is likely involved in a compensatory mechanism of muscle atrophy in response to IL-6.

The Atrophic Effect of 1,25(OH)2 Vitamin D3 (Calcitriol) on C2C12 Myotubes Depends on Oxidative Stress

Dysfunctional mitochondrial metabolism has been linked to skeletal muscle loss in several physio-pathological states. Although it has been reported that vitamin D (VD) supports cellular redox homeostasis by maintaining normal mitochondrial functions, and VD deficiency often occurs in conditions associated with skeletal muscle loss, the efficacy of VD supplementation to overcome muscle wasting is debated. Investigations on the direct effects of VD metabolites on skeletal muscle using C2C12 myotubes have revealed an unexpected pro-atrophic activity of calcitriol (1,25VD), while its upstream metabolites cholecalciferol (VD3) and calcidiol (25VD) have anti-atrophic effects. Here, we investigated if the atrophic effects of 1,25VD on myotubes depend on its activity on mitochondrial metabolism. The impact of 1,25VD and its upstream metabolites VD3 and 25VD on mitochondria dynamics and the activity of C2C12 myotubes was evaluated by measuring mitochondrial content, architecture, metabolism, and reactive oxygen species (ROS) production. We found that 1,25VD induces atrophy through protein kinase C (PKC)-mediated ROS production, mainly of extramitochondrial origin. Consistent with this, cotreatment with the antioxidant N-acetylcysteine (NAC), but not with the mitochondria-specific antioxidant mitoTEMPO, was sufficient to blunt the atrophic activity of 1,25VD. In contrast, VD3 and 25VD have antioxidant properties, suggesting that the efficacy of VD supplementation might result from the balance between atrophic pro-oxidant (1,25VD) and protective antioxidant (VD3 and 25VD) metabolites.

Garcinol Promotes the Formation of Slow-Twitch Muscle Fibers by Inhibiting p300-Dependent Acetylation of PGC-1α

Background The conversion of skeletal muscle fiber from fast twitch to slow-twitch is crucial for sustained contractile and stretchable events, energy homeostasis, and anti-fatigue ability. The purpose of our study was to explore the mechanism and effects of garcinol on the regulation of skeletal muscle fiber type transformation. Methods Forty 21-day-old male C57/BL6J mice (n = 10/diet) were fed a control diet or a control diet plus garcinol at 100 mg/kg (Low Gar), 300 mg/kg (Mid Gar), or 500 mg/kg (High Gar) for 12 weeks. The tibialis anterior (TA) and soleus muscles were collected for protein and immunoprecipitation analyses. Results Dietary garcinol significantly downregulated (P<0.05) fast MyHC expression and upregulated (P<0.05) slow MyHC expression in the TA and soleus muscles. Garcinol significantly increased (P<0.05) the activity of PGC-1α and markedly decreased (P<0.05) the acetylation of PGC-1α. In vitro and in vivo experiments showed that garcinol decreased (P<0.05) lactate dehydrogenase activity and increased (P<0.05) the activities of malate dehydrogenase and succinic dehydrogenase. In addition, the results of immunostaining C2C12 myotubes showed that garcinol treatment increased (P<0.05) the transformation of glycolytic muscle fiber to oxidative muscle fiber by 45.9%. Garcinol treatment and p300 interference reduced (P<0.05) the expression of fast MyHC but increased (P<0.05) the expression of slow MyHC in vitro. Moreover, the acetylation of PGC-1α was significantly decreased (P<0.05). Conclusion Garcinol promotes the transformation of skeletal muscle fibers from the fast-glycolytic type to the slow-oxidative type through the p300/PGC-1α signaling pathway in C2C12 myotubes.

Downregulation of miR-29c promotes muscle wasting by modulating the activity of leukemia inhibitory factor in lung cancer cachexia

Background Cancer cachexia is a wasting disorder characterized by significant weight loss, and is attributed to skeletal muscle weakness. In the process of cancer development, microRNAs act as oncogenes or tumor suppressors. Moreover, they are implicated in muscle development and wasting. This study sought to explore the mechanisms and correlation between miR-29c and muscle wasting in lung cancer cachexia. Methods Data for expression analysis were retrieved from the Cancer Genome Atlas (TCGA) database. qRT-PCR analyses were performed to explore the expression levels of miR-29c and Leukemia Inhibitory Factor (LIF). Lewis lung carcinoma (LLC) cell line was used to establish a cachexia model to explore the functions of miR-29c and LIF in lung cancer cachexia. Furthermore, in vitro (in C2C12 myotubes) and in vivo (in LLC tumor-bearing mice) experiments were performed to explore the mechanisms of miR-29c and LIF in lung cachexia. Results Analysis of the lung cancer cachexia model showed that miR-29c was down-regulated, and its expression was negatively correlated with muscle catabolic activity. Overexpression of miR-29c mitigated the cachectic phenotype. Mechanistic studies showed that LIF was a direct target gene of miR-29c, and LIF was upregulated in vitro and in vivo. Analysis showed that LIF promoted muscle wasting through the JAK/STAT and MAP-kinase pathways. Conclusions The findings indicated that miR-29c was negatively correlated with the cachectic phenotype, and the miR-29c-LIF axis is a potential therapeutic target for cancer cachexia.

Pharmacological Inhibition of HMGB1 Prevents Muscle Wasting

Background: Cachexia is a multifactorial disorder characterized by weight loss and muscle wasting, making up for about 20% of cancer-related death. However, there are no effective drugs to combat cachexia at present.Methods: In this study, the effect of CT26 exosomes on C2C12 myotubes was observed. We compared serum HMGB1 level in cachexia and non-cachexia colon cancer patients. We further explored HMGB1 expression level in CT26 exosome. We added recombinant HMGB1 to C2C12 myotubes to observe the effects of HMGB1 on C2C12 myotubes and detected the expression level of the muscle atrophy-related proteins. Then, we used the HMGB1 inhibitor glycyrrhizin to reverse the effects of HMGB1 on C2C12 myotubes. Finally, HMGB1 inhibitor glycyrrhizin was utilized to relieve cachexia in CT26 cachexia mouse model.Results: Exosomes containing HMGB1 led to muscle atrophy with significantly decreased myotube diameter and increased expression of muscle atrophy-related proteins Atrogin1 and MuRF1. Further, we detected that HMGB1 induced the muscle atrophy mainly via TLR4/NF-κB pathway. Administration of the HMGB1 inhibitor glycyrrhizin could relieve muscle wasting in vitro and attenuate the progression of cachexia in vivo.Conclusion: These findings demonstrate the cachectic role of HMGB1, whether it is soluble form of HMGB1 or secreted from tumor cells as part of exosomes. HMGB1 inhibitor glycyrrhizin might be a promising drug in colon cancer cachexia.

Anti-Hyperglycemic Effects of Refined Fractions from Cyclocarya Paliurus Leaves on Streptozotocin-Induced Diabetic Mice

To identify the chemical components responsible for the anti-hyperglycemic effect of Cyclocarya paliurus (Batal.) Iljinsk (Juglandaceae) leaves, an ethanol extract (CPE) and a water extract (CPW) of C. paliurus leaves, as well as their total flavonoids (CPF), triterpenoids (CPT) and crude polysaccharides (CPP), were prepared and assessed on streptozotocin (STZ)-induced diabetic mice. After being orally administrated once a day for 24 days, CPF (300 mg/kg), CPP (180 mg/kg), or CPF+CPP (300 mg/kg CPF + 180 mg/kg CPP) treatment reversed STZ-induced body weight and muscle mass losses. The glucose tolerance tests and insulin tolerance tests suggested that CPF, CPP, and CPF+CPP showed anti-hyperglycemic effect in STZ-induced diabetic mice. Furthermore, CPF enhances glucose-stimulated insulin secretion in MIN6 cells and insulin-stimulated glucose uptake in C2C12 myotubes. CPF and CPP suppressed inflammatory cytokine levels in STZ-induced diabetic mice. Additionally, CPF and CPP improved STZ-induced diabetic nephropathy assessed by H&E staining, blood urea nitrogen content, and urine creatinine level. The molecular networking and Emperor analysis results indicated that CPF showed potential anti-hyperglycemic effects, and HPLC–MS/MS analysis indicated that CPF contains 3 phenolic acids and 9 flavonoids. In contrast, CPT (650 mg/kg) and CPC (300 mg/kg CPF + 180 mg/kg CPP + 650 mg/kg CPT) did not show anti-hyperglycemic effect. Taken together, polysaccharides and flavonoids are responsible for the anti-hyperglycemic effect of C. paliurus leaves, and the clinical application of C. paliurus need to be refined.