Myogenic Differentiation of Human Myoblasts and Mesenchymal Stromal Stem Cells under GDF11 on Poly-ɛ-Caprolactone-Collagen I-Polyethylene-Nanofibers

Primary myoblasts (Mb) and adipose derived mesenchymal stromal cells (ADSC) can be co-cultured and myogenically differentiated in the process of skeletal muscle tissue engineering. Electrospun composite nanofiber scaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining biocompatibility and stability. Although growth differentiation factor 11 (GDF11) has been proposed as a rejuvenating circulating factor, restoring skeletal muscle function in aging mice, some studies have also described a harming effect of GDF11.Therefore the aim of the study was to analyze the effect of GDF11 on co-cultures of Mb and ADSC on poly-ε-caprolacton (PCL)-collagen I-polyethylene oxide (PEO)-nanofibers.Human Mb were co-cultured with ADSC two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-PEO-nanofibers. Differentiation media were either serum-free with or without GDF11, or serum containing as in a conventional differentiation medium. Cell viability was higher after conventional myogenic differentiation compared to serum-free and serum-free + GDF11 differentiation as was creatine kinase activity. Immunofluorescence staining showed myosin heavy chain expression in all groups after 28 days of differentiation. Gene expression of myosin heavy chain (MYH2) increased after serum-free + GDF11 stimulation compared to serum-free stimulation alone. The results of this study show that PCL-collagen I-PEO-nanofibers represent a suitable matrix for 3D myogenic differentiation of Mb and ADSC. In this context, GDF11 seems to promote myogenic differentiation of Mb and ADSC co-cultures compared to serum-free differentiation without any evidence of a harming effect.

circTAF8 Regulates Myoblast Development and Associated Carcass Traits in Chicken

Recent studies have shown that circular RNAs (circRNAs) play important roles in skeletal muscle development. CircRNA biogenesis is dependent on the genetic context. Single-nucleotide polymorphisms in the introns flanking circRNAs may be intermediate-inducible factors between circRNA expression and phenotypic traits. Our previous study showed that circTAF8 is an abundantly and differentially expressed circRNA in leg muscle during chicken embryonic development. Here, we aimed to investigate circTAF8 function in muscle development and the association of the SNPs in the circTAF8 flanking introns with carcass traits. In this study, we observed that overexpression of circTAF8 could promote the proliferation of chicken primary myoblasts and inhibit their differentiation. In addition, the SNPs in the introns flanking the circTAF8 locus and those associated with chicken carcass traits were analyzed in 335 partridge chickens. A total of eight SNPs were found associated with carcass traits such as leg muscle weight, live weight, and half and full-bore weight. The association analysis results of haplotype combinations were consistent with the association analysis of a single SNP. These results suggest that circTAF8 plays a regulatory role in muscle development. These identified SNPs were found correlated with traits to muscle development and carcass muscle weight in chickens.

Improved CRISPR/Cas9 gene editing in primary human myoblasts using low confluency cultures on Matrigel

Background CRISPR/Cas9 is an invaluable tool for studying cell biology and the development of molecular therapies. However, delivery of CRISPR/Cas9 components into some cell types remains a major hurdle. Primary human myoblasts are a valuable cell model for muscle studies, but are notoriously difficult to transfect. There are currently no commercial lipofection protocols tailored for primary myoblasts, and most generic guidelines simply recommend transfecting healthy cells at high confluency. This study aimed to maximize CRISPR/Cas9 transfection and editing in primary human myoblasts. Methods Since increased cell proliferation is associated with increased transfection efficiency, we investigated two factors known to influence myoblast proliferation: cell confluency, and a basement membrane matrix, Matrigel. CRISPR/Cas9 editing was performed by delivering Cas9 ribonucleoprotein complexes via lipofection into primary human myoblasts, cultured in wells with or without a Matrigel coating, at low (~ 40%) or high (~ 80%) confluency. Results Cells transfected at low confluency on Matrigel-coated wells had the highest levels of transfection, and were most effectively edited across three different target loci, achieving a maximum editing efficiency of 93.8%. On average, editing under these conditions was >4-fold higher compared to commercial recommendations (high confluency, uncoated wells). Conclusion This study presents a simple, effective and economical method of maximizing CRISPR/Cas9-mediated gene editing in primary human myoblasts. This protocol could be a valuable tool for improving the genetic manipulation of cultured human skeletal muscle cells, and potentially be adapted for use in other cell types.

Dexamethasone Inhibits the Pro-Angiogenic Potential of Primary Human Myoblasts

Tissue regeneration depends on the complex processes of angiogenesis, inflammation and wound healing. Regarding muscle tissue, glucocorticoids (GCs) inhibit pro-inflammatory signalling and angiogenesis and lead to muscle atrophy. Our hypothesis is that the synthetic GC dexamethasone (dex) impairs angiogenesis leading to muscle atrophy or inhibited muscle regeneration. Therefore, this study aims to elucidate the effect of dexamethasone on HUVECs under different conditions in mono- and co-culture with myoblasts to evaluate growth behavior and dex impact with regard to muscle atrophy and muscle regeneration. Viability assays, qPCR, immunofluorescence as well as ELISAs were performed on HUVECs, and human primary myoblasts seeded under different culture conditions. Our results show that dex had a higher impact on the tube formation when HUVECs were maintained with VEGF. Gene expression was not influenced by dex and was independent of cells growing in a 2D or 3D matrix. In co-culture CD31 expression was suppressed after incubation with dex and gene expression analysis revealed that dex enhanced expression of myogenic transcription factors, but repressed angiogenic factors. Moreover, dex inhibited the VEGF mediated pro angiogenic effect of myoblasts and inhibited expression of angiogenic inducers in the co-culture model. This is the first study describing a co-culture of human primary myoblast and HUVECs maintained under different conditions. Our results indicate that dex affects angiogenesis via inhibition of VEGF release at least in myoblasts, which could be responsible not only for the development of muscle atrophy after dex administration, but also for inhibition of muscle regeneration after vascular damage.

ERK3-MK5 signaling regulates myogenic differentiation and muscle regeneration by promoting FoxO3 degradation

ABSTRACTThe physiological functions and downstream effectors of the atypical mitogen-activated protein kinase ERK3 remain to be characterized. We recently reported that mice expressing catalytically-inactive ERK3 (Mapk6KD/KD) exhibit a reduced post-natal growth rate as compared to control mice. Here, we show that genetic inactivation of ERK3 impairs post-natal skeletal muscle growth and adult muscle regeneration after injury. Loss of MK5 phenocopies the muscle phenotypes of Mapk6KD/KD mice. At the cellular level, genetic or pharmacological inactivation of ERK3 or MK5 induces precocious differentiation of C2C12 or primary myoblasts, concomitant with MyoD activation. Reciprocally, ectopic expression of activated MK5 inhibits myogenic differentiation. Mechanistically, we show that MK5 directly phosphorylates FoxO3, promoting its degradation and reducing its association with MyoD. Depletion of FoxO3 rescues in part the premature differentiation of C2C12 myoblasts observed upon inactivation of ERK3 or MK5. Our findings reveal that ERK3 and its substrate MK5 act in a linear signaling pathway to control post-natal myogenic differentiation.

Urolithin A improves muscle function by inducing mitophagy in muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy, and despite advances in genetic and pharmacological disease-modifying treatments, its management remains a major challenge. Mitochondrial dysfunction contributes to DMD, yet the mechanisms by which this occurs remain elusive. Our data in experimental models and patients with DMD show that reduced expression of genes involved in mitochondrial autophagy, or mitophagy, contributes to mitochondrial dysfunction. Mitophagy markers were reduced in skeletal muscle and in muscle stem cells (MuSCs) of a mouse model of DMD. Administration of the mitophagy activator urolithin A (UA) rescued mitophagy in DMD worms and mice and in primary myoblasts from patients with DMD, increased skeletal muscle respiratory capacity, and improved MuSCs’ regenerative ability, resulting in the recovery of muscle function and increased survival in DMD mouse models. These data indicate that restoration of mitophagy alleviates symptoms of DMD and suggest that UA may have potential therapeutic applications for muscular dystrophies.

MicroRNA-27b-3p Targets the Myostatin Gene to Regulate Myoblast Proliferation and Is Involved in Myoblast Differentiation

microRNAs play an important role in the growth and development of chicken embryos, including the regulation of skeletal muscle genesis, myoblast proliferation, differentiation, and apoptosis. Our previous RNA-seq studies showed that microRNA-27b-3p (miR-27b-3p) might play an important role in regulating the proliferation and differentiation of chicken primary myoblasts (CPMs). However, the mechanism of miR-27b-3p regulating the proliferation and differentiation of CPMs is still unclear. In this study, the results showed that miR-27b-3p significantly promoted the proliferation of CPMs and inhibited the differentiation of CPMs. Then, myostatin (MSTN) was confirmed to be the target gene of miR-27b-3p by double luciferase reporter assay, RT-qPCR, and Western blot. By overexpressing and interfering with MSTN expression in CPMs, the results showed that overexpression of MSTN significantly inhibited the proliferation and differentiation of CPMs. In contrast, interference of MSTN expression had the opposite effect. This study showed that miR-27b-3p could promote the proliferation of CPMs by targeting MSTN. Interestingly, both miR-27b-3p and MSTN can inhibit the differentiation of CPMs. These results provide a theoretical basis for further understanding the function of miR-27b-3p in chicken and revealing its regulation mechanism on chicken muscle growth.