Nano-sized graphene oxide coated nanopillars on microgroove polymer arrays that enhance skeletal muscle cell differentiation

The degeneration or loss of skeletal muscles, which can be caused by traumatic injury or disease, impacts most aspects of human activity. Among various techniques reported to regenerate skeletal muscle tissue, controlling the external cellular environment has been proven effective in guiding muscle differentiation. In this study, we report a nano-sized graphene oxide (sGO)-modified nanopillars on microgroove hybrid polymer array (NMPA) that effectively controls skeletal muscle cell differentiation. sGO-coated NMPA (sG-NMPA) were first fabricated by sequential laser interference lithography and microcontact printing methods. To compensate for the low adhesion property of polydimethylsiloxane (PDMS) used in this study, graphene oxide (GO), a proven cytophilic nanomaterial, was further modified. Among various sizes of GO, sGO (< 10 nm) was found to be the most effective not only for coating the surface of the NM structure but also for enhancing the cell adhesion and spreading on the fabricated substrates. Remarkably, owing to the micro-sized line patterns that guide cellular morphology to an elongated shape and because of the presence of sGO-modified nanostructures, mouse myoblast cells (C2C12) were efficiently differentiated into skeletal muscle cells on the hybrid patterns, based on the myosin heavy chain expression levels. Therefore, the developed sGO coated polymeric hybrid pattern arrays can serve as a potential platform for rapid and highly efficient in vitro muscle cell generation.

ViaFuse: Fiji macros to calculate skeletal muscle cell viability and fusion index

Background Measuring biological features of skeletal muscle cells is difficult because of their unique morphology and multinucleate nature upon differentiation. Here, we developed a new Fiji macro package called ViaFuse (that stands for viability and fusion) to measure skeletal muscle cell viability and differentiation. To test ViaFuse, we utilized immunofluorescence images of differentiated myotubes where the capping actin protein of muscle z-line subunit beta (CAPZB) was depleted in comparison with control cells. Results We compared the values achieved using the ViaFuse macros first with manual quantification performed by researchers and second with those obtained utilizing the MATLAB muscle-centric software MyoCount. We observed a high degree of correlation between all methods of quantification. Conclusions ViaFuse can detect the borders of myotubes and identify nuclear clumps which have been limitations of previous muscle-centric imaging software. The ViaFuse macros require little computer power or space to run and user inputs to the ViaFuse macros are minimal, thereby automating the analysis process in a quick, easy, and accurate fashion. Additionally, the ViaFuse macros work with Fiji, an existing imaging software widely used by skeletal muscle researchers. Furthermore, ViaFuse is compatible with many computer systems, has a very intuitive interface, and does not require prior complex mathematical knowledge. Therefore, we propose ViaFuse as a robust and meticulous method to quantify skeletal muscle cell viability and differentiation.

Conditionally Immortalised Equine Skeletal Muscle Cell Lines for in Vitro Analysis

BackgroundThoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C>T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype.ResultsPrimary myoblasts conditionally immortalized with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q10 (CoQ10) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ10 in the diet. ConclusionsThe observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine.

Human and rat skeletal muscle single-nuclei multi-omic integrative analyses nominate causal cell types, regulatory elements, and SNPs for complex traits

Skeletal muscle accounts for the largest proportion of human body mass, on average, and is a key tissue in complex diseases and mobility. It is composed of several different cell and muscle fiber types. Here, we optimize single-nucleus ATAC-seq (snATAC-seq) to map skeletal muscle cell–specific chromatin accessibility landscapes in frozen human and rat samples, and single-nucleus RNA-seq (snRNA-seq) to map cell-specific transcriptomes in human. We additionally perform multi-omics profiling (gene expression and chromatin accessibility) on human and rat muscle samples. We capture type I and type II muscle fiber signatures, which are generally missed by existing single-cell RNA-seq methods. We perform cross-modality and cross-species integrative analyses on 33,862 nuclei and identify seven cell types ranging in abundance from 59.6% to 1.0% of all nuclei. We introduce a regression-based approach to infer cell types by comparing transcription start site–distal ATAC-seq peaks to reference enhancer maps and show consistency with RNA-based marker gene cell type assignments. We find heterogeneity in enrichment of genetic variants linked to complex phenotypes from the UK Biobank and diabetes genome-wide association studies in cell-specific ATAC-seq peaks, with the most striking enrichment patterns in muscle mesenchymal stem cells (∼3.5% of nuclei). Finally, we overlay these chromatin accessibility maps on GWAS data to nominate causal cell types, SNPs, transcription factor motifs, and target genes for type 2 diabetes signals. These chromatin accessibility profiles for human and rat skeletal muscle cell types are a useful resource for nominating causal GWAS SNPs and cell types.

The Effect of Acute and Chronic Thermotherapy on Type 2 Diabetic Skeletal Muscle Gene Expression and Inflammatory Markers

Background: Type 2 diabetes (T2D) is a chronic illness associated with resistance to or defective insulin secretion. This study investigates the effects of thermotherapy on cell viability, gene expression and inflammation in skeletal muscle cell lines. Methods: Healthy and T2D human skeletal muscle cell lines (HSMM and D-HSMM, respectively) were subjected to acute or chronic thermo-therapy (AT or CT, respectively). CT consisted of a 30 min exposure to 40 °C, three times a week for three weeks; AT was a one-time exposure. Results: A significant decrease in D-HSMM cell viability percentage followed AT; however, no significant change occurred in CT. HSMM yielded the highest elevations of genes following CT. In D-HSMM, both treatments yielded gene upregulation. Both treatments significantly down-regulated IL-1β, IL-6, IL-10 and TNF-α in HSMM. AT significantly decreased IL-1β, IL-6 and upregulated IL-10 and TNF-α levels in D-HSMM, while CT yielded a reduction in IL-4, TNF-α and an upregulation of IL-6 and IL-10. Conclusions: An increase in gene expression indicates actin activity and cellular responses, suggesting an increase in transcriptional regulation. The upregulation of IL-6 and IL-10 in D-HSMM negatively correlated with a decrease in TNF-α and IL-1β, indicating improved adverse inflammatory effects associated with the disease.

A xanthene derivative, DS20060511, attenuates glucose intolerance by inducing skeletal muscle-specific GLUT4 translocation in mice

Reduced glucose uptake into the skeletal muscle is an important pathophysiological abnormality in type 2 diabetes, and is caused by impaired translocation of glucose transporter 4 (GLUT4) to the skeletal muscle cell surface. Here, we show a xanthene derivative, DS20060511, induces GLUT4 translocation to the skeletal muscle cell surface, thereby stimulating glucose uptake into the tissue. DS20060511 induced GLUT4 translocation and stimulated glucose uptake into differentiated L6-myotubes and into the skeletal muscles in mice. These effects were completely abolished in GLUT4 knockout mice. Induction of GLUT4 translocation by DS20060511 was independent of the insulin signaling pathways including IRS1-Akt-AS160 phosphorylation and IRS1-Rac1-actin polymerization, eNOS pathway, and AMPK pathway. Acute and chronic DS20060511 treatment attenuated the glucose intolerance in obese diabetic mice. Taken together, DS20060511 acts as a skeletal muscle-specific GLUT4 translocation enhancer to facilitate glucose uptake. Further studies of DS20060511 may pave the way for the development of novel antidiabetic medicines.

Skeletal Muscle Cell Growth Alters the Lipid Composition of Extracellular Vesicles

We sought to characterize the lipid profile of skeletal muscle cell-derived Extracellular Vesicles (EVs) to determine if a hypertrophic stimulus would affect the lipid composition of C2C12 myotube-derived EVs. Analyses included C2C12 murine myoblasts differentiated into myotubes and treated with Insulin-Like Growth Factor 1 (IGF-1) for 24 h to induce hypertrophic growth. EVs were isolated from cell culture media, quantified using Nanoparticle Tracking Analysis (NTA) and analyzed using Transmission Electron Microscopy (TEM). EVs were homogenized and lipids extracted for quantification by Mass Spectrometry followed by downstream lipid class enrichment and lipid chain analysis. IGF-1 treatment elicited an increase in CD63 and CD81 levels (39% and 21%) compared to the controls (16%), respectively. Analysis revealed that skeletal muscle-derived EVs are enriched in bioactive lipids that are likely selectively incorporated into EVs during hypertrophic growth. IGF-1 treatment of myotubes had a significant impact on the levels of diacylglycerol (DG) and ceramide (Cer) in secreted EVs. Specifically, the proportion of unsaturated DG was two- to three-fold higher in EVs derived from IGF-treated cells, as compared to those from control cells. The levels of saturated DG were unaffected. Selective increases were similarly seen in C16- and C24-Cer but not in other species. Levels of free sphingoid bases tended to decrease, while those of sphingosine-1-phosphate was unaffected. Our results suggest that the lipid composition and biogenesis of skeletal muscle-derived EVs, are specific and highly selective during hypertrophic growth.

Skin and Bones—and Muscle Too

This chapter recounts bioprinting studies of skin, bone, skeletal muscle, and neuromuscular junctions. The chapter begins with a study of bioprinted skin designed to enable the creation of skin with a uniform pigmentation. The chapter relates two very different approaches to bioprinted bone: a synthetic bone called hyperelastic bone and a strategy that prints cartilage precursors to bone and then induces the conversion of the cartilage to bone by judicious choice of bioinks. Muscles move bone, and the chapter discusses an investigation of bioprinted skeletal muscle. Finally, the chapter considers an attempt to bioprint a neuromuscular junction, a synapse—a minute gap—of about 20 billionths of a meter between a motor neuron and the cell membrane of a skeletal muscle cell. A motor neuron is a nerve in the central nervous system that sends signals to the muscles of the body.