Stimulation of glucose uptake by glycosides from Alectra parasitica subsp. chitrakutensis (M.A. Rau) K.K. Khanna & An. Kumar: An in-vitro study of TNF-α induced insulin resistance in L6 myoblasts.

Alectra parasitica subsp. chitrakutensis (M.A. Rau) K.K. Khanna & An. Kumar (Orobanchaceae) is a parasitic plant indigenous to India. Locally, the plant is known as 'Midaki and Nirgundikand'. It is used to treat fever, piles, cardiovascular disorders, and blood-borne non-infectious diseases by ethnic communities. The phytochemical investigation of A. parasitica subsp. chitrakutensis rhizome led to the isolation of azafrin (1), rehmaionoside-C (2), and mussaenoside (3). Compounds (2) and (3) are being reported for the first time from this plant. Compounds were evaluated for their intercellular glucose uptake activity in basal and insulin-TNF-α-stimulated L6 muscle cells. In particular, rehmaionoside C exhibited activity comparative to metformin, increasing uptake by basal- and insulin-TNF-α-stimulated cells by 4.88- and 3.90-fold and 5.04- and 4.04-fold. While azafrin and mussaenoside have produced 3.03- and 2.36-fold; 4.03- and 3.22-fold increase in intercellular glucose uptake. Compounds did not show toxicities in rat L6 myoblast cells. The study suggests that rehmaionoside-C from A. parasitica subsp. chitrakutensis might activate glucose uptake by insulin mimics and could be a nontoxic anti-diabetes lead for drug discovery.

Immature adipocyte-derived exosomes inhibit expression of muscle differentiation markers.

Exosomes are released from a variety of cells to communicate with recipient cells. Exosomes contain microRNAs (miRNAs), which are noncoding RNAs that suppress target genes. Our previous proteomic study (FEBS Open Bio 2016, 6, 816-826) demonstrated that 3T3-L1 adipocytes secrete exosome components as well as growth factors, inspiring us to investigate what type of miRNA is involved in adipocyte-secreted exosomes and what functions they carry out in recipient cells. Here, we profiled miRNAs in 3T3-L1 adipocyte-secreted exosomes and revealed suppression of muscle differentiation by adipocyte-derived exosomes. Through our microarray analysis, we detected over 300 exosomal miRNAs during adipocyte differentiation. Exosomal miRNAs present during adipocyte differentiation included not only pro-adipogenic miRNAs but also miRNAs associated with muscular dystrophy. Gene ontology analysis predicted that the target genes of miRNAs are associated primarily with transcriptional regulation. To further investigate whether adipocyte-secreted exosomes regulate the expression levels of genes involved in muscle differentiation, we treated cultured myoblasts with adipocyte-derived exosome fractions. Intriguingly, the expression levels of myogenic regulatory factors, Myog and Myf6, and other muscle differentiation markers, myosin heavy-chain 3 and insulin-like growth factor 2, were significantly downregulated in myoblasts treated with adipocyte-derived exosomes. Immature adipocyte-derived exosomes exhibited a stronger suppressive effect than mature adipocyte-derived exosomes. Our results suggest that adipocytes suppress the expression levels of muscle differentiation-associated genes in myoblasts via adipocyte-secreted exosomes containing miRNAs.

MicroRNA­23a­5p mediates the proliferation and differentiation of C2C12 myoblasts.

Skeletal myogenesis is a highly ordered and complex biological process that is mediated by numerous regulatory factors. In previous studies, it has been demonstrated that microRNAs (miRs) and long non­coding RNAs (lncRNAs) serve key roles in skeletal myogenesis. The present study showed that the expression levels of miR­23a­5p showed a dynamic change from decrease to increase during C2C12 myoblast proliferation and differentiation. Functional analysis using 5­ethynyl­2'­deoxyuridine proliferation and Cell Counting Kit­8 detection assays indicated that overexpression of miR­23a­5p significantly promoted C2C12 myoblast proliferation compared with the negative control. In addition, in C2C12 myoblasts transfected with miR­23a­5p mimics, increased expression levels of regulators associated with cell proliferation (Cyclin E, CCND1 and Cyclin B) were observed compared with the negative control. By contrast, overexpression of miR­23a­5p decreased the expression levels of specific­myogenesis factors (MyoD, MyoG and Myf5) and decreased C2C12 myoblast differentiation. Luciferase activity assays indicated that miR­23a­5p suppressed the luciferase activity of lncDum. Further analysis demonstrated that miR­23a­5p not only showed an opposite expression level pattern compared with lncDum, which was first increased and then decreased, but also had an opposite effect on the proliferation and differentiation of C2C12 myoblasts compared with lncDum which inhibited cell proliferation and promoted cell differentiation. Taken together, these results indicated that miR­23a­5p may mediate the proliferation and differentiation of C2C12 myoblasts, which may be involved in lncDum regulation.

Theaflavin Promotes Myogenic Differentiation by Regulating the Cell Cycle and Surface Mechanical Properties of C2C12 Cells.

Aging and muscle diseases often lead to a decline in the differentiation capacity of myoblasts, which in turn results in the deterioration of skeletal muscle (SkM) function and impairment of regeneration ability after injury. Theaflavins, the "gold molecules" found in black tea, have been reported to possess various biological activities and have a positive effect on maintaining human health. In this study, we found that among the four theaflavins (theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B), and theaflavin-3,3'-digallate (TF3) monomers), TF1 (20 µM) significantly promoted the fusion index of myoblasts, number of mature myotubes, and degree of myotube development. By combining transcriptomics, bioinformatics, and molecular biology experiments, we showed that TF1 may promote myoblast differentiation by (1) regulating the withdrawal of myoblasts from the cell cycle, inducing the release of myogenic factors (MyoD, MyoG, and MyHC) and accelerating myogenic differentiation and (2) regulating the adhesion force of myoblasts and mechanical properties of mature myotubes and promoting the migration, fusion, and development of myoblasts. In conclusion, our study outcomes show that TF1 can promote myoblast differentiation and regulate myotube mechanical properties. It is a potential dietary supplement for the elderly. Our findings provide a new scientific basis for the relationship between tea drinking and aging.

3D Bioprinting and Differentiation of Primary Skeletal Muscle Progenitor Cells.

Volumetric loss of skeletal muscle can occur through sports injuries, surgical ablation, trauma, motor or industrial accident, and war-related injury. Likewise, massive and ultimately catastrophic muscle cell loss occurs over time with progressive degenerative muscle diseases, such as the muscular dystrophies. Repair of volumetric loss of skeletal muscle requires replacement of large volumes of tissue to restore function. Repair of larger lesions cannot be achieved by injection of stem cells or muscle progenitor cells into the lesion in absence of a supportive scaffold that (1) provides trophic support for the cells and the recipient tissue environment, (2) appropriate differentiational cues, and (3) structural geometry for defining critical organ/tissue components/niches necessary or a functional outcome. 3D bioprinting technologies offer the possibility of printing orientated 3D structures that support skeletal muscle regeneration with provision for appropriately compartmentalized components ranging across regenerative to functional niches. This chapter includes protocols that provide for the generation of robust skeletal muscle cell precursors and methods for their inclusion into methacrylated gelatin (GelMa) constructs using 3D bioprinting.

Modeling the transport of nuclear proteins along single skeletal muscle cells.

Skeletal muscle cells contain hundreds of myonuclei within a shared cytoplasm, presenting unique challenges for regulating gene expression. Certain transcriptional programs (e.g., postsynaptic machinery) are segregated to specialized domains, while others (e.g., contractile proteins) do not show spatial confinement. Furthermore, local stimuli, such as denervation, can induce transcriptional responses that are propagated along the muscle cells. Regulated transport of nuclear proteins (e.g., transcription factors) between myonuclei represents a potential mechanism for coordinating gene expression. However, the principles underlying the transport of nuclear proteins within multinucleated cells remain poorly defined. Here we used a mosaic transfection model to create myotubes that contained exactly one myonucleus expressing a fluorescent nuclear reporter and monitored its distribution among all myonuclei. We found that the transport properties of these model nuclear proteins in myotubes depended on molecular weight and nuclear import rate, as well as on myotube width. Interestingly, muscle hypertrophy increased the transport of high molecular weight nuclear proteins, while atrophy restricted the transport of smaller nuclear proteins. We have developed a mathematical model of nuclear protein transport within a myotube that recapitulates the results of our in vitro experiments. To test the relevance to nuclear proteins expressed in skeletal muscle, we studied the transport of two transcription factors-aryl hydrocarbon receptor nuclear translocator and sine oculis homeobox 1-and found that their distributions were similar to the reporter proteins with corresponding molecular weights. Together, these results define a set of variables that can be used to predict the spatial distributions of nuclear proteins within a myotube.

Selective clearance of the inner nuclear membrane protein emerin by vesicular transport during ER stress.

The inner nuclear membrane (INM) is a subdomain of the endoplasmic reticulum (ER) that is gated by the nuclear pore complex. It is unknown whether proteins of the INM and ER are degraded through shared or distinct pathways in mammalian cells. We applied dynamic proteomics to profile protein half-lives and report that INM and ER residents turn over at similar rates, indicating that the INM's unique topology is not a barrier to turnover. Using a microscopy approach, we observed that the proteasome can degrade INM proteins in situ. However, we also uncovered evidence for selective, vesicular transport-mediated turnover of a single INM protein, emerin, that is potentiated by ER stress. Emerin is rapidly cleared from the INM by a mechanism that requires emerin's LEM domain to mediate vesicular trafficking to lysosomes. This work demonstrates that the INM can be dynamically remodeled in response to environmental inputs.

4D biofabrication of skeletal muscle microtissues.

Skeletal muscle is one of the most abundant tissues in the body. Although it has a relatively good regeneration capacity, it cannot heal in the case of disease or severe damage. Many current tissue engineering strategies fall short due to the complex structure of skeletal muscle. Biofabrication techniques have emerged as a popular set of methods for increasing the complexity of tissue-like constructs. In this paper, 4D biofabrication technique is introduced for fabrication of the skeletal muscle microtissues. To this end, a bilayer scaffold consisting of a layer of anisotropic methacrylated alginate fibers (AA-MA) and aligned polycaprolactone (PCL) fibers were fabricated using electrospinning and later induced to self-fold to encapsulate myoblasts. Bilayer mats undergo shape-transformation in an aqueous buffer, a process that depends on their overall thickness, the thickness of each layer and the geometry of the mat. Proper selection of these parameters allowed fabrication of scroll-like tubes encapsulating myoblasts. The myoblasts were shown to align along the axis of the anisotropic PCL fibers and further differentiated into aligned myotubes that contracted under electrical stimulation. Overall the significance of this approach is in the fabrication of hollow tubular constructs that can be further developed for the formation of a vascularized and functional muscle.

Calcineurin Broadly Regulates the Initiation of Skeletal Muscle-Specific Gene Expression by Binding Target Promoters and Facilitating the Interaction of the SWI/SNF Chromatin Remodeling Enzyme.

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. During skeletal muscle differentiation, Cn activates the nuclear factor of activated T-cell (NFAT) transcription factor but also promotes differentiation by counteracting the negative influences of protein kinase C beta (PKCß) via dephosphorylation and activation of Brg1, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Here we identified four major temporal patterns of Cn-dependent gene expression in differentiating myoblasts and determined that Cn is broadly required for the activation of the myogenic gene expression program. Mechanistically, Cn promotes gene expression through direct binding to myogenic promoter sequences and facilitating the binding of Brg1, other SWI/SNF subunit proteins, and MyoD, a critical lineage determinant for skeletal muscle differentiation. We conclude that the Cn phosphatase directly impacts the expression of myogenic genes by promoting ATP-dependent chromatin remodeling and formation of transcription-competent promoters.

Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide.

Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M-1 s-1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.

A novel 3D bioprinted flexible and biocompatible hydrogel bioelectronic platform.

Bioelectronics platforms are gaining widespread attention as they provide a template to study the interactions between biological species and electronics. Decoding the effect of the electrical signals on the cells and tissues holds the promise for treating the malignant tissue growth, regenerating organs and engineering new-age medical devices. This work is a step forward in this direction, where bio- and electronic materials co-exist on one platform without any need for post processing. We fabricate a freestanding and flexible hydrogel based platform using 3D bioprinting. The fabrication process is simple, easy and provides a flexible route to print materials with preferred shapes, size and spatial orientation. Through the design of interdigitated electrodes and heating coil, the platform can be tailored to print various circuits for different functionalities. The biocompatibility of the printed platform is tested using C2C12 murine myoblasts cell line. Furthermore, normal human dermal fibroblasts (primary cells) are also seeded on the platform to ascertain the compatibility.

Combining De Novo Peptide Sequencing Algorithms, A Synergistic Approach to Boost Both Identifications and Confidence in Bottom-up Proteomics.

Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms. Results were validated using state-of-the art database search algorithms as well specifically synthesized reference peptides. Thus, we could increase the number of PSMs meeting a stringent FDR of 5% more than 3-fold compared to the single best de novo sequencing algorithm alone, accounting for an average of 11 120 PSMs (combined) instead of 3476 PSMs (alone) in triplicate 2 h LC-MS runs of tryptic HeLa digestion.

Macroporous mesh of nanoporous gold in electrochemical monitoring of superoxide release from skeletal muscle cells.

Real-time monitoring of metabolically relevant biochemicals released in minuscule amounts is of utmost diagnostic importance. Superoxide anion as a primary member of reactive oxygen species, has physiological and pathological effects that depend on its concentration and release rate. Here we present fabrication and successfully testing of a highly sensitive electrochemical biosensor featuring a three-dimensional macroporous mesh of nanoporous gold tailored to measure the dynamics of extracellular superoxide concentration. Wide and accessible surface of the mesh combined with high porosity of the thin nanoporous gold coating enables capturing the analyte in pico- to nano-molar ranges. The mesh is functionalized with cytochrome-c (cyt-c) and incorporated as a working electrode to measure the release rate of drug-induced superoxides from C2C12 cells through a porous membrane. The device displays a considerably improved superoxide sensitivity of 7.29nAnM-1cm-2 and a low level of detection of 70pM. Such sensitivity is orders of magnitude higher than any similar enzyme-based electrochemical superoxide sensor and is attributed to the facile diffusion of the analyte through the well-spread nanofeatured gold skin. Superoxide generation rates captured from monolayer myoblast cultures containing about 4×104 cells, varied from 1.0 to 9.0nMmin-1 in a quasi-linear fashion as a function of drug concentration. This work provides a platform for the development of highly sensitive molecular electrochemical biosensors.

Analysis and quantification of in vitro myoblast fusion using the LADD Multiple Stain.

Myoblast fusion, which is essential for muscle development, regeneration, and repair, can be assessed in vitro via the calculation of a fusion index. Traditionally, this requires use of either immunocytochemistry or fluorescently-labeled cytoskeletal staining, followed by microscopy and laborious analysis. The expense and time-consuming nature of the optimization and application of antibody-based techniques such as immunocytochemistry, as well as the need for specialized analytical equipment such as fluorescence microscopes, presents a barrier to the routine analysis of this crucial step during terminal differentiation. Here, we describe (i) a novel use of the commonly available LADD Multiple Stain for visualization of myoblast fusion in vitro; (ii) the optimization of a simple image analysis method to generate quick, quantifiable data representative of a fusion index; and (iii) the use of a protocol combining these two procedures to investigate in vitro myoblast fusion in a simple and efficient manner as proof-of-concept.

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance.

Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5-25 µM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr-Purcell-Meiboom-Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.

Creating Hierarchical Topographies on Fibrous Platforms Using Femtosecond Laser Ablation for Directing Myoblasts Behavior.

Developing an artificial extracellular matrix that closely mimics the native tissue microenvironment is important for use as both a cell culture platform for controlling cell fate and an in vitro model system for investigating the role of the cellular microenvironment. Electrospinning, one of the methods for fabricating structures that mimic the native ECM, is a promising technique for creating fibrous platforms. It is well-known that align or randomly distributed electrospun fibers provide cellular contact guidance in a single pattern. However, native tissues have hierarchical structures, i.e., topographies on the micro- and nanoscales, rather than a single structure. Thus, we fabricated randomly distributed nanofibrous (720 ± 80 nm in diameter) platforms via a conventional electrospinning process, and then we generated microscale grooves using a femtosecond laser ablation process to develop engineered fibrous platforms with patterned hierarchical topographies. The engineered fibrous platforms can regulate cellular adhesive morphology, proliferation, and distinct distribution of focal adhesion proteins. Furthermore, confluent myoblasts cultured on the engineered fibrous platforms revealed that the direction of myotube assembly can be controlled. These results indicate that our engineered fibrous platforms may be useful tools in investigating the roles of nano- and microscale topographies in the communication between cells and ECM.

Chitooligomer-Immobilized Biointerfaces with Micropatterned Geometries for Unidirectional Alignment of Myoblast Cells.

Skeletal muscle possesses a robust capacity to regenerate functional architectures with a unidirectional orientation. In this study, we successfully arranged skeletal myoblast (C2C12) cells along micropatterned gold strips on which chitohexaose was deposited via a vectorial chain immobilization approach. Hexa-N-acetyl-D-glucosamine (GlcNAc6) was site-selectively modified at its reducing end with thiosemicarbazide, then immobilized on a gold substrate in striped micropatterns via S-Au chemisorption. Gold micropatterns ranged from 100 to 1000 µm in width. Effects of patterning geometries on C2C12 cell alignment, morphology, and gene expression were investigated. Unidirectional alignment of C2C12 cells having GlcNAc6 receptors was clearly observed along the micropatterns. Decreasing striped pattern width increased cell attachment and proliferation, suggesting that the fixed GlcNAc6 and micropatterns impacted cell function. Possibly, interactions between nonreducing end groups of fixed GlcNAc6 and cell surface receptors initiated cellular alignment. Our technique for mimicking native tissue organization should advance applications in tissue engineering.

Lateral Chain Length in Polyalkyl Acrylates Determines the Mobility of Fibronectin at the Cell/Material Interface.

Cells, by interacting with surfaces indirectly through a layer of extracellular matrix proteins, can respond to a variety of physical properties, such as topography or stiffness. Polymer surface mobility is another physical property that is less well understood but has been indicated to hold the potential to modulate cell behavior. Polymer mobility is related to the glass-transition temperature (Tg) of the system, the point at which a polymer transitions from an amorphous solid to a more liquid-like state. This work shows that changes in polymer mobility translate to interfacial mobility of extracellular matrix proteins adsorbed on the material surface. This study has utilized a family of polyalkyl acrylates with similar chemistry but different degrees of mobility, obtained through increasing length of the side chain. These materials are used, in conjunction with fluorescent fibronectin, to determine the mobility of this interfacial layer of protein that constitutes the initial cell-material interface. Furthermore, the extent of fibronectin domain availability (III9, III10, - the integrin binding site), cell-mediated reorganization, and cell differentiation was also determined. A nonmonotonic dependence of fibronectin mobility on polymer surface mobility was observed, with a similar trend noted in cell-mediated reorganization of the protein layer by L929 fibroblasts. The availability of the integrin-binding site was higher on the more mobile surfaces, where a similar organization of the protein into networks at the material interface was observed. Finally, differentiation of C2C12 myoblasts was seen to be highly sensitive to surface mobility upon inhibition of cell contractility. Altogether, these findings show that polymer mobility is a subtle influence that translates to the cell/material interface through the protein layer to alter the biological activity of the surface.

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis(1). Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate(2), the same is not true for extracellular acid production and its relationship to glycolytic rate (3-6). Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate(-) + H(+) and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3(-) + H(+) is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification (6). Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.