Histone Lysine Methylation and Long Non-Coding RNA: The New Target Players in Skeletal Muscle Cell Regeneration

Satellite stem cell availability and high regenerative capacity have made them an ideal therapeutic approach for muscular dystrophies and neuromuscular diseases. Adult satellite stem cells remain in a quiescent state and become activated upon muscular injury. A series of molecular mechanisms succeed under the control of epigenetic regulation and various myogenic regulatory transcription factors myogenic regulatory factors, leading to their differentiation into skeletal muscles. The regulation of MRFs via various epigenetic factors, including DNA methylation, histone modification, and non-coding RNA, determine the fate of myogenesis. Furthermore, the development of histone deacetylation inhibitors (HDACi) has shown promising benefits in their use in clinical trials of muscular diseases. However, the complete application of using satellite stem cells in the clinic is still not achieved. While therapeutic advancements in the use of HDACi in clinical trials have emerged, histone methylation modulations and the long non-coding RNA (lncRNA) are still under study. A comprehensive understanding of these other significant epigenetic modulations is still incomplete. This review aims to discuss some of the current studies on these two significant epigenetic modulations, histone methylation and lncRNA, as potential epigenetic targets in skeletal muscle regeneration. Understanding the mechanisms that initiate myoblast differentiation from its proliferative state to generate new muscle fibres will provide valuable information to advance the field of regenerative medicine and stem cell transplant.

Multiple roles of syndecan-4 during myoblast migration

Background and purpose: Cell migration is one of the cornerstones of regeneration processes, as it is necessary for wound healing, and also required for embryonic development, immune system activation, or tumor metastasis formation. Skeletal muscle has a special, advanced dynamism that allows it to adapt to various impacts and recover successfully after an injury, exercise, or muscle disease. Satellite stem cells are activated by local damage during muscle regeneration, and after asymmetric division, myoblasts (i.e., activated satellite cells) migrate to the site of injury, differentiate, and fuse to form muscle fibers. Migration of the cells requires cellular polarization, the creation of leading and trailing edges, as well as the proper orientation and positioning of organelles inside the cell. Efficient migration also requires the presence of an asymmetrical front-to-rear calcium (Ca2+) gradient to regulate focal adhesion assembly and actomyosin contractility. The transmembrane proteoglycan syndecan-4 (SDC4), which is one of the cell surface markers of resting and activated satellite stem cells, is involved in the formation of focal adhesions. Furthermore, SDC4 plays a variety of roles in signal transduction processes, including controlling the function of the small GTPase Rac1 by binding to and inhibiting the activity of T-lymphoma invasion and metastasis-1 (Tiam1), a guanine nucleotide exchange factor for Rac1 (Ras-related C3 botulinum toxin substrate 1) GTPase. Cell migration also requires Rac1-mediated actin remodeling. SDC4 knockout mice are unable to regenerate damaged muscle; however, its underlying precise mechanism is unclear; therefore, our aim was to analyze the role of SDC4 in myoblast migration. Experimental approaches: To achieve SDC4 knockdown, C2C12 murine myoblast cells were transfected stably with plasmids expressing short hairpin RNAs (shRNAs) specific for mouse SDC4 (shSDC4#1 and shSDC4#2) or a scrambled target sequence. To study cell migration, time-lapse images were captured at 37 °C and 5% CO2 using a high-content imaging system for single-cell tracking or wound scratch assay was performed. To evaluate the movement of the single cells, the cell nuclei were tracked with ImageJ and CellTracker software. Super-resolution direct stochastic optical reconstruction microscopy (dSTORM) measurements were performed for the nanoscale analysis of the lamellipodial actin network of the migrating cells. To study the intracellular Ca2+ level, Fluo-4 and Fura Red indicators were applied. Immunofluorescence cytochemistry was performed to analyze the distribution of SDC4, Tiam1, centrosomes, FAK (focal adhesion kinase) or GM130 (anti- Golgi matrix protein of 130 kDa) followed by wide-field fluorescence or confocal microscopy. Image analysis was performed with ImageJ. Rac1 was inhibited by NSC23766 treatment during the measurements (50 µM). Key results: Silencing of SDC4 disrupts the correct polarization of migrating mammalian myoblasts. SDC4 knockdown completely abolished the intracellular Ca2+ gradient, abrogated centrosome reorientation, and thus decreased cell motility, demonstrating the role of SDC4 in cell polarity. Additionally, SDC4 exhibited a polarized distribution during migration. SDC4 knockdown cells exhibited decreases in the total movement distance during migration, maximum and vectorial distances from the starting point, as well as average and maximum cell speeds. Analysis of the dSTORM images of SDC4 knockdown cells revealed nanoscale changes in the actin cytoskeletal architecture, such as decreases in the numbers of branches and individual branch lengths in the lamellipodia of the migrating cells. The Rac1 inhibitor NSC23766 did not restore the migration capacity of SDC4 silenced cells; in fact, it reduced it further. SDC4 knockdown decreased the directional persistence of migration, abrogated the polarized, asymmetric distribution of Tiam1, and reduced the total Tiam1 level of the cells. Conclusion: According to our results, SDC4 affects the migration of C2C12 myoblasts and modulates cell polarity by influencing centrosome positioning, intracellular Ca2+ and Tiam1 distribution. These findings may promote greater understanding the essential role of SDC4 in the embryonic development and postnatal regeneration of skeletal muscle. Given the ubiquitous expression and crucial role of SDC4 in cell migration, we conclude that our findings can facilitate understanding the general role of SDC4 during cell migration.

Agent-based model provides insight into the mechanisms behind failed regeneration following volumetric muscle loss injury

Skeletal muscle possesses a remarkable capacity for repair and regeneration following a variety of injuries. When successful, this highly orchestrated regenerative process requires the contribution of several muscle resident cell populations including satellite stem cells (SSCs), fibroblasts, macrophages and vascular cells. However, volumetric muscle loss injuries (VML) involve simultaneous destruction of multiple tissue components (e.g., as a result of battlefield injuries or vehicular accidents) and are so extensive that they exceed the intrinsic capability for scarless wound healing and result in permanent cosmetic and functional deficits. In this scenario, the regenerative process fails and is dominated by an unproductive inflammatory response and accompanying fibrosis. The failure of current regenerative therapeutics to completely restore functional muscle tissue is not surprising considering the incomplete understanding of the cellular mechanisms that drive the regeneration response in the setting of VML injury. To begin to address this profound knowledge gap, we developed an agent-based model to predict the tissue remodeling response following surgical creation of a VML injury. Once the model was able to recapitulate key aspects of the tissue remodeling response in the absence of repair, we validated the model by simulating the tissue remodeling response to VML injury following implantation of either a decellularized extracellular matrix scaffold or a minced muscle graft. The model suggested that the SSC microenvironment and absence of pro-differentiation SSC signals were the most important aspects of failed muscle regeneration in VML injuries. The major implication of this work is that agent-based models may provide a much-needed predictive tool to optimize the design of new therapies, and thereby, accelerate the clinical translation of regenerative therapeutics for VML injuries.

Building the case for the calcitonin receptor as a viable target for the treatment of glioblastoma

Researchers are actively seeking novel targeted therapies for the brain tumour glioblastoma (GBM) as the mean survival is less than 15 months. Here we discuss the proposal that the calcitonin receptor (CT Receptor), expressed in 76–86% of patient biopsies, is expressed by both malignant glioma cells and putative glioma stem cells (GSCs), and therefore represents a potential therapeutic target. Forty-two per cent (42%) of high-grade glioma (HGG; representative of GSCs) cell lines express CT Receptor protein. CT Receptors are widely expressed throughout the life cycle of organisms and in some instances promote apoptosis. Which of the common isoforms of the CT Receptor are predominantly expressed is currently unknown, but a functional response to cell stress of the insert-positive isoform is hypothesised. A model for resistant malignancies is one in which chemotherapy plays a direct role in activating quiescent stem cells for replacement of the tumour tissue hierarchy. The putative role that the CT Receptor plays in maintenance of quiescent cancer stem cells is discussed in view of the activation of the Notch–CT Receptor–collagen V axis in quiescent muscle (satellite) stem cells. The pharmacological CT response profiles of four of the HGG cell lines were reported. Both CT responders and non-responders were sensitive to an immunotoxin based on an anti-CT Receptor antibody. The CALCR mRNA exhibits alternative splicing commonly associated with cancer cells, which could result in the atypical pharmacology exhibited by CT non-responders and an explanation of tumour suppression. Due to the inherent instability of CALCR mRNA, analysis of CT Receptor protein in patient samples will lead to improved data for the expression of CT Receptor in GBM and other cancers, and an understanding of the role and activity of the splice variants. This knowledge will aid the effective targeting of this receptor for treatment of GBM.

Computational Modeling of Muscle Regeneration and Adaptation to Advance Muscle Tissue Regeneration Strategies

Skeletal muscle has an exceptional ability to regenerate and adapt following injury. Tissue engineering approaches (e.g. cell therapy, scaffolds, and pharmaceutics) aimed at enhancing or promoting muscle regeneration from severe injuries are a promising and active field of research. Computational models are beginning to advance the field by providing insight into regeneration mechanisms and therapies. In this paper, we summarize the contributions computational models have made to understanding muscle remodeling and the functional implications thereof. Next, we describe a new agent-based computational model of skeletal muscle inflammation and regeneration following acute muscle injury. Our computational model simulates the recruitment and cellular behaviors of key inflammatory cells (e.g. neutrophils and M1 and M2 macrophages) and their interactions with native muscle cells (muscle fibers, satellite stem cells, and fibroblasts) that result in the clearance of necrotic tissue and muscle fiber regeneration. We demonstrate the ability of the model to track key regeneration metrics during both unencumbered regeneration and in the case of impaired macrophage function. We also use the model to simulate regeneration enhancement when muscle is primed with inflammatory cells prior to injury, which is a putative therapeutic intervention that has not yet been investigated experimentally. Computational modeling of muscle regeneration, pursued in combination with experimental analyses, provides a quantitative framework for evaluating and predicting muscle regeneration and enables the rational design of therapeutic strategies for muscle recovery.

Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength

Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle.