Temporal Adaptive Changes in Contractility and Fatigability of Diaphragm Muscles from Streptozotocin-Diabetic Rats

Diabetes is characterized by ventilatory depression due to decreased diaphragm (DPH) function. This study investigated the changes in contractile properties of rat DPH muscles over a time interval encompassing from 4 days to 14 weeks after the onset of streptozotocin-induced diabetes, with and without insulin treatment for 2 weeks. Maximum tetanic force in intact DPH muscle strips and recovery from fatiguing stimulation were measured. An early (4-day) depression in contractile function in diabetic DPH was followed by gradual improvement in muscle function and fatigue recovery (8 weeks). DPH contractile function deteriorated again at 14 weeks, a process that was completely reversed by insulin treatment. Maximal contractile force and calcium sensitivity assessed in Triton-skinned DPH fibers showed a similar bimodal pattern and the same beneficial effect of insulin treatment. While an extensive analysis of the isoforms of the contractile and regulatory proteins was not conducted, Western blot analysis of tropomyosin suggests that the changes in diabetic DPH response depended, at least in part, on a switch in fiber type

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7

Multi-Staged Regulation of Lipid Signaling Mediators during Myogenesis by COX-1/2 Pathways

Cyclooxygenases (COXs), including COX-1 and -2, are enzymes essential for lipid mediator (LMs) syntheses from arachidonic acid (AA), such as prostaglandins (PGs). Furthermore, COXs could interplay with other enzymes such as lipoxygenases (LOXs) and cytochrome P450s (CYPs) to regulate the signaling of LMs. In this study, to comprehensively analyze the function of COX-1 and -2 in regulating the signaling of bioactive LMs in skeletal muscle, mouse primary myoblasts and C2C12 cells were transfected with specific COX-1 and -2 siRNAs, followed by targeted lipidomic analysis and customized quantitative PCR gene array analysis. Knocking down COXs, particularly COX-1, significantly reduced the release of PGs from muscle cells, especially PGE2 and PGF2α, as well as oleoylethanolamide (OEA) and arachidonoylethanolamine (AEA). Moreover, COXs could interplay with LOXs to regulate the signaling of hydroxyeicosatetraenoic acids (HETEs). The changes in LMs are associated with the expression of genes, such as Itrp1 (calcium signaling) and Myh7 (myogenic differentiation), in skeletal muscle. In conclusion, both COX-1 and -2 contribute to LMs production during myogenesis in vitro, and COXs could interact with LOXs during this process. These interactions and the fine-tuning of the levels of these LMs are most likely important for skeletal muscle myogenesis, and potentially, muscle repair and regeneration

Sarcopenia: pharmacology of today and tomorrow

ABSTRACT Sarcopenia remains largely undiagnosed and undertreated because of the lack of a universally accepted definition, effective ways to measure it, and identification of the outcomes that should guide treatment efficacy. An ever-growing number of clinicians and researchers along with funding and regulatory agencies have gradually recognized that sarcopenia is a human condition that requires both prevention and treatment. In this article, we review sarcopenia and its common and less known pharmacological treatments, attempt to define sarcopenia in its broader context, and present some new ideas for potential future treatment for this devastating condition

Detrimental effects of malaria, toxoplasmosis, leishmaniosis and Chagas disease on cardiac and skeletal muscles

The pathogenic mechanisms of several diseases triggered by protozoan parasites, such as the causative agents of Chagas disease, toxoplasmosis, leishmaniosis, and malaria, have demonstrated to cause direct detrimental effect on cardiac and skeletal muscle. These are amongst the most prevalent and epidemiologically relevant protozoan infections worldwide and infecting millions of people per year. As such, this review focuses on the current knowledge on the pathogenic mechanisms of these diseases on muscles. Case studies and original research addressing the mechanisms of action for direct and indirect damage to cardiac and skeletal muscle were analyzed and the main findings summarized. Importantly, all diseases reviewed here produce an intense inflammatory response, with the associated oxidative stress and pro-inflammatory cytokine production leading to or furthering these detrimental effects. Critically, the disruption of cardiac muscle function can lead to minor arrhythmias and even death, and skeletal muscle damage can result in homeostatic imbalances serving to further morbidity and mortality. Strategies for preventing complications and determining the effectiveness of interventions designed with antioxidant and anti-inflammatory molecules to minimize muscle injury and help the millions of people with these diseases are an urgent need

Visual gene-network analysis reveals the cancer gene co-expression in human endometrial cancer

Abstract Background Endometrial cancers (ECs) are the most common form of gynecologic malignancy. Recent studies have reported that ECs reveal distinct markers for molecular pathogenesis, which in turn is linked to the various histological types of ECs. To understand further the molecular events contributing to ECs and endometrial tumorigenesis in general, a more precise identification of cancer-associated molecules and signaling networks would be useful for the detection and monitoring of malignancy, improving clinical cancer therapy, and personalization of treatments. Results ECs-specific gene co-expression networks were constructed by differential expression analysis and weighted gene co-expression network analysis (WGCNA). Important pathways and putative cancer hub genes contribution to tumorigenesis of ECs were identified. An elastic-net regularized classification model was built using the cancer hub gene signatures to predict the phenotypic characteristics of ECs. The 19 cancer hub gene signatures had high predictive power to distinguish among three key principal features of ECs: grade, type, and stage. Intriguingly, these hub gene networks seem to contribute to ECs progression and malignancy via cell-cycle regulation, antigen processing and the citric acid (TCA) cycle. Conclusions The results of this study provide a powerful biomarker discovery platform to better understand the progression of ECs and to uncover potential therapeutic targets in the treatment of ECs. This information might lead to improved monitoring of ECs and resulting improvement of treatment of ECs, the 4th most common of cancer in women.Peer Reviewe

A Proposed Nanocomposite Hydrogel for Promoting Reinnervation Following Volumetric Muscle Loss

Skeletal muscle possesses an innate capacity for regeneration following damage, however, severe injuries resulting in significant tissue loss, categorized as volumetric muscle loss (VML) can overwhelm endogenous repair mechanisms. Recent evidence suggests that nanocomposite silica-based hydrogels (NC gels) possess rheological properties necessary for precise 3D bioprinting and promote secretion of endogenous neurotrophic factors for tissue repair. A shear behavior index between 0.2-0.6 is indicative of shear thinning behavior and describes the ability of a bioink to flow under a certain amount of shear stress. Bioinks displaying shear thinning behavior have pseudoplasticity, an essential property of muscle viscoelasticity, and are optimal for printing constructs with highly precise micro-architecture. PURPOSE: To determine the effect of ionic silicon (Si4+) on neurotrophic factor release in C2C12 murine myoblasts and determine the effects of modified silica nanoparticles (SiOx-np) on the rheological properties of an established Gelatin Methacrylate-Sodium Alginate (GelMA-Alg) hydrogel for applications in skeletal muscle tissue engineering. METHODS: To determine the effects of Si4+ on neurotrophic signaling, C2C12 murine myoblasts were differentiated for 3 days in media containing 0.1mM, 0.5mM, and 1.0mM Si4+ and a silicon free control. An Enzyme-limited Immunosorbent Assay (ELISA) was performed to determine the level of the neurotrophic factor NRTN, a myokine released from skeletal muscle that enhances motor neuron recruitment and neuromuscular junction remodeling. The data was normalized to standard curves and origin was used to analyze the results. To determine the effects of SiOx-np on the rheological properties of GelMA-Alg, a 10% GelMA-2% Alg bioink was fabricated along with a 10% GelMA-2%Alg-2% SiOx-np (0.6mM Si4+) bioink. Flow curve tests were performed using an MCR702 multidrive rheometer from Anton Paar at 26°C and shear rate of 0.001-500s-1. The results were analyzed based on a power law regression model using RheoCompass to determine the shear behavior index and the effects of SiOx-np on printability of the bioink. RESULTS: Following 3 days of differentiation it was determined that there was a significant increase in neurotrophic factor release in the 0.1mM group (1.20±0.04ng/mL, p\u3c0.05), 0.5mM group (1.42±0.07ng/mL, p\u3c0.001), and 1.0mM group (1.28±0.05ng/mL, p\u3c0.01) compared to the silicon free control (1.14±0.06ng/mL). The shear behavior index was found to be similar between GelMA-Alg (0.3887) and GelMA-Alg-SiOx-np (0.37434) suggesting that the bioink displayed shear thinning behavior for precise 3D printing. CONCLUSION: The results of this study indicate that neurotrophic factor release in C2C12 myoblasts has an apical dose response to Si4+ in vitro. The shear thinning behavior of GelMA-Alg-SiOx-np is indicative of the pseudoplasticity of the material, suggesting constructs can be bioprinted with precise micro-architecture similar to skeletal muscle. These results suggest that GelMA-Alg-SiOx-np constructs may provide a way to promote reinnervation following VML

Reliable, Semi-Automated Wound Healing Rate Determination in Muscle

In the field of Regenerative and Sport Medicine, there is great interest in the development and validation of compounds and devices with the potential to accelerate wound healing and muscle regeneration. In vitro, this effect can be evaluated in a scratch test model, in which a pipette removes a line of cells from a confluent monolayer of cells with high regenerative capacity and the time to close this injury measured. PURPOSE: To develop a reliable, dynamic, and quantitative process with a shorter duty cycle and semi-automatic operation for the determination of wound healing rate, as compared to fully manual operation. METHODS: C2C12 murine myoblasts were cultured to confluence under standard conditions. A 200 µL pipette tip was used to make a scratch across each well, and 0 and 0.5mM of pro-myogenic Si-ions were added to the media. A Keyence BZX-710 microscope was used to capture images every 183 seconds over 36 hours at 10x magnification with 0.7 pixels/μm and 4 µm pitch. An enclosed cell culture stage contained a cell incubator system keeping cells at 37°C with a 5% CO2 humidified air. For the manual operation, one image was randomly selected from the automated images every 12hr. ImageJ Macro WH_NJ was used to quantify the percent area of the field (scratched) of interest without cells and was normalized as needed per experimental conditions. RESULTS: The manual and automatic slopes for the 0 and 0.5mM Si-ion treatments were -4.87E-06, -4.84E-06, -6.01E-06, and -5.98E-06, respectively, for the full 0-36hr. There was a high degree of correlation between the manual and semi-automatic rates for both the 0 and 0.5mM Si-ions, at r=.84 and .98, respectively. There were no statistically significant differences between healing rates (i.e., closure times) for the automatic or manual 0 or 0.5mM Si-ions, or within either method, following a two-tailed student’s t-test with alpha level of p\u3c.05. Within 12hr periods, the semi-automatic method provided greater detail for the healing rate, such as the faster initial rate seen in 0.5mM Si-ion, not discernible in 12-hour increments for the manual method. CONCLUSION: These data support the functionality of our new methodology described here. The descriptive and inferential statistics shown here demonstrate agreement between the two analyses, while the semi-automated method presented additional dynamics and kinetics information beyond the manual method in early-test behavior that could not be measured manually. Further development in this area will focus on continuing to shorten duty cycles for higher fidelity and the quantitative analysis of dynamic behaviors. Potential clinical-translational applications of our new method are to screen libraries of compounds with putative muscle regeneration capacity using human muscle cells. We also plan to test basal differences in muscle cells from biopsies of sedentary and active individuals, as well as healthy individuals vs. those with various metabolic and musculoskeletal and cardiovascular disorders, and aging sarcopenia. Our new methodology coupled with these translational studies will help advance new compounds and devices with early promise for the field of Regenerative and Sport Medicine into the pre-clinical animal phases of validation