Hematopoietic-Derived Galectin-3 Causes Cellular and Systemic Insulin Resistance.

In obesity, macrophages and other immune cells accumulate in insulin target tissues, promoting a chronic inflammatory state and insulin resistance. Galectin-3 (Gal3), a lectin mainly secreted by macrophages, is elevated in both obese subjects and mice. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. In vitro treatment with Gal3 directly enhanced macrophage chemotaxis, reduced insulin-stimulated glucose uptake in myocytes and 3T3-L1 adipocytes and impaired insulin-mediated suppression of glucose output in primary mouse hepatocytes. Importantly, we found that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These observations elucidate a novel role for Gal3 in hepatocyte, adipocyte, and myocyte insulin resistance, suggesting that Gal3 can link inflammation to decreased insulin sensitivity. Inhibition of Gal3 could be a new approach to treat insulin resistance.

Protection of dystrophic muscle cells using Idebenone correlates with the interplay between calcium, oxidative stress and inflammation.

There is strong cross-talk between abnormal intracellular calcium concentration, high levels of reactive oxygen species (ROS) and an exacerbated inflammatory process in the dystrophic muscles of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). In this study, we investigated effects of Idebenone, a potent anti-oxidant, on oxidative stress markers, the anti-oxidant defence system, intracellular calcium concentrations and the inflammatory process in primary dystrophic muscle cells from mdx mice. Dystrophic muscle cells were treated with Idebenone (0.05 µM) for 24 h. The untreated mdx muscle cells were used as controls. The MTT assay showed that Idebenone did not have a cytotoxic effect on the dystrophic muscle cells. The Idebenone treatment was able to reduce the levels of oxidative stress markers, such as H2 O2 and 4-HNE, as well as decreasing intracellular calcium influx in the dystrophic muscle cells. Regarding Idebenone effects on the anti-oxidant defence system, an up-regulation of catalase levels, glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity was observed in the dystrophic muscle cells. In addition, the Idebenone treatment was also associated with reduction in inflammatory molecules, such as nuclear factor kappa-B (NF-κB) and tumour necrosis factor (TNF) in mdx muscle cells. These outcomes supported the use of Idebenone as a protective agent against oxidative stress and related signalling mechanisms involved in dystrophinopathies, such as DMD.

KLF15 overexpression in myocytes fails to ameliorate ALS-related pathology or extend the lifespan of SOD1G93A mice.

Amyotrophic Lateral Sclerosis (ALS) is a currently incurable disease that causes progressive motor neuron loss, paralysis and death. Skeletal muscle pathology occurs early during the course of ALS. It is characterized by impaired mitochondrial biogenesis, metabolic dysfunction and deterioration of the neuromuscular junction (NMJ), the synapse through which motor neurons communicate with muscles. Therefore, a better understanding of the molecules that underlie this pathology may lead to therapies that slow motor neuron loss and delay ALS progression. Kruppel Like Factor 15 (KLF15) has been identified as a transcription factor that activates alternative metabolic pathways and NMJ maintenance factors, including Fibroblast Growth Factor Binding Protein 1 (FGFBP1), in skeletal myocytes. In this capacity, KLF15 has been shown to play a protective role in Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), however its role in ALS has not been evaluated. Here, we examined whether muscle-specific KLF15 overexpression promotes the health of skeletal muscles and NMJs in the SOD1G93A ALS mouse model. We show that muscle-specific KLF15 overexpression did not elicit a significant beneficial effect on skeletal muscle atrophy, NMJ health, motor function, or survival in SOD1G93A ALS mice. Our findings suggest that, unlike in mouse models of DMD and SMA, KLF15 overexpression has a minimal impact on ALS disease progression in SOD1G93A mice.

DUX4 expressing immortalized FSHD lymphoblastoid cells express genes elevated in FSHD muscle biopsies, correlating with the early stages of inflammation.

Facioscapulohumeral muscular dystrophy (FSHD) is an incurable disorder linked to ectopic expression of DUX4. However, DUX4 is notoriously difficult to detect in FSHD muscle cells, while DUX4 target gene expression is an inconsistent biomarker for FSHD skeletal muscle biopsies, displaying efficacy only on pathologically inflamed samples. Immune gene misregulation occurs in FSHD muscle, with DUX4 target genes enriched for those associated with inflammatory processes. However, there lacks an assessment of the FSHD immune cell transcriptome, and its contribution to gene expression in FSHD muscle biopsies. Here, we show that EBV-immortalized FSHD lymphoblastoid cell lines express DUX4 and both early and late DUX4 target genes. Moreover, a biomarker of 237 up-regulated genes derived from FSHD lymphoblastoid cell lines is elevated in FSHD muscle biopsies compared to controls. The FSHD Lymphoblast score is unaltered between FSHD myoblasts/myotubes and their controls however, implying a non-myogenic cell source in muscle biopsies. Indeed, the FSHD Lymphoblast score correlates with the early stages of muscle inflammation identified by histological analysis on muscle biopsies, while our two late DUX4 target gene expression biomarkers associate with macroscopic inflammation detectable via MRI. Thus, FSHD lymphoblastoid cell lines express DUX4 and early and late DUX4 target genes, therefore, muscle-infiltrated immune cells may contribute the molecular landscape of FSHD muscle biopsies.

Lysine methyltransferase 2D regulates muscle fiber size and muscle cell differentiation.

Kabuki syndrome (KS) is a rare genetic disorder caused primarily by mutations in the histone modifier genes KMT2D and KDM6A. The genes have broad temporal and spatial expression in many organs, resulting in complex phenotypes observed in KS patients. Hypotonia is one of the clinical presentations associated with KS, yet detailed examination of skeletal muscle samples from KS patients has not been reported. We studied the consequences of loss of KMT2D function in both mouse and human muscles. In mice, heterozygous loss of Kmt2d resulted in reduced neuromuscular junction (NMJ) perimeter, decreased muscle cell differentiation in vitro and impaired myofiber regeneration in vivo. Muscle samples from KS patients of different ages showed presence of increased fibrotic tissue interspersed between myofiber fascicles, which was not seen in mouse muscles. Importantly, when Kmt2d-deficient muscle stem cells were transplanted in vivo in a physiologic non-Kabuki environment, their differentiation potential is restored to levels undistinguishable from control cells. Thus, the epigenetic changes due to loss of function of KMT2D appear reversible through a change in milieu, opening a potential therapeutic avenue.

Muscle-tendon cross talk during muscle wasting.

In organisms from flies to mammals, the initial formation of a functional tendon is completely dependent on chemical signals from muscles (myokines). However, how myokines affect the maturation, maintenance, and regeneration of tendons as a function of age is completely unstudied. Here we discuss the role of four myokines-fibroblast growth factors (FGF), myostatin, the secreted protein acidic and rich in cysteine (SPARC) miR-29-in tendon development and hypothesize a role for these factors in the progressive changes in tendon structure and function as a result of muscle wasting (disuse, aging, and disease). Because of the close relationship between mechanical loading and muscle and tendon regulation, disentangling muscle-tendon cross talk from simple mechanical loading is experimentally quite difficult. Therefore, we propose an experimental framework that hopefully will be useful in demonstrating muscle-tendon cross talk in vivo. Though understudied, the promise of a better understanding of muscle-tendon cross talk is the development of new interventions that will improve tendon development, regeneration, and function throughout the lifespan.

Autophagy displays divergent roles during intermittent amino acid starvation and toxic stress-induced senescence in cultured skeletal muscle cells.

Due to the ever-expanding functions attributed to autophagy, there is widespread interest in understanding its contribution to human physiology; however, its specific cellular role as a stress-response mechanism is still poorly defined. To investigate autophagy's role in this regard, we repeatedly subjected cultured mouse myoblasts to two stresses with diverse impacts on autophagic flux: amino acid and serum withdrawal (Hank's balanced salt solution [HBSS]), which robustly induces autophagy, or low-level toxic stress (staurosporine, STS). We found that intermittent STS (int-STS) administration caused cell cycle arrest, development of enlarged and misshapen cells/nuclei, increased senescence-associated heterochromatic foci and senescence-associated ß-galactosidase activity, and prevented myogenic differentiation. These features were not observed in cells intermittently incubated in HBSS (int-HB). While int-STS cells displayed less DNA damage (phosphorylated H2A histone family, member X content) and caspase activity when administered cisplatin, int-HB cells were protected from STS-induced cell death. Interestingly, STS-induced senescence was attenuated in autophagy related 7-deficient cells. Therefore, while repeated nutrient withdrawal did not cause senescence, autophagy was required for senescence caused by toxic stress. These results illustrate the context-dependent effects of different stressors, potentially highlighting autophagy as a distinguishing factor.

Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease.

The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1-/-, alias Thbs1-/-) mice and from mice expressing α-smooth muscle actin-green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α1-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca2+] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca2+]I with VIP and UTP was significantly smaller in MECs from TSP1-/- compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1-/- mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1-/- compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1-/- mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1-/- compared with WT mice.

Pathological features of COVID-19-associated myocardial injury: a multicentre cardiovascular pathology study.

AIMS: Coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been associated with cardiovascular features of myocardial involvement including elevated serum troponin levels and acute heart failure with reduced ejection fraction. The cardiac pathological changes in these patients with COVID-19 have yet to be well described. METHODS AND RESULTS: In an international multicentre study, cardiac tissue from the autopsies of 21 consecutive COVID-19 patients was assessed by cardiovascular pathologists. The presence of myocarditis, as defined by the presence of multiple foci of inflammation with associated myocyte injury, was determined, and the inflammatory cell composition analysed by immunohistochemistry. Other forms of acute myocyte injury and inflammation were also described, as well as coronary artery, endocardium, and pericardium involvement. Lymphocytic myocarditis was present in 3 (14%) of the cases. In two of these cases, the T lymphocytes were CD4 predominant and in one case the T lymphocytes were CD8 predominant. Increased interstitial macrophage infiltration was present in 18 (86%) of the cases. A mild pericarditis was present in four cases. Acute myocyte injury in the right ventricle, most probably due to strain/overload, was present in four cases. There was a non-significant trend toward higher serum troponin levels in the patients with myocarditis compared with those without myocarditis. Disrupted coronary artery plaques, coronary artery aneurysms, and large pulmonary emboli were not identified. CONCLUSIONS: In SARS-CoV-2 there are increased interstitial macrophages in a majority of the cases and multifocal lymphocytic myocarditis in a small fraction of the cases. Other forms of myocardial injury are also present in these patients. The macrophage infiltration may reflect underlying diseases rather than COVID-19.

Histopathological changes of myocytes in restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare primary myocardial disease, and its pathological features are yet to be determined. Restrictive cardiomyopathy with MHY7 mutation was diagnosed in a 65-year-old Japanese woman. Electron microscopy of a myocardial biopsy revealed electron-dense materials resulting from focal myocyte degeneration and necrosis as well as tubular structures and pseudo-inclusion bodies in some nuclei. These features may be associated with the pathogenesis of RCM.

Identification and characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase.

Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.

Dual-specificity phosphatase 29 is induced during neurogenic skeletal muscle atrophy and attenuates glucocorticoid receptor activity in muscle cell culture.

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Newly named dual-specificity phosphatase 29 (Dusp29) has been identified as a novel neurogenic atrophy-induced gene in skeletal muscle. Quantitative PCR analysis revealed that Dusp29 expression is significantly higher in differentiated myotubes compared with proliferating myoblasts. To determine how Dusp29 is transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dusp29 were cloned, fused to a reporter gene, and found to be highly inducible in response to ectopic expression of the myogenic regulatory factors (MRF), MyoD and myogenin. Furthermore, site-directed mutagenesis of conserved E-box elements within the proximal promoter of Dusp29 rendered a Dusp29 reporter gene unresponsive to MRF overexpression. Dusp29, an atypical Dusp also known as Dupd1/Dusp27, was found to attenuate the ERK1/2 branch of the MAP kinase signaling pathway in muscle cells and inhibit muscle cell differentiation when ectopically expressed in proliferating myoblasts. Interestingly, Dusp29 was also found to destabilize AMPK protein while simultaneously enriching the phosphorylated pool of AMPK in muscle cells. Additionally, Dusp29 overexpression resulted in a significant increase in the glucocorticoid receptor (GR) protein and elevation in GR phosphorylation. Finally, Dusp29 was found to significantly impair the ability of the glucocorticoid receptor to function as a transcriptional activator in muscle cells treated with dexamethasone. Identifying and characterizing the function of Dusp29 in muscle provides novel insights into the molecular and cellular mechanisms for skeletal muscle atrophy.

Nucleotide excision repair capacity increases during differentiation of human embryonic carcinoma cells into neurons and muscle cells.

Embryonic stem cells can self-renew and differentiate, holding great promise for regenerative medicine. They also employ multiple mechanisms to preserve the integrity of their genomes. Nucleotide excision repair, a versatile repair mechanism, removes bulky DNA adducts from the genome. However, the dynamics of the capacity of nucleotide excision repair during stem cell differentiation remain unclear. Here, using immunoslot blot assay, we measured repair rates of UV-induced DNA damage during differentiation of human embryonic carcinoma (NTERA-2) cells into neurons and muscle cells. Our results revealed that the capacity of nucleotide excision repair increases as cell differentiation progresses. We also found that inhibition of the apoptotic signaling pathway has no effect on nucleotide excision repair capacity. Furthermore, RNA-Seq-based transcriptomic analysis indicated that expression levels of four core repair factors, xeroderma pigmentosum (XP) complementation group A (XPA), XPC, XPG, and XPF-ERCC1, are progressively up-regulated during differentiation, but not those of replication protein A (RPA) and transcription factor IIH (TFIIH). Together, our findings reveal that increase of nucleotide excision repair capacity accompanies cell differentiation, supported by the up-regulated transcription of genes encoding DNA repair enzymes during differentiation of two distinct cell lineages.

Peripheral EphrinB1/EphB1 signalling attenuates muscle hyperalgesia in MPS patients and a rat model of taut band-associated persistent muscle pain.

BACKGROUND: Myofascial pain syndrome (MPS) is an important clinical condition that is characterized by chronic muscle pain and a myofascial trigger point (MTrP) located in a taut band (TB). Previous studies showed that EphrinB1 was involved in the regulation of pathological pain via EphB1 signalling, but whether EphrinB1-EphB1 plays a role in MTrP is not clear. METHODS: The present study analysed the levels of p-EphB1/p-EphB2/p-EphB3 in biopsies of MTrPs in the trapezius muscle of 11 MPS patients and seven healthy controls using a protein microarray kit. EphrinB1-Fc was injected intramuscularly to detect EphrinB1s/EphB1s signalling in peripheral sensitization. We applied a blunt strike to the left gastrocnemius muscles (GM) and eccentric exercise for 8 weeks with 4 weeks of recovery to analyse the function of EphrinB1/EphB1 in the muscle pain model. RESULTS: P-EphB1, p-EphB2, and p-EphB3 expression was highly increased in human muscles with MTrPs compared to healthy muscle. EphB1 (r = 0.723, n = 11, P < 0.05), EphB2 (r = 0.610, n = 11, P < 0.05), and EphB3 levels (r = 0.670, n = 11, P < 0.05) in the MPS group were significantly correlated with the numerical rating scale (NRS) in the MTrPs. Intramuscular injection of EphrinB1-Fc produces hyperalgesia, which can be partially prevented by pre-treatment with EphB1-Fc. The p-EphB1 contents in MTrPs of MPS animals were significantly higher than that among control animals (P < 0.01). Intramuscular administration of the EphB1 inhibitor EphB1-Fr significantly suppressed mechanical hyperalgesia. CONCLUSIONS: The present study showed that the increased expression of p-EphB1/p-EphB2/p-EphB3 was related to MTrPs in patients with MPS. This report is the first study to examine the function of EphrinB1-EphB1 signalling in primary muscle afferent neurons in MPS patients and a rat animal model. This pathway may be one of the most important and promising targets for MPS.

A patient-derived iPSC model revealed oxidative stress increases facioscapulohumeral muscular dystrophy-causative DUX4.

Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient-derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2-derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS-induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS-induced DUX4 increase and identified ataxia-telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS-induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.

Divergent effects of myogenic differentiation and diabetes on the capacity for muscle precursor cell adipogenic differentiation in a fibrin matrix.

The development of ectopic adipose tissue in skeletal muscle is associated with several skeletal muscle and metabolic pathologies, including Type II Diabetes Mellitus. The adipogenic differentiation of muscle precursor cells (MPCs) has been postulated to occur in skeletal muscle in vivo in a three-dimensional (3-D) configuration; therefore, it is appropriate to investigate this phenomenon using 3-D matrices in vitro. The capacity for MPC adipogenic differentiation in a 3-D environment was investigated in fibrin hydrogels by treating MPCs derived from healthy or diabetic animals with adipogenic induction medias that differed in their ability to increase lipid accumulation and activate the expression of genes associated with adipogenic differentiation (peroxisome proliferator-activated receptor gamma (PPARG), adiponectin (ADIPOQ), and fatty acid synthase (FAS)). The capacity for adipogenic differentiation was diminished, but not prevented, if myogenic differentiation preceded MPC exposure to adipogenic induction conditions. Conversely, adipogenic differentiation was greater in hydrogels containing MPCs from diabetic rats as compared to those derived from lean rats, as evidenced by an increase in lipid accumulation and adipogenic gene expression. Collectively, the data herein support a role for the MPCs in adipogenesis in a 3-D environment and that they may contribute to the ectopic accumulation of adipose tissue. The observation that the potential for adipogenic differentiation is maintained even after a period of myogenic differentiation alludes to the possibility that adipogenesis may occur during different phases of muscle development. Finally, the increase in adipogenic differentiation in hydrogels containing MPCs derived from diabetic animals provides strong evidence that a pathological environment in vivo increases their capacity for adipogenesis.

Functional significance of post-myocardial infarction inflammation evaluated by 18F-fluorodeoxyglucose imaging in swine model.

BACKGROUND: The aim of the study was to investigate the relationship between post-myocardial infarction (MI) inflammation and left ventricular (LV) remodeling in a swine model by 18F-fluorodeoxyglucose (FDG) imaging. METHODS: MI was induced in swine by balloon occlusion of the left anterior descending coronary artery. A series of FDG positron emission tomography (PET) images were taken within 2 weeks post-MI, employing a comprehensive strategy to suppress the physiological uptake of cardiomyocytes. Echocardiography was applied to evaluate LV volume, global and regional function. CD68+ macrophage and glucose transporters (GLUT-1, -3 and -4) were investigated by immunostaining. RESULTS: The physiological uptake of myocardium was adequately suppressed in 92.3% of PET scans verified by visual analysis, which was further confirmed by the minimal expression of myocardial GLUT-4. Higher FDG uptake was observed in the infarct than in the remote area and persisted within the observational period of 2 weeks. The FDG uptake of infarcted myocardium on day 1 post-MI was correlated with LV global remodeling, and the FDG uptake of infarcted myocardium on days 1 and 8 post-MI had a trend of correlating with regional remodeling of the infarct area. CONCLUSIONS: We here report a feasible swine model for investigating post-MI inflammation. FDG signal in the infarct area of swine persisted for a longer duration than has been reported in small animals. FDG activity in the infarct area could predict LV remodeling.

Photobiomodulation reduces cell death and cytokine production in C2C12 cells exposed to Bothrops venoms.

Snakebites caused by the genus Bothrops are often associated with severe and complex local manifestations such as edema, pain, hemorrhage, and myonecrosis. Conventional treatment minimizes the systemic effects of venom; however, their local action is not neutralized. The purpose of this study was to evaluate the effect of photobiomodulation (PBM) on C2C12 muscle cells exposed to B. jararaca, B. jararacussu, and B. moojeni venoms on events involved in cell death and the release of inflammatory mediators. Cells were exposed to venoms and immediately irradiated with low-level laser (LLL) application in continuous wave at the wavelength of 660 nm, energy density of 4.4 J/cm2, power of 10 mW, area of 0.045 cm2, and time of 20 s. Cell integrity was analyzed by phase contrast microscope and cell death was performed by flow cytometry. In addition, interleukin IL1-ß, IL-6, and IL-10 levels were measured in the supernatant. Our results showed that the application of PBM increases cell viability and decreases cell death by apoptosis and necrosis. Moreover, the release of pro-inflammatory interleukins was also reduced. The data reported here indicate that PBM resulted in cytoprotection on myoblast C2C12 cells after venom exposure. This protection involves the modulation of cell death mechanism and decreased pro-inflammatory cytokine release.

Cellular Stress in the Pathogenesis of Muscular Disorders-From Cause to Consequence.

Cellular stress has been considered a relevant pathogenetic factor in a variety of human diseases. Due to its primary functions by means of contractility, metabolism, and protein synthesis, the muscle cell is faced with continuous changes of cellular homeostasis that require rapid and coordinated adaptive mechanisms. Hence, a prone susceptibility to cellular stress in muscle is immanent. However, studies focusing on the cellular stress response in muscular disorders are limited. While in recent years there have been emerging indications regarding a relevant role of cellular stress in the pathophysiology of several muscular disorders, the underlying mechanisms are to a great extent incompletely understood. This review aimed to summarize the available evidence regarding a deregulation of the cellular stress response in individual muscle diseases. Potential mechanisms, as well as involved pathways are critically discussed, and respective disease models are addressed. Furthermore, relevant therapeutic approaches that aim to abrogate defects of cellular stress response in muscular disorders are outlined.

Active Compound of Pharbitis Semen (Pharbitis nil Seeds) Suppressed KRAS-Driven Colorectal Cancer and Restored Muscle Cell Function during Cancer Progression.

Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The seeds of Pharbitis nil, also known as morning glory, have been used as traditional medicine in East Asia. We focused on whether Pharbitis nil seeds have a suppressive effect on mutated KRAS-driven CRC as well as reserving muscle cell functions during CRC progression. Seeds of Pharbitis nil (Pharbitis semen) were separated by chromatography and the active compound of Pharbitis semen (PN) was purified by HPLC. The compound PN efficiently suppressed the proliferation of mutated KRAS-driven CRC cells and their clonogenic potentials in a concentration-dependent manner. It also induced apoptosis of SW480 human colon cancer cells and cell cycle arrest at the G2/M phase. The CRC related pathways, including RAS/ERK and AKT/mTOR, were assessed and PN reduced the phosphorylation of AKT and mTOR. Furthermore, PN preserved muscle cell proliferation and myotube formation in cancer conditioned media. In summary, PN significantly suppressed mutated KRAS-driven cell growth and reserved muscle cell function. Based on the current study, PN could be considered as a promising starting point for the development of a nature-derived drug against KRAS-mutated CRC progression.