Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

Maladaptive endoplasmic reticulum (ER) stress is associated with modified reactive oxygen species (ROS) generation and mitochondrial abnormalities; and is postulated as a potential mechanism involved in muscle weakness in myositis, an acquired autoimmune neuromuscular disease. This study investigates the impact of ROS generation in an in vitro model of ER stress in skeletal muscle, using the ER stress inducer tunicamycin (24 h) in the presence or absence of a superoxide dismutase/catalase mimetic Eukarion (EUK)-134. Tunicamycin induced maladaptive ER stress, which was mitigated by EUK-134 at the transcriptional level. ER stress promoted mitochondrial dysfunction, described by substantial loss of mitochondrial membrane potential, as well as a reduction in respiratory control ratio, reserve capacity, phosphorylating respiration, and coupling efficiency, which was ameliorated by EUK-134. Tunicamycin induced ROS-mediated biogenesis and fusion of mitochondria, which, however, had high propensity of fragmentation, accompanied by upregulated mRNA levels of fission-related markers. Increased cellular ROS generation was observed under ER stress that was prevented by EUK-134, even though no changes in mitochondrial superoxide were noticeable. These findings suggest that targeting ROS generation using EUK-134 can amend aspects of ER stress-induced changes in mitochondrial dynamics and function, and therefore, in instances of chronic ER stress, such as in myositis, quenching ROS generation may be a promising therapy for muscle weakness and dysfunction

A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.

Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and βIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy

Cross-talk between motor neurons and myotubes via endogenously secreted neural and muscular growth factors.

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular patho-physiology and development of novel therapies for diseases associated with NM dysfunction. In vivo, the micro-environment surrounding the NMJ has a significant impact on NMJ formation and maintenance via neurotrophic and differentiation factors that are secreted as a result of cross-talk between muscle fibers and motor neurons. Recently we showed the formation of functional NMJs in vitro in a co-culture of immortalized human myoblasts and motor neurons from rat-embryo spinal-cord explants, using a culture medium free from serum and neurotrophic or growth factors. The aim of this study was to assess how functional NMJs were established in this co-culture devoid of exogenous neural growth factors. To investigate this, an ELISA-based microarray was used to compare the composition of soluble endogenously secreted growth factors in this co-culture with an a-neural muscle culture. The levels of seven neurotrophic factors brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-1 (IGF-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and vascular endothelial growth factor (VEGF) were higher (p < 0.05) in the supernatant of NMJ culture compared to those in the supernatant of the a-neural muscle culture. This indicates that the cross-talk between muscle and motor neurons promotes the secretion of soluble growth factors contributing to the local microenvironment thereby providing a favourable regenerative niche for NMJs formation and maturation

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction.

BACKGROUND:In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. OBJECTIVE:to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. METHODS:an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. RESULTS:blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. CONCLUSION:a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions

Omega-3 fatty acid EPA improves regenerative capacity of mouse skeletal muscle cells exposed to saturated fat and inflammation

© 2016 The Author(s) Sarcopenic obesity is characterised by high fat mass, low muscle mass and an elevated inflammatory environmental milieu. We therefore investigated the effects of elevated inflammatory cytokine TNF-α (aging/obesity) and saturated fatty acid, palmitate (obesity) on skeletal muscle cells in the presence/absence of EPA, a-3 polyunsaturated fatty acid with proposed anti-inflammatory, anti-obesity activities. In the present study we show that palmitate was lipotoxic, inducing high levels of cell death and blocking myotube formation. Cell death under these conditions was associated with increased caspase activity, suppression of differentiation, reductions in both creatine kinase activity and gene expression of myogenic factors; IGF-II, IGFBP-5, MyoD and myogenin. However, inhibition of caspase activity via administration of Z-VDVAD-FMK (caspase-2), Z-DEVD-FMK (caspase-3) and ZIETD-KMK (caspase 8) was without effect on cell death. By contrast, lipotoxicity associated with elevated palmitate was reduced with the MEK inhibitor PD98059, indicating palmitate induced cell death was MAPK mediated. These lipotoxic conditions were further exacerbated in the presence of inflammation via TNF-α co-administration. Addition of EPA under cytotoxic stress (TNF-α) was shown to partially rescue differentiation with enhanced myotube formation being associated with increased MyoD, myogenin, IGF-II and IGFBP-5 expression. EPA had little impact on the cell death phenotype observed in lipotoxic conditions but did show benefit in restoring differentiation under lipotoxic plus cytotoxic conditions. Under these conditions Id3 (inhibitor of differentiation) gene expression was inversely linked with survival rates, potentially indicating a novel role of EPA and Id3 in the regulation of apoptosis in lipotoxic/cytotoxic conditions. Additionally, signalling studies indicated the combination of lipo- and cyto-toxic effects on the muscle cells acted through ceramide, JNK and MAPK pathways and blocking these pathways using PD98059 (MEK inhibitor) and Fumonisin B1 (ceramide inhibitor) significantly reduced levels of cell death. These findings highlight novel pathways associated with in vitro models of lipotoxicity (palmitate-mediated) and cytotoxicity (inflammatory cytokine mediated) in the potential targeting of molecular modulators of sarcopenic obesity

Dysregulation of CXC motif ligand 10 during aging and association with cognitive performance

Chronic low-grade inflammation during aging (inflammaging) is associated with cognitive decline and neurodegeneration, however, the mechanisms underlying inflammaging are unclear. We studied a population (n = 361) of healthy young and old adults from the MyoAge cohort. Peripheral levels of C-X-C motif chemokine 10 (CXCL10) was found to be higher in older adults, compared with young, and negatively associated with working memory performance. This coincided with an age-related reduction in blood DNA methylation at specific CpGs within the CXCL10 gene promoter. In vitro analysis supported the role of DNA methylation in regulating CXCL10 transcription. A polymorphism (rs56061981) that altered methylation at one of these CpG sites further associated with working memory performance in two independent aging cohorts. Studying prefrontal cortex samples, we found higher CXCL10 protein levels in those with Alzheimer’s disease, compared to aged controls. These findings support the association of peripheral inflammation, as demonstrated by CXCL10, in aging and cognitive decline. We reveal age-related epigenetic and genetic factors which contribute to the dysregulation of CXCL10

Investigation of the molecular pathways controlling the differentiation and proliferation of human CD8&quot;+T cells in and ex vivo expansion model

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PD98059 enhances C2 myoblast differentiation through p38 MAPK activation: a novel role for PD98059.

Cell differentiation is usually accompanied by irreversible cell cycle exit, which is a critical step for skeletal muscle differentiation. We therefore hypothesise that PD98059 that blocks the MAP kinase kinase (MEK) pathway (proliferation pathway) when administrated to murine C2 skeletal myoblasts will arrest cell cycle and, consequently, enhances differentiation relative to untreated controls. In this study, we aimed to examine this hypothesis using phenotypic differentiation, biochemical assays, flow cytometry and real-time PCR in C2 cells cultured for 48 h in differentiation media only (untreated) or supplemented with either a single dose of 10 ng/ml IGF-I or 20 muM PD98059 for 48 h. Creatine kinase (CK) activity was increased by 7.5-fold (P<0.05) in the presence of PD98059, whereas untreated and IGF-I-treated cells induced 4.5- and 4-fold increase respectively when compared with baseline controls. Increased CK values in the presence of PD98059 were not only associated with myotube formation but also associated with cell cycle arrest in G1 phase (86+/-3.2%; P<0.05). Moreover, the expression of myogenic-specific transcriptional factor mRNAs (MyoD and myogenin) was significantly higher in PD-treated cells (4.7+/-0.15 and 314+/-10.2 ng/reaction respectively; P<0.05) than untreated (2.0+/-0.2 and 233+/-11 ng/reaction respectively) or IGF-treated cells (3.2+/-0.24 and 296+/-16.2 ng/reaction respectively). Unexpectedly, Id3 mRNA, the potent negative regulator of muscle differentiation, was also expressed at significantly higher levels in PD-treated cells (77+/-0.346 ng/reaction; P<0.05) than untreated (49+/-7.7 ng/reaction) or IGF-I-treated cells (47+/-0.7 ng/reaction). Furthermore, expression of the muscle differentiation-specific genes (IGF-binding protein-5, IGF-II receptor and IGF-II) was also increased significantly in PD-treated cells when compared with untreated cells. Phosflow analysis showed a significant increase in the levels of phosphorylation of p38 mitogen-activated protein kinase (49.0+/-6.7%, P<0.05) in PD-treated cells when compared with DM-treated cells (31.7+/-5.7%). These findings uncover a previously unconsidered positive effect of PD98059 on C2 myoblast differentiation and identify the pathway(s) underlying PD-induced C2 myoblast differentiation

C2 skeletal myoblast survival, death, proliferation and differentiation: regulation by Adra1d

IGF-I positively impacts on muscle anabolism/regeneration. Using C2 skeletal myoblasts, we previously reported high dose TNF-α -induced (10 ng.ml-1) cell death is rescued by IGF-I. However, non-myotoxic low dose TNF-α (1.25 ng.ml-1) elicits a MAPK-mediated apoptotic response when co-incubated with IGF-I (1.5 ng.ml-1). Our aim was to investigate these conflicting roles of IGF-I in our model. Insulin array and qRT-PCR identified Adra1d as a potential regulatory gene that was up-regulated in survival and down-regulated under apoptotic conditions. TNF-α administration (1.25 or 10 ng.ml-1) induced significant decreases (∼50% both incubations) in Adra1d expression relative to DM. IGF-I addition to high dose TNF-α (10 ng.ml-1) induced myoblast survival and matched a significant (P < 0.05) increase in Adra1d expression. By contrast, IGF-I addition to low dose TNF-α (1.25 ng.ml-1) induced elevated death resulting in a significant (P < 0.05) decline (∼55%) in Adra1d expression. Pre-administration of PD98059 (20 uM), which rescues death induced by co-incubation of low dose TNF-α with IGF-I, Adra1d levels were again comparable to DM control. Since Adra1d was elevated following incubations that induced myoblast survival, we investigated effects of Adra1d siRNA gene silencing under these conditions. Adra1d knockdown resulted in significantly higher levels of cell death under all incubations suggesting Adra1d expression is essential for skeletal muscle cell survival

Waste management—cytokines, growth factors and cachexia

Muscle damage with a lack of regeneration, manifests itself in several life-threatening diseases, including cancer cachexia, congestive heart failure, AIDS and sepsis. Often misdiagnosed as a condition simply of weight loss, cachexia is actually a highly complex metabolic disorder involving features of anorexia, anaemia, lipolysis and insulin resistance. A significant loss of lean body mass arises from such conditions, resulting in wasting of skeletal muscle. Unlike starvation, the weight loss seen in chronic illnesses arises equally from loss of muscle and of fat. The cachectic state is particularly problematic in cancer, typifying poor prognosis and often lowering responses to chemotherapy and radiation treatment. More than half of cancer patients suffer from cachexia, and strikingly, nearly one-third of cancer deaths are related to cachexia rather than the tumour burden. In considering this disorder, we are faced with a conundrum; how is it possible for uncontrolled growth to prevail in the tumour, in the face of unrestrained tissue loss in our muscles? Consistently, the catabolic state has been associated with a shift in the homeostatic balance between muscle synthesis and degradation mediated by the actions of growth factors and cytokines. Indeed, tumour necrosis factor-alpha (TNF-α) levels are raised in several animal models of cachectic muscle wasting, whereas the insulin-like growth factor (IGF) system acts potently to regulate muscle development, hypertrophy and maintenance. This concept of skeletal muscle homeostasis, often viewed as the net balance between two separate processes of protein synthesis and degradation has however changed. More recently, the view is that these two biochemical processes are not occurring independently of each other but in fact are finely co-ordinated by a web of intricate signalling networks. This review, therefore, aims to discuss data currently available regarding the mechanisms of degeneration and regeneration with specific emphasis on the potential and controversial cross-talk which may exist between anabolic growth factors (e.g. IGF-I) and catabolic cytokines (e.g. TNF-α). Also importantly, the potential impact at a cellular level of exercise, diet and age will be addressed. Finally, the ability to ‘hi-jack’ signalling pathways traditionally believed to be for growth and survival or death will be reviewed. It is anticipated that such a review will highlight significant gaps in our knowledge of the cachectic state as well as provide caution with regards to therapeutics suggesting total block on inflammatory processes such as that associated with TNF-α action