Project description:Muscle contraction during exercise is the major stimulus for the release of peptides and proteins (myokines) that are supposed to take part in the benefical adaptation to exercise. We hypothesize that application of an in vitro exercise stimulus as electric pulse stimulation (EPS) to human myotubes enables the investigation of the human muscle secretome in a clearly defined model. We applied EPS for 24 h to primary human myotubes and studied the whole genome-wide transcriptional response and as well as the release of candidate myokines. We observed 183 differentially regulated transcripts with fold-changes > 1.3. The transcriptional response resembles several properties of the in vivo situation in the skeletal muscle after endurance exercise, namely significant enrichment of pathways associated with interleukin and chemokine signaling, lipid metabolism, and anti-oxidant defense; notably without increased release of creatin kinase. Multiplex immunoassays verified the translation of the transcriptional response of several myokines into high secretion levels (IL6, IL8, CXCL1, LIF, CSF3, IL1B, TNF) and identified an increased secretion of additional cytokines (IL2, IL4, IL13, IL17A). Inhibitor studies and immunoblotting revealed the participation of ERK1/2 and AMPK dependent pathways in the upregulation of myokines. To conclude our data highlight the importance of skeletal muscle cells per se as endocrine cells. This in vitro exercise model is not only suitable to identify known and novel exercise-regulated myokines but it might be applied to primary human myotubes obtained from different muscle biopsy donors to study molecular mechanisms of the individual response to exercise. We performed gene expression microarray analysis of myotubes in vitro stimulated by EPS and control myotubes
Project description:Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses (i.e. myokine secretion). We herein describe an "in vitro exercise model", using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the "in vitro exercise model".
Project description:PURPOSE:Myokines have been shown to affect muscle physiology and exert systemic effects. We endeavored to investigate a panel of myokine mRNA expression after a single exercise bout (studies 1 and 2) to measure myokine mRNA in primary human myotubes in an in vitro exercise model (study 2). METHODS:Vastus lateralis muscle biopsies were obtained from 20 healthy males (age, 24.0 ± 4.5 yr; BMI, 23.6 ± 1.8 kg·m)(-2) before and after a single exercise bout (650 kcal at 50% V˙O2max). Primary myotubes from active and sedentary male donors were treated with a pharmacological cocktail (palmitate, forskolin, and ionomycin (PFI)) to mimic exercise-stimulated contractions in vitro. RESULTS:Interleukin 6 and 8 (IL-6 and IL-8), leukocyte-inducing factor, and connective tissue growth factor (CTGF) mRNA levels increased approximately 10-fold after a single exercise bout (all P < 0.001), whereas myostatin levels decreased (P < 0.05). Key correlations between myokine expression and parameters of muscle and whole-body physiology were found: myostatin versus skeletal muscle citrate synthase activity (r = -0.69, P < 0.001), V˙O2max (r = -0.64, P = 0.002) and the percentage of Type I fibers (r = -0.55, P = 0.01); IL-6 versus the RER (r = 0.45, P = 0.04), homeostatic model assessment of insulin resistance (r = 0.44, P = 0.05), and serum lactate (r = 0.50, P = 0.02). Myokine expressions in myotubes from sedentary donors for CTGF and myostatin decreased, whereas IL-6 and IL-8 increased after PFI treatment. In myotubes from active donors, myokine expression increased for IL-6, CTGF, and myostatin but decreased for IL-8 after PFI treatment. CONCLUSION:These data offer insight into the differences in regulation of myokine expression and their possible physiologic relationships.
Project description:OBJECTIVE:We tested the hypothesis that exercise training would attenuate metabolic impairment in a model of severe cancer cachexia. METHODS:We used multiple in vivo and in vitro methods to explore the mechanisms underlying the beneficial effects induced by exercise training in tumor-bearing rats. RESULTS:Exercise training improved running capacity, prolonged lifespan, reduced oxidative stress, and normalized muscle mass and contractile function in tumor-bearing rats. An unbiased proteomic screening revealed COP9 signalosome complex subunit 2 (COPS2) as one of the most downregulated proteins in skeletal muscle at the early stage of cancer cachexia. Exercise training normalized muscle COPS2 protein expression in tumor-bearing rats and mice. Lung cancer patients with low endurance capacity had low muscle COPS2 protein expression as compared to age-matched control subjects. To test whether decrease in COPS2 protein levels could aggravate or be an intrinsic compensatory mechanism to protect myotubes from cancer effects, we performed experiments in vitro using primary myotubes. COPS2 knockdown in human myotubes affected multiple cellular pathways, including regulation of actin cytoskeleton. Incubation of cancer-conditioned media in mouse myotubes decreased F-actin expression, which was partially restored by COPS2 knockdown. Direct repeat 4 (DR4) response elements have been shown to positively regulate gene expression. COPS2 overexpression decreased the DR4 activity in mouse myoblasts, and COPS2 knockdown inhibited the effects of cancer-conditioned media on DR4 activity. CONCLUSIONS:These studies demonstrated that exercise training may be an important adjuvant therapy to counteract cancer cachexia and uncovered novel mechanisms involving COPS2 to regulate myotube homeostasis in cancer cachexia.
Project description:To evaluate transcriptomic changes induced by in vitro exercise, we established two in vitro exercise models; EPS (electrical pulse stimulation and clenbuterol treatment). As for clen-buterol treatment, differentiated C2C12 myotubes were treated by 30 ng/ml clenbuterol for 1 hour and control and clenbuterol treated C2C12 myotubes were analyzed by RNA-sequencing. As for an EPS model, EPS was applied to differentiated C2C12 myotubes for 24 hours and control and EPS applied C2C12 myotubes were analyzed by RNA-sequencing. Overall design: Examination of 2 different in vitro exercise models (EPS and clenbuterol) in C2C12 myotubes.
Project description:Regular exercise has emerged as one of the best therapeutic strategies to prevent and treat type-2-diabetes. Exercise-induced changes in the muscle secretome, consisting of myokines and metabolites, may underlie the inter-organ communication between muscle and other organs. To investigate this crosstalk, we developed an in vitro system in which mouse C2C12 myotubes underwent electric pulse stimulation (EPS) to induce contraction. Subsequently the effects of EPS-conditioned media (EPS-CM) on hepatocytes were investigated. Here, we demonstrate that EPS-CM induces Metallothionein 1/2 and Slc30a2 gene expression and reduces Cyp2a3 gene expression in rat hepatocytes. When testing EPS-CM that was generated in the absence of C2C12 myotubes (non-cell EPS-CM) no decrease in Cyp2a3 expression was detected. However, similar inductions in hepatic Mt1/2 and Slc30a2 expression were observed. Non-cell EPS-CM were also applied to C2C12 myotubes and compared to C2C12 myotubes that underwent EPS: here changes in AMPK phosphorylation and myokine secretion largely depended on EPS-induced contraction. Taken together, these findings indicate that EPS can alter C2C12 myotube function and thereby affect gene expression in cells subjected to EPS-CM (Cyp2a3). However, EPS can also generate non-cell-mediated changes in cell culture media, which can affect gene expression in cells subjected to EPS-CM too. While EPS clearly represents a valuable tool in exercise research, care should be taken in experimental design to control for non-cell-mediated effects.
Project description:BACKGROUND AND PURPOSE:Endothelial progenitor cells (EPCs) have been extensively investigated as a therapeutic approach for repairing the vascular system in cerebrovascular diseases. Beyond vascular regeneration per se, EPCs may also release factors that affect the entire neurovascular unit. Here, we aim to study the effects of the EPC secretome on oligovascular remodeling in a mouse model of white matter injury after prolonged cerebral hypoperfusion. METHODS:The secretome of mouse EPCs was analyzed with a proteome array. In vitro, the effects of the EPC secretome and its factor angiogenin were assessed on primary oligodendrocyte precursor cells and mature human cerebral microvascular endothelial cells (hCMED/D3). In vivo, mice were subjected to permanent bilateral common carotid artery stenosis, then treated with EPC secretome at 24 hours and at 1 week, and cognitive outcome was evaluated with the Y maze test together with oligodendrocyte precursor cell proliferation/differentiation and vascular density in white matter at 4 weeks. RESULTS:Multiple growth factors, cytokines, and proteases were identified in the EPC secretome, including angiogenin. In vitro, the EPC secretome significantly enhanced endothelial and oligodendrocyte precursor cell proliferation and potentiated oligodendrocyte precursor cell maturation. Angiogenin was proved to be a key factor since pharmacological blockade of angiogenin signaling negated the positive effects of the EPC secretome. In vivo, treatment with the EPC secretome increased vascular density, myelin, and mature oligodendrocytes in white matter and rescued cognitive function in the mouse hypoperfusion model. CONCLUSIONS:Factors secreted by EPCs may ameliorate white matter damage in the brain by boosting oligovascular remodeling.
Project description:Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO2max of 70%. Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines.
Project description:BACKGROUND AND AIMS:Physical activity has preventive as well as therapeutic benefits for overweight subjects. In this study we aimed to examine effects of in vivo exercise on in vitro metabolic adaptations by studying energy metabolism in cultured myotubes isolated from biopsies taken before and after 12 weeks of extensive endurance and strength training, from healthy sedentary normal weight and overweight men. METHODS:Healthy sedentary men, aged 40-62 years, with normal weight (body mass index (BMI) < 25 kg/m2) or overweight (BMI ? 25 kg/m2) were included. Fatty acid and glucose metabolism were studied in myotubes using [14C]oleic acid and [14C]glucose, respectively. Gene and protein expressions, as well as DNA methylation were measured for selected genes. RESULTS:The 12-week training intervention improved endurance, strength and insulin sensitivity in vivo, and reduced the participants' body weight. Biopsy-derived cultured human myotubes after exercise showed increased total cellular oleic acid uptake (30%), oxidation (46%) and lipid accumulation (34%), as well as increased fractional glucose oxidation (14%) compared to cultures established prior to exercise. Most of these exercise-induced increases were significant in the overweight group, whereas the normal weight group showed no change in oleic acid or glucose metabolism. CONCLUSIONS:12 weeks of combined endurance and strength training promoted increased lipid and glucose metabolism in biopsy-derived cultured human myotubes, showing that training in vivo are able to induce changes in human myotubes that are discernible in vitro.
Project description:Eccentric exercise leads to focal disruptions in the myofibrils, referred to as "lesions". These structures are thought to contribute to the post-exercise muscle weakness, and to represent areas of mechanical damage and/or remodelling. Lesions have been investigated in human biopsies and animal samples after exercise. However, this approach does not examine the mechanisms behind lesion formation, or their behaviour during contraction. To circumvent this, we used electrical pulse stimulation (EPS) to simulate exercise in C2C12 myotubes, combined with live microscopy. EPS application led to the formation of sarcomeric lesions in the myotubes, resembling those seen in exercised mice, increasing in number with the time of application or stimulation intensity. Furthermore, transfection with an EGFP-tagged version of the lesion and Z-disc marker filamin-C allowed us to observe the formation of lesions using live cell imaging. Finally, using the same technique we studied the behaviour of these structures during contraction, and observed them to be passively stretching. This passive behaviour supports the hypothesis that lesions contribute to the post-exercise muscle weakness, protecting against further damage. We conclude that EPS can be reliably used as a model for the induction and study of sarcomeric lesions in myotubes in vitro.