Night work, season and alertness as occupational safety hazards in the Arctic: protocol for the Noralert observational crossover study among Norwegian process operators.

INTRODUCTION: The objective of this study is to determine the effects of night work, Arctic seasonal factors and cold working environments on human functions relevant to safety. The study aims to quantify the contribution of (1) several consecutive night shifts, (2) seasonal variation on sleepiness, alertness and circadian rhythm and (3) whether a computational model of sleep, circadian rhythms and cognitive performance can accurately predict the observed sleepiness and alertness. METHODS AND ANALYSIS: In an observational crossover study of outdoor and indoor workers (n=120) on a three-shift schedule from an industrial plant in Norway (70 °N), measurements will be conducted during the summer and winter. Sleep duration and quality will be measured daily by smartphone questionnaire, aided by actigraphy and heart rate measurements. Sleepiness and alertness will be assessed at regular intervals by the Karolinska Sleepiness Scale and the psychomotor vigilance test, respectively. Saliva samples will assess melatonin levels, and a blood sample will measure circadian time. Thermal exposures and responses will be measured by sensors and by thermography. ETHICS AND DISSEMINATION: All participants will give written informed consent to participate in the study, which will be conducted in accordance with the Declaration of Helsinki. The Norwegian Regional Committee for Medical Research Ethics South-East D waivered the need for ethics approval (reference 495816). Dissemination plans include academic and lay publications, and partnerships with national and regional policymakers.

Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes.

Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging. TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found. In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 are expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.

Antidepressants escitalopram and venlafaxine up-regulate BDNF promoter IV but down-regulate neurite outgrowth in differentiating SH-SY5Y neurons.

Antidepressants are used to treat depression and some anxiety disorders, including use in pregnant patients. The pharmacological actions of these drugs generally determine the uptake and metabolism of a series of neurotransmitters, such as serotonin, norepinephrine, or dopamine, along with an increase in BDNF expression. However, many aspects of antidepressant action remain unknown, particularly whether antidepressants interfere with normal neurodevelopment when taken by pregnant women. In order to reveal cellular and molecular implications crucial to the functioning of pathways related to antidepressant effects, we performed an investigation on neuronally differentiating human SH-SY5Y cells. To our knowledge, this is the first time human SH-SY5Y cells in cultures of purely neuronal cells induced by controlled differentiation with retinoic acid are followed by short-term 48-h exposure to 0.1-10 µM escitalopram or venlafaxine. Treatment with antidepressants (1 µM) did not affect the electrophysiological properties of SH-SY5Y cells. However, the percentage of mature neurons exhibiting voltage-gated sodium currents was substantially higher in cultures pre-treated with either antidepressant. After exposure to escitalopram or venlafaxine, we observed a concentration-dependent increase in activity-dependent BDNF promoter IV activation. The assessment of neurite metrics showed significant down-regulation of neurite outgrowth upon exposure to venlafaxine. Identified changes may represent links to molecular processes of importance to depression and be involved in neurodevelopmental alterations observed in postpartum children exposed to antidepressants antenatally.

Comparison of young male mice of two different strains (C57BL/6J and the hybrid B6129SF1/J) in selected behavior tests: a small scale study.

BACKGROUND: All mouse strains are different, before choosing a strain for a large study, a small scale study should be done. In this study, we compared young males of two mouse strains, C57BL/6J and the hybrid B6129SF1/J, and gained knowledge on their performance in three different behavioral tests; open field (OF) test, Barnes maze (BM) test and a restraint stress test. RESULTS: We found that the young males of the C57BL/6J strain spent more time moving in the OF. In the BM, the hybrid covered less ground before reaching the goal box during the first three sessions, than the C57BL/6J. The hybrid left more fecal pellets than C57BL/6J both in OF and BM. During the stress test, the C57BL/6J had a lower corticosterone response than the hybrid. CONCLUSIONS: Our findings indicate that the C57BL/6J has a presumably higher locomotor activity and/or explorative behavior than the hybrid, while the hybrid appeared more sensitive to stress.

Experimental Models for Cold Exposure of Muscle in vitroand in vivo.

Work in cold environments may have a significant impact on occupational health. In these and similar situations, cold exposure localized to the extremities may reduce the temperature of underlying tissues. To investigate the molecular effects of cold exposure in muscle, and since adequate methods were missing, we established two experimental cold exposure models: 1) In vitroexposure to cold (18°C) or control temperature (37°C) of cultured human skeletal muscle cells (myotubes); and 2) unilateral cold exposure of hind limb skeletal muscle in anesthetized rats (intramuscular temperature 18°C), with contralateral control (37°C). This methodology enables studies of muscle responses to local cold exposures at the level of gene expression, but also other molecular outcomes. Graphical abstract.

Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome.

Electrical pulse stimulation (EPS) has proven to be a useful tool to interrogate cell-specific responses to muscle contraction. In the present study, we aimed to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes from young male subjects were exposed to EPS protocol 1 (i.e. 2 ms, 10 V, and 0.1 Hz for 24 h), whereas myotubes from middle-aged women and men were exposed to protocol 2 (i.e. 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling as well as the transcriptome, cellular proteome, and secreted proteins of EPS-treated myotubes from young male subjects were analyzed using a combination of high-throughput RNA sequencing, high-resolution liquid chromatography-tandem mass spectrometry, oxidation assay, and immunoblotting. The data showed that oxidative metabolism was enhanced in EPS-exposed myotubes from young male subjects. Moreover, a total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in these cells after EPS protocol 1. We also found 61 overlapping genes while comparing the DEGs to mRNA expression in myotubes from the middle-aged group exposed to protocol 2, assessed by microarray. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Additionally, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes from young male subjects exposed to EPS protocol 1. Selected putative myokines were measured using ELISA or multiplex assay to validate the results. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.

Paracetamol perturbs neuronal arborization and disrupts the cytoskeletal proteins SPTBN1 and TUBB3 in both human and chicken in vitro models.

Epidemiological studies have linked long-term/high-dose usage of paracetamol (N-acetyl-para-aminophenol, APAP) during pregnancy to adverse neuropsychiatric outcomes, primarily attention-deficit hyperactive disorder (ADHD), in the offspring. Though variable, ADHD has been associated with phenotypic alterations characterized by reductions in grey matter densities and aberrations in structural connectivity, effects which are thought to originate in neurodevelopment. We used embryonic chicken cerebellar granule neurons (CGNs) and neuronally differentiating human NTERA2 cells (NT2Ns) to investigate the in vitro effects of APAP on cell viability, migration, neuritogenesis, and the intracellular levels of various proteins involved in neurodevelopment as well as in the maintenance of the structure and function of neurites. Exposure to APAP ranging from 100 to 1600 µM yielded concentration- and time-dependent reductions in cell viability and levels of neurite arborization, as well as reductions in the levels of the cytoskeletal protein ß2-spectrin, with the highest APAP concentration resulting in between 50 and 75% reductions in the aforementioned metrics over the course of 72 h. Exposure to APAP also reduced migration in the NT2Ns but not CGNs. Moreover, we found concentration- and time-dependent increases in punctate aggregation of the cytoskeletal protein ß3-tubulin following exposure to APAP in both cell model systems, with the highest APAP concentration approximately doubling the number of aggregates over 72-120 h. Our findings demonstrate that APAP negatively perturbs neurite arborization degree, with concurrent reductions in the protein levels of ß2-spectrin and disruption of the integrity of ß3-tubulin, both proteins of which play important roles in neuronal structure and function.

SARS-CoV-2 serological findings and exposure risk among employees in school and retail after first and second wave COVID-19 pandemic in Oslo, Norway: a cohort study.

OBJECTIVES: The objective was to characterize and compare SARS-CoV-2 serology among Norwegian school employees and retail employees, and describe preventive measures taken at the workplaces. MATERIAL AND METHODS: A cohort of 238 school and retail employees was enrolled to an ambidirectional cohort study after the first COVID-19 pandemic wave. Self-reported exposure history and serum samples were collected at 10 schools and 15 retail stores in Oslo, Norway, sampled at 2 time-points: baseline (May-July 2020); and follow-up (January-March 2021). SARS-CoV-2 antibodies targeting both spike and nucleocapsid were detected by multiplex microsphere-based serological methods. RESULTS: At baseline, 6 enrolled workers (5 in retail) presented with positive SARS-CoV-2 serology, higher than the expected 1% prevalence (3%, 95% CI: 1-6, p = 0.019). At followup, school and retail groups presented 11 new seropositive cases altogether, but groups were not significantly different, although exposure and preventive measures against viral transmission at workplaces were different between groups. Self-reported medical history of COVID-19 infection showed that all but one positive SARS-CoV-2 serological findings arising between baseline and follow-up had been diagnosed with virus testing. CONCLUSIONS: Distribution of SARS-CoV-2 positive serology after the first wave was slightly higher than expected. Distribution of infection was not significantly different between the groups at baseline nor at follow-up, despite difference in exposure and protective measures. Nearly all new seropositive cases discovered between baseline and follow-up, had already been diagnosed, highlighting the importance of extensive viral testing among workers. Int J Occup Med Environ Health. 2022;35(5):537-47.

The effect of toll-like receptor ligands on energy metabolism and myokine expression and secretion in cultured human skeletal muscle cells.

Skeletal muscle plays an important role in glycaemic control and metabolic homeostasis, making it a tissue of interest with respect to type 2 diabetes mellitus. The aim of the present study was to determine if ligands of Toll-like receptors (TLRs) could have an impact on energy metabolism and myokine expression and secretion in cultured human skeletal muscle cells. The myotubes expressed mRNA for TLRs 1-6. TLR3, TLR4, TLR5 and TLR6 ligands (TLRLs) increased glucose metabolism. Furthermore, TLR4L and TLR5L increased oleic acid metabolism. The metabolic effects of TLRLs were not evident until after at least 24 h pre-incubation of the cells and here the metabolic effects were more evident for the metabolism of glucose than oleic acid, with a shift towards effects on oleic acid metabolism after chronic exposure (168 h). However, the stimulatory effect of TLRLs on myokine expression and secretion was detected after only 6 h, where TLR3-6L stimulated secretion of interleukin-6 (IL-6). TLR5L also increased secretion of interleukin-8 (IL-8), while TLR6L also increased secretion of granulocyte-macrophage colony stimulating factor (GM-CSF). Pre-incubation of the myotubes with IL-6 for 24 h increased oleic acid oxidation but had no effect on glucose metabolism. Thus IL-6 did not mimic all the metabolic effects of the TLRLs, implying metabolic effects beyond the actions of this myokine.

Diminished Cold Avoidance Behaviours after Chronic Cold Exposure - Potential Involvement of TRPM8.

Ambient temperature changes trigger plastic biological responses. Cold temperature is detected by the somatosensory system and evokes perception of cold together with adaptive physiological responses. We addressed whether chronic cold exposure induces adaptive adjustments of (1) thermosensory behaviours, and (2) the principle molecular cold sensor in the transduction machinery, transient receptor potential melastatin subtype 8 (TRPM8). Mice in two groups were exposed to either cold (6 °C) or thermoneutral (27 °C) ambient temperatures for 4 weeks and subjected to thermosensory behavioural testing. Cold group mice behaved different from Thermoneutral group in the Thermal Gradient Test: the former occupied a wider temperature range and was less cold avoidant. Furthermore, subcutaneous injection of the TRPM8 agonist icilin, enhanced cold avoidance in both groups in the Thermal Gradient Test, but Cold group mice were significantly less affected by icilin. Primary sensory neuron soma are located in dorsal root ganglia (DRGs), and western blotting showed diminished TRPM8 levels in DRGs of Cold group mice, as compared to the Thermoneutral group. We conclude that acclimation to chronic cold altered thermosensory behaviours, so that mice appeared less cold sensitive, and potentially, TRPM8 is involved.

Novel methods for cold exposure of skeletal muscle in vivo and in vitro show temperature-dependent myokine production.

Proteins secreted from skeletal muscle serving a signalling role have been termed myokines. Many of the myokines are exercise factors, produced and released in response to muscle activity. Cold exposures affecting muscle may occur in recreational, occupational and therapeutic settings. Whether muscle temperature independently affects myokine profile, is still to be elucidated. We hypothesized that manipulating muscle temperature by means of external cooling would change myokine production and release. In the present study we have established new models for cold exposure of muscle in vivo and in vitro where rat hind limb or cultured human myotubes were cooled to 18 °C. After a recovery period, muscle tissue, cells and culture media were harvested for further analysis by qPCR and immunoassays. Expression of several myokine genes were significantly increased after cold exposure in both models: in rat muscle, mRNA levels of CCL2 (p = 0.04), VEGFA (p = 0.02), CXCL1 (p = 0.02) and RBM3 (p = 0.02) increased while mRNA levels of IL-6 (p = 0.03) were decreased; in human myotubes, mRNA levels of IL6 (p = 0.01), CXCL8 (p = 0.04), VEGFA (p = 0.03) and CXCL1 (p < 0.01) were significantly increased, as well as intracellular protein levels of IL-8 (CXCL8 gene product; p < 0.01). The corresponding effect on myokine secretion was not observed, on the contrary, IL-8 (p = 0.02) and VEGF (VEGFA gene product) p < 0.01) concentrations in culture media were reduced after cold exposure in vitro. In conclusion, cold exposure of muscle in vivo and in vitro had an effect on the production and release of several known exercise-related myokines. Myokine expression at the level of mRNA and protein was increased by cold exposure, whereas secretion tended to be decreased.

Innervation and electrical pulse stimulation - in vitro effects on human skeletal muscle cells.

Contraction-induced adaptations in skeletal muscles are well characterized in vivo, but the underlying cellular mechanisms are still not completely understood. Cultured human myotubes represent an essential model system for human skeletal muscle that can be modulated ex vivo, but they are quiescent and do not contract unless being stimulated. Stimulation can be achieved by innervation of human myotubes in vitro by co-culturing with embryonic rat spinal cord, or by replacing motor neuron activation by electrical pulse stimulation (EPS). Effects of these two in vitro approaches, innervation and EPS, were characterized with respects to the expression of myosin heavy chains (MyHCs) and metabolism of glucose and oleic acid in cultured human myotubes. Adherent human myotubes were either innervated with rat spinal cord segments or exposed to EPS. The expression pattern of MyHCs was assessed by quantitative polymerase chain reaction, immunoblotting, and immunofluorescence, while the metabolism of glucose and oleic acid were studied using radiolabelled substrates. Innervation and EPS promoted differentiation towards different fiber types in human myotubes. Expression of the slow MyHC-1 isoform was reduced in innervated myotubes, whereas it remained unaltered in EPS-treated cells. Expression of both fast isoforms (MyHC-2A and MyHC-2X) tended to decrease in EPS-treated cells. Both approaches induced a more oxidative phenotype, reflected in increased CO2 production from both glucose and oleic acid. Novelty: Innervation and EPS favour differentiation into different fiber types in human myotubes. Both innervation and EPS promote a metabolically more oxidative phenotype in human myotubes.

A human relevant mixture of persistent organic pollutants (POPs) and perfluorooctane sulfonic acid (PFOS) enhance nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells.

Disruption of neurite outgrowth is a marker for neurotoxicity. Persistent organic pollutants (POPs) are potential developmental neurotoxicants. We investigated their effect on neurite outgrowth in PC12 rat pheochromocytoma cells, in absence or presence of nerve growth factor (NGF), an inducer of neuronal differentiation. Cells were exposed for 72 h to a defined mixture of POPs with chemical composition and concentrations based on blood levels in the Scandinavian population. We also evaluated perfluorooctane sulfonic acid (PFOS) alone, the most abundant compound in the POP mixture. Only higher concentrations of POP mixture reduced tetrazolium salt (MTT) conversion. High-content analysis showed a decrease in cell number, but no changes for nuclear and mitochondrial cellular health parameters. Robust glutathione levels were observed in NGF-differentiated cells. Live imaging, using the IncuCyte ZOOM platform indicated ongoing cell proliferation over time, but slower in presence of NGF. The pollutants did not inhibit neuritogenesis, but rather increased NGF-induced neurite length. PFOS induced neurite outgrowth to about 50 % of the level seen with the POP mixture. Neither the POP mixture nor PFOS affected neurite length in the absence of NGF. Our observations indicate that realistic complex mixtures of environmental pollutants can affect neuronal connectivity via NGF-induced neurite outgrowth.

Epiregulin is released from intervertebral disks and induces spontaneous activity in pain pathways.

INTRODUCTION: Lumbar radicular pain after disk herniation is associated with local release of many inflammatory molecules from nucleus pulposus (NP) cells leaking out of the intervertebral disk. Here, we have used a rat model to investigate the role of epiregulin (EREG), a member of the epidermal growth factor (EGF) family, in this process. METHODS: A protein immunoassay was chosen to confirm the release of EREG from the NP tissue. Single unit recordings were used to demonstrate the effect of recombinant EREG applied onto the dorsal nerve roots in vivo. Intracellular responses induced by recombinant EREG were studied in cultured dorsal root ganglion (DRG) cells by phosphoprotein assay. Changes in EGF receptor expression induced by NP in the DRG were examined by quantitative polymerase chain reaction. RESULTS: The protein immunoassay showed that EREG was released from the NP tissue. Moreover, application of EREG onto the spinal dorsal nerve roots induced a decrease in the evoked responses, but an increase in spontaneous activity in the dorsal horn neurons. Interestingly, the EREG activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the DRG, a pathway previously linked to cellular growth, proliferation, and tissue regeneration. An NP-induced upregulation of the EGF receptor HER3 in the DRG was also revealed. CONCLUSION: Taken together, the present observations indicate that EREG may induce changes in the DRG and spontaneous activity in the pain pathways. We suggest that EREG signaling may be involved in the pathophysiological process leading to sensory deficits and neuropathic pain in patients after disk herniation.

Deficiency in perilipin 5 reduces mitochondrial function and membrane depolarization in mouse hearts.

Myocardial triglycerides stored in lipid droplets are important in regulating the intracellular delivery of fatty acids for energy generation in mitochondria, for membrane biosynthesis, and as agonists for intracellular signaling. Previously, we showed that deficiency in the lipid droplet protein perilipin 5 (Plin5) markedly reduces triglyceride storage in cardiomyocytes and increases the flux of fatty acids into phospholipids. Here, we investigated whether Plin5 deficiency in cardiomyocytes alters mitochondrial function. We found that Plin5 deficiency reduced mitochondrial oxidative capacity. Furthermore, in mitochondria from Plin5-/- hearts, the fatty acyl composition of phospholipids in mitochondrial membranes was altered and mitochondrial membrane depolarization was markedly compromised. These findings suggest that mitochondria isolated from hearts deficient in Plin5, have specific functional defects.

MicroRNA-223 demonstrated experimentally in exosome-like vesicles is associated with decreased risk of persistent pain after lumbar disc herniation.

BACKGROUND: Previous findings have demonstrated that lumbar radicular pain after disc herniation may be associated with up-regulation of inflammatory mediators. In the present study we examined the possible role of extracellular microRNAs (miRs) in this process. METHODS: Single unit recordings, isolation of exosome-like vesicles, electron microscopy, nanoparticle tracking analysis, western blot analysis and qPCR were used in rats to demonstrate the effect of nucleus pulposus (NP) applied onto the dorsal nerve roots. ELISA and qPCR were used to measure the level of circulating IL-6 and miRs in a 1-year observational study in patients after disc herniation. RESULTS: In the rats, enhanced spinal cord nociceptive responses were displayed after NP applied onto the dorsal nerve roots. An increased release of small non-coding RNAs, including miR-223, miR-760 and miR-145, from NP in exosome-like vesicles was demonstrated. In particular, the NP expression of miR-223, which inhibited the nociceptive spinal signalling, was increased. In the patients, increased extracellular miR-223 was also verified in the acute phase after disc herniation. The increased miR-223 expression was, however, only observed in those who recovered (sex, age and smoking were included as covariates). CONCLUSIONS: Our findings suggest that miR-223, which can be released from the NP after disc herniation, attenuates the neuronal activity in the pain pathways. Dysregulation of miR-223 may predict chronic lumbar radicular pain. Trial registration/ethics REK 2014/1725.

Perilipin 5 is protective in the ischemic heart.

BACKGROUND: Myocardial ischemia is associated with alterations in cardiac metabolism, resulting in decreased fatty acid oxidation and increased lipid accumulation. Here we investigate how myocardial lipid content and dynamics affect the function of the ischemic heart, and focus on the role of the lipid droplet protein perilipin 5 (Plin5) in the pathophysiology of myocardial ischemia. METHODS AND RESULTS: We generated Plin5(-/-) mice and found that Plin5 deficiency dramatically reduced the triglyceride content in the heart. Under normal conditions, Plin5(-/-) mice maintained a close to normal heart function by decreasing fatty acid uptake and increasing glucose uptake, thus preserving the energy balance. However, during stress or myocardial ischemia, Plin5 deficiency resulted in myocardial reduced substrate availability, severely reduced heart function and increased mortality. Importantly, analysis of a human cohort with suspected coronary artery disease showed that a common noncoding polymorphism, rs884164, decreases the cardiac expression of PLIN5 and is associated with reduced heart function following myocardial ischemia, indicating a role for Plin5 in cardiac dysfunction. CONCLUSION: Our findings indicate that Plin5 deficiency alters cardiac lipid metabolism and associates with reduced survival following myocardial ischemia, suggesting that Plin5 plays a beneficial role in the heart following ischemia.

Extracellular mtDNA activates NF-κB via toll-like receptor 9 and induces cell death in cardiomyocytes.

Acute myocardial infarction (AMI) causes sterile inflammation, which exacerbates tissue injury. Elevated levels of circulating mitochondrial DNA (mtDNA) have been associated with AMI. We hypothesized that mtDNA triggers an innate immune response via TLR9 and NF-κB activation, causing cardiomyocyte injury. Murine cardiomyocytes express TLR9 mRNA and protein and were able to internalize fluorescently labeled mouse mtDNA. Incubation of human embryonic kidney cells with serum from AMI patients containing naturally elevated levels of mtDNA induced TLR9-dependent NF-κB activity. This effect was mimicked by isolated mtDNA. mtDNA activated NF-κB in reporter mice both in vivo and in isolated cardiomyocytes. Moreover, incubation of isolated cardiomyocytes with mtDNA induced cell death after 4 and 24 h. Laser confocal microscopy showed that incubation of cardiomyocytes with mtDNA accelerated mitochondrial depolarization induced by reactive oxygen species. In contrast to mtDNA, isolated total DNA did not activate NF-κB nor induce cell death. In conclusion, mtDNA can induce TLR9-dependent NF-κB activation in reporter cells and activate NF-κB in cardiomyocytes. In cardiomyocytes, mtDNA causes mitochondrial dysfunction and death. Endogenous mtDNA in the extracellular space is a danger signal with direct detrimental effects on cardiomyocytes.

Hyperexcitability in Spinal WDR Neurons following Experimental Disc Herniation Is Associated with Upregulation of Fractalkine and Its Receptor in Nucleus Pulposus and the Dorsal Root Ganglion.

Introduction. Lumbar radicular pain following intervertebral disc herniation may be associated with a local inflammatory response induced by nucleus pulposus (NP) cells. Methods. In anaesthetized Lewis rats, extracellular single unit recordings of wide dynamic range (WDR) neurons in the dorsal horn and qPCR were used to explore the effect of NP application onto the dorsal nerve roots (L3-L5). Results. A clear increase in C-fiber response was observed following NP conditioning. In the NP tissue, the expression of interleukin-1ß (IL-1ß), colony stimulating factor 1 (Csf1), fractalkine (CX3CL1), and the fractalkine receptor CX3CR1 was increased. Minocycline, an inhibitor of microglial activation, inhibited the increase in neuronal activity and attenuated the increase in IL-1ß, Csf1, CX3L1, and CX3CR1 expression in NP tissue. In addition, the results demonstrated an increase in the expression of TNF, CX3CL1, and CX3CR1 in the dorsal root ganglions (DRGs). Conclusion. Hyperexcitability in the pain pathways and the local inflammation after disc herniation may involve upregulation of CX3CL1 signaling in both the NP and the DRG.

Expression of bone morphogenetic protein 4 and its receptors in the remodeling heart.

AIMS: Heart failure is associated with activation of fetal gene programs. Bone morphogenetic proteins (BMPs) regulate embryonic development through interaction with BMP receptors (BMPRs) on the cell surface. We investigated if the expression of BMP4 and its receptors BMPR1a and BMPR2 were activated in post-infarction remodeling and heart failure. MAIN METHODS: Left ventricular biopsies were taken from explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy (CMP; n=15) or ischemic heart disease (CAD; n=9), and compared with homograft control preparations from organ donors deceased due to non-cardiac causes (n=7). Other samples were taken from patients undergoing coronary artery bypass grafting (CABG; n=11). Mice were subjected to induced infarction by permanent coronary artery ligation or sham operation, and hearts were sampled serially thereafter (n=7 at each time point). KEY FINDINGS: Human and mouse hearts expressed BMP4 and both receptor subtypes. CABG and CMP patients had increased expression of mRNA encoding for BMP4, but unchanged protein. Mouse hearts had increased BMP4 precursor protein 24h after infarction. BMPR1a protein decreased in CAD patients and initially in postinfarcted mouse hearts, but increased again in the latter after two weeks. Human recombinant BMP4 promoted survival after H2O2 injury in HL-1 cells, and also protected adult mouse cardiomyocytes against hypoxia-reoxygenation injury. SIGNIFICANCE: Adult hearts express BMP4, the mRNA increasingly so in patients with coronary artery disease with good cardiac function. BMPRs are downregulated in cardiac remodeling and failure. Recombinant BMP4 has protective effects on cultured cardiomyocytes.