Antiarrhythmiás hatású mechanizmusok in vivo és in vitro tanulmányozása = In vivo and in vitro analysis of antiarrhythmic mechanisms

Az aritmiák csökkentésére irányuló korábbi munkánk folytatásaként a 2008-2012 kutatási periódusban a következő eredményeket értük el. A gap junkciós (GJ) csatornák szerepét vizsgálva kimutattuk, hogy ezen csatornák iszkémia alatti nyitása és zárása is, paradox módon, csökkenti az iszkémia/reperfúzió okozta kamrai aritmiákat. Elsőként közöltük, hogy a prekondicionálás egyik fő mediátoraként tekintett nitrogén monoxid (NO), a szívizom GJ csatornáit is módosítani képes; in vivo és in vitro kísérleteink eredményei igazolták, hogy a szerves és szervetlen nitritekből származó NO gátolja a GJ csatornák szétkapcsolását és az aritmiák kialakulását. Igazoltuk a GJ csatornák szerepét a szívingerléssel kiváltott késői védőhatásban rámutatva arra, hogy a gyors szívingerlés időfüggő változást okoz mind a GJ csatornákat felépítő Cx43 gén és fehérje, mind más olyan egyéb gének expressziójában, amelyek a késői védőhatás kialakulásáért felelős fehérjéket kódolnak. A NO és a ROS kardioprotekcióban játszott szerepét vizsgáló kísérleteinkben kimutattuk, hogy az iszkémia okozta csökkent NO képződés fokozza az aritmiákat és bármilyen olyan beavatkozás, amely a NO hozzáférhetőségét helyreállítja védőhatású lehet. Kísérleteinkben a nátrium nitrit infúziója jelentős mértékben csökkentette az aritmiákat feltehetően a fehérjék S-nitrozilációja és a ROS-képződés NO általi gátlása révén. A projekt sikeres teljesítését Ph.D. hallgatók és asszisztensek munkája, és az OTKA támogatása tette lehetővé. | Continuing our previous research on protection against arrhythmias our achievements between 2008 and 2012 were as the follows. In studies, aiming to explore the role of gap junctions (GJ), we have shown that the pharmacological modulation of GJs using either GJ openers or blockers reduces arrhythmias, resulting from ischaemia and reperfusion. We were the first to propose that nitric oxide (NO), a key mediator of the preconditioning-induced protection modifies myocardial GJ function. Studies in vivo and in vitro have revealed that both organic and inorganic NO donors attenuate the uncoupling of GJs and this effect plays a role in the protection. We have also shown that GJs are involved in the delayed antiarrhythmic effect of cardiac pacing. Rapid pacing induces time-dependent changes in Cx43 gene and protein expressions, and in the translation of many other genes, encoding proteins involved in the late phase of the protection. In studies, aiming to explore the role of NO and reactive oxygen species (ROS) in cardioprotection, we have found that the ischaemia-induced loss in NO production leads to increased arrhythmia generation and that any manoeuvre which increases NO availability, would result in protection. Sodium nitrite has been found to reduce arrhythmias via the regulation of ROS production and protein S-nitrosylation. The work of young scientists and technical assistants, and the support of the OTKA are largely contributed to the successful completion of the project

High Mobility Group Box 1 Protein Induction by Mycobacterium Bovis BCG

High mobility group box 1 protein (HMGB1), a nuclear protein, is a critical cytokine that mediates the response to infection, injury, and inflammation. The aim of our study was to elaborate a reliable in vitro model to investigate whether Mycobacterium bovis BCG is able to induce HMGB1 secretion from the monocytic U-937 cells. Western blot technique was applied for the detection of HMGB1 from supernatants of cells, following induction with Mycobacterium bovis BCG. Densitometric analysis revealed higher concentrations of HMGB1 in cell supernatants stimulated with BCG than in the supernatants of the control, nonstimulated cells. Further quantitation of the secreted HMGB1 was performed by ELISA. The BCG strain resulted in a higher amount of secreted HMGB1 (450 ± 44 ng/mL) than that of LPS (84 ± 12 ng/mL) or Staphylococcus aureus (150 ± 14 ng/mL). BCG and Phorbol −12-myristate −13 acetate (PMA), added together, resulted in the highest HMGB1 secretion (645 ± 125 ng/mL). The translocation of the HMGB1 towards the cytoplasm following infection of cells with BCG was demonstrated by immunofluorescence examinations. Conclusion: Our pilot experiments draw attention to the HMGB1 inducing ability of Mycobacterium bovis. Assesment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations

Propionibacterium acnes induces autophagy in keratinocytes: involvement of multiple mechanisms

Propionibacterium acnes is a dominant member of the cutaneous microbiota. Herein, we evaluate the effects of different P. acnes strains and propionic acid on autophagy in keratinocytes. Our results showed that P. acnes strain 889 altered the architecture of the mitochondrial network, elevated the levels of LC3B-II, Beclin-1 and phospho-AMPKalpha, stimulated autophagic flux, facilitated intracellular redistribution of LC3B, increased average number of autophagosomes per cell, and enhanced development of acidic vesicular organelles in the HPV-KER cell line. Propionic acid increased the level of phospho-AMPKalpha, enhanced lipidation of LC3B, stimulated autophagic flux, as well as facilitated translocation of LC3B into autophagosomes in HPV-KER cells. P. acnes strains 889, 6609 and heat-killed strain 889 also stimulated autophagosome formation in primary keratinocytes to varying degrees. These results indicate that cell wall components and secreted propionic acid metabolite of P. acnes evoke mitochondrial damage successively, thereby trigger AMPK-associated activation of autophagy, which in turn facilitates the removal of dysfunctional mitochondria and promotes survival of keratinocytes. Thus, we suggest that low-level colonization of hair follicles with non-invasive P. acnes strains, by triggering a local increase in autophagic activity, might exert a profound effect on several physiological processes responsible for the maintenance of skin tissue homeostasis

IL-36α and Lipopolysaccharide Cooperatively Induce Autophagy by Triggering Pro-Autophagic Biased Signaling

Autophagy is an intracellular catabolic process that controls infections both directly and indirectly via its multifaceted effects on the innate and adaptive immune responses. It has been reported that LPS stimulates this cellular process, whereas the effect of IL-36α on autophagy remains largely unknown. We therefore investigated how IL-36α modulates the endogenous and LPS-induced autophagy in THP-1 cells. The levels of LC3B-II and autophagic flux were determined by Western blotting. The intracellular localization of LC3B was measured by immunofluorescence assay. The activation levels of signaling pathways implicated in autophagy regulation were evaluated by using a phosphokinase array. Our results showed that combined IL-36α and LPS treatment cooperatively increased the levels of LC3B-II and Beclin-1, stimulated the autophagic flux, facilitated intracellular redistribution of LC3B, and increased the average number of autophagosomes per cell. The IL36α/LPS combined treatment increased phosphorylation of STAT5a/b, had minimal effect on the Akt/PRAS40/mTOR pathway, and reduced the levels of phospho-Yes, phospho-FAK, and phospho-WNK1. Thus, this cytokine/PAMP combination triggers pro-autophagic biased signaling by several mechanisms and thus cooperatively stimulates the autophagic cascade. An increased autophagic activity of innate immune cells simultaneously exposed to IL-36α and LPS may play an important role in the pathogenesis of Gram-negative bacterial infections

Sarcolemmal Ca2+-entry through L-type Ca2+ channels controls the profile of Ca2+-activated Cl- current in canine ventricular myocytes

Ca2+-activated Cl- current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca2+]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca2+]i was monitored using Fura-2-AM. Setting [Ca2+]i to the systolic level measured in the bulk cytoplasm (1.1muM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca2+-entry through L-type Ca2+ channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca2+-entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA

Cerebrovascular Pathology in Hypertriglyceridemic APOB-100 Transgenic Mice

Hypertriglyceridemia is not only a serious risk factor in the development of cardiovascular diseases, but it is linked to neurodegeneration, too. Previously, we generated transgenic mice overexpressing the human APOB-100 protein, a mouse model of human atherosclerosis. In this model we observed high plasma levels of triglycerides, oxidative stress, tau hyperphosphorylation, synaptic dysfunction, cognitive impairment, increased neural apoptosis and neurodegeneration. Neurovascular dysfunction is recognized as a key factor in the development of neurodegenerative diseases, but the cellular and molecular events linking cerebrovascular pathology and neurodegeneration are not fully understood. Our aim was to study cerebrovascular changes in APOB-100 transgenic mice. We described the kinetics of the development of chronic hypertriglyceridemia in the transgenic animals. Increased blood-brain barrier permeability was found in the hippocampus of APOB-100 transgenic mice which was accompanied by structural changes. Using transmission electron microscopy, we detected changes in the brain capillary endothelial tight junction structure and edematous swelling of astrocyte endfeet. In brain microvessels isolated from APOB-100 transgenic animals increased Lox-1, Aqp4, and decreased Meox-2, Mfsd2a, Abcb1a, Lrp2, Glut-1, Nos2, Nos3, Vim, and in transgenic brains reduced Cdh2 and Gfap-σ gene expressions were measured using quantitative real-time PCR. We confirmed the decreased P-glycoprotein (ABCB1) and vimentin expression related to the neurovascular unit by immunostaining in transgenic brain sections using confocal microscopy. We conclude that in chronic hypertriglyceridemic APOB-100 transgenic mice both functional and morphological cerebrovascular pathology can be observed, and this animal model could be a useful tool to study the link between cerebrovascular pathology and neurodegeneration