Glucocorticoid-Induced Cardioprotection: A Novel Role for Autophagy

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Cell death in hyppxic injury : signaling mechanisms and dynamics in the decision making process

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2009ENGLISH ABSTRACT: Three main morphologies of cell death have been described in the diseased myocardium, type I, better known as apoptotic cell death, which is characterized by cell shrinkage and chromatin condensation, type II, or cell death with autophagy, presents a morphology with intracellular accumulation of autophagic vacuoles and type III, better known as necrosis, is characterized by cellular swelling and rapid loss in cellular membrane integrity. However, recent literature strongly argues against rigid classifications in the context of cell death mechanisms but rather suggests to adopt a view of cell death as a dynamic and integrative cellular response. Furthermore, the contribution of autophagy in cell death or cell survival is still poorly understood. Therefore the aims of this study were twofold: (i) to characterize the contribution of each cell death type in context of the severity and duration of an ischaemic insult and (ii) to determine whether manipulation of the autophagic pathway affects the contribution of cell death and translates into protection of the heart. Rodent derived cardiac myoblast cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions. Cells were submitted to protocols of 2, 4 and 8 hrs of simulated ischaemia (SI) under hypoxic conditions in a humidified environment containing 0.1% O2, 5% CO2 and the balance N2, followed by 1 hr of reperfusion respectively. We employed a modified ischaemic buffer containing either 2-deoxy- D-glucose, sodium dithionate or both, with the aim to create an ischaemic insult of mild (mild SI), moderate (moderate SI) and severe (severe SI) character respectively. We evaluated the contribution of each cell death mode using a combination of viability- and ATP assays. Molecular markers for each cell death process such as LC3, PARP and HMGB1 were evaluated using 3-dimensional fluorescence techniques as well as western blot analysis and flow cytometry. Next, autophagy was induced or inhibited prior to the ischaemic insult, using rapamycin and 3MA respectively, and similar parameters were evaluated after 2 hours of mild or moderate SI. Propidium Iodide exclusion and Fluorescence Resonance Energy Transfer (FRET) in combination with mitochondrial inner membrane depolarization were employed to assess the onset of cell death dynamically. Flow cytometry was employed to evaluate the degree of protection. In addition, the ATP levels and reactive oxygen species (ROS) were evaluated. Our results strongly indicate a differential induction of cell death, which is dependent on the severity and duration of the ischaemic insult. Mild SI led to the induction of autophagy and apoptosis, whilst moderate or severe SI induced both apoptotic and necrotic cell death without an indication of autophagy. Only mild SI, but not moderate and severe SI, resulted in an ATP surge. Moreover, our data provide direct evidence that increased autophagy delays the loss of cellular membrane integrity and delays caspase-3 activation as well as mitochondrial depolarization in ischaemic cardiomyocytes. Our results show a profound effect of increased autophagy on the onset of apoptosis as well as necrosis under simulated ischaemic conditions, providing cellular protection. This ATP surge observed during mild SI was abolished with increased autophagy. Furthermore, our results indicate a profound effect of autophagy on ROS generation. Under normoxic conditions, increased autophagy induced a significant decrease in ROS while the inhibition of autophagy significantly increased ROS generation. However, when increasing or decreasing autophagy prior to the ischaemic insult, ROS increased significantly in both scenarios. The results suggest that the severity of ischaemia determines the mode of cell death differentially. An increase in autophagic responsiveness and flux, as induced through rapamycin treatment, provides a selective advantage for tissue against injury, possibly by maintaining intracellular ATP levels through the provision of metabolic substrates. Autophagy is described as an inherent cellular mechanism v which affects the onset of cell death and exhibits protective effects in the ischaemic myocardium when upregulated prior to the ischaemic insult. The protective effect of increased autophagy was mirrored in the isolated perfused rat heart model, reflected by improved functional recovery during ischaemia/reperfusion.AFRIKAANSE OPSOMMING: Die drie belangrikste morfologiese beskrywings van seldood in die hart sluit die volgende in: tipe I, beter bekend as apoptose wat gekenmerk word deur selkrimping en chromatienkondensering, tipe II, of seldood deur middel van autofagie wat gekenmerk word deur die intrasellulêre versameling van autofagiese vakuole en tipe III, beter bekend as nekrose wat gekenmerk word deur sel swelling en ‘n vinnige verlies aan membraanintegriteit. Onlangse literatuur waarsku egter teen die onbuigsame klassifikasie van seldoodmeganismes en stel voor dat seldood as ‘n dinamiese proses met integrerende sellulêre meganismes beskou moet word. Die bydrae van autofagie in seldoodmeganismes word ook nog nie goed verstaan nie. Die doel van hierdie studie is dus tweevoudig: (i) om die bydrae van elke tipe seldood te bepaal in konteks van die felheid en tydperk van die iskemiese ingryping en (ii) om te bepaal of the manupilering van autofagie ‘n betekenisvolle bydrae lewer in seldoodmeganismes en sodoende tot beskerming van die hart kan lei. Kardiale mioblaste wat van rotweefsel afkomstig is, is in Dulbecco se gemodifiseerde Eagle medium (DMEM), waarby daar 10% fetale kalfserum gevoeg is en wat onderhewig was aan 5% CO2 toestande, onderhou. Selle was onderhewig aan protokolle van 2, 4 en 8 ure gesimuleerde iskemie (SI) onder hipoksiese toestande in ‘n humiditeitsomgewing wat 0.1% O2, 5% CO2 en die balans N2 bevat. Daarna was die selle onderhewig aan 1 uur reperfusie. ‘n Gemodifiseerde iskemiese buffer wat óf 2-deoksie-D-glukose óf natriumdithionaat, of beide bevat, is gebruik om lig, matig en strawwe iskemiese toestande na te boots. Die bydrae van elke tipe seldood is geëvalueer tydens bogenoemde toestande deur gebruik te maak van ‘n kombinasie van sellewensvatbaarheid- en ATP tegnieke. Molekulêre merkers, wat LC3, PARP en HMGB1 insluit, is gebruik om deur middel van 3-dimensionele fluoresensie tegnieke, westelike kladtegnieke en vii vloeisitometrie die verskillende vorme van seldood te ondersoek. Autofagie is ook geïnduseer en geïnhibeer voor die iskemiese ingryping, deur middel van rapamycin en 3MA, respektiewelik om die rol van autofagie tydens seldood te bepaal. Propidium iodite uitluiting en fluoresensie resonansie energie oordrag (FRET) in kombinasie met ‘n merker vir mitochondriale binneste membraan depolarisasie is gebruik om die aanvang van seldood dinamies te ondersoek. Vloeisitometrie is gebruik om die graad van beskerming aan te dui, terwyl intrasellulêre ATP vlakke en reaktiewe suurstof spesies (ROS) ook gemeet is. Ons resultate het getoon dat daar ‘n differensiële indusering van seldood plaasvind wat afhanklik is van die felheid en tydsduur van die iskemiese ingryping. ‘n Ligte graad van iskemie lei tot die indusering van autofagie en apoptose, terwyl matige en strawwe iskemie beide apoptose en nekrose induseer sonder autofagie. Verder het slegs ‘n ligte graad van iskemie ‘n skerp styging in ATP tweeggebring, terwyl dit nie die geval was tydens matige en strawwe iskemie nie. Ons data verskaf ook direkte bewyse dat ‘n toename in autofagie die verlies van sellulêre membraanintegriteit vertraag. Dit het ook ‘n vermindering in caspase-3 aktivering en mitochondriale depolarisasie in iskemiese kardiomiosiete teweegebring. Die data dui aan dat ‘n toename in autofagie ‘n beduidende effek op apoptose en nekrose tydens gesimuleerde iskemiese toestande het om sodoende selbeskerming te verskaf. Die skerp styging in ATP wat tydens die ligte graad van iskemie teweeggebring is, is opgehef met ‘n toename in autofagie. Ons resultate dui ook daarop dat autofagie ‘n beduidende rol in ROS generering speel. Onder normoksiese omstandighede veroorsaak ‘n toename in autofagie ‘n insiggewende afname in ROS generering, terwyl die inhibisie van autofagie ROS generering insiggewend laat toeneem. Wanneer autofagie egter voor die iskemiese ingryping verhoog of verlaag word, vermeerder ROS generering in beide gevalle. Hierdie resultate bewys dat die graad van iskemie ‘n invloed het op die tipe seldood wat geïnduseer word. ‘n Toename in autofagie reaksietyd en vloei, soos viii bewerkstellig deur rapamycin, verskaf ‘n selektiewe voordeel vir weefsel teen beskadiging, heel waarskynlik deur die handhawing van intrasellulêre ATP-vlakke deur die verskaffing van metaboliese substrate. Autofagie word beskryf as ‘n inherente sellulêre meganisme wat seldood beïnvloed en die iskemiese miokardium beskerm wanneer dit opgereguleer word voor die iskemiese ingryping. Hierdie beskermende rol van autofagie wat in die weefselkultuur waargeneem is, is ook in die geïsoleerde geperfuseerde rot hart model waargeneem, waar funksionele herstel verbeter is tydens iskemie/reperfusie

Stochastic Thermodynamics of a Probe in a Fluctuating Correlated Field

We develop a framework for the stochastic thermodynamics of a probe coupled to a fluctuating medium with spatio-temporal correlations, described by a scalar field. For a Brownian particle dragged by a harmonic trap through a fluctuating Gaussian field, we show that near criticality the spatially-resolved heat flux develops an unexpected dipolar structure, where on average heat is absorbed in front, and dissipated behind, the particle. Moreover, a perturbative calculation reveals that the dissipated power displays three distinct dynamical regimes depending on the drag velocity.Comment: 7 pages, 3 figures (main) + 21 pages, 1 figure (SM

St John's Wort (Hypericum perforatum L.) photomedicine: hypericin-photodynamic therapy induces metastatic melanoma cell death

Hypericin, an extract from St John's Wort ( Hypericum perforatum L. ), is a promising photosensitizer in the context of clinical photodynamic therapy due to its excellent photosensitizing properties and tumoritropic characteristics. Hypericin-PDT induced cytotoxicity elicits tumor cell death by various mechanisms including apoptosis, necrosis and autophagy-related cell death. However, limited reports on the efficacy of this photomedicine for the treatment of melanoma have been published. Melanoma is a highly aggressive tumor due to its metastasizing potential and resistance to conventional cancer therapies. The aim of this study was to investigate the response mechanisms of melanoma cells to hypericin-PDT in an in vitro tissue culture model. Hypericin was taken up by all melanoma cells and partially co-localized to the endoplasmic reticulum, mitochondria, lysosomes and melanosomes, but not the nucleus. Light activation of hypericin induced a rapid, extensive modification of the tubular mitochondrial network into a beaded appearance, loss of structural details of the endoplasmic reticulum and concomitant loss of hypericin co-localization. Surprisingly the opposite was found for lysosomal-related organelles, suggesting that the melanoma cells may be using these intracellular organelles for hypericin-PDT resistance. In line with this speculation we found an increase in cellular granularity, suggesting an increase in pigmentation levels in response to hypericin-PDT. Pigmentation in melanoma is related to a melanocyte-specific organelle, the melanosome, which has recently been implicated in drug trapping, chemotherapy and hypericin-PDT resistance. However, hypericin-PDT was effective in killing both unpigmented (A375 and 501mel) and pigmented (UCT Mel-1) melanoma cells by specific mechanisms involving the externalization of phosphatidylserines, cell shrinkage and loss of cell membrane integrity. In addition, this treatment resulted in extrinsic (A375) and intrinsic (UCT Mel-1) caspase-dependent apoptotic modes of cell death, as well as a caspase-independent apoptotic mode that did not involve apoptosis-inducing factor (501 mel). Further research is needed to shed more light on these mechanisms

How do taxes affect the trading behavior of private investors? Evidence from individual portfolio data

We exploit a large reform of capital-gains taxation in Germany combined with portfolio-level daily panel data to study the causal effect of taxes on individual stock-trading behavior and the disposition effect. We find substantial spikes in selling probabilities around an intertemporal tax discontinuity, and no such spikes after the abolishment of the discontinuity. Using difference-in-bunching methods, non-parametric regressions and effective tax rates, we quantify the tax effect and identify interesting patterns of heterogeneity. We further find evidence that the well-established disposition effect is strongly affected by the tax discontinuity through tax motivated selling of both gains and losses

Flexible State-Merging for learning (P)DFAs in Python

We present a Python package for learning (non-)probabilistic deterministic finite state automata and provide heuristics in the red-blue framework. As our package is built along the API of the popular \texttt{scikit-learn} package, it is easy to use and new learning methods are easy to add. It provides PDFA learning as an additional tool for sequence prediction or classification to data scientists, without the need to understand the algorithm itself but rather the limitations of PDFA as a model. With applications of automata learning in diverse fields such as network traffic analysis, software engineering and biology, a stratified package opens opportunities for practitioners

Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

<p>Abstract</p> <p>Background</p> <p>Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation.</p> <p>Results</p> <p>During a yeast 2-hybrid (Y2H) library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL) as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP).</p> <p>To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor.</p> <p>We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by <it>in vivo </it>co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under β-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage.</p> <p>Conclusions</p> <p>This study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL is an important regulator of cardiac contractility. This has further implications for understanding the patho-aetiology of HCM-causing mutations in the genes encoding cMyBPC and cTNI, and raises the question of whether MMGL might itself be considered a candidate HCM-causing or modifying factor.</p

Electrolysis in reduced gravitational environments: current research perspectives and future applications

Electrochemical energy conversion technologies play a crucial role in space missions, for example, in the Environmental Control and Life Support System (ECLSS) on the International Space Station (ISS). They are also vitally important for future long-term space travel for oxygen, fuel and chemical production, where a re-supply of resources from Earth is not possible. Here, we provide an overview of currently existing electrolytic energy conversion technologies for space applications such as proton exchange membrane (PEM) and alkaline electrolyzer systems. We discuss the governing interfacial processes in these devices influenced by reduced gravitation and provide an outlook on future applications of electrolysis systems in, e.g., in-situ resource utilization (ISRU) technologies. A perspective of computational modelling to predict the impact of the reduced gravitational environment on governing electrochemical processes is also discussed and experimental suggestions to better understand efficiency-impacting processes such as gas bubble formation and detachment in reduced gravitational environments are outlined