Search

The present study investigates the flexibility of statistically learned distractor suppression between different contexts. Participants performed the additional singleton task searching for a unique shape, while ignoring a uniquely colored distractor. Crucially, we created two contexts within the experiments, and each context was assigned its own high-probability distractor location, so that the location where the distractor was most likely to appear depended on the context. Experiment 1 signified context through the color of the background. In Experiment 2, we aimed to more strongly differentiate between the contexts using an auditory or visual cue to indicate the upcoming context. In Experiment 3, context determined the appropriate response ensuring that participants engaged the context in order to be able to perform the task. Across all experiments, participants learned to suppress both high-probability locations, even if they were not aware of these spatial regularities. However, these suppression effects occurred independent of context, as the pattern of suppression reflected a de-prioritization of both high-probability locations which did not change with the context. We employed Bayesian analyses to statistically quantify the absence of context-dependent suppression effects. We conclude that statistically learned distractor suppression is robust and generalizes across contexts.

Introduction. Septic shock is a systemic inflammatory response syndrome associated with organ failures. Earlier clinical diagnosis would be of benefit to a decrease in the mortality rate. However, there is currently a lack of predictive biomarkers. The secretome is the set of proteins secreted by a cell, tissue, or organism at a given time and under certain conditions. The plasma secretome is easily accessible from biological fluids and represents a good opportunity to discover new biomarkers that can be studied with nontargeted “omic” strategies. Aims. To identify relevant deregulated proteins (DEP) in the secretome of a rat endotoxemic shock model. Methods. Endotoxemic shock was induced in rats by intravenous injection of lipopolysaccharides (LPS, S. enterica typhi, 0.5 mg/kg) and compared to controls (Ringer Lactate, iv). Under isoflurane anesthesia, carotid cannulation allowed mean arterial blood pressure (MAP) and heart rate (HR) monitoring and blood sampling at different time points (T0 and T50 or T0 and T90, with EDTA and protease inhibitor). Samples were prepared for large-scale tandem mass spectrometry (MS-MS) based on a label-free quantification to allow identification of the proteins deregulated upon endotoxemic conditions. A Gene Ontology (GO) analysis defined several clusters of biological processes (BP) in which the DEP are involved. Results. Ninety minutes after shock induction, the LPS group presents a reduction in MAP (-45%, p < 0.05 ) and increased lactate levels (+27.5%, p < 0.05 ) compared to the control group. Proteomic analyses revealed 10 and 33 DEP in the LPS group, respectively, at 50 and 90 minutes after LPS injection. At these time points, GO-BP showed alterations in pathways involved in oxidative stress response and coagulation. Conclusion. This study proposes an approach to identify relevant DEP in septic shock and brings new insights into the understanding of the secretome adaptations upon sepsis.

Introduction Mutations in KCNH2 (coding for hERG) cause long or short QT syndromes (LQTS or SQTS), predisposing to life-threatening arrhythmias. More than 1000 variations in hERG sequences have been described, most of them are to be characterized. Objective The objective is to standardize and accelerate the entire process necessary to phenotype hERG variants. An in silico evaluation was also included to characterize the structural impact of the variants. Methods We selected 12 variants from patients with LQTS, and 1 with SQTS. We optimized the protocol to efficiently introduce mutations in hERG cDNA despite GC-rich sequences, using the Gibson assembly strategy. A pH-sensitive fluorescent tag was fused to hERG for fast-track evaluation of hERG cell trafficking. An optimized patch-clamp protocol of 35 sec was developed to evaluate hERG channel activity in transfected cells. R software was used to speed up analyses. Results In the present work, we observed a good correlation between cell surface expression, assessed by the pH-sensitive tag, and current densities. Also, we showed that the new biophysical protocol allows a significant gain of time in recording ion channel properties and provides extensive information on WT and variant channel biophycical parameters, that can all be recapitulated in a single parameter defined herein as the repolarization power. The impact of the variants on channel structure were also reported where structural information was available. These three readouts (trafficking, repolarization power and structural impact) define 3 pathogenicity indexes that may help clinical diagnosis. Conclusion Fast track characterization of KCNH2 genetic variants shows its relevance to discriminate mutants that affect hERG channel activity from variants with undetectable effects. This information is meant to fill a patient database, as a basis for personalized medicine. The next steps will be to further accelerate the process using an automated patch-clamp system.

Background We have shown that increase in O-GlcNAc levels, a post-translational modification, at the early phase of septic shock in adult (84 days old rats) resulted in a 3-fold increase in survival time. Most studies focus only in adults, and the population most affected by septic shock, young children, is rarely studied. Sepsis, with a mortality rate of 29%, is an important but preventable cause of pediatric death worldwide. Considering that basal O-GlcNAc levels are higher in the young, the potential impact of O-GlcNAc stimulation at the early phase of septic shock in the young should be evaluated. Purpose Evaluate if O-GlcNAc stimulation could improve sepsis outcomes in young. Methods Endotoxemic shock was induced in 28 days old rats with an i.v. injection of saline (CTRL) or LPS (O111:B4, 20 mg.kg−1 – LPS). 1 hour after LPS rats were randomly assigned to no therapy (LPS), fluidotherapy (saline, 10 mL.kg−1 – LPS + R) ± NButGT (10 mg.kg−1 – NButGT) to increase O-GlcNAc levels. 2 hours later, physiological functions and plasmatic markers were measured and used in adapted Pediatric RISk of Mortality score (PRISM score). The impact of treatment on survival was evaluated on n = 64. Results LPS induced a shock (mean arterial pressure (MAP): CTRL: 67.2 ± 1.9; LPS: 50.7 ± 2.1; mmHg; P Conclusion Despite higher O-GlcNAc levels in young population, we demonstrate that O-GlcNAc stimulation is able to improve pups’ response to endotoxemic shock. Our results show that it is the difference in O-GlcNAcylation between the basal levels and the post-stimulation levels that induces protection against septic shock.

Background Mitochondria is a key cellular component. During pathophysiological stress mitochondrial function is regulated dynamically to provide cell with sufficient energy. O-GlcNAcylation, a post-translational modification associated with stress response, has been linked to beneficial effect when stimulated in shock situation, yet the link between O-GlcNAc and mitochondrial function remains poorly understood. Aim To evaluate mitochondrial impact of O-GlcNAc modulation. Methods Hearts were harvested from Wistar rat from Ctrl, shocked (LPS) and treated by NButGT, a pharmacological O-GlcNAcylation inducer. Respiration rate was assessed polarographically in isolated mitochondria. O-GlcNAcylomic was performed by mass spectrometry on cardiac tissue from rats at D0 (after birth) and D28 (suckling weaning) as well as from LPS and NButGT treated animals. Gene Ontology (GO) for cellular components (CC) of identified cardiac O-GlcNAcylated proteins from both groups were obtained by running the clusterProfileR package. Results Within D0/28 groups, 647 O-GlcNAcylated proteins were found and 1012 in Ctrl/LPS/NButGT groups. Protein for subsequent analysis were selected by the log(FoldChange) > 2 or Conclusion Mitochondrial O-GlcNAcylation appears to play a major role in pathophysiology. Targeting mitochondrial O-GlcNAcylation could represent a new therapeutic challenge and should be further explored.

Introduction Understanding endothelial dysfunction is at the heart of current concerns due to its involvement in many cardiovascular diseases such as heart failure (HF). The β3-adrenergic receptor (β3-AR) could be at the origin of this endothelial dysfunction. Indeed, it is expressed at the level of the endothelial cell and induces vasorelaxation by the release of nitric oxide (NO), a central actor in endothelial dysfunction. Objective Our objective was therefore to study the overexpression of the receptor in endothelial cells to understand its involvement in endothelial dysfunction. Method We used the Tgβ3 rat at 45 weeks of age, which overexpresses the human β3-AR gene in the endothelium. Endothelium-dependent aortic relaxation versus isoproterenol was measured with or without L-VNIO an inhibitor of nNOS at 10 μM. The protein expression of NOS was evaluated by Western-Blot and the production of NO and O2- was evaluated by EPR on aorta. Results Vascular reactivity was significantly reduced in response to isoproterenol in Tgβ3 aorta (−10% P Conclusion The increase in β3-AR expression induces endothelial dysfunction with a key role of eNOS and nNOS. Further studies will be conducted to understand the causal links between endothelial dysfunction and the alteration of the cardiac function.

Cardio-metabolic diseases (CMD; cardiovascular disease, type 2 diabetes, chronic kidney disease) represent a global public health problem. Worldwide, nearly half a billion people are currently diagnosed with diabetes, and cardiovascular disease is the leading cause of death. Most of these diseases can be assuaged/prevented through behavior change. However, the best way to implement preventive interventions is unclear. We aim to fill this knowledge gap by creating an evidence-based and adaptable “toolbox” for the design and implementation of selective prevention initiatives (SPI) targeting CMD. We built our toolbox based on evidence from a pan-European research project on primary-care SPIs targeting CMD. The evidence includes (1) two systematic reviews and two surveys of patient and general practitioner barriers and facilitators of engaging with SPIs, (2) a consensus meeting with leading experts to establish optimal SPI design, and (3) a feasibility study of a generic, evidence-based primary-care SPI protocol in five European countries. Our results related primarily to the five different national health-care contexts from which we derived our data. On this basis, we generated 12 general recommendations for how best to design and implement CMD-SPIs in primary care. We supplement our recommendations with practical, evidence-based suggestions for how each recommendation might best be heeded. The toolbox is generic and adaptable to various national and systemic settings by clinicians and policy makers alike. However, our product needs to be kept up-to-date to be effective and we implore future research to add relevant tools as they are developed.

Introduction The secretome is a set of proteins secreted by a cell at a given time and under certain conditions. The secretome, easily accessible from plasma and analyzable by a proteomic approach, represents a good opportunity to identify biomarkers in septic shock. This pathology corresponds to a systemic inflammatory response syndrome related to an infection and associated with cardiocirculatory failure leading to organ dysfunction with a mortality rate of 40%. The identification of diagnostic/prognostic biomarkers is crucial to improve patient outcome. Objective Study the secretome of a rat model of endotoxemic shock to identify relevant deregulated proteins (DEP). Methods Endotoxemic shock was induced by lipopolysaccharides injection (LPS, S. enterica typhi, 0.5 mg/kg, iv) and compared to controls (Ringer lactate, iv). Under isoflurane anesthesia, carotid cannulation allowed evaluation of mean arterial blood pressure (MAP), heart rate and blood sampling (T0, and T50 or T90 min on EDTA and protease inhibitor). Plasma was analyzed with tandem MS based on a label free quantification. Gene ontology (GO) analyses defined several groups of biological process (BP) in which the DEP were involved. Results Ninety minutes after shock induction, the LPS group present a reduction in MAP (−45%, P Conclusion This study proposes an approach to identify DEP in septic shock. DEP need to be tested in cellular models in order to understand their role in the physiopathology of shock and to improve understanding of adaptations of the secretome during sepsis. The link between our results and patient prognosis will need to be studied for clinical use.

Introduction During post-natal development, the organism is subjected to 2 metabolic transitions. The weaning transition is particularly interesting as it is associated with a change in glucose metabolism. O-GlcNAcylation is an ubiquitous post-translational modification, described as a nutrient sensor. The GlcNAc moiety is added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Interestingly, cardiac O-GlcNAc levels decrease throughout post-natal development but its regulatory mechanism and impact on proteins have never been studied under this condition. Objective Study the regulatory mechanism involved in O-GlcNAc regulation and its potential impact on proteins implied in cardiac metabolism. Method Hearts from 28 day-olds (D28) Wistar rats fed with a standard or low-carbohydrate (low-CHO) diet (D28F: 50% fat, 30% protein, 3% carbohydrates) during the weaning period (D12 to D28) were studied. O-GlcNAc levels and regulatory enzymes expression were evaluated by western blot. The OGA activity was evaluated with a spectrophotometric assay. The impact of selected protein expression in response to O-GlcNAc levels variations (Thiamet-G, OGA inhibitor) was evaluated on cell culture (HEK and COS). Results No change was reported on O-GlcNAc levels between the diets (D28: 1.00 ± 0.06; D28F: 0.99 ± 0.08). Cardiac OGA expression was 5 times lower in D28F however OGA activity remained stable (D28: 1.00 ± 0.03; D28F: 0.83 ± 0.16 AU). Protein expression, acetyl-CoA transferase 1 (ACAT1) and acetyl-CoA synthetase 1 (ACSS1), was reduced both in HEK and COS cells in responses to O-GlcNAc stimulation with Thiamet-G. Conclusion While OGA expression is lower with the low-CHO diet, OGA activity and O-GlcNAc levels remain stable. This indicates a strong regulatory mechanism to control O-GlcNAc levels throughout post-natal development. This regulatory mechanism could help to modulate the expression of key metabolic enzymes such as ACAT1 or ACSS1.

Introduction The young population, particularly at risk of septic shock, is rarely studied. The O-GlcNAcylation is a post-translational modification involved in cell survival, metabolism and stress response. We have shown that O-GlcNAc stimulation at the early phase of septic shock is beneficial in adult rats. Considering that O-GlcNAc levels are higher in the young, the impact of O-GlcNAc stimulation on sepsis in the young should be tested. Objective Evaluate if O-GlcNAc stimulation could improve sepsis outcomes in young. Method Endotoxemic shock was induced in 28 days old rats by injection of LPS (O111 :B4, 20 mg.kg−1–LPS) and compared to control rats (injection of saline–CTRL). One hour after LPS injection rats were randomly assigned to no therapy (LPS), fluidotherapy (saline, 10 mL.kg−1–LPS + R) ± NButGT (10 mg.kg−1–NButGT) to increase O-GlcNAc levels (n = 11 per group). Physiological functions and plasmatic markers were evaluated 2 hours later. Impact of treatment was evaluated with an adapted Pediatric RISk of Mortality score (PRISM score) and mortality was evaluated over 36 hours (n = 16 per group). Untargeted mass spectrometry was performed to identify cardiac O-GlcNAcylated proteins. Results LPS induced a shock (mean arterial pressure (MAP): CTRL: 67.2 ± 1.9; LPS: 50.7 ± 2.1 mmHg; P Conclusion O-GlcNAc stimulation acutely improves outcome in young septic shock rat. The impact of O-GlcNAcylation on ACLY is a potential issue to decipher the role of this protein during sepsis.

Introduction Kv11.1 channel (hERG) and the corresponding IKr current is a major component of cardiac repolarization and sinoatrial pacemaking. Dysfunctions in hERG channels are associated with cardiac arrhythmias and remains a driving force to study the structure, biophysics and pharmacology of this channel. Several compounds that block hERG channels have been developed. In this study, we focused on BeKm-1, a toxin which is highly selective for the hERG channel. Objective To study hERG channels at high temporal and spatial resolution, we developed a photo-protected analog of BeKm-1 that enables control of hERG using light. Method We developed a photo-protected BeKm-1 analog (BeKm-1-Lys18Nvoc) which is cleaved by UV-light at 365 nm. Using a hERG stable cell line and high-throughput automated patch-clamp (Syncropatch 384, Nanion) we validated several properties of the BeKm-1 analog blockage. To demonstrate spatial control of cardiomyocyte function, hiPS-derived cardiomyocytes were plated on CardioExcyte96 to record extrafield potentials. Results We first validated physico-chemical properties of BeKm-1-Nvoc compared to BeKm-1 using HPLC analyses. Patch-clamp experiments displayed no inhibition of hERG current by BeKm-1-Nvoc at concentrations for which wild-type Bekm-1 is maximally inhibiting. Illumination of BeKm-1-Nvoc (100 nM) at 365 nm allows cleavage of the protecting group and strong inhibition of hERG current. The block of hERG is reversible after washes. Illumination of BeKm-1-Nvoc on hiPS-derived cardiomyocytes induces prolongation of extrafied potential which reflects an increase in repolarization time. Study of effects of photoactivation of BeKm-1 on mouse and zebrafish heart rates are ongoing. Conclusion We developed for the first time a photo-sensitive blocker of endogenous hERG channel which allows control of its activity with the spatiotemporal precision of light. Studies of implication of hERG on arrhythmias using this compound are ongoing.

Background O-GlcNAcylation is a well-preserved, ubiquitous, post-translational modification involved in response to stress, considered as a nutrient sensor. GlcNAc moiety is added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Metabolic sources vary from carbohydrate in utero, to fatty acid after birth then a mix once adult. Purpose Study the evolution of O-GlcNAc levels during the first period of life and decipher the impact of O-GlcNAc on cardiac proteins. Methods Heart and brain were harvested from Wistar rats before and after birth (D-1 and D0), in suckling animals (D12), after weaning (D28) with a standard or a carbohydrate free diet, and when adults (D84). O-GlcNAc levels, GFAT, OGT and OGA protein expression were evaluated by western blot. Mass spectrometry approaches were performed to (i) quantify known regulator of O-GlcNAc and (ii) identify cardiac O-GlcNAcylated proteins at D0 and D28. Results Cardiac protein O-GlcNAc levels decrease progressively from D-1 to D84 (13 fold, P Conclusion We demonstrate for the first time that O-GlcNAc levels are not linked to the dietary sources. Our results suggest that glutamine and UDP-GlcNAc are not the main regulatory element of O-GlcNAc levels. Proteins identified by untargeted mass spectrometry will need to be specifically studied to better understand the impact of O-GlcNAcylation during development, growth or stress response.

Introduction hERG potassium channels are essential for normal cardiac electrical activity. Mutations in the hERG gene (KCNH2) cause long QT syndrome, a disorder that predisposes individuals to life-threatening arrhythmias. More than 1000 mutations in hERG sequence are described in databases and most of them remains to be characterized. Objective The objective of the work is to determine the consequences of the variants identified in LQT2 patients on the expression and function of the hERG channel. Methods An optimized sequence hERG fused to pHluorin tag was synthesized. By amplifying mutated overlapping fragments, and using the Gibson assembly strategy, we constructed hERG-pHluorin plasmids with missense variations. CHO cells were transfected by electroporation with the Maxcyte system or Fugene method, and studied after 40 hours. Confocal images and flow cytometry analysis were used to evaluate the percentage of transfected cells and to quantify the membrane channel expression, through pH sensitive pHluorin tag. Conventional or automated patch clamp were used to evaluate the hERG activity. Results In the present work, we constructed hERG plasmids carrying 48 variations identified in patients in France. Higher efficiency of transfection was observed with electroporation as compared to the Fugene method. The activity of hERG-pHluorin was validated in manual patch-clamp, the pHluorin tag does not interfere with channel activity. Characterization of several mutations in conventional patch clamp showed difference in current amplitude. Automated patch-clamp data was compared to manual patch-clamp, as an initial validation for high throughput screening of all constructed mutations. Conclusion High-throughput characterization of KCNH2 genetic variants is relevant to discriminate mutants that affect hERG channel activity from variants with undetectable effects. This information will be indicated in a patient database, as a basis of personalized medicine.

Introduction Septic shock is a systemic inflammatory response syndrome associated with circulation failure leading to organ failure with a mortality rate of 40%. Early diagnosis and prognosis of septic shock are necessary for timely and specific treatment but no predictive biomarkers are available. The plasma proteome is easily accessible and may contain biomarkers that can be studied with non-targeted “omic” strategies. Aims We aim to decipher a relevant proteome signature through a non-targeted mass spectrometry (MS) analysis in a rat septic shock model. Methods In 10-week-old Wistar rats (n = 11/group), 30 min post-buprenorphine (0.05 mg/kg, s) endotoxemic shock was induced by lipopolysaccharides injection (LPS, S. enterica typhi, 0.5 mg/kg, iv) and compared to controls (NaCl 0.9%, iv). Under isoflurane anesthesia, carotid cannulation allowed mean arterial blood pressure (MAP), heart rate (HR) monitoring and blood sampling (T0, 50 and 150 min-EDTA with protease inhibitor). Each plasma was prepared for RPHPLC/MS by performing Proteominer, dialysis and trypsin digestion. RPHPLC-MS analysis was performed on a ESI-Q-Exactive mass spectrometer (ThermoFisher) according to a non-targeted label-free MS analysis. Data were analyzed with PEAKS and R software. Results LPS group present circulation failure (−40% MAP, +20% of HR vs Ctrl, P Conclusion This preliminary study revealed propose a new approach to identify relevant deregulated proteins in septic shock. The investigation of the link between our results and patients’ clinical features is in progress.

Background The systemic inflammation response syndrome (SIRS) is a consequence of the cardiopulmonary bypass (CPB) in cardiac surgery that can trigger multiple organ dysfunction (MOD). Pulmonary, renal and cerebral dysfunctions are the most common post-operative complications of CPB. The sustained of activation of integrated stress response (ISR) pathway could impact patient outcomes. Aim This project aims to decipher pathophysiological process in SIRS and organ dysfunction after recovery of CPB in a rat model. Methods After isoflurane induction (2%, O2: 0.5 L/min) surgical procedure for CPB or Sham was performed under propofol-fentanyl infusion (30–4 μg/kg/h) on mechanically ventilated 15-week old rats. The CPB circuit allows blood to be drawn by the vena cava and reperfused in the aorta after oxygenation. Hemodynamic and biochemical parameters were monitored during the procedure. Mean Arterial Pressure (MAP) was maintained to a minimum of 60 mmHg with a FiO2 1.0 for 1 h. Five hours post-recovery, behavior, respiratory rate and hemodynamic were measured. Blood and organs were collected to evaluate the integrated stress response pathway. Results Compared to Sham, CPB leads to a decrease of MAP (88.3 ± 2.6 vs 60.0 ± 9.4 mmHg, P Conclusion On our CPB rat model, we highlight organs dysfunctions associated with an activation of the ISR pathway. We will now evaluate the potential impact of ISR activation on MOD.

Background The systemic inflammation response syndrome (SIRS) is a common side-effect of the cardiopulmonary bypass (CPB) in cardiac surgery. SIRS can trigger multiple organ dysfunction which can lead to patient death. Pulmonary dysfunction is the most common post-operative complication of CPB. However, the lack of animal models recapitulating the disease prevents the investigation of pathophysiological mechanisms of CPB's complications. Aim This project aims to develop a robust rat model recapitulating the SIRS during an extracorporeal circulation. This model mimics the clinical procedure and will be used to investigate the activation of stress response pathway (ISR). Methods Surgery was made under isoflurane (3%, 02: 0.4L/min) and CPB was performed under propofol infusion (5μg/kg/hr) on mechanically ventilated 12 weeks old rats. Blood was drawn from cava vein then reinjected in the aorta. Hemodynamic parameters were measured in femoral artery. The circuit was primed with Ringer Lactate ( Results Mean blood pressure was maintained at 50mmHg with a PaO2 > 380mmHg. Lactate and kaliemia were increased after CPB (pre: 1.31 ± 0.49 vs post: 5.32 ± 3.67 and pre: 4.73 ± 0.15 vs post: 6.83 ± 2.22, respectively, N = 3). An activation of the ISR pathway was demonstrated by western blot on lung from CPB rats compared to Sham rats, with a 10-fold increase in p(ser51)-eIF2α/eIF2α ratio, a 2-fold increase in CHOP and GADD34 protein levels. Conclusion We have developed a pre-clinical rat model of CPB in which preliminary data highlight an early activation of ISR pathway in lung. This model will be used to decipher the pathophysiological mechanisms of CPB's complications.

Background Voltage-gated potassium channel Kv4.2 (encoded by KCND2 gene) contributes to the cardiac transient outward potassium current (Ito1). This current is the main contributor to the repolarisation phase 1 of the cardiac action potential. The toxin AmmTx3, identified from the venom of the scorpion Androctonus mauretanicus, is a blocker of Kv4.x channels, and have interesting therapeutic potential for neurological disorders due to its effect in cerebellar granule neurons. Its effects on cardiac Kv4.2 channels remains unclear. Objective The aim of this study was to: – produce a synthetic AmmTx3 (AmmTx3-NCL); – evaluate in vitro and in vivo, effects of this toxin on Kv4.2 channels compared to native toxin. Method We did chemical synthesis of AmmTx3 using the native chemical ligation strategy (NCL). We characterized its biological activity compared to native AmmTx3 using an automated patch-clamp system (Syncropatch 384 PE, Nanion) on HEK cells co-expressing Kv4.2 α subunit and two auxiliaries subunits (KChIP and DPP6). Effects of AmmTx3 and AmmTx3-NCL on cardiac cellular electrophysiology were studied by patch clamp on adult rat cardiomyocytes and hiPSC-CMs. Integrated cardiac effects of AmmTx3 were studied on ECG of wild-type mice. Results In this study, we report the total chemical synthesis of AmmTx3 and 3D structure was also determined. As expected, biological activity of folded AmmTx3-NCL is coherent to native toxin with a similar time course of Kv4.2 current inhibition without any change of activation kinetics. Characterisation of AmmTx3 effects on isolated cardiomyocytes, hiPSC-CMs and in vivo by ECG is ongoing. Conclusion AmmTx3 toxin can be chemically synthesized and used as a Kv4.2 channel inhibitor to contributed to the better understanding of the exact role of Ito1 in cardiac electrophysiology. Those first results seem to be a promising evidence that AmmTx3 could a potential inhibitor of Ito current in early repolarisation syndrome.

Introduction Heart failure with preserved ejection fraction (HFpEF) is a public health burden associated to diastolic dysfunction with no effective treatment mostly affecting elderly women. We use the Tgβ3 rats to explore the potential impact of phytoestrogen in the development of this pathology as Tgβ3 females does not develop HFpEF with ageing while Tgβ3 male with a phytoestrogen-free diet does. Objective The aim of our study was to evaluate phytoestrogen impact on HFpEF development. Method HFpEF development has been evaluated in males and females, ovariectomized or not, with or without a phytoestrogen-free diet. HFpEF was validated by echography study (E/A ratio), invasive hemodynamic study (LVEDP) and fibrosis. Then the heart was harvested to evaluated HFpEF impact on protein expression involved on cardiac metabolism and endothelial function. Results HFpEF was not established in rats with phytoestrogen diet (males and females, ovariectomized or not) and also in female's groups feed with phytoestrogen-free diet. In males with phytoestrogen-free diet, phenotype evaluation revealed diastolic dysfunction with significant modification of E/A ratio and LVEDP in Tgβ3 rats with ageing (E/A: + 15% vs WT and LVEDP: + 109% vs WT; P Conclusion We demonstrated that long term exposure to oestrogen or phytoestrogen in our model is protective against the development of HFpEF but the mechanism remains unclear. Our results highlight the potential role of eNOS in HFpEF development.

Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease exposing afflicted patients to sudden cardiac death. Most CPVT cases have been linked to mutations of the gene encoding ryanodine receptor type 2 (RYR2). RyR2 is a calcium channel located in the membrane of the sarcoplasmic reticulum (SR). It mediates release of calcium stocks from the SR to the cytosol, playing a major role in excitation-contraction coupling. Although several studies have investigated mechanisms underlying CPVT in mice or in vitro models, little has been done concerning functional assessment of CPVT physiopathology in human cardiomyocytes. Objective We aimed at characterizing, at cellular level, CPVT phenotype of patients carrying two new RyR2 mutations. Method Human induced pluripotent stem cells (hiPSCs) were derived from three CPVT patients. Two patients belonging to the same family carry the R4959Q RyR2 pore mutation. The third patient harbors the Y2476D mutation located in the cytosolic domain of the protein. hiPSCs were differentiated into cardiomyocytes for functional studies. Action potentials were recorded using patch-clamp technique. Calcium handling and contractility were investigated using Ionoptix and CardioExcyte technologies. Results hiPS-derived cardiomyocytes (hiPS-CMs) from CPVT patients displayed impaired beating properties. Calcium handling properties were also disrupted with changes in calcium transients, accompanied with increased diastolic calcium leak. At last, CPVT hiPS-CMs showed characteristics changes in action potential and arrhythmic events. Coherently with the patients’ phenotype, arrhythmic events were more frequent under beta-adrenergic stress. Conclusion This work enabled us to characterize two novel CPVT mutations, providing more insight into functional mechanisms involved in this pathology, and further validating the use of human pluripotent stem cells for investigating complex cardiac rhythm disorders.

Background Ryanodine receptors type 2 (RyR2) the intracellular channels that control excitation-contraction (EC) coupling in cardiomyocytes represent an important target for treatment of arrhythmias accompanying cardiac pathologies. Maurocalcine (MCa), a peptide isolated from scorpion venom, has been described as a high affinity activator of RyR in skeletal muscle. Phosphorylation of MCa has been shown to induce a complete remodelling of its effect on RyR, making it a negative allosteric modulator of RyR. Objective The purpose of this study is to characterize the effects of MCa and different analogues on EC coupling in isolated adult ventricular cardiomyocytes and hiPS-derived cardiomyocytes (hiPS-CMs). Methods Adult ventricular myocytes were isolated from 3–6 months-old Sprague Dawley rats with conventional collagenase type II and protease type XIV mix, and hiPS-CMs were obtained from urine cells of healthy volunteers. Changes of cytoplasmic calcium concentration were measured using Fura-2 and IonOptix software. hiPS-CMs contractions and extrafield potentials were recorded with CardioExcyte system (Nanion). Results We first confirmed binding between MCa and RyR2. Next, we showed that MCa (500nM) induces asynchronous calcium release and contractions of isolated adult rat cardiomyocytes under electrical stimulation at 1Hz. Similar results were observed on hiPS-CMs in the presence of MCa. Effects of native MCa and analogues on isoprenaline-mediated stimulation of RyR2 are under investigation. Conclusion Our results demonstrate that MCa penetrates adult cardiomyocytes as well as human iPS derived cardiomyocytes and modulates RyR2 activity. MCa analogues exhibit different effects on RyR2, from positive to negative allosteric modulation. These results provide promising evidence that MCa could be used as an in cellulo modulator of RyR2 to control abnormal RyR activity in pathological situations.

intellectual history remains one of the least studied of the various forms of historiography practiced by the Victorians, certainly when compared to political, constitutional, or literary history, or to the historical novel when considered as a historiographical genre. Yet intellectual-historical inquiry formed a vital strand in the Victorian discourse on the past, particularly in liberal quarters, where a belief in ideas as the engines of change reinforced the prevailing doctrine of progress in history as shaped by human agency, volition, and “char acter.” s ome of the more popular intellectual histories were epic in intention and scale: Henry Thomas Buckle’s History of Civilization in England (1857–61), John William Draper’s History of the Intellectual Devel opment of Europe (1863), and W. E. H. Lecky’s History of the Rise and Influ ence of the Spirit of Rationalism in Europe (1865) presented the march of mind in terms of a steady, incremental victory of reason over supersti tion and of heroic human effort over the unruly forces of nature. i n a more scholarly (and more self-searching) key, historians like Mark pattison, John Morley, and Leslie s tephen saw intellectual history as a cornerstone of the historiography of civilization or—in the fashion able parlance of mid-Victorian sociology—of the study of the laws of social change. For them, publishing their major works of intellectual

Introduction Heart failure (HF) with preserved ejection fraction (pEF) is mainly associated with diastolic function. It affects mostly elderly patients with cardiac and non-cardiac comorbidities and is considered as a public health burden. So far, no treatment is available to reduce mortality and morbidity due to lack of knowledge of HFpEF pathophysiology. Our team developed an HFpEF transgenic rat (Tgβ3) overexpressing the human β3-adrenoreceptor in endothelium. Objective The aim of our study is to identify new targets involved in the development of the pathology. Methods Cardiac function was characterized by echocardiography and invasive hemodynamic studies at 45 and 60 weeks of age in male rats (n = 21/11). Transcriptome analysis was performed on left ventricle (LV) tissue at 45 weeks old rats. Among altered genes we focused on metabolic targets by RT-qPCR and Western-blot. Results At 45 and 60 weeks, Tgβ3 rats presented a significant increase in LV end diastolic pressure (+109% and +175% vs. WT P Conclusion Our results demonstrate a close link between HFpEF development and cardiac remodelling. Understanding the implication of the metabolic target 1 in the development of HFpEF in our model could lead to the identification of new biomarkers or treatment for the patient.

Introduction Endothelial has been suggested to play an important role in the development of heart failure with preserved ejection fraction (HFpEF), but there is no real link so far. Mechanism could involve alteration in vascular structure and reactivity. •NO bioavailability has been suggested to play a central role in this phenomenon and •NO production is partly mediated through β3-adrenoreceptor (β3-AR) activation. Our team developed a transgenic rat (Tgβ3) overexpressing the human β3-AR in the endothelium leading with ageing to HFpEF. Objective The aim of our study was to understand the implication of the endothelium in HFpEF development. Methods Aorta from 45 weeks old rat have been used to evaluate arterial reactivity (aorta and mesenteric rings). Endothelium-dependent relaxation to isoproterenol (0.1 nM to 100 μM) was measured in the presence or absence of •NO synthase (NOS) inhibitors: L-VNIO (NOS-1), 1400 W (NOS-2) and L-NIO (NOS-3) at 10 μM. •NO production in aorta has been evaluated by electron paramagnetic resonance (EPR). Results Concentration-dependent vasodilation to isoproterenol was significantly reduced in Tgβ3 aorta and mesenteric arteries (−10%, P Conclusion Our animal model demonstrates for the first time that alteration of the endothelial function could lead to HFpEF development. This observation could lead a better understanding of HFpEF pathophysiology, yet evolution of vascular function and •NO production need to be evaluated at different time of HFpEF development.

Introduction It is estimated that there are about 170,000 venomous species throughout the world. The venom produced by their glands contains hundreds of bioactive compounds. These natural libraries open complete new therapeutic avenues. Heart failure is a cardiac disease associated with morbidities. Recent progress in medicine allowed to develop treatments, yet they are often not highly efficient and possess harmful side effects. Given that venom compounds are pharmacologically active, they can be considered as a source of life-saving therapeutics. Objective The aim of our study is to find therapeutic effects of venom compounds to treat hypertension which can lead to heart failure. Method Black Mamba snake venom fractionations were performed by using cation exchange and RP-HPLC techniques. The obtained fractions and purified compounds were investigated for their ability to displace 3H-NMS from muscarinic type 2 receptors (M2R). One particular snake peptide, found to be active, was de novo sequenced and synthesized. This peptide was studied in vivo by invasive hemodynamics to evaluate the main cardiovascular parameters (n = 5, 100 μg/kg) and ex vivo on isolated mesenteric and aorta arteries (n = 6; 0.8 nM, 8 nM, 80 nM and 8 μM). Wistar rats that were 10–12 weeks old were used for this study. Results After consecutive fractionations of the venom, we succeeded to identify a unique peptide with major hypotension effects. Once injected intravenously in vivo, invasive hemodynamic studies highlighted a significant arterial blood pressure decrease (−65%, P Conclusion In this study, we demonstrated the hypotensive effect of an isolated peptide from Black Mamba snake. These results confirm that venom compounds represent a promising opportunity to develop new drugs.

Introduction Septic shock is a worldwide burden, without efficient treatments, killing one person every 3 s. Recently, O-GlcNAc stimulation (a post-translational modification) appeared to be beneficial in acute pathologies. Objective This study has been designed to evaluate if O-GlcNAc stimulation shows the same benefits in two relevant models of septic shock. Method Impact of NButGT (an OGlcNAcase inhibitor – 10 mg/kg) has been evaluated in LPS or CLP (caecal ligation and puncture) and their respective controls with or without fluidotherapy (FR). Cardiovascular function (mean arterial blood pressure (MAP), echocardiography) and biochemical markers (creatinine, lactates) were measured at 3 h and 24 h after shock induction respectively, then heart and blood were harvested to evaluate impact of the treatment. Also, O-GlcNAc impact on calcium actor was evaluated by western blot. LPS study was completed with a Kaplan–Meier mortality evaluation. Results NButGT treatments efficiently increased cardiac O-GlcNAc in both model (+300% vs. Ctrl or Sham). In both group, NButGT treatment induced an amelioration of the outcome and organs function specifically: cardiac (heart rate −9% vs. LPS; −10% vs. CLP), renal (creatinine −35% vs. LPS; −27% vs. CLP) and beneficial impact on blood pressure (MAP + 10% vs. CLP). Treatment had a significant impact on lactate in LPS group (−36% vs. LPS) consistent with a 3 times higher survival rate. Treatment impacted on SERCA2a protein (−150% vs. LPS, NS vs. ctrl) without modification of its regulator (phospholamban) or ryanodine receptor. Conclusion Our results support the fact that acute O-GlcNAc stimulation is a potential new therapeutic target at the early phase of septic shock. Potentially through improvement of cardiomyocytes’ calcium handling. We are currently validating our approach at different dose and age to be able to transfer our results to the clinic.

Introduction Pacemaker activity of the sino-atrial node (SAN) generates the heart rate. SAN dysfunction leads to sick sinus syndrome (SSS) characterized by abnormally low heart rate (HR). The only currently available therapy for SSS is electronic pacemaker implantation. Mice lacking SAN L-type Cav1.3 Ca2+ channels (Cav1.3−/−) present symptoms similar to those observed of SSS in humans especially SAN bradycardia and atrioventricular (AV) block. Cardiac voltage-gated Na+ channels (Nav) are essential for SAN impulse conduction through the AV node and the working myocardium. Mutations in the α-subunit of the cardiac Nav1.5 channel isoform are associated with inherited dysfunction of heart impulse conduction. We recently showed that genetic ablation of muscarinic-gated K+ channels (IKAch) rescues bradycardia of Cav1.3−/− mice. Purpose We tested pharmacologic inhibition of IKAch by the peptide tertiapine-Q to rescue SSS and conduction dysfunction in Cav1.3−/− mice having concurrent ablation of Cav1.3 and T-type Cav3.1 channels (Cav1.3−/−/Cav3.1−/−) and Nav1.5± mice, which present ECG profiles similar to individuals carrying congenital bradycardia and conduction defects. Methods We employed telemetric ECG recordings of SAN activity, HR and AV dysfunction in mice before and after administration of different doses of tertiapin-Q. Results Tertiapin-Q significantly improves the HR of Cav1.3−/− (19%), Cav1.3−/−/Cav3.1−/− (23%) from doses of 0.1 to 5 mg/kg. HRs of tertiapine-Q-treated mice were similar to those recorded in untreated wild-type mice. Tertiapin-Q also improved cardiac conduction of Nav1.5± mice by 24%. Conclusion Pharmacological inhibition of IKAch by tertiapin-Q prevents SAN dysfunction and improves conduction in three models of congenital bradycardia and impulse conduction defects suggesting the possibility of IKACh pharmacologic targeting to manage SAN and conduction disease.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfide- bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1^C5, C2^ C6, C3^C4 and C7^C8, versus C1^C5, C2^C6, C3^C7 and C4^ C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys13) and C4 (Cys19), results in conventional Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, (A12,A20)MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog. fl 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Ca2+channel inactivation is a key element in controlling the level of Ca2+entry through voltage-gated Ca2+channels. Interaction between the pore-forming α1subunit and the auxiliary β subunit is known to be a strong modulator of voltage-dependent inactivation. Here, we demonstrate that an N-terminal membrane anchoring site (MAS) of the β2asubunit strongly reduces α1A(CaV2.1) Ca2+channel inactivation. This effect can be mimicked by the addition of a transmembrane segment to the N terminus of the β2asubunit. Inhibition of inactivation by β2aalso requires a link between MAS and another important molecular determinant, the β interaction domain (BID). Our data suggest that mobility of the Ca2+channel I–II loop is necessary for channel inactivation. Interaction of this loop with other identified intracellular channel domains may constitute the basis of voltage-dependent inactivation. We thus propose a conceptually novel mechanism for slowing of inactivation by the β2asubunit, in which the immobilization of the channel inactivation gate occurs by means of MAS and BID.

Item does not contain fulltext

International audience; Ochratoxin A (OTA) is a widespread and abundant natural carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium fungi. Due to the ubiquitous presence of these fungi in food and potential risk for human health, a rapid and sensitive in vitro detection assay is required. Analytical methods for OTA detection/identification are generally based on liquid-liquid extraction, clean-up using an immunoaffinity column (IAC), and identification by reversed-phase high pressure liquid chromatography with fluorescence detection (HPLC-FLD). However, IACs are costly and have a short lifespan. Therefore, an interesting approach would appear to be the design and chemical synthesis of a mimotope peptide simulating mycotoxin-specific antibodies. We have developed a promising alternative method that is based on the use of peptides which are able to bind to specific chemical functions and/or molecular structures. Accordingly, a number of peptides (derived from the structures of major redox proteins) were selected and produced by chemical solid phase syntheses. The ability of such peptides to bind to ochratoxin A was evaluated by HPLC. The peptide NF04 (structurally derived from an oxidoreductase enzyme), which was found to be the sole potently reactive compound among tested molecules, was further evaluated in a peptide-based enzyme-linked immunosorbent assay (peptide-based ELISA), thus confirming its specific interaction with ochratoxin A.

1. Voltage-dependent Ca2+ channels are multi-protein complexes composed of at least three subunits: alpha 1, alpha 2 delta and beta. Ba2+ currents were recorded in Xenopus oocytes expressing the neuronal alpha 1A Ca2+ channel, using the two-electrode voltage-clamp technique. Various subunit combinations were studied: alpha 1A, alpha 1A alpha 2 delta b, alpha 1A beta or alpha 1A alpha 2 delta b beta. 2. The alpha 1A subunit alone directs the expression of functional Ca2+ channels. It carries all the properties of the channel: gating, permeability, voltage dependence of activation and inactivation, and pharmacology. The alpha 1A channel is activated by low voltages when physiological concentrations of the permeant cation are used. Both ancillary subunits alpha 2 delta and beta induced considerable changes in the biophysical properties of the alpha 1A current. The subunit specificity of the changes in current properties was analysed for all four beta gene products by coexpressing beta 1b, beta 2a, beta 3 and beta 4. 3. All beta subunits induce a stimulation in the current amplitude, a change in inactivation kinetics, and two hyperpolarizing shifts--one in the voltage dependence of activation and a second in the voltage dependence of steady-state inactivation. The most significant difference in regulation among beta subunits is the induction of variable rate constants of current inactivation. Rates of inactivation were induced in the following order (fastest to slowest): beta 3 > beta 1b = beta 4 > beta 2a. 4. The alpha 2 delta b subunit does not modify the properties of alpha 1A Ca2+ channels in the absence of beta subunits. However, this subunit increases the beta-induced stimulation in current amplitude and also regulates the beta-induced change in inactivation kinetics. 5. Of all the subunit combinations tested, Ca2+ channels that included a beta subunit were the most prone to decrease in activity. It is concluded that beta subunits are the primary target for the inhibitory mechanisms involved in Ca2+ channel run-down. 6. Both alpha 2 delta b and beta 1 b subunits slightly modified the sensitivity of the alpha 1A subunit to the snail peptide omega-conotoxin MVIIC. 7. The subunit-induced changes in properties of the alpha 1A channel are surprisingly similar to changes reported for other alpha 1 subunits. These modifications in channel activity should therefore represent important functional landmarks in the on-going characterization of subunit-subunit interactions.

Cytomegalovirus is an important cause of sensorineural hearing loss in children. In contrast to congenitally infected infants, little is known about hearing in preterm infants with postnatal cytomegalovirus infection. We studied the hearing in 64 preterm infants during the first year of life and in 18 during the second year of life. None of the infants developed sensorineural hearing loss.

N-type Ca2+ channels control a variety of key neuronal functions including transmitter release at synaptic terminals. The purified omega-conotoxin receptor from rabbit brain is a multisubunit complex composed of alpha 1B, alpha 2 delta, beta 3, and 95-kDa subunits. Immunoadsorption experiments confirm that the purified preparation does not contain alpha 1 subunits other than the omega-conotoxin-sensitive class B isoform. The functional properties of the purified channel have been analyzed further in lipid bilayers, and similarities to or differences from the native N-type Ca2+ channel have been outlined. Conserved properties include ion selectivity, open-time duration, and pharmacology (insensitivity to drugs affecting skeletal muscle L-type Ca2+ channels). Observed properties of the reconstituted channel which differ from the native channel include (a) sustained channel activity without Ca(2+)- or voltage-induced inactivation; (b) examples of extremely high open-state probability; (c) the absence of "run-down"; and (d) voltage independence of the Ca2+ channel gating. In addition, the conductance of the purified receptor is comprised between 7 and 27 picosiemens. Our results suggest that cellular components may play critical roles in the regulation of several biophysical properties and neuronal function of the native N-type Ca2+ channel.

The properties and characteristics of calcium channel openings in cerebellar granule cells were analysed by the cell-attached patch-clamp technique. At depolarized potentials, with 110 mM Ba2+ as the divalent charge carrier, 36% of the patches displayed activity that consisted of elementary events whose amplitude ranged from -0.3 to -1.75 pA at 0 mV, giving rise to a high threshold current. In this population of events at least four different types of channel openings were identified by their distinct biophysical and pharmacological properties. Two types of channel openings, with conductances around 24 and 7 pS, had similar characteristics in that both opened following two modes of gating characterized by brief (approximately 2 ms) and longer openings (approximately 8 ms) and both were sensitive to dihydropyridines. A further type of channel opening, with a conductance around 11 pS gated mainly with brief openings (approximately 1 ms), was shown to be insensitive to dihydropyridines but was undetectable in recordings from the cells that had been treated with omega-conotoxin. The last type of event was revealed after treatment of the cell with nicardipine or nifedipine and omega-conotoxin. The corresponding channel had a conductance of 19 pS and opened in one dominant mode characterized by brief openings (approximately 1 ms). The data obtained on single-channel activity of cerebellar granule cells are compared with the properties of the total current recorded in whole-cell conditions.

Calcium channels play an important role in regulating various neuronal processes, including synaptic transmission and cellular plasticity. The N-type calcium channels, which are sensitive to omega-conotoxin, are involved in the control of transmitter release from neurons. A functional N-type calcium channel complex was purified from rabbit brain. The channel consists of a 230-kilodalton subunit (alpha 1B) that is tightly associated with a 160-kilodalton subunit (alpha 2 delta), a 57-kilodalton subunit (beta 3), and a 95-kilodalton glycoprotein subunit. The complex formed a functional calcium channel with the same pharmacological properties and conductance as those of the native omega-conotoxin-sensitive calcium channel in neurons.

International audience; The neuropharmacological effects of the decoction of Nauclea latifolia Smith (Rubiaceae) roots were studied in mice Different experimental models (forced swimming test, horizontal wire test and hole-board test) were used for detecting antidepressant, myorelaxant and anxiolytic properties The results revealed that Nauclea latifolia induced a reduction of immobility, in a similar way to that of fluoxetine, along with a significant increase in the percentage of spent time in swimming behavior Nauclea latifolia displayed a myorelaxant activity in the horizontal wire test In the hole-board test, Nauclea laufolta significantly increased the number and duration of head-dips In addition, anxiolytic-like properties of Nauclea laufolta were blocked by anxiogenic agents as examined in the hole-board test This was the case for N-methyl-beta-carboline-3-carboxamide (FG7142), a partial Inverse agonist at the benzodiazepine site of the GABA(A) receptor complex, flumazenil (RO151788), a central benzodiazepine receptor antagonist and bicuculline, a light-sensitive competitive antagonist of GABA(A) receptors These results suggest that Nauclea latifolia roots decoction possess antidepressant, myorelaxant and anti-anxiety-like properties in the models employed The extracts might potentially act by GABAergic activation and/or by modulating the serotoninergic levels in the central nervous system However, further studies were still required

Background Recent advances in the form of foam sclerotherapy have extended the indications for sclerotherapy in the treatment of varicose veins. Objectives (1) To see if perforator veins can be closed by using foam sclerotherapy; (2) to evaluate whether using foam sclerotherapy for treatment of incompetent perforator veins has an influence on venous leg ulcer healing. Methods Incompetent perforator veins above bilateral venous leg ulcers were treated with duplex ultrasonography-guided sclerotherapy using polidocanol foam. Results After 4 days, significant improvement in the ulcers was visible; after 10 days, one ulcer had closed; and within 3 weeks, both ulcers were closed. Control duplex ultrasonography showed closed perforator veins. Conclusion This case illustrates that duplex ultrasonography-guided foam sclerotherapy is an effective and minimally invasive way of treating incompetent perforator veins. The fast healing of the venous leg ulcers in this case report indicates that foam sclerotherapy could be a promising addition in the treatment of venous leg ulceration with incompetent perforator veins.

The developmental changes of intracellular calcium release channels of mouse neocortex were studied at the onset of neurogenesis, which occurs between embryonic days E11 and E17. The three main isoforms of the two families of intracellular calcium release channels, namely the inositol trisphosphate receptors (IP3R) and the ryanodine receptors (RyR), were detected by their transcripts in the cerebral hemispheres, as early as stage E11. The major isoforms of each family, IP3R-1 and RyR-2, were found at the protein level by Western blot analysis. Expression of these proteins increases progressively throughout brain development. Their localization in coronal sections of cortex has been observed by immunodetection from E12, and compared to the TuJ1 (anti-class III beta-tubulin antibody) neuronal specific labelling. The expression of both channels is greatly enhanced after E12, and both were seen to be present in most of the proliferative and neuronal cells of the slice. Between E12 and E13, there is a striking transition in the pattern of calcium release elicited by specific agonists of these channels, thimerosal for IP3R and caffeine for RyR. The signals induced by thimerosal were not zone-specific, while the observed calcium release signals induced by caffeine were predominantly restricted out of the ventricular zone. This zone-specific caffeine sensitivity is consistent with the main RyR localization immunodetected at E13. Our results indicate that there is a time lag of several days between the molecular detection of calcium release channels and their functional expression, around the time of neuronal differentiation. Altogether, they provide a molecular basis for analyzing the developmental modulation of calcium signals useful for neurogenesis progression.

Purpose: Cardio-protective effects of physical exercise training are generally recognized. However, previously we demonstrated that the beneficial effects of exercise critically depend on the underlying cause of cardiac dysfunction and showed that unlike following myocardial infarction (MI), exercise did not improve but rather tended to aggravate cardiac dysfunction and remodeling following mild and severe pressure-overload induced by a trans-aortic constriction (TAC). The mechanism behind the etiology-dependent effects of exercise in cardiac disease remains unclear but we hypothesize that nitric oxide synthases (NOS) dependent regulation of the balance between nitric oxide and superoxide (O2•−) is critically involved. Methods: Mice were exposed to 8 weeks of voluntary wheel running exercise training (EX) or sedentary housing (SED) immediately following MI, TAC or sham (SH) surgery. At the end of the 8 weeks training period cardiac function was studied and mice were sacrificed for cardiac tissue analysis. O2•− was measured using lucigenin-enhanced chemiluminescence. NOS, NADPH and xanthine oxidase (XO)-dependent O2•− production were measured using the NOS inhibitor L-NAME; NADPH substrate and inhibitor VAS2870, and XO substrate and inhibitor oxypurinol. eNOS uncoupling was evaluated with western blot. Results: Cardiac dysfunction and pulmonary congestion were ameliorated by exercise in MI but not in TAC mice. Thus fractional shortening increased from 8±1% in MISED to 12±1% in MIEX (p

Two C-terminal splice variants (BI-1 and BI-2, now termed Ca(v)2.1a and Ca(v)2.1b) of the neuronal voltage-gated P/Q-type Ca(2+) channel alpha(1A) pore-forming subunit have been cloned (Mori et al., 1991, Nature, 350, 398-402). BI-1 and BI-2 code for proteins of 2273 and 2424 amino acids, respectively, and differ only by their extreme carboxyl-termini sequences. Here, we show that, in Xenopus oocytes, the two isoforms direct the expression of channels with different properties. Electrophysiological analysis showed that BI-1 and BI-2 have peak Ba(2+) currents (I(Ba)) at a potential of +30 and +20 mV, respectively. The different C-terminal sequence (amino acids 2229-2273) of BI-1 caused a shift in steady-state inactivation by +10 mV and decreased the proportion of fast component of current inactivation twofold. Likewise, the biophysical changes in I(Ba) caused by coexpression of the beta(4) auxiliary subunit were substantially different in BI-1- and BI-2-containing channels in comparison to those induced by beta(3). Several of these differences in beta regulation were abolished by deleting the carboxyl-terminal splicing region. By creating a series of GST fusion proteins, we identified two locations in the C-terminal (Leu2090-Gly2229 for BI-1 and BI-2, and Arg2230-Pro2424 for BI-2 only) that determine the differential interaction of beta(4) with the distinct alpha(1A) isoforms. These interactions appear to favour the binding of beta(4) to the AID site, and also the plasma membrane expression of BI-2. These results demonstrate that the final segment of the C-terminal affects alpha(1A) channel gating, interaction and regulation with/by the beta subunits. The data will have several implications for the understanding of the biophysical effects of many channelopathies in which the carboxyl-termini of alpha(1A) and beta(4) are affected.

SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.