Marion Constantin

The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors

CXCR3 plays important roles in angiogenesis, inflammation, and cancer. However, the precise mechanism of regulation and activity in tumors is not well known. We focused on CXCR3-A conformation and on the mechanisms controlling its activity and trafficking and investigated the role of CXCR3/LRP1 cross talk in tumor cell invasion. Here we report that agonist stimulation induces an anisotropic response with conformational changes of CXCR3-A along its longitudinal axis. CXCR3-A is internalized via clathrin-coated vesicles and recycled by retrograde trafficking. We demonstrate that CXCR3-A interacts with LRP1. Silencing of LRP1 leads to an increase in the magnitude of ligand-induced conformational change with CXCR3-A focalized at the cell membrane, leading to a sustained receptor activity and an increase in tumor cell migration. This was validated in patient-derived glioma cells and patient samples. Our study defines LRP1 as a regulator of CXCR3, which may have important consequences for tumor biology.The chemokine receptor CXCR3-A is expressed in high grade glioma. Here the authors show that CXCR3-A interacts with LPR1, a protein highly expressed in glioma, and this interaction regulates the trafficking of the receptor ultimately impacting on tumour cell migration.

Proarrhythmic remodelling of the right ventricle in a porcine model of repaired tetralogy of Fallot

Objective The growing adult population with surgically corrected tetralogy of Fallot (TOF) is at risk of arrhythmias and sudden cardiac death. We sought to investigate the contribution of right ventricular (RV) structural and electrophysiological remodelling to arrhythmia generation in a preclinical animal model of repaired TOF (rTOF). Methods and results Pigs mimicking rTOF underwent cardiac MRI functional characterisation and presented with pulmonary regurgitation, RV hypertrophy, dilatation and dysfunction compared with Sham-operated animals (Sham). Optical mapping of rTOF RV-perfused wedges revealed a significant prolongation of RV activation time with slower conduction velocities and regions of conduction slowing well beyond the surgical scar. A reduced protein expression and lateralisation of Connexin-43 were identified in rTOF RVs. A remodelling of extracellular matrix-related gene expression and an increase in collagen content that correlated with prolonged RV activation time were also found in these animals. RV action potential duration (APD) was prolonged in the epicardial anterior region at early and late repolarisation level, thus contributing to a greater APD heterogeneity and to altered transmural and anteroposterior APD gradients in rTOF RVs. APD remodelling involved changes in Kv4.3 and MiRP1 expression. Spontaneous arrhythmias were more frequent in rTOF wedges and more complex in the anterior than in the posterior RV. Conclusion Significant remodelling of RV conduction and repolarisation properties was found in pigs with rTOF. This remodelling generates a proarrhythmic substrate likely to facilitate re-entries and to contribute to sudden cardiac death in patients with rTOF.

Identification of Region-Specific Myocardial Gene Expression Patterns in a Chronic Swine Model of Repaired Tetralogy of Fallot

Surgical repair of Tetralogy of Fallot (TOF) is highly successful but may be complicated in adulthood by arrhythmias, sudden death, and right ventricular or biventricular dysfunction. To better understand the molecular and cellular mechanisms of these delayed cardiac events, a chronic animal model of postoperative TOF was studied using microarrays to perform cardiac transcriptomic studies. The experimental study included 12 piglets (7 rTOF and 5 controls) that underwent surgery at age 2 months and were further studied after 23 (+/- 1) weeks of postoperative recovery. Two distinct regions (endocardium and epicardium) from both ventricles were analyzed. Expression levels from each localization were compared in order to decipher mechanisms and signaling pathways leading to ventricular dysfunction and arrhythmias in surgically repaired TOF. Several genes were confirmed to participate in ventricular remodeling and cardiac failure and some new candidate genes were described. In particular, these data pointed out FRZB as a heart failure marker. Moreover, calcium handling and contractile function genes (SLN, ACTC1, PLCD4, PLCZ), potential arrhythmia-related genes (MYO5B, KCNA5), and cytoskeleton and cellular organization-related genes (XIRP2, COL8A1, KCNA6) were among the most deregulated genes in rTOF ventricles. To our knowledge, this is the first comprehensive report on global gene expression profiling in the heart of a long-term swine model of repaired TOF.

0307 : Acute activation of the hexosamine biosynthetic pathway promotes electrical instability

Background The hexosamine biosynthetic pathway (HBP) is heightened in cardiomyopathies. We aimed at understanding how over-activation of HBP impacts on cardiac electrophysiology, and acts as a trigger or a substrate of cardiac arrhythmias. Methods Overactivation of the HBP by injection of glucosamine (GlcN 270mg/kg) was assessed under basal conditions and during beta-adrenergic stress on the myocardial energetic status (phosphocreatine to ATP ratio) of rats by 31 P nuclear magnetic resonance spectroscopy. Rat hearts were perfused ex vivo to evaluate the effects of the HBP activation (physiologically: glutamine 0.5mM – gln; overactivated: GlcN 1mM) on: cardiac function, MVO 2 , and arrhythmia score (AS) under basal (spontaneous arrhythmias: sAS) and pacing (pAS) conditions. At the cellular level (HL1 cells), HBP overactivation (GlcN) was assessed on conduction velocity characterized by micro-electrode arrays recordings, and properties of voltage gated ionic channels involved in cellular excitability and conduction by patch clamp experiments. Results In vivo GlcN did not modify PCr/ATP of the left ventricle under basal conditions. This ratio was significantly decreased during beta-adrenergic stress, while a similar increased heart rate was reported. When hearts were isolated and perfused ex vivo , GlcN induced a decrease of the heart rate and MVO 2 temporarily, and an increase of the sAS and pAS. Physiological activation of HBP with gln did not impact the ex vivo cardiac function and the sAS, but further increased the probability of atrial arrhythmias during pacing. In HL1 cells, GlcN reduced the rate of spontaneous depolarization, modified the direction and the velocity of the impulse propagation, and altered activation and inactivation properties of the sodium channel. Conclusion Acute HBP activation is associated with an increased vulnerability for arrhythmias and alteration of impulse conduction, suggesting its potential role in the atrial and ventricular arrhythmias. The author hereby declares no conflict of interest

January 16th, Friday 20150244: Pro-arrhythmic ventricular remodeling in a porcine model of repaired tetralogy of Fallot

Ventricular arrhythmias are frequent in patients with repaired tetralogy of Fallot but their underlying mechanisms remain unclear. In this study, ventricular electrical and structural remodelling was assessed in an animal model that mimics postoperative tetralogy of Fallot. Piglets underwent a tetralogy of Fallot repair-like surgery (rTOF N=6) or were sham-operated (Sham N=5). Following cardiac function assessment in vivo by MRI 3-4 months after surgery, pigs were euthanized and their hearts rapidly excised. Electrophysiological properties of right (RV) and left ventricles (LV) were obtained by optical mapping. Fibrosis was assessed histologically. RV dysfunction was evident while LV function remained unaltered in rTOF pigs. LV action potential duration (APD) was significantly longer on the epicardium (Sham 280±50ms; rTOF 390±76ms) and endocardium (Sham 301±20ms; rTOF 403±34ms) of rTOF animals (P

0305 : Heterogeneous conduction properties in the pig right ventricle

The right ventricular outflow tract (RVOT) has a distinct embryological origin and is a common anatomical source of arrhythmias in the healthy and diseased myocardium. We hypothesised that specific RVOT activation and conduction properties may underlie the preferential RVOT origin of arrhythmias. Pig right ventricular (RV) wedge preparations were perfused via the left anterior descending and right coronary arteries. Electrical activation and conduction properties were obtained by optical mapping of the epicardial surface (di-4- ANEPPS 10μM) upon electrical stimulation of the preparation. Transmural needles were inserted in the RV free wall and RVOT and unipolar electrograms (EGMs) were recorded. Fiber orientation was obtained by diffusion tensor MRI. Regional mRNA expression was determined by RT-PCR and fibrosis was assessed histologically. Longitudinal and transverse conduction velocities were significantly reduced in RVOT compared to RV free wall (P Conduction is slower in the pig RVOT and is associated with fractionated unipolar electrograms. Conduction slowing was related to (i) reduced connexin and sodium channel expression and (ii) region-specific structural properties which may generate a substrate for RVOT arrhythmias.

0179 : Specific down-regulation of cardiac connexin and pro-arrhythmic impulse propagation under electrical pacing

The rhythmic and coordinated propagation cardiac of the action potential is ensured by gap junction channels (GJCs) that are composed of connexin40 (Cx40), Cx43, Cx45 and Cx30.2, which present specific patterns of expression and electrical properties in different functionally specialized cardiac tissues. This study aims to determine the distinct contribution of Cxs in regulating the action potential propagation in mimicked healthy and diseased cardiac rates. The mouse atrial phenotypic HL-1 cell line that co-expresses Cx40, Cx43 and Cx45 was seeded on microelectrodes arrays to characterize the intrinsic electrical conduction and during pacing frequency from 2 to 30Hz. Specific SiRNA transfection of each Cx is used to determine the contribution of Cxs on regulating the conduction. We previously showed that the higher the frequency, the slower the conduction velocity (CV), up to a pro-arrhythmic frequency (10Hz). After downregulation of Cx40 (controlled by immunofluorescence), cells display a similar intrinsic coordinated activity to non-transfected cells (≈1Hz). However, the action potential propagates slower than non-transfected cells (≈60% at maximum downregulation of Cx40). Importantly, we observed that the arrhythmic conduction occurs at lower frequency after down-regulation of Cx40. These results indicate that Cx40 ensures a positive regulation of the action potential propagation under an increase of the cardiac rates, which suggests a specific GJCs make-up and electrical properties. Ongoing siRNA transfection for Cx43 and Cx45 will permit to distinguish the contribution of each Cxs in such regulation. Complementary biochemical characterization and patch clamp recordings on cell pairs will correlate the GJCs make-up and electrical properties to the changes of CV. This will improve our understanding on the specific role of each type of Cxs in regulating the impulse propagation in the healthy heart and their pro-arrhythmic dysfunction.

0167 : Dependence of the ratio of co-expressed connexin43 and connexin45 on the connexin composition of gap junction channels

In the heart, the propagation of the action potential (AP) is regulated by several factors, including gap junction channels (GJCs) made of connexins (Cxs). The ventricular myocardium expresses large amount of Cx43 and traces of Cx45 at distinct co-expression ratios that change in physiological and pathological conditions. This study aims to understand whether and how the ratio Cx43:Cx45 regulates the formation of GJCs. Rat Liver Epithelial cells that express endogenous Cx43 and stably transfected with Cx45 to induce accurate Cx43:Cx45 ratios are used. Electrical macroscopic and unitary properties of GJCs are determined by applying the dual voltage clamp method. We previously showed that induction of Cx45 decrease the electrical coupling, increase the voltage dependence and accelerate or decelerate the kinetics of deactivation and recovery. To date, our single channel recordings permit to estimate the Cxs composition of GJCs. Induction of Cx45 leads to the formation of one dominant GJCs population made of mixed Cx43/Cx45 composition, whereas GJCs composed with only Cx43 or Cx45 are present in minor proportions. Interestingly, this finely regulated GJCs make-up with Cx43 and Cx45 seems to be independent on the ratios Cx43:Cx45. Further experiments will be performed to mimic cardiac border zone which have different ratios of Cxs co-expression. This will provide a better understanding of the regulation of the cardiac impulse propagation in the healthy heart, and the pro-arrhythmic behavior of Cxs dysfunction and remodeling in the diseased heart.

0184 : Distinct ratios of co-expressed Cx40 and Cx43 regulate a fine gap junction channel make-up and properties

Co-expressed connexins (Cxs) can co-assemble to form gap junction channels (GJCs) of mixed Cxs composition characterized by specific electrical properties. To date the detailed GJCs make up in the distinct cardiac tissues that coexpress distinct gradients of Cxs stays poorly understood. We aim to investigate whether and how the GJCs make-up depends on the level and ratios of coexpressed connexin 40 (Cx40) and Cx43. Rat Liver Epithelial cells that endogenously express Cx43 and stably transfected to induce accurate ratios Cx43:Cx40 were used to perform electrical recordings on cell pairs by applying the dual voltage clamp method. We previously showed that the induction of Cx40 decreases the electrical coupling at high ratio Cx43:Cx40 while increasing at low ratios, decreases the voltage-dependence and slows the kinetics of deactivation and of recovery of GJCs. A deeper investigation at the single channel level permits to evaluate the Cxs composition and the distribution of different kinds of GJCs. Whereas non-induced cells, are coupled by homomeric-homotypic Cx43 GJCs, induction of Cx40 modifies the GJCs population in function of the ratio Cx43:Cx40: at high ratio GJCs made of Cx43 dominate the population, whereas lower ratios favor the Cx43/Cx40 heteromerization and the contribution of the Cx40. Our data show that the GJCs make-up is finely regulated in function on the ratio Cx43:Cx40 to form functional homotypic or heteromeric GJCs with distinct contributions of Cx40 and Cx43 properties and function in regulating the impulse propagation in the healthy heart and the pro-arrhythmic Cxs dysfunction. To correlate these results with the relative cardiac Cxs expression profiles, cell pairs of different Cxs expression pattern will be studied.

0106: Influence of the ratio of co-expressed cardiac connexins Cx43 and Cx45 in the formation of gap junction channels and their electrical properties

The cardiac action potential (AP) propagation is regulated to permit the coordinated and rhythmic atrial and ventricular contractions. This regulation requires several factors, especially gap junctions, which ensure a direct pathway for electrical and biochemical signaling. They are clusters of few to hundred intercellular gap junction channels (GJC) made of two hemichannels docked in the membrane of adjacent cells, which are composed of six connexins (Cxs). Their distinct electrical properties are a key factor regulating the propagation of the AP. Four cardiac Cxs, Cx40, Cx43, Cx45 and Cx30.2, exhibit specific patterns of expression that change in the healthy and diseased heart, which leads to different possible configurations of GJC. The aim of this study is to investigate the function of the distinct ratio of co-expressed Cxs in regulating the formation and function of GJC. Electrical properties of GJC (junctional coupling, voltage dependence, unitary conductance) are determined by performing electrical recordings on cell pair by applying the dual voltage-clamp method. Rat Liver Epithelial cells stably transfected to induce accurate Cx43:Cx45 ratios of 0 (single Cx43 expression), 0.5, 1 and 2, are used. The ongoing recordings show distinct electrical properties before and after the induction of Cx45: induction of Cx45 decreases the cell-to-cell coupling and rectifies the voltage dependence of GJC. Preliminary unitary recordings suggest a distinct formation of GJC of mixed Cx43/Cx45 composition in function of the Cx43:Cx45 ratio. Further investigations will provide better understanding on the distinct contributions of Cx43 and Cx45 in the GJC make-up, electrical properties and function of the Cx43/Cx45 expression pattern in regulating the cardiac impulse propagation in the healthy heart, and the pro-arrhythmic behavior in the diseased heart.