Chronic pain is a debilitating disease with a very important socio-economic burden. The objective of this thesis was to contribute to our understanding of the normal organization of the dorsal horn of the spinal cord and its changes in chronic neuropathic pain, a form of chronic pain that sometimes follows lesions of the nervous system. Our studies focused on two important components of spinal cord pain-related circuitry, the projection neurons and their innervation by the small diameter nociceptive afferents. Spinal lamina I projection neurons have been classified, based on their morphology into fusiform, multipolar and pyramidal neurons. The two former types have been shown to respond to noxious stimuli and express the substance P receptor (NK-1r), while pyramidal neurons seldom express the NK-1r and respond to innocuous cooling only. The two main populations of small diameter nociceptive afferents are the peptidergic, which expresses the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the non-peptidergic, which is mostly devoid of neuropeptides but binds the plan lectin IB4. In the first experimental chapter, we investigated the changes that occur at the level of the spinal dorsal horn in an animal model of chronic neuropathic pain. We demonstrated a de novo expression of NK-1r by pyramidal neurons, similar to that previously observed in our laboratory in a chronic arthritis model. This phenotypic switch was associated with a dramatic increase in the peptidergic (SP-immunoreactive) innervation of this cell population, which normally is sparsely innervated by these fibers. To assess whether pyramidal neurons responded to noxious stimuli in neuropathic animals, we injected capsaicin in the hind paw, which induced a massive internalization of NK-1r on these neurons, an indication of cell activation. To assess whether a chronic pain state was needed to trigger the expression of NK-1r by pyramidal neurons, in the second experimental chapter of this thesis we used a model in which there is no chronic pain but in which previous work from our laboratory had revealed a marked increase in NK-1r in the dorsal horn. In this model, the non-peptidergic population of nociceptive afferents is specifically ablated by the injection of the neurotoxin saporin conjugated to the lectin IB4 (IB4-SAP) into the sciatic nerve. The animals did not display any pain-related behavioral changes. However, we observed a significant upregulation of NK-1r in lamina I, in neuronal types that normally expressed it (i.e. fusiform and multipolar cells), with no de novo expression by pyramidal neurons. In the third experimental chapter, we addressed the issue of whether lamina I projection neurons which express the NK-1r are innervated by non-peptidergic nociceptive afferents, as a study in a transgenic mouse model had provided data suggesting that non-peptidergic afferents had connections with deep dorsal horn neurons but not with lamina I NK-1r-expressing cells. We performed a systematic study aimed at identifying the normal connections of the non-peptidergic nociceptive fibers with lamina I neurons using both confocal and electron microscopy and we found a considerable innervation by non-peptidergic afferents on all three types of lamina I projection neurons. The results of this thesis, taken together with previous data from our lab, suggest that a chronic pain state, such as neuropathic pain, seems necessary to trigger a de novo expression of NK-1r in pyramidal neurons and their increased innervation by peptidergic afferents. Further studies are required to clarify the role, in normal nociception and chronic pain states, of the significant direct innervation of lamina I projection neurons by non-peptidergic afferents which we revealed for the first time. / La douleur chronique est une condition débilitante ayant de sérieuses répercussions socio-économiques. L'objectif de cette thèse était de mieux comprendre l'organisation de la corne dorsale de la moelle épinière et les changements qui s'y produisent dans les cas de douleurs chronique neuropathique suite à une lésion du système nerveux. Nos études se sont concentrées sur deux composantes importantes des circuits de la douleur: les neurones de projection et leur innervation par les afférents nociceptifs de petit diamètre. Les neurones de projection de la couche 1 de la moelle épinière sont classées selon leur morphologie en 3 types: les neurones fusiformes, multipolaires et pyramidaux. Les deux premiers répondent aux stimuli douloureux et expriment le récepteur de la substance P (NK-1r), alors que les neurones pyramidaux n'expriment ce récepteur qu'occasionellent et répondent au froid non-douloureux. Les deux populations d'afférents principales sont les fibres de petit diamètre peptidergiques, qui expriment la substance P et le "calcitonin gene-related peptide" (CGRP), et les non-peptidergiques, qui sont dépourvues de neuropeptides et qui s'associent avec la lectine IB4. Lors du premier chapitre expérimental, nous avons étudié les changement qui se produisent dans la corne dorsale de la moelle épinière dans un modèle de douleur neuropathique chronique. Nous avons démontré une expression de novo du NK-1r sur les neurones pyramidales, un changement similaire à celui se produisant dans un modèle d'arthrite chronique. Ce changement de phénotype était associé à une augmentation significative du nombre d'appositions peptidergiques faites sur cette population neuronale, qui reçoit habituellement très peu de ces entrées. Afin de vérifier si ces récepteurs sont fonctionnels et répondent aux stimuli douloureux, nous avons injecté de la capsaicine dans la patte arrière, ce qui a mené à une internalisation du récepteur, marquant l'activation de celui-ci. Le deuxième chapitre de cette thèse vérifie si un état de douleur chronique est nécéssaire pour ce changement phénotypique, utilisant une lésion non douloureuse qui cause une augmentation signnificative du NK-1r dans la corne dorsale. Dans ce modèle, une population de nocicepteurs non peptidergiques est excise par une injection dans le nerf sciatique de la toxine saporine conjuguée à la lectine IB4 (IB4-SAP). En absence de symptômes douloureux, la couche 1 de la corne dorsale des animaux lésés a subi une augmentation générale du NK-1r mais sa distribution cellulaire est restée normale, sans expression de novo sur les cellules pyramidales.Lors du troisième chapitre de cette thèse, nous avons vérifié si les neurones de projection de la couche 1 exprimant le NK-1r recevaient des entrées des fibres nociceptives non peptidergiques, comme ce sujet était controversé suite à une publication utilisant des souris transgéniques démontrant une absence de connections de la sorte. Nous avons fait une étude systématique utilisant la microscopie confocale et électronique et avons démontré que les 3 types morphologiques de cellules de projection reçoivent des entrées non peptidergiques directes.Pris ensembles, les résultats de cette thèse suggèrent qu'une condition de douleur chronique est nécessaire pour l'expression du NK-1r sur les neurones pyramidaux et l'augmentation des entrées peptidergiques faites sur celles-ci. D'autres études seront nécessaires pour clarifier l'implication des entrées non peptidergiques faites sur les neurones de projection dans la nociception normale et dans la douleur chronique.

Animal studies have shown that duloxetine is a dual inhibitor of norepinephrine (NE) and serotonin (5-HT) reuptake. The aim of this study was to assess the effects of duloxetine on the 5-HT and NE reuptake processes in healthy human volunteers. Twenty-seven healthy young males were randomly assigned to one of four groups, receiving one of the following daily drug regimens: placebo, clomipramine (a potent 5-HT/NE reuptake blocker) 100 mg/day, duloxetine 20 mg/day, or duloxetine 60 mg/day. In order to assess the NE reuptake process, the pressor response to intravenous tyramine was measured. Determination of the whole blood 5-HT content was used to evaluate the 5-HT reuptake blockade. These measurements were performed at baseline and repeated after 7 and 14 days of drug intake. Both duloxetine, at doses of 20 to 60 mg/day, and clomipramine significantly interfered with the 5-HT reuptake process, as demonstrated by marked decreases in blood 5-HT concentrations. However, the same doses of duloxetine, unlike clomipramine, failed to impede the usual increase in blood pressure that follows a tyramine intravenous infusion, indicating that clomipramine, but not duloxetine, blocked the NE reuptake process. At doses tested in healthy volunteers, duloxetine acted as a selective 5-HT reuptake inhibitor, having no clear effect on the NE reuptake process

In this thesis, 5-azacytidine, a drug which is incorporated into DNA and blocks DNA methylation, was administrated to male rats to determine its effects on male germ cell development and function. Chronic treatment at doses of 0 to 5.0 mg/kg for four, six, eleven or sixteen weeks were designed to expose different populations of germ cells. Males were mated with normal females to evaluate progeny outcome. Treatment of rats for four weeks, which exposes postmeiotic germ cells only, resulted in no changes in male reproductive organ weights, or abnormal progeny outcome. Treatment for eleven and sixteen weeks which exposes germ cells throughout spermatogenesis, resulted in dose-dependent decreases in testis and epididymal weights, and sperm counts, and increased preimplantation loss when embryos were examined at day twenty of gestation. Examination of embryos at day two of gestation confirmed that embryos sired by males treated with 5-azacytidine were abnormal and died prior to implantation. Six weeks of treatment, which exposes meiotic and post-meiotic germ cells, had an effect on male reproductive organs and progeny outcome intermediate between that of four and eleven weeks of treatment. 5-Azacytidine exposure resulted in abnormalities in testicular histology after eleven, but not six weeks, however closer evaluation using terminal deoxynucleotidyl transferase-mediated nick end-labeling detection in situ showed that germ cells were undergoing apoptosis after both exposure times. Analysis of DNA methylation levels in isolated germ cells and sperm indicated that 5-azacytidine caused decreases in DNA methylation in spermatozoa after 6 and 11 weeks of treatment, and 6 weeks of treatment caused decreases in DNA methylation of round spermatids, but not pachytene spermatocytes. Preimplantation embryos sired by males treated with 5-azacytidine became developmentally arrested in culture. These embryos, however, were partially rescued by the addition of the RNA transcription

The effects of age on the structure and functions of the epididymis were studied using the Brown Norway rat model. Striking quantitative and qualitative changes during aging in the histology of the epididymis were observed. Characteristic features of aging, such as increases in basement membrane thickness and massive accumulation of lipofuscin, were found. In addition, epididymis-specific signs of aging such as polymorphism of lysosomes, presence of giant vacuoles and spermiophagy, and a major increase in the number of halo cells were also found. The precise nature of halo cells was demonstrated by using specific antibodies. In epididymides from young adult animals, halo cells are composed of three types of immune cells: helper T cells, cytotoxic T cells and monocytes. The three immune cell types present increased with age in the epididymal epithelium in a segment specific manner. In contrast their numbers did not increase significantly in the interstitial tissue. Furthermore, the concentration of cytotoxic T cells and monocytes-macrophages was enhanced in the epididymal epithelium of aged rats whose epididymal lumen contained few spermatozoa. The recruitment of immune cells might be triggered by the accumulation of damaged epithelial cells, or by sperm antigens leaking through the blood epididymis barrier. Thus, the epididymal microenvironment that is essential for the maturation of spermatozoa could be altered in old age. The effect of advancing paternal age on pregnancy and progeny outcomes was assessed by mating Brown Norway male rats of increasing age with young Sprague-Dawley females. The effects of advancing paternal age on the offspring included an increase in preimplantation loss, a decrease in the average fetal weight on day 20 of gestation, and an increase in early neonatal death. These results indicated that the quality of spermatozoa decreases as males age.

Previous work demonstrated that influx of (2-chloroethyl)-1-sarcosinamide-1-nitrosourea (SarCNU) may be altered from the known influx of clinically available chloroethylnitrosoureas which occurs through passive diffusion. The objective of this thesis was to determine the exact mode of influx of SarCNU into human glioma cell lines SK-MG-1 and SKI-1 which are sensitive and resistant to SarCNU, respectively. The influx of SarCNU into SK-MG-1 cells was shown to be mediated by a saturable, energy and sodium independent epinephrine sensitive carrier system. Analysis of influx of SarCNU into SKI-1 cells demonstrated a technically non-saturable mechanism of entry consistent with passive diffusion. Steady-state accumulation of SarCNU was demonstrated to be greater in SK-MG-1 cells versus SKI-1 cells at 37$ sp circ$C whereas there was similar accumulation at 22$ sp circ$C. Differences in steady-state accumulation were not attributable to altered metabolism or efflux. Increased accumulation of SarCNU in SK-MG-1 cells at 37$ sp circ$C was identified to be a consequence of an increased initial rate of influx at 37$ sp circ$C in SK-MG-1 cells versus SKI-1 cells with no significant difference at 22$ sp circ$C. Analysis of chloroethylnitrosourea sensitivity revealed that SKI-1 cells were 3 fold resistant versus SK-MC-1 to SarCNU at 37$ sp circ$C but only 2 fold resistant at 22$ sp circ$C with no temperature shift effect on the 2 fold level of resistance to BCNU. A more detailed analysis of the SarCNU carrier involving the analysis of norepinephrine influx revealed that SarCNU influx into SK-MG-1 cells occurs through the extraneuronal catecholamine uptake$ sb2$ transporter which is not detectable in SKI-1 cells. This is the first direct demonstration of the presence of the uptake$ sb2$ transporter in a human glioma cell line. These findings suggest that increased sensitivity of SK-MG-1 cells to SarCNU is secondary to enhanced accumulation of SarCNU mediated via the uptake$ sb2$ t

The responses of the atrioventricular node (AVN) to changes in activation rate is complex. A variety of approaches have been used to explain the different responds of AVN, but none has been able to describe fully nodal behavior. Three rate-dependent nodal properties referred to as recovery, facilitation, and fatigue contribute to rate-induced changes in nodal conduction time. We developed a model incorporating a quantitative description of these properties that was able to accurately predict AV node conduction during steady state atrial pacing. This model provides a mathematical framework through which interventions such as changes in the autonomic tone may affect AV nodal conduction. This was evaluated by studying effects of vagal stimulation (VS), isoproterenol (IP), and beta blockade (BB) an AVN conduction. Experimental results and mathematical modelling revealed that these interventions alter the ways in which the AVN responds to changes in activation rate. These changes result in enhanced rate-dependent AVN conduction slowing with VS and BB, and reduced rate-dependent slowing with IP. / The potential implications of the rate-dependent properties of the AVN were studied by evaluating whether the depressant effects of diltiazem (D) on the AVN are enhanced during arrhythmias which increase AVN activation rate. The relative magnitude of D's effects on the AVN during tachycardia (atrial fibrillation, and circus movement tachycardia) were significantly magnified compared to the effects at races comparable to sinus rhythm in man. Rate-dependent calcium channel-blocking properties of D were an intrinsic property, though their intensity was modulated by autonomic tone. This thesis provides a link between basic theories of rate dependent properties and physiologic and pharmacologic implications.

With over 4000 chemicals in tobacco, nicotine is thought to account for the primary reinforcing effects that lead to tobacco dependence. However, there are indications that both environmental stimuli and non-nicotine components in tobacco smoke may also contribute to smoking behaviour. In an attempt to identify other reinforcing compounds present in tobacco, we tested a number of tobacco-related chemicals, either independent of nicotine (i.e. harman) or in combination with nicotine (i.e. tobacco smoke condensate), using the intravenous self-administration paradigm in adult rats. For this purpose, we used a novel procedure of self-administration that is intended to mimic the pharmacokinetics of nicotine delivery seen in human smokers. At what we initially inferred to be smoking-relevant doses, neither harman nor tobacco smoke condensate was detectably reinforcing. Additionally, we observed that a motivationally neutral cue can facilitate intravenous nicotine self-administration. The work described in this thesis also supports the view that nicotine is a weak primary reinforcer. / Avec plus de 4 000 substances chimiques entrant dans la constitution du tabac, la nicotine est soupçonnée comme étant le principal composant responsable des effets de renforcement positif qui mènent à la dépendance tabagique. Toutefois, il y a des indications que des stimuli présents dans l'environnement ainsi que des éléments chimiques autre que la nicotine peuvent aussi contribuer au comportement des fumeurs. Afin d'identifier d'autres constituants du tabac impliqués dans les effets de renforcement, nous avons examiné quelques produits chimiques liés au tabac, indépendants de la nicotine (c.-à-d. Harman) et en combinaison avec la nicotine (c.-à-d. condensat de fumée de tabac). Cela a été effectué en utilisant le paradigme de l'auto-administration par voie intra-veineuse chez le rat adulte. À cette fin, nous avons utilisé une nouvelle procédure d'auto-administration qui suit la pharmacocinétique de la distribution de la nicotine chez les fumeurs. À des doses que nous avons déduit d'être pertinentes pour le fumeur au commencement, ni Harman ni le condensat de fumée de tabac n´ont eu un effet détectable de renforcement. En outre, nous avons aussi observé qu'un signal de motivation neutre peut faciliter l'auto-administration de la nicotine par voie intra-veineuse. Le travail dépeint dans cette thèse supporte l'idée que la nicotine est un faible renforçateur primaire.

The epididymis functions in the transport, maturation, storage and protection of spermatozoa. The structure and functions of the epididymis are dependent on androgens, primarily dihydrotestosterone (DHT). 5a-Reductases (types 1 and 2) are thus key enzymes in this tissue because of their role in converting testosterone to DHT. The goal of this thesis was to examine the consequences on the rat epididymis and epididymal sperm of inhibiting DHT formation using novel dual 5alpha-reductase inhibitors (i.e. inhibitors specific to both isoforms of 5alpha-reductase). The first objective was to analyze the effects of 5alpha-reductase inhibitor treatment on gene expression in the four epididymal segments (initial segment, caput, corpus and cauda epididymidis) using cDNA arrays. Treatment had highly segment specific effects on epididymal gene expression. Affected genes included those involved in fatty acid and lipid metabolism, regulation of ion and fluid transport, luminal acidification, oxidative defense and protein processing and degradation; these are essential processes contributing to the formation of an optimal luminal microenvironment required for proper sperm maturation. The next objective was to determine whether the observed changes in gene expression actually translated into effects on epididymal sperm functions. Fertility and several key facets of epididymal sperm maturation were analyzed. The percentages of motile and progressively motile sperm from the cauda epididymidis decreased and characteristic sperm motion parameters were altered. An elevated proportion of sperm from this region also retained their cytoplasmic droplet. Matings with treated males resulted in fewer successful pregnancies and a higher rate of preimplantation loss. Lastly, cDNA arrays and quantitative real-time RT-PCR were used to elucidate potential signaling mechanisms via which DHT mediates and/or regulates its differential effects on epididymal gene expression and

Ventricular arrhythmia is the main leading cause of sudden cardiac death, which remains a major public health problem. To date, no available antiarrhythmic drug therapies have been proven mortality benefit. Abnormalities in cardiac repolarization are important contributors to the development of lethal ventricular arrhythmias. The basic ion-channel control mechanisms for ventricular repolarization are still poorly understood. This thesis addressed potential mechanisms responsible for ion channel regulation involved in three clinically relevant paradigms of ventricular repolarization control. / I first characterized in detail the ionic currents controlling repolarization across the ventricular wall in female/male canine left ventricles. I found intrinsic sex-related differences in overall inward L-type calcium current (ICa,L) and transmural repolarizing K+ currents, including the slowly-activated delayed rectifier K+ current (IKs) and transient outward potassium current (Ito). Offsetting effects of larger female IKs and ICa,L explain similar male/female APDs in epicardium and endocardium. However, I found larger ICa,L with similar repolarizing K+ currents between female and male in the midmyocardium, which may account for the observed longer female action potential duration (APD) in this specific cell-type. The reduced repolarization reserve in the female midmyocardium explains the female sensitivity to QT-prolonging drugs and Torsades de pointes (TdP). / Chronic tachycardia produces ventricular electrical remodeling, heart failure (HF) and susceptibility to cardiac arrhythmia. I assessed the hypothesis that increased heart rate per se regulates ion channel expression. I found that the Ito and its subunit Kv4.3 are directly regulated by increased electrical firing rate. I established the signaling pathways transducing changes in cardiac firing rate into downregulation of Kv4.3 transcription. I determined that Ca2+/calmodulin-dependent CaMKII and calcineurin/NFAT systems play key Ca2+-sensing and signal-transducing roles in rate-dependent Ito control, identifying molecular mechanisms contributing to cardiac excitation-transcription coupling. / I identified a novel mechanism potentially governing repolarization reserve, feedback regulation of ion channel expression. Traditionally, cardiac repolarization reserve refers to functionally-based compensatory increases in repolarizing currents, usually IKs, that minimize changes in APD caused by dysfunction or inhibition of a single K+-current. I found that chronic action potential prolongation, induced by inhibiting the rapidly-activated delayed rectifier K+ current (IKr), increases repolarization reserve through compensatory upregulation of IKs and the protein levels of its molecular components KvLQT1 and minK. The feedback regulation of KvLQT1 expression is partly mediated by downregulating the inhibitory microRNA (miRNA) miR-133. Impairment of this feedback system could contribute to the occurrence of cardiac arrhythmias in repolarization dysfunction paradigms like congenital and acquired long-QT syndromes. / In conclusion, my findings in this thesis highlight the delicate and complex control of ventricular repolarization through homeostatic regulation of underlying ion-channel function and expression under physiological and pathophysiological conditions. / L'arythmie ventriculaire est la principale cause principale de mort subite d'origine cardiaque et demeure un problème de santé majeur. À date, aucun traitement médicamenteux antiarythmique a démontré utile dans la prévention de la mort subite. Les anomalies de la répolarisation cardiaque contribuent d'une façon importante à l'apparition d'arythmies ventriculaires létales. Les mécanismes impliqués dans la régulation des canaux ioniques qui contrôlent la répolarisation ventriculaire sont encore mal compris. L'objectif central de cette thèse était de déterminer les mécanismes responsables de la régulation des canaux ioniques dans trois paradigmes cliniquement significative du contrôle de la repolarisation ventriculaire. fr / Nous avons d'abord caractérisé en détail les courants ioniques en relation avec la répolarisation à travers la paroi ventriculaire chez les ventricules gauches canins selon le sexe. Nous avons noté des différences selon le sexe des animaux dans les courants calciques de type L (ICaL) et l'ensemble des courants potassiques, incluant le « slow delayed rectifier » IKs et le courant transitoire sortant Ito. L'augmentation parallèle de IKs et ICa chez les femmes aux niveaux sous épicardique et endocardique n'a pas amené à un changement dans la répolarisation. Par contre, au niveau du milieu du myocarde, le ICaL a été augmenté chez les femmes sans changement dans les courants potassiques, ce qui a provoqué une augmentation dans la durée du potentiel d'action (DPA). Le réduction du « repolarization reserve » à ce niveau peut expliquer la sensibilités des femmes aux molécules qui prolongent le DPA et leur prédilection pour les TdPs. / La tachycardie chronique produit un remodelage électrique cardiaque, une insuffisance cardiaque et une susceptibilité à l'arythmie cardiaque. Nous avons évalué l'hypothèse qu'une augmentation de la fréquence cardiaque peut, en soi, affecter l'expression des canaux ioniques. Nous avons constaté que l'expression de l'Ito et de sa sous unité sous jacente Kv4.3 sont supprimé une augmentation dans la fréquence d'activation des cellules cardiaques. Nous avons également établi les voies de signalisation sous jacentes, qui impliquent le CaMKII Ca2+/calmoduline-dépendent et le système calcineurine/NFAT, qui ressentient le taux cellulaire de Ca2+ et ajustent l'expression de Kv4.3 en conséquence. Il s'agit d'une découverte des mécanismes moléculaires qui contribuent au couplage entre l'excitation cardiaque et la transcription génique. fr / Nous avons identifié un nouveau mécanisme qui la réserve de la répolarisation (« repolarization reserve »), médié par la régulation de l'expression des canaux ioniques. Traditionnellement, la réserve de la répolarisation cardiaque fait référence à des augmentations fonctionnelles compensatoires dans les courants répolarisants, en particulier IKs, qui minimisent les changements dans la DPA causés par un dysfonctionnement d'un simple courant potassique. Nous avons alors constaté que la prolongation chronique de la DPA induite par l'inhibition du courant « rapid delayed rectifier » IKr de la réserve rapidement activé redresseur retardé courant K+ (IKr), entraine une augmentation de la réserve de la répolarisation par une augmentation dans l'expression de l'IKs qui est réalisé par une augmentation de l'expression des taux de protéines de ses composants moléculaires KVLQT1 et minK. Cette régulation semble produit par une diminution dans l'expression d'une petite molécule ARN inhibitrice, le micro-ARN (miRNA) miR-133. Une déficience de ce système de contrôle peut contribuer à la survenue d'arythmies cardiaques chez les patients atteints d'une dysfonction de répolarisation. fr / En conclusion, nos résultats dans cette thèse soulignent le contrôle délicat et complexe de la répolarisation ventriculaire par la régulation homéostatique du fonctionnement des canaux ioniques dans les conditions physiologiques et physiopathologiques. fr

A downregulation of the transient outward K+ current (Ito) is a common finding in hypertrophy; tachypacing induced heart failure, which can lead to lethal arrhythmias and sudden death as a result of action potential prolongation.To better understand how variations in heart rate may affect the electrical properties of the heart, based on an in vitro tachypacing model, we compared frequency-dependent changes in adult epicardial (Epi) versus endocardial (Endo) canine cells. Thus, Ito was recorded with whole-cell patch-clamp in Endo and Epi cardiomyocytes after 24-hr culture under continuous electrical stimulation at either 1-Hz (normal rate) or 3-Hz (tachycardia). Ca2+ transient amplitudes (Indo1-AM) were recorded from En-do and Epi cells at 0hr, 2hr, 4hr, 8hr and 24hr after continuous electrical stimulation at either 1-Hz or 3-Hz. Analysis of inactivation/reactivation kinetics, activation/inactivation voltage dependence properties showed no difference between 1- and 3-Hz paced cells for either cell type. Tachypacing (3Hz pacing) reduced Ito density and Kv4.3 mRNA ex-pression in Epi cells but not in Endo. Ca2+-chelation by BAPTA-AM, prevented rate-dependent Ito downregulation in Epi cells, increasing Ito in tachypaced cells by ~47%. In Endo cells, BAPTA-AM increased Ito by ~200% in a rate-independent way. Con-sistent with the larger Endo response to Ca2+-chelation, Ca2+ transient amplitudes were greater in Endo (85±4 nM) than Epi (46±2 nM) cells. Ca2+ transient amplitude increased at early stages of tachypacing in Epi cells (i.e. 64.2±4.2 from 40.7±3.5 nM), in contrast to Endo cells. Inhibition of the calcineurin/NFAT pathway prevented rate-dependent Ito decrease in tachypaced Epi cells, without affecting Endo Ito.Our findings show that fast firing rate induces a different response in the regulation of Ito in adult Epi and Endo cells, which might be related to differential control by calcium-dependent signaling systems. / Il est admit que le courant potassique sortant Ito est diminué de façon importante dans l'insuffisance cardiaque induite par un contexte d'hypertrophie ventriculaire et de tachystimulation. Plus précisément, des travaux précédents de notre laboratoire montrent que l'insuffisance cardiaque induite par tachystimulation ventriculaire chez le chien favorise les arythmies ventriculaires létales et la mort subite. Une réduction du courant Ito dans les ventricules a été démontrée dans ce modèle. Afin de mieux comprendre comment la tachystimulation (tachycardie ventricu-laire soutenue) affecte les propriétés électriques des cardiomyocytes ventriculaires, nous avons comparé l'effet de la tachystimulation in vitro sur le courant potassique Ito de cardiomyocytes de l'épicarde et de l'endocarde ventriculaire de chien. Le courant Ito de cardiomyocytes de l'epicarde et de l'endocarde à été enregistré à l'aide de la technique de patch-clamp en configuration cellule entière après 24h de culture sous tachystimulation continue a 1-Hz (rythme normal) ou 3-Hz (tachycardie). Les transi-toires calciques ont été étudiés (Indo1-AM) après 0, 2, 4, 8 et 24hr de stimulation a 1-Hz ou 3-Hz. L'analyse de la dépendance au voltage des processus d'inactivation et de réactivation du courant Ito n'a démontré aucune différence entre les cellules de l'épicarde et de l'endocarde, ni même entre 1-Hz et 3-Hz. De façon intéressante, la tachystimulation (3-Hz) réduisait la densité du courant Ito et l'expression des ARNms codant pour le canal Kv4.3 dans les cellules de l'épicarde mais pas dans l'endocarde. La chélation du calcium intracellulaire à l'aide du BAPTA-AM, prévenait cette diminution de Ito dans les cellules de l'épicarde entrainant une augmentation de ~47% du courant. Dans les cellules de l'endocarde, le BAPTA-AM provoquait une augmentation de ~200%. De façon cohérente avec une sensibilité des cellules de l'endocarde plus importante à la chélation de calcium, l'amplitude des transitoires calciques était plus importante dans les cellules de l'endocarde (85±4 nM) que dans les cellules de l'épicarde (46±2 nM). Alors que l'amplitude des transitoires calciques n'était pas modifiée par la tachystimulation dans les cellules de l'endocarde, une augmentation précoce était me-surée dans les cellules de l'épicarde (i.e. 64.2±4.2 vs 40.7±3.5 nM). L'inhibition de la voie de signalisation NFAT/calcineurine prévenait la diminution d' Ito dans les cellules de l'épicarde soumises à la tachystimulation et restait sans effet sur le courant Ito des cellules de l'endocarde.Nos résultats montrent que les cellules adultes de l'épicarde et de l'endocarde répondent de façon différente suite à une tachystimulation. Ce processus est possiblement lié à un contrôle différentiel de la signalisation induite par le calcium.