Ca channels induced in Xenopus oocytes by rat brain mRNA

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2–5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline. I_(Ba) in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 ± 4 nA, n = 21). In contrast, injected oocytes showed a peak I_(Ba) of 339 ± 42 nA (n = 33). The threshold for activation of I_(Ba) was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (τ = 650 ± 50 msec at +20 mV). The maintained current was 67 ± 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward I_(Ba) could be observed in BaCl₂ solutions at potentials positive to E_(Cl) and also in Na-free salines, indicating that neither Cl⁻ nor Na⁺ was carrying the inward current. Although I_(Ba) displayed voltage- independent blockade by Cd (50% inhibition at 6 µM), the peptide Ca channel antagonist, ω-CgTX (1 µM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 µM, or when cells were held at -40 mV) or agonist Bay K-8644. However, I_(Ba) was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT₂). In contrast, use of forskolin to activate protein kinase A did not alter I_(Ba). However, experiments in the presence of Cd revealed that forskolin decreased I_K. Ca channels produced by rat brain mRNA were thus in contrast to the nifedipine-sensitive, Bay K-8644- and forskolin-enhanced Ca channels observed after injection of rat heart mRNA (Dascal et al., 1986)

Ca channels induced in Xenopus oocytes by rat brain mRNA

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2–5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline. I_(Ba) in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 ± 4 nA, n = 21). In contrast, injected oocytes showed a peak I_(Ba) of 339 ± 42 nA (n = 33). The threshold for activation of I_(Ba) was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (τ = 650 ± 50 msec at +20 mV). The maintained current was 67 ± 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward I_(Ba) could be observed in BaCl₂ solutions at potentials positive to E_(Cl) and also in Na-free salines, indicating that neither Cl⁻ nor Na⁺ was carrying the inward current. Although I_(Ba) displayed voltage- independent blockade by Cd (50% inhibition at 6 µM), the peptide Ca channel antagonist, ω-CgTX (1 µM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 µM, or when cells were held at -40 mV) or agonist Bay K-8644. However, I_(Ba) was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT₂). In contrast, use of forskolin to activate protein kinase A did not alter I_(Ba). However, experiments in the presence of Cd revealed that forskolin decreased I_K. Ca channels produced by rat brain mRNA were thus in contrast to the nifedipine-sensitive, Bay K-8644- and forskolin-enhanced Ca channels observed after injection of rat heart mRNA (Dascal et al., 1986)

Aldosterone increases T-type calcium channel expression and in vitro beating frequency in neonatal rat cardiomyocytes

Objective: Although aldosterone has been implicated in the pathogenesis of cardiac hypertrophy and heart failure, its cellular mechanism of action on cardiomyocyte function is not yet completely elucidated. This study was designed to investigate the effect of aldosterone on calcium channel expression and cardiomyocyte contraction frequency. Methods: Cultured neonatal rat ventricular cardiomyocytes were stimulated in vitro with 1 μmol/L aldosterone for 24 h. Calcium currents were then measured with the patch clamp technique, while calcium channel expression was assessed by real-time RT-PCR. Results: In the present study, we show that aldosterone increases Ca2+ currents by inducing channel expression. Indeed, aldosterone led to a substantial increase of L- and T-type Ca2+ current amplitudes, and we found a concomitant 55% increase of the mRNA coding for α1C and β2 subunits of cardiac L channels. Although T-type currents were relatively small under control conditions, they increased 4-fold and T channel α1H isoform expression rose in the same proportion after aldosterone treatment. Because T channels have been implicated in the modulation of membrane electrical activity, we investigated whether aldosterone affects the beating frequency of isolated cardiomyocytes. In fact, aldosterone dose-dependently increased the spontaneous beating frequency more than 4-fold. This effect of aldosterone was prevented by actinomycin D and spironolactone and reduced by RU486, suggesting a mixed mineralocorticoid/glucocorticoid receptor-dependent transcriptional mechanism. Moreover, inhibition of T currents with Ni2+ or mibefradil significantly reduced beating frequency towards control values, while conditions affecting L-type currents completely blocked contractions. Conclusion: Aldosterone modulates the expression of cardiac voltage-operated Ca2+ channels and accelerates beating in cultured neonatal rat ventricular myocytes. This chronotropic action of aldosterone appears to be linked to increased T channel activity and could contribute to the deleterious effect of an excess of this steroid in vivo on cardiac functio

The G-protein–gated K+ channel, IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K+ current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4−/− mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4−/− mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4−/− animals. Although the relative extent of heart rate regulation of Girk4−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4−/− animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation

Clonage et caractérisation fonctionnelle de deux canaux calciques de type T humains "alpha"1G et "alpha"1I

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Cardioprotection au cours de l'ischémie-reperfusion myocardique chez la souris (modèle transgénique Daxx-dominant négatif et postconditionnement ischémique)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Identification de nouvelles cibles thérapeutiques potentielles parmi les récepteurs couplés aux protéines G des muscles striés

Les Récepteurs Couplés aux Protéines G (RCPGs) sont la plus grande et la plus diversifiée des familles de récepteurs membranaires. Environ un tiers des molécules thérapeutiques actuellement disponibles sur le marché ciblent un nombre très limité des membres de cette famille. Une meilleure connaissance de l'expression des RCPGs dans divers tissus et contextes physiopathologiques devrait permettre d'identifier de nouvelles cibles thérapeutiques. Les pathologies cardiaques sont la première cause de mortalité dans le monde occidental et sont souvent associées à une dysfonction des muscles squelettiques. L'objectif du travail rapporté dans cette thèse était d'analyser l'expression des endoRCPGs (RCPGs ayant des ligands endogènes) dans le cœur et le muscle squelettique afin d'identifier de nouveaux récepteurs, dont la fonction dans ces tissus est inconnue, et évaluer leur potentiel en tant que cibles thérapeutiques. Nous avons tout d'abord établi le répertoire de l'ensemble des endoRCPGs dans les quatre chambres cardiaques de souris adultes et mis en évidence un récepteur atypique, le récepteur métabotropique du glutamate, mGluR1. Des expériences préliminaires suggèrent une implication de mGluR1 dans la cardioprotection lors de l'ischémie/reperfusion. Nous avons aussi établi le répertoire des endoRCPGs du muscle squelettique issu d'un modèle murin d'atrophie réversible. L'analyse de ce répertoire nous a permis d'identifier des nouveaux récepteurs impliqués dans l'atrophie musculaire. Parmi ces récepteurs, nous nous focalisons sur le récepteurs béta2-adrénergique et le récepteur Frizzled 9. Ce dernier a particulièrement attiré notre attention car il est peu exprimée dans le cœur et pourrait ainsi être ciblé sans avoir d'incidence cardiaqueG protein-coupled receptors (GPCRs) are the largest and most diverse family of membrane receptors. Approximately one third of currently available pharmaceutical drugs target a small number of these receptors. New information on the expression of GPCRs in various tissues and physiopathological contexts should enable the identification of new therapeutic targets. Cardiac pathologies are the leading cause of morbidity in the Western world and are often associated to skeletal muscle dysfunction. The aim of the work described in this thesis was to analyse the expression of all endoGPCRs (GPCRs with endogenous ligands) in heart and skeletal muscle in order to identify new receptors, with no known function in these tissues, and evaluate there potential as therapeutic targets. We first established the endoGPCR repertoire of the four cardiac chambers and identified an atypical cardiac receptor, mGluR1. Preliminary results suggest that mGluR1 is involved in cardioprotection in the context of ischemia/reperfusion. We also established the repertoire of endoGPCRs in skeletal muscle using a reversible mouse atrophy model. The analysis of this repertoire enabled us to identify new receptors involved in the development of atrophy. Among these, we focused on the beta2-adrenergic and Frizzled 9 receptors. The latter particularly interests us as it is very lowly expressed in the heart, suggesting that targeting this receptor would have little incidence on cardiac functionMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Développement et caractérisation de nouveaux outils pour l'étude de l'adressage membranaire des canaux calciques voltage-dépendants

MONTPELLIER-BU Médecine UPM (341722108) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

Rôle fonctionel des canaux ioniques dans la génération de l'automatisme et de la conduction cardiaque

La génération et la régulation du rythme cardiaque nécessite une coordination précise entre l'activité électrique du nœud sinoatrial (NSA), à l'origine de l'activité pacemaker cardiaque, et le tissu de conduction (TC), composé du nœud atrioventriculaire (NAV) et du réseau de His-Purkinje. Lors de pathologies cardiaques, il arrive que le centre primaire pacemaker (NSA) soit défaillant et ne génère plus d'impulsions électriques. Le NAV, en plus de sa fonction de conduction, est doté d'automatisme et impose sa fréquence au cœur. La connaissance de la physiologie du rythme cardiaque nécessite l'identification et la description du rôle des gènes des différents canaux ioniques impliqués dans la génération et la régulation de l'activité pacemaker. L'approche via les souris génétiquement modifiées nous donnent l'opportunité de mieux comprendre le mécanisme exact de génération de l'automatisme cardiaque. L'approche expérimentale était jusqu'à présent considérée comme difficile du fait du manque d'accessibilité à ce type de cellules chez la souris. Pendant ma thèse, j'ai développé une technique d'isolement des différentes cellules automatiques de la souris adulte (cellules du NSA, cellules du NAV et cellules des FP). Une comparaison des différents courants ioniques dépendants du voltage sur ces cellules automatiques montrent que le courant pacemaker (If) et les courants calciques (ICa,L et ICa,T) sont présents dans les cellules de conduction en moins forte densité que dans les cellules du SAN. Les souris invalidées pour les gènes Cav1.3 codant pour ICa,L et Cav3.1 codant pour ICa,T montrent une absence totale de ces courants dans les cellules du NAV et du SAN. Au niveau cellulaire, les cellules du SAN et du NAV présentent une bradycardie associée à des dysrythmies impliquant que ces courants calciques sont primordiaux pour l'activité pacemaker. Par contre, les courants potassiques (IK,r et IK1) sont davantage exprimés dans les cellules du NAV et les cellules de Purkinje que dans les cellules du SAN. Cette forte expression de ces courants pourrait expliquer ce ralentissement de l'automatisme des cellules pacemaker du NAV et des cellules des FPThe generation and regulation of the heartbeat is a phenomenon, which requires the precise coordination between the electrical activity of the sino-atrial node (SAN) and the specialized conduction tissue. The heart conduction system (CS) is composed by the atrioventricular node (AVN) and the his-Purkinje fibers (PFs) network system. Automaticity is initiated in the SAN by primary pacemaker cells which generate spontaneous periodic oscillation of their membrane potential. The AVN is also able to generate automaticity , and can drive the heartbeat in case of SAN failure. The PFs network can pace the heart, in case of atrioventricular block. The spontaneous activity of the pacemaker cells is due to the diastolic depolarisation, a slow phase of the pacemaker cycle which drives the membrane voltage from the end of repolarisation phase of an action potential toward the threshold of the following action potential. At the cellular level, pacemaker activity requires the intervention of several families of ionic channels. The molecular basis of AVN and PF automaticity are poorly understood and the role of different ionic channel genes in controlling the diastolic depolarisation phase (DD) in CS have not been investigated. We have isolated spontaneously beating cells from the mouse AVN region and studied ionic channels and pacemaker activity by employing the whole-cell configuration of the patch-clamp technique. ). Compared to SAN, the AVN cells (AVNC) were characterized by a higher expression of the inward rectifier current (IK1) and of the fast delayed-rectifier current (IKr). In contrast, the densities of the hyperpolarization-activated current (If), that of the T- (ICa,T) and L-type (ICa,L) Ca2+ currents were lower AVNC than in SAN cells (SANC). Inhibition of If by 3 M ZD-7228 slowed automaticity of AVNC indicating that If participates to AVNC automaticity in spite of a more negative activation curve in AVNC. The Cav1.3 mediated ICa,L plays an obligatory role in automaticity of AVNC and accounted for almost the totality of ICa,L. TTX-sensitive (INas) and TTX-resistant (INar) Na+ currents had a differential role in automaticity of mouse AVNC, because block of INas slowed pacemaker activity by specifically reducing the rate of the diastolic depolarization (DD), while inhibition of INar showed that this current is the key contributor of the action potential (AP) threshold and upstroke velocity. Modelling of AVN cell automaticity indicates that the lower densities of If, ICa,L, ICa,T, and INas, as well as the higher expression of IK1 and IKr in AVNC than in SANC can explain the intrinsically slower automaticity of AVNCMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF