Simultaneous assessment of mechanical and electrical function in Langendorff-perfused ex-vivo mouse hearts

Background: The Langendorff-perfused ex-vivo isolated heart model has been extensively used to study cardiac function for many years. However, electrical and mechanical function are often studied separately—despite growing proof of a complex electro-mechanical interaction in cardiac physiology and pathology. Therefore, we developed an isolated mouse heart perfusion system that allows simultaneous recording of electrical and mechanical function. Methods: Isolated mouse hearts were mounted on a Langendorff setup and electrical function was assessed via a pseudo-ECG and an octapolar catheter inserted in the right atrium and ventricle. Mechanical function was simultaneously assessed via a balloon inserted into the left ventricle coupled with pressure determination. Hearts were then submitted to an ischemia-reperfusion protocol. Results: At baseline, heart rate, PR and QT intervals, intra-atrial and intra-ventricular conduction times, as well as ventricular effective refractory period, could be measured as parameters of cardiac electrical function. Left ventricular developed pressure (DP), left ventricular work (DP-heart rate product) and maximal velocities of contraction and relaxation were used to assess cardiac mechanical function. Cardiac arrhythmias were observed with episodes of bigeminy during which DP was significantly increased compared to that of sinus rhythm episodes. In addition, the extrasystole-triggered contraction was only 50% of that of sinus rhythm, recapitulating the “pulse deficit” phenomenon observed in bigeminy patients. After ischemia, the mechanical function significantly decreased and slowly recovered during reperfusion while most of the electrical parameters remained unchanged. Finally, the same electro-mechanical interaction during episodes of bigeminy at baseline was observed during reperfusion. Conclusion: Our modified Langendorff setup allows simultaneous recording of electrical and mechanical function on a beat-to-beat scale and can be used to study electro-mechanical interaction in isolated mouse hearts

Gene- and variant-specific efficacy of serum/glucocorticoid-regulated kinase 1 inhibition in long QT syndrome types 1 and 2.

AIMS Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. METHODS AND RESULTS Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300 nM-10 µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3 µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10 µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3 µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10 µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10 µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3 µM. CONCLUSION A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS

KCNQ1 suppression-replacement gene therapy in transgenic rabbits with type 1 long QT syndrome.

BACKGROUND AND AIMS Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded Kv7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. METHODS KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. RESULTS KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD90 behaviour. CONCLUSIONS This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD90 to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1

Rôle des canaux calciques de type L Cav1.3 dans le contrôle de la fréquence cardiaque par les catécholamines

The heart is a spontaneously active organ whose contractile activity is dependent of a spontaneous intrinsic electrical activity, which refers to one of the fundamental cardiac properties: cardiac automaticity. The electrical signal is generated in the sino-atrial node tissue (SAN) and is then propagated throughout the heart, triggering cardiomyocytes contraction. Cardiac automaticity relies on the capacity of pacemaker sino-atrial node cells (SANC) to spontaneously depolarize (diastolic depolarization, DD) which brings membrane potential to the threshold of the sinoatrial action potential (AP). Complex and not entirely understood yet, diastolic depolarization results from a robust interplay of membrane ion channels activity (hyperpolarization-activated HCN4 channels, L-type Cav1.3 and T-type Cav3.1 calcium channels) and intracellular calcium dynamics with spontaneous local calcium releases (LCRs) from the sarcoplasmic reticulum via the ryanodine receptors (RyR). By triggering cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) catecholaminergic stimulation of pacemaker activity by sympathetic autonomic nervous system accelerates the firing frequency of SANC.The objective of my PhD was to determine the role of L-type Cav1.3 calcium channels in the catecholaminergic regulation of SANC. Patch-clamp experiments on isolated mouse SANC showed that pharmacologic inhibition of Cav1.3 channels completely reversed the effects of adrenergic stimulation on the frequency. Imaging of intracellular calcium further revealed that LCRs synchronization under activation of adrenergic receptors was dependent of Cav1.3 channels activity. Hence, L-type Cav1.3 calcium channels seem to be the main mediator of the positive chronotropic response of pacemaker activity in mouse SANC.Le cœur est un organe autonome dont l’activité contractile est dépendante d’une activité électrique intrinsèque spontanée. Au niveau de l’oreillette droite se situe le nœud sinoatrial (NSA) au sein duquel une impulsion électrique spontanée est initiée puis propagée à l’ensemble du tissu cardiaque provoquant alors la contraction des cardiomyocytes. La clé de l’automaticité cardiaque réside dans le fait que les cellules sinusales (SANC) ou « pacemaker » sont capables de se dépolariser spontanément (dépolarisation diastolique, DD) ce qui amène progressivement leur potentiel de membrane jusqu’au seuil de déclenchement du potentiel d’action (PA) sinusal. Complexe et encore non entièrement comprise, la dépolarisation diastolique est due à une robuste interaction entre l’activation séquentielle de canaux ioniques de la membrane cellulaire (canaux s’ouvrant par hyperpolarisation HCN4, canaux calciques de type L Cav1.3 et de type T Cav3.1) et une dynamique calcique intracellulaire avec des relargages calciques spontanés (LCRs) diastoliques par le réticulum sarcoplasmique via les récepteurs à la ryanodine (RyR). En déclenchant la voie de signalisation adénosine monophosphate cyclique/protéine kinase A (AMPc/PKA) la régulation adrénergique par le système nerveux autonome sympathique permet d’accélérer la fréquence de dépolarisation des SANC.L’objectif de ma thèse était de déterminer le rôle des canaux calciques de type L Cav1.3 dans la régulation adrénergique des SANC. La technique de patch-clamp sur SANC isolées de souris a montré que l’inhibition pharmacologique des canaux Cav1.3 inversait complètement les effets de la stimulation adrénergique sur la fréquence de dépolarisation des SANC. Les expérimentations d’imagerie calcique ont permis de révéler que la synchronisation des LCRs sous stimulation adrénergique était dépendante des canaux Cav1.3. Ainsi, les canaux calciques de type L Cav1.3 semblent être les régulateurs majoritaires de la réponse chronotrope positive chez la souris

Rôle des canaux calciques de type L Cav1.3 dans le contrôle de la fréquence cardiaque par les catécholamines

The heart is a spontaneously active organ whose contractile activity is dependent of a spontaneous intrinsic electrical activity, which refers to one of the fundamental cardiac properties: cardiac automaticity. The electrical signal is generated in the sino-atrial node tissue (SAN) and is then propagated throughout the heart, triggering cardiomyocytes contraction. Cardiac automaticity relies on the capacity of pacemaker sino-atrial node cells (SANC) to spontaneously depolarize (diastolic depolarization, DD) which brings membrane potential to the threshold of the sinoatrial action potential (AP). Complex and not entirely understood yet, diastolic depolarization results from a robust interplay of membrane ion channels activity (hyperpolarization-activated HCN4 channels, L-type Cav1.3 and T-type Cav3.1 calcium channels) and intracellular calcium dynamics with spontaneous local calcium releases (LCRs) from the sarcoplasmic reticulum via the ryanodine receptors (RyR). By triggering cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) catecholaminergic stimulation of pacemaker activity by sympathetic autonomic nervous system accelerates the firing frequency of SANC.The objective of my PhD was to determine the role of L-type Cav1.3 calcium channels in the catecholaminergic regulation of SANC. Patch-clamp experiments on isolated mouse SANC showed that pharmacologic inhibition of Cav1.3 channels completely reversed the effects of adrenergic stimulation on the frequency. Imaging of intracellular calcium further revealed that LCRs synchronization under activation of adrenergic receptors was dependent of Cav1.3 channels activity. Hence, L-type Cav1.3 calcium channels seem to be the main mediator of the positive chronotropic response of pacemaker activity in mouse SANC.Le cœur est un organe autonome dont l’activité contractile est dépendante d’une activité électrique intrinsèque spontanée. Au niveau de l’oreillette droite se situe le nœud sinoatrial (NSA) au sein duquel une impulsion électrique spontanée est initiée puis propagée à l’ensemble du tissu cardiaque provoquant alors la contraction des cardiomyocytes. La clé de l’automaticité cardiaque réside dans le fait que les cellules sinusales (SANC) ou « pacemaker » sont capables de se dépolariser spontanément (dépolarisation diastolique, DD) ce qui amène progressivement leur potentiel de membrane jusqu’au seuil de déclenchement du potentiel d’action (PA) sinusal. Complexe et encore non entièrement comprise, la dépolarisation diastolique est due à une robuste interaction entre l’activation séquentielle de canaux ioniques de la membrane cellulaire (canaux s’ouvrant par hyperpolarisation HCN4, canaux calciques de type L Cav1.3 et de type T Cav3.1) et une dynamique calcique intracellulaire avec des relargages calciques spontanés (LCRs) diastoliques par le réticulum sarcoplasmique via les récepteurs à la ryanodine (RyR). En déclenchant la voie de signalisation adénosine monophosphate cyclique/protéine kinase A (AMPc/PKA) la régulation adrénergique par le système nerveux autonome sympathique permet d’accélérer la fréquence de dépolarisation des SANC.L’objectif de ma thèse était de déterminer le rôle des canaux calciques de type L Cav1.3 dans la régulation adrénergique des SANC. La technique de patch-clamp sur SANC isolées de souris a montré que l’inhibition pharmacologique des canaux Cav1.3 inversait complètement les effets de la stimulation adrénergique sur la fréquence de dépolarisation des SANC. Les expérimentations d’imagerie calcique ont permis de révéler que la synchronisation des LCRs sous stimulation adrénergique était dépendante des canaux Cav1.3. Ainsi, les canaux calciques de type L Cav1.3 semblent être les régulateurs majoritaires de la réponse chronotrope positive chez la souris

Role of L-type Cav1.3 calcium channels in the control of cardiac frequency by catecholamines

Le cœur est un organe autonome dont l’activité contractile est dépendante d’une activité électrique intrinsèque spontanée. Au niveau de l’oreillette droite se situe le nœud sinoatrial (NSA) au sein duquel une impulsion électrique spontanée est initiée puis propagée à l’ensemble du tissu cardiaque provoquant alors la contraction des cardiomyocytes. La clé de l’automaticité cardiaque réside dans le fait que les cellules sinusales (SANC) ou « pacemaker » sont capables de se dépolariser spontanément (dépolarisation diastolique, DD) ce qui amène progressivement leur potentiel de membrane jusqu’au seuil de déclenchement du potentiel d’action (PA) sinusal. Complexe et encore non entièrement comprise, la dépolarisation diastolique est due à une robuste interaction entre l’activation séquentielle de canaux ioniques de la membrane cellulaire (canaux s’ouvrant par hyperpolarisation HCN4, canaux calciques de type L Cav1.3 et de type T Cav3.1) et une dynamique calcique intracellulaire avec des relargages calciques spontanés (LCRs) diastoliques par le réticulum sarcoplasmique via les récepteurs à la ryanodine (RyR). En déclenchant la voie de signalisation adénosine monophosphate cyclique/protéine kinase A (AMPc/PKA) la régulation adrénergique par le système nerveux autonome sympathique permet d’accélérer la fréquence de dépolarisation des SANC.L’objectif de ma thèse était de déterminer le rôle des canaux calciques de type L Cav1.3 dans la régulation adrénergique des SANC. La technique de patch-clamp sur SANC isolées de souris a montré que l’inhibition pharmacologique des canaux Cav1.3 inversait complètement les effets de la stimulation adrénergique sur la fréquence de dépolarisation des SANC. Les expérimentations d’imagerie calcique ont permis de révéler que la synchronisation des LCRs sous stimulation adrénergique était dépendante des canaux Cav1.3. Ainsi, les canaux calciques de type L Cav1.3 semblent être les régulateurs majoritaires de la réponse chronotrope positive chez la souris.The heart is a spontaneously active organ whose contractile activity is dependent of a spontaneous intrinsic electrical activity, which refers to one of the fundamental cardiac properties: cardiac automaticity. The electrical signal is generated in the sino-atrial node tissue (SAN) and is then propagated throughout the heart, triggering cardiomyocytes contraction. Cardiac automaticity relies on the capacity of pacemaker sino-atrial node cells (SANC) to spontaneously depolarize (diastolic depolarization, DD) which brings membrane potential to the threshold of the sinoatrial action potential (AP). Complex and not entirely understood yet, diastolic depolarization results from a robust interplay of membrane ion channels activity (hyperpolarization-activated HCN4 channels, L-type Cav1.3 and T-type Cav3.1 calcium channels) and intracellular calcium dynamics with spontaneous local calcium releases (LCRs) from the sarcoplasmic reticulum via the ryanodine receptors (RyR). By triggering cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) catecholaminergic stimulation of pacemaker activity by sympathetic autonomic nervous system accelerates the firing frequency of SANC.The objective of my PhD was to determine the role of L-type Cav1.3 calcium channels in the catecholaminergic regulation of SANC. Patch-clamp experiments on isolated mouse SANC showed that pharmacologic inhibition of Cav1.3 channels completely reversed the effects of adrenergic stimulation on the frequency. Imaging of intracellular calcium further revealed that LCRs synchronization under activation of adrenergic receptors was dependent of Cav1.3 channels activity. Hence, L-type Cav1.3 calcium channels seem to be the main mediator of the positive chronotropic response of pacemaker activity in mouse SANC

Dispositif endoscopique à fibre optique pour l'imagerie polarimétrique distale

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