Rôle de l interleukine 6 dans les interations cardiomyocytes-fibroblastes murins au cours du remodelage myocardique in vitro

L hypertrophie cardiaque est un processus adaptatif du cœur caractérisé par une augmentation de la taille des cardiomyocytes (CMs), une prolifération des fibroblastes (FCs) et le développement d une fibrose. Ce remodelage myocardique est régulé par des facteurs qui ont une action autocrine et /ou paracrine sur les CMs et les FCs. Parmi ces facteurs, les cytokines de la famille de l interleukine 6 (IL-6) semblent jouer un rôle important. De ce fait, la caractérisation des interactions entre les CMs et les FCs in vitro et l étude du rôle de l IL-6 ont constitué les objectifs de ce travail. Afin de réaliser cette étude, nous avons développé un modèle de dédifférenciation de CMs murins adultes en culture primaire et en coculture avec des FCs, modèle reconnu pour l étude de l hypertrophie cardiaque cellulaire in vitro. Sur ce modèle, nous avons montré que l hypertrophie des CMs est significativement améliorée en présence de FCs, et que les CMs modulent aussi l adhésion et/ou la prolifération des FCs. Dans ces conditions de coculture, de fortes concentrations en IL-6 sont détectées, supérieures à celles mesurées dans les milieux des cultures de CMs et de FCs. Nous avons alors cherché à préciser le rôle de l IL-6 dans les interactions entre les CMs et les FCs, et à vérifier si les effets induits par l IL-6 impliquent l angiotensine II (Ang II). Pour cela nous avons traité les milieux de culture avec des antagonistes de l IL-6, de la gp130, sous-unité transductrice commune aux cytokines de la famille de l interleukine-6, et du récepteur AT1 de l angiotensine II. Nos résultats montrent que l IL-6 et la gp130 sont exprimées dans les CMs et les FCs en culture et que l IL-6, secrétée par les CMs, induit l hypertrophie des CMs selon un mode autocrine et la prolifération des FCs selon un mode paracrine; l Ang II, principalement produite par les FCs, serait impliquée dans ce processus. Par ailleurs, afin d analyser le mode d action de l IL-6 dans l induction du remodelage des cardiomyocytes, nous avons analysé les effets de l IL-6 sur le courant calcique de type L et la concentration en calcium intracellulaire des CMs murins adultes. Nos résultats montrent que l IL-6 n a pas d effet à court terme mais après 24 heures de traitement, elle augmente significativement la densité du courant calcique de type L mesuré dans la configuration cellule entière du patch-clamp ainsi que le rapport de la fluorescence mesurée avec la sonde indo-1. En conclusion, l ensemble de nos résultats montre que l IL-6 est une cytokine très fortement impliquée dans les interactions complexes qui s établissent entre myocytes et non-myocytes au cours du remodelage myocardique en contrôlant à la fois le processus hypertrophique des cellules contractiles et le processus de prolifération des fibroblastes.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Electrophysiological characterization of mouse intracardiac calbindin neurons

International audienceNeural control of the heart involves central and peripheral neurons that act interdependently to modulate cardiac parameters such as heart rate, conduction velocity or contractility. Within this cardiac neuronal regulation, the intrinsic cardiac nervous system, which correspond to clusters of neurons found on the dorsal atrial surface of the heart, is receiving growing attention. Indeed, whereas they were initially considered as simple parasympathetic postganglionic neurons, studies conducted over the past 30 years suggested a more complex organization, involving the existence of sensory, local regulatory and motor neurons within intracardiac ganglia. Moreover, growing evidence suggest the implication of this neural network in the initiation and maintenance of cardiac arrhythmias. However, the functional organization of this intracardiac neural network, as well as its involvement in cardiac diseases have not been fully elucidated.Therefore, this study aims to decipher the complexity of this mouse cardiac nervous system by examining the electrophysiological properties of intracardiac neurons. The characterization of passive and active electrical membrane properties of these neurons gave rise to the identification of two distinct neuronal profiles displaying different firing characteristics. The first group was classified as phasic due to its limited firing activity while the second was defined as adapting. Phasic neurons were also characterized by a higher rheobase as well as higher AHP amplitude and duration compared to the adapting one. By using cre transgenic mice and targeted viral transduction strategy, we identified calbindin expressing neurons as a population of neurons with a distinct electrophysiological signature. This could be explained by the differential expression of several ionic channels including sodium and calcium channels and will be further investigated in the future

Bioactive Natural Product and Superacid Chemistry for Lead Compound Identification: A Case Study of Selective hCA III and L-Type Ca2+ Current Inhibitors for Hypotensive Agent Discovery

International audienc

Hydrocotyle bonariensis Comm ex Lamm (Araliaceae) leaves extract inhibits IKs not IKr potassium currents: Potential implications for anti-arrhythmic therapy

International audienc

The hypotensive agent dodoneine inhibits L-type Ca2+ current with negative inotropic effect on rat heart.

International audienceAgelanthus dodoneifolius is one of the medicinal plants used in African pharmacopeia and traditional medicine for the treatment of cardiovascular diseases. A chemical analysis has identified one of the active principles: Dodoneine (Ddn). It is a new dihydropyranone which exerts hypotensive and vasorelaxant effects on rat. Since the mechanism of the hypotensive effect is unknown, we performed a variety of preclinical and mechanistic studies to characterize the specific cardiac effect of Ddn at tissue (ex-vivo) and cellular levels (in-vitro) in order to determine a molecular target. Ddn effects were evaluated in an isolated rat heart preparation using Langendorff retrograde perfusion and then, the effects of Ddn were characterized in freshly dissociated cardiac ventricular myocytes using the whole-cell patch-clamp configuration. Ex-vivo, Ddn produced a dose-dependent negative inotropic effect with an IC50 value of 10 µM without changed heart rate. 100 µM Ddn decreased left ventricular developed pressure of about 40%. In isolated cardiac myocytes, Ddn reduced I(Ca),L density of about 30% with an IC50 value estimated at 3 µM. Ddn did not change current-voltage relation but it shifted the inactivation curve toward negative potentials and modified the half inactivation potentials. Furthermore, Ddn induced a phasic-dependent blocking on ICa,L. This study demonstrates that the hypotensive property of dodoneine is likely associated with a negative inotropic effect and the blockade of the L-type calcium channels

Polyunsaturated Phospholipids Increase Cell Resilience to Mechanical Constraints

International audienceIf polyunsaturated fatty acids (PUFAs) are generally accepted to be good for health, the mechanisms of their bona fide benefits still remain elusive. Membrane phospholipids (PLs) of the cardiovascular system and skeletal muscles are particularly enriched in PUFAs. The fatty acid composition of PLs is known to regulate crucial membrane properties, including elasticity and plasticity. Since muscle cells undergo repeated cycles of elongation and relaxation, we postulated in the present study that PUFA-containing PLs could be central players for muscle cell adaptation to mechanical constraints. By a combination of in cellulo and in silico approaches, we show that PUFAs, and particularly the ω-3 docosahexaenoic acid (DHA), regulate important properties of the plasma membrane that improve muscle cell resilience to mechanical constraints. Thanks to their unique property to contortionate within the bilayer plane, they facilitate the formation of vacuole-like dilation (VLD), which, in turn, avoid cell breakage under mechanical constraints

Functional BKCa channel in human resident cardiac stem cells expressing W8B2

International audienc

Vasorelaxation induced by dodoneine is mediated by calcium channels blockade and carbonic anhydrase inhibition on vascular smooth muscle cells.

publicationInternational audienceDodoneine (Ddn) is one of the active compounds identified from Agelanthus dodoneifolius (DC.) Polhill and Wiens, a medicinal plant used in traditional medicine for the treatment of hypertension. This dihydropyranone exerts hypotensive and vasorelaxant effects on rats, and two molecular targets have been characterized: the carbonic anhydrase and the L-type calcium channel in cardiomyocytes with biochemical and electrophysiological techniques, respectively. To further evaluate the involvement of these two molecular targets in vasorelaxation, the effect of Ddn on rat vascular smooth muscle was investigated. The effects of Ddn on L-type calcium current and on resting membrane potential were characterized in A7r5 cell line using the whole-cell patch-clamp configuration. The molecular identities of carbonic anhydrase isozymes in smooth muscle cells were examined with RT-PCR. Vascular response was measured on rat aortic rings in an organ bath apparatus and the effect of Ddn on intracellular pH was determined by flow cytometry using the pH-sensitive fluorescent probe BCECF-AM [2,7-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester]. 100µM Ddn reduced calcium current density of about 30%. In addition, carbonic anhydrase II, III, XIII and XIV were shown to be expressed in rat aorta and inhibited in smooth muscle cells by Ddn. This inhibition resulted in a rise in pHi of about 0.31, leading to KCa channel activation, thereby inducing membrane hyperpolarization and vasorelaxation. The results of vascular reactivity experiments obtained with pharmacological tools acting on the L-type calcium current and carbonic anhydrase suggest that Ddn produces its vasorelaxant effect via the inhibition of these two molecular targets. This study demonstrates that Ddn induced vasorelaxation by targeting two proteins involved in the modulation of excitation-contraction coupling: L-type calcium channels and carbonic anhydrase

From the vasodilator and hypotensive effects of an extract fraction from Agelanthus dodoneifolius (DC) Danser (Loranthaceae) to the active compound dodoneine

IF (2,26)International audienceAim of the study: Effects of the different fractions obtained by partition of ethanolic extract (EE) of Agelanthus dodoneifolius through column chromatography were investigated on rat blood pressure and aortic relaxation and compared to those observed in the presence of crude EE. Materials and methods: The acute hypotensive activity of EE, fractions and dodoneine, administrated intravenously, was evaluated in anaesthetized rats using the invasive method of blood pressure recording. Bioassay-guided fractionation using rat aorta pre-contracted by norepinephrine to monitor the relaxant activity led to the isolation of dodoneine. Results: In normotensive rats, injection of EE (0.01–10 mg/kg) produced a dose-dependent decrease in both systolic and diastolic blood pressure without any significant change in heart rate. In a similar way, the EE (0.001–3 mg/mL) caused relaxation of rat pre-contracted aorta in a concentration-dependent manner. Fractionation of the EE afforded 14 fractions, F1–F14, that were tested on rat precontracted aortic rings. At the concentration level of 1 mg/mL, a maximum relaxation effect was observed for fractions F2–F5. F4 was the most effective to elicit a concentration-dependent relaxation effect with an ED50 = 160±1.1 g/mL (n = 5) and to decreased systolic and diastolic control pressure by 56.9% and 81.6% respectively. F4 contains most of the dihydropyranone dodoneine, with 93% of the sample mass. Dodoneine separated from this fraction was also able to decrease both systolic and diastolic arterial pressure by 32.5% and 38.7% at 100g/kg, respectively. Conclusion: For the first time, this study demonstrates the hypotensive property of the dodoneine present in Agelanthus dodoneifolius