Rôle physiopathologique du récepteur minéralocorticoïde dans le rein et dans le coeur (approches utilisant des modèles conditionnels cellulaires et animaux)

Le récepteur minéralocorticoïde est un facteur de transcription dépendant de sa liaison à l'hormone, l'aldostérone, qui régule la réabsorption terminale de sodium dans le néphron distal (tubule collecteur cortical), participant au contrôle de la volémie et de la pression artérielle. Les cibles moléculaires et voies de signalisation impliquées dans ce processus n'ont pas toutes été déterminées. De plus ce récepteur s'exprime dans d'autres tissus, dont le cœur, où sa fonction reste largement inconnue. A partir d'une lignée cellulaire de rat, nous avons établi un clone permettant l'expression inductible (Cre-lox) d'une protéine de fusion entre le récepteur minéralocorticoïde humain et un marqueur eGFP fluorescent (Ouvrard-Pascaud et al. 2004), qui a été utilisé dans une étude proposant que la translocation nucléaire du récepteur ne soit pas requise pour stimuler la réabsorption du sodium au cours de la phase précoce de la réponse hormonale (1 à 2 heures) (Le Moellic et al. 2004). Nous avons développé des souris transgéniques pour l'expression inductible (tet-OFF) du récepteur minéralocorticoïde humain spécifiquement dans les cardiomyocytes. 50% de ces animaux meurent au cours du développement embryonnaire sans défaut de cardiogenèse. L'histologie cardiaque adulte est normale. Des études d'électrophysiologie sur cellules isolées montrent un allongement du potentiel d'action. Des électrocardiogrammes montrent des extrasystoles et tachycardies ventriculaires. Ce phénotype évoque la survenue de morts subites par troubles du rythme, suggérant une fonction du récepteur dans la signalisation électrique cardiaque et sa possible implication dans des pathologies intégrant des arythmies.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Mineralocorticoid receptor antagonism improves diastolic dysfunction in chronic kidney disease in mice

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Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis

International audienceFunctional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells

Protein tyrosine phosphatase 1B inactivation limits aging-associated heart failure in mice

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Smooth Muscle Cell Mineralocorticoid Receptors Are Mandatory for Aldosterone\textendashSalt to Induce Vascular Stiffness

International audienceArterial stiffness is recognized as a risk factor for many cardiovascular diseases. Aldosterone via its binding to and activation of the mineralocorticoid receptors (MRs) is a main regulator of blood pressure by controlling renal sodium reabsorption. Although both clinical and experimental data indicate that MR activation by aldosterone is involved in arterial stiffening, the molecular mechanism is not known. In addition to the kidney, MR is expressed in both endothelial and vascular smooth muscle cells (VSMCs), but the specific contribution of the VSMC MR to aldosterone-induced vascular stiffness remains to be explored. To address this question, we generated a mouse model with conditional inactivation of the MR in VSMC (MRSMKO). MRSMKO mice show no alteration in renal sodium handling or vascular structure, but they have decreased blood pressure when compared with control littermate mice. In vivo at baseline, large vessels of mutant mice presented with normal elastic properties, whereas carotids displayed a smaller diameter when compared with those of the control group. As expected after aldosterone/salt challenge, the arterial stiffness increased in control mice; however, it remained unchanged in MRSMKO mice, without significant modification in vascular collagen/elastin ratio. Instead, we found that the fibronectin/α5-subunit integrin ratio is profoundly altered in MRSMKO mice because the induction of α5 expression by aldosterone/salt challenge is prevented in mice lacking VSMC MR. Altogether, our data reveal in the aldosterone/salt hypertension model that MR activation specifically in VSMC leads to the arterial stiffening by modulation of cell-matrix attachment proteins independent of major vascular structural changes

Enhanced angiogenesis and increased cardiac perfusion after myocardial infarction in protein tyrosine phosphatase 1B-deficient mice

International audienceThe protein tyrosine phosphatase 1B (PTP1B) modulates tyrosine kinase receptors, among which is the vascular endothelial growth factor receptor type 2 (VEGFR2), a key component of angiogenesis. Because PTP1B deficiency in mice improves left ven-tricular (LV) function 2 mo after myocardial infarction (MI), we hypothesized that enhanced angiogenesis early after MI via activated VEGFR2 contributes to this improvement. At 3 d after MI, capillary density was increased at the infarct border of PTP1B ؊/؊ mice [؉7؎2% vs. wild-type (WT), P ‫؍‬ 0.05]. This was associated with increased extracellular signal-regulated kinase 2 phosphorylation and VEGFR2 activation (i.e., phosphorylated-Src/Src/VEGFR2 and dissocia-tion of endothelial VEGFR2/VE-cadherin), together with higher infiltration of proangiogenic M2 macro-phages within unchanged overall infiltration. In vitro, we showed that PTP1B inhibition or silencing using RNA interference increased VEGF-induced migration and proliferation of mouse heart microvascular endo-thelial cells as well as fibroblast growth factor (FGF)-induced proliferation of rat aortic smooth muscle cells. At 8 d after MI in PTP1B ؊/؊ mice, increased LV capillary density (؉21؎3% vs. WT; P<0.05) and an increased number of small diameter arteries (15-50 m) were likely to participate in increased LV perfu-sion assessed by magnetic resonance imaging and improved LV compliance, indicating reduced diastolic dysfunction. In conclusion, PTP1B deficiency reduces MI-induced heart failure promptly after ischemia by enhancing angiogenesis, myocardial perfusion, and di-astolic function S., Richard, V., Ouvrard-Pascaud, A. Enhanced angiogenesis and increased cardiac perfusion after myocardial infarction in protein tyrosine phosphatase 1B-deficient mice. FASEB J. 28, 3351-3361 (2014). www.fasebj.or

Vascular Smooth Muscle Mineralocorticoid Receptor Contributes to Coronary and Left Ventricular Dysfunction After Myocardial Infarction

International audienceC hronic heart failure (HF) after myocardial infarction (MI) is steadily increasing worldwide and remains a major cause of death. Mineralocorticoid receptor (MR) antagonists (MRAs) improve survival in patients with HF as illustrated by spironolactone in the RALES (Randomized Aldactone Evaluation Study) 1 trial and by eplerenone in the EPHESUS (Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) trial, 2 which only included patients with post-MI. Recently, the EMPHASIS (Eplerenone in Mild Patients Hospitalization and Survival Study in Heart Failure) study even showed that eplerenone is effective in slowing down the progression of mild-to-moderate HF. 3 However, MRAs are associated with side effects, such as hyperkalemia or gynecomastia; the latter is caused by the antiandrogenic properties of their steroidal structure. These adverse effects are responsible for the underuse of MRAs. 4 Understanding of the cell-specific contribution of MR to HF and of the effects of MRA on various cell subtypes will be useful for the future development of tissue-selective MR targeting approaches that would improve the benefit/risk ratio. 5 A crucial role of cardiomyocyte MR has been demonstrated in mice with cardiomyocyte-specific MR deletion, which allows improving left ventricular (LV) function after either MI 6 or pressure overload induced by transverse aortic constriction. 7 The deletion of MR specifically in fibroblasts does not affect cardiac failure after aortic constriction. 7 The role of vascular MR has been underlined recently: the MR expressed in vas-cular smooth muscle cells (VSMCs) is involved in age-related Abstract-Mineralocorticoid receptor (MR) antagonists slow down the progression of heart failure after myocardial infarction (MI), but the cell-specific role of MR in these benefits is unclear. In this study, the role of MR expressed in vascular smooth muscle cells (VSMCs) was investigated. Two months after coronary artery ligation causing MI, mice with VSMC-specific MR deletion (MI-MR SMKO) and mice treated with the MR antagonist finerenone (MI-fine) had improved left ventricular compliance and elastance when compared with infarcted control mice (MI-CTL), as well as reduced interstitial fibrosis. Importantly, the coronary reserve assessed by magnetic resonance imaging was preserved (difference in myocardial perfusion before and after induction of vasodilatation, mL mg −1 min −1 : MI-CTL: 1.1±0.5, nonsignificant; MI-MR SMKO : 4.6±1.6 [P<0.05]; MI-fine: 3.6±0.7 [P<0.01]). The endothelial function, tested on isolated septal coronary arteries by analyzing the acetylcholine-induced nitric oxide-dependent relaxation, was also improved by MR deletion in VSMCs or by finerenone treatment (relaxation %: MI-CTL: 36±5, MI-MR SMKO : 54±3, and MI-fine: 76±4; P<0.05). Such impairment of the coronary endothelial function on MI involved an oxidative stress that was reduced when MR was deleted in VSMCs or by finerenone treatment. Moreover, short-term incubation of coronary arteries isolated from noninfarcted animals with low-dose angiotensin-II (10 −9 mol/L) induced oxidative stress and impaired acetylcholine-induced relaxation in CTL but neither in MR SMKO nor in mice pretreated with finerenone. In conclusion, deletion of MR in VSMCs improved left ventricular dysfunction after MI, likely through maintenance of the coronary reserve and improvement of coronary endothelial function. MR blockage by finerenone had similar effects

Short- and long-term administration of the non-steroidal mineralocorticoid receptor antagonist finerenone opposes metabolic syndrome-related cardio-renal dysfunction

International audienceAim: To determine whether non-steroidal mineralocorticoid receptor (MR) antagonists oppose metabolic syndrome-related end-organ, i.e. cardiac, damage. Materials and methods: In Zucker fa/fa rats, a rat model of metabolic syndrome, we assessed the effects of the non-steroidal MR antagonist finerenone (oral 2 mg/kg/day) on left ventricular (LV) function, haemodynamics and remodelling (using echocardiography, magnetic resonance imaging and biochemical methods). Results: Long-term (90 days) finerenone modified neither systolic blood pressure nor heart rate, but reduced LV end-diastolic pressure and LV end-diastolic pressure-volume relationship, without modifying LV end-systolic pressure and LV end-systolic pressure-volume relationship. Simultaneously , long-term finerenone reduced both LV systolic and diastolic diameters, associated with reductions in LV weight and LV collagen density, while proteinuria and renal nGAL expression were reduced. Short-term (7 days) finerenone improved LV haemodynamics and reduced LV systolic diameter, without modifying LV diastolic diameter. Moreover, short-term finerenone increased myocardial tissue perfusion and reduced myocardial reactive oxygen species, while plasma nitrite levels, an indicator of nitric oxide (NO) bio-availability, were increased. Conclusions: In rats with metabolic syndrome, the non-steroidal MR antagonist finerenone opposed metabolic syndrome-related diastolic cardiac dysfunction and nephropathy. This involved acute effects, such as improved myocardial perfusion, reduced oxidative stress/ increased NO bioavailability, as well as long-term effects, such as modifications in the myocar-dial structure. K E Y W O R D S animal pharmacology, cardiovascular disease, diabetes complications, drug development, experimental pharmacology, type 2 diabete

Reduction of heart failure by pharmacological inhibition or gene deletion of protein tyrosine phosphatase 1B

International audienceProtein tyrosine phosphatase 1B (PTP1B) regulates tyrosine kinase receptor-mediated responses, and especially negatively influences insulin sensitivity, thus PTP1B inhibitors (PTP1Bi) are currently evaluated in the context of diabetes. We recently revealed another important target for PTP1Bi, consisting in endothelial protection. The present study was designed to test whether reduction of PTP1B activity may be beneficial in chronic heart failure (CHF). We evaluated the impact of either a 2 month pharmacological inhibition, or a gene deletion of PTP1B (PTP1B −/−) in CHF mice (2 months post-myocardial infarction). PTP1Bi and PTP1B deficiency reduced adverse LV remodeling, and improved LV function, as shown by the increased LV fractional shortening and cardiac output (measured by echocardiography), the increased LV end systolic pressure, and the decreased LV end diastolic pressure, at identical infarct sizes. This was accompanied by reduced cardiac fibrosis, myocyte hypertrophy and cardiac expression of ANP. In vitro vascular studies performed in small mesenteric artery segments showed a restored endothelial function (i.e. improved NO-dependent, flow-mediated dilatation, increased eNOS phosphorylation) after either pharmacological inhibition or gene deletion. PTP1B −/− CHF also displayed an improved insulin sensitivity (assessed by euglycemic-hyperinsulinemic clamp studies), when compared to wild-type CHF associated with an increased insulin mediated mesenteric artery dilation. Thus, chronic pharmacological inhibition or gene deletion of PTP1B improves cardiac dysfunction and cardiac remodeling in the absence of changes in infarct size. Thus this enzyme may be a new therapeutic target in CHF. Diabetic patients with cardiac complications may potentially benefit from PTP1B inhibition via two different mechanisms, reduced diabetic complications, and reduced heart failure