Early nongenomic events in aldosterone action in renal collecting duct cells: PKCalpha activation, mineralocorticoid receptor phosphorylation, and cross-talk with the genomic response

Effects of aldosterone on its target cells have long been considered to be mediated exclusively through the genomic pathway; however, evidence has been provided for rapid effects of the hormone that may involve nongenomic mechanisms. Whether an interaction exists between these two signaling pathways is not yet established. In this study, the authors show that aldosterone triggers both early nongenomic and late genomic increase in sodium transport in the RCCD(2) rat cortical collecting duct cell line. In these cells, the early (up to 2.5 h) aldosterone-induced increase in short-circuit current (Isc) is not blocked by the mineralocorticoid receptor (MR) antagonist RU26752, it does not require mRNA or protein synthesis, and it involves the PKCalpha signaling pathway. In addition, this early response is reproduced by aldosterone-BSA, which acts at the cell surface and presumably does not enter the cells (aldo-BSA is unable to trigger the late response). The authors also show that MR is rapidly phosphorylated on serine and threonine residues by aldosterone or aldosterone-BSA. In contrast, the late (4 to 24 h) aldosterone-induced increase in ion transport occurs through activation of the MR and requires mRNA and protein synthesis. Interestingly, nongenomic and genomic aldosterone actions appear to be interdependent. Blocking the PKCalpha pathway results in the inhibition of the late genomic response to aldosterone, as demonstrated by the suppression of aldosterone-induced increase in MR transactivation activity, alpha1 Na(+)/K(+)/ATPase mRNA, and Isc. These data suggest cross-talk between the nongenomic and genomic responses to aldosterone in renal cells and suggest that the aldosterone-MR mediated increase in mRNA/protein synthesis and ion transport depends, at least in part, upon PKCalpha activation.Fil: Le Moëllic, Cathy. Inserm; FranciaFil: Ouvrard Pascaud, Antoine. Inserm; FranciaFil: Capurro, Claudia Graciela. Inserm; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cluzeaud, Francoise. Inserm; FranciaFil: Fay, Michel. Inserm; FranciaFil: Jaisser, Frederic. Inserm; FranciaFil: Farman, Nicolette. Inserm; FranciaFil: Blot Chabaud, Marcel. Inserm; Franci

BPIFB4 and its longevity-associated haplotype protect from cardiac ischemia in humans and mice

Long-living individuals (LLIs) escape age-related cardiovascular complications until the very last stage of life. Previous studies have shown that a Longevity-Associated Variant (LAV) of the BPI Fold Containing Family B Member 4 (BPIFB4) gene correlates with an extraordinarily prolonged life span. Moreover, delivery of the LAV-BPIFB4 gene exerted therapeutic action in murine models of atherosclerosis, limb ischemia, diabetic cardiomyopathy, and aging. We hypothesize that downregulation of BPIFB4 expression marks the severity of coronary artery disease (CAD) in human subjects, and supplementation of the LAV-BPIFB4 protects the heart from ischemia. In an elderly cohort with acute myocardial infarction (MI), patients with three-vessel CAD were characterized by lower levels of the natural logarithm (Ln) of peripheral blood BPIFB4 (p = 0.0077). The inverse association between Ln BPIFB4 and three-vessel CAD was confirmed by logistic regression adjusting for confounders (Odds Ratio = 0.81, p = 0.0054). Moreover, in infarcted mice, a single administration of LAV-BPIFB4 rescued cardiac function and vascularization. In vitro studies showed that LAV-BPIFB4 protein supplementation exerted chronotropic and inotropic actions on induced pluripotent stem cell (iPSC)-derived cardiomyocytes. In addition, LAV-BPIFB4 inhibited the pro-fibrotic phenotype in human cardiac fibroblasts. These findings provide a strong rationale and proof of concept evidence for treating CAD with the longevity BPIFB4 gene/protein

Cardiomyopathy and Response to Enzyme Replacement Therapy in a Male Mouse Model for Fabry Disease

Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, (predominately globotriaosylceramide; GL-3) in lysosomes, as well as other cellular compartments and the extracellular space. Our aim was to characterize the cardiac phenotype of male knock-out mice that are deficient in alpha-galactosidase A activity, as a model for Fabry disease and test the efficacy of Enzyme Replacement Therapy with agalsidase-beta. Male mice (3–4 months of age) were characterized with awake blood pressure and heart rate measurements, cardiac echocardiography and electrocardiography measurements under light anesthesia, histological studies and molecular studies with real-time polymerase chain reaction. The Fabry knock-out mouse has bradycardia and lower blood pressure than control wild type (CB7BL/6J) mice. In Fabry knock-out mice, the cardiomyopathy associated mild hypertrophy at echography with normal systolic LV function and mild diastolic dysfunction. Premature atrial contractions were more frequent in without conduction defect. Heart weight normalized to tibial length was increased in Fabry knock-out mice. Ascending aorta dilatation was observed. Molecular studies were consistent with early stages of cardiac remodeling. A single dose of agalsidase-beta (3 mg/kg) did not affect the LV hypertrophy, function or heart rate, but did improve the mRNA signals of early cardiac remodeling. In conclusion, the alpha-galactosidase A deficient mice at 3 to 4 months of age have cardiac and vascular alterations similar to that described in early clinical stage of Fabry disease in children and adolescents. Enzyme replacement therapy affects cardiac molecular remodeling after a single dose

Why are mineralocorticoid receptor antagonists cardioprotective?

Two clinical trials, the Randomized ALdosterone Evaluation Study (RALES) and the EPlerenone HEart failure and SUrvival Study (EPHESUS), have recently shown that mineralocorticoid receptor (MR) antagonists reduce mortality in patients with heart failure on top of ACE inhibition. This effect could not be attributed solely to blockade of the renal MR-mediated effects on blood pressure, and it has therefore been proposed that aldosterone, the endogenous MR agonist, also acts extrarenally, in particular in the heart. Indeed, MR are present in cardiac tissue, and possibly aldosterone synthesis occurs in the heart. This review critically addresses the following questions: (1) is aldosterone synthesized at cardiac tissue sites, (2) what agonist stimulates cardiac MR normally, and (3) what effects are mediated by aldosterone/MR in the heart that could explain the beneficial effects of MR blockade in heart failure? Conclusions are that most, if not all, of cardiac aldosterone originates in the circulation (i.e., is of adrenal origin), and that glucocorticoids, in addition to aldosterone, may serve as the endogenous agonist of cardiac MR. MR-mediated effects in the heart include effects on endothelial function, cardiac fibrosis and hypertrophy, oxidative stress, cardiac inotropy, coronary flow, and arrhythmias. Some of these effects occur via or in synergy with angiotensin II, and involve a non-MR-mediated mechanism. This raises the possibility that aldosterone synthase inhibitors might exert beneficial effects on top of MR blockade

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