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, Annexe 1 : Le dosage de l'activité adenylyl cyclase par radio-détection
, Dans la plupart des cas, les dosages d'activité cyclase reposent sur l'isolement et la quantification de la radioactivité émise par un produit AMPc radiomarqué, l'AMPc, vol.32
, L'échantillon est incubé, pour un intervalle de temps fixé, 1974.
, en quantité limitée dans une solution de substrat ATP froid
, ii) du cofacteur enzymatique Mg
, P] synthétisé ; iv) des couples creatine phosphate / creatine phosphokinase ou phosphoénolpyruvate / pyruvate kinase qui assurent la régénération continue de l'ADP en ATP, v) d'éventuels composés physiologiques ou pharmacologiques régulant l'activité cyclase. La réaction est stoppée par l'addition d'acide trichloroacétique puis une solution d'AMPc [ 3 H] est ajoutée au mélange réactionnel. Les molécules d'AMPc contenues dans l'échantillon sont ensuite séparées des autres nucléotides dérivés de l'adenine (ATP, ADP, AMP) par chromatographie séquentielle (Figure 34). L'échantillon est d'abord déposé sur une colonne DOWEX échangeuse de cations. Suite à l'addition d'un premier volume d'eau distillée, la majorité des composés dérivés de l'adénine sont éliminés tandis que les molécules d'AMPc sont sélectivement retenues dans la colonne DOWEX, ADP ou AMP résiduel est retenu tandis que les molécules d'AMPc sont éluées dans le tube à scintillation, 1968.
, H] récoltées sont mesurés grâce à un compteur à scintillation. En parallèle, on mesure également la radioactivité émise par les solutions stocks d'ATP
, La concentration de ces solutions étant connue, le rayonnement des molécules d'AMPc radiomarquées peut être converti en nombre de moles d'AMPc récoltées. Le calcul du rendement d
, évaluer précisément l'activité cyclase en nombre de moles d'AMPc synthétisées / unité de temps / quantité d'échantillon testé
, Le dosage non-compétitif de l'accumulation d'AMPc Le dosage non-competitif de l'accumulation d'AMPc repose, dans la plupart des cas, sur l'isolement et la quantification de la radioactivité émise par un produit AMPc radiomarqué, Annexe, vol.3
, Celui-ci est synthétisé à partir d'un pool d'adenine
, , 1984.
, L'échantillon est d'abord incubé dans une solution d'adenine, 1969.
, La réaction est initiée par l'ajout d'éventuels composés physiologiques ou pharmacologiques régulant l'activité cyclase. A l'issue d'un intervalle de temps fixé, la réaction est stoppée et l'échantillon lysé par l'addition d'acide trichloroacétique. Le mélange réactionnel est supplémenté par une solution d'AMPc [ 14 C] puis divisé en deux aliquots. Les molécules d'AMPc contenues dans le premier aliquot sont purifiées par chromatographie séquentielle sur colonne DOWEX échangeuse de cation et colonne d'aluminium activé. Les rayonnements émis par les molécules d'AMPc [ 3 H] et d'AMPc [ 14 C] ainsi récoltées sont mesurés grâce à un compteur à scintillation. La mesure de la radioactivité émise par la solution stock d, Après élimination du précurseur adénine en excès, l'échantillon est mis en présence d'IBMX et/ou de théophylline pour inhiber les PDE et/ou de probénécide pour inhiber les transporteurs MRP
, On mesure enfin la radioactivité émise par l'ensemble des molécules dérivées de l'adenine
, H] contenues dans l'aliquot du mélange réactionnel qui n'a pas été purifié. Ainsi, on peut évaluer l'accumulation d'AMPc
, Bien qu'il soit très sensible et présente un très bon ratio signal sur bruit, le dosage non-competitif de l'accumulation d'AMPc reste dépendant de la pénétration et de l
, Annexe 4 : Les dosages de l'accumulation d'AMPc par compétition de liaison
, Cette approche est moins sensible mais autorise une quantification rapide et absolue de l'AMPc accumulé sans ou avec peu d'étapes de purification préalable de l'échantillon. La réaction se fait exclusivement en présence i) d'inhibiteurs pharmacologiques des PDE et/ou des transporteurs MRP (IBMX, théophylline, probénécide) et ii) d'éventuels composés régulant l'activité des AC. A l'issue d'un intervalle de temps fixé, la réaction est stoppée et l'échantillon lysé. On ajoute successivement au mélange un traceur AMPc exogène puis un ligand doté d'une haute affinité pour l'AMPc. On évalue alors la capacité de l'AMPc endogène à déplacer la liaison du traceur sur le ligand. En parallèle, une gamme étalon est établie en répétant l'opération à partir de solutions d'AMPc de concentrations connues. Ainsi, l'accumulation d'AMPc peut être quantifiée, de façon absolue, en nombre de moles d'AMPc synthétisé / unité de temps / quantité d'échantillon. Diverses techniques de dosage par compétition de liaison ont été développées. Elles diffèrent par la nature du traceur AMPc et du ligand utilisé et par la méthode de détection associée
, Dans le cadre des immunoassays, le traceur est une molécule d'AMPc radio-marquée (RIA) ou couplée à la peroxydase de Raifort (EIA) et le ligand est un anticorps dirigé contre l'AMPc (Harper and Brooker, 1971.
, On quantifie ainsi l'accumulation d'AMPc endogène en mesurant directement la lumière émise par l'échantillon. Une méthode dérivée du dosage RIA-SPA et reposant sur le principe du transfert d'énergie de fluorescence par résonnance (FRET) permet d'éviter l'utilisation de composés radioactifs. Le traceur utilisé est une molécule d'AMPc couplée au fluorophore d2 (donneur), le ligand est un anticorps anti-AMPc couplé au fluorophore cryptate d'europium (accepteur), Lorsque le dosage RIA est couplé à une méthode de « scintillation proximity assay » (SPA), il est possible de quantifier l'accumulation d'AMPc sans étape préalable de purification du complexe traceur-ligand, 1999.
, Formation à l'expérimentation animale de niveau 1, Master de Biologie Intégrative et Physiologie, spécialité Physiologie et Physiopathologie, Université Pierre et Marie Curie (UPMC), 1990.
, Situation actuelle Doctorant en sciences à l'ED394 Physiologie, Physiopathologie et Thérapeutique, équipe Contrôle phénotypique des cellules musculaires lisses vasculaires, Licence de Sciences et Technologies mention Sciences de la Vie, vol.8256, 2008.
, Biochimie (fractionnement cellulaire, immunoprécipitation, GST Pull-down, western-blot, zymographie, ELISA, promoteur-gène rapporteur Luciférase, Pr Isabelle Limon et Dr Régis Blaise. Sujet d'étude : Caractérisation fonctionnelle de nouvelles isoformes d'Adénylyl Cyclase 8 identifiées dans les cellules musculaires lisses vasculaires trans-différenciées. Compétences techniques : Biologie moléculaire (clonage, extraction d'ADN/ARN, PCR)
, Sujet de stage : Etude de la régulation de l'expression de l'Adénylyl Cyclase 8 dans les cellules musculaires lisses vasculaires trans-différenciées, Stagiaire M2, équipe Contrôle phénotypique des cellules musculaires lisses vasculaires, vol.8256, 2013.
, Encadrant : Pr P. Le Rouzic, Stagiaire M1, équipe Maladies du tissu adipeux et du système respiratoire, 2012.
, Myriam Moumni, master Biologie Intégrative (UPMC). Intitulé du stage : « Etude des phosphodiestérases exprimées dans les cellules musculaires lisses vasculaires pathologiques, 2014.
, 11 ème congrès annuel de la Nouvelle Société Francophone d'Athérosclérose, ème Journées de Recherche Respiratoire (J2R), 2014.
, 12 ème congrès annuel de la Nouvelle Société Francophone d'Athérosclérose, 2016.
, Communications orales Séminaire interne de l'UMR 8256 Adaptation biologique et vieillissement, ème congrès annuel du Latinorum Investigatorum de Arteriis Colloquium, vol.30, 2014.
, Séminaire du « club AMPc » à l'UPMC, campus Jussieu, 2016.
, Adénylyl cyclases et transdifférenciation des cellules musculaires lisses vasculaires : rôle dans le remodelage vasculaire pathologique
, Revue de la Société de Biologie 05, 2016.
,
, Prochainement sousmis à, Nature Chemical Biology
, Creteil, 2017.
, apparent beneficial effect of adenylyl cyclase overexpression in young mice (Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase" by myself, Circ Res, vol.86, pp.795-801, 2000.
, This manuscript, or part of it, has neither been published nor is currently under consideration for publication by any other journal. All the co-authors have read the manuscript and approved its submission to Cardiovascular Research
,
,
,
,
, Taking into account the modifications of cAMP signaling in AC8TG, we can conclude that the modifications of cAMP signaling in AC8TG and old NTG mice described here enhance the stress-response of the heart. As expected, normal aging was accompanied by an alteration of cardiac function with a progressive increase in LVESD (Figure 3C) and a deterioration of LVEF (Figure 3D). However, LVEDD (Figure 3B) and LVFS (Figure 3E) were unaltered in old NTG mice. By contrast, in AC8TG mice, we observed a concentric pattern of hypertrophy (decrease in end-diastolic LV diameter and increase in wall thickness) in middle age followed by eccentric LV remodeling (increase in end-diastolic LV diameter) in old age (Figure 3B & C). LVEF and LVFS were lower in AC8TG than in NTG mice only for the oldest animals (Figure 3D & E). Taken together, conventional echocardiography parameters such as LVEF and LVFS did not reveal any alteration in basal cardiac function within young AC8TG and NTG, but accelerated deterioration of cardiac function in AC8TG with aging. The new Doppler-derived technique Tissue-Doppler Imaging (TDI) and its derived parameters strain and strain rate provide more accurate and sensitive information not only about regional and global LV function, but also about the current tissue condition (stiffness), indicating alterations in myocardial architecture (e.g. the degree of fibrosis) and molecular substructure (e.g. strain difference in the molecular structure of actin, myosin or titin). Particularly, systolic strain rate indicates how fast the myocardial tissue is shortening and is closely related to invasive parameters of contractility such as elastance and dP/dtmax. As previously shown 19 , normal aging was accompanied by decrease of myocardial systolic strain rate in NTG animals (Figure 4A & B). Surprisingly, strain rate imaging identified alteration in myocardial contractility in young AC8TG mice, and a progressive decline with age compared with NTG group (Figure 4A & B), title: Cardiac adenylyl cyclase overexpression and aging for AC8TG mice, by performing cross-sectional echocardiography in conscious mice from three age groups, young (2-4 months old)
, At 12 to 14 months, cardiac function further decreased, and mortality rates were already high in AC8TG mice. Finally, the LV thickness/radius ratio (h/r ratio) gradually decreased during aging in AC8TG mice, reflecting inadequate hypertrophic remodeling of the LV (Figure 5). We also performed in vivo hemodynamics studies in young and old NTG and AC8TG mice sedated with sodium pentobarbital (Figure 6), However, progressive LV parietal hypertrophy and LV cavity dilation became apparent in the AC8TG mice at 8 months (Figure 5), and led to the development of dilated cardiomyopathy, with decrease in LVFS (Figure 5)
, Whereas in NTG mice the RVSP slightly increased in the course of aging, in AC8TG the higher RVSP was maintained throughout life until the last measurement made at 12 to 14-month age. Consistent with the HF phenotype, we observed RV hypertrophy in old AC8TG mice (RV weight was 24±2 mg (n=7) in NTG vs. 31±2 mg (n=10) in AC8TG (P<0.01)) and a progressive muscularization of the distal pulmonary arteries in AC8TG mice between the ages of 2 to 14 months (Figure 7B & C). The degree of muscularization in old AC8TG animals was twice that of old NTG animals, NTG mice, ?-adrenergic stimulation (ISO) induced a classical dose-dependent stimulation of HR and LV contractility; however his effect was slightly blunted in old NTG animals (Figure 6A,C & D), vol.18
, P<0.05) weights were higher in the old AC8TG mice (n=10) than in the old NTG mice (n=10), suggesting respective dysfunction of both LV and RV. Thus, consistent with the HF phenotype, old AC8TG mice also developed an early trend towards pulmonary hypertension (PH), ± 98 mg, P<0.05) and lung (204 ± 11 vs. 257 ± 13 mg
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, animals. B & C, Pulmonary vessel muscularization (B) and representative micrographs showing low-resistance pulmonary vessels stained with hematoxylin/eosin (C) in NTG ( ) and AC8TG ( ) animals. Bar=50 ?m. D, RV remodeling in old AC8TG mice: RV hypertrophy index, NTG ( ) and AC8TG (
, The horizontal line indicates the mean value for each group