Activation De La Phosphodiestérase De Type 2 Pour Traiter L'insuffisance Cardiaque
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NNT : 2016SACL336 THESE DE DOCTORAT DE L’UNIVERSITE PARIS-SACLAY PREPAREE A UNIVERSITE PARIS SUD ECOLE DOCTORALE N° 569 ITFA Innovation thérapeutique: du fondamental à l’appliqué Spécialité de doctorat: Physiologie, physiopathologie Par Marta Lindner Activation de la phosphodiestérase de type 2 pour traiter l'insuffisance cardiaque Thèse présentée et soutenue à Châtenay-Malabry, le 12 Octobre 2016 Composition du Jury : M. Christian POÜS Professeur, UPSud Président Mme Claire LUGNIER DR Emérite CNRS Rapporteur Mme Catherine PAVOINE CR INSERM Rapporteur Mme Liliana CASTRO Maître de Conférence, UPMC Examinateur M. Ali EL-ARMOUCHE Professeur, Univ. Dresden Examinateur M. Grégoire VANDECASTEELE DR INSERM Examinateur M. Rodolphe FISCHMEISTER DR INSERM Directeur de thèse Table of contents Sommaire Introduction ______________________________________________________________ 13 I. The heart _______________________________________________________________ 14 I.1 Anatomy of the heart ________________________________________________________ 14 I.2 Cardiac excitation-contraction coupling __________________________________________ 15 I.2.1 Structures involved in cardiac ECC _____________________________________________________ 15 I.2.1.1 Sarcomere and T-tubules _______________________________________________________ 15 I.2.1.2 Sarcoplasmic reticulum ________________________________________________________ 17 I.2.1.3 Myofilaments ________________________________________________________________ 17 I.2.1.4 Mitochondria ________________________________________________________________ 18 I.2.2 Excitation _________________________________________________________________________ 19 I.2.3 Contraction _______________________________________________________________________ 21 I.2.4 Relaxation ________________________________________________________________________ 22 I.3 Regulation of ECC ____________________________________________________________ 22 I.3.1 Frank-Starling law __________________________________________________________________ 22 I.3.2 Neurohormonal regulation of heart function ____________________________________________ 23 II. Cyclic AMP signaling in the heart ___________________________________________ 24 II.1 G-protein coupled receptors (GPCRs) ___________________________________________ 24 II.2 β-adrenergic receptors _______________________________________________________ 25 II.2.1 β1-adrenergic receptor ______________________________________________________________ 27 II.2.2 β2-adrenergic receptors _____________________________________________________________ 28 II.2.3 β3-adrenergic receptors _____________________________________________________________ 29 II.2.4 Desensitization of β-adrenergic receptors ______________________________________________ 29 II.3 Adenylyl cyclases ___________________________________________________________ 31 II.4 Effectors of cAMP ___________________________________________________________ 34 II.4.1 Protein kinase A ___________________________________________________________________ 34 II.4.1.1 Structure and regulation _______________________________________________________ 34 II.4.1.2 Targets of PKA in the heart _____________________________________________________ 35 L-type Ca2+ channels (LTTCs) ____________________________________________________ 35 Ryanodine receptor ___________________________________________________________ 37 Phospholamban ______________________________________________________________ 39 Contractile proteins___________________________________________________________ 39 II.4.1.3 Subcellular targeting of PKA ____________________________________________________ 40 II.4.2 Other effectors of cAMP ____________________________________________________________ 40 II.4.2.1 Exchange factor Epac _________________________________________________________ 40 II.4.2.2 CNG and HCN channels ________________________________________________________ 42 II.4.2.3 Popeye domain containing protein family _________________________________________ 43 II.5 cAMP degradation __________________________________________________________ 43 II.5.1 Structure, localization and regulation of phosphodiesterases _______________________________ 45 II.5.1.1 Phosphodiesterase type 1 ______________________________________________________ 45 II.5.1.2 Phosphodiesterase type 2 ______________________________________________________ 47 II.5.1.3 Phosphodiesterase type 3 ______________________________________________________ 48 1 II.5.1.4 Phosphodiesterase type 4 ______________________________________________________ 49 II.5.2 Pharmacological inhibition of phosphodiesterases _______________________________________ 50 II.5.3 cAMP compartmentation ____________________________________________________________ 51 III. Cyclic GMP signaling in the heart ___________________________________________ 54 III.1 Cyclic GMP production _______________________________________________________ 54 III.1.1 Soluble guanylyl cyclase ____________________________________________________________ 55 III.1.2 Particulate guanylyl cyclase _________________________________________________________ 56 III.2 cGMP targets in cardiac myocytes _____________________________________________ 57 III.2.1 Protein kinase G __________________________________________________________________ 57 III.2.1.1 LTCCs ______________________________________________________________________ 59 III.2.1.2 Troponin I __________________________________________________________________ 59 III.2.1.3 Phospholamban _____________________________________________________________ 60 III.2.2 Phosphodiesterase ________________________________________________________________ 60 III.3 cGMP signaling in cardiac hypertrophy__________________________________________ 62 IV. cAMP and cGMP signaling crosstalk ________________________________________ 63 V. Heart failure ____________________________________________________________ 66 V.1 Pathophysiology of heart failure _______________________________________________ 66 V.2 Treatment of heart failure ____________________________________________________ 67 V.2.1 β-blockers ________________________________________________________________________ 67 V.2.2 Inhibitors of phosphodiesterases _____________________________________________________ 68 V.2.3 Vasodilators ______________________________________________________________________ 69 V.2.4 Ca2+ channel blockers _______________________________________________________________ 69 V.2.5 ACE inhibitors and angiotensin receptor blockers ________________________________________ 70 V.2.6 LCZ 696 __________________________________________________________________________ 70 VI. Phosphodiesterase type 2 _________________________________________________ 71 VI.1 Structure, function and subcellular localization of PDE2 ____________________________ 71 VI.2 Regulation of expression and activity of PDE2 ____________________________________ 73 VI.3 PDE2 as potential target in heart failure ________________________________________ 74 Objectives ________________________________________________________________ 76 Materials and methods _____________________________________________________ 79 I. Animal model ____________________________________________________________ 80 I.1 Transgenic mice with PDE2 overexpression _______________________________________ 80 I.2 Experimental model of hypertrophy _____________________________________________ 80 II. Exercise capacity test _____________________________________________________ 81 III. Langendorff perfused heart _______________________________________________ 82 III.1 Principle __________________________________________________________________ 82 III.2 Experimental set-up _________________________________________________________ 82 III.3 Experimental protocol _______________________________________________________ 83 IV. Isolation of adult mouse ventricular cardiomyocytes ___________________________ 84 2 V. Sarcomere shortening and Ca2+ transient measurements ________________________ 85 V.1 Principle __________________________________________________________________ 85 V.2 Fluorescent Ca2+ indicator Fura-2-AM ___________________________________________ 85 V.3 Experimental set-up _________________________________________________________ 86 V.4 Experimental protocols ______________________________________________________ 87 V.5 Data processing _____________________________________________________________ 88 V.6 Data analysis _______________________________________________________________ 88 VI. Fluorescence resonance energy transfer (FRET) imaging ________________________ 89 VI.1 Principle __________________________________________________________________ 89 VI.2 Experimental set-up ________________________________________________________ 90 VI.3 FRET sensor – H187 _________________________________________________________ 90 VI.4 Adenoviral infection ________________________________________________________ 91 VI.5 Experimental protocol _______________________________________________________ 91 VI.6 Data analysis ______________________________________________________________ 91 VII. L type Ca2+ current measurement – the patch clamp technique __________________ 91 VII.1 Principle _________________________________________________________________ 91 VII.2 Solutions _________________________________________________________________ 92 VII.3 Experimental setup _________________________________________________________ 92 VII.4 Experimental protocol ______________________________________________________