Compartmentalized cGMP signalling in cardiomyocyte hypertrophy and heart failure

Viacheslav Nikolaev, Hamburg

The ubiquitous second messenger cGMP regulates multiple cellular processes including smooth and heart muscle contractility, pathological cardiac growth (hypertrophy) and remodelling. In our previous work, we uncovered that hypertrophy is associated with relocation of the cGMP-regulated phosphodiesterases (PDEs) 2 and 3 between b-adrenoceptor (b-AR)-associated submembrane microdomains. This newly identified molecular mechanism allows augmentation of catecholamine-stimulated cardiac contractility by atrial natriuretic peptide (ANP) through its receptor GC-A located in T-tubules of cardiomyocytes, where we could also localise functional b3-ARs. This microdomain might bring together distinct guanylyl cyclases (GC-A/B and NO-GC) to generate an important local pool of cGMP which regulates contraction and pathological remodelling. cGMP microdomain remodelling is a stage-dependent process which involves dynamic changes of cGMP/cAMP cross-talk due to PDE and b-AR redistribution. Based on our previous work and recently established mouse lines, we will now continue to analyse changes of submembrane cGMP signalling in hypertrophy and heart failure. Using Förster resonance energy transfer and scanning ion conductance microscopy in pm-DE5 mouse cells (expressing a cGMP sensor targeted to T-tubules and caveolin-rich membrane microdomains), we will study whether hypertrophy leads to changes of PDE2-dependent GC-A/cGMP and PDE3-dependent GC-B/cGMP microdomain regulation in the T-tubules vs cell crests.  Potentially, GC-A desensitisation and redistribution at the later transition to chronic disease will be analysed together with other FOR 2060 projects. Secondly, we will use pm-DE5 and pm-Epac1-camps animals bred with b3-AR transgenic mice to study functional interactions of b3-AR and GC-A receptors in the T-tubular compartment and the regulation of cGMP/cAMP cross-talk by their respective, presumably distinct cGMP pools in the context of disease. Finally, findings on cGMP dynamics obtained in single isolated cardiomyocytes will be further verified using newly established FRET imaging in intact Langendorff perfused hearts with transgenic sensor expression. In this experimental setting, cell type-specific effects in cardiomyocytes, cardiac fibroblasts and potentially also in endothelial cells can be studied and the role of cell-cell communication in the context of pharmacological treatments (such as by sildenafil or drugs acting via NO-GC) can be evaluated. The long-term goal of this project is to identify new druggable targets at the level of subcellular cGMP microdomains, which can be used to prevent cardiac hypertrophy and heart failure progression.

Fig. 1. Visualization of cGMP in adult mouse CMs. (A) Schematics of the redDE5 sensor containing T-Sapphire (Sapp) and Dimer2 (RFP) fluorescent proteins as donor and acceptor fluorophores. Biding of cGMP to the sensor leads to a decrease of FRET. (B) Confocal images of CMs isolated from adult mice transgenically expressing redDE5 in CMs. (C, D, E) These cells respond to beta3-AR stimulation (combination of the beta-adrenergic agonist isoprenaline ISO with selective beta1- and beta2-AR receptor blockers CGP20712A and ICI118551, respectively), to ANP and CNP with a decrease of FRET which has different amplitudes. (F) Quantification of FRET signals recorded from CMs treated as mentioned above or with various PDE inhibitors: 100 µM 8-MMX (PDE1), 100 nM BAY 60-7550 (PDE2), 1 µM cilostamide (PDE3) or 100 nM tadalafil (PDE5). (C) Viacheslav Nikolaev
Fig. 2. cGMP-FRET measurements in intact working Langendorff hearts transgenically expressing redDE5 sensor. (A) Fluorescence in the FRET channel. (B) FRET response to CNP (100 nM) application shows a rapid increase in cardiomyocyte cGMP. (C) Viacheslav Nikolaev

Project-related publications

PI of this project; PIs of other FOR 2060 projects are in bold.

1.    Perera RK, Sprenger JU, Steinbrecher JH, Hubscher D, Lehnart SE, Abesser M, Schuh K, El-Armouche A, Nikolaev VO. Microdomain switch of cGMP-regulated phosphodiesterases leads to ANP-Induced augmentation of beta-adrenoceptor-stimulated contractility in early cardiac hypertrophy. Circ Res. 2015;116:1304-11. [Project 4] [pubmed]

2.    Straubinger J, Schöttle V, Bork N, Subramanian H, Dünnes S, Russwurm M, Gawaz M, Friebe A, Nemer A, Nikolaev VO, Lukowski R. Sildenafil does not prevent heart hypertrophy and fibrosis induced by cardiomyocyte AT1R signaling. J Pharmacol Exp Ther. 2015;354:406-16. [Project 3Project 4, and Project 5] [pubmed]

3.    Shuhaibar LC, Egbert JR, Norris RP, Lampe PD, Nikolaev VO, Thunemann M, Wen L, Feil R, Jaffe LA. Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles. Proc Natl Acad Sci U S A. 2015;112:5527-32. [Project 1Project 4, and Project Z] [pubmed]

4.    Götz KR, Sprenger JU, Perera RK, Steinbrecher JH, Lehnart SE, Kuhn M, Gorelik J, Balligand JL, Nikolaev VO. Transgenic mice for real-time visualization of cGMP in intact adult cardiomyocytes. Circ Res. 2014;114:1235-45. [Project 4] [pubmed]

5.    Belge C, Hammond J, Dubois-Deruy E, Manoury B, Hamelet J, Beauloye C, Markl A, Pouleur AC, Bertrand L, Esfahani H, Jnaoui K, Götz KR, Nikolaev VO, Vanderper A, Herijgers P, Lobysheva I, Iaccarino G, Hilfiker-Kleiner D, Tavernier G, Langin D, Dessy C, Balligand JL. Enhanced expression of beta3-adrenoceptors in cardiac myocytes attenuates neurohormone-induced hypertrophic remodeling through nitric oxide synthase. Circulation. 2014;129:451-62. [Project 4] [pubmed]

6.    Nakagawa H, Oberwinkler H, Nikolaev VO, Gassner B, Umbenhauer S, Wagner H, Saito Y, Baba HA, Frantz S, Kuhn M. Atrial natriuretic peptide locally counteracts the deleterious effects of cardiomyocyte mineralocorticoid receptor activation. Circ Heart Fail. 2014;7:814-21. [Project 4] [pubmed]

7.    Götz KR, Nikolaev VO. Advances and techniques to measure cGMP in intact cardiomyocytes. Methods Mol Biol. 2013;1020:121-9. [Project 4] [pubmed]

8.    Sprenger JU, Nikolaev VO. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells. Int J Mol Sci. 2013;14:8025-46. [Project 4] [pubmed]

9.    Klaiber M, Dankworth B, Kruse M, Hartmann M, Nikolaev VO, Yang RB, Volker K, Gassner B, Oberwinkler H, Feil R, Freichel M, Groschner K, Skryabin BV, Frantz S, Birnbaumer L, Pongs O, Kuhn M. A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide. Proc Natl Acad Sci U S A. 2011;108:18500-5. [Project 1 and Project 4] [pubmed]

10.  Nikolaev VO, Moshkov A, Lyon AR, Miragoli M, Novak P, Paur H, Lohse MJ, Korchev YE, Harding SE, Gorelik J. Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation. Science. 2010;327:1653-7. [Project 4] [pubmed]