Transgenic Mice for Real-Time Visualization of cGMP in Intact Adult Cardiomyocytes

Abstract

1235 3ʹ,5ʹ-Cyclic guanosine monophosphate (cGMP) is one of the ubiquitous second messengers, which is critically involved in the regulation of cardiac contractility and pathological hypertrophy.1,2 In cardiomyocytes, 2 classes of guanylyl cyclases (GCs) are responsible for cGMP synthesis. The first class, particulate GCs (pGCs) represented by GC-A and GC-B, are plasma membrane receptors for atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), respectively. 3,4 Second, the so-called soluble or NO-sensitive GCs (NO-GCs) are also functionally present in cardiomyocytes.5-7 cGMP levels are negatively regulated by cGMP-hydrolyzing enzymes phosphodiesterases (PDEs) with at least 4 families (PDE1, 2, 3, and 5) expressed in cardiac myocytes, whereby the first 3 PDEs can degrade both cGMP and cAMP. [8][9][10][11] cGMP is generally considered as a cardioprotective second messenger, 12 because pharmacological elevation of cGMP levels either by inhibition of cGMP-hydrolyzing PDE5 and PDE1 or by activation of NO-GC and pGC prevents pathological cardiomyocyte growth in vitro 13 and cardiac remodeling in vivo. 2,14-16In This Issue, see p 1221Reliable cGMP measurements in adult cardiomyocytes have been challenging. 17 In adult heart, cGMP is present at much lower concentrations than cAMP and acts in a compartmentalized New Methods in Cardiovascular Biology© 2014 American Heart Association, Inc. Rationale: 3ʹ,5ʹ-Cyclic guanosine monophosphate (cGMP) is an important second messenger that regulates cardiac contractility and protects the heart from hypertrophy. However, because of the lack of real-time imaging techniques, specific subcellular mechanisms and spatiotemporal dynamics of cGMP in adult cardiomyocytes are not well understood.Objective: Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes. Methods and Results:We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Förster resonance energy transfer-based cGMP biosensor red cGES-DE5 and performed the first Förster resonance energy transfer measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (≈10 nmol/L) basal cytosolic cGMP levels, which can be markedly increased by natriuretic peptides (C-type natriuretic peptide >> atrial natriuretic peptide) and, to a much smaller extent, by the direct stimulation of soluble guanylyl cyclase. Constitutive activity of this cyclase contributes to basal cGMP production, which is balanced by the activity of clinically established phosphodiesterase (PDE) families. The PDE3 inhibitor, cilostamide, showed especially strong cGMP responses. In a mild model of cardiac hypertrophy after transverse aortic constriction, PDE3 effects were not affected, whereas the contribution of PDE5 was increased. In addition, after natriuretic peptide stimulation, PDE3 was also involved in cGMP/cAMP crosstalk. Conclusions:

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

“…Adult ventricular cardiomyocytes were isolated via the Langendorff perfusion 18,38 . Mice were euthanized and the hearts were quickly transferred into a chamber filled with ice-cold PBS (Biochrom) where the aorta was connected to a 21G cannula.…”

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis

“…It was reported that cardiac cGMP can affects cardiac properties by modulating cAMP levels, bypassing again cGKI (16,17). Thus, the site(s) of action of cGMP/cGKI are not entirely clear (18) and interpretation of cGMP/cGKI effects on cardiac hypertrophy may be quite complicated.…”

Sildenafil Does Not Prevent Heart Hypertrophy and Fibrosis Induced by Cardiomyocyte Angiotensin II Type 1 Receptor Signaling

“…Using a genetically encoded cGMP FRET-based sensor in intact adult CMs (Götz et al, 2014), we observed that CNP induced an ∼2-fold-higher cGMP elevation in AT 1 R tg/1 cells (isolated from 120-day-old double-transgenic mice) compared with control mice that carried only the FRET-based sensor (Fig. 1A).…”

“…To analyze CM-specific changes in cGMP levels, we crossed aMHC-AT 1 R tg/1 animals with red cGES-DE5 transgenic cGMP sensor mice (Götz et al, 2014). Adult CMs were isolated from double-transgenic animals versus littermate controls expressing the sensor without AT 1 R overexpression and subjected to cGMP-Förster resonance energy transfer (FRET) measurements under stimulation with C-type natriuretic peptide (CNP) (1 mM) and 3-isobutyl-1-methylxanthine (IBMX) (100 mM) as previously described (Götz et al, 2014).…”