Transgenic Mice for cGMP Imaging

Thunemann, Martin; Wen, Lai; Hillenbrand, Matthias; Vachaviolos, Angelos; Feil, Susanne; Ott, Thomas; Han, Xiaoxing; Fukumura, Dai; Jain, Rakesh K.; Russwurm, Michael; Wit, Cor de; Feil, Robert

doi:10.1161/circresaha.113.301063

Cited by 69 publications

(139 citation statements)

References 30 publications

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“…1A) (25), we measured cGMP levels in mural granulosa, cumulus, and oocyte regions before and after addition of LH. Binding of cGMP to cGi500 decreases FRET between CFP and YFP, such that the CFP/YFP emission ratio measured after CFP excitation indicates cGMP concentration; the EC 50 of cGi500 for cGMP is 500 nM (25)(26)(27), which is appropriate for detection of cGMP in the range of concentrations in mouse follicles before and after LH treatment. ELISA measurements of the cGMP content of follicles from cGi500-expressing mice showed an LH-induced decrease (Fig.…”

Section: Resultsmentioning

confidence: 99%

Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles

Shuhaibar

Egbert

Norris

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.cyclic GMP | gap junctions | ovarian follicle | oocyte meiosis | luteinizing hormone M eiosis in mammalian oocytes begins during embryonic development and then arrests in late prophase, for up to 50 y in women and for many months in mice. At the time of ovulation, luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to release the arrest and restart meiosis in preparation for fertilization (1-3). In the mouse preovulatory follicle, inhibition of meiotic progression is dependent upon the cyclic nucleotide cyclic GMP (cGMP), which diffuses from the granulosa cells into the oocyte through gap junctions that connect all cells of the follicle (4-6). The cGMP is produced by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2, also known as guanylyl cyclase B), which is present in all of the granulosa cells, but not in the oocyte (7-11). In the oocyte, cGMP inhibits the degradation of another cyclic nucleotide, cAMP, which depends primarily on the phosphodiesterase PDE3A, an enzyme whose activity is antagonized by cGMP (4, 5). The resulting high level of cAMP, through a series of intermediate steps, inhibits meiotic progression (2, 3, 12).LH signaling is initiated in the outer (mural) layers of granulosa cells; receptors for LH are absent in the oocyte and in the granulosa cells that directly surround it (the cumulus cells) (13-15). Ensuing events cause meiosis to resume by reducing cGMP in the oocyte (4, 5), but how LH receptor activation up to 10 cell layers away lowers oocyt...

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Section: Resultsmentioning

confidence: 99%

Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles

Shuhaibar

Egbert

Norris

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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140

155

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“…Some other biosensors, such as the A-kinase activity reporter fused to PLN have recently been introduced into adult cardiomyocytes via adenoviral vectors to monitor local kinase activity 21 . Although we and others have successfully generated TG mice expressing some of the cytosolic FRET biosensors 19,[22][23][24] , it remained unclear whether targeted, microdomain-specific biosensors, which act in the functionally relevant subcellular locations, are also compatible with in vivo animal models. Here, we describe the generation and validation of the first TG mouse model, which expresses a targeted cAMP biosensor designed to dissect the cAMP signalling in the vicinity of an important calcium-handling protein SERCA2a.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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3 0 ,5 0 -cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced b-adrenergic receptor-cAMP signalling to SERCA.

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“…Therefore, we hypothesized that only a highly sensitive FRET sensor can be used for cGMP imaging in these cells, whereas other biosensors, including cGi500 (affinity ≈0.5 μmol/L), for which an ubiquitous transgenic mouse has recently been published, 37 would not pick up low cGMP levels typical for adult cardiomyocytes. Here we generated transgenic mice expressing a highly sensitive cGMP biosensor red cGES-DE5 and used them for FRET measurements.…”

Section: Discussionmentioning

confidence: 99%

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

Götz

Sprenger

Perera³

et al. 2014

Circulation Research

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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:

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Transgenic Mice for cGMP Imaging

Cited by 69 publications

References 30 publications

Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles

Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles

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

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

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