Divergent off-target effects of RSK N-terminal and C-terminal kinase inhibitors in cardiac myocytes.

P90 ribosomal S6 kinases (RSK) are ubiquitously expressed and regulate responses to neurohumoral stimulation. To study the role of RSK signalling on cardiac myocyte function and protein phosphorylation, pharmacological RSK inhibitors were tested. Here, the ATP competitive N-terminal kinase domain-targeting compounds D1870 and SL0101 and the allosteric C-terminal kinase domain-targeting FMK were evaluated regarding their ability to modulate cardiac myocyte protein phosphorylation. Exposure to D1870 and SL0101 significantly enhanced phospholamban (PLN) Ser16 and cardiac troponin I (cTnI) Ser22/23 phosphorylation in response to D1870 and SL0101 upon exposure to phenylephrine (PE) that activates RSK. In contrast, FMK pretreatment significantly reduced phosphorylation of both proteins in response to PE. D1870-mediated enhancement of PLN Ser16 phosphorylation was also observed after exposure to isoprenaline or noradrenaline (NA) stimuli that do not activate RSK. Inhibition of ß-adrenoceptors by atenolol or cAMP-dependent protein kinase (PKA) by H89 prevented the D1870-mediated increase in PLN phosphorylation, suggesting that PKA is the kinase responsible for the observed phosphorylation. Assessment of changes in cAMP formation by FRET measurements revealed increased cAMP formation in vicinity to PLN after exposure to D1870 and SL0101. D1870 inhibited phosphodiesterase activity similarly as established PDE inhibitors rolipram or 3-isobutyl-1-methylxanthine. Assessment of catecholamine-mediated force development in rat ventricular muscle strips revealed significantly reduced EC50 for NA after D1870 pretreatment (DMSO/NA: 2.33 µmol/L vs. D1870/NA: 1.30 µmol/L). The data reveal enhanced cardiac protein phosphorylation by D1870 and SL0101 that was not detectable in response to FMK. This disparate effect might be attributed to off-target inhibition of PDEs with impact on muscle function as demonstrated for D1870.

Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET.

Calcium (Ca2+) and 3',5'-cyclic adenosine monophosphate (cAMP) play a critical role for cardiac excitation-contraction-coupling. Both second messengers are known to interact with each other, for example via Ca2+-dependent modulation of phosphodiesterase 1 (PDE1) and adenylyl cyclase 5/6 (AC 5/6) activities, which is supposed to occur especially at the local level in distinct subcellular microdomains. Currently, many studies analyze global and local cAMP signaling and its regulation in resting cardiomyocytes devoid of electrical stimulation. For example, Förster resonance energy transfer (FRET) microscopy is a popular approach for visualization of real time cAMP dynamics performed in resting cardiomyocytes to avoid potential contractility-related movement artifacts. However, it is unknown whether such data are comparable with the cell behavior under more physiologically relevant conditions during contraction. Here, we directly compare the cAMP-FRET responses to AC stimulation and PDE inhibition in resting vs. paced adult mouse ventricular cardiomyocytes for both cytosolic and subsarcolemmal microdomains. Interestingly, no significant differences in cAMP dynamics could be detected after ß-adrenergic (isoproterenol) stimulation, suggesting low impact of rapidly changing contractile Ca2+ concentrations on cytosolic cAMP levels associated with AC activation. However, the contribution of the calcium-dependent PDE1, but not of the Ca2+-insensitive PDE4, to the regulation of cAMP levels after forskolin stimulation was significantly increased. This increase could be mimicked by pretreatment of resting cells with Ca2+ elevating agents. Ca2+ imaging demonstrated significantly higher amplitudes of Ca2+ transients in forskolin than in isoproterenol stimulated cells, suggesting that forskolin stimulation might lead to stronger activation of PDE1. In conclusion, changes in intracellular Ca2+ during cardiomyocyte contraction dynamically interact with cAMP levels, especially after strong AC stimulation. The use of resting cells for FRET-based measurements of cAMP can be justified under ß-adrenergic stimulation, while the reliable analysis of PDE1 effects may require electric field stimulation.

Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII.

AIMS: Enhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca(2+)-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca(2+)-leak in atrial cardiomyocytes (CMs). METHODS AND RESULTS: In murine atrial CMs, SR-Ca(2+)-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca(2+)/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca(2+)-leak. The SR-Ca(2+)-leak induction by ATX-II was not detected when either the Na(+)/Ca(2+) exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca(2+)-transient amplitude or SR-Ca(2+)-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca(2+)-transient amplitude and SR-Ca(2+)-load were increased, whereas PKA inhibition reduced Ca(2+)-transient amplitude and load and additionally slowed Ca(2+) elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca(2+)-leak. CONCLUSION: Late INa exerts distinct effects on Ca(2+) homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca(2+)-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late INa inhibition.

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

3',5'-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 ß-adrenergic receptor-cAMP signalling to SERCA.

Microdomain switch of cGMP-regulated phosphodiesterases leads to ANP-induced augmentation of ß-adrenoceptor-stimulated contractility in early cardiac hypertrophy.

RATIONALE: Cyclic nucleotides are second messengers that regulate cardiomyocyte function through compartmentalized signaling in discrete subcellular microdomains. However, the role of different microdomains and their changes in cardiac disease are not well understood. OBJECTIVE: To directly visualize alterations in ß-adrenergic receptor-associated cAMP and cGMP microdomain signaling in early cardiac disease. METHODS AND RESULTS: Unexpectedly, measurements of cell shortening revealed augmented ß-adrenergic receptor-stimulated cardiomyocyte contractility by atrial natriuretic peptide/cGMP signaling in early cardiac hypertrophy after transverse aortic constriction, which was in sharp contrast to well-documented ß-adrenergic and natriuretic peptide signaling desensitization during chronic disease. Real-time cAMP analysis in ß1- and ß2-adrenergic receptor-associated membrane microdomains using a novel membrane-targeted Förster resonance energy transfer-based biosensor transgenically expressed in mice revealed that this unexpected atrial natriuretic peptide effect is brought about by spatial redistribution of cGMP-sensitive phosphodiesterases 2 and 3 between both receptor compartments. Functionally, this led to a significant shift in cGMP/cAMP cross-talk and, in particular, to cGMP-driven augmentation of contractility in vitro and in vivo. CONCLUSIONS: Redistribution of cGMP-regulated phosphodiesterases and functional reorganization of receptor-associated microdomains occurs in early cardiac hypertrophy, affects cGMP-mediated contractility, and might represent a previously not recognized therapeutically relevant compensatory mechanism to sustain normal heart function.

Transgenic mice for real-time visualization of cGMP in intact adult cardiomyocytes.

RATIONALE: 3',5'-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: The new sensor model allows visualization of real-time cGMP dynamics and pharmacology in intact adult cardiomyocytes. Förster resonance energy transfer imaging suggests the importance of well-established and potentially novel PDE-dependent mechanisms that regulate cGMP under physiological and pathophysiological conditions.

Biophysical techniques for detection of cAMP and cGMP in living cells.

Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains.

FRET microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors.

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium, cAMP, inositol phosphates and cGMP. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a ß-adrenergic receptor agonist and blocker.