Anna Zoccarato

Protein kinase A type I and type II define distinct intracellular signaling compartments.

Protein kinase A (PKA) is a key regulatory enzyme that, on activation by cAMP, modulates a wide variety of cellular functions. PKA isoforms type I and type II possess different structural features and biochemical characteristics, resulting in nonredundant function. However, how different PKA isoforms expressed in the same cell manage to perform distinct functions on activation by the same soluble intracellular messenger, cAMP, remains to be established. Here, we provide a mechanism for the different function of PKA isoforms subsets in cardiac myocytes and demonstrate that PKA-RI and PKA-RII, by binding to AKAPs (A kinase anchoring proteins), are tethered to different subcellular locales, thus defining distinct intracellular signaling compartments. Within such compartments, PKA-RI and PKA-RII respond to distinct, spatially restricted cAMP signals generated in response to specific G protein–coupled receptor agonists and regulated by unique subsets of the cAMP degrading phosphodiesterases. The selective activation of individual PKA isoforms thus leads to phosphorylation of unique subsets of downstream targets.

cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes.

Rationale: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). Objective: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. Methods and Results: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. Conclusions: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

Control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals through PKA and PDE4D3 interaction with the A kinase anchoring protein AKAP9.

Measuring Spatiotemporal Dynamics of Cyclic AMP Signaling in Real-Time Using FRET-Based Biosensors

Cyclic AMP governs many fundamental signaling events in eukaryotic cells. Although cAMP signaling has been a major research focus for a long time, recent technological developments are revealing novel aspects of this paradigmatic pathway. In this chapter, we give an overview over current fluorescence resonance energy transfer (FRET)-based sensors for detection of cAMP dynamics, and their application in monitoring local, compartmentalized cAMP signals within living cells. A basic step-by-step protocol is given for conducting a FRET experiment in primary cells with a unimolecular cAMP sensor, which can easily be adapted to a users specific requirements.

Analysis of Compartmentalized cAMP: A Method to Compare Signals from Differently Targeted FRET Reporters

Förster resonance energy transfer (FRET)-based reporters are important tools to study the spatiotemporal compartmentalization of cyclic adenosine monophosphate (cAMP) in living cells. To increase the spatial resolution of cAMP detection, new reporters with specific intracellular targeting have been developed. Therefore it has become critical to be able to appropriately compare the signals revealed by the different sensors. Here we illustrate a protocol to calibrate the response detected by different targeted FRET reporters involving the generation of a dose-response curve to the cAMP raising agent forskolin. This method represents a general tool for the accurate analysis and interpretation of intracellular cAMP changes detected at the level of different subcellular compartments.

Glycoproteomics reveals decorin peptides with anti-myostatin activity in human atrial fibrillation

Background: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). Methods: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. Results: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. Conclusions: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.

Response to Wagner et al.: phosphodiesterase-2—anti-adrenergic friend or hypertrophic foe in heart disease?

In Zoccarato et al. (Zoccarato et al. 2015), we provide extensive evidence that inhibition of PDE2 counteracts cardiac myocyte hypertrophic growth. By measuring cell size, nuclear NFAT-translocation, protein synthesis, protein to DNA ratio and expression of hypertrophic markers, we demonstrate that pharmacological inhibition of PDE2, unlike inhibition of PDE3 or PDE4, inhibits the hypertrophy induced by norepinephrine (NE); and that overexpression of PDE2, but not overexpression of PDE4 or PDE3 isoforms, is sufficient to induce hypertrophic growth. We show that the effect of pharmacological inhibition of PDE2 is recapitulated by its genetic knock down (KD) using siRNA. We further demonstrate that the effect of PDE2 KD is specific as the phenotype can be rescued by overexpression of a siRNA-insensitive PDE2 construct. In addition, we find that overexpression of a catalytically inactive version of PDE2 also counteracts hypertrophy, an effect that is due to displacement of localised active endogenous PDE2. We confirm the anti-hypertrophic effect of PDE2 inhibition both in neonatal and adult ventricular myocytes and both in rat and mouse models. Crucially, we demonstrate in vivo that pharmacological inhibition of PDE2 counteracts hypertrophy in a mouse model of pressure overload and that in vivo overexpression of PDE2-mCherry, but not of its catalytically inactive mutant, induces cardiac hypertrophic growth in rat cardiac myocytes. Based on the above evidence, we suggest that inhibition of PDE2 may represent a novel therapeutic approach to control cardiac hypertrophy.

Relax It’s Not All About Degradation

The second messenger cyclic guanosine monophosphate (cGMP) is involved in a variety of physiological processes, such as phototransduction,1 regulation of synaptic plasticity,2 metabolism,3 and vascular relaxation.4 The physiological effects of cGMP are regulated by a balanced control of its synthesis and its degradation, which in turn regulates the activity of downstream targets via protein kinase G–mediated phosphorylation. cGMP is synthesized by 2 types of cyclases, the soluble guanylyl cyclase, activated by nitric oxide (NO), and the particulate guanylyl cyclase, which responds to natriuretic peptides. cGMP is degraded by the enzymes phosphodiesterases (PDEs).5 Mammalian cells express 5 cGMP-degrading PDEs (PDE1, -2, -3, -5, -9) that differ by substrate specificity, catalytic properties, and intracellular localization.5 Compartmentalization of cyclic nucleotide signaling is an important aspect of cellular biology because it allows, on specific stimuli, for selective activation of downstream targets.6 PDEs play a key role in this process not only by terminating the cyclic nucleotide signal, but also by determining the amplitude and the specific subcellular site where the signal operates.6 In addition to degradation, export of cyclic nucleotides via the multidrug resistance protein 4 (MRP4)7 can also contribute to cyclic nucleotide signaling modulation.8 MRP4 is a member of the C subfamily of ATP-binding cassette transporters and is capable of pumping out of the cells xenobiotics and endogenous molecules like the cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cGMP.7 Interestingly, a recent study describes that …

Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation

Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4–dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial.

C Nox4-dependent Reprogramming of Glucose Metabolism and Fatty Acid Oxidation Facilitates Cardiac Adaption to Chronic Pressure-Overload

Introduction Increased reactive oxygen species (ROS) production is involved in the pathophysiology of cardiac hypertrophy and failure. Interestingly, a specialised ROS-generating enzyme NADPH oxidase-4 (Nox4) was previously found to have beneficial effects by promoting adaptive remodelling during pressure-overload cardiac hypertrophy. Nox4 modulates intracellular signalling cascades but how it achieves beneficial effects in the chronically overloaded heart remains unclear. Methods and results To obtain an unbiased global overview of putative Nox4-mediated changes, the proteome of cardiac-specific Nox4 transgenic (TG) and wild-type (WT) mouse hearts was first characterised through a 2D-DIGE approach. TG hearts had a significant over-representation of changes in protein levels of enzymes involved in glucose and fatty acid utilisation. We therefore analysed the metabolome using 1H-NMR and targeted LC-MS approaches. This identified a differential accumulation of glycolytic intermediates in the proximal part of glycolysis both in unstressed and pressure-overloaded TG hearts, as well as an increase in alanine levels (1.4 fold, p = 0.05), confirming significant alterations to metabolism. To specifically quantify glucose uptake, glycolysis, glucose oxidation and fatty acid oxidation rates, ex vivo working heart studies were conducted. TG hearts had a marked increase cf. WT in palmitate oxidation rate in the unstressed as well as pressure-overloaded heart (3.6 fold increase; n = 6/group; p = 0.01). Glucose uptake was unaltered but glycolysis and oxidation rates were decreased, suggesting diversion of glucose away from oxidation. Importantly, an increase in palmitate oxidation was not detrimental either for in vivo cardiac energetics (31P-NMR) or contractile function during pressure-overload hypertrophy. We found that activity of the hexosamine biosynthesis pathway (HBP), an alternative route for glucose metabolism, was increased in TG hearts as assessed by the O-GlcNAc post-translational modification of cardiac proteins by N-acetylglucosamine, the end-product of HBP. O-GlcNAc levels were 2.4 fold higher in TG cf. WT (n = 4/group; p = 0.02). In cultured cardiomyocytes, endogenous Nox4 induced similar changes in HBP and palmitate oxidation (extracellular flux analysis), and it was found that changes in O-GlcNAcylation regulated fatty acid oxidation. Discussion These results show that Nox4 reprograms substrate utilisation in the heart by directing glucose towards the HBP and inducing a linked increase in fatty acid oxidation. These changes appear to enable the heart to better adapt to chronic pressure overload and may be important in the beneficial effects of Nox4 on cardiac remodelling. These data identify a novel redox mechanism that drives beneficial metabolic reprogramming in the heart and suggest potential new therapeutic approaches to promote adaptation to chronic overload stress.