Publications by authors named "Anna Zoccarato"

13 Publications

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In vivo [U-C]glucose labeling to assess heart metabolism in murine models of pressure and volume overload.

Am J Physiol Heart Circ Physiol 2020 08 10;319(2):H422-H431. Epub 2020 Jul 10.

King's College London British Heart Foundation Centre of Excellence, School of Cardiovascular Medicine & Sciences, London, United Kingdom.

Alterations in the metabolism of substrates such as glucose are integrally linked to the structural and functional changes that occur in the remodeling heart. Assessment of such metabolic changes under in vivo conditions would provide important insights into this interrelationship. We aimed to investigate glucose carbon metabolism in pressure-overload and volume-overload cardiac hypertrophy by using an in vivo [U-C]glucose labeling strategy to enable analyses of the metabolic fates of glucose carbons in the mouse heart. Therefore, [U-C]glucose was administered in anesthetized mice by tail vein infusion, and the optimal duration of infusion was established. Hearts were then excised for C metabolite isotopomer analysis by NMR spectroscopy. [U-C]glucose infusions were performed in mice 2 wk following transverse aortic constriction (TAC) or aortocaval fistula (Shunt) surgery. At this time point, there were similar increases in left ventricular (LV) mass in both groups, but TAC resulted in concentric hypertrophy with impaired LV function, whereas Shunt caused eccentric hypertrophy with preserved LV function. TAC was accompanied by significant changes in glycolysis, mitochondrial oxidative metabolism, glucose metabolism to anaplerotic substrates, and de novo glutamine synthesis. In contrast to TAC, hardly any metabolic changes could be observed in the Shunt group. Taken together, in vivo [U-C]glucose labeling is a valuable method to investigate the fate of nutrients such as glucose in the remodeling heart. We find that concentric and eccentric cardiac remodeling are accompanied by distinct differences in glucose carbon metabolism. This study implemented a method for assessing the fate of glucose carbons in the heart in vivo and used this to demonstrate that pressure and volume overload are associated with distinct changes. In contrast to volume overload, pressure overload-induced changes affect the tricarboxylic acid cycle, glycolytic pathways, and glutamine synthesis. A better understanding of cardiac glucose metabolism under pathological conditions in vivo may provide new therapeutic strategies specific for different types of hemodynamic overload.
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http://dx.doi.org/10.1152/ajpheart.00219.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473922PMC
August 2020

Cardioprotective Effect of the Mitochondrial Unfolded Protein Response During Chronic Pressure Overload.

J Am Coll Cardiol 2019 04;73(14):1795-1806

School of Cardiovascular Medicine & Sciences, King's College London British Heart Foundation Centre, London, United Kingdom. Electronic address:

Background: The mitochondrial unfolded protein response (UPR) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPR in the heart is unclear.

Objectives: The purpose of this study was to: 1) identify conditions that activate the UPR in the heart; and 2) study the relationship among the UPR, mitochondrial function, and cardiac contractile function.

Methods: Cultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis.

Results: Diverse neurohumoral or mitochondrial stresses transiently induced the UPR in cultured cardiomyocytes. The UPR was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPR with nicotinamide riboside (which augments NAD pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPR activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction (N-terminal pro-B-type natriuretic peptide).

Conclusions: These results identify the induction of the UPR in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPR ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.
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http://dx.doi.org/10.1016/j.jacc.2018.12.087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456800PMC
April 2019

RORα nuclear receptors in protection against angiotensin II-induced cardiac hypertrophy.

Am J Physiol Heart Circ Physiol 2019 02 7;316(2):H357-H359. Epub 2018 Dec 7.

King's College London British Heart Foundation Centre, School of Cardiovascular Medicine and Sciences , London , United Kingdom.

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http://dx.doi.org/10.1152/ajpheart.00732.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397382PMC
February 2019

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

JCI Insight 2017 12 21;2(24). Epub 2017 Dec 21.

Cardiovascular Division, King's College London British Heart Foundation Centre of Excellence, London, United Kingdom.

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.
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http://dx.doi.org/10.1172/jci.insight.96184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752273PMC
December 2017

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

Naunyn Schmiedebergs Arch Pharmacol 2016 Nov 27;389(11):1143-1145. Epub 2016 Sep 27.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

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http://dx.doi.org/10.1007/s00210-016-1301-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059414PMC
November 2016

Glycoproteomics Reveals Decorin Peptides With Anti-Myostatin Activity in Human Atrial Fibrillation.

Circulation 2016 Sep 24;134(11):817-32. Epub 2016 Aug 24.

From King's British Heart Foundation Centre, King's College London, United Kingdom (J.B.-B., A. Zoccarato, R.K.-T., M.F., X.Y., A. Zampetaki, M.C., P.W., A.M.S., K.O., M.M.); Institute for Molecular and Translational Therapeutic Strategies, MH-Hannover, Germany (S.K.G., T.T.); St George's Hospital, NHS Trust, London, United Kingdom (M.F., A.V., A.K., M.J.); University Medical Center Hamburg-Eppendorf, Germany (T.W., M.N.H.); Protein Metrics, San Carlos, CA (M.B.); Biobanco A Coruña, INIBIC-Complexo Hospitalario Universitario de A Coruña, Spain (N.D.); Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt am Main, Germany (L.S.); Institute for Pharmacology and Clinical Pharmacology, Heinrich-Heine-University, Düsseldorf, Germany (J.W.F.); Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA (R.V.I.); Thermo Fisher Scientific, San Jose, CA (R.V.); Experimental Cardiology, Department of Cardiology and Angiology, MH-Hannover, Germany (J.H.); and Laboratoire Vecteurs: Synthèse et Applications Thérapeutiques, UMR 7199 CNRS Université de Strasbourg, Illkirch, France (A.K.).

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081096PMC
http://dx.doi.org/10.1161/CIRCULATIONAHA.115.016423DOI Listing
September 2016

Relax: It's Not All About Degradation.

Arterioscler Thromb Vasc Biol 2015 Sep;35(9):1907-9

From the WellcomeTrust-MRC Institute of Metabolic Science, University of Cambridge, Department of Clinical Neurosciences, Addenbrooke's Hospital, Cambridge, United Kingdom (A.S.); Department of Cardiology, Cardiovascular Division, King's College, London, British Heart Foundation Centre, London, United Kingdom (A.Z.).

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http://dx.doi.org/10.1161/ATVBAHA.115.306217DOI Listing
September 2015

Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

Circ Res 2015 Sep 4;117(8):707-19. Epub 2015 Aug 4.

From the Institute of Neuroscience and Psychology (A.Z., L.A.F., A.S., C.L., H.J., F.G., A.T., G.H., M.Z.) and Institute of Cardiovascular and Medical Sciences (Y.Y.S., G.S.B., S.A.N., D.G.), University of Glasgow, Glasgow, UK; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK (N.C.S., K.L., M.Z.); Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway (J.M.A., I.S.); Venetian Institute of Molecular Medicine, University of Padova, Padova, Italy (L.M., G.D., A.T., L.F., V.C.); Cardiology Section, VA Salt Lake City Health Care System and Cardiovascular Medicine Division, University of Utah School of Medicine, Salt Lake City, UT (N.S.-F., J.K., F.V., M.M.); Bjorknes College, Oslo, Norway (J.M.A.); and BHF Centre of Research Excellence, Oxford, UK (K.L., M.Z.).

Rationale: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains.

Objective: How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth.

Methods And Results: Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells.

Conclusions: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.
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http://dx.doi.org/10.1161/CIRCRESAHA.114.305892DOI Listing
September 2015

Analysis of compartmentalized cAMP: a method to compare signals from differently targeted FRET reporters.

Methods Mol Biol 2014 ;1071:59-71

College of Medical, Veterinary & Life Sciences, Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, UK.

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.
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http://dx.doi.org/10.1007/978-1-62703-622-1_5DOI Listing
April 2014

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

J Cell Biol 2012 Aug;198(4):607-21

Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK.

Previous work has shown that the protein kinase A (PKA)-regulated phosphodiesterase (PDE) 4D3 binds to A kinase-anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA-PDE4D3-AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals.
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http://dx.doi.org/10.1083/jcb.201201059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514031PMC
August 2012

Measuring spatiotemporal dynamics of cyclic AMP signaling in real-time using FRET-based biosensors.

Methods Mol Biol 2011 ;746:297-316

Neuroscience and Molecular Pharmacology, Biomedical & Life Sciences, University of Glasgow, Glasgow, UK.

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 user's specific requirements.
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http://dx.doi.org/10.1007/978-1-61779-126-0_16DOI Listing
September 2011

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

Circ Res 2011 Apr 17;108(8):929-39. Epub 2011 Feb 17.

Institute of Neuroscience & Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, United Kingdom.

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.
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http://dx.doi.org/10.1161/CIRCRESAHA.110.230698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083836PMC
April 2011

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

Circ Res 2008 Oct 28;103(8):836-44. Epub 2008 Aug 28.

Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, Padova, Italy.

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.
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http://dx.doi.org/10.1161/CIRCRESAHA.108.174813DOI Listing
October 2008