Sofia-Iris Bibli1,2,3, Zongmin Zhou2, Sven Zukunft3, Beate Fisslthaler3, Ioanna Andreadou1, Csaba Szabo4, Peter Brouckaert5,6, Ingrid Fleming3, Andreas Papapetropoulos1,2,7. 1. Laboratory of Pharmacology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupolis, Zografou 15771, Greece. 2. "George P. Livanos and Marianthi Simou" Laboratories, First Department of Pulmonary and Critical Care Medicine, Evangelismos Hospital, Faculty of Medicine, National and Kapodistrian University of Athens, Ipsilantou 45-47, Athens, 10675 Greece. 3. Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Theodor-Stern-Kai 7, Frankfurt am Main, D-60590, Germany. 4. Department of Anesthesiology, University of Texas Medical Branch, 601 Harborside Drive, Galveston, 77555 TX, USA. 5. Department of Biomedical Molecular Biology, Ghent University, Ghent B-9052, Belgium. 6. Laboratory for Molecular Pathology and Experimental Therapy, Inflammation Research Center, VIB, Ghent B-9052, Belgium. 7. Clinical, Experimental Surgery and Translational Research Center, Biomedical Research Foundation of the Academy of Athens, Soranou Ephessiou 4, Athens 11527, Greece.
Abstract
AIMS: Endothelial nitric oxide (NO) synthase (eNOS) is known to play a cardioprotective protective. However, the molecular mechanisms regulating eNOS activity during ischaemia/reperfusion (I/R) injury are incompletely understood. eNOS is a substrate for several kinases that positively or negatively affect its enzymatic activity. Herein, we sought to correlate eNOS phosphorylation status with cardiomyocyte survival and we investigated the contribution of the proline-rich tyrosine kinase 2 (PYK2)/eNOS axis to the regulation of myocardial infarct size in vivo. METHODS AND RESULTS: Exposure of H9c2 cardiomyocytes to H2O2 lead to PYK2 phosphorylation on its activator site (Y402) and eNOS phosphorylation on the inhibitor site Y656 and the activator site S1176. Both H2O2-induced eNOS phosphorylation events were abolished by PYK2 pharmacological inhibition or gene knockdown. Activity assays demonstrated that phosphorylation of the tyrosine inhibitory site exerts a dominant effect over S1176. In cardiomyocytes subjected to oxidative stress or oxygen-glucose deprivation, inhibition of PYK2 limited cell injury; this effect was prevented by inhibition of NO production. In vivo, ischaemia-reperfusion induced an early activation of PYK2, leading to eNOS phosphorylation on Y656, which, in turn, reduced NO output, as judged by the low tissue levels of its downstream effector cGMP. Moreover, pharmacological blockade of PYK2 alleviated eNOS inhibition and prevented cardiac damage following I/R injury in wild-type, but not in eNOS KO mice. CONCLUSION: The current studies demonstrate that PYK2 is a pivotal regulator of eNOS function in myocardial infarction and identify PYK2 as a novel therapeutic target for cardioprotection. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: Endothelial nitric oxide (NO) synthase (eNOS) is known to play a cardioprotective protective. However, the molecular mechanisms regulating eNOS activity during ischaemia/reperfusion (I/R) injury are incompletely understood. eNOS is a substrate for several kinases that positively or negatively affect its enzymatic activity. Herein, we sought to correlate eNOS phosphorylation status with cardiomyocyte survival and we investigated the contribution of the proline-rich tyrosine kinase 2 (PYK2)/eNOS axis to the regulation of myocardial infarct size in vivo. METHODS AND RESULTS: Exposure of H9c2 cardiomyocytes to H2O2 lead to PYK2 phosphorylation on its activator site (Y402) and eNOS phosphorylation on the inhibitor site Y656 and the activator site S1176. Both H2O2-induced eNOS phosphorylation events were abolished by PYK2 pharmacological inhibition or gene knockdown. Activity assays demonstrated that phosphorylation of the tyrosine inhibitory site exerts a dominant effect over S1176. In cardiomyocytes subjected to oxidative stress or oxygen-glucose deprivation, inhibition of PYK2 limited cell injury; this effect was prevented by inhibition of NO production. In vivo, ischaemia-reperfusion induced an early activation of PYK2, leading to eNOS phosphorylation on Y656, which, in turn, reduced NO output, as judged by the low tissue levels of its downstream effector cGMP. Moreover, pharmacological blockade of PYK2 alleviated eNOS inhibition and prevented cardiac damage following I/R injury in wild-type, but not in eNOS KO mice. CONCLUSION: The current studies demonstrate that PYK2 is a pivotal regulator of eNOS function in myocardial infarction and identify PYK2 as a novel therapeutic target for cardioprotection. Published on behalf of the European Society of Cardiology. All rights reserved.
Authors: Sofia-Iris Bibli; Andreas Papapetropoulos; Efstathios K Iliodromitis; Andreas Daiber; Voahanginirina Randriamboavonjy; Sebastian Steven; Peter Brouckaert; Athanasia Chatzianastasiou; Kyriakos E Kypreos; Derek J Hausenloy; Ingrid Fleming; Ioanna Andreadou Journal: Cardiovasc Res Date: 2019-03-01 Impact factor: 10.787