Molecular and functional
compartmentation of cGMP in the heart
Gaia Calamera
Department of Pharmacology Institute of Clinical Medicine
University of Oslo and Oslo University Hospital
2020
© Gaia Calamera, 2020
Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-8377-720-8
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
2
Table of contents
1 AcknowledgementsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶ 2 AbbreviationsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ
3 PrefaceǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͲ 4 List of papers includedǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͳ 5 IntroductionǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳʹ 5.1 The heartǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳʹ The contractile machinery in the heartǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳʹ 5.2 MitochondriaǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳ
StructureǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳ
Mitochondrial dynamicsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͺ ApoptosisǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͲ 5.3 Cyclic nucleotide signalingǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹʹ cGMPǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹʹ Natriuretic PeptidesǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹʹ Nitric OxideǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͶ PKGǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͶ PKG IǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹ
PKG IIǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹ
PKG from Plasmodium falciparumǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹ
PhosphodiesterasesǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͺ Anchoring proteinsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͳ A-kinase anchoring proteins (AKAPs)ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͳ G-kinase anchoring proteins (GKAPs)ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͳ 5.4 Compartmentation of cyclic nucleotide signaling in cardiomyocytesǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͳ Cyclic GMP compartments and related cGMP-cAMP crosstalk affecting contractility͵ͳ Regulation of cGMP compartments by PDEsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵͵
Cyclic GMP compartmentation regulating mitochondriaǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵͵
5.5 Förster resonance energy transfer (FRET) technologyǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͷ FRET-based cGMP biosensorsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵
6 Aims of the present studyǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͺ 7 Main findingsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͻ 8 Methodological considerationsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͳ Cell linesǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͳ
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In vitro FRET assayǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶʹ Left ventricular muscle stripsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶʹ Isolation of rat left ventricular cardiomyocytesǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶ͵
Confocal microscopyǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͶ Förster resonance energy transfer biosensors for cGMPǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͷ Scanning ion conductance microscopy (SICM) in hopping modeǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͻ Cytotoxicity and apoptosis measurementsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷͲ 9 General discussionǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷʹ GCs/cGMP compartmentation affecting contractilityǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷʹ GCs/cGMP compartmentation regulating mitochondrialǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷ
NPs as potential drug treatmentǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷͻ FRET-based biosensors to investigate cGMP compartmentationǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͲ Comparison between biosensorsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵
Biosensor affinity for cGMPǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵
Targeted FRET-based biosensorsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷ Biosensors dynamic rangeǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͺ Biosensors selectivity for cGMP vs. cAMPǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵
NO/sGC/cGMP compartmentǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷ 10 Main conclusionsǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ
11 Future perspectivesǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ
12 ReferencesǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͻ
4
1 Acknowledgements
The present work was carried out at the Department of Pharmacology, Institute of Clinical Medicine, University of Oslo and Oslo University Hospital. My PhD fellowship was granted by the South-Eastern Norway Regional Health Authority (Helse Sør-Øst RHF). My gratitude goes to all the people that made this thesis possible.
First to my supervisors who believed in me throughout these years. Kjetil Wessel Andressen who has been my main guide and who has taught me most of the skills needed in this study. I am thankful for your invaluable support and dedication; your great enthusiasm has not only inspired me but has also helped me to stay motivated. Thanks to Lise Román Moltzau who was an important guide for me during my PhD; I am grateful for your instantaneous willingness to help, for the encouragement and kind support, it has been a pleasure working on the mitochondria project with you. A special thanks to my co-supervisor Finn Olav Levy for awarding me the opportunity to work in his group, first as a masters student and then as PhD student. Your scientific knowledge and guidance has been invaluable me.
I am grateful to all my colleagues at the Department of Pharmacology for their help and constructive talks: Vladimir, Monica, Iwona, Selene, Marianne, Tor, Jan-Bjørn, Magnus, Cecilie, Soheil, Kurt, Halvard, Henriette, Andrea, Ornella, Kristin, Ben, Marie, Ana, Nicole and Dulasi. In particular thanks to the members of the “Failed experiments support group.”
Kristin, who has been an irreplaceable support in the lab and a kind, funny friend to me.
Henriette, thank you for the interesting discussions and priceless fun moments that we have had in and outside the lab. Ben, who has been a great professional role model to me; thanks for the deep and thought-provoking discussions along with the amazing and most memorable parties we had. Nicole and Dulasi, you both brought fresh energy, positivity and great amounts of laughter; I hope you girls always stay like this!
A huge thank you to Andrea who was my first guide during my masters, you have been the kindest and the most patient person with me, I would not have survived in the lab without your help! I am enormously thankful to Iwona, your contribution in the lab was priceless and your personal support has been irreplaceable!
A special thanks to Ornella who was my point of reference! You have been the best in understanding me and advising me when I was at my worst. But you have especially brought
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me joy and so much laughter in these years that made me feel at home! Can’t wait till you’re back -
I cannot express enough how thankful I am to Marie who has been by my side since the start of this journey! Thanks for making these years the most enjoyable, thank you for the stupid jokes, for the parties, for understanding my italian-english words when nobody else could, and mostly thanks for your never ending support, you pushed me forward when I couldn’t do it alone!
Thanks to my friends in Oslo who reminded me that research was not the only thing; among them Lila, Meredith, the sweet Franci and “dulcis in fundo” Alessandra, thanks for your empathy, you have been a wise, fun and trustful friend!
I would also like to thank Nicolas who has been my rock, especially during the hardest times and throughout my many ups and downs, you have always motivated me and this was invaluable towards the end of my PhD!
My deepest gratitude and love goes to my family, who made me achieve all my goals!
Grazie mamma papà e Calos, per avermi aiutato a inseguire i miei obiettivi e per avermi supportato nonostante la distanza, e a volte la mia assenza. Un mega grazie alla mia sorellona, senza di te non riuscirei a superare ogni minimo ostacolo! Sei stata ciò che piu mi e mancato in questi anni. Vi voglio bene!
Gaia Calamera Oslo, May 2020
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2 Abbreviations
5-HT 5-Hydroxytryptamine
ABCB ATP-binding cassette transporter, subfamily B
ACE Angiotensin-converting-enzyme
ADP Adenosine diphosphate
AFP Aequorea victoria-derived fluorescent proteins
AKAP A-kinase anchoring proteins
ANP Atrial natriuretic peptide
ATP Adenosine 5'-triphosphate
BNP Brain natriuretic peptide
CaMKI-II Ca2+/calmodulin-dependent protein kinase I or II
CFP Cyan fluorescent protein
cGMP Cyclic guanosine 3’,5’-monophosphate
CL Cardiolipin
CM Cardiomyocyte
CN Cyclic nucleotide
CNBD Cyclic nucleotide binding domain CNGC Cyclic nucleotide gated ion channel
CNP C-type natriuretic peptide
co-IP Co-immunoprecipitation
COX Cytochrome c oxidase
cpEGFP Circularly permuted enhanced GFP
CRC Contraction-relaxation cycle
DAF-FM DA Diaminofluorescein-FM diacetate DAPI 4′,6-diamidino-2-phenylindole DD domains Dimerization/docking-domain
DEA-NONOate Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate DISC Death-inducing signaling complexes
EC50 Half maximal effective concentration ECFP Enhanced cyan fluorescent protein EHNA Erythro-9-(2-hydroxy-3-nonyl)adenine
FAD Flavin adenine dinucleotide
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FMN Flavin mononucleotide
FP Fluorescent protein
FRET Fluorescence resonance energy transfer
GAF cGMP-binding PDEs, Anabaena adenylyl cyclase and E. coli Fh1A
GC Guanylyl cyclase
GED GTPase effector domain
GFP Green fluorescent protein
GKAP G kinase anchoring proteins GSK-3β Glycogen synthase kinase 3 beta
GSNO S-Nitrosoglutathione
GTP Guanosine triphosphate
GTPase Guanosine triphosphate hydrolase enzymes HAP1 Huntingtin associated protein 1
HEK293 cells Human embryonic kidney 293 cells
HF Heart failure
HFpEF Heart failure with preserved ejection fraction HFrEF Heart failure with reduced ejection fraction
IBMX 3-isobutyl-1-methylxanthine
ICa,L L-type calcium current
IMM Inner mitochondrial membrane
IMS Intermembrane space
IP3 Inositol trisphosphate or 1,4,5-trisphosphate
IPC Ischemic preconditioning
IR injury Ischemia-reperfusion injury
IRAG Inositol trisphosphate receptor-associated cGMP-kinase substrate
LDH Lactate dehydrogenase
LR Lusitropic response
LTCC L-type calcium channel
MCU Mitochondrial calcium uniport channel
mitoKATP Mitochondrial adenosine triphosphate-dependent potassium channels
MOI Multiplicity of infection
MPTP Mitochondrial permeability transition pore MRP Multidrug resistance-associated proteins
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NAD Nicotinamide adenine dinucleotide
NADH Nicotinamide adenine dinucleotide +hydrogen
NADPH Reduced form of Nicotinamide adenine dinucleotide phosphate
NCX Na+/Ca2+ exchanger
NEP Neutral endopeptidase
NHE Sodium-hydrogen exchanger
NIR Negative inotropic response
NO Nitric oxide
NOC7 3-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine
NOS Nitric oxide synthase
NP Natriuretic peptide
ODQ 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
OMM Outer mitochondrial membrane
OPA-1 Optic atrophy1
PAPA NONOate (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate
PARP Poly ADP-ribose polymerase
PDE Phosphodiesterase
PfPKG Plasmodium falciparium PKG
PGC-1α Peroxisome proliferator-activated receptor-gamma coactivator
PGE1 Prostaglandin E1
PKA cAMP-dependent protein kinase
PKG cGMP-dependent protein kinase, cyclic GMP kinase (cGK)
PLB Phospholamban
PPARδ Peroxisome proliferator-activated receptor delta PROLI 1-(hydroxy-NNO-azoxy)-L-proline, disodium salt
ROS Reactive oxygen species
SERCA Sarcoplasmic reticulum Ca2+-ATPase SICM Scanning ion conductance microscopy SHAM Normal animal operated as control SNAP S-Nitroso-N-acetylpenicillamine
SR Sarcoplasmic reticulum
TAC Transverse aortic constriction TEV protease Tobacco Etch Virus protease
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TnI Troponin I
TRPC Transient receptor potential cation channels
TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
YFP Yellow fluorescent protein
β-AR Beta adrenergic receptor
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3 Preface
The research group of Finn Olav Levy focuses on intracellular signaling in the heart. Previously the group worked on cAMP signaling and found evidences on different compartmentation of cAMP between the β-AR stimulation and the 5-HT4 serotonin receptorstimulation ,which both increase cAMP but have different downstream effects. Similarly, cGMP signaling seems to compartmentalize and while BNP showed no effect on contractility, CNP was found to cause a negative inotropic response and a lusitropic response, despite both increasing cGMP levels.
Our aim is to uncover the intracellular mechanisms behind downstream effects such as contractility and we want to investigate cGMP compartmentation by applying targeted FRET biosensors. This will allow us to link cGMP increases in specific compartments with functional responses. In Paper I we clarify the CNP/cGMP functional compartment in adult cardiomyocytes. Using FRET biosensors targeted in the sarcoplasmic reticulum and myofilaments, we measure cGMP increase after receptor stimulation and evaluate the role of PDEs by stimulating with selective PDE inhibitors. We also show receptor localization at the cell surface by applying SICM technology together with FRET. To do so, I had the opportunity to visit VO Nikolaev`s lab at the University of Hamburg, where they already established such method.
Although new tools for real-time monitoring of cGMP are available and allowed us to understand the role of cGMP compartmentation, such studies remain challenging. It requires more effort in the development of better techniques and better biosensors. Therefore we constructed improved FRET biosensors for cGMP in Paper II and present a new high affinity- high dynamic range FRET biosensor that can be used in different cell types.
In Paper III we show the application of yet another novel FRET biosensor in a less known cGMP compartment: the mitochondria. We first elucidated NPs effects on cardioprotection, their role on mitochondria and then measured cGMP by mitochondria targeted FRET biosensors.
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4 List of papers included
Paper I
CNP regulates cardiac contractility and increases cGMP near both SERCA and TnI- difference from BNP visualized by targeted cGMP biosensors.
Ornella Manfra, Gaia Calamera, Alexander Froese, Nicoletta C. Surdo, Silja Meier, Monica Aasrum,Viacheslav O. Nikolaev, Manuela Zaccolo,Lise Román Moltzau, Finn Olav Levy and Kjetil Wessel Andressen. Submitted manuscript
Paper II
New FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons.
Gaia Calamera, Dan Li, Andrea Hembre Ulsund, Jeong Joo Kim, Oliver C. Neely, Lise Román Moltzau, Marianne Bjørnerem, David Paterson, Choel Kim, Finn Olav Levy, Kjetil Wessel Andressen. Commun Biol. 2:394, 2019
Paper III
Natriuretic peptides protect against apoptosis and increase cGMP around cardiomyocyte mitochondria.
Gaia Calamera, Bernadin Dongmo Ndongson, Jeong Joo Kim, Dulasi Arunthavarajah, Choel Kim, Kjetil Wessel Andressen and Lise Román Moltzau. Manuscript
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5 Introduction
5.1 The heart
The heart is a muscular organ that pumps blood into the circulatory system. It is organized in four chambers: the left atria and left ventricle receive oxygenated blood from the pulmonary circulation and pump it into the systemic circulation; the right chambers receive the blood from the systemic veins and pump it into the pulmonary circulation where blood is oxygenated. The heart tissue is formed of cardiomyocytes (CMs), endothelial cells (ECs), fibroblast (FBs) and peri-vascular cells1. The present study focus on cardiomyocytes which are contractile cells, mainly responsible for the contraction of the heart.
The contractile machinery in the heart
The excitation-contraction coupling is an important mechanism where the production of an electrical impulse, defined as an action potential, is translated into muscle contraction and followed by relaxation. The efficiency of this machinery is in part granted by the highly organized structures of the cell, confined by the sarcolemma. The sarcolemma is crucial in forming the cell-cell junctions (intercalated disk), which guarantee the synchronized contraction throughout the cardiac tissue. In addition, the sarcolemma forms invaginations at the Z-line, called T-tubules, with transversal and longitudinal orientation (Figure 1). These structures are important for conduction of membrane potential trough the cell and are close to the cellular region where the mechanical structures contract and relax, and several ion channels are localized there2.
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Figure 1. Cardiac muscle anatomy. a) Photomicrograph of myocardial tissue. b) Cardiac muscle fibers with the intercalated discs components and cardiomyocyte intracellular organization. From slideplayer.com
The contractile structures are composed of myofilaments, myosin and actin, and organized in units called sarcomeres. Along the myofilaments, actin monomers are localized with tropomyosin and the complex of troponin T, troponin I and troponin C (TnT, TnI and TnC, respectively)3. Depolarization of cardiomyocytes triggers L-type Ca2+ channels (LTCC) opening and influx of intracellular Ca2+, which in turn triggers the opening of ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR) for even more Ca2+ release into the cytosol.
Intracellular Ca2+ binds to TnC, leading to its conformational change and binding to TnI; this in turn moves its inhibitory domain away from actin and promotes the displacement of tropomyosin, so that the actin surface is free to bind the myosin heads. These changes in conformation all together lead to formation of cross-bridges along the myosin and actin myofilament. However, the transduction of the chemical stimulus in mechanical work occurs also thanks to the ATPase function at the myosin heads. When the cross-bridges are formed, ATP binding to myosin and its hydrolysis into ADP+Pi with release of Pi, brings the myosin heads to produce the power stroke, and the myofilaments will slide past each other. This triggers muscle shortening and ultimately to contraction of the heart, called systole3. After the contraction, the cytosolic Ca2+ levels drop, due to the activation of the Na+/Ca2+ exchanger (NCX), the sarcolemmal ATPase (PMCA), the mitochondrial Ca2+ uniporter (MCU) and
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sarcoplasmatic reticulum Ca2+ ATPase (SERCA). Subsequently, calcium bound to TnC is released, and TnI and tropomyosin are back in a closed state that prevent actin to interact with myosin, thus, relaxation (heart diastole) can occur (Figure 2)4.
Figure 2. Excitation-contraction-relaxation cycle machinery. Left panel shows the excitation- contraction coupling with calcium fluxes; right panel shows the myofilaments organization and cross- bridges formation. Used with permission from4 (left) and with permission from5, copyright Massachussetts Medical Society (right).
The contraction-relaxation cycle (CRC), as described above, can be modulated by stimuli which can produce an inotropic response, positive or negative (PIR and NIR respectively), affecting the contractility, and some stimuli that can trigger a lusitropic response (LR), accelerating the relaxation. These stimuli involve modulation, mainly by phosphorylation, of contractile proteins. The proteins involved in CRC modulation that are particularly relevant for this thesis are TnI and phospholamban (PLB).
TnI was first described in 1973 by Greaser and Gergely as a 24kDa protein with inhibitory effect on actin-myosin interaction6. As mentioned before, TnI is part of a complex together with TnC and TnT. As shown by Taked et al. this complex is highly flexible, and binding of Ca2+ to TnC moves away the C-terminus of TnI from actin, thereby abolishing its inhibitory function7. Phosphorylation of TnI seems to affect the kinetics of the conformational changes that occur within the troponins complex8, 9 and it is reported that different protein kinases can phosphorylate several TnI sites10. While the effects of some of these phosphorylations need still to be elucidated, studies on intracellular signaling and contractility
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show that TnI represent an important element for CRC modulation11. Hence, phosphorylation of TnI at Ser23/24 by PKA or PKG can decrease the Ca2+ sensitivity of the myofilaments and increase the Ca2+ removal rate from TnC, thus leading to an accelerated relaxation or positive LR12, 13. Further in this thesis, we explain how cGMP mediated signaling can modulate TnI (see “Cyclic GMP contractility compartments and related cGMP-cAMP crosstalk affecting contractility”), and in Paper I we measure cGMP increase around the troponins complex by linking the TnI protein sequence to a FRET-based biosensor for cGMP.
PLB is a 24-27 kDa protein that acts as an inhibitor of SERCA preventing Ca2+ flux in the SR14-17. β-adrenergic stimulation is found to induce PLB phosphorylation through the AKAP18δ /PKA/PLB complex18, 19. Phosphorylation of PLB relieves the inhibitory action on SERCA, thus allowing activation of the SERCA pump and cytosolic Ca2+ flux into the SR 20,
21. Therefore, phosphorylation of PLB is also thought to be contribute to a LR. Phosphorylation of PLB can be mediated also by PKG22, 23 and to further study the cGMP signaling in the SR compartment, we constructed a FRET-based biosensor that measure cGMP around SERCA by using AKAP18δ as targeting.
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5.2 Mitochondria
Mitochondria are organelles with cylindric-like shape and diameter of 0.5-1μm, found in eukaryotic organisms24. Mitochondria are mainly involved in cellular metabolism, but extensive research has elucidated other important roles in cell differentiation, motility, intracellular Ca2+ signaling and apoptosis25-27. In the heart, the excitation-contraction coupling requires high energy supply in form of ATP, which is mainly provided by the mitochondria through the electron transport chain. Moreover, heart failure (HF) is associated with energy unbalance, changes in the substrate source of energy and oxidative stress, suggesting an important role of mitochondria in cardiovascular pathophysiology28-30.
In adult cardiac cells approximately 30% of the volume is occupied by mitochondria.
These are tightly organized in a complex cytoarchitecture and can be classified as 1) perinuclear 2) subsarcolemmal and 3) intermyofibrillar (Figure 3)31. This classification reflects a difference in the function and in the morphology. In particular, the intermyofibrillar mitochondria seem to locate between sarcomeres and are in contact with myofibrils and SR, thus they are mainly involved in the energy supply of myosin and SERCA. The subsarcolemmal mitochondria are less organized and they might be more involved in ions homeostasis, while the perinuclear mitochondria form clusters that probably act in transcriptional and translational signaling32-34.
Figure 3. Electron micrograph of adult murine heart. SSM: subsarcolemmal mitochondria; IF:
intermyofibrillar mitochondria; PN: perinuclear mitochondria. Used with permission from35.
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Structure
The mitochondrion structure (Figure 4) includes two phospholipid bilayers: the outer mitochondrial membrane (OMM), which delimits the organelle, and the inner mitochondrial membrane (IMM) which faces the mitochondrial matrix. Between the two membranes there is an intermembrane space (IMS, with about 20 nm gap). This space has membrane invaginations, called cristae, which make up a third mitochondrial compartment where cytochrome c can accumulate, and the electron transport chain takes place36, 37. Mitochondrial membranes contain lipids, such as cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidic acid (PA) and diacylglycerol (DAG) which are regulated by phosphatases, phospholipases, and acyltransferases38, 39.
Figure 4. Mitochondria structure. Used with permission from40.
The OMM is permeable to proteins less than 5 kDa and due to its porosity, there is no membrane potential. However, it contains voltage-dependent anion channels (VDACs/porines)41, 42 and two main translocation and insertion pore complexes (translocase of the outer membrane, TOM43,44, 45 and the sorting and assembly machinery of the outer membrane, SAM46). The intramembrane space function involves bioenergetics processes, transfer of metabolites and regulation of apoptosis. The IMM is less permeable due to its membrane potential (180 mV) that limits the diffusion of ions47. It comprises a series of transporters such as those carrying out the oxidation products and ATP and allows the transport
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of metabolites. The mitochondrial permeability transition pore (MPTP) is the channel responsible for the change in the IMM permeability, and while they are normally closed, in pathological situations, such as cytosolic Ca2+ overload, Ca2+ accumulate in the matrix leading to pore opening48. This opening reduces the membrane potential by pumping Ca2+ out from the matrix, gives uncoupling of the oxidative phosphorylation and reduced ATP production that mediates apoptosis49, 50. Their opening is also associated with cytochrome c release, OMM disruption and matrix swelling51-53. MPTP is a focus of research for potential treatments against cardiovascular diseases and ischemia-reperfusion (IR) injury54, 55. In fact, during ischemic events oxygen levels are low and this cause reduced intracellular ATP and pH which consequently increase in Ca2+ levels and thus ROS production, MPTP opening and mitochondrial dysfunction50, 56.
Mitochondrial dynamics
Mitochondrial dynamics include the ability of these organelles to move inside the cell, ensuring the energy supply distribution, and the changes in their morphology by fusion and fission processes (Figure 5). Both fusion and fission are important mechanisms to meet cell metabolic requests and respond to stress conditions, as well as maintaining the correct distribution and integrity of the mtDNA31, 57. A common view is that the fusion process is related to active metabolic cells and that fragmented mitochondria are found in quiescent cells57-59, but this is not a fixed rule since cardiomyocytes that are metabolically active contain a fragmented mitochondrial network33, 35.
The fission proteins include Drp1 (dynamin-related peptide 1)60, 61 and Fis1 (mitochondrial fission protein 1)62. Drp1 is a cytosolic protein that has a GTPase activity, a GED domain (GTPase effector domain) and a central domain; it is recruited from the cytosol to the OMM, where it oligomerize and form scission sites in the mitochondria. Drp1 binds the OMM trough the Fis1 protein, which is in the OMM transmembrane and face the IMS63. The function of Drp1 can be modulated through sumoylation, ubiquitination, S-nitrosylation or phosphorylation/dephosphorylation. It has been suggested that the Drp1 phosphorylation by CaMKIα and CaMKII induce fission64, 65, similarly to calcineurin dephosphorylation66, 67, while phosphorylation by PKA or PKG can result in opposite effects66, 68, 69.
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Fusion of mitochondria is regulated by proteins in the OMM like Mitofusin (Mfn) 1 and 270 and Opa1 (optic atrophy1)71 in the IMM. Mfn1 and 2 are transmembrane proteins of the OMM with GTPase activity and mediate the fusion of OMM by forming dimers of Mitofusins from two adjacent mitochondria. Opa1 is a GTPase protein of the dynamin family and it is transmembrane in the IMM; like the Mfns, it binds another Opa1 promoting the fusion of two different mitochondrial membranes72, 73.
Alteration of the mitochondria morphology due to abnormal regulation of the proteins involved, might be sign of cell dysfunction and apoptotic cell death74-76. HF has been associated with an increased number of mitochondria, suggesting the activation of the fission process, as well as Drp1 translocation to the mitochondria with fragmentation, during ischemia35. There are more and more evidences that mitochondrial dynamics are very important processes in adult cardiomyocytes77, 78 and Beraud N. et al. described mitochondria dynamics in adult cardiac cells as very low amplitude-high frequency fluctuations79. It appears overall, that mitochondrial dynamics in adult cardiomyocytes are important when the heart requires a new turnover of mitochondria, for example during ischemia or pressure overload31, 35.
Figure 5. Mitochondrial dynamics. A shows the fusion mechanisms involving Mitofusins (Mfns) and OPA-1. B shows the fission mechanism where phosphorylation of Drp1 causes its recruitment to the OMM and through Fis-1, induces mitochondrial fragmentation. Figure from80.
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Apoptosis
Cellular homeostasis is based on proliferation/differentiation processes counteracted by cell death mechanisms. Even though cell death is physiologically useful, it has been linked to many cardiac diseases such as reperfusion injury, heart failure and ischemic heart pathologies 81,
82.The Nomenclature Committee on Cell Death (NCCD) defines different mechanisms of cell death based on the signaling that is involved. Apoptosis is regulated by caspase signaling;
autophagy is modulated by lipidation of microtubule-associated protein light chain 3 and degradation of sequestosome 1, and necroptosis is a form of necrosis linked to receptor- interacting protein kinase 1 and 383-85.
Apoptosis can be regulated by an extrinsic pathway or an intrinsic pathway (Figure 6).
The extrinsic pathway is triggered by extracellular inputs like TNF-α (Tumor necrosis factor alpha), FasL (Fas ligand), TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) which bind to specific cell surface receptors. These receptors contain death domains which allow protein-protein interaction and formation of death-inducing signaling complexes (DISC).
The intrinsic pathway, or mitochondrial pathway, is instead initiated by intracellular events such as DNA damage, oxidative stress or calcium overload86. Even though these pathways have different stimuli they can have similar signaling steps and “interconnect” to each other (Figure 6). The main regulators of apoptosis are cysteine proteases called caspases. These enzymes cleave substrates C-terminally of aspartic acid residues and are themselves activated after cleavage, thus they function within a cascade process of several caspases87, 88. The initiator caspases possess death effector domains (caspase 8 and 10) or recruitment domains (caspase 2); these interact with other proteins that help for the auto proteolysis. These initiators cleave the effector caspases (caspase 3, 6 and 7) which will start a proteolytic cascade. In the extrinsic pathway the DISC allows the activation of procaspase 8 to caspase 8 that cleaves procaspase 3 and Bid (BH3 interacting-domain death agonist). Once Bid is cleaved, it can be inserted in the OMM and recruit Bax and Bak (pro-apoptotic proteins of the Bcl-2 family) to stimulate mitochondrial response to apoptosis86. The intrinsic pathway instead leads to cytochrome c release from mitochondria in the cytosol, where, together with the adapter Apaf-1, forms a complex (apoptosome) that activate procaspase 9. Caspase 9 in turn activates procaspase 389 (Figure 6). The ultimate step for both pathways is the cleavage of nuclear PARP (Poly ADP- ribose polymerase) by caspase 3. This cleavage inactivates PARP which normally induces DNA repair, and this leads to apoptosis90.
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The consequences of apoptosis are chromatin condensation in nuclear bodies, cytoplasm condensation, and cell shrinkage. Hence, cells undergo fragmentation and the apoptotic bodies will be phagocytized by other cells. During apoptosis endonucleases cleave the DNA, generating double stranded fragments. In order to detect apoptotic cells, the TUNEL assay can be used; here, fluorescently labeled nucleotides are added to the 3`-OH termini in single stranded breaks of DNA fragments by terminal deoxynucleotidyl transferase (TdT). In combination with a nuclear staining (like DAPI) it is possible to determine the number of apoptotic cells relative to the total number of cells91.
Figure 6. Extrinsic and intrinsic apoptotic pathways. Schematic of the apoptotic signaling showing how the extrinsic (left) and the intrinsic (right) pathway can be differently triggered and which steps are in common. Figure from92.
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5.3 Cyclic nucleotide signaling
cGMP
Cyclic guanosine 3’,5’-monophosphate (cGMP) is a second messenger discovered about 60 years ago in rat urine93. The cGMP pathway can regulate the cardiovascular system by mediating vasodilation, natriuresis, antifibrotic and antihypertrophic effects and can modulate cardiac contractility94-97. Moreover, augmentations of cGMP levels have been shown to be beneficial in pathologies like heart failure98-100 and pulmonary arterial hypertension101. Cyclic GMP is synthesized by NO-dependent activation of soluble guanylyl cyclase (sGC) and natriuretic peptide (NP)-dependent activation of particulate guanylyl cyclases102. In order to mediate its related downstream effects, cGMP activates protein kinase G (PKG), which can phosphorylate several targets, and cyclic nucleotide-gated ion channels (CNGC) and regulates phosphodiesterases (PDEs) 103, 104.
Natriuretic Peptides
Natriuretic peptides act on the renal system mediating natriuresis, diuresis, suppression of the renin-angiotensin-aldosterone system and on the vasculature mediating vasodilation105. They also modulate metabolism, promoting lipolysis and regulating mitochondrial respiration106, 107; moreover, low NP levels in the plasma are found in obesity, metabolic syndrome and diabetes type 2108-110.
There are three types of natriuretic peptides (NPs): atrial natriuretic peptide (ANP) 111, brain natriuretic peptide (BNP)112 and C-type natriuretic peptide (CNP)113. ANP is mainly produced in atria and BNP is found normally in the left ventricle, but both are released from the left ventricle during HF, resulting in high ANP and BNP plasma levels114. Consequently, they are used as biomarkers for heart failure and NT-proBNP is commonly measured for diagnosis and monitoring of the therapy115. CNP is produced in endothelial cells, rat cardiac fibroblasts and cardiomyocytes116-119; moreover, CNP expression is also abundant in the brain, chondrocytes and kidney120-122. In HF patients, increased CNP was detected in the heart and plasma123, 124.
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The receptors for NPs are called GC-A (or NPR-A), GC-B (or NPR-B) and NPR-C. Whereas the latter can bind all three NPs, GC-A binds ANP and BNP, and GC-B is selective for CNP;
however high BNP concentrations can also activate GC-B125-127. These receptors are single transmembrane receptors that contain a guanylyl cyclase in the intracellular domain of the receptor. However, NPR-C does not have GC activity and is involved in clearance of NPs and alternative signaling128, 129. The NP effects can also be terminated by the neutral endopeptidase (NEP), neprilysin130. The GC-A is expressed in vascular smooth muscle, endothelium, heart, the nervous system, adrenals and kidney, mediating effects such as decrease in arterial pressure, blood volume and inhibition of cardiomyocyte growth131. Stimulation of GC-A has been reported to prevent hypertrophy and mediate antifibrotic effects132. The GC-B is mainly distributed in vascular smooth muscle, endothelium and heart, mediating vasodilation more efficiently than GC-A, and mainly in the peripheral vasculature133; CNP can promotes anti- fibrotic effects and modulates heart contractility117, 134-136, but it also promotes bone growth and reduced CNP/GC-B signaling is found to causes dwarfism137, 138. Even though ANP and BNP levels are increased in HF, receptor density is downregulated and GC activity seem to be reduced139, 140. Therefore, augmentation of the natriuretic peptide signaling has been considered in the treatment for heart failure, either by synthesis of NP analogues, or by using neprilysin inhibitors. The ANP analogues, anaritide and carperitide, did not show hemodynamic or renal effects except in one study, however carpertide is approved in Japan only for acute HF141-143. Neseritide, the synthetic BNP analogue, was initially approved for acute decompensated HF for the natriuretic and hypotensive effects 144, 145. Subsequent studies were required to verify the absence of renal side effects, however in those studies no lasting beneficial effect was found, instead the possibility of symptomatic hypotension was increased146. A chimeric CNP analogue (centeritide) was synthetized in 2008 which can activate both NP receptors and is resistant to neprilysin, combining the renal effect with the antifibrotic response 147, 148. More promising results have been shown with neprilysin inhibitors. This class of drugs was tested alone149, 150 and in combination with ACE inhibitors151, 152. However, more successful studies were obtained by combination with angiotensin receptor blockers, and the sacubitril-valsartan combination is approved as Ernesto® for heart failure treatment 99.
24 Nitric Oxide
NO was first discovered as endothelium-derived relaxing factor, thus mediating smooth muscle relaxation, but it is also involved in neurotransmission. NO is produced by the enzyme NOS (nitric oxide synthase) which exists in three main isoforms (nNOS, eNOS and iNOS) all found in cardiomyocytes153, 154. NOSs are oxidoreductase homodimer enzymes with an oxygenase domain and a reductase domain divided by a linker that binds calcium-complexed calmodulin.
Activation of nNOS and eNOS by this complex occurs when there is an increase in calcium concentration while the iNOS binds the complex at basal Ca2+ concentration155. Nitric oxide has lipophilic properties and can therefore diffuse through membranes, acting as a paracrine signal from endothelial cells to vascular smooth muscle cells or CMs, or as an autocrine signal in CMs156. NO can signal through activation of sGC or through S-nitrosylation of proteins.
Soluble GC consists of an α(α1 or α2) and a β(β1 or β2) subunit connected by a ferrous heme and when NO binds to the heme, it activates the enzyme and cGMP is synthetized from GTP155.
PKG
Cyclic GMP-dependent protein kinases (PKGs) are homodimeric serine/threonine kinases157. In mammals, the prkg1 gene is expressed as two splice variants, PKG Iα and PKG Iβ, and the prkg2 gene is expressed as PKG II158-160. The PKG Is are expressed in smooth muscle, cardiac muscle, vascular endothelium, platelets, nerve cells and kidney. The Iα isoform, specifically, is found in vascular smooth muscle cells and in the heart. The PKG II is found mostly in kidney, intestinal mucosa, and brain nuclei161. PKGs are sensitive to nano to micromolar concentrations of cGMP and the Iβ isoform is 10-fold less sensitive than the Iα isoform due to the different N- terminal domain which is responsible for regulating the cooperativity in the cGMP-binding sites of PKG Iα104.
PKGs are holoenzymes organized with an N-terminal regulatory (R) domain and a catalytic (C) domain in a single protein (Figure 7). N-terminally there is a leucine zipper motif important for the enzyme dimerization and, by interacting with anchoring proteins (like huntingtin associated protein 1, HAP1), it dictates subcellular localization162. After this motif there is an autoinhibitory domain (AI), followed by the regulatory domain. The latest is
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characterized by two cyclic nucleotide binding domains (CNBD): CNBD-A and CNBD-B. As showed in Figure 8, in the cGMP unbound inactive state, the R and C domains in PKG I are folded. Binding of cGMP induces a conformational change that unfolds the R and C domains and thus activates the enzyme163, 164. Moreover, the binding of cGMP to the four CNBD sites is necessary for full activation of PKG164.
Figure 7. PKG Iα domain structure. D/D: docking and dimerization domain with the Leucine Zipper, which is responsible for binding to the complementary side of the homodimer; AI: auto-inhibitory domain; cGMP-A/B: cGMP binding sites A and B; SW: switch helix; red bars indicate sites of homodimerization. Reproduced with permission from 165.
Figure 8. Mechanism of PKG activation by cGMP binding to the regulatory domains. Reproduced with permission from166.
The following subparagraphs focus on the PKGs binding sites used in this thesis to develop novel FRET-based biosensors for cGMP.
26 PKG I
In the regulatory domain of PKG I, the CNBD- A and B have similar structure, but they have different affinity and selectivity (CNBD-A: EC50cGMP 12 nM and EC50cAMP 27nM; CNBD-B:
EC50cGMP 215nM and EC50cAMP 52μM)167, 168 and different cGMP binding kinetics, where the A domain displays slow disassociation and B domain displays fast disassociation169. Each binding pocket can be defined by three main parts, described here for the A domain (Figure 9):
Site 1, that captures the cyclic phosphate and interacts with the ribose by hydrogen bonds and electrostatic interactions; Site 2 is important for the selectivity and has the Thr193 residue that bridges the cyclic phosphate with the guanine ring of cGMP. Importantly, mutations of Thr193 decrease cGMP affinity169. This, together with other side chain residues, forces cGMP to bind in a syn conformation 167, 168. Site 3 involve the β5-strand where Leu172 and Cys173 dock the purine of cGMP, where the first involve non-polar interactions while the second residue interacts via hydrogen bond. These contacts can occur only with cGMP in syn conformation
167, 168.
Figure 9. cGMP and cAMP binding pockets of PKG I show cyclic nucleotide-aa residue interactions and the cGMP syn conformation (left) and the cAMP anti conformation (right). Reproduced from167.
The differences between cGMP and cAMP binding are mainly seen in the β4 and β5 strands where interaction of cAMP with Site 2 and Site 3 residues allow cAMP to arrange in an anti conformation (residues involved: Thr193, that interacts with the 2-NH group of cGMP, while it cannot interact with the -H that is in the same position in the cAMP molecule, Leu172 and
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Cys173)167, 168. Similar interactions are seen in the CNBD-B. However, while the selectivity of the CNBD-A is only two-fold, that of the B domain is 240-fold. In this domain cGMP also binds in a syn conformation, however in Site 3 there is an arginine (Arg297) instead of the cysteine (Cys173) found in the A domain. This arginine does not form Van der Waals interactions but two hydrogen bonds168. In PKA RIα and RIIβ binding pockets, the analogue residues of Leu296 and Arg297, do not interact with cAMP or show only Van der Waals interactions168, 170. Mutating the Arg297 to alanine improves cAMP affinity, suggesting that these residues in the β5 strand of the PKG I CNBD-B are important for the selectivity of PKG.
Another important contribution to the binding of cGMP in the B domain is the presence of the Tyr351 residue that interacts with the guanine moiety driving it against the Leu296 and providing a “capping” for the cGMP binding pocket 168.
PKG II
The PKG II structure was solved in 2016 and the binding affinity was measured in the two isolated domains (CNBD-A: EC50cGMP 43.8 nM and EC50cAMP 418nM; CNBD-B: EC50cGMP 31.4 nM and EC50cAMP 15.6 μM)171. PKG II has a similarity of around 50% with the PKG I protein and the main difference is at the αC-helix in the CNBD-B, which interacts with the guanine moiety through hydrogen bonds of Asp412 or Arg415, and in the β5 strand, where there is a Lys347 instead of the Arg297. While in PKG Iβ the Tyr351 serves as a cap, in PKG II the Leu408 gives a hydrophobic cap and Lys358 with the Asn409 shield the sugar phosphate through hydrogen bonds. These features create a less open pocket compared to CNBD-B of PKG I and might explain the higher affinity to cGMP171, 172.
PKG from Plasmodium falciparum
The protozoan parasite Plasmodium falciparum (Pf) causes the most severe form of malaria compared to the other four species of Plasmodium173 and thus it is the subject of several studies with the aim to find a treatment against this infection. Studying the cell reproduction cycle of Plasmodium, it was seen that an important role is covered by several protein kinases, including cyclic nucleotide-dependent protein kinases174. The reported protein sequence of PfPKG has a similarity of ~30-33% with the mammalian PKGs, showing an N-terminal regulatory domain and a C-terminal catalytic domain. The most relevant difference, compared to the mammalian
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PKGs, is in the regulatory domain. Here, the PfPKG has three cGMP-binding domains and an additional fourth degenerate cGMP-binding domain175, 176, shown as the “C” domain in Figure 10. This fourth degenerate motif shows more different amino acid sequence from the other three domains and was reported to influence the PfPKG maximal activity but not the Ka value, suggesting that it is probably not binding cGMP but is rather important for a full activation of the enzyme176. On the other hand, the third cGMP binding domain (domain D in Figure 10) has an affinity of 37.8 nM for cGMP177 and seems to have a crucial role in binding the cyclic nucleotide and therefore in the enzyme activity; this reveals that the more C-terminal binding domains are more relevant for the PfPKG function compared to the mammalians PKGs, where the N-terminal binding domain has a higher affinity for cGMP176.
Figure 10. PKG from Plasmodium falciparum. The N-terminal regulatory domain includes binding domains A, B, C and D, while the catalytic domain is C-terminal. Numbers indicate amino acid sequence.
Phosphodiesterases
Phosphodiesterases (PDEs) are enzymes that degrade cyclic nucleotides (CNs), cAMP and cGMP, by hydrolysis to nucleoside phosphates (5´AMP and 5´GMP). Since these enzymes were first found, researchers have categorized 11 families with subfamilies and splice variants178, 179. Based on the different affinities and activities towards cAMP and cGMP they can be classified as PDE1, 2, 3, 10 and 11 that hydrolyze both cAMP and cGMP, PDE4, 7 and 8 that are selective for cAMP and PDE5, 6 and 9 that are selective for cGMP. PDE1, 2, 3, 4, 5, 8, 9 and 10 are expressed in the heart180, 181. PDEs contain a conserved catalytic domain and a variable N-terminal domain. The catalytic site binds the CNs or the PDE inhibitors, and differences in single amino acids at this binding site allow development of specific inhibitors for PDE isoforms. The N-terminal region, instead, determine functional features and
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localization of the enzyme180.Since the focus of this thesis is around cGMP, the cGMP- degrading PDEs that are expressed in the heart are described below.
PDE1. PDE1A and 1B catalyze cGMP degradation with higher Km than for cAMP, while 1C has equal affinity182, 183. The regulatory domain includes two binding sites for Ca2+/CaM which activate the enzyme and a phosphorylation site, that can be regulated by PKA or CaMKII ,which reduce the sensitivity to Ca2+/CaM for 1A and 1B respectively184-186. PDE1 can be inhibited by the following: vinpocetine187, nimodipine188, IC224189, the novel Lu AF41228 and Lu AF58027190.
PDE2. PDE2 has three splice variants PDE2A1-3. The regulatory region contains two GAF (cGMP-binding PDEs, Anabaena adenylyl cyclase and Escherichia coli Fh1A) domains, A and B191. PDE2 has similar Vmax and Km for cAMP and cGMP but it has been reported that cGMP can enhance cAMP degradation by binding to the GAF-B domain192. Thus, this PDE is a key factor in the cAMP/cGMP cross-talk signaling193. Commonly used PDE2 inhibitors are BAY 60-7550194 and EHNA195.
PDE3. PDE3 has two isoforms 3A and 3B. PDE3 is also an important key factor in the cAMP/cGMP cross-talk signaling; in fact, PDE3 has a similar Km for cAMP and cGMP but the Vmax is 10-fold higher for cAMP, hence, cGMP binding inhibits cAMP degradation by PDE3196. PDE3 inhibitors, such as cilostamide, are used for their positive inotropic effects197. Other PDE3 inhibitors have been studied for chronic heart failure treatment, like milrinone, amrinone, enoximone and cilostazol; however the initial promising results failed in the clinical studies198, 199. Nevertheless, they could be beneficial in acute treatments for example during cardiac surgery or in the cases where the β-agonists did not show improvements200, 201.
PDE5. PDE5 has three isoforms. The regulatory domain contains GAF domains and a phosphorylation site for PKG, here PKG phosphorylation can increase the enzymatic activity and thus cGMP degradation202. In normal adult cardiomyocytes PDE5 is expressed at low levels and localized to the Z-disc203 while its expression is increased in heart failure with a more diffuse localization204-208. Different PDE5 inhibitors are currently available, among them sildenafil, tadalafil and vardenafil. They are mostly used for erectile dysfunction and pulmonary arterial hypertension209 and they also showed beneficial effect in heart failure with reduced ejection fraction (HFrEF) treatment, but failed to show clinical benefits in heart failure with preserved ejection fraction (HFpEF)210-214.
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PDE9. PDE9A is the PDE with the lowest Km for cGMP and it was recently discovered to be expressed in the heart, localized to the SR and regulating cGMP from NP stimulations180, 215. The expression in the heart is low, but it was upregulated in human left ventricular biopsies from HFpEF patients, opening the possibility of using PDE9 inhibitors for HFpEF treatment.
Beneficial effect have also been shown against hypertrophy and fibrosis, which are found in HFpEF215, 216. Few PDE9 inhibitors have been developed: BAY 73-6691, which showed improvements in learning and memory processes217 and PF-04447943218 which showed beneficial effects on heart failure215.
Anchoring proteins
CNs signaling is spatially and temporally restricted by organized complexes called signalosomes where anchoring proteins are involved. While PDEs regulate CNs signaling in terms of duration, amplitude, spatial extent and cross-talk, the anchoring proteins function to tether the regulator and the effector, and sometimes also tether those to a specific location.
A-kinase anchoring proteins (AKAPs)
Currently more than 50 different A-kinase anchoring proteins (AKAPs) are known and around 15 are expressed in the heart; these have a common domain that can bind the R-subunit of the cAMP-dependent protein kinase A (PKA)219. In paper I we use AKAP18δ to target our biosensors to the SR compartment; here, AKAP18δ is found to form a complex with SERCA, PLB and PKA RII, which is important for regulation of cardiac contractility220. In Paper III, we use part of AKAP1 (aa 1-30) to target a different biosensor to the OMM. Previously, others have developed a cAMP sensor tagged in the mitochondria using the same tag sequence221. The full length AKAP1 contains the transmembrane N terminus sequence which targets the protein to the OMM or the ER, depending on the splicing variant, and the C-terminus facing the cytosol222-224. AKAP1 can bind either PKA I or PKA II promoting Drp1 phosphorylation leading to mitochondria elongation225; other studies where AKAP1 localization signal or PKA binding is lost show apoptosis226, 227. In cardiomyocytes, AKAP1 is found to interact with the calcium-responsive phosphatase calcineurin (CaN) which mediates Drp1 dephosphorylation and mitochondrial fission67, 228.
