Targeted disruption of PDE3B, but not PDE3A, protects PNAS PLUS murine heart from ischemia/reperfusion injury Youn Wook Chunga,b,1,2, Claudia Lagranhac,1, Yong Chend, Junhui Sunc, Guang Tongc,e, Steven C. Hockmana, Faiyaz Ahmada, Shervin G. Esfahanif, Dahae H. Baef, Nazari Polidovitchg, Jian Wug, Dong Keun Rheea, Beom Seob Leeb,h, Marjan Gucekd, Mathew P. Danielsf, Christine A. Brantnerf, Peter H. Backxg,i, Elizabeth Murphyc, and Vincent C. Manganielloa,2 aCardiovascular and Pulmonary Branch, cSystems Biology Center, dProteomics Core Facility, and fElectron Microscopy Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; bYonsei Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul 120-752, Korea; eDepartment of Cardiovascular Surgery, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong 510010, China; gDepartments of Physiology and Medicine, University of Toronto, Toronto, ON M5S 3A8, Canada; hGraduate Program in Science for Aging, Yonsei University, Seoul 120-752, Korea; and iDivision of Cardiology, University Health Network, Toronto, ON M5S 3E2, Canada Edited by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, and approved March 13, 2015 (received for review August 29, 2014) Although inhibition of cyclic nucleotide phosphodiesterase type 3 PDE3 inhibitors, e.g., milrinone, are thought to enhance (PDE3) has been reported to protect rodent heart against ischemia/ myocardial inotropic responses via cAMP/PKA regulation of + reperfusion (I/R) injury, neither the specific PDE3 isoform involved Ca2 cycling in the sarcoplasmic reticulum (SR) (1, 5). The nor the underlying mechanisms have been identified. Targeted PDE3 inhibitor cilostazol (6–9) and the PDE5 inhibitor sildenafil disruption of PDE3 subfamily B (PDE3B), but not of PDE3 sub- (10, 11) have been reported to protect hearts against ischemia/ family A (PDE3A), protected mouse heart from I/R injury in vivo reperfusion (I/R) injury in various species. Fukasawa et al. (8) and in vitro, with reduced infarct size and improved cardiac − − have suggested that cilostazol exerts its cardioprotective effect by function. The cardioprotective effect in PDE3B / heart was re- 2+ + activating mitochondrial Ca -activated K (mitoKCa) channels, versed by blocking cAMP-dependent PKA and by paxilline, an in- whose opening protects hearts against infarction (12). Further- hibitor of mitochondrial calcium-activated K channels, the opening more, studies have shown that the opening of mitoKCa channels of which is potentiated by cAMP/PKA signaling. Compared with WT is potentiated by cAMP-dependent PKA signaling (13), whereas mitochondria, PDE3B−/− mitochondria were enriched in antiapop- + PKC potentiates mitochondrial ATP-sensitive K (mitoKATP) totic Bcl-2, produced less reactive oxygen species, and more fre- channel activation (14). Kukreja and his associates have sug- quently contacted transverse tubules where PDE3B was localized − − gested that the cardioprotective effects of sildenafil are mediated with caveolin-3. Moreover, a PDE3B / mitochondrial fraction con- + by activation of both mitoK (10) and mitoK channels (11). taining connexin-43 and caveolin-3 was more resistant to Ca2 - ATP Ca Ischemic preconditioning (PreC), a process in which brief in- induced opening of the mitochondrial permeability transition pore. − − termittent episodes of ischemia and reperfusion protect the heart Proteomics analyses indicated that PDE3B / heart mitochondria from subsequent prolonged ischemic injury (15), initiates a number fractions were enriched in buoyant ischemia-induced caveolin-3– enriched fractions (ICEFs) containing cardioprotective proteins. Ac- of cardioprotective signaling pathways at the plasma membrane, which are transduced to mitochondria (16). According to the “sig- cumulation of proteins into ICEFs was PKA dependent and was ” achieved by ischemic preconditioning or treatment of WT heart nalosome hypothesis, cardioprotective [e.g., G protein-coupled with the PDE3 inhibitor cilostamide. Taken together, these find- ings indicate that PDE3B deletion confers cardioprotective effects Significance because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in By catalyzing the destruction of cAMP and cGMP, cyclic nucleo- ICEFs. To our knowledge, our study is the first to define a role for tide phosphodiesterases (PDEs) regulate their intracellular con- PDE3B in cardioprotection against I/R injury and suggests PDE3B as a centrations and biological actions. Eleven distinct gene families target for cardiovascular therapies. (PDE1–PDE11) define the PDE superfamily. Most families contain several PDE genes. Two separate but related genes generate − − PDE3B / mice | protein kinase A | ischemia/reperfusion injury | PDE3 subfamilies PDE3A and PDE3B. Although inhibition of PDE3 signalosome | membrane repair protects rodent heart against ischemia/reperfusion (I/R) injury, the specific PDE3 isoform involved is undetermined. Using he two cyclic nucleotide phosphodiesterase type 3 (PDE3) PDE3A- and PDE3B-KO mice, we report that deletion of PDE3B, PHYSIOLOGY Tsubfamilies PDE3A and PDE3B are products of separate but but not PDE3A, protected mouse heart from I/R injury in vivo homologous genes. PDE3 isoforms hydrolyze both cAMP and cGMP and in vitro, via cAMP-induced preconditioning. To our knowl- edge, our study is the first to define a role for PDE3B in car- with high affinity (K <1 μM) in a mutually competitive manner and m dioprotection against I/R injury and suggests PDE3B as a target are important regulators of cyclic nucleotide signaling pathways for cardiovascular therapies. and responses in cardiomyocytes and vascular smooth muscle (1). PDE3A and PDE3B exhibit different patterns of expression. PDE3A Author contributions: Y.W.C., C.L., and V.C.M. designed research; Y.W.C., C.L., Y.C., J.S., G.T., is more abundant in platelets, airway and vascular smooth muscle, F.A., S.G.E., D.H.B., N.P., J.W., and B.S.L. performed research; S.C.H., D.K.R., M.G., and M.P.D. contributed new reagents/analytic tools; Y.W.C., Y.C., J.S., G.T., F.A., S.G.E., D.H.B., N.P., J.W., and cardiovascular tissues, whereas PDE3B is relatively more highly M.P.D., C.A.B., P.H.B., E.M., and V.C.M. analyzed data; and Y.W.C. and V.C.M. wrote expressed in tissues that are important in regulating energy metab- the paper. olism, including liver, pancreatic β cells, brown adipose tissue (BAT), The authors declare no conflict of interest. and white adipose tissue (WAT) (2). Little is known about their This article is a PNAS Direct Submission. 1 differential localization and functions when PDE3A and PDE3B are Y.W.C. and C.L. contributed equally to this work. 2To whom correspondence may be addressed. Email: [email protected] or manganiv@ presentinthesamecell.TogainfurtherinsightintospecificPDE3A nhlbi.nih.gov. and PDE3B functions in physiological contexts, we have generated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. −/− −/− and studied PDE3A and PDE3B mice (3, 4). 1073/pnas.1416230112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1416230112 PNAS | Published online April 15, 2015 | E2253–E2262 Downloaded by guest on September 23, 2021 receptor (GPCR)-induced or ouabain-induced] signals are de- A SvJ129 WT PDE3B KO C57BL/6J WT PDE3A KO livered to mitochondria by specialized caveolae-derived vesicular structures, signalosomes, which contain a wide variety of receptors (e.g., GPCRs) and signaling molecules (e.g., Akt, Src, eNOS, and PKCe) that are assembled in lipid rafts and caveolae (17). In recent years, the role of lipid rafts and caveolae in cardiovascular signaling has attracted much attention (18), and adenylyl cyclases and PDEs have emerged as key players in shaping and organizing intracellular BC 80 *** 80 signaling microdomains (19-21). 70 70 Accumulating evidence implicates the mitochondrial perme- 60 60 ability transition (MPT) pore as a key effector of cardioprotection 50 50 against I/R injury, and reperfusion-induced elevation of reactive 40 40 30 30 oxygen species (ROS) can trigger the opening of the MPT pore, 20 20 resulting in ischemic injury, apoptosis, and cell death (16). A wide 10 Zone/ Ischemic Risk 10 range of cardioprotective signaling pathways converge on glycogen 0 Area Ventricular Total (%) 0 synthase kinase-3β (GSK-3β), and its inhibition directly and/or Zone(%) Infarct/Ischemic Risk indirectly regulates MPT pore-regulatory factors (e.g., cyclophilin DE7070 7070 D and voltage-dependent anion channels) and antiapoptotic Bcl-2 6060 † 6060 family members (22). Physical association between mitochondria 50 50 50 * 50 and the endoplasmic reticulum (ER) [via mitochondria-associated 4040 *‡ 4040 ER membranes (MAMs)] (23) or the SR (24) also may reduce 3030 *†‡ *†‡ 3030 2+ 2020 2020 reperfusion-induced mitochondrial Ca overload and consequent (%) size AAR Infarct size (%) 1010 1010 oxidative stress and thus block MPT pore opening (25). 00 00 In this study, we report that, 24 h after in vivo coronary artery Milrinone - + - + - + Milrinone - + - + - + ligation, I/R or, in a Langendorff cardiac I/R model system, infarct Saline+-+-+- Saline+-+-+- −/− −/− size is reduced in PDE3B heart, but not in PDE3A heart, WT 3A KO 3B KO WT 3A KO 3B KO compared with WT heart. This protective effect is most likely + −/− −/− caused by reduced production of ROS and reduced Ca2 -induced Fig. 1. PDE3B