Inhibiting Protease-Activated Receptor 4 Limits Myocardial Ischemia/Reperfusion Injury in Rat Hearts by Unmasking Adenosine Signaling

Harnessing endogenous cardioprotectants is a novel therapeutic strategy to combat ischemia/reperfusion (I/R) injury. Thrombin causes I/R injury, whereas exogenous adenosine prevents I/R injury. We hypothesized that blocking thrombin receptor activation with a protease-activated receptor (PAR) 4 antagonist would unmask the cardioprotective effects of endogenous adenosine. The protective role of two structurally unrelated PAR4 antagonists, trans-cinnamoyl-YPGKF-amide (tc-Y-NH2) and palmitoyl-SGRRYGHALR-amide (P4pal10), were evaluated in two rat models of myocardial I/R injury. P4pal10 (10 μg/kg) treatment before ischemia significantly decreased infarct size (IS) by 31, 21, and 19% when given before, during, and after ischemia in the in vivo model. tc-Y-NH2 (5 μM) treatment before ischemia decreased IS by 51% in the in vitro model and increased recovery of ventricular function by 26%. To assess whether the cardioprotective effects of PAR4 blockade were due to endogenous adenosine, isolated hearts were treated with a nonselective adenosine receptor blocker, 8-sulfaphenyltheophylline (8-SPT), and tc-Y-NH2 before ischemia. 8-SPT abolished the protective effects of tc-Y-NH2 but did not affect IS when given alone. Adenosine-mediated survival pathways were then explored. The cardioprotective effects of tc-Y-NH2 were abolished by inhibition of Akt (wortmannin), extracellular signal-regulated kinase 1/2 [PD98059 (2′-amino-3′-methoxyflavone)], nitric-oxide synthase [NG-monomethyl-l-arginine (l-NMA)], and KATP channels (glibenclamide). PD98059, l-NMA, and glibenclamide alone had no effect on cardioprotection in vitro. Furthermore, inhibition of mitochondrial KATP channels [5-hydroxydecanoic acid (5-HD)] and sarcolemmal KATP channels (sodium (5-(2-(5-chloro-2-methoxybenzamido)ethyl)-2-methoxyphenylsulfonyl)(methylcarbamothioyl)amide; HMR 1098) abolished P4pal10-induced cardioprotection in vivo. Thrombin receptor blockade by PAR4 inhibition provides protection against injury from myocardial I/R by unmasking adenosine receptor signaling and supports the hypothesis of a coupling between thrombin receptors and adenosine receptors.

[1]  A. Hsu,et al.  SCH 79797, a selective PAR1 antagonist, limits myocardial ischemia/reperfusion injury in rat hearts , 2007, Basic Research in Cardiology.

[2]  K. Minneman,et al.  Heterodimerization and surface localization of G protein coupled receptors. , 2007, Biochemical pharmacology.

[3]  S. P. Srinivas,et al.  Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells. , 2007, Investigative ophthalmology & visual science.

[4]  A. Malik,et al.  Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization , 2007, Proceedings of the National Academy of Sciences.

[5]  R. Touyz.  Regulation of endothelial nitric oxide synthase by thrombin. , 2007, Hypertension.

[6]  M. Matsunami,et al.  [Proteinase-activated receptor]. , 2006, Nihon yakurigaku zasshi. Folia pharmacologica Japonica.

[7]  M. Hirano,et al.  Involvement of Gi/o in the PAR-4-induced NO production in endothelial cells. , 2006, Biochemical and biophysical research communications.

[8]  C. Derian,et al.  Blocking the Protease-Activated Receptor 1-4 Heterodimer in Platelet-Mediated Thrombosis , 2006, Circulation.

[9]  R. Germack,et al.  Adenosine triggers preconditioning through MEK/ERK1/2 signalling pathway during hypoxia/reoxygenation in neonatal rat cardiomyocytes. , 2005, Journal of molecular and cellular cardiology.

[10]  R. Gibbons,et al.  A randomized, double-blinded, placebo-controlled multicenter trial of adenosine as an adjunct to reperfusion in the treatment of acute myocardial infarction (AMISTAD-II). , 2005, Journal of the American College of Cardiology.

[11]  T. Wieland,et al.  Regulation of the extracellular signal-regulated kinase pathway in adult myocardium: differential roles of G(q/11), Gi and G(12/13) proteins in signalling by alpha1-adrenergic, endothelin-1 and thrombin-sensitive protease-activated receptors. , 2005, Cellular signalling.

[12]  G. Gross,et al.  Cardioprotection following adenosine kinase inhibition in rat hearts , 2005, Basic Research in Cardiology.

[13]  D. Yellon,et al.  The reperfusion injury salvage kinase pathway: a common target for both ischemic preconditioning and postconditioning. , 2005, Trends in cardiovascular medicine.

[14]  J. Downey,et al.  Acetylcholine and bradykinin trigger preconditioning in the heart through a pathway that includes Akt and NOS. , 2004, American journal of physiology. Heart and circulatory physiology.

[15]  N. Bunnett,et al.  Protease-activated receptors: contribution to physiology and disease. , 2004, Physiological reviews.

[16]  J. Downey,et al.  NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO. , 2004, Journal of molecular and cellular cardiology.

[17]  K. Reymann,et al.  Transient focal ischemia in rat brain differentially regulates mRNA expression of protease‐activated receptors 1 to 4 , 2004, Journal of neuroscience research.

[18]  J. Headrick,et al.  Acute adenosinergic cardioprotection in ischemic-reperfused hearts. , 2003, American journal of physiology. Heart and circulatory physiology.

[19]  G. Gross,et al.  Adenosine and opioid receptor-mediated cardioprotection in the rat: evidence for cross-talk between receptors. , 2003, American journal of physiology. Heart and circulatory physiology.

[20]  A. Sabri,et al.  Mechanisms of Protease-activated Receptor-4 Actions in Cardiomyocytes , 2003, The Journal of Biological Chemistry.

[21]  Michael V. Cohen,et al.  Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase. , 2003, American journal of physiology. Heart and circulatory physiology.

[22]  N. Mackman,et al.  Tissue factor and thrombin mediate myocardial ischemia-reperfusion injury. , 2003, The Annals of thoracic surgery.

[23]  Y. Kuroda,et al.  Hetero‐oligomerization of adenosine A1 receptors with P2Y1 receptors in rat brains , 2002, FEBS letters.

[24]  G. Gross,et al.  Sarcolemmal and mitochondrial KATP channels and myocardial ischemic preconditioning , 2002, Journal of cellular and molecular medicine.

[25]  A. Kuliopulos,et al.  Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation , 2002, Nature Medicine.

[26]  Y. Li,et al.  Dual signaling via protein kinase C and phosphatidylinositol 3'-kinase/Akt contributes to bradykinin B2 receptor-induced cardioprotection in guinea pig hearts. , 2001, Journal of molecular and cellular cardiology.

[27]  Lakshmi A. Devi,et al.  G protein coupled receptor dimerization: implications in modulating receptor function , 2001, Journal of Molecular Medicine.

[28]  M. Hollenberg,et al.  Proteinase-activated receptor 4 (PAR4): activation and inhibition of rat platelet aggregation by PAR4-derived peptides. , 2001, Canadian journal of physiology and pharmacology.

[29]  K. A. Kirkebøen,et al.  Adenosine and cardioprotection during ischaemia and reperfusion – an overview , 2000, Acta anaesthesiologica Scandinavica.

[30]  B. O'dowd,et al.  Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties. , 2000, The Journal of biological chemistry.

[31]  E I Canela,et al.  Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[32]  R. Califf,et al.  Myocardial Infarction Adenosine as an Adjunct to Thrombolytic Therapy for Acute Myocardial Infarction , 1999 .

[33]  E. Gabazza,et al.  Adenosine regulates tissue factor expression on endothelial cells. , 1998, Thrombosis research.

[34]  E. Jackson,et al.  Exogenous and endogenous adenosine inhibits fetal calf serum-induced growth of rat cardiac fibroblasts: role of A2B receptors. , 1997, Circulation.

[35]  J. Shryock,et al.  Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology. , 1997, The American journal of cardiology.

[36]  K. Ytrehus,et al.  Blockade of the KATP-channel by glibenclamide aggravates ischemic injury, and counteracts ischemic preconditioning , 1996, Basic Research in Cardiology.

[37]  D. Yellon,et al.  Ischaemic preconditioning limits infarct size in the rat heart. , 1992, Cardiovascular research.

[38]  R. Mentzer,et al.  The A2a/A2b receptor antagonist ZM-241385 blocks the cardioprotective effect of adenosine agonist pretreatment in in vivo rat myocardium. , 2007, American journal of physiology. Heart and circulatory physiology.

[39]  J. Downey,et al.  Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection. , 2004, American journal of physiology. Heart and circulatory physiology.

[40]  P. Schwartz,et al.  Adult Ventricular Rat Heart Muscle Cells , 1990 .

[41]  Hans Michael Piper,et al.  Cell Culture Techniques in Heart and Vessel Research , 1990, Springer Berlin Heidelberg.