RAGE modulates hypoxia/reoxygenation injury in adult murine cardiomyocytes via JNK and GSK-3beta signaling pathways.
BACKGROUND
Advanced glycation end-products (AGEs) have been implicated in diverse pathological settings including diabetes, inflammation and acute ischemia/reperfusion injury in the heart. AGEs interact with the receptor for AGEs (RAGE) and transduce signals through activation of MAPKs and proapoptotic pathways. In the current study, adult cardiomyocytes were studied in an in vitro ischemia/reperfusion (I/R) injury model to delineate the molecular mechanisms underlying RAGE-mediated injury due to hypoxia/reoxygenation (H/R).


RESULTS
Cardiomyocytes isolated from adult wild-type (WT), homozygous RAGE-null (RKO), and WT mice treated with soluble RAGE (sRAGE) were subjected to hypoxia for 30 minutes alone or followed by reoxygenation for 1 hour. In specific experiments, RAGE ligand carboxymethyllysine (CML)-AGE (termed "CML" in this manuscript) was evaluated in vitro. LDH, a marker of cellular injury, was assayed in the supernatant in the presence or absence of signaling inhibitor-treated cardiomyocytes. Cardiomyocyte levels of heterogeneous AGEs were measured using ELISA. A pronounced increase in RAGE expression along with AGEs was observed in H/R vs. normoxia in WT cardiomyocytes. WT cardiomyocytes after H/R displayed increased LDH release compared to RKO or sRAGE-treated cardiomyocytes. Our results revealed significant increases in phospho-JNK in WT cardiomyocytes after H/R. In contrast, neither RKO nor sRAGE-treated cardiomyocytes exhibited increased phosphorylation of JNK after H/R stress. The impact of RAGE deletion on GSK-3beta phosphorylation in the cardiomyocytes subjected to H/R revealed significantly higher levels of phospho-GSK-3beta/total GSK-3beta in RKO, as well as in sRAGE-treated cardiomyocytes versus WT cardiomyocytes after H/R. Further investigation established a key role for Akt, which functions upstream of GSK-3beta, in modulating H/R injury in adult cardiomyocytes.


CONCLUSIONS
These data illustrate key roles for RAGE-ligand interaction in the pathogenesis of cardiomyocyte injury induced by hypoxia/reoxygenation and indicate that the effects of RAGE are mediated by JNK activation and dephosphorylation of GSK-3beta. The outcome in this study lends further support to the potential use of RAGE blockade as an adjunctive therapy for protection of the ischemic heart.
Shang
Linshan
L
Division of Surgical Science, Department of Surgery, College of Physicians and Surgeons, Columbia University, New York, New York, United States of America.
Ananthakrishnan
Radha
R
Li
Qing
Q
Quadri
Nosirudeen
N
Abdillahi
Mariane
M
Zhu
Zhengbin
Z
Qu
Wu
W
Rosario
Rosa
R
Touré
Fatouma
F
Yan
Shi Fang
SF
Schmidt
Ann Marie
AM
Ramasamy
Ravichandran
R
D000818:Animals; D017127:Glycation End Products, Advanced / Q000032:analysis; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000860:Hypoxia; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D032383:Myocytes, Cardiac / Q000473:pathology*; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000032:analysis; D015427:Reperfusion Injury / Q000188:drug therapy*; D015398:Signal Transduction
D017127:Glycation End Products, Advanced; D008024:Ligands; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D048031:JNK Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3
J Cell Physiol. 2003 Apr;195(1):27-37
12599206
Proteomics. 2003 Apr;3(4):513-26
12687618
Circ Res. 2002 Mar 8;90(4):377-9
11884365
FASEB J. 2004 Jul;18(10):1060-70
15226266
Mol Pharmacol. 2001 Apr;59(4):867-74
11259632
Diabetes. 2008 Jul;57(7):1941-51
18420491
J Clin Invest. 2004 Jun;113(11):1535-49
15173880
Circulation. 2006 Mar 7;113(9):1226-34
16505177
J Cell Sci. 2003 Apr 1;116(Pt 7):1175-86
12615961
J Biol Chem. 2000 Nov 17;275(46):36108-15
10945990
FASEB J. 2004 Dec;18(15):1818-25
15576485
Neuropsychopharmacology. 2005 Jul;30(7):1223-37
15827567
Nat Med. 1998 Sep;4(9):1025-31
9734395
Curr Drug Targets. 2006 Nov;7(11):1377-88
17100578
J Biol Chem. 2002 Mar 22;277(12):10244-50
11786558
Chem Biol. 2000 Oct;7(10):793-803
11033082
Circulation. 2000 Feb 15;101(6):660-7
10673259
J Exp Med. 2005 Feb 7;201(3):473-84
15699076
Circ Res. 1997 Aug;81(2):137-44
9242174
J Clin Invest. 2003 Apr;111(7):959-72
12671045
J Neurochem. 2001 Sep;78(6):1219-32
11579131
Circ Res. 2001 Feb 16;88(3):305-12
11179198
Biochem J. 2002 Mar 15;362(Pt 3):561-71
11879182
Circ Res. 1998 Jul 13;83(1):1-14
9670913
J Biol Chem. 2005 Apr 1;280(13):12944-55
15668244
Sci STKE. 2001 Sep 18;2001(100):re12
11579232
Lab Invest. 1996 Jan;74(1):86-107
8569201
Am J Physiol Heart Circ Physiol. 2008 Apr;294(4):H1823-32
18245563
Circ Res. 2006 Jan 6;98(1):111-8
16306447
Circ Res. 1998 Jul 13;83(1):15-26
9670914
Cell Death Differ. 1999 Oct;6(10):987-91
10556976
J Biol Chem. 2005 Sep 23;280(38):32602-8
16043490
Carcinogenesis. 2009 Mar;30(3):529-37
19037093
Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):15127-32
10611349
Cell Signal. 2008 Jan;20(1):206-18
17993264