Therapeutic hypothermia cardioprotection via Akt- and nitric oxide-mediated attenuation of mitochondrial oxidants.
Therapeutic hypothermia (TH) is a promising cardioprotective treatment for cardiac arrest and acute myocardial infarction, but its cytoprotective mechanisms remain unknown. In this study, we developed a murine cardiomyocyte model of ischemia-reperfusion injury to better determine the mechanisms of TH cardioprotection. We hypothesized that TH manipulates Akt, a survival kinase that mediates mitochondrial protection by modulating reactive oxygen species (ROS) and nitric oxide (NO) generation. Cardiomyocytes, isolated from 1- to 2-day-old C57BL6/J mice, were exposed to 90 min simulated ischemia and 3 h reperfusion. For TH, cells were cooled to 32 degrees C during the last 20 min of ischemia and the first hour of reperfusion. Cell viability was evaluated by propidium iodide and lactate dehydrogenase release. ROS production was measured by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate and mitochondrial membrane potential (DeltaPsim) by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazoly-carbocyanine iodide (JC-1). Phospho (p)-Akt (Thr308), p-Akt (Ser473), and phosphorylated heat shock protein 27 (p-HSP27) (Ser82) were analyzed by Western blot analysis. TH attenuated reperfusion ROS generation, increased NO, maintained DeltaPsim, and decreased cell death [19.3 + or - 3.3% (n = 11) vs. 44.7 + or - 2.7% (n = 10), P < 0.001]. TH also increased p-Akt during ischemia before reperfusion. TH protection and attenuation of ROS were blocked by the inhibition of Akt and NO synthase but not by a cGMP inhibitor. HSP27, a regulator of Akt, also exhibited increased phosphorylation (Ser82) during ischemia with TH. We conclude that TH cardioprotection is mediated by enhanced Akt/HSP27 phosphorylation and enhanced NO generation, resulting in the attenuation of ROS generation and the maintenance of DeltaPsim following ischemia-reperfusion.
Shao
Zuo-Hui
ZH
Section of Emergency Medicine, Department of Medicine, Emergency Resuscitation Center, University of Chicago, Chicago, IL, USA.
Sharp
Willard W
WW
Wojcik
Kimberly R
KR
Li
Chang-Qing
CQ
Han
Mei
M
Chang
Wei-Tien
WT
Ramachandran
Srinivasan
S
Li
Jing
J
Hamann
Kimm J
KJ
Vanden Hoek
Terry L
TL
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004195:Disease Models, Animal; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism; D007036:Hypothermia, Induced / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008929:Mitochondria, Heart / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors
D055551:HSP27 Heat-Shock Proteins; D016877:Oxidants; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D051057:Proto-Oncogene Proteins c-akt
J Cell Biochem. 2009 Jul 1;107(4):697-705
19388003
Am J Physiol. 1996 Apr;270(4 Pt 2):H1334-41
8967373
J Thorac Cardiovasc Surg. 2006 May;131(5):969-74
16678577
Shock. 2009 Aug;32(2):210-6
19060788
J Mol Cell Cardiol. 2006 Jan;40(1):16-23
16288777
Mol Cell. 2010 Jan 15;37(1):1-2
20129048
Biochem J. 1980 Nov 1;191(2):421-7
6263247
N Engl J Med. 2007 Sep 13;357(11):1121-35
17855673
Free Radic Biol Med. 2007 Aug 15;43(4):590-9
17640569
Resuscitation. 2008 Feb;76(2):311-3
17765383
N Engl J Med. 2001 Apr 26;344(17):1304-13
11320390
Am J Physiol Heart Circ Physiol. 2010 Jan;298(1):H152-7
19880666
Circulation. 2000 Feb 15;101(6):660-7
10673259
J Biol Chem. 2006 Aug 11;281(32):22943-52
16754658
Antioxid Redox Signal. 2008 Mar;10(3):579-99
18052718
Cancer Lett. 1999 Feb 8;136(1):41-6
10211937
FEBS Lett. 1997 Jun 30;410(2-3):493-8
9237690
Circulation. 2008 Nov 4;118(19):1970-8
18936326
Crit Care Med. 2009 Jan;37(1):256-62
19050616
Circulation. 2009 Sep 8;120(10):897-905
19704094
Crit Care Med. 2006 Dec;34(12 Suppl):S474-82
17114980
Cell. 2007 Jun 29;129(7):1261-74
17604717
Crit Care Med. 2005 Sep;33(9):2043-8
16148478
J Physiol. 2003 Oct 15;552(Pt 2):335-44
14561818
Cardiovasc Res. 2003 Sep 1;59(3):715-22
14499873
Am J Physiol Heart Circ Physiol. 2007 Aug;293(2):H903-4
17513492
Cardiovasc Res. 1998 Dec;40(3):502-7
10070490
Am J Physiol Heart Circ Physiol. 2007 Apr;292(4):H1995-2003
17172266
Crit Care Med. 2008 Jun;36(6):1780-6
18496378
Circulation. 2008 Nov 4;118(19):1915-9
18981312
Circ Res. 2000 Oct 27;87(9):781-8
11055982
Am J Physiol Heart Circ Physiol. 2010 Jun;298(6):H1761-8
20363892
Cancer Res. 2004 Jul 1;64(13):4394-9
15231645
Chin Med J (Engl). 2007 Dec 20;120(24):2271-7
18167217
Crit Care Med. 2007 Jul;35(7):1709-16
17522572
Cardiovasc Res. 2004 Feb 15;61(3):580-90
14962488
Cardiovasc Res. 2009 Jul 15;83(2):345-53
19196828
J Neurosci. 2005 Oct 19;25(42):9794-806
16237183
Resuscitation. 2003 Jun;57(3):231-5
12858857
Circulation. 2002 Mar 26;105(12):1497-502
11914261
Circulation. 2004 Jun 8;109(22):2786-91
15159295