Oxidative Stress Stimulates Autophagic Flux During Ischemia/Reperfusion

Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, UMDNJ, New Jersey Medical School, New Jersey.

^{Corresponding author.}

Address correspondence to: Prof. Junichi Sadoshima, Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Medical Science Building G-609, Newark, NJ 07103. E-mail:ude.jndmu@ujhsodaS

Received 2010 Aug 16; Accepted 2010 Sep 2.

Abstract

Autophagy is a bulk degradation process in which cytosolic proteins and organelles are degraded through lysosomes. To evaluate autophagic flux in cardiac myocytes, we generated adenovirus and cardiac-specific transgenic mice harboring tandem fluorescent mRFP-GFP-LC3. Starvation significantly increased the number of mRFP-GFP-LC3 dots representing both autophagosomes and autolysosomes per cell, suggesting that autophagic flux is increased in cardiac myocytes. H₂O₂ significantly increased autophagic flux, which was attenuated in the presence of N-2-mercaptopropionyl glycine (MPG), an antioxidant, suggesting that oxidative stress stimulates autophagy in cardiac myocytes. Myocardial ischemia/reperfusion (I/R) increased both autophagosomes and autolysosomes, thereby increasing autophagic flux. Treatment with MPG attenuated I/R-induced increases in oxidative stress, autophagic flux, and Beclin-1 expression, accompanied by a decrease in the size of myocardial infarction (MI)/area at risk (AAR), suggesting that oxidative stress plays an important role in mediating autophagy and myocardial injury during I/R. MI/AAR after I/R was significantly reduced in beclin1^+/− mice, whereas beclin1^{mice treated with MPG exhibited no additional reduction in the size of MI/AAR after I/R. These results suggest that oxidative stress plays an important role in mediating autophagy during I/R, and that activation of autophagy through oxidative stress mediates myocardial injury in response to I/R in the mouse heart.}Antioxid. Redox Signal. 14, 2179–2190.

Abstract

Abbreviations Used

AAD	amino acid deprivation
AAR	area at risk
Ad	adenovirus
DAPI	4′,6-diamidino-2-phenylindole
DHE	dihydroethidium
GFP	green fluorescent protein
H₂O₂	hydrogen peroxide
I/R	ischemia/reperfusion
MI	myocardial infarction size
MOI	multiplicities of infection
MPG	N-2-mercaptopropionyl glycine
mPTP	mitochondrial permeability transition pore
mRFP	monomeric red fluorescent protein
NTg	non-transgenic
PBS	phosphate-buffered saline
ROS	reactive oxygen species
tf-LC3	tandem fluorescent mRFP-GFP-LC3
Tg	transgenic
TTC	triphenyltetrazolium chloride

Abbreviations Used

References

1. Ago T. Kuroda J. Pain J. Fu C. Li H. Sadoshima J. Upregulation of Nox4 by hypertrophic stimuli promotes apoptosis and mitochondrial dysfunction in cardiac myocytes. Circ Res. 2004;106:1253–1264.
2. Bjorkoy G. Lamark T. Brech A. Outzen H. Perander M. Overvatn A. Stenmark H. Johansen T. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol. 2005;171:603–614.
3. Bopassa JC. Michel P. Gateau-Roesch O. Ovize M. Ferrera R. Low-pressure reperfusion alters mitochondrial permeability transition. Am J Physiol Heart Circ Physiol. 2005;288:H2750–H2755.[PubMed]
4. Carreira RS. Lee Y. Ghochani M. Gustafsson AB. Gottlieb RA. Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells. Autophagy. 2010;6 [epub ahead of print.].
5. Garcia-Dorado D. Agullo L. Sartorio CL. Ruiz-Meana M. Myocardial protection against reperfusion injury: the cGMP pathway. Thromb Haemost. 2009;101:635–642.[PubMed]
6. Gurusamy N. Das DK. Autophagy, redox signaling, and ventricular remodeling. Antioxid Redox Signal. 2009;11:1975–1988.
7. Gustafsson AB. Gottlieb RA. Autophagy in ischemic heart disease. Circ Res. 2009;104:150–158.
8. Gustafsson AB. Gottlieb RA. Eat your heart out: role of autophagy in myocardial ischemia/reperfusion. Autophagy. 2008;4:416–421.
9. Halestrap APCalcium, mitochondria and reperfusion injury: a pore way to die. Biochem Soc Trans. 2006;34:232–237.[PubMed][Google Scholar]
10. Hamacher-Brady A. Brady NR. Logue SE. Sayen MR. Jinno M. Kirshenbaum LA. Gottlieb RA. Gustafsson AB. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death Differ. 2007;14:146–157.[PubMed]
11. Horwitz LD. Fennessey PV. Shikes RH. Kong Y. Marked reduction in myocardial infarct size due to prolonged infusion of an antioxidant during reperfusion. Circulation. 1994;89:1792–1801.[PubMed]
12. Kiffin R. Bandyopadhyay U. Cuervo AM. Oxidative stress and autophagy. Antioxid Redox Signal. 2006;8:152–162.[PubMed]
13. Kimura S. Noda T. Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007;3:452–460.[PubMed]
14. Klionsky DJ. Abeliovich H. Agostinis P. Agrawal DK. Aliev G. Askew DS. Baba M. Baehrecke EH. Bahr BA. Ballabio A. Bamber BA. Bassham DC. Bergamini E. Bi X. Biard-Piechaczyk M. Blum JS. Bredesen DE. Brodsky JL. Brumell JH. Brunk UT. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin LS. Choi A. Chu CT. Chung J, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008;4:151–175.
15. Levine B. Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42.
16. Li W. Yuan XM. Ivanova S. Tracey KJ. Eaton JW. Brunk UT. 3-Aminopropanol, formed during cerebral ischaemia, is a potent lysosomotropic neurotoxin. Biochem J. 2003;371:429–436.
17. Matsui Y. Kyoi S. Takagi H. Hsu CP. Hariharan N. Ago T. Vatner SF. Sadoshima J. Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion. Autophagy. 2008;4:409–415.
18. Matsui Y. Takagi H. Qu X. Abdellatif M. Sakoda H. Asano T. Levine B. Sadoshima J. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res. 2007;100:914–922.[PubMed]
19. Misra MK. Sarwat M. Bhakuni P. Tuteja R. Tuteja N. Oxidative stress and ischemic myocardial syndromes. Med Sci Monit. 2009;15:RA209–RA219.[PubMed]
20. Mitsos SE. Askew TE. Fantone JC. Kunkel SL. Abrams GD. Schork A. Lucchesi BR. Protective effects of N-2-mercaptopropionyl glycine against myocardial reperfusion injury after neutrophil depletion in the dog: evidence for the role of intracellular-derived free radicals. Circulation. 1986;73:1077–1086.[PubMed]
21. Mitsos SE. Fantone JC. Gallagher KP. Walden KM. Simpson PJ. Abrams GD. Schork MA. Lucchesi BR. Canine myocardial reperfusion injury: protection by a free radical scavenger, N-2-mercaptopropionyl glycine. J Cardiovasc Pharmacol. 1986;8:978–988.[PubMed]
22. Mizushima NAutophagy: process and function. Genes Dev. 2007;21:2861–2873.[PubMed][Google Scholar]
23. Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell. 2004;15:1101–1111.
24. Mizushima N. Yoshimori T. Levine B. Methods in mammalian autophagy research. Cell. 2006;140:313–326.
25. Nishida K. Kyoi S. Yamaguchi O. Sadoshima J. Otsu K. The role of autophagy in the heart. Cell Death Differ. 2009;16:31–38.[PubMed]
26. Pankiv S. Clausen TH. Lamark T. Brech A. Bruun JA. Outzen H. Overvatn A. Bjorkoy G. Johansen T. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem. 2007;282:24131–24145.[PubMed]
27. Scherz-Shouval R. Shvets E. Fass E. Shorer H. Gil L. Elazar Z. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J. 2007;26:1749–1760.
28. Sinha S. Levine B. The autophagy effector Beclin 1: a novel BH3-only protein. Oncogene. 2008;27(suppl 1):S137–S148.
29. Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO J. 2001;20:5971–5981.
30. Takagi H. Matsui Y. Sadoshima J. The role of autophagy in mediating cell survival and death during ischemia and reperfusion in the heart. Antioxid Redox Signal. 2007;9:1373–1381.[PubMed]
31. Terada M. Nobori K. Munehisa Y. Kakizaki M. Ohba T. Takahashi Y. Koyama T. Terata Y. Ishida M. Iino K. Kosaka T. Watanabe H. Hasegawa H. Ito H. Double transgenic mice crossed GFP-LC3 transgenic mice with alphaMyHC-mCherry-LC3 transgenic mice are a new and useful tool to examine the role of autophagy in the heart. Circ J. 2008;74:203–206.[PubMed]
32. Yan L. Sadoshima J. Vatner DE. Vatner SF. Autophagy in ischemic preconditioning and hibernating myocardium. Autophagy. 2009;5:709–712.[PubMed]
33. Yan L. Vatner DE. Kim SJ. Ge H. Masurekar M. Massover WH. Yang G. Matsui Y. Sadoshima J. Vatner SF. Autophagy in chronically ischemic myocardium. Proc Natl Acad Sci U S A. 2005;102:13807–13812.