Enhancing Macroautophagy Protects against Ischemia/Reperfusion Injury in Cardiac Myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against I/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell. Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against I/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell. Autophagy involves processes for the turnover of long lived macromolecules and organelles via the lysosomal degradative pathway (1Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar, 2Cuervo A.M. Mol. Cell. Biochem. 2004; 263: 55-72Crossref PubMed Scopus (395) Google Scholar). Macroautophagy (referred to hereafter as autophagy) is a specific mode of autophagy in which isolation membranes envelop a portion of the cytosol, containing nonspecific cytosolic components, selectively targeted toxic protein aggregates (3Ravikumar B. Vacher C. Berger Z. Davies J.E. Luo S. Oroz L.G. Scaravilli F. Easton D.F. Duden R. O'Kane C.J. Rubinsztein D.C. Nat. Genet. 2004; 36: 585-595Crossref PubMed Scopus (1990) Google Scholar), intracellular pathogens (4Gutierrez M.G. Master S.S. Singh S.B. Taylor G.A. Colombo M.I. Deretic V. Cell. 2004; 119: 753-766Abstract Full Text Full Text PDF PubMed Scopus (1769) Google Scholar), or organelles such as mitochondria (5Xue L. Fletcher G.C. Tolkovsky A.M. Curr. Biol. 2001; 11: 361-365Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 6Priault M. Salin B. Schaeffer J. Vallette F.M. di Rago J.P. Martinou J.C. Cell Death Differ. 2005; 12: 1613-1621Crossref PubMed Scopus (242) Google Scholar). The autophagosomes are then delivered to the lysosome, forming the autophagolysosome, for subsequent degradation of their contents by lysosomal hydrolases (Fig. 10). Interest in autophagy has increased recently, because of the recognition of its involvement in caspase-independent programmed cell death (PCD 2The abbreviations used are: PCD, programmed cell death; AVs, autophagic vacuoles; GFP, green fluorescent protein; KH, Krebs-Henseleit; 3-MA, 3-methyladenine; PI3K, phosphatidylinositol 3-kinase; I/R, ischemia/reperfusion; sI/R, simulated ischemia/reperfusion; RNAi, RNA interference; PI3P, phosphatidylinositol 3-phosphate; mTOR, mammalian target of rapamycin; ER, endoplasmic reticulum. type II) and its regulation by components of the apoptotic death pathway (PCD type I) (7Saeki K. Yuo A. Okuma E. Yazaki Y. Susin S.A. Kroemer G. Takaku F. Cell Death Differ. 2000; 7: 1263-1269Crossref PubMed Scopus (172) Google Scholar, 8Yanagisawa H. Miyashita T. Nakano Y. Yamamoto D. Cell Death Differ. 2003; 10: 798-807Crossref PubMed Scopus (91) Google Scholar, 9Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol. 2004; 6: 1221-1228Crossref PubMed Scopus (1192) Google Scholar). Anti-apoptotic Bcl-2 and Bcl-xL have been linked to the autophagic pathway via an interaction with Beclin1, a key mediator of autophagic activity (9Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol. 2004; 6: 1221-1228Crossref PubMed Scopus (1192) Google Scholar, 10Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. J. Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar). Autophagy is a vital process in the heart, presumably participating in the removal of dysfunctional cytosolic components and serving as a catabolic energy source during times of starvation. For example, autophagy in cardiac myocytes has been suggested to provide a necessary source of energy between birth and suckling (11Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2405) Google Scholar), and in a GFP-LC3 transgenic mouse, cardiac myocytes from starved animals displayed high numbers of autophagosomes, some of which contained mitochondria (12Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1942) Google Scholar). On the other hand, impaired autophagy a in cardiac autophagic removal of mitochondria the source of a toxic that during the A. J. Biochem. PubMed Scopus Google Scholar), and of the results in reduced Y. R. K. Mol. 2001; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). of the autophagic pathway to cardiac cell death lysosomal is and lysosomal are the K. J. 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D. 2005; PubMed Scopus Google Scholar). The target and to other as determined by The used to which were the which has expression of for of by The and were and cells were with both to To for nonspecific the used as a were in and by a to and the in with of by a to in and at in were in for for of that with of the cell not were a with a a and a and a a and and by a by an for and via a and an for and were from were of a to are of with cellular of and are known to by the Y. Mizushima N. Yamamoto A. S. Ohsumi Y. Yoshimori T. J. Cell 2004; PubMed Scopus Google Scholar). in of in HL-1 cells not cellular contents of autophagosomal were quantified via of GFP-LC3 Y. Mizushima N. T. Yamamoto A. T. T. E. Ohsumi Y. Yoshimori T. J. 2000; PubMed Scopus Google or To from the Nat. 2004; PubMed Scopus Google and with enhanced of the Y. Mizushima N. T. Yamamoto A. T. T. E. Ohsumi Y. Yoshimori T. J. 2000; PubMed Scopus Google HL-1 cells were with and cells were to as were with in for To the autophagic in a of cells were at and as or as autophagic cells were in of or For of the autophagic of of of cells in were the with the of the cell. were and and of the autophagosome cell were determined of autophagic cells were to the with and a of the lysosomal to A. Y. Yoshimori T. Y. R. Y. Cell 1998; PubMed Scopus Google Scholar), of and to lysosomal of GFP-LC3 used to cellular autophagosomal as of the is a that selectively with and used to C. J. B. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar). and cells were with for in the then with and cells were by and intracellular of a lysosomal were to the cells during the of an to the of A. J. Cell Biol. PubMed Scopus Google or A. PubMed Scopus Google used as a to cellular injury. were with and the and to for were then to of in by of and cells were by were as cells with or cells were at in of were by and at for at and with To cell cell were in and and for The cell were at for to cellular of and were at for and to membranes against Bcl-2 Bcl-xL Beclin1 and fluorescent protein to and and and were because of in and to the that are used against are of at of between determined by the are as of at Cell Death in HL-1 Cardiac HL-1 cell is an model for of cardiac cell J. 2004; PubMed Scopus Google Scholar). HL-1 cells in to simulated I/R via in cardiac I/R injury A. PubMed Scopus Google Scholar). One key feature of cell death is the of the pro-apoptotic Bcl-2 protein Bax in the death Bax a in the pathway, is by a from the to at the mitochondria and quantified via fluorescent of a protein (Fig. A. J. Cell Biol. 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