The interplay between pro-death and pro-survival signaling pathways converging at mitochondria in myocardial Ischemia/Reperfusion : Apoptosis meets Autophagy

Apoptotic cell death of cardiac myocytes contributes to cardiovascular disease. Increased levels of autophagosomes have been reported during myocardial ischemia/reperfusion (I/R) injury, however, whether autophagy functions as a protective or lethal response is unclear. Thus, the main objective of this thesis was to investigate the nature of the relationship between I/R-activated mitochondrial (apoptotic) death pathways and autophagy. Multidimensional fluorescence deconvolution microscopy of specific fluorescent dyes and GFP-based biosensors allowed for the quantification of spatial and temporal events in the single cell. Simulated I/R (sI/R) of cardiac HL-1 cells was employed as an in vitro model of I/R injury to the heart. To assess macroautophagy autophagosome generation and degradation (autophagic flux) was quantified, as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. Apoptotic and autophagic pathways are linked through the interaction of anti-apoptotic Bcl-2 proteins with the autophagy protein Beclin1. In this thesis a two-tiered relationship between autophagic activity and anti-apoptotic Bcl-2 was revealed: under normal conditions Bcl-2 is a necessary participant in autophagy via its interaction with Beclin1, yet under conditions in which Bcl-2 is artificially concentrated at the sarco/endoplasmic reticulum, the consequent depletion of lumenal Ca2+ results in an overriding inhibition of autophagy. Subsequently, the dynamics and role of autophagy during sI/R were investigated. It was found that autophagic flux was null during the ischemic period, and increased at reperfusion, but not to the same degree as under normoxic conditions. sI/R impaired both formation and downstream lysosomal degradation of autophagosomes. Both rapamycin and overexpression of Beclin1 enhanced autophagic flux following sI/R and significantly reduced activation of Bax, while 3-methyladenine, wortmannin, and RNAi knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against sI/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against sI/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, while 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. Residual levels of autophagy, even though not maximal, functioned to preserve cell viability following sI/R and enhancing autophagy constituted a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell. Based on the obtained results the autophagic removal of dysfunctional mitochondria was investigated as a potential mechanism of protection. During sI/R fragmented mitochondria were frequently sequestered by autophagosomes (mitophagy). Bnip3, a mitochondrial pro-apoptotic Bcl-2 family member which mediates I/R-induced mitochondrial dysfunction in the heart and has been shown to activate autophagy, was used to simulate I/R-activated mitochondrial dysfunction. Overexpression of Bnip3 induced fragmentation of the mitochondrial network and cell death in the absence of sI/R. Moreover, Bnip3 expression enhanced both steady-state levels of autophagosomes and autophagic flux. Many of the Bnip3-induced autophagosomes contained fragmented mitochondria. Overexpression of both Beclin1 and Atg5 protected against Bnip3-mediated cell death, while blocking autophagy with dominant negative mutants of Beclin1 or Atg5 enhanced cell death. Thus, the autophagic pathway may oppose the mitochondrial apoptotic pathway, in that autophagy of dysfunctional mitochondria counteracts Bnip3-mediated mitochondrial death signaling.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Hamacher, Anne

Contributors dc:contributor

• Kreuzaler, Fritz

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Apoptosis
• Ischämie
• Mitochondrium
• Autophagie
• Ischemia
• Mitochondria
• Autophagy
• cardiac

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123434%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61812