Abstract
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-x(L) were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x(L) 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.
Highlights
Interest in autophagy has increased recently, because of the recognition of its involvement in caspase-independent programmed cell death (PCD2 type II) and its regulation by components of the apoptotic death pathway (PCD type I) [7,8,9]
Our results indicate that in HL-1 cardiac myocytes subjected to simulated I/R (sI/R), autophagic flux is impaired at the level of both induction and degradation, yet remains a vital underlying protective response against sI/R injury
SI/R Induces Programmed Cell Death in HL-1 Cardiac Myocytes—The HL-1 cell line is an excellent model for studying many aspects of cardiac cell physiology [26]
Summary
Interest in autophagy has increased recently, because of the recognition of its involvement in caspase-independent programmed cell death (PCD2 type II) and its regulation by components of the apoptotic death pathway (PCD type I) [7,8,9]. Quantitative analysis per- the use of inhibitors; based on GFP-LC3 imaging alone (Fig. 2), formed on Z-stacks of GFP-LC3 fluorescence revealed that sI/R low autophagic activity in KH solution would be incorrectly significantly increased the number of AVs per cell and, likewise, assumed.
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