Enhancing Macroautophagy Protects against Ischemia/Reperfusion Injury in Cardiac Myocytes

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.Autophagy involves processes for the turnover of long lived macromolecules and organelles via the lysosomal degradative pathway (1, 2). 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 (3), intracellular pathogens (4), or organelles such as mitochondria (5, 6). 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 2 type II) and its regulation by components of the apoptotic death pathway (PCD type I) (7-9). Anti-apoptotic Bcl-2 and Bcl-x L have been linked to the autophagic pathway via an interaction with Beclin1, a key mediator of autophagic activity (9, 10).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 (11), and in a GFP-LC3 transgenic mouse, cardiac myocytes from starved animals displayed high numbers of autophagosomes, some of...

Targeting the Autophagy Pathway Using Ectopic Expression of Beclin 1 in Combination with Rapamycin in Drug-Resistant v-Ha-ras-Transformed NIH 3T3 Cells

“…However, overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect autophagy induction, although there is a report that enfor-ced expression of Atg5 sensitized tumour cells to anticancer drug treatment both in vitro and in vivo (Yousefi et al, 2006). Consistent with our results, other laboratories also reported the lack of effect of Atg5 overexpression in the Atg5 +/+ cells (Hamacher-Brady et al, 2006;King et al, 2008). Thus, this result implies that the autophagic machinery downstream of Beclin1-dependent induction was not a rate-limiting factor.…”

Recycle or die: The role of autophagy in cardioprotection

“…Upregulation of autophagy has also been shown to be protective in vitro. Inhibition of autophagy in HL-1 myocytes caused increased cell death in response to simulated I/R (sI/R), whereas enhancement of the autophagic response was protective [4]. Similarly, Dosenko et al reported that inhibiting autophagy in isolated cardiac myocytes during exposure to anoxia/reoxygenation caused an increase in cell death [42].…”

“…Macroautophagy is the most common form of autophagy in mammalian cells and will be herein referred to as autophagy. Autophagy occurs at low levels under normal conditions, but is also upregulated in response to stress such as nutrient deprivation, hypoxia, mitochondrial dysfunction, and infection [1][2][3][4]. Autophagy can promote cell survival by generating free amino acids and fatty acids required to maintain function during nutrientlimiting conditions, or by removing damaged organelles and intracellular pathogens.…”

“…It occurs at low basal levels under normal conditions and is important for the turnover of organelles [7]. Many studies have demonstrated that autophagy is upregulated in the heart in response to stress such as ischemia/reperfusion [4,[8][9][10][11][12]. Increased numbers of autophagosomes are a prominent feature in many cardiovascular diseases such as cardiac hypertrophy and heart failure [13][14][15][16][17][18].…”

The role of autophagy in the heart

“…For example, autophagy is important for the turnover of organelles at low basal levels under normal conditions. 2 In the heart, the level of autophagy is altered not only in response to stress such as ischemia/reperfusion 14,15 but also to stress triggered by cardiovascular diseases such as cardiac hypertrophy 16 and heart failure. 17,18 Heart failure is preceded by a process termed cardiac remodeling, which encompasses the aggregate of compensatory structural and functional changes in response to stress on the chamber wall.…”