Anne Hamacher-Brady

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.

Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3ΔTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3ΔTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.

The interplay between pro-death and pro-survival signaling pathways in myocardial ischemia/reperfusion injury: apoptosis meets autophagy.

IntroductionProgrammed cell death of cardiac myocytes occurs following a bout of ischemia/reperfusion (I/R), which results in reduced function of the heart. Numerous studies, including in vivo, have shown that cell death occurs via necrosis and apoptosis following I/R. Recently, autophagy has emerged as a powerful mediator of programmed cell death, either opposing or enhancing apoptosis, or acting as an alternative form of programmed cell death distinct from apoptosis.AimHere we review the apoptotic and autophagic signaling pathways, their influences on each other, and we discuss the relevance of autophagy in the heart.

The autophagic response to nutrient deprivation in the hl-1 cardiac myocyte is modulated by Bcl-2 and sarco/endoplasmic reticulum calcium stores.

Macroautophagy is a vital process in the cardiac myocyte: it plays a protective role in the response to ischemic injury, and chronic perturbation is causative in heart disease. Recent findings evidence a link between the apoptotic and autophagic pathways through the interaction of the antiapoptotic proteins Bcl‐2 and Bcl‐XL with Beclin 1. However, the nature of the interaction, either in promoting or blocking autophagy, remains unclear. Here, using a highly sensitive, macroautophagy‐specific flux assay allowing for the distinction between enhanced autophagosome production and suppressed autophagosome degradation, we investigated the control of Beclin 1 and Bcl‐2 on nutrient deprivation‐activated macroautophagy. We found that in HL‐1 cardiac myocytes the relationship between Beclin 1 and Bcl‐2 is subtle: Beclin 1 mutant lacking the Bcl‐2‐binding domain significantly reduced autophagic activity, indicating that Beclin 1‐mediated autophagy required an interaction with Bcl‐2. Overexpression of Bcl‐2 had no effect on the autophagic response to nutrient deprivation; however, targeting Bcl‐2 to the sarco/endoplasmic reticulum (S/ER) significantly suppressed autophagy. The suppressive effect of S/ER‐targeted Bcl‐2 was in part due to the depletion of S/ER calcium stores. Intracellular scavenging of calcium by BAPTA‐AM significantly blocked autophagy, and thapsigargin, an inhibitor of sarco/endoplasmic reticulum calcium ATPase, reduced autophagic activity by ∼ 50%. In cells expressing Bcl‐2–ER, thapsigargin maximally reduced autophagic flux. Thus, our results demonstrate that Bcl‐2 negatively regulated the autophagic response at the level of S/ER calcium content rather than via direct interaction with Beclin 1. Moreover, we identify calcium homeostasis as an essential component of the autophagic response to nutrient deprivation.

Autophagy as a Protective Response to Bnip3-Mediated Apoptotic Signaling in the Heart

Bnip3 is a member of the ‘BH3-only’ Bcl-2 subfamily which has been implicated in apoptotic, necrotic, and autophagic cell death. We recently reported that Bnip3 is a key mediator of mitochondrial dysfunction and cell death in the ex vivo heart following ischemia/reperfusion (I/R). Moreover, we found that Bnip3 was involved in upregulation of autophagy in I/R and that Bnip3-mediated mitochondrial dysfunction correlated with upregulation of autophagy. Using a model of simulated I/R and overexpression of Bnip3 in HL-1 cardiac myocytes, we determined that Bnip3-mediated upregulation of autophagic activity constituted a protective response against Bnip3 death signaling. Here we present additional evidence that enhanced autophagic activity functions as a cytoprotective pathway to oppose ischemia/reperfusion-related apoptosis.