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,(1) necrotic(2) and autophagic cell death.(3,4) 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).(5) 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.     

Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca²⁺ have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca²⁺ chelator BAPTA-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition, and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes, and that Bnip3 triggers induction of autophagy independent of Ca²⁺, ROS generation, and mPTP opening.     

Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca²⁺ have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca²⁺ chelator BAPTA-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes and that Bnip3 triggers induction of autophagy independent of Ca²⁺, ROS generation and mPTP opening.Key words: Bnip3, autophagy, cardiac myocytes, mitochondria, permeability transition pore, cyclophilin D     

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.     

Mitochondrial autophagy by Bnip3 involves Drp1-mediated mitochondrial fission and recruitment of Parkin in cardiac myocytes

The Bcl2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) is an atypical BH3-only protein that is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of mitochondrial autophagy, and in this study we have investigated the mechanisms by which Bnip3 induces autophagy in cardiac myocytes. We found that Bnip3 induced mitochondrial translocation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission in adult myocytes. Drp1-mediated mitochondrial fission correlated with increased autophagy, and inhibition of Drp1 reduced Bnip3-mediated autophagy. Overexpression of Drp1K38E, a dominant negative of Drp1, or mitofusin 1 prevented mitochondrial fission and autophagy by Bnip3. Also, inhibition of mitochondrial fission or autophagy resulted in increased death of myocytes overexpressing Bnip3. Moreover, Bnip3 promoted translocation of the E3 ubiquitin ligase Parkin to mitochondria, which was prevented in the presence of a Drp1 inhibitor. Interestingly, induction of autophagy by Bnip3 was reduced in Parkin-deficient myocytes. Thus our data suggest that induction of autophagy in response to Bnip3 is a protective response activated by the cell that involves Drp1-mediated mitochondrial fission and recruitment of Parkin.     

Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. Bnip3 is an atypical BH3-only protein that is known to cause mitochondrial dysfunction and cell death. Interestingly, Bnip3 can also protect against cell death by inducing mitochondrial autophagy. The mechanism for this process, however, remains poorly understood. Bnip3 contains a C-terminal transmembrane domain that is essential for homodimerization and proapoptotic function. In this study, we show that homodimerization of Bnip3 is also a requirement for induction of autophagy. Several Bnip3 mutants that do not interfere with its mitochondrial localization but disrupt homodimerization failed to induce autophagy in cells. In addition, we discovered that endogenous Bnip3 is localized to both mitochondria and the endoplasmic reticulum (ER). To investigate the effects of Bnip3 at mitochondria or the ER on autophagy, Bnip3 was targeted specifically to each organelle by substituting the Bnip3 transmembrane domain with that of Acta or cytochrome b(5). We found that Bnip3 enhanced autophagy in cells from both sites. We also discovered that Bnip3 induced removal of both ER (ERphagy) and mitochondria (mitophagy) via autophagy. The clearance of these organelles was mediated in part via binding of Bnip3 to LC3 on the autophagosome. Although ablation of the Bnip3-LC3 interaction by mutating the LC3 binding site did not impair the prodeath activity of Bnip3, it significantly reduced both mitophagy and ERphagy. Our data indicate that Bnip3 regulates the apoptotic balance as an autophagy receptor that induces removal of both mitochondria and ER.     

Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling

The traditional Chinese medicine Danshensu (DSS) has a protective effect on cardiac ischaemia/reperfusion (I/R) injury. However, the molecular mechanisms underlying the DSS action remain undefined. We investigated the potential role of DSS in autophagy and apoptosis using cardiac I/R injury models of cardiomyocytes and isolated rat hearts. Cultured neonatal rat cardiomyocytes were subjected to 6 hrs of hypoxia followed by 18 hrs of reoxygenation to induce cell damage. The isolated rat hearts were used to perform global ischaemia for 30 min., followed by 60 min. reperfusion. Ischaemia/reperfusion injury decreased the haemodynamic parameters on cardiac function, damaged cardiomyocytes or even caused cell death. Pre‐treatment of DSS significantly improved cell survival and protected against I/R‐induced deterioration of cardiac function. The improved cell survival upon DSS treatment was associated with activation of mammalian target of rapamycin (mTOR) (as manifested by increased phosphorylation of S6K and S6), which was accompanied with attenuated autophagy flux and decreased expression of autophagy‐ and apoptosis‐related proteins (including p62, LC3‐II, Beclin‐1, Bax, and Caspase‐3) at both protein and mRNA levels. These results suggest that alleviation of cardiac I/R injury by pre‐treatment with DSS may be attributable to inhibiting excessive autophagy and apoptosis through mTOR activation.     

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-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.     

Gadd45b prevents autophagy and apoptosis against rat cerebral neuron oxygen-glucose deprivation/reperfusion injury

Autophagic (type II) cell death has been suggested to play pathogenetic roles in cerebral ischemia. Growth arrest and DNA damage response 45b (Gadd45b) has been shown to protect against rat brain ischemia injury through inhibiting apoptosis. However, the relationship between Gadd45b and autophagy in cerebral ischemia/reperfusion (I/R) injury remains uncertain. The aim of this study is to investigate the effect of Gadd45b on autophagy. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) model of rat primary cortex neurons, and lentivirus interference used to silence Gadd45b expression. Cell viability and injury assay were performed using CCK-8 and LDH kit. Autophagy activation was monitored by expression of ATG5, LC3, Beclin-1, ATG7 and ATG3. Neuron apoptosis was monitored by expression of Bcl-2, Bax, cleaved caspase3, p53 and TUNEL assay. Neuron neurites were assayed by double immunofluorescent labeling with Tuj1 and LC3B. Here, we demonstrated that the expression of Gadd45b was strongly up-regulated at 24 h after 3 h OGD treatment. ShRNA-Gadd45b increased the expression of autophagy related proteins, aggravated OGD/R-induced neuron cell apoptosis and neurites injury. ShRNA-Gadd45b co-treatment with autophagy inhibitor 3-methyladenine (3-MA) or Wortmannin partly inhibited the ratio of LC3II/LC3I, and slightly ameliorated neuron cell apoptosis under OGD/R. Furthermore, shRNA-Gadd45b inhibited the p-p38 level involved in autophagy, but increased the p-JNK level involved in apoptosis. ShRNA-Gadd45b co-treatment with p38 inhibitor obviously induced autophagy. ShRNA-Gadd45b co-treatment with JNK inhibitor alleviated neuron cell apoptosis. In conclusion, our data suggested that Gadd45b inhibited autophagy and apoptosis under OGD/R. Gadd45b may be a common regulatory protein to control autophagy and apoptosis.     

The EphB2 tumor suppressor induces autophagic cell death via concomitant activation of the ERK1/2 and PI3K pathways

EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2-induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.     

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Phosphorylation by Protein Kinase Cδ (PKCδ) Inhibits Mitochondria Elimination by Lysosomal-like Structures following Ischemia and Reoxygenation-induced Injury

After cardiac ischemia and reperfusion or reoxygenation (I/R), damaged mitochondria propagate tissue injury by promoting cell death. One possible mechanism to protect from I/R-induced injury is the elimination of damaged mitochondria by mitophagy. Here we identify new molecular events that lead to mitophagy using a cell culture model and whole hearts subjected to I/R. We found that I/R induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) association with mitochondria and promotes direct uptake of damaged mitochondria into multiorganellar lysosomal-like (LL) structures for elimination independently of the macroautophagy pathway. We also found that protein kinase C δ (PKCδ) inhibits GAPDH-driven mitophagy by phosphorylating the mitochondrially associated GAPDH at threonine 246 following I/R. Phosphorylated GAPDH promotes the accumulation of mitochondria at the periphery of LL structures, which coincides with increased mitochondrial permeability. Either inhibition of PKCδ or expression of a phosphorylation-defective GAPDH mutant during I/R promotes a reduction in mitochondrial mass and apoptosis, thus indicating rescued mitophagy. Taken together, we identified a GAPDH/PKCδ signaling switch, which is activated during oxidative stress to regulate the balance between cell survival by mitophagy and cell death due to accumulation of damaged mitochondria.     

Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy

Loss of cardiomyocytes through programmed cell death is a key event in the development of heart failure, but the inciting molecular mechanisms are largely unknown. We used microarray analysis to identify a genetic program for myocardial apoptosis in Gq-mediated and pressure-overload cardiac hypertrophy. A critical component of this apoptotic program was Nix/Bnip3L. Nix localized to mitochondria and caused release of cytochrome c, activation of caspase-3 and apoptotic cell death, when expressed in HEK293 fibroblasts. A previously undescribed truncated Nix isoform, termed sNix, was not targeted to mitochondria but heterodimerized with Nix and protected against Nix-mediated apoptosis. Forced in vivo myocardial expression of Nix resulted in apoptotic cardiomyopathy and rapid death. Conversely, sNix protected against apoptotic peripartum cardiomyopathy in G(alpha)q-overexpressors. Thus, Nix/Bnip3L is upregulated in myocardial hypertrophy, and is both necessary and sufficient for Gq-mediated apoptosis of cardiomyocytes and resulting hypertrophy decompensation.     

Proapoptotic BCL-2 family members and mitochondrial dysfunction during ischemia/reperfusion injury, a study employing cardiac HL-1 cells and GFP biosensors

The objective of this study was to evaluate mitochondrial alterations in a cell-based model of myocardial ischemia/reperfusion (I/R) injury. Using GFP-biosensors and fluorescence deconvolution microscopy, we investigated mitochondrial morphology in relation to Bax and Bid activation in the HL-1 cardiac cell line. Mitochondria underwent extensive fragmentation during ischemia. Bax translocation from cytosol to mitochondria was initiated during ischemia and proceeded during reperfusion. However, Bax translocation was not sufficient to induce cell death or mitochondrial dysfunction. Bid processing was caspase-8 dependent, and Bid translocation to mitochondria occurred after Bax translocation and clustering, and minutes before cell death. Clustering of Bax into distinct regions on mitochondria could be prevented by CsA, an inhibitor of the mitochondrial permeability transition pore, and also by SB203580, an inhibitor of p38 MAPK. Surprisingly, mitochondrial fragmentation which occurred during ischemia and before Bax translocation could be reversed by the addition of the p38 inhibitor SB203580 at reperfusion. Taken together, these results implicate p38 MAPK in the mitochondrial remodeling response to I/R that facilitates Bax recruitment to mitochondria.     

Mitophagy: mechanisms, pathophysiological roles, and analysis

Abstract Mitochondria are essential organelles that regulate cellular energy homeostasis and cell death. The removal of damaged mitochondria through autophagy, a process called mitophagy, is thus critical for maintaining proper cellular functions. Indeed, mitophagy has been recently proposed to play critical roles in terminal differentiation of red blood cells, paternal mitochondrial degradation, neurodegenerative diseases, and ischemia or drug-induced tissue injury. Removal of damaged mitochondria through autophagy requires two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagic recognition. Recent progress in mitophagy studies reveals that mitochondrial priming is mediated either by the Pink1-Parkin signaling pathway or the mitophagic receptors Nix and Bnip3. In this review, we summarize our current knowledge on the mechanisms of mitophagy. We also discuss the pathophysiological roles of mitophagy and current assays used to monitor mitophagy.     

Bnip3-mediated defects in oxidative phosphorylation promote mitophagy

The Bcl-2 proteins are best known as regulators of the intrinsic mitochondrial pathway of apoptosis. However, recent studies have demonstrated that they can also regulate autophagy. For many years, autophagy was considered to be a nonselective process where the autophagosomes randomly sequestered contents in the cytosol to supply the cells with amino acids and fatty acids during nutrient deprivation. However, it is now clear that autophagy is important for cellular homeostasis under normal conditions, and that it can be a selective process where specific protein aggregates or organelles, such as mitochondria, are targeted for removal by the autophagosomes. Removal of damaged mitochondria is essential for cellular survival, and defects in this process lead to accumulation of dysfunctional mitochondria and cell death. However, the molecular mechanism underlying the selective removal of mitochondria in cells is still poorly understood. A recent study from our laboratory demonstrates that the BH3-only protein Bnip3 is a specific activator of mitochondrial autophagy (mitophagy) and that this process is independent of its role in apoptotic signaling. Here, we discuss how Bnip3-mediated impairment of mitochondrial oxidative phosphorylation facilitates mitochondrial turnover via autophagy in the absence of permeabilization of the mitochondrial membrane and apoptosis.     

Mitophagy in yeast occurs through a selective mechanism

The regulation of mitochondrial degradation through autophagy is expected to be a tightly controlled process, considering the significant role of this organelle in many processes ranging from energy production to cell death. However, very little is known about the specific nature of the degradation process. We developed a new method to detect mitochondrial autophagy (mitophagy) by fusing the green fluorescent protein at the C terminus of two endogenous mitochondrial proteins and monitored vacuolar release of green fluorescent protein. Using this method, we screened several atg mutants and found that ATG11, a gene that is essential only for selective autophagy, is also essential for mitophagy. In addition, we found that mitophagy is blocked even under severe starvation conditions, if the carbon source makes mitochondria essential for metabolism. These findings suggest that the degradation of mitochondria is a tightly regulated process and that these organelles are largely protected from nonspecific autophagic degradation.     

Bnip3-mediated defects in oxidative phosphorylation promote mitophagy

The Bcl-2 proteins are best known as regulators of the intrinsic mitochondrial pathway of apoptosis. However, recent studies have demonstrated that they can also regulate autophagy. For many years, autophagy was considered to be a nonselective process where the autophagosomes randomly sequestered contents in the cytosol to supply the cells with amino acids and fatty acids during nutrient deprivation. However, it is now clear that autophagy is important for cellular homeostasis under normal conditions, and that it can be a selective process where specific protein aggregates or organelles, such as mitochondria, are targeted for removal by the autophagosomes. Removal of damaged mitochondria is essential for cellular survival, and defects in this process lead to accumulation of dysfunctional mitochondria and cell death. However, the molecular mechanism underlying the selective removal of mitochondria in cells is still poorly understood. A recent study from our laboratory demonstrates that the BH3-only protein Bnip3 is a specific activator of mitochondrial autophagy (mitophagy) and that this process is independent of its role in apoptotic signaling. Here, we discuss how Bnip3-mediated impairment of mitochondrial oxidative phosphorylation facilitates mitochondrial turnover via autophagy in the absence of permeabilization of the mitochondrial membrane and apoptosis.