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The interplay between cell death signaling pathways in the heart
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Available online at www.sciencedirect.com
www.elsevier.com/locate/tcm
The interplay between cell death signaling pathways in the heart Agnieszka K. Bialaa,b and Lorrie A. Kirshenbauma,b,c,n a
The Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of Manitoba, Centre Rm. 3016, 351 TachéAvenue, Winnipeg, Manitoba, Canada R2H 2A6 b Department of Physiology, College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada c Department of Pharmacology and Therapeutics, College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
abstra ct To date, one of the most intriguing and compelling concepts to impact contemporary cell biology is the notion that cell fate is “programmed” or genetically controlled. Indeed, the regulation of cell fate is crucial for embryonic development, and tissue homeostasis. Given the importance of removing damaged or irreversibly injured cells from the body, it is not surprising that defects in the regulatory mechanisms that govern cell death and/or survival more generally have been implicated in a number of human pathologies including cancer, neurodegenerative diseases, and cardiac failure. Several processes involved in the regulation of cell fate through apoptosis, necrosis, and autophagy are commonly linked through the actions of certain Bcl-2 proteins that act on the mitochondrion. For example, the Bcl-2 protein Beclin-1 is actively involved in the clearance of damaged mitochondria via mitophagy, while other Bcl-2 proteins such as Bax/Bak can initiate apoptosis or necrotic signaling pathways. The overlapping and redundant nature of these proteins highlights their evolutionary importance for regulating cardiac cell survival and death during normal and disease states. Here, we explore the interrelationship between these signaling pathways and the cellular effectors that influence cardiac cell fate. & 2014 Published by Elsevier Inc.
Introduction Despite the significant advances in cardiovascular research over the past two decades, heart disease still remains a significant cause of morbidity and mortality and is reaching pandemic proportions worldwide. Indeed, the combinations of the Western diet, sedentary lifestyles, and genetic predispositions have been considered the prevailing underlying risk factors for cardiovascular disease, including diabetes, hypertension, atherosclerosis, ischemic heart disease, and heart failure. In contrast to other cells of the body that retain the ability for regeneration and self-renewal, cardiac myocytes
lose this property shortly after birth. Hence, the loss of functional cardiac cells has been suggested as an underlying feature of ventricular remodeling and diminished cardiac pump performance after injury. Therefore, one unifying theme toward the ultimate therapeutic goal of preventing heart failure would be to suppress the inappropriate loss of cardiac cells after myocardial injury. It is now appreciated that cardiac cells die during ischemic injury or postmyocardial infarction by highly orchestrated genetically programmed cell death pathways involving apoptosis, necrosis, and in some instances autophagy. Notably, while autophagy is generally considered a catabolic survival mechanism,
The authors have indicated there are no conflicts of interest. This work was supported by Grants to L.A.K. from the Canadian Institutes of Health Research (MOP42402, MOP74456), Canada. A.K.B. holds a post-doctoral fellowship from the Manitoba Health Research Council and IMPACT-CIHR. n Corresponding author at: Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre Rm. 3016, 351 Taché Avenue, Winnipeg, Manitoba, Canada, R2H 2A6. E-mail address: [email protected] (L.A. Kirshenbaum). http://dx.doi.org/10.1016/j.tcm.2014.08.002 1050-1738/& 2014 Published by Elsevier Inc.
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de-regulated autophagy can be maladaptive and promote cell death by the novel process recently described in neuronal tissue termed “autosis” [1]. Indeed, the evidence for apoptosis, necrosis, and autophagy has been detected in a number of cardiac pathologies including, ischemia followed by reperfusion, after myocardial infarction, and in the failing human heart. Interestingly, in some instances, apoptosis, necrosis and autophagy have been detected in the heart under the same pathological conditions, suggesting that these signaling pathways may be commonly linked through overlapping cellular effectors that regulate cell fate. Insights into the signaling mechanisms that underlie cell death have revealed apoptosis can occur via an extrinsic pathway involving death receptors and intrinsic pathway involving the mitochondrion. Although these pathways were first considered to be functionally independent, there is emerging evidence that these pathways are commonly linked to the mitochondrion by certain Bcl-2 proteins. Indeed, cellular defects associated with apoptosis have classically involved mitochondrial cytochrome c release, caspase activation, and DNA fragmentation with the loss of outer cell membrane integrity. This, on the other hand, contrasts necrosis, which is typified by the rupture of the outer cell membrane and liberation of intracellular constituents into the interstitial spaces provoking inflammation. However, because not all forms of cell death involved cytochrome c release, or were independent of caspase activation, it hinted at the possibility that other modes of cell death may be operational. This led to the suggestion that “necroptosis”, a hybrid of apoptosis and necrosis, may be involved. Not surprisingly, apoptosis and necrosis appear to share common overlapping signaling pathways that link to the mitochondrion [2]. While the mitochondrion was first identified as the “powerhouse” of the cell for ATP synthesis, fatty acid oxidation, Ca2þ homeostasis, and other vital cellular process, it is now well appreciated that the mitochondrion plays a central and vital role in integrating signals for apoptosis, necrosis, and autophagy/mitophagy, respectively reviewed in Refs. [2,3]. From a biochemical perspective, it is difficult to reconcile how a given mitochondrial perturbation can drive apoptosis or necrosis. The answer to this conundrum stems from intricate studies that have revealed the initial determining factor for apoptosis or necrosis involves permeability changes to the outer mitochondrial membrane (OMM) by channel-forming Bax/Bak proteins for inducing apoptosis or early permeability changes to the inner mitochondrial membrane (IMM) that triggers permeability transition pore opening (mPTP) [4,5]. Alternatively, secondary necrosis resulting from late OMM permeability defects can trigger loss of mitochondrial ΔΨm, IMM swelling, and cell death [2]. Based on the overlapping features of apoptosis and necrosis that are linked to mitochondrial injury albeit OMM or IMM, the question arises: What is the ultimate mitochondrial event that determines whether the cell will undergo apoptosis, necrosis, or autophagy? What are the determinant factors that drive one pathway versus another? How are these pathways regulated and under what circumstances? Herein, we provide a brief overview of apoptosis, necrosis, and autophagy; their signaling pathways; and molecular effectors that commonly regulate the fate of cardiac myocytes.
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Apoptosis Extrinsic pathway Apoptosis is governed by two central pathways, an extrinsic pathway involving death receptors and an intrinsic pathway involving the mitochondrion (Fig). The extrinsic pathway is associated with death receptor signaling and has been studied classically in the context of Fas/CD95/Apo-1- and/or TNFα-mediated cell death. In brief, ligation of Fas/TNF receptor initiates a series of intracellular events that recruit a number of adapter proteins to the cytoplasmic face of the receptor. These proteins form homotypic and heterotypic interactions via conserved Caspase Activation and Recruitment Domain (CARD) and Death Domain (DD), which are domains that initiate a caspase signaling cascade and apoptosis. For example, Fas-associated death domain (FADD), TNFα Receptor Activator Death Domain (TRADD), and pro-caspase8 constitute the Death-Inducing Signaling Complex (DISC), which leads to the autocatalytic activation of caspase-8 and subsequent activation of downstream death effector caspases such as caspase-3, -6, and -7 [6,7]. In addition, caspase-8 and the DISC can be regulated by cellular inhibitors of apoptosis (c-IAP1/2) and anti-death proteins such as apoptosis repressor with CARD domain (ARC) [8,9]. Notably, proteolytic cleavage of the Bcl-2 protein Bid to t-Bid by caspase-8 during death receptor signaling targets t-Bid to mitochondria where it disrupts OMM to promote apoptosis [10]—thereby providing a molecular bridge between the extrinsic and intrinsic cell death pathways.
Intrinsic pathway The intrinsic or mitochondrial death pathway is regulated by members of the Bcl-2 gene family. These proteins are categorized by their multidomain structure and functional ability to prevent or promote cell death (Fig). For example, Bcl-2, Bcl-xL, and Mcl-1 are archetypic members of this group that promote cell survival while other family members including Bax and Bak and others promote cell death. Moreover, a subclass of Bcl-2 proteins known as “BH3”-only proteins which include Bid, Bad, Bim, Bmf, Noxa, BNip3, Nix/BNip3L, and Puma promote cell death by either directly or indirectly altering activity of Bax/Bak proteins or mitochondria following a specific death signal, reviewed in Refs. [2,11,12]. Recruitment and oligomerization of Bax/Bak to OMM promotes permeabilization of the OMM, which ultimately leads to the loss of mitochondrial ΔΨm and the release of apoptogenic proteins including cytochrome c, Smac, AIF-1, Endo-G, and others that promote apoptosis [5]. Bcl-2 suppresses death by sequestering Bax and Bak proteins, thereby preventing OMM permeabilization [13,14]. Notably, Bcl-2 can reportedly suppress cell death by inhibiting endoplasmic reticulum (ER) Ca2þ release by IP-3 receptor [15]. Whether Bax or Bak proteins are involved in ER-mediated Ca2þ release or are inhibited by Bcl-2 at the ER is currently unknown. Recently, defects associated with permeability changes to the IMM have been postulated to underlie mitochondrial permeability
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Fig – Interplay of cell death signaling pathways. Apoptosis and necrosis are mediated by death receptor (extrinsic) and mitochondrial (intrinsic). In death receptor pathway, TNF-α stimulates formation of complex I comprising TRADD, TRAF2, TRAF5, RIP1, and cIAPs. Complex I activates NF-κB signaling and promotes cell survival. Dissociation of complex I from TNFα receptor, results in the deubiquitination of RIP1, which together with FADD–RIP3 forms complex II. Active caspase-8 can either trigger downstream death effector capsases-3, -6, and -7 or inactivate RIP1/RIP3 complex through RIP1 cleavage. This releases caspase-8 from complex II triggering activation of the intrinsic mitochondrial apoptosis pathway. Apoptotic signaling in response to caspase-8 activation promotes proteolytic cleavage of Bid to t-Bid and recruitment of Bax/Bak proteins to mitochondrial outer membrane (OMM). Oligomerization of Bax/Bak provokes OMM permeabilization, resulting in the release of cytochrome c and other apoptotic proteins from mitochondria, resulting in further caspase activation and apoptosis. Inhibition of caspase-8 results in the formation of complex III, which contains phosphorylated RIP1 and RIP3 and adapter proteins for programmed necrosis. Mitochondrial-associated RIP1/RIP3 promotes glycolysis and glutaminolysis, ROS production, mitochondrial Ca2þ overload, loss of the inner mitochondrial membrane (IMM) integrity, permeability transition pore opening (mPTP), and necrosis. Mst-1 provides a molecular switch for apoptosis or autophagy through phosphorylation of Beclin-1. During cellular stress, phosphorylation of Beclin-1 by Mst-1 increases Beclin-1–Bcl-2 complexes displacing Bax from Bcl-2 resulting in apoptosis; alternatively, phosphorylation of Beclin-1 diminishes Beclin-1–Vps34–Atg14L complexes and inhibits autophagy. transition pore (mPTP) opening [16,17]. In this regard, mPTP opening leads to large amplitude mitochondrial swelling of the IMM and eventual rupture of the OMM [18]. While the structure of mPTP has not been resolved and several candidate proteins have been postulated to underlie the mPTP, including cyclophilin D, F-ATPase and inorganic phosphate transporter, adenine nucleotide transporter (ANT), and voltage-dependent ion channel (VDAC) proteins, the structure of the mPTP remains undetermined, as reviewed in Refs. [2,19–26]. Interestingly, ablation of ppif, the gene that encodes cyclophilin D, was sufficient to suppress not only mPTP opening but also necrotic cell death following myocardial infarction [24]. These findings strongly suggest that cyclophilin D is a component of mPTP and supports the notion
that IMM defects trigger necrosis. Because apoptosis generally involves the activation of intracellular proteases, DNAses, and mitochondrial injury, it can be detected biochemically in cells by assessing single-stranded nuclear DNA breaks, cytochrome c release, loss of mitochondrial membrane potential, and permeability changes to OMM with a variety of immunocytochemical probes reviewed in Refs. [27–29]. Notably, however, based on the temporal and spatial activation and subsequent detection of apoptosis, it is possible that early apoptotic events may coincide with events associated with necrosis. Hence, it is therefore, imperative that careful discrimination between early and late events associated with apoptotic and necrotic cell death (discussed below) be considered during analysis.
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Necrosis Molecular regulation of necrosis Necrotic cell death is characterized by the rupture of the cell membrane and liberation of intracellular contents into extracellular space. In contrast to apoptosis, which is an ATP-driven process, necrotic death typically occurs under conditions of low cellular ATP. As stated above, necrosis was initially viewed as an accidental or uncontrolled form of cell death; however, new emerging evidence suggests that necrosis like apoptosis is a regulated event. In fact, programmed necrosis or “necroptosis” involves several overlapping features with apoptosis that converge upon the mitochondrion. The best example of programmed necrosis is illustrated by the signaling cascade involving the serine/ threonine kinases—Receptor-Interacting Proteins (RIP1 and RIP3) [30–32]. Initially, the RIP proteins were first identified as signaling adapters that were linked to the TNFα receptor for downstream NF-κB activation. It is now well appreciated that these proteins play dual roles not only in regulating NF-κB for cell survival but also in promoting mitochondrial injury and necrotic cell death. The differential outcome vis a vis cell life or death is likely dependent upon wiring of the cell and propensity for a given signaling pathway downstream of TNFα receptor to activate NF-κB or necrosis. For example, cardiac myocytes are relatively resistant to the cytotoxic effects of TNFα because the dominant signaling pathway activated by TNFα leads to NF-κB activation, which promotes survival [33]. However, rendering cardiac myocytes deficient for TNFα receptor signaling or for NF-κB activation unmasks the cytotoxic effects of TNFα and triggers necrotic cell death [34]. In contrast to other cells, certain cancer cells are sensitive to TNFα-stimulated necrosis because the dominant signaling pathway through CD95/Apo-1 predisposes cells to necrosis and not apoptosis. This raises the question: How do cells activate a given cell death pathway? Using a death receptor model (Fig), stimulation of cells with TNFα results in the recruitment and formation of a series of signaling complexes or “necrosomes” at the TNFα receptor reviewed in [32]. Complex I contains TRADD along with RIP1, TRAF2, and c-IAP1/2, and it leads to NF-κB activation. Following endocytosis and dissociation of complex I from TNFα receptor, RIP1 becomes de-ubiquitinylated by cylindromatosis and A20, a crucial step for the formation of complex II. In complex II, association of FADD along with de-ubiquitinated RIP1, RIP3, and TRADD triggers caspase-8 activation, which then proteolytically cleaves and inactivates RIP1, blocking necrosis while permitting activation of death effector caspase-3 and caspase-7 and apoptosis. However, in the absence of caspase-8, activated RIP1 kinase associates with activated RIP3 (complex II) at the mitochondrion increasing metabolic utilization of carbohydrates and glutamate [30,35]. Notably, mitochondrial activated RIP3 promotes metabolic enzyme activity of cytosolic glycogen phosphorylase (PYGL), glutamate ammonia ligase (GLUL), and the glutaminolysisinitiating enzyme (GLUD1), which ultimately increases glutamine production and glycogenolysis [30]. This in turn stimulates IMM defects leading mPTP, ROS production,
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which presumably triggers necrosis [36]. Pharmacological inhibition of RIP1 activity with Necrostatin-1 reduced myocardial infarct size in a model following ischemia–reperfusion, presumably by preventing RIP1-dependent targeting of RIP3 to mitochondria [37,38]—supporting the notion that RIP1/ RIP3 signaling induces IMM defects and necrosis. The exact mode by which RIP1/RIP3 drives mitochondrial IMM defects and necrosis remains to be completely worked out since inhibition of RIP1 with Necrostatin-1 was also shown to block autophagy, suggesting a tentative link between these processes.
Mitochondrial dynamics and cell death signaling Another interesting relationship involves mitochondrial dynamics through altered fission and fusion events and necrotic cell death. RIP3 can reportedly interact with the mitochondrial protein phosphatase (PGAM5) [39]. PGAM5 activates the mitochondrial fission protein Dynamin-related protein 1 (Drp1) at the OMM by dephosphorylation of Ser637, triggering mitochondrial fission [39]. These findings support a model whereby RIP3–Drp1 interaction regulates mitochondrial fission events linked to programmed necrosis. Indeed, alterations to IMM and ΔΨm have been postulated as central underlying events of necrosis. However, the relationship between OMM and IMM defects for inducing mitochondrial fission/fusion events and necrosis is less clear. A recent study showed combined deletion of Bax/Bak proteins reduced necrotic injury following myocardial infarction in vivo, which was reduced further by combined deletion of cyclophilin D [28]. Similarly, Mitofusin 2 (Mfn2) ablation was also found to be cardioprotective, presumably by preventing mitochondrial fusion. In that study, mitochondrial fusion induced necrotic cell death secondary to MPTP opening. These findings highlight the interrelationship of Bak/Bax proteins in apoptosis and necrosis linked through mitochondrial fission/fusion events [28]. Whether RIP1 or RIP3 is involved in mitochondrial fission/fusion and IMM defects leading to necrosis in cells deficient for Bax/Bak is undetermined. While apoptotic cell death is typified by cellular abnormalities that lead to the biochemical demise independent of cell membrane rupture, necrosis on the other hand has classically been associated with the rupture of the outer cell membrane and release of cytoplasmic enzymes including LDH and cTnT and loss of nuclear High-Mobility Group Box 1 (HMGB1) protein [40,41]. As stated above, early permeability changes to the mitochondrial inner membrane leading to permeability transition pore opening are considered to be more contemporary events associated with necrotic cell death [2]. Hence, early versus late changes in OMM or IMM integrity may reflect facets of common overlapping signaling pathways that link apoptosis and necrosis, respectively.
Autophagy Molecular regulation of autophagy Autophagy is an evolutionarily conserved catabolic process by which macromolecules, mis-folded proteins, and damaged organelles (e.g., mitochondria) are discarded (mitophagy) for
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maintaining quality control or used as a nutrient source for ATP production during cellular stress [42]. The autophagic pathway mediated mainly by Beclin-1 autophagy-related gene (Atg 6) comprises four steps; initiation, engulfment of cargo by a double-membrane structure, formation of autophagolysosome via the fusion of autophagosome and lysosome, and the final process of lysosomal degradation by acid hydrolases resulting in the recycling of lysosomal contents reviewed in Refs. [43–46]. Because microtubule Light Chain 3-I (LC3-I) protein conjugated to phosphatidylethanolamine (LC3-I-phosphatidylethanolamine) is recruited to autophagosomes and is subsequently proteolytically cleaved to LC3-II, the turnover of LC3-II along with another autophagosome membrane associated protein, p62, can be monitored and used as an index of autophagosome formation and clearance. Hence, detection of LC3-II, p62, or Beclin-1 can be used as indices of autophagy [47]. Autophagy is essential for maintaining tissue homeostasis and is generally viewed as an important cellular process vital for cell survival. However, there are examples where deregulated autophagy beyond a certain threshold can be detrimental and can promote cell death [3,48]. Indeed, although a defined genetic pathway for autophagic cell death has not been identified, it is well appreciated that at least in the context of the heart there are several examples where either too little or too much autophagy has been associated with cardiac dysfunction and heart failure. This “Goldilocks” phenomenon highlights the intricate balance between autophagy activation and inhibition for normal cardiac function [43,49]. For example, in the context of ischemia–reperfusion, activation of autophagy during early ischemia is protective, whereas delayed or late activation of autophagy during reperfusion is detrimental and provokes death [46,50]. Hence, it is not surprising that several of the factors that regulate apoptosis have overlapping roles with autophagy. In particular, the Bcl-2 protein Beclin-1 plays a crucial role in initiating the early steps in autophagosome formation. Notably, Atg5-deficient mice exhibit impaired removal of apoptotic bodies during development [51,52]. While inhibiting autophagy by Beclin-1 or Atg7 knock-down suppressed apoptotic cell death [51]—demonstrating a functional relationship between these cellular processes. Another interesting feature linking apoptosis and autophagy involves the coordinated clearance of dysfunctional mitochondria during cellular stress. In this regard, the clearance of damaged mitochondria by mitophagy ensures the cell to maintain adequate mitochondrial quality control [53,54]. Moreover, given the involvement of mitochondria in regulating intrinsic cell death signaling, mitophagy can also be seen as an important survival mechanism for removing mitochondria that would otherwise drive cell death. The underlying mechanisms by which mitochondria are removed by cells are an active area of investigation and believed to involve subset of BH3-only proteins [11,55]. For example, mitochondrial clearance by mitophagy has been shown to be crucial for reticulocyte maturation. Here, it was demonstrated that the assembly of Nix/Bnip3L–Atg8–LC3-II complexes on reticulocyte mitochondria was crucial for efficient mitochondrial clearance and maturation of red blood cells [56]. Mitochondrial clearance is regulated by the phosphatase and tensin homolog deleted in chromosome 10 (PTEN)-induced
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putative kinase 1 (PINK1) and the E3 ligase Parkinson protein-2 (Parkin) [57–59]. Phosphorylation of Mitochondrial fusion 2 protein (Mfn2) by PINK recruits Parkin to mitochondria, resulting in the ubiquitination and degradation of Mfn2 activity reviewed in Refs. [59,60]. Presumably, increased mitochondrial fission from loss of Mfn2 is critical for efficient packaging and digestion of autophagosome containing mitochondria. Interestingly, conditional cardiac knockout of Mfn2 gene impaired Parkin-mediated mitophagy, increasing ROS production, cardiac dysfunction, and heart failure [57]. Though proteins such as Bnip3 can reportedly trigger mitochondrial fission, this likely occurs as an indirect effect from the loss of mitochondrial ΔΨm, which would recruit Drp1 and Parkin to mitochondria and mitophagy [61]. These findings highlight the signaling pathways that link mitochondrial dynamics to apoptosis and mitophagy for regulating cell fate. As stated earlier, autophagy and apoptosis share common regulatory pathways that converge upon the mitochondrion. Apoptosis may be initiated by the same signals that trigger autophagy and vice versa. Again, this raises the question: How does the cell evoke a different cellular response apoptosis or autophagy through the same signaling proteins? The answer to this question comes from recent study by the Sadoshima laboratory that sheds light on this issue, see the Fig. Maejima et al. [62] described a novel mechanism that involves the intracellular Mst-1 kinase to explain how cells differentially regulate autophagy and apoptosis during cellular stress. In this report, Maejima et al. [62] demonstrated that Mammalian Sterile 20- like kinase-1 (Mst-1) dually regulates apoptosis or autophagy simply by altering molecular complexes between Bcl-2 and Beclin-1 and Beclin-1–Atg14L– Vsp34. Notably, phosphorylation of Beclin-1 by Mst-1 during cellular stress increased Beclin-1's affinity for Bcl-2, which displaced Bax from Bcl-2 resulting in apoptosis; at the same time, decreasing Beclin-1's affinity for Atg14L–Vsp34 thereby inhibiting autophagy. Hence, the major discovery of this work demonstrates that Mst-1 provides a molecular switch that dually regulates two major cellular processes involved in regulating cell fate through the phosphorylation of Beclin-1. Interestingly, a recent report by Liu et al. [1] demonstrated that suppression by inhibition of autophagy signaling resulted in an alternative form of cell death distinct from apoptosis and necrosis referred to as “autosis” in hippocampal neuronal cells subjected to hypoxic/ischemic injury. These interesting findings suggest that autophagy under certain conditions is maladaptive and promotes cell death. At present, it is currently unknown whether autosis is a restricted feature of the hippocampus or also occurs in the heart.
Conclusion In this review, we provide an overview of the interplay between the signaling pathways that govern cell death in the heart. While apoptosis, necrosis, and autophagy have been traditionally viewed as distinct signaling entities, several lines of investigation have revealed these pathways are biochemically and functionally linked by common proteins. Even more profound are recent studies where apoptosis,
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necrosis, and autophagy appear to be mutually dependent and obligatorily linked to mitochondrial function. However, despite the current literature and presented work, several questions remain unanswered. For example, how does the cell decide to switch from apoptosis or necrosis? Is this strictly a temporal event linking common Bcl-2 proteins to a common cell death pathway? For that matter, at what point does the cell switch from adaptive autophagy to maladaptive autophagy (autosis) resulting in death? What are the breaks on autophagy or mitochondrial-regulated programmed necrosis in the heart? A better understanding of the signaling pathways that govern cell fate more generally will lead to the development of new therapies to activate or suppress these pathways in the treatment of human diseases. These questions and several others will undoubtedly provide the fodder for future investigations into this very exciting and dynamic field.
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www.elsevier.com/locate/tcm
The interplay between cell death signaling pathways in the heart Agnieszka K. Bialaa,b and Lorrie A. Kirshenbauma,b,c,n a
The Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of Manitoba, Centre Rm. 3016, 351 TachéAvenue, Winnipeg, Manitoba, Canada R2H 2A6 b Department of Physiology, College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada c Department of Pharmacology and Therapeutics, College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
abstra ct To date, one of the most intriguing and compelling concepts to impact contemporary cell biology is the notion that cell fate is “programmed” or genetically controlled. Indeed, the regulation of cell fate is crucial for embryonic development, and tissue homeostasis. Given the importance of removing damaged or irreversibly injured cells from the body, it is not surprising that defects in the regulatory mechanisms that govern cell death and/or survival more generally have been implicated in a number of human pathologies including cancer, neurodegenerative diseases, and cardiac failure. Several processes involved in the regulation of cell fate through apoptosis, necrosis, and autophagy are commonly linked through the actions of certain Bcl-2 proteins that act on the mitochondrion. For example, the Bcl-2 protein Beclin-1 is actively involved in the clearance of damaged mitochondria via mitophagy, while other Bcl-2 proteins such as Bax/Bak can initiate apoptosis or necrotic signaling pathways. The overlapping and redundant nature of these proteins highlights their evolutionary importance for regulating cardiac cell survival and death during normal and disease states. Here, we explore the interrelationship between these signaling pathways and the cellular effectors that influence cardiac cell fate. & 2014 Published by Elsevier Inc.
Introduction Despite the significant advances in cardiovascular research over the past two decades, heart disease still remains a significant cause of morbidity and mortality and is reaching pandemic proportions worldwide. Indeed, the combinations of the Western diet, sedentary lifestyles, and genetic predispositions have been considered the prevailing underlying risk factors for cardiovascular disease, including diabetes, hypertension, atherosclerosis, ischemic heart disease, and heart failure. In contrast to other cells of the body that retain the ability for regeneration and self-renewal, cardiac myocytes
lose this property shortly after birth. Hence, the loss of functional cardiac cells has been suggested as an underlying feature of ventricular remodeling and diminished cardiac pump performance after injury. Therefore, one unifying theme toward the ultimate therapeutic goal of preventing heart failure would be to suppress the inappropriate loss of cardiac cells after myocardial injury. It is now appreciated that cardiac cells die during ischemic injury or postmyocardial infarction by highly orchestrated genetically programmed cell death pathways involving apoptosis, necrosis, and in some instances autophagy. Notably, while autophagy is generally considered a catabolic survival mechanism,
The authors have indicated there are no conflicts of interest. This work was supported by Grants to L.A.K. from the Canadian Institutes of Health Research (MOP42402, MOP74456), Canada. A.K.B. holds a post-doctoral fellowship from the Manitoba Health Research Council and IMPACT-CIHR. n Corresponding author at: Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre Rm. 3016, 351 Taché Avenue, Winnipeg, Manitoba, Canada, R2H 2A6. E-mail address: [email protected] (L.A. Kirshenbaum). http://dx.doi.org/10.1016/j.tcm.2014.08.002 1050-1738/& 2014 Published by Elsevier Inc.
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de-regulated autophagy can be maladaptive and promote cell death by the novel process recently described in neuronal tissue termed “autosis” [1]. Indeed, the evidence for apoptosis, necrosis, and autophagy has been detected in a number of cardiac pathologies including, ischemia followed by reperfusion, after myocardial infarction, and in the failing human heart. Interestingly, in some instances, apoptosis, necrosis and autophagy have been detected in the heart under the same pathological conditions, suggesting that these signaling pathways may be commonly linked through overlapping cellular effectors that regulate cell fate. Insights into the signaling mechanisms that underlie cell death have revealed apoptosis can occur via an extrinsic pathway involving death receptors and intrinsic pathway involving the mitochondrion. Although these pathways were first considered to be functionally independent, there is emerging evidence that these pathways are commonly linked to the mitochondrion by certain Bcl-2 proteins. Indeed, cellular defects associated with apoptosis have classically involved mitochondrial cytochrome c release, caspase activation, and DNA fragmentation with the loss of outer cell membrane integrity. This, on the other hand, contrasts necrosis, which is typified by the rupture of the outer cell membrane and liberation of intracellular constituents into the interstitial spaces provoking inflammation. However, because not all forms of cell death involved cytochrome c release, or were independent of caspase activation, it hinted at the possibility that other modes of cell death may be operational. This led to the suggestion that “necroptosis”, a hybrid of apoptosis and necrosis, may be involved. Not surprisingly, apoptosis and necrosis appear to share common overlapping signaling pathways that link to the mitochondrion [2]. While the mitochondrion was first identified as the “powerhouse” of the cell for ATP synthesis, fatty acid oxidation, Ca2þ homeostasis, and other vital cellular process, it is now well appreciated that the mitochondrion plays a central and vital role in integrating signals for apoptosis, necrosis, and autophagy/mitophagy, respectively reviewed in Refs. [2,3]. From a biochemical perspective, it is difficult to reconcile how a given mitochondrial perturbation can drive apoptosis or necrosis. The answer to this conundrum stems from intricate studies that have revealed the initial determining factor for apoptosis or necrosis involves permeability changes to the outer mitochondrial membrane (OMM) by channel-forming Bax/Bak proteins for inducing apoptosis or early permeability changes to the inner mitochondrial membrane (IMM) that triggers permeability transition pore opening (mPTP) [4,5]. Alternatively, secondary necrosis resulting from late OMM permeability defects can trigger loss of mitochondrial ΔΨm, IMM swelling, and cell death [2]. Based on the overlapping features of apoptosis and necrosis that are linked to mitochondrial injury albeit OMM or IMM, the question arises: What is the ultimate mitochondrial event that determines whether the cell will undergo apoptosis, necrosis, or autophagy? What are the determinant factors that drive one pathway versus another? How are these pathways regulated and under what circumstances? Herein, we provide a brief overview of apoptosis, necrosis, and autophagy; their signaling pathways; and molecular effectors that commonly regulate the fate of cardiac myocytes.
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Apoptosis Extrinsic pathway Apoptosis is governed by two central pathways, an extrinsic pathway involving death receptors and an intrinsic pathway involving the mitochondrion (Fig). The extrinsic pathway is associated with death receptor signaling and has been studied classically in the context of Fas/CD95/Apo-1- and/or TNFα-mediated cell death. In brief, ligation of Fas/TNF receptor initiates a series of intracellular events that recruit a number of adapter proteins to the cytoplasmic face of the receptor. These proteins form homotypic and heterotypic interactions via conserved Caspase Activation and Recruitment Domain (CARD) and Death Domain (DD), which are domains that initiate a caspase signaling cascade and apoptosis. For example, Fas-associated death domain (FADD), TNFα Receptor Activator Death Domain (TRADD), and pro-caspase8 constitute the Death-Inducing Signaling Complex (DISC), which leads to the autocatalytic activation of caspase-8 and subsequent activation of downstream death effector caspases such as caspase-3, -6, and -7 [6,7]. In addition, caspase-8 and the DISC can be regulated by cellular inhibitors of apoptosis (c-IAP1/2) and anti-death proteins such as apoptosis repressor with CARD domain (ARC) [8,9]. Notably, proteolytic cleavage of the Bcl-2 protein Bid to t-Bid by caspase-8 during death receptor signaling targets t-Bid to mitochondria where it disrupts OMM to promote apoptosis [10]—thereby providing a molecular bridge between the extrinsic and intrinsic cell death pathways.
Intrinsic pathway The intrinsic or mitochondrial death pathway is regulated by members of the Bcl-2 gene family. These proteins are categorized by their multidomain structure and functional ability to prevent or promote cell death (Fig). For example, Bcl-2, Bcl-xL, and Mcl-1 are archetypic members of this group that promote cell survival while other family members including Bax and Bak and others promote cell death. Moreover, a subclass of Bcl-2 proteins known as “BH3”-only proteins which include Bid, Bad, Bim, Bmf, Noxa, BNip3, Nix/BNip3L, and Puma promote cell death by either directly or indirectly altering activity of Bax/Bak proteins or mitochondria following a specific death signal, reviewed in Refs. [2,11,12]. Recruitment and oligomerization of Bax/Bak to OMM promotes permeabilization of the OMM, which ultimately leads to the loss of mitochondrial ΔΨm and the release of apoptogenic proteins including cytochrome c, Smac, AIF-1, Endo-G, and others that promote apoptosis [5]. Bcl-2 suppresses death by sequestering Bax and Bak proteins, thereby preventing OMM permeabilization [13,14]. Notably, Bcl-2 can reportedly suppress cell death by inhibiting endoplasmic reticulum (ER) Ca2þ release by IP-3 receptor [15]. Whether Bax or Bak proteins are involved in ER-mediated Ca2þ release or are inhibited by Bcl-2 at the ER is currently unknown. Recently, defects associated with permeability changes to the IMM have been postulated to underlie mitochondrial permeability
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Fig – Interplay of cell death signaling pathways. Apoptosis and necrosis are mediated by death receptor (extrinsic) and mitochondrial (intrinsic). In death receptor pathway, TNF-α stimulates formation of complex I comprising TRADD, TRAF2, TRAF5, RIP1, and cIAPs. Complex I activates NF-κB signaling and promotes cell survival. Dissociation of complex I from TNFα receptor, results in the deubiquitination of RIP1, which together with FADD–RIP3 forms complex II. Active caspase-8 can either trigger downstream death effector capsases-3, -6, and -7 or inactivate RIP1/RIP3 complex through RIP1 cleavage. This releases caspase-8 from complex II triggering activation of the intrinsic mitochondrial apoptosis pathway. Apoptotic signaling in response to caspase-8 activation promotes proteolytic cleavage of Bid to t-Bid and recruitment of Bax/Bak proteins to mitochondrial outer membrane (OMM). Oligomerization of Bax/Bak provokes OMM permeabilization, resulting in the release of cytochrome c and other apoptotic proteins from mitochondria, resulting in further caspase activation and apoptosis. Inhibition of caspase-8 results in the formation of complex III, which contains phosphorylated RIP1 and RIP3 and adapter proteins for programmed necrosis. Mitochondrial-associated RIP1/RIP3 promotes glycolysis and glutaminolysis, ROS production, mitochondrial Ca2þ overload, loss of the inner mitochondrial membrane (IMM) integrity, permeability transition pore opening (mPTP), and necrosis. Mst-1 provides a molecular switch for apoptosis or autophagy through phosphorylation of Beclin-1. During cellular stress, phosphorylation of Beclin-1 by Mst-1 increases Beclin-1–Bcl-2 complexes displacing Bax from Bcl-2 resulting in apoptosis; alternatively, phosphorylation of Beclin-1 diminishes Beclin-1–Vps34–Atg14L complexes and inhibits autophagy. transition pore (mPTP) opening [16,17]. In this regard, mPTP opening leads to large amplitude mitochondrial swelling of the IMM and eventual rupture of the OMM [18]. While the structure of mPTP has not been resolved and several candidate proteins have been postulated to underlie the mPTP, including cyclophilin D, F-ATPase and inorganic phosphate transporter, adenine nucleotide transporter (ANT), and voltage-dependent ion channel (VDAC) proteins, the structure of the mPTP remains undetermined, as reviewed in Refs. [2,19–26]. Interestingly, ablation of ppif, the gene that encodes cyclophilin D, was sufficient to suppress not only mPTP opening but also necrotic cell death following myocardial infarction [24]. These findings strongly suggest that cyclophilin D is a component of mPTP and supports the notion
that IMM defects trigger necrosis. Because apoptosis generally involves the activation of intracellular proteases, DNAses, and mitochondrial injury, it can be detected biochemically in cells by assessing single-stranded nuclear DNA breaks, cytochrome c release, loss of mitochondrial membrane potential, and permeability changes to OMM with a variety of immunocytochemical probes reviewed in Refs. [27–29]. Notably, however, based on the temporal and spatial activation and subsequent detection of apoptosis, it is possible that early apoptotic events may coincide with events associated with necrosis. Hence, it is therefore, imperative that careful discrimination between early and late events associated with apoptotic and necrotic cell death (discussed below) be considered during analysis.
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Necrosis Molecular regulation of necrosis Necrotic cell death is characterized by the rupture of the cell membrane and liberation of intracellular contents into extracellular space. In contrast to apoptosis, which is an ATP-driven process, necrotic death typically occurs under conditions of low cellular ATP. As stated above, necrosis was initially viewed as an accidental or uncontrolled form of cell death; however, new emerging evidence suggests that necrosis like apoptosis is a regulated event. In fact, programmed necrosis or “necroptosis” involves several overlapping features with apoptosis that converge upon the mitochondrion. The best example of programmed necrosis is illustrated by the signaling cascade involving the serine/ threonine kinases—Receptor-Interacting Proteins (RIP1 and RIP3) [30–32]. Initially, the RIP proteins were first identified as signaling adapters that were linked to the TNFα receptor for downstream NF-κB activation. It is now well appreciated that these proteins play dual roles not only in regulating NF-κB for cell survival but also in promoting mitochondrial injury and necrotic cell death. The differential outcome vis a vis cell life or death is likely dependent upon wiring of the cell and propensity for a given signaling pathway downstream of TNFα receptor to activate NF-κB or necrosis. For example, cardiac myocytes are relatively resistant to the cytotoxic effects of TNFα because the dominant signaling pathway activated by TNFα leads to NF-κB activation, which promotes survival [33]. However, rendering cardiac myocytes deficient for TNFα receptor signaling or for NF-κB activation unmasks the cytotoxic effects of TNFα and triggers necrotic cell death [34]. In contrast to other cells, certain cancer cells are sensitive to TNFα-stimulated necrosis because the dominant signaling pathway through CD95/Apo-1 predisposes cells to necrosis and not apoptosis. This raises the question: How do cells activate a given cell death pathway? Using a death receptor model (Fig), stimulation of cells with TNFα results in the recruitment and formation of a series of signaling complexes or “necrosomes” at the TNFα receptor reviewed in [32]. Complex I contains TRADD along with RIP1, TRAF2, and c-IAP1/2, and it leads to NF-κB activation. Following endocytosis and dissociation of complex I from TNFα receptor, RIP1 becomes de-ubiquitinylated by cylindromatosis and A20, a crucial step for the formation of complex II. In complex II, association of FADD along with de-ubiquitinated RIP1, RIP3, and TRADD triggers caspase-8 activation, which then proteolytically cleaves and inactivates RIP1, blocking necrosis while permitting activation of death effector caspase-3 and caspase-7 and apoptosis. However, in the absence of caspase-8, activated RIP1 kinase associates with activated RIP3 (complex II) at the mitochondrion increasing metabolic utilization of carbohydrates and glutamate [30,35]. Notably, mitochondrial activated RIP3 promotes metabolic enzyme activity of cytosolic glycogen phosphorylase (PYGL), glutamate ammonia ligase (GLUL), and the glutaminolysisinitiating enzyme (GLUD1), which ultimately increases glutamine production and glycogenolysis [30]. This in turn stimulates IMM defects leading mPTP, ROS production,
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which presumably triggers necrosis [36]. Pharmacological inhibition of RIP1 activity with Necrostatin-1 reduced myocardial infarct size in a model following ischemia–reperfusion, presumably by preventing RIP1-dependent targeting of RIP3 to mitochondria [37,38]—supporting the notion that RIP1/ RIP3 signaling induces IMM defects and necrosis. The exact mode by which RIP1/RIP3 drives mitochondrial IMM defects and necrosis remains to be completely worked out since inhibition of RIP1 with Necrostatin-1 was also shown to block autophagy, suggesting a tentative link between these processes.
Mitochondrial dynamics and cell death signaling Another interesting relationship involves mitochondrial dynamics through altered fission and fusion events and necrotic cell death. RIP3 can reportedly interact with the mitochondrial protein phosphatase (PGAM5) [39]. PGAM5 activates the mitochondrial fission protein Dynamin-related protein 1 (Drp1) at the OMM by dephosphorylation of Ser637, triggering mitochondrial fission [39]. These findings support a model whereby RIP3–Drp1 interaction regulates mitochondrial fission events linked to programmed necrosis. Indeed, alterations to IMM and ΔΨm have been postulated as central underlying events of necrosis. However, the relationship between OMM and IMM defects for inducing mitochondrial fission/fusion events and necrosis is less clear. A recent study showed combined deletion of Bax/Bak proteins reduced necrotic injury following myocardial infarction in vivo, which was reduced further by combined deletion of cyclophilin D [28]. Similarly, Mitofusin 2 (Mfn2) ablation was also found to be cardioprotective, presumably by preventing mitochondrial fusion. In that study, mitochondrial fusion induced necrotic cell death secondary to MPTP opening. These findings highlight the interrelationship of Bak/Bax proteins in apoptosis and necrosis linked through mitochondrial fission/fusion events [28]. Whether RIP1 or RIP3 is involved in mitochondrial fission/fusion and IMM defects leading to necrosis in cells deficient for Bax/Bak is undetermined. While apoptotic cell death is typified by cellular abnormalities that lead to the biochemical demise independent of cell membrane rupture, necrosis on the other hand has classically been associated with the rupture of the outer cell membrane and release of cytoplasmic enzymes including LDH and cTnT and loss of nuclear High-Mobility Group Box 1 (HMGB1) protein [40,41]. As stated above, early permeability changes to the mitochondrial inner membrane leading to permeability transition pore opening are considered to be more contemporary events associated with necrotic cell death [2]. Hence, early versus late changes in OMM or IMM integrity may reflect facets of common overlapping signaling pathways that link apoptosis and necrosis, respectively.
Autophagy Molecular regulation of autophagy Autophagy is an evolutionarily conserved catabolic process by which macromolecules, mis-folded proteins, and damaged organelles (e.g., mitochondria) are discarded (mitophagy) for
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maintaining quality control or used as a nutrient source for ATP production during cellular stress [42]. The autophagic pathway mediated mainly by Beclin-1 autophagy-related gene (Atg 6) comprises four steps; initiation, engulfment of cargo by a double-membrane structure, formation of autophagolysosome via the fusion of autophagosome and lysosome, and the final process of lysosomal degradation by acid hydrolases resulting in the recycling of lysosomal contents reviewed in Refs. [43–46]. Because microtubule Light Chain 3-I (LC3-I) protein conjugated to phosphatidylethanolamine (LC3-I-phosphatidylethanolamine) is recruited to autophagosomes and is subsequently proteolytically cleaved to LC3-II, the turnover of LC3-II along with another autophagosome membrane associated protein, p62, can be monitored and used as an index of autophagosome formation and clearance. Hence, detection of LC3-II, p62, or Beclin-1 can be used as indices of autophagy [47]. Autophagy is essential for maintaining tissue homeostasis and is generally viewed as an important cellular process vital for cell survival. However, there are examples where deregulated autophagy beyond a certain threshold can be detrimental and can promote cell death [3,48]. Indeed, although a defined genetic pathway for autophagic cell death has not been identified, it is well appreciated that at least in the context of the heart there are several examples where either too little or too much autophagy has been associated with cardiac dysfunction and heart failure. This “Goldilocks” phenomenon highlights the intricate balance between autophagy activation and inhibition for normal cardiac function [43,49]. For example, in the context of ischemia–reperfusion, activation of autophagy during early ischemia is protective, whereas delayed or late activation of autophagy during reperfusion is detrimental and provokes death [46,50]. Hence, it is not surprising that several of the factors that regulate apoptosis have overlapping roles with autophagy. In particular, the Bcl-2 protein Beclin-1 plays a crucial role in initiating the early steps in autophagosome formation. Notably, Atg5-deficient mice exhibit impaired removal of apoptotic bodies during development [51,52]. While inhibiting autophagy by Beclin-1 or Atg7 knock-down suppressed apoptotic cell death [51]—demonstrating a functional relationship between these cellular processes. Another interesting feature linking apoptosis and autophagy involves the coordinated clearance of dysfunctional mitochondria during cellular stress. In this regard, the clearance of damaged mitochondria by mitophagy ensures the cell to maintain adequate mitochondrial quality control [53,54]. Moreover, given the involvement of mitochondria in regulating intrinsic cell death signaling, mitophagy can also be seen as an important survival mechanism for removing mitochondria that would otherwise drive cell death. The underlying mechanisms by which mitochondria are removed by cells are an active area of investigation and believed to involve subset of BH3-only proteins [11,55]. For example, mitochondrial clearance by mitophagy has been shown to be crucial for reticulocyte maturation. Here, it was demonstrated that the assembly of Nix/Bnip3L–Atg8–LC3-II complexes on reticulocyte mitochondria was crucial for efficient mitochondrial clearance and maturation of red blood cells [56]. Mitochondrial clearance is regulated by the phosphatase and tensin homolog deleted in chromosome 10 (PTEN)-induced
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putative kinase 1 (PINK1) and the E3 ligase Parkinson protein-2 (Parkin) [57–59]. Phosphorylation of Mitochondrial fusion 2 protein (Mfn2) by PINK recruits Parkin to mitochondria, resulting in the ubiquitination and degradation of Mfn2 activity reviewed in Refs. [59,60]. Presumably, increased mitochondrial fission from loss of Mfn2 is critical for efficient packaging and digestion of autophagosome containing mitochondria. Interestingly, conditional cardiac knockout of Mfn2 gene impaired Parkin-mediated mitophagy, increasing ROS production, cardiac dysfunction, and heart failure [57]. Though proteins such as Bnip3 can reportedly trigger mitochondrial fission, this likely occurs as an indirect effect from the loss of mitochondrial ΔΨm, which would recruit Drp1 and Parkin to mitochondria and mitophagy [61]. These findings highlight the signaling pathways that link mitochondrial dynamics to apoptosis and mitophagy for regulating cell fate. As stated earlier, autophagy and apoptosis share common regulatory pathways that converge upon the mitochondrion. Apoptosis may be initiated by the same signals that trigger autophagy and vice versa. Again, this raises the question: How does the cell evoke a different cellular response apoptosis or autophagy through the same signaling proteins? The answer to this question comes from recent study by the Sadoshima laboratory that sheds light on this issue, see the Fig. Maejima et al. [62] described a novel mechanism that involves the intracellular Mst-1 kinase to explain how cells differentially regulate autophagy and apoptosis during cellular stress. In this report, Maejima et al. [62] demonstrated that Mammalian Sterile 20- like kinase-1 (Mst-1) dually regulates apoptosis or autophagy simply by altering molecular complexes between Bcl-2 and Beclin-1 and Beclin-1–Atg14L– Vsp34. Notably, phosphorylation of Beclin-1 by Mst-1 during cellular stress increased Beclin-1's affinity for Bcl-2, which displaced Bax from Bcl-2 resulting in apoptosis; at the same time, decreasing Beclin-1's affinity for Atg14L–Vsp34 thereby inhibiting autophagy. Hence, the major discovery of this work demonstrates that Mst-1 provides a molecular switch that dually regulates two major cellular processes involved in regulating cell fate through the phosphorylation of Beclin-1. Interestingly, a recent report by Liu et al. [1] demonstrated that suppression by inhibition of autophagy signaling resulted in an alternative form of cell death distinct from apoptosis and necrosis referred to as “autosis” in hippocampal neuronal cells subjected to hypoxic/ischemic injury. These interesting findings suggest that autophagy under certain conditions is maladaptive and promotes cell death. At present, it is currently unknown whether autosis is a restricted feature of the hippocampus or also occurs in the heart.
Conclusion In this review, we provide an overview of the interplay between the signaling pathways that govern cell death in the heart. While apoptosis, necrosis, and autophagy have been traditionally viewed as distinct signaling entities, several lines of investigation have revealed these pathways are biochemically and functionally linked by common proteins. Even more profound are recent studies where apoptosis,
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necrosis, and autophagy appear to be mutually dependent and obligatorily linked to mitochondrial function. However, despite the current literature and presented work, several questions remain unanswered. For example, how does the cell decide to switch from apoptosis or necrosis? Is this strictly a temporal event linking common Bcl-2 proteins to a common cell death pathway? For that matter, at what point does the cell switch from adaptive autophagy to maladaptive autophagy (autosis) resulting in death? What are the breaks on autophagy or mitochondrial-regulated programmed necrosis in the heart? A better understanding of the signaling pathways that govern cell fate more generally will lead to the development of new therapies to activate or suppress these pathways in the treatment of human diseases. These questions and several others will undoubtedly provide the fodder for future investigations into this very exciting and dynamic field.
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