Fundamental Mechanisms of Regulated Cell Death and Implications for Heart Disease

Abstract

Twelve regulated cell death programs have been described. We review in detail the basic biology of nine including death receptor-mediated apoptosis, death receptor-mediated necrosis (necroptosis), mitochondrial-mediated apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, ferroptosis, pyroptosis, parthanatos, and immunogenic cell death. This is followed by a dissection of the roles of these cell death programs in the major cardiac syndromes: myocardial infarction and heart failure. The most important conclusion relevant to heart disease is that regulated forms of cardiomyocyte death play important roles in both myocardial infarction with reperfusion (ischemia/reperfusion) and heart failure. While a role for apoptosis in ischemia/reperfusion cannot be excluded, regulated forms of necrosis, through both death receptor and mitochondrial pathways, are critical. Ferroptosis and parthanatos are also likely important in ischemia/reperfusion, although it is unclear if these entities are functioning as independent death programs or as amplification mechanisms for necrotic cell death. Pyroptosis may also contribute to ischemia/reperfusion injury, but potentially through effects in non-cardiomyocytes. Cardiomyocyte loss through apoptosis and necrosis is also an important component in the pathogenesis of heart failure and is mediated by both death receptor and mitochondrial signaling. Roles for immunogenic cell death in cardiac disease remain to be defined but merit study in this era of immune checkpoint cancer therapy. Biology-based approaches to inhibit cell death in the various cardiac syndromes are also discussed.

Keywords: apoptosis; cell death; heart disease; necroptosis; necrosis.

Graphical abstract

FIGURE 1.

Overview of apoptosis and necrosis pathways. Both apoptosis and necrosis can be mediated through pathways that involve death receptors or mitochondria, resulting in four distinct pathways: 1) death receptor-mediated apoptosis, 2) death receptor-mediated necrosis (necroptosis), 3) mitochondrial-mediated apoptosis, and 4) mitochondrial-mediated necrosis. Regardless of upstream signaling, apoptosis in both pathways leads to activation of the cysteine-dependent proteases, called caspases. In death receptor-mediated apoptosis, binding of death ligands to their cognate receptors results in the formation of sequential protein complexes (complexes I and II). In the latter, caspase-8 (or -10, not shown) is activated. These caspases then cleave and activate downstream caspases, such as -3 and -7. In mitochondrial-mediated apoptosis, caspases are activated in a cytosolic complex (not shown) whose formation is triggered by cytochrome c. Cytochrome c gains access to the cytosol following permeabilization of the outer mitochondrial membrane, which is mediated by B cell lymphoma-2 (BCL-2)-associated X protein (BAX) and BCL-2 antagonist/killer 1 (BAK). In contrast to caspases in apoptosis, a single unifying mechanism has not yet been identified for downstream necrosis signaling that is common to both death receptor and mitochondrial pathways. In necroptosis, activation of the serine/threonine kinases receptor interacting protein kinase 1 (RIPK1) and RIPK3 is critical. RIPK3 then phosphorylates and activates a pseudokinase called mixed lineage kinase-like domain (MLKL), which oligomerizes and permeabilizes the plasma membrane to induce necroptosis. The critical event in the induction of mitochondrial necrosis is Ca²⁺-triggered opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane. This opening causes rapid dissipation of the proton gradient across the inner membrane that drives ATP synthesis leading to energetic deficits. However, the precise mechanism leading to plasma membrane dysfunction in this pathway is unclear (see text).

FIGURE 2.

Death receptor apoptosis and necroptosis pathways. Death receptor signaling is initiated by death ligand binding to its specific plasma membrane death receptor. Shown here is the binding of tumor necrosis factor-α (TNFα) to TNF receptor 1 (TNFR1). This initiates assembly of complex I on the cytoplasmic tail of the receptor. Outcomes downstream of complex I assembly include cell survival, apoptosis, or necroptosis. In the formation of complex I, TNFR1 recruits the adaptor protein TNFR1-associated death domain (TRADD) which, in turn, recruits receptor interacting protein kinase 1 (RIPK1). TRADD recruits the adaptor proteins TNF receptor-associated factor 2 and 5 (TRAF2/5) and cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2), which attach lysine 63 (K63)-linked ubiquitin chains onto RIPK1. The K63-linked ubiquitin chains promote the recruitment of the linear ubiquitin chain assembly complex (LUBAC), which catalyzes the attachment of linear [or methionine 1 (M1)-linked] ubiquitin chains onto RIPK1. K63-linked chains on RIPK1 promote the activation of transforming growth factor-β-activated kinase 1 (TAK1) through recruitment of TAK1 binding proteins 2 and 3 (TAB2/3). TAK1 [acting through inhibitor of κB kinases (IKKs) and nuclear factor-κB (NF-κB) essential modulator (NEMO) recruited to the linear ubiquitin chains, not shown] promotes the activation of NF-κB. NF-κB is a transcription factor whose targets include multiple genes that promote cell survival and inflammation. In addition, TAK1 also activates mitogen-activated protein kinases (MAPKs), which provide additional survival signals. Cell death initiation requires the transition from complex I to cytosolic complex II. This transition is promoted by decreases in RIPK1 ubiquitination and specifically a decrease in the ratio of linear/K63-linked ubiquitin chains. Cylindromatosis (CYLD) and A20 remove primarily linear- and K63-linked ubiquitin chains, respectively. Complex IIa can take two forms. In one, the binding of TRADD-FADD-procaspase-8 signals RIPK1-independent apoptosis. In the other, binding of activated RIPK1-FADD-procaspase-8 signals RIPK1-dependent apoptosis. Furthermore, caspase-8 inhibits necroptosis by cleaving RIPK1 and RIPK3 (not shown). However, if caspase-8 is inhibited (see text), the binding of activated RIPK1 and RIPK3 in complex IIb leads to RIPK3 activation. RIPK3 then phosphorylates and activates a pseudokinase called mixed lineage kinase-like domain (MLKL), which translocates to and permeabilizes the plasma membrane to cause necroptosis.

FIGURE 3.

Mitochondrial apoptosis pathway. The mitochondrial apoptosis pathway can be triggered by diverse extracellular and intracellular stress stimuli. The central event in the execution of this pathway is permeabilization of the outer mitochondrial membrane (OMM), which is tightly regulated by the B cell lymphoma-2 (BCL-2) family proteins. These are divided into three subfamilies according to their function and BCL-2 homology (BH) domains: 1) pro-survival proteins, including BCL-2, BCL-2-like-1 long form (BCL-xL), and myeloid cell leukemia-1 (MCL-1); 2) multidomain pro-cell death proteins, including BCL-2-associated X protein (BAX) and BCL-2 antagonist/killer 1 (BAK); and 3) pro-cell death BH3-only proteins, which are further subdivided into “activators” and “sensitizers.” Activator BH3-only proteins [BCL-2-interacting domain death agonist (BID), BCL-2-interacting mediator of cell death (BIM), p53 upregulated modulator of apoptosis (PUMA), and perhaps phorbol-12-myristate-13-acetate-induced protein 1 (NOXA)] directly bind and conformationally activate BAX and BAK. Conversely, the pro-survival BCL-2 proteins oppose these events both through sequestering the activator BH3-only proteins and through interacting with and inhibiting BAX and BAK. Sensitizer BH3-only proteins [BCL-2 antagonist of cell death (BAD), BCL-2-interacting killer (BIK), BCL-2-modifying factor (BMF), and Harakiri (HRK)] indirectly activate BAX and BAK through binding the pro-survival BCL-2 proteins, which both displaces the activator BH3-only proteins as well as prevents the pro-survival proteins from binding and neutralizing BAX and BAK. Activation of BAX and BAK drives their homo- and hetero-oligomerization within the OMM. This results in membrane permeabilization and release of apoptogenic factors from mitochondria, including cytochrome c, second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis (IAP) binding protein with low PI (DIABLO), and OMI/high temperature requirement protein A2 (OMI/HtrA2). The binding of cytochrome c and dATP to Apaf-1 promotes formation of the apoptosome, in which procaspase-9 is activated. Caspase-9 subsequently activates downstream procaspases including -3 and -7. IAPs bind and inhibit procaspase-9 and active caspases-3 and -7. SMAC/DIABLO and OMI/HtrA2 bind and inhibit IAPs resulting in activation of caspases. Caspase-8 can also cleave and activate BID (tBID), allowing tBID to bind and activate BAX, thereby linking the death receptor and mitochondrial apoptosis pathways.

FIGURE 4.

Mitochondrial necrosis pathway. The mitochondrial necrosis pathway can be triggered by diverse stimuli including loss of nutrient/survival factors, increase in intracellular calcium (Ca²⁺), reactive oxygen species (ROS), ischemia, and ischemia/reperfusion. The central event in the execution of this pathway is opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane (IMM). Ischemia deprives the myocardium of oxygen (O₂), resulting in anaerobic metabolism and intracellular acidosis. In response, the cell pumps out the excess proton (H⁺) and accumulates sodium (Na⁺) by reverse operation of the Na⁺/H⁺ ion exchanger (NHE). The excess Na⁺ is, in turn, pumped out by the Na⁺/Ca²⁺ exchanger (NCX), which leads to an increase in intracellular Ca²⁺. Further increases in Ca²⁺ levels result from Ca²⁺-induced Ca²⁺ release from the endo/sarcoplasmic reticulum (ER/SR), resulting in intracellular Ca²⁺ overload. Increased concentrations of Ca²⁺ in the mitochondrial matrix promote mPTP opening during reperfusion which neutralizes intracellular acidosis and stimulates ROS production. The pro-cell death protein BCL-2-associated X protein (BAX) also sensitizes Ca²⁺-induced mPTP opening and necrosis. The consequences of mPTP opening are 1) loss of the H⁺ gradient across the IMM (mitochondrial membrane potential, Δψ_m), which leads to cessation of ATP synthesis, and 2) influx of water into the hyperosmolar mitochondrial matrix leading to mitochondrial swelling and eventual rupture of the outer mitochondrial membrane (OMM).

FIGURE 5.

Autophagy-dependent cell death. The “conventional” view of autophagy-dependent cell death is that increases in rates of macroautophagy induce cell death through excessive catabolism of cellular components. Specific death machinery and specific autophagy-dependent cell death markers have not been identified. In contrast, autosis is a form of autophagy-dependent cell death that is characterized by a specific cellular morphology (see text) and mediated by the plasma membrane Na⁺-K⁺-ATPase.

FIGURE 6.

Overview of ferroptosis. Ferroptosis is an iron-dependent form of regulated cell death mediated by lipid peroxidation of cellular membranes, although which damaged membranes induce cell death is not yet clear. Ferroptosis can be initiated by iron overload in which Fenton reaction-produced Fe³⁺ activates lipoxygenases or by mechanisms that inactivate glutathione peroxidase 4 (GPX4), which opposes lipid peroxidation. GPX4 can be inactivated by extracellular glutamate overload, which inhibits antiporter system x_c⁻ . System x_c⁻ normally imports cystine, which is converted to cysteine that subsequently generates glutathione (GSH), a cofactor for GPX4. Fe³⁺ imported through the transferrin receptor (TR) is converted to Fe²⁺ in endosomes by the metalloreductase six-transmembrane epithelial antigen of prostate 3 (Steap3) and released from endosomes by divalent metal transporter 1 (DMT1). Erastin, sulfasalazine, and sorafenib are small molecules that inhibit system x_c⁻ to induce ferroptosis. Ras synthetic lethal 3 (RSL3) and FIN56 are small molecules that inhibit GPX4 also inducing ferroptosis. Ferrostatin-1, liproxstatin-1, vitamin E, and CoQ10 are lipid antioxidants that inhibit ferroptosis. In addition, FIN56 also inhibits CoQ10 through the mevalonate pathway. Deferoxamine (DFO) binds Fe²⁺ mainly extracellularly, while dexrazoxane and ciclopirox (CPX) bind Fe²⁺ intracellularly.

FIGURE 7.

Overview of pyroptosis. The sentinel event in pyroptosis is the creation of pores in the plasma membrane by gasdermin D (GSDMD). GSDMD is activated through cleavage by inflammatory caspases (caspases-1, -4, and -5 in humans). Procaspase-1 is activated in response to diverse stresses including infection, toxic insults impinging on the cell, and multiple intracellular stresses [e.g., reactive oxygen species (ROS), cytosolic DNA, and others]. Pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) resulting from these stresses promote assembly of inflammasomes, which are subcellular multiprotein complexes in which procaspase-1 activation takes place. This involves the sensing of PAMPs and DAMPs by pattern recognition receptors such as NOD-like receptor (NLR) pyrin domain (PYD) containing receptor 3 (NLRP3). NLRP3 subsequently recruits ASC, which recruits and activates procaspase-1 (see text for details). Other proteins besides NLRP3 can also function as the “apical” protein in inflammasome assembly. Inflammasome formation is “primed” through transcriptional and nontranscriptional mechanisms (see text for latter). Transcriptional priming occurs downstream of Toll-like receptors (TLRs) and TNFR1 through NF-κB-mediated increases in NLRP3, ASC, and procaspase-1 expression. In addition, the expression of downstream effectors in this pathway including the cytokines pro-interleukin (IL)-1β and pro-IL-18 are also transcriptionally upregulated. Noncanonical activation of procaspase-4 and -5 can also take place through their binding to lipopolysaccharide (LPS) in the cytoplasm. Once activated, inflammatory caspases cleave and activate GSDMD as noted above and process pro-IL-1β and pro-IL-18 to their mature forms, which escape through GSDMD-generated pores. Interaction of NLRP3 with mitochondrial antiviral signaling protein (MAVS) and cardiolipin facilitate inflammasome assembly and activation.