Introduction
Significant progress has been made in demonstrating the role of apoptosis in various heart diseases, and in elucidating the molecular mechanisms of cardiac apoptosis. Apoptosis has been attributed an essential role in cardiomyopathy. The progressive loss of cardiac myocytes is one of the most important pathogenic components of the heart failure. While initial studies reported unrealistically high levels of cell death, probably due to methodological problems, later work has consistently shown that approximately 80-250 heart muscle cells per 105 cardiac nuclei commit suicide at any given time in patients with late-stage dilated cardiomyopathy. In contrast, the base-line rate of apoptosis in healthy human hearts is only one to ten cardiac myocytes per 105 nuclei (Anversa & Kajstura, 1998; Yue et al., 1998). Even though the rate of apoptosis in heart failure is relatively low in absolute numbers, it is significantly higher than that in the normal heart, which has essentially negligible baseline apoptosis. Recently, animal models of heart failure incorporating transgenic technology have confirmed that myocyte apoptosis itself is sufficient to induce heart failure. Apoptosis has been implicated in a wide variety of physiological and pathological processes. The importance of apoptosis in cardiovascular system is also becoming increasingly clear, and the inhibition of apoptosis is emerging as a potential therapeutic tool for various forms of cardiovascular disease. In this topic, we examine the evidence for apoptosis in cardiovascular disease and the molecular mechanisms of cardiac apoptosis.
Apoptosis
Concept and morphologic characteristics of apoptosis
There are two primary pathways by which cells die. The path for accidental cell death is called necrosis. Accidental cell death occurs when cells receive a structural or chemical insult from which they cannot recover. Examples of such insults include ischemia, extremes of temperature, and physical trauma. The hallmark of necrosis is that cells die because they are damaged. In contrast, cells that die by programmed cell death commit suicide actively as the results of activation of a dedicated intracellular program. For programmed cell death, the most commonly described pathway is apoptosis. Apoptosis begins with a signal that can come from within the cell (e.g., detection of radiation-induced DNA breaks) or from without (e.g., a decrease in the level of an essential growth factor or hormone). This pro-apoptotic signal induces the cell to make a decision to commit suicide. Initially, cells that are committed to undergo programmed cell death are in a latent phase of apoptosis. The latent phase can be subdivided into two stages: a condemned stage, during which the cell is proceeding on a pathway toward death but can still be rescued if it is exposed to anti-apoptotic activities, and a committed stage, beyond which rescue is impossible. Ultimately, the cells enter the execution phase of apoptosis, in which they undergo the dramatic morphologic and physiological changes (Pollard & Earnshaw, 2004). Apoptosis is characterized by a reproducible pattern of structural alterations of both the nucleus and cytoplasm. In order of appearance, these include: (1) Loss of microvilli and intercellular junctions. (2) Shrinkage of the cytoplasm. The cell is smaller in size. The cytoplasm is dense. The organelles, although relatively normal, are more tightly packed. (3) Dramatic changes in cytoplasmic motility with activation of a violent program of blebbing. (4) Loss of plasma membrane asymmetry, with the distribution of phosphatidyl serine being randomized so that it appears in the outer membrane leaflet. (5) Changes in the organization of the cell nucleus, typically involving the hypercondensation of the chromatin. This is the most characteristic feature of apoptosis. The chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. The nucleus itself may break up, producing two or more fragments. (6) Formation of cytoplasmic blebs and apoptotic bodies. The apoptotic cell first shows extensive surface blebbing, then undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragments. (7) Phagocytosis of apoptotic cells or cell bodies, usually by macrophages. The apoptotic bodies are rapidly degraded within lysosomes, and the adjacent healthy cells migrate or proliferate to replace the space occupied by the now deleted apoptotic cell. Because the vesicles remain membrane bound, the cellular contents are never released into the environment. As a result, apoptotic death does not lead to an inflammatory response. (Kumar et al., 2005; Pollard & Earnshaw, 2004).
Molecular mechanism and pathway description
The process of apoptosis may divided into an initiation phase, during which caspases become catalytically active, and an execution phase, during which these enzymes act to cause cell death. Initiation of apoptosis occurs principally by signals from two distinct but convergent pathways: the extrinsic, or receptor-initiated, pathway and the intrinsic or mitochondrial pathway. Both pathways converge to activate caspases and they may be interconnected at numerous steps.
The extrinsic (death receptors) pathway
This pathway is initiated by engagement of cell surface death receptors on a variety of cells. Death receptors are members of the tumor necrosis factor receptor family (Fas, TNFaR, DR3, DR4, DR5) that contain a cytoplasmic domain involved in protein-protein interactions that is called death domain because it is essential for delivering apoptotic signals.. Death receptor ligands characteristically initiate signaling via receptor oligomerization, which in turn results in the recruitment of specialized adaptor proteins and activation of caspase cascades. Binding of FasL induces Fas trimerization, which recruits initiator caspase-8 via the adaptor protein FADD. Caspase-8 then oligomerizes and is activated via autocatalysis. Activated caspase-8 stimulates apoptosis via two parallel cascades: it can directly cleave and activate caspase-3, or alternatively, it can cleave Bid, a pro-apoptotic Bcl-2 family protein. Truncated Bid (tBid) translocates to mitochondria, inducing cytochrome c release, which sequentially activates caspase-9 and -3. TNF-a and DR-3L can deliver pro- or anti-apoptotic signals. TNFaR and DR3 promote apoptosis via the adaptor proteins TRADD/FADD and the activation of caspase-8. Interaction of TNF-a with TNFaR may activate the NF-kB pathway via NIK/IKK. The activation of NF-kB induces the expression of pro-survival genes including Bcl-2 and FLIP, the latter can directly inhibit the activation of caspase-8. Some viruses and normal cells produce FLIP, which binds to pro-caspase-8 but cannot cleave and activate the enzyme because it lacks enzymatic activity, and use this inhibitor to protect infected and normal cells from Fas-mediated apoptosis (Kumar et al., 2005). FasL and TNF-a may also activate JNK via ASK1/MKK7. Activation of JNK may lead to the inhibition of Bcl-2 by phosphorylation. In the absence of caspase activation, stimulation of death receptors can lead to the activation of an alternative programmed cell death pathway termed necroptosis by forming complex IIb. (Humphreys & Halpern, 2008; Logue & Martin, 2008; Yuan, 2010)
The intrinsic (mitochondrial) pathway
This pathway of apoptosis is the result of increased mitochondrial permeability and release of pro-apoptotic molecules into the cytoplasm, without a role for death receptors. Growth factors and other survival signals stimulate the production of anti-apoptotic members of the Bcl-2 family of proteins. The Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial permeability. The anti-apoptotic proteins Bcl-2 and Bcl-xL reside in the outer mitochondrial wall and inhibit cytochrome c release. The proapoptotic Bcl-2 proteins Bad, Bid, Bax, and Bim may reside in the cytosol but translocate to mitochondria following death signaling, where they promote the release of cytochrome c. Bad translocates to mitochondria and forms a pro-apoptotic complex with Bcl-xL. This translocation is inhibited by survival factors that induce the phosphorylation of Bad, leading to its cytosolic sequestration. Cytosolic Bid is cleaved by caspase-8 following signaling through Fas; its active fragment (tBid) translocates to mitochondria. Bax and Bim translocate to mitochondria in response to death stimuli, including survival factor withdrawal. Activated following DNA damage, p53 induces the transcription of Bax, Noxa, and PUMA. Upon release from mitochondria, cytochrome c binds to Apaf-1 and forms an activation complex with caspase-9. Although the mechanism(s) regulating mitochondrial permeability and the release of cytochrome c during apoptosis are not fully understood, Bcl-xL, Bcl-2, and Bax may influence the voltage-dependent anion channel (VDAC), which may play a role in regulating cytochrome c release. Mule/ ARF-BP1 is a DNA damage activated E3 ubiquitin ligase for p53, and Mcl-1, an anti-apoptotic member of Bcl-2 (Brenner & Mak, 2009; Chalah & Khosravi-Far, 2008; Yuan 2010). The essence of this intrinsic pathway is a balance between pro-apoptotic and protective molecules that regulate mitochondrial permeability and the release of death inducers that are normally sequestered within the mitochondria (Kumar et al., 2005).
The execution phase
The final phase of apoptosis is mediated by a proteolytic cascade, toward which the various initiating mechanisms converge. Caspases, a family of cysteine proteases, are the central regulators of apoptosis. They are mammalian homologues of the ced-3 in the nematode Caenorhabditis elegans. The caspase family, now including more than 10 members, can be divided functionally into two basic groups – initiator and executioner – depending on the order in which they are activated during apoptosis. Caspases exist as inactive pro-enzymes, or zymogens, and must undergo an activating cleavage for apoptosis to be initiated. Caspases have their own cleavage sites that can be hydrolyzed not only by other caspases but also autocatalytically. After an initiator caspase is cleaved to generate its active form, the enzymatic death program is set in motion by rapid and sequential activation of other caspases. Executioner caspases act on many cellular components. They cleave cytoskeletal and nuclear matrix proteins and thus disrupt the cytoskeleton and lead to breakdown of the nucleus. In the nucleus, the targets of caspase activation include proteins involved in transcription, DNA replication, and DNA repair (Haunstetter & Izumo, 1998; Kumar et al., 2005) (Table 1). Initiator caspases (including caspase-2, -8, -9, -10, -11, and -12) are closely coupled to pro-apoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including caspase-3, -6, and -7), which in turn execute apoptosis by cleaving cellular proteins following specific Asp residues. Activation of Fas and TNFR by FasL and TNF, respectively, leads to the activation of caspase-8 and -10. DNA damage induces the expression of PIDD which binds to RAIDD and caspase-2 and leads to the activation of caspase-2. Cytochrome c released from damaged mitochondria is coupled to the activation of caspase-9. XIAP inhibits caspase-3, -7, and -9. Mitochondria release multiple pro-apoptotic molecules, such as Smac/ Diablo, AIF, HtrA2 and EndoG, in addition to cytochrome c. Smac/Diablo binds to XIAP which prevents it from inhibiting caspases. Caspase-11 is induced and activated by pathological proinflammatory and pro-apoptotic stimuli and leads to the activation of caspase-1 to promote inflammatory response and apoptosis by directly processing caspase-3. Caspase-12 and caspase-7 are activated under ER stress conditions. Anti-apoptotic ligands including growth factors and cytokines activate Akt and p90RSK. Akt inhibits Bad by direct phosphorylation and prevents the expression of Bim by phosphorylating and inhibiting the Forkhead family of transcription factors (Fox0). Fox0 promotes apoptosis by upregulating pro-apoptotic molecules such as FasL and Bim (Degterev & Yuan, 2008; Kurokawa & Kornbluth, 2009; Yuan 2010).
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Nuclear proteins |
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Lamin, Rb protein, DNA-dependent protein kinase, 70-kDa subunit of U1 small nuclear ribonucleoprotein, Poly (ADP)-ribosylating protein (PARP), Mdm2 Regulatory proteins |
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MAPK/ERK kinase kinase 1 (MEKK1), Protein Kinase C8, G4-GDI GDP dissociation inhibitor, Sterol regulatory element binding protein, DNA fragmentation factor/inhibitor of caspase-activated DNAse |
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Cytoskeletal proteins |
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Fodrin, Gelsolin, Actin, Gas2 |
Table 1. Downstream targets of Caspases.
Study of apoptosis
Light and electron microscopy are two of the classical techniques for the study of this process. Because of the lack of cellular synchronization in apoptosis and of the fact that the apoptotic cell is rapidly disposed of through phagocytosis, study methods based on morphologic criteria are adequate for the demonstration of the process, but are not useful for quantifying it. Further to these procedures, the study of DNA fragmentation in agarose gels has been considered to be identificative for apoptosis. A number of techniques take advantage of this DNA fragmentation for labelling the fragments and thus for quantifying the proportion of apoptotic cells. Each DNA fragment has a 3′OH terminal portion. This terminal fragment can be labelled in various ways (for instance, with the help of a modified terminal deoxynucleotidyl transferase), so that the labelling rate is proportional to the degree of DNA fragmentation. In TUNEL assay (terminal deoxyribonucleotidyl transferase [TdT]-mediated dUTP-digoxigeninnick end labeling), TdT transfers a fluorescent nucleotide to exposed breakpoints of DNA. Apoptotic cells that have incorporated the labeled nucleotide are then visualized by fluorescence microscopy or flow cytometry. Apoptotic cells that have extruded some of the DNA have less than their normal diploid content. Automated measurement of the amount of DNA in individual cells by flow cytometry thus produces a population distribution according to DNA content (cytofluorograph) (Rubin et al., 2005). At present, the most widely accepted and standardized technique takes advantage of the changes in the membrane phospholipids that occur early in apoptotic cells (Vermes et al., 1995). The negatively charged membrane phospholipids exposed to the external environment by the apoptotic cell are labeled with fluorochrome-conjugated molecules, and the percentage of fluorescent cells can be easily quantified (Chamond et al., 1999).
Apoptosis in developmental and physiological processes
Cell death is extremely important in embryonic development, maintenance of tissue homeostasis, establishment of immune self-tolerance, killing by immune effector cells, and regulation of cell viability by hormones and growth factors. Apoptosis is a normal phenomenon that serves to eliminate cells that are not longer needed and to maintain a steady number of various cell populations in tissues. It is important in the following physiologic situations. During molecular maturation of T-cell antigen receptors, immature T cells in the thymus rearrange the genes encoding the receptor a and p chains. Cells with receptors recognizing self-antigens are potentially harmful and are eliminated through programmed cell death. Cells with damaged DNA tend to accumulate mutations, and they are potentially harmful to the organism. DNA damage induces programmed cell death in many cell types. Furthermore one of the mechanisms to eliminate infected cells is through the action of cytotoxic T lymphocytes, which kill cells, by inducing them to undergo programmed cell death. T lymphocytes whose T-cell receptors cannot interact with the spectrum of MHC glycoproteins expressed in a given individual are ineffective in the immune response. Up to 95% of immature T cells die by apoptosis without leaving the thymus. Fetal development involves the sequential appearance and regression of many anatomical structures: some aortic arches do not persist, the mesonephros regresses in favor of the metanephros, interdigital tissues disappear to allow discrete fingers and toes, and excess neurons are pruned from the developing brain. Cells in these conditions serve no purpose in humans and are eliminated by programmed cell death. Excess neurons that do not make appropriate connections have no function and so are eliminated by apoptosis. Up to 80%of neurons in certain developing ganglia die this way (Rubin et al., 2005). Apoptosis also eliminates the constituent cells of mullerian ducts in males. Epithelial cells must die to allow fusion of palate and mammary and prostate cells die when deprived of hormones (Pollard & Earnshaw, 2004).
Apoptosis and disease
Death by apoptosis is also responsible for loss of cells in a variety of pathologic states. The diseases in which apoptosis has been involved can be divided into two groups: those in which there is an increase in cell survival (or diseases associated to inhibition of apoptosis), and those in which there is an increase in cell death (and hence hyperactive apoptosis) (Chamond et al., 1999). The group of diseases associated to apoptosis inhibition includes those diseases in which an excessive accumulation of cells occurs (neoplastic diseases, autoimmune diseases) (Table 2). It was classically believed that the excessive accumulation of cells in these diseases occurred because of an increased cell proliferation. In recent years, the study of apoptosis in these patients has led to a new and different approach, according to which this accumulation of cells would be due to defective apoptosis. Increased cell apoptosis has also been implicated in the aetiopathogenesis of a number of diseases. A large group of neurodegenerative diseases, among them Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and retinitis pigmentosa, are associated to selective apoptosis of the neurones. This neuronal death appears to be associated to increases susceptibility to apoptosis in these cells. Oxidative stress, mitochondrial defects and neurotoxic agents have been postulated as the inductors of neuronal death. The disease associated to infection by the Human Immunodeficiency Virus (HIV) has been defined as an imbalance between the number of CD4+ lymphocytes and the ability of the bone marrow to generate new mature cells. The CD4+ cells of the HIV(+) patients die through apoptosis when stimulated in vitro. Also, HIV infection of cells from healthy subjects induces apoptosis of CD4+ cells. However, further to this, not only the infected cells but also non-infected cells undergo apoptosis. Replicative exhaustion of the responding cell clones has been demonstrated in a number of diseases (among them AIDS, in which the responding clone is the CD4+ one); in these diseases the responding clone, after an initial phase of intense response to the stimulus, become exhausted and undergo apoptosis (Ameisen, 1995).
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Through inhibition of apoptosis |
Through excess apoptosis |
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Cancer |
AIDS |
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Colorectal |
T lymphocytes |
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Glioma |
Neurodegenerative diseases |
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Hepatic |
Alzheimer’s disease |
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Neuroblastoma |
Amyotrophic lateral sclerosis |
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Leukaemias and lymphomas |
Parkinson’s disease |
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Prostate |
Retinitis pigmentosa |
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Autoimmune diseases |
Epilepsy |
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Myastenia gravis |
Haematologic diseases |
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Systemic lupus erythematosus |
Aplastic anaemia |
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Inflammatory diseases |
Myelodysplastic syndrome |
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Bronchial asthma |
T CD4+ lymphocytopenia |
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Inflammatory intestinal disease |
G6PD deficiency |
|
Pulmonary inflammation |
Tissue damage |
|
Viral infections |
Myocardial infarction |
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Adenovirus |
Cerebrovascular accident |
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Baculovirus |
Ischaemic renal damage Polycystic kidney |
Table 2. Diseases associated to apoptosis
Apoptosis and cardiovascular disease
The myocardial cells allocate a limited faculty of proliferation and correspondingly, apoptosis is observed infrequently in adult hearts. On the contrary at the duration of organogenesis and in the formation of heart the apoptosis plays an important role, as an example in the formation of septa between the cardiac chambers and the valves. As consequence the defects in apoptosis can constitute basic causative factor of relatives of congenital heart disease. Apoptosis of myocardial cells is also observed afterwards the birth and concretely in the interventricular septum and right ventricular wall, at the duration of passage from the fetal circulation in the adult circulation. Moreover the phenomenon of apoptosis is also distinguished in the conducting system and can lead to congenital heart block, syndrome of long QT, and the existence of accessory pathways (Table 3) (Bennett, 2002). Until recently, the loss of myocytes was attributed to necrosis; however, it is now clear that apoptosis may play an important role in the pathogenesis of a variety of cardiovascular diseases. For instance, apoptosis has been detected in myocardial samples obtained from patients with end-stage congestive heart failure, arrhythmogenic right ventricular dysplasia and myocardial infraction. In addition, apoptosis has been detected in cardiac myocytes under hypoxia/reoxygenetion, mechanical stretch and in animal models of cardiac ischemia/reperfusion injury (Table 4) (Gustafsson & Gottlieb, 2003).
|
Myocyte |
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Idiopathic dilated cardiomyopathy |
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Ischaemic cardiomyopathy |
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Acute myocardial infarction |
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Arrhythmogenic right ventricular dysplasia |
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Myocarditis |
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Conducting tissues |
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Pre-excitations syndromes |
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Congenital complete atrioventricular heart |
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Long QT syndromes |
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Vascular |
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Atherosclerosis |
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Restenosis after angioplasty / stenting |
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Vascular graft rejection |
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Arterial aneurysm formation |
Table 3. Apoptosis and cardiovascular disease
|
Stimulus |
Signaling pathway |
Potential inhibitor |
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ERK/SARK |
Activation of ERK, inhibition of SARK signaling |
|
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Ischaemia/reperfusion |
ERK/SARK |
Activation of ERK, inhibition of SARK |
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Pressure overload |
G protein coupling |
signaling |
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Neurohormonal factors |
Lack of growth factor signaling |
P blockers Activation of Akt/ERK pathways |
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Ischaemia |
Adapter molecules / |
Decoy receptors / receptor |
|
Death receptor ligands |
caspases |
antagonists IAPs / caspase inhibitors |
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ERK, extracellular signal related kinase; IAP, inhibitor of apoptosis protein; SAPK, stress activated protein kinase |
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Table 4. Potential inhibitors and signaling pathways of cardiomyocyte apoptosis.
