Basic Concepts and Definitions
The ultimate functional effect of cardiac toxic manifestations is decreased cardiac output and peripheral tissue hypoperfusion, resulting from alterations in biochemical pathways, energy metabolism, cellular structural and function, electrophysiology, and contractility of the heart. These morphological and functional alterations induced by toxic exposure are referred to as toxicological cardiomyopathy. The critical cellular event leading to toxicological cardiomyopathy is myocardial cell death and extracellular matrix (ECM) remodeling. The recognition of the role of apoptosis in the development of heart failure during the last decade has significantly enhanced our knowledge of myocardial cell death (James, 1994; Haunstetter and Izumo, 1998; Sabbah and Sharov, 1998).
Manipulation of genes responsible for cardiac function began in the mid-1990s (Robbins, 2004). The most important conclusion of these studies is that a sustained expression of any single mutated functional gene, either in the form of gain-of-function or loss-of-function, can lead to a significant phenotype, often in the form of cardiac hypertrophy and heart failure (Robbins, 2004; Olson, 2004). However, it is difficult to apply this knowledge to patients: first, acquired cardiac disease such as heart failure is the result of interaction between environmental factors and genetic susceptibility, indicating the role of polymorphisms. Second, extrinsic and intrinsic stresses produce lesions that cannot be explained by a single gene or a single pathway, suggesting complexity between deleterious factors and the heart. Cardiac toxicity is the critical link between environmental factors and myocardial pathogenesis.
For a better understanding of cardiac toxicology, a triangle model of cardiac toxicity is presented in Fig. 18-8. In this model, complexity of the interaction between environmental stresses and the heart, and the balance between myocardial protection and deleterious dose and time effects are considered. First, it is important to recognize that chemicals can lead to heart failure without heart hypertrophy. Second, a chemical can lead to activation of both protective and destructive responses in the myocardium. Third, long-term toxicological responses often result in maladaptive hypertrophy, which primes the heart for malignant arrhythmia, leading to sudden cardiac death or transition to heart failure.
Triangle analytical model of cardiac responses to drugs and xenobiotics. Drugs or xenobiotics can directly cause both heart failure and heart hypertrophy. Under severe acute toxic insults, myocardial cell death becomes the predominant response leading to cardiac dilation and heart failure. In most cases, myocardial survival mechanisms can be activated so that myocardial apoptosis is inhibited. The survived cardiomyocytes often become hypertrophy through activation of calcium-mediated fetal gene expression and other hypertrophic program. If toxic insult continues, the counter-regulatory mechanisms against heart hypertrophy such as activation of cytokine-medicated pathways eventually lead to myocardial cell death through apoptosis or necrosis, dilated cardiomyopathy, and heart failure.
In the study of cardiac toxicology, the manifestations of cardiac toxicity in human patients and animal models are critical parameters serving as indices of cardiac toxicity. These manifestations are expressed in the forms of cardiac arrhythmia, hypertrophy, and heart failure. These abnormal changes reflect myocardial functional alterations resulting from both acute and chronic cardiac toxicity. Although some changes including cardiac hypertrophy were viewed as a compensatory response to hemodynamic changes in the past, more recent studies suggest that cardiac hypertrophy is a maladaptive process of the heart in response to intrinsic and extrinsic stresses (van Empel and De Windt, 2004; Berenji et al., 2005; Dorn and Force, 2005). Cardiac hypertrophy is a risk factor for sudden cardiac death and has a high potential to progress to overt heart failure. Therefore, a distinction between compensatory and maladaptive responses is critical for treatment of patients with toxicological cardiomyopathy.
Cardiac rhythms under physiological conditions are set by pacemaker cells that are normally capable of developing spontaneous depolarization and responsible for generating the cardiac rhythm, the so-called automatic rhythm. A cardiac rhythm that deviates from the normal automatic rhythm is called cardiac arrhythmia, often manifested in the form of tachycardia (fast heart rate). There are several classes of tachycardia, including sinus tachycardia, atrial tachycardia, ventricular tachycardia, and torsades de pointes (TdP) (a life-threatening ventricular tachycardia). In addition, subclasses such as atrial fibrillation, atrial flutter, and accelerated idioventricular rhythm provide further description of the manifestations of arrhythmia. Mechanisms for different classes of arrhythmia will be discussed in the section “QT prolongation and sudden cardiac death.” According to the cause of the tachycardia, it is divided into abnormal automatic arrhythmia and triggered arrhythmia, which will be discussed in other sections.
There are two basic forms of cardiac hypertrophy: concentric hypertrophy, which is often observed during pressure overload and is characterized by new contractile-protein units assembled in parallel resulting in a relative increase in the width of individual cardiac myocytes (De Simone, 2003). By contrast, eccentric hypertrophy is characterized by the assembly of new contractile-protein units in series resulting in a relatively greater increase in the length than in the width of individual myocytes, occurring in human patients and animal models with dilated cardiomyopathy (Kass et al., 2004). Toxicological cardiomyopathy is often manifested in the form of eccentric hypertrophy. The development of cardiac hypertrophy can be divided into three stages: developing hypertrophy, during which period the cardiac workload exceeds cardiac output; compensatory hypertrophy, in which the workload/mass ratio is normalized and normal cardiac output is maintained; decompensatory hypertrophy, in which ventricular dilation develops and cardiac output progressively declines, and overt heart failure occurs (Richey and Brown, 1998).
A traditional definition of heart failure is the inability of the heart to maintain cardiac output sufficient to meet the metabolic and oxygen demands of peripheral tissues. This definition has been modified to include changes in systolic and diastolic function that reflect specific alterations in ventricular function and abnormalities in a variety of subcellular processes (Piano et al., 1998). Therefore, a detailed analysis to distinguish right ventricular from left ventricular failure can provide a better understanding of the nature of the heart failure and predicting the prognosis.
Acute cardiac toxicity is referred to as cardiac response to a single exposure to a high dose of cardiac toxic chemicals. It is often manifested by cardiac arrhythmia. However, myocardial apoptosis is also involved in acute cardiac toxicity. It is not difficult to define acute cardiac toxicity; however, it sometimes is technically difficult to measure acute cardiac toxicity. In particular, the impact of acute cardiac toxicity on the ultimate outcome of cardiac function is not often easily recognized. For instance, a single high dose of arsenic can lead to cardiac arrhythmia and sudden cardiac death, which is easy to measure (Goldsmith and From, 1980). However, that a single oral dose of monensin (20 mg/kg) leads to a diminished cardiac function progressing to heart failure in calves requires a long-term observation; often a few months for clinical signs of heart failure (van Vleet, et al., 1983; Litwak et al., 2005), which is difficult to measure. As shown in Fig. 18-8, toxic exposure can directly lead to heart failure, which is different from an often-observed hypertrophic response, which may or may not progress to heart failure.
Chronic cardiac toxicity is the cardiac response to long-term exposure to chemicals, which is often manifested by cardiac hypertrophy and the transition to heart failure. About 25% of human patients with cardiomyopathy are categorized as having idiopathic cardiomyopathy. At least a portion of these patients with idiopathic cardiomyopathy are due to chemical exposure. Environmental exposure to particulate matters (PMs) in the air can lead to cardiomyopathy, which has only been recognized a decade ago (Dockery, 2001; Gordon and Reibman, 2000). Recognition of chronic cardiac toxicity in the pathogenesis of cardiomyopathy is of clinical relevance, and this knowledge can be used to prevent and treat patients with toxicological cardiomyopathy.
Myocardial Degeneration and Regeneration
Myocardial degeneration is the ultimate response of the heart to toxic exposure, which can be measured by both morphological and functional degenerative phenotypes. However, myocardial degeneration should not be considered an irreversible toxic response. In the past, the heart has been considered incapable of regenerating, so that cardiac injury in the form of cell loss or scar tissue formation was considered permanent damage to the heart. However, evidence now indicates myocardial regeneration and recovery from cardiomyopathy. Cardiac toxic responses or damage are now divided into reversible and irreversible.
Myocardial Degenerative Responses
Myocardial cell death, fibrosis (scar tissue formation), and contractile dysfunction are considered as degenerative responses, which can result in cardiac arrhythmia, hypertrophy, and heart failure. If acute cardiac toxicity does not affect the capacity of myocardial regeneration, the degenerative phenotype is reversible. Both acute and chronic toxic stresses can lead to irreversible degeneration, depending on whether or not the cardiac repair mechanisms are overwhelmed. Cell death is the most common phenotype of myocardial degeneration. Both apoptosis and necrosis occur in the process of myocardial cell death, which will be discussed in the next section. Myocardial cell death is accompanied by hypertrophy of the remaining cardiac myocytes so that in the hypertrophic heart, the total number of cardiac myocytes is reduced but the size or volume of individual cells is increased.
During myocardial remodeling after cell death, not only is there an increase in the size of cardiac myocytes, but also cardiac fibrosis occurs. Myocardial fibrosis results from excess accumulation of ECM, which is mainly composed of collagens. The net accumulation of ECM connective tissue results from enhanced synthesis or diminished break down of the matrix, or both. Collagens, predominately types I and III, are the major fibrous proteins in ECM and their synthesis may increase in response to toxic insults. The degradation of ECM is dependent on the activity of matrix metalloproteinases (MMPs). According to their substrate specificity, MMPs fall into five categories: collagenases (MMP-1, MMP-8, and MMP-13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-7, MMP-10, and MMP-11), membrane-type MMPs (MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, and MMP-25), and metalloelastase (MMP-12). These MMPs are organ specific so that not all are present in the heart. The activities of these enzymes are altered during the processes of fibrogenesis and fibrinolysis. Under toxic stress conditions, the imbalance between fibrogenesis and fibrinolysis leads to enhanced fibrogenesis and excess collagen accumulation—fibrosis.
The MMPs are inhibited by specific endogenous tissue inhibitor of metalloproteinases (TIMPs), which comprise a family of four protease inhibitors: TIMP-1, TIMP-2, TIMP-3, and TIMP-4. In general, during heart remodeling the concentration of TIMPs increases. Synthetic inhibitors generally contain a chelating group that binds the catalytic zinc atom at the MMP active site tightly. Common chelating groups include hydroxamates, carboxylates, thiols, and phosphinyls. Hydroxymates are particularly potent inhibitors of MMPs and other zinc-dependent enzymes, due to their bidentate chelation of the zinc atom. Other substituents of TIMPs are usually designed to interact with various binding pockets on the MMP of interest, making the inhibitor more or less specific for a given MMP.
Toxic Effect on Myocardial Regeneration
The mainstay of cardiac medicine and therapy has centered on the concept that the heart is a terminally differentiated organ and that cardiac myocytes are incapable of proliferating. Thus, cell death would lead to a permanent loss of the total number of cardiac myocytes. However, this view has been challenged recently due to the identification of cardiac progenitor cells (Anversa et al., 2006). These cells are characterized and proposed to be responsible for cardiac repair because these cells can make myocytes and vascular structures. These cells possess the fundamental properties of stem cells; therefore, they are also called cardiac stem cells. They are self-renewing, clonogenic, and multipotent, as demonstrated by reconstitution of infarcted heart by intramyocardial injection of cardiac progenitor cells or the local activation of these cells by growth factors. It is important to note that toxicological studies of the cardiac progenitor cells have not been done, and it is important to determine the potential of cardiac stem cells to help recover from toxic insults. The effect of chemicals on the cardiac progenitor cells is unknown. One speculation is that when severe damage to cardiac progenitor cells occurs, the potential for recovery from severe cardiac injury would be limited.
The removal of scar tissue or fibrosis in the myocardium in the past has been considered impossible. Although there are no studies that have shown scar tissue is removable in humans, there are observations in animal models of hypertensive heart disease that myocardial fibrosis is recoverable (Weber, 2005).
Myocardial vascularization is required for myocardial regeneration. Many toxic insults affect the capacity of angiogenesis in the myocardium, so that cardiac ischemia occurs. The combination of cardiac ischemia and the direct toxic insults to cardiomyocytes constitute synergistic damage to the heart. During regeneration, coronary arterioles and capillary structures are formed to bridge the dead tissue (scar tissue) and supply nutrients for the survival of the regenerated cardiomyocytes. There is an orderly organization of myocytes within the myocardium and a well-defined relationship between the myocytes and the capillary network. This proportion is altered under cardiac toxic conditions; either toxicological hypertrophy or diminished capillary formation can lead to hypoperfusion of myocytes in the myocardium. Unfortunately, our understanding of the toxic effects on myocardial angiogenesis is limited.
Reversible and Irreversible Toxic Responses
Cardiomy-opathy was viewed not to be recoverable in the past, but there is cumulative evidence that demonstrates reversibility of cardiomyopathy. The issue related to whether or not toxicological cardiac lesions are reversible has not been explored. However, it can be speculated that there would be reversible and irreversible manifestations of the cardiac response to toxic insults. With regard to this, toxic effect on the capacity of myocardial regeneration is a major concern and myocardial regenerative toxicity determines the fate of toxicological cardiomyopathy reversible or irreversible.
Myocardial Cell Death and Signaling Pathways
Toxic insults trigger a series of reactions in cardiac cells leading to measurable changes. Mild injuries can be repaired. However, severe injuries will lead to cell death in the modes of apoptosis and necrosis. If the cell survives the insults, structural and functional adaptations will take place.
Apoptosis was found to be involved in cardiomyopathy in 1994 (Gottlieb et al., 1994). The loss of cardiac myocytes is a fundamental component of myocardial injury, which initiates and aggravates cardiomyopathy. An important mode of myocardial cell loss is apoptosis, which has been demonstrated in heart failure patients (Olivetti et al., 1997). Myocardial apoptosis has been shown to play an important role in cardiac toxic effects induced by Adriamycin (Kang et al., 2000a; Wang et al., 2001a), an important anticancer drug whose clinical application is limited by its cardiotoxicity. Exposure of primary cultures of cardiomyocytes to cadmium also induces apoptosis (El-Sherif et al., 2000).
Many in vivo studies have shown that only a very small percentage of myocardial cell populations undergo apoptosis under pathological conditions. For example, less than 0.5% of cells appeared apoptotic in myocardial tissue under the stress of dietary copper deficiency in mice (Kang et al., 2000b). At first glance, this number seems to be too insignificant to account for myocardial pathogenesis. In a carefully designed time-course study (Kajstura et al., 1996), it was estimated that cardiomyocyte apoptosis is completed in less than 20 hours in rats. Myocytes that undergo apoptosis are lost and may not be replaced under toxicological conditions. Although the possibility of myocardial regeneration has been identified (Anversa et al., 2006), xenobiotics often cause degenerative effect through apoptosis as well as inhibitory effect on regeneration. Adriamycin-induced cardiomyopathy is a good example for the pathogenesis resulting from both degeneration and inhibition of regeneration. If apoptosis occurs at a constant rate of about 0.5% myocytes a day (Kang et al., 2000b), the potential contribution of apoptosis to the overall loss of myocytes over a long period of time is significant under Adrimycin toxic exposure.
Necrosis is a term that had been widely used to describe myocardial cell death in the past. Myocardial infarction, in particular, had been considered as a consequence of necrosis (Eliot et al., 1977). It is now recognized that apoptosis contributes significantly to myocardial infarction (Yaoita et al., 2000). However, the importance of necrosis in myocardial pathogenesis cannot be underestimated. The contribution of necrosis to cardiomyopathy induced by environmental toxicants and pollutants is particularly important. A critical issue is how to distinguish apoptosis from necrosis.
Apoptosis and necrosis were originally described as two distinct forms of cell death that can be clearly distinguished (Wyllie, 1994). However, these two modes of cell death can occur simultaneously in tissues and cultured cells. The intensity and duration of insults may determine the outcome. Triggering events can be common for both types of cell death. A downstream controller, however, may direct cells toward a programmed execution of apoptosis. If the apoptotic program is aborted before this control point and the initiating stimulus is severe, cell death may occur by necrosis (Leist et al., 1997).
To distinguish apoptosis from necrosis, specific oligonucleotide probes have been developed to recognize different aspects of DNA damage (Didenko et al., 1998), and have been successfully applied, in combination with confocal microscopy, to identify apoptotic and necrotic cell death in the heart with different pathogenic challenges.
A monoclonal mouse anti-ssDNA antibody has been developed that is specifically reactive with ssDNA, but does not recognize dsDNA. An immunohistochemical assay for detection of ssDNA using this antibody in combination with a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay can distinguish repairable ssDNA breaks from apoptotic DNA damage in the heart.
This produces end products that are fragments of double-strand DNA cleavage with three overhangs (Didenko et al., 1998), which can be specifically identified by Tag polymerase-generated probe (Didenko et al., 1998). The specificity of this molecular probe to identify apoptosis has been confirmed by other methods such as dual labeling of TdT and caspase-3 (Frustaci et al., 2000). In addition, this apoptotic specific probe in combination with fluorescence labeling of different cellular components allows quantitative detection of apoptotic cells, with the possibility of identifying the origin of the apoptotic cells, such as myocytes (stained with α-sarcomeric actin), endothelial cells (stained with factor VIII), and fibroblasts (stained with vimentin) in the heart (Anversa, 2000).
This is characterized by double-strand DNA cleavage with blunt ends. That is because during necrosis, the release of lysosomal proteases degrades histones, resulting in loss of DNA protection and exposure to endonucleases and exonucleases. Endonucleases produce double-strand DNA cleavage with three overhangs, but exonucleases remove terminal nucleotides, leading to a blunt end of the damaged DNA. A probe generated by pfu polymerase can specifically recognize these blunt-end DNAs (Anversa, 2000). Its specific reaction with necrotic DNA has been confirmed by other methods such as the permeability of myosin antibody into necrotic cells (Guerra et al., 1999), and the disruption of the sarcolemma by vinculin staining, which can clearly define the continuity of the sarcolemmal surface (Yamashita et al., 2001).
Proportion of Apoptotic and Necrotic Cell Death in the Heart
This can be estimated by the combination of the above procedures. First, a conventional TUNEL procedure can be used to identify the total TUNEL-positive cells. Second, the procedure to define double-strand DNA breaks with blunt ends can be used to quantify the proportion of necrotic cells in the total TUNEL-positive population. Finally, the combination of the procedure to identify double-strand DNA breaks with 3′ overhangs, and the specific antibody to identify total ssDNA breaks can distinguish the proportion of apoptotic cells from those with ssDNA breaks only.
Distinguishing Apoptotic Myocytes from Nonmyocytes in the Myocardium
This is another problem to overcome. An in situ TUNEL assay in combination with a dual immunohistochemical detection of α-sarcomeric actin has been used to distinguish apoptotic myocytes from nonmyocytes (Kang et al., 2000a). Apoptotic myocytes are dually stained by TUNEL and α-sarcomeric actin, and apoptotic nonmyocytes are stained only by TUNEL. Another procedure is immuno-gold TUNEL and electron microscopic examination of the apoptotic cells (Kang et al., 2000a). The gold standard for identification of apoptotic cells is morphological examination by electron microscopy. The immuno-gold TUNEL and electron microscopic procedure defines the cell type and morphological characteristics of apoptotic cells.
Mitochondrial Control of Cell Death
The role of mitochondria in myocardial response to toxicants as well as therapeutic drugs has long been a focus of investigation. Mitochondrial control of cell death is an important topic of apoptotic research. Factors affecting mitochondrial control of cell death are presented in Fig. 18-9. These factors have the same target effect: modification of mitochondrial permeability transition (MPT).
Major factors affecting mitochondrial MPTP and myocardial cell death. Mitochondrial cytochrome c release is a critical factor controlling cardiomyocyte apoptosis. Mitochondrial permeability transition pore (MPTP) opening is a determinant factor for cytochrome c release, as well as for electron transport collapsing leading to decreases in ATP production. The factors affecting MPTP thus are classified as pro-cell death and anti-cell death. Many other factors also affect cell death programs such as apoptosis inducing factor (AIF) released from mitochondria, but the involvement of MPTP is not evidenced.
Mitochondrial Permeability Transition
MPT occurs under toxic insults (Kroemer et al., 1997). This MPT behaves like a membrane pore that allows diffusion of solutes <1500 Da in size. Although MPT can occur as a temporary event, it can rapidly become irreversible, with the resulting loss of mitochondrial homeostasis and high-amplitude mitochondrial swelling. Because the inner membrane has a larger surface area than the outer membrane, mitochondrial swelling can cause the rupture of the outer membrane, releasing intermembrane proteins into the cytosol (Reed et al., 1998). Among the intermembrane proteins is cytochrome c. Another possible mechanism that leads to mitochondrial cytochrome c release is the action of Bax, a proapoptotic protein of the Bcl-2 family (Adams and Cory, 1998). Overexpression of Bax under oxidative stress conditions has been observed in a number of studies in different tissues including the heart (Cook et al., 1999). It has been shown that Bax is translocated from cytosol to mitochondria and forms pores in mitochondrial outer membranes, leaving the inner membranes intact (Jurgensmeier et al., 1998). This mechanism implies that Bax-mediated cytochrome c release is independent of MPT (Saikumar et al., 1998). The release of cytochrome c from mitochondria into the cytosol is a critical initiation step in myocardial apoptosis. Cytochrome c aggregates with apoptotic protease activating factor-1 (apaf-1, another factor released from mitochondria under oxidative stress), procaspase-9, and dATP, and subsequently activates caspase-9, which activates caspase-3. The apoptotic pathway involving mitochondrial cytochrome c release and caspase-3 activation is presented in Fig. 18-10. To determine the significance of the caspase-3-activated apoptotic pathway in the pathogenesis of toxicological cardiomyopathy, a caspase-3-specific inhibitor, Ac-DEVD-cmk, is often used. For instance, treatment of cultured cardiomyocytes isolated from neonatal mice with Ac-DEVD-cmk efficiently suppressed caspase-3 activity and reduced the number of apoptotic cells in cultures under treatment with Adriamycin (Wang et al., 2001a).
Mitochondrial control of myocardial apoptotic pathway. Exposure to drugs or xenobiotics can cause mitochondrial MPTP opening and cytochrome c release, which in association with apaf-1 and in the presence of dATP activates caspase-9. Caspase-9 consequently activates caspase-3 leading to apoptosis. Factors regulating the apoptotic pathway include those listed in Fig. 18-9, such as Bax and Bcl-2. Ac-DEVD-cml is a selective inhibitor of caspase-3.
Defective Mitochondrial Oxidative Phosphorylation
This has been extensively investigated ever since the identification of mitochondrial oxidative phosphorylation. The link between defective oxidative phosphorylation and pathogenesis of cardiomyopathy has been revealed recently. The early phase of defects in oxidative phosphorylation increases mitochondrial outer membrane permeability, leading to cytochrome c release, thus resulting in cytochrome c-mediated caspase-9 activation and thereby caspase-3 activation, leading to apoptosis (Fosslien, 2001). The defective oxidative phosphorylation also leads to depletion of cellular ATP levels, resulting in necrosis (Fosslien, 2001). Fig. 18-11 presents a generalized mitochondrial oxidative phosphorylation process, including electron-transferring complexes and ATP production. Detection of mutated or otherwise defective components in oxidative phosphorylation is important for understanding the myocardial cell death by xenobiotics.
Diagram of the electron-transferring complexes I, II, III, and IV and the ATPase (V) present in the inner mitochondrial membrane. The respective complexes are I, NADH: ubiquinone oxidoreductase; II, succinate: ubiquinone oxidoreductase; III, ubiquinol: ferrocytochrome c oxidoreductase; IV, ferrocytochrome c: oxygen oxidoreductase; V, ATP synthesase. UQ 5 ubiquinone or coenzyme Q. The mitochondrial electron transport chain is coupled at three points so that the electron transfer between carriers is sufficiently exergonic to drive the transport of protons and to establish an electrochemical proton gradient upon which ATP formation depends. Various inhibitors and their sites are also noted.
Abnormal Mitochondrial Biosynthesis
This is also linked to myocardial pathogenesis. Both nuclear and mitochondrial DNA encode mitochondrial proteins. Therefore, nuclear DNA damage can lead to mutated products and abnormal mitochondrial biosynthesis. However, mitochondrial DNA encodes essential elements for mitochondrial function. Mitochondrial DNA is subjected to far more oxidative injury than nuclear DNA due to the lack of histones and high exposure to reactive oxygen species (ROS) generated by the electron transport chain. Mitochondrial DNA repair mechanism exists, although the repair is not as efficient as that of nuclear DNA repair. Due to these unique characteristics of mitochondrial DNA, cumulative mitochondrial DNA damage under oxidative stress conditions such as Adriamycin treatment leads to irreversible mitochondrial dysfunction in the heart (Zhou et al., 2001). This cumulative and relatively irreversible oxidative mitochondrial dysfunction concept has an important impact on our understanding of chronic as well as late-onset cardiomyopathy of anthracyclines. These drugs cause cardiomyopathy sometimes months to years after cessation of the drug therapy. During this time period, subtle pathological changes that may not be detectable but may continue to accumulate and lead to an overt toxic event. The cumulative and irreversible mitochondrial dysfunction might explain such a phenomenon, and may contribute to the delayed myocardial pathogenesis by Adriamycin.
Generation of Reactive Oxygen Species
Generation of ROS has been ascribed as an “unwanted” function of mitochondria. Drugs and other chemicals have been studied individually to determine how they produce ROS; however, debate continues regarding the importance of each identified pathway as well as the site of ROS generation in mitochondria. In general, it is accepted that changes in mitochondrial membrane potential are critically involved in ROS generation. There are two important potassium channels that play important roles in mitochondrial membrane permeability. The first is the mitochondrial ATP-sensitive potassium channel (Akao et al., 2001), and the second is the Ca2+-activated potassium channel in the cardiac inner mitochondrial membrane (Xu et al., 2002). It has been shown that diazoxide opens mitochondrial ATP-sensitive potassium channels and preserves mitochondrial integrity, as well as suppresses hydrogen peroxide-induced apoptosis in cardiomyocytes (Akao et al., 2001). The Ca2+-activated potassium channels, in contrast, contribute to mitochondrial potassium uptake of myocytes, and opening of these channels protects the heart from infarction (Xu et al., 2002).
Death Receptors and Signaling Pathways
Death receptor-mediated apoptotic signaling pathway has been one of the focuses of cardiotoxicity research. In this regard, cytokines that trigger the death receptor signaling pathways have been studied. Among these cytokines is tumor necrosis factor-α (TNF-α) (Kubota et al., 2001), the most studied cytokine in myocardial cell death signaling pathways. The pathway leading to TNF-α-induced myocardial apoptosis is mediated by TNF receptors, TNFR1, and TNFR2. TNF-α-binding of these receptors leads to activation of caspase 8, which in turn cleaves BID, a BH3 domain-containing proapoptotic Bcl2 family member. The truncated BID is translocated from cytosol to mitochondria, inducing first the clustering of mitochondria around the nuclei and release of cytochrome c, and then the loss of mitochondrial membrane potential, cell shrinkage, and nuclear condensation, that is, apoptosis. Caspase 8 also directly activates caspase-3, leading to apoptosis (Fig. 18-12). Besides TNF-α, Fas ligand is also able to induce apoptosis of cardiomyocytes through the death receptor-mediated signaling pathway (Hayakawa et al., 2002).
Simplified presentation of death receptors-mediated myocardial apoptosis. The cardiotoxicity-related death receptors include Fas and tumor necrotic factor receptor 1 (TNFR1), as shown. Other death receptors such as DR3 and DR4/5 are not included in the figure. Both Fas- and TNFR1-activated apoptotic pathways can be altered by drugs and xenobiotics directly or indirectly through changes in the production of Fas ligand (FasL) or TNF-α. Fas activation activates caspase-8 through Fas-associated death domain (FADD). TNFR1 activates TNF-R1-associated death domain (TRADD), leading to activation of FADD. TNFR1 also activates a receptor-interacting protein (RIP), which activates caspase-2 through the adaptor protein, RIP-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD). Both caspase-8 and caspase-3 activate the apoptosis executor proteins, caspase-3, -6, and -7.
The death receptor-mediated apoptosis thus can be divided into mitochondrion-dependent and mitochondrion-independent signaling pathways. There is a bewildering diversity of programmed cell death paradigms related to the mitochondrion-controlled process, eventually leading to caspase-3 activation and apoptosis. Under chronic toxic insults, the relative importance of mitochondrial MPT pore opening, cytochrome c release, and electron transport defects needs to be critically examined in order to understand the process or mode of cell death.
There are two important questions that need to be carefully addressed. First, how important is apoptosis in the overall pathogenesis of cardiomyopathy under different conditions? It essentially is a universal observation that apoptosis is involved in the cardiotoxicity of drugs and chemicals; however, there is very limited information regarding its significance in a quantitative manner in contribution to cardiomyopathy. This concern is followed by the second important question: can caspase inhibitors offer long-term protection against myocardial cell death leading to prevention of cardiomyopathy? It is important to note that apoptosis and necrosis are linked phenomena sharing common triggers, such as MPT pore opening, as depicted in Fig. 18-13. For instance, some studies have shown that caspase inhibitors effectively inhibit apoptosis, but cell death occurred by necrosis instead of apoptosis (Suzuki et al., 2001). It is thus important to define the most efficient approach to blocking myocardial cell death rather than inhibiting a particular cell death program (Kang, 2001).
Interchange between apoptotic and necrotic cell death pathways regulated by mitochondrial MPTP. Mitochondrial MPTP opening leads to cytochrome c-mediated activation of apoptotic pathway, which is ATP dependent. However, MPTP formation also leads to electron transport collapse and reduced ATP production, which eventually leads to necrosis. Inhibition of caspase-3 also potentially switches the cell death program from apoptotic to necrotic if the exposure to drugs or xenobiotics is persistent.
It is important to note that apoptosis is an energy-dependent process and that the switch in the decision between apoptosis and necrosis depends on ATP concentrations (Eguchi et al., 1997), as depicted in Fig. 18-13. In particular, conditions causing ischemia by many drug and chemical exposures to myocardial cells result in significant reduction and eventual depletion of adenine nucleotides. Loss of more than 70% of the total ATP pool present in myocardial cells results in a switch from apoptosis to necrosis (Leist et al., 1999). This extent of ATP depletion is observed in myocardial infarction.
Mitochondrial Dynamics and Autophagy
The importance of mitochondria in cardiac response to toxic insults and in the process of toxicological cardiomyopathy is related not only to the control of cell death, but also to autophagy, the tightly regulated cellular “housekeeping” process responsible for the degradation of damaged and dysfunctional cellular organelles and protein aggregates.
Autophagy occurs in all eukaryotic cells under the stress of starvation, hypoxia, and toxic insults, as well as under physiological stimulation such as hormones and developmental signals. There are selective autophagy and nonselective autophagy. Selective autophagy of mitochondria is termed mitophagy, which is triggered by MPT pore opening and loss of mitochondrial membrane potential (Tolkovsky, 2009). In cardiomyocytes and other terminally differentiated cells, mitophagy is a continuous process of mitochondrial turnover, but the rate of this turnover is influenced by stresses and makes a critical contribution to myocardial pathogenesis. Nonselective autophagy has been observed in response to nutrient starvation; the degradation of cytosolic components including mitochondria via autophagy provides amino acids and lipid substrates for intermediate metabolism (Tolkovsky, 2009).
The well-known and comprehensively depicted function of mitochondria is the generation of adenosine triphosphate (ATP) through oxidative phosphorylation. However, it has been known that mitochondria also carry out other critical functions, including regulation of programmed cell death or apoptosis, the synthesis and degradation of essential metabolites, heme and steroid synthesis, regulation of cell proliferation, maintenance of plasma membrane potential, and calcium signaling. These diverse functions demand mitochondrial biogenesis and turnover highly regulated. In cardiomyocytes and other terminally differentiated cells, this regulation is far more important for the vital function of the cell.
Mitochondria are dynamic organelles and the morphology of mitochondria undergoes interchange between two distinct arrangements: elongated interconnected mitochondrial networks and discrete fragments. The former is accomplished by mitochondrial fusion and the latter by mitochondrial fission. In the heart, mitochondrial fusion and fission constitute a major response of cardiomyocytes to stresses. For instance, sustained pressure overload promotes structural, functional and metabolic remodeling of cardiomyocyte mitochondria to compensate for changes in energy demands and to remove damaged organelles. This process ultimately determines the fate of a cardiomyocyte: survival versus death. The changes in mitochondrial morphology are orchestrated by a group of mitochondrial fusion and fission proteins. These proteins were first identified in yeast or Drosophila, and the function of these proteins has been a major focus in the study of mitochondrial dynamics. But the identification of these proteins in the mammalian heart has made a significant impact on the role of mitochondrial dynamics in the pathogenesis of cardiac disease.
Mitochondrial fusion is a process of an elongated interconnected mitochondrial network formation. This is a fundamental process in the life of eukaryotic cells. Mitochondrial fusion requires coordinated joining of both the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). In this process, the constituents in the matrix and intermembrane space are secured—not to be released or mixed. Therefore, the fusion machinery involves OMM and IMM fusion, and their coordination.
OMM fusion is directed by mitofusins 1 and 2 (MFN1 and MFN2), large GTPase located in the OMM. They contain two transmembrane regions in the OMM, with a short loop in the intermembrane space and the major parts of the protein facing the cytosol. In mammalian cells, early during fusion two mitochondria approach each other in a tethered docking step. The carboxy-terminal heptad repeats of MFN1 have been shown to form an intermolecular antiparallel coiled coil that may tether adjacent mitochondria before fusion (Koshiba et al., 2004). The coiled coil formation by mitofusins thus draws the membranes close together and initiates lipid bilayer mixing, and the GTPase provides biomechanical energy for outer membrane fusion. It has been shown that mutation of the GTPase domain of MFN2 results in an accumulation of tethered but unfused mitochondria (Eura et al., 2003).
IMM fusion is governed by Mgm1, a dynamin-related large GTPase that is essential for inner membrane fusion in yeast (Meeusen et al., 2006). The mammalian Mgm1 ortholog, optic atrophy 1 (OPA1) protein, is tethered to the IMM and in the intermembrane space (Olichon et al., 2002). Differential splicing of Opa1 transcripts generates multiple variants (Song et al., 2007). A number of proteases localized in mitochondria cleave OPA1 disrupting OPA1 function and inhibiting fusion. Mutations in OPA1 selectively block inner membrane fusion. After the completion of outer membrane fusion, OPA1 is required in trans on both inner membranes of the fusion parteres. A normal membrane potential is required for fusion of IMM. Loss of membrane potential promotes cleavage of OPA1, causing that outer membrane fusion proceeds in the absence of inner membrane fusion.
Mitochondrial fusion is a process of the fragmented discrete mitochondrial morphogenesis. This process is regulated by dynamin-related protein 1 (DRP1) in mammals. This protein is soluble and contains an N-terminal GTPase, a middle domain, and a C-terminal GTPase effector, which is involved in self-assembly. Cells lacking DRP1 contain highly interconnected mitochondrial networks (Smirnova et al., 1998). During fission, DRP1 is recruited from the cytoplasm to mitochondria. Mammalian fission protein 1 (FIS1) in the OMM interacts with DRP1. FIS1 overexpression promotes mitochondrial fragmentation and FIS1 depletion stimulates interconnected mitochondrial networks (Yoon et al., 2003; James et al., 2003). In yeast, a mitochondrial division protein 1 (Mdv1) has been shown to coexist with Fis1 and interact with DRP1, but its metazoan homologs have not been identified, indicating a significant difference between metazoan and yeast mitochondrial fission machineries.
The function of mitochondrial fission machinery has been extensively understood in yeast, but less comprehensively in metazoans. In yeast, the fis1–Mdv1–Dnm1 complex is important for mitochondrial fission, but the lack of Mdv1 in metazoans makes a different mitochondrial fission machinery. It has been shown that knockdown of human FIS1 does not affect the distribution of DRP1 in mitochondria (Lee et al., 2004). A mitochondrial fission factor (MFF) has been found in mammalians, but not in yeast (Gandre-Babbe and van der Bliek, 2008). MFF is a tail-anchored protein containing heptad repeats and a C-terminal transmembrane domain that is embedded in the OMM. Depletion of MFF suppresses mitochondrial fission in mammalian cells. However, MFF and FIS1 exist in separate complexes, suggesting that they may act differently in mitochondrial fission (Gandre-Babbe and van der Bliek, 2008).
Regulation of Mitochondrial Dynamics
This is subjected to multiple factors that affect mitochondrial fusion and fission. However, a single factor-regulated mitochondrial dynamics rarely occurs because under most circumstances several factors are often coexisting. Therefore, whether or not mitochondria eventually undergo fusion or fission depends on the ultimate balance between the actions induced by several factors. On the other hand, it cannot be judged whether or not mitochondrial fusion or fission is beneficial or detrimental; only can the ultimate effect of mitochondrial fusion or fission be the judgment under certain conditions.
Cell cycle control of mitochondrial dynamics is often observed in mitotically active cells. Mitochondrial fusion is found during G1-S phase, but mitochondrial fission is necessary in cell division. In differentiated cells such as cardiomyocytes, the cell cycle control of mitochondrial dynamics is less important. However, reactivation of fetal gene program in adult cardiomyocytes in cardiac hypertrophy and heart failure would affect mitochondrial dynamics, which has not been understood.
Oxidative and nitrosative stress often induces mitochondrial fission. Mitochondria are the major site for the production of ROS in the heart. The process not only affects mitochondrial dynamics, but generates ROS-induced damage to proteins, lipids, and DNA. Studies have shown overexpression of MFN inhibits ROS-induced mitochondrial fission and protects cardiomyocytes from oxidative injury (Yu et al., 2011).
Ca2+ regulation of mitochondrial dynamics is more related to the role of mitochondria in Ca2+ transport and homeostasis. Activation of calcineurin (CaN) and calmodulin-dependent protein kinase 1 (CaMK1) by Ca2+ affects the recruitment of DRP1 and mitochondrial translocation. Inhibition of mitochondrial Ca2+ uptake attenuates mitochondrial fission, indicating the importance of intermitochondrial Ca2+ concentrations in the regulation of mitochondrial dynamics.
Metabolic regulation of mitochondrial dynamics is the fundamental process of mitochondrial biological function. Mitochondrial dynamics and energy substrate metabolism are tightly linked. Increased metabolic rates upregulate mitochondrial fusion and dense packing of cristae. Downregulation of OPA1 or MFN leads to fragmented mitochondria with reduced membrane potential and oxygen consumption. Glucose concentrations play a critical role in the regulation of mitochondrial dynamics. Changes in fusion-related proteins have been observed in animal models and patients with type 2 diabetes (Zorzano et al., 2010). High glucose levels stimulate mitochondrial fragmentation in H9c2 cardiomyoblasts and neonatal rat cardiomyocytes (Yu et al., 2008).
Toxicological Significance of Mitochondrial Dynamics
Toxico-logical significance of mitochondrial dynamics in the heart can be observed under a diversity of exogenous and endogenous stress conditions. Mitochondrial dynamics is a physiological process as well as a feature of pathogenesis. Under physiological conditions, mitochondrial fusion and fission are essential to maintain metabolic homeostasis and the balance between energy production and consumption. Disruption of this physiological process under stress conditions or toxicological exposures leads to overwhelming of mitochondrial fusion or fission. Under these conditions, cell death and maladaptation occur, leading to toxicological pathogenesis.
Intricately regulated degradation and turnover of subcellular components ensure normal cellular function, growth, and development. The major catabolic pathway responsible for the disposal of damaged organelles and protein aggregates is autophagy, is a lysosome-dependent proteolytic pathway capable of processing cellular components, including damaged organelles and protein aggregates. During this process, damaged organelles and proteins are encircled in a double-membrane vesicle, so-called autophagosome, delivered to lysosomes. The contents in the autophagosome are hydrolyzed to yield amino acids, fatty acids, and substrates for ATP generation that can be recycled to synthesize new proteins, high energy phosphates, and renewed cellular components. Autophagy is a conserved mechanism for cell survival under conditions of starvation and stress. Therefore, autophagy has been recognized to play an important role in maintaining cellular homeostasis, and upregulation of autophagy under stress conditions may serve as an adaptive process.
Autophagy can be subdivided into three different processes: macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy is often referred to as autophagy that is characterized by the sequestration of organelles and proteins within an autophagosome, as described above. Microautophagy refers to protrusion of the lysosomal membranes per se around a region of cytoplasm. Chaperone-mediated autophagy is defined as that degradation is restricted only to those proteins with a consensus peptide sequence recognized by specific chaperone complexes. Current mechanistic understanding mostly focuses on macroautophagy or autophagy.
There are multiple proteins and interrelated signaling pathways involved in autophagy. This complex process can be divided into four distinct but consecutive steps: (1) induction of autophagy; (2) assembly of autophagosomes; (3) docking and fusion of autophagosome with lysosomal membrane; and (4) breakdown of the autophagic body.
The induction of autophagy requires the participation of three different protein complexes: autophagy-specific gene 1 (Atg1) complex, target of rapamycin (TOR) complex 1 (TORC1), and class III phosphatidylinositol-1 kinase (PI3K) complex. In this step, TORC1 acts as a sensor of cellular nutrient status. Nutrient stress leads to the partial dephosphorylation of Atg13, which in turn increases the binding affinity of Atg13 to Atg1, a pivotal precursor for subsequent autophagosome formation.
The autophagosome assembly and formation is the most complex phase of autophagy. The Atg5 complex and microtubule-associated protein 1 light chain 1 (LC3)-II work in concert to initiate elongation or expansion of the isolated membrane that will eventually form the matured autophagosome. There are several Atg genes involved in this phase and they work sequentially. It has been shown that Atg5 is essential for autophagy. Mice with a cardiac-specific disruption of Atg-5 develop premature age-related heart failure, indicating that autophagy is an essential regulatory mechanism for normal cardiac function over the course of a life (Taneike et al., 2010).
Autophagosome docking and fusion have been studied in yeast and found to involve multiple vesicle trafficking and membrane fusion proteins. The final stage of autophagy, breakdown of autophagic body and its content, generates substances for protein synthesis, lipid turnover, and ATP production.
Cardiac Hypertrophy and Heart Failure
Adaptive and Maladaptive Responses
Myocardial adaptation refers to the general process by which the ventricular myocardium changes in structure and function. This process is often referred to as “remodeling.” During maturation, myocardial remodeling is a normal feature for adaptation to increased demands. However, in response to pathological stimuli, such as exposure to environmental toxicants, myocardial remodeling is adaptive in the short term, but is maladaptive in the long term, and often results in further myocardial dysfunction. The central feature of myocardial remodeling is an increase in myocardial mass associated with a change in the shape of the ventricle (Frey and Olson, 2003).
At the cellular level, the increase in myocardial mass is reflected by cardiac myocyte hypertrophy, which is characterized by enhanced protein synthesis, heightened organization of the sarcomere, and the eventual increase in cell size. At the molecular level, the phenotype changes in cardiac myocytes are associated with reintroduction of the so-called fetal gene program, characterized by the patterns of gene expression mimicking those seen during embryonic development. These cellular and molecular changes are observed in both adaptive and maladaptive responses, thus distinguishing adaptive from maladaptive responses is difficult.
There are both physiological hypertrophy and pathological hypertrophy of the heart. Physiological hypertrophy is considered an adaptive response, which is an adjustment of cardiac function for an increased demand of cardiac output. Such an adaptive hypertrophy is the increase in cardiac mass after birth and in response to exercise. A biochemical distinction of the adaptive hypertrophy is that myocardial accumulation of collagen does not accompany the hypertrophy. Functionally, the increased mass is associated with enhanced contractility and cardiac output. In response to toxicological stresses, the heart also often increases its mass, which has been viewed as an adaptive response as well. However, most recent evidence suggests that cardiac hypertrophy is a maladaptive process of the heart in response to intrinsic and extrinsic stresses.
Although toxic stress-induced hypertrophy can normalize wall tension, it is a risk factor for sudden cardiac death and has a high potential to progress to overt heart failure. A distinction between adaptive and maladaptive hypertrophy is whether the hypertrophy is necessary for the compensatory function of the heart under physiological and pathological stress conditions. Many studies using genetically manipulated mouse models, either in the form of gain-of-function or loss-of-function, have supported the hypothesis that cardiac hypertrophy is neither required nor necessarily compensatory. For instance, forced expression of a dominant-negative calcineurin mutant confers protection against hypertrophy and fibrosis after abdominal aortic construction (Zou et al., 2001). Also, the elimination of hypertrophy in animals by calcineurin suppression did not cause compromised hemodynamic changes over a period of several weeks (Hill et al., 2000). Therefore, in these experimental approaches, hypertrophic growth could be abolished in the presence of continuous pressure overload, but the compensatory response could not be compromised. An interesting observation is that an almost complete lack of cardiac hypertrophy in response to aortic banding in a transgenic mouse model was accompanied with a significant slower pace of deterioration of systolic function (Esposito et al., 2002). These observations indicate that cardiac hypertrophy in response to extrinsic and intrinsic stresses is not a compensatory response. However, cardiac hypertrophy increases the risk for malignant arrhythmia and heart failure, and thus is now viewed as a maladaptive response.
Hypertrophic Signaling Pathways
Extrinsic and intrinsic stresses activate signaling transduction pathways leading to fetal gene program activation, enhanced protein synthesis of adult cardiomyocytes, and the eventual hypertrophic phenotype. The signaling pathways include several components: G-protein-coupled receptors, protein kinases including MAPK, PKC, and AMPK, calcium and calcineurin, and phosphoinositide 3-kinase (PI3K)/glycogen synthase kinase 3β (GSK3β), and transcription factors. Activation of each of the components is sufficient to induce myocardial hypertrophic growth. These components also affect each other through cross talk. The diagram presented in Fig. 18-14 briefly summarizes these pathways and their interactions. Among these pathways, protein kinases, calcium/calcineurin, and transcription factors have been discussed above. A brief summary for the G-protein-coupled receptors and the PI3K/GSK3 pathway is presented as follows.
Overview of signaling transduction pathways involved in cardiac hypertrophic growth and their cross-talk interactions. The signaling that occurs at the sarcolemmal membrane is shown at the top and the intermediate transduction of signals by various kinases and phosphatases is shown in the middle. The nucleus is shown at the bottom. ANP, atrial natriuretic peptide; Ang II, angiotensin II; BNP, B-type natriuretic peptide; CaMK, calmodulin-dependent kinase; CDK, cyclin-dependent kinase; DAG, diacylglycerol; EGF, epidermal growth factor; Endo-1, endothelin-1; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; FGFR, FGF receptor; GC-A, guanyl cyclase-A; GPCR, G-protein-coupled receptors; GSK3β, glycogen synthase kinase-3β; HDAC, histone deacetylases; IκB, inhibitor of NF-κB; IGF-I, insulin-like growth factor-I; IKK; inhibitor of NF-κB kinase; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; JNK, c-Jun N-terminal kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; MAPKKKK, MAPKKK kinase; MEF, myocyte-enhancer factor; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; NIK, NF-κB-inducing kinase; PDK, phosphoinositide-dependent kinase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PKC, protein kinase C; PKD, protein kinase D; PLA2, phospholipase A2; PLC, phospholipase C; Pol II, RNA polymerase II; RTK, receptor tyrosine kinase; TAK, TGFβ-activated kinase; TGF-β, transforming growth factor-beta; TGFR, TGF receptor; TNFα, tumor necrosis factor-α; TNFR, TNFα receptor (Copied from Heineke, 2006).
Myocardial adrenergic, angiotensin, and endothelin (ET-1) receptors belong to G-protein-coupled receptors, which are coupled to three major classes of heterotrimeric GTP-binding proteins, Gαs, Gαq/Gα11, and Gαi. Activation of Gαq-coupled receptors is sufficient to induce myocyte hypertrophy in vitro (Adams et al., 1998). Cardiac-specific ablation of Gαq/Gα11 in adult animals causes an almost complete lack of cardiac hypertrophy in response to aortic banding (Wettschureck et al., 2001). Overexpression of a dominant-negative mutant of Gαq in transgenic mouse hearts suppresses pressure-overload hypertrophy (Akhter et al., 1998). Cardiac overexpression of Gαs, the downstream effector of β1-adenergic receptors in the heart, initially increases contractility, but eventually results in cardiac hypertrophy, fibrosis, and heart failure (Bisognano et al., 2000).
Phosphoinositide 3-Kinase/Glycogen Synthase Kinase 3β Pathway
Activation of PI3K is found in both physiological and pathological hypertrophy. Insulin-like growth factor (IGF) is involved in the growth of the heart after birth (Shioi et al., 2002). Overexpression of IGF induces cardiac hypertrophy (Delaughter et al., 1999). IGF signals through PI3K to the serine/threonine kinase Akt or protein kinase B. Both PI3K and the Akt induce hypertrophic growth of adult hearts. Overexpression of constitutively active PI3K mutant in the heart leads to increased heart size in mice, and expression of dominant-negative PI3K results in a small heart (Shioi et al., 2000). Overexpression of Akt induces cardiac hypertrophy in transgenic mice without adverse effects on systolic function (Matsui et al., 2002). Akt phosphorylates GSK3β, and thus inhibits the activation of GSK3β. Otherwise, the activated GSK3β phosphorylates transcription factors of the NFAT family (Fig. 18-14). As discussed above, activation of calcineurin dephosphorylates NFAT3 in the cytoplasm, which enables NFAT3 to translocate to the nucleus where it can activate hypertrophic gene expression dependent on or independent of GATA4. Phosphorylation of NFAT3 in the nucleus by GSK3β promotes NFAT3 translocation to the cytoplasm, becoming inactive. Hypertrophic stimuli such as β-adrenergic agonist isoproterenol, ET-1, and phenylepherine all induce GSK3β phosphorylation in a PI3K-dependent fashion, indicating possible requirement of inactivation of GSK3β through phosphorylation in hypertrophic growth of the heart.
Transition from Cardiac Hypertrophy to Heart Failure
Pathological hypertrophy is a risk factor for malignant arrhythmia and heart failure. The link of heart hypertrophy to malignant arrhythmia will be discussed in the next section. The critical cellular event of this transition is myocardial apoptosis triggered by inflammatory cytokines, such as TNF-α, and neurohormonal factors, such as atrial natriuretic peptide (ANP), which leads to dilated cardiomyopathy and deterioration of cardiac function. Toxicological exposures may cause dilated cardiomyopathy or heart failure without an intermediate hypertrophic stage. Myocardial cell death also plays an essential role in direct cardiac dilation pathogenesis. Fig. 18-15 illustrates the process of xenobiotic-induced transition from cardiac hypertrophy to heart failure.
Acute and chronic toxic exposure-induced heart failure and the transition from heart hypertrophy to heart failure. Acute exposure to drugs or xenobiotics can cause cardiac arrhythmia, which is often observed. But if the toxic insult is so severe, myocardial apoptosis and necrosis become predominant leading to dilated cardiomyopathy and heart failure. However, the heart often survives from toxic insults through adaptive mechanisms involving upregulation of hypertrophic genes and heart hypertrophy. Heart hypertrophy increases the risk for QT prolongation and sudden cardiac death, and also activates neurohormonal regulatory mechanisms including elevation of plasma concentration of sympathetic neural transmitters and angiotensins. These compensatory mechanisms in turn activate counter-regulatory mechanisms such as ANP, BNP, and TNF-α. A long-term action of the counter-regulatory mechanisms leads to myocardial remodeling and the transition from heart hypertrophy to heart failure.
Alterations of biochemical reactions in the myocardium are often seen soon after exposure to environmental toxicants. These include alterations in ionic homeostasis, such as changes in intracellular calcium concentrations, which occur in most exposures to environmental toxicants (Symanski and Gettes, 1993). Aberrant energy metabolism is another early response to environmental toxicants in the heart, resulting in decreased production and/or enhanced consumption of ATP (Abas et al., 2000). Alterations in enzymatic reactions are often described in cardiac toxic responses (Depre and Taegtmeyer, 2000). The early signaling pathways leading to myocardial toxic responses are the focus of current cardiac toxicology research (Piano, 1994). Detailed descriptions of these pathways and their role in cardiotoxicity are yet to be explored. It is likely that activation of signaling pathways is a critical response of myocardial cells to environmental toxic insults (Cheng et al., 1999a). The cross talk between signaling pathways determines the ultimate outcome of myocardial responses to chemicals.
Physiological alterations occur both as early responses to environmental toxicants and as subsequent events in the late development of cardiomyopathy. The most obvious myocardial dysfunction that occurs in the early responses to toxicants is cardiac arrhythmia (Peters et al., 2000), which often results from the changes in intracellular calcium concentrations and other biochemical alterations, leading to miscommunication between cells and misconduction of electricity (Rosen, 1995). These changes, if not accompanied by cardiomyopathy, do not involve myocardial cell death and are reversible. In contrast, the late phase of cardiac dysfunction and arrhythmia, however, often result from cardiomyopathy.
Changes in myocardial morphology take place when extensive toxic insults are imposed on the heart and/or toxic exposures persist (He et al., 1996). Cardiac hypertrophy is often observed as a consequence of long-term toxic insults. From cardiac hypertrophy to heart failure, activation of compensatory mechanisms, including the sympathetic nervous system and the renin–angiotensin system, occurs (Holtz, 1993). The compensatory response in turn activates counter-regulatory mechanisms such as upregulation of ANP expression (Francis and Chu, 1995) and increases in cytokines, such as TNF-α production. Extensive biochemical, physiological, and molecular changes result in myocardial remodeling (Swynghedauw, 1999) and remarkable cell death, ultimately leading to heart failure.
QT Prolongation and Sudden Cardiac Death
Recognition of QT prolongation and its associated adverse effects on the heart has been a major focus in drug discovery and development during the last decade. A number of drugs have been found to cause QT prolongation and TdP, and thus were removed from the market or relabeled for restricted use. It has been known for a long time that quinidine causes sudden cardiac death; however, the severe and lethal side effect of QT prolongation did not draw sufficient attention until the last decade, due to the lack of knowledge and experimental approaches to obtain a comprehensive understanding of QT prolongation. Knowledge on QT prolongation has accumulated and regulatory guidelines for a battery of preclinical tests to assess QT liability of a potential drug are recommended.
Definition of QT Prolongation
A simple definition for QT prolongation is that the length of QT interval observed from a typical electrocardiogram is prolonged. Clinically, long QT syndrome is defined when the QT interval is longer than 460 milliseconds. However, TdP occurs with an average increase in QT interval by approximately 200 milliseconds (a normal QT interval is about 300 milliseconds). A human study has found that TdP does not occur with a QT interval shorter than 500 milliseconds (Joshi et al., 2004). In general, the long QT syndrome can be divided into two classes: congenital and acquired. Congenital long QT syndrome is rare and acquired is the major concern of drug cardiac toxicity in pharmaceutical discovery and development.
Molecular Basis of QT Prolongation
Prolongation of the QT interval on the electrocardiogram is caused by prolongation of the action potential of ventricular myocytes. In cardiac action potential, phase 0 represents depolarization of myocytes and the depolarization of all ventricular myocytes is measurable as the QRS complex on the electrocardiogram. Phase 1 of the cardiac action potential is recognized as a partial repolarization of the membrane due to inactivation of cardiac sodium channels, and activation of transit outward potassium channels. Phase 2 of the action potential is generated primarily by slowly decreasing inward calcium currents through L-type calcium channels, and gradually increasing outward currents through several types of potassium channels. This phase is sensitive to small changes in ion currents and is a critical determinant of the duration of the action potential. At this point, the cardiac cycle of the electrocardiogram has returned to baseline. Phase 3 of the cardiac action potential represents myocardial cell repolarization due to outward potassium currents. There are two critical potassium channels that terminate the plateau phase (phase 2) and initiate the final repolarization phase 3: IKr and IKs. IKr is the rapidly activating delayed rectifier potassium current, and IKs is the slowly activating delayed rectifier potassium current. The repolarization phase correlates with the T wave on the electrocardiogram. Therefore, the duration of the QT interval is related to the length of the ventricular action potentials.
A reduction in net outward current and/or an increase in inward current are potential contributors to the prolongation of cardiac action potential, thereby QT prolongation on the electrocardiogram. Although many channels are potentially involved in the prolongation of the cardiac action potential, current studies have identified three important channels that play a critical role in the plateau phase (phase 2) of the cardiac action potential, sodium inward channels, and potassium outward channels (IKr and IKs).
Sodium Channel Dysfunction
Sodium channel dysfunction in congenital long QT syndrome is related to mutations in SCN5A gene that encodes the α-subunits of sodium channels. Mutational analyses have found 14 distinct mutations of SCN5A associated with long QT syndrome (Splawski et al., 2000). It has been hypothesized that gain-of-function mutations in SCN5A would cause long QT syndrome because reopening of the sodium channels during the plateau phase of action potential, even in a small inward current, would lengthen the duration of the cardiac action potential. Sodium channel inactivation immediately following depolarization (phase 1) is important for the transition to phase 2 of the action potential. A mutation of SCN5A has been found to destabilize the inactivation gate (Bennett et al., 1995). Activation of these mutant sodium channels is normal and the rate of inactivation appears slightly faster than normal, but these mutant channels can reopen during the plateau phase of the action potential, leading to a prolonged plateau phase.
The IKr potassium channels critically affect the length of the plateau phase of the cardiac action potential. The human ether-à-go-go -related gene (HERG) is expressed primarily in the heart and encodes the α-subunit of the cardiac IKr potassium channel. There are 94 mutations of HERG, which have been identified to represent 45% of the total number of mutations related to long QT syndrome to date (Splawski et al., 2000). The HERG α-subunits assemble with MiRP1 β-subunits to form cardiac IKr channels. The IKr potassium channel is one of the two channels that are primarily responsible for termination of the plateau phase of the action potential. During the repolarization of the action potential, the IKr channels open, resulting in an increase in the magnitude of IKr current during the first-half of phase 3 repolarization. Many HERG mutations occur around the membrane-spanning domains and the pore region of the channel. Most of these mutations have a loss-of-function effect and many long QT syndrome-associated mutations in HERG are missense mutations, which lead to a dominant-negative effect on IKr channels because the functional IKr potassium channels are composed of heteromultimers including several HERG subunits. Therefore, the loss-of-function mutations in HERG make a critical contribution to the long QT syndrome due to the prolonged plateau phase of cardiac potential.
The IKs potassium channel is the other one of the two channels primarily responsible for the termination of the plateau phase of the action potential. The IKs potassium channel is assembled from KVLQT1 α-subunits and the minK β-subunits. There are two molecular mechanisms that possibly account for reduced KVLQT1 function in the long QT syndrome (Wollnik et al., 1997). First, intragenic deletions of one KVLQT1 allele result in synthesis of abnormal α-subunits that do not assemble with normal subunits, leading to a 50% reduction in the number of the functional channels. Second, missense mutations result in synthesis of KVLQT1 subunits with structural abnormalities, which can assemble with normal subunits. Channels formed from the mutant KVLQT1 subunits have reduced or no function. Both of these mutations result in a dominant-negative effect. Interestingly, both KVLQT1 and minK are expressed in the inner ear, in which the channels function to produce a potassium-rich fluid known as endolymph that bathes the organ of Corti, the cochlear organ responsible for hearing. Individuals with Jervell and Lange-Nielsen syndrome have homozygous mutations of KVLQT1 or minK, thus having no functional IKs channels. These individuals have severe arrhythmia susceptibility and congenital neural deafness.
Therefore, the molecular basis of the QT prolongation on the electrocardiogram is the prolongation of cardiac action potential. In this regard, the inward sodium channels and outward potassium channels play an important role in increasing the length of the plateau phase of action potentials. Congenital long QT syndrome is related to gain-of-function mutations in sodium channels and/or loss-of-function mutations in potassium channels. Acquired long QT syndrome is also related to altered function of these channels; however, many other factors that affect the phenotype of long QT syndrome and the clinical manifestations.
Torsade De Pointes and Sudden Cardiac Death
The abnormalities of different channels in different regions of the heart at varying levels result in channel dysfunction with regional variability. The regional abnormalities of cardiac repolarization or conductance provide a substrate for arrhythmia. Under these conditions, arrhythmia is induced if a trigger mechanism is implanted. The trigger for arrhythmia in the long QT syndrome is believed to be spontaneous secondary depolarization that arises during or just following the plateau of the action potential. This small action potential is the so-called early afterdepolarization, which occurs preferentially in M cells and Purkinje cells due to reactivation of the L-type calcium channels and/or activation of the sodium–calcium exchange current. When the spontaneous depolarization is accompanied by a marked increase in dispersion of repolarization, the likelihood to trigger an arrhythmia is increased. Once triggered, the arrhythmia is maintained by a regenerative circuit of electrical activity around relatively inexcitable tissue, a phenomenon known as reentry. The development of multiple reentrant circuits within the heart causes ventricular arrhythmia, or TdP, leading to sudden cardiac death. Drugs causing TdP are considered severe cardiac toxic agents. There are several drugs that were removed from the market due to their TdP effect. These drugs include those recently removed cyclooxygenase-2 (COX-2) inhibitors, Vioxx and Bextra, which will be discussed in the “Cardiac Toxic Chemicals” section. QT prolongation effect is now a required test by the US Food and Drug Administration for drug development.
Parameters Affecting QT Prolongation and Torsadogenesis
Alterations in the function of cardiac channels or “cardiac channelopathies” occur at the cellular level. However, electrotonic cell-to-cell coupling influences the dispersion of repolarization. If myocardial cells with intrinsically different duration of action potential are well coupled, electrotonic current flow attenuates the differences in action potential duration in individual cardiomyocytes. Therefore, torsadogenesis results from not only cardiomyocytes, but also other types of cells and the interaction among these cells. There are many factors that affect the clinical manifestations of QT prolongation and torsadogenesis. Genetic polymorphisms and female gender are two distinct risk factors. The mechanism of the polymorphisms and the rationale for high susceptibility of females to QT prolongation and torsadogenesis are yet to be determined.
Drugs and Environmental Toxicants
Drug-induced QT prolongation is a major acquired long QT syndrome. Selective blockers of potassium channels, including the drugs so-called class III antiarrhythmics, have been developed for the treatment of various atrial arrhythmias. However, these drugs predictably produce long QT syndrome, which is sufficient to cause TdP in 5%–7% of recipients. Environmental exposure to PM pollution in air is a risk factor for QT prolongation in elders, children, and individuals with compromised hearts.
Disturbances in Ion Homeostasis
Hypokalemia in combination with torsadogenic drugs is a most recognized risk factor for QT prolongation and TdP. It is also shown that sodium supplementation can diminish the long QT syndrome due to the gain-of-function mutations in sodium channels. Stress-induced Ca2+ overload in myocardial cells increases the likelihood of arrhythmia. The electrode imbalance exerts more effect on compromised hearts.
Gap junction-mediated intercellular communication is essential in the propagation of electrical impulse in the heart. The gap junction is composed of connexons, as described in the overview of cardiac structural and physiological features section. Under normal conditions, the gap junction electrotonic current flow attenuates the differences in action potential duration of myocardial cells. Toxicological exposures cause damage to connexons leading to disruption of electrotonic cell-to-cell coupling, thus the differences in the action potential duration would be dominant, in particular under the influence of torsadogenic drugs or conditions.
Myocardial Ischemic Injury
Acute myocardial ischemia can cause immediate arrhythmia due to disturbance in ionic homeostasis, which is often transient. However, acute ischemia induces myocardial infarction that can lead to the block of cardiac conductance. Under the myocardial infarction, the areas separated by the scar tissue would be uncoupled, making the differences in the duration of action potential of myocardial cells in different regions apparent. The infarct heart thus is more susceptible to drug-induced QT prolongation and TdP.
Purkinje fibers are derived from myogenic precursors during embryonic development. The normal distribution of Purkinje fibers in the myocardium is proportional to the mass of the heart. Cardiac hypertrophy resulting from the hypertrophic growth of cardiac myocytes would lead to unbalanced distribution of Purkinje fibers in the remodeling heart. The conduction of pacemaker potentials would thus be interrupted.
Dilated cardiomyopathy in alcoholics often involves myocardial fibrosis, which simulates the effect of myocardial infarction on the electrical conduction in the heart and block of cardiac conductance.
Most individuals with failing hearts die suddenly of cardiac arrhythmias. Heart failure presents a common, acquired form of the long QT syndrome. In human heart failure, selective downregulation of two potassium channels, Ito1 and IK1, has been shown to be involved in action potential prolongation. The Ito1 current is involved in phase 1 of the action potential and opposes the depolarization. The increase in depolarization may be adaptive in the short term because it provides more time for excitation–contraction coupling, mitigating the decrease in cardiac output. However, downregulation of potassium channels becomes maladaptive in the long term because it predisposes the individual to early afterdepolarization, inhomogeneous repolarization, and polymorphic ventricular tachycardia.
Biomarkers for Cardiac Toxicity
Myocardial injury can be divided into two major classes: structural and nonstructural injuries. The structural damage of the heart includes cell death and the associated histopathological changes such as myocardial infarction. Functional deficits often accompany the structural injury. Nonstructural damage represents functional deficits without apparent structural alterations. Myocardial adaptation to intrinsic and extrinsic stresses leading to myocardial structural changes such as hypertrophy should be in the category of structural damage because the progression of hypertrophy leads to heart failure in which cell death is a major determinant factor. Myocardial structural changes and functional alterations can be indirectly measured by echocardiography and electrocardiogram in combination with stress testing. The data generated from these measurements can be considered in a broad sense as biomarkers. However, in clinical practice and experimental approach, biomarkers are referred to as indexes of myocardial injury measured from blood samples. The fundamental principle of the biomarkers is that molecules that are released from the myocardium under various injury conditions are readily detectable from blood samples.
For a biomarker to be indicative of myocardial damage, an important question needs to be addressed is what characteristics are required for a valid biomarker. In 2000, an Expert Working Group (EWG) on biomarkers of drug-induced cardiac toxicity was established under the Advisory Committee for Pharmaceutical Sciences of the Center for Drug Evaluation and Research of the US Food and Drug Administration. The report from this EWG has summarized the characteristics of ideal cardiac toxic injury biomarkers (Wallace et al., 2004). These characteristics include cardiac specificity, sensitivity, predictive value, robust, bridge preclinical to clinical, and noninvasive procedure/accessibility. These characteristics are adapted as a standard for development and validation of a biomarker of myocardial injury.
Availability of Biomarkers
Currently, validated biomarkers that are included in clinical diagnostic testing guidelines are all related to myocardial structural injury. Developing biomarkers for nonstructural injury is most challenging and demands implantation of more advanced technologies such as functional genomics and proteomics. In addition, currently available biomarkers have limitations, although they are useful.
There are three major CK isoenzymes identified; CK-MM is the principal form in skeletal muscle, CK-MB presents in myocardium in which CK-MM is also found, and CK-BB is the predominant form in brain and kidney. Elevation of serum CK-MB is considered a reasonably specific marker of acute myocardial infarction.
Myoglobin is found in all muscle types and its value as a biomarker of myocardial injury is based on the fact that serum concentrations of myoglobin increase rapidly following myocardial tissue injury, with peak values observed one to four hours after acute myocardial infarction. Elevation of serum myoglobin is likely reflective of the extent of myocardial damage.
B-Type Natriuretic Peptide
BNP is a cardiac neurohormone secreted by the ventricular myocardium in response to volume and pressure overload, and the release of BNP appears to be directly correlated with the degree of ventricular wall tension. BNP is now accepted as a biomarker for congestive heart failure and is included in the European guidelines for the diagnosis of chronic heart failure.
The acute phase reactant CRP is a marker of systemic and vascular inflammation, which appears to predict future cardiac events in asymptomatic individuals. In particular, inflammation has been shown to play a pivotal role in the inception, progression, and destabilization of atheromas. A predictive value of CRP for the prognosis of coronary heart disease is thus proposed. The measurement of CRP appears to provide additional prognostic information when cTnT is measured at the same time.
Cardiac troponin T (cTnT) and I (cTnI) are constituents of the myofilaments and expressed exclusively in cardiomyocytes. It is thus of absolute myocardial tissue specificity. In healthy persons, serum cTnT or cTnI are rarely detectable. Therefore, any measurable concentrations of serum cTnT or cTnI reflect irreversible myocardial injury such as myocardial infarction. The clinical experience has arrived at a recommendation that cTn measurement becomes the “gold standard” for diagnosis of acute myocardial infarction.
Biomarker Applications and Limitations
All the biomarkers described above have been used as indices of myocardial injury in clinical practice and experimental studies. The major concern of most of the biomarkers is their specificity. CK-MB is present in small quantities in skeletal muscle and other tissues, thus elevations of CK-MB occur in some diseases involving skeletal muscle injury. Myoglobin is found in all muscle types and its concentrations vary significantly between species and even within species. BNP has been proposed to use as a prognostic indicator of disease progression and outcome of congestive heart failure. However, the actual utility of this biomarker is untested. BNP is involved in the counter-regulation of heart hypertrophy, thus the changes in serum BNP concentrations as a function of time in the transition from cardiac hypertrophy to heart failure need to be understood comprehensively. Higher levels of BNP may not necessarily indicate more severity of the heart disease, indicating that more scrutiny to analysis is needed. CRP is a biomarker of inflammation, and its use in myocardial injury is more supplementary to other tests than having independently predictive value.
Considering all of the limitations above, more reliable biomarkers are needed. A significant advance in the development and validation of biomarkers for myocardial injury is the promising clinical experience with cTn, which has absolute myocardial tissue specificity and high sensitivity. cTn is now accepted by the clinical community as the biomarker of choice for assessing myocardial damage in humans. Its preclinical value for monitoring drug cardiac toxicity and in drug development needs to be evaluated.