Molecular and cellular mechanisms of cardiotoxicity.

Cardiotoxicity resulting from detrimental environmental insults has been recognized for a long time. However, extensive studies of the mechanisms involved had not been undertaken until recent years. Advances in molecular biology provide powerful tools and make such studies possible. We are gathering information about cellular events, signaling pathways, and molecular mechanisms of myocardial toxicologic responses to environmental toxicants and pollutants. Severe acute toxic insults cause cardiac cell death instantly. In the early response to mild environmental stimuli, biochemical changes such as alterations in calcium homeostasis occur. These may lead to cardiac arrhythmia, which most often is reversible. Prolonged stimuli activate transcription factors such as activator protein-1 through elevation of intracellular calcium and the subsequent activation of calcineurin. Upregulation by activated transcription factors of hypertrophic genes results in heart hypertrophy, which is a short-term adaptive response to detrimental factors. However, further development of hypertrophy will lead to severe and irreversible cardiomyopathy, and eventually heart failure. From cardiac hypertrophy to heart failure, myocardial cells undergo extensive biochemical and molecular changes. Cardiac hypertrophy causes tissue hypoperfusion, which activates compensatory mechanisms such as production of angiotensin II and norepinephrine. Both further stimulate cardiac hypertrophy and, importantly, activate counterregulatory mechanisms including overexpression of atrial natriuretic peptide and b-type natriuretic peptide, and production of cytokines such as tumor necrosis factor-alpha. This counterregulation leads to myocardial remodeling as well as cell death through apoptosis and necrosis. Cell death through activation of mitochondrial factors and other pathways constitutes an important cellular mechanism of heart failure. Our current knowledge of cardiotoxicity is limited. Further extensive studies are warranted for a comprehensive understanding of this field.

Cardiotoxicity resulting from exposure to environmental toxicants and pollutants has been known for a long time (1). However, a detailed examination of cardiac toxic effects and investigations of the mechanisms of cardiotoxicity had not been undertaken until recently. In fact, cardiotoxicology was not a defined discipline in the past. Two major advances in biomedical research and practice have made an urgent need for cardiotoxicologic studies. First, it is now an exciting time in the history of cardiovascular medicine. New drugs and devices are invented for the treatment of heart disease on a weekly basis (2). Undoubtedly some of these drugs and procedures will have cardiotoxic effects. For every new treatment, it will be essential to thoroughly assess toxic effects on the heart. Second, the application of cutting-edge molecular biology approaches has provided significant and novel insights into cardiac toxicity and its mechanisms (3,4). Mechanistic studies on cardiovascular effects of environmental toxicants and pollutants have emerged at a very fast pace. We have just begun to understand cellular events and molecular mechanisms of cardiotoxicity. A survey of our current knowledge in this exciting field will help to identify gaps that need to be filled. There are excellent textbooks (5-7) describing basic anatomic, biochemical, and physiologic principles of the heart from humans to experimental animals. Ultrastructural and histologic changes of the heart in toxic injury have also been elaborated in detail (8)(9)(10). This review thus only focuses on cellular events, signaling pathways, and molecular mechanisms of cardiotoxicity, with a brief review, if necessary, of bridging knowledge leading to the focused discussion.

Myocardial Responses to Environmental Toxicants
The foremost changes in the early phase of responses of myocardium to environmental toxicants involve alterations in biochemical reactions. These include the most often described alterations in ionic homeostasis such as changes in intracellular calcium concentrations, which occur in almost all examined exposures to environmental toxicants to date (11,12). Aberrant energy metabolism is another early response to environmental toxicants in the heart, resulting in decreased production and/or enhanced consumption of adenosine triphosphate (ATP) (13,14). Alterations in enzymatic reactions are often described in cardiac toxic responses (15), although we know very little about these alterations. The early signaling pathways leading to myocardial toxic responses are the recent focus of cardiotoxicologic research (16,17). Detailed descriptions of these pathways and their roles in cardiotoxicity are yet to be explored. It is likely that activation of early genes and signaling pathways is a critical response of myocardial cells to environmental toxic insults (18). The cross-talk between signaling pathways determines the ultimate outcome of myocardial responses to environmental toxicants and pollutants.
Physiologic 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 (19), which often results from the changes in intracellular calcium concentrations and other biochemical alterations, leading to miscommunication between cells and misconduction of electricity (20). These changes, if not accompanied by cardiomyopathy, do not involve myocardial cell death and are reversible. The late phase of cardiac dysfunction, however, often results from cardiomyopathy.
Changes in myocardial morphology take place when extensive toxic insults are imposed on the heart and/or toxic exposures persist on a long-term basis (21)(22)(23). Cardiac hypertrophy is often observed as a consequence of long-term toxic insults. The hypertrophy is considered a protective and adaptive response. However, further hypertrophy leads to severe and irreversible cardiomyopathy, eventually resulting in heart failure (24)(25)(26).
From cardiac hypertrophy to heart failure, activation of compensatory mechanisms including the sympathetic nerve system and the renin-angiotensin system takes place Cardiotoxicity resulting from detrimental environmental insults has been recognized for a long time. However, extensive studies of the mechanisms involved had not been undertaken until recent years. Advances in molecular biology provide powerful tools and make such studies possible. We are gathering information about cellular events, signaling pathways, and molecular mechanisms of myocardial toxicologic responses to environmental toxicants and pollutants. Severe acute toxic insults cause cardiac cell death instantly. In the early response to mild environmental stimuli, biochemical changes such as alterations in calcium homeostasis occur. These may lead to cardiac arrhythmia, which most often is reversible. Prolonged stimuli activate transcription factors such as activator protein-1 through elevation of intracellular calcium and the subsequent activation of calcineurin. Upregulation by activated transcription factors of hypertrophic genes results in heart hypertrophy, which is a short-term adaptive response to detrimental factors. However, further development of hypertrophy will lead to severe and irreversible cardiomyopathy, and eventually heart failure. From cardiac hypertrophy to heart failure, myocardial cells undergo extensive biochemical and molecular changes. Cardiac hypertrophy causes tissue hypoperfusion, which activates compensatory mechanisms such as production of angiotensin II and norepinephrine. Both further stimulate cardiac hypertrophy and, importantly, activate counterregulatory mechanisms including overexpression of atrial natriuretic peptide and b-type natriuretic peptide, and production of cytokines such as tumor necrosis factor-α. This counterregulation leads to myocardial remodeling as well as cell death through apoptosis and necrosis. Cell death through activation of mitochondrial factors and other pathways constitutes an important cellular mechanism of heart failure. Our current knowledge of cardiotoxicity is limited. Further extensive studies are warranted for a comprehensive understanding of this field.  (27,28). The compensatory response in turn activates counterregulatory mechanisms such as upregulation of atrial natriuretic peptide (ANP) expression (29,30). These responses collectively lead to extensive biochemical, physiologic, and molecular changes, eventually myocardial remodeling (31)(32)(33), and remarkable cell death. These changes ultimately result in heart failure. Figure 1 depicts these mechanisms. Most cardiotoxicologic studies address fundamental and characteristic changes during this stage of myocardial responses to toxic insults. The ultimate targets and the determining units of cardiotoxicity are cardiac cells. In this context, the following sections will be devoted to discussion of the cellular events, molecular mechanisms, and signaling pathways that are involved in the myocardial responses to toxic insults.

Cellular Events Involved in Cardiotoxicity
Toxic insults trigger a series of reactions in cardiac cells leading to measurable changes in myocardial morphology, biochemistry, and physiology. Mild injuries can be repaired; however, severe injuries lead to cell death in the form of apoptosis and necrosis. If the cell survives the insults, structural and functional adaptations take place.

Myocardial Apoptosis
Myocardial apoptosis, which is involved in cardiomyopathy, was first recognized in 1994 (34). Recent progress in myocardial research has provided significant insights into the cellular mechanisms of cardiotoxicity. It has been recognized that the loss of cardiac myocytes is a fundamental part of the myocardial injury that initiates or aggravates cardiomyopathy and leads to premature death (35)(36)(37). An important mode of myocardial cell loss is apoptosis (38)(39)(40), which has been demonstrated in the myocardium of heart failure patients (41,42). In our early studies, we have found that dietary copper restriction causes cardiomyopathy, which is accompanied with apoptosis in rat hearts (43). Myocardial apoptosis has been shown to play an important role in cardiac toxic effects induced by adriamycin (44), an important anticancer agent whose clinical application is limited by its major side effect, cardiotoxicity (45). Exposure of primary cultures of cardiomyocytes to cadmium also induces apoptosis (46).
Many in vivo studies have shown that only a very small percentage of myocardial cell populations undergo apoptosis under pathologic conditions (47,48). For example, we have observed that less than 0.5% of cells appeared apoptotic in myocardial tissue obtained from copper-deficient mice (49). At first glance, this number seems to be too insignificant to account for myocardial pathogenesis. However, this is a false assessment. In a carefully designed time course study (50), it has been estimated that cardiomyocyte apoptosis may be completed in less than 20 hr in rats. Because the heart is a terminally differentiated organ, myocytes undergoing apoptosis are lost and are not replaced. Thus, the total cell loss can simply be accounted for by the rate of apoptosis plus necrosis. If apoptosis occurs at a constant rate of about 0.5% myocytes a day (49), the potential contribution of apoptosis to the overall loss of myocytes over a long period of time is significant.

Myocardial Necrosis
The term myocardial necrosis has been widely used to describe myocardial cell death in the past. Myocardial infarction, in particular, was considered a consequence of necrosis (51). It is now recognized that apoptosis contributes significantly to the formation of myocardial infarction (52), although the consequence of apoptosis in this pathogenesis is yet to be defined. However, the significance 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 between apoptosis and necrosis.
Apoptosis and necrosis were originally described as two distinct forms of cell death that can be clearly distinguished (53).
However, these two modes of cell death can occur simultaneously in tissues and cultured cells (54). The intensity and duration of insults may decide the outcome (55). Thus, 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 (56). Alternatively, in acute injury, apoptotic cells can progress along a continuum to eventual necrosis.
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 (57). In particular, conditions causing ischemia to myocardial cells result in significant reduction and eventual depletion of adenine nucleotides. ATP loss of more than 70% of the total pool present in myocardial cells has been shown to cause a switch from apoptosis to necrosis (56,58). This extent of depletion of the ATP pool is often observed in myocardial infarction.
Identification of apoptotic and necrotic cell death processes requires more rigorous methods than have been generally used. Artifacts inherent in techniques that detect double-stranded DNA breaks as well as overlaps between apoptotic and necrotic death programs account for these problems (59,60). Current assays are limited in their capacities to distinguish between apoptosis and necrosis in myocytes. In addition, distinguishing between myocyte and nonmyocyte apoptosis in the myocardium is another problem to overcome. In our recent studies examining adriamycininduced myocardial apoptosis (61), we used an in situ terminal deoxynucleotidyl-transferasemediated dUTP nick end labeling (TUNEL) assay in combination with a dual immunohistochemical detection of α-sarcomeric actin, a specific protein present in cardiomyocytes. This procedure thus identified whether the TUNEL-positive cells were myocytes. Another procedure we used was immuno-gold TUNEL and electron microscopic examination of the apoptotic cells (61). It is well known that the gold standard for identification of apoptotic cells is morphologic examination by electron microscopy. This immuno-gold TUNEL and electron microscopic procedure thus provides information about cell type and morphologic characteristics of apoptotic cells.

Myocardial Adaptation
Myocardial adaptation is 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 that is a useful adaptation to   increased demands. However, in response to pathologic stimuli such as exposure to environmental toxicants, myocardial remodeling is adaptive in the short term but maladaptive in the long term and often eventuates 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 (62).

Molecular Mechanisms Involved in Cardiotoxicity
The most severe and early response of myocardium to overwhelming environmental toxic insults is cell death by apoptosis and necrosis. Myocardial cells, like other cells, are equipped with cell death programs and contain preexisting death machinery. In response to environmental toxic insults, these programs are activated. At the same time, cytoprotective mechanisms are also turned on, such as activation of antiapoptotic factors including the Bcl-2 family. Therefore, whether a cell will die depends on the balance between the activities of cell death programs and the cytoprotective mechanisms. The signaling pathways leading to activation of cell death program are discussed below. Although cell death is the ultimate end point that is often measured in assessing myocardial responses to environmental toxic insults (63,64), cellular and molecular events leading to myocardial remodeling are critical parameters of cardiotoxicity. The foremost observation of myocardial morphologic change in response to stimuli is heart hypertrophy. Although the hypertrophic response initially is a compensatory mechanism that augments cardiac output, sustained hypertrophy can lead to dilated cardiomyopathy, heart failure, and sudden death (65,66). A series of prototypical molecular responses of cardiomyocytes to environmental stimuli includes an increase in cell size and protein synthesis (67), enhanced sarcomeric organization (68), upregulation of fetal cardiac genes (69,70), and induction of immediate-early genes (71,72). These responses collectively lead to cardiac hypertrophy.

Myocardial Gene Regulation
Myocardial gene regulation in response to toxic insults is not well understood. However, extensive studies on the activation of transcription factors in cardiac hypertrophy have been undertaken. Because cardiac hypertrophy is the most common response to environmental stimuli, the molecular mechanism involved in gene regulation in cardiac hypertrophy would have implications in myocardial responses to environmental toxicants and pollutants. The transcription factors involved in cardiac hypertrophy include activator protein-1 (AP-1), transcriptional enhancer factor-1 (TEF-1), serum response factor (SRF), nuclear factors of activated T cells (NFATs), and GATA4 . Most of these transcription factors function coordinately in mediating extracellular signaling to regulate hypertrophic gene expression.
AP-1 is a transcription factor composed of Jun and Fos gene family members (73). The AP-1 binding site is the TRE (12-O-tetradecanoylphorbol-13-acetate response element), and the binding of AP-1 to the TRE initiates transcription of the target genes (74). In recent studies, it has been shown that elevated levels of c-Jun are associated with the stress induced by ischemia/reperfusion in cardiomyocytes (75). In volume-overload hypertrophy, AP-1 plays an important role in the regulation of Fas and FasL activities (76). Overstretching of myocardium induces Fas expression (77). Fas-dependent signaling pathways are coupled to the activation of AP-1 in isolated cardiomyocytes (76,78). These are pathways that can lead to myocardial cell apoptosis (76)(77)(78)(79). However, there are studies showing that activation of AP-1 is independent of the induction of apoptosis (80). AP-1 has been implicated in transcriptional regulation of several genes associated with a hypertrophic response (81,82).
TEF-1 is a transcription factor that has been shown to be activated in α 1 -adrenergicstimulated hypertrophy of cultured cardiomyocytes (83). It is involved in induction of β-myosin heavy chain (MHC) and α-skeletal actin fetal isogenes in cardiac hypertrophy (84). SRF has been demonstrated to be both necessary and sufficient to mediate stretch responses in myocardium, leading to induction of c-fos expression (85). This stretchinduced activation of SRF was also observed in hypotonic cell swelling, leading to c-fos induction (86). Thus, activation of SRF may be the general mechanism of c-fos activation in response to increased membrane tension in cardiac myocytes.
NFAT3 is a member of a multigene family that contains four members, NFATc, NFATp, NFAT3, and NFAT4 (87). These factors bind the consensus DNA sequence GGAAAAT as monomers or dimers through a Rel homology domain (88). Unlike the other three members that are restricted in their expression to T cells and skeletal muscle, NFAT3 is expressed in a variety of tissues including the heart (87,89). The role of NFAT3 in cardiac hypertrophy has been demonstrated recently in an elegant study (90). Hypertrophic stimuli such as angiotensin II and phenylephrine cause an increase in intracellular Ca 2+ levels in myocardial cells. This elevation in turn results in activation of calcineurin (discussed below). NFAT3 is localized within the cytoplasm and is dephosphorylated by the activated calcineurin. This dephosphorylation enables NFAT3 to translocate to the nucleus where it can interact with GATA4. NFAT3 can also activate some hypertrophic responsive genes through mechanisms independent of GATA4.
GATA factors are a family of nuclear transcriptional regulatory proteins that are related structurally within a central DNA-binding domain but are restricted in expression to distinct sets of cell types (91). Currently, six different family members have been characterized in vertebrate species. They are GATA1, 2, 3, 4, 5, and 6. Each protein contains two similar repeats of a highly conserved zinc finger of the form CXNCX 6 LWRRX 7 CNAC. The c-terminal repeat constitutes a minimal DNAbinding domain sufficient for sequence-specific recognition of a "GATA" cis-element, usually (A/T)GATA(A/G) or a related DNA sequence, present in promoters and/or enhancers of target genes (92). It has been shown that GATA1, 2, and 3 mainly regulate various aspects of hematopoiesis (93), whereas the GATA4, 5, and 6 factors are involved in regulation of cardiogenesis (91). The significance of GATA4 in regulation of hypertrophic response in myocardial cells has been demonstrated recently (94). Cardiac hypertrophy induced by angiotensin II is mediated by an angiotensin II type 1a receptor (AT 1a R). A GATA motif was identified in the AT 1a R promoter. Mutations introduced into the consensus binding site for GATA factor abolished the pressure overload response (94). Moreover, it has been demonstrated that the interactions between AP-1 and GATA4 and between NFAT3 and GATA4 are essential in myocardial hypertrophic responses (90,94).

Molecular Basis for Alterations in Myocardial Structure and Function
It has been demonstrated that reprogramming of gene expression of β-MHC and α-skeletal actin is associated with myocardial hypertrophy (69)(70)(71)(72)95). Upregulation of these fetal protein isoforms is associated with downregulation of the corresponding adult isoforms, α-MHC and α-cardiac actin (96). These molecular processes are correlated with the development of hypertrophy and the thickness of the ventricular wall, which has been shown to result from an increase in cell size due to an increase in the number of sarcomeres and mitochondria within the cell (97). This hypertrophic development leads to tissue hypoperfusion, which in turn results in the activation of compensatory mechanisms such as the sympathetic nerve system and the renin-angiotensin system.
The neurohumoral compensatory response activates counter regulatory hormones such as ANP and b-type natriuretic peptide (BNP) (98). The long-term expression of these hormones, particularly ANP and Environmental Health Perspectives • VOLUME 109 | SUPPLEMENT 1 | March 2001 BNP, is associated with numerous detrimental biological effects that eventually lead to cardiac failure by way of myocyte dysfunction, apoptosis, and cell loss (99). For instance, the ANP gene is expressed in both atrium and ventricle during embryonic development, but its expression is downregulated in the ventricle shortly after birth, leaving the atrium as the primary site of ANP synthesis within the mature myocardium (100). During myocardial pathologic remodeling, reexpression of ANP in myocytes of the left ventricle takes place (99)(100)(101). Recent studies have demonstrated that ANP is a factor responsible for myocardial apoptosis (49,102). Moreover, apoptosis plays a critical role in the development of heart failure (38)(39)(40)(41)(42)103,104). Therefore, serum concentrations of ANP have been monitored experimentally and clinically and serve as important indices of heart failure (99-101,105).

Mitochondrial Factors in Myocardial Cell Death
Recent studies (106,107) have focused on mitochondrial factors in myocardial cell death. Current knowledge obtained from cutting-edge experimental approaches suggests that signaling pathways mediating oxidative stress-induced myocardial cell death can be divided into those that trigger the early events of cell loss and those that result in severe, endstage myocardial cell death. Cytochrome c and pro-caspase-3 are preexisting factors that are activated by environmental toxic insults.
Mitochondrial permeability transition (MPT) occurs under toxic insults (108). This MPT behaves like a membrane pore that allows diffusion of solutes < 1,500 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 rupture of the outer membrane, releasing intermembrane proteins into the cytosol (109). 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 (110). Overexpression of Bax under oxidative stress conditions has been observed in a number of studies using different organs including the heart (111,112). It has been shown that Bax is translocated from cytosol to mitochondria and forms pores in mitochondrial outer membranes, leaving the inner membranes intact (113).
This mechanism implies that Bax-mediated cytochrome c release is independent of MPT (114). 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 (another factor released from mitochondria under oxidative stress), procaspase-9, and deoxyadenosine triphosphate (dATP), and subsequently activates caspase-9, which activates caspase-3. In our recent studies, we detected a significant decrease in cytochrome c concentrations in mitochondria and a concurrent increase in the cytosol by Western blot analysis in adriamycin-treated cardiomyocytes (115).
To determine the significance of the caspase-3-activated apoptotic pathway in the pathogenesis of cardiomyopathy, we have used a caspase-3-specific inhibitor, Ac-DEVDcmk, to determine its effect on adriamycininduced myocardial cell death. Cultured cardiomyocytes isolated from neonatal mice were treated with Ac-DEVD-cmk before adriamycin treatment. This inhibitor efficiently suppressed caspase-3 activity and reduced the number of apoptotic cells in cultures (115).
Inhibition of caspase-3-activated apoptotic pathway, however, may not abort ultimate cell death because of necrosis that can also be triggered by MPT and mitochondrial cytochrome c release. The loss of cytochrome c blocks electron transport, leading to decreased production of ATP and eventually ATP depletion, which results in necrosis. Enhanced necrotic cell death in the presence of caspase-3 inhibitor has been observed in our recent studies examining adriamycin cardiotoxicity. There is another group of proapoptotic factors that are released from mitochondria, but the mechanisms of action of these factors are unclear. These are apoptosis-inducing factors (AIF). It has been shown that AIF may directly translocate to the nucleus after they are released from mitochondria, causing DNA framentation (116). These multiple pathways mediated by mitochondrial factors suggest that there is a bewildering diversity of cell death paradigms. In particular, under chronic environmental toxic insults, the relative importance of mitochondrial electron transport defects, MPT, cytochrome c leakage, and nonmitochondrial factors needs to be carefully examined. An important clinical consideration is whether caspase inhibitors can offer long-term protection against cell death, as delineated in Figure 2. It is thus important to define the most efficient therapeutic strategy in blocking myocardial cell death rather than in inhibiting a particular cell death program.

Cytokines and Other Factors in Myocardial Death Signaling
Cytokines have been shown to be associated with the pathogenesis of acute coronary syndromes (117) and chronic heart failure (118). In both cases, myocardial cell loss is the essential feature of the disease. Increased serum concentrations of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) have been found in chronic heart failure (119). Another biomarker of chronic heart failure is the increased serum concentrations of Fas ligand (120). Both TNF-α and Fas ligand induce apoptosis of cardiomyocytes in various experimental studies (120,121). Other cytokines such as interleukin-6 are also found in the serum of patients with failing hearts (122)(123)(124).
TNF-α is the most studied cytokine in myocardial cell death signaling pathways. Cardiomyocytes are both the source and the target of this cytokine (125). The proinflammatory cytokines interleukin-1, interleukin-2 and interferon-γ can induce TNF production from target cells, including cardiomyocytes (116). The most important role of TNF-α in chronic heart failure is the induction of myocardial cell apoptosis (125)(126)(127). The pathway leading to TNF-α-induced myocardial apoptosis is mediated by TNF receptors, TNFR1 and TNFR2 (128). Activation of these receptors leads to activation of caspase-8 (129). A BH3 domain-containing proapoptotic Bcl2 family member, BID, is then cleaved by caspase-8 (130). 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, i.e., apoptosis (130). Caspase-8 also directly activates caspase-3, leading to apoptosis (131). Interleukin-6 is a multifunctional proinflammatory cytokine that mediates both immune and inflammatory responses. Elevated serum concentrations of interleukin-6 have been observed in patients with heart failure (126,132). A new member of the interleukin-6 receptor family of cytokines, cardiotrophin-1, has been cloned recently (133). This is added to the list of other members of this family, including interleukin-6, interleukin-11, leukemia inhibitory factor, oncostatin, and ciliary neurotrophic factor. Cardiotrophin-1 acts as a nerve growth factor and also induces hypertrophy of cardiac myocytes (134,135). Interestingly, cardiotrophin-1 has been shown to inhibit cytokine-induced apoptosis in vitro (136). Further studies are needed to investigate its role in myocardial disease conditions. Endothelin-1 is importantly involved in cardiomyopathy. It is produced within both failing and nonfailing hearts from several types of cells, including endothelial cells (137), endocardium, and myocytes (138). It has been considered that limited expression of endothelin-1 within the myocardium is an adaptive response to stress, providing increased inotropic support for the cardiac myocyte (139,140) as well as increasing the rate of myocyte protein synthesis (141). Overexpression of endothelin-1, however, eventually becomes maladaptive by producing focal vasospasm, myocytosis, and increased myocardial fibrosis (142). In a rat model, it has been shown that myocardial infarction is accompanied with overexpression of endothelin-1 and that selective inhibition of the endothelin-A receptor with a specific antagonist (BQ-123) improved long-term survival of these animals (143).

Sarcoplasmic Reticulum Pathway Leading to Apoptosis
This pathway was identified recently (144). It was shown that caspase-12 is localized in the endoplasmic reticulum (ER) and activated by ER stress. This stress includes disruption of ER calcium homeostasis and accumulation of excessive proteins in the ER. Mice deficient in caspase-12 are resistant to ER stressinduced apoptosis. Therefore, caspase-12 mediates an ER-specific apoptotic pathway. This pathway would have a large impact on myocardial cells and should be investigated in the future.

Role of Calcium in Cardiotoxicity
This topic has been the most discussed and investigated in cardiotoxicologic research. However, our understanding of the role of calcium in cardiotoxicity is rather superficial. When carefully examining the current literature, one finds that there are few mechanistic studies that specifically probe the role of calcium in cardiotoxicity. Yet, numerous studies have implicated intracellular Ca 2+ as a signal for cardiac responses to environmental toxic insults (145)(146)(147). In response to myocardial stress by environmental stimuli, calcium concentrations are increased in the myocardial cells (148). This is consistent with the speculation that Ca 2+ coordinates physiologic responses to stresses. Many other speculations are derived from the studies examining the role of calcium in toxicologic responses in other systems. The unique action of calcium in cardiotoxicity, however, has to be studied specifically.
Recently there has been a breakthrough in understanding the role of calcium in mediating myocardial hypertrophic signals (90). A sustained increase in intracellular Ca 2+ concentrations activates calcineurin. Calcineurin is a ubiquitously expressed serine/threonine phosphatase that exists as a heterodimer, comprised of a 59-kDa calcium-binding catalytic A subunit and a 19-kDa Ca 2+ -binding regulatory B subunit (149). Activation of calcineurin is mediated by binding of Ca 2+ and calmodulin to the regulatory and catalytic subunits, respectively. A toxicologic significance of calcineurin is that it is activated by a sustained Ca 2+ elevation and is insensitive to transient Ca 2+ fluxes such as those that occur in response to cardiomyocyte contraction (90).
Numerous studies have demonstrated important roles for Ras, mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) signaling pathways in myocardial responses to hypertrophic stimuli (150,151). All these signal transduction pathways are associated with an inotropic increase in intracellular Ca 2+ concentrations (152). The coordinating role of calcium in cardiac hypertrophic response was speculated in a recent study (90) as follows. Hypertrophic stimuli such as angiotensin II and phenylephrine cause an elevation of intracellular Ca 2+ that results in activation of calcineurin. A series of reactions occurs through the activated calcineurin, including dephosphorylation of NFAT3 and its translocation to nucleus where it can interact with GATA4 (90). Calcineurin could also act through an NFAT-independent mechanism to regulate myocardial hypertrophy.

Protein Kinase C and the Myocardial Signaling Pathway
PKC and the myocardial signaling pathway are among the most extensively studied topics in cardiac research. Most cardiotoxicologic studies have adapted the concept of cardiac physiologic studies in examining the role of PKC in mediating toxic signals. Several excellent reviews are available on PKC in myocardial signaling pathways leading to cardiac hypertrophy and heart failure (153-157). Therefore, this topic will not be included in this review.

Oxidative Stress and Mitogen-Activated Protein Kinases
Oxidative stress and MAPKs are involved in myocardial remodeling and play a major role in the development of cardiotoxicity. Among the MAPKs, p38 MAPK has been extensively studied in myocardial apoptosis. The p38 MAPK is a subfamily of the MAPK superfamily and is stress responsive. This subfamily consists of p38α, p38β, p38γ, and p38δ (158,159). Recent studies have identified p38 MAPK as an important group of signaling molecules that mediate environmental stress responses in various cell types (160,161). In noncardiac cells, p38 MAPK has been implicated in gene expression, morphologic changes, and cell death in response to endotoxin, cytokines, physical stress, and chemical insults (162)(163)(164). In cardiac cells, it has been reported that p38 MAPK is associated with the onset of apoptosis in ischemia-reperfusion-treated hearts (165,166). In particular, transfection experiments using primary cultures of neonatal rat cardiomyocytes have shown that p38α is critically involved in myocyte apoptosis (167). In any event, the common observation is that p38 MAPK activation is associated with accumulation of reactive oxygen species generated under stress conditions. In this context, we have examined the possible role of p38 MAPK in mediating adriamycin-induced myocardial apoptosis.
Treatment with adriamycin significantly induced apoptosis in neonatal cardiomyocyte cultures and activated p38 MAPK (61). That p38 MAPK was involved at least in part in the adriamycin-induced myocyte apoptosis was demonstrated by two important observations (61). First, a time-course analysis revealed that p38 MAPK activation preceded the onset of apoptosis. A sensitive and early apoptosis detection method of Annexin V-FITC has been used to detect the onset of myocyte apoptosis. It was demonstrated that as early as 30 min after adriamycin treatment, myocyte apoptosis occurred. The early detection of p38 MAPK activation by a sensitive FITC-conjugated anti-phospho-p38 antibody and confocal microscopy was observed 20 min after adriamycin treatment. Second, application of SB203580, a specific inhibitor of p38 MAPK, significantly inhibited adriamycin-induced myocyte apoptosis. Because SB203580 acts as a specific inhibitor of p38α and p38β but not p38γ and p38δ, the involvement of the former specific isoforms of p38 MAPK in the adriamycininduced myocyte apoptosis are implicated. Recent studies have identified that p38α is specifically involved in apoptosis of neonatal rat cardiomyocytes in primary cultures and Environmental Health Perspectives • VOLUME 109 | SUPPLEMENT 1 | March 2001 VOLUME 109 | SUPPLEMENT 1 | March 2001 • Environmental Health Perspectives p38β mediates hypertrophy of these cells (167). Further studies are required to determine which specific isoform(s) of p38 MAPK are essential in the signaling pathway of adriamycin-induced apoptosis.
An important novel observation in our recent studies is that metallothionein (MT), an important antioxidant, inhibited both apoptosis and p38 MAPK activation by adriamycin in cardiomyocytes (61). Although adriamycin-induced apoptosis was partially inhibited (by 50%), the activation of p38 MAPK was almost completely blocked by MT. Taken together, these observations suggest that oxidative stress-activated p38 MAPK plays a crucial role in myocardial apoptosis.

Conclusion
Myocardial responses to detrimental environmental insults lead to myocardial apoptosis or necrosis, remodeling, and heart failure. The current understanding of the cardiac toxic effects is, however, superficial. Most of the cardiotoxicologic studies have been descriptive. Any agent, either physical or chemical, can trigger myocardial responses. However, these responses may not necessarily be toxic. On the other hand, there is no single agent that only triggers toxic responses. Of note, cellular responses to toxic insults always start with initiation of protective mechanisms. The toxic end points are the ultimate outcome of the balance between cytoprotection and the insult. Therefore, it is critical to identify the true toxic end point of cardiotoxicity. Many intermediate responses, however, can lead to either protective or injurious responses. For example, p38 MAPK activation can lead to cytoprotection against oxidative stress by activating transcription factors that upregulate protective mechanisms or cause cell death by activating apoptotic programs. It is well known that multiple signaling pathways exist and are activated in response to stresses. One cannot define which pathway mediates the toxic response or the protective action. In this context, inhibition of one pathway may activate the other. For instance, inhibition of caspase-3 can abort apoptosis if caspase-3 is a key element in a particular apoptotic signaling pathway. This inhibition may shift the apoptotic program to necrotic if mitochondrial cytochrome c release is a critical upstream event that leads to caspase-3 activation. Thus, an important consideration in preventing myocardial cell death induced by environmental toxicants and pollutants is not an inhibition of a particular cell death program but rather the ultimate survival of a cell. Further extensive studies of the molecular mechanisms and signaling transduction pathways of cardiotoxicity are warranted. Advances in molecular biology and the available cutting-edge experimental approaches will provide powerful tools for such studies. It is probable that significant insights into cardiotoxicity will be obtained in the near future. A comprehensive understanding of cardiotoxicity will greatly contribute to the advance of cardiac medicine.