Cardiac Hypertrophy: A Risk Factor for QT-Prolongation and Cardiac Sudden Death

Abstract

Cardiac hypertrophy was viewed as a compensatory response to hemodynamic stress. However, cumulative evidence obtained from studies using more advanced technologies in human patients and animal models suggests that cardiac hypertrophy is a maladaptive process of the heart in response to intrinsic and extrinsic stimuli. Although hypertrophy can normalize wall tension, it is a risk factor for QT-prolongation and cardiac sudden death. Studies using molecular biology techniques such as transgenic and knockout mice have revealed many important molecules that are involved in the development of heart hypertrophy and have demonstrated signaling pathways leading to the pathogenesis. With the same approach, the consequence of heart hypertrophy has been examined. The significance of hypertrophy in the development of overt heart failure has been demonstrated and several critical molecular pathways involved in the process were revealed. A comprehensive understanding of the threats of heart hypertrophy to patients has helped to develop novel treatment strategies. The recognition of hypertrophy as a major risk factor for QT-prolongation and cardiac sudden death is an important advance in cardiac medicine. Cellular and molecular mechanisms of this risk aspect are currently under extensively exploring. These studies would lead to more comprehensive approaches to prevention of potential life threatening arrhythmia and cardiac sudden death. The adaptation of new approaches such as functional genomics and proteomics will further advance our knowledge of heart hypertrophy.

Introduction

Cardiac hypertrophy was viewed as a compensatory response to hemodynamic changes, however, cumulative evidence obtained from studies using more advanced technologies in human patients and animal models suggests that cardiac hypertrophy is a maladaptive process of the heart in response to intrinsic and extrinsic stresses (Berenji et al., 2005; Dorn and Force, 2005; van Empel and De Windt, 2004). There are two basic forms of cardiac hypertrophy: Concentric hypertrophy, which is often observed during pressure overload and new contractile-protein units are assembled in parallel resulting in a relative increase in the width of individual cardiac myocytes (de Simone, 2003). By contrast, eccentric hypertrophy results from the assembly of contractile-protein units in series, which represents a relatively greater increase in the length than in the width of individual myocytes (Kass et al., 2004). 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.

Cardiac hypertrophy is a risk factor for QT-prolongation and cardiac sudden death. Recent studies in human patients and animal models have demonstrated that cardiac hypertrophy significantly affects myocardial electrotonic cell-to-cell coupling, leading to disturbance in action potential duration and potential malignant arrhythmia and cardiac sudden death (ten Eick et al., 1992; Frenneaux, 2004; Kahan and Bergfeldt, 2005). Electrocardiography recoding has shown that heart hypertrophy posses a high risk for QT-prolongation and higher sensitivity to torsadogenic drugs (Kozhevnikov et al., 2002; Schoenmakers et al., 2003; Swynghedauw et al., 2003; Schreiner et al. 2004; Eghbali et al., 2005). This review will briefly discuss the signaling pathways leading to cardiac hypertrophy and the link between cardiac hypertrophy and QT-prolongation and cardiac sudden death.

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 that is a useful adaptation to increased demands. However, in response to pathological stimuli such as exposure to cardiac toxic drugs, myocardial remodeling is adaptive in the short term, but is 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 (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, making the distinguishing of adaptive response from maladaptive response a difficult task in cardiac toxicological studies.

Adaptive Response

There are physiological hypertrophy and pathological hypertrophy of the heart. The physiological hypertrophy is considered as an adaptive response, which is accompanied by an adjustment of the cardiac function with an increased demand of cardiac output. Such an adaptive hypertrophy is observed in an 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 with 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 was viewed in the past as an adaptive response as well. However, cumulative evidence obtained from studies using more advanced technologies in human patients and animal models suggests that cardiac hypertrophy is a maladaptive process of the heart in response to intrinsic and extrinsic stresses.

Maladaptive Response

Although toxic stress-induced hypertrophy can normalize wall tension, it is a risk factor for malignant arrhythmia and cardiac sudden death. 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 through an observation of 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 by a significant slower pace of deterioration of systolic function (Esposito et al., 2002). These observations thus indicate that cardiac hypertrophy in response to extrinsic and intrinsic stress is not a compensatory response. However, hypertrophy makes the heart of high risk for malignant arrhythmia and heart failure, 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 and 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 synthesis kinase-3 (GSK3), and transcription factors. Activation of each of the components is sufficient to induce myocardial hypertrophic growth. These components also affect each other through crosstalk. A brief summary of these signaling pathways is presented as follows:

G-Protein-Coupled Receptors

Myocardial adrenergic, angiotensin, and endothelin (ET-1) receptors belong to G-protein-coupled receptors, which are coupled to 3 major classes of heterotrimeric GTP-binding proteins, G _{α s}, G _{α q}/G _{α 11}, and G _{α i}. Activation of G _{α q}-coupled receptors is sufficient to induce myocyte hypertrophy in vitro (Adams et al., 1998). Cardiac-specific ablation of G _{α q}/G _{α 11} 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 G _{α q} in transgenic mouse hearts suppresses pressure-overload hypertrophy (Akhter et al., 1998). Cardiac overexpression of G _{α s}, 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).

Calcium and Calcineurin

The role of calcium in cardiac toxic responses has been extensively investigated. However, our understanding of the role of calcium in cardiac toxicity remains superficial. When carefully examining the current literature, one can find that there are very few mechanistic studies that specifically probe the role of calcium in cardiac toxicity, although numerous studies have implicated intracellular Ca²⁺ as a signal for cardiac response to toxic insults (Shier and DuBourdieu, 1992; Buck et al., 1999; Toraason et al., 1997). In response to myocardial stress by environmental toxic exposures, calcium concentrations are increased in the myocardial cells (Sleight, 1996). This is consistent with the speculation that Ca²⁺ coordinates physiological responses to stresses. The unique action of calcium in cardiac toxicity, however, has to be studied specifically.

The role of calcium in mediating myocardial hypertrophic signals has been extensively studied and postulated (Stemmer and Klee, 1994). A sustained increase in intracellular Ca²⁺ concentrations activates calcineurin. Calcineurin is a ubiquitously expressed serine/threonine phosphatase that exists as a heterodimer, comprised of a 59 kDa calmodulin-binding catalytic A subunit and a 19 kDa Ca²⁺-binding regulatory B subunit (Molkentin et al., 1998). Activation of calcineurin is mediated by binding of Ca²⁺ and calmodulin to the regulatory and catalytic subunits, respectively. A toxicological significance of calcineurin is that it is activated by a sustained Ca²⁺ elevation and is insensitive to transient Ca²⁺ fluxes such as that occur in response to cardiomyocyte contraction (Stemmer and Klee, 1994).

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 (Jalili et al., 1999). All of these signal transduction pathways are associated with an increase in intracellular Ca²⁺ concentrations (Ho et al., 1998). The coordinating role of calcium in cardiac hypertrophic response has been demonstrated (Stemmer and Klee, 1994). Hypertrophic stimuli such as angiotensin II and phenylephrine cause an elevation of intracellular Ca²⁺ that results in activation of calcineurin. A series of reactions occur through the activated calcineurin, including dephosphorylation of nuclear factor of activated T-cell (NFAT) and its translocation to nucleus, where it can interact with GATA4. Calcineurin could also act through an NFAT-independent mechanism to regulate hypertrophic gene expression.

AMP-Activated Protein Kinase (AMPK)

Activation of AMPK under the condition of myocardial metabolic shift to fetal phenotype is often observed. It is predictable for the activation of AMPK in association with the changes in high-energy phosphate metabolism in hypertrophic and failing hearts. In particular, the rise of AMP/ATP ratio due to decreased concentrations in phosphocreatine (PCr) with or without a concomitant decrease in ATP reducing the PCr/ATP ratio. The decrease in PCr/ATP ratio, being an index of decreased energy reserve, was reported to correlate well with the severity of heart failure and is of prognostic value (Neubauer et al., 1997). Activation of AMPK leads to the translocation of insulin-dependent glucose transporter (GLUT4) from intracellular stores to the sarcolemma (Russell et al., 1999). Mice overexpressing an active AMPK mutant form suffer from pathological cardiac glucogen accumulation (Arad et al., 2003). Furthermore, the AMPK-dependent phosphorylation of the enzyme 6-phosphofructo-2-kinase stimulates glycolysis (Marsin et al., 2000). These studies indicate the importance of AMPK activation in cardiac metabolic shift to reliance on glucose metabolism.

Mitogen-Activated Protein Kinases (MAPKs)

MAPKs play a major role in cardiac response to toxic insults. 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 (Sugden and Clerk, 1998). Recent studies have identified p38 MAPK as an important group of signaling molecules that mediate toxic stress responses in various cell types (Tan et al., 1996). In non-cardiac cells, p38 MAPK has been implicated in gene expression, morphological changes, and cell death in response to endotoxin, cytokines, physical stress, and chemical insults (Tan et al., 1996; Wang and Ron, 1996). In cardiac cells, it has been reported that p38 MAPK is associated with the onset of apoptosis in ischemia-reperfusion-treated hearts (Yin et al., 1997). In particular, transfection experiments using primary cultures of neonatal rat cardiomyocytes have shown that p38α is critically involved in myocyte apoptosis (Wang et al., 1998). In any event, the common observation is that p38 MAPK activation is associated with accumulation of reactive oxygen species generated under stress conditions.

Treatment with Adriamycin significantly induced apoptosis in primary cultures of neonatal mouse cardiomyocytes and activated p38 MAPK (Kang et al., 2000). That p38 MAPK was involved at least in part in the Adriamycin-induced myocyte apoptosis was demonstrated by two important observations (Kang et al., 2000). 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 Adriamycin-induced myocyte apoptosis are implicated. The p38α is specifically involved in apoptosis of neonatal rat cardiomyocytes in primary cultures and p38β mediates hypertrophy of these cells (Wang et al., 1998).

Protein Kinase C (PKC)

PKC is among the most extensively studied signaling molecules in the heart. Most cardiac toxicologic studies have adapted the concept of cardiac physiological studies in examining the role of PKC in mediating toxic signals. Several excellent reviews on PKC in myocardial signaling pathways leading to cardiac hypertrophy and heart failure are available (Puceat and Vassort, 1996). PKC is a ubiquitously expressed serine/threonine kinase, which is activated predominantly by G_q/G₁₁-coupled receptors. The PKC family consists of 11 isoforms, which are divided into three subgroups: conventional PKCs (cPKCs) including α, β (I and II) and γ, novel PKCs (nPKCs) including ɛ, δ, η, ζ and θ, and atypical PKCs (aPKCs) including ι, λ, and μ (Newton, 1995). The cPKCs are activated by Ca²⁺ and di-acylglycerol (DAG) and phorbol esters. The nPKCs do not bind Ca²⁺, but respond to DAG and phorbol ester stimulation. The aPKCs respond neither Ca²⁺, DAG, nor phorbol esters. PKC has been demonstrated to participate in the regulation of transcription, the maintenance of cell growth and membrane structure, and modulation of immune responses. Disturbances in PKC signaling pathways lead to cardiac hypertrophy and heart failure, which is of cardiac toxicologic significance.

PI3K/GSK3 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 have been demonstrated to be sufficient to induced hypertrophic growth of adult hearts. Overexpression of constitutively active PI3K mutant in the heart leads to increased heart size in mice and by contrast, forced 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 phsophorylates GSK3β, thus inhibits the activation of GSK3β. Otherwise, GSK3β phosphorelates transcription factors of the NFAT family. 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 in the fashion of dependent on or independent of GATA4. Phosphorylation of NFAT3 in the nucleus by GSK3β promotes NFAT3 translocation to 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.

Transcription Factors

Transcription factors are critical group of molecules that control the phenotype switch of cardiac morphology and functional alteration of the heart through activation or deactivation of myocardial gene expression. There are many transcription factors that have been studied in the myocardial tissue. Several of them are of cardiac toxicologic significance as described below:

Activator protein-1 (AP-1) is a transcription factor composed of Jun and Fos gene family members (McMahon and Monroe, 1992). The AP-1 binding site is the TRE (12-O-tetradecanoyl phorbol 13-acetate response element), and the binding of AP-1 to the TRE initiates transcription of the target genes (Diamond et al., 1990). In recent studies, it has been shown that elevated levels of c-Jun are associated with the stress induced by ischemia/reperfusion in cardiomyocytes (Brand et al., 1992). In volume-overload hypertrophy, AP-1 plays an important role in the regulation of Fas and FasL activities (Wollert et al., 2000). Overstretching of myocardium induces Fas expression (Cheng et al., 1995). Fas-dependent signaling pathways are coupled to the activation of AP-1 in isolated cardiomyocytes. These are pathways that can lead to myocardial cell apoptosis. However, there are studies showing that activation of AP-1 is independent of the induction of apoptosis (Lenczowski et al., 1997). AP-1 has been implicated in transcriptional regulation of several genes associated with a hypertrophic response (Paradis et al., 1996).

Myocyte enhancer factor-2 (MEF-2) is a transcription factor that binds to A/T-rich DNA sequences within the promoter regions of a number of cardiac genes, including muscle creatine kinase gene, β-myosin heavy chain (MHC), MLC1/3, MLC2v, skeletal α-actin, SR Ca²⁺-ATPase, cardiac troponin T, C, and I, desmin, and dystrophin. (Black and Olson, 1998). MEF2 is critically involved in the regulation of inducible gene expression during myocardial hypertrophy. The activation of MEF2 involves phosphorylation of the transcription factor by p38 MAPK or ERK5-MAPK. The ERK5-MEF2 pathway has been observed in the generation of cardiac hypertrophy. An important function of MEF2 is the converge in the binary downstream pathway of Ca²⁺ signaling. Increased intracellular Ca²⁺ binds to and activates Ca²⁺-binding proteins including calmodulin (CaM), which regulates calcineurin and Ca²⁺/CaM-dependent protein kinase (CaMKs). Activation of either calcineurin or CaMKs induces cardiac hypertrophy. The MEF2 activity is stimulated by CaMKs through the phosphorylation of the transcriptional suppressor, histone deacetylases (HDACs). CaMK is considered as HDAC kinase whose activity is enhanced by calcineurin. Thus MEF2 converges the stimulating signaling of both CaMKs and calcineurin leading to activation of hypertrophic gene expression.

Nuclear factor of activated T cells 3 (NFAT3) is a member of a multigene family that contains 4 members, NFATc, NFATp, NFAT3 and NFAT4 (Rao et al., 1997). These factors bind to the consensus DNA sequence GGAAAAT as monomers or dimers through a Rel homology domain (Rooney et al., 1994). 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. The role of NFAT3 in cardiac hypertrophy has been demonstrated (Pu et al., 2003). Hypertrophic stimuli such as angiotensin II and phenylephrine cause an increase in intracellular Ca²⁺ levels in myocardial cells. This elevation in turn results in activation of calcineurin. 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 (Yamamoto et al., 1990). 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 CXNCX₆LWRRX₇CNAC. The c-terminal repeat constitutes a minimal DNA-binding 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 (Evans et al., 1988). It has been shown that GATA-1/2/3 mainly regulate various aspects of hematopoiesis (Orkin, 1992), whereas the GATA 4/5/6 factors are involved in regulation of cardiogenesis (Yamamoto et al., 1990). The significance of GATA-4 in regulation of hypertrophic response in myocardial cells has been demonstrated recently (Evens, 1997). Cardiac hypertrophy induced by angiotensin II is mediated by an angiotensin II type_{1 α} receptor (AT_{1 α} R). A GATA motif was identified in the AT_{1 α} R promoter. Mutations introduced to the consensus-binding site for GATA factor abolished the pressure overload response .(Evens, 1997). Moreover, it has been demonstrated that the interactions between AP-1 and GATA-4 and between NFAT3 and GATA-4 are essential in myocardial hypertrophic responses.

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 transition from cardiac hypertrophy to heart failure is presented in Figure 1. The critical cellular event of this transition is myocardial apoptosis triggered by inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and neurohormonal factors such as atrial natriuretic peptide (ANP), leading to dilated cardiomyopathy and deterioration of cardiac function. Toxicological exposures may cause dilated cardiomyopahty or heart failure without an intermediate hypertrophic stage. Myocardial cell death also plays an essential role in the direct cardiac dilation pathogenesis.

The 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 (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 cardiac toxicological 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., 1999). The crosstalk between signaling pathways determines the ultimate outcome of myocardial responses to environmental toxicants and pollutants.

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 a long-term (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 nerve system and the renin-angiotensin system takes place (Holtz, 1993). The compensatory response in turn activates counterregulatory mechanisms such as up-regulation of ANP expression (Francis and Chu, 1995) and increase in cytokine 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 Cardiac Sudden Death

The recognition of QT prolongation and its associated adverse effects on the heart has been a major focus in drug discovery and development and environmental cardiac toxicity in the past decade. Many cardiac and non-cardiac drugs have been found to cause QT prolongation and torsade de pointes (TdP), thus were removed from the market or relabeled for restricted use. It has been known for a long time that quinidine causes cardiac sudden death, however, the severe and lethal side effect of QT prolongation was not drawn sufficient attention until the last decade due to the lack of knowledge and experimental approaches to a comprehensive understanding of QT prolongation. A great deal of understanding of QT prolongation is now achieved and a new regulatory guideline for a battery of preclinical tests to assess a new drug for the QT liability in humans is 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 ms. However, TdP occurs with an average increase in QT interval by approximately 200 ms (a normal QT interval is about 300 ms). A human study has found that TdP did not occur with a QT interval shorter than 500 ms in the cases studied (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 action potential. At this potent, the cardiac cycle of 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, I_kr and I_ks. I_kr is the rapidly activating delayed rectifier potassium current and I_ks is the slowly activating delayed rectifier potassium current. The repolarization phase correlates with the T wave on electrocardiogram. Therefore, the duration of the QT interval is related to the length of 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 (I_kr and I_ks).

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 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 I _kr potassium channels critically affect the length of the plateau phase of the cardiac action potential. The human ether-á-go-go-related gene (HERG) is expressed primarily in the heart and encodes the α-subunit of the cardiac I_kr 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 I_kr channels. The I_kr 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 I_kr channels open, resulting in an increase in the magnitude of I_kr 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 I_kr channels because the functional I_kr 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 I _ks potassium channel is the other one of the two channels primarily responsible for the termination of the plateau phase of the action potential. The I_ks potassium channel is assembled from KVLQT1 α-subunits and the minK β-subunits. There are 2 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 I_ks channels. These individuals have severe arrhythmia susceptibility and congenital neural deafness.

The molecular basis of QT prolongation on electrocardiogram is the prolongation of cardiac action potential. In this regard, the inward sodium channels and outward potassium channels play important role in increasing the length of the plateau phase of action potential. 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 (TdP) and Cardiac Sudden 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 action potential. This small action potential is 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 cardiac sudden death.

The Link Between Cardiac Hypertrophy and Cardiac Sudden Death

Cardiac hypertrophy is a phenotype resulting from a diversity of etiologies and the merging point of myocardial pathological changes and the transition point of myocardial decompensatory remodeling. Therefore, in the hypertrophic phase of cardiac pathogenesis there exist multiple cellular and molecular alterations: each making a distinction contribution to the risk of QT-prolongation and cardiac sudden death. Alterations in the function of cardiac channels, or “cardiac channelopathies” occur at the cellular level in cardiac hypertrophy. 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. However, in the hypertrophic myocardium, electrotonic cell-to-cell coupling is disturbed so that the differences in action potential duration become dominant. In addition, in the hypertrophic myocardium, multiple pathological changes occur such as myocardial fibrosis, myocyte hypertrophy, cell death, and disturbance in neurohormonal regulation. All of these pathological changes have an important impact on QT-prolongation and cardiac sudden death. The factors that can be identified in hypertrophic myocardium and importantly affect the clinical manifestations of QT prolongation and cardiac sudden death are briefly described as follows:

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.

Abnormal Gap Junction

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 cardiac physiology and structural 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 can lead to 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.

Myocyte Hypertrophy

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.

Myocardial Fibrosis

Dilated cardiomyopathy in alcoholics often involves myocardial fibrosis, which simulates the effect of myocardial infarction on the electrical conduction in the heart and the block of cardiac conductance.

Heart Failure

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, I_to1 and I_k1, has been shown to be involved in action potential prolongation. The I_to1 current is involved in phase 1 of 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.

Looking to the Future

Cumulative evidence obtained from more advanced studies in human patients and animal models suggests that cardiac hypertrophy is a maladaptive process of the heart in response to intrinsic and extrinsic stresses. An important advance in the understanding of heart hypertrophy is the recognition of heart hypertrophy as a risk factor for QT-prolongation and cardiac sudden death. Studies using molecular biology techniques such as transgenic and knockout mice have begun to determine molecular mechanisms of QT-prolongation and cardiac sudden death and their link to heart hypertrophy. Results obtained from these studies will lead to more comprehensive understanding of the threats of heart hypertrophy to patients and novel treatment strategies. The adaptation of new approaches such as functional genomics and proteomics will further advance our knowledge of heart hypertrophy and its risk consequences.