Journal of Interventional Cardiac Electrophysiology 2000;4:7–16 © Kluwer Academic Publishers. Boston. Printed in The Netherlands

Impact of Recent Molecular Studies on Evaluation of Ventricular Arrhythmias Impact of Recent Molecular Studies Roden

Dan M. Roden Professor of Medicine and Pharmacology and Director, Division of Clinical Pharmacology and Arrhythmia Unit, Vanderbilt University School of Medicine, Nashville, TN

The increasing understanding of molecular mechanisms in disease has important implications for the clinician. As mechanisms are better understood, patients can be subsetted into groups with homogeneous, and predictable, responses to therapy. Further, it may be possible to develop new therapies, based on a better understanding of such underlying mechanisms. These powerful molecular techniques are now being applied to the understanding of normal and abnormal electrophysiology, and hold the promise of advanced diagnostics and therapeutics. A Molecular View of Normal Cardiac Electrophysiology A fundamental “unit” of basic cardiac electrophysiology is the action potential, the representation of the time-dependent changes in voltage across the membrane of a cardiac cell (Figure 1) [1–3]. The shape and duration of the action potential are determined, in turn, by the amplitudes of individual ionic currents _owing through ion speci~c, pore-forming protein complexes, termed “ion channels,” in the cell membrane. The magnitude of a speci~c ion current is determined by the number of channels for that current, the probability that a single channel is open at a given instant in time, and the amplitude of the current _owing through individual open channels. Open channel probability and single channel amplitude, in turn, are determined by factors such as time after initial stimulation, voltage, and (for some channels) activation of intracellular signaling systems, such as the b-adrenergic cascade. This time- and voltage-dependance of the opening and closing of individual ion channels confers a high degree of interdependence on the behavior of individual ion channels, with resulting important implications for the action potential as a whole. For example, inhibition of function of the transient outward current (ITO) will have the readily-predictable effect of reducing the size of the “notch” of phase 1 of the action potential. However, this reduction in the notch also resets the voltage at which the plateau (phase 2) of the action potential is initiated. This resetting, in turn, alters the amplitude of the L-type calcium channel, of IKs, or of IKr. Hence, a mutation in a gene encoding ITO, or the use of drugs

that speci~cally block ITO, would also modify the behavior of drug-unmodi~ed and genetically-normal currents (ICa-L, IKs, IKr, etc) to produce changes in individual action potentials. The extent of such changes in individual cell thus depends not only on the magnitude of ITO, but also on the other currents whose behavior depends on that of ITO. A similar mechanism seems to operate in long QT-related arrhythmias discussed below. One characteristic of cardiac action potentials is that, even under physiologic conditions, they display considerable cell-to-cell variability, as a function of factors such as rate, stage of development, or speci~c location within the heart (e.g., atrium vs. ventricle). This heterogeneity has been recognized for many decades, and it is now generally held that it must re_ect variability in the individual ionic currents that determine the shape and duration of the action potential in an individual cell. This translates into a more contemporary molecular view, that variability in the expression (i.e., number) or function of individual ion channels is the fundamental determinant of this heterogeneity. Thus, the crucial ~rst step in creating a molecular understanding of normal cardiac electrophysiology has been the cloning of genes that encode ion channel proteins. While detailed discussion of the methods used in these experiments is beyond the scope of this review, several comments on the approaches are relevant. For example, one important result of the application of genetic approaches in the long QT syndrome has been the identi~cation of genes whose protein products underlie

Supported in part by grants from the United States Public Health Service (HL46681 and HL49989). Dr. Roden is the holder of the William Stokes Chair in Experimental Therapeutics, a gift from the Daiichi Corporation.

Address for correspondence: Dan M. Roden, M.D., Director, Division of Clinical Pharmacology, Vanderbilt University School of Medicine, 532C Medical Research Building-I, Nashville, TN 37232-6602. Phone: (615) 322-0067; Fax: (615) 343-4522; Email: [email protected]

Received 2 December 1998; accepted 19 March 1999 7

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Table 1. Cardiac ion channels and their genes

Inward currents INa ICa-T ICa-T If Na-Ca exchanger2 Outward currents IK1 IK-Ach IK-ATP ITO IKs IKr IKur ICl-cAMP

a-subunit

Subunits

SCN5A a1C a1H BCNG

b1,b2 a1-d,b

Kir 2.1 Kir 3.1/3.4 Kir 6.1/6.2 Kv4.3 KvLQT1 HERG Kv1.5 CFTR

SUR minK m:RP1

1

Only subunits de~nitely associated with their a partners are listed. For many other channels, subunits have been inferred or even identi~ed in other tissues but not (yet) demonstrated to play a role in heart. 3 The exchanger can operate in either the forward or reverse mode, and thus provide inward or outward current. The gene has been cloned.

Fig. 1. Stylized ECG, ventricular action potential and cell membrane. The phases of the action potential are shown in bold. The onset of the QRS corresponds to the onset of phase 0, the phase 1 notch is determined by the potassium current ITO, and slow repolarization during phases 2 and 3 represents a balance between inward current, primarily through calcium channels, and outward current primarily through potassium channels (IKr, IKs, IK1, and others not shown). Any intervention (drug, ion channel mutation) that alters this balance in favor of inward current prolongs action potentials in individual cells, and QT interval on the ECG.

important cardiac ion currents, such as IKs and IKr, that are a crucial component of normal electrophysiology [4–7]. Another common technique that has been applied very successfully to the cloning of cardiac ion channel genes is to isolate the cardiac homolog of a gene that is known to encode an ion channel protein in extracardiac tissue, such as brain or skeletal muscle. Examples of the success of this approach are the cloning of the genes encoding the cardiac isoforms of the sodium channel [8], L- and T-type calcium channels [9,10], the ATP-inhibited potassium channel [11] and, most recently, the channel that likely underlies pacemaker activity [12–14]. As shown in Table 1, these and other molecular approaches have succeeded in cloning the genes encoding virtually all pore-forming ion channel proteins (“asubunits”) in the heart. However, despite this success,

it is also increasingly apparent that expression of genes encoding a multitude of other proteins is required to recapitulate normal cardiac electrophysiology. Connexins, the structures that determine cell-cell communications, are one example [15]. Another is b subunits that associated with a-subunits to modify functions such as ion current amplitude, opening or closing probability, or transport to the cell surface [16–18]. An emerging theme in neurobiology, and in the heart, is that ion channel proteins, once expressed within a cell, do not simply meander to the cell surface in a random fashion, but rather are anchored at speci~c sites, and very often in association with important modulatory proteins such as protein kinase A [19]. Indeed, a whole new family of proteins, termed A kinase anchoring proteins (AKAPs) have been cloned from heart and other tissues, and normal AKAP function seems required for b-adrenergic modulation of ion currents [20].

Examples of a Molecular View of Normal Cardiac Electrophysiology Development-dependent action potential changes In the mouse and in the rat, action potentials in the late fetus are quite long, resembling those in larger mammmals, such as adult dogs. However, shortly after birth, action potential durations markedly shorten. Developmental studies have indicated that the major inward current in the developing mouse or rat heart is

Impact of Recent Molecular Studies

the L-type calcium current, while the ~rst repolarizing current to be observed is IKr [21]. At birth, these animals begin to express a very large transient outward current, with resultant marked shortening of action potential duration, presumably to accommodate the very fast rates that these hearts exhibit [22,23]. If an IKr-speci~c blocker is administered to a pregnant rat, there is a very high incidence of embryolethality at certain stages of development [24]. This is now thought to re_ect the fact that, because IKr is the dominant (or in fact sole) repolarizing current in the early heart, disruption of its function leads to marked action potential prolongation and the development of afterdepolarizations, as in the long QT syndrome (LQTS). Thus, the developmental information not only provides an explanation for why action potentials vary as a stage of development, but also suggests a mechanism for one form of animal drug toxicity. The pattern of expression of individual ion channel genes during development in the human heart is not yet known, and it is not known whether drugs that block IKr (quinidine, sotalol, dofetilide) are associated with increased fetal loss.

Rate-dependence of cardiac action potential duration Repolarization in the adult ventricle is determined by a balance between inward (depolarizing) current, primarily carried by L-type calcium current, and outward (repolarizing) current, carried by a current known as the delayed recti~er. Studies in the late 1980s made it clear that the delayed recti~er in many species actually consists of at least two components, IKr and IKs [25]. These components are products of separate genes, display distinctive and different pharmacologies, and activate and deactivate during an action potential at different rates. IKr activates and deactivates rapidly, and is blocked by a variety of antiarrhythmic and other drugs, including quinidine, sotalol, dofetilide, ibutilide, terfenadine, and cisapride. Many of these drugs are associated with Torsades de Pointes, and mutations in HERG, the gene that underlies IKr, are now recognized as a cause of the congenital long QT syndrome (as discussed further below) [26]. IKs activates and deactivates slowly, and its amplitude is markedly increased by b-adrenergic stimulation [27,28]. Unlike IKr, speci~c blockers for IKs have only very recently been synthesized, and the effects of IKs block in human subjects is not well-understood. However, it is known that mutations in the genes underlying IKs (KvLQT1 and minK) also cause the congenital long QT syndrome [5,29]. so it must be considered that IKs-speci~c blockers may also be associated with Torsades de Pointes. Current views indicate that, because of the differences in activation and deactivation rates of the two components, IKr plays a relatively more important role in determining repolarization at slow rates, whereas IKs is relatively more important than fast rates [30]. Stated in another way, the extent of QT prolongation with an IKr-speci~c

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blocker is greater at slow rates than at fast rates. This phenomenon is termed “reverse use-dependence” and a marked action potential at slow rates likely helps explain IKr-related bradycardia-dependent Torsades de Pointes [31]. On the other hand, the effects of IKs block are predicted to be greater at fast, rather than slow rates. While this was viewed as a desirable antiarrhythmic action, the identi~cation of mutations in the genes underlying IKs as a cause of LQTS makes this drug effect seem less desirable. The mechanisms whereby marked action potential prolongation at fast rates might be arrhythmogenic, as in IKs-related LQTS, have not been completely worked out.

Regional heterogeneity of action potential duration and con~guration Since the advent of microelectrode recordings of action potentials in heart, it has been recognized that upstroke slope in sinus and AV nodal tissue is much slower than that in atrium, ventricle, or the Purkinje network. This likely re_ects lack of a prominent inward sodium current in these tissues. Similarly, atrial action potentials have long been recognized as shorter than those in the ventricle or Purkinje network. This is now understood to re_ect, at least in part, a very prominent acetylcholine-activated outward (repolarizing) potassium current IK-ACh, recorded in atrium, but not in ventricle. Indeed, it can be shown that the genes whose expression results in IK-ACh are expressed in the atrium, but not in the ventricle [32]. It is also increasingly appreciated that action potentials may vary among cells in the atrium, and variability in expression of components of a transient outward current, or components of delayed recti~er (IKr or IKs) likely play a role [33]. Action potentials also vary as a function of region within the ventricle [34]. Epicardial action potentials display a prominent phase 1 notch, while those in the endocardium do not. This is though to re_ect expression of the channels that underlie ITO in the epicardium, but not the endocardium. Cells from the mid-myocardium (“M-cells”) display longer action potential durations than those in the endocardium or epicardium, possibly as a result of reduced IKs in this region [35]. These regional differences across the wall of the heart may be especially important in promoting action potential prolongation at slow rates selectively in the M-cell layer, a phenomenon that appears to underlie long QT-related arrhythmias.

Response of the cardiac action potential to b-adrenergic stimulation Probably the most important electrophysiologic effect of b-adrenergic stimulation is augmentation of the Ltype calcium current [36]. This is one factor underlying the increased force of contraction seen with b-adrenergic stimulation, a crucial element in such phenomena as the primitive “~ght or _ight” reaction. However, unbridled L-type calcium channel stimulation will result in

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marked action potential prolongation. Ordinarily, this effect is opposed by simultaneous b-adrenergic stimulation of repolarizing currents such as IKs. Thus, the usual response of the QT interval to b-adrenergic stimulation is QT shortening, and QT response to adrenergic stimulation is abnormal when genes encoding IKs are mutated, as described below.

Altered Ion Channel Function in Long QT Syndromes Subtypes of LQTS The most obvious example of the way in which altered function of cardiac ion channels can result in arrhythmias is LQTS. This will be brie_y discussed here, and the reader is referred to recent comprehensive reviews for further information [37–40]. To date, mutations in four genes have been identi~ed as the cause of LQTS. Three of these, KvLQT1 [5], HERG [4], and SCN5A [41], encode ion channels, while the fourth, KCNE1 (or minK) [29] encodes an ancillary (␤ subunit for KvLQT1. The mutations in genes encoding potassium channel proteins (KvLQT1, HERG, and minK) all result in decreased potassium current, producing prolongation of individual action potential durations and hence the QT interval on the electrocardiogram [42]. The SCN5A mutations, on the other hand, result in a “gain of function” of the sodium channel: mutant sodium channels display a small non-inactivating component of current which persists during the plateau [43,44]. Since action potential duration depends on a balance between inward and outward currents, this persistent inward current, like the decrease in outward current seen with mutant K⫹ channel genes, also results in action potential prolongation. Mutations in KvLQT1 are the commonest cause of LQTS (Table 2) and those in HERG are the second commonest cause. Mutations in SCN5A are rare, and those in minK have only been reported in two families. In addition, other families in which LQTS has not been linked to these four loci have also been reported. Multiple mutations in KvLQT1, HERG, and SCN5A have been reported to cause LQTS. Each case

identi~ed to date results in a mutation in the ion channel protein. It is theoretically possible that other mutations in these genes could result in abnormal levels of expression of functionally normal proteins, but such mutations have not been reported. The identi~cation of multiple reported mutations in each of these genes has proven a gold mine for the molecular biophysicist and electrophysiologist because they have identi~ed speci~c protein domains in these complex channel structures that are important for speci~c functions. For example, the failure of mutant SCN5A channels to inactivate normally during the plateau provides some insight into normal inactivation mechanisms, and the identi~cation of speci~c mutations then provides clues to speci~c regions that are important to determining normal inactivation. In fact, some of these mutations have been identi~ed in regions that were not thought to be important in normal inactivation [45]. Another example is the identi~cation of mutations in the C-terminus of HERG as causing LQTS. Further studies have suggested that the mechanism whereby these mutations cause IKr dysfunction is likely lack of a C-terminal region of the protein that is important for channel assembly or transport to the cell surface [46]. Thus, studies of LQTS had been important not only in understanding the molecular basis of the disease itself, but have also provided tremendous advances in our understanding of normal cardiac electrophysiology. It now appears that the clinical characteristics of patients with the various subtypes of LQTS may vary, as a function of the speci~c gene affected. These differences are likely related in some way to the transmural heterogeneity of action potential durations, and the variability in rate- or adrenergic-dependence of the individual ion currents that underlie this heterogeneity discussed above. For example, patients with SCN5A-related mutations have long isoelectric ST segments and tall peaked T waves, while those with IKr and IKs lesions have longer broader T waves, often with notches [47]. Arrhythmias or sudden death in patients with KvLQT1-associated mutations almost inevitably occur with adrenergic stimulation [48]. The likely explanation is failure to develop adrenergic-dependent

Table 2. The long QT syndrome Name1

Mutant gene

Occurrence

Episodes

ECG

LQT1 LQT2 LQT3 LQT4 LQT5 JLN1 JLN2

KvLQT1 HERG SCN5A unknown minK KvLQT1 minK

commonest 2nd commonest rare one family very rare very rare very rare

adrenergic stress sleep or stress sleep

broad T notched T long isolectric ST

stress stress

TU alterans TU alterans

1 The LQT variants refer to the Romano-Ward syndrome of autosomal dominant inheritance without deafness. The JLN variants refer to the Jervell-Lange-Nielsen syndrome of autosomal recessive inheritance, with deafness. JLN parents carry LQT mutations (by de~nition), but are usually asymptomatic.

Impact of Recent Molecular Studies

IKs activation to limit action potential prolongation caused by increased ICa-L. Subjects with KvLQT1 LQTS mutations do in fact display failure of QT interval to shorten appropriately with exercise. On the other hand syncope and death in patients with SCN5A mutations almost inevitably occurs at night or during periods of bradycardia and these patients shorten QT normally, or even super-normally, with exercise. Patients with HERG-associated mutations causing LQTS display either activity-related or rest-related cardiovascular events, and near-normal QT shortening with exercise. These clinical and molecular ~ndings have important potential implications for the clinician managing patients with LQTS. First, the standard of therapy has been b-adrenergic inhibition, and since KvLQT1associated lesions are the commonest cause of LQTS, the mechanism whereby b-adrenergic inhibition is effective is now more readily understood. As a corollary, sympathetic inhibition may be less effective in other forms of LQTS, and other therapies may be considered. The one that has received the most attention to date is mexiletine, which in in vitro studies can inhibit the late openings of mutant SCN5A channels at concentrations that are readily achieved in human subjects [49]. Preliminary data do show that mexiletine markedly shortens QT interval in patients with SCN5A-associated mutations [50]. However, mexiletine is also effective in shortening action potential duration, and preventing experimental arrhythmias, in the setting of IKr block [51–53]. As well, since the development of arrhythmias with action potential prolongation may (as discussed below) be independent of the speci~c ionic lesion causing action potential prolongation, adrenergic inhibition is still the gold standard against which other therapies in LQTS should probably be measured.

Mechanisms underlying arrhythmias related to action potential prolongation When cardiac Purkinje ~bers are exposed to conditions mimicking those observed in clinical Torsades de Pointes (low drive rates, low extracellular potassium, QT prolonging drug), distinctive abnormalities in the trajectory of repolarization, termed early afterdepolarizations (EADs), and spontaneous beats rising from EADs are frequently observed [54–56]. Contemporary mapping studies support the view that triggered beats arising from EADs initiate Torsades de Pointes [57]. Under normal conditions, beats arising in the subendocardial Purkinje network propagate very rapidly to the epicardium. However, in the presence of low drive rates, low extracellular potassium, and QT prolonging drug, individual cell layers across the wall of the heart display marked differences in action potential prolongation. Speci~cally, action potentials in the M-cell layer markedly prolong, compared to those in endocardium and epicardium. Thus, impulses initiated in the suben-

11

docardial Purkinje network may successfully propagate across the M-cell layer to the epicardium in some regions, but fail to propagate, because of block by very long action potentials, in others. This represents a form of unidirectional block, setting up reentrant excitation in spirals, or scrolls, across the heart. Mapping studies have supported this mechanism as underlying maintenance of Torsades de Pointes, and thus explain why heterogeneity of QT interval durations (i.e., regional heterogeneity in repolarization) represents an additional risk factor for the development of Torsades de Pointes [58,59]. One interesting question that has not yet been satisfactorily answered is the mechanism underlying the EAD and triggered beats that initiate the arrhythmia. What is clear is that the ion channel lesion that results in action potential prolongation (be it in IKs, IKr, or INa) cannot, in general, be implicated in the development of EADs and triggered beats. Rather, it seems likely that action potential prolongation facilitaties the development of inward current through drug-unmodi~ed and genetically-normal channels that then produces EADs and triggered beats. The most likely candidate for this arrhythmogenic inward current is the L-type calcium current, although under different conditions, sodiumcalcium exchange, the T-type calcium current or even the sodium current may contribute [49,60,61]. The well-known effect of b-adrenergic stimulation to augment the L-type calcium current explains why b-blockers and other forms of adrenergic inhibition are effective in LQTS, and suggest ef~cacy independent of the speci~c gene affected.

Phenotype as a function of speci~c mutation? In vitro studies strongly suggest that the functional consequences of LQTS-associated lesions will depend not only on the speci~c gene affected, but also on the speci~c mutation. Indeed, several lines of evidence suggest that there are speci~c mutations in KvLQT1 that carry a low risk of causing arrhythmias, whereas there are others that are more arrhythmogenic [62,63]. An interesting example is the Jervell-Lange-Nielsen syndrome of autosomal recessive deafness, QT prolongation and risk for sudden death. It is now apparent that consanguinity is common in these families, and affected children inherit abnormal IKs-encoding alleles (KvLQT1 or minK) from both parents [29,64,65]. Thus, the parents have (by de~nition) LQT1 or LQT5 but they only very rarely develop symptoms. Deafness appears attributable to the loss of IKs function in the inner ear [64,66]. While the concept of relating genotype to phenotype is very attractive, there are major gaps in our knowledge at this point: the clinical presentations in all patients with LQTS have not been completely characterized (with respect to inciting factors, QT response to exercise, etc.), and the individual mutations have not

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all been well-characterized at the level of the individual ion channel and at the level of the action potential. Overriding these concerns, however, are the problems already apparent that even among patients with the same genetic abnormality, the clinical presentation may vary widely and that large numbers of patients with identical mutations have not been identi~ed.

Genetic testing Patients who clearly have the long QT syndrome do not need a speci~c molecular diagnosis. There may be some interest on the part of some research laboratories in identifying new mutations and in pursuing genotypephenotype correlations, but these are not particularly germane to the individual patient known to have LQTS, at this point. The clinician may be faced with diagnostic uncertainty in an individual member of a family known to have LQTS. Here, diagnostic maneuvers, such as response of the QT interval to exercise, can be helpful. If the speci~c mutation is known, many laboratories have the capability of determining whether a speci~c individual also carries the known mutation, or not. Finally, the clinician occasionally faces a patient with a clinical presentation that is in some way reminiscent of the long QT syndrome. Here, the preferred approach is to ~rst screen the family by history and perhaps ECGs, to determine if there are other affected individuals. If there are not, then the diagnosis is certainly not excluded, because LQTS is now well-recognized to arise because of de novo mutations. However, since dozens of mutations have now been associated with LQTS, testing for each of these in an individual suspect subject is not currently within the capability of most commercial or research laboratories. It is likely, however, that as DNA chip technology moves forward, this aspect of molecular diagnosis may become a reality. Another question that frequently arises with respect to molecular diagnostics is the question of whether genetic testing could identify patients at risk for drug-associated Torsades de Pointes. A number of groups have screened for mutations in LQTS disease genes in patients who present with drug-associated Torsades de Pointes. One Italian family with mutation in KvLQT1 but near-normal QT intervals, and who came to attention only after exposure of the proband to cisapride, has been described. Similarly, a family with a mutation in HERG that came to attention after exposure of a proband to quinidine has also been described. Finally, a mutation in the C-terminus of KvLQT1 associated with less QT interval prolongation than other mutations and a high incidence of presentation after challenge with QT prolonging drugs, has also been described. However, such cases appear to be relatively unusual, and a common molecular basis whereby some patients appear particularly susceptible to QT prolonging drugs has not yet been identi~ed.

The Molecular Genetics of Other Arrhythmia-Associated Diseases Modern molecular approaches have identi~ed speci~c mutations causing a wide range of other cardiovascular diseases associated with arrhythmias. Examples include mutations causing hypertrophic cardiomyopathy (HCM) [67], some forms of dilated cardiomyopathy [68], and those causing various forms of muscular dystrophy [37]. In addition, linkage to speci~c loci has been identi~ed in familial atrial ~brillation [69], dilated cardiomyopathy [70], and arrhythmogenic right ventricular dysplasia [71], although the speci~c disease genes have not yet been identi~ed. The issues with respect to molecular genetic testing and implications of new molecular genetic knowledge for the clinician are quite similar to those with LQTS. For example, mutations in seven different genes have been associated with HCM, and the clinical presentations of individual mutations do seem to vary among disease gene, and perhaps as a function of location within speci~c genes. Issues of incomplete penetrance complicate the phenotypic diagnosis. Interestingly, in HCM, mutations that are associated with minimal hypertrophy and yet a high incidence of sudden death have been described, particularly in the troponin-T gene [72,73]. On the other hand, other disease genes associated with a more benign prognosis have been identi~ed [74]. It seems likely that increased understanding of the molecular mechanisms whereby mutations in HCM disease genes cause myo~brillar disarray, thought to be the arrhythmogenic substrate in this disease, will improve diagnostic methods, and conceivably molecular geneticbased approaches to therapy.

The Brugada Syndrome, Another Ion Channel Disease In the early 1990s, Brugada and Brugada described the syndrome of unusual J-point elevation and right bundle branch block-like ECGs, associated with sudden death [75]. This entity, since termed the “Brugada syndrome,” may also underlie the relatively common development of sudden death in young men in the Far East [76]. Interestingly, mutations in SCN5A have been identi~ed in some patients with the Brugada syndrome [77]. Unlike those associated with LQTS, however, the Brugada syndrome-associated mutations likely result in a loss of sodium channel function. At least one in vitro study suggests that the mechanism underlying the unusual ECG pattern, and the arrhythmias, is loss of sodium current in the epicardium, which then results in a marked heterogeneity of epicardial action potentials and increased risk of reentrant excitation [78]. Unraveling the relationship between clinical presentation and genetic abnormality in the Brugada syndrome is only now beginning, and it is likely that further important insights into the normal physi-

Impact of Recent Molecular Studies

ology of the sodium channel will be one important spinoff. Since patients with the Brugada syndrome may have normal ECGs and yet have a high risk of sudden death due to ventricular ~brillation, the role that sodium channel mutations might play in conferring risk for sudden death, in general, remains to be determined.

A Molecular View of Ventricular Arrhythmias in Acquired Cardiovascular Disease In congestive heart failure (CHF), it is now increasingly well-recognized that QT interval is prolonged in an inhomogenous fashion, and the notion has been advanced that this failure of normal action potential control may be arrhythmogenic in much the same fashion as in LQTS [79]. In humans with CHF as well as in animal models, action potential prolongation seems likely attributable to decreased transient outward current [80], and decreased expression of an ITO-encoding gene has been found in dogs with rapid pacing-induced CHF. Decreases in other potassium currents have also been described [81]. In addition, abnormalities in control of intracellular calcium, a crucial component to maintenance of normal cardiac electrophysiology, also occur and may be arrhythmogenic [82]. Prolongation of the cardiac action potential is also a characteristic ~nding in various models of cardiac hypertrophy, and has been linked to arrhythmias. Either increases in L-type calcium current or decreases in a range of potassium currents have been described, and the extent to which these changes occur and modulate arrhythmias in the hypertrophied heart remains to be determined [83]. In hearts that have been previously subjected to an ischemic insult, a variety of electrophysiologic changes have been described, dependent upon the time after the ischemic insult. These include not only changes in amplitude or function of speci~c ion currents, such as INa, ITO, or IKr [84,85], but also changes in the distribution of ~brous tissue between cells [86], in the distribution of connexins between cells [87], and in the distribution of ion channels within cells [88]. Atrial ~brillation has been associated with a similar range of changes in cellular electrophysiology as discussed elsewhere in this monograph. The extent to which each of these changes mediates speci~c arrhythmias seen in the post-MI patient, or the maintenance of atrial ~brillation, and the extent to which they might be targets for the development of entirely new therapies for the treatment or prevention of these arrhythmias remains to be determined.

Summary It is thus apparent that identi~cation of the molecules whose normal function underlies cardiac electrophysi-

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ology provides a crucial new tool for the experimentalist studying the electrical behavior of the heart. This applies not only to physiologic behaviors, but also pathological behaviors, that may be tightly linked to the development of arrhythmias observed in disease. For the biophysicist, cloning of ion channel genes has provided remarkable new insights into the molecular basis of fundamental properties of ion channels whose underlying mechanisms were only inferred previously. This includes a better understanding of phenomena such as activation, inactivation, drug block, and modulation of function by intracellular signaling systems. Indeed, a crystal structure of a potassium channel has now been produced and is providing molecular biophysicists with entirely new insights into the way in which these fundamental units of excitability operate at the level of the individual atom. Implicit throughout this discussion has been the idea that it is variability in function or expression of individual genes that encode ion channels (and other important proteins) that determines the normal electrophysiology of the heart and its perturbations in disease. Molecular biologists have made tremendous strides in understanding the primary cast of characters in this complex biological system. However, it is apparent that many other proteins do contribute to normal function and these are only now being identi~ed. Importantly, studies to understand fundamental mechanism that control expression of these genes are only now being undertaken. For the clinician, such advances may seem of only academic interest. However, it is the great hope of molecular medicine that understanding the basic mechanisms underlying disease will lead to improved subsetting of patients (in whom speci~cally targeted therapies will be predicted to the effective, or not) and to the development of entirely new molecularly-based therapies.

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