Toward a molecular view of cardiac arrhythmogenesis

Walsh PN: 1994. Platelet-coagulant protein interactions. In Colman RW, Hirsh J, Marder VJ, SaIzrnan EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd ed. Philadelphia, JB Lippincott, pp 629-65 1.

in intrinsic factor Xa formation. Haematol22:743-760.

Br J

Zur M, Nemerson Y: 1980. Kinetics of factor IX activation via the extrinsic pathway dependence of Km on tissue factor. J Biol Chem 255:5703-5707.

Walsh PN, Biggs R: 1972. The role of platelets

TCM

Toward a Molecular View of Cardiac Arrhythmogenesis Dan M. Roden and Michael M. Tamkun

Clinical evidence strongly suggests that the electrophysiologic behavior of the heart, and its response to drugs, is a highly dynamic process. Ion channels are the fundamental molecular units determining cardiac excitability. The cloning of ion channels that are responsible for the generation and maintenance of the cardiac action potential will enable studies of the molecular mechanisms underlying the highly heterogeneous nature of cardiac electrophysiology. The molecular bases of important pathophysiologic events, such as acute regulation of channel function by phosphorylation or drug block, or the long-term electrophysiologic changes accompanying diseases such as hypertension, are now being elucidated. Identification of these molecular mechanisms may point to novel approaches to the treatment of cardiac arrhythmias. (Trends

Cardiovasc

Abnormalities common

Med

of cardiac

clinical problems

1994;4:278-285)

rhythm

are

whose mani-

excitation dependent on scarring due to remote myocardial infarction. Clinicians

festations vary from incidental and asymp-

must distinguish

between

tomatic arrhythmias

tives to develop

appropriate

plans. Moreover,

any treatment

detected during elec-

trocardiography

to catastrophic

such

cardiac

as sudden

ventricular clinical

fibrillation.

aspect

arrhythmias cardiogram)

One

that superficially can

arise

diverse mechanisms. tricular fibrillation regional

due to

intriguing is that resemble

on the electroas a result

For example,

of ven-

can result from acute

myocardial

ously normal

death

of this problem

each other (for example,

events

heart,

ischemia or from

in a previreentrant

Dan M. Roden and Michael M. Tamkun are at the Departments of Medicine, Pharmacology, and Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA.

278

to prevent tricular

these alterna-

an arrhythmia

fibrillation

treatment designed

such as ven-

may be effective

in

only some patient groups, depending

on

the mechanism

targeted.

The study of the mechanisms ing cardiac

arrhythmias

underly-

at the whole

heart and action potential levels has been an area of fruitful collaboration between

basic

scientists

The fundamental study of cardiac

and clinicians.

molecular

unit in the

electrophysiology

is the

ion channel. Block of specific ion channels is the major mode of action of most currently available antiarrhythmics. (For a glossary of relevant terms, see the Appendix.) Currently, two complemen-

01994, ElsevierScience Inc., lOSO-1738/94/$7.00

tary technological advances, the cloning of ion channel cDNAs and the application of voltage-clamp techniques to single cells and to small patches of cell membrane containing as few as one ion channel, are enabling a detailed understanding of the molecular biophysics underlying gating of individual ion channel proteins, which make up the cardiac action potential. New information continues to become available regarding the electrophysiologic mechanisms underlying cardiac arrhythmias, both in experimental animals and in patients. This review first describes how recent advances in the clinical care of patients with arrhythmias have changed philosophies about how to approach treating arrhythmias. Then, information on the electrophysiology and molecular biology of individual ion channels is reviewed, and the ways in which advances at the molecular level may impact our understanding of arrhythmogenesis and its treatment are discussed.

l

Contemporary An&r-rhythmic Therapy: The Problem of the “Moving Target”

It is now increasingly

recognized that the electrophysiologic behavior of the heart is highly heterogeneous. This heterogeneity may be physiologic, as occurs during development, or as a function of location within the heart (atrium vs ventricle; epicardium vs endocardium), or pathologic. Moreover, cardiac electrophysiology may be highly dynamic, so an intervention that is beneficial at one point in time may be ineffective, or even harmful, at another. This was well exemplified by the results of the Cardiac Arrhythmia Suppression Trial (CAST). In CAST, patients convalescing from acute myocardial infarction first received open-label therapy, with the potent sodium channel blockers encainide or flecainide, to suppress premature ventricular complexes (PVCS), a generally asymptomatic arrhythmia whose presence has been linked to increased mortality in some patients. Then, once a drug regimen that effectively suppressed PVCs was identified, patients were randomly assigned in a double-blind fashion to continue that drug therapy or placebo (Echt et al. 1991). Over 1 year of followup, mortality due to arrhythmias, as well as all-cause mortality, was unexpectedly higher among patients treated with the

TCM Vol. 4, No. 6, 1994

drugs than it was among those patients randomized to receive placebo. The terminal event in many of these patients is actually unknown, but ventricular fibrillation was commonly observed. Importantly, the mortality rate was higher among drug-treated patients for the entire year of follow-up. This persistent increase in mortality rates indicates that the mere presence of drug was insufficient to increase mortality; had this been the case, the mortality rate would have been higher among drug-treated patients early in therapy, and then the survival curves would have been parallel. Rather, it seems likely that the presence of drug acted in some way to sensitize the recently ischemic myocardium to the development of fatal arrhythmias (Ruskin 1989, Roden 1991a). A reasonable view of an-hythmogenesis requires both an appropriate electrophysiologic substrate-such as an area of slowed conduction that might facilitate the development of reentrant excitation, or an area of injury in which various forms of normal or abnormal automaticity might occur-and the appropriate trigger(s). As the electrophysiologic behavior of the heart changes over time, new substrates or triggers for arrhythmias may evolve. This can occur in a matter of seconds, as when a coronary artery is occluded, or over years, as exemplified by the structural and electrophysiologic changes that occur with diseases such as hypertension. The factor(s) that sensitized the myocardium to fatal a~h~hmias in CAST has not been identified with certainty, but changes in autonomic tone, the slow healing process following a myocardial infarction, myocardial stretch, and recurrent myocardial ischemia are all reasonable candidates. In experimental animals, pretreatment with sodium channel blockers increased the incidence of ventricular fibrillation developing with acute coronary occlusion (Nattel et al, 1981, Dawson et al. 1984). One likely explanation is conduction slowing due to sodium channel blockade and the attendant enabling of previously latent reentrant circuits. In CAST, mortality was especially high among patients thought to be at risk for recurrent episodes of myocardial ischemia (Echt et al. 1991, Akiyama et al. 1991). These results of the CAST have had a number of important consequences. First, they serve to emphasize the fact that

arrhythmias arise as a consequence of multiple mechanisms: although the drugs tested in CAST were highly effective in suppressing PVCs, they nevertheless facilitated the development of more serious arrhythmias, such as ventricular fibrillation. Second, these results have served as an impetus to reevaluate the safety of other forms of antiarrhythmic therapy. Although most other antiarrhythmic drugs have not been tested in a CAST-like design, suggestive data are nevertheless available that other sodium channel blockers may also increase mortality in some settings (IMPACT Research Group 1984, UK Rythmodan Multicentre Study Group 1984, Cardiac Arrhythmia Suppression Trial II Investigators 1991). In addition, the widely used agent quinidine (both a sodium and potassium channel blocker) may also be associated with an increased mortality rate during chronic treatment (Coplen et al. 1990). Quinidine, along with many other drugs that prolong cardiac repolarization, is well recognized to have the potential to elicit polymorphic ventricular tachycardia when repolarization is markedly prolonged, the “Torsades de Pointes” syndrome. Risk factors for

developing

this arrhythmia,

which

can

be lethal, are incompletely understood, but bradycardia and hypokalemia seem important (Roden and Hoffman 1985, Jackman et al. 1988, Roden 1991b). Thus, as in CAST, the notion that changes in the electrophysiologic milieu in which drugs act (the “moving target”) may serve to facilitate the development of drug-induced arrhythmias seems likely. Third, since a major electrophysiologic property of encainide and flecainide is to block cardiac sodium channels, the results of CAST have driven antiarrhythmic treatment to alternate strategies. One such strategy, supported as well by extensive preclinical data, is action potential prolongation, which is most readily accomplished by potassium channel block. A number of compounds that prolong action potentials without blocking sodium channels (for example, E403 1, dofetilide, almokalant, d-sotalol, and ibutilide) are in clinical trials (Roden 1993). It is unclear which of the many channels active during cardiac repolarization are best targeted in the implementation of this strategy; moreover, Torsades de Pointes may be a problem with this approach. In addition, “nonpharma-

Figure 1. The major ion currents contributing to the generation of a (hypothetical) cardiac action potential. Inward currents are depicted as downward deflections from their respective baselines, and outward currents are upward deflections (amplitudes are not to scale). The currents are listed on the left, and the cDNAs that are known or thought to encode the ion currents are listed on the right (-- indicates that no cDNA has been reported). Adapted, with permission, from the Task Force of the Working Group on Arrhythmias of the European Society of Cardiology (1991).

Probableclone

Current sodium current L-type calcium current T-type calcium current Na-Ca exchange I,,, (4-AP-sensitive) 7 ITo (Ca*-activated) _

, T-

I, 1,

Science Inc.,

sodium-calcium exchanger Kv1.2, 1.4, 1.5, 2.1 +lor 4.2 minK (IsK)

IKW I,, or 1, _ I, (inward rectifier) __

01994, Elsevier

ill dihydropyridine receptor

possibly Kvl.5 CFTR (and possibly others) ROMKlI(G)IRKI family

1050-1?38/94/$7.~

279

O;a

N-linked glycosylation

L

C-type inactivation

involves these

These regions require for subunit assembly

ples include an alternatively spliced form of the cystic fibrosis transport regulator (CFTR), which appears to encode a cardiac chloride current (Levesque et al. 1992); a family of inward rectifiers, which may have evolved from “truncated” versions of Shaker-type channels

N-type inactivation “ball” Protein kinase A and C sites Amino acid sequence conserved between isoforms

(Kubo et al. 1993, Ho et al. 1993); and minK, a cDNA, which is thought to encode a slowly activating delayed rectifier, and which has been discussed in detail in this journal (Kass and Freeman 1993).

Amino acid sequence varying between isoforms

-

Beta subunits, if present. alter function

Figure 2. A summary of structure-function relationships among cloned Shaker-like K+ channels. The predicted channel proteins are -600 amino acids long and share six hydrophobic segments that are thought to span the lipid bilayer. Important functional sites (for example, the voltage sensor), which have been tentatively identified, are indicated. As indicated on the bortom, functional potassium channels are thought to be tetrameric, and different isoforms can assemble to form functionally heteromeric channels.

cologic” (catheter ablation or implanted defibrillator) treatments now appear to be the approach of choice for some arrhythmias. Fourth, the results of CAST have emphasized the highly heterogeneous and dynamic electrophysiologic behavior of the heart. It is in this area that advances in understanding ion currents at the whole cell, single channel, and molecular levels are most likely to have an impact.

l

A Multiplicity of Cardiac Ion Channels

The cardiac action potential is the timedependent voltage signature of the behavior of multiple individual ion channel proteins in a single cardiac cell (Figure 1). The development of voltage-clamp technology over 40 years ago enabled an initial definition of the ion currents, including a resting potassium conductance, the fast inward sodium current, and a repolarizing potassium current, which underlie action potentials in many

280

tissues. With the application clamp technique,

of the patch-

has come the identifi-

cation of the multiple ion channels whose integrated behavior makes up the cardiac action potential. By using this approach, electrophysiologists have been able to predict that ion channel proteins would have domains responsible for specific tion,

functions

such as ion permea-

inactivation,

binding

and ligand

or drug

(Figure 2). The nucleotide

and

predicted amino acid sequence of a complementary DNA encoding the sodium channel of the eel electroplax reported Since

in 1984 (Noda

then,

cDNAs

was

et al. 1984).

encoding

other ion channel proteins cloned, and some predicted

multiple have been functional

domains have been identified using mutagenesis approaches. Proteins encoded by sodium,

calcium,

and many potas-

sium channel cDNAs (of the Shaker superfamily) all share a distinctive sixmembrane spanning segment motif, and are thought to assemble as tetramers, in the case of potassium

01994,

channels

2), or as four roughly homologous domains, in the case of the larger sodium and calcium channel proteins (Catterall 1988, Miller 1990, Roberds et al. 1993). cDNAs encoding other voltage- or ligandgated ion channels in the heart, and which bear no structural similarity to the eel electroplax sodium channel or to Shaker potassium channels, have also been isolated. Interesting recent exam-

(Figure

Elsevier Science Inc., 1050-1738/94/$7.00

The major events in a single action potential are sodium and calcium entry and potassium egress. However, other ion movements are equally important in the maintenance of cardiac homeostasis. For example, contraction is initiated by calcium entry, which serves as the signal for calcium-induced calcium release from sarcoplasmic reticulum via calcium release channels (Marks et al. 1989, Otsu et al. 1990). This entry is usually thought to occur through L-type calcium channels, but recent evidence also supports an important role for the sodium-calcium exchanger (Chin et al. 1993). It is vital that sodium, potassium, and calcium homeostasis be reestablished after each action potential; this is accomplished by the sodium-potassium pump (Young and Lingrel 1987), by the sodiumcalcium exchanger (Nicoll et al. 1990), and the Ca-dependent pump in sarcoplasmic reticulum (Carafoli and Guerini 1993). Certain ion currents present only in some tissues: examples include the pacemaker current I,, which mediates normal automaticity [for a review, see DiFrancesco (1993)] and the ATPinhibited K+ channel, which may become especially important in ischemia (Weiss and Venkatesh 1993). Finally, propagation of electrical impulses from one cell to the next appears to be critically dependent on the expression of connexins to form gap junctions (Beyer et al. 1990).

TCM Vol.4,No. 6,1994

The expectation that the cloning of ion channel cDNAs would allow precise identification of single molecules whose expression gives rise to single ion currents has not been realized. That is, expression of single ion channel cDNAs in heterologous expression systems such as Xenopus oocytes or mammalian cells may give rise to currents that do not precisely replicate behaviors observed in native myocytes. One reason for this dichotomy is that increasingly detailed electrophysiologic approaches have identified multiple components of individual ion current species. For example, the cardiac delayed rectifier (I,) is a current that develops and is then maintained during depolarization. It includes at least four separate ion channel species: a very slowly activating delayed rectifier, I,, thought to be encoded by the minK gene (Takumi et al. 1988, Folander et al. 1990, Honor6 et al. 1991); a more rapidly activating delayed rectifier, I,,, which is the target of a number of new methanesulfonanilide antiarrhythmic compounds (such as E4031, almokalant, and dofetilide) and for which a clone has not yet been identified (Sanguinetti and Jurkiewicz 1990, Roden 1993, Carmeliet 1993); IKur, an ultrarapidly activating delayed rectifier, which may be encoded by the Kv1.5 cDNA (Snyders et al. 1993, Wang et al. 1993); and I,,, a virtually time-independent (on the time scale of an action potential) outward current active at plateau potentials (Yue and Marban 1988). The role of the minK gene in the genesis of I,, has recently become quite controversial. The deduced protein is only 129-130 amino acids long, with a single hydrophobic (possibly membrane-spanning) domain. It is unclear how this small protein can form a potassiumselective pore; nevertheless, mutagenesis at critical amino acids does appear to alter the permeation characteristics or kinase sensitivity of the current expressed in Xenoptcs oocytes by the minK cRNA (Goldstein and Miller 199 1, Blumenthal and Kaczmarek 1992). On the other hand, Attall et al. (1993) have recently suggested that minK actually serves as a regulator of ordinarily “silent” potassium and chloride channels native to Xenopus oocytes. It is clear that there are at least two different types of calcium currents in heart, carried by L-type and T-type channels; cDNAs encoding L-type channels

TCM Vol.4,No. 6,1994

have been isolated from multiple tissues (Collin et al. 1993), whereas a clone for the T-type channel has not yet been identified. Similarly, the transient outward current in cardiac myocyteswhose amplitude first increases and then decreases (inactivates) with sustained depolarization-appears to include not only a potassium channel (IToI) sensitive to 4-aminopyridine, but also, under some experimental conditions, a calciumactivated chloride channel, I,,, (Tseng and Hoffman 1989, Zygmunt and Gibbons 1991). The magnitude and gating kinetics of each ion current described here may be acutely modified by factors such as activation of intracellular signaling systems or membrane depolarization. These changes, then, presumably also may occur under analogous clinical conditions, such as an acute increase in neurohormones like norepinephrine or acute ischemia. The study of molecular mechanisms whereby these signals alter channel function (for example, by phosphylating the channel protein) is an area of active investigation. Recently, data have been presented from both cardiac and brain preparations indicating that coexpression of potassium channel cDNAs can lead to heterotetramerit assembly of ion channels (PO et al. 1993, Sheng et al. 1993). That is, functional ion channels may arise as the result of assembly of multiple different proteins. An extension of this concept is the identification of multiple small subunit proteins associated with some ion channels. For example, expression of brain sodium channel cDNA in Xenopus oocytes gives rise to a sodium current that inactivates far slower than does current observed in brain tissue (Isom et al. 1992). Coexpression of the sodium channel cDNA with its fi subunit, however, restores normal inactivation kinetics. Moreover, some data indicate that subunits can dramatically change the phenotype of an expressed channel: for example, a potassium channel fi subunit changes an expressed delayed rectifier to an inactivating phenotype (Trimmer 199 1, Scott et al. 1994, Heinemann et al. 1994). The role of subunits in modulation of cardiac ion channels is an area that remains largely unexplored. In addition, it has been reported that the phenotype of expressed ion channel cDNA can depend on the amount of mRNA present (Honor6 et al. 1992). Thus, the way in which ion

01994,

Elsevier Science Inc., 1050-l 738/94/$7.00

channel cDNA protein products assemble to form ion channels may be another major mechanism defining variability in whole cell electrophysiology.

l

Ion Channel Expression Is a Dynamic Process

Cloning of multiple ion channel isoforms and increasingly sophisticated electrophysiologic methods have established that the cardiac action potential is the summation of activity of multiple ion channels. Thus, heterogeneity among cardiac action potentials, as a consequence of development, region within the heart, or disease should be viewed as a reflection of diversity in expression of individual ion channel genes, an area of investigation that has become quite active. For example, mRNAs encoding some potassium channel isoforms cannot be isolated in fetal tissue, and only appear postnatally, whereas others appear to be expressed at a relatively constant level, even in early embryogenesis (Roberds and Tamkun 1991). Sodium channel mRNA and protein, assessed by Northern analysis and radioligand binding, have been modulated in vitro or in vivo by calcium ionophore, 8-bromo-CAMP, potassium depolarization, or sodium channel block (Sherman and Catterall 1984, Offord and Catterall 1989, Duff et al. 1992). One proposed mechanism is that alterations in intracellular calcium regulate sodium channel gene expression. Similarly, in vitro and in vivo studies indicate that potassium depolarization, glucocorticoids, increased intracellular calcium, or activation of protein kinase A (Levitan et al. 1991, Matsubara et al. 1993, Mori et al. 1993) can increase mRNA encoding the potassium channels Kv1.4 and Kv1.5. In hypertension and hypertrophy, action potentials are prolonged, an effect that may be linked to arrhythmogenesis; alterations in potassium or calcium channel number or function seem likely (Aronson 1991). Similarly, in patients with advanced heart failure, action potentials are prolonged, an effect that has been attributed to reduction in I,, (the cardiac inward rectifier) and I,,, (Beuckelmann et al. 1993). In other systems, anoxia or repetitive activity have been implicated in chronic regulation of ion channel number or function (Pkrez-Pinz6n et al. 1992, Tsaur et al. 1992).

281

Innervation appears to be another important modulator of ion channel expression: denervation of skeletal muscle alters the pattern of expression of sodium channel isoforms (Yang et al. 199 l), and coculture of cardiac myocytes with sympathetic ganglia increased the number of myocyte calcium channels (Ogawa et al. 1992). A number of laboratories have reported that interference with cardiac innervation during development results in prolonged repolarization (Christiansen et al. 1989, Malfatto et al. 1990). Certainly, one possible explanation for the genetic defect in the congenital long QT syndromes is absent or defective sympathetic regulation of ion channel gene expression (Keating et al. 1991, Vincent 1992).

l

A Molecular Approach Arrhythmogenesis

to

The tools now exist to localize channel mRNAs or the proteins they encode to individual cells. Techniques such as in situ hybridization and immunolocalization are likely to provide major advances in the cellular localization of cardiac ion channels. These tools have been difficult to apply in cardiac ion channel biology because of the relatively low number of molecules per cell, as well as the potential for crossreactivity among highly similar isoforms. Application of mutagenesis strategies will continue to evolve our understanding of the detailed biophysics (gating, inactivation, modification by phosphorylation or other posttranslational events, and ion selectivity and permeation) underlying channel function. Also, the site(s) that is important for binding of an&x-rhythmic drugs to ion channel proteins has been tentatively identified (Snyders et al. 1992); further details of the determinants of ion channel block by drugs may well lead to the development of compounds with desired selectivity and/or affinity for specific ion channels, or for specific ion channel conformations (in electrophysiologic jargon, for specific ion channel “states”). Most ant&rhythmic drugs act by inhibiting ion permeation through ion channels. The ability to express individual channel cDNAs in heterologous systems such as Xenopus oocytes or nonexcitable mammalian cells will facilitate the development of drugs that specifically target individual ion channels. In

282

fact, a number of such compounds are available: the methanesulfonanilide I, blockers and digitalis glycosides targeting Na-K ATPase are examples (and these were developed prior to the availability of individual channel clones). It is important to point out, however, that drugs such as these act in the context of the cardiac action potential, and specific block of a single membrane protein may nevertheless produce substantial secondary alterations in overall cardiac electrophysiology. For example, by inhibiting Na-K ATPase, digitalis glycosides are thought to increase intracellular sodium, with the inotropic effects of the drugs then actually due to secondary activation of Na-Ca exchange. Similarly, drugs such as dofetilide and almokalant appear to block only I,,, an effect that increases action potential durations. Under appropriate in vitro conditions, however, the drugs also produce early afterdepolarizations and triggered activity (Tande et al. 1990); the mechanisms underlying this arrhythmogenic effect are uncertain, but inward current flow through L- or T-type calcium channels or the Na-Ca exchanger (which might occur with action potential prolongation) seems possible. This form of triggered automaticity likely plays a role in the development of Torsades de Pointes during treatment with action potential prolonging agents (Roden 1993). This argument can be extended to predict that specific blockers of other ion channels may exert multiple (and sometimes unanticipated) effects on the action potential. For example, block of ITo would allow the plateau of the action potential to originate at more positive potentials. This, in turn, would alter the magnitude of at least I ca_,_,I,,, I,,, IKr, and I,,; thus, a potent and specific blocker of I, might have highly variable effects, depending on heart rate (a major determinant of the magmtude of I, itself), and the magnitude of other ion currents, itself a function of cell type and factors such as exogenous neurohormones. It should also be recognized that absolutely no epidemiologic evidence supports the idea that drugs whose major effect is inhibition of ion permeation through ion channel(s) reduce cardiovascular morbidity in large populations. The only “antiarrhythmic” drugs that consistently reduce the incidence of serious arrhythmias in patients convalesc-

01994, Elsevier Science Inc., IOSO-1738/94/$7.00

ing from myocardial infarction are pblockers (Beta-Blocker Heart Attack Trial Research Group 1982), and perhaps amiodarone (Ceremuzynski et al. 1992, Pfisterer et al. 1992), a drug that exerts a multiplicity of actions including antiadrenergic effects. Thus, interference with activation of intracellular signaling systems (presumably the mechanism of action of p-blockers) may be at least one alternate antiarrhythmic strategy. Clinicians and basic scientists interested in ion channel biology should retain a very open mind about the way in which further understanding of the molecular mechanisms of cardiac electrogenesis might lead to identification of completely novel targets for antiarrhythmic drug action. These include the mechanisms whereby channel proteins assemble with each other or important subunits, posttranslational changes such as glycosylation, the mechanisms whereby channel protein complexes are anchored to the cytoskeleton, the role of intracellular calcium in modulating cardiac electrophysiology, or ion channels which become active only in disease. Finally, a number of lines of evidence now strongly support the concept that ion channel gene expression can be modulated by factors such as repetitive activity, depolarization, protein kinase activation, or elevation of intracellular calcium. To the extent that such modulation may serve to create an “arrhythmogenic” substrate in diseases such as hypertension or ischemic heart disease, targeting such modulatory mechanisms in an attempt to achieve an antiarrhythmic effect seems like an additional reasonable strategy.

l

Acknowledgments

This work was supported in part by grants from the US Public Health Service (HL47599, HL46681, and HL49989). M.M.T. is an Established Investigator of the American Heart Association. D.M.R. currently holds the William Stokes Chair in Experimental Therapeutics, a gift of the Daiichi Corporation.

l

Appendix

Action potential is the transmembrane voltage recorded as a function of time in a cardiac or other excitable cell (for example, Figure 1). Action potential configuration and duration

TCM Vol. 4, No. 6, 1994

are determined by the activities of the individual ion currents activated, and then inactivated and deactivated, in an individual cell. Afterdepolarizations are abnormalities in the repolarization pattern of an action potential and may be involved in the genesis of some arrhythmias. Early afterdepolarizations interrupt the terminal repolarization process, whereas delayed afterdepolarizations occur after action potential repolarization is complete. Multiple mechanisms may underlie a~e~epola~zations, and they may display specific sensitivity to exogenous factors such as potassium or heart rate. If afterdepolarizations are appropriately timed and are sufficiently large, they may actually produce secondary action potentials, termed triggered beats. Antiarrhythmicdrugs. Most available drugs act by decreasing permeation through one or more ion channel. A common feature of antiarrhythmic drug action is the apparent state dependence of drug block, that is, many ~tia~hy~mic drugs appear to bind preferentially to open or inactivated ion channels. This state dependence has been formalized as the Modulated Receptor Hypothesis (Hille 1977, Hondeghem and Katzung 1977). a framework that has been useful in understanding rate- or voltage-dependent drug effects. Automatic&y refers to the property of some cardiac cells to spontaneously depolarize between action potentials. When membrane potential in such a cell reaches a threshold, a new action potential occurs. This property underlies the normal pacemaker activity characteristic of some cardiac cells, such as those in the sinus node. In addition, automatic behavior may also account for arrhythmias. Calcium channels are responsible for the maintenance of the plateau of the action potential; calcium entry through these channels provides a signal for calcium-induced calcium release from sarcoplasmic reticulum to initiate contraction. In some tissues, such as the sinus and AV nodes, the inward movement of calcium is responsible for the initial upstroke of the action potential. In these tissues, the inward current is smaller, and impulse propagation is slow. Blockers of these cardiac *L-type” calcium channels, such as verapamil, are useful in treatment of arrhythmias involving the AVnode. cDNA refers to either single- or doublestranded DNAthat is complementary to mRNA. This term does not necessarily refer to cloned DNA molecules: pieces of cloned genomic DNA fragments are not cDNAs. cRlUA refers to in vitro synthesized RNA, which is complementary to a cDNA template. Conduction velocity is the rate at which excitation, that is, membrane depolarization, spreads through the myocardium. The magnitude of the inward (depolarizing) current

TCM Vol.4,No. 6,1994

initiating the action potential is a major determinant of conduction velocity. Hence, conductionvelocity is slowest in the sinus and AV nodes, faster in the atrium and ventricle, and fastest in the Purkinje system. Gating is used to describe the changes in ion channel conformation that then result in changes in function (for example, movement from closed to open states). Heterologous expression is the directed synthesis of a protein using its cDNA or cRNA in a cell type not normally expressing the protein. Examples include the injection of cI2NA into a Xenopus oocyte to induce synthesis of the desired ion channel or the insertion of channel cDNA into the genome of a fibroblast under conditions whereby the cell produces the channel mRNA, which directs channel synthesis. inactivation. Some ion currents increase rapidly during a step depolarization but, with maintained depolarization, current magnitude decreases. This decrease during maintained depolarization is referred to as ‘inactivation.“ Channel opening and channel inactivation are separate phenomena, and so are usually thought of as involving different molecular mechanisms. Sodium channels, calcium channels, and transient outward potassium channels all undergo inactivation. Deactivation refers to channel closing with depolarization. Ion channel state is used to describe the functional properties of a channel at any point in time. Different ion channel states likely reflect different ion channel protein conformations. Major ion channel states are closed, open, and inactivated. Permeation does not occur through closed or inactivated channels. Closed channels may open, but (in general) inactivated channels must first shuttle to the closed state prior to opening. ion channels are pore-forming proteins embedded in the lipid bilayer of the cell surface or of intracellular structures such as sarcoplasmic reticulum or the nuclear envelope. With an appropriate stimulus, such as a change in transmembrane voltage or the binding of a ligand on or near the channel protein, the conformation of the protein changes, enabling the pore to open and pass ions across the cell membrane. Ion channels may display selectivity, that is, they permit movement of only one ion species (sodium, calcium, or potassium channel), or may be nonselective, as in the case of some stretchtransduced channel openings. mRNA refers to the fully processed cytoplasmic RNA molecules that contain the genetic code for a specific protein and direct synthesis of the protein on the ribosome. Permeation refers to the movement of ions through ion channels. Potassium channels are a highly heterologous group of channels that have in common

01994,EisevierScience Inc., lOSO-1738/94/$7.00

the ability to pass potassium across cell membranes. Important potassium channel functions include repolarizing cells following depolarization due to sodium or calcium entry (Ix), mediating pacemaker activity (I,), setting resting potential (I& and initial plateau potential (ITo), and controlling the action potential during ischemia (I,,,). Block of repolarizing potassium currents will result in prolongation of the action potential and of the QT interval on the surface electrocardiogram. Rectification is the property of some ion channels to conduct ions more effectively in one direction than in the other. For example, the potassium channel that helps to set the resting potential is an inward rectifier: it conducts potassium ions far more readily from the outside of the cell to the inside (inward) than vice versa. Another class of potassium channels are those that open with a step depolarization, but reach their peak only after a delay, and so are referred to as delayed rectifiers. Reentrant excitation is a mechanism underlying some arrhythmias. Under pathologic conditions, the characteristics of cardiac cells may become sufficiently heterogeneous so that an impulse may not uniformly propagate, but rather may block in some areas but not in others. Under these conditions, it is possible for a propagating wavefront to reenter an area in which excitation had previously been blocked and thereby initiate premature beats or sustained arrhythmias. Repolarization is the process whereby the action potential returns to its basal state following a depolarizing upstroke. Changes in ventricular repolarization are often reflected as changes in the QT interval on the surface electrocardiogram. Sodium channels are responsible for the initial upstroke in atrial, ventricular, and conducting tissue. The large depolarizing sodium current also is responsible for propagation of impulses from one cell to the next. Sodium channels usually undergo rapid inactivation. Sodium channel blockers may suppress arrhythmias by multiple mechanisms such as altering threshold for excitation or decreasing conduction velocity.

T&I currents are the decreasing ionic currents observed during a voltage clamp experiment when a depolarizing stimulus is removed and the potential is returned toward rest. The current is decreasing due to channel closing or deactivation. Voltage clamp is a common experimental technique whereby voltage across a cell membrane is controlled, and current measured. The current represents ion flow across the membrane. By a combination of specific voltage clamp pulse patterns, ion substitutions in extracellular or intracellular solutions, and the use of specific blockers, the characteristics of individual ion currents can

283

be studied. studied

Individual

ion channels

can be

using the patch-clamp mode of the

voltage-clamp

technique.

Akiyama T, Pawitan Y, Greenberg Reynolds-Haertle gators:

1991.

cardiac

H, Kuo CS,

RA, and the CAST investi-

Increased

arrest

from

risk of death encainide

and

and

fle-

cainide in patients after non-Q-wave acute myocardial infarction in the Cardiac Arrhythmia Suppression

Trial. Am J Cardiol68:1551-

1555. Aronson

RS:

199 1. Mechanisms

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