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Studying Cardiac Arrhythmias in the Mouse—A Reasonable Model for Probing Mechanisms?
BRIEF REVIEWS Studying Cardiac Arrhythmias in the Mouse—A Reasonable Model for Probing Mechanisms? Jeanne M. Nerbonne* The normal mechanical functioning of the heart depends on proper electrical functioning, reflected in the sequential activation of pacemaker cells, and the normal propagation of activity through the ventricles. Myocardial electrical activity is evident in the form of action potentials, reflecting the activation (and inactivation) of depolarizing (Na+, Ca2+) and repolarizing (K+) current channels. There are multiple types of myocardial K+ channels, contributing to regional differences in action potential waveforms and to the generation of normal cardiac rhythms. The conduction and propagation of activity through the myocardium depends on electrical coupling between cells, mediated by gap junction channels. In the diseased myocardium, action potential waveforms and conduction are affected markedly, owing to changes in the functional expression of repolarizing K+ and other channels. These changes can lead to desynchronization of the heart and to arrhythmia generation. There is presently great interest in defining the cellular, molecular, and systemic mechanisms contributing to the generation and the maintenance of cardiac arrhythmias. Although a variety of experimental (animal) model systems have been (and are being) exploited in these efforts, the mouse is being used increasingly, due to the ease with which molecular genetic strategies can be applied. The important issue is whether the mouse is an appropriate model system to explore arrhythmia mechanisms. (Trends Cardiovasc Med 2004;14:83–93) n 2004, Elsevier Inc. Jeanne M. Nerbonne is at the Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri, USA. * Address correspondence to: Jeanne M. Nerbonne, Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA. Tel.: (+1) 314362-2564; fax: (+1) 314-362-7058; e-mail: [email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter
TCM Vol. 14, No. 3, 2004
The normal mechanical (pump) functioning of the mammalian heart depends on proper electrical functioning, reflected in the sequential activation of cells in specialized regions of the heart, and the normal propagation of activity through the ventricles. Myocardial electrical activity is evident in the form of action potentials, reflecting the activation (and inactivation) of depolarizing (Na+, Ca2+) and repolarizing (K+)
current channels (Figure 1). There are multiple types of myocardial K+ channels (Figure 1), contributing to regional differences in action potential waveforms and to the generation of normal cardiac rhythms (Antzelovitch and Dumaine 2002, Nerbonne and Kass 2003). The conduction and propagation of activity through the myocardium, in contrast, depend on electrical coupling between cells, mediated by gap junction channels (Kanno and Saffitz 2001). In the diseased myocardium, action potential waveforms and conduction can change markedly, owing to changes in the functional expression of repolarizing K+ (and other) channels (Peters and Witt 1998, Tomaselli and Marban 1999). These changes can lead to desynchronization and arrhythmias, and there is considerable interest in defining the cellular, molecular, and systemic mechanisms contributing to the generation and the maintenance of potentially life-threatening cardiac arrhythmias. Although a variety of experimental (animal) model systems have been exploited in these efforts over the years, the mouse is being used increasingly, due to the ease with which molecular genetic strategies can be applied. The question then arises whether the mouse is an appropriate model system to explore arrhythmia mechanisms. This article considers this question, with a particular focus on mouse models in which the expression of repolarizing K+ channels has been altered.
Myocardial Action Potential Waveforms and Underlying Ionic Currents
There are marked differences in the waveforms of action potentials recorded in different species, as well as in myocardial cells in different regions of the heart in the same species (Antzelevitch and Dumaine 2002, Nerbonne and Kass 2003). Action potential waveforms in
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Figure 1. Schematic of action potentials and underlying ionic currents in adult human (left) and mouse (right) ventricular myocytes. As illustrated, the diversity of outward K+ currents in myocardial cells is greater than that for the inward Na+ and Ca2+ currents, and the various K+ currents play distinct roles in action potential repolarization in human and mouse ventricular cells.
mouse ventricular (Figure 1) and atrial (Figure 2) myocytes, for example, as well as the electrical properties of the intact murine heart (London 2001, Nerbonne et al. 2001), are really quite different from those of larger animals, particularly humans (Figure 2). In addition, the resting heart rate in the adult mouse is 600 to 700 beats per minute, f10 times faster than in the normal adult human (Figure 2). Furthermore, although the action potential upstroke (phase 0)—reflecting inward current through voltage-gated Na+ channels— is similar in human and mouse ventricular myocytes, repolarization is distinct (Figure 1). In humans, the upstroke is followed by a transient repolarization (phase 1) to a plateau phase (phase 2). The driving force for K+ efflux is high during the plateau and, as Ca2+ channels inactivate, outward K+ currents predominate, resulting in a second, rapid phase (phase 3) of repolarization
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back to the resting potential (Figure 1). In mouse ventricular cells, there is no clear plateau phase, and repolarization is rapid (Figure 1). Although there are multiple types of myocardial K+ currents (Antzelevitch and Dumaine 2002, Nerbonne and Kass 2003), the properties of the various K+ currents in different cell types and species are similar, suggesting that the molecular correlates of the underlying K+ channels are also the same. A rather large number of K+ channel pore forming (a) and accessory (h) subunits (Figures 3 and 4) have now been identified. Exploiting transgenic and gene-targeting strategies in mice— considerable progress has been made in defining the relationships between these subunits and functional cardiac K + channels (Kuo et al. 2001, Nerbonne and Kass 2003, Nerbonne et al. 2001). Although these efforts have clearly demonstrated the power of in vivo genetic
approaches in mice for molecular studies, it is of interest to consider whether the mouse is suitable for functional studies focused on exploring the cellular and systemic mechanisms underlying arrhythmia generation and propagation.
Repolarizing K+ Currents in the Mammalian Myocardium
Two types of repolarizing voltage-gated K + (Kv) currents have been distinguished: transient outward K+ currents (Ito) and delayed, outwardly rectifying K+ currents (IK) (Nerbonne and Kass 2003) (Figure 1). These are broad classifications, however, and there are multiple (transient and delayed rectifier) Kv currents expressed in myocardial cells (Nerbonne and Kass 2003). For example, two transient outward K+ currents—Ito, fast (Ito,f) and Ito,slow (Ito,s)—with distinct time- and voltagedependent properties and pharmacologic TCM Vol. 14, No. 3, 2004
the genes encoding Kv channels, as well as changes in Kv channel properties and/or expression patterns, are linked to cardiac arrhythmias (Chen et al. 2003, Keating and Sanguinetti 1999, Tomaselli and Marban 1999). As a result, considerable effort has been directed towards defining the molecular correlates and the functional roles of myocardial K+ channels, as well as in exploring the molecular mechanisms controlling the properties and the expression of these channels.
Figure 2. Comparison of human and mouse ventricular (V) and atrial (A) action potentials and electrocardiogram (ECG). Representative transmembrane action potentials, recorded using intracellular microelectrodes, are plotted above representative surface ECG recordings. Note the waveform and time-scale differences in the two sets of records. (Adapted from Nerbonne JM, Nichols CG, Schwarz TL, Escande D: 2001. Genetic manipulations of cardiac K+ channel function in mice. What have we learned and where do we go from here? Circ Res 89:944 – 956, with permission. Copyright 2001, Lippincott, Williams & Wilkins.)
sensitivities have been distinguished (Nerbonne and Kass 2003). Although Ito,f is expressed in cells isolated from right and left ventricles and from the interventricular septum, Ito,s is only detected in septum cells (Xu et al. 1999b). Multiple components of IK have also been distinguished. In human ventricular myocytes, two prominent delayed rectifiers—IKr and IKs (Figure 1)—have been identified (Li et al. 1996), although neither IKr nor IKs is a prominent repolarizing current in the adult mouse (Xu et al. 1999b). Rather, there are three distinct delayed rectifier currents—I K,slow1 , I K,slow2 and I ss — expressed in mouse ventricular myocytes (Xu et al. 1999b, Zhou et al. 2003). The inwardly rectifying K+ channel currents (Figure 1) can also contribute to myocardial action potential repolarization (Babenko et al. 1998, Flagg and Nichols 2001, Lopatin and Nichols 2001). These (K+) channels are encoded by a subfamily of inward-rectifier K+ (Kir) channel pore-forming a subunits TCM Vol. 14, No. 3, 2004
(Figure 4), producing proteins with two transmembrane domains. The Kir2 a subunits encode the strong inwardly rectifying IK1 channels (Lopatin and Nichols 2001), whereas Kir6.2 underlies the weakly inwardly rectifying I KATP channels (Babenko et al. 1998, Bolli and Marban 1999, Flagg and Nichols 2001). In human and mouse ventricular myocytes, IK1 channels determine resting membrane potentials and I KATP channels are thought to provide a link between cellular metabolism and membrane potential (Babenko et al. 1998, Flagg and Nichols 2001, Lopatin and Nichols 2001, Nerbonne et al. 2001). In large mammals, IK1 also contributes the plateau potential (phase 2) and shapes phase 3 repolarization. The primary determinants of cardiac repolarization, therefore, are the Kv channels, and these channels are also the most diverse with respect to function and expression (Nerbonne and Kass 2003). In addition, mutations in
Molecular Analysis of Myocardial Kv a Subunit Diversity and Functioning
Kv channel pore-forming (a) subunits are six transmembrane-spanning domain proteins that belong to the ‘‘S4’’ superfamily of voltage-gated channels, and functional Kv channels comprise four a subunits (Nerbonne and Kass 2003). Multiple Kv a-subunit subfamilies and a large number of Kv accessory (h) subunits have been identified, and many of these are expressed in the heart (Figure 3). There are also many nonvoltage-gated K+ channel a-subunit subfamilies (Figure 4) with two or four transmembrane domains, and several of these are expressed in heart (Nerbonne and Kass 2003). The multiplicity of Kv a and h subunits (Figure 3), suggests considerable potential for generating functionally diverse cardiac Kv channels (Nerbonne and Kass 2003). Two Kv a-subunit genes—KCNQ1 and KCNH2—have been identified as loci of mutations in familial long QT syndromes 1 and 2, and shown to encode the Kv a subunits important in the generation of cardiac IKr and IKs channels (Barhanin et al. 1996, Sanguinetti et al. 1995 and 1996). Considerable evidence suggests that Kv4 a subunits encode myocardial Ito,f channels. In transgenic mice expressing a pore mutant of Kv4.2 (Kv4.2W362F) that functions as a dominant negative (Kv4.2DN) (Barry et al. 1998, Xu et al. 1999c), Ito,f is eliminated (Figure 5). Although action-potential durations and QT intervals are prolonged (Figure 5), Kv4.2 DN mice do not display spontaneous arrhythmias or increased susceptibility to arrhythmia induction (Barry et al. 1998, Brunner et al. 2001, Guo et al. 2000b). Ventricular Ito,f densities are also 85
Figure 3. Kv a and Kv accessory subunits and voltage-gated cardiac K+ channels. The boxes indicate subunits that are expressed in mammalian heart; gene designations are in italics. In the column labeled ‘‘cardiac current,’’ the channel type encoded by each Kv subunit is provided; ?? indicates current uncertain or unknown.
reduced (but not eliminated) in transgenic mice expressing a truncated Kv4.2 a subunit, Kv4.2N (Figure 5), that also functions as a dominant negative (Wickenden et al. 1999). In contrast to the Kv4.2DN mice, cardiac hypertrophy, chamber dilation, fibrosis, and sudden death are observed in Kv4.2N mice (Wickenden et al. 1999). The pathology evident in Kv4.2N mice cannot reflect the loss of Ito,f, because the elimination of Ito,f in Kv4.2 DN mice (Barry et al. 1998, Brunner et al. 2001, Guo et al. 2000b) and in mice with a targeted deletion in the
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Kv4.2 gene (Kv4.2 / ) (Guo et al. 2000a) has no detectable pathophysiologic consequences (Figure 5). Nevertheless, the disparate experimental observations reported in these studies illustrate one of the potential problems with transgenic approaches, which, in most cases (but see Robbins 2000), involve the ‘‘overexpression’’ of transgenic proteins. The problem is that there may be pathophysiologic effects resulting directly from protein overexpression per se. In addition, the mouse heart may adapt and/or remodel following genetic mani-
pulation. In efforts to probe K+ (and other) channel functioning, therefore, the study of multiple lines of mice in complementary model systems would seem to be a reasonable goal. Biochemical studies have revealed that mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 (Guo et al. 2002). It is somewhat surprising, therefore, that Ito,f is undetectable in mice lacking Kv4.2, even though Kv4.3 expression appears to be unaffected (Guo et al. 2000a). In human ventricular myocytes, however, TCM Vol. 14, No. 3, 2004
Subfamily
Protein
Gene
Cardiac current
Kir1
Subfamily
Protein
Gene
Cardiac current
TWIK-1
KCNK1
??
TWIK-2
KCNK6
??
TWIK-3
KCNK7
TWIK-4
KCNK8
TREK-1
KCNK2
TREK-2
KCNK10
TASK-1
KCNK3
TASK-2 TASK-3 TASK-4 TASK-5
KCNK5 KCNK9 KCNK14 KCNK15
TRAAK-1
KCNK4
THIK-1
KCNK13
THIK-2
KCNK12
TALK-1
KCNK16
TALK-2
KCNK17
TWIK Kir1.1
KCNJ1
??
Kir2 Kir2.1
KCNJ2
IK1
Kir2.2
KCNJ12
IK1
Kir2.3
KCNJ4
??
Kir2.4
KCNJ14
??
Kir3.1
KCNJ3
IKACh
Kir3.2 Kir3.3
KCNJ6 KCNJ7 KCNJ9
Kir3.4
KCNJ5
TREK ??
TASK
Kir3
IKp ??
TRAAK IKACh
Kir4
THIK Kir4.1 Kir4.2
KCNJ10 KCNJ15
Kir5.1
KCNJ16
Kir5 Kir6 Kir6.1
KCNJ8
Kir6.2
KCNJ11
??
TALK ??
IKATP
Figure 4. Kir a and KTP a subunits in the mammalian myocardium. The boxes indicate subunits that are expressed in heart; gene designations are in italics. In the column labeled ‘‘cardiac current,’’ the likely functional correlate of the a subunit is listed; ?? indicates current uncertain or unknown.
functional I t o, f channels are likely Kv4.3 homotetramers, because Kv4.2 is not expressed (Dixon et al. 1996). In addition to revealing that the molecular correlates of mouse and human ventricular Ito,f channels are distinct, these observations suggest that novel mechanisms have evolved to control the cellsurface expression of homotetrameric Kv4.3 channels—likely with associated Kv accessory subunits (Rosati et al. 2001 and 2003)—in humans (and other large mammals). The observations that Ito,s is undetectable (Figure 5) and Ito,f is unaffected in interventricular septum myocytes (Guo et al. 1999) from mice with a targeted deletion in the Kv1.4 gene, Kv1.4 / (London et al. 1998b), clearly demonstrate that the molecular correlates of Ito,s and Ito,f channels are distinct. It seems likely that Kv1.4 also encodes slow transient outward K + currents (Ito,s) in human ventricular myocytes (Nerbonne and Kass 2003). Although TCM Vol. 14, No. 3, 2004
there are no electrocardiographic (ECG) changes (Figure 5) and no pathophysiology is evident in Kv1.4 / mice (Guo et al. 1999), it is certainly possible that alterations in Ito,s expression/properties in human ventricles could impact the normal transmural gradient of repolarization and, therefore, have profound electrophysiologic consequences (Antzelevich and Dumaine 2002, Nerbonne and Guo 2002). Unexpectedly, Ito,s is unregulated in the right and left ventricles of Kv4.2DN mice (Figure 5), reducing the normal, albeit small, dispersion of ventricular repolarization in the mouse (Guo et al. 2000b). The loss of both Ito,f and Ito,s in Kv4.2DN/Kv1.4 / mice (Figure 5) eliminates the regional differences in outward K+ current densities and action potential durations (Guo et al. 2000b). In addition, in spite of the marked QT prolongation in Kv1.4 / /Kv4.2DN mice (Figure 5), ventricular arrhythmias, although occasionally observed, are really quite rare
(Guo et al. 2000b). These observations suggest that reduced dispersion of repolarization in the mouse is antiarrhythmic (Guo et al. 2000b, Nerbonne et al. 2001) and this is likely also the case in larger mammals, including humans (Antzelevich and Dumaine 2002, Nerbonne and Guo 2002). Transgenic and targeted deletion strategies in mice have also been exploited to explore the diversity and the functioning of cardiac delayed rectifier K + currents. The functional consequences of the in vivo manipulation of the subunit genes (including KCNQ1, KCNH2, and KCNE1) encoding cardiac IKr and IKs channels, for example, have been explored (Figure 5). Mice with targeted deletions in KCNE1 (KCNE1 / ) or KCNQ1 (KCNQ1 / ) display shaker/Waltzer behavior, attributed to loss of transepithelial K+ secretion and collapse of the spaces in the inner ear that normally contain K + -rich endolymph (Casimiro et al. 2001, Charpentier
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Figure 5. Genetically engineered mice with altered K+ current expression. Phenotypic consequences of transgenic or gene-targeted alterations in the functioning of myocardial K+ currents in the mouse are noted. APD, action potential duration; ATP, adenosine triphosphate; ND, not determined; Tg, transgenic.
et al. 1998, Drici et al. 1998, Kupershmidt et al. 1999, Lee et al. 2000, Vetter et al. 1996). Although there are, as yet, unexplained phenotypic differences in the consequences of the deletion of the KCNE1 and KCNQ1 genes in different mouse models (Casimiro et al. 2001, Charpentier et al. 1998, Drici et al. 1998, Kupershmidt et al. 1998, Lee et al. 2000, Warth and Barhanin 2002), the cardiac effects are really quite subtle (Figure 5), as might have been expected, given that IKs is not a prominent repolarizing K+ current in the mouse (Figure 1). Transgenic strategies have also been exploited to explore the functional impact of expression of known long QT
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mutations in KCNH2 and KCNQ1. ECG recordings from mice expressing a long QT2 (KCNH2) mutation (Babij et al. 1998), and whole-cell recordings from ventricular myocytes isolated from these animals, are indistinguishable from wildtype animals/cells (Figure 5). In contrast, animals expressing a truncated splice variant of KCNQ1—KvLQT1-isoform 2 (Demolombe et al. 2001)—exhibit sinus bradycardia, QT prolongation (Figure 5), abnormal P wave morphology, and intranodal conduction block. Unexpectedly, electrophysiologic recordings from KvLQT1-isoform2-expressing ventricular myocytes revealed that action potentials are prolonged and that outward (Ito) and inward (IK1) K+ current
densities are reduced (Figure 5). In addition, the severity of the cardiac phenotype was directly correlated with the amount of the mutant protein in different KvLQT1-isoform2-expressing transgenic lines (Demolombe et al. 2001). Taken together, these observations again raise concerns that there are nonspecific in vivo cardiac effects of transgenic protein ‘‘overexpression’’ (Nerbonne et al. 2001, Robbins 2000). Additional studies have focused directly on determining the molecular identity of the delayed rectifier K+ currents expressed in the mouse myocardium and on exploring the functional consequences of the manipulation of these currents in vivo (Brunner et al. 2001, Jeron et al. TCM Vol. 14, No. 3, 2004
Figure 6. Electrocardiographic (ECG) abnormalities seen in genetically engineered mice (left) are displayed alongside corresponding ECG abnormalities seen in humans (right). (LQT) QT prolongation evident in LQT1 and LQT2 patients and in a KCNQ1-isoform2-expressing transgenic (TG) mouse; a wild-type (WT) mouse ECG is also shown (Demolombe et al. 2001). (AVb) High-degree atrioventricular (AV) block in humans and in Kv4.2DN/Kv1.4 / mice (Guo et al. 2000); P waves are indicated by the arrowheads. (TdP) Torsade de Pointes, a potentially lifethreatening arrhythmia in humans is detected in KCNQ1-isoform2-expressing transgenics (Demolombe et al. 2001). (We) Wenckebach AV block in KCNQ1-isoform2-expressing mice (Demolombe et al. 2001); P waves are indicated by the arrowheads. (Adapted from Nerbonne JM, Nichols CG, Schwarz TL, Escande D: 2001. Genetic manipulations of cardiac K+ channel function in mice. What have we learned and where do we go from here? Circ Res 89:944 – 956, with permission. Copyright 2001, Lippincott, Williams & Wilkins.)
2000, Kodirov et al. 2004, Li et al. 2004, London et al. 1998a and 2001, Xu et al. 1999a, Zhou et al. 2003). Although the molecular identity of mouse ventricular I ss remains to be defined, dominant negative and targeted deletion strategies have revealed that there are actually two molecularly distinct components of mouse ventricular IK,slow: IK,slow1, which is encoded by Kv1.5, and IK,slow2, which is encoded by Kv2.1 (Li et al. 2004, London et al. 1998a and 2001, Xu et al. 1999a, Zhou et al. 2003). Interestingly, ventricular arrhythmias are observed in Kv1.1DN animals but not in Kv2.1DN animals, in spite of the fact that IK,slow densities and QT intervals are similarly affected (Figure 5). Also, in contrast to Kv1.1DN mice, there is also no evidence of arrhythmias in mice expressing a pore mutant of Kv1.5 (Kv1.5W461F) that also functions as a dominant negative (Kv1.5DN) (Li et al. 2004), or in mice TCM Vol. 14, No. 3, 2004
with a targeted deletion of the Kv1.5 a subunit (Kv1.5 / ; SWAP) mice (London et al. 2001). The observation of spontaneous arrhythmias and the increased susceptibility to arrhythmia induction only in Kv1.1DN mice appears to reflect increased dispersion (of repolarization) in these animals (Baker et al. 2000, Jeron et al. 2000). The increased dispersion may reflect the fact that IK,slow2 current remodeling is evident, but only in the apex of Kv1.1DN mice (Zhou et al. 2003). Consistent with this interpretation, there are marked differences in the extent of electrical remodeling seen in Kv1.1DN, Kv2.1DN, Kv1.5DN, and Kv1.5 / (SWAP) mice (Figure 5). It has also been reported that the additional elimination of Ito,f suppresses arrhythmias in Kv1.1DN mice in spite of the fact that QT intervals are prolonged more in Kv1.1 DN/Kv4.2DN (and Kv4.2 DN) ani-
mals than in Kv1.1 DN animals (Brunner et al. 2001). These observations reinforce the hypothesis that neither action potential nor QT prolongation, per se (Figure 6), is a good predictor of arrhythmia incidence or susceptibility, at least in mice. Rather, the dispersion of ventricular repolarization appears to be the more important parameter in considering arrhythmia susceptibility. If correct, then it seems reasonable to suggest that expression of Kv4.2DN and the additional loss of I to,f (in Kv1.1DN/ Kv4.2DN mice) reduces the dispersion of repolarization sufficiently to suppress the arrhythmogenic effects of Kv1.1 DN expression (Brunner et al. 2001). Elimination of the Kv2.1-encoded IK,slow2 in Kv1.1 DN-expressing animals, in contrast, does not measurably affect spontaneous arrhythmias or inducibility (Figure 5) relative to mice expressing Kv1.1 DN alone (Kodirov et al. 2004).
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Molecular Analysis of Functional Kv Channel Accessory Subunit Diversity
A variety of cardiac Kv channel accessory subunits—including Kvh, MiRP, KChAP ,and KChIPs (Abbott and Goldstein 1998, An et al. 2000, Pongs et al. 1999, Wible et al. 1998)—have been identified (Figure 3). In contrast to the Kv a subunits, the roles of these (accessory) subunits in the generation of functional cardiac K + channels are not presently very well understood. One potential complication is the growing awareness that these subunits can, at least in heterologous expression studies, interact with several different Kv a, as well as Kv h, subunits (Abbott and Goldstein 1998, Pongs et al. 1999). In addition to interacting with the N termini of Kv4 and Kv2 a subunits, for example, KChAP binds to the C termini of Kv h subunits (Wible et al. 1998). Nevertheless, some progress has been made. Biochemical studies, for example, have shown that KChIP2—originally identified in a yeast two hybrid screen using the N terminus of Kv4x as bait (An et al. 2000)—co-immunoprecipitates with Kv4.2 and Kv4.3 from adult mouse ventricles, consistent with a role for the KChIP2 subunit in the generation of Ito,f channels (Guo et al. 2002). Interestingly, there are marked variations in KChIP2 expression in canine and human ventricles, suggesting that regional differences in KChIP2 expression underlie the observed gradients in Ito,f densities observed in these species (Rosati et al. 2001 and 2003). In mouse, however, there is no gradient in KChIP2 expression, and the differential expression of Kv4.2 underlies the regional differences in Ito,f densities (Guo et al. 2002). Although both human and mouse I to,f channels are encoded by Kv4 a subunits and the KChIP2 accessory subunit, both the molecular compositions of the underlying channels (Kv4.2 and Kv4.3 in mouse versus Kv4.3 in human) and the molecular determinants of the regional differences in the functional cell surface expression of these channels (Kv4.2 in mouse versus KChIP2 in human) are distinct (Dixon et al. 1996, Guo et al. 2002, Rosati et al. 2001 and 2003). In ventricular myocytes isolated from mice with a targeted deletion in the KChIP2 gene (KChIP2 / ), Ito,f is elimi-
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nated (Kuo et al. 2001). In contrast to the observations in Kv4.2DN-expressing animals (Barry et al. 1998, Brunner et al. 2001, Guo et al. 2000b) and Kv4.2 / (Guo et al. 2000a) animals, and in spite of a marked increase in ventricular action potential durations, however, QT prolongation is not evident in KChIP2 / mice. Also, in contrast to the findings in Kv4.2DN-expressing animals and Kv4.2 / animals, KChIP2 / hearts display increased susceptibility to ventricular tachycardia and sudden cardiac death (Kuo et al. 2001). Although the arrhythmia susceptibility in KChIP2 / mice is attributed to the loss of Ito,f, it is not clear why (or how) cardiac rhythms are affected in this model, but not in Kv4.2DN, Kv4.2 / , Kv4.2DN/Kv1.4 / , or Kv4.2DN/Kv1.1DN mice (Figure 5); and the molecular mechanisms underlying these profound phenotypic differences in the different mouse models with altered Ito,f expression remain to be elucidated.
Overview and Summary
Myocardial Kv channels are primary determinants of action potential repolarization, and differences in Kv current
expression are largely responsible for the observed (regional and species) differences in action potential waveforms. There are marked differences in Kv channel expression patterns in humans and mice, and the functional roles of the various K+ currents in action potential repolarization in humans and mice are distinct (Figure 1). In contrast to large mammals, for example, neither IKr nor IKs contributes in action potential repolarization in the adult mouse (Figure 1). In addition, although Ito,f plays a prominent role in action potential repolarization in mouse atrial and ventricular myocytes (Barry et al. 1998, Guo et al. 1999, 2000a and 2000b, and 2003, Xu et al. 1999c), Ito,f primarily contributes to phase 1 repolarization in humans (Figure 1). It is also interesting to note that action potential repolarization remains fast in mice lacking individual and multiple Kv currents, suggesting additional important pathways for repolarization, perhaps indicating Na+ channel inactivation. Consistent with this hypothesis, ventricular action potential durations are prolonged in transgenic mice heterozygous for the voltage-gated Na+ channel (long QT3), DKPQ mutation (Fabritz et al. 2003, Nuyens et al. 2001).
Figure 7. Ventricular arrhythmias are common in MHC-CKO mice in which Cx-43 has been (conditionally) deleted only in the myocardium. Continuous telemetric ECG recordings from 5-(top), 8- (middle), and 7- (bottom) week-old MHC-CKO mice. In each mouse, the recordings show normal sinus rhythm, followed by ventricular tachycardia (arrowheads), which rapidly degenerates to ventricular fibrillation. (Adapted from Gutstein DE, Morley GE, Tamaddon H, et al.: 2001. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ Res 88:333 – 339, with permission. Copyright 2001, Lippincott, Williams & Wilkins.)
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Important insights into the relationships between Kv a subunits and functional Kv channels have been provided through the application of molecular genetic techniques in mice in vivo. Exploiting dominant negative strategies in transgenic animals, for example, has revealed that Kv4 subunits encode Ito,f and that there are two, molecularly distinct components of IK,slow, IK,slow1, and IK,slow2. In addition, the targeted deletion of individual Kv a and h subunits has revealed the essential subunits required for the generation of (mouse ventricular) Ito,f (Kv4.2 and KChIP2), Ito,s (Kv1.4), and IK,slow1 (Kv1.5) channels (Figure 5). Gene-targeting approaches have also revealed roles for Kir2.1 and Kir 6.2 in the generation of cardiac IK1 and IKATP channels, respectively (Figure 5) (Babenko et al. 1998, Flagg and Nichols 2001, Lopatin and Nichols 2001). In contrast, the molecular identity of noninactivating, steady state, outward K + current (Iss) has not been determined. One possibility is that Iss is encoded by two-pore domain (KTP) K+ channel a subunits (Goldstein et al. 2001). Experiments that focus on defining the functional roles of KTP channels, as well as the functional consequences of the targeted deletion of these subunits, are needed to test this hypothesis. Similarly, further studies that focus on exploring the functioning of KChIP2 and the other Kv accessory subunits are needed to define the roles of these subunits, and it seems certain that the mouse will be the model of choice. In several transgenic and gene-targeted mice with altered Kv channel expression, ECG abnormalities are evident (Baker et al. 2000, Barry et al. 1998, Brunner et al. 2001, Demolombe et al. 2001, Guo et al. 2000b, Jeron et al. 2000, Kodirov et al. 2004, Kuo et al. 2001, Li et al. 2004, London et al. 1998a). Prominent among these are QT prolongation, the magnitude of which can usually be correlated with the attenuation of repolarizing K+ currents and prolongation of ventricular action potentials (Barry et al. 1998, Brunner et al. 2001, Demolombe et al. 2001, Guo et al. 2000b, Jeron et al. 2000, Kodirov et al. 2004, Li et al. 2004, London et al. 1998a). Nevertheless, in some cases, the phenotype consequences of manipulating K+ channel subunit expression are model dependent (Figure 5). TCM Vol. 14, No. 3, 2004
Although most mice with altered K+ channel expression do not display increased incidence of arrhythmias and/or increased inducibility, ventricular arrhythmias can be observed in mice and, in some cases, these structurally resemble those seen in humans (Figure 6). Ventricular tachyarrhythmias, for example, can be induced in Kv1.1DN and Kv1.1DN/Kv2.1DN, but not in Kv4.2DN, Kv1.4 / , Kv1.5 / , Kv4.2DN/Kv1.4 / , or Kv4.2DN/Kv1.1DN mice (Brunner et al. 2001, Kodirov et al. 2004, London et al. 2001). The arrhythmia susceptibility in Kv1.1DN mice has been attributed to increased dispersion of repolarization and refractoriness, postulated to reflect the loss of IK,slow1 and regional variations in IK,slow2 (Baker et al. 2000, Brunner et al. 2001, Zhou et al. 2003). Consistent with this interpretation, the expression of Kv4.2DN and the elimination of Ito,f in Kv4.2DN/Kv1.1DN mice reduces dispersion, and is antiarrhythmic (Brunner et al. 2001). In contrast, the increased arrhythmia susceptibility and sudden death in KChIP2 / mice has been attributed to the loss of the transmural gradient in I to,f . This explanation seems unlikely to be correct, however, given that the loss of Ito,f reduces dispersion and is antiarrhythmic (Brunner et al. 2001, Guo et al. 2000b, not proarrhythmic, in the mouse. In addition, it seems likely that this general principle (i.e., that reduced dispersion of repolarization is antiarrhythmic, whereas increased dispersion is proarrhythmic) is also applicable to large mammals, including humans (Antzelevitch and Dumaine 2002, Nerbonne and Guo 2002). There are K+ channel transgenic mice that manifest quite dramatic ECG phenotypes and spontaneous arrhythmias (Figure 6). In the KvLQT1-isoform2expressing transgenics (Demolombe et al. 2001), for example, QT prolongation, P wave abnormalities, and Torsade de Pointes are observed (Figure 7). Ventricular arrhythmias, however, are rare in normal and K+ channel-modified mice and, when observed, are typically not sustained (Figure 5). Nevertheless, arrhythmias can be induced in wild-type mice and susceptibility to induction is strain dependent (Maguire et al. 2003). The relatively low incidence of (spontaneous and/or inducible) arrhythmias— particularly lethal arrhythmias that mimic those in humans—may reflect an
inherent limitation of the mouse, perhaps due to the small size of the heart and the rapid heart rate. This seems unlikely, however, because ventricular arrhythmias and sudden death (Figure 7) are observed in mice with cardiacrestricted deletion of the cardiac gap junction channel protein, Cx-43 (Gutstein et al. 2001a and 2001b). Perhaps conduction defects—alone or in combination with altered repolarization—are more influential in inducing arrhythmias than are changes in repolarizing K+ currents alone. In several of the K+ channel mouse models, electrical remodeling is evident (Figure 5). In Kv4.2DN and in Kv4.2 / animals, for example, Ito,s is selectively upregulated in (right and left) ventricular myocytes that do not normally express this current. Remodeling is also seen in Kv1.1DN- and Kv2.1DN-expressing, and in Kv1.5 / ventricular cells, in which IK,slow1 or IK,slow2 is selectively attenuated (London et al. 2001, Xu et al. 1999a, Zhou et al. 2003). The mouse, therefore, should also be useful as a model system to probe the molecular mechanisms underlying cardiac remodeling. In this context, it would clearly be of interest to control the level—and perhaps the timing—of gene manipulation. It seems reasonable to suggest that the next generation of transgenic and genetargeted animals to be generated for mechanistic studies should be designed with inducible and cardiac-specific promoters to allow endogenous and transgene expression levels to be tightly controlled (Gutstein et al. 2001a and 2001b). This would certainly make the mouse an important experimental tool in studies aimed at detailing the molecular mechanisms involved in controlling the expression and the properties of functional cardiac K+ (and other) channels in the normal, the remodeled, and the diseased myocardium.
References Abbott GW, Goldstein SA: 1998. A superfamily of small potassium channel subunits: form and function of the MinK-related peptides (MiRPs). Q Rev Biophys 31:357 – 398. An WF, Bowlby MR, Betty M, et al.: 2000. Modulation of A-type potassium channels by a family of calcium sensors. Nature 403:553 – 556.
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Antzelevitch C, Dumaine R: 2002. Electrical heterogeneity in the heart: physiological, pharmacological and clinical implications. In Page E, Fozzard HA, Solaro RJ eds. Handbook of Physiology. Vol. 1. New York, Oxford, pp. 654 – 692. Babenko AP, Aguilar-Bryan L, Bryan J: 1998. A view of sur/KIR6.X, KATP channels. Annu Rev Physiol 60:667 – 687. Babij P, Askew GR, Nieuwenhuijsen B, et al.: 1998. Inhibition of cardiac delayed rectifier K+ current by overexpression of the longQT syndrome HERG G628S mutation in transgenic mice. Circ Res 83:668 – 678. Baker LC, London B, Choi BR, et al.: 2000. Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia. Circ Res 86:396 – 407. Barhanin J, Lesage F, Guillemare E, et al.: 1996. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature 384:78 – 80. Barry DM, Xu H, Schuessler RB, Nerbonne JM: 1998. Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit. Circ Res 83:560 – 567. Bolli R, Marban E: 1999. Molecular and cellular mechanisms of myocardial stunning. Physiol Rev 79:609 – 634. Brunner M, Guo W, Mitchell GF, et al.: 2001. Characterization of mice with a combined suppression of I(to) and I(K,slow). Am J Physiol 281:H1201 – H1209. Casimiro MC, Knollmann BC, Ebert SN, et al.: 2001. Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange-Nielsen Syndrome. Proc Natl Acad Sci USA 98:2526 – 2531. Charpentier F, Merot J, Riochet D, et al.: 1998. Adult KCNE1-knockout mice exhibit a mild cardiac cellular phenotype. Biochem Biophys Res Commun 251:806 – 810. Chen YH, Xu SJ, Bendahhou S, et al.: 2003. KCNQ1 gain-of-function mutation in familial atrial fibrillation. Science 299: 251 – 254. Demolombe S, Lande G, Charpentier F, et al.: 2001. Transgenic mice overexpressing human KvLQT1 dominant-negative isoform. Part I: Phenotypic characterisation. Cardiovasc Res 50:314 – 327. Dixon JE, Shi W, Wang HS, et al.: 1996. Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ Res 79: 659 – 668. Drici MD, Arrighi I, Chouabe C, et al.: 1998. Involvement of IsK-associated K+ channel in heart rate control of repolarization in a murine engineered model of Jervell and
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Kuo HC, Cheng CF, Clark RB, et al.: 2001. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to ventricular tachycardia. Cell 107: 801 – 813.
Flagg TP, Nichols CG: 2001. Sarcolemmal K(ATP) channels in the heart: molecular mechanisms brought to light, but physiological consequences still in the dark. J Cardiovasc Electrophysiol 12: 1195 – 1198. Goldstein SA, Bockenhauer D, O’Kelly I, Zilberberg N: 2001. Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci 2:175 – 184. Guo W, Xu H, London B, Nerbonne JM: 1999. Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes. J Physiol (Lond) 521:587 – 599. Guo W, Jung WE, Schwarz TL, Nerbonne JM: 2000a. Ito,f is eliminated in mouse ventricular myocytes isolated from mice lacking Kv4.2: what is the role of Kv4.3? Biophys J 78:451A. Guo W, Li H, London B, Nerbonne JM: 2000b. Functional consequences of elimination of Ito,f and Ito,s: early afterdepolarizations, atrioventricular block, and ventricular arrhythmia in mice lacking Kv1.4 and expressing a dominant-negative Kv4 alpha subunit. Circ Res 87:73 – 79. Guo W, Li H, Aimond F, et al.: 2002. Role of heteromultimers in the generation of myocardial transient outward K+ currents. Circ Res 90:586 – 593. Gutstein DE, Morley GE, Tamaddon H, et al.: 2001a. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ Res 88:333 – 339. Gutstein DE, Morley GE, Vaidya D, et al.: 2001b. Heterogeneous expression of gap junction channels in the heart leads to conduction defects and ventricular dysfunction. Circulation 104:1194 – 1199. Jeron A, Mitchell GF, Zhou J, et al.: 2000. Inducible polymorphic ventricular tachyarrhythmias in a transgenic mouse model with a long Q-T phenotype. Am J Physiol 278:H1891 – H1898. Kanno S, Saffitz JE: 2001. The role of myocardial gap junctions in electrical conduction and arrhythmogenesis. Cardiovasc Pathol 10:169 – 177. Keating MT, Sanguinetti MC: 1999. Molecular and cellular mechanisms of cardiac arrhythmias. Cell 104:569 – 580. Kodirov SA, Brunner M, Nerbonne JM, et al.: 2004. Attenuation of IK,slow1 and IK,slow2 in Kv1/Kv2DN mice prolongs the APD and QT intervals but does not abolish spontaneous
Kupershmidt S, Yang T, Anderson ME, et al.: 1999. Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system. Circ Res 84:146 – 152. Lee MP, Ravenel JD, Hu RJ, et al.: 2000. Targeted disruption of the KvLQT1 gene causes deafness and gastric hyperplasia in mice. J Clin Invest 106:1447 – 1455. Li GR, Feng J, Yue L, et al.: 1996. Evidence for two components of delayed rectifier K+ current in human ventricular myocytes. Circ Res 78:689 – 696. Li H, Guo W, Yamada K, Nerbonne JM: 2004 Selective elimination of the 4-AP-sensitive component of delayed rectification, IK,slow1, in mouse ventricular myocytes expressing a dominant negative Kv1.5 a subunit. Am J Physiol 286:H319 – H328. London B: 2001. Cardiac arrhythmias: from (transgenic) mice to men. J Cardiovasc Electrophysiol 12:1089 – 1092. London B, Jeron A, Zhou J, et al.: 1998a. Long QT and ventricular arrhythmias in transgenic mice expressing the N terminus and first transmembrane segment of a voltagegated potassium channel. Proc Natl Acad Sci 95:2926 – 2931. London B, Wang DW, Hill JA, Bennett PB: 1998b. The transient outward current in mice lacking the potassium channel gene Kv1.4. J Physiol (Lond) 509:171 – 182. London B, Guo W, Pan XH, et al.: 2001. Targeted replacement of Kv1.5 in the mouse leads to loss of the 4-aminopyridine-sensitive component of IK,slow and resistance to drug-induced QT prolongation. Circ Res 88:940 – 946. Lopatin AN, Nichols CG: 2001. Inward rectifiers in the heart: an update on IK1. J Mol Cell Cardiol 33:625 – 638. Maguire CT, Wakimoto H, Patel VV, et al.: 2003. Implications of ventricular arrhythmia vulnerability during murine electrophysiology studies. Physiol Genomics 15: 84 – 91. Nerbonne JM, Guo W: 2002. expression of voltage-gated the heart: roles in normal arrhythmias. J Cardiovasc 13:406 – 409.
Heterogeneous K+ channels in excitation and Electrophysiol
Nerbonne JM, Kass RS: 2003. Physiology and molecular biology of ion channels contributing to ventricular repolarization.
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In Gussak IB, Antzelevitch C eds. Cardiac repolarization. bridging basic and clinical science. Totowa, NJ, Humana Press, pp. 25 – 62. Nerbonne JM, Nichols CG, Schwarz TL, Escande D: 2001. Genetic manipulations of cardiac K+ channel function in mice. What have we learned and where do we go from here? Circ Res 89:944 – 956.
Rosati B, Grau F, Rodriguez S, et al.: 2003. Concordant expression of KChIP2 mRNA, protein and transient outward current throughout the canine ventricle. J Physiol 548:815 – 822.
Wickenden AD, Lee P, Sah R, et al.: 1999. Targeted expression of a dominant negative Kv4.2 K+ channel subunit in mouse heart. Circ Res 85:1067 – 1076.
Sanguinetti MC, Jiang C, Curran ME, Keating MT: 1995. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the I Kr potassium channel. Cell 81:299 – 307.
Xu H, Barry DM, Li H, et al.: 1999a. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ Res 85:623 – 633.
Sanguinetti MC, Curran ME, Zou A, et al.: 1996. Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel. Nature 384:80 – 83.
Xu H, Guo W, Nerbonne JM: 1999b. Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes. J Gen Physiol 113:661 – 678.
Peters NS, Wit AL: 1998. Myocardial architecture and ventricular arrhythmogenesis. Circulation 97:1746 – 1755.
Tomaselli GF, Marban E: 1999. Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc Res 42:270 – 283.
Pongs O, Leicher T, Berger M, et al.: 1999. Functional and molecular aspects of voltage-gated K+ channel beta subunits. Ann NY Acad Sci 868:344 – 355.
Vetter DE, Mann JR, Wangemann P, et al.: 1996. Inner ear defects induced by null mutation of the isk gene. Neuron 17: 1251 – 1264.
Xu H, Li H, Nerbonne JM: 1999c. Elimination of the transient outward current and action potential prolongation in mouse atrial myotcytes expressing a dominant negative Kv4 a subunit. J Physiol (Lond) 519: 11 – 21.
Robbins J: 2000. Remodeling the cardiac sarcomere using transgenesis. Ann Rev Physiol 62:261 – 287.
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Zhou J, Kodirov S, Murata M, et al.: 2003. Regional upregulation of Kv2.1-encoded current, IK,slow2, in Kv1DN mice is abolished by crossbreeding with Kv2DN mice. Am J Physiol 284:H491 – H500.
Wible BA, Yang Q, Kuryshev YA, et al.: 1998. Cloning and expression of a novel K+ channel regulatory protein, KChAP. J Biol Chem 273:11,745 – 11,751.
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TCM Vol. 14, No. 3, 2004
The normal mechanical (pump) functioning of the mammalian heart depends on proper electrical functioning, reflected in the sequential activation of cells in specialized regions of the heart, and the normal propagation of activity through the ventricles. Myocardial electrical activity is evident in the form of action potentials, reflecting the activation (and inactivation) of depolarizing (Na+, Ca2+) and repolarizing (K+)
current channels (Figure 1). There are multiple types of myocardial K+ channels (Figure 1), contributing to regional differences in action potential waveforms and to the generation of normal cardiac rhythms (Antzelovitch and Dumaine 2002, Nerbonne and Kass 2003). The conduction and propagation of activity through the myocardium, in contrast, depend on electrical coupling between cells, mediated by gap junction channels (Kanno and Saffitz 2001). In the diseased myocardium, action potential waveforms and conduction can change markedly, owing to changes in the functional expression of repolarizing K+ (and other) channels (Peters and Witt 1998, Tomaselli and Marban 1999). These changes can lead to desynchronization and arrhythmias, and there is considerable interest in defining the cellular, molecular, and systemic mechanisms contributing to the generation and the maintenance of potentially life-threatening cardiac arrhythmias. Although a variety of experimental (animal) model systems have been exploited in these efforts over the years, the mouse is being used increasingly, due to the ease with which molecular genetic strategies can be applied. The question then arises whether the mouse is an appropriate model system to explore arrhythmia mechanisms. This article considers this question, with a particular focus on mouse models in which the expression of repolarizing K+ channels has been altered.
Myocardial Action Potential Waveforms and Underlying Ionic Currents
There are marked differences in the waveforms of action potentials recorded in different species, as well as in myocardial cells in different regions of the heart in the same species (Antzelevitch and Dumaine 2002, Nerbonne and Kass 2003). Action potential waveforms in
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Figure 1. Schematic of action potentials and underlying ionic currents in adult human (left) and mouse (right) ventricular myocytes. As illustrated, the diversity of outward K+ currents in myocardial cells is greater than that for the inward Na+ and Ca2+ currents, and the various K+ currents play distinct roles in action potential repolarization in human and mouse ventricular cells.
mouse ventricular (Figure 1) and atrial (Figure 2) myocytes, for example, as well as the electrical properties of the intact murine heart (London 2001, Nerbonne et al. 2001), are really quite different from those of larger animals, particularly humans (Figure 2). In addition, the resting heart rate in the adult mouse is 600 to 700 beats per minute, f10 times faster than in the normal adult human (Figure 2). Furthermore, although the action potential upstroke (phase 0)—reflecting inward current through voltage-gated Na+ channels— is similar in human and mouse ventricular myocytes, repolarization is distinct (Figure 1). In humans, the upstroke is followed by a transient repolarization (phase 1) to a plateau phase (phase 2). The driving force for K+ efflux is high during the plateau and, as Ca2+ channels inactivate, outward K+ currents predominate, resulting in a second, rapid phase (phase 3) of repolarization
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back to the resting potential (Figure 1). In mouse ventricular cells, there is no clear plateau phase, and repolarization is rapid (Figure 1). Although there are multiple types of myocardial K+ currents (Antzelevitch and Dumaine 2002, Nerbonne and Kass 2003), the properties of the various K+ currents in different cell types and species are similar, suggesting that the molecular correlates of the underlying K+ channels are also the same. A rather large number of K+ channel pore forming (a) and accessory (h) subunits (Figures 3 and 4) have now been identified. Exploiting transgenic and gene-targeting strategies in mice— considerable progress has been made in defining the relationships between these subunits and functional cardiac K + channels (Kuo et al. 2001, Nerbonne and Kass 2003, Nerbonne et al. 2001). Although these efforts have clearly demonstrated the power of in vivo genetic
approaches in mice for molecular studies, it is of interest to consider whether the mouse is suitable for functional studies focused on exploring the cellular and systemic mechanisms underlying arrhythmia generation and propagation.
Repolarizing K+ Currents in the Mammalian Myocardium
Two types of repolarizing voltage-gated K + (Kv) currents have been distinguished: transient outward K+ currents (Ito) and delayed, outwardly rectifying K+ currents (IK) (Nerbonne and Kass 2003) (Figure 1). These are broad classifications, however, and there are multiple (transient and delayed rectifier) Kv currents expressed in myocardial cells (Nerbonne and Kass 2003). For example, two transient outward K+ currents—Ito, fast (Ito,f) and Ito,slow (Ito,s)—with distinct time- and voltagedependent properties and pharmacologic TCM Vol. 14, No. 3, 2004
the genes encoding Kv channels, as well as changes in Kv channel properties and/or expression patterns, are linked to cardiac arrhythmias (Chen et al. 2003, Keating and Sanguinetti 1999, Tomaselli and Marban 1999). As a result, considerable effort has been directed towards defining the molecular correlates and the functional roles of myocardial K+ channels, as well as in exploring the molecular mechanisms controlling the properties and the expression of these channels.
Figure 2. Comparison of human and mouse ventricular (V) and atrial (A) action potentials and electrocardiogram (ECG). Representative transmembrane action potentials, recorded using intracellular microelectrodes, are plotted above representative surface ECG recordings. Note the waveform and time-scale differences in the two sets of records. (Adapted from Nerbonne JM, Nichols CG, Schwarz TL, Escande D: 2001. Genetic manipulations of cardiac K+ channel function in mice. What have we learned and where do we go from here? Circ Res 89:944 – 956, with permission. Copyright 2001, Lippincott, Williams & Wilkins.)
sensitivities have been distinguished (Nerbonne and Kass 2003). Although Ito,f is expressed in cells isolated from right and left ventricles and from the interventricular septum, Ito,s is only detected in septum cells (Xu et al. 1999b). Multiple components of IK have also been distinguished. In human ventricular myocytes, two prominent delayed rectifiers—IKr and IKs (Figure 1)—have been identified (Li et al. 1996), although neither IKr nor IKs is a prominent repolarizing current in the adult mouse (Xu et al. 1999b). Rather, there are three distinct delayed rectifier currents—I K,slow1 , I K,slow2 and I ss — expressed in mouse ventricular myocytes (Xu et al. 1999b, Zhou et al. 2003). The inwardly rectifying K+ channel currents (Figure 1) can also contribute to myocardial action potential repolarization (Babenko et al. 1998, Flagg and Nichols 2001, Lopatin and Nichols 2001). These (K+) channels are encoded by a subfamily of inward-rectifier K+ (Kir) channel pore-forming a subunits TCM Vol. 14, No. 3, 2004
(Figure 4), producing proteins with two transmembrane domains. The Kir2 a subunits encode the strong inwardly rectifying IK1 channels (Lopatin and Nichols 2001), whereas Kir6.2 underlies the weakly inwardly rectifying I KATP channels (Babenko et al. 1998, Bolli and Marban 1999, Flagg and Nichols 2001). In human and mouse ventricular myocytes, IK1 channels determine resting membrane potentials and I KATP channels are thought to provide a link between cellular metabolism and membrane potential (Babenko et al. 1998, Flagg and Nichols 2001, Lopatin and Nichols 2001, Nerbonne et al. 2001). In large mammals, IK1 also contributes the plateau potential (phase 2) and shapes phase 3 repolarization. The primary determinants of cardiac repolarization, therefore, are the Kv channels, and these channels are also the most diverse with respect to function and expression (Nerbonne and Kass 2003). In addition, mutations in
Molecular Analysis of Myocardial Kv a Subunit Diversity and Functioning
Kv channel pore-forming (a) subunits are six transmembrane-spanning domain proteins that belong to the ‘‘S4’’ superfamily of voltage-gated channels, and functional Kv channels comprise four a subunits (Nerbonne and Kass 2003). Multiple Kv a-subunit subfamilies and a large number of Kv accessory (h) subunits have been identified, and many of these are expressed in the heart (Figure 3). There are also many nonvoltage-gated K+ channel a-subunit subfamilies (Figure 4) with two or four transmembrane domains, and several of these are expressed in heart (Nerbonne and Kass 2003). The multiplicity of Kv a and h subunits (Figure 3), suggests considerable potential for generating functionally diverse cardiac Kv channels (Nerbonne and Kass 2003). Two Kv a-subunit genes—KCNQ1 and KCNH2—have been identified as loci of mutations in familial long QT syndromes 1 and 2, and shown to encode the Kv a subunits important in the generation of cardiac IKr and IKs channels (Barhanin et al. 1996, Sanguinetti et al. 1995 and 1996). Considerable evidence suggests that Kv4 a subunits encode myocardial Ito,f channels. In transgenic mice expressing a pore mutant of Kv4.2 (Kv4.2W362F) that functions as a dominant negative (Kv4.2DN) (Barry et al. 1998, Xu et al. 1999c), Ito,f is eliminated (Figure 5). Although action-potential durations and QT intervals are prolonged (Figure 5), Kv4.2 DN mice do not display spontaneous arrhythmias or increased susceptibility to arrhythmia induction (Barry et al. 1998, Brunner et al. 2001, Guo et al. 2000b). Ventricular Ito,f densities are also 85
Figure 3. Kv a and Kv accessory subunits and voltage-gated cardiac K+ channels. The boxes indicate subunits that are expressed in mammalian heart; gene designations are in italics. In the column labeled ‘‘cardiac current,’’ the channel type encoded by each Kv subunit is provided; ?? indicates current uncertain or unknown.
reduced (but not eliminated) in transgenic mice expressing a truncated Kv4.2 a subunit, Kv4.2N (Figure 5), that also functions as a dominant negative (Wickenden et al. 1999). In contrast to the Kv4.2DN mice, cardiac hypertrophy, chamber dilation, fibrosis, and sudden death are observed in Kv4.2N mice (Wickenden et al. 1999). The pathology evident in Kv4.2N mice cannot reflect the loss of Ito,f, because the elimination of Ito,f in Kv4.2 DN mice (Barry et al. 1998, Brunner et al. 2001, Guo et al. 2000b) and in mice with a targeted deletion in the
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Kv4.2 gene (Kv4.2 / ) (Guo et al. 2000a) has no detectable pathophysiologic consequences (Figure 5). Nevertheless, the disparate experimental observations reported in these studies illustrate one of the potential problems with transgenic approaches, which, in most cases (but see Robbins 2000), involve the ‘‘overexpression’’ of transgenic proteins. The problem is that there may be pathophysiologic effects resulting directly from protein overexpression per se. In addition, the mouse heart may adapt and/or remodel following genetic mani-
pulation. In efforts to probe K+ (and other) channel functioning, therefore, the study of multiple lines of mice in complementary model systems would seem to be a reasonable goal. Biochemical studies have revealed that mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 (Guo et al. 2002). It is somewhat surprising, therefore, that Ito,f is undetectable in mice lacking Kv4.2, even though Kv4.3 expression appears to be unaffected (Guo et al. 2000a). In human ventricular myocytes, however, TCM Vol. 14, No. 3, 2004
Subfamily
Protein
Gene
Cardiac current
Kir1
Subfamily
Protein
Gene
Cardiac current
TWIK-1
KCNK1
??
TWIK-2
KCNK6
??
TWIK-3
KCNK7
TWIK-4
KCNK8
TREK-1
KCNK2
TREK-2
KCNK10
TASK-1
KCNK3
TASK-2 TASK-3 TASK-4 TASK-5
KCNK5 KCNK9 KCNK14 KCNK15
TRAAK-1
KCNK4
THIK-1
KCNK13
THIK-2
KCNK12
TALK-1
KCNK16
TALK-2
KCNK17
TWIK Kir1.1
KCNJ1
??
Kir2 Kir2.1
KCNJ2
IK1
Kir2.2
KCNJ12
IK1
Kir2.3
KCNJ4
??
Kir2.4
KCNJ14
??
Kir3.1
KCNJ3
IKACh
Kir3.2 Kir3.3
KCNJ6 KCNJ7 KCNJ9
Kir3.4
KCNJ5
TREK ??
TASK
Kir3
IKp ??
TRAAK IKACh
Kir4
THIK Kir4.1 Kir4.2
KCNJ10 KCNJ15
Kir5.1
KCNJ16
Kir5 Kir6 Kir6.1
KCNJ8
Kir6.2
KCNJ11
??
TALK ??
IKATP
Figure 4. Kir a and KTP a subunits in the mammalian myocardium. The boxes indicate subunits that are expressed in heart; gene designations are in italics. In the column labeled ‘‘cardiac current,’’ the likely functional correlate of the a subunit is listed; ?? indicates current uncertain or unknown.
functional I t o, f channels are likely Kv4.3 homotetramers, because Kv4.2 is not expressed (Dixon et al. 1996). In addition to revealing that the molecular correlates of mouse and human ventricular Ito,f channels are distinct, these observations suggest that novel mechanisms have evolved to control the cellsurface expression of homotetrameric Kv4.3 channels—likely with associated Kv accessory subunits (Rosati et al. 2001 and 2003)—in humans (and other large mammals). The observations that Ito,s is undetectable (Figure 5) and Ito,f is unaffected in interventricular septum myocytes (Guo et al. 1999) from mice with a targeted deletion in the Kv1.4 gene, Kv1.4 / (London et al. 1998b), clearly demonstrate that the molecular correlates of Ito,s and Ito,f channels are distinct. It seems likely that Kv1.4 also encodes slow transient outward K + currents (Ito,s) in human ventricular myocytes (Nerbonne and Kass 2003). Although TCM Vol. 14, No. 3, 2004
there are no electrocardiographic (ECG) changes (Figure 5) and no pathophysiology is evident in Kv1.4 / mice (Guo et al. 1999), it is certainly possible that alterations in Ito,s expression/properties in human ventricles could impact the normal transmural gradient of repolarization and, therefore, have profound electrophysiologic consequences (Antzelevich and Dumaine 2002, Nerbonne and Guo 2002). Unexpectedly, Ito,s is unregulated in the right and left ventricles of Kv4.2DN mice (Figure 5), reducing the normal, albeit small, dispersion of ventricular repolarization in the mouse (Guo et al. 2000b). The loss of both Ito,f and Ito,s in Kv4.2DN/Kv1.4 / mice (Figure 5) eliminates the regional differences in outward K+ current densities and action potential durations (Guo et al. 2000b). In addition, in spite of the marked QT prolongation in Kv1.4 / /Kv4.2DN mice (Figure 5), ventricular arrhythmias, although occasionally observed, are really quite rare
(Guo et al. 2000b). These observations suggest that reduced dispersion of repolarization in the mouse is antiarrhythmic (Guo et al. 2000b, Nerbonne et al. 2001) and this is likely also the case in larger mammals, including humans (Antzelevich and Dumaine 2002, Nerbonne and Guo 2002). Transgenic and targeted deletion strategies in mice have also been exploited to explore the diversity and the functioning of cardiac delayed rectifier K + currents. The functional consequences of the in vivo manipulation of the subunit genes (including KCNQ1, KCNH2, and KCNE1) encoding cardiac IKr and IKs channels, for example, have been explored (Figure 5). Mice with targeted deletions in KCNE1 (KCNE1 / ) or KCNQ1 (KCNQ1 / ) display shaker/Waltzer behavior, attributed to loss of transepithelial K+ secretion and collapse of the spaces in the inner ear that normally contain K + -rich endolymph (Casimiro et al. 2001, Charpentier
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Figure 5. Genetically engineered mice with altered K+ current expression. Phenotypic consequences of transgenic or gene-targeted alterations in the functioning of myocardial K+ currents in the mouse are noted. APD, action potential duration; ATP, adenosine triphosphate; ND, not determined; Tg, transgenic.
et al. 1998, Drici et al. 1998, Kupershmidt et al. 1999, Lee et al. 2000, Vetter et al. 1996). Although there are, as yet, unexplained phenotypic differences in the consequences of the deletion of the KCNE1 and KCNQ1 genes in different mouse models (Casimiro et al. 2001, Charpentier et al. 1998, Drici et al. 1998, Kupershmidt et al. 1998, Lee et al. 2000, Warth and Barhanin 2002), the cardiac effects are really quite subtle (Figure 5), as might have been expected, given that IKs is not a prominent repolarizing K+ current in the mouse (Figure 1). Transgenic strategies have also been exploited to explore the functional impact of expression of known long QT
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mutations in KCNH2 and KCNQ1. ECG recordings from mice expressing a long QT2 (KCNH2) mutation (Babij et al. 1998), and whole-cell recordings from ventricular myocytes isolated from these animals, are indistinguishable from wildtype animals/cells (Figure 5). In contrast, animals expressing a truncated splice variant of KCNQ1—KvLQT1-isoform 2 (Demolombe et al. 2001)—exhibit sinus bradycardia, QT prolongation (Figure 5), abnormal P wave morphology, and intranodal conduction block. Unexpectedly, electrophysiologic recordings from KvLQT1-isoform2-expressing ventricular myocytes revealed that action potentials are prolonged and that outward (Ito) and inward (IK1) K+ current
densities are reduced (Figure 5). In addition, the severity of the cardiac phenotype was directly correlated with the amount of the mutant protein in different KvLQT1-isoform2-expressing transgenic lines (Demolombe et al. 2001). Taken together, these observations again raise concerns that there are nonspecific in vivo cardiac effects of transgenic protein ‘‘overexpression’’ (Nerbonne et al. 2001, Robbins 2000). Additional studies have focused directly on determining the molecular identity of the delayed rectifier K+ currents expressed in the mouse myocardium and on exploring the functional consequences of the manipulation of these currents in vivo (Brunner et al. 2001, Jeron et al. TCM Vol. 14, No. 3, 2004
Figure 6. Electrocardiographic (ECG) abnormalities seen in genetically engineered mice (left) are displayed alongside corresponding ECG abnormalities seen in humans (right). (LQT) QT prolongation evident in LQT1 and LQT2 patients and in a KCNQ1-isoform2-expressing transgenic (TG) mouse; a wild-type (WT) mouse ECG is also shown (Demolombe et al. 2001). (AVb) High-degree atrioventricular (AV) block in humans and in Kv4.2DN/Kv1.4 / mice (Guo et al. 2000); P waves are indicated by the arrowheads. (TdP) Torsade de Pointes, a potentially lifethreatening arrhythmia in humans is detected in KCNQ1-isoform2-expressing transgenics (Demolombe et al. 2001). (We) Wenckebach AV block in KCNQ1-isoform2-expressing mice (Demolombe et al. 2001); P waves are indicated by the arrowheads. (Adapted from Nerbonne JM, Nichols CG, Schwarz TL, Escande D: 2001. Genetic manipulations of cardiac K+ channel function in mice. What have we learned and where do we go from here? Circ Res 89:944 – 956, with permission. Copyright 2001, Lippincott, Williams & Wilkins.)
2000, Kodirov et al. 2004, Li et al. 2004, London et al. 1998a and 2001, Xu et al. 1999a, Zhou et al. 2003). Although the molecular identity of mouse ventricular I ss remains to be defined, dominant negative and targeted deletion strategies have revealed that there are actually two molecularly distinct components of mouse ventricular IK,slow: IK,slow1, which is encoded by Kv1.5, and IK,slow2, which is encoded by Kv2.1 (Li et al. 2004, London et al. 1998a and 2001, Xu et al. 1999a, Zhou et al. 2003). Interestingly, ventricular arrhythmias are observed in Kv1.1DN animals but not in Kv2.1DN animals, in spite of the fact that IK,slow densities and QT intervals are similarly affected (Figure 5). Also, in contrast to Kv1.1DN mice, there is also no evidence of arrhythmias in mice expressing a pore mutant of Kv1.5 (Kv1.5W461F) that also functions as a dominant negative (Kv1.5DN) (Li et al. 2004), or in mice TCM Vol. 14, No. 3, 2004
with a targeted deletion of the Kv1.5 a subunit (Kv1.5 / ; SWAP) mice (London et al. 2001). The observation of spontaneous arrhythmias and the increased susceptibility to arrhythmia induction only in Kv1.1DN mice appears to reflect increased dispersion (of repolarization) in these animals (Baker et al. 2000, Jeron et al. 2000). The increased dispersion may reflect the fact that IK,slow2 current remodeling is evident, but only in the apex of Kv1.1DN mice (Zhou et al. 2003). Consistent with this interpretation, there are marked differences in the extent of electrical remodeling seen in Kv1.1DN, Kv2.1DN, Kv1.5DN, and Kv1.5 / (SWAP) mice (Figure 5). It has also been reported that the additional elimination of Ito,f suppresses arrhythmias in Kv1.1DN mice in spite of the fact that QT intervals are prolonged more in Kv1.1 DN/Kv4.2DN (and Kv4.2 DN) ani-
mals than in Kv1.1 DN animals (Brunner et al. 2001). These observations reinforce the hypothesis that neither action potential nor QT prolongation, per se (Figure 6), is a good predictor of arrhythmia incidence or susceptibility, at least in mice. Rather, the dispersion of ventricular repolarization appears to be the more important parameter in considering arrhythmia susceptibility. If correct, then it seems reasonable to suggest that expression of Kv4.2DN and the additional loss of I to,f (in Kv1.1DN/ Kv4.2DN mice) reduces the dispersion of repolarization sufficiently to suppress the arrhythmogenic effects of Kv1.1 DN expression (Brunner et al. 2001). Elimination of the Kv2.1-encoded IK,slow2 in Kv1.1 DN-expressing animals, in contrast, does not measurably affect spontaneous arrhythmias or inducibility (Figure 5) relative to mice expressing Kv1.1 DN alone (Kodirov et al. 2004).
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Molecular Analysis of Functional Kv Channel Accessory Subunit Diversity
A variety of cardiac Kv channel accessory subunits—including Kvh, MiRP, KChAP ,and KChIPs (Abbott and Goldstein 1998, An et al. 2000, Pongs et al. 1999, Wible et al. 1998)—have been identified (Figure 3). In contrast to the Kv a subunits, the roles of these (accessory) subunits in the generation of functional cardiac K + channels are not presently very well understood. One potential complication is the growing awareness that these subunits can, at least in heterologous expression studies, interact with several different Kv a, as well as Kv h, subunits (Abbott and Goldstein 1998, Pongs et al. 1999). In addition to interacting with the N termini of Kv4 and Kv2 a subunits, for example, KChAP binds to the C termini of Kv h subunits (Wible et al. 1998). Nevertheless, some progress has been made. Biochemical studies, for example, have shown that KChIP2—originally identified in a yeast two hybrid screen using the N terminus of Kv4x as bait (An et al. 2000)—co-immunoprecipitates with Kv4.2 and Kv4.3 from adult mouse ventricles, consistent with a role for the KChIP2 subunit in the generation of Ito,f channels (Guo et al. 2002). Interestingly, there are marked variations in KChIP2 expression in canine and human ventricles, suggesting that regional differences in KChIP2 expression underlie the observed gradients in Ito,f densities observed in these species (Rosati et al. 2001 and 2003). In mouse, however, there is no gradient in KChIP2 expression, and the differential expression of Kv4.2 underlies the regional differences in Ito,f densities (Guo et al. 2002). Although both human and mouse I to,f channels are encoded by Kv4 a subunits and the KChIP2 accessory subunit, both the molecular compositions of the underlying channels (Kv4.2 and Kv4.3 in mouse versus Kv4.3 in human) and the molecular determinants of the regional differences in the functional cell surface expression of these channels (Kv4.2 in mouse versus KChIP2 in human) are distinct (Dixon et al. 1996, Guo et al. 2002, Rosati et al. 2001 and 2003). In ventricular myocytes isolated from mice with a targeted deletion in the KChIP2 gene (KChIP2 / ), Ito,f is elimi-
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nated (Kuo et al. 2001). In contrast to the observations in Kv4.2DN-expressing animals (Barry et al. 1998, Brunner et al. 2001, Guo et al. 2000b) and Kv4.2 / (Guo et al. 2000a) animals, and in spite of a marked increase in ventricular action potential durations, however, QT prolongation is not evident in KChIP2 / mice. Also, in contrast to the findings in Kv4.2DN-expressing animals and Kv4.2 / animals, KChIP2 / hearts display increased susceptibility to ventricular tachycardia and sudden cardiac death (Kuo et al. 2001). Although the arrhythmia susceptibility in KChIP2 / mice is attributed to the loss of Ito,f, it is not clear why (or how) cardiac rhythms are affected in this model, but not in Kv4.2DN, Kv4.2 / , Kv4.2DN/Kv1.4 / , or Kv4.2DN/Kv1.1DN mice (Figure 5); and the molecular mechanisms underlying these profound phenotypic differences in the different mouse models with altered Ito,f expression remain to be elucidated.
Overview and Summary
Myocardial Kv channels are primary determinants of action potential repolarization, and differences in Kv current
expression are largely responsible for the observed (regional and species) differences in action potential waveforms. There are marked differences in Kv channel expression patterns in humans and mice, and the functional roles of the various K+ currents in action potential repolarization in humans and mice are distinct (Figure 1). In contrast to large mammals, for example, neither IKr nor IKs contributes in action potential repolarization in the adult mouse (Figure 1). In addition, although Ito,f plays a prominent role in action potential repolarization in mouse atrial and ventricular myocytes (Barry et al. 1998, Guo et al. 1999, 2000a and 2000b, and 2003, Xu et al. 1999c), Ito,f primarily contributes to phase 1 repolarization in humans (Figure 1). It is also interesting to note that action potential repolarization remains fast in mice lacking individual and multiple Kv currents, suggesting additional important pathways for repolarization, perhaps indicating Na+ channel inactivation. Consistent with this hypothesis, ventricular action potential durations are prolonged in transgenic mice heterozygous for the voltage-gated Na+ channel (long QT3), DKPQ mutation (Fabritz et al. 2003, Nuyens et al. 2001).
Figure 7. Ventricular arrhythmias are common in MHC-CKO mice in which Cx-43 has been (conditionally) deleted only in the myocardium. Continuous telemetric ECG recordings from 5-(top), 8- (middle), and 7- (bottom) week-old MHC-CKO mice. In each mouse, the recordings show normal sinus rhythm, followed by ventricular tachycardia (arrowheads), which rapidly degenerates to ventricular fibrillation. (Adapted from Gutstein DE, Morley GE, Tamaddon H, et al.: 2001. Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ Res 88:333 – 339, with permission. Copyright 2001, Lippincott, Williams & Wilkins.)
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Important insights into the relationships between Kv a subunits and functional Kv channels have been provided through the application of molecular genetic techniques in mice in vivo. Exploiting dominant negative strategies in transgenic animals, for example, has revealed that Kv4 subunits encode Ito,f and that there are two, molecularly distinct components of IK,slow, IK,slow1, and IK,slow2. In addition, the targeted deletion of individual Kv a and h subunits has revealed the essential subunits required for the generation of (mouse ventricular) Ito,f (Kv4.2 and KChIP2), Ito,s (Kv1.4), and IK,slow1 (Kv1.5) channels (Figure 5). Gene-targeting approaches have also revealed roles for Kir2.1 and Kir 6.2 in the generation of cardiac IK1 and IKATP channels, respectively (Figure 5) (Babenko et al. 1998, Flagg and Nichols 2001, Lopatin and Nichols 2001). In contrast, the molecular identity of noninactivating, steady state, outward K + current (Iss) has not been determined. One possibility is that Iss is encoded by two-pore domain (KTP) K+ channel a subunits (Goldstein et al. 2001). Experiments that focus on defining the functional roles of KTP channels, as well as the functional consequences of the targeted deletion of these subunits, are needed to test this hypothesis. Similarly, further studies that focus on exploring the functioning of KChIP2 and the other Kv accessory subunits are needed to define the roles of these subunits, and it seems certain that the mouse will be the model of choice. In several transgenic and gene-targeted mice with altered Kv channel expression, ECG abnormalities are evident (Baker et al. 2000, Barry et al. 1998, Brunner et al. 2001, Demolombe et al. 2001, Guo et al. 2000b, Jeron et al. 2000, Kodirov et al. 2004, Kuo et al. 2001, Li et al. 2004, London et al. 1998a). Prominent among these are QT prolongation, the magnitude of which can usually be correlated with the attenuation of repolarizing K+ currents and prolongation of ventricular action potentials (Barry et al. 1998, Brunner et al. 2001, Demolombe et al. 2001, Guo et al. 2000b, Jeron et al. 2000, Kodirov et al. 2004, Li et al. 2004, London et al. 1998a). Nevertheless, in some cases, the phenotype consequences of manipulating K+ channel subunit expression are model dependent (Figure 5). TCM Vol. 14, No. 3, 2004
Although most mice with altered K+ channel expression do not display increased incidence of arrhythmias and/or increased inducibility, ventricular arrhythmias can be observed in mice and, in some cases, these structurally resemble those seen in humans (Figure 6). Ventricular tachyarrhythmias, for example, can be induced in Kv1.1DN and Kv1.1DN/Kv2.1DN, but not in Kv4.2DN, Kv1.4 / , Kv1.5 / , Kv4.2DN/Kv1.4 / , or Kv4.2DN/Kv1.1DN mice (Brunner et al. 2001, Kodirov et al. 2004, London et al. 2001). The arrhythmia susceptibility in Kv1.1DN mice has been attributed to increased dispersion of repolarization and refractoriness, postulated to reflect the loss of IK,slow1 and regional variations in IK,slow2 (Baker et al. 2000, Brunner et al. 2001, Zhou et al. 2003). Consistent with this interpretation, the expression of Kv4.2DN and the elimination of Ito,f in Kv4.2DN/Kv1.1DN mice reduces dispersion, and is antiarrhythmic (Brunner et al. 2001). In contrast, the increased arrhythmia susceptibility and sudden death in KChIP2 / mice has been attributed to the loss of the transmural gradient in I to,f . This explanation seems unlikely to be correct, however, given that the loss of Ito,f reduces dispersion and is antiarrhythmic (Brunner et al. 2001, Guo et al. 2000b, not proarrhythmic, in the mouse. In addition, it seems likely that this general principle (i.e., that reduced dispersion of repolarization is antiarrhythmic, whereas increased dispersion is proarrhythmic) is also applicable to large mammals, including humans (Antzelevitch and Dumaine 2002, Nerbonne and Guo 2002). There are K+ channel transgenic mice that manifest quite dramatic ECG phenotypes and spontaneous arrhythmias (Figure 6). In the KvLQT1-isoform2expressing transgenics (Demolombe et al. 2001), for example, QT prolongation, P wave abnormalities, and Torsade de Pointes are observed (Figure 7). Ventricular arrhythmias, however, are rare in normal and K+ channel-modified mice and, when observed, are typically not sustained (Figure 5). Nevertheless, arrhythmias can be induced in wild-type mice and susceptibility to induction is strain dependent (Maguire et al. 2003). The relatively low incidence of (spontaneous and/or inducible) arrhythmias— particularly lethal arrhythmias that mimic those in humans—may reflect an
inherent limitation of the mouse, perhaps due to the small size of the heart and the rapid heart rate. This seems unlikely, however, because ventricular arrhythmias and sudden death (Figure 7) are observed in mice with cardiacrestricted deletion of the cardiac gap junction channel protein, Cx-43 (Gutstein et al. 2001a and 2001b). Perhaps conduction defects—alone or in combination with altered repolarization—are more influential in inducing arrhythmias than are changes in repolarizing K+ currents alone. In several of the K+ channel mouse models, electrical remodeling is evident (Figure 5). In Kv4.2DN and in Kv4.2 / animals, for example, Ito,s is selectively upregulated in (right and left) ventricular myocytes that do not normally express this current. Remodeling is also seen in Kv1.1DN- and Kv2.1DN-expressing, and in Kv1.5 / ventricular cells, in which IK,slow1 or IK,slow2 is selectively attenuated (London et al. 2001, Xu et al. 1999a, Zhou et al. 2003). The mouse, therefore, should also be useful as a model system to probe the molecular mechanisms underlying cardiac remodeling. In this context, it would clearly be of interest to control the level—and perhaps the timing—of gene manipulation. It seems reasonable to suggest that the next generation of transgenic and genetargeted animals to be generated for mechanistic studies should be designed with inducible and cardiac-specific promoters to allow endogenous and transgene expression levels to be tightly controlled (Gutstein et al. 2001a and 2001b). This would certainly make the mouse an important experimental tool in studies aimed at detailing the molecular mechanisms involved in controlling the expression and the properties of functional cardiac K+ (and other) channels in the normal, the remodeled, and the diseased myocardium.
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