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(Circulation Research. 2001;89:944.)
© 2001 American Heart Association, Inc.

Reviews

Genetic Manipulation of Cardiac K⁺ Channel Function in Mice

What Have We Learned, and Where Do We Go From Here?

Jeanne M. Nerbonne, Colin G. Nichols, Thomas L. Schwarz, Denis Escande

From the Departments of Molecular Biology and Pharmacology (J.M.N.) and Cell Biology and Physiology (C.G.N.), Washington University Medical School, St Louis, Mo; the Division of Neuroscience (T.L.S.), Children’s Hospital and Harvard Medical School, Boston, Mass; and INSERM U533 (D.E.), Hôpital Hotel-Dieu, Nantes, France.

Correspondence to Denis Escande, Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, INSERM U533, 1 rue Gaston Veil, Faculté de Médecine, 44035 Nantes, France. E-mail denis.escande{at}nantes.inserm.fr

Abstract

In the mammalian myocardium, potassium (K⁺) channels control resting potentials, action potential waveforms, automaticity, and refractory periods and, in most cardiac cells, multiple types of K⁺ channels that subserve these functions are expressed. Molecular cloning has revealed the presence of a large number of K⁺ channel pore forming () and accessory (ß) subunits in the heart, and considerable progress has been made recently in defining the relationships between expressed K⁺ channel subunits and functional cardiac K⁺ channels. To date, more than 20 mouse models with altered K⁺ channel expression/functioning have been generated using dominant-negative transgenic and targeted gene deletion approaches. In several instances, the genetic manipulation of K⁺ channel subunit expression has revealed the role of specific K⁺ channel subunit subfamilies or individual K⁺ channel subunit genes in the generation of myocardial K⁺ channels. In other cases, however, the phenotypic consequences have been unexpected. This review summarizes what has been learned from the in situ genetic manipulation of cardiac K⁺ channel functioning in the mouse, discusses the limitations of the models developed to date, and explores the likely directions of future research.

Key Words: I_to • I_K • I_K1 • mouse models • cardiac remodeling

Myocardial K⁺ channels control resting membrane potentials, the amplitudes and the duration of action potentials, refractoriness, and automaticity. These channels are also targets for the actions of neurotransmitters, neurohormones, intracellular mediators, and exogenous drugs that modulate cardiac function. In addition, the properties and/or expression of these K⁺ channels are altered with myocardial disease,^1–6 changes that affect the propagation of electrical activity and increase the propensity to develop and sustain arrhythmias. Most cardiac cells express multiple voltage-gated transient outward (I_to) and delayed rectifier (I_K) K⁺ channels, as well as inward rectifier (I_K1) and ligand-gated (I_KATP and I_Kach) K⁺ channels (Figure 1). Electrophysiological studies suggest that these currents are differentially distributed,^6–8 contributing to regional differences in action potential waveforms, transmitter-mediated responses, and the impact of myocardial damage and/or disease.

View larger version (18K): [in this window] [in a new window]   Figure 1. Schematic of action potentials and underlying ionic currents in adult human (left) and mouse (right) ventricular myocytes. Although numerous K+ currents are expressed, there are differences in the contributions of these currents to action potential repolarization in human and mouse ventricular cells.

A large number of K⁺ channel pore forming () (Tables 1 and 2) and accessory (ß) (Table 3) subunit genes have now been identified.^8,9 All K⁺ channel subunits are transmembrane proteins (Figure 2)A that assemble as tetramers or dimers (Figure 2)B to form K⁺ selective pores. Importantly, the presence of multiple (Tables 1 and 2) and ß (Table 3) subunit genes in each subfamily, alternative splicing of transcripts,⁹ and coassembly with accessory subunits (Figure 2)C suggests great potential for generating K⁺ channel diversity. To probe the relationships between K⁺ channel subunit genes and functional cardiac K⁺ channels, in vitro and in vivo experimental approaches have been developed and exploited, and considerable progress has been made recently in defining the molecular correlates of a number of cardiac K⁺ channels.^10–29

View this table: [in this window] [in a new window]   Table 1. Diversity of Voltage-Gated K+ Channel (Kv) Subunits

View this table: [in this window] [in a new window]   Table 3. Auxiliary K+ Channel Subunits

View larger version (49K): [in this window] [in a new window]   Figure 2. Molecular composition of cardiac K+ channels. Kv, Kir, and two-pore subunits (A) are integral membrane proteins with six, two, or four, respectively, membrane spanning domains that assemble as tetramers or dimers to form K+ selective pores (B). Accessory subunits also contribute to the formation of functional cardiac K+ channels (C).

View this table: [in this window] [in a new window]   Table 2. Diversity of Inward Rectifier and Two-Pore K+ Channel Subunits

To manipulate cardiac K⁺ channel expression in vivo, either the -myosin heavy chain (-MHC) promoter has been used to direct the cardiac specific expression^30,31 of mutant K⁺ channel subunits in transgenic mice,^{13,15,22,24,26,32–36} or homologous recombination has been used to disrupt the expression of individual K⁺ channel subunit genes.^{14–18,20,26–28,37–44} The in vivo molecular genetic manipulation of cardiac K⁺ channel expression holds promise for studies focused on exploring the functional roles of specific K⁺ channels because the lack of selective tools to discriminate individual K⁺ channel subunits has impeded detailed analysis of K⁺ channel functioning in situ. The development of in vivo genetic models should also allow direct testing of the predictions of computer models of cardiac action potentials. It is important to note, however, that the waveforms of action potentials in murine myocytes are distinct from those in larger animals, including humans (Figure 1), suggesting species-specific differences in the expression and/or the functional roles of individual K⁺ (and other) currents. There are also marked differences in ECG waveforms and in resting heart rates in mice compared with humans (Figure 3), differences that need to be considered when evaluating the utility of the mouse as a model system.

View larger version (13K): [in this window] [in a new window]   Figure 3. Comparison of human (H) and mouse (M) atrial (A) and ventricular (V) action potentials and ECGs. Transmembrane action potentials, recorded from endocardial myocytes using intracellular microelectrodes, are plotted in relation to the normal surface ECGs. Vertical bars: 50 mV; horizontal bars: 250 ms (human) and 25 ms (mouse).

This review summarizes the progress made in defining the subunits contributing to the formation of the various types of myocardial K⁺ channels, discusses the phenotypic consequences of manipulating K⁺ channel expression in the mouse myocardium in situ, and explores the advantages, disadvantages, and limitations of the various mouse models developed. Finally, the questions that remain, the likely future areas of research, and the technical advances that will be necessary are discussed.

Molecular Dissection of Cardiac Transient Outward K⁺ Currents, I_to,f and I_to,s

Voltage-gated K⁺ channels open in response to membrane depolarization and contribute to determining the amplitudes and the durations of cardiac action potentials. In most cells, transient outward (I_to) and delayed rectifier (I_K) K⁺ currents (Figure 1) have been distinguished. These are broad classifications, however, and at least two transient K⁺ currents, I_to,f and I_to,s, and multiple components of delayed rectification, including I_Kr, I_Ks, and I_Kur, have been described and shown to be differentially expressed.⁸ These observations suggest that distinct molecular entities underlie the various K⁺ currents distinguished electrophysiologically, and considerable evidence has now accumulated to support this hypothesis.⁸

In ventricular myocytes from transgenic mice expressing a pore mutant of Kv4.2 (Kv4.2W362F) that functions as a dominant negative, for example, I_to,f is selectively eliminated¹³ (Table 4). Subsequent studies revealed that I_to,f is eliminated in all left and right ventricular, as well as atrial, Kv4.2W362F-expressing cells.^18,19,22 Action potentials and QT intervals are prolonged in Kv4.2W362F animals (Table 4), ^13,19 demonstrating a prominent role for I_to,f in ventricular repolarization in the mouse. In contrast, I_to,f density is reduced (but not eliminated) in a subset of ventricular myocytes isolated from young (2- to 3-week-old) animals expressing a truncated Kv4.2 subunit, Kv4.2N, that also functions as a dominant negative³³ (Table 4). As the Kv4.2N transgenics age, hypertrophy, chamber dilation, and interstitial fibrosis are evident and, at 10 to 12 weeks, these animals develop congestive heart failure, and the incidence of sudden death is increased.³³ Recordings from myocytes isolated from older Kv4.2N animals revealed marked reductions in I_K1, as well as I_to,f, densities.³³ Although the pathological effects of Kv4.2N expression were attributed to attenuation of I_to,f,³³ this interpretation is inconsistent with the results obtained in the Kv4.2W362F transgenics, which lack I_to,f and are phenotypically normal.^13,19,24 In addition, I_to,f is selectively eliminated in ventricular myocytes isolated from mice with a targeted deletion of Kv4.2 (Kv4.2^-/-) (Table 4) ²⁰. The Kv4.2^-/- mice are indistinguishable from Kv4.2W362F transgenics (Table 4), and there is no evidence of pathology in these animals.²⁰

View this table: [in this window] [in a new window]   Table 4. Summary of Transgenic and Targeted Deletion Mice With Altered K+ Current Expression Circa 2001

Although it was initially reported that deletion of the Kv1.4 subunit (Kv1.4^-/-) has no detectable effects on outward K⁺ currents or action potential waveforms in mouse ventricular myocytes,⁴⁴ subsequent studies revealed that I_to,s is eliminated in myocytes isolated from the septum of Kv1.4^-/- animals¹⁸ (Table 4). A role for Kv1.4 in the generation of rabbit atrial I_to has also been proposed,²¹ and Kv1.4 has been suggested to underlie I_to,s in ferret left ventricular endocardial myocytes.⁴⁵

In myocytes from Kv4.2W362F transgenics, a novel rapidly activating, slowly inactivating current, similar to I_to,s in wild-type septum cells,²² is upregulated.¹³ Interestingly, when Kv4.2W362F was expressed in the Kv1.4^-/- background, both I_to,f and I_to,s were eliminated, revealing that Kv1.4 (I_to,s) is indeed upregulated in the ventricles of Kv4.2W362F trnsgenics.¹⁹ Consistent with the absence of I_to,f and I_to,s, action potentials and QT intervals are prolonged markedly in Kv4.2W362F/Kv1.4^-/- animals (Table 4) ¹⁹. In addition, ventricular arrhythmias were seen in Kv4.2W362F/Kv1.4^-/- mice,¹⁹ suggesting that upregulation of Kv1.4 (I_to,s) in Kv4.2W362F transgenics protects the heart from the arrhythmogenic effects of the loss of I_to,f. Atrioventricular (AV) block is also seen in Kv4.2W362F/Kv1.4^-/- animals (Figure 4), ¹⁹ which may reflect secondary effects due to loss of atrial I_to,f and ventricular I_to,f and I_to,s or, alternatively, direct effects on AV nodal cells. In addition, the finding that AV block is only prominent in the Kv4.2W362F/Kv1.4^-/- animals suggests that electrical remodeling may also occur in the conducting system. In this model, the upregulation of I_to,s in AV nodal cells is protective, and only when both I_to,f and I_to,s are eliminated is conduction measurably affected.

View larger version (30K): [in this window] [in a new window]   Figure 4. ECG anomalies in humans and in genetically engineered mice. LQT: prolonged QT interval in a KCNQ1-isoform 2 transgenic (TG)35 compared with a wild-type (WT) mouse; human recordings are from LQT1 (KCNQ1) and LQT2 (KCNH2) patients. TdP: torsade-de-pointe episode recorded in a KCNQ1-isoform 2 transgenic challenged with erythromycin (courtesy of Dr Alain Corbier, Avantis); human recording is from a LQT2 patient. We: Wenckebach AV block in KCNQ1-isoform 2 mouse35 and in human. AVb: High degree AV block in a Kv4.2W362F/Kv1.4-/- mouse19 and in human. The human recordings are courtesy of Dr Hervé Le Marec (Nantes University Hospital).

Molecular Dissection of Delayed Rectifier K⁺ Currents, I_K,slow, in Mouse

A role for Kv1 subunits in the generation of I_K,slow²³ was revealed in studies on ventricular myocytes isolated from transgenic mice expressing a truncated Kv1.1 subunit, Kv1.1N206Tag, which functions as a dominant negative.¹⁵ Unexpectedly, I_K,slow is also affected in transgenic mice expressing a truncated Kv2.1 subunit, Kv2.1N216 (Table 4) ^22,46. Pharmacological experiments revealed that the TEA sensitivity of I_K,slow is selectively eliminated in Kv2.1N216-expressing cells, whereas the sensitivity of I_K,slow to micromolar 4-AP^15,22,47,48 is unaffected.²⁴ The simplest interpretation of these combined observations is that there are two molecularly distinct components of mouse I_K,slow, one (the µmol/L 4-AP–sensitive) encoded by Kv1 subunits and the other (the TEA-sensitive) by Kv2 subunits.²⁴ Support for this hypothesis was provided with the demonstration that the µmol/L-AP–sensitive component of I_K,slow is eliminated (Table 4) in ventricular myocytes isolated from (SWAP) mice in which the Kv1.5 gene has been replaced by Kv1.1.¹⁶

The attenuation of I_K,slow in the Kv1.1N260Tag and the Kv2.1N216 transgenics is accompanied by action potential and QT prolongation (Table 4). ^15,24 In addition, ECG recordings from Kv1.1N260Tag animals revealed an increase in the frequency of premature ventricular beats and ventricular arrhythmias,^15,26,49,50 whereas spontaneous arrhythmias are not seen in Kv2.1N216 or SWAP mice.^16,22

In Vivo Manipulation of Voltage-Gated LQT K⁺ Channel Genes

Three K⁺ channel subunit genes, KCNQ1, KCNH2, and KCNE1, have been identified as loci of mutations underlying inherited Long QT (LQT) syndromes, LQT1, LQT2 and LQT5,⁵¹ all of which are characterized by QT prolongation and abnormal T wave morphology in surface ECGs (Figure 4). KCNQ1 encodes KvLQT1 (Table 1), which underlies the slow component of delayed rectification, I_Ks, in human heart (Figure 1) whereas KCNH2 encodes ERG1 (Table 1), which underlies the rapid component of human I_K, I_Kr (Figure 1) ⁵¹. KCNE1, in contrast, encodes the accessory subunit, minK (Table 3), that coassembles with KvLQT1.^52,53 Additional minK-related peptides (MiRPs) have been identified (Table 3), and one of these, MiRP1, was suggested to combine with ERG1 to generate cardiac I_Kr channels.⁵⁴ More recent studies, however, suggest that MiRP1 interacts with pore-forming subunits in addition to ERG1.^55–57

In adult mouse heart, KCNQ1 message expression is high,^58,59 whereas KCNE1 message expression is low,⁶⁰ and electrophysiological studies have failed to detect I_Ks in adult ventricular cells.^23,32,34,61 Similarly, KCNH2 protein expression is high in adult mouse ventricles,⁶² although I_Kr density is low.^32,34 Although these observations suggest that I_Kr and I_Ks are not prominent repolarizing currents in the adult mouse heart, the finding of high KCNQ1 and KCNH2 expression raises the interesting possibility that KvLQT1 and/or ERG1 encode K⁺ currents distinct from I_Kr and I_Ks and/or subserve functional roles other than the formation of K⁺ channels in mouse.

Mice with targeted deletions in KCNE1 (KCNE1^-/-) and KCNQ1 (KCNQ1^-/-) have been generated.^37,39–41 In one KCNE1^-/- model, the entire KCNE1 coding sequence was deleted³⁷ whereas in the other, KCNE1 was replaced by LacZ.³⁹ Two KCNQ1^-/- mouse models have also been generated, in which exon 1⁴⁰ or exon 2⁴¹ of KCNQ1 was deleted. The most striking phenotype of the KCNE1^-/- and the KCNQ1^-/- mice is a shaker/Waltzer behavior (Table 4) attributed to a lack of transepithelial K⁺ secretion in the inner ear and the collapse of the spaces normally containing the K⁺-rich endolymph.³⁷ The deafness and inner ear defects in these animals are reminiscent of the sensorineural deficits observed in patients with Jervell and Lange-Nielsen syndrome.⁶³ The KCNQ1^-/- mice also exhibit gastric hyperplasia and hypochloridic gastric fluid,⁴⁰ anomalies not previously noted in LQT patients.

The two KCNE1^-/- mice express distinct cardiac phenotypes (Table 4). In the mice generated by Vetter and colleagues,³⁷ for example, the baseline ECG is normal.⁵⁸ Under bradycardic conditions, however, QT intervals are longer in KCNE1^-/- than in wild-type mice, whereas at fast heart rates, QT intervals are shorter.⁵⁸ These mice, therefore, display an accentuated QT adaptation to heart rate, as is observed in LQT1 patients.⁶⁴ Paradoxically, endocardial action potentials show a normal adaptation to rate.⁶⁵ In the LacZ-KCNE1^-/- mice,³⁹ in contrast, both the baseline ECG and the QT adaptation to rate are normal (Table 4). Phenotypic differences were also seen in the KCNQ1^-/- animals.^40,41 In the exon1-KCNQ1^-/- mice,⁴⁰ for example, heart rate, heart rate variability, ventricular repolarization, and AV conduction are normal. In contrast, prolonged QT intervals, abnormal P and T wave morphologies, and delayed AV conduction are seen in exon 2-KCNQ1^-/- animals.⁴¹ Unexpectedly, QT intervals, as well as endocardial and epicardial monophasic action potentials recorded in isolated, perfused exon 2-KCNQ1^-/- hearts are indistinguishable from those in wild-type hearts.⁴¹

Transgenic mice expressing a splice variant of KCNQ1 (isoform 2), which is expressed in the human heart and functions as a dominant negative,⁶⁶ have also been described.³⁵ The phenotype of the KCNQ1-isoform 2 mice is quite dramatic including sinus bradycardia, QT prolongation (Figure 4), abnormal P wave morphology, and intranodal conduction block (Table 4).³⁵ Recordings from KCNQ1-isoform 2–expressing ventricular myocytes revealed that action potentials are prolonged, and that outward (I_to) and inward (I_K1) K⁺ current densities are reduced.³⁵ Interestingly, the severity of the cardiac phenotype observed in the KCNQ1-isoform 2 transgenics, as well as the magnitude of the reductions in current densities, is directly correlated with the amount of the transgenic protein produced, ie, the higher the protein expression, the more severe the phenotype.³⁵ The line with the lowest expression is phenotypically similar to the exon 2-KCNQ1^-/- mice.⁴¹ In vitro studies revealed that KCNQ1-isoform 2 functions as a subfamily specific dominant negative.⁶⁶ Nevertheless, it is possible that there are nonspecific effects of overexpression of the truncated protein in vivo. Although the marked reductions in I_to and I_K1 are reminiscent of changes seen in cardiac hypertrophy and failure,³ there is no histological or functional evidence of cardiac hypertrophy or compromised cardiac functioning evident in KCNQ1-isoform 2 mice.³⁵

Transgenic mice expressing a dominant-negative long QT KCNH2 mutation have also been described.³² ECG recordings from these animals were indistinguishable from wild-type control animals, as were the properties of ventricular action potentials (Table 4). Although these observations are consistent with the suggestion that I_Kr is not an important repolarizing K⁺ current in adult mouse heart,⁶¹ QT intervals are prolonged in heterozygous ERG1^+/- animals.³⁸ Homozygous ERG1^-/- mice, however, die early in fetal development (Table 4), precluding determination of the electrophysiological consequences of eliminating I_Kr. Selective deletion of the N terminal splice variant of ERG1, ERG1B, in contrast, is not lethal, although ERG1B^-/- animals are bradycardic with QT prolongation (Table 4).⁶⁷

Molecular Dissection of Cardiac Inward Rectifiers, I_K1 and I_KACh

In contrast to the voltage-gated K⁺ channels, the conductance of inward rectifying K⁺ channels is highest near the equilibrium potential for K⁺, suggesting that these channels function primarily during diastole (Figure 1) to reduce automaticity. The inward rectifiers are generated by a large and diverse subfamily of K⁺ channel (Kir) subunit genes (Table 2) encoding proteins with two transmembrane domains (Figure 2)) that assemble as tetramers to form K⁺ selective pores (Figure 2)B).

Based on the properties of the currents produced in heterologous expression systems, Kir2 subunits have been suggested to encode cardiac I_K1 channels and, recently, mice with a targeted deletion of the coding region of Kir2.1 (Kir2.1^-/-) were described.⁴² These mice have a cleft palate and die shortly after birth, probably due to respiratory complications and the inability to suckle. Although the premature death of the Kir2.1^-/- animals has precluded characterization of adult animals, recordings from isolated newborn Kir2.1^-/- ventricular myocytes revealed that I_K1 is absent (Table 4).²⁸ Interestingly, a small, slowly activating inward rectifier current, distinct from I_K1, is evident in Kir2.1^-/- myocytes, although it is unclear if this current is expressed in wild-type cells.²⁸ Newborn Kir2.1^-/- ventricular myocytes fire action potentials spontaneously and contract to a greater extent than wild-type cells. In the intact animal, however, the expected functional consequences of this cellular phenotype are not evident. Indeed, despite the loss of I_K1, the only abnormality seen in ECG recordings from neonatal Kir2.1^-/- animals is pronounced bradycardia (Table 4).

The phenotype of Kir2.1^-/- mice provides an interesting comparison to individuals with Andersen’s syndrome, recently shown to arise from loss of function mutations in Kir2.1 that reduce I_K1.⁶⁸ Individuals bearing Andersen’s syndrome mutations can display QT prolongation, skeletal and craniofacial abnormalities, and periodic paralysis, alone or in combination.^68,69 Similar to other LQT syndromes, some carriers display little or no phenotype.⁶⁸ These effects are likely correlates of the slowing of ventricular repolarization and the cleft palate seen in the Kir2.1^-/- mice. As in the mouse, however, the most striking feature of Andersen’s syndrome may be the mildness of the cardiac phenotype in spite of the fact that the Kir 2.1 mutations likely suppress I_K1 markedly.

Other members of the Kir2 subfamily are also expressed in the heart⁷⁰ and, interestingly, deletion of the open reading frame of Kir2.2 results in a quantitative reduction in I_K1.²⁸ Although this finding suggests that Kir2.2 also contributes to I_K1, the absence of I_K1 in Kir2.1^-/- cells suggests that Kir2.2 does not give rise to functional channels in the absence of Kir2.1.²⁸ Biochemical studies aimed at determining if Kir2.x subunits⁷⁰ coassemble in the (mouse) heart will clearly be of interest.

Based on studies in heterologous systems, the ACh-regulated current, I_KACh, was predicted to arise from heteromeric assembly of Kir3.1 and Kir3.4.⁷¹ Channels encoded by these subunits are modulated by activation of muscarinic and purinergic receptors through the action of trimeric G-proteins and the binding of G-protein ß subunits.^72–74 In mice with a targeted deletion of the Kir3.4 gene (Kir3.4^-/-), the essential role of this subunit in the generation of atrial I_KACh was confirmed.¹⁷ Although the Kir3.4^-/- animals are overtly normal, recordings from Kir3.4^-/- atrial myocytes revealed the absence of the GTPS-activated (I_KACh) channels (Table 4) that are prominent in wild-type atrial cells. Interestingly, the Kir3.1 protein is made in Kir3.4^-/- cells, although it is not trafficked to the cell surface or appropriately glycosylated in the absence of Kir3.4.⁷⁵

Resting heart rates in Kir3.4^-/- and wild-type animals are not significantly different,¹⁷ suggesting that steady-state activation of I_KACh channels is not required for establishing resting heart rate in the mouse. Heart rate variability in response to stimulation of the vagus or A1 receptors, however, is reduced in Kir3.4^-/- hearts (Table 4).¹⁷ Approximately half of the bradycardic response to vagal stimulation is suppressed in Kir3.4^-/- animals, and the remaining response appears with a longer latency, revealing a role for I_KACh in the early stage of vagal modulation.¹⁷ In addition, atrial fibrillation does not arise in Kir3.4^-/- animals challenged with carbachol,⁷⁶ confirming that I_KACh activation is a critical step in the cholinergic induction of atrial fibrillation.

Molecular Analysis and Manipulation of Cardiac I_KATP Channels

ATP inhibited (I_KATP) channels⁷⁷ are expressed in many cells and have been implicated in the regulation of insulin secretion in pancreatic ß-cells⁷⁸ and in the vasodilation observed in response to K⁺ channel openers.⁷⁹ In the heart, I_KATP channels are thought to be involved in in myocardial ischemia and preconditioning.⁸⁰ I_KATP channels are essentially voltage-independent and, when open, function to stabilize resting potentials and shorten action potentials (Figure 1).⁸¹ In heterologous systems, I_KATP channels are reconstituted by coexpression of Kir6.x subunits with ATP-binding cassette proteins that encode sulfonylurea receptors, SURx.⁸² Coexpression of SUR1 and Kir6.2 generates channels similar to pancreatic ß-cell I_KATP channels, and a clear genetic link was established by the demonstration that loss of function SUR1 and Kir6.2 mutations abolish I_KATP in ß-cells and cause persistent hypoglycemic hyperinsulinemia of infancy,^83,84 whereas gain of function mutations in Kir6.2 cause hypoinsulinemic diabetes.⁸⁵

Although mRNA expression and pharmacological data suggested that cardiac sarcolemmal I_KATP channels are likely encoded by Kir6.2 and SUR2A, Kir6.1 is also expressed in heart,⁸⁶and antisense oligodeoxynucleotides against SUR1 reduce I_KATP in ventricular myocytes.²⁵ An essential role for Kir6.2 was demonstrated with the finding that cardiac I_KATP channel activity is absent in Kir6.2^-/- animals.^43,88 Although no cardiac effects of deleting SUR1⁸⁷ have been reported, I_KATP channel density is reduced (Table 4) in SUR2^-/- animals and the properties of the residual channels are similar to those seen on coexpression of Kir6.2 and SUR1.²⁷ It is possible that the targeting construct used²⁷ permits expression of (truncated) SUR2 and that this underlies the aberrant channel activity in SUR2^-/- cells. Alternatively, because Kir6.2 subunits can traffic alone to the cell surface, albeit inefficiently,⁸⁹ the residual I_KATP channel activity in SUR2^-/- cells might reflect homomeric Kir6.2 channels.²⁵

Although the S-T segment of the ECG is poorly resolved in mouse (Figure 3), there is a region following the QRS complex that is typically triangular with respect to the baseline. During ischemia, this segment can show elevation reminiscent of the ST elevation seen in larger animals and presumed to result from dispersion of ventricular repolarization. In Kir6.2^-/- animals, ST elevation is absent,⁸⁸ supporting the hypothesis that it results from variable I_KATP activation and consequent heterogeneities in ventricular repolarization. Although action potentials in Kir6.2^-/- myocytes appear normal,⁴³ action potential shortening during ischemia or metabolic blockade is abolished (Table 4). These observations are consistent with previous voltage-clamp studies,⁹⁰ demonstrating that twitch shortening is maintained in metabolic blockade longer than when I_KATP channels are activated. Action potential shortening and the consequent protection from Ca²⁺ loading have been proposed to underlie a cardioprotective effect of I_KATP channels.⁸⁰ Consistent with this hypothesis, recovery of Langendorff-perfused hearts from 20 minutes of global ischemia is reduced from 50% to 10% in Kir6.2^-/- animals (H. Nakaya, MD, oral communication, June 2001).

Although inhibited under normal physiological conditions, I_KATP channels are expressed at much higher density than other sarcolemmal K⁺ channels.^80,91,92 Given the high channel density, action potentials are expected to shorten by 50% if I_KATP channels are activated to only 1% of the maximal conductance.^92–96 Surprisingly, however, action potential durations are largely unaffected in transgenic animals expressing mutant I_KATP channels with reduced ATP sensitivity.³⁶ In spite of a 40-fold reduction in ATP sensitivity, the mutant channels remain inactive, suggesting that additional inhibitory mechanisms may regulate cardiac I_KATP channel activity in vivo.

The availability of mice with altered cardiac I_KATP channel function has also enabled in situ studies focused on exploring the roles of these channels in ischemic preconditioning, in which a brief period of ischemia protects the heart from subsequent prolonged ischemia. Pharmacological studies suggest a role for I_KATP channels,^97–99 although the consensus has been that preconditioning results from activation of mitochondrial,¹⁰⁰ not sarcolemmal, I_KATP channels.^101–104 In Langendorff perfused Kir 6.2^-/- hearts, however, the protective effect of preconditioning is abolished in spite of the fact that the infarction resulting from (30 minutes) global ischemia is similar in wild-type and Kir6.2^-/- hearts (H. Nakaya, MD, oral communication, June 2001). Although it is possible that Ca²⁺ loading during ischemic intervals overcomes preconditioning in Kir6.2^-/- hearts, these results are consistent with direct involvement of sarcolemmal I_KATP channels in preconditioning.

Summary, Conclusions, and Future Directions

Relation Between K⁺ Channel Subunit Genes and Functional Cardiac K⁺ Channels
The application of transgenic and targeted deletion strategies in mouse has led to the identification of the subunits contributing to the formation of most of the K⁺ channels expressed in cardiac cells (Tables 1 and 2). Exploiting dominant negative strategies in transgenic animals, for example, has revealed that distinct Kv subfamilies encode I_to,f (Kv4)¹³ and I_K,slow (Kv1 and Kv2)^15,22 and has led to the identification of two molecularly-distinct (ie, Kv1- and Kv2-encoded) components of I_K,slow^15,22 that could not be distinguished unequivocally using conventional electrophysiological approaches.²³ The targeted deletion of individual subunits, however, has revealed the essential subunits required for I_to,f (Kv4.2),²⁰ I_to,s (Kv1.4),¹⁸ the µmol/L 4-AP–sensitive I_K,slow (Kv1.5),¹⁶ I_K1 (Kir2.1),²⁸ I_KACh (Kir3.4),¹⁷ and I_KATP (Kir6.2)^14,43 (Table 4). The manipulation of K⁺ channel subunit expression in mice, therefore, has identified the molecular correlates of most cardiac K⁺ channels (Tables 1 and 2), in spite of the marked differences in heart rates, action potential, and ECG waveforms in mice and larger mammals (Figures 1 and 3) that limit the usefulness of the mouse as an electrophysiological model of (human) cardiac functioning.

The in vivo molecular genetic analysis of cardiac K⁺ channels has also provided insights into the functional roles of these channels in (mouse) myocardium (Table 4). The results obtained with the Kv4.2W362F and Kv4.2^-/- animals, for example, reveal a prominent role for I_to,f in action potential repolarization in mouse atrial and ventricular myocytes.^13,18,19,24 This contrasts markedly with large mammals, in which I_to,f contributes primarily to early, phase 1 repolarization (Figure 1). Interestingly, the extent of action potential and QT prolongation in animals lacking I_to,f^13,19,20 is greater than when either component of I_K,slow is eliminated.^15,22 Nevertheless, action potential repolarization remains fast in animals lacking I_to,f,^13,19,20 as well as in animals in which both I_to,f and I_to,s are eliminated.¹⁹ These observations implicate an important role for the noninactivating K⁺ current, I_ss, in mouse. Clearly, it will be of interest to determine the molecular identity of I_ss and to explore the functional consequences of manipulating I_ss in vivo.

The lack of pronounced electrophysiological effects of deletion of either KCNE1^37,39 or KCNQ1^40,41 is consistent with previous suggestions^24,61 that, in contrast to large mammals, I_Ks does not play a major role in repolarization in the adult mouse (Figure 1). In contrast, there are marked phenotypic consequences of the cardiac-specific expression of a truncated KCNQ1 (isoform 2) transgene,³⁵ including QT prolongation and sinus bradycardia (Table 4). Unexpectedly, recordings from KCNQ1-isoform 2–expressing ventricular cells revealed marked reductions in I_to,f and I_K1.³⁵ Although it is unclear why these currents are affected, the attenuation of I_to,f (and I_K1?) likely underlies the QT prolongation observed in KCNQ1-isoform 2–expressing mice.³⁵ Interestingly, the attenuation of I_Kr in KCNH2G628S-expressing ventricular cells has no measurable effects on QT intervals, revealing that, in contrast to large animals, I_Kr is also not a prominent repolarizing K⁺ current in the adult mouse³² (Figure 1).

The observations that I_to,s is unaffected in septum cells from SWAP mice (lacking Kv1.5)¹⁶ and that I_K,slow is unaffected in Kv1.4^-/- septum cells¹⁸ reveal that Kv1.4 and Kv1.5 encode distinct populations of voltage-gated cardiac K⁺ channels, I_to,s and I_K,slow (I_Kur) (Table 1), and that the Kv1.4 and Kv1.5 proteins do not coassemble in the (mouse) myocardium.¹⁶ In contrast, recent biochemical studies reveal that Kv4.2 and Kv4.3 are associated in vivo (W. Guo, MD, PhD, and J.M. Nerbonne, PhD, oral communication, August 2001) and that functional mouse ventricular I_to,f channels are heteromeric.³⁴ In contrast, Kv4.2 homomultimers reportedly encode I_to,f channels in adult rat atrial myocytes.¹⁰⁵ Although it seems reasonable to speculate that Kv4 subunits also underlie I_to,f in other animals, Kv4.2 appears not to be expressed in non-rodent species,¹⁰⁶ suggesting that functional I_to,f channels reflect the homomeric assembly of Kv4.3 subunits. Consistent with this hypothesis, I_to,f densities are reduced in human atrial myocytes exposed to antisense oligodeoxynucleotides targeted against Kv4.3.²¹ Interestingly, it has also been suggested that coassembly of Kv4.3 with the Kv channel accessory subunit, KChiP2, contributes the generation of functional I_to,f channels and underlies the regional heterogeneity in I_to,f expression in canine and human heart.¹⁰⁷ Very little is presently known, however, about the roles of KChiP2 and/or of accessory Kv ß subunits (Table 3) in the generation and/or the functioning of myocardial K⁺ channels. Exploring the roles of these proteins, as well as of the family of two pore K⁺ channel subunits (Table 2), will likely be important areas of future research, and it seems certain that the mouse will also be the model of choice for these studies.

Electrophysiological Consequences of Altered K⁺ Channel Expression In Vivo
In several of the K⁺ channel mouse models (Table 4), QT prolongation is evident,^{13,15,19,22,26,35,41,67} consistent with the attenuation of repolarizing K⁺ currents. In other models, however, QT prolongation is not seen,^18,32,42 in spite of measured changes in K⁺ current densities and action potential durations. Although the reasons for these apparent discrepancies have not been determined, some of the differences likely reflect the experimental conditions. Voltage- and current-clamp recordings, for example, are often obtained at room (rather than at physiological) temperature and at low stimulation frequencies (typically 1 Hz), ie, experimental conditions that are quite distinct from those in the mouse heart in situ. Consistent with this hypothesis, marked differences in K⁺ current and action potential waveforms are evident at physiological temperatures and stimulation frequencies.^16,19,48 For direct comparison of in vivo and in vitro data, therefore, it is imperative that similar experimental conditions be used.

Rather dramatic cardiac phenotypes have been seen in some of the K⁺ channel mouse models (Table 4). Spontaneous ventricular arrhythmias, for example, are recorded in Kv1.1N206-expressing transgenics,^15,26 which lack the µmol/L 4-AP–sensitive component of I_K,slow, although the reductions in outward K⁺ currents and the action potential prolongation in these animals are less than in Kv4.2W362F mice.^13,19,26 These observations suggest that factors in addition to prolonged repolarization play important roles in determining the propensity to develop and to sustain arrhythmias. Consistent with this hypothesis, spontaneous ventricular arrhythmias are not evident in mice expressing both the Kv4.2W362F and Kv1.1N216 dominant negative constructs, although ventricular action potential durations and QT intervals are prolonged more in these (crossed) animals than in animals expressing either dominant negative construct alone (Table 4).²⁶ The simplest interpretation of these findings is that the spontaneous ventricular arrhythmias seen in the Kv1.1N206 mice reflect more than the increased action potential durations resulting from attenuation of I_K,slow.

Interestingly, sinus bradycardia is also evident in telemetric ECG recordings from Kv2.1^-/-28 and KCNQ1-isoform 2–expressing³⁵ animals. In addition, AV block is observed in Kv4.2W362F/Kv1.4^-/- animals (Figure 4), which lack I_to,f and I_to,s.²⁴ Taken together, these results suggest that there are differences in the electrical properties of cells in the conducting system of the mouse myocardium. Consistent with this hypothesis, it was recently reported that action potential waveforms in mouse His Purkinje fibers are markedly different than in ventricular cells.¹⁰⁸ It will clearly be of considerable interest to explore the functional consequences of altered K⁺ channel expression in Purkinje and other cells of the conducting system in genetically engineered animals.

Cardiac Remodeling in Mice With Altered K⁺ Channel Subunit Expression
In several of the mouse models described, remodeling of cardiac K⁺ currents is evident (Table 4). In Kv4.2W362F and Kv4.2^-/- animals, for example, I_to,s is upregulated in (right and left) ventricular myocytes that do not normally express this current.^18,20 In septum cells, which normally express I_to,s, in contrast, I_to,s density is unaffected by the elimination of I_to,f.¹⁸ In addition, the upregulation of I_to,s is specific; the densities of I_K,slow and I_ss in Kv4.2W362F and Kv4.2^-/- cells are indistinguishable from those in wild-type cells.^18–20 In addition, no changes in I_K,slow and/or I_ss densities are evident in Kv4.2W362FxKv1.4^-/- cells, which lack both I_to,f and I_to,s.¹⁹ Remodeling is also seen in Kv2.1N206 and SWAP ventricular cells, in which one component of I_K,slow is selectively attenuated.^16,24 In both the Kv2.1N206 and SWAP cells, the density of the component of I_K,slow remaining is increased, whereas I_to,f and I_ss are unaffected.^16,24

Although the results obtained in the Kv4.2W362F,^13,19 Kv4.2^-/-,20 SWAP,¹⁶ and Kv2.1N206²⁴ suggest rather remarkable specificity in K⁺ current remodeling, there are other cases in which multiple current systems appear to be affected by the (over)expression of dominant negative K⁺ channel subunits.^33,35 In Kv4.2N and in KCNQ1-isoform 2 ventricular cells, for example, I_to,f and I_K1 are attenuated.^33,35 It is presently unclear, however, whether these changes reflect cardiac remodeling similar to that seen in the Kv4.2W362F,^13,19 Kv4.2^-/-,20 SWAP,¹⁶ and Kv2.1N206²⁴ animals or if the unexpected effects on I_to,f and I_K1 simply reflect protein overexpression. Clearly, it would be of interest to be able to control the level, as well perhaps as the timing, of transgene expression. Given the progress made in developing inducible promoters,^109–111 it seems likely that these will be exploited increasingly, and that this will facilitate studies focused on exploring the molecular mechanisms underlying cardiac remodeling in some detail.

Pathophysiological Consequences of Altered K⁺ Channel Functioning
In contrast to the marked electrophysiological consequences of the expression of mutant K⁺ channel subunits or the deletion or specific K⁺ channel subunit genes (Table 4), in most cases, there appear not to be any detectable cardiac (or other) effects.^{13,15,16,18,19,23,24,26,28,35,37,39–41,44} Heart weights and body weights, for example, appear to be well within normal ranges and, like the animals themselves, the hearts of Kv1.1N206Tag-, Kv4.2W362F-, Kv2.1N216-, Kv4.2W362F-xKv1.1N206Tag-, KCNQ1-isoform 2–, or Kv4.2W362FxKv1.4^-/-–expressing transgenics are normal.^{13,15,19,23,24,26,35} Similarly, no pathology is evident in the hearts of adult mice with targeted deletions in Kv1.4,¹⁵ Kv4.2,²⁰ Kv1.5,¹⁶ KCNE,^37,39 KCNQ1,^40,41 Kir 3.4,¹⁷ Kir6.2,^14,43 or Kir2.2.²⁸ In addition, although the deletion of ERG1³⁸ or Kir2.1⁴² has profound functional consequences, the observed lethality does not reflect compromised cardiac function. Taken together, therefore, the results obtained with the vast majority of the models reveal that it is possible to markedly alter K⁺ channel expression, action potential waveforms, and ECG patterns without measurably affecting the overall physiology, behavior, or survival of the animal. In other systems, age-dependent, as well as gender-dependent, effects of transgene expression have been documented.^112,113 Clearly, experiments aimed at examining the impact of gender and aging on the phenotypic consequences of manipulating cardiac K⁺ channel expression are warranted.

In contrast to most of the transgenic and targeted deletion animals generated to date, the expression of the truncation mutant, Kv4.2N, has profound pathophysiological consequences (Table 4), including congestive heart failure and sudden death.³³ The phenotype of these animals, therefore, is quite different from both the Kv4.2W362F transgenics and the Kv4.2^-/- animals (Table 4), in which I_to,f is eliminated and action potential durations and QT intervals are prolonged.¹³ Although the underlying molecular mechanisms have not been determined, the pathology evident in the Kv4.2N animals cannot simply result from the attenuation of I_to,f, as originally suggested.³³ Rather, it seems more likely that these effects reflect the deleterious effects of the (over)expression of the truncated Kv4.2N protein and/or the transgene insertion site. In this context, it is of interest to note that it has also been reported that GFP expression (alone) can result in dilated cardiac myopathy.¹¹⁴ In contrast, no pathology is seen in transgenic mice expressing a GFP-tagged Kv1.5 subunit.¹¹⁵ In addition, in the GFP-expressing transgenics, the cardiomyopathy was seen in the 2 (of 4 generated) lines with the highest protein (GFP) expression.¹¹⁴ Taken together, these results suggest that the transgene expression level could play an important role in mediating the unexpected pathology seen in the Kv4.2N animals.³³

Limitations of Existing Mouse Models and Future Directions
In some cases, phenotypic differences are evident in the K⁺ channel mouse models, in spite of the fact that the same subunit was targeted (Table 4). It is presently unclear, however, whether some or all of these differences reflect the nature of the genetic manipulation (ie, expression of dominant-negative transgenes versus targeted deletion or replacement), strain-dependent (ie, C57BL6, FVB or 129Sv) effects, complex and variable cardiac remodeling, and/or secondary effects due to the elimination of K⁺ channels in the heart or in the other tissues. Given that the -MHC promoter is quite robust,^30,31 some of the differences between transgenic models or between transgenic and targeted deletion animals seem likely to reflect transgene overexpression. Clear support for this hypothesis is provided with the KCNQ1-isoform 2–expressing transgenics,³⁵ in which the severity of the cardiac phenotype was correlated with the amount of the mutant protein produced. It seems reasonable to suggest that in the future, transgenic animals should be generated using inducible (and cardiac specific) promoters to allow transgene expression levels to be tightly controlled.^109,110

There are also limitations with the use of targeted deletion strategies, as is evident in the results obtained with the ERG1^-/- and the Kir2.1^-/- mice.^38,42 These deletions are lethal either during fetal (ERG1^-/-) or early during postnatal (Kir2.1^-/-) development (Table 4) precluding functional studies of the roles of the channels encoded by these subunits in the adult (or developing) heart. These observations suggest that alternative experimental strategies, such as the use of methods to permit the cardiac-specific deletion of these genes,^111,116 will be necessary to explore directly the functional roles of I_K1 and I_Kr, as well as of other cardiac K⁺ channels.

The combined application of inducible promoters and cardiac-specific gene ablation holds particular promise for functional studies, assuming that spatial and temporal control of gene expression is realized. These advanced methods will also enable future studies aimed at exploring the molecular mechanisms underlying cardiac remodeling. Although there are numerous possible (transcriptional, translational, and posttranslational) regulatory steps that could be involved in controlling the expression and the properties of functional cardiac K⁺ channels, little is known about the mechanisms that mediate changes in channel expression during remodeling, as well as in conjunction with myocardial damage or disease. Mice provide an excellent experimental system for exploring the underlying molecular mechanisms in detail.

Acknowledgments

Research in the authors’ laboratories has been supported by the National Institutes of Health (J.M.N., C.G., and T.L.S.), INSERM (D.E.), and by GIP "fonds de recherche" Hoechst Marion Roussel (D.E.). The authors are grateful to Peter Backx, Jacques Barhanin, Sophie Demolombe, Milou Drici, Steven Ebert, Sabina Kupershmidt, Barry London, Matteo Mangoni, Haruaki Nakaya, Guy Salama, and Dirk Snyders for stimulating discussions.

Received September 7, 2001; revision received October 8, 2001; accepted October 8, 2001.

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