Electrophysiological phenotyping in genetically engineered mice1
Charles I. Berul
Department of Cardiology, Childrens Hospital-Boston, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT
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Advances in transgene and gene targeting technology have enabled sophisticated manipulation of the mouse genome, providing important insights into the molecular mechanisms underlying cardiac conduction, arrhythmogenesis, and sudden cardiac death. The mouse is currently the principal mammalian model for studying biological processes, particularly related to cardiac pathophysiology. Murine models have been engineered harboring gene mutations leading to inherited structural and electrical disorders of the heart due to transcription factor mutations, connexin protein defects, and G protein and ion channelopathies. These mutations lead to phenotypes reminiscent of human clinical disease states including congenital heart defects, cardiomyopathies, and long-QT syndrome, creating models of human electrophysiological disease. Functional analyses of the underlying molecular mechanisms of resultant phenotypes require appropriate and sophisticated experimental methodology. This paper reviews current in vivo murine electrophysiology study techniques and genetic mouse models pertinent to human arrhythmia disorders.
electrophysiology; molecular biology; arrhythmias; animal model
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INTRODUCTION
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ANIMAL MODELS OF ELECTROPHYSIOLOGICAL disorders have contributed to the understanding of the mechanisms associated with human disease. The genetic basis of inherited cardiac arrhythmias is becoming clearer (1, 14, 60). Although selective genome manipulation is possible in several species, the mouse has become the principal mammalian species for transgenic technology (3). Several murine models consequential to human electrophysiological diseases have been developed, including familial conduction disorders (42, 65), familial hypertrophic cardiomyopathies (28, 80), congenital long-QT syndromes (LQTS) (23, 49), and gap junction defects (35, 62, 66). These murine models display particular electrophysiological abnormalities that can be characterized using ex vivo and in vivo electrophysiological techniques. The physiological significance of genetic modifications that were previously characterized only at the cellular or molecular level can now be studied in whole organs or in the intact animal. We developed a mouse cardiac electrophysiology technique to directly assess the in vivo role of specific gene products in cardiac conduction (5). These models are based on clinical protocols and allow assessment of the conduction characteristics of the murine heart via an epicardial or endocardial approach, including evaluation of electrophysiological responses to programmed stimulation and pharmacological agents. The use of implantable telemetry systems makes noninvasive, conscious heart rate analysis feasible in mice (29, 45). These systems are particularly useful in the study of sympathetic and parasympathetic signal transduction pathways, such as the GTP-binding proteins or ion channel systems.
Reviews of cardiac physiology in transgenic mice (38), animal models of cardiac arrhythmias (39), and cardiovascular phenotyping in mice (22) have recently been published. This paper reviews the in vivo mouse electrophysiology methods and their application to transgenic murine models of human electrophysiological disease at the whole animal level.
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EX VIVO ELECTROPHYSIOLOGICAL STUDY
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Langendorff-perfused heart.
Langendorff first described the isolated perfused heart procedure in 1895, and since then this technique has been contributing importantly to the understanding of cardiovascular hemodynamics. This method has been described elsewhere in detail (70). Briefly, the coronary arteries are continuously perfused via a cannula in the aortic root with an oxygenated Tyrodes solution under constant flow and perfusion pressure. The stability of electrophysiological variables in such an ex vivo isolated heart model has been previously reported in small mammals such as rabbits (24) and more recently in mice (34, 73). Pharmacological interventions are performed by direct cardiac injection by a microinfusion pump. Electrocardiogram (ECG) recordings are obtained from needle electrodes placed into the myocardium, and hearts can be instrumented with atrial and ventricular recording and pacing electrodes. Electrophysiology (EP) studies can be performed using a multiple electrode array to record spontaneous and paced electrical activity.
High-resolution optical mapping.
Electrophysiological heterogeneities in ventricular myocardium may be caused by spatial dispersion of ionic membrane properties, which produces regional differences in action potential duration (APD) and refractoriness. Structural heterogeneities, such as tissue anisotropy, or changes in the geometry of a propagating reentrant wave front produced by conduction through a narrow isthmus, or pivoting around obstacles may introduce changes in conduction and refractoriness that are expected to additionally influence path length. The utility of conventional recording techniques for mapping reentrant wave fronts is limited by the inability to take simultaneous measurements of membrane repolarization. Estimations of path length are calculated (conduction velocity x refractoriness) at single recording sites. High-resolution optical mapping with voltage-sensitive dye is an important technique for studying arrhythmia mechanisms in perfused hearts and has been used to map the transmembrane potential changes during electrical stimulation (43, 79). Several studies have been done in different mammalian species with various types of arrhythmias (2, 21, 53, 67). This technique has the capacity to record transmembrane action potentials and provides quantitative analysis of action potential duration restitution kinetics and repolarization patterns in intact hearts. Gray et al. (33) nicely described the technique in a study of ventricular tachycardia in the isolated rabbit heart, using a combination of high-resolution video imaging, ECG, and image processing together with mathematical modeling to characterize the dynamics of epicardial transmembrane potentials.
Mouse ex vivo EP study applications.
We previously demonstrated that electrophysiological data of sinus node function, AV nodal function, and ventricular effective refractory periods can be obtained from the isolated perfused heart of normal (C57BL/6J) mice, comparable to those reported in studies employing the in vivo technique (5, 30). Among early electrophysiological studies in genetically engineered animals were mice with a targeted ablation of a connexin gene. Cardiac gap junctions enable electrical coupling of adjacent cells and play a role in the propagation and maintenance of arrhythmias (18, 48, 68). At least three different connexin isoforms are expressed in the mouse cardiac myocytes. In mice, connexin-43 (Cx43), the most abundant cardiac connexin, and connexin-45 (Cx45) are predominantly seen in atrial and contractile ventricular myocytes, whereas connexin-40 (Cx40) is expressed in atrial myocytes and the His-Purkinje system, but not in ventricular contractile myocytes (12, 17). Alterations in intercellular conduction are critical in the pathogenesis of sudden cardiac death due to reentrant ventricular arrhythmias and in the pathogenesis of conduction abnormalities and lethal arrhythmias (20, 50, 58).
To delineate the functional role of Cx43, the predominant cardiac connexin in ventricular conduction, ex vivo electrophysiological studies were performed in mice heterozygous for a Cx43 null mutation (Cx43+/-) (34). Ventricular epicardial conduction of paced beats was slower in neonatal and adult Cx43+/- mouse hearts, compared with control hearts, indicating that Cx43 is a principal conductor of ventricle intercellular current.
In a subsequent study combining the ex vivo and in vivo approach, Thomas et al. (73) demonstrated that a reduction of Cx43 in atrial myocytes had no effect on atrial conduction, suggesting that Cx40, which is expressed in atrial but not in ventricular myocytes, may be a major electrical coupling protein in the atrium. Thus these studies showed that Cx43 and Cx40 are molecular chamber-specific determinants of ventricular and atrial myocardial conduction in mice (8).
Baker et al. (2) studied Langendorff-perfused hearts from transgenic mice that express an NH2-terminal fragment of the K+ channel Kv1.1 in the heart and lack a rapidly activating slowly inactivating, 4-aminopyridine-sensitive current (Islow) encoded by Kv1.5. Phenotypically, these mice have prolonged action potential durations, prolonged QT intervals on ECG, and spontaneous ventricular tachycardia (VT). With optical mapping techniques, they demonstrated increased action potential duration and abnormal repolarization contributing to inducible reentrant ventricular tachycardia in affected mice.
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IN VIVO ELECTROPHYSIOLOGICAL STUDY
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Epicardial approach.
Mice are anesthetized, and a multilead surface ECG is obtained by subcutaneous electrode placement in each extremity. For the open-chest epicardial approach, the mouse is intubated with a 3/4-in., 24-gauge catheter and ventilated with a rodent respirator. Mechanical ventilation is performed at 130 breaths/min with 1-ml tidal volume, using a mixture of air and supplemental oxygen. In the epicardial procedure, a midline sternotomy is performed, and epicardial pacing wires are placed directly on the surfaces of the right and left ventricles and right atrium. Pacing electrodes are Teflon-coated wire, with the end stripped away of insulating material. The wires are externalized through the skin at the posterior neck, the lungs are re-expended with positive end-expiratory pressure, and the sternotomy incision is suture closed. Mice recover from surgery prior to EP study.
Endocardial approach.
Intubation is not required for the closed-chest endocardial approach. For intracardiac studies, a single-pass octapolar mouse 2-French EP catheter (Fig. 1) with precise interelectrode distances (0.5 mm) is advanced from the right internal jugular vein through the right atrium to the right ventricle. The distal electrodes pace and record from the right ventricle, while the proximal electrodes pace and record from the right atrium. The middle electrode pairs can record a distinct triphasic His bundle electrogram (52). An end-hole lumen (0.009 in.) is utilized for infusion of catecholamine and/or antiarrhythmic medication. The ECG channels are filtered between 1 and 50 Hz, and intracardiac ECGs are amplified and filtered between 5 and 400 Hz. Recordings are displayed continuously on an oscilloscope and simultaneously acquired to a computer via an analog-to-digital converter and stored on disc for later analysis and measurement with online calipers (Fig. 2).

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Fig. 1. A mouse 2-French octapolar electrophysiology catheter (bottom) is shown compared with a standard 5-French bipolar electrophysiology catheter (top) used in human clinical studies.
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Fig. 2. An example of prolonged AV conduction: surface ECG (top) and an intracardiac ECG (bottom) from a catheter positioned across the anterior tricuspid valve. During sinus rhythm, the PR interval on surface ECG is prolonged, as is the AV interval on intracardiac ECG, due to supra-Hisian (AH) delay. The ordinate is in millivolts; the abscissa is 1 s.
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EP study protocol.
In murine studies, similar for the ex vivo or in vivo techniques, standard clinical EP pacing protocols are used to determine basic electrophysiological parameters. Uni- and bipolar ECG and intracardiac recordings are obtained from right atrium, right and left ventricles via the epicardial, and from right atrium and ventricle via the endocardial route. Pacing thresholds are determined and stimulation is performed at twice the diastolic capture threshold. All ECG intervals [sinus cycle lengths (SCL), PR, QRS, QT, JT, QTc] and axes (P and QRS) are measured. Basic electrophysiological parameters, including sinus node function, AV nodal conduction properties, and refractory periods are determined. Programmed extra-stimulation techniques are used to attempt induction of potential arrhythmias (Figs. 3 and 4). To test for pharmacological effects on basal ECG and EP parameters, intracardiac or intraperitoneal drug injections are given and the EP protocols are repeated as in the baseline state to determine changes in arrhythmia inducibility, conduction, or refractoriness.

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Fig. 3. An example of ventricular tachycardia induced during a typical programmed stimulation protocol. Programmed extrastimulus testing at cycle length 50 ms induces a 19-s episode of ventricular tachycardia at a rate of 750 beats/min. In each tracing, the top recording is surface ECG lead I, the middle section is the right ventricular intracardiac electrogram, and the bottom section is the right atrial/tricuspid annular intracardiac electrogram (in mV). A: the entire duration from induction to spontaneous termination; dotted vertical lines represent the sections enlarged in B and C. B: a magnified segment from the induction sequence, illustrating the programmed stimulation sequence (50/30/30/30/30 ms). C: a magnified segment of the termination portion, showing spontaneous conversion back to normal sinus rhythm.
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Fig. 4. Induction of atrial fibrillation following carbamylcholine administration. The right atrium is rapidly paced until provocation of sustained atrial fibrillation, with variable AV conduction. The atrial cycle length varies between 20 and 40 ms, and the ventricular cycle length varies between 250 and 330 ms. Shown are a surface ECG (top tracing), an intracardiac right atrial ECG (middle tracing), and an intracardiac right ventricular ECG (bottom tracing). The ordinate is in millivolts; the abscissa is in seconds.
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Mouse in vivo EP study applications.
Berul et al. (5) initially reported the feasibility of assaying conduction properties of the mouse heart in vivo and characterized standard electrophysiological parameters in normal (C57BL/6J) mice, first describing an epicardial approach, followed by endocardial transcatheter techniques (6, 7).
Familial hypertrophic cardiomyopathy.
Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant inherited disease caused by specific mutations in the genes encoding the sarcomeric structural components, including myosin, tropomyosin, and myosin binding proteins. Affected patients are at risk for ventricular and atrial arrhythmias and sudden cardiac death (15). Genetically engineered mice with a missense mutation (
-MHC403/+) in the
-myosin heavy chain gene Arg403Gln provide a genetic model of FHC and display the characteristic histopathological and hemodynamic phenotype. FHC mice with this
-myosin mutation had distinct electrophysiological abnormalities, including rightward frontal plane axis, sinus node dysfunction, and prolonged ventricular repolarization (6, 7, 10). During programmed ventricular stimulation, 62% of male and 28% of female transgenic mice had inducible ventricular tachycardia, exemplifying the malignant nature of this specific mutation and sex-specific electrophysiological abnormalities, consistent with the histological and hemodynamic alterations.
Another mouse model of hypertrophic cardiomyopathy carrying a defect in the gene encoding myosin binding protein C (MyBP-C) has been created. Mutations in MyBP-C gene lead to an early stop codon resulting in truncated proteins. Histologically these mice display atrial and ventricular cardiomyocyte pathology with hypertrophy, fibrosis, and myofibrillar disarray (6, 80). As in the
-myosin heavy chain mutation, a malignant electrophysiological phenotype was found in these mice with 50% having inducible VT (9).
Congenital long-QT syndrome.
The congenital LQTS is a group of genetic disorders characterized by prolonged ventricular repolarization and risk of ventricular tachyarrhythmias, caused by mutations in genes encoding for cardiac ion channels. Considerable genetic and phenotypic heterogeneity contributes to a variable clinical presentation (25, 61). London et al. (49) generated a transgenic mouse model of LQTS by overexpressing the NH2 terminus and first transmembrane segment of the voltage-gated potassium channel Kv1.1. They postulated that overexpression of this truncated potassium channel in the hearts of transgenic mice would reduce outward potassium currents. On surface ECG, heterozygote mice expressing the Kv1.1N206Tag transcript had prolonged corrected QT (QTc) intervals. Conscious ECG analysis was also performed in these mice using telemetry (described below).
Drici et al. (23) developed a mouse model of Jervell and Lange-Nielsen syndrome, in humans characterized by severe bilateral deafness and long-QT interval. Mutations in KVLQT1 and IsK (minK) genes forming the K+ channel are responsible for the cardiac and inner ear slowly activating component of the delayed rectifier K+ channel current (Iks). They engineered transgenic mice with a null mutation of the isk gene and determined the effect of this mutation on cardiac parameters associated with LQTS. On ECG, isk-/- mice had a longer QT interval in bradycardic conditions, a shorter QT interval at fast heart rates, and an abnormal QT-heart rate adaptation. The authors speculated that the change in QT-RR adaptability and consequent triggering of early after-depolarizations, rather than the QT duration, may increase the propensity for torsades de pointes.
The minK (or IsK) gene encodes a protein that modulates the function of cardiac delayed rectifier currents Ikr and Iks resulting from expression of HERG or KvLQT1. Mutations in this gene constitute one cause of the congenital LQTS in humans (69). Kupershmidt et al. (47) generated mice with a targeted disruption in the minK gene and demonstrated absence of Iks in addition to reduction and slowed deactivation of Ikr in minK-/- myocytes. Despite these findings, mice did not display QT prolongation or other ECG phenotype, suggesting a restricted minK expression. In fact, they demonstrated cardiac conduction system-specific minK expression in postnatal and embryonic mice, implicating minK expression in the developing conduction system. Thus, by the time of birth, Iks and Ikr do not have a clear role for repolarization in mice.
OBrien et al. (57) identified phenotypic features of human LQTS in transgenic mice overexpressing the human
3-isoform of the Na-K-ATPase in the heart. Na-K-ATPase is an enzyme critical to the normal mechanical and electrical function of the heart and comprises of two (
and ß) protein subunits. They hypothesized that upregulated
3-isoform expression could prolong the QT interval by several potential mechanisms: a decrease in net electrogenic Na+ flux, an increase in intracellular Na+ concentration, a decrease in the concentration gradient favoring potassium extrusion, or by alteration in other ion channels or transporters. In vivo experiments revealed differences in the QT-SCL relationship between the groups indicating a steeper dependence of QT on SCL in the transgenic mice. Mice were atrially paced to eliminate SCL variability as confounding factor, and QT interval remained significantly longer in the transgenic mice. In addition, was increased in transgenic mice. Reproducible ventricular tachycardia, QT dispersion and T wave alternans, features of human LQTS, were also seen in some of the transgenic mice. Clinical mutations in Na-K-ATPase pump genes have not been identified, although these transgenic mice showed several phenotypic features similar to human LQTS.
Congenital heart disease (familial atrial septal defect).
Atrial septal defects (ASD) are usually sporadic, but some individuals with ASD and AV conduction abnormalities have a family history (4, 54). Mutations in the gene encoding the homeobox transcription factor Nkx2.5 were found to cause this nonsyndromic, human congenital heart disease. A dominant disease locus was mapped to chromosome 5, where Nkx2.5 is located. Nkx2.5 is important for the regulation of septation during cardiac morphogenesis and for maturation and maintenance of A-V node function throughout life (4, 65). Targeted disruption of Nkx2.5 in a mouse model causes early embryonic lethality, with arrest of cardiac development at the linear heart tube stage prior to looping in homozygous mice, whereas heterozygous mice survive (4). We characterized in vivo the electrophysiological properties in Nkx2.5 haploinsufficient mice and transgenic overexpressing mice. All Nkx2.5 haploinsufficient mice had PR interval prolongation, analogous to 1° AV block. Both atrial and ventricular arrhythmia vulnerability were provoked with programmed electrical stimulation. Atrial fibrillation was observed in 8 of 10 transgenic mice, compared with 0 of 9 wild-type (WT) controls; ventricular tachycardia was inducible in 6 of 10 heterozygote vs. 0 of 9 WT mice (71). Interestingly, the transgenic mutant mice had progressive AV conduction defects, including complete AV block due to supra-Hisian block, and increased AV nodal refractoriness (76). These findings may help elucidate the pathogenesis of human inherited arrhythmia disorders.
Connexin protein defects.
As outlined earlier, Cx40 is mainly expressed in atrial tissue and His-Purkinje cells. Mice with a targeted disruption of the Cx40 gene were interbred to generate mice homozygous for the Cx40 deletion (Cx40-/-) (66). Bevilacqua et al. (11) performed detailed intracardiac EP studies in Cx40-/- mice and found that Cx40 deficiency resulted in abnormal electrical coupling in the specialized conduction system. Significant differences in AV conduction were found in Cx40-/- mice compared with WT controls. The PR interval was prolonged in Cx40-/- mice, with no difference in P wave duration, consistent with His bundle conduction delay. As expected, no differences in QRS duration or QT interval were seen, since Cx40 is not expressed in ventricular myocytes. EP findings included significant prolongation of AV refractoriness, Wenckebach and 2:1 block cycle lengths in Cx40-/- mice compared with WT controls, indicating the specialized conduction tissue as the site of the block, as Cx40 is not seen in the compact AV node. Similar findings have been verified using a transesophageal pacing approach in Cx40-deficient mice (35).
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AMBULATORY ELECTROCARDIOGRAPHIC TELEMETRY
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For ambulatory long-term ECG analysis in the conscious state, telemetry devices can be implanted in mice. Thus noninvasive assessment of heart rate dynamics is feasible in unrestrained, freely moving mice without the need for physical contact. In the anesthetized animal, under sterile conditions, an implantable 3.5-g wireless radio frequency transmitter is inserted into a subcutaneous tissue pocket or in the peritoneal cavity, allowing for chronic implantation and long-term telemetric ECG monitoring (Fig. 5).

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Fig. 5. Telemetered single-lead ECG in normal mouse, showing the fidelity of the implantable telemetry system for ECG transmission in conscious, exercising mice. The transmitters are implanted subcutaneously in the back with antennae secured at both shoulders, and a receiver is positioned under the multilane treadmill. The heart rate, cardiac rhythm, and conduction intervals can be easily measured from a lead I vector ECG. In this example, the sinus cycle length (SCL) = 74 ms, the PR interval = 25 ms, QRS = 13 ms, and QT = 24 ms.
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Ambulatory long-term ECG and exercise ECG analysis.
Following recovery from surgery, ECG information from a single lead is monitored by placing mice in a cage on a receiver connected to a data acquisition system. The analog ECG data are digitized using a 16-bit analog-to-digital converter. Measurements of beat-to-beat heart rate, ECG intervals, and heart rate variability (HRV) parameters can be obtained at rest and during normal activity. Stored recordings can be screened for arrhythmias. The effects of acute or chronic exercise stress on arrhythmia induction can be evaluated by subjecting the mouse to a standard graded exercise protocol (e.g., swimming or treadwheel) with the implantable transmitter chronically in place.
Mouse telemetry study applications.
Telemetric ECG recordings, combined with statistical and computational analysis of the obtained data, were used in several recent studies of different genetically manipulated mouse strains. In particular, HRV analysis led to a detailed understanding of the molecular regulatory mechanisms that determine the signal transduction pathways within the heart on the G protein-ion channel level. Telemetry implant devices have also been used as an adjunct to treadmill exercise protocols to detect cardiac abnormalities that are not easily apparent at rest. Mitchell et al. (56) measured heart rate and QT intervals in conscious mice, and they introduced a formula for appropriate rate correction of the QT interval.
Congenital LQTS.
As outlined above, telemetry was used to determine phenotypic expression in transgenic mice expressing the NH2 terminus and first transmembrane segment of the voltage-gated potassium channel Kv1.1N206Tag (49). Significant prolongation of the QT interval was found in LQT mice (59.9 ± 2.5 ms) compared with control mice (54.2 ± 1.8 ms), whereas no differences were found for RR intervals between groups. LQT mice also had a significant increase in the frequency of ventricular premature beats and spontaneous episodes of ventricular tachycardia. Taken together, the in vivo studies confirmed observations made in the in vitro experiments (24). Telemetry monitoring of LQT mice is consistent with the human clinical phenotype of ambient ventricular ectopy, paroxysmal ventricular tachycardia, and inconstant QT prolongation.
Atrial ß1-adrenoceptor overexpression.
Experimental evidence supports the role of G protein-linked ß-adrenergic receptor distribution within the sinoatrial node on heart rate regulation. To test this hypothesis, Mansier et al. (55) utilized telemetry and analyzed heart rate dynamics in transgenic mice with a targeted atrial overexpression of ß1-adrenoceptors. They demonstrated a pronounced attenuation of HRV. As these mice exhibited no modification of central or baroreflexes, it was concluded that 1) the myocardial phenotype (ß1-adrenoceptors density) was a major determinant of HRV and 2) saturation of the sympathetic input was the causative pathophysiological principle in HRV reduction. Since these mice had a normal lifespan despite severe reduction of HRV, the authors suggested that HRV might only be of prognostic value in the diseased heart.
Cardiac Gs
overexpression.
The
-subunit of GTP-binding protein (Gs
) stimulates ß-adrenergic signal transduction and consequently heart rate control through activation of L-type Ca2+ channels. Uechi et al. (74) demonstrated that overexpression of cardiac atrial Gs
in transgenic mice resulted in enhanced ß-adrenergic signal transduction, characterized by permanent elevations in heart rate, but depressed HRV and circadian variation of heart rate. It was hypothesized that these findings constituted the critical mechanism for the later development of cardiomyopathy in these animals.
GIRK4 knockout mice.
The muscarinic-gated potassium channel (IKACh) is involved in vagally mediated heart rate regulation (63), but the extent of its contribution was unclear. In a study conducted in a mouse line deficient in IKACh by targeted disruption of the gene encoding one of the channel subunits, GIRK4, Wickman et al. (78) demonstrated that IKACh is critical for effective parasympathetic heart rate regulation. They found that IKACh mediates approximately half of the negative chronotropic effects of vagal stimulation and adenosine, and that IKACh is responsible for beat-to-beat-fluctuations in heart rate. Kovoor and colleagues (44) then showed that these mice were fully resistant to vagally mediated atrial fibrillation induction.
Neuronal nitric oxide synthase-deficient mice.
Inhibitory G protein activity and nitric oxide (NO) synthesized within mammalian sinoatrial cells have been shown to provide parallel pathways in cholinergic control of heart rate (36). It was, however, unknown whether neuronally derived NO played a role in heart rate regulation. Jumrussirikul et al. (41) evaluated heart rate dynamics in transgenic mice in which the gene for neuronal NO synthase (nNOS) had been disrupted. They found that absence of nNOS activity lead to reduced parasympathetic heart rate modulation at baseline but did not prevent a baroreflex-mediated rise in parasympathetic tone. These findings provide evidence for parallel cardioinhibitory pathways consisting of nNOS activity on the one hand and cardiac inhibitory G protein activity on the other.
Reduced G protein ß
-subunit in cardiocyte membranes.
Acetylcholine released upon parasympathetic stimulation of the vagal nerve slows heart rate through activation of muscarinic receptors and subsequent opening of the atrial muscarinic IKACh channel. This channel is activated by ß
-subunits released from G proteins, which couple to the M2-muscarinic receptor (16, 37, 46). Gehrmann et al. (32) evaluated heart rate regulation in transgenic mice with a reduction of membrane-bound G protein ß-subunits in atrial cardiomyocytes. Compared with WT controls, in transgenic mice the parasympathetic branch of signal transduction was impaired, as reflected by markedly depressed HRV parameters. These data provide evidence that G protein ß-subunits are integral components in parasympathetic heart rate regulation and physiological IKACh channel function, consistent with prior single cell experiments.
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POTENTIAL LIMITATIONS OF MURINE CARDIAC EP STUDIES
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The limitations of murine cardiac electrophysiology must be recognized. In general, electrophysiological properties are subject to species variability. In addition, the high basal heart rate of mice might make accurate delineation of timing intervals more difficult. Differences in the mouse and human action potential duration, which are predominantly due to differences in heart size, body mass, oxygen consumption, and heart rate may have an impact on data interpretation and on the extrapolation of mouse data for human electrophysiology. For example, the short action potential duration (<30 ms) in mice (13) may limit studies of the potential contribution of the dispersion of repolarization to arrhythmogenesis. The ionic currents determining repolarization time in adult mice were shown to be different from those in humans (77). Previously published work indicates that murine models of human electrophysiological disease, although exhibiting important phenotypic features, fail to demonstrate a complete spectrum of the characteristic human pathologies. For example, in humans, mutations in the minK gene have been associated with prolongation of the QT interval; in a study in mice with this defect (47) no QT prolongation was observed, suggesting species-specific roles of this gene product in mediating the electrophysiological properties of the heart (see above).
Using the mouse as an experimental animal raises the question whether heart size is a problem in terms of arrhythmogenesis. In 1914, Garrey (27) established that a critical mass of tissue is needed to sustain fibrillation. We demonstrated reproducible atrial fibrillation in mice with a targeted deletion of the homeobox transcription factor Csx/Nkx2.5 (71). In addition, we have produced a model of vagally mediated atrial fibrillation induction by administration of the cholinergic agonist carbamylcholine during electrical stimulation (75). These examples further underscore the relevancy of the mouse as a model of disease.
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SUMMARY AND FUTURE DIRECTIONS
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This review summarizes current technology available to characterize the electrophysiological phenotype in genetically manipulated murine models of human cardiovascular disease. Considering the expanding knowledge of the mouse genome, gestation times, technical feasibility, expense, and ethics, the mouse has become the species of choice in this area of molecular research (64). The concern that mice may not approximate human cardiovascular electrophysiology as closely as larger mammals has to be considered, although human and mouse proteins show >80% homology. The electrophysiological studies conducted so far demonstrate the scientific relevance of the mouse model. Previous technical difficulties were overcome by downsizing technology and miniaturizing investigational electrophysiological tools and techniques, allowing these studies to become practicable as assays for characterization of cardiac conduction abnormalities and arrhythmia phenotypes.
With the advent of transgenic and gene-targeted manipulations of cardiac electrophysiological diseases, genetic systems are emerging with the potential to provide definite insights into the genetic pathogenesis of electrophysiological diseases and conduction abnormalities critical in the initiation and maintenance of arrhythmias. There is now a rich collection of mouse strains harboring targeted gene mutations substantial to human arrhythmogenic disorders. It became obvious that analyses of whole organ and whole animal function would be needed to investigate important aspects of the resultant phenotypes.
Isolated perfused heart systems have been employed in many species to address numerous physiological, pharmacological, and pathophysiological questions. More recently, electrophysiological parameters comparable to those found in in vivo studies could be reliably obtained in normal and engineered mice. This technique permits the study of electrophysiology under regulated conditions, without influences of the autonomic nervous system (19) and distributional effects, minimizing the variability of numerous undesirable effectors. Jones et al. (40) demonstrated the feasibility of an isolated working murine heart preparation, which is particularly useful in the study of cardiomyopathy models. A further advantage of the Langendorff-perfused heart technique is the feasibility of optical mapping studies in mice. A limitation of isolated heart perfusion techniques is that the interdependent physiology of the neural-humoral-vascular system is not considered. Additionally, extended periods of tissue exposure in an ex vivo artificial environment may alter electrophysiological properties and results. His bundle electrograms have not been reported using this technique. For the aforementioned reasons and since excised cardiac preparations and isolated whole heart systems do not represent the complexity of an in vivo physiological system, intact animal experimental techniques may have distinct advantages.
The in vivo technique is capable of obtaining electrophysiological parameters from intact genetically altered mice. The epicardial, open-chest approach overcomes the disadvantages of the isolated heart technique, since neural-humoral-vascular influences can be taken into account; the influences of intrathoracic pressure and pericardial restraint, however, are lost. The endocardial, closed-chest approach allows for minimally invasive acquisition of electrophysiological data under more normal physiological conditions in the intact organism. This technology may also be applied to mouse models (transgenic and nontransgenic) of ischemia and infarction, since the feasibility of modeling myocardial infarction in mice has been demonstrated (31, 51).
The use of implantable telemetry devices permits electrophysiological studies at the conscious animal level. Thus the confounding effects of restraints and anesthesia are removed, and heart rate, as an important parameter of cardiovascular physiology, is now amenable to noninvasive assessment at rest, during normal activity and during exercise protocols (26). This way, exercise stress may reveal subtle cardiac phenotypes not apparent under resting conditions. Because mean heart rate is subject to many different control mechanisms and pathological phenomena, and mice are physiologically operating at their upper heart rate limit, it cannot be used as a reliable estimator of autonomic activity and tone. Therefore HRV analysis constitutes an alternate possibility to explore the influence of the autonomic nervous system on the murine heart and is a powerful tool in the quantitative dissection of complex and convergent signaling pathways.
Promising future directions of cardiac electrophysiological research in transgenic mice involve the study of ion channel genes and channel biophysics, the prediction and prevention of arrhythmias, the design of potential therapies, and ultimately the development of gene-specific therapies. Murine models of myocardial infarction and other disease states may be useful to analyze the contributions of myocardial cell decoupling and gap-junctional distribution to arrhythmogenesis. Additional inherited arrhythmogenic human disorders will be manipulated in the mouse genome. The expanding knowledge of genomic and genetic resources will make the mouse an even more useful model in arrhythmia research.
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ACKNOWLEDGMENTS
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This work could not have been completed without the skilled technical assistance of Colin Maguire, Joseph Gehrmann, and Hiroko Wakimoto; their superb commitment to developing mouse cardiac electrophysiology is greatly appreciated.
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FOOTNOTES
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1 This review article was based on work originally presented at the "NHLBI Symposium on Phenotyping: Mouse Cardiovascular Function and Development" held at the Natcher Conference Center, NIH, Bethesda, MD, on October 1011, 2002. 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: C. I. Berul, Dept. of Cardiology, Childrens Hospital-Boston, 300 Longwood Ave., Boston, MA 02115 (E-mail: charles.berul{at}cardio.chboston.org).
10.1152/physiolgenomics.00183.2002.
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