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2Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida. 3Department of Genetics, Harvard Medical ...
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genesis 37:144 –150 (2003)

ARTICLE

Cardiac Electrophysiological Phenotypes in Postnatal Expression of Nkx2.5 Transgenic Mice Hiroko Wakimoto,1 Hideko Kasahara,2 Colin T. Maguire,1 Ivan P.G. Moskowitz,3 Seigo Izumo,4 and Charles I. Berul1* 1

Department of Cardiology, Children’s Hospital, Boston, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 2 Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida 3 Department of Genetics, Harvard Medical School, Howard Hughes Medical Institute, Department of Pathology and Cardiac Registry, Children’s Hospital, Boston, Massachusetts 4 Cardiovascular Division, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts Received 7 March 2003; Accepted 29 August 2003

Summary: Nkx2.5 is a conserved homeodomain (HD) containing a transcription factor essential for early cardiac development. We generated several mutations modeling some patients with congenital heart disease. Transgenic mice (tg) expressing the wildtype Nkx2.5 under ␤-myosin heavy chain (MHC) promoter died during the embryonic stage. However, tg mice expressing this mutation under ␤-MHC promoter (␤-MHC-TG(I183P)), the wildtype Nkx2.5 (␣-MHC-TG(wild)), and a putative transcriptionally active mutant (carboxyl-terminus deletion, ␣-MHC-TG(⌬C)) under ␣-MHC promoter showed postnatal lethal heart failure. Given the profound atrioventricular conduction abnormalities we recently demonstrated in ␤-MHC-TG(I183P) mice, the aim of this study was to determine whether ␣-MHCTG(wild) and ␣-MHC-TG(⌬C) mutant mice display similar cardiac electrophysiological phenotypes. Surface ECG recordings and in vivo electrophysiology studies were performed in ␣-MHC-TG(wild) mice and controls at 6 weeks of age, and in ␣-MHC-TG(⌬C) mice and controls at 10 weeks of age. Ambulatory ECG recordings in ␣-MHC-TG(wild) and controls were obtained using an implantable radiofrequency telemetry system. PR prolongation and atrioventricular nodal dysfunction were detected in ␣-MHCTG(wild) and ␣-MHC-TG(⌬C) mice. Bradycardia and prolonged PR interval were seen in ambulatory ECG of ␣-MHC-TG(wild) mice compared to controls. Several ␣-MHC-TG(wild) mice died of bradycardia. Fetal and neonatal mutant Nkx2.5 expression causes severe cardiac conduction failure. Postnatal overexpression of nonmutant (wild) Nkx2.5 also causes conduction abnormalities, although the onset is after the neonatal stage. Bradycardia and AV conduction failure may contribute to the lethal heart failure and early mortality. genesis 37:144 –150, 2003. © 2003 Wiley-Liss, Inc.

early cardiac development, including regulation of septation during cardiac morphogenesis and also for maturation and maintenance of the conduction system. Nkx2.5 expression starts on 7.5 days postcoitum (dpc) in the precardiac mesoderm and its expression continues until adulthood (Lints et al., 1993; Komuro and Izumo, 1993; Kasahara et al., 1998). In humans, 26 different heterozygous NKX2.5 mutations have been identified in patients with congenital heart disease, and these appear inherited in an autosomal dominant fashion (Schott et al., 1998; Benson et al., 1999; Goldmuntz et al., 2001; Gutierrez-Roelens et al., 2002; Watanabe et al., 2002; McElhinney et al., 2003). Common cardiac phenotypes in patients include secundum atrial septal defect (ASD) and progressive atrioventricular (AV) conduction failure. Progressive heart failure was also reported (Schott et al., 1998; Benson et al., 1999). In mice, Nkx2.5 is also important in early cardiac development. Nkx2.5-targeted homozygous mutant mice form normal heart tubes, however, they die during the period of looping morphogenesis, which suggests that NKX2.5 is also important in the later stages of heart development and maturation (Lyons et al., 1995; Tanaka et al., 1999). Among heterozygous mutations found in humans, six are single missense mutations within the HD (Schott et al., 1998; Benson et al., 1999; Gutierrez-Roelens et al., 2002). All four mutants previously examined showed reduce DNA binding but preserved homodimerization ability (Kasahara et al., 2000). We generated a similar

Key words: cardiac conduction; transgenic mice

HW is currently at Tokyo Medical and Dental University, Tokyo, Japan. CTM is currently at Baylor College of Medicine, Houston, TX. * Correspondence to: Charles I. Berul, M.D., Associate Professor of Pediatrics, Department of Cardiology–Children’s Hospital, Boston, 300 Longwood Avenue, Boston, MA 02115. E-mail: [email protected]

INTRODUCTION Nkx2.5 is an evolutionarily conserved homeodomain (HD)-containing transcription factor that is essential for

DOI: 10.1002/gene.10236

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Table 1 ECG Data in ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C) Mice

␣-MHC-TG(wild) (6 weeks old) ␣-MHC-TG(⌬C) (6 weeks old) ␣-MHC-TG(⌬C) (10 weeks old)

tg (n ⫽ 7) wt (n ⫽ 9) tg (n ⫽ 5) wt (n ⫽ 8) tg (n ⫽ 7) wt (n ⫽ 8)

SCL (ms)

HR (bpm)

PR (ms)

QRS (ms)

QT (ms)

QTc (ms)

154 ⫾ 35 128 ⫾ 17 143 ⫾ 24 128 ⫾ 18 136 ⫾ 9 142 ⫾ 24

405 ⫾ 80 474 ⫾ 51 428 ⫾ 66 474 ⫾ 55 442 ⫾ 30 431 ⫾ 66

59 ⫾ 22* 40 ⫾ 5 44 ⫾ 2 41 ⫾ 5 52 ⫾ 10** 38 ⫾ 2

12 ⫾ 1 13 ⫾ 1 13 ⫾ 2 13 ⫾ 1 12 ⫾ 1 12 ⫾ 1

34 ⫾ 7 30 ⫾ 2 32 ⫾ 9 29 ⫾ 2 25 ⫾ 2 25 ⫾ 3

27 ⫾ 4 26 ⫾ 2 27 ⫾ 3 26 ⫾ 3 22 ⫾ 2 21 ⫾ 3

SCL ⫽ sinus cycle length; HR ⫽ heart rate; PR, QRS, QT, QTc ⫽ standard ECG intervals; ms ⫽ milliseconds; bpm ⫽ beats per minute; tg ⫽ transgenic; wt ⫽ wild-type. *P ⬍ 0.05, **P ⬍ 0.01.

single missense mutation, Ile183Pro in the HD (Kasahara et al., 2001). For further investigation of Nkx2.5 cardiac developmental and maturational function during either the embryonic or postnatal stage, transgenic mice (tg) were generated by using two different promoter/enhancer transgenic constructs; ␤-myosin heavy chain (␤MHC) promoter, which expresses transgene mainly in embryonic heart, and ␣-MHC promoter, which expresses transgene mainly in the postnatal period. Five different transgenic mice were designed, expressing either: the wildtype (nonmutant) Nkx2.5 under ␤-MHC promoter (␤-MHC-TG(wild)); non-DNA binding mutant Nkx2.5 under ␤-MHC promoter (␤-MHC-TG(I183P)); the wildtype Nkx2.5 under ␣-MHC promoter (␣-MHC-TG(wild)); a putative transcriptionally active mutant (⌬C, deletion of 88 amino acids of the carboxyl-terminus of Nkx2.5) under ␣-MHC promoter (␣-MHC-TG(⌬C)); and non-DNA binding mutant Nkx2.5 under ␣-MHC promoter (␣-MHC-TG(I183P)). ␤-MHC-TG(wild) died during the embryonic or early postnatal stage but ␤-MHCTG(I183P) survived but showed a postnatal lethal phenotype with heart failure (Kasahara et al., 2001). Surface electrocardiography (ECG) revealed a progressive dysfunction of the AV conduction system. ␤-MHCTG(I183P) mice had significantly slower heart rates at 1 week of age and, after 2 weeks, demonstrated bradycardia, 1st degree AV block, wide QRS, low voltage, and left axis deviation. They progressively deteriorated to complete AV block by 4 weeks of age (Wakimoto et al., 2002). The hearts of these ␤-MHC-TG(I183P) mice displayed strikingly reduced expression of Connexin 40 and 43. In addition, mice heterozygous for a null Nkx2.5 mutation had atrial septal defects, abnormal AV conduction, and arrhythmia susceptibility (Tanaka et al., 2003). The ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C)) mice also have a postnatal lethal phenotype with heart failure (Kasahara et al., 2003). Given the marked electrophysiological abnormalities identified in ␤-MHC-TG(I183P) and haploinsufficient mice, we sought to evaluate the detailed cardiac electrophysiological phenotypes in these tg mice. Thus, to further explore Nkx2.5 function in postnatal cardiac conduction, in this present report we studied the in vivo cardiac electrophysiological characteristics of ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C) transgenic mice.

RESULTS ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C) Surface ECG The ECG of TG(wild) at 6 weeks demonstrated AV conduction defects evidenced by prolongation of the PR interval, while TG-⌬C did not show apparent PR prolongation or other ECG abnormalities at the age of 6 weeks but they later developed AV conduction delay by 10 weeks of age. Surface ECG data in ␣-MHC-TG(wild) (7 tg, 9 wt) and ␣-MHC-TG(⌬C) (7 tg, 8 wt) are summarized in Table 1. The PR interval was significantly prolonged both in 6-week-old ␣-MHC-TG(wild) (59 ⫾ 22 ms) and 10 week-old ␣-MHC-TG(⌬C) mice (52 ⫾ 10 ms) compared to their own controls (40 ⫾ 5 ms, P ⬍ 0.05, 38 ⫾ 2 ms, P ⬍ 0.01, respectively). All other ECG properties showed no significant differences between both tg and their own age-matched control groups (Fig. 1). In Vivo Endocardial Electrophysiological Study In ␣-MHC-TG(wild) (7 tg, 8 wt) and ␣-MHC-TG(⌬C) (6 tg, 7 wt), in vivo electrophysiological (EP) studies were successfully performed (Table 2), while 3/31 mice in the initial cohorts died prior to completion of the entire EP study protocol. The ␣-MHC-TG(⌬C) mice had normal EP parameters at 6 weeks of age. However, both the 6-week-old ␣-MHC-TG(wild) cohort and the 10-week-old ␣-MHC-TG(⌬C) tg mice had an increased AH interval compared to their controls at similar ages (48 ⫾ 14 ms versus 35 ⫾ 5 ms, P ⬍ 0.05; 54 ⫾ 8 ms versus 33 ⫾ 3 ms, P ⬍ 0.0001, respectively), while both groups had preserved normal HV interval timing (13 ⫾ 4 ms versus 10 ⫾ 2 ms, P ⬎ 0.05; 9 ⫾ 2 ms versus 10 ⫾ 2 ms, P ⬎ 0.05, respectively). The ␣-MHC-TG(wild) mice presented altered AV conduction capability, as AVWBCL (134 ⫾ 28 ms) and AV2:1CL (104 ⫾ 15 ms) were prolonged compared to controls (105 ⫾ 10 ms, P ⬍ 0.05; 86 ⫾ 8 ms, P ⬍ 0.05, respectively). Retrograde 1:1 V-A conduction was intact in only 2 of 7 tg, whereas 1:1 V-A conduction was present in 6 of 8 wt controls. ␣-MHCTG(⌬C) mice also revealed abnormal AV conduction, with longer AVWBCL in tg than wt (135 ⫾ 26 ms versus 106 ⫾ 11 ms, P ⬍ 0.05). Retrograde VAWBCL were also prolonged in tg mice (151 ⫾ 11 ms versus 127 ⫾ 9 ms, P ⬍ 0.01). ␣-MHC-TG(wild) mice showed significant

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of fatal stage, which indicated that bradycardia, not tachyarrhythmia, induced death in these tg mice (Fig. 2). Histological Analysis of Transgenic Hearts The AV canal of ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C) transgenic mice was analyzed to determine whether structural changes within the heart may be responsible for the demonstrated conduction abnormalities. Hearts were excised after EP testing and sagittal sections through the AV canal were analyzed. Sections were carefully inspected for conduction system tissue and identification of the AV node. On histological examination, no structural defects were identified in the AV canal region of 2-week-old or 6-week-old tg mice. DISCUSSION

FIG. 1. Prolonged atrioventricular conduction in Nkx2.5 transgenic mice. Displayed from top to bottom are: surface ECG lead I, intracardiac right atrial electrogram, and His-bundle, right ventricular electrogram in a transcriptionally active Nkx2.5 mouse. A triphasic His bundle recording (A,H,V) is visible in the lower tracing. The PR interval is prolonged on ECG, concordant with a long atrial-His (AH) interval on the intracardiac His electrogram, with a normal His bundle-ventricle (HV) activation time.

increased VERP (81 ⫾ 26 ms versus 45 ⫾ 15 ms, P ⬍ 0.05), so did ␣-MHC-TG(⌬C) mice (61 ⫾ 23 ms versus 41 ⫾ 4 ms, P ⬍ 0.05). Parameters of sinus nodal and atrial electrical function were not significantly different between genotype groups. Ambulatory ECG in Conscious ␣-MHC-TG(wild) Mice To obtain the ECG in physiological condition and to exclude the influence of anesthesia on SCL, HR, or PR interval, telemetry devices were implanted and ambulatory ECG recordings under the conscious unrestrained state were performed in seven ␣-MHC-TG(wild) and six wt mice at 7 weeks of age. Compared to wt mice, conscious, unrestrained tg mice showed remarkably slow SCL (108 ⫾ 19 ms versus 79 ⫾ 5 ms, P ⬍ 0.01), slow HR (570 ⫾ 80 ms versus 767 ⫾ 53 ms, P ⬍ 0.001), and prolonged PR interval (35 ⫾ 5 ms versus 26 ⫾ 2 ms, P ⬍ 0.001). In ␣-MHC-TG(wild), the ambulatory ECG was continuously monitored until their fatal event. In two mice, we successfully documented ECG recordings

In this study, we clarify the EP characteristics in two types of Nkx2.5-overexpressing transgenic mice. In ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C), which overexpress wildtype and a putative transcriptionally active mutant Nkx2.5, respectively, during postnatal stage, EP testing revealed AV nodal dysfunction in both groups of mutant mice, with preservation of sinus nodal, atrial, and distal conduction system function. Ventricular refractoriness was also prolonged in both groups of transgenic mice. Cellular ionic or whole-cell patch-clamp studies were not performed as part of this series of experiments; therefore, we cannot determine whether potassium channel function was affected by these mutations. It appears, however, that alteration of connexin isoform function is involved, at least in part, in the EP manifestations of Nkx2.5 mutations. Connexin Modulation in Transgenic Mice Gap junctions play an important role in impulse conduction in the heart. In the heart, three major connexin (Cx) isotypes have been identified. Cx43 expresses mainly in ventricular myocytes, while Cx45 is found in the AV node and His bundles. In contrast, Cx40 is expressed in the atrium and His-Purkinje system (Lo et al., 2000). In our previous studies, we demonstrated that embryonic overexpression of DNA nonbinding mutant protein downregulates the expression of Cx40 and Cx43 (Kasahara et al., 2001), and postnatal overexpression of wildtype and transcriptionally active mutant Nkx2.5 also reduced the expression of Cx40 and Cx43 (Kasahara et al., 2003). Homozygous Cx40 (Cx40-/-) mice display PR prolongation with AH and HV delay, AV nodal dysfunction, wide QRS, and abnormal QRS axis (Kirchhoff et al., 1998; Hagendorff et al., 1999; Bevilacqua et al., 2000). However, no effect was seen on ventricular refractoriness with Cx40 deficiency (Bevilacqua et al., 2000), but impaired bundle branch conduction has been shown in Cx40-/- mice (van Rijen et al., 2001). Wide QRS duration with normal PR interval has been described in adult Cx43 heterozygote (Cx43⫹/-) mice (Guerrero et al., 1997; Thomas et al., 1998). As in Cx40-/- mice, Cx43⫹/mice did not demonstrate increased ventricular refracto-

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Table 2 EP Study Data in ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C) Mice ␣-MHC-TG(wild) (6 weeks old) tg (n ⫽ 7) wt (n ⫽ 8) Body weight (g) AH (ms) HV (ms) SNRT (ms) cSNRT (ms) AERP (ms) VERP (ms) AVERP (ms) AVWBCL (ms) AV2:1CL (ms) VAWBCL (ms)

18.4 ⫾ 2.4 48 ⫾ 14* 13 ⫾ 4 257 ⫾ 84 76 ⫾ 64 36 ⫾ 17 81 ⫾ 26* 97 ⫾ 6 134 ⫾ 28* 104 ⫾ 15*

20.2 ⫾ 2.1 35 ⫾ 5 10 ⫾ 2 215 ⫾ 46 68 ⫾ 42 39 ⫾ 9 45 ⫾ 15 88 ⫾ 8 105 ⫾ 10 86 ⫾ 8

␣-MHC-TG(⌬C) (10 weeks old) tg (n ⫽ 6) wt (n ⫽ 7) 21.1 ⫾ 1.6 54 ⫾ 8*** 9⫾2 235 ⫾ 43 77 ⫾ 44 55 ⫾ 21 61 ⫾ 23* 96 ⫾ 25 135 ⫾ 26* 103 ⫾ 23 151 ⫾ 11**

21.7 ⫾ 0.5 33 ⫾ 3 10 ⫾ 2 211 ⫾ 37 60 ⫾ 35 37 ⫾ 8 41 ⫾ 4 86 ⫾ 9 106 ⫾ 11 87 ⫾ 6 127 ⫾ 9

␣-MHC-TG(⌬C) data at 6 weeks old—no significant differences (data not shown). AH ⫽ atrial-His timing; HV ⫽ His-ventricular timing; SNRT ⫽ sinus node recovery time; cSNRT ⫽ rate-corrected SNRT; AERP ⫽ atrial effective refractory period; VERP ⫽ ventricular effective refractory period; AVERP ⫽ atrioventricular effective refractory period; AVWBCL ⫽ atrioventricular Wenckebach block cycle length; AV2:1CL ⫽ atrioventricular 2:1 conduction block cycle length; VAWBCL ⫽ retrograde ventriculo-atrial Wenckebach block cycle length. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.

riness. Homozygous Cx43-/- mice die shortly after birth with right ventricle outflow tract obstruction (Reaume et al., 1995). Surface ECG data in 5-day-old Cx43-/- mice exhibit advanced AV block (Vaidya et al., 2001), while double-deficient Cx43/40 mice have more diffuse conduction system disease and morphological abnormalities (Kirchhoff et al., 2000). Both ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C) also presented a reduced expression of Cx43 and Cx40. However, connexin 43 downregulation was already evident at 3 weeks of age in ␣-MHC-TG(wild), while it was at ⬃6 weeks of age in ␣-MHC-TG(⌬C) (Kasahara et al., 2003). These observations are congruent with the cardiac electrophysiological phenotypes in these two different tg mice, as both demonstrate AV conduction defects, but ␣-MHC-TG(wild) manifest the phenotype earlier than those of ␣-MHC-TG(⌬C) mice. Histopathological Analysis of Nkx2.5 Mutant Hearts On morphologic and histologic examination, no structural defects were identified in hearts of 2-week-old tg mice, and 6-week-old hearts had left atrial thrombi but no obvious structural defects. Although the conduction tissue appeared histologically normally organized and positioned, immunohistochemical techniques or specific markers of the conduction system such as lacZ-minK (Arad et al., 2003) were not utilized in this series. Therefore, subtle AV nodal histopathological deficiencies cannot be fully excluded. Postnatal Overexpression of Nonmutant and Transcriptionally Active Mutant Nkx2.5 Both transgenic mice with postnatal overexpression of wildtype nonmutant and transcriptionally active mutant Nkx2.5 under ␣-MHC promoter exhibited almost identical cardiac EP features, that is, prolonged AV conduction

interval due to supraHisian delay and prolonged AV nodal conduction properties. The expression of transgene protein in the murine heart is heterogeneous, and the reduced expression of Cx43 was seen in the transgene protein positive cells. The later onset of reduced Cx43 expression in the transcriptionally active mutant protein-overexpressing mice leads to delayed onset of AV conduction block compared to the wildtype proteinoverexpressing mice. Cx40 expression was also affected by the overexpression of these proteins. Both connexin 40 and 43 downregulation was confirmed in cultured cardiomyocytes infected with adenovirus encoding wildtype Nkx2.5 (Kasahara et al., 2003). Thus, the postnatal overexpression of nonmutant wildtype and transcriptionally active mutant Nkx2.5 may also play an important role in inhibition of conduction system with downregulation of Cx43 and Cx40. However, as these tg genes are expressed heterogeneously in the murine heart, the reduced expression of Cx43 and Cx40 is only partial. Therefore, they may alter the other connexins or these tg proteins could have other still unidentified functions. Limitations and Summary Currently, there is not a known human disease that is thought to be caused by overexpression or constitutively active mutations of Nkx2.5. However, human Nkx2.5 mutations are associated with specific congenital heart diseases and AV conduction abnormalities. These transgenic mouse models and in vivo mouse EP testing may not be relevantly extrapolated to human clinical diseases, but provide a mechanism for hypothesis-testing and experimental design of genetically modeled pathophysiology. Postnatal overexpression of wildtype Nkx2.5 or transcriptionally active mutant Nkx2.5 in murine hearts is associated with distinct abnormalities in cardiac conduction properties, potentially modulated at least in part via

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with that of endogenous Nkx2.5 expression was reported previously (Kasahara et al., 2003). As described, we have a single transgenic line for each construct that is transmitted germline and has a lower mosaicism. Female ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C) mice and their age-matched littermate wildtypes (wt), all FVB background, underwent surface ECG and in vivo endocardial electrophysiology (EP) study (Berul et al., 1996). In ␣-MHC-TG(wild) and wt mice, ambulatory ECG recordings were also performed with wireless implantable telemetry (“Holter ECG”) recordings. Mice were anesthetized by intraperitoneal administration of chloral hydrate (0.4 mg/g). Bupivacaine 0.25% was infiltrated subcutaneously for local anesthesia. All animal care protocols conformed to the Association for the Assessment and Accreditation of Laboratory Animal Care, with approvals from the Institutional Animal Care and Use Committees.

FIG. 2. Death electrocardiographically correlated with bradycardia. Segments of the telemetered surface ECG of a wildtype transgenic Nkx2.5 mouse that died suddenly during continuous ambulatory monitoring, illustrating progressive bradycardia leading to asystole. The lead I ECG is continuously recorded and representative samples 5 min apart are displayed. This reveals junctional bradycardia associated with sudden death rather than progressive atrioventricular conduction block or tachyarrhythmia as the mechanism for death.

downstream effects on connexin isoform inhibition. These defects in AV conduction persist postnatally and into adulthood. MATERIALS AND METHODS Mouse Preparation We generated transgenic mice expressing FLAG-tagged wildtype (TG-wild), a putative gain-of-function mutant of Nkx2.5 (TG-⌬C: deletion of 88 amino acids of the carboxyl-terminus of Nkx2.5) (Chen and Schwartz, 1995; Lee et al., 1998; Kasahara and Izumo, 1999) under control of the cardiac-specific ␣-MHC promoter (Gulick et al., 1991). The levels of transgene expression compared

Surface 6-Lead ECG and In Vivo Electrophysiological Study Protocols The surface frontal-plane 6-lead ECG recordings and the in vivo EP studies were performed at 6 weeks of age in ␣-MHC-TG(wild) (7 tg, 9 wt), and serially at 6 and 10 weeks of age in ␣-MHC-TG(⌬C) (7 tg, 8 wt) and their age-matched littermate controls. ECG recordings were obtained from 25 gauge subcutaneous electrodes in each limb. Rate-corrected QT interval (QTc) was calculated by a formula appropriate for mice HR (Mitchell et al., 1998). After surface ECG recordings, in vivo EP studies were performed in each mouse. A similar protocol described previously was used for the in vivo EP studies (Berul et al., 1997; Maguire et al., 2000; Wakimoto et al., 2001). Briefly, for simultaneous atrial and ventricular pacing and recording, a 2 French octapolar catheter with an interelectrode interval of 0.5 mm (CIBer mouse EP; NuMed, Nicholville, NY) was inserted via a jugular vein cutdown approach. Standard pacing protocols were used to determine the EP parameters as described below. For sinus nodal evaluation, sinus node recovery time (SNRT) and rate-corrected SNRT (CSNRT) were determined. Tissue refractoriness data were obtained from atrium, AV node, and ventricle (AERP, AVERP, and VERP). AV nodal conduction properties evaluated were the longest pacing cycle length for Wenckebach type and 2:1 AV conduction (AVWBCL, AV2:1CL) and the retrograde Wenckebach ventriculoatrial (V-A) conduction (VAWBCL). Telemetry Implantation and Ambulatory ECG Recordings in Conscious Mice For ambulatory ECG analysis in conscious unrestrained mice, 3.5-g wireless radiofrequency telemetry devices (DataSciences International, St. Paul, MN) were implanted using a similar technique as previously reported (Gehrmann et al., 2000) to acquire ECG analogous to a surface lead I recording. After 72 h recovery time from surgical instrumentation, ECG recordings were performed in each mouse placed in a separate cage overlying a receiver (DataSciences International).

CARDIAC CONDUCTION IN Nkx2.5 TRANSGENIC MICE

Data Acquisition ECG channels were filtered between 0.5 and 400 Hz. Intracardiac electrograms were filtered between 5 and 400 Hz, at an acquisition rate of 2,000 samples per second. Surface ECG and the intracardiac recordings were displayed on an oscilloscope and simultaneously recorded to computer through an analog to digital converter (MacLab Systems, Milford, MA) for detailed analysis and measurement. Two independent observers, who did not know the genotype of the mouse, performed all ECG and EP parameter measurements. Statistical Analysis Values are presented as the mean ⫾ 1 standard deviation (SD). Surface ECG and intracardiac conduction parameters were measured by two independent observers and compiled for statistical interpretation. A two-tailed t-test was used in comparisons of EP data between the tg mice and wt mice, with a P value ⬍0.05 considered significant. Histology and Morphology Hearts were excised from 2-week-old and 8-week-old ␣-MHC-TG(wild) and ␣-MHC-TG(⌬C) transgenic mice, washed in 37°C Dulbecco’s 1⫻PBS and fixed in 4% paraformaldehyde/PBS. Intact hearts were sectioned in the sagittal plane in 5 ␮M sections through the atrioventicular canal. Slides were stained with hematoxolin and eosin and analyzed on a Leica DMR compound microscope. Hearts were analyzed by an experienced cardiac pathologist blinded to genotype. LITERATURE CITED Arad M, Moskowitz IPG, Patel VV, Ahmad F, Perez AR, Sawyer DB, Walter M, Li GH, Burgon PG, Maguire CT, Stapleton D, Schmitt JP, Guo XX, Pizard A, Kupershmidt S, Roden DM, Berul CI, Seidman CE, Seidman JG. 2003. Transgenic mice overexpressing mutant PRKAG2 define the cause of Wolff-Parkinson-White syndrome in glycogen storage cardiomyopathy. Circulation 107:2850 –2856. Benson W, Silberbach GM, Kavanaugh-McHugh A, Cottrill C, Zhang Y, Riggs S, Smalls O, Johnson MC, Watson MS, Seidman JG, Seidman CE, Plowden J, Kugler JD. 1999. Mutations in the cardiac transcription factor Nkx2-5 affect diverse cardiac developmental pathways. J Clin Invest 104:1567–1573. Berul CI, Aronovitz MJ, Wang PJ, Mendelsohn ME. 1996. In vivo cardiac electrophysiology studies in the mouse. Circulation 94:2641– 2648. Berul CI, Christe ME, Aronovitz MJ, Seidman CE, Seidman JG, Mendelsohn ME. 1997. Electrophysiological abnormalities and arrhythmias in alpha MHC mutant familial hypertrophic cardiomyopathy mice. J Clin Invest 99:570 –576. Bevilacqua LM, Simon AM, Maguire CT, Gehrmann J, Wakimoto H, Paul DL, Berul CI. 2000. A targeted disruption in connexin40 leads to distinct atrioventricular conduction defects. J Interv Card Electrophysiol 4:459 – 467. Chen CY, Schwartz RJ. 1995. Identification of novel DNA binding targets and regulatory domains of a murine tinman homeodomain factor, Nkx-2.5. J Biol Chem 270:15628 –15633. Gehrmann J, Hammer PE, Maguire CT, Wakimoto H, Triedman JK, Berul CI. 2000. Phenotypic screening for heart rate variability in the mouse. Am J Physiol Heart Circ Physiol 279:H733–740. Goldmuntz E, Geiger E, Benson DW. 2001. NKX2.5 mutations in patients with tetralogy of Fallot. Circulation 104:2565–2568.

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