Cardiomyocyte-specific overexpression of the ubiquitin ligase Wwp1 contributes to reduction in Connexin 43 and arrhythmogenesis.

Gap junctions (GJ) are intercellular channels composed of connexin subunits that play a critical role in a diverse number of cellular processes in all tissue types. In the heart, GJs mediate electrical coupling between cardiomyocytes and display mislocalization and/or downregulation in cardiac disease (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is Connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a ß-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and -independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.

Dual-source CT imaging to plan transcatheter aortic valve replacement: accuracy for diagnosis of obstructive coronary artery disease.

PURPOSE: To assess the accuracy of computed tomographic (CT) examinations performed for the purpose of transcatheter aortic valve replacement (TAVR) planning to diagnose obstructive coronary artery disease (CAD). MATERIALS AND METHODS: With institutional review board approval, waivers of informed consent, and in compliance with HIPAA, 100 consecutive TAVR candidates (61 men, mean age 79.6 years ± 9.9) who underwent both TAVR planning CT (with a dual-source CT system) and coronary catheter (CC) angiographic imaging were retrospectively analyzed. At both modalities, the presence of stenosis in the native coronary arteries was assessed. Additionally, all coronary bypass grafts were rated as patent or occluded. With CC angiographic imaging as the reference standard, the accuracy of CT for lesion detection on a per-vessel and per-patient basis was calculated. The accuracy of CT for the assessment of graft patency was also analyzed. RESULTS: For per-vessel and per-patient analysis for the detection of stenosis that was 50% or more in the native coronary arteries, CT imaging had, respectively, 94.4% and 98.6% sensitivity, 68.4% and 55.6% specificity, 94.7% and 93.8% negative predictive value (NPV), and 67.0% and 85.7% positive predictive value. Per-patient sensitivity of stenosis 50% or greater with CT for greater than 70% stenosis at CC angiographic imaging was 100%. All 12 vessels in which percutaneous coronary intervention was performed were correctly identified as demonstrating stenosis 50% or greater with CT. There was agreement between CT and CC angiographic imaging regarding graft patency in 114 of 115 grafts identified with CC angiographic imaging. CONCLUSION: TAVR planning CT has high sensitivity and NPV in excluding obstructive CAD. An additional preprocedural CC angiographic examination may not be required in TAVR candidates with a CT examination that does not show obstructive CAD.

Incremental value of pharmacological stress cardiac dual-energy CT over coronary CT angiography alone for the assessment of coronary artery disease in a high-risk population.

OBJECTIVE: The purpose of this article is to prospectively determine the value of stress dual-energy CT (DECT) myocardial perfusion imaging to coronary CT angiography (CTA) for the assessment of coronary artery disease (CAD) in a high-risk population. SUBJECTS AND METHODS: We prospectively enrolled 29 consecutive patients who were referred for cardiac SPECT examinations for known or suspected CAD to also undergo pharmacologic stress cardiac DECT. In 25 patients, cardiac catheterization was available as the reference standard for morphologically significant stenosis. The performance of coronary CTA alone, DECT myocardial perfusion alone, and the combination of both was assessed by calculating sensitivity, specificity, and AUC values. RESULTS: For morphologically significant stenosis, coronary CTA alone and myocardial DECT assessment alone had 95% sensitivity and 50% specificity. The combined approach yielded 100% sensitivity and 33% specificity if either was positive and 90% sensitivity and 67% specificity if both were positive. The AUC value was highest (0.78) if both were positive. For hemodynamically significant lesions, coronary CTA alone had 91% sensitivity and 38% specificity, and DECT alone had 95% sensitivity and 75% specificity. The combined approach yielded 100% sensitivity and 38% specificity if either was positive and 86% sensitivity and 75% specificity if both were positive. AUC values were highest for DECT alone (0.85) and the "both positive" evaluation (0.80). CONCLUSION: The combined analysis of coronary CTA and DECT myocardial perfusion reduces the number of false-positives in a high-risk population for CAD and outperforms the purely anatomic test of coronary CTA alone for the detection of morphologically and hemodynamically significant CAD.

Image quality and radiation dose of low tube voltage 3rd generation dual-source coronary CT angiography in obese patients: a phantom study.

OBJECTIVES: To assess the influence of tube potential on radiation dose and image quality of third-generation dual-source coronary CT angiography (CTA) in a phantom simulating an obese patient. METHODS: A thoracic phantom was equipped with tubular inserts containing iodine solution and water. A soft-tissue-equivalent ring around the phantom simulated an obese patient. Images were acquired at tube potentials of 80, 100, 120 and 140 kV with second-generation dual-source CT (DSCT) and 70-150 kV (in 10-kV increments) with third-generation DSCT. Contrast-to-noise ratio (CNR) was calculated and CT dose index was recorded. RESULTS: With second-generation DSCT, CNR was highest for 120 kV (19.0) and decreased with lower tube potential (12.0 at 80 kV) owing to disproportionately increased image noise. With third-generation DSCT, 70- and 80-kV acquisitions showed a smaller increase in noise. CNRs for third-generation DSCT were highest for 70 and 80 kV (21.1 and 21.2, respectively). Compared to 120 kV, radiation dose was 68% and 49% lower at 70 kV and 80 kV, respectively. CONCLUSION: Third-generation DSCT enables one to perform coronary CTA at 70-80 kV in obese patients without compromising CNR and thus reduces radiation dose by 49-68%. KEY POINTS: • Low tube potential CT angiography is currently not suitable for obese patients. • Third-generation DSCT offers substantially increased tube power at low tube potential. • This enables one to perform coronary CT angiography at 70-80 kV in obese patients. • Signal-to-noise ratio is maintained owing to increased tube current. • This approach can be expected to reduce radiation dose by 49-68%.

Developmental basis for filamin-A-associated myxomatous mitral valve disease.

AIMS: We hypothesized that the structure and function of the mature valves is largely dependent upon how these tissues are built during development, and defects in how the valves are built can lead to the pathological progression of a disease phenotype. Thus, we sought to uncover potential developmental origins and mechanistic underpinnings causal to myxomatous mitral valve disease. We focus on how filamin-A, a cytoskeletal binding protein with strong links to human myxomatous valve disease, can function as a regulatory interface to control proper mitral valve development. METHODS AND RESULTS: Filamin-A-deficient mice exhibit abnormally enlarged mitral valves during foetal life, which progresses to a myxomatous phenotype by 2 months of age. Through expression studies, in silico modelling, 3D morphometry, biochemical studies, and 3D matrix assays, we demonstrate that the inception of the valve disease occurs during foetal life and can be attributed, in part, to a deficiency of interstitial cells to efficiently organize the extracellular matrix (ECM). This ECM organization during foetal valve gestation is due, in part, to molecular interactions between filamin-A, serotonin, and the cross-linking enzyme, transglutaminase-2 (TG2). Pharmacological and genetic perturbations that inhibit serotonin-TG2-filamin-A interactions lead to impaired ECM remodelling and engender progression to a myxomatous valve phenotype. CONCLUSIONS: These findings illustrate a molecular mechanism by which valve interstitial cells, through a serotonin, TG, and filamin-A pathway, regulate matrix organization during foetal valve development. Additionally, these data indicate that disrupting key regulatory interactions during valve development can set the stage for the generation of postnatal myxomatous valve disease.

Remodeling of the peripheral cardiac conduction system in response to pressure overload.

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% (P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression (P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS (P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS.

GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling.

This study examined transgenic mice whose expression of a ß-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that <1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial ß-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states.

SPARC regulates collagen interaction with cardiac fibroblast cell surfaces.

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

Inhibition of renin-angiotensin system (RAS) reduces ventricular tachycardia risk by altering connexin43.

Renin-angiotensin system (RAS) activation is associated with arrhythmias. We investigated the effects of RAS inhibition in cardiac-specific angiotensin-converting enzyme (ACE) overexpression (ACE 8/8) mice, which exhibit proclivity to ventricular tachycardia (VT) and sudden death because of reduced connexin43 (Cx43). ACE 8/8 mice were treated with an ACE inhibitor (captopril) or an angiotensin receptor type-1 blocker (losartan). Subsequently, electrophysiological studies were performed, and the hearts were extracted for Cx43 quantification using immunoblotting, immunohistochemistry, fluorescent dye spread method, and sodium current quantification using whole cell patch clamping. VT was induced in 12.5% of captopril-treated ACE 8/8 and in 28.6% of losartan-treated mice compared to 87.5% of untreated mice (P < 0.01). Losartan and captopril treatment increased total Cx43 2.4-fold (P = 0.01) and the Cx43 phosphorylation ratio 2.3-fold (P = 0.005). Treatment was associated with a recovery of gap junctional conductance. Survival in treated mice improved to 0.78 at 10 weeks (95% confidence interval 0.64 to 0.92), compared to the expected survival of less than 0.50. In a model of RAS activation, arrhythmic risk was correlated with reduced Cx43 amount and phosphorylation. RAS inhibition resulted in increased total and phosphorylated Cx43, decreased VT inducibility, and improved survival.

A peptide mimetic of the connexin43 carboxyl terminus reduces gap junction remodeling and induced arrhythmia following ventricular injury.

RATIONALE: Remodeling of connexin (Cx)43 gap junctions (GJs) is linked to ventricular arrhythmia. OBJECTIVES: A peptide mimetic of the carboxyl terminal (CT) of Cx43, incorporating a postsynaptic density-95/disks-large/ZO-1 (PDZ)-binding domain, reduces Cx43/ZO-1 interaction and GJ size remodeling in vitro. Here, we determined: (1) whether the Cx43-CT mimetic αCT1 altered GJ remodeling following left ventricular (LV) injury in vivo; (2) whether αCT1 affected arrhythmic propensity; and (3) the mechanism of αCT1 effects on arrhythmogenicity and GJ remodeling. METHODS AND RESULTS: A cryoinjury model generating a reproducible wound and injury border zone (IBZ) in the LV was used. Adherent methylcellulose patches formulated to locally release αCT1 (< 48 hours) were placed on cryoinjuries. Relative to controls, Cx43/ZO-1 colocalization in the IBZ was reduced by αCT1 by 24 hours after injury. Programmed electric stimulation ex vivo and optical mapping of voltage transients indicated that peptide-treated hearts showed reduced inducible arrhythmias and increased ventricular depolarization rates 7 to 9 days after injury. At 24 hours and 1 week after injury, αCT1-treated hearts maintained Cx43 in intercalated disks (IDs) in the IBZ, whereas by 1 week after injury, controls demonstrated Cx43 remodeling from IDs to lateralized distributions. Over a postinjury time course of 1 week, αCT1-treated IBZs showed increased Cx43 phosphorylation at serine368 (Cx43-pS368) relative to control tissues. In biochemical assays, αCT1 promoted phosphorylation of serine368 by protein kinase (PK)C-ε in a dose-dependent manner that was modulated by, but did not require ZO-1 PDZ2. CONCLUSIONS: αCT1 increases Cx43-pS368 in vitro in a PKC-ε-dependent manner and in the IBZ in vivo acutely following ventricular injury. αCT1-mediated increase in Cx43-pS368 phosphorylation may contribute to reductions in inducible-arrhythmia following injury.

Notch signaling regulates murine atrioventricular conduction and the formation of accessory pathways.

Ventricular preexcitation, which characterizes Wolff-Parkinson-White syndrome, is caused by the presence of accessory pathways that can rapidly conduct electrical impulses from atria to ventricles, without the intrinsic delay characteristic of the atrioventricular (AV) node. Preexcitation is associated with an increased risk of tachyarrhythmia, palpitations, syncope, and sudden death. Although the pathology and electrophysiology of preexcitation syndromes are well characterized, the developmental mechanisms are poorly understood, and few animal models that faithfully recapitulate the human disorder have been described. Here we show that activation of Notch signaling in the developing myocardium of mice can produce fully penetrant accessory pathways and ventricular preexcitation. Conversely, inhibition of Notch signaling in the developing myocardium resulted in a hypoplastic AV node, with specific loss of slow-conducting cells expressing connexin-30.2 (Cx30.2) and a resulting loss of physiologic AV conduction delay. Taken together, our results suggest that Notch regulates the functional maturation of AV canal embryonic myocardium during the development of the specialized conduction system. Our results also show that ventricular preexcitation can arise from inappropriate patterning of the AV canal-derived myocardium.

ZO-1 determines adherens and gap junction localization at intercalated disks.

The disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.

Cardiac expression patterns of endothelin-converting enzyme (ECE): implications for conduction system development.

The spatiotemporal distribution of the endothelin-converting enzyme (ECE) protein in the embryonic chick heart and the association of this polypeptide with the developing cardiac conduction system is described here for the first time. Further, we show how cardiac hemodynamic load directly affects ECE level and distribution. Endothelin (ET) is a cytokine involved in the inductive recruitment of Purkinje fibers. ET is produced by proteolytic cleavage of Big-ET by ECE. We generated an antibody against chick ECE recognizing a single band at approximately 70 kD to correlate the cardiac expression of this protein with that reported previously for its mRNA. ECE protein expression was more widespread compared to its mRNA, being present in endothelial cells, mesenchymal cells, and myocytes, and particularly enriched in the trabeculae and nascent ventricular conduction system. The myocardial expression was significantly modified under experimentally altered hemodynamic loading. In vivo, ET receptor blockade with bosentan delayed activation sequence maturation. These data support a role for ECE in avian cardiac conduction system differentiation and maturation.

Differentiation of cardiac Purkinje fibers requires precise spatiotemporal regulation of Nkx2-5 expression.

Nkx2-5 gene mutations cause cardiac abnormalities, including deficits of function in the atrioventricular conduction system (AVCS). In the chick, Nkx2-5 is elevated in Purkinje fiber AVCS cells relative to working cardiomyocytes. Here, we show that Nkx2-5 expression rises to a peak as Purkinje fibers progressively differentiate. To disrupt this pattern, we overexpressed Nkx2-5 from embryonic day 10, as Purkinje fibers are recruited within developing chick hearts. Overexpression of Nkx2-5 caused inhibition of slow tonic myosin heavy chain protein (sMHC), a late Purkinje fiber marker but did not affect Cx40 levels. Working cardiomyocytes overexpressing Nkx2-5 in these hearts ectopically up-regulated Cx40 but not sMHC. Isolated embryonic cardiomyocytes overexpressing Nkx2-5 also displayed increased Cx40 and suppressed sMHC. By contrast, overexpression of a human NKX2-5 mutant did not effect these markers in vivo or in vitro, suggesting one possible mechanism for clinical phenotypes. We conclude that a prerequisite for normal Purkinje fiber maturation is precise regulation of Nkx2-5 levels.

Transcriptional regulation of cardiac conduction system development: 2004 FASEB cardiac conduction system minimeeting, Washington, DC.

The development of the complex network of specialized cells that form the atrioventricular conduction system (AVCS) during cardiac morphogenesis occurs by progressive recruitment within a multipotent cardiomyogenic lineage. Understanding the molecular control of this developmental process has been the focus of recent research. Transcription factors representative of multiple subfamilies have been identified and include members of zinc-finger subfamilies (GATA4, GATA6 HF-1b), skeletal muscle transcription factors (MyoD), T-box genes (Tbx5), and also homeodomain transcription factors (Msx2 and Nkx2.5). Mutations in some of these transcription factors cause congenital heart disease and are associated with cardiac abnormalities, including deficits within the AVCS. Mouse models that closely phenocopy known human heart disease provide powerful tools for the study of molecular effectors of AVCS development. Indeed, investigations of the Nkx2.5 haploinsufficient mouse have shown that peripheral Purkinje fibers are significantly underrepresented. This piece of data corroborates our previous work showing in chick, mouse, and humans that Nkx2.5 is elevated in the differentiating AVCS relative to adjacent working ventricular myocardial tissues. Using the chick embryo as a model, we show that this elevation of Nkx2.5 is transient in the network of conduction cells comprising the peripheral Purkinje fiber system. Functional studies using defective adenoviral constructs, which disrupt the normal variation in level of this gene, result in perturbations of Purkinje fiber phenotype. Thus, the precise spatiotemporal regulation of Nkx2.5 levels during development may be required for the progressive emergence of gene expression patterns specific to differentiated Purkinje fiber cells.

Function follows form: cardiac conduction system defects in Nkx2-5 mutation.

Mutations of Nkx2-5 cause congenital heart disease and atrioventricular block in man. The altered expression of an electrophysiologic protein regulated by Nkx2-5 was originally presumed to cause the conduction defect, but when no such protein was found, an alternative hypothesis was considered. In pediatric patients, the association of certain cardiac malformations with congenital atrioventricular block suggests that errors in specific developmental pathways could cause both an anatomic and a physiologic defect. We therefore hypothesized that Nkx2-5 insufficiency perturbs the conduction system during development, which in turn manifests as a postnatal conduction defect. Experimental results from Nkx2-5 knockout mouse models support the developmental hypothesis. Hypoplasia of the atrioventricular node, His bundle, and Purkinje system can explain in whole or in part specific conduction and electrophysiologic defects present in Nkx2-5 haploinsufficiency.

Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system.

Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause atrioventricular conduction defects in humans by unknown mechanisms. We show in KO mice that the number of cells in the cardiac conduction system is directly related to Nkx2-5 gene dosage. Null mutant embryos appear to lack the primordium of the atrioventricular node. In Nkx2-5 haploinsufficiency, the conduction system has half the normal number of cells. In addition, an entire population of connexin40(-)/connexin45(+) cells is missing in the atrioventricular node of Nkx2-5 heterozygous KO mice. Specific functional defects associated with Nkx2-5 loss of function can be attributed to hypoplastic development of the relevant structures in the conduction system. Surprisingly, the cellular expression of connexin40, the major gap junction isoform of Purkinje fibers and a putative Nkx2-5 target, is unaffected, consistent with normal conduction times through the His-Purkinje system measured in vivo. Postnatal conduction defects in Nkx2-5 mutation may result at least in part from a defect in the genetic program that governs the recruitment or retention of embryonic cardiac myocytes in the conduction system.