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Originally Published 29 May 2012
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Epistatic Rescue of Nkx2.5 Adult Cardiac Conduction Disease Phenotypes by Prospero-Related Homeobox Protein 1 and HDAC3

Catherine A. Risebro, Louisa K. Petchey, Nicola Smart, John Gomes, James Clark, Joaquim M. Vieira, Joseph Yanni, … Show All … , Halina Dobrzynski, Sean Davidson, Zia Zuberi, Andrew Tinker, Bo Shui, Yvonne I. Tallini, Michael I. Kotlikoff, Lucile Miquerol, Robert J. Schwartz, and Paul R. RileyAuthor Info & Affiliations
Circulation Research
Volume 111, Number 2
https://doi.org/10.1161/CIRCRESAHA.111.260695
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Abstract

Rationale:

Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias (“sudden cardiac death”) that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5.

Objective:

To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5-dependent adult congenital heart disease.

Methods and Results:

A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5Cre/+; Prox1loxP/+ animals versus Nkx2.5Cre/+ controls. Anatomic studies, on a Cx40EGFP background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5Cre/+ littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca2+ handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction.

Conclusions:

Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.

Introduction

The cardiac conduction system comprises a network of specialized cardiomyocytes that generate and propagate electric impulses throughout the muscle of the heart, punctuated by nodal tissue, to collectively pace and stimulate the synchronized contraction of the atria and ventricles and facilitate unidirectional blood flow. A number of cardiac transcription factors have been implicated in the maintenance of conduction system structure and function (reviewed in Ref. 1), of which Nkx2.5 is arguably the most well studied. However, despite a causative role for NKX2.5 in conduction disease and a substantial focus on Nkx2.5 in both the developing2–5 and postnatal conduction system,6–8 there have been no reports yielding insight into upstream modifier effects on Nkx2.5 activity or physiologically relevant factors identified to rescue Nkx2.5/NKX2.5 function in disease.
The adult Nkx2.5 heterozygous mouse (Nkx2.5Cre/+) presents with background-dependent phenotypes, including atrial septal defects and atrioventricular (AV) conduction delay, which precisely mimic the clinical symptoms in humans with pathological mutations in NKX2.5.9 Thus the mouse, in this instance, represents a bona fide model to study the manifestation of NKX2.5-derived human adult congenital heart disease. As such we focused on candidate factors that may regulate Nkx2.5 function in this model, in the context of modifying the disease phenotype, to provide insight into pathways for potential therapeutic targeting. Prox1 is a homeobox factor we have previously shown plays a critical role in maintaining muscle structure in the developing heart.10 Here we show that Prox1 is coexpressed with Nkx2.5 in the specialized cardiomyocytes of the conduction system and negatively regulates Nkx2.5 expression to ensure the anatomic integrity of mature atrial and ventricular conduction components, notably the atrioventricular node (AVN) and His-Purkinje network. Functional electrocardiography (ECG) parameters indicated a Prox1 mediated compensatory response to overcome PR delay indicative of AV block and chromatin immunoprecipitation (ChIP) with cotransfection reporter assays revealed Prox1 and HDAC3 as direct corepressors of Nkx2.5. These data define Prox1 as a novel modifier of Nkx2.5 function, acting in epistasis to maintain adult cardiac conduction preventing arrhythmia and conduction system disease.

Methods

An expanded Methods section is provided in the online-only Data Supplement.
Briefly, measurements of left ventricular function were attained by pressure-volume loop catheterization and micromanometry. Macroscopic examination of the internal surface of the ventricles and quantification of the number of enhanced green fluorescent protein (EGFP)+ cells was described previously.7 The location of the AVN was confirmed by immunofluorescence on adjacent heart sections to those used for Picro-Sirius red staining through the AVN and its surrounding atrial and ventricular muscle; AV node size was quantified by measuring relative hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) expression. A lead II ECG was recorded using Powerlab and Chart software v5.0 (AD Instruments, Oxford, UK) and the PR interval was calculated by averaging 2 minutes of steady state anesthetized ECGs. Cx40BAC-GCaMP2 mice were used to obtain atrial and ventricular Ca2+ transient recordings in the Purkinje fibers of the heart. Total RNA was extracted using Trizol and cDNA was synthesized using SuperscriptIII reverse transcriptase (Invitrogen). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using SYBR Green (Applied Biosystems) and Applied Biosystems 7900 Real-Time PCR System. Chromatin immunoprecipitation was carried out on adult hearts as described previously10 with minor modifications. NIH3T3 and H9c2 cell culture was carried out under standard conditions and transfections were carried out using Effectene (Promega) or Lipofectamine 2000 (Invitrogen).

Results

Defects in Cardiac Function in Nkx2.5 Heterozygotes Are Rescued by Prox1 Haploinsufficiency

In our previous study we demonstrated that cardiac-specific loss-of-function of Prox1 in Nkx2.5Cre/+;Prox1loxP/loxP mice resulted in embryonic muscle ultrastructure defects and specifically failed maintenance of sarcomeric integrity during heart development.10 Given the loss-of-function phenotype, we subsequently went on to examine cardiac function in Nkx2.5Cre/+;Prox1loxP/+ compound adult heterozygote mice to determine whether they might be compromised by loss of a single copy of Prox1. Here we used pressure-volume loop catheterization and micromanometry11 and observed that although Nkx2.5Cre/+ mice had significantly altered cardiac output there was little difference between the compound heterozygotes and the floxed-control animals (Online Table I). A potential caveat was that Nkx2.5Cre/+ mice had significantly reduced heart rates that might be symptomatic of overanesthesia in this group leading to adverse effects on the pressure-volume relationship. However, repeat pressure-volume analyses, on 6 to 8 mice per genotype over time, revealed heart rate to be significantly lower in Nkx2.5Cre/+ mice and, moreover, all functional parameters assessed including heart rate were rescued in Nkx2.5Cre/+;Prox1loxP/+ mice. This result led us to speculate that the loss of a single copy of Prox1 may rescue defects arising from Nkx2.5 haploinsufficiency. Importantly, to rule out genetic background effects as attributable to phenotypic variation, the Prox1loxP/+ and Nkx2.5Cre/+ parent strains were maintained on identical genetic backgrounds and the pressure-volume study and all subsequent analyses across the 3 genotypes (Prox1loxP/+, Nkx2.5Cre/+, and Nkx2.5Cre/+;Prox1loxP/+) were carried out on littermates within each experiment.

Hypoplasia of the Purkinje Fiber Network in Nkx2.5 Heterozygotes Is Rescued by Prox1 Haploinsufficiency

To investigate the potential for epistasis at a phenotypic level, we initially examined adult hearts for atrial septal and valvular defects, associated with Nkx2.5 heterozygosity (Nkx2.5 targeting generated by insertion of GFP into exon 1)12 but failed to identify any structural anomalies of this type (not shown), presumably because of genetic background differences as previously reported.12 In addition, we also checked for any evidence of ventricular myocardial defects and/or progressive cardiomyopathy in our Nkx2.5Cre/+ mice as has been previously reported (ventricular-restricted Nkx2.5 homozygous knock-down by MLC2vCre)4 alongside potential rescue in the Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes. No obvious myocardial defects or myopathy were observed within the ventricles of the Nkx2.5Cre/+ or Nkx2.5Cre/+;Prox1loxP/+ mice at the stages (12–16 weeks of age) examined throughout this study (data not shown).
Given that Nkx2.5 has also been shown to function in both the developing and adult cardiac conduction system we initially confirmed that Prox1 is expressed in cardiomyocytes of the conduction system in both the AVN and His-Purkinje fibers (PF) (Online Figure IA–ID) and subsequently sought to determine an effect of Prox1 on Nkx2.5-mediated cardiac conduction as underpinning the functional epistasis, observed from the pressure-volume micromanometry studies (Online Table I). To visualize the His-PF network, we crossed the Prox1loxP/+ and Nkx2.5Cre/+ strains with a Cx40EGFP strain.13 Comparisons across Nkx2.5Cre/+ or Nkx2.5Cre/+;Prox1loxP/+ genotypes, following crosses with Cx40EGFP mice, were made between littermates to ensure identical genetic background. The PFs form a network of elliptical arrangements over the internal surface of the ventricles. In both the left and right ventricles, Nkx2.5Cre/+ hearts displayed hypoplasia of the network demonstrated by fewer EGFP+ fibers compared to Prox1loxP/+ controls and Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes (Figure 1A–1F). To quantify the PF hypoplasia in Nkx2.5Cre/+ and rescue in Nkx2.5Cre/+;Prox1loxP/+ hearts, transverse sections at 3 levels through the heart (apex, middle, and base; n=4 hearts per genotype) were counterstained with wheat germ agglutinin-Alexa594 to demarcate cell membranes and the number of EGFP+ cells counted.7 In accordance with previous studies on Nkx2.5 heterozygous mice, in which the entire Nkx2.5 coding sequence was replaced with LacZ,7 we observed fewer EGFP+ cells in Nkx2.5Cre/+ hearts compared to controls at all 3 levels (percentage decrease: base, 50%; mid, 65%; apex, 75%), whereas in the Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes there was only a 30% decrease in EGFP+ cells at all 3 levels relative to controls (Figure 1G–1I; P≤0.05, P≤0.001, and P≤0.0001 for base, mid, and apex, respectively; Figure 1J). Furthermore, when branch points were analyzed within the His-Purkinje system there was a significant reduction in branching in the Nkx2.5Cre/+ hearts relative to Prox1loxP/+ control (P≤0.005), which was rescued in the Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes (P≤0.02; Figure 1K). Importantly, we determined the specificity of the knock-down of Prox1 with respect to Nkx2.5 heterozygosity by examining the hearts of Mlc2vCre/+;Prox1loxP/+ mice against the respective control Prox1loxP/+ and Mlc2vCre/+ strains. Mlc2vCre/+ mice express Cre recombinase throughout the ventricular cardiomyocytes from E8.5 onwards and also throughout the AV canal.14 No defects were observed either in the Mlc2vCre/+ or Mlc2vCre/+;Prox1loxP/+ mice at the level of the His-Purkinje conduction system (Online Figure IIA–IID), consistent with a restriction of the effects of Prox1 haploinsufficiency to the Nkx2.5 domain within the adult conduction system. This was confirmed by qRT-PCR analyses, which revealed no changes in Nkx2.5 or Cx40 (Gja5) expression between Mlc2vCre/+ or Mlc2vCre/+;Prox1loxP/+ mice (Online Figure IIE). Furthermore, a lack of phenotype in the Mlc2vCre/+;Prox1loxP/+ mice confirmed that the conduction system defects were not secondary to ventricular cardiomyocyte knock-down of Prox1 and associated ventricular dysfunction or cardiomyopathy.10
Figure 1. Hypoplasia of the Purkinje fiber network in Nkx2.5 heterozygotes is rescued by Prox1 haploinsufficiency. Whole mount Cx40-enhanced green fluorescent protein (EGFP) epifluorescence highlighting the Purkinje fiber (PF) network in Prox1loxP/+ (co; A, D), Nkx2.5Cre/+ (B, E) and Nkx2.5Cre/+;Prox1loxP/+ (C, F) hearts. The left and right bundle branches (LBB and RBB, respectively) and PF network in Nkx2.5Cre/+ hearts is hypoplastic in both left and right ventricles (B, E) compared to controls (A, D); however, in compound heterozygote hearts there is rescue of both the LBB and RBB and PF network (C, F; white arrowhead in (F) indicates the atrioventricular bundle absent in (E). Transverse cryosections through control (G), Nkx2.5Cre/+ (H), and Nkx2.5Cre/+; Prox1loxP/+ (I) hearts were stained with wheat germ agglutinin (WGA)-Alexa594 that demarcates cell membranes enabling determination of the number of EGFP+ cells. Fewer EGFP+ cells were seen in Nkx2.5Cre/+ hearts (H; white arrowheads) compared to controls (G) and Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes (I). J, Quantification at 3 distinct levels through the heart of rescue of Nkx2.5 haploinsufficiency by loss of Prox1 revealed significantly fewer EGFP+ cells in Nkx2.5 heterozygote hearts compared to controls that were significantly restored following knock-down of Prox1. K, Branch point quantification using Image J (inset panel for assigned branching) revealed a significant restoration in branching of the PF network following knock-down of Prox1 relative to Nkx2.5 heterozygotes. *P≤0.05, **P≤0.001, ***P≤0.0001; 1-way ANOVA with Bonferroni's multiple comparison test. Frontal sections at anatomically equivalent planes per genotype through adult hearts immunostained for Cx40 in Prox1loxP/+ (co; L), Nkx2.5Cre/+ (M) and Nkx2.5Cre/+;Prox1loxP/+ (N) hearts. Immunostaining of Prox1loxP/+ (co; L), Nkx2.5Cre/+ (M) and Nkx2.5Cre/+;Prox1loxP/+ (N) on a Cx40EGFP/+ background, revealed endogenous Cx40 protein expression was reduced in Nkx2.5Cre/+ hearts relative to controls (L, M) and this was rescued in Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes, consistent with rescue of the number of EGFP+ cells within the PF network (N). Higher magnification views of Cx40 staining confirmed rescue of endogenous protein levels (O, P, Q). co, control; My, myocardium. VS, ventricular septum. Scale bars: A–F, 500 μm; G–I, 50 μm; L–N, 100 μm; O–Q, 50 μm.

Atrioventricular Node Hypoplasia in Nkx2.5 Heterozygotes Is Rescued by Prox1 Haploinsufficiency

An additional, well-studied, conduction system defect caused by Nkx2.5 haploinsufficiency is hypoplasia of the AVN as documented following ventricular-restricted MLC2vCre homozygous knock-down and in animals in which Nkx2.5 was targeted by insertion of neomycin resistance cassette into intron 1 or replacement of the entire Nkx2.5 coding sequence with LacZ.4,5,15 The AVN is ovoid, located between the atria and the ventricles and isolated from chamber myocardium by a fibrous tissue layer. It is responsible for the delay (pacing) between atrial and ventricular excitation allowing complete filling of the ventricles for efficient cardiac function. HCN4 is the predominant pacemaking channel expressed in the cardiac conduction system including the AVN16 and is responsible for the hyperpolarization-activated current, If. A combination of positive HCN4 and negative Cx40 (not shown) and Cx43 immunofluorescence (HCN4+, Cx40−, Cx43−) staining17 (Figure 2) was used to precisely map the AVN within equivalent plane of heart sections across littermates of each genotype. We observed hypoplasia of the AVN and reduced expression of HCN4 in Nkx2.5Cre/+ hearts compared to controls (Figure 2A–2G) whereas in compound heterozygotes the AVN was similar to controls in size (relative area as determined by Image J measurements, n=3; Figure 2G), shape, and expression of HCN4 albeit with an apparent increase in myocyte density and more tightly packed collagen matrix (data not shown).
Figure 2. Hypoplasia of the atrioventricular node (AVN) in Nkx2.5 heterozygotes is rescued by Prox1 haploinsufficiency. Frontal sections through adult hearts, at equivalent anatomic planes, immunostained for HCN4 (A, C, E) highlighting the AVN in Prox1loxP/+ (co; A, B), Nkx2.5Cre/+ (C, D) and Nkx2.5Cre/+; Prox1loxP/+ (E, F) hearts (n=4 per genotype). Precise location of the AVN was confirmed by immunostaining of serial sections for Cx43 (B, D, F) and Cx40 (negative staining; not shown) as the major cardiac gap junction proteins highlighting surrounding non-nodal cells of the atrioventricular (AV) bundle. The AVN (HCN4+, Cx43−) is hypoplastic in Nkx2.5 heterozygote hearts (C) compared to controls (A); however, in compound heterozygote hearts there is significant rescue of the AVN in terms of both HCN4 expression (E) and size (relative area as determined by Image J assessment of HCN4 expression at equivalent plane of sections, n=3 per genotype; **P≤0.01, ***P≤0.001; 1-way ANOVA with Bonferroni's multiple comparison test (G). as, atrial septum; AU, arbitrary units; co, control; vs, ventricular septum. Scale bars: C, F, I, 200 μm.
These data collectively demonstrate that the deficient AVN morphology and anatomic hypoplasia of the His-Purkinje system in Nkx2.5 heterozygotes was rescued by a corresponding reduction in Prox1 expression; which, in turn, is indicative of a role for Prox1 in regulating Nkx2.5 activity during AV pacing and the maintenance of an integral adult AV conduction system.

Compensatory Electrocardiographical Parameters Following Knock-Down of Prox1

Given the anatomic phenotypes in our Nkx2.5Cre/+ mice and apparent rescue in the compound heterozygotes, we analyzed their respective conduction system function by surface ECG. We observed a prolonged PR interval consistent with 1st-degree AV block in the Nkx2.5Cre/+ mice and individual animals with Moblitz type 1 3:2 heart block (Wenkebach; Figure 3) both of which have specifically been documented as progressive clinical manifestation of NKX2.5 mutations in human patients.9 Other ECG parameters, such as QRS and QT intervals, were normal (Table), consistent with the specificity of the NKX2.5 electrophysiological phenotype at the level of the PR interval.9 No incidence of AV block was observed in either Prox1loxP/+ control, nor Nkx2.5Cre/+;Prox1loxP/+ compound heterozygote littermates (Figure 3A–3C). Statistical analysis across each genotype group revealed a significantly delayed PR interval (mean 0.0396 s; n=13; Figure 3D and Table) in the Nkx2.5Cre/+ animals compared to littermate controls (mean 0.0341s; n=13; P≤0.05; 1-way ANOVA) consistent with the individual examples of PR delay, AV block (Figure 3B, 3E), and extensive hypoplasia of both the PFs and AVN (Figure 1 and 2). In accordance with functional rescue, we observed PR intervals in compound heterozygotes (mean 0.0351 s; n=13) that were statistically equivalent to that of control animals and significantly shortened relative to the Nkx2.5Cre/+ animals (Figure 3D; P≤0.05; 1-way ANOVA). Although we noted some variation in ECG parameters in control and Nkx2.5cre/+ mice consistent with previous reports,4,12,18 the restoration of a normal PR interval in the Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes suggested a Prox1-induced compensatory effect on Nkx2.5-dependent electrophysiological function. Therefore, we propose that release of Prox1 inhibition of the single Nkx2.5 allele maintains the overall duration of the cardiac cycle, overriding AV block, and facilitating transmission to coordinate ventricular contraction.
Figure 3. Compensatory atrioventricular (AV) function via shortened PR interval in Nkx2.5 heterozygotes following knock-down of Prox1. Electrocardiogram (ECG) traces from Prox1loxP/+ control (A), Nkx2.5Cre/+ (B), and Nkx2.5Cre/+;Prox1loxP/+ (C) animals. There was an increased incidence of AV delay (B) resulting in 1st-degree AV block in the Nkx2.5Cre/+ mice that was not observed in either Prox1loxP/+ control (A) or Nkx2.5Cre/+; Prox1loxP/+ compound heterozygote (C) littermates. PR interval (from the start of the P wave to the start of the QRS complex) is highlighted by red line (A–C). Statistical analysis across the respective genotype groups revealed significant PR interval delay in Nkx2.5Cre/+ animals compared to controls, with a significantly shorter PR interval observed in Nkx2.5Cre/+;Prox1loxP/+ as compared to Nkx2.5Cre/+, approaching that of control animals; n=13 littermates per genotype, * P≤0.05; 1-way ANOVA with Bonferroni's multiple comparison test (D). E, ECG trace showing Moblitz type 1 3:2 heart block (Wenkebach) in the adult hearts of a subset of Nkx2.5Cre/+ mice, revealing complete dissociation between atrial and ventricular activity. Top trace (intracardiac electrograms, A=atrial signal and V=ventricular signal and corresponds to the P wave and QRS complexes on the surface ECG below) illustrates no relationship between the A and V signals indicating AV dissociation and Moblitz type 1 3:2 heart block (Wenkebach). AV block is never observed in the Nkx2.5Cre/+;Prox1loxP/+ rescued adult hearts.
Table. Surface ECG Recording in Prox1loxP/+, Nkx2.5Cre/+, and Nkx2.5Cre/+;Prox1loxP/+ Mice
GenotypeRR Interval (s)PR Interval (s)QRS Interval (s)QT Interval (s)
Prox1loxP/+0.113±0.014*0.0341±0.0020.009±0.00050.0187±0.002
Nkx2.5Cre/+0.108±0.0040.0396±0.0009†0.0091±0.00030.0177±0.001
Prox1loxP/+;Nkx2.5Cre/+0.111±0.0090.0351±0.0020.0095±0.00050.0202±0.004
*
Values are all mean±SEM for 13 mice/genotype.
†
Values in red for the Nkx2.5Cre/+ mice are significantly different from Prox1loxP/+ and Nkx2.5Cre/+;Prox1loxP/+ (P≤0.05 1-way ANOVA with Bonferroni's multiple comparison test).
For all other interval comparisons between genotypes there are no significant differences.
Because excitation and contraction are tightly coupled, we assessed Ca2+ handling in atrial and Purkinje-network myocytes, within the ventricles of Cx40BAC-GCaMP2 mice19 crossed onto the Nkx2.5 and Prox1 backgrounds. High speed imaging of isolated perfused mouse hearts revealed Ca2+ transients in the atrium that were synchronized with transients in ventricular PFs in Prox1loxP/+ control hearts (Figure 4A), whereas in Nkx2.5Cre/+ mice Ca2+ transients could be detected in the atrium but not in the ventricular myocardium (Figure 4B). In contrast to the mutants, Nkx2.5Cre/+;Prox1loxP/+ heterozygote animals exhibited clearly detectable PF transients of equivalent amplitude to control animals, although partially asynchronous with respect to the corresponding atrial signal (Figure 4C). Representative traces, as shown in Figure 4, were supported by comparing the mean peak amplitude of PF transients, across n=3 mice per genotype on the Cx40BAC-GCaMP2 background. This revealed no significant differences between Prox1loxP/+ control and Nkx2.5Cre/+;Prox1loxP/+ mice (2.100±0.12 versus 1.849±0.07; n=mean ΔFo/F±SEM) but significant rescue of Ca2+ handling within the ventricles of the Nkx2.5Cre/+;Prox1loxP/+ mice relative to the Nkx2.5Cre/+ (1.239±0.09; P≤0.01; 1-way ANOVA). Thus Prox1 knock-down partially restored PF Ca2+ handling in line with the rescue of His-PF excitation.
Figure 4. Rescue of Ca2+ transients and ventricular contraction in Nkx2.5 heterozygotes following knock-down of Prox1. High speed imaging of isolated perfused mouse hearts expressing the fluorescent Ca2+ reporter construct, GCaMP2, revealed Ca2+ transients in the atrium (within the red dotted line; highlighted by red arrow), which were well-synchronized with transients in ventricular Purkinje fibers in Prox1loxP/+ control hearts (blue arrow) (A). The time difference (x-axis offset) is due to the signal delay as it is transmitted through the atrioventricular junction. In Nkx2.5Cre/+ mice, Ca2+ transients could be detected in the atrium but not in the ventricular myocardium (B). In contrast, Nkx2.5Cre/+;Prox1loxP/+ heterozygotes, a Ca2+ transient can be detected in a Purkinje fiber, although not completely normalized relative to the atrial signal (C). Representative traces and internal images of the atrial and ventricular transients are shown (A–C); n=4 hearts were analyzed per genotype.

Rescue of Nkx2.5 Expression and Downstream Targets and Effectors

In light of the potential for epistatic interaction between Prox1 and Nkx2.5 in the cardiac conduction system, we sought to investigate whether Prox1 might regulate Nkx2.5 gene expression. Both total RNA and protein were extracted from whole adult hearts for quantitative RT-PCR (qRT-PCR) and western blot analysis, respectively. As expected, we observed a decrease in Nkx2.5 expression in Nkx2.5Cre/+ hearts and a decrease in Prox1 expression in Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes, compared to controls (Figure 5A and Online Figure III). However, Nkx2.5 expression in Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes was equivalent to control hearts, at both the RNA and protein level (Figure 5A and Online Figure III). This demonstrated that a reduction in Prox1 expression resulted in a concomitant increase in Nkx2.5 expression, suggesting that in the wild type situation Prox1 negatively regulates Nkx2.5 at the gene expression level.
Figure 5. Rescue of cardiac conduction system gene and protein expression. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using RNA extracted from Prox1loxP/+ control, Nkx2.5Cre/+, and Nkx2.5Cre/+;Prox1loxP/+ whole adult hearts. Expression of Prox1, Nkx2.5 and ion channel genes shown to be downstream of Nkx2.5 revealed Scn5a, Kcne1, and Cacna1g to be significantly downregulated in Nkx2.5Cre/+ adult hearts and rescued in the Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes (A). Immunostaining on Prox1loxP/+ control, Nkx2.5Cre/+, and Nkx2.5Cre/+;Prox1loxP/+ adult heart sections for Nav1.5 protein (B–J) encoded by the misregulated/rescued gene (Scn5a,) revealed the Nav1.5 ion channel protein was down regulated in Nkx2.5Cre/+ adult hearts (specifically in conduction system cells, B–G, and within the myocardium, H–J) and rescued in the Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes consistent with the gene expression data (white arrowheads E, G highlight membrane staining for Nav1.5; H-J, white box insets highlight expression at higher magnification; all sections were processed together and images captured using identical brightness settings). Quantitative RT-PCR for hyperpolarization-activated cyclic nucleotide-gated pacemaker channel genes (K), connexin genes (L), and cardiac conduction system transcription factors (M) were examined to reveal Hcn4 and Cx40 gene misregulation in Nkx2.5Cre/+ hearts, which was normalized following knock-down of Prox1 in Nkx2.5Cre/+;Prox1loxP/+ hearts; * P≤0.05; ** P≤0.01; 1-way ANOVA with Bonferroni's multiple comparison test. See Figure 1L–1Q and Figure 2A, 2E, and 2C for corresponding downregulation and rescue of Cx40 and HCN4 protein expression, respectively. Scale bars B (as applies to B–D), 50 μm; D (as applies to D–F), 50 μm; H (as applies to H–J), 100 μm.
We subsequently examined rescue of expression of downstream ion channels previously implicated as potential targets of Nkx2.5 (following tamoxifen-induced perinatal knock-down of Nkx2.5)20 and for which specific variants (SCN5A, KCNE1, and KCNQ) have been associated with sudden cardiac death in primary arrhythmic syndromes.21 In accordance with the earlier study,20 qRT-PCR analyses revealed a downregulation of Scn5a, Kcne1, and Cacna1g in Nkx2.5Cre/+ adult hearts; yet, in contrast, we observed an increase in Cacna1h and no change in Ryr2 (Figure 5A). We attribute this discrepancy to the fact that we analyzed adult heterozygote mice whereas previously the focus was exclusively on postnatal Nkx2.5-null mice. However, in line with our anatomic and functional results, we observed expression of Scn5a, Kcne1, Cacna1g, and Cacna1h return to control levels in the Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes (Figure 5A). This was further supported by analyses of corresponding protein expression for Nav1.5 (Scn5a) in adult heart sections, which revealed spatial rescue of the Nav1.5 ion channel in Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes (Figure 5B–5J). The normalization of downstream effectors of Nkx2.5 function such as Kcne1 and Scn5a/Nav1.5 reflected the rescue of AV block and intraventricular conduction defects and implies that a reduction of Prox1 can rescue aberrant genetic pathways within the conduction system associated with Nkx2.5 haploinsufficiency.
Given the importance of Nkx2.5 in the developing cardiac conduction system, we investigated the expression of those genes mentioned above which were significantly rescued in the adult Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes at E13.5; a stage consistent with functional maturation of the conduction system.22 Interestingly, despite appropriate rescue of Nkx2.5 levels between Nkx2.5Cre/+ and Nkx2.5Cre/+;Prox1loxP/+ embryos, we did not observe significant differences in expression between these 2 genotypes for Scn5a, Kcne1, or Cacna1g (Online Figure IV). Furthermore, it has been shown previously that the peripheral conduction system develops normally in Nkx2.5 heterozygous embryos in which the entire Nkx2.5 coding sequence was replaced with LacZ.7 Therefore, we examined E18.5 hearts from Prox1loxP/+, Nkx2.5Cre/+, and Nkx2.5Cre/+;Prox1loxP/+ genotypes (following crosses with Cx40EGFP mice) and saw no significant difference between the 3 genotypes (Online Figure V). Together, these results suggest that Prox1 does not regulate downstream targets of Nkx2.5 during conduction system development and that epistasis is restricted to maintenance of conduction in the adult heart.

Rescue of Generic Conduction System Genes Following Knock-Down of Prox1

In addition to known downstream targets and effectors of Nkx2.5, we determined the expression of genes that have previously been implicated as generically important for cardiac conduction. Having observed an effect on HCN4 protein expression (Figure 2) we further examined all HCN pacemaker channel genes (Hcn1, Hcn2, and Hcn4) that are expressed in the heart. As predicted, from the reduced AV node HCN4 immunostaining (Figure 2A, 2C, and 2E), we observed significantly reduced Hcn4 expression in Nkx2.5Cre/+ hearts, compared to controls, which was restored on the Nkx2.5Cre/+;Prox1loxP/+ background (Figure 5K). However, both Hcn1 and Hcn2 were increased in Nkx2.5Cre/+ hearts compared to controls and further increased in Nkx2.5Cre/+;Prox1loxP/+, with Hcn1 showing the largest fold change (Figure 5K). A compensatory increase in the expression of Hcn1 and Hcn2 in the rescue animals is potentially significant in that they encode HCN channels that are faster than voltage-gated variants and consequently contribute to the spontaneous rhythmic activity in the heart.23 We next examined expression of the connexins (Cxs), which encode gap junction proteins that play a role in cardiac electric conduction and have been shown to be affected by Nkx2.5 levels.4,24,25 We focused on 4 of the 5 Cxs that are expressed in the heart: Cx43 (Gja1), Cx40 (Gja5), Cx45 (Gjc1), and Cx30.2 (Gjd3), each with different expression patterns; excluding Cx30, which is expressed exclusively in the sinoatrial node,26 anatomically and functionally upstream of the AVN and His-PF axis. Cx43 is predominantly expressed in the working cardiomyocytes of the heart and its expression was unchanged in all 3 groups (Figure 5L). Cx40 is strongly expressed in the atria and the ventricular conduction system and, consistent with our observed hypoplasia of the Cx40-expressing His-Purkinje system and reduced endogenous Cx40 protein expression (Figure 1) along with previous reports that Cx40 is reduced in Nkx2.5 heterozygotes,18 we observed a significant decrease at the gene expression level in Nkx2.5Cre/+ hearts, which was returned to normal in the Nkx2.5Cre/+;Prox1loxP/+ background (Figure 5L). Both Cx45 and Cx30.2 are more strongly expressed in the sinoatrial node and AVN and in line with the hypoplasia of the AVN (Figure 2) we observed a slight decrease in expression of both genes in Nkx2.5Cre/+, relative to controls, and, in this instance, a trend toward restoration in Nkx2.5Cre/+;Prox1loxP/+ compound heterozygotes (Figure 5L).
To investigate whether there were any effects of Prox1 on genes classically associated with the developing conduction system, we assessed a cohort of factors in adult hearts in an Nkx2.5 haploinsufficient background. We observed very little difference in the expression of Tbx2, Tbx3, Tbx5, Id2, and Irx3 between control, Nkx2.5Cre/+, and Nkx2.5Cre/+;Prox1loxP/+ hearts (Figure 5M). Hopx, a non-DNA binding homeobox factor thought to be downstream of Nkx2.5 during development, and which also functions in the adult conduction system, was significantly reduced in Nkx2.5Cre/+ but failed to be rescued following the loss of a copy of Prox1 (Figure 5D). In the adult heart, Hopx functions below the AVN. Hopx-homozygous mutant mice show widening of the QRS-complex and QT-interval prolongation,27 as such Hopx may reflect downstream function independent of the Prox1-Nkx2.5 axis. Collectively these data suggest that Nkx2.5 only regulates this cohort of factors during conduction system development and not during adulthood and is consistent with the gene expression data from E13.5 hearts (Online Figure IV) inferring a restricted role for Prox1 in maintaining the homeostatic function of the adult cardiac conduction system.

Prox1 Directly Interacts With an Upstream Nkx2.5 Enhancer

Since our anatomic, functional, and gene expression data collectively pointed to a role for Prox1 regulating Nkx2.5 in the adult conduction system, we sought to determine whether the mechanism of epistasis might be direct. We carried out in vivo ChIP experiments on adult wild type hearts using a Prox1 antibody previously used successfully for ChIP10 and PCR detection against a well-characterized 2.1 kb enhancer sequence for Nkx2.5 located approximately 9 kb upstream of the Nkx2.5 gene.28 The enhancer contained regulatory elements sufficient to drive transgene (lacZ or EGFP) expression in the developing heart of transgenic mice28,29 and when enhancer-driven EGFP+ (Nkx2.5+) derivatives were isolated by FACs, cultured, and ECG profiled, they were shown to differentiate into both contractile cardiomyocytes and conduction system cells.29 ChIP was carried out on overlapping 100 to 300 bp fragments of the enhancer region, in line with the fact that no known consensus binding sequence for Prox1 currently exists.10 A single fragment was enriched relative to a no-antibody control and quantified by real time qPCR as 0.034% of the input chromatin (Figure 6A–6C); indicative of direct Prox1 binding to the Nkx2.5 enhancer. In the absence of available Prox1-deficient adult heart tissue, as a negative control, because of the embryonic lethality of Prox1-null mutants,10,30 we carried out ChIP on flanking genomic fragments within the enhancer to confirm specificity of Prox1 binding to the region defined across −8943 to −9210 (Figure 6A and 6B).
Figure 6. Prox1 and HDAC3 directly repress Nkx2.5. Prox1 and HDAC3 directly bound to a fragment of an Nkx2.5 cardiac enhancer (A) in vivo (adult heart) as revealed by a representative chromatin immunoprecipitation (ChIP) from adult hearts (B). Positive fragment is indicated in red and 2 negative control flanking regions are indicated in blue (A, B). Prox1 and HDAC3 ChIPs were enriched relative to a no antibody control (B), confirmed by qPCR for Ct values expressed as a percentage of input (C; quantification of ChIP is shown across replicates within a single immunoprecipitation; repeat ChIP experiments n=3 were carried out to ensure reproducible enrichment of Prox1 and HDAC3 bound fragments, data not shown). HDAC3 immunostaining of control adult hearts revealed expression in the atrioventricular node (AVN) (D, E; white box area highlighted in D is shown at higher magnification in E; white arrowheads in E highlight HDAC3+ AVN cells) as determined by costaining for HCN4. In Cx40EGFP/+ hearts, HDAC3 was also expressed in the EGFP+ His-Purkinje fiber (PF) network (F, G; white box area highlighted in F is shown at higher magnification in G; white arrowheads in G highlight HDAC3+ PF cells). Cotransfection reporter assays in NIH3T3 cells (H) revealed that Prox1 significantly activated a luciferase reporter (A) downstream of the Nkx2.5 enhancer (compare lanes 1 and 2) whereas, both HDAC2 and HDAC3 significantly repressed reporter activity (compare lane 2 with both lanes 3 and 4). In combination, HDAC3 inhibited the Prox1 activation, whereas a similar repression was not observed with cotransfection of HDAC2 (lanes 5 and 6) as an indication of the specificity of the Prox1-HDAC3 interaction. An N-terminal deletion of Prox1 that does not interact with HDAC335 activated the Nkx2.5 enhancer driven reporter but was immune to repression by either HDAC2 or HDAC3 (for statistical purposes, compare lanes 6 and 9). Repeat NIH3T3 reporter transfection assays (I) revealed that HDAC3 inhibited Prox1 activation of the reporter in a dose-dependent manner (lanes 4–6; 150 ng, 300 ng, and 600 ng HDAC3, respectively) whereas treatment with trichostatin A (TSA), a known inhibitor of histone deacetylase activity (class I and II;38), restored Prox1 activation in the presence of 300 ng of HDAC3 at concentrations of 10 pM and 100 pM (lanes 8 and 9; for statistical purposes compare lanes 4 and 8; full TSA concentration range: lanes 7–10 1 pM, 10 pM, 100 pM, and 100 nM, respectively). Neither, increasing concentrations of TSA nor EtOH vehicle had any effect on basal reporter activity (lanes 11–14). Dose-dependent knock-down of Prox1 by siRNA (10 nmol/L and 15 nmol/L), in the rat ventricular cell line H9c2, resulted in significant upregulation of Nkx2.5 expression, relative to a scrambled siRNA oligo control (Co), as determined by qRT-PCR (J). Specific knock-down of Prox1 was confirmed by western analyses and scanning densitometry (K). HDAC3 siRNA-mediated knock-down resulted in a significant increase in Nkx2.5 expression in H9c2 cells, relative to a scrambled control; specificity was confirmed by the lack of effect of HDAC2 knock-down (L). Reciprocal overexpression of Prox1 in H9c2 cells resulted in a significant knock-down of Nkx2.5 protein expression compared to untransfected controls; as confirmed by western analyses in triplicate and scanning densitometry (M). *P≤0.05, **P≤0.01, ***P≤0.001; Student t test. Scale bars D and F, 100 μm; E and G, 20 μm.

Prox1 Recruits the Transcriptional Corepressor HDAC3 to Directly Inhibit Nkx2.5 Expression

To establish how Prox1 might negatively regulate Nkx2.5 expression we carried out further ChIP and reporter-based corepression assays. Prox1 has previously been shown to act as both an activator and repressor of gene transcription.10,31,31–34 Repression by Prox1 was dependent on recruitment of corepressors to target promoters and one of these corepressors, HDAC3,35,36 has previously been implicated in regulating cardiac function.37 We initially confirmed HDAC3 was expressed in the AVN and PF network (Figure 6D–6G) and to investigate whether Prox1 might act with HDAC3 to repress Nkx2.5, we determined that αHDAC3 immunoprecipitated the enhancer fragment, previously pulled down by Prox1, enriched to 0.141% of the input adult heart chromatin (Figure 6A–6C). Cotransfection experiments in NIH3T3 cells, with a luciferase reporter downstream of the full length Nkx2.5 enhancer plus a 500-bp base promoter,29 (Figure 6A and 6H) revealed that addition of ectopic Prox1 alone activated the enhancer-driven reporter (2.5-fold; P≤0.01), whereas cotransfection of HDAC3 significantly repressed both nascent reporter activity (−10-fold; P≤0.01) and that stimulated by Prox1 (−2.0-fold; P≤0.05). Importantly, whereas the related class I HDAC protein, HDAC2, inhibited reporter activity alone, it did not inhibit Prox1-induced activation as a negative control for specificity. Addition of a Prox1 construct in which the HDAC3 interaction domain at the N-terminus was deleted35 in combination with HDAC3, or HDAC2, failed to repress Prox1 activation of the reporter (Figure 6H). Treatment with Trichostatin A (10 pM), an inhibitor of class I and II HDAC activity38 significantly enhanced reporter activation (2.5-fold relative to Prox1 plus 150 ng of HDAC3 and 10-fold relative to Prox1 plus 600 ng HDAC3; P≤0.01 and P≤0.001, respectively; Figure 6I). Thus, although Prox1 alone activated the Nkx2.5 enhancer in the artificial context of single transfection and absence of cofactors, cotransfection and the recruitment of HDAC3 as a strong corepressor resulted in a net inhibition in line with a prerequisite for fine tuning of Nkx2.5 expression to ensure appropriate homeostatic conduction. To confirm that Prox1 inhibits Nkx2.5 expression, in a more physiologically relevant setting, we transfected a Prox1 siRNA into the H9c2 myogenic cell line derived from embryonic rat heart ventricle39 and observed that endogenous levels of Nkx2.5 were significantly elevated following knock-down of Prox1 (Figure 6J and 6K). Further, siRNA experiments to knock-down HDAC3 in the same H9c2 model system confirmed a corresponding increase in Nkx2.5 expression, which was not observed for HDAC2 as a control for specificity (Figure 6L); western analysis confirmed significant siRNA mediated knock-down of HDAC2 and HDAC3 protein levels (data not shown). Finally, reciprocal experiments in which we overexpressed Prox1 in H9c2 cells resulted in significant downregulation of Nkx2.5 protein as confirmed by western analysis and scanning densitometry (Figure 6M).
Collectively, these data are consistent with Prox1 acting in conjunction with HDAC3 to directly repress Nkx2.5 in the adult conduction system.

Discussion

We identify Prox1 as an upstream modifier of Nkx2.5 function. Nkx2.5 is a conserved, critical cardiac transcription factor extensively characterized in terms of a role in cardiac morphogenesis (reviewed in Ref. 40) and more recently as an early commitment marker for embryonic cardiovascular progenitors.12,29,41–43 In addition, its elevated expression in the developing cardiac conduction system provoked a number of studies, reviewed in Ref.18, including a combinatorial role with Tbx5 and Id2 in the specification of the ventricular conduction system lineage.6 Dominant mutations in Nkx2.5 have been shown to cause congenital conduction system anomalies, most notably AV block in both mouse4 and man,9 and NKX2.5 was identified as a genetic determinant of PR interval and risk factor for atrial fibrillation by genome-wide association study.44
Previous studies have highlighted Gata4 and Smad cofactors as important in the control of Nkx2.5 expression, via evolutionary conserved sites within an upstream enhancer.28,45–47 These studies are restricted to developmental stages and it remains unclear as to the physiological relevance of the cofactor interactions. What has not hitherto been described is epistatic rescue of Nkx2.5 biological activity during any aspect of cardiac development, function, or relevance to human disease. Genetic modifiers for Nkx2.5 were recently implied, arising from a large scale survey of Nkx2.5 heterozygous animals and, although specific variants were not identified, this study revealed extensive background-dependent buffering of Nkx2.5 haploinsufficiency underlying pleiotropic heart defects.48 Pleiotropy also importantly holds true in humans, where patients with pathogenic NKX2.5 mutations present with atrial-septal defects but without characteristic conduction block.49 Here we reveal how Prox1 can modulate Nkx2.5 function in the adult conduction system, such that Cre-mediated knock-down of Prox1 rescues conduction anomalies, arising because of Nkx2.5 haploinsufficiency, which phenocopy human NKX2.5 conduction disease. Importantly, use of congenic parent strains (Nkx2.5Cre/+ and Prox1loxP/+) and littermates for within experimental comparisons ensured the interaction of Prox1 and Nkx2.5 was not dependent on genetic background, as has previously been attributed to phenotypic variation across different lines of Nkx2.5 heterozygote mice.12 Consequently, we excluded potential artifacts associated with unknown modifier activity. Moreover, the modulation of Nkx2.5 by Prox1 was confirmed as a primary effect at the level of the conduction system and not secondary to ventricular knock-down of Prox1 or Nkx2.5-related ventricular dysfunction and/or progressive cardiomyopathy. Multiple lines of evidence are presented to suggest that Prox1 and Nkx2.5 act in epistasis to maintain cardiac conduction. First functional parameters showing cardiac output deficits in Nkx2.5 heterozygotes were returned to control values following knock-down of Prox1. Second, anatomic defects in both atrial (AVN) and ventricular (His bundle branches and PFs) conduction components in Nkx2.5 heterozygous animals were significantly attenuated by a reduction in Prox1. Third, surface electrocardiograms documented evidence of AV delay with AV block in Nkx2.5 heterozygous animals, which was not only absent with Prox1 knock-down in Nkx2.5 heterozygotes but compensated for by normalized PR intervals to ensure AV conduction. Fourth, PF Ca2+ transients, both duration and amplitude, were significantly restored in Nkx2.5Cre/+ animals following knock-down of Prox1. Fifth, Nkx2.5 gene expression and that of a cohort of key conduction-specific genes was restored to control levels in a reduced Prox1 background relative to Nkx2.5Cre/+ mutants. Finally, ChIP and siRNA loss-of-function experiments revealed Prox1 bound to a region of Nkx2.5 conduction system enhancer which, in combination with HDAC3, repressed Nkx2.5 expression and reporter gene activity.
Thus we propose a model whereby Prox1 regulates Nkx2.5 expression and activity in a stochastic manner to ensure the integrity of atrial and ventricular components and electrophysiological homeostasis in the adult heart (Figure 7). During normal contractile function Prox1 represses Nkx2.5 to an optimal level for maintenance of adult cardiac conduction, thus avoiding compounding effects of “unconstrained” Nkx2.5 activity, as observed during development, such as myocyte hyperplasia, conduction lineage specification and morphogenetic remodeling.6 Haploinsufficiency for Nkx2.5, although still under the inhibitory influence of Prox1, results in conduction system defects due to enforced Nkx2.5 levels below the threshold for normal anatomic and electrophysiological maintenance (Figure 7A). Previous reports describe that Nkx2.5 acts cell autonomously to maintain conductive phenotype in Purkinje cells and that in Nkx2.5 haploinsufficient mice conductive myocytes lose Cx40 expression and change phenotype,7 which suggests a defect in retention of conduction system cells. Knock-down of Prox1 restores Nkx2.5 activity, in a haploinsufficient background, to a level which facilitates rescue of the morphology and function of the cardiac conduction system, potentially by maintaining appropriate levels of Cx40 expression (as shown herein) preventing conduction cell loss. The mechanism for Prox1 inhibition of Nkx2.5 appears direct, whereby Prox1 recruits the transcriptional corepressor HDAC3 to bind and negatively regulate Nkx2.5 at a proximal cardiac enhancer (Figure 7B). There remain some outstanding questions, including a requirement for further characterization of the Nkx2.5 conduction system enhancer we identify herein, and specifically to investigate in vivo activity within the adult conduction system. Thus far, this enhancer lies within a proximal fragment upstream of Nkx2.5, known to be active during development; however, it has also been documented to drive reporter expression within young adult hearts, active in cells that are not differentiated cardiomyocytes but have yet to be determined as components of the conduction system (Sean Wu, personal communication). The ChIP data demonstrates binding of both Prox1 and HDAC3 to this enhancer in adult heart, with associated transcriptional activity via in vitro reporter assays, suggesting the enhancer is functional in the adult setting. Although Prox1 and HDAC3 are both expressed in the AVN and His-PF network and bind the same regulatory region upstream of Nkx2.5, we do not currently have insight into the Prox1-HDAC3 DNA binding complex or whether the targeting of the complex to the Nkx2.5 enhancer requires the involvement of cofactors such as NCoR/SMRT (reviewed in Ref. 50). It will also be of interest to look in HDAC3 mutants for Nkx2.5 misregulation within the conduction system, although global knock-outs of HDAC3 are embryonic lethal and to-date conditional targeting of HDAC3 is restricted to cardiomyocyte-specific deletion.37 A further possibility is that, in the absence of HDAC3, Prox1 may recruit an alternative corepressor given its known association with LRH1 and COUP-TFII33–36; follow-up studies to determine involvement of these factors in the conduction system are ongoing.
Figure 7. Model of Prox1 regulation of Nkx2.5 activity in the adult cardiac conduction system. In the wild type (control) setting (Nkx2.5+/+;Prox1loxP/+) Prox1 represses Nkx2.5 to an optimal level for maintenance of adult cardiac conduction (green solid shaded area). Haploinsufficiency for Nkx2.5 (Nkx2.5Cre/+;Prox1+/+), although still under the inhibitory influence of Prox1, results in conduction system defects because of repressed Nkx2.5 levels below the threshold (red dashed line) for “normal” anatomic and electrophysiological maintenance (A). Knock-down of Prox1 (Nkx2.5Cre/+;Prox1loxP/+) restores Nkx2.5 activity, in an Nkx2.5 haploinsufficient background. Incomplete rescue, falling short of levels equivalent to that in the control animals, is subject to tolerance (dashed line and green dashed shaded region) such that rescue is still able to maintain the morphology and function of the conduction system (A). Prox1 (blue diamond) recruits the transcriptional corepressor HDAC3 (orange circle) to bind and negatively regulate Nkx2.5 at a proximal cardiac enhancer. The balance between Prox1 nascent activation and HDAC3 inhibition results in a partial net repression across both alleles in a wild type background to fine tune Nkx2.5 activity in maintaining the integrity of the AVN and His-Purkinje network and PR-interval (B, i). This level of Nkx2.5 activity is virtually recapitulated in the compound heterozygote background by the loss of Prox1, and accompanying HDAC3, at the single allele (B, ii). Whereas, in Nkx2.5 heterozygotes wild type for Prox1 repression of Nkx2.5 at the single allele enforces Nkx2.5 activity below the threshold for maintaining appropriate conduction system structure-function (B, iii).
In conclusion, this study reveals novel upstream regulation and rescue of Nkx2.5 function, highlights the essential role for Nkx2.5 in maintaining the integrity of the adult cardiac conduction system and reports a hitherto unknown function for Prox1 in this regard. Since NKX2.5 mutations underlie AV delay and block in humans an understanding of how NKX2.5 activity is regulated in the arrhythmic setting could promote further molecular insights into both cardiac electrophysiology and the underlying pathology of conduction disease.

Acknowledgments

The authors thank Dr Eckardt Treuter for Prox1 and Dr Ed Seto for HDAC2 and HDAC3 expression plasmids. We thank Dr Sean Wu for provision of the Nkx2.5 enhancer element. We thank Karina Dubé for technical assistance and Vincent Christoffels for critical comments on the manuscript.

Novelty and Significance

What Is Known?

•
Nkx2.5 is an essential cardiac transcription factor with conserved roles in the developing and adult cardiac conduction system.
•
Dominant mutations in NKX2.5 in humans result in cardiac defects which include delayed or blocked atrioventricular conduction.

What New Information Does This Article Contribute?

•
Nkx2.5 plays a critical role in maintaining the adult conduction system.
•
The homeobox factor Prox1, acting with HDAC3, acts as a direct negative regulator of Nkx2.5 and haploinsufficiency for Prox1 rescues anatomical, molecular, and functional conduction defects in Nkx2.5 heterozygous mutant mice.
•
Prox1 is a direct upstream regulator of Nkx2.5 and the Prox1-HADC3=>NKX2.5 signaling pathway may be a novel therapeutic target in human conduction disease.
The molecular basis of human cardiac conduction disease is not well understood and yet lethal arrhythmia is prevalent worldwide. Mutations in the transcription factor NKX2.5 have been implicated in atrioventricular delay and block in humans. Here we demonstrate that the homeobox factor Prox1 acts in epistasis to negatively regulate Nkx2.5 function in the adult cardiac conduction system. In compound Nkx2.5 and Prox1 heterozygous mice, we describe anatomical rescue of the atrioventricular node and His-Purkinje system, normalized atrioventricular conduction and Ca2+ handling and rescue of ion channel and generic conduction genes downstream of Nkx2.5. Mechanistically, Prox1 acts with the canonical transcriptional repressor HDAC3 directly on an upstream enhancer to regulate NKX2.5 transcriptional activity. These findings suggest that direct upstream modification and regulation of Nkx2.5 function maintains the integrity of the adult conduction system and reveal a hitherto unknown role of the essential transcription factor Prox1, which may be important in ensuring electrophysiological cardiac function and could be a potential modifier in human conduction disease.

Footnote

Non-standard Abbreviations and Acronyms
AV
atrioventricular
AVN
atrioventricular node
ChIP
chromatin immunoprecipitation
ECG
electrocardiography
EGFP
enhanced green fluorescent protein
HCN4
hyperpolarization-activated cyclic nucleotide-gated channel 4
PF
Purkinje fiber
qRT-PCR
quantitative reverse transcription polymerase chain reaction

Supplemental Material

File (res201281supplementmaterial.pdf)

Sources of Funding

This study was generously supported by the British Heart Foundation (PG/09/043/27565).

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On the cover: C57BL6 mice were injected with the cell cycle marker EDU and simultaneously the carotids were exposed to PDGF for 48 hours. Harvested carotids were counterstained with DAPI (blue) to mark nuclei and EDU-Alexa594 (red). The intact smooth muscle cell layers were viewed en face on an Olympus Fluoview 1000 confocal microscope. See related article, page 201.

Circulation Research
Volume 111 • Number 2 • 6 July 2012
Pages: e19 - e31
PubMed: 22647876

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© 2012 American Heart Association, Inc.

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History

Received: 11 November 2011
Revision received: 16 May 2012
Accepted: 21 May 2012
Published online: 29 May 2012
Published in print: 6 July 2012

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Keywords

  1. Nkx2.5
  2. Prox1
  3. atrioventricular node
  4. Purkinje fiber

Authors

Affiliations

Catherine A. Risebro
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Louisa K. Petchey
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Nicola Smart
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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John Gomes
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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James Clark
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Joaquim M. Vieira
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Joseph Yanni
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Halina Dobrzynski
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Sean Davidson
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Zia Zuberi
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
View all articles by this author
Andrew Tinker
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
View all articles by this author
Bo Shui
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Yvonne I. Tallini
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
View all articles by this author
Michael I. Kotlikoff
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Lucile Miquerol
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
View all articles by this author
Robert J. Schwartz
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
View all articles by this author
Paul R. Riley
From the Institute of Child Health (C.A.R., L.K.P.), University College London, London, UK; Department of Physiology, Anatomy and Genetics (N.S., J.M.V., P.R.R.),University of Oxford, Oxford, UK; Department of Metabolism and Experimental Therapeutics (J.G., Z.Z.), Rayne Institute, University College London, London, UK; King's College London (J.C.), BHF Centre, St Thomas' Hospital, London, UK; Cardiovascular Medicine (J.Y., H.D.), University of Manchester, Manchester, UK; The Hatter Cardiovascular Institute (S.D.), University College London, London, UK; William Harvey Research Institute (A.T.), Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK; Biomedical Sciences Department (B.S., Y.I.T., M.I.K), Cornell University, Ithaca, USA.; Developmental Biology (L.M.), Institute of Marseilles—Luminy, Université de la Méditerranée, Campus de Luminy, Marseille, France; and Texas Heart Institute/St. Luke Episcopal Hospital (R.J.S.), Houston, TX.
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Notes

In April 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.79 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.260695/-/DC1.
Correspondence to Paul R. Riley, Department of Physiology, Anatomy and Genetics, Sherrington Building,University of Oxford, Oxford, UK. E-mail [email protected]

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Epistatic Rescue of Nkx2.5 Adult Cardiac Conduction Disease Phenotypes by Prospero-Related Homeobox Protein 1 and HDAC3
Circulation Research
  • Vol. 111
  • No. 2

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  • Vol. 111
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