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Electrophysiological patterning of the heart
Boukens, B.J.D.
Publication date 2012
Link to publication
Citation for published version (APA):
Boukens, B. J. D. (2012). Electrophysiological patterning of the heart.
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Bas Boukens
Mathilda Mommersteeg
Saskia van de Velden
Corrie de Gier-de Vries
Richard Harvey
Antoon Moorman
Ruben Coronel
Vincent Christoffels
Complementary roles of Tbx3 and Nkx2-5 in
the structure and function of the adult
atrioventricular conduction system
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Abstract
Rationale: Dysfunction of the atrioventricular (AV) node or AV bundle can cause AV block,
which requires implantation of an electronic pacemaker. Genome-wide association studies have revealed that genetic variation in multiple cardiac transcription factor loci, including NKX2-5 and
TBX3, influence the function of the conduction system. Objective: In this study we investigated
the role of Tbx3 and Nkx2-5 in the formation and the functional maintenance of the AV conduction system. Methods and results: We studied 4 groups of mice: wildtype, haploinsufficient for
Tbx3, for Nkx2-5 or for both Tbx3 and Nkx2-5. Their age was 2 months or 16 months. In these
groups, AV conduction parameters were measured, and the structure and size of the AV node and bundle were assessed using 3D reconstruction of gene expression patterns. In addition, we determined the expression pattern of proteins relevant for conduction. At 2 month of age, the AV bundle of Tbx3, Nkx2-5 and Tbx3/Nkx2-5 haploinsufficient mice was significantly smaller than that of wild type mice, whereas the size of the AV node was not different. AV delay was prolonged only in the Tbx3 haploinsufficient mutant mice. At 16 months of age, the AV bundle of Tbx3, Nkx2-5 and Tbx3/Nkx2-5 haploinsufficient mice was smaller than that of wildtype mice. The AV node size was not different between the groups but the AV delay was shorter in both
Tbx3 and Tbx3/Nkx2-5 haploinsufficient mice than in wildtype and Nkx2-5 haploinsufficient
mice. Furthermore, both Nkx2-5 and Tbx3/Nkx2-5 haploinsufficient mice developed AV block.
Conclusions: Normal levels of both Tbx3 and Nkx2-5 are required for AV bundle formation. The
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Introduction
The cardiac conduction system enables rhythmic and coordinated activation of the heart. The development of the conduction system is guided by temporal and spatial expression of transcription factors.1 Genome-wide association studies show that variations (single
nucleotide polymorphisms; SNPs) in multiple loci containing cardiac transcription factors, including NKX2-5 and TBX3, relate to the function of the conduction system in the adult human population.2-4 These variations are likely to influence the expression level or function
of these factors, thereby modifying their action.5 On the other hand, heterozygous mutations
causing loss of function of transcription factors including NKX2-5 and TBX5 can lead to severe conduction system abnormalities like 2nd or 3rd degree AV block.6-8 Thus, the development of
the conduction system is regulated by cardiac transcription factors in a dose-dependent manner. Tbx3 is essential for conduction system development and function.9, 10 Heterozygous
mutations in TBX3 cause ulnar-mammary syndrome of congenital defects in humans. However, the consequences of heterozygous loss of function for conduction system development are unknown.11 Tbx3 has been shown to physically and functionally interact
with Nkx2-5 to regulate target gene expression in the developing cardiac conduction system.12 Heterozygous loss of Nkx2-5 results in a hypoplastic AV conduction system and AV
conduction defects in mice13 and mutations in NKX2-5 are found in patients with AV conduction
disturbances.6, 7 Despite these insights, little is known about the regulation of conduction
system homeostasis, and the roles of these factors during aging have not been explored. The AV node forms from the slowly conducting primary myocardium of the AV canal and the AV bundle forms from the initially slowly but eventually fast-conducting myocardium of the crest of the interventricular septum.14 In the AV node and AV bundle, transcription factors
including Tbx2 and Tbx3 repress the working myocardial gene program,15 whereas factors like
Tbx5 activate the gene program for fast conduction in the AV bundle.6, 16, 17 Nkx2-5 plays an
important role in cardiac and conduction system gene regulation, and synergizes with both repressing and activating T-box transcription factors.12, 17 We hypothesize that a disturbed
balance in levels of these transcription factors affect the gene programs and function of the AV node and AV bundle. We addressed this hypothesis by investigating the structure and the function of the AV conduction system in young and old mice that were haploinsufficient for
Tbx3, for 5 or for both genes. Our data indicate that normal levels of Tbx3 and of
Nkx2-5 are required for the AV bundle formation and function in young and old mice, but that both factors to a large extent have distinguishable functions and act in non-redundant manners.
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Material and Methods
Animals
The investigation conforms with the local Animal Experiments Committee of the Academic Medical Center and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) or the European Commission Directive 2010/63/EU. Nkx2-5-GFP and Tbx3-cre mouse lines have been described previously.18, 19 All mice had a FVB/N background.
Immunohistochemistry and in situ hybridization
In situ hybridization was performed according to a previously described method.20 Probes have
been described previously.21, 22 For immunohistochemistry, rehydration, unmasking, blocking
and washing steps were performed according to the protocol of the tetramethylrhodamide based amplification kit (Perkin Elmer). Primary antibodies used for mouse sections were: cTnI rabbit polyclonal (1:250; Hytest Ltd); Tbx3 goat polyclonal (1:500; Santa Cruz Biotechnology); Cx43 and Cx40 mouse monoclonal (1:250; US Biological); Hcn4 Goat polyclonal (1:250 Santa Cruz Biotechnology). Secondary antibodies when using amplification were: Biotinylated donkey-anti-goat (1:250; Jackson Immunology); biotinylated goat-anti-rabbit (1:250; DAKO); biotinylated goat-anti-mouse (1:250; DAKO). For visualization without the amplification step, secondary antibodies coupled to an Alexa fluorescent (1:250; Invitrogen) were used.
3D reconstruction
In short, 10 µm serial sections of the AV junction and of ventricular septum were stained for Hcn4 and Cx40 and reconstructed. Subsequently, 3D reconstruction were performed as described previously using Amira 5.2 software.23 We defined the AV node
and the AV bundle based on the expression of hyperpolarization-activated channel (Hcn4), which encodes the subunit that form the channels carrying the If current, and connexin (Cx40), which forms gap junctions with high conductance. Cx40 is expressed in the AV bundle but not in the AV node whereas Hcn4 is expressed in both structures.14
ECG
Animals were anaesthetized with 1.5% isoflurane. Electrodes were placed at the right (R) and left (L) armpit and the left groin (F). A reference electrode was placed at the right groin. ECGs were recorded (Biosemi, Amsterdam, The Netherlands, sampling rate 2,048 Hz, filtering DC 400 kHz [3dB]) for a period of 5 minutes. From these leads a standard six lead ECG was calculated as follows: I = L-R, II = F-R, III = F-L, aVR = R-(L+F)/2, aVL = L-(R+F)/2 and aVF = F-(L+R)/2.24
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Langendorff experiments
Mice were stunned by inhalation of CO and killed by cervical dislocation. Then the heart was excised, the aorta was cannulated, mounted on a Langendorff perfusion set-up, and perfused at 37°C with Tyrode’s solution ((in mmol/L) 128 NaCl, 4.7 KCl, 1.45 CaCl2, 0.6 MgCl2, 27 NaHCO3, 0.4 NaH2PO4, and 11 glucose (pH maintained at 7.4 by equilibration with a mixture of 95% O2 and 5% CO2)). Local electrograms were acquired using a custom-built 256 channel data acquisition (Biosemi) system and analyzed using custom made software. Local electrograms were recorded from the right atrium and left ventricle during sinus rhythm and right atrial pacing at a basic cycle length of 120 ms, and twice the diastolic stimulus. The AV conduction delay was defined as the time between the start of the stimulus and the start of the first deflection on the ventricular electrogram. The refractory period of the AV node was determined by reducing the coupling interval of a premature stimulus on the right atrium (after 10 stimuli at basic cycle length 120 ms) in steps of 5 ms until activation of the ventricle failed. His potentials were measured during sinus rhythm and premature stimulation on the right atrium with a unipolar electrode that was placed on the His bundle after a small incision in the right atrium.
Statistics
Group comparisons were performed using one-way ANOVA with Bonferroni test for post hoc matched pairs. Values are given as mean ± SEM. A p-value < 0.05 was considered statistically significant.
Results
Normally sized AV node but hypoplastic AV bundle in mice haploinsufficient for Tbx3 or Nkx2-5
To study the requirement for normal levels of Tbx3 and of Nkx2-5 in the formation of the conduction system, we determined the size of the AV node and AV bundle in mice haploinsufficient for Tbx3 and/or Nkx2-5 at 2 months of age. Figure 1A shows the expression pattern of Hcn4, Cx43 and Cx40 in the AV junction of a wildtype mouse. We used Hcn4 as a marker for all AV conduction system components and Cx40 to discriminate between the AV
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node (Cx40-negative) and the AV bundle (Cx40-positive) (Figure 1B). In order to compare the size of the AV node, bundle and bundle branches between mice haploinsufficient for Tbx3 and/or Nkx2-5 we reconstructed the expression patterns of Hcn4 and Cx40 in the AV junction and the ventricular septum. The AV node was not different in size, which contrasts previously published data in which Nkx2-5 haploinsufficiency been associated with AV node hypoplasia.13, 25 However, the AV bundle and the bundle branches were hypoplastic in mice haploinsufficient
for Tbx3 and/or Nkx2-5 (Figure 1D, n=5 for each group). The size of the AV bundle and bundle branches in mice haploinsufficient for both Tbx3 and Nkx2-5 was not different from those in mice haploinsufficient for either Tbx3 or Nkx2-5. The latter indicates that reduction of both Tbx3 and Nkx2-5 does not have an additive effect on the size of the conduction system.
Figure 1. The AV bundle and bundle branches are hypoplastic in mice haploinsufficient for Tbx3 and/
or Nkx2-5. Panel A shows Immunohistochemistry staining for Cx43, Cx40 and Hcn4 in the AV junction of a wildtype mouse. Panel B shows the transition of the AV node in the AV bundle. Not that the lower nodal cells are part of the AV bundle. Panel C shows the reconstructed expression patter of Cx40 and Hcn4 in the AV junction and the ventricular septum in wildtype mice and mice haploinsufficient for Tbx3 and/ or Nkx2-5. The bar graph in panel D shows the average size of the AV node, bundle and bundle branches. AS, atrial septum; AVN, atrioventricular node; AVB, atrioventricular bundle; VS, ventricular septum.
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Haploinsufficiency for Tbx3 results in AV delay prolongation by an Nkx2-5-dependent mechanism
To determine the consequence of AV bundle hypoplasia observed in Tbx3, Nkx2-5 and compound heterozygotes for AV conduction, we recorded electrocardiograms. The RR, PR and QRS intervals were not different between groups (Table 1, n=6 for each group). Furthermore, the morphology of the QRS complex was not different between groups indicating that ventricular activation was not affected.
Next, we studied the properties of the AV conduction system in a Langendorff set-up to allow programmed stimulation. The refractory period of the AV node (at a basic cycle length of 120 ms) was not different across groups (Table 2). However, at each pacing interval the AV delay was larger in the Tbx3 heterozygotes than in mice from the other groups (Figure 2A). To investigate whether the increase in AV delay was the result of slower conduction in the AV node or in the ventricular conduction system, we subsequently measured HV intervals.
Figure 2. AV conduction is delayed in mice haploinsufficient for Tbx3. Panel A shows local eletrogram recordings
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haploinsufficient for both Tbx3 and Nkx2-5, AV delay was not prolonged. This suggests that the effect of loss of one allele of Tbx3 on AV delay is attenuated by removing one allele of Nkx2-5.
Figure 3. Haploinsufficiency for Tbx3 prevents age-mediated prolongation of atrioventricular delay. The
graph in panel A shows the average conduction delay of wildtype mice and mice haploinsufficient for Tbx3 and/or Nkx2-5 at 2 and 16 months of age. Panel B shows In-situ hybridizations for Cx40 in the His bundle. The bar graph in panel C shows the average size of the atrioventricular node and ventricular conduction system in wildtype mice and mice haploinsufficient for Tbx3 and/or Nkx2-5. Panel D shows the relation between the atrioventricular delay and the size of the connection between the atrioventricular node and His bundle. AS, atrial septum; AVN, atrioventricular node; AVB, atrioventricular bundle; VS, ventricular septum
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Tbx3 haploisufficiency prevents age-mediated AV delay prolongation
Heterozygous loss of Nkx2-5 can lead to AV delay prolongation and eventually AV block in aging mice.13 We wanted to investigate the role of Tbx3 in age-mediated AV delay prolongation
and AV block in mice. Therefore, we recorded ECGs and measured the properties of the AV node in a Langendorff set up. At 9 months we recorded ECGs to check whether AV delay had changed compared to 2 months. Although the PR interval increased somewhat compared to 2 months in all groups (compare Tables 1 and 3), the RR, QRS, PR intervals were not different
across groups at 9 months (Table 3). At 16 months, however, in explanted hearts of wildtype mice, the AV delay was significantly larger than at 2 months of age (44.8±2.3 vs 33.5±0.6, n=8 and n=6, respectively, p<0.05, Figure 3A). The refractory period of the AV node was not different between ages (Table 2). In Nkx2-5 heterozygotes, the AV delay was also larger at 16 months than at 2 months of age (40.5±2.4 vs 33.3±1.0, n=4 and n=10, respectively) but the AV delay was not different from wildtype mice at both ages. In contrast, in Tbx3 heterozygotes the AV delay was not different at 16 months of age compared to 2 months of
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A small AV node-His connection in Tbx3 heterozygotes correlates with a short AV delay
Next, we reconstructed the AV node and the ventricular conduction system based on the expression pattern of Hcn4 and Cx40. In Figure 3B representative sections of the AV bundle of the four groups of mice are shown. Similar to the situation at 2 months of age, a hypoplastic AV bundle was observed also at 16 months of age in mice haploinsufficient for Tbx3 and/or Nkx2-5. Furthermore, the AV node was not different in size in the transgenic mice compared to in the wildtype mice, resembling what we found in the younger mice. To understand why the AV delay in aged mice was not different from young mice that missed one allele of Tbx3 we correlated the diameter of the AV bundle and the AV nodal -AV bundle transition with the AV delay within the same mouse. The diameter of the AV bundle did not correlate with the AV delay (r=0.43, p=0.25). However, the size of the transition between the AV node and the AV bundle (averaged ranking from small to large by two independent observers), indicated by the contact surface of the Cx40+/Hcn4+
myocardium with the Cx40-/Hcn4+ myocardium, significantly correlated with the AV delay
(Figure 3D). The latter suggests that both Tbx3 and Nkx2-5 haploinsufficiency affects the AV delay by affecting the structural transition between the AV node and the AV bundle.
A Tbx3-independent mechanism underlies AV conduction block in aging Nkx2-5 heterozygous mice
At 16 months, we observed 2nd or 3rd degree AV block in 3 of 8 explanted hearts (38%) from
Nkx2-5 heterozygous mice, and 3rd degree AV block in 1 of 4 hearts from mice that were
haploinsufficient for both Nkx2-5 and Tbx3. The hearts that did not have 2nd or 3rd degree AV
block had normal AV conduction (Figure 4). In hearts of wildtype (n=6) or Tbx3 haploinsufficient (n=4) mice we did not observe AV block. The percentage of hearts with AV block was not significantly different between groups. However, if we selected by haploinsufficiency for Nkx2-5, significantly more AV block occurred when compared to hearts of mice with two Nkx2-5 alleles.
Discussion
Our results show that haploinsufficiency of Tbx3, Nkx2-5 or both causes hypoplasia of the AV bundle and bundle branches by, most likely, two independent mechanisms that are, however, not additive in effect. Furthermore, in young animals, Tbx3-haploinsufficiency lead to prolonged AV delay, which can be attenuated by Nkx2-5-haploinsufficiency, whereas in old animals Tbx3-haploinsufficiency lead to a shorting of AV delay independent of
Nkx2-5 haploinsufficiency. Thus, although Tbx3 and Nkx2-Nkx2-5 interact with each other in young
mice, they seem to act in parallel in old mice. This suggests that, as expected, Tbx3 and Nkx2-5 are part of a more complex transcriptional network that regulates the formation and function of the AV conduction system. We also describe that small differences in the transition between AV node and AV bundle, independent of genotype, appear to influence
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AV conduction. It is unclear how the transcriptional balance regulates the shaping of this structural transition, and whether other factors including like Irx326 and Tbx517, are involved.
Prolonged AV delay in Tbx3 haploinsufficient mice
The structure of the AV conduction system was similarly affected in Tbx3-, Nkx2-5- and Tbx3/
Nkx2-5-haploinsufficient mice. However, the AV conduction times were different. The latter
suggest that it is not the AV conduction structure that underlies these functional differences. In Tbx3 haploinsufficient mice, conduction in the AV node was only slowed in the isolated and denervated heart but not in the innervated heart in vivo, which is in line with previously published results of our group.9 When ectopically expressed in the heart, Tbx3 directly or
indirectly induces the expression of Cav3.1 (alpha subunit of T-type Calcium channels),19
which therefore may be downregulated in Tbx3-haploinsufficient mice. As a consequence, the expression of T-type calcium channels may be reduced in Tbx3 mutants but provide sufficient inward current for normal AV delay upon a certain degree of autonomic stimulation. When autonomic stimulation is absent, like in a Langendorff set-up, the inward calcium current may not be sufficient for normal conduction and AV delay prolongs.27 The expression of the
T-type calcium channels is also downregulated in the hearts of mice haploinsufficient for Nkx2-5.25 Therefore, the mechanism by which haploinsufficiency for Nkx2-5 counteracts
the prolonged AV delay in mice that are haploinsufficient for Tbx3 remains unclear.
Molecular mechanism by which Nkx2-5 and Tbx3 affect structure and function of the AV conduction system
Our data indicate that Tbx3 affects AV delay in a mechanism that is attenuated by a reduction in Nkx2-5. It has been shown that Tbx3 and Nkx2-5 can physically interact.12 However, to
our knowledge, a direct regulation of Nkx2-5 by Tbx3, or vice versa, has never been reported. Tbx3 may indirectly reduce Nkx2-5 expression via Prospero-Related Homeobox Protein (Prox) 1, which inhibits Nkx2-5 expression and is upregulated in Tbx3 overexpression mice.19, 25
However, the latter rather suggests an additive effect of a reduction of Tbx3 and Nkx2-5 than a counteractive effect, which is what our data indicate. Therefore, Nkx2-5 and Tbx3 most likely regulate the expression of certain genes by co-occupying the same genomic regions28 similar to
how Nkx2-5 and Tbx5 synergistically regulate the expression of inhibitor of differentiation (Id) 2 in the conduction system.17 The expression of Id2, however, is not affected in mice deficient
for either Nkx2-5 or Tbx3.9, 10 Gene expression analysis will reveal whether the expression of
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number of mice lacking one functional Nkx2-5 allele develop AV block. In contrast to our data, other studies reported that mice haploinsufficient for Nkx2-5 have a hypoplastic AV node and prolonged PR intervals already at 2-4 months of age.13, 25 An explanation for this discrepancy
may be found in differences in genetic background. We used FVB/N mice whereas other studies were performed in C57Bl/6 or 129/SvJ mice. It has been shown that the functional consequences of a mutation can differ between mouse strains.31 Another major discrepancy is the definition
of the AV node and AV bundle, respectively. In contrast to other studies, we have used Cx40 to discriminate between the AV node (negative) and AV bundle cells (positive) in the AV junction.14
We, therefore, defined the AV node based on the presence of Hcn4 and absence of Cx40 and the AV bundle and branches on the presence of both Hcn4 and Cx40. Furthermore, instead of comparing sections we reconstructed the conduction system and compared volumes, which is more accurate. These differences most likely explain the difference in size of the AV node and bundle in Nkx2-5 haploinsufficient mice observed in our study compared to in other studies.13, 25
The role of Tbx3 in prevention of age-mediated prolongation of AV delay
Our data indicate that Tbx3 is involved in age-mediated prolongation of the AV delay. In mice, and also in human, AV conduction becomes slower during ageing whereas the conduction in the ventricular conduction system remains the same.13, 32 Not much is known about the
underlying mechanism in mice, but in rat altered collagen deposition in the AV node is thought to contribute to AV delay prolongation.33 In the atria of mice that overexpress Tbx3 the
expression of Procollagen, type VI, alpha 2 (col6a2) and Procollagen, type I, alpha 1 (col1a1) are upregulated.12 We surmise that haploinsufficiency of Tbx3 results in less collagen in the AV
node during ageing and thereby affecting AV conduction. This, however, remains to be tested. Alternatively, ion channel remodeling may be involved in AV delay prolongation during
Figure 4. Aged mice that are haploinsufficient for Nkx2-5 develop atrioventricular block. Panel A
shows local eletrogram recordings from mice haploinsufficient for Nkx2-5 with normal atrioventricular delay (upper) and atriovenricular block (lower). The bar graph in panel B shows at the right the amount atrioventricular block in wildtype mice and mice deficient for Tbx3 and/or Nkx2-5 and. At the left the amount of atrioventricular block is shown in mice that Nkx2-5 haploinsufficiency or not.
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ageing. For the sinus node it has been reported that ageing leads to sinus node dysfunction independent of structural changes, which may be the case for the AV node as well.34
However, the role of transcription factors, like Tbx3, in maintaining the nodal gene program at older age is not known. We speculate that haploinsufficiency of Tbx3 results in a reduced nodal gene program during ageing and to faster conduction in the AV node.
Conclusion
We conclude that normal levels of Tbx3 and Nkx2-5 are required for correct formation of the AV bundle. However, in the young heart, Tbx3 and Nkx2-5 regulate AV conduction via a similar mechanism whereas in the old heart they regulate AV conduction independently.
Funding
This work was supported by grants from the Netherlands Heart Foundation (2008B062 to V.M.C. and R.C.); the European Community’s Seventh Framework Programme contract (‘CardioGeNet’ 223463 to V.M.C.).
Acknowledgements
We thank Raoul Freitas Vale Germano for his assistance.
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