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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
Marc Sylva
Corrie de Gier-de Vries
Carol Ann Remme
Connie Bezzina
Vincent Christoffels
Ruben Coronel
Reduced sodium channel function unmasks
slow conduction in the adult right ventricular
outflow tract as part of the maintained
embryonic gene program
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Abstract
Background: In patients with the Brugada Syndrome, arrhythmias typically originate in the
right ventricular outflow tract (RVOT). The RVOT is formed from the slowly conducting embryonic outflow tract that expresses Tbx2, a transcriptional repressor of genes important for rapid conduction. We hypothesize that aspects of the embryonic phenotype are maintained in the fetal and adult RVOT, and contribute to conduction slowing when cardiac sodium channel function is reduced. Furthermore, we investigated whether the slow conduction in the RVOT is regulated by Tbx2. Methods: We determined the expression of embryonic outflow tract and working myocardial genes by in situ hybridization and immunohistochemistry in mouse hearts. We performed activation mapping in fetal hearts (embryonic day 14.5 and 17.5), in adult hearts, in hearts of mice that were deficient for Tbx2, and in hearts from adult mice heterozygous for the Scn5a1798insD/+ mutation equivalent to a mutation found in some Brugada syndrome patients.
Results: In the RVOT of hearts in the early and late fetal stage, the mRNA for Cx43 (principal
gap-junction subunit for ventricular conduction) was absent and conduction was slower than in the right ventricular free wall. Tbx2-deficiency did not affect Cx43 expression and conduction velocity in the RVOT. In the adult heart, Cx43 and Scn5a mRNA was lower in the myocardium of the RVOT, but conduction velocity was not decreased in the RVOT in comparison with the right ventricular free wall. However, in hearts of heterozygous Scn5a1798insD/+ mutant mice,
conduction was slower in the RVOT than in the right ventricular wall. Conclusion: Aspects of the slowly conducting embryonic phenotype are retained in the fetal and adult RVOT through a Tbx2-independent mechanism, particularly when cardiac sodium channel function is reduced. This may have important consequences for arrhythmias emanating from the RVOT.
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Introduction
The right ventricular outflow tract (RVOT) is the main origin of arrhythmias in the Brugada syndrome and to a lesser extent also in arrhythmogenic right ventricular cardiomyopathy (ARVC). 1-3 The mechanism underlying arrhythmias in Brugada syndrome is debated but most likely involves
conduction delay or block in the presence of subtle structural discontinuities and is modulated by variations or mutations in ion channel and other genes.4-7 The electrocardiographic signs and
arrhythmias often only become evident after the application of sodium channel blockers.8 Why
arrhythmias in patients with these syndromes preferentially originate in the RVOT is unclear. The RVOT has a developmental history that is different from that of the right and left ventricle9 and therefore developmental aspects at least in part may explain this
idiosyncrasy of the RVOT.10 The RVOT is formed from the embryonic outflow tract
which is composed of slowly conducting myocardium.11, 12 We hypothesize that the adult
RVOT myocardium has retained aspects of this embryonic outflow tract phenotype and that the embryonic aspects contribute to conduction delay and block in the RVOT. During embryogenesis the chamber myocardium is typified by an increase in conduction velocity and in the expression of the gap junction subunits alpha-1 and -5 protein (Gja1 and Gja5), encoding connexin 43 (Cx43), and Cx40, respectively, and in the cardiac sodium channel protein type 5 subunit alpha (Scn5a), encoding Nav1.5.12, 13 In the embryonic outflow tract, however, the
expression of Cx40 and Cx43 are not initiated, and slow conduction is maintained.12, 14 Instead,
the embryonic outflow tract expresses T-box transcription factor (Tbx) 2 which is a repressor of the gap-junction subunit-encoding genes.15 At embryonic day (E) 14.5 in mice, the heart is fully
septated and the embryonic outflow tract has been incorporated into the right ventricular wall, thus forming the RVOT.11, 16, 17 Whether the expression of Cx43 is low and conduction velocity is
slow in the RVOT at this stage is unknown. Also, the role of Tbx2 in this process has never been addressed. Furthermore, it is also unknown whether the expression levels of Cx43 are lower and conduction velocity is slower in the adult RVOT in comparison with the right ventricular free wall. In this study we investigated the expression pattern of genes associated with conduction in the mouse RVOT in relation to conduction velocity during development and in the adult heart. In addition, we addressed whether nodal like cells are present in the RVOT. Furthermore, we investigated the functional consequences of reduced sodium current on RVOT conduction in a mouse model with a cardiac sodium channel mutation.
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Methods
Transgenic mice
The investigation conforms with 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 and was approved by the institutional review board. Tbx2cre transgenic mice and Scn5a1798insD/+ mice have been
described previously.18, 19 In this study we used three E14.5 and E17.5 and adult FVB/N
wildtype mice. Furthermore, we used five E14.5 Tbx2cre/cre mice and five littermate controls,
and five adult Scn5a1798insD/+ (129P2-OlaHsd strain) mice and three littermate controls.
Immunohistochemistry and in situ hybridization
Mice were stunned by inhalation of CO and killed by cervical dislocation, after which the adult heart or the embryos were collected. The adult heart or and whole embryos were fixed in 4% PBS buffered formaldehyde, embedded in paraplast® and sectioned at 7-8 μm for immunohistochemistry and at 10-14 μm for RNA in situ hybridization. RNA In situ hybridization was performed according to a previously described method.20 Probes for selected genes have
been described previously.18 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); Cx40 mouse monoclonal (1:250; US Biological); Cx43 mouse monoclonal (1:250; BD Transduction). 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.
Preparation of the hearts and recording of optical action potentials
Adult hearts
Mice were stunned by inhalation of CO and killed by cervical dislocation, after which the heart was excised, 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)). The hearts were placed in 10 ml Tyrode’s solution containing 15 μM Di-4 ANEPPS and subsequently in an optical mapping setup. Excitation light was provided by a 5 Watt power LED (filtered 510 +/- 20 nm). Fluorescence (filtered > 610 nm) was transmitted through a tandem lens system on CMOS sensor (100 x 100 elements, MICAM Ultima). Activation patterns were measured during sinus rhythm and ventricular pacing at a basic cycle length of 120 ms (twice the diastolic stimulation threshold).
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Fetal hearts
The hearts where removed from the fetus and incubated for 5 minutes with Tyrode’s solution containing 5 μM Di-4 ANEPPS at 37 oC. After incubation, fetal hearts were superfused with
Tyrode’s solution and placed on the stage of an inverted microscope set-up for recording optical signals. The size of the adult heart and the intrinsic heterogeneity of the outflow tract myocardium did not allow measurement of conduction velocity following central stimulation. Therefore, we analyzed isochronal patterns during sinus rhythm instead.
Quantitative PCR
RNA was isolated from dissected hearts using the NucleoSpin® RNA II (Marchery Nagel Cat:740955.50). From 1 μg of RNA, single stranded cDNA was made using SuperScript™ II Reverse Transcriptase (Invitrogen Cat: 18064-071) with Oligo(dT) primers, according to manufacture’s protocol. Quantitative PCR (qPCR) was performed using the LightCycler® 480 (Roche) and the LightCycler® 480 SYBR Green I Master solution (Roche Cat: 04887352001). Primer sequences for the gene products Scn5a, Cx43, Tnni3 and Hprt are available on request. qPCR data was analyzed using LinRegPCR program.21
Analysis and Statistics
Optical action potentials were analyzed using custom-made software based on MATLAB R2006b (MathsWorks Inc., Natick, MA).22 The local moment of activation was defined as the maximum
positive dV/dt of the action potential. Group comparisons were performed using (repeated) ANOVA. Values are given as mean +/- SEM. A P-value of 0.05 was considered statistically significant.
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Results
The working myocardial gene program is established in the RVOT just before birth
The embryonic outflow tract is a slow conducting structure marked by the expression of Tbx2, and absence of Cx43 and hairy/enhancer-of-split related with YRPW motif 2 (Hey2). To investigate whether the embryonic signature is maintained in the RVOT, we investigated the patterns of these markers in the middle fetal (E14.5) and late fetal (E17.5) stages of the heart. We used Cardiac muscle troponin I (Tnni3) to mark myocardium. At E14.5, the expression of
Cx43 was absent from the myocardium of the RVOT but present in that of the right ventricle.
This pattern was similar to that of Hey2. The expression of Tbx2 was maintained in the RVOT,
Figure 1. In the fetal heart the RVOT is composed of slowly conducting primary myocardium. Panel A and
B show in-situ hybridizations in E14.5 (A) and E17.5 (B) wildtype hearts of expression of Tnni3, Cx43,
Tbx2 and Hey2 in the right ventricle and RVOT. Note that Cx43 expression is not present in the RVOT
(black arrow). Panel 1C shows the expression of Scn5a in the RVOT at E12.5. The black bar indicates 0.1 mm. Ao, aorta; rv, right ventricle, RVOT, right ventricular outflow tract; ra, right atrium; la, left atrium.
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in a pattern complementary to that of Cx43 and Hey2 (Figure 1A). These data indicate that at stage E14.5 the RVOT had maintained the signature of the embryonic outflow tract. At stage E17.5, Cx43 and Hey2 were still absent from the RVOT region that, however, had relatively decreased in size. In contrast to stage E14.5, the expression of Tbx2 was absent from the Tnni3 positive section of the RVOT (Figure 1B). Scn5a, unlike Cx43, was already expressed in the outflow tract at E12.5 (Figure 1C) and remained expressed in the RVOT throughout later stages.
Tbx2-independent conduction slowing in the RVOT
Because the absence of Cx43 is maintained in the RVOT during the middle and late fetal stages, we assessed whether this structure also maintained its slowly conducting phenotype. To address this question, we measured conduction velocity in the right ventricular myocardium and RVOT at E14.5. Figure 2A shows the activation pattern of a wildtype heart (left). Note that the distance between isochrones decreases from the right ventricular free wall towards the RVOT. The conduction velocity was lower in the RVOT than in the right ventricular myocardium (0.075±0.013 m/s vs 0.385±0.065 m/s, respectively, p<0.05). The area of conduction slowing co-localized with the Cx43-negative myocardial (Tnni3-positive) RVOT domain (Figure 1A).
To determine whether Tbx2 regulates the expression of Cx43 and slow conduction in the RVOT, as it does in the AV canal myocardium15, we measured conduction in hearts that
were deficient for Tbx2.18 The RVOT of the hearts of Tbx2-deficient fetuses was normally
formed and expressed Cre, a reporter inserted into the Tbx2 gene to inactivate Tbx2 function (Figure 2).18 As in wildtype mice, expression of Cx43 was absent from the Cre expression
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Figure 3. The expression of Cx43 is lower in the RVOT than in right ventricle. Panel A and B show in situ
hybridizations of adult wildtype heart showing the expression of cTnI, Cx43, Tbx2 and Hey2 in the right ventricle and RVOT. Panel B illustrates the part of the right ventricle that was defined as RVOT. The bar graph in panel C shows the expression levels (qPCR, corrected for HPRT and Tnni3) of Cx43 and Scn5a in the RVOT and right and left ventricle. Panel D shows the activation patterns during central stimulation. The bar graph in panel E shows the average longitudinal and transversal conduction velocity in the right and left ventricle and the RVOT (n=3). In the RVOT no longitudinal conduction velocity could be measured. Ao, aorta; tp, truncus pulmonalis; rv, right ventricle, RVOT, right ventricular outflow tract.
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domain. The conduction velocity in the RVOT was lower than in the right ventricular myocardium (0.09±0.024 m/s vs 0.33±0.037 m/s, respectively, p<0.05), but not significantly different from the corresponding values in wildtype mice (Figure 2, bar graph) These results show that Tbx2 does not regulate the expression of Cx43 and conduction in the RVOT.
Low expression of Cx43, but not slow conduction, is maintained in the adult RVOT
Figure 3A shows the expression pattern of the Tnni3, Cx43, Tbx2 and Hey2 in hearts of adult mice. The expression of Cx43 was lower in a subepicardial region of the RVOT myocardium compared
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to the right or left ventricular myocardium. The expression of Tbx2 and Hey2 was absent in the ventricular myocardium (Figure 3A). To quantify the reduced Cx43 expression in the myocardium of the RVOT we measured Cx43 mRNA levels by quantitative RT-PCR in the entire RVOT. We also measured the expression level of Scn5a. For this purpose, the RVOT was defined as the myocardium located at the base of the right ventricle just below the pulmonary valves (Figure 3B). As control for the amount of myocardial tissue we determined the expression of Tnni3, which was not different between the RVOT and the right and left ventricle. The expression levels of both Cx43 and Scn5a were lower in the RVOT than in the right and left ventricle (Figure 3C).
We then measured conduction velocity in the RVOT and right and left ventricle (Figure 3D/E). Longitudinal conduction velocity did not differ between the right and left ventricle. There was no anisotropy present in the RVOT so we defined transverse fiber direction by the slowest conduction velocity. Transversal conduction velocity was not different across the right and left ventricle and the RVOT, but was significantly lower than longitudinal conduction velocity in the right and left ventricle (n=3).
Sodium channel dysfunction results in conduction slowing in preferential in the RVOT
Conduction velocity in the RVOT of wildtype mice was not significantly lower than in the other regions of the heart. However, the smooth walled tissue architecture of the adult RVOT and the lower level of Cx43 and Scn5a suggest that some aspects of the fetal RVOT phenotype have been retained in the adult RVOT. Therefore, we challenged conduction with reduced sodium current in order to unmask an intrinsic regional difference in conduction velocity. We measured mice heterozygous for the Scn5a-1798insD mutation, which is the murine equivalent of the human SCN5A-1795insD mutation found in patients with Brugada syndrome.23 This mutation leads to a reduced peak sodium current resulting in right ventricular
conduction slowing in mice.23 Figure 5A shows the activation pattern during sinus rhythm in
hearts from wildtype (upper panel) and Scn5a1798insD/+ (lower panel) mice. During sinus rhythm,
crowding of isochrones indicated that conduction was delayed in the right ventricle and RVOT of Scn5a1798insD/+ mice in comparison with wildtype animals. We subsequently measured
conduction velocity during central stimulation. Conduction slowing in the longitudinal direction is evident during stimulation on the RV of mutant mice in comparison with wildtype mice. Because the activation maps of the RVOT did not show a clear anisotropic pattern we only analyzed conduction velocity in a line along the outflow tract in comparison with transversal conduction velocity in the right ventricle (Figure 4C). In the wildtype mice the conduction velocity in the RVOT was not different from conduction in the right ventricle. In Scn5a1798insD/+
mice, transversal conduction velocity was lower in both the right ventricle and RVOT when compared to wildtype mice (Figure 4C). However, conduction slowing was also significantly more pronounced in the RVOT than in the right ventricle of Scn5a1798insD/+ mice (Figure 4D).
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Tbx3-positive pacemaker cells could not be detected in the RVOT
To determine whether nodal cells are present in the myocardium of the RVOT, as was suggested previously by the ventriculo-atrial ring hypothesis24, we studied the expression
of Tbx3 that delineates the pacemaker and conduction system cells.25, 26 Figure 5A shows
that at E14.5, Tbx3 mRNA and protein was expressed in the cushions of the RVOT but not in the myocardium. Also in the adult heart we could not detect Tbx3 expression in the myocardium of the RVOT, whereas we did detect, as control, Tbx3 expression in the left atrio-ventricular ring bundle (Figure 5B, black arrow). Tbx3 protein was also not detected in the RVOT, as was Hcn4, another marker for pacemaker and conduction system (data not shown). These data indicate that nodal cells are absent from the adult RVOT myocardium.
Discussion
Our current findings indicate that the Tbx2-positive, Cx43-negative myocardial gene program and
Figure 5. Tbx3 positive pacemaker cells are not present in the myocardium of the RVOT. Panel A shows at the
right In-situ hybridizations for Tnni3 and Tbx3 and at the left an immunohistochemistry staining for Tbx3, cTnI and nuclei. Panel B shows at the right In-situ hybridizations for Tnni3 and Tbx3 in the adult RVOT. Note that Tbx3 is absent in the RVOT myocardium whereas it is present in the ventricular septum (black arrow). Ao, aorto; tp, truncus pulmonalis; rv, right ventricle, RVOT, right ventricular outflowt tract; vs, ventricular septum; av, atrioventricular.
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‘nodal’ pacemaker tissue in the RVOT. Our data provide an explanation why conduction delay occurs preferentially in the RVOT, as has been observed in patients with Brugada syndrome.27
Developmental gene program and differentiation of the RVOT
In the adult heart, the RVOT is defined as the smooth-walled right ventricular myocardium that connects the base of the right ventricle with the pulmonary trunk.28 In contrast to the
embryonic outflow tract,29 the pre- and postnatal RVOT has not been well studied at the
molecular and functional level. We show that the fetal RVOT is marked by Tbx2 expression but specifically lacks expression of Hey2 and Cx43. Consistently, conduction was relatively slow in the fetal RVOT. In the adult mouse, RVOT expression levels of Cx43 were low, as was also previously observed in rabbit30, and in situ hybridization revealed a subepicardial
region in the RVOT where transcript levels were very low. These data suggest that aspects of the embryonic program are, to some extent, maintained in the adult RVOT. Nevertheless, conduction velocities are similar in the adult RVOT and right ventricle, indicating that the RVOT largely acquired a ventricular program that is sufficient for normal function.
The expression of Tbx2 disappears from the RVOT during the fetal period indicating differentiation of the RVOT into a more working myocardial phenotype. Tbx2 is a transcription factor that directly represses the chamber myocardial genes (Cx43, Cx40, Nppa) in the embryonic atrioventricular canal, and suppresses the differentiation of this component to chamber myocardium.15 Therefore, we hypothesized that Tbx2 is also involved in the repression of these
genes in the outflow tract and RVOT. However, the RVOT of homozygous Tbx2 mutants was normal and Cx43 expression was not upregulated. The related transcriptional regulator and functional homologue of Tbx2, Tbx3 is not likely to compensate for loss of Tbx2. as it is not expressed in the outflow tract (Figure 5A).25 Alternatively, as yet unidentified repressors of the
chamber genes and of differentiation are expressed in the outflow tract, or crucial activators are not expressed. One of these absent activators could be Tbx5, an obligatory activator of
Cx40 and Nppa and of chamber differentiation that is not expressed in the outflow tract or
RVOT.31 On the other hand, although Cx43 expression is strictly controlled (repressed) by
Tbx2 and Tbx315, 26, it is not dependent on Tbx531, indicating that yet other activators control
the process of gene expression and of differentiation of the outflow tract into the RVOT.
Mechanism underlying arrhythmias originating in the RVOT
To date, the mechanism underlying arrhythmias that originate in the RVOT, as seen in Brugada syndrome patients, is debated.4, 6 In particular, it has not been elucidated why the RVOT is
preferentially affected by this syndrome, that has been associated with sodium channel mutations.4 Our group has recently proposed a unifying mechanism explaining arrhythmias
in Brugada syndrome patients.27 The hypothesis involves small structural discontinuities in
the RVOT myocardium, but does not offer an explanation for the preferential location of these abnormalities in the RVOT.6 Our data indicate that the expression levels of Cx43 and
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Scn5a in the RVOT are lower than in the right and left ventricular myocardium. This suggest
that electrical coupling and sodium channel availability is intrinsically less in the RVOT than in the right ventricle and, therefore, may explain why conduction preferentially delays or blocks in the RVOT of Brugada syndrome patients. However, the effect of uncoupling on safety of conduction in discontinuous myocardium, like in the RVOT of Brugada syndrome patients, is not completely clear.32 Heterogeneous uncoupling may cause conduction
block and increase arrhythmogenesis32, 33 whereas homogeneous reduction in coupling in
discontinuous myocardium may increase the safety factor of conduction.32 In the normal
myocardium the safety of conduction is high 34-36 and reduction in electrical coupling is not
linearly related to conduction velocity.36, 37 The latter most likely explains why in wildtype
mice we did not observe slower conduction in the RVOT than in the right ventricle. We thus surmise that genes of the ventricular working myocardial gene program are less active in the RVOT, leading to less conduction reserve. Indeed, also Scn5a expression levels were lower in the RVOT than in the ventricles. This implies that conduction is more slowed in the RVOT than in the right or left ventricle when conduction reserve is challenged by a decrease in Na+ channel
function, by for example a premature beat, pharmacologic sodium channel block, or due to a mutation in Scn5a. This hypothesis is supported by our observations in the Scn5a1798insD/+ mice.
Nodal cells in the RVOT
The expression pattern of Tbx3 defines the conduction system of the heart, and is required and sufficient for pacemaker and conduction system development.25, 26, 38 Its overexpression
in adult working myocardium induces a pacemaker-like gene program upon differentiated cardiomyoctyes.25, 39 Therefore, Tbx3 expression in myocardium is a functional and strict
marker for cardiomyocytes with pacemaker properties. The presence of ‘nodal’ or ‘conduction system cells’ in the outflow tract has been suggested.24 It would imply that slow conduction in
the region is secondary to the ‘nodal’ cell type, which typically depends on the Ca-current for conduction. However, although Tbx3 is required for formation of the outflow tract, it is not expressed in the myocardium of the embryonic outflow tract.25, 38 Furthermore, we could also
not detect Tbx3-positive cardiomyocytes in the RVOT of the fetal and adult heart.25, 26 Finally,
the conduction velocity observed in the RVOT is not compatible with Ca-channel dependent conduction. Therefore, pacemaker cells are not present in the myocardium of the RVOT.
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Sources of 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.); C.R. Bezzina is supported by an Established Investigator Grant from the Netherlands Heart Foundation (NHS 2005T025). C.A. Remme is supported by the Division for Earth and Life Sciences (ALW; project 836.09.003) with financial aid from the Netherlands Organization for Scientific Research (NWO).
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