Published online before print June 4, 2009, doi: 10.1161/CIRCRESAHA.108.192443
© 2009 American Heart Association, Inc.
Cellular Biology |
Gene Expression Profiling of the Forming Atrioventricular Node Using a Novel Tbx3-Based Node-Specific Transgenic Reporter
From the Heart Failure Research Center (T.H., J.F.B., A.O.V., M.L.B., V.W., D.E.W.C., A.F.M.M., V.M.C.), Academic Medical Center, University of Amsterdam; and Center for Human and Clinical Genetics (H.P.J.B., P.A.C.H.), Leiden University Medical Center, The Netherlands.
Correspondence to Vincent M. Christoffels, Heart Failure Research Center, AMC, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail v.m.christoffels{at}amc.uva.nl
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Abstract |
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The atrioventricular (AV) node is a recurrent source of potentially life-threatening arrhythmias. Nevertheless, limited data are available on its developmental control or molecular phenotype. We used a novel AV nodal myocardium–specific reporter mouse to gain insight into the gene programs determining the formation and phenotype of the developing AV node. In this reporter, green fluorescent protein (GFP) expression was driven by a 160-kbp bacterial artificial chromosome with Tbx3 and flanking sequences. GFP was selectively active in the AV canal of embryos and AV node of adults, whereas the Tbx3-positive AV bundle and sinus node were devoid of GFP, demonstrating that distinct regulatory sequences and pathways control expression in the components of the conduction system. Fluorescent AV nodal and complementary Nppa-positive chamber myocardial cell populations of embryonic day 10.5 embryos and of embryonic day 17.5 fetuses were purified using fluorescence-activated cell sorting, and their expression profiles were assessed by genome-wide microarray analysis, providing valuable information concerning their molecular identities. We constructed a comprehensive list of sodium, calcium, and potassium channel genes specific for developing nodal or chamber myocardium. Furthermore, the data revealed that the AV node and the chamber (working) myocardium phenotypes diverge during development but that the functional gene classes characterizing both subtypes are maintained. One of the repertoires identified in the AV node–specific gene profiles consists of multiple neurotrophic factors and semaphorins, not yet appreciated to play a role in nodal development, revealing shared characteristics between nodal and nervous system development.
Key Words: cardiac development • gene expression • gene regulation • ion channels • transgenic mice
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Introduction |
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The electric impulse is initiated in the sinus node, propagated rapidly through the muscular tissues of the atria, delayed in the atrioventricular (AV) node, and then rapidly propagated through the ventricular conduction system, activating the ventricles from apex to base. The slow conduction through the AV node ensures sequential, synchronized contractions of the atria and ventricles (reviewed elsewhere1,2). This guarantees the ventricles to benefit from the atrial contraction, adding 15% to cardiac output, and prevents one-to-one conduction of potentially life-threatening supraventricular arrhythmias. Furthermore, the AV node is the major subsidiary pacemaker in case of failure of the sinus node (reviewed elsewhere1,3). However, the AV node is also a recurrent source of tachy- and bradyarrhythmias, notably AV node reentrant tachycardia and AV block, requiring lifelong medication, ablation, or electronic pacemaker implantation. Knowledge regarding development,4–8 anatomy,2 and molecular background of the physiological function9–13 of the AV node has increased gradually. Nevertheless, insights into the transcriptional pathways regulating its development, and the gene expression profiles underlying its phenotype and function are limited.
The T-box transcription factor Tbx3 belongs to an evolutionary conserved family of factors, plays key roles in development and cancer, and is mutated in the ulnar mammary syndrome of congenital defects.14 Tbx3 is specifically expressed in the components of the cardiac conduction system throughout development and in the adult and plays important roles in their formation.15–17 We identified a large regulatory fragment driving green fluorescent protein (GFP) reporter gene expression selectively in the Tbx3-positive, Cx40/Cx43-negative nodal myocardium of the AV canal of embryos and in the AV canal–derived AV node and AV ring bundle in fetuses and adults. Subsequently, this unique AV nodal marker model was used to study the transcription profiles of the developing AV node through microarray analysis, providing new insights into the molecular pathways underlying differentiation and function of the embryonic AV canal and maturing AV node.
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Materials and Methods |
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Transgenic Mice
The transgenic working myocardium (WM)-specific promoter–reporter line NppaBAC336-Egfp has been described previously.18 BAC clone 366H17, containing genomic sequences from –83 to +77 kbp relative to the transcription start site of Tbx3, was obtained from a C57BL/6J mouse BAC/PAC library (CHORI, Oakland, Calif). To generate the Tbx3BAC-Egfp construct, the sequence ATG.agc.ctc.t was replaced by GFP using the BAC modification method kindly provided by S. Gong and N. Heintz.19
Fluorescent-Activated Cell Sorting and RNA Preparation and Handling
Hearts from transgenic stage-matched embryos were dissected, pooled, and single-cell fluorescent-activated cell sorting (FACS) samples were prepared using a protocol kindly provided by Masino and Garry.20 Cells were sorted on a FacsAria flow cytometer (BD Biosciences) using FacsDIVA software. Samples from wild-type age-matched hearts were used for gating. Samples were gated to exclude debris and cell clumps. Fluorescent cells comprised, on average, 1% to 4% of live-gated AV nodal, and 3% to 8% of live-gated WM cell populations, whereas postsort analysis revealed 85% to 90% purity of GFP+ cell populations after flow cytometry. The number of embryonic day (E)10.5 AV canal GFP+ cells per embryo obtained were typically 600 to 900. RNA was subjected to 2 rounds of linear amplification and biotin labeling. Per sample, 5000 live-gated cells were collected directly in RA1 Nucleospin RNA cell lysis buffer and RNA was isolated following the protocol of the manufacturer (Machery-Nagel). RNA integrity was analyzed on an Agilent Bioanalyzer 2100, using RNA 6000 pico chips. The RIN numbers for the E10.5 AVC samples were: 10, 8.9, 9.0, 9.0, 8.9, 8.3; for the E10.5 WM samples: 7.5, 9.8, 9.0, 7.2, 8.6, 9.9; for the E17.5 AVN samples: 8.0, 8.0, 9.0, 7.7, 9.0, 6.8; and for the E17.5 WM samples: 8.6, 8.0, 7.7, 8.0, 7.8, 7.3. Samples were subjected to 2 rounds of linear amplification, using the MessageAmp II aRNA Amplification Kit (Ambion) for the first round and the Illumina TotalPrep RNA Amplificaton Kit (Ambion) for the second round and biotin-16-UTP labeling. For each group, 6 labeled antisense RNA samples were hybridized to separate Illumina Mouse-Ref-6 BeadChips following the instructions of the manufacturer (Illumina Inc).
An expanded Materials and Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
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Results |
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Tbx3 Activity Is Driven by Distal Regulatory Sequences
To assess the spatiotemporal activity of the putative regulatory sequences of Tbx3, transgenic mice were generated in which an 8-kbp DNA fragment around the transcription start site drives LacZ reporter gene expression (Figure 1). Five of 6 independently obtained lines expressed LacZ in a pattern unlike that of Tbx3. In 4 of these lines, LacZ was expressed mainly ectopically in the caudal part of the embryo and the forelimbs, whereas no expression was detected in the Tbx3+ tissues such as the mammary glands or heart (Figure 1D through 1F and 1H through 1J). These observations indicate that the regulatory sequences driving Tbx3 expression are located outside the 8-kbp region.
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-BsteII marker; lv, left ventricle; fl, forelimb; ra, right atrium; la, left atrium; rv, right ventricle; lsh, left sinus horn; rsh, right sinus horn.
A BAC clone containing Tbx3 flanked by 83-kbp upstream sequences and 77-kbp downstream sequences (Figure 1A through 1C) was modified by inserting the enhanced (E)GFP encoding reporter gene at the translation start site of Tbx3, thereby functionally inactivating the gene in the BAC to avoid gain of Tbx3 function in transgenics. This BAC contains the gene for AK048257, upstream of Tbx3 (Figure 1A). However, this transcript was not detectable in the prenatal heart by quantitative RT-PCR (not shown), indicating that this gene would not interfere with heart development when introduced in transgenic mice. Next, 6 independent transgenic mouse lines were generated with the Tbx3BAC-Egfp construct randomly integrated in the genome. Four independent mouse lines carrying Tbx3BAC-Egfp showed comparable spatiotemporal EGFP patterns (Figures 1G, 1K, 2B, and 2C) highly similar to the pattern of Tbx3 (1D,H and 2A). In situ hybridizations on serial sections confirmed that in the eye, snout, mammary glands and limbs Tbx3BAC-Egfp mimicked Tbx3 expression (Figures 1G and 2A through 2
I, Online Table I). However, the construct appeared inactive in the ear (Figures 1G, 2B, and 2C) and the lung mesenchyme (Figure 2K and 2L), showing that not all DNA sequences regulating Tbx3 expression were present in the 160-kbp BAC.
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Tbx3BAC Drives AV Node Myocardium–Specific Expression
Tbx3 is expressed in the sinus node, AV node, AV bundle, and bundle branches,15 but Tbx3BAC-Egfp was not active in the Tbx3-positive, Cx43/Nppa-negative sinus node, AV bundle, and bundle branches (Figure 2J through 2L). In contrast, Tbx3BAC-Egfp was active in the nodal (Tbx3-positive, Cx40/Cx43/Nppa-negative) myocardium of the AV canal from E9 onwards (Figure 3). No activity was detected in the Tbx3-positive left AV canal. Also, the Tbx3-positive AV mesenchymal cushions were devoid of EGFP (Figure 4A). EGFP expression was found in the AV node, right AV ring bundle, and retroaortic root branch, structures derived from the AV canal that have a nodal phenotype (Figure 2K, 2L, and 3A through 3F).4,21–23 Immunohistochemistry revealed that EGFP expression was limited to Tbx3-positive myocardium of the these structures, whereas expression was not observed in the Tbx3-positive AV bundle or in nonmyocardial cells, such as the valve mesenchyme, fibrous body, and epicardium (Figure 3G through 3I and Online Figure I). In the adult, we detected EGFP expression strictly limited to the Tbx3-positive, Hcn4-positive muscle tissue of the AV node and the anterior node, complementary to the Cx40-positive AV bundle (Online Figure II).
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To further verify AV nodal specificity of Tbx3BAC-Egfp, we performed patch-clamp experiments on single EGFP-positive and EGFP-negative cardiac cells of E17.5 embryos. Importantly, all EGFP-positive cells (n=11) showed diastolic depolarization, resulting in spontaneous activity, and exhibited the hyperpolarization-activated current (If), whereas all EGFP-negative cells (n=13) had a stable resting membrane potential, were quiescent, and lacked If. EGFP-negative cells could be stimulated by current pulses through the patch pipette. All action potential parameters, except overshoot, differed significantly between both groups (Figure 3J and 3K and Online Table II). In conclusion, EGFP-positive cells had electric properties typical for nodal cells and EGFP-negative cells for WM cells. The combined data of the marker expression analysis and patch-clamp experiments indicate that the 160-kbp Tbx3BAC-Egfp construct contains regulatory sequences driving specific AV nodal expression but lacks additional enhancers driving expression in the sinus node, AV bundle, and bundle branches.
Gene Expression Profiles of the Developing AV Node and WM
The property of Tbx3BAC-Egfp to drive EGFP expression specifically in the developing and mature AV nodal myocardium allowed monitoring the changes in genome-wide expression profiles during their differentiation. Nppa, the gene encoding atrial natriuretic factor, is specifically expressed in developing WM, complementary to the Tbx3-positive conduction system myocardium.5,15–18 The expression of EGFP in the NppaBAC336-Egfp transgene recapitulates the Nppa expression pattern precisely.18 Patch-clamp experiments on single EGFP-positive cells of E17.5 NppaBAC336-Egfp fetuses confirmed that these cells had a stable resting membrane potential, were quiescent, and lacked If (Online Figure III).
E10.5 Tbx3BAC-Egfp cells (E10.5 AV canal) were compared with age-matched pooled atrial and ventricular cardiomyocytes expressing NppaBAC336-Egfp (E10.5 WM). Fetal E17.5 Tbx3BAC-Egfp (E17.5 AV nodal) cells were compared with E17.5 NppaBAC336-Egfp atrial WM (E17.5 WM) (Figures 4A and 5A). The cell purification procedure has been outlined in the Methods and Figure 4B and 4C. Microarray data have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE13614.
All genes known to be specific to the AV myocardium, including Tbx3 itself, were highly enriched in both E10.5 and E17.5 AV myocardial groups (Table). Also the array data regarding Tbx20 were consistent with its initial broad and later AV canal-enriched pattern. All genes known to be specific to developing chambers/WM were strongly enriched in chamber myocardium at both stages (Table). Furthermore, of 27 of 29 genes the differences in expression between E17.5 AV nodal and WM samples were confirmed by quantitative RT-PCR (Online Table III). Together, these data validate the microarray analysis and indicate that it efficiently identified genes differentially expressed between the developing AV node and age-matched WM.
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Diverging Phenotypes of the Developing AV Node and WM
Of 46 643 transcripts present on the array, 1259 (3%) were over- and 856 (2%) were underrepresented in the E10.5 AV canal compared to E10.5 WM (Figure 5A and 5B). Another 12 593 (27%) had signals above threshold and were considered to be expressed, but not differentially. In the E17.5 AV node, 3182 (7%) transcripts were overrepresented and 3370 (7%) were underrepresented compared to the E17.5 WM, revealing an evident divergence of gene profiles of the differentiating AV node and WM. To assess this deviation in gene profiles we used the GLOBAL test.24 Using standardized test conditions, we identified 124 differentially expressed Gene Ontology (GO) categories at E10.5, and 431 terms at E17.5 (Online Table IV). These data confirmed divergence of gene expression profiles and phenotypes between maturing AV nodal and WM.
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Early Differentiation and Further Specialization of AV Nodal Cells During Development
To evaluate AV nodal gene expression during development independently of developmental changes in WM gene expression, the expression profile data of the E10.5 WM cell population were used as common reference for the three other data sets. We calculated genes differentially expressed between E10.5 WM and E17.5 AV nodal, and between E10.5 WM and E17.5 WM, respectively, and combined these data with the values found comparing both E10.5 groups. Whereas 2115 transcripts (1259 up, 856 down) were expressed differentially between E10.5 WM and AV canal myocardium (Figure 6A), the difference between E10.5 WM and E17.5 AV nodal myocardium had increased to 11 388 (5366 up, 6022 down), implying an increase in differential expression of 9273 transcripts in the maturing AV nodal cells (Figure 6A). Of these 9273 transcripts, 4611 transcripts appeared differentially expressed between E10.5 WM and E17.5 WM as well, indicating these were not node-specific, but characteristic of myocardial maturation in general (Figure 6B). To gain insight into nodal differentiation, we assessed transcripts differentially expressed between E10.5 WM and E10.5 and/or E17.5 nodal tissue (Figure 6C), excluding the 4611 transcripts associated with myocardial maturation. Of the 1092 transcripts differentially expressed at E10.5, 249 (23%) transcripts were differentially expressed only early in AV canal development, whereas 77% (843) were still differentially expressed at E17.5 (Figure 6C). A total of 5013 transcripts were found to be differentially expressed only in the E17.5 AV nodal cells. These transcript lists are available on request. Together, these data indicate that the AV node differentiates considerably during development but that the late fetal AV node largely maintains the AV canal program acquired at E10.5.
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To gain insight into the 3 groups of differentially expressed genes in the developing AV node, we used the Database for Annotation, Visualization and Integrated Discovery (http://david.abcc.ncifcrf.gov) bioinformatics tool to functionally categorize the transcripts, using GO annotations (Online Table V). Found percentages were compared to the relative presence of specific GO terms on the Illumina Mouse-Ref-6 BeadChip. Among transcripts enriched in both the AV canal and AV node, GO categories associated with "nervous system development" were overrepresented. Among nervous system development, neurotrophic factors Nrn1, Ntng1, Ntn2l, Gdnf, Slit2, and Rtn4, the receptors for these neurotrophic factors Unc5b, Unc5c, Ret, and Robo2, and several semaphorins, including Sema3f, Sema3b, Sema4c, Sema4g, and Sema6a, were found to be enriched in the AV canal and/or node. Contamination of nervous tissue at E10.5 can be excluded as the innervation of the heart has not yet taken place at this stage. Therefore, the latter finding reveals a substantial overlap in the gene expression signature between developing nodal myocardium and nervous tissue. Among transcripts expressed at a lower level in nodal tissue at both stages, GO terms associated with contraction, energy and electrophysiology were overrepresented ("muscle development," "mitochondrion," "contractile fiber," "cell junction," "ion channel activity"). These processes and structures are typically more developed in the WM,25 further elaborating the notion that although AV canal cells substantially differentiate to form AV nodal cells, their primitive, nonworking properties are maintained.
The AV canal/node population could be contaminated with copurified mesenchymal cells of the AV cushions/fibrous insulation that are in close association with these nodal cells. Resorting of purified EGFP-positive cells revealed 
85% to 90% pure cell populations. Indeed, in both E10.5 and E17.5 AV nodal groups, mesenchymal transcripts like Postn26 that are highly expressed specifically in the cushion mesenchyme were found to be significantly enriched, whereas the signals for these transcripts was low or absent in both WM groups. It is difficult to estimate contamination, because of most transcripts the precise spatial distribution has not been established.
Genome-Wide Profile of Ion Channel Families Expressed in AV Nodal Tissue
The AV myocardium has highly specific electrophysiological properties. To gain insight into the associated gene profiles, we examined ion channel 
- and β-subunit and accessory gene expression profiles of the E10.5 and E17.5 AV nodal myocardium relative to age-matched WM. In addition to previously established differences,9,12 we noted several novel channel transcripts to be enriched in the developing AV or WM (Online Figure IV and Online Table VI). For example, both the cardiac -subunit Scn5A (Nav1.5) and fibroblast growth factor Fgf12, a protein shown to interact with Nav1.5 and modulate its properties,27 were expressed high in WM throughout development. In addition to transient receptor potential (Trp) cation channels Trpc2, Trpc6, and Trpm8, known to function in calcium homeostasis, several novel members of this family were enriched in AV nodal (Trpc1, Trpm5, Trpv4) or WM tissue (Trpm2). Inositol 1,4,5-triphosphate receptor 3 (Itpr3), previously associated with pacemaker activity in differentiating cardiomyocytes,28 and Trpv4 were enriched only in the AV canal. The genes encoding -subunits Kv1.4, Kv3.2, Kv4.2, Kv4.3, Kv6.2, erg6, Kcnq1 (KvLQT1), and channel modifier genes Dpp6 and Kcmf1 (Pmcf) predominated in WM, of which several already at E10.5. Overviews are given in Online Figure IV and Online Table VI.
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Discussion |
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Assessing the Unique Gene Profile of the Developing AV Node Myocardium
The adult AV node is a morphologically complex structure with heterogeneous molecular and functional characteristics, containing not only cardiomyocytes but also mesenchymal cells, fibroblasts, smooth muscle, innervating neurons, and extracellular matrix. Here, we identified novel Tbx3 sequences that in the heart drive reporter gene expression exclusively in the AV canal and node myocardium, allowing their identification and purification. The specificity of EGFP expression in Tbx3-expressing AV nodal myocardium was assessed by detailed expression analysis and patch-clamp analyses of isolated fluorescent cells. Hence, we have generated a unique transgenic reporter model for developing and adult AV nodal myocardium that may represent a useful tool to dissect the mechanisms of its formation and to assess its phenotype during development or pathology.
Using FACS, we obtained purified AV myocardial cell populations largely free of other cell types, and their expression profiles were compared to those of age-matched WM cells obtained from NppaBAC336-Egfp mice.18 Transcripts associated with mitochondria and contractile fibers were underrepresented in both the AV canal and AV node, whereas those associated with nervous system development (especially a subclass of neurotrophic genes and semaphorins) were prominently overrepresented in both the AV canal and AV node. These data not only confirm previous findings4,21,25,29,30 but provide a much more elaborate picture of the transcriptional landscape of the developing AV node and WM. The transcripts differentially expressed only in developing AV canal myocardium at E10.5 are interesting because they probably reveal gene programs involved in the early differentiation of the AV canal but not in the maintenance of its phenotype (Figure 6C). The latter genes are represented by the substantial overlap in transcripts between the E10.5 and E17.5 AV nodal myocardium. Furthermore, the large number of transcripts uniquely differentially expressed in the E17.5 AV nodal myocardium represents a gene program associated with further specialization of the AV myocardium. The transcripts of the different AV nodal groups are interesting candidates for investigation in models for AV conduction defects and structural abnormalities, including mutants for Nkx2-5, Tbx5, and Gata4.31 These transcription factor genes appeared to be not enriched in the AV canal but likely have target genes among the differentially expressed genes identified here. Taken together, we have generated a potentially valuable transgenic tool to study AV node development and function in vivo.
To gain insight into the molecular underpinning of the physiologically complex AV node, we analyzed the expression profile data sets for expression of "electrophysiological" transcripts (ion channel, accessory subunit, etc). Previous expression studies of the adult AV node have provided valuable data on the expression distribution of those genes.9 However, those were performed on dissected adult tissues contaminated with adjacent WM and the nonmyocardial cell populations, most likely affecting the observed expression profiles. Furthermore, they examined expression of 82 predefined genes.9 The microarray approach presented here largely confirmed these findings and, in addition, identified a considerable number of novel cardiac transcripts enriched in either AV nodal or WM that may be important for understanding AV node function (Online Figure IV and Online Table VI).
The Origin and Modular Development of the AV Node
Histological studies and marker analyses revealed that the conduction system (sinus node, AV node, AV bundle, and bundle branches) is formed from a contiguous network of myocardial cells distinctive from the surrounding atrial and ventricular (working) myocardium.4–6,29 This network can be discriminated already early in development by poorly developed sarcomeres and sarcoplasmatic reticulum, high automaticity, low proliferation rate, sparse mitochondria, selective expression of markers (eg, Gln2, HNK-1, Tbx3, minK-lacZ, CCS-lacZ), and absence of high-conductance gap junctions (eg, Cx43).4,21,25,29,32 These properties, which are controlled by Tbx316,17 and other factors, are largely maintained in the adult components. The existence of this contiguous precursor network with shared properties suggests a common regulatory mechanism for conduction system gene expression and formation. On the other hand, arguments in favor of a modular composition have been put forward as well. During midfetal stages, the initially "nodal" AV bundle acquires rapid conduction and Cx40 expression.17,33 In Tbx5/Nkx2-5 and Tbx5/Id2 double heterozygous mutants and in Tbx3 homozygous mutants, which develop severe AV bundle defects, the AV node seems less affected,17,34 suggestive of differential sensitivity of these components to loss of transcription factor function. In the present study, we found that 160 kbp of Tbx3 and flanking sequences drive expression highly selectively in the AV canal of embryos and AV node of adults but not in the Tbx3-expressing AV bundle or sinus node. These data unambiguously demonstrate that separable regulatory sequences and regulatory pathways (regulatory modules) control gene expression and formation of the AV node, AV bundle, and other conduction system components.
The AV node may be derived from the embryonic AV canal or AV ring or may form de novo from other sources.4,29,35 From E9 onward, the expression of EGFP was observed selectively in the myocardium of the AV canal and gradually became confined to the AV node and anterior node–like structure.22 Furthermore, the expression profile of the embryonic AV canal showed a large overlap with the profile of the late fetal AV node, whereas both profiles were found to be consistently distinctive from both E10.5 and E17.5 WM. These findings indicate early segregation of AV canal cells and adjacent WM cells from E10.5 or earlier, arguing the AV node arises from myocardium in the dorsal wall of the AV canal as proposed by Virágh and Challice.4,29
Specialization of the AV Nodal Myocardium During Development
The embryonic AV canal and the derived AV node maintain many properties (see above) found already in the embryonic heart tube, whereas the surrounding myocardium acquires WM properties.5 These observations have led to the idea that suppression of (WM) differentiation in the AV canal domain is an important contributing mechanism to AV node formation, an idea that gained strong support by the functional identification of suppressors Tbx2 and Tbx3 in the AV canal and the conduction system.5 However, the AV myocardium itself is likely to also specialize during development,36 an aspect that had not been thoroughly examined thus far. When comparing the AV gene expression profiles at an early (E10.5) and late (E17.5) time point of development with the common reference, 249 transcripts were found to be differentially expressed only at E10.5. These are interesting candidates potentially involved in the initiation and early differentiation of the AV node. A total of 77% of transcripts (843 transcripts) differentially expressed at E10.5 still are differentially expressed in the fetal AV node, indicative for maintenance of properties during AV node development. On the other hand, 5000 transcripts not differentially expressed at E10.5 were differentially expressed in the E17.5 AV node, indicating that the AV node undergoes substantial specialization during development.
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Acknowledgments |
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We thank Berend Hooibrink, Corrie de Gier-de Vries, Wim Aanhaanen, Chantal Schreurs and Floor Hageman for excellent technical support, Daniel J Garry for providing protocols, and Marian van Roon of the Academic Medical Center Genetisch Gemodificeerde Muizen facility for generating transgenic mice.
Sources of Funding
This work was supported by grants from The Netherlands Heart Foundation (96.002 to V.M.C. and A.F.M.M.); European Community’s Sixth Framework Programme contract ("HeartRepair") LSHM-CT-2005-018630 to the Academic Medical Center (V.M.C., A.F.M.M.) and Leiden University Medical Center (P.A.C.H.).
Disclosures
None.
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Footnotes |
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*Authors contributed equally to this work.
Original received December 10, 2008; revision received May 15, 2009; accepted May 21, 2009.
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References |
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1. Meijler FL, Janse MJ. Morphology and electrophysiology of the mammalian atrioventricular node. Physiol Rev. 1988; 68: 608–647.
2. Anderson RH, Becker AE, Tranum-Jensen J, Janse MJ. Anatomico-electrophysiological correlations in the conduction system–a review. Br Heart J. 1981; 45: 67–82.
3. Mangoni ME, Nargeot J. Genesis and regulation of the heart automaticity. Physiol Rev. 2008; 88: 919–982.
4. Virágh S, Challice CE. The development of the conduction system in the mouse embryo heart. II. Histogenesis of the atrioventricular node and bundle. Dev Biol. 1977; 56: 397–411.[CrossRef][Medline] [Order article via Infotrieve]
5. Moorman AFM, Christoffels VM. Cardiac chamber formation: development, genes and evolution. Physiol Rev. 2003; 83: 1223–1267.
6. Gourdie RG, Harris BS, Bond J, Justus C, Hewett KW, O'Brien TX, Thompson RP, Sedmera D. Development of the cardiac pacemaking and conduction system. Birth Defects Res Part C Embryo Today. 2003; 69: 46–57.[CrossRef][Medline] [Order article via Infotrieve]
7. Myers DC, Fishman GI. Molecular and functional maturation of the murine cardiac conduction system. Trends Cardiovasc Med. 2003; 13: 289–295.[CrossRef][Medline] [Order article via Infotrieve]
8. Stroud DM, Gaussin V, Burch JB, Yu C, Mishina Y, Schneider MD, Fishman GI, Morley GE. Abnormal conduction and morphology in the atrioventricular node of mice with atrioventricular canal-targeted deletion of Alk3/Bmpr1a receptor. Circulation. 2007; 116: 2535–2543.
9. Marionneau C, Couette B, Liu J, Li H, Mangoni ME, Nargeot J, Lei M, Escande D, Demolombe S. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol. 2005; 562: 223–234.
10. Li J, Greener ID, Inada S, Nikolski VP, Yamamoto M, Hancox JC, Zhang H, Billeter R, Efimov IR, Dobrzynski H, Boyett MR. Computer three-dimensional reconstruction of the atrioventricular node. Circ Res. 2008; 102: 975–985.
11. Boyett MR, Harrison SM, Janvier NC, McMorn SO, Owen JM, Shui Z. A list of vertebrate cardiac ionic currents nomenclature, properties, function and cloned equivalents. Cardiovasc Res. 1996; 32: 455–481.
12. Schram G, Pourrier M, Melnyk P, Nattel S. Differential distribution of cardiac ion channel expression as a basis for regional specialization in electrical function. Circ Res. 2002; 90: 939–950.
13. Nerbonne JM, Kass RS. Molecular physiology of cardiac repolarization. Physiol Rev. 2005; 85: 1205–1253.
14. Naiche LA, Harrelson Z, Kelly RG, Papaioannou VE. T-box genes in vertebrate development. Annu Rev Genet. 2005; 39: 219–239.[CrossRef][Medline] [Order article via Infotrieve]
15. Hoogaars WMH, Tessari A, Moorman AFM, de Boer PAJ, Hagoort J, Soufan AT, Campione M, Christoffels VM. The transcriptional repressor Tbx3 delineates the developing central conduction system of the heart. Cardiovasc Res. 2004; 62: 489–499.
16. Hoogaars WMH, Engel A, Brons JF, Verkerk AO, de Lange FJ, Wong LYE, Bakker ML, Clout DE, Wakker V, Barnett P, Ravesloot JH, Moorman AFM, Verheijck EE, Christoffels VM. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev. 2007; 21: 1098–1112.
17. Bakker ML, Boukens BJ, Mommersteeg MTM, Brons JF, Wakker V, Moorman AFM, Christoffels VM. Transcription factor Tbx3 is required for the specification of the atrioventricular conduction system. Circ Res. 2008; 102: 1340–1349.
18. Horsthuis T, Houweling AC, Habets PEMH, de Lange FJ, el Azzouzi H, Clout DEW, Moorman AFM, Christoffels VM. Distinct regulation of developmental and heart disease induced atrial natriuretic factor expression by two separate distal sequence. Circ Res. 2008; 102: 849–859.
19. Gong S, Yang XW, Li C, Heintz N. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication. Genome Res. 2002; 12: 1992–1998.
20. Masino AM, Gallardo TD, Wilcox CA, Olson EN, Williams RS, Garry DJ. Transcriptional regulation of cardiac progenitor cell populations. Circ Res. 2004; 95: 389–397.
21. Moorman AFM, de Jong F, Denyn MMFJ, Lamers WH. Development of the cardiac conduction system. Circ Res. 1998; 82: 629–644.
22. Blom NA, Gittenberger-de Groot AC, deRuiter MC, Poelmann RE, Mentink MMT, Ottenkamp J. Development of the cardiac conduction tissue in human embryos using HNK-1 antigen expression: possible relevance for understanding of abnormal atrial automaticity. Circulation. 1999; 99: 800–806.
23. Anderson RH, Yanni J, Boyett MR, Chandler NJ, Dobrzynski H. The anatomy of the cardiac conduction system. Clin Anat. 2009; 22: 99–113.[CrossRef][Medline] [Order article via Infotrieve]
24. Goeman JJ, van de Geer SA, de Kort F, van Houwelingen HC. A global test for groups of genes: testing association with a clinical outcome. Bioinformatics. 2004; 20: 93–99.
25. de Haan RL. Differentiation of the atrioventricular conducting system of the heart. Circulation. 1961; 24: 458–470.
26. Kruzynska-Frejtag A, Wang J, Maeda M, Rogers R, Krug E, Hoffman S, Markwald RR, Conway SJ. Periostin is expressed within the developing teeth at the sites of epithelial-mesenchymal interaction. Dev Dyn. 2004; 229: 857–868.[CrossRef][Medline] [Order article via Infotrieve]
27. Liu CJ, Dib-Hajj SD, Renganathan M, Cummins TR, Waxman SG. Modulation of the cardiac sodium channel Nav1.5 by fibroblast growth factor homologous factor 1B. J Biol Chem. 2003; 278: 1029–1036.
28. Kapur N, Banach K. Inositol-1,4,5-trisphosphate-mediated spontaneous activity in mouse embryonic stem cell-derived cardiomyocytes. J Physiol. 2007; 581: 1113–1127.
29. Virágh S, Challice CE. The development of the conduction system in the mouse embryo heart. I. The first embryonic A-V conduction pathway. Dev Biol. 1977; 56: 382–396.[CrossRef][Medline] [Order article via Infotrieve]
30. Walls EW. The development of the specialized conducting tissue of the human heart. J Anat. 1947; 81 (pt 1): 93–110.6.[Medline] [Order article via Infotrieve]
31. Bruneau BG. The developmental genetics of congenital heart disease. Nature. 2008; 451: 943–948.[CrossRef][Medline] [Order article via Infotrieve]
32. de Jong F, Opthof T, Wilde AAM, Janse MJ, Charles R, Lamers WH, Moorman AFM. Persisting zones of slow impulse conduction in developing chicken hearts. Circ Res. 1992; 71: 240–250.
33. Chuck ET, Freeman DM, Watanabe M, Rosenbaum DS. Changing activation sequence in the embryonic chick heart: implications for the development of the His-Purkinje system. Circ Res. 1997; 81: 470–476.
34. Moskowitz IP, Kim JB, Moore ML, Wolf CM, Peterson MA, Shendure J, Nobrega MA, Yokota Y, Berul C, Izumo S, Seidman JG, Seidman CE. A molecular pathway including id2, tbx5, and nkx2-5 required for cardiac conduction system development. Cell. 2007; 129: 1365–1376.[CrossRef][Medline] [Order article via Infotrieve]
35. Arguëllo C, Alanis J, Valenzuela B. The early development of the atrioventricular node and bundle of His in the embryonic chick heart. An electrophysiological and morphological study. Development. 1988; 102: 623–637.[Abstract]
36. Milan DJ, Giokas AC, Serluca FC, Peterson RT, MacRae CA. Notch1b and neuregulin are required for specification of central cardiac conduction tissue. Development. 2006; 133: 1125–1132.
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