Transcriptional regulation of cardiac conduction system development2004 FASEB cardiac conduction system minimeeting Washington DC.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 280A:1036 –1045 (2004) Transcriptional Regulation of Cardiac Conduction System Development: 2004 FASEB Cardiac Conduction System Minimeeting, Washington, DC BRETT S. HARRIS,1,2* PATRICK Y. JAY,3 MARY S. RACKLEY,2,4 SEIGO IZUMO,5 TERRENCE X. O’BRIEN,2,4 AND ROBERT G. GOURDIE1 1 Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 2 Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina 3 Developmental Biology Unit, Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 4 Medical Research Service, Ralph H. Johnson Department of Veteran Affairs Medical Center, Medical University of South Carolina, Charleston, South Carolina 5 Cardiology Division, Beth Israel Deaconess Medical Center, Boston, Massachusetts ABSTRACT The development of the complex network of specialized cells that form the atrioventricular conduction system (AVCS) during cardiac morphogenesis occurs by progressive recruitment within a multipotent cardiomyogenic lineage. Understanding the molecular control of this developmental process has been the focus of recent research. Transcription factors representative of multiple subfamilies have been identiﬁed and include members of zinc-ﬁnger subfamilies (GATA4, GATA6 HF-1b), skeletal muscle transcription factors (MyoD), T-box genes (Tbx5), and also homeodomain transcription factors (Msx2 and Nkx2.5). Mutations in some of these transcription factors cause congenital heart disease and are associated with cardiac abnormalities, including deﬁcits within the AVCS. Mouse models that closely phenocopy known human heart disease provide powerful tools for the study of molecular effectors of AVCS development. Indeed, investigations of the Nkx2.5 haploinsufﬁcient mouse have shown that peripheral Purkinje ﬁbers are signiﬁcantly underrepresented. This piece of data corroborates our previous work showing in chick, mouse, and humans that Nkx2.5 is elevated in the differentiating AVCS relative to adjacent working ventricular myocardial tissues. Using the chick embryo as a model, we show that this elevation of Nkx2.5 is transient in the network of conduction cells comprising the peripheral Purkinje ﬁber system. Functional studies using defective adenoviral constructs, which disrupt the normal variation in level of this gene, result in perturbations of Purkinje ﬁber phenotype. Thus, the precise spatiotemporal regulation of Nkx2.5 levels during development may be required for the progressive emergence of gene expression patterns speciﬁc to differentiated Purkinje ﬁber cells. Published 2004 Wiley-Liss, Inc.† Key words: heart conduction system; Nkx2-5; Purkinje ﬁbers; development; atrioventricular block The atrioventricular conduction system (AVCS) is essential for the coordination of normal contractile cycles of the heart (Pennisi et al., 2002; Gourdie et al., 2003; Moorman and Christoffels, 2003). The cardiac activation is initiated in the sinoatrial node (SAN) located at the junction of the sinus venosus and right atrium and spreads through the atria to the atrioventricular junction. Here, activation coalesces into a single specialized central structure, the atrioventricular node (AVN). The AVN acts as a delay generator and forms the gateway to a fast conduction tissue pathway that networks through the ventricles. From the AVN, fast conduction propagates through a sinPublished 2004 WILEY-LISS, INC. †This article is a U.S. Government work and, as such, remains in the public domain of the United States of America. Seigo Izumo’s present address is Novartis Institutes for Biomedical Research, Cambridge, MA. *Correspondence to: Brett S. Harris, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425. Fax: 843-792-0664. E-mail: email@example.com Received 3 June 2004; Accepted 18 June 2004 DOI 10.1002/ar.a.20101 Published online 14 September 2004 in Wiley InterScience (www.interscience.wiley.com). 1037 TRANSCRIPTIONAL REGULATION TABLE 1. Transcription factors identiﬁed within the atrioventricular conduction system* Transcription factor family Zinc-ﬁnger bHLH T-box Homeodomain Transcription factor name Cardiac expression Conduction tissue GATA4 GATA6 HF-1b MyoD Tbx-5 Msx2 PCM, WM PCM RA, WM Nkx2-5 PCM, WM PFs AVN, His AVN, His, PFs His, PFs AVN, His AVN, His, LBB, RBB AVN, His, LBB, RBB, PFs PCM WM Organism Stage Human, mouse, chicken Mouse Human, mouse Chicken Human, mouse, chicken Mouse, chicken E9 adult E9 E17.5 E16 adult HH14 Human, mouse, chicken 10.1 W E9 HH24-P5 *PCM, precardiac mesoderm; WM, working myocardium; AVN, atrioventricular node; His, His bundle; LBB, left bundle branch; RBB, right bundle branch; Pfs, Purkinje ﬁbers; W, weeks of human gestation; E, embryonic day; HH, Hamburger and Hamilton staging of the chick. gle His bundle located at the crest of the interventricular septum, which bifurcates into the left and right bundle branches, directing signals to the left and right ventricles, respectively. These large conduction bundles then expand into an extensive network of peripheral Purkinje ﬁbers that terminate onto the working cardiomyocytes of the ventricles. Previously, we have shown that the elaboration of the AVCS during cardiac morphogenesis occurs by a process of progressive recruitment within a multipotent cardiomyogenic lineage, rather than from proliferative outgrowth of already differentiated conduction cells (Gourdie et al., 1995; Cheng et al., 1999). Our knowledge of the molecular cues that determine the development of this complex set of specialized cardiac tissues has undergone signiﬁcant advances in the last decade. The discovery of speciﬁc transcription factors present within and important for conduction cell development is ongoing (Bruneau, 2002; Myers and Fishman, 2003). Transcription factors representative of multiple subfamilies have been identiﬁed and include members of zinc-ﬁnger subfamilies, skeletal muscle transcription factors, T-box genes, and also homeodomain transcription factors (Table 1). It is noteworthy that no single transcription factor deﬁnes the entire conduction system either spatially or temporally during development and subsequent maturation. ZINC-FINGER TRANSCRIPTION FACTORS The GATA family of transcription factors is relatively small in size, with only six vertebrate members currently identiﬁed (Patient and McGhee, 2002). Members of this family possess a binding domain consisting of either one or two zinc-ﬁnger motifs coupled to a region rich in basic residues and derive their name from the consensus sequence to which they bind, (A/T) GATA (A/G). Of the six known vertebrate GATA transcription factors, three have been implicated in cardiogenesis: GATA4, -5, and -6 (Molkentin, 2000). GATA4 is expressed in the embryonic and adult murine heart and targeted disruption results in a lethal phenotype (Kuo et al., 1997; Narita et al., 1997). These mice die at around embryonic day 8 due to compound abnormalities of foregut closure and heart dismorphogenesis characterized as cardiac biﬁda. Further significance for a cardiovascular function of this gene comes from the ﬁnding that in humans GATA4 is located on chromosome 8p22-23 and that deletion of this region may cause congenital heart disease (CHD) (Bhatia et al., 1999; Pehlivan et al., 1999). Speciﬁc mutations located within the human GATA4 gene have now been identiﬁed in large kindreds presenting with CHD (Garg et al., 2003). These cardiac defects frequently include atrial septal defects (ASDs) and, to a lesser degree, ventricular septal defects (VSDs) and valvular abnormalities. Noticeably absent from the recorded cardiac defects resulting from these GATA4 mutations are abnormalities of the cardiac conduction system itself. Although GATA4 has not been described within the mammalian conduction system, its presence has been identiﬁed within the peripheral Purkinje ﬁbers of the adult chicken heart (Takebayashi-Suzuki et al., 2001). In this case, GATA4 mRNA has been shown to be localized to the mature periarterial Purkinje ﬁbers at much higher levels than the surrounding working myocardium. We have previously characterized an in vitro model of conduction cell development in which dissociated embryonic cardiomyocytes can be induced into a Purkinje ﬁber phenotype by protracted exposure to the shearstress-induced cytokine, endothelin-1 (ET-1) (Gourdie et al., 1998; Takebayashi-Suzuki et al., 2000). Using this paradigm, it has been shown that ET-1-induced embryonic Purkinje ﬁbers maintain expression of GATA4, leading to the suggestion that this gene may be important in the maintenance of conduction cell phenotype (Takebayashi-Suzuki et al., 2001). In contrast to GATA4, relatively little is known about both GATA5 and GATA6 function, which is partly due to the complication that these genes are involved in the early development of endoderm. GATA5 knockout mice survive to term but female mice experience genitourinary abnormalities (Molkentin, 2000). Surprisingly, the targeted disruption of zebraﬁsh GATA5 (faust mutant) results in embryonic lethality due to abnormal heart development and a reduction in cardiomyocyte numbers (Reiter et al., 1999), similar to the GATA4 null allele in mice, suggesting that the function of GATA4 and -5 in the ﬁsh may be reversed to that of mouse. Inactivation of the GATA6 gene in mice, as with GATA4, results in an embryonic lethal phenotype but differs in that the lethality occurs even earlier in development caused by visceral endoderm abnormalities, making study of the heart in these mutants impossible (Morrisey et al., 1998; Koutsourakis et al., 1999). Although GATA6 has not been shown to be present in cells of the AVCS, an upstream enhancer element of the proximal GATA6 promoter, derived from the chick genome, directs LacZ marker expression in a portion of these 1038 HARRIS ET AL. Fig. 1. Nkx2-5 expression localizes to the AVCS of the embryonic chick heart. Nkx2-5 protein expression in the developing chick heart at E19 detected by immunohistochemistry with Nkx2-5 antibodies and antibodies labeling the AVCS. A and B: Nkx2-5 expression is present within the left bundle branch (LBB; arrows) located in the interventricular septum. The corresponding phase contrast image shows the position of the LBB (dashed lines in B). The point at which the LBB breaks through into a subendocardial location is indicated by the arrowhead. C and D: Double immunohistochemistry shows Nkx2-5 within periarterial Purkinje ﬁbers (PPFs; C) and subendocardial Purkinje ﬁbers (SPFs; D), respectively. Here antibodies against sMHC (shown in green; FITC) label fully differentiated conduction cells and nuclear Nkx2-5 protein can be seen at higher levels in these specialized cells as compared to surrounding myocardium (shown in red; TRITC). Hoechst counterstain (blue) labels the nuclei in C. Scale bars ⫽ 100 m (A, B), 50 m (C, D). TRANSCRIPTIONAL REGULATION 1039 Fig. 2. Schematic diagram of Nkx2-5 detailing known human mutations. Protein schema of Nkx2-5 with conserved regions shown as gray boxes. Overlayed are the known human NKX2-5 mutations identiﬁed in families with congenital heart disease TN, TN domain; HB, homeodomain or DNA binding domain; NK, NK domain; GIRAW, GIRAW sequence. specialized cells (Davis et al., 2001). These authors have demonstrated, using this promoter fragment termed cGATA6, expression within the developing elements of the central conduction system. Given the identiﬁed expression patterns within the AVN and proximal His bundle exclusive of peripheral Purkinje ﬁbers, the cGATA6 enhancer has become an important tool for the transgenic analysis of subcompartments of the atrioventricular conduction system. HF-1b is also a zinc-ﬁnger transcription factor family member, but is from a different subclass from that of the GATA family being closely related to Sp-1 from Xenopus. The targeted disruption of the HF-1b gene by insertion of a LacZ reporter construct has enabled the cardiac expression pattern of this gene to be analyzed in mouse (NguyenTran et al., 2000). Although HF-1b localizes to the developing working myocardium at E17, relatively high expression levels were also noted in both central (AVN) and peripheral (His bundle and Purkinje ﬁbers) conduction tissues. Further immunohistochemical analysis of individual peripheral Purkinje ﬁbers revealed that the normal patterns of connexin40 (Cx40) typically present in these specialized cells was reduced and disorganized. The functional consequences of such perturbations were analyzed electrophysiologicaly (using telemetry) showing that ﬁrst and second AV block together with tachycardia were indeed present. In fact, this latter phenomenon frequently preceded sudden cardiac death. Although abnormal conduction in the HF-1b null mice is likely due to disrupted Cx40 patterns, it is not yet clear if there is a myocardial component to the electrophysiology or perhaps a dysfunction of the developmental transition between Purkinje ﬁbers and working cardiomyocytes. BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTORS The recent and intriguing ﬁnding that conduction cells express MyoD suggests that these specialized cardiac cells share some phenotypic commonalities with skeletal muscle (Takebayashi-Suzuki et al., 2001). MyoD is an example of a basic helix-loop-helix transcription factor (bHLH). As this name implies, they are characterized by a conserved DNA binding motif that recognizes a six nucleotide DNA sequence known as an E-box (Olson, 1993). Although this transcription factor has not been described in cardiac muscle, it has been shown to be present and control skeletal muscle phenotype through the transcription of proteins that are expressed similarly in both cardiac and skeletal muscle. MyoD was detected in isolated embryonic chick cardiomyocytes and expression was further increased by treatment of these cultures with ET-1, known to induce Purkinje ﬁber phenotype in vitro (Takebayashi-Suzuki et al., 2001). Investigation of MyoD expression in vivo revealed that mRNA expression was detected as early as E16 in chick periarterial Purkinje ﬁbers from E16 while showing that MyoD protein localized to the mature periarterial Purkinje ﬁbers present in the adult chick heart. 1040 HARRIS ET AL. Fig. 3. AdNkxHA perturbs AVCS markers in vivo. Sections of embryonic chick hearts harvested at E18 either microinjected at E10 with AdNkxHA (A and B) or with vehicle control (C) were immunolabeled for AVCS markers. A: Immunolabeling for HA-tag reveals abundant Nkx2-5 overexpression within the myocardium and subendocardium of this section through the right ventricular myocardium (shown in red; TRITC). B: A sister section to that shown in A, immunolabeled for sMHC, shows loss of sMHC protein within this domain of AdNkxHA-infected tissue (shown in green FITC). Arrows indicate subendocardial region where presumptive Purkinje ﬁbers are found. C: Control tissue shows typical bright sMHC staining of Purkinje ﬁbers of the subendocardium in age-matched control tissue (arrowheads). Scale bars ⫽ 100 m. TRANSCRIPTIONAL REGULATION Figure 4 (legend on overleaf) 1041 1042 HARRIS ET AL. T-BOX TRANSCRIPTION FACTORS The T-mutant is a naturally occurring genetic mutation in mice consisting of multiple alleles present in the T/t complex on chromosome 17 that cause a short tail phenotype (Chesley, 1935). Mutation of the T gene results in early developmental abnormalities of the primitive streak, which in turn causes perturbations of gastrulation with a resulting mesodermal cell insufﬁciency. The T-mutant, also known as Brachyury, was cloned in 1990, becoming the ﬁrst T-box transcription factor to be characterized (Herrmann et al., 1990; Wilkinson et al., 1990). T-box genes recognize and bind to a 20 base pair palindromic DNA consensus sequence in a speciﬁc fashion, where contacts with the DNA are made in both major and minor grooves (Muller and Herrmann, 1997; Papaioannou, 2001). This family of transcription factors is large with 20 or so members but continues to grow rapidly with the ongoing discovery and cloning of novel T-box genes. These transcription factors are emerging as important effectors of normal patterning processes associated with organ development (Papaioannu, 2001). A number of T-box genes have been identiﬁed in the developing vertebrate heart, including Tbx1, Tbx2, Tbx3, Tbx5, Tbx12, Tbx18, and Tbx20 (Horb and Thomsen, 1999; Carson et al., 2000; Yamada et al., 2000; Merscher et al., 2001; Takeuchi et al., 2003; Tanaka and Tickle, 2004). So far, in human populations, CHD has been found to be attributable to mutations within two family members of these cardiac associated T-box genes, Tbx1 and Tbx5 (Basson et al., 1997; Li et al., 1997; Merscher et al., 2001). Tbx1 is localized to chromosome 22q11 and a 1.5 Mb deletion within this locus may cause velocardiofacial/DiGeorge syndrome (Merscher et al., 2001); a similar deletion in mouse also causes a comparable phenotype to that observed in humans and such a phenotype may be rescued in the mouse by expression of human Tbx1. A second human syndrome associated with another T-box gene is known as Holt-Oram syndrome (HOS), an autosomal-dominant condition characterized by limb defects and congenital heart disease (Mori and Bruneau, 2004). In this case, Tbx5 is the mutated gene and the cardiovascular abnormalities include most commonly ASDs and, to a lesser extent, VSDs, patent ductus arteriosus (PDA), and rarely double-outlet right ventricle (DORV) and tetralogy of Fallot (TOF). Pertinent to this review is the ﬁnding that perturbations of cardiac electrophysiology may also occur, indicative of abnormalities localizing to the conduction system (Basson et al., 1994). To date, some 37 Tbx5 mutations have been identiﬁed and the majority result in truncated proteins, which may be nonfunctional, indicating that the disease state is likely due to Tbx5 haploinsufﬁciency. A mouse model of Tbx5 haploinsufﬁciency has been generated, Fig. 4. Nkx.2-5 haploinsufﬁciency causes a reduction in Purkinje ﬁber number. Peripheral Purkinje ﬁbers are underrepresented in Nkx2-5 heterozygous mice. Immunohistochemistry was carried out on multiple short-axis sections from 3-month-old wild-type and Nkx2-5 heterozygous mouse hearts. Confocal images of the right ventricle and surrounding myocardium are shown labeled with antibodies to Cx40 in red as punctuate staining, in both wild-type (A) and Nkx2-5 ⫹/⫺ tissue (B). The extent and distribution of Cx40 labeling in the Nkx2-5 ⫹/⫺ myocardium (B) is considerably less when compared to the wild type (A). C and D: which closely phenocopies the human conditions detailed above, including the abnormal cardiac electrophysiology (Bruneau et al., 2001). Heterozygous null Tbx5 mice have conduction system abnormalities revealed on electrocardiogram (ECG) as PR and PQ interval delays as well as ﬁrst- and also second-degree atrioventricular heart block (AVB). The precise nature of this defect within the conduction system of Tbx5 heterozygous mice is currently under investigation and will lead to a more complete picture of how Tbx5 may be involved in the patterning of these specialized cardiac tissues. HOMEODOMAIN TRANSCRIPTION FACTORS These transcription factors are characterized by a single homeodomain DNA binding motif and so far two members of this class have been identiﬁed in the cardiac conduction system: Msx-2 and Nkx2-5. Three Msx genes closely related to Drosophila msh have been identiﬁed in mammalian tissues: Msx-1, Msx-2, and Msx-3 (Davidson, 1995). Within murine cardiac tissues, Msx-2 has been shown to be downstream target of Pax-3/splotch (Kwang et al., 2002), a key player within early cardiac neural crest development (Conway et al., 2000; Epstein et al., 2000); Pax3 directly represses Msx-2. No cardiac defects were observed in Msx-2 knockout mice, suggesting that some redundancy may be present across murine Msx genes (Satokata et al., 2000). In the chick, Msx-2 mRNA expression is found with the developing conduction system as early as HH stage 15⫹, where it is found in the lesser curvature of the tubular heart (Chan-Thomas et al., 1993). Expression of Msx-2 continues at HH stage 27 within the developing AV junction, AV rings, and at the crest of the interventricular septum, regions where the specialized conduction system is thought to develop (Wessels et al., 1992). By HH stage 29 and 34, Msx-2 labels the developing His bundle and bundle branches within the interventricular septum while expression is noticeably absent from the developing peripheral Purkinje ﬁber network (Thomas et al., 2001). These data suggest that Msx-2 may delineate a population of cardiomyocytes destined to be recruited to and form the central conduction system elements such as the AV node and proximal His bundle. A second and arguably more important member of this transcription factor subclass is the NK2 class homeodomain transcription factor, Nkx2-5, alternately named Csx or Tinman (Evans, 1999). In vertebrate embryos, Nkx2-5 is localized within cells that include the cardiogenic mesoderm and as such it is accepted as one of the earliest markers of heart-forming potential. Functional studies have shown that postgastrulation expression of Nkx2-5 is required for the continued maintenance or speciﬁcation of cardiomyogenic cell fate. Absence of Nkx2-5 expression Higher-magniﬁcation sample images are shown of sections from another pair of mice hearts similarly immunolabeled for Cx40. These images formed the basis of a quantitation carried out using NIH image. Counts of cells were made within Cx40-positive domains using WGA to label cell membranes and a DNA intercalating dye to label nuclei. A greater than 50% reduction in the average number of Purkinje ﬁber cells was found in Nkx2-5 ⫹/⫺ ventricles compared to wild-type littermates (P ⬍ 0.05). RV, right ventricle. Scale bars ⫽ 100 m (A, B), 50 m (C, D). 1043 TRANSCRIPTIONAL REGULATION leads to abnormal heart formation in Drosophila, Xenopus, and mouse (Grow and Krieg, 1998). Homozygous null Nkx2-5 mice are not viable and do not survive past 10 d.p.c. in utero (Lyons et al., 1995). The majority die shortly after looping morphogenesis and the hearts of these animals display altered gene expression patterns. Our previous work and that of others has shown that Nkx2-5 is expressed at elevated levels in the forming AVCS of higher vertebrates, such as chick, mouse, and even humans (Fig. 1) (Takebayashi-Suzuki et al., 2001; Thomas et al., 2001). Indeed, the discovery of Nkx2-5 heterozygous mutations in populations of humans that result in functional and structural cardiac abnormalities including AV block provides yet further evidence of the key role that this transcription factor may play in the development of specialized cardiac tissues (Fig. 2) (Schott et al., 1998; Benson et al., 1999; Goldmuntz et al., 2001; Gutierrez-Roelens et al., 2002; Ikeda et al., 2002; Watanabe et al., 2002; McElhinney et al., 2003). Of the 25 NKX2-5 mutations currently identiﬁed in humans, the presence of AV block has been noted to be particularly associated with those mutations present within the DNA binding homeodomain (Benson et al., 1999; GutierrezRoelens et al., 2002). Interestingly, the AV block phenotype emerges over the period of postnatal maturation and it has been suggested that this time course results from progressive disease of the cardiac conduction system. Similar cardiac abnormalities have been noted in transgenic mouse models of known human Nkx2-5 mutations (Biben et al., 2000; Kasahara et al., 2001; Wakimoto et al., 2002). In one study, conduction defects were more pronounced when mutant Nkx2-5 protein was expressed during fetal and neonatal life in contrast to later postnatal time periods (Wakimoto et al., 2002). Subsequent work has shown that the spatiotemporal pattern of expression of Nkx2-5 in the developing AVCS demonstrates a striking correlation with the timing and location of recruitment of cells to this network of specialized tissues (Thomas et al., 2001). In particular, a wave of upregulated Nkx2-5 expression coincides with the most active phase of cellular conscription to the central AVCS, including the His bundle and its proximal branches. Our most recent data indicate that recruitment to the peripheral AVCS is also associated with a transient rise and fall in relative levels of Nkx2-5. Moreover, disruption of this wave-like pattern by constitutive expression of Nkx2-5 leads to differential effects on genes expressed early and late during the developmental emergence of a terminally differentiated Purkinje ﬁber cell (Fig. 3). These data in chick would be consistent with our recent study in mouse, where we found that Nkx2-5 haploinsufﬁciency caused a dramatic reduction in Purkinje ﬁber numbers (Jay et al., 2004). We hypothesized that in these animals, there was a failure to delineate a large enough population of cells that could be recruited to the developing conduction system (Fig. 4). The key theme linking these data is that Nkx2-5 underexpression due to heterozygous mutation or overexpression may perturb differentiation of specialized conduction cells. Thus, precise regulation of Nkx2-5 expression levels through a critical time period, encompassing late embryonic and early postnatal development, has the potential to cause conduction system disease in the mature animal. CONCLUSIONS Multiple transcription factors from different families contribute to the formation of the cardiac conduction system. While no single transcription factor describes this complex specialized network of cells in its entirety, we now have molecular markers of all subcompartments. The overlapping expression patterns of these genes and commonalities of both transgenic and disease phenotypes suggest that these factors participate cooperatively to deﬁne specialized tissues. 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