Published online before print February 21, 2008, doi: 10.1161/CIRCRESAHA.107.163188
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Molecular Medicine |
Myocardial Pitx2 Differentially Regulates the Left Atrial Identity and Ventricular Asymmetric Remodeling Programs
From the CNR Institute of Neurosciences (A.T., M.P., M.C.), Department of Biomedical Sciences, University of Padova, Italy; the Department of Angio-Cardio-Neurology (A.N., G.C., G.L.), IRCCS Neuromed, Polo Molise "Sapienza" University of Rome, Pozzilli (Isernia), Italy; Ophthalmology & Visual Sciences (P.J.G.), Cell & Developmental Biology, University of Michigan Medical School; and the Department of Cardiovascular Sciences (M.D.S.), National Heart and Lung Institute, Imperial College, London.
Correspondence to Marina Campione, CNR Institute of Neurosciences, Department of Biomedical Sciences, University of Padova, Italy. E-mail campione{at}bio.unipd.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Pitx2 gene regulates left-right (L/R) asymmetrical cardiac morphogenesis. Constitutive Pitx2 knock out (ko) mice die before birth and display, among other defects, right atrial isomerism, atrial and ventricular septal defects, and double outlet right ventricle. The myocardial role of the gene has not been dissected. In particular, how Pitx2 regulates the differential L/R cardiac identity program is not clear. Additionally, the relation between Pitx2 ko ventricular defects and the gene expression pattern is not understood. In this article we analyze Pitx2 myocardial function during mouse heart development. By in situ hybridization analysis we show that myocardial Pitx2 expression delineates the remodeling of the left atrioventricular canal, the inner curvature, the ventral part of the interventricular ring, and the ventral portion of the right and left ventricle. By genetic analysis using an allelic series of Pitx2 mutants, among which a myocardial specific ko (komyo) we show it has a crucial role in this process. Pitx2 komyo mutants survive to adulthood, when they present strong cardiac morphological and functional defects. Confocal analysis of embryonic Pitx2 komyo hearts reveals delayed cardiomyocyte development in the ventricular but not in the atrial Pitx2 null areas. Conversely, selective left atrial BMP10 mRNA downregulation which normally occurs at fetal stages is not found in the Pitx2 komyo mice. This is the first evidence for distinct Pitx2 action in mediating L/R atrial identity and asymmetrical ventricular remodeling.
Key Words: Pitx2 • heart development • conditional transgenic mouse • BMP10 • cardiomyocyte maturation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cardiac morphogenesis is an extremely complex process, as reflected by the high frequency of congenital cardiovascular malformations.1 Cardiogenic precursors of the first and second heart field (SHF)2 dynamically integrate into a primitive heart tube, which rapidly grows within the pericardial cavity and undergoes rightwards looping. Concomitantly, cardiac chamber growth and remodeling along the inner curvature, accompanied by septation, leads to the formation of the 4-chambered heart. To date, the molecular and cellular bases driving this process have only partially been elucidated.3
The Pitx2 gene generates 3 isoforms, Pitx2 a, b, c,4 but only Pitx2c is expressed in the heart and drives asymmetrical cardiac morphogenesis.5–7 Pitx2 and Pitx2c-isoform specific knock out (ko) mice die before birth and display right atrial isomerism (RAI), atrial (ASD) and ventricular (VSD) septal defects, double inlet left ventricle, transposition of the great arteries (TGA), persistent truncus arteriosus (PTA), and abnormal aortic arch remodeling.8–11 Pitx2c is asymmetrically expressed within the left lateral plate mesoderm (LPM), in the cardiogenic precursors of the left SHF, both at the venous and the arterial pole, and the developing heart.7,12,13 Therefore, this role in asymmetrical cardiac morphogenesis may result from its early requirement in the LPM, in the cardiogenic precursors or in the developing myocardium. The early role of the Pitx2 gene at the venous14 and the arterial poles15–17 has recently been highlighted. However, its myocardial function is not completely understood. In this study we show with a genetic approach a dosage sensitivity and continuous requirement for Pitx2 both in the cardiogenic precursors and in the developing heart. Furthermore, we show that myocardial presence of Pitx2 is required for cardiac function in adult mice. By using combined expression analysis and a myocardial ko approach we unravel a novel role of the gene for venticular asymmetrical remodeling around the inner curvature, interventricular septum (IVS) growth and ventricular chamber expansion, which is associated with a selective cardiomyocyte maturation defect phenotype in the ventricular but not the atrial Pitx2 null cells. In addition, we show that Pitx2 is required for BMP10 selective downregulation in the left atrium (LA). Our results underscore for the first time regional differences in the mechanisms by which Pitx2 acts in asymmetrical patterning of the developing heart.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mouse Lines
The Pitx2 floxed and the
-MHC Cre transgenic mouse line have previously been described.8,18 Mice have been bred into a pure C57Bl/6J genetic background, and offspring from mouse lines matings were screened for the presence of the Pitx2 floxed allele and the Cre sequence. Animals were kept in accordance with the institutional guidelines. An expanded Material and Methods section is available in the supplemental materials (available online at http://circres. ahajournals.org).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Expression of Pitx2 mRNA in the Developing Heart
We began our work by refining Pitx2 mRNA cardiac expression analysis by section ISH, by using the full length Pitx2 riboprobe which in the heart detects the Pitx2c isoform.7 At E10.5 Pitx2 expression (Figure 1A, 1B, 1G) comprises the (LA), the forming interatrial septum (IAS), the left part of the atrio-ventricular canal (AVC), the inner curvature (red asterisk), the ventral portion of the right ventricle (RV), and more weakly of the left ventricular (LV) compact myocardium. Notably, Pitx2 is expressed in the ventral part of the interventricular (IV) ring (Figure 1A and 1G), which surrounds the IV foramen and demarcates both the RV inlet and the LV outlet. At E12.5, Pitx2 expression outside the LA marks sites of ongoing cardiac remodeling (Figure 1C). Interestingly, Pitx2 additionally delineates the right part of the growing IVS. By E 14.5, IV ring remodeling has given rise to the right atrioventricular junction and the myocardium surrounding the LV outlet19 (Figure 1D, 1E, 1H). At this stage, Pitx2 mRNA is found in the pulmunary veins (PVs), left superior caval vein (LSCV), LA, IAS, and the muscularized atrial spine (AS), more strongly on the left side (Figure 1D). Additionally, the dynamic Pitx2 expression profile underlies remodeling in the AVC, in the ventricles and the ventral IV ring (Figure 1D, 1E, 1H), which has become the LV outlet. Therefore Pitx2 distinguishes in the IV ring, for the first time, the myocardium surrounding the LV outlet. Pitx2 is also still visible at the ventral portion of the IVS and on the ventral side of the LV and the RV, confined to the compact layer (Figure 1E, 1F, 1H). At E17.5 selective Pitx2 downregulation has occurred in the ventricles, with the exception of the LV outlet (supplemental Figure I).
|
Generation of the Allelic Series of Pitx2 Myocardial Mutants
To generate the Pitx2 myocardial-specific ko we crossed the Pitx2 floxed mice (Pitx2 loxP/loxP)8 with the -myosin heavy chain (-MHC) Cre deletor mice,18,20 which display efficient myocardial Cre activity from E8.521 (supplemental Figure II). Selective myocardial deletion of one loxP allele (-MHC Cre; Pitx2loxP/wt=Pitx2 hetmyo), resulted in viable and fertile offspring. Spontaneous transient ectopic activity of the -MHC Cre promoter in germ cell precursors, previously reported,22 was happening in our mice. This led, in the Pitx2 hetmyo, to the production of the deleted allele (Pitx2del), which was meiotically segregated and transmitted to the offspring (Figure 2A). Therefore, crossing of the Pitx2loxP/loxP with the Pitx2 hetmyo (Figure 2B), resulted in the following genotypes, screenable by PCR analysis (supplemental Figure II): -MHC Cre; Pitx2loxP/loxP (herein referred as Pitx2 komyo, bearing the myocardial specific ko), -MHC Cre; Pitx2loxP/wt (=Pitx2 hetmyo), -MHC Cre; Pitx2loxP/del (=Pitx2 komyoenh, bearing constitutive Pitx2 deletion in one allele, and the myocardial ko in the second), Pitx2loxP/del (=Pitx2 hetcost, bearing constitutive Pitx2 deletion only in one allele), and wild-type (wt) pups (Pitx2loxP/loxP and Pitx2loxP/wt). Additional animals were subsequently generated by crossing the Pitx2 komyo with the Pitx2loxP/loxP mice (supplemental Figure II). Their genotype distribution at postnatal day (P)15, was according to the expected Mendelian ratio (supplemental Figure II).
|
The allelic series of newborn mice were analyzed histologically at P2 (Figure 3 and supplemental Table I). Pitx2 hetmyo pups (n=6) had an almost normal heart (Figure 3C and 3D), however presenting incomplete closure of the IAS (ASD type1) (Figure 3C) and some mild malformation of the IVS (Figure 3D). Constitutive loss of one Pitx2 allele (Pitx2 hetcost, n=6) led to stronger morphogenetic defects (Figure 3E). All the mutant hearts presented ASD, either mild (type I) or strong (type II). The IVS was malformed or, in one case, incomplete, resulting in VSD accompanied by DORV (not shown). Furthermore, both the LV inlet and outlet were broader than in the wt hearts. The stronger phenotype of the Pitx2 hetcost thus revealed a role of the gene on cardiac morphogenesis before -MHC Cre activity.
|
In the Pitx2 komyo pups (n=7), hearts had an almost squared appearance, lacking the leftwards apex (Figure 3F and 3G), and almost always presented with ASD type I or II (Figure 3F and 3G and supplemental Table I). The LV inlet and outlet were broader (Figure 3F and 3G), and occasionally the left ventricular myocardium had a looser appearance (not shown). Additionally, the IVS was always malformed, and it could be also abnormally connected with the base of the IAS (Figure 3F), or grossly enlarged and misshaped (Figure 3G), which was associated with lack of ventral expansion of the ventricles and a strong reduction of ventricular chamber volume. No anomalies were found in the AV or semilunar valves (not shown). All the described defects were also observed in the Pitx2 komyoenhpups (n=7), with higher incidence (Figure 3H and 3I and supplemental Table I). In particular, the IVS was always maldeveloped, and in 2/7 samples there was VSD accompanied by DORV (Figure 3I).
In conclusion, these results revealed a dosage sensitivity and continuous requirement for Pitx2 both before Cre activity and in the developing heart.
Adult Pitx2 komyo Animals Display Cardiac Abnormalities
We focused on the Pitx2 komyo and hetmyo mice and followed their viability for 12 months, which was comparable to that of wt animals. We then analyzed their cardiac morphology and function by ecocardiographic analysis. In 2-month-old Pitx2 komyo mice (n=7), echo imaging showed IAS defects, abnormal right chambers enlargement (Figure 4C and 4F), an asymmetrical hypertrophy of the IVS (Figure 4F) and overall cardiac dysfunction. Morphometric echocardiographic analysis (Table) revealed a marked increase in RV end diastolic diameter (RVIDd), RA, and pulmonary artery (PA) internal diameters (ID) and IVS diastolic thickness (IVSd). The LV was differently affected, showing a significant reduction in LVIDd, associated with a slight increase in LAID. This asymmetrical remodeling with a selective enlargement of right chambers could be dependent on the right ventricular overload due because of IAS defect. Finally, analysis of cardiac systolic function revealed an impairment of both ventricles. This phenomenon was however more evident on the LV, likely depending on the fact that IVS contraction in the LV was limited by the concomitant systolic excursion of overloaded dilated RV. These structural and functional abnormalities were substantially confirmed at 6 months, with noticeable increase in RV dilatation, and slight reduction in LV ID, accompanied by further IVS thickening (not shown).
|
|
Histological analysis of 2-month-old Pitx2 komyo hearts additionally revealed increased intercellular space (supplemental Figure III). Furthermore, several foci of NppA mRNA expression were found, indicating cardiac disease23 however not accompanied by fibrosis (not shown).
On the other hand, Pitx2 hetmyo mice (n=10) showed by echo a completely closed IAS (Figure 4B), in line with the notion that mild ASD can spontaneously close postnatally.24 Additionally, they presented normal diameters of the RA and RV (Figure 4B and 4E), associated with a slight but not significant reduction of systolic function (Table). Interestingly, Pitx2 hetmyo mice presented an increased LVIDd associated with a significant reduction of left ventricular systolic function (Table).
Developmental Defects in the Pitx2 komyo Embryos
To get insights on the onset of cardiac defects, Pitx2 komyo embryos were isolated. At E9.5 the komyo embryos presented slightly less expanded ventricular chambers (supplemental Figure IV). However, an abnormally enlarged AVC and lack of ventricular chamber expansion were clearly visible only at E10.5 (Figure 5A through 5D) and E14.5 (Figure 5E through 5L). Abnormal inner curvature remodeling and IVS shape were additionally observed at E14.5. In 2/4 samples this defect was more pronounced, resulting in a still partially unclosed IVS (Figure 5L) and DORV (Figure 5E and 5H). All samples displayed looser IVS, especially on the right side, also abnormally connected to the endocardial cushions (not shown).
|
No increased apoptosis (assessed by cleaved caspase-3 staining) or obvious differences in proliferation (assessed by phospho-hystone H3 staining) were found in the komyo hearts at E9.5, the stage of phenotype onset (supplemental Figure IV), or at later stages (E11.5) (not shown), if compared to wt.
Section ISH analysis on E12.5 Pitx2 komyo embryo to assess expression of cardiac patterning genes, markers of primary and chamber myocardium, compact and trabeculated myocardium markers did not reveal any obvious differences in their expression profile (supplemental Figure V and not shown). A noticeable exception was BMP10, which in wt hearts is expressed in the trabeculated ventricular myocardium and in the atrial chambers, being progressively downregulated in the LA from E12.525 (Figure 6A and 6D). Interestingly, in the komyo embryos, LA expression was retained (Figure 6C), also at newborn stage (Figure 6F) and in the adult heart (not shown), whereas ventricular expression was unaffected (Figure 6C). In the hetmyo hearts, a weak atrial BMP10 expression could be detected, thus indicating a Pitx2 dosage dependent effect (Figure 6B and 6E).
|
Delayed Ventricular Cardiomyocyte Maturation in the Pitx2 komyo Hearts
In vitro studies have previously suggested a role for Pitx2 in promoting changes in cell morphology and cytoskeletal reorganization,26 therefore we decided to explore this issue by confocal analysis of embryonic heart sections.
In E10.5 wt hearts, Pitx2 positive cardiomyocytes in the ventral outer layer of the RV appeared polyhedrical (Figure 7Ba). Actinin staining outlined their contractile apparatus consisting of premyofibrils anchored to the membrane, visualized by the extracellular matrix marker (ECM) laminin, through spot-like Z-bodies (Figure 7Ba), in line with published findings.27 Analysis of additional regions, either Pitx2 positive (not shown) or Pitx2 negative, eg, the dorsal wall of the LV (Figure 7Bb) led to similar results.
|
In E10.5 komyo hearts, Pitx2-null cardiomyocytes in the regions at corresponding levels in the ventral RV (Figure 7Bc), which have been shown to be still colonized by Pitx2-null daughter cells,10 did not differ from that of wt samples. The same was true for the Pitx2 negative areas (not shown).
With progression of development, cardiomyocyte maturation occurs.27 At E14.5, in wt hearts, Pitx2-positive cardiomyocytes in the ventral RV wall (Figure 7Ca), in the right IVS (Figure 7Cc), and at the IVS base (not shown) presented clear features of cellular maturation, as shown by their elongated shape and myofibrillar alignment with formed Z-disks. In the komyo embryos, Pitx2 null cardiomyocytes in the same regions were still polyedrically shaped, with mostly spotted and perimembranous actinin organization, revealed by close localization to laminin (Figure 7Cb and 7Cd, and not shown). We therefore concluded that the Pitx2-null cells were retaining a still immature phenotype. Additionally, in the Pitx2 null regions (Figure 7Cb and 7Cd and not shown), a clear cellular alignment was not visible.
Interestingly, in E14.5 wt hearts, Pitx2-negative cardiomyocytes in the dorsal wall of the LV (Figure 7Da) or the left part of the IVS (Figure 7Dc), appeared less mature that those in the Pitx2-positive areas. In the komyo, Pitx2 negative cardiomyocytes(Figure 7Db and 7Dd) were comparable to those of the wt embryos at the same level, as well as to the Pitx2 null cells (Figure 7Cb and 7Cd). Hence, Pitx2-negative and null cardiomyocyte were comparable (Rotation movies of the presented regions are available online; see also supplemental Figure VI). Interestingly, in the komyo embryos at all stages, cardiomyocyte cytoarchitecture and ECM deposition in the LA and RA were normal and comparable to that of the wt samples (supplemental Figure VII). We therefore concluded that cardiomyocyte maturation was a selective phenotype of the ventricular but not the atrial Pitx2 null cells.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this study we have analyzed Pitx2 role in the developing and adult heart. A myocardial-specific ko together with detailed expression analysis has uncovered a novel crucial role of myocardial Pitx2 in inner curvature remodeling, IVS growth, and ventricular chamber expansion. We provide for the first time evidence for a distinct Pitx2 action in directing the L/R atrial identity and asymmetrical ventricular remodeling processes. Furthermore, the viability of our mutant mice has allowed to explore the role of Pitx2 for adult cardiac function. The implications of these findings will be discussed below.
Pitx2 Expression in the Developing Heart
In this article we have highlighted some novel Pitx2 myocardial expression domains, in particular in the remodeling ventral part of the IV ring, where Pitx2 distinguishes, for the first time, the myocardium surrounding the LV outlet. Additionally, Pitx2 expression extends into the right ventral part of the growing IVS. At early stages symmetrical components from the left and right ventricles contribute to the IVS, whereas from E12.5 the myocardium with left ventricular identity dominates dorsally.28 In line, Pitx2 delineates the right ventricular component of the IVS ventrally, and therefore results complementary to Tbx18, which delineates the LV derived transcriptional domain.28
Overall, Pitx2 expressing areas outside the LA are crucial regions for heart morphogenesis. In particular, IV ring remodeling and IVS growth are responsible for the correct AVC expansion to the right, ventricle inlet and outlet positioning, and ventricular septation. Noticeably, all these processes are affected in the Pitx2 komyo hearts that display enlarged LV inlet and outlet, defects in AO, and PT alignment and VSD.
Pitx2 Role in Cardiac Atrial and Ventricular Identity
It is known that Pitx2 is required to confer the differential cardiac L/R identity8–11 recognizable at the sinoatrial (SA) region29,30 and to suppress a SA node transcriptional program in the left side.31 In line, here we have shown that Pitx2 activity is required for BMP10 repression which occurs selectively in the LA at mid gestation. These combined evidences suggest that Pitx2 represses a default right transcriptional pathway which differentiates molecularly and morphologically the left and right SA regions. Additionally, the absence of RAI in our conditional model supports the idea that the molecular and morphological actions of Pitx2 can be separable. Notably, Pitx2 is a transcriptional activator,32,33 therefore these actions might be mediated by selective activation of one or more repressive molecular pathways.
On the other hand, Pitx2 role in ventricular remodeling is unknown. We have shown here that in the komyo, E14.5 Pitx2 null ventricular cardiomyocytes present a delay in maturation and lack oriented directionality. Additionally, in wt hearts, Pitx2-positive cardiomyocytes appear more differentiated, with respect to myofiber organization and cellular elongation those Pitx2 negative. Together, these results suggest that Pitx2 may contribute to promote the process of cardiomyocyte maturation.
Some morphological defects were present at E9.5-E10.5 in the mutant hearts, before any obvious differences in cellular maturation could be scored. However, we cannot rule out that minor differences in cardiomyocyte cytoarchitecture might be present already at this early stage. Cardiac morphogenesis requires regulated control of directional cell division, growth, and of regional cell proliferation.34,35 However we did not observe any obvious differences in ventricular cell proliferation or death at phenotype onset. Therefore, Pitx2 could modulate still unknown cellular mechanisms in the ventricles, whose at least one final readout is the specific delay in cardiomyocyte maturation at E14.5 if the gene action is lost.
Taking into account the Pitx2 expression profile and the observed phenotype of the Pitx2 komyo embryos and newborn mice, we propose that role of this gene in the ventricles is to provide the cellular maturation and organization framework that is crucial for their asymmetrical remodeling.
Overall, the observed different cellular and molecular action in the atrial and ventricular chambers lead us to conclude that cell type-specific cofactors are needed to modulate Pitx2 action in the heart.
A Time- and Dose-Dependent Role of Pitx2 in Cardiac Morphogenesis
Our allelic series of conditional mutant Pitx2 mice, as a consequence of the homeodomain deletion, have lost activity of all 3 isoforms. However, given the exclusive expression of Pitx2c in the left cardiogenic precursors and during heart development,12,13 it is reasonable to conclude the observed cardiac phenotype is due to the loss of Pitx2c action.
In general, cardiac defects in the mutants were overlapping, however their higher incidence in the komyo than the hetmyo, indicates a dose dependent role of Pitx2 for cardiac morphogenesis. In particular, incomplete closure of the IAS was already visible in the Pitx2 hetmyo newborns, which indicates a strong sensitivity to Pitx2 dosage reduction for this process. Interestingly, cardiac defects were not observed in Pitx2c heterozygotes mice in a mixed 129/SvxC57BL/6J genetic background,9 whereas our lines were bred into a pure C57BL/6J background. The milder phenotype might be attributable to the "hybrid vigor" effect, which suppresses the action of genomic modifier genes that may influence phenotypic penetrance.36
Cardiac malformations were more pronounced when the Pitx2del allele was present, additionally uncovering a continuous temporal requirement of the gene for this process. The cardiac phenotype of the Pitx2 komyo or the Pitx2 komyoenh did not recapitulate entirely that of the constitutive Pitx2 null mice.8,9,11 In particular RAI, abnormal venous return, TGA, PTA, and aortic arch patterning defects were not found. Because clear Pitx2 gene deletion with the -MHC Cre line is visible from E9.5 and is completed at E10.5 (supplemental Figure II), we cannot rule out that an earlier activated myocardial Cre might result in a stronger phenotype.
Recent data have shown an early requirement of Pitx2 at the arterial pole of the heart to direct, via Wnt11 signaling, extracellular matrix composition, cytoskeletal rearrangments, polarized cell movements,16 and regional proliferation15 required for OFT morphogenesis, which is additionally driving asymmetrical aortic arch patterning.17 Additionally, at the venous pole, a prior presence of Pitx2 in the SHF derived pulmonary mesenchymal cells is required for their subsequent differentiation into PV myocardium.14 Taking into account these data, the phenotype of the Pitx2 null mice, and our results, we suggest that LA morphological identity as well as aortic arch patterning must be driven by an earlier role of the Pitx2 gene in the left LMP or the SHF, or by a very early myocardial requirement at the venous and arterial poles. Our result additionally indicate that Pitx2 presence in the developing myocardium is crucial for the formation of a complete IAS.
Pitx2 Role for Adult Heart Function
Pitx2 komyo adult mice develop a severe cardiomyopathy, accompanied by contractility defects and reduced cardiac performance, possibly secondary to volume overload, thus to ASD. However, the left ventricular dilation and dysfunction displayed by adult hetmyo in the absence of IAS defects suggests that genetic causes, related to reduced Pitx2 gene dosage, might also play a role in onset of the adult komyo mice disease. Thus, maturation of cardiomyocyte regulated by Pitx2 could play a main role in cardiac contractile function.
Together, the Pitx2 hetmyo and komyo mice represent unique mouse models that show the impact of both Pitx2 gene reduction or absence and the structural and functional abnormalities consequent to the hemodynamic overload originated by the interatrial defect.
|
Acknowledgments |
|---|
We thank Diego Franco and Stefano Schiaffino for critical discussion and comments on the manuscript, Andrea Mattiotti for technical assistance, and Alessandra Gastaldello for initial contribution to the experiments.
Sources of Funding
M.C. is supported by the European Community’s Sixth Framework Programme contract ("HeartRepair") LSHM-CT-2005-018630. G.L. is supported by PRIN 06 and FIRB 04 interational Projects. P.J.G. is supported by the National Eye Institute at the U.S. National Institutes of Health (EY07003, EY014126).
Disclosures
None.
|
Footnotes |
|---|
*Both authors contributed equally to this study.
Original received August 30, 2007; revision received February 11, 2008; accepted February 13, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Hoffman JI. Incidence of congenital heart disease: II. Prenatal incidence. Pediatr Cardiol. 1995; 16: 155–165.[Medline] [Order article via Infotrieve]
2. Buckingham M, Meilhac S, Zaffran S. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet. 2005; 6: 826–835.[CrossRef][Medline] [Order article via Infotrieve]
3. Srivastava D. Making or breaking the heart: from lineage determination to morphogenesis. Cell. 2006; 126: 1037–1048.[CrossRef][Medline] [Order article via Infotrieve]
4. Schweickert A, Campione M, Steinbeisser H, Blum M. Pitx2 isoforms: involvement of Pitx2c but not Pitx2a or Pitx2b in vertebrate left-right asymmetry Mech Dev. 2000; 90: 41–51.[CrossRef][Medline] [Order article via Infotrieve]
5. Logan M, Pagan-Westphal SM, Smith DM, Paganessi L, Tabin CJ. The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell. 1998; 94: 307–317.[CrossRef][Medline] [Order article via Infotrieve]
6. Piedra ME, Icardo JM, Albajar M, Rodriguez-Rey JC, Ros MA. Pitx2 participates in the late phase of the pathway controlling left-right asymmetry. Cell. 1998; 94: 319–324.[CrossRef][Medline] [Order article via Infotrieve]
7. Campione M, Steinbeisser H, Schweichert A, Deissler K, van Bebber F, Lowe LA, Nowotschin S, Viebahn C, Kuehn MR, Blum M. The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. Development. 1999; 126: 1225–1234.[Abstract]
8. Gage PJ, Suh H, Camper SA. Dosage requirement of Pitx2 for development of multiple organs. Development. 1999; 126: 4643–4651.[Abstract]
9. Liu C, Liu W, Lu MF, Brown NA, Martin JF. Regulation of left-right asymmetry by thresholds of Pitx2c activity. Development. 2001; 128: 2039–2048.
10. Liu C, Liu W, Palie J, Lu MF, Brown NA, Martin JF. Pitx2c patterns anterior myocardium and aortic arch vessels and is required for local cell movement into atrioventricular cushions. Development. 2002; 129: 5081–5091.
11. Lu MF, Pressman C, Dyer R, Johnson RL, Martin JF. Function of Rieger syndrome gene in left-right asymmetry and craniofacial development. Nature. 1999; 401: 276–278.[CrossRef][Medline] [Order article via Infotrieve]
12. Campione M, Ros MA, Icardo JM, Piedra E, Christoffels VM, Schweichert A, Blum M, Franco D, Moorman AFM. Pitx2 expression defines a left lineage of cells: evidence for atrial and ventricular molecular isomerism in the iv/iv mice. Dev Biol. 2001; 231: 252–264.[CrossRef][Medline] [Order article via Infotrieve]
13. Nowotschin S, Liao J, Gage PJ, Epstein JA, Campione M, Morrow BE. Tbx1 affects asymmetric cardiac morphogenesis by regulating Pitx2 in the secondary heart field. Development. 2006; 133: 1565–1573.
14. Mommersteeg MT, Brown NA, Prall OW, de Gier-de Vries C, Harvey RP, Moorman AF, Christoffels VM Pitx2c and Nkx2–5 are required for the formation and identity of the pulmonary myocardium. Circ Res. 2007; 101: 902–909.
15. Ai D, Liu W, Ma L, Dong F, Lu MF, Wang D, Verzi MP, Cai C, Gage PJ, Evans S, Black BL, Brown NA, Martin JF. Pitx2 regulates cardiac left-right asymmetry by patterning second cardiac lineage-derived myocardium. Dev Biol. 2006; 296: 437–449.[CrossRef][Medline] [Order article via Infotrieve]
16. Zhou W, Lin L, Majumdar A, Li X, Zhang X, Liu W, Etheridge L, Shi Y, Martin J, Van deVen W, Kaartinen V, WynshawBoris A, McMahon AP, Rosenfeld MG, Evans SM. Modulation of morphogenesis by noncanonical Wnt signaling requires ATF/CREB family-mediated transcriptional activation of TGFbeta2. Nat Genet. 2007; 39: 1225–1234.[CrossRef][Medline] [Order article via Infotrieve]
17. Yashiro K, Shiratori H, Hamada H. Haemodinamics determined by a genetic programme govern asymmetric development of the aortic arch. Nature. 2007; 450: 285–288.[CrossRef][Medline] [Order article via Infotrieve]
18. Agah R, Frenkel PA, French BA, Michael LH, Overbeek PA, Schneider MD. Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J Clin Invest. 1997; 100: 169–179.[Medline] [Order article via Infotrieve]
19. 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.
20. Gaussin V, Van de Putte T, Mishina Y, Hanks MC, Zwijsen A, Huylebroeck D, Behringer RR, Schneider MD. Endocardial cushion and myocardial defects after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK3. Proc Natl Acad Sci U S A. 2002; 99: 2878–2883.
21. McFadden DG, Barbosa AC, Richardson JA, Schneider MD, Srivastava D, Olson EN. The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner. Development. 2005; 132: 189–201.
22. Eckart D, Theis M, Doring B, Speidel D Willecke K, and Ott T. Spontaneous ectopic recombination in cell-type specific Cre mice removes loxP-flanked marker cassettes in vivo. Genesis. 2004; 38: 159–165.[CrossRef][Medline] [Order article via Infotrieve]
23. Vikstrom KL, Bohlmeyer T, Factor SM, Leinwand LA. Hypertrophy, pathology, and molecular markers of cardiac pathogenesis. Circ Res. 1998; 82: 773–778.
24. Helgason H, Jonsdottir G. Spontaneous closure of atrial septal defects. Pediatr Cardiol. 1999; 20: 195–199.[CrossRef][Medline] [Order article via Infotrieve]
25. Chen H, Shi S, Acosta L, Li W, Lu J, Bao S, Chen Z, Yang Z, Schneider MD, Chien KR, Conway SJ, Haneline LS, Franco D, Shou W. BMP10 is essential for maintaining cardiac growth during murine cardiogenesis. Development. 2004; 131: 2219–2231.
26. Wei Q, Adelstein RS. Pitx2a expression alters actin-myosin cytoskeleton and migration of HeLa cells through Rho GTPase signaling. Mol Biol Cell. 2002; 13: 683–697.
27. Hirshy A, Schatzmann F, Ehler E, Perriard JC. Extablishent of cardiac cytoarchitecture in developing mouse heart. Dev Biol. 2006; 289: 430–441.[CrossRef][Medline] [Order article via Infotrieve]
28. Franco D, Meilhac SM, Christoffels VM, Kispert A, Buckingham M, Kelly RG. Left and right ventricular contributions to the formation of the interventricular septum in the mouse heart. Dev Biol. 2006; 294: 366–375.[CrossRef][Medline] [Order article via Infotrieve]
29. Van Mierop LHS, and Wigglesworth FW. Isomerism of the cardiac atria in asplenia syndrome. Lab Invest. 1962; 11: 1303–1315.[Medline] [Order article via Infotrieve]
30. Seo JW, Brown NA, Ho SY, and Anderson RH. Abnormal laterality and congenital cardiac anomalies: Relations of visceral and cardiac morphogenesis in iv/iv mouse. Circulation. 1992; 86: 642–650.
31. Mommersteeg MT, Hoogaars WM, Prall OW, de Gier-de Vries C, Wiese C, Clout DE, Papaioannou VE, Brown NA, Harvey RP, Moorman AF, Christoffels VM. Molecular pathway for the localized formation of the sinoatrial node. Circ Res. 2007; 100: 354–362.
32. Vadlamudi U, Espinoza HM, Ganga M, Martin DM, Liu X, Engelhardt JF, Amendt BA. PITX2, beta-catenin and LEF-1 interact to synergistically regulate the LEF-1 promoter. J Cell Sci. 2005; 118: 1129–1137.
33. Toro R, Saadi I, Kuburas A, Nemer M, Russo AF. Cell-specific activation of the atrial natriuretic factor promoter by PITX2 and MEF2A. J Biol Chem. 2004; 279: 52087–55294.
34. Meilhac SM, Esner M, Kerszberg M, Moss JE, Buckingham ME. Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis. J Cell Biol. 2004; 164: 97–109.
35. Soufan AT, van den Berg G, Ruijter JM, de Boer PA, van den Hoff MJ, Moorman AF. Regionalized sequence of myocardial cell growth and proliferation characterizes early chamber formation. Circ Res. 2006; 99: 545–552.
36. Birchler JA, Riddle NC, Auger DL, Veitia RA. Dosage balance in gene regulation: biological implications. Trends Genet. 2005; 21: 219–226.[CrossRef][Medline] [Order article via Infotrieve]
Related Article:
Circ. Res. 2008 102: 749-751.
This article has been cited by other articles:
 |
|
 |
|
  S. C. Body, C. D. Collard, S. K. Shernan, A. A. Fox, K.-Y. Liu, M. D. Ritchie, T. E. Perry, J. D. Muehlschlegel, S. Aranki, B. S. Donahue, et al. Variation in the 4q25 Chromosomal Locus Predicts Atrial Fibrillation After Coronary Artery Bypass Graft Surgery Circ Cardiovasc Genet, October 1, 2009; 2(5): 499 - 506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. MacRae Symptoms in Atrial Fibrillation: Why Keep Score? Circ Arrhythm Electrophysiol, June 1, 2009; 2(3): 215 - 217. [Full Text] [PDF]
|
|
|
|
 |
|
 B. J.D. Boukens, V. M. Christoffels, R. Coronel, and A. F.M. Moorman Developmental Basis for Electrophysiological Heterogeneity in the Ventricular and Outflow Tract Myocardium As a Substrate for Life-Threatening Ventricular Arrhythmias Circ. Res., January 2, 2009; 104(1): 19 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Poelmann, M. R.M. Jongbloed, and A. C. Gittenberger-de Groot Pitx2: A Challenging Teenager Circ. Res., April 11, 2008; 102(7): 749 - 751. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||