Published online before print October 10, 2008, doi: 10.1161/01.RES.0000338613.89841.c1
© 2008 American Heart Association, Inc.
Molecular Medicine |
Essential Role of Developmentally Activated Hypoxia-Inducible Factor 1
for Cardiac Morphogenesis and Function
From the Institute of Cell Biology (J.K., P.A., S.B., E.P., W.K., J.-C.P.), Eidgenössische Technische Hochschule, Zürich, Switzerland; and VisualSonics BV (D.K.), Amsterdam, The Netherlands. Present address for P.A.: Department of Medicine and Physiology, School of Medicine, University of California, Los Angeles. Present address for S.B.: Institute of Pharmacology and Toxicology, University of Zurich, Switzerland.
Correspondence to Wilhelm Krek, ETH Zurich, Institute of Cell Biology, Schafmattstrasse 18, Zurich 8093, Switzerland. E-mail wilhelm.krek{at}cell.biol.ethz.ch
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Development of the mammalian heart is governed by precisely orchestrated interactions between signaling pathways integrating environmental cues and a core cardiac transcriptional network that directs differentiation, growth and morphogenesis. Here we report that in mice, at about embryonic day (E)8.5 to E10.0, cardiac development proceeds in an environment that is hypoxic and characterized by high levels of hypoxia-inducible factor (HIF)1
protein. Mice lacking HIF1 in ventricular cardiomyocytes exhibit aborted development at looping morphogenesis and embryonic lethality between E11.0 to E12.0. Intriguingly, HIF1-deficient hearts display reduced expression of the core cardiac transcription factors Mef2C and Tbx5 and of titin, a giant protein that serves as a template for the assembly and organization of the sarcomere. Chromatin immunoprecipitation experiments revealed that Mef2C, Tbx5, and titin are direct target genes of HIF1 in vivo. Thus, hypoxia signaling controls cardiac development through HIF1-mediated transcriptional regulation of key components of myofibrillogenesis and the cardiac transcription factor network, thereby providing a mechanistic basis of how heart development, morphogenesis, and function is coupled to low oxygen tension during early embryogenesis.
Key Words: cardiac development • hypoxia • transcription • myofibrillogenesis • HIF1
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cardiac development is a multi-stage process governed by a network of transcription factors including Nkx2.5, Tbx5, Mef2C, and GATA4 that connect signaling pathways with the regulation of gene expression for the specification of cardiac cell fate, differentiation and morphogenesis. These transcription factors act in an interconnected and coordinated manner at specific stages of cardiogenesis. Although Nkx2.5 and GATA4 activity is predominantly associated with cardiac lineage specification and differentiation, it is also required for induction of downstream cardiogenic transcription factors such as Tbx5 and Mef2C, whose function is primarily linked to looping morphogenesis and chamber formation.1 Thus specific transcription factors induce the expression of genes necessary for progression through the respective stages of cardiogenesis. How the expression and activity of these transcription factors are coordinated spatially and temporally in the course of cardiogenesis remains less clear.
A central feature of mammalian embryogenesis is the development of intraembryonic hypoxia.2 As the embryo increases in size, oxygenation of the avascular early postimplantation embryo by diffusion alone becomes limited, leading to a state of regional embryonic hypoxia. In normal development, regional hypoxia has been implicated to serve as a stimulus for the tissue/region-specific induction of genes required for yolk sac vasculogenesis,3 the establishment of the maternal-fetal circulatory network4 and the initiation of intraembryonic circulation and cardiac function.5,6 Thus, the embryonic hypoxic phase is characterized by the coordinated development of multiple components of the embryonic cardiovascular system with the aim of facilitating intraembryonic oxygenation and relieving the hypoxic stress. Because of the critical role of the heart in oxygen and nutrient conduction, it has been hypothesized that adaptive processes to low oxygen tension occurring early in development could potentially be critical for cardiac development by serving as a stimulus for the induction of transcription factors and structural proteins required for development of the mature heart and the initiation of rhythmic contraction.7
A central mediator of adaptive responses to low oxygen tension is HIF, a heterodimeric transcription factor composed of HIF1 or HIF2 and HIFβ/ARNT subunits. The HIF subunits are under exquisite oxygen control in that they accumulate rapidly in response to hypoxia to induce an adaptive transcription program. Complete deficiency of HIF1 in mice results in embryonic lethality around embryonic day (E)10.0, characterized by a broad spectrum of abnormalities including striking retarded development, neural tube defects, dysfunctional vasculogenesis and angiogenesis, a reduction in somite numbers and cardiovascular malformations.8–10 The main defect observed in HIF1 deficient hearts relates to the absence of a patent ventricular lumen and outflow tract caused by hyperproliferation of myocardial cells. In contrast, mouse embryos lacking HIF2 die between E16.5 and postnatally, and do not display early cardiac developmental or morphological defects11–13
Although one might infer from these studies a possible role specifically for HIF1 in early cardiogenesis, the detailed molecular mechanism underlying a requirement for HIF1 in cardiac developmental processes remains to be defined. Therefore, we generated a ventricular-restricted conditional disruption of HIF1 in the mouse to study its impact on cardiac development. Our results suggest a key role for HIF1 in the regulation of core cardiac transcription factor network and the process of myofibrillogenesis through direct HIF1-mediated activation of the Mef2C, Tbx5, and titin genes, respectively.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mice and Physiological Measurements
C57/Black6 mice were from Elevage Janvier and the ROSA26 Cre reporter (R26R) line was from The Jackson Laboratory. HIF1
f/f and HIF1+/– mice were obtained from Randall S. Johnson (University of California, San Diego) and Gregg L. Semenza (Johns Hopkins University School of Medicine, Baltimore, Md), respectively, whereas the MLC2vcre/+ line was from Ju Chen (University of California, San Diego). All mice were maintained at the Institute of Cell Biology, ETH-Zurich specific pathogen-free facility in full compliance with guidelines approved by the Swiss Federal Veterinary Office (SFVO). Embryonic echocardiography measurements were performed on the VisualSonics Vevo 770 machine essentially as recommended by the manufacturer.
Antibodies
Myomesin and EH-myomesin antibodies were generated in-house.14 The MyBPC antibody was a kind gift from Mathias Gautel (King’s College, London, UK). Antibodies against sarcomeric -actinin, titin and Ser10-phosphorylated histone H3 were from Sigma, LGC Promochem and Upstate Biotechnology, respectively. Antibodies used in the ChIP assay against HIF1 were from Santa Cruz Biotechnology.
Luciferase Promoter Assays
One kb of the Mef2C and 2kb of the Tbx5 promoter was amplified from mouse genomic DNA and cloned into the pGL3 luciferase reporter vector (Stratagene). The 5.87 kb titin promoter was a kind gift from Gregory A. Cox (The Jackson Laboratory) and was subcloned in its entirety into pGL3. HRE- mutants of the respective promoters were generated by recombinant PCR.15 Wild-type and mutant CXCR4 reporters were generated as described,16 and subcloned into pGL3. The ANF-Luc and the ANF 
TRE-Luc reporters were generously provided by David Brook (University of Nottingham, UK), whereas the 3xMEF reporter was a kind gift from Eric N. Olson (UT Southwestern Medical Center at Dallas). The HIF1 (P402A/P577A) expression construct was generated as described,16 whereas the HIF1 (ODD) expression construct was kindly provided by H Franklin Bunn (Harvard Medical School).
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Developmental Myocardial Hypoxia Induces HIF1
AccumulationPimonidazole, a nitroimidazole-derivative that incorporates in hypoxic cells below a PO2 of 10 mm Hg was used to assess the oxygenation state of E9.5 wild-type mouse embryos. The specificity of pimonidazole incorporation under conditions of hypoxia was confirmed by subjecting primary neonatal mouse cardiomyocytes (NMCs) to 1% oxygen and assessing levels of pimonidazole adduct formation (Figure I in the online data supplement). Incorporation of pimonidazole in the whole embryo was restricted to the developing limb buds and the heart, where colocalization was observed with the embryonic cardiomyocyte marker EH-myomesin (Figure 1A).14 Sections of embryos revealed myocardial pimonidazole incorporation at E9.0 and E10.0 (supplemental Figure II). Negligible incorporation was observed beyond E11.0 (data not shown). Thus the early embryonic heart develops in hypoxia.
|
To ascertain if myocardial hypoxia was associated with HIF1 accumulation, whole mount immunostaining of wild-type hearts between E8.0 to E11.5 were performed. HIF1 was first detectable in nuclei of cardiomyocytes at E8.0. Its levels increased dramatically at embryonic days E8.5 and E9.5 and diminished thereafter (E10.5) to become undetectable by E11.5 (Figure 1B). Specificity of the HIF1 antibody was confirmed by whole-mount immunostaining of HIF1–/–9 hearts and by subjecting NMCs to hypoxia (supplemental Figure III, top and bottom).
Although levels of HIF1 mRNA remained relatively constant at the respective time points as analyzed by semiquantitative (Figure 1C) and quantitative (Figure 1D) RT-PCR, immunoblotting of myocardial extracts revealed a similar pattern of HIF1 protein abundance changes as seen in immunohistochemical sections (Figure 1E). These results suggest a close temporal link between a specific window of time during heart development where the developing myocardium is hypoxic and the consequent accumulation of HIF1.
HIF1 Is Required for Cardiac Morphogenic and Functional Development
Next we generated ventricular-restricted HIF1-deficient mice by mating HIF1 floxed allele mice (HIF1f/f)17,18 with the well-characterized myosin light chain-2v (MLC2v) cre recombinase knock-in mouse line (MLC2v-cre/+).19 Consistent with a previous study20 HIF1f/f/cre+ embryos were viable and did not exhibit any overt phenotype. Analysis of hearts at E10.0 of HIF1f/f/cre+ mice however revealed inefficient excision of the HIF1 gene (data not shown) suggesting that although MLC2v-driven cre expression is initiated at E8.2521 early expression of cre recombinase is low. To circumvent the low efficiency of cre recombinase activity and achieve efficient deletion of the HIF1 gene by around E8.5 to E10.5, the time during which myocardial hypoxia is apparent, we generated mice that are floxed at 1 HIF1 allele and null on the other (referred to as HIF1f/–) on the MLC2v-cre/+ background (referred to as HIF1f/–/cre+). This strategy permitted efficient excision of the HIF1 floxed allele and ablation of HIF1 mRNA (Figure 3A). Specificity and efficiency of MLC2v-cre/+-mediated recombination for the myocardium was confirmed by crossing MLC2v-cre/+ mice with the R26R reporter mouse line (Figure III, middle, and supplemental Figure IV).22 Whereas HIF1f/–/cre offspring were phenotypically normal and present at the expected Mendelian frequency, HIF1f/–/cre+ neonates were not found among 148 offspring.
At the linear heart tube stage, around E8.0 to E8.5, HIF1f/–/cre+ and control HIF1f/–/cre embryos were indistinguishable and exhibited the characteristic peristaltic-like contractions (data not shown). Around E8.5 to E9.5 the heart initiates rightward looping, to orientate the atrial and ventricular chambers and to align the outflow tract with the vasculature. At E9.5 to E10.5 the looped cardiac tube matures to give rise to the future right and left ventricles, and the common atrial chambers.23 Although the initial stages of cardiac looping occurred normally in HIF1f/–/cre+ embryos, progression of the looped heart tubes toward chamber formation failed to occur in HIF1f/–/cre+ embryos. Instead, these hearts developed a single ventricular chamber fused directly to the atria (Figure 2A and 2B), with little evidence of distinct right and left ventricles. Moreover, at E9.5 to E10.5 when the heart of HIF1f/–/cre– control embryos exhibited rhythmic contractions, the hearts of HIF1f/–/cre+ littermates did not and continued to display peristaltic-like contractions reminiscent of the immature tubular heart (supplemental Movies I and II and expanded Materials and Methods section). HIF1f/–/cre+ embryos died between E11.5 to E12.0 as a result of cardiac contractile dysfunction. These findings suggest that HIF1 function is essential for normal heart development, in particular for the progression from the looping heart tube stage to ventricular and atrial chamber formation.
|
Hearts of HIF1f/–/cre+ embryos also exhibited hyperplasia of the ventricular myocardium resulting in the complete absence of a ventricular cavity (Figure 2B and supplemental Figure V). At E10.0, the ventricular myocardium is normally comprised of 2 to 3 cell layers enveloping the ventricular cavity in which blood is collected and channeled to the atria. Consistent with the above-noted observation, we found a significant higher fraction of actively proliferating ventricular cardiomyocytes in hearts of HIF1f/–/cre+ embryos at E10.0 than in hearts of HIF1f/–/cre– control embryos as indicated by the increased fraction of cells positive for serine10-phosphorylated histone H3, a marker of mitotically active cells, and sarcomeric -actinin (Figure 2C and 2D). Thus, accumulation of HIF1 during heart development appears to be also critical for constraining uncontrolled ventricular cardiomyocyte proliferation
As hearts of HIF1f/–/cre+ embryos lacked the characteristic contractile profile of a functional heart, we asked if the contractile defect occurred as a result of impaired cardiomyocyte myofibrillogenesis. Proper expression and assembly of myofibrillar proteins at the sarcomere is a prerequisite for cardiac contractility and morphological integrity of cardiomyocytes. We assessed the expression of 3 essential sarcomeric components - titin, EH-myomesin and -actinin. Titin is a giant protein that anchors in the Z-disk and extends to the M-line region of the sarcomere forming a continuous filament along the entire length of the myofibril to provide the structural foundation on which all other myofibrillar proteins including myomesin and -actinin attach and are assembled in the sarcomere. Histological examination of HIF1f/–/cre+ embryos indicated a profound decrease in titin protein levels (Figure 2E). No such decrease was seen in HIF1f/–/cre– embryos. The decrease in titin expression was restricted to the ventricular myocardium as the atria of both control HIF1f/–/cre– and HIF1f/–/cre+ embryos exhibited normal titin expression and organization (Figure 2E). The expression of 2 other sarcomeric proteins, EH-myomesin (Figure 2E) and -actinin (supplemental Figure VI), were not dramatically affected in ventricular cardiomyocytes. However, the staining patterns of EH-myomesin and -actinin unveiled disorganized myofibrils and the complete absence of sarcomere assembly (Figure 2E and supplemental Figure VI). Again, cardiomyocytes of the atria of HIF1f/–/cre+ embryos displayed properly organized myofibrils as evidenced by staining for EH-myomesin and -actinin. Together, these results suggest that HIF1 acts to maintain the expression of the sarcomeric protein titin, but not that of the sarcomeric proteins EH-myomesin and -actinin, during cardiac development. The reduced expression of titin likely contributes to impaired myofibrillogenesis and failure of HIF1-deficient hearts to complete looping morphogenesis and exhibit normal contractility.
HIF1 Regulates the Expression of Core Cardiogenic Transcription Factors, Cell Cycle Mediators, and Structural Proteins
To identify the molecular basis underlying the effects of HIF1 on myofibrillogenesis, cell proliferation and looping morphogenesis, we compared the expression of a set of genes encoding sarcomeric components, cell cycle regulatory proteins and core cardiac transcription factors in the hearts of HIF1f/–/cre– control embryos and HIF1f/–/cre+ littermates by semiquantitative RT-PCR (Figure 3A) and quantitative RT-PCR (Figure 3B). Whereas expression of the developmentally predominant cardiac titin N2B isoform24 was present in HIF1f/–/cre– control ventricles, it was markedly reduced in HIF1-deficient ventricles. To rule out the possibility that in HIF1f/–/cre+ ventricles the lack of titin N2B isoform expression is compensated by that of other titin isoforms, HIF1f/–/cre control and HIF1f/–/cre+ ventricles were analyzed for expression of the titin protein kinase domain, a region ubiquitous to all titin isoforms (Figure 3A and 3B).25
|
Expression of the titin protein kinase domain was similarly reduced in HIF1-deficient hearts. Although mRNA levels of -actinin, myomesin and myosin binding protein C (MyBPC) genes remained unaffected by lack of HIF1 (Figure 3A and 3B), accumulation of myomesin and -actinin protein was decreased (Figure 3C) which may relate to posttranslational mechanisms regulating the stability of sarcomeric proteins in the absence of its myofibril incorporation26 because of lack of normal titin expression. Genes associated with myocardial cell proliferation such as cyclin D1 and CDK4 were significantly upregulated, whereas genes encoding the cyclin-dependent kinase inhibitors p27 and p21 were downregulated (Figure 3D and 3E). This expression pattern is in accord with the hyperplasia observed in the ventricular myocardium of HIF1-deficient embryonic hearts (Figure 2B through 2D), the effect of HIF1 deficiency during chondrogenesis18 and hypoxia-induced growth arrest.27 Intriguingly, transcript levels of the cardiogenic factors Nkx2.5, Tbx5, Mef2C were almost absent in HIF1f/–/cre+ ventricles compared to control HIF1f/–/cre– ventricles (Figure 3D and 3E). The expression of GATA4, another member of the core cardiac transcription factor network implicated in cell specification and determination28 was however not changed in the mutant hearts, suggesting that its expression is not dependent on HIF1 (Figure 3D and 3E). These analyses suggest that HIF1-deficiency affects, directly or indirectly, the expression of at least 1 core structural gene as well as genes orchestrating the core cardiac transcription program.
HIF1 Directly Interacts With the Mef2C, Tbx5, and Titin Promoters In Vivo
The dramatic differences in Tbx5, Mef2C, and titin gene transcription in HIF1 deficient and wild-type embryonic hearts, and the fact that HIF1f/–/cre– and HIF1f/–/cre+ exhibit comparable levels of Tbx5, Mef2C, and titin transcription before initiation of MLC2vdriven Cre recombinase expression (supplemental Figure VII) suggests that these genes might be direct targets of HIF1 in vivo. Moreover, Tbx5, Mef2C, and titin transcription is dysregulated post-MLC2v-driven Cre recombinase expression but before development of defective cardiac morphogenesis (supplemental Figure VIII), further supporting the notion that downregulation of Tbx5, Mef2C, and titin expression observed at E9.5 to 10.0 occurred as a result of Cre recombinase-mediated HIF1 excision and not as a consequence of the morphologically defective state of the HIF1f/–/cre+ hearts. Analysis of the promoters of mouse Tbx5, Mef2C, and titin genes revealed the existence of potential hypoxia response elements (HREs).29 These putative HREs were present at sites –293 and –1514 in Tbx5, 445 in Mef2C and –37 and –462 in titin relative to the transcription start site (supplemental Figure IX). The HREs were contained within promoter regions of Tbx5, Mef2C, and titin that support transcription of the respective genes in vivo.30–32 HREs were similarly conserved in the respective promoters of the human Tbx5, Mef2C, and titin genes (supplemental Figure IX). We performed Chromatin Immunoprecipitation (ChIP) experiments on nuclear extracts of E10.0 control HIF1f/–/cre– ventricles using antibodies against HIF1 or its heterodimerizing partner HIFβ/ARNT. Strikingly, ChIP assays showed that both HIF1 and its heterodimerization partner HIFβ associated with certain HREs in the Mef2C (Figure 4A), Tbx5 (Figure 4B), and titin promoters (Figure 4C) in native chromatin. Although the expression of Nkx2.5 changed as a function of HIF1, we failed repeatedly to detect HIF1 binding to the Nkx2.5 promoter (data not shown). These data support the conclusion that the Tbx5, Mef2C, and titin genes are direct transcriptional targets of HIF1 in vivo.
|
To corroborate the above finding for Mef2C, Tbx5, and titin, the corresponding regulatory sequences encompassing HRE elements occupied by HIF1 in vivo (as evidenced by ChIP assays) were fused to the luciferase reporter gene and transfected into NMCs and in C2C12 myoblasts (data not shown). When these reporter constructs were cotransfected with an expression plasmid harboring the HIF1 mutant species, HIF1(P402A/P577A) (which contains alanine substitutions at the proline hydroxylation sites Pro402 and Pro577 and thus escapes prolyl-hydroxylation dependent ubiquitin-mediated degradation33) or subjected to hypoxia (1% O2), the promoter activity of Mef2C, Tbx5, and titin increased (Figure 4D through 4F, respectively). Mutation of the HRE element in the respective promoters caused significantly reduced responsiveness of the luciferase reporter to HIF1(P402A/P577A) or hypoxia (Figure 4D through 4F, respectively). Similar results were obtained on transfection with the constitutively active HIF1(ODD) mutant lacking the oxygen-dependent degradation domain (Figure 4F). To further confirm a direct link between HIF1 levels with induction of Mef2C, Tbx5, and titin promoter activation, increasing amounts of the HIF1(ODD) expression construct was cotransfected into NMCs in the presence of wild-type Mef2C, Tbx5 or titin luciferase reporter constructs, respectively (Figure 4G through 4I). As shown, a dose-dependent increase in Mef2C, Tbx5, and titin reporter activity was observed in response to increasing amounts of HIF1(ODD).
In addition, ectopic expression of HIF1 induced the activation a synthetic promoter containing Mef2 response elements (MREs) (Figure 5A). HIF1(P402A/P577A) also activated the promoter of the ANF gene, an established Tbx5 target34 in a Tbx5 response element (TRE)-dependent manner (Figure 5B). Wild-type and HRE-mutated luciferase reporters of CXCR416 were used to confirm the activity and specificity of the respective HIF1 constructs and of the hypoxic stimulation (Figure 5C). Together, these results strengthen the view that the Tbx5, Mef2C, and titin represent novel direct transcriptional target genes of HIF1.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Here we have demonstrated in the context of heart development how embryonic hypoxia spatially effects cardiac oxygenation and serves as a stimulus for the accumulation and transcriptional activity of the oxygen sensitive factor, HIF1
. Loss of HIF1 in ventricular cardiomyocytes leads to severe cardiac-specific developmental and morphological defects. In contrast to the systemic HIF1 null phenotype where a dramatic reduction in embryonic growth, cardiovascular and neural tube development defects were reported, ventricular-specific deletion of HIF1 did not lead to these defects. Rather, the restricted loss of HIF1 in ventricular cardiomyocytes led specifically to defective cardiac development. Although profound cardiac developmental defects were reported in HIF1 knockout embryos, these studies suffer from the fact that the cardiac phenotype was observed in the context of a systemic lack of HIF1. The additional defects in embryonic angiogenesis and neural tube morphogenesis in these embryos makes interpretation of the heart phenotype difficult because of the contribution of the vascular and neural cell population to embryonic circulation and development of the secondary heart field, respectively. Thus, by specifically deleting HIF1 in ventricular cardiomyocytes we uncoupled the effects of other components of the embryonic cardiovascular system and specifically addressed the contribution of HIF1 in the development of the primary heart field.
On the basis of our findings, we propose now a model of how the hypoxic environment in the developing heart contributes to cardiac morphogenesis and function. It involves tightly coupled direct interactions between an oxygen-sensitive transcription factor, HIF1, key genes of the core cardiac transcription factor network including Tbx5 and Mef2C and the gene encoding titin, a sarcomeric protein critical for myofibrillogenesis. According to our model, developmentally triggered hypoxia acts as a physiological signal that induces the accumulation of HIF1, which directly contributes to maintain the expression of the Tbx5, Mef2C, and titin genes between E8.5 to E10.0 by binding to and activating these genes. This model is supported by the fact that null mutants of Tbx534 and Mef2C35 and partial-deletion mutants of titin26,36 all exhibit cardiac phenotypes similar to those observed in the conditional HIF1 mutant and result in aborted cardiac development during progression to chamber formation, thus being consistent with the fact that HIF1 activity may indeed be critical to integrate hypoxic stress with the coordinated induction of the embryonic transcription factor gene program and that of titin, a fundamentally critical component of the cardiomyocyte structural and contractile apparatus.
Existing evidence suggests that these core cardiac transcription factors act in a functionally interconnected network to cooperatively direct downstream target gene activation.1 Therefore, regulating the expression of 1 or more of the core genes in this network is likely to have profound effects on the transcriptional output of the network. Indeed, despite decreased Nkx2.5 expression in HIF1-deficient hearts, we failed to detect a direct binding of HIF1 to Nkx2.5 promoter in vivo. The finding that Tbx5 is a direct a target gene of HIF1 combined with the fact that Tbx5 can activate the Nkx2.5 gene,37 argue that the lack of Nkx2.5 expression in HIF1-deficient heart may well be a consequence of decreased Tbx5 expression. Therefore, developmentally regulated HIF1 activation appears to be critical for maintaining the proper operation of the core cardiac gene network, an essential prerequisite for the formation of the 4-chambered heart.
Finally, our data also uncovered an unexpected direct connection between hypoxia-mediated HIF1 induction, titin gene activation, the consequent assembly of a functional sarcomere and development of the capacity for rhythmic cardiac contraction. Thus, HIF1 regulates both, the expression of selected core cardiac regulatory as well as structural genes, which implies that cardiac morphogenesis and contractility are coordinated by a common signaling pathway whose activity is exquisitely regulated by oxygen levels. In summary, the results presented here suggest that the low oxygen environment early in heart development initiates a HIF1-mediated transcriptional program that facilitates the making of a functional heart, which in turn, is vital for the distribution of oxygen in the embryo and further development of the organism.
|
Acknowledgments |
|---|
We thank all members of the laboratory for discussion. We are particularly grateful to R. Johnson (University of California, San Diego) for providing floxed HIF1
and G. Semenza (Johns Hopkins, Baltimore, Md) for HIF1+/– mice; J. Chen (University of California, San Diego) and K. Chien (Harvard, Boston, Mass) for MLC2v-cre/+ mice; M. Gassmann (University of Zürich) for reagents; T. Pedrazzini (University Lausanne, Lausanne, Switzerland) for providing access to echocardiography; and E. Olson (University of Texas Southwestern Medical Center, Dallas) and R. Eckner (New Jersey Medical School, Newark, NJ) for critical reading of the manuscript. We also thank Tatiana Krebs, Elisabeth Ehler, Stephan Keller, and Alain Hirschy for advice and help. Sources of Funding
This work was supported by the Swiss Cardiovascular Research & Teaching Network, sponsored by the Swiss University Conference; Swiss National Science Foundation grant 3100-063486 (to J.-C.P.); Gebert-Rüf Foundation grant P038/01 (to J.-C.P.); a Novartis Foundation grant (to J.K.); and the Dr Josef Steiner Cancer Grant (to W.K.).
Disclosures
None.
|
Footnotes |
|---|
*Both authors contributed equally to this work.
Original received February 20, 2008; revision received September 29, 2008; accepted September 29, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Dunwoodie SL. Combinatorial signaling in the heart orchestrates cardiac induction, lineage specification and chamber formation. Semin Cell Dev Biol. 2007; 18: 54–66.[CrossRef][Medline] [Order article via Infotrieve]
2. Lee YM, Jeong CH, Koo SY, Son MJ, Song HS, Bae SK, Raleigh JA, Chung HY, Yoo MA, Kim KW. Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: a possible signal for vessel development. Dev Dyn. 2001; 220: 175–186.[CrossRef][Medline] [Order article via Infotrieve]
3. Palis J, Robertson S, Kennedy M, Wall C, Keller G. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development. 126: 5073–5084.
4. Adelman DM, Gertsenstein M, Nagy A, Simon MC, Maltepe E. Placental cell fates are regulated in vivo by HIF-mediated hypoxia responses. Genes Dev. 2000; 14: 3191–3203.
5. Ji RP, Phoon CKL, Aristizabal O, McGrath KE, Palis J, Turnbull DH. Onset of cardiac function during early mouse embryogenesis coincides with entry of primitive erythroblasts into the embryo proper. Circ Res. 2003; 92: 133–135.
6. Chen EY, Fuginaga M, Giaccia AJ. Hypoxic microenvironment within an embryo induces apoptosis and is essential for proper morphological development. Teratology. 1999; 60: 215–225.[CrossRef][Medline] [Order article via Infotrieve]
7. MacLellan WR, Schneider MD. Genetic dissection of cardiac growth control pathways. Annu Rev Physiol. 2000; 62: 289–319.[CrossRef][Medline] [Order article via Infotrieve]
8. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J. 1998; 17: 3005–3015.[CrossRef][Medline] [Order article via Infotrieve]
9. Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, Wenger RH, Gassmann M, Gearhart JD, Lawler AM, Yu AY, Semenza GL. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev. 1998; 12: 149–162.
10. Compernolle V, Brusselmans K, Franco D, Moorman A, Dewerchin M, Collen D, Carmeliet P. Cardia bifida, defective heart development and abnormal neural crest migration in embryos lacking hypoxia-inducible factor-1alpha. Cardiovasc Res. 2003; 60: 569–579.
11. Tian H, Hammer RE, Matsumoto AM, Russell DW, McKnight SL. The hypoxia- responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development. Genes Dev. 1998; 12: 3320–3324.
12. Compernolle V, Brusselmans K, Acker T, Hoet P, Tjwa M, Beck H, Plaisance S, Dor Y, Keshet E, Lupu F, Nemery B, Dewerchin M, Van Veldhoven P, Plate K, Moons L, Collen D, Carmeliet P. Loss of HIF-2alpha and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med. 2002; 8: 702–710.[Medline] [Order article via Infotrieve]
13. Scortegagna M, Ding K, Oktay Y, Gaur A, Thurmond F, Yan LJ, Marck BT, Matsumoto AM, Shelton JM, Richardson JA, Bennett MJ, Garcia JA. Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice. Nat Genet. 2003; 35: 331–340.[CrossRef][Medline] [Order article via Infotrieve]
14. Agarkova I, Auerbach D, Ehler E, Perriard JC. A novel marker for vertebrate embryonic heart, the EH-myomesin isoform. J Biol Chem. 2000; 275: 10256–10264.
15. Elion EA, Marina P, Yu L. Constructing recombinant DNA molecules by PCR. In Ausubel FM, ed. Current Protocols in Molecular Biology. Hoboken, NJ: John Wiley & Sons; 2007; Chapter 3: Unit 3.17.
16. Staller P, Sulitkova J, Lisztwan J, Moch H, Oakeley EJ, Krek W. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature. 2003; 425: 307–311.[CrossRef][Medline] [Order article via Infotrieve]
17. Ryan HE, Poloni M, McNulty W, Elson D, Gassmann M, Arbeit JM, Johnson RS. Hypoxia-inducible factor-1alpha is a positive factor in solid tumor growth. Cancer Res. 2000; 60: 4010–4015.
18. Schipani E, Ryan HE, Didrickson S, Kobayashi T, Knight M, Johnson RS. Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev. 2001; 15: 2865–2876.
19. Chen J, Kubalak SW, Chien KR. Ventricular muscle-restricted targeting of the RXRalpha gene reveals a non-cell-autonomous requirement in cardiac chamber morphogenesis. Development. 1998; 125: 1943–1949.[Abstract]
20. Huang Y, Hickey RP, Yeh JL, Liu D, Dadak A, Young LH, Johnson RS, Giordano FJ. Cardiac myocyte-specific HIF-1alpha deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart. FASEB J. 2004; 18: 1138–1140.
21. O'Brien TX, Lee KJ, Chien KR. Positional specification of ventricular myosin light chain 2 expression in the primitive murine heart tube. Proc Natl Acad Sci U S A. 1993; 90: 5157–5161.
22. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999; 21: 70–71.[CrossRef][Medline] [Order article via Infotrieve]
23. Moorman AF, Christoffels VM. Cardiac chamber formation: development, genes, and evolution. Physiol Rev. 2003; 83: 1223–1267.
24. Opitz CA, Linke WA. Plasticity of cardiac titin/connectin in heart development. J Muscle Res Cell Motil. 2005; 26: 333–342.[CrossRef][Medline] [Order article via Infotrieve]
25. Lange S, Xiang F, Yakovenko A, Vihola A, Hackman P, Rostkova E, Kristensen J, Brandmeier B, Franzen G, Hedberg B, Gunnarsson LG, Hughes SM, Marchand S, Sejersen T, Richard I, Edstrom L, Ehler E, Udd B, Gautel M. The kinase domain of titin controls muscle gene expression and protein turnover. Science. 2005; 308: 1599–1603.
26. Weinert S, Bergmann N, Luo X, Erdmann B, Gotthardt M. M line-deficient titin causes cardiac lethality through impaired maturation of the sarcomere. J Cell Biol. 2006; 173: 559–570.
27. Goda N, Ryan HE, Khadivi B, McNulty W, Rickert RC, Johnson RS. Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during hypoxia. Mol Cell Biol. 2003; 23: 359–369.
28. Molkentin JD, Lin Q, Duncan SA, Olson EN. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev. 1997; 11: 1061–1072.
29. Wenger RH, Stiehl DP, Camenisch G Integration of oxygen signaling at the consensus HRE. Sci STKE. 2005; 2005: re12.
30. Breckenridge R, Kotecha S, Towers N, Bennett M, Mohun T. Pan-myocardial expression of Cre recombinase throughout mouse development. Genesis. 2007; 45: 135–144.[CrossRef][Medline] [Order article via Infotrieve]
31. Dodou E, Verzi MP, Anderson JP, Xu SM, Black BL. Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development. Development. 2004; 131: 3931–3942.
32. Maddatu TP, Garvey SM, Schroeder DG, Zhang W, Kim SY, Nicholson AI, Davis CJ, Cox GA. Dilated cardiomyopathy in the nmd mouse: transgenic rescue and QTLs that improve cardiac function and survival. Hum Mol Genet. 2005; 14: 3179–3189.
33. Pereira T, Zheng X, Ruas JL, Tanimoto K, Poellinger L. Identification of residues critical for regulation of protein stability and the transactivation function of the hypoxia- inducible factor-1alpha by the von Hippel-Lindau tumor suppressor gene product. J Biol Chem. 2003; 278: 6816–6823.
34. Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA, Gessler M, Nemer M, Seidman CE, Seidman JG. A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell. 2001; 106: 709–721.[CrossRef][Medline] [Order article via Infotrieve]
35. Lin Q, Schwarz J, Bucana C, Olson EN. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science. 1997; 276: 1404–1407.
36. Radke MH, Peng J, Wu Y, McNabb M, Nelson OL, Granzier H, Gotthardt M. Targeted deletion of titin N2B region leads to diastolic dysfunction and cardiac atrophy. Proc Natl Acad Sci U S A. 2007; 104: 3444–3449.
37. Hiroi Y, Kudoh S, Monzen K, Ikeda Y, Yazaki Y, Nagai R, Komuro I. Tbx5 associates with Nkx2–5 and synergistically promotes cardiomyocyte differentiation. Nat Genet. 2001; 28: 276–280.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  C. Bianco, C. Cotten, E. Lonardo, L. Strizzi, C. Baraty, M. Mancino, M. Gonzales, K. Watanabe, T. Nagaoka, C. Berry, et al. Cripto-1 Is Required for Hypoxia to Induce Cardiac Differentiation of Mouse Embryonic Stem Cells Am. J. Pathol., November 1, 2009; 175(5): 2146 - 2158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. L. Semenza Regulation of Oxygen Homeostasis by Hypoxia-Inducible Factor 1 Physiology, April 1, 2009; 24(2): 97 - 106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Lavine and D. M. Ornitz Shared Circuitry: Developmental Signaling Cascades Regulate Both Embryonic and Adult Coronary Vasculature Circ. Res., January 30, 2009; 104(2): 159 - 169. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||