- Short report
- Open Access
Deletion of Cdc42 in embryonic cardiomyocytes results in right ventricle hypoplasia
© The Author(s) 2017
- Received: 4 October 2017
- Accepted: 17 October 2017
- Published: 3 November 2017
Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cytoskeleton remodeling and cell polarity establishment. Inactivating Cdc42 in cardiomyocytes resulted in embryonic lethality with heart developmental defects, including ventricular septum defects and thin ventricle wall syndrome.
In this study, we have generated a Cdc42 cardiomyocyte knockout mouse line by crossing Cdc42/flox mice with myosin light chain 2a (MLC2a)-Cre mice. We found that the deletion of Cdc42 in embryonic cardiomyocytes resulted in an underdeveloped right ventricle. Microarray analysis and real-time PCR data analysis displayed that the deletion of Cdc42 decreased dHand expression level. In addition, we found evaginations in the ventricle walls of Cdc42 knockout hearts.
We concluded that Cdc42 plays an essential role in right ventricle growth.
- Heart development
- Right ventricle development
The mammalian heart is the first functional organ formed during embryogenesis, and it uninterruptedly pumps blood throughout adulthood. Interfering with the process of cardiac development results in congenital heart diseases, which affect 40,000 newborns per year in the USA [1–3]. Cardiac morphogenesis and maturation require the deployment of multiple cell lineages that are derived from the primary heart field, secondary heart field, and neural crest cells [4–6]. Initially, the cardiac progenitors that derive from the primary heart field migrate toward the midline of the embryo to fuse and form the primary heart tube. Subsequently, the primary heart tube is elongated by recruiting cells that are generated from the second heart field. Finally, the heart tube undergoes rightward looping and chamber formation [5, 6].
The cardiac progenitors of the right and left ventricles come from different sources . The left ventricle cardiac progenitors are mainly derived from the primary heart field, and the right ventricle cardiomyocytes are from the secondary heart field . Development of the right and left ventricles is regulated by distinct developmental programs. Interfering ventricle-specific transcription factors and/or their effectors can cause sole ventricle developmental defects . Hypoplastic right heart syndrome is a rare congenital heart disease that is characterized by an underdeveloped right ventricle [10, 11]. However, the molecular mechanisms that regulate right ventricle development remain elusive. Three papers published in 2001 reported that cardiac progenitors deriving from the secondary heart field contributed to heart tube elongation and right ventricle development in chicks and mice [12–14]. Interfering with cardiac progenitor recruitment from the secondary heart field resulted in a small right ventricle. Insulin gene enhancer protein-1 (Isl-1) is expressed in the entire secondary heart field and then in the bilateral pharyngeal mesoderm. The inactivation of Isl-1 prevented formation of the right ventricle and outflow track . Right ventricle development is dependent on ventricle-specific transcriptional factors, including myocyte enhancer factor 2c (MEF2c) and heart- and neural crest derivatives-expressed protein 2 (dHand), as well as the recruitment of cardiac progenitors from the secondary heart field . MEF2c is required for right ventricle formation, and its promoter region contains Isl-1 and GATA binding sites . dHand is a home box transcription factor that is predominantly expressed in the right ventricle . dHand cannot be directly regulated by Isl-1, but can be regulated by BOP, a downstream effector of MEF2c. Inactivation of dHAND resulted in right ventricle hypoplasia. Furthermore, fibroblast growth factor 10 (FGF10) as well as bone morphogenetic protein (BMP)-initiated signal transduction are involved in right ventricle development . However, the role of signal transduction in regulating right ventricle development remains elusive.
Cdc42 is a Ras GTPase superfamily member that is essential for regulating establishment of actin polymerization . In response to extracellular stimulation (growth factors, shear stress, etc.), Cdc42’s activity is tightly controlled by GTPase-activating proteins (GAPs), guanine nucleotide exchange factors (GEF) and guanine nucleotide dissociation inhibitors (GDI) [19, 20]. Recently, we and others have reported that Cdc42 plays an important role in heart development in both mice and drosophila [21, 22]. In addition, the deletion of Cdc42 resulted in small right ventricles and enlarged right atria in the adult mice . To further investigate the role of Cdc42 in heart development, we examined the role of Cdc42 in right ventricle development and found that Cdc42 is required for right ventricle growth.
Generation of Cdc42 cardiomyocyte knockout mice
Cdc42 cardiomyocyte knockout mice were generated by crossing Cdc42/flox mice with MLC-2a Cre mice, as previously reported . The protocol of animal studies was reviewed and approved by the Animal Care and Use Committee of Texas A&M Health Science Center.
E14.5 embryos were harvested and fixed with 4% paraformaldehyde. After dehydration, the embryos were embedded in paraffin and sectioned in 4 micrometers. Hematoxylin and eosin (H&E) staining was then performed.
Microarray and real-time PCR analysis
E14.5 embryonic hearts were harvested and RNA was isolated with the Qiagen RNeasy mini kit (QIAGEN, CA). cRNA was synthesized using standard protocols and then applied to an Affymetrix Gene Chip Mouse Exon 1.0 ST Array (Affymetrix, CA). cDNA was synthesized with SuperScript® III Reverse Transcriptase (Invitrogen, CA). Real-time PCR analysis was performed as previously reported . We harvested six control and knockout embryonic hearts for real-time PCR analysis.
Student’s T test was used to determine statistical significance. P values < 0.05 were considered significant. Values for all measurements were expressed as mean ± standard deviation.
Congenital heart disease is the most common birth defect in the world and can be induced by genetic mutations and/or exposures to environmental risk factors . In our previous studies, we have reported that deleting Cdc42 in cardiomyocytes resulted in thin ventricle walls and ventricular septum defects. In addition, we also observed dilated right atria and small right ventricles in Cdc42 knockout mice . In the current study, we analyzed Cdc42 knockout embryonic hearts using histology analysis and found that the deletion of Cdc42 resulted in small right ventricles. Additionally, the deletion of Cdc42 in the embryonic hearts decreased dHand expression.
One of the most dramatic phenotypes of the Cdc42 knockout mice were the underdeveloped right ventricles. The size difference between the left and right ventricles cannot be explained by cardiomyocyte proliferation defects because the deletion of Cdc42 affected cardiomyocyte proliferation in both left and right ventricles. In addition, it was documented that many other genes, including focal adhesion kinase, etc., are important for cardiomyocyte proliferation. However, inactivation of those genes did not affect the right ventricle size . Therefore, the ventricle size and ventricle cardiomyocyte proliferation may be controlled by independent regulatory mechanisms. In our case, we inactivated Cdc42 in cardiomyocytes using MLC2a-Cre. Thus, it was impossible to affect cardiac progenitor recruitment from the secondary heart field because MLC2a was expressed in differentiated cardiomyocytes instead of in cardiac progenitors of the secondary heart field.
It was documented that the regulatory transcription factors in the left and right ventricles are different . For example, dHand is specifically expressed in the right ventricle and is required for right ventricle and cardiac outflow formation . In contrast, eHand is expressed in the left ventricle and inactivation of eHand impaired left ventricle growth and maturation . In our study, we found that the inactivation of Cdc42 impaired dHand expression and resulted in a small right ventricle. Therefore, it is possible that Cdc42 controls right ventricle growth through dHand. However, it remains unclear how Cdc42 regulates dHand expression during heart development.
FGF10 was detected only in the right ventricle during embryonic heart development . Consistently, the inactivation of FGF10 resulted in an underdeveloped right ventricle. It was reported that FGF stimulation induced BNIP-2 (a member of the BCL2/adenovirus E1B 19 kd-interacting protein) phosphorylation. The phosphorylation of BNIP-2 decreased its binding and GAP-like activity toward Cdc42, keeping Cdc42 in its active status . Therefore, it is possible that Cdc42 is involved in FGF10-initiated signal transduction and that the inactivation of Cdc42 impaired FGF10-induced right ventricle formation.
In addition to the smaller right ventricles found in Cdc42 knockout mice, we also noted that the inactivation of Cdc42 resulted in ventricle wall evagination. Previously, we reported that Cdc42 is required for maintaining cardiomyocyte cell–cell adhesion and that the inactivation of Cdc42 impaired cardiomyocyte-adherent junction formation . It was reported that the activated Cdc42 can prevent IQGAP1 from binding to β-catenin, weakening the Cadherin-Catenin-actin-based cell–cell adherent junctions . Therefore, it is possible that the inactivation of Cdc42 increased the binding between β-catenin and IQGAP1 and disrupted N-cadherin–catenin–actin-based cell–cell adherent junctions. Because of the increased pressure inside the ventricle during heart development, the weakest section of the ventricle wall may balloon out to form abnormal evaginations.
YL and XP designed research. YL, JW, RW, JL, XP, performed research. BT, SLZ, CWT contributed material and critical discussion. YL, and XP, wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
Available from corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
American Heart Association (13BGIA14470002) to X.P.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Hoffman JI, Kaplan S (2002) The incidence of congenital heart disease. J Am Coll Cardiol 39(12):1890–1900View ArticlePubMedGoogle Scholar
- Reller MD, Strickland MJ, Riehle-Colarusso T, Mahle WT, Correa A (2008) Prevalence of congenital heart defects in metropolitan Atlanta, 1998–2005. J Pediatr 153(6):807–813View ArticlePubMedPubMed CentralGoogle Scholar
- Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R et al (2017) Heart disease and stroke statistics-2017 update: a report from the American Heart Association. Circulation 135(10):e146–e603View ArticlePubMedPubMed CentralGoogle Scholar
- Bruneau BG (2008) The developmental genetics of congenital heart disease. Nature 451(7181):943–948View ArticlePubMedGoogle Scholar
- Rochais F, Mesbah K, Kelly RG (2009) Signaling pathways controlling second heart field development. Circ Res 104(8):933–942View ArticlePubMedGoogle Scholar
- Srivastava D (2006) Making or breaking the heart: from lineage determination to morphogenesis. Cell 126(6):1037–1148View ArticlePubMedGoogle Scholar
- Farrell MJ, Kirby ML (2001) Cell biology of cardiac development. Int Rev Cytol 202:99–158View ArticlePubMedGoogle Scholar
- Moon A (2008) Mouse models of congenital cardiovascular disease. Curr Top Dev Biol 84:171–248View ArticlePubMedGoogle Scholar
- Bruneau BG (2013) Signaling and transcriptional networks in heart development and regeneration. Cold Spring Harb Perspect Biol 5(3):a008292View ArticlePubMedPubMed CentralGoogle Scholar
- Dib C, Araoz PA, Davies NP, Dearani JA, Ammash NM (2012) Hypoplastic right-heart syndrome presenting as multiple miscarriages. Tex Heart Inst J 39(2):249–254PubMedPubMed CentralGoogle Scholar
- Dimopoulos A, Sicko RJ, Kay DM, Rigler SL, Druschel CM, Caggana M et al (2017) Rare copy number variants in a population-based investigation of hypoplastic right heart syndrome. Birth Defects Res 109(1):8–15View ArticlePubMedGoogle Scholar
- Kelly RG, Brown NA, Buckingham ME (2001) The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev Cell 1(3):435–440View ArticlePubMedGoogle Scholar
- Waldo KL, Kumiski DH, Wallis KT, Stadt HA, Hutson MR, Platt DH et al (2001) Conotruncal myocardium arises from a secondary heart field. Development (Cambridge, England) 128(16):3179–3188Google Scholar
- Mjaatvedt CH, Nakaoka T, Moreno-Rodriguez R, Norris RA, Kern MJ, Eisenberg CA et al (2001) The outflow tract of the heart is recruited from a novel heart-forming field. Dev Biol 238(1):97–109View ArticlePubMedGoogle Scholar
- Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J et al (2003) Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell 5(6):877–889View ArticlePubMedPubMed CentralGoogle Scholar
- Srivastava D, Olson EN (2000) A genetic blueprint for cardiac development. Nature 407(6801):221–226View ArticlePubMedGoogle Scholar
- Lin Q, Srivastava D, Olson EN (1997) A transcriptional pathway for cardiac development. Cold Spring Harb Symp Quant Biol 62:405–411View ArticlePubMedGoogle Scholar
- Srivastava D, Thomas T, Lin Q, Kirby ML, Brown D, Olson EN (1997) Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND. Nat Genet 16(2):154–160View ArticlePubMedGoogle Scholar
- Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology. Nature 420(6916):629–635View ArticlePubMedGoogle Scholar
- Cerione RA (2004) Cdc42: new roads to travel. Trends Cell Biol 14(3):127–132View ArticlePubMedGoogle Scholar
- Vogler G, Liu J, Iafe TW, Migh E, Mihaly J, Bodmer R (2014) Cdc42 and formin activity control non-muscle myosin dynamics during Drosophila heart morphogenesis. J Cell Biol 206(7):909–922View ArticlePubMedPubMed CentralGoogle Scholar
- Li J, Liu Y, Jin Y, Wang R, Wang J, Lu S et al (2017) Essential role of Cdc42 in cardiomyocyte proliferation and cell-cell adhesion during heart development. Dev Biol 421(2):271–283View ArticlePubMedGoogle Scholar
- Lage K, Greenway SC, Rosenfeld JA, Wakimoto H, Gorham JM, Segre AV et al (2012) Genetic and environmental risk factors in congenital heart disease functionally converge in protein networks driving heart development. Proc Natl Acad Sci USA 109(35):14035–14040View ArticlePubMedPubMed CentralGoogle Scholar
- Peng X, Wu X, Druso JE, Wei H, Park AY, Kraus MS et al (2008) Cardiac developmental defects and eccentric right ventricular hypertrophy in cardiomyocyte focal adhesion kinase (FAK) conditional knockout mice. Proc Natl Acad Sci USA 105(18):6638–6643View ArticlePubMedPubMed CentralGoogle Scholar
- McFadden DG, Barbosa AC, Richardson JA, Schneider MD, Srivastava D, Olson EN (2005) The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner. Development (Cambridge, England) 132(1):189–201View ArticleGoogle Scholar
- Togi K, Kawamoto T, Yamauchi R, Yoshida Y, Kita T, Tanaka M (2004) Role of Hand1/eHAND in the dorso-ventral patterning and interventricular septum formation in the embryonic heart. Mol Cell Biol 24(11):4627–4635View ArticlePubMedPubMed CentralGoogle Scholar
- Low BC, Lim YP, Lim J, Wong ES, Guy GR (1999) Tyrosine phosphorylation of the Bcl-2-associated protein BNIP-2 by fibroblast growth factor receptor-1 prevents its binding to Cdc42GAP and Cdc42. J Biol Chem 274(46):33123–33130View ArticlePubMedGoogle Scholar
- Kaibuchi K, Kuroda S, Fukata M, Nakagawa M (1999) Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases. Curr Opin Cell Biol 11(5):591–596View ArticlePubMedGoogle Scholar