Congenital heart defect causing mutation in Nkx2.5 displays in vivo functional deficit

Zakariyah, Abeer; Rajgara, Rashida; Veinot, John P.; Skerjanc, Ilona S.; Burgon, Patrick G.

doi:10.1016/j.yjmcc.2017.03.003

Cited by 24 publications

(23 citation statements)

References 35 publications

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“…The gene has also been shown to play a role in the heart's conduction system postnatally. Therefore, reduction of NKX2‐5 expression by cigarette smoke components may cause abnormal looping morphogenesis during early embryonic development, resulting in heart development defects and congenital heart malformations …”

Section: Discussionmentioning

confidence: 99%

Effects of cigarette smoke components on myocardial differentiation of mouse embryonic stem cells

Kim

et al. 2019

Environmental Toxicology

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The heart is the first organ formed in the developing fetus, and abnormal development of the heart is a major cause of fetal death. The adverse effects of cigarette smoke on the heart have been well established, but it is not well understood how cigarette smoke components regulate signaling molecules and cardiac specific functions during the early differentiation stage of the embryonic heart. In this study, we identified changes in the size of mouse embryoid bodies (mEBs) in response to treatment with cigarette smoke extract (CSE) via regulation of HDAC2, p53, p21, and cyclin D1 protein expression, which are cardiac differentiation and cell-cycle markers, respectively.In addition, exposure of mouse embryonic stem cells (mESCs) to cigarette smoke components inhibited myocardial differentiation and development through the expression of HDAC1, HDAC2, GATA4, NKX2-5, TBX5, HAND1, and Troponin I. Long-term exposure studies showed that CSE and nicotine may delay the development of mouse cardiomyocytes from mESCs and inhibit the contractibility, which is a fundamental function of the heart. Taken together, these findings suggest that cigarette smoke components, including nicotine, may affect abnormal myocardial differentiation and development. K E Y W O R D S cardiomyocyte, cell cycle, cigarette smoke extract, HDACs, mouse embryonic stem cells, myocardial differentiation, nicotine 1 | INTRODUCTION Embryonic stem cells isolated from the inner cell mass in the blastocyst stage can differentiate into three germ layers, the endoderm, mesoderm, and ectoderm. 1 The mesoderm layer forms connective tissues and muscles including cardiac muscle throughout the body. The heart is the first organ formed and differentiated in the developing embryo and fetus. 2,3 In early embryonic development, cardiac cells begin to develop through specific protein signaling. During embryogenesis, NK2 homeobox 5 (NKX2-5) is expressed in early cardiac mesoderm cells throughout the left ventricle and atrial chambers. 4,5 NKX2-5 activates a number of downstream transcription factors (such as myocyte enhancer factor-2 [MEF2] and GATA), which activate the expression of cardiac muscle specific proteins. Mutations in NKX2-5 result in heart development defects and congenital heart malformations. 6-8 The heart tube undergoes right-ward looping to change from anterior/posterior polarity to left/right polarity. 9 Looping alsodepends on heart specific proteins activated by NKX2-5 such as heartand neural crest derivatives-expressed protein (HAND) 1 and HAND2. 10,11 HAND1 is localized to the left ventricle while HAND2 is localized to the right ventricle. 12 When the heart is developed through this specific protein signaling, it starts to beat and pump blood at about 22-23 days, with blood flow beginning in week 4. 2,3 It is well known that cigarette smoke adversely affects the development of early embryos. [13][14][15] Cigarette smoke contains over 4000

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Section: Discussionmentioning

confidence: 99%

Effects of cigarette smoke components on myocardial differentiation of mouse embryonic stem cells

Kim

et al. 2019

Environmental Toxicology

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“…Cre treatment should be applied to both the knock-in and control cell lines. Generation of the knock-in Nkx2.5 R141C/+ mESCs by this method is described in Zakariyah et al (2017Zakariyah et al ( , 2018. The mutation was introduced into the Nkx2.5 locus, and the targeted construct was linearized and electroporated into R1 mESCs.…”

Section: Strategic Planningmentioning

confidence: 99%

“…Therefore, we aim to create an in vitro model of CHD that will allow us to observe and investigate the abnormalities that occur at early stages of heart development. To validate this approach, we generated in vivo (mouse) and in vitro (mESC) models that have a human knock-in heterozygous mutation in the Nkx2.5 gene (Zakariyah et al, 2017(Zakariyah et al, , 2018, which causes CHD in humans.…”

Section: Background Informationmentioning

confidence: 99%

“…We have generated and characterized novel heterozygous Nkx2.5 R141C/+ mESCs that harbor the CHD-causing mutation (Zakariyah et al, 2018). Our recent study in the mouse model shows that the homozygous mutation causes lethality at embryonic day 10.5 (E10.5), whereas the heterozygous mutation results in septal and conduction defects (Zakariyah, Rajgara, Veinot, Skerjanc, & Burgon, 2017). The molecular mechanism of how this mutation leads to CHD is still unknown.…”

Section: Introductionmentioning

confidence: 99%

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Combinatorial Utilization of Murine Embryonic Stem Cells and In Vivo Models to Study Human Congenital Heart Disease

Zakariyah

Rajgara

Shelton

et al. 2018

CP Stem Cell Biology

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We have established an in vitro model of the human congenital heart defect (CHD)-associated mutation NKX2.5 R141C. We describe the use of the hanging drop method to differentiate Nkx2.5 R141C/+ murine embryonic stem cells (mESCs) along with Nkx2.5 +/+ control cells. This method allows us to recapitulate the early stages of embryonic heart development in tissue culture. We also use qRT-PCR and immunofluorescence to examine samples at different time points during differentiation to validate our data. The in vivo model is a mouse line with a knock-in of the same mutation. We describe the isolation of RNA from embryonic day 8.5 (E8.5) embryos and E9.5 hearts of wild-type and mutant mice. We found that the in vitro model shows reduced cardiomyogenesis, similar to Nkx2.5 R141C/+ embryos at E8.5, indicating a transient loss of cardiomyogenesis at this time point. These results suggest that our in vitro model can be used to study very early changes in heart development that cause CHD. C 2018 by John Wiley & Sons, Inc.Keywords: cardiac differentiation r embryoid bodies r embryos r hanging drops r mouse embryonic stem cells

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“…In vitro experiments demonstrated that NKX2‐5 R142C protein has a reduced DNA binding affinity, attenuated transcriptional activity and reduced physical interaction with wild‐type NKX2‐5, GATA4, or TBX5 proteins . We have recently reported the generation and characterization of a heterozygous Nkx2‐5 R141C/+ mouse model that presented with a high penetrance of septal and conduction heart defects, whereas an early embryonic lethality was observed for the Nkx2‐5 R141C/R141C homozygous mice .…”

Section: Introductionmentioning

confidence: 99%

In Vitro Modeling of Congenital Heart Defects Associated with an NKX2-5 Mutation Revealed a Dysregulation in BMP/Notch-Mediated Signaling

et al. 2018

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The Nkx2-5 gene codes for a transcription factor that plays a critical role in heart development. Heterozygous mutations in NKX2-5 in both human and mice result in congenital heart defects (CHDs). However, the molecular mechanisms by which these mutations cause the disease are still unknown. Recently, we have generated the heterozygous mouse model of the human CHDs associated mutation NKX2-5 R142C (Nkx2-5 mouse ortholog of human NKX2-5 R142C variant) that developed septal and conduction defects. This study generated a heterozygous Nkx2-5 R141C mouse embryonic stem cell line (Nkx2-5 mESCs) to model CHDs in vitro. We observed that Nkx2-5 mESCs display an alteration in the expression of genes that are essential for normal heart development. Furthermore, the reduced cardiomyogenesis is paralleled by a reduction in nuclear import of Nkx2-5 protein. Examination of the Nkx2-5 embryos at E8.5 revealed a transient loss of cardiomyogenesis, which is consistent with the phenotype observed in vitro. Moreover, gene expression profiling of Nkx2-5 cells at an early stage of cardiac differentiation revealed pronounced deregulation of several cardiac differentiation and function genes. Collectively, our data showed that heterozygosity for the R141C mutation results in disruption of the cellular distribution of Nkx2-5 protein, a transient reduction in cardiomyogenesis that may disrupt the early patterning of the heart, and this, in turn, affects the intricate orchestration of signaling pathways leading to downregulation of Bone morphogenetic protein (BMP) and Notch signaling. Therefore, we have developed mESCs model of a human CHD, providing an in vitro system to examine early stages of heart development, which are otherwise difficult to study in vivo. Stem Cells 2018;36:514-526.

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Congenital heart defect causing mutation in Nkx2.5 displays in vivo functional deficit

Cited by 24 publications

References 35 publications

Effects of cigarette smoke components on myocardial differentiation of mouse embryonic stem cells

Effects of cigarette smoke components on myocardial differentiation of mouse embryonic stem cells

Combinatorial Utilization of Murine Embryonic Stem Cells and In Vivo Models to Study Human Congenital Heart Disease

In Vitro Modeling of Congenital Heart Defects Associated with an NKX2-5 Mutation Revealed a Dysregulation in BMP/Notch-Mediated Signaling

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