Cardiac Developmental Defects Research Articles

Bisphenol C Induces Cardiac Developmental Defects by Disrupting m6A Homeostasis.

Bisphenol A (BPA) is a commonly used plastic additive. Since BPA has been banned in maternal and infant food containers in many countries, BPA substitutes have been widely introduced to replace it. By systematically assessing the potential developmental toxicity of BPA substitutes, we observed that the 41-150 nM in vivo BPC exposure (around the reported concentration detected in infant urine: 6-186 nM) induced cardiac defects in zebrafish. Mechanistically, BPC disrupted m6A homeostasis by downregulation of the key m6A methyltransferase, Mettl3, thereby causing the m6A reader, Igf2bp2b, to fail in recognizing and stabilizing the inefficiently m6A-modified acox1 and tnnt2d mRNA. Then, downregulation of Acox1 (a regulator in cardiac fatty acid metabolism) and Tnnt2d (a component of cardiac troponin for muscle contraction) led to cardiac defects. Indeed, the dual cardiac functional axes regulated by the same m6A reader in response to BPC provided new insight into the regulatory mechanisms of epitranscriptomics and cardiac development. Collectively, our study not only presented evidence showing that the internal exposure levels of BPC in humans could lead to cardiac developmental defects but also demonstrated the underlying mechanism of BPC-mediated defects by disrupting the Mettl3-m6A-Igf2bp2b-Acox1/Tnnt2d pathways, which provided potential molecular markers associated with BPC exposure.

Microcystin-LR prenatal exposure induces coronary heart disease through macrophage polarization imbalance mediated by trophoblast-derived extracellular vesicles

Microcystin-leucine arginine (MC-LR) has been reported to exhibit placental toxicity, leading to potential adverse pregnancy outcomes. Placental abnormalities often coincide with congenital heart defects (CHD). However, the extent to which MC-LR–induced placental abnormalities contribute to CHD and the cellular mechanisms underlying this association remain unknown. In this study, we observed abnormal polarization of placental macrophages in pregnant mice exposed to MC-LR during pregnancy, and the embryos developed cardiac developmental defects that persisted into adulthood. Trophoblast-derived extracellular vesicles (T-EVs) increase in number during pregnancy and act as a critical signal in macrophage polarization. However, MC-LR significantly affected the miRNA expression profile of T-EVs. Upon internalization into macrophages, T-EV-derived miR-377-3p specifically targets the 3’UTR region of NR6A1 to inhibit gene expression. Silencing of transcription suppressor NR6A1 leads to abnormal activation of the downstream mTOR/S6K1/SREBP pathway, inducing metabolic reprogramming and ultimately leading to M1 polarization of macrophages. This study elucidated the placental mechanism underlying MC-LR–induced CHD for the first time, providing insights into the environmental risks associated with CHD.

Resveratrol ameliorates cyprodinil-induced zebrafish cardiac developmental defects as an aryl hydrocarbon receptor antagonist.

Cyprodinil, a globally utilized broad-spectrum pyrimidine amine fungicide, has been observed to elicit cardiac abnormality. Resveratrol (RSV), a naturally occurring polyphenolic compound, showcases remarkable defensive properties in nurturing cardiac development. To investigate whether RSV could protect against cyprodinil-induced cardiac defects, we exposed zebrafish embryos to cyprodinil (500μg/L) in the presence or absence of RSV (1μM). Our results showed that RSV significantly mitigated the decrease of survival rate and embryo movement and the hatching delay induced by cyprodinil. In addition, RSV also improved cyprodinil-induced zebrafish cardiac developmental toxicity, including pericardial edema and cardiac function impairment. In mechanism, RSV attenuated the cyprodinil-induced changes in mRNA expression involved in cardiac development, such as myh6, myl7, tbx5, and gata4, and calcium ion channels, such as ncx1h, slc8a4a, and atp2a2b. We further showed that RSV might inhibit the activity of aryl hydrocarbon receptor (AhR) signaling pathways induced by cyprodinil. In summary, our findings establish that the protective effects of RSV against the cardiac developmental toxicity are induced by cyprodinil due to its remarkable ability to inhibit AhR activity. Our findings not only shed light on a new avenue for regulating and ensuring the safe utilization of cyprodinil but also presents a novel concept to promote its responsible use.

(-)-Epicatechin gallate ameliorates cyprodinil-induced cardiac developmental defects through inhibiting aryl hydrocarbon receptor in zebrafish.

Cyprodinil is a widely used fungicide with broad-spectrum activity, but it has been associated with cardiac abnormalities. (-)-Epicatechin gallate (ECG), a natural polyphenolic compound, has been shown to possess protective properties in cardiac development. In this study, we investigated whether ECG could mitigate cyprodinil-induced heart defects using zebrafish embryos as a model. Zebrafish embryos were exposed to cyprodinil with or without ECG. Our results demonstrated that ECG significantly improved the survival rate, embryo movement, and hatching delay induced by cyprodinil. Furthermore, ECG effectively ameliorated cyprodinil-induced cardiac developmental toxicity, including pericardial anomaly and impairment of cardiac function. Mechanistically, ECG attenuated the cyprodinil-induced alterations in mRNA expression related to cardiac development, such as amhc, vmhc, tbx5, and gata4, as well as calcium ion channels, such as ncx1h, atp2a2a, and cdh2. Additionally, ECG was found to inhibit the activity of the aryl hydrocarbon receptor (AhR) signaling pathways induced by cyprodinil. In conclusion, our findings provide evidence for the protective effects of ECG against cyprodinil-induced cardiac developmental toxicity, mediated through the inhibition of AhR activity. These findings contribute to a better understanding of the regulatory mechanisms and safe utilization of pesticide, such as cyprodinil.

Early life stage exposure to fenbuconazole causes multigenerational cardiac developmental defects in zebrafish and potential reasons

With the increasing use of triazole fungicides in agriculture, triazole pesticides have aroused great concern about their toxicity and ecological risk. The current study investigated the impairments of embryonic exposure to fenbuconazole (FBZ) on cardiac transgenerational toxicity and related mechanisms. The fertilized eggs were exposed to 5, 50 and 500 ng/L FBZ for 72 h, and the larvae were then raised to adulthood in clean water. The adult fish were mated with unexposed fish to produce maternal and paternal F1 and F2 embryos, respectively. The results showed that increased arrhythmia were observed in F0, F1 and F2 larvae. Transcriptome sequencing indicated that the pathway of adrenergic signaling in cardiomyocytes was enriched in F0 and F2 larvae. In both F0 and F1 adult zebrafish hearts, ADRB2 protein expression decreased, and transcription of genes related to cardiac development and Ca2+ homeostasis was downregulated. These alterations might cause cardiac developmental defects. Significantly decreased protein levels of H3K9Ac and H3K14Ac might be linked with the downregulation in transcription of cardiac development genes. Protein‒protein interaction analysis exhibited that the pathway affecting the heart was well inherited in the paternal line. These results provide new ideas for the analysis and prevention of congenital heart disease.

On the involvement of the second heart field in congenital heart defects.

Congenital heart defects (CHD) affect 1 in 100 live births and result from defects in cardiac development. Growth of the early heart tube occurs by the progressive addition of second heart field (SHF) progenitor cells to the cardiac poles. The SHF gives rise to ventricular septal, right ventricular and outflow tract myocardium at the arterial pole, and atrial, including atrial septal myocardium, at the venous pole. SHF deployment creates the template for subsequent cardiac septation and has been implicated in cardiac looping and in orchestrating outflow tract development with neural crest cells. Genetic or environmental perturbation of SHF deployment thus underlies a spectrum of common forms of CHD affecting conotruncal and septal morphogenesis. Here we review the major properties of SHF cells as well as recent insights into the developmental programs that drive normal cardiac progenitor cell addition and the origins of CHD.

Ca2+ homeostasis imbalance induced by Pparg: A key factor in di (2-ethylhexyl) phthalate (DEHP)-induced cardiac dysfunction in zebrafish larvae

Widespread application of the typical phthalate plasticizers, di (2-ethylhexyl) phthalate (DEHP), poses a serious potential threat to the health of animals and even humans. Previous studies have confirmed the mechanism of DEHP-induced cardiac developmental defects in zebrafish larvae. However, the mechanism of cardiac dysfunction is still unclear. Thus, this work aimed to comprehensively investigate the mechanisms involved in DEHP-induced cardiac dysfunction through computational simulations, in vivo assays in zebrafish, and in vitro assays in cardiomyocytes. Firstly, molecular docking and western blot initially investigated the activating effect of DEHP on Pparg in zebrafish. Although GW9662 (PPARG antagonist) effectively alleviated DEHP-induced cardiac dysfunction and lipid metabolism disorders, it did not restore significant decreases in mitochondrial membrane potential and ATP levels. In vitro assays in cardiomyocytes, DEHP caused overexpression of PPARG and proteins involved in the regulation of Ca2+ homeostasis, and the above abnormalities were effectively alleviated by GW9662, suggesting that the Ca2+ homeostatic imbalance caused by activation of PPARG by DEHP seems to be the main cause of DEHP-induced cardiac dysfunction. To sum up, this work not only refines the mechanism of toxic effects of cardiotoxicity induced by DEHP, but provides an important theoretical basis for enriching the toxicological effects of DEHP.

Abstract 13987: RBX1-CRLs Are Essential for Perinatal Heart Development by Regulating Hippo-YAP Signaling

Defects in cardiac development can lead to congenital heart diseases (CHD) or predispose the heart to cardiomyopathies in adult life. The molecular mechanisms that regulate this process remain elusive. Cullin-RING E3 ligases (CRLs) are multi-subunit complexes that are critical to maintain protein homeostasis by targeting a variety of protein substrates for proteasomal degradation. In the CRLs, RBX1 serves as the catalytic subunit that promotes the transfer of ubiquitin to the target protein. Previous animal studies uncovered a crucial role for RBX1 in development. However, its importance in the developing heart has not been explored. Here, we report for the first time the indispensable role of RBX1 in perinatal cardiac development. We created mice with cardiac-specific deletion of RBX1 driven by αMHC Cre . Compared with littermate control mice, RBX1-deficient mouse embryos exhibited no gross morphological abnormalities at E12.5 and E14.5, displayed peripheral edema at E16.5 indicative of heart failure, and eventually died around E18.5. Histological analysis revealed myocardial hypoplasia and a substantially thinner compact layer in E16.5 mutant hearts. This event was preceded by a remarkable decrease in cardiomyocyte proliferation and diminished nuclear localization of YAP, a powerful regulator of cardiomyocyte proliferation, at E14.5. In cultured cardiomyocytes, depletion of RBX1 inhibited proliferation and induced profound transcriptomic changes like the upregulation of cardiomyopathy-related genes. Mechanistically, RBX1 deficiency destabilized multiple CRLs and resulted in the accumulation of Hippo kinases and adaptor proteins such as SAV1, LATS1/2, and MOB1 leading to the inactivation of YAP by phosphorylation. We further demonstrated that RBX1 degrades the Hippo adaptors SAV1 and MOB1. Collectively, we propose that the fine-tuned, RBX1-mediated removal of Hippo components serves as a critical checkpoint to activate YAP signaling ensuring cardiac ventricular chamber development and morphogenesis.

Crosstalk between aryl hydrocarbon receptor and Wnt/β-catenin signaling pathway: Possible culprit of di (2-ethylhexyl) phthalate-mediated cardiotoxicity in zebrafish larvae

Typical plasticizer di (2-ethylhexyl) phthalate (DEHP) has been demonstrated to induce cardiotoxicity in zebrafish, but the potential molecular mechanisms involved have not been fully elucidated. Aryl hydrocarbon receptor (AhR), an essential protein for inducing developmental abnormalities, has been demonstrated to be activated by DEHP in other species, but whether the AhR signaling pathway also contributes to DEHP-mediated cardiac developmental toxicity in zebrafish remains unclear. Firstly, molecular docking simulations initially confirmed the possibility that DEHP has AhR agonistic activity. To further confirm this conjecture, this work analyzed the changes of cardiac-related indexes in zebrafish stressed by DEHP at individual, protein, and gene levels. The results showed that DEHP mediated cardiac phenotypic developmental defects, increased CYP1A1 activity, and oxidative stress as well as significant changes in the expression levels of key proteins and genes of AhR, Wnt/β-catenin, and Nrf2-Keap1 signaling pathways. Notably, the addition of AhR inhibitors effectively alleviated the above negative effects, indicating that the AhR signaling pathway and its crosstalk with the Wnt/β-catenin signaling pathway is an essential pathway for DEHP-mediated cardiac developmental toxicity. Overall, this work enriches the molecular mechanism of DEHP-mediated cardiac developmental defects in zebrafish and provides a reliable biomarker for future environmental risk assessment of DEHP.

Abstract P2020: Binding Of Filamin C With Actin Is Essential For Cardiac Development And Function

Filamin C (FLNC), a filamin family member predominantly expressed in striated muscles, is crucial for bridging the extracellular matrix and the cytoskeleton in cardiomyocytes (CMs), and the maintenance of heart integrity and function. FLNC consists of an N-terminal actin-binding domain (ABD) and 24 immunoglobulin (Ig)-like repeat domains at C-terminus. Many pathogenic mutations on N-terminal ABD have been found in patients with cardiomyopathies. However, little is known about how the actin-binding activity intrinsically contributes to the function of FLNC in mammalian hearts. Using structure-based design, we mutated two key actin binding residues F93 and L98 into Ala and Glu respectively in FLNC ABD (F93A/L98E) and show that the mutations completely disrupted the FLNC-actin interaction. We then generated a knock-in mouse model, demonstrating that loss of FLNC-actin interaction in embryonic CMs led to embryonic lethality and cardiac developmental defects displayed by ventricular rupture and reduced CM proliferation. Moreover, disruption of FLNC-actin interaction in adult CMs resulted in severe DCM phenotypes and reduced survival rate, accompanied with aberrant levels of key cytoskeleton components. These phenotypes resemble those of FLNC knockout mouse models, demonstrating a vital role of the FLNC-actin binding in the function of CMs. Our results also provide a foundation for understanding the mutation-induced cardiomyopathies associated with dysregulation of FLNC ABD-actin interaction.

Interaction of Filamin C With Actin Is Essential for Cardiac Development and Function.

FLNC (filamin C), a member of the filamin family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular matrix) in cardiomyocytes, thereby maintaining heart integrity and function. Although genetic variants within the N-terminal ABD (actin-binding domain) of FLNC have been identified in patients with cardiomyopathy, the precise contribution of the actin-binding capability to FLNC's function in mammalian hearts remains poorly understood. We conducted in silico analysis of the 3-dimensional structure of mouse FLNC to identify key amino acid residues within the ABD that are essential for FLNC's actin-binding capacity. Subsequently, we performed coimmunoprecipitation and immunofluorescent assays to validate the in silico findings and assess the impact of these mutations on the interactions with other binding partners and the subcellular localization of FLNC. Additionally, we generated and analyzed knock-in mouse models in which the FLNC-actin interaction was completely disrupted by these mutations. Our findings revealed that F93A/L98E mutations completely disrupted FLNC-actin interaction while preserving FLNC's ability to interact with other binding partners ITGB1 (β1 integrin) and γ-SAG (γ-sarcoglycan), as well as maintaining FLNC subcellular localization. Loss of FLNC-actin interaction in embryonic cardiomyocytes resulted in embryonic lethality and cardiac developmental defects, including ventricular wall malformation and reduced cardiomyocyte proliferation. Moreover, disruption of FLNC-actin interaction in adult cardiomyocytes led to severe dilated cardiomyopathy, enhanced lethality and dysregulation of key cytoskeleton components. Our data strongly support the crucial role of FLNC as a bridge between actin filaments and ECM through its interactions with actin, ITGB1, γ-SAG, and other associated proteins in cardiomyocytes. Disruption of FLN-actin interaction may result in detachment of actin filaments from the extracellular matrix, ultimately impairing normal cardiac development and function. These findings also provide insights into mechanisms underlying cardiomyopathy associated with genetic variants in FLNC ABD and other regions.

Mechanisms regarding cardiac toxicity triggered by up-regulation of miR-144 in larval zebrafish upon exposure to triclosan.

Although triclosan (TCS) is ubiquitously detected in environmental media and organisms, little information is available on its cardiotoxicity and underlying mechanisms. Herein, acute TCS exposure (0.69-1.73μM) to zebrafish from embryos (6 hpf) to larvae (72 hpf) resulted in cardiac development defects, including increased angle between atrium and ventricle, prolonged SV-BA distance, linearized heart and pericardial cyst in 72-hpf larvae. These malformations resulted from interfered oxidative-stress pathways, reflecting in accumulated ROS and MDA and inhibited SOD and CAT activities. By RT-qPCR, the transcription levels of four cardiac development-related marker genes were significantly up-regulated except for gata4. Besides, miR-144 was identified as a regulatory molecule of TCS-induced cardiac defects by integrating analyses of artificial intervene expression and RNA-Seq data. Interestingly, the target genes of miR-144 were found and interacted with the above marker genes through constructing protein-protein interaction networks. After intervening the expression of miR-144 by microinjecting and activating Wnt pathway by an agonist BML-284, we confirmed that up-regulated miR-144 suppressed the expression of angiogenesis-related genes and negatively regulated Wnt pathway, further triggering angiogenesis disorders and cardiac phenotypic malformation. These findings unravel the underlying molecular mechanisms regarding TCS-induced cardiac development toxicity, and contribute to early warning and risk management of TCS.

The valence state of iron-based nanomaterials determines the ferroptosis potential in a zebrafish model

Many nanomaterials containing different valences of iron have been designed for applications in biomedicine, energy, catalyzers, nanoenzymes, and so on. However, the toxic effects of the valence state of iron in iron-based nanomaterials are still unclear. Here, three different-valence iron-based nanomaterials (nFe@Fe3O4, nFe3O4 and nFe2O3) were synthesized and exposed to zebrafish embryos and mammalian cardiomyocytes. All of them induced ferroptosis along with an increase in valence through iron overload and the Fenton reaction. Specifically, we exposed Tg (cmlc2:EGFP) zebrafish to the three iron-based nanomaterials and found that nFe@Fe3O4 treatments led to enlarged ventricles, while nFe3O4 and nFe2O3 increased atrial size, which was consistent with the results from hematoxylin-eosin staining and in situ hybridization. Moreover, we used ferroptosis inhibitors (ferrostatin-1 or deferoxamine) to treat zebrafish along with nanoparticles exposure and found that the cardiac developmental defects caused by nFe3O4 and nFe2O3, but not nFe@Fe3O4, could be completely rescued by ferroptosis inhibitors. We further found that nFe@Fe3O4, rather than nFe3O4 and nFe2O3, reduced the dissolved oxygen in the medium, which resulted in hypoxia and acceleration of heart tube formation and ventricular enlargement, and both were fully rescued by oxygen donors combined with ferroptosis inhibitors. Consistently, these findings were also observed in mammalian cardiomyocytes. In summary, our study demonstrates that the valence state of iron-based nanomaterials determines the ferroptosis potential. Our study also clarifies that high-valence iron-based nanomaterials induce an enlarged atrium via ferroptosis, while low-valence ones increase the ventricular size through both hypoxia and ferroptosis, which is helpful to understand the potential adverse effects of different valences of iron-based nanomaterials on environmental health and assure the responsible and sustainable development of nanotechnology.

Transcriptional responses of fluxapyroxad-induced dysfunctional heart in zebrafish (Danio rerio) embryos.

Fluxapyroxad (FLU) is a succinate dehydrogenase inhibitor (SDHI) fungicide used in controlling crop diseases. Potential toxicity to aquatic organisms is not known. We exposed zebrafish to 1, 2, and 4 μM FLU for 3 days. The embryonic zebrafish showed developmental cardiac defects, including heart malformation, pericardial edema, and heart rate reduction. Compared with the controls, cardiac-specific transcription factors (nkx2.5, myh7, myl7, and myh6) exhibited dysregulated expression patterns after FLU treatment. We next used transcriptome and qRT-PCR analyses to explore the molecular mechanism of FLU cardiotoxicity. The transcriptome analysis and interaction network showed that the downregulated genes were enriched in calcium signaling pathways, adrenergic signaling in cardiomyocytes, and cardiac muscle contraction. FLU exposure repressed the cardio-related calcium signaling pathway, associated with apoptosis in the heart and other manifestations of cardiotoxicity. Thus, the findings provide valuable evidence that FLU exposure causes disruption of cardiac development in zebrafish embryos. Our findings will help to promote a better understanding of the toxicity mechanisms of FLU and act as a reference to explore the rational use and safety of FLU in agriculture.

MO047: Biallelic pathogenic variants in ROBO1 associate with syndromic CAKUT

Abstract BACKGROUND AND AIMS Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. METHOD We collected phenotypic data of all participants. Genetic analysis was conducted by either exome or whole genome sequencing, respectively. We used Sanger sequencing for variant confirmation and segregation analysis and performed in-silico analysis of identified missense variants. In an ENU-induced mouse model, we examined mice with the mutation Robo1Ile270Thr/Ile270Thr. RESULTS By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses from Germany, France, Turkey, Japan and the USA with biallelic truncating or combined missense and truncating variants in ROBO1. Renal and genitourinary manifestation included unilateral or bilateral renal agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation and increased renal echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects (Figure 1). By in-silico analysis, we determined the functional significance of identified missense variants and observed absence of renal ROBO1 expression in both human and murine mutant tissues (Figure 2). CONCLUSION This study describes six live-born and two non-viable individuals with syndromic CAKUT due to probably deleterious ROBO1 variants. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. In conclusion, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Conversely, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for renal involvement is indicated.

Biallelic pathogenic variants in roundabout guidance receptor 1 associate with syndromic congenital anomalies of the kidney and urinary tract

Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses with biallelic truncating or combined missense and truncating variants in ROBO1. Kidney and genitourinary manifestation included unilateral or bilateral kidney agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation, and increased kidney echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects. By in silico analysis, we determined the functional significance of identified missense variants and observed absence of kidney ROBO1 expression in both human and murine mutant tissues. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of the kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. Thus, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Hence, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for kidney involvement is indicated.

Lenvatinib induces cardiac developmental toxicity in zebrafish embryos through regulation of Notch mediated-oxidative stress generation.

Due to an increasing number of abused drugs dumped into the wastewater, more and more drugs are detected in the water environment, which may affect the survival of aquatic organisms. Lenvatinib is a multi-targeted tyrosine kinase inhibitor, and is clinically used to treat differentiated thyroid cancer, renal epithelial cell carcinoma and liver cancer. However, there are few reports on the effects of lenvatinib in embryos development. In this study, zebrafish embryos were used to evaluate the effect of lenvatinib on cardiovascular development. Well-developed zebrafish embryos were selected at 6h post fertilization (hpf) and exposed to 0.05 mg/L, 0.1mg/L and 0.2mg/L lenvatinib up to 72 hpf. The processed embryos demonstrated cardiac edema, decreased heart rate, prolonged SV-BA distance, inhibited angiogenesis, and blocked blood circulation. Lenvatinib caused cardiac defects in the whole stage of cardiac development and increased the apoptosis of cardiomyocyte. Oxidative stress in the processed embryos was accumulated and inhibiting oxidative stress could rescue cardiac defects induced by lenvatinib. Additionally, we found that lenvatinib downregulated Notch signaling, and the activation of Notch signaling could rescue cardiac developmental defects and downregulate oxidative stress level induced by lenvatinib. Our results suggested that lenvatinib might induce cardiac developmental toxicity through inducing Notch mediated-oxidative stress generation, raising concerns about the harm of exposure to lenvatinib in aquatic organisms.

The role of polypeptide PDTLN1 in suppression of PI3K/AKT signaling causes cardiogenetic disorders in vitro and in vivo

AimsA new polypeptide, PDTLN1, derived from the human Talin-1 protein, which is highly expressed in both myocardial tissue and maternal peripheral blood of aborted fetuses with congenital heart disease (CHD). However, its role in cardiac developmental disorders has not been disclosed till now. In the present study, we aim to assess the functions of PDTLN1 in heart development of zebrafish and cellular viability, proliferation, and apoptosis of P19 cells. Main methodsCellular viability was assessed by Cell Counting Kit-8, the EdU Kit was used to evaluate cellular proliferation, and apoptosic rate of P19 was examined using FITC Annexin-V staining followed by flow cytometry. The zebrafish embryos were divided into three groups: PEP group and NC group were microinjected with polypeptides, WT group without any intervention. The protein expression of PI3K/AKT were evaluated by western blotting. Key findingsPDTLN1 could suppress the proliferation, and facilitate apoptosis. PDTLN1 caused abnormal heart development of zebrafish embryos and the PDTLN1 (50 μM)-injected group showed an aberrant expression pattern of vmhc, amhc and cmlc2. Compared to the CTL group and SC79 group of P19 cells, the PDTLN1 group had a lower phosphorylated PI3K/AKT proteins level, decreased cellular viability and lower proliferation activity. SignificancePDTLN1 caused cardiac developmental defects in zebrafish, inhibited cellular viability, proliferation, and promoted apoptosis of P19 cells via suppressing the PI3K/AKT signaling pathway. Our findings provide a fresh perspective on the functional mechanism of human-derived peptides and may promote novel diagnostic biomarkers detection and therapeutic targets in CHD.

Effects of low-concentration glyphosate and aminomethyl phosphonic acid on zebrafish embryo development

Glyphosate (GLY) is the most widely used broad-spectrum, non-selective herbicide in the world, whose main degradation product is aminomethyl phosphonic acid (AMPA). Because of long-term and large-scale use, residual GLY and AMPA in the environment pose great environmental and human health threats. The purpose of this study is to evaluate the effects and mechanism of residual low-concentrations of GLY and AMPA in the environment on the development of zebrafish embryos. Zebrafish embryos were exposed to 0, 1, 10, 100, and 700 ng·mL−1 GLY and AMPA for 72 h (from 2 to 74 h post-fertilization). With increasing exposure dose, heart rates of both embryos and larvae showed a rising trend and obvious arrhythmia appeared. Defects in cardiac development and function of zebrafish juveniles may be related to altered transcription levels of cardiac development genes (TBX5, NKX2.5, BMP4) and apoptosis genes (Bcl-2, Bax). In addition, pericardial edema and bone deformation of zebrafish embryos may be caused by inhibition of Na+/K+-ATPase and Ca2+-ATPase after exposure to GLY and AMPA. The present results demonstrated that at typical environmental residual concentrations of GLY and AMPA had similar developmental toxicity in zebrafish embryos.

Inhibition of the ­ miR‐200 family results in cardiac developmental defects

Cardiac development is a multifaceted process involving a plethora of genes and pathways.Errors in cardiac development are classified under the umbrella of Congenital Cardiovascular Defects (CCDs), which occur in 6.9 of 1000 live births, making CCDs the most common birth defect.microRNAs (miRs) have been shown to contribute to proper cardiac development by regulating cardiomyocyte differentiation and mature cardiomyocyte function. Furthermore, ectopic expression of miRs have been found in patients with CCDs. In recent years the miR-200 family, has been proposed to play a critical role in cardiac development and CCDs. Though miRs have a proposed role in cardiac development, current methods of miR inhibition cannot be studied ­in vivo. Our lab has been able to overcome this obstacle by utilizing the Plasmid-based MicroRNA Inhibitor System (PMIS) where we are able to inhibit the miR-200 family in vivo. Analysis of hearts from PMIS miR-200 inhibitor mice, show a range of defects including ventricle wall development and abnormal cardiomyocyte maturation.PMIS inhibitor mice present with imperfections in trabecular and compact myocardium development.Moreover, ultrasound analysis of PMIS inhibitor mice displayed a slower heart rate and hemorrhaging in the pericardial cavity.Immunofluorescent analysis of cardiac troponin showed PMIS cardiomyocytes have expression of sarcomere bands at earlier stages in development.Additionally, these mice have defects in the both non-muscle layers of the heart, the epicardium and endocardium, which appear to have lack of EMT into the myocardium.Our model offers a unique and novel approach to studying the precise role of the ­miR-200 family in cardiac development and CCDs progression.