Affinity chromatography reveals direct binding of the GATA4-NKX2-5 interaction inhibitor (3i-1000) with GATA4.

Heart failure is a serious medical condition with a poor prognosis. Current treatments can only help manage the symptoms and slow the progression of heart failure. However, there is currently no cure to prevent and reverse cardiac remodeling. Transcription factors are in a central role in various cellular processes, and in the heart, GATA4 and NKX2-5 transcription factors mediate hypertrophic responses and remodeling. We have identified compounds that modulate the synergistic interaction of GATA4 and NKX2-5 and shown that the most promising compound (1, 3i-1000) is cardioprotective in vitro and in vivo. However, direct evidence of its binding site and mechanism of action has not been available. Due to the disordered nature of transcription factors, classical target engagement approaches cannot be utilized. Here, we synthesized a small-molecule ligand-binding pulldown probe of compound 1 to utilize affinity chromatography alongside CETSA, AlphaScreen, and molecular modeling to study ligand binding. These results provide the first evidence of direct physical binding of compound 1 selectively to GATA4. While developing drugs that target transcription factors presents challenges, advances in technologies and knowledge of intrinsically disordered proteins enable the identification of small molecules that can selectively target transcription factors.

The generation and validation of two NKX2-5 fluorescent reporter human embryonic stem cell lines: UMANe002-A-1 and UMANe002-A-2.

The transcription factor NKX2-5 is a highly conserved master regulator of heart development which is widely expressed in cardiac progenitors and cardiomyocytes. Fluorescent reporters of NKX2-5 that minimally perturb normal protein expression can enable the identification, quantification and isolation of NKX2-5-expressing cells in a normal physiological state. Here we report the generation of two new hESC lines with eGFP inserted upstream (5') or downstream (3') of NKX2-5, linked by a cleavable T2A peptide. These complementary reporters produce a robust fluorescent signal in cardiac cells and have wide utility particularly for research on developmental biology and disease modelling.

Prioritizing cardiovascular disease-associated variants altering NKX2-5 and TBX5 binding through an integrative computational approach.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies have mapped >90% of CVD-associated variants within the noncoding genome, which can alter the function of regulatory proteins, such as transcription factors (TFs). However, due to the overwhelming number of single-nucleotide polymorphisms (SNPs) (>500,000) in genome-wide association studies, prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1535 CVD-associated SNPs within TF footprints and putative cardiac enhancers plus 14,218 variants in linkage disequilibrium with genotype-dependent gene expression in cardiac tissues. Using ChIP-seq data from two cardiac TFs (NKX2-5 and TBX5) in human-induced pluripotent stem cell-derived cardiomyocytes, we trained a large-scale gapped k-mer SVM model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay. Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and are in cardiac tissue eQTLs. All five variants altered NKX2-5 and TBX5 DNA binding. We present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro analysis.

The multi-lineage transcription factor ISL1 controls cardiomyocyte cell fate through interaction with NKX2.5.

Congenital heart disease often arises from perturbations of transcription factors (TFs) that guide cardiac development. ISLET1 (ISL1) is a TF that influences early cardiac cell fate, as well as differentiation of other cell types including motor neuron progenitors (MNPs) and pancreatic islet cells. While lineage specificity of ISL1 function is likely achieved through combinatorial interactions, its essential cardiac interacting partners are unknown. By assaying ISL1 genomic occupancy in human induced pluripotent stem cell-derived cardiac progenitors (CPs) or MNPs and leveraging the deep learning approach BPNet, we identified motifs of other TFs that predicted ISL1 occupancy in each lineage, with NKX2.5 and GATA motifs being most closely associated to ISL1 in CPs. Experimentally, nearly two-thirds of ISL1-bound loci were co-occupied by NKX2.5 and/or GATA4. Removal of NKX2.5 from CPs led to widespread ISL1 redistribution, and overexpression of NKX2.5 in MNPs led to ISL1 occupancy of CP-specific loci. These results reveal how ISL1 guides lineage choices through a combinatorial code that dictates genomic occupancy and transcription.

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

BACKGROUND: Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. METHODS: We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Nr2f1a maintains atrial nkx2.5 expression to repress pacemaker identity within venous atrial cardiomyocytes of zebrafish.

Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5, a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in nr2f1a mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative nkx2.5 enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.

Activation of Nkx2.5 transcriptional program is required for adult myocardial repair.

The cardiac developmental network has been associated with myocardial regenerative potential. However, the embryonic signals triggered following injury have yet to be fully elucidated. Nkx2.5 is a key causative transcription factor associated with human congenital heart disease and one of the earliest markers of cardiac progenitors, thus it serves as a promising candidate. Here, we show that cardiac-specific RNA-sequencing studies reveal a disrupted embryonic transcriptional profile in the adult Nkx2.5 loss-of-function myocardium. nkx2.5-/- fish exhibit an impaired ability to recover following ventricular apex amputation with diminished dedifferentiation and proliferation. Complex network analyses illuminate that Nkx2.5 is required to provoke proteolytic pathways necessary for sarcomere disassembly and to mount a proliferative response for cardiomyocyte renewal. Moreover, Nkx2.5 targets embedded in these distinct gene regulatory modules coordinate appropriate, multi-faceted injury responses. Altogether, our findings support a previously unrecognized, Nkx2.5-dependent regenerative circuit that invokes myocardial cell cycle re-entry, proteolysis, and mitochondrial metabolism to ensure effective regeneration in the teleost heart.

Computational profiling of hiPSC-derived heart organoids reveals chamber defects associated with NKX2-5 deficiency.

Heart organoids have the potential to generate primary heart-like anatomical structures and hold great promise as in vitro models for cardiac disease. However, their properties have not yet been fully studied, which hinders their wide spread application. Here we report the development of differentiation systems for ventricular and atrial heart organoids, enabling the study of heart diseases with chamber defects. We show that our systems generate chamber-specific organoids comprising of the major cardiac cell types, and we use single cell RNA sequencing together with sample multiplexing to characterize the cells we generate. To that end, we developed a machine learning label transfer approach leveraging cell type, chamber, and laterality annotations available for primary human fetal heart cells. We then used this model to analyze organoid cells from an isogeneic line carrying an Ebstein's anomaly associated genetic variant in NKX2-5, and we successfully recapitulated the disease's atrialized ventricular defects. In summary, we have established a workflow integrating heart organoids and computational analysis to model heart development in normal and disease states.

Tissue-specific multi-omics analysis of atrial fibrillation.

Genome-wide association studies (GWAS) for atrial fibrillation (AF) have uncovered numerous disease-associated variants. Their underlying molecular mechanisms, especially consequences for mRNA and protein expression remain largely elusive. Thus, refined multi-omics approaches are needed for deciphering the underlying molecular networks. Here, we integrate genomics, transcriptomics, and proteomics of human atrial tissue in a cross-sectional study to identify widespread effects of genetic variants on both transcript (cis-eQTL) and protein (cis-pQTL) abundance. We further establish a novel targeted trans-QTL approach based on polygenic risk scores to determine candidates for AF core genes. Using this approach, we identify two trans-eQTLs and five trans-pQTLs for AF GWAS hits, and elucidate the role of the transcription factor NKX2-5 as a link between the GWAS SNP rs9481842 and AF. Altogether, we present an integrative multi-omics method to uncover trans-acting networks in small datasets and provide a rich resource of atrial tissue-specific regulatory variants for transcript and protein levels for cardiovascular disease gene prioritization.

The G4 resolvase RHAU regulates ventricular trabeculation and compaction through transcriptional and post-transcriptional mechanisms.

The G-quadruplex (G4) resolvase RNA helicase associated with AU-rich element (RHAU) possesses the ability to unwind G4 structures in both DNA and RNA molecules. Previously, we revealed that RHAU plays a critical role in embryonic heart development and postnatal heart function through modulating mRNA translation and stability. However, whether RHAU functions to resolve DNA G4 in the regulation of cardiac physiology is still elusive. Here, we identified a phenotype of noncompaction cardiomyopathy in cardiomyocyte-specific Rhau deletion mice, including such symptoms as spongiform cardiomyopathy, heart dilation, and death at young ages. We also observed reduced cardiomyocyte proliferation and advanced sarcomere maturation in Rhau mutant mice. Further studies demonstrated that RHAU regulates the expression levels of several genes associated with ventricular trabeculation and compaction, including the Nkx2-5 and Hey2 that encode cardiac transcription factors of NKX2-5 and Hey2, and the myosin heavy chain 7 (Myh7) whose protein product is MYH7. While RHAU modulates Nkx2-5 mRNA and Hey2 mRNA at the post-transcriptional level, we uncovered that RHAU facilitates the transcription of Myh7 through unwinding of the G4 structures in its promoter. These findings demonstrated that RHAU regulates ventricular chamber development through both transcriptional and post-transcriptional mechanisms. These results contribute to a knowledge base that will help to understand the pathogenesis of diseases such as noncompaction cardiomyopathy.

lncENST Suppress the Warburg Effect Regulating the Tumor Progress by the Nkx2-5/ErbB2 Axis in Hepatocellular Carcinoma.

The therapeutic efficacy of radiofrequency ablation (RFA) against liver cancer is often limited by proliferation and metastasis of residual tumor cells. These phenomena are closely associated with the Warburg effect, wherein ErbB2 is activated. While RFA inhibits the Warburg effect of residual tumor cells at the early stage, the specific mechanisms remain unclear. We explored the regulatory relationship between the long noncoding RNA ENST00000570843.1 (lncENST) and ErbB2 using lentiviral transfection of lncENST and ErbB2 overexpression/interference vectors in in vitro and in vivo models of hepatocellular carcinoma in the presence of sublethal heat at 50°C. ErbB2-mediated Warburg effect was suppressed by lncENST, as manifested by reduced glucose uptake and lactic acid production in SMMC-7721 cells. lncENST also increased tumor apoptosis and inhibited tumor progression in nude Balb/c mice for up to 28 days after RFA. Additionally, we predicted through bioinformatic analysis that the promoter of ErbB2 binds to the transcription factor Nkx2-5, resulting in a negative regulatory effect. This speculation was confirmed by chromatin immunoprecipitation of the Nkx2-5 protein and ErbB2, indicating that ErbB2 transcription was curbed by Nkx2-5. We propose that lncENST downplays the Warburg effect in residual tumor cells by downregulating ErbB2 via Nkx2-5 activation. This study is aimed at providing molecular targets that can prevent residual tumor cell proliferation after RFA, with clinical significance in hepatocellular carcinoma treatment.

Stage-specific regulation of DNA methylation by TET enzymes during human cardiac differentiation.

Changes in DNA methylation are associated with normal cardiogenesis, whereas altered methylation patterns can occur in congenital heart disease. Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA demethylation. Here, we characterize stage-specific methylation dynamics and the function of TETs during human cardiomyocyte differentiation. Human embryonic stem cells (hESCs) in which all three TET genes are inactivated fail to generate cardiomyocytes (CMs), with altered mesoderm patterning and defective cardiac progenitor specification. Genome-wide methylation analysis shows TET knockout causes promoter hypermethylation of genes encoding WNT inhibitors, leading to hyperactivated WNT signaling and defects in cardiac mesoderm patterning. TET activity is also needed to maintain hypomethylated status and expression of NKX2-5 for subsequent cardiac progenitor specification. Finally, loss of TETs causes a set of cardiac structural genes to fail to be demethylated at the cardiac progenitor stage. Our data demonstrate key roles for TET proteins in controlling methylation dynamics at sequential steps during human cardiac development.

Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Noncanonical Notch signals have opposing roles during cardiac development.

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.

H3K9me2 regulates early transcription factors to promote mesenchymal stem­cell differentiation into cardiomyocytes.

Studies have shown that histone H3 at lysine 9 (H3K9me2) is an important epigenetic modifier of embryonic development, cell reprogramming and cell differentiation, but its specific role in cardiomyocyte formation remains to be elucidated. The present study established a model of 5­Azacytidine­induced differentiation of rat bone mesenchymal stem cells (MSCs) into cardiomyocytes and, on this basis, investigated the dimethylation of H3K9me2 and its effect on cardiomyocyte formation by knockdown of H3K9me2 methylase, euchromatic histone­lysine N­methyltransferase 2 (G9a) and H3K9me2 lysine demethylase 3A (KDM3A). The results demonstrated that, in comparison with the normal induction process, the knockdown of G9a could significantly reduce the H3K9me2 level of the MSCs in the induced model. Reverse transcription­quantitative (RT­q) PCR demonstrated that the expression of cardiac troponin T(cTnT) was significantly increased. In addition, flow cytometry demonstrated that the proportion of cTnT­positive cells was significantly increased on day 21. With the knockdown of KDM3A, the opposite occurred. In order to explore the specific way of H3K9me2 regulating cardiomyocyte formation, western blotting and RT­qPCR were used to detect the expression of key transcription factors including GATA binding protein 4 (GATA­4), NK2 Homeobox 5 (Nkx2.5) and myocyte enhancer factor 2c (MEF2c) during cardiomyocyte formation. The decrease of H3K9me2 increased the expression of transcription factors GATA­4, Nkx2.5 and MEF2c in the early stage of myocardial development while the increase of H3K9me2 inhibited the expression of those transcription factors. Accordingly, it was concluded that H3K9me2 is a negative regulator of cardiomyocyte formation and can participate in cardiomyocyte formation by activating or inhibiting key transcription factors of cardiomyocytes, which will lay the foundation for the optimized induction efficiency of cardiomyocytes in in vitro and clinical applications.

TBX3 induces biased differentiation of human induced pluripotent stem cells into cardiac pacemaker-like cells.

BACKGROUND: TBX3 plays a critical role in the formation of the sinoatrial node (SAN) during embryonic heart development. However, the contribution of TBX3 in driving the differentiation of human induced pluripotent stem cells (hiPSC)into pacemaker cells remains to be explored. RESULTS: Using the pan-cardiomyocyte differentiation protocol of human induced pluripotent stem cells (hiPSC)，TBX3 gene was introduced into the differentiating hiPSC on day 5 post-differentiation, and the differentiation of pacemaker-like cardiomyocytes was evaluated on day 21. The results showed that TBX3 significantly induced biased differentiation of hiPSC into pacemaker-like cells as judged by significantly increased expression of SAN-specific marker gene, SHOX2, and slightly decreased expression of SAN-detrimental transcription factor, NKX2-5. CONCLUSION: Our results suggest that TBX3 plays an important role in driving the differentiation of hiPSC into pacemaker-like cells, and manipulation of TBX3 expression during pan-cardiomyocyte differentiation may lead to the development of therapeutic pacemaker cells.

Cardioids reveal self-organizing principles of human cardiogenesis.

Organoids capable of forming tissue-like structures have transformed our ability to model human development and disease. With the notable exception of the human heart, lineage-specific self-organizing organoids have been reported for all major organs. Here, we established self-organizing cardioids from human pluripotent stem cells that intrinsically specify, pattern, and morph into chamber-like structures containing a cavity. Cardioid complexity can be controlled by signaling that instructs the separation of cardiomyocyte and endothelial layers and by directing epicardial spreading, inward migration, and differentiation. We find that cavity morphogenesis is governed by a mesodermal WNT-BMP signaling axis and requires its target HAND1, a transcription factor linked to developmental heart chamber defects. Upon cryoinjury, cardioids initiated a cell-type-dependent accumulation of extracellular matrix, an early hallmark of both regeneration and heart disease. Thus, human cardioids represent a powerful platform to mechanistically dissect self-organization, congenital heart defects and serve as a foundation for future translational research.

Prediction of single-cell mechanisms for disease progression in hypertrophic remodelling by a trans-omics approach.

Heart failure is a heterogeneous disease with multiple risk factors and various pathophysiological types, which makes it difficult to understand the molecular mechanisms involved. In this study, we proposed a trans-omics approach for predicting molecular pathological mechanisms of heart failure and identifying marker genes to distinguish heterogeneous phenotypes, by integrating multiple omics data including single-cell RNA-seq, ChIP-seq, and gene interactome data. We detected a significant increase in the expression level of natriuretic peptide A (Nppa), after stress loading with transverse aortic constriction (TAC), and showed that cardiomyocytes with high Nppa expression displayed specific gene expression patterns. Multiple NADH ubiquinone complex family, which are associated with the mitochondrial electron transport system, were negatively correlated with Nppa expression during the early stages of cardiac hypertrophy. Large-scale ChIP-seq data analysis showed that Nkx2-5 and Gtf2b were transcription factors characteristic of high-Nppa-expressing cardiomyocytes. Nppa expression levels may, therefore, represent a useful diagnostic marker for heart failure.

Bacterial Lipase Neutralized Toxicity of Lipopolysaccharide on Chicken Embryo Cardiac Tissue.

It has been shown that near all organs, especially the cardiovascular system, are affected by bacterial lipopolysaccharide via the activation of Toll-like receptor signaling pathways. Here, we tried to find the blunting effect of bacterial lipase on lipopolysaccharide (LPS)-induced cardiac tissue toxicity in chicken embryos. 7-day fertilized chicken eggs were divided randomly into different groups as follows; Control, Normal Saline, LPS (0.1, 0.5 and 1 mg/kbw), and LPS (0.1, 0.5 and 1 mg/kbw) plus 5 mg/ml Lipase. On day 17, the hearts were sampled. The expression of genes such as GATA4, NKX2.5, EGFR, TRIF, and NF-ƙB was monitored using real-time PCR analysis. Using western blotting, we measured NF-ƙB protein level. Total antioxidant capacity, glutathione peroxidase, and Catalase activity were also studied. Microvascular density and anterior wall thickness were monitored in histological samples using H&E staining. High dose of LPS (1 mg/kbw) increased the expression of TRIF but not NF-ƙB compared to the control group (p < 0.05). We found a statistically significant reduction in groups that received LPS + Lipase compared to the control and LPS groups (p < 0.05). Western blotting revealed that the injection of Lipase could reduce LPS-induced NF-ƙB compared to the control group (p < 0.05). The expression of GATA4, NKx2.5, and EGFR was not altered in the LPS group, while the simultaneous application of LPS and Lipase significantly reduced GATA4, NKx2.5, and EGFR levels below the control (p < 0.05). We found non-significant differences in glutathione peroxidase, and Catalase activity in all groups (p > 0.05), while total antioxidant capacity was increased in groups that received LPS + Lipase. Anterior wall thickness was diminished in LPS groups and the use of both lipase and LPS returned near-to-control values (p < 0.05). Despite a slight increase in microvascular density, we found statistically non-significant differences in all groups (p > 0.05). Bacterial lipase reduces detrimental effects of LPS on chicken embryo heart induced via Toll-like receptor signaling pathway.

Protective Effects of Spermidine and Melatonin on Deltamethrin-Induced Cardiotoxicity and Neurotoxicity in Zebrafish.

Increased application of the pyrethroid insecticide deltamethrin has adverse effects on the cardiac system and neurobehavior on the non-target organisms, which has raised the public's attention. Because of spermidine and melatonin considered to have cardioprotective and neuroprotective characteristics, zebrafish were utilized as the model organism to explore the protective effects of spermidine and melatonin against deltamethrin-induced toxicity. We tested the neurobehavior of zebrafish larvae through a rest/wake behavior assay, and evaluated the levels of the expression of Scn5lab, gata4, nkx2.5, hcrt, hcrtr, and aanat2 by qRT-PCR. Besides that cmlc2 was evaluated by whole-mount in situ hybridization. Results have shown that compared with control group, 0.025 mg/L deltamethrin could significantly disturb the cardiac development, downregulating the expression of Scn5lab and transcriptional factors gata4 and nkx2.5, disturbing cardiac looping, resulting in defects in cardiac morphology and function. Moreover, deltamethrin could alter the expression levels of rest/wake genes and cause hyperactivity in zebrafish larvae. Besides, compared with deltamethrin group, the exogenous 0.01 mg/L spermidine and 0.232 mg/L melatonin could significantly rescue the adverse effects of deltamethrin on the cardiac system and neurobehavior in zebrafish. This indicated that spermidine and melatonin have neuroprotective and cardioprotective effects against deltamethrin-induced adverse effects in zebrafish.