Cardiac Developmental Defects Research Articles

The role of histone methyltransferase SMYD4 in cardioprogenitor cell specification and differentiation

SMYD4 belongs to a SET and MYND domain-containing lysine methyltransferase. In zebrafish, smyd4 is maternally and ubiquitously expressed in early embryos, and becomes more restricted in the developing cardiovascular system at 48 hours post-fertilization (hpf). We have generated a smyd4 knockout zebrafish line. The maternal and zygotic knockout mutants (zmsmyd4-/-) demonstrate that majority of mutant embryos (62%) halt the development at late gastrula (8 hpf) and about 20% mutant embryos can survive to later stage, showing a series of cardiac developmental defects, including left/right patterning and heart looping defect, and hypoplastic ventricle. To determine the underlying molecular events, we performed transcriptomic analysis of zmsmyd4-/- embryos at blastula and gastrula stages. Our data demonstrate that the genes involved in the mesendodermal and cardioprogenitor cell specification and differentiation are significantly altered; brachyury, gata5, mespbb, mespaa, mespab, and nkx2.5 are downregulatedin zmsmyd4-/- embryos. Consistent with this finding, the key signaling pathways that regulate early embryonic pattern, such as TGFβ/NODAL/BMP signaling and Wnt signaling, are also significantly altered, suggesting that smyd4 is an important epigenetic regulator in mesendodermal and cardioprogenitor cells specification and differentiation. Additionally, our biochemical analyses suggest that SMYD4 is a positive epigenetic regulator for transcription by promoting trimethylation of H3K4 and inhibiting HDAC1. Importantly, two rare genetic variants of SMYD4 are found in CHDs. Both biochemical and functional analyses indicated that SMYD4(G345D) was highly pathogenic. Taken together, our data suggest that smyd4 is a key epigenetic regulator of cardiogenesis, likely via its regulation of early cardiogenic lineage specification and differentiation.

MRNA decapping factor Dcp1a is essential for embryonic growth in mice

mRNA decapping is a critical step in posttranscriptional regulation of gene expression in eukaryotes. Although Dcp1a is a well characterized and widely conserved mRNA decapping factor, little is known about its physiological function. To extend our understanding of Dcp1a function in vivo, we employed a transgenic rescue strategy to produce Dcp1a-deficient mice using the CRISPR/Cas9 system. This approach arrowed us to generate heterozygous Dcp1a mice and define the phenotype of Dcp1a-deficient embryos. We found that expression of Dcp1a protein, which is detectable in most mouse tissues, was developmentally regulated through embryonic growth, and that depletion of the Dcp1a gene resulted in embryonic lethality around embryonic day 10.5 (E10.5) concomitant with massive growth retardation and cardiac developmental defects. Moreover, the embryonic lethality was fully rescued by transgenic expression of exogenous human Dcp1a. Together, our results suggest that Dcp1a is required for embryonic growth.

Multifaceted Function of Myosin-18, an Unconventional Class of the Myosin Superfamily.

Myosin is a diverse superfamily of motor proteins responsible for actin-based motility and contractility in eukaryotic cells. Myosin-18 family, including myosin-18A and myosin-18B, belongs to an unconventional class of myosin, which lacks ATPase motor activity, and the investigations on their functions and molecular mechanisms in vertebrate development and diseases have just been initiated in recent years. Myosin-18A is ubiquitously expressed in mammalian cells, whereas myosin-18B shows strong enrichment in striated muscles. Myosin-18 family is important for cell motility, sarcomere formation, and mechanosensing, mostly by interacting with other cytoskeletal proteins and cellular apparatus. Myosin-18A participates in several intracellular transport processes, such as Golgi trafficking, and has multiple roles in focal adhesions, stress fibers, and lamellipodia formation. Myosin-18B, on the other hand, participates in actomyosin alignment and sarcomere assembly, thus relating to cell migration and muscle contractility. Mutations of either Myo18a or Myo18b cause cardiac developmental defects in mouse, emphasizing their crucial role in muscle development and cardiac diseases. In this review, we revisit the discovery history of myosin-18s and summarize the evolving understanding of the molecular functions of myosin-18A and myosin-18B, with an emphasis on their separate yet closely related functions in cell motility and contraction. Moreover, we discuss the diseases tightly associated with myosin-18s, especially cardiovascular defects and cancer, as well as highlight the unanswered questions and potential future research perspectives on myosin-18s.

Effects of miR-155 on High Glucose Induced-Cardiac Developmental Defects of Zebrafish Embryos

Background: Gestational diabetes mellitus (GDM) is known to have a teratogenic effect on heart development. Former studies determined that miR-155 is elevated in GDM patients. In the present study, we explored the potential role of miR-155 on high glucose-induced cardiac developmental defects. Methods: Serum miR-155 of GDM patients was determined by using ELISA. Fetal cardiac characteristics were collected by echocardiography. Zebrafish embryos were exposed to 2% D-Glucose in a fluctuating manner. The expression levels of miR-155, Ets1 and cardiac specific genes were evaluated by real-time PCR or western blot. Reactive oxygen species (ROS) were detected by DCFH-DA. Findings: High glucose exposure in zebrafish embryos altered the morphology of the heart, increased the expression of miR-155 and cardiac specific genes. Upregulation of miR-155 activated Igf1-Akt-Gsk3β pathway by targeting Ets1 and increased the production of ROS and may thereby exert teratogenic effect on cardiac development. Knockdown of miR-155 could rescue high glucose-induced zebrafish heart developmental defects. Besides, miR-155 levels are increased in serum from GDM patients and are correlated with fetal cardiac structural changes. Moreover, loss of miR-155 blocked Igf1 survival pathway and induced apoptosis and may thus cause zebrafish cardiac developmental defects. Interpretation: miR-155 is a key molecule for heart development and is involved in high glucose-induced cardiac malformation and it might be a novel biomarker as well as a potential drug target of high glucose-induced cardiac defects. Funding：This work was supported by grants from: (1) the Youth Program of National Natural Science Foundation (NSF)of China (81200125) to LY; (2) the Priority Academic Program Development (PAPD) and the Key Project of Natural Science Foundation of the Higher Education Institutions (17KJA310001) to LY; (3) the Six Talent Peaks Project (STPP) of Jiangsu Province (2015-WSN-032) to ZXT. Declaration of Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Approval Statement: The studies involving human participants were reviewed and approved by Ethical Committee, Jiangsu province Hospital. The patients/participants provided their written informed consent to participate in this study. Zebrafish maintenance and procedures were conducted in accordance with the medical ethics committee of Nanjing Medical University. All experiments were approved by the medical ethics committee of Nanjing Medical University.

Zebrafish ambra1a and ambra1b Silencing Affect Heart Development.

In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)-regulated autophagy ambra1a and ambra1b, both required for the autophagic process and during development, encode the protein AMBRA1, a positive regulator of early steps of autophagosome formation. As transcripts for both genes are expressed during embryogenesis in the heart region, in this work, we investigated the effects of ambra1a and ambra1b knockdown on heart development by means of morpholino oligonucleotides (MOs). Silencing of the two proteins by MOs directed against the ATG translation initiation codon affects cardiac morphogenesis, resulting in a small, string-like heart with pericardial edema, whereas treatment with splice-blocking MOs does not lead to overt cardiac phenotypes, thus revealing the relevance of maternally supplied ambra1 transcripts for heart development. Co-injection of both ATG-MOs determines a more severe cardiac phenotype, with prominent pericardial edema. Whole-mount in situ hybridization (WMISH) for myosin light chain 7 (myl7), as well as ambra1 ATG-MO microinjection in zebrafish transgenic line expressing green fluorescent protein in the heart, revealed defects with the heart jogging process followed by imperfect cardiac looping. Moreover, WMISH of homeodomain transcription factor 2 isoform c (pitx2c) transcripts showed both bilateral and reversed pitx2c expression in morphants. The morphants' cardiac phenotypes were effectively rescued by co-injection of MOs with human AMBRA1 (hAMBRA1) messenger RNA (mRNA), pointing at the conservation of Ambra1 functions during evolution. Co-injections of ambra1 ATG-MOs with a hAMBRA1 mRNA mutated in the protein phosphatase 2a (PP2A) binding sites (hAMBRA1PXP) were not able to rescue the cardiac phenotypes, at the difference from wild-type hAMBRA1 mRNA, and treatment of zebrafish embryos with the specific PP2A inhibitor cantharidin resulted in similar developmental cardiac defects. These results suggest a critical role for AMBRA1 in vertebrate heart development, likely involving the binding site for the PP2A phosphatase.

RNA‐Binding Protein QKI is a critical pre‐RNA splicing regulator for cardiac development and function

RNA‐binding protein QKI (also known as Quaking) belongs to hnRNP K‐homology (KH)‐domain protein family and is implicated a novel regulator in pre‐RNA splicing. As previously shown, the QKI homolog how potentially regulates heart lumen formation and function in fruit fly. However, QKI’s function in mammalian heart development is largely unknown. We found that Qki specifically expressed in mouse developing hearts as early as E7.0 and this high‐level expression was maintained in the hearts to adulthood. Consistent with this finding, by using human embryonic stem cells (hESCs)‐cardiomyocyte differentiation system, QKI expression was found up‐regulated at Day 6, a critical time frame that cardiogenic progenitor transition into early cardiomyocytes. To determine the biological function of QKI in cardiogenesis, we generated hESCsQKIdef by using CRISPR/Cas9 gene editing technology and analyzed the physiological impact of QKI‐deficiency to cardiomyocyte differentiation and maturation. As expected, we did not find defect in pluripotency in hESCsQKIdef, but the contractile function was dramatically reduced in hESCsQKIdef‐derived cardiomyocytes. Based on scRNA‐seq and clustering analyses, there was no significant difference in the distribution of the clusters when compared hESCsQKIdef‐cardiomyocyte group to normal control group, suggesting that the cardiomyocyte differentiation was largely unaffected in hESCsQKIdef. However, scRNA‐seq demonstrated a handful of genes encoding sarcomere proteins were significantly altered in cardiomyocyte clusters, which included ACTN2, ACTA1, MYH7, and MYL2, etc., indicating a significant defect in cardiomyocyte maturation. Bulk RNA‐seq data not only further confirmed this notion, but also provided additional depth for detailed analysis on the QKI‐mediated pre‐RNA splicing events. In conjunction with QKI‐binding motif search and RNA immunoprecipitation (RIP) analysis, we were able to demonstrate that QKI was a key pre‐RNA splicing regulator for sets of genes involved in cardiac myofibrillogenesis and cardiac excitation‐contraction (E‐C) coupling, which ultimately impacted on cardiomyocyte maturation and function. Additional analysis of cardiac developmental defects in Qkibgeo mutant mice further validated that QKI was an indispensable splicing regulator in cardiovascular development. Our results not only revealed a novel mechanism by which QKI regulates pre‐mRNA splicing in cardiogenesis, but also implied that QKI was likely involved in the pathogenesis of certain forms of congenital heart defects and cardiomyopathies.Support or Funding InformationThis work was supported in part by National Institute of Health P01HL134599

Chromosome 15q13 microduplication in a fetus with cardiac rhabdomyoma: a case report

BackgroundCopy number variation (CNV) is a complex genomic rearrangement that has been linked to a large number of human diseases. Chromosome 15q13 microduplication is a rare form of CNV, which has been proved to be associated with multiple human disorders; however, the association between chromosome 15q13 microduplication and cardiac disorders has not been fully understood.Case presentationA fetus with fetal cardiac developmental defects was detected by Color Doppler ultrasound imaging; however, further chromosomal G-banding revealed no abnormal karyotype. Then, chromosomal microarray analysis (CMA) was performed and revealed a 1.8 Mb-duplication of the chromosome 15q13.2q13.3 region containing 7 genes (TRPM1, KLF13, OTUD7A, CHRNA7, FAN1, MIR211 and RAHGAP11A). Cardiac ultrasound follow-up displayed significant enlargement of the space-occupying lesion in the fetal heart with extension of the gestational age, and the space-occupying lesion was finally pathologically diagnosed as cardiac rhabdomyoma. Next-generation sequencing revealed no mutations in the TSC1 or TSC2 gene in the fetus, the mother or the father.ConclusionsThis is the first report to demonstrate the potential association between chromosome 15q13 microduplication and fetal cardiac rhabdomyoma. It is recommended that CMA be employed in fetuses with abnormal cardiac development diagnosed by routine cardiac color Doppler ultrasound imaging for early detection of congenital genetic abnormality, which may provide valuable information for prenatal diagnostic consultation and the decision on pregnancy termination.

Exposure to low-level metalaxyl impacts the cardiac development and function of zebrafish embryos

Metalaxyl is an anilide pesticide that is widely used to control plant diseases caused by Peronosporales species. In order to study the toxic effects, zebrafish embryos were exposed to metalaxyl at nominal concentrations of 5, 50 and 500 ng/L for 72 hr, and the cardiac development and functioning of larvae were observed. The results showed that metalaxyl exposure resulted in increased rates of pericardial edema, heart hemorrhage and cardiac malformation. The distance between the sinus venosus and bulbus arteriosus, stroke volume, cardiac output and heart rate were significantly increased in larvae exposed to 50 and 500 ng/L metalaxyl compared to solvent control larvae. Significant upregulation in the transcription of tbx5, gata4 and myh6 was observed in the 50 and 500 ng/L treatments, and that of nkx2.5 and myl7 was observed in the 5, 50 and 500 ng/L groups. These disturbances may be related to cardiac developmental and functional defects in the larvae. The activity of Na+/K+-ATPase and Ca2+-ATPase was significantly increased in zebrafish embryos exposed to 500 ng/L metalaxyl, and the mRNA levels of genes related to ATPase (atp2a11, atp1b2b, and atp1a3b) (in the 50 and 500 ng/L groups) and calcium channels (cacna1ab) (in the 500 ng/L group) were significantly downregulated; these changes might be associated with heart arrhythmia and functional failure.

Deletion of Pr72 causes cardiac developmental defects in Zebrafish.

The alpha regulator subunit B'' of protein phosphatase 2 (PPP2R3A), a regulatory subunit of protein phosphatase 2A (PP2A), was reported to present a special subcellular localization in cardiomyocytes and elevate in non-ischemia failing hearts. PPP2R3A has two transcriptions PR72 and PR130. PR72 acts as a negative regulator of the Wnt signaling cascade, while the Wnt signaling cascade plays a pivotal role in cardiac development. And PR130 was found to be involved in cardiac development of zebrafish in our previous study. Thus, to investigate the function of PR72 in heart, two stable pr72 knockout (KO) zebrafish lines were generated using Transcription Activator-Like Effector Nuclease (TALEN) technology. Homozygous pr72 KO fish struggled to survive to adulthood and exhibited cardiac developmental defects, including enlarged ventricular chambers, reduced cardiomyocytes and decreased cardiac function. And the defective sarcomere ultrastructure that affected mitochondria, I bands, Z lines, and intercalated disks was also observed. Furthermore, the abnormal heart looping was detected in mutants which could be rescued by injection with wild type pr72 mRNA. Additionally, it was found that Wnt effectors were elevated in mutants. Those indicated that deletion of pr72 in zebrafish interrupted cardiac development, probably through activation of the Wnt pathway.

CRELD1 is an evolutionarily-conserved maturational enhancer of ionotropic acetylcholine receptors.

The assembly of neurotransmitter receptors in the endoplasmic reticulum limits the number of receptors delivered to the plasma membrane, ultimately controlling neurotransmitter sensitivity and synaptic transfer function. In a forward genetic screen conducted in the nematode C. elegans, we identified crld-1 as a gene required for the synaptic expression of ionotropic acetylcholine receptors (AChR). We demonstrated that the CRLD-1A isoform is a membrane-associated ER-resident protein disulfide isomerase (PDI). It physically interacts with AChRs and promotes the assembly of AChR subunits in the ER. Mutations of Creld1, the human ortholog of crld-1a, are responsible for developmental cardiac defects. We showed that Creld1 knockdown in mouse muscle cells decreased surface expression of AChRs and that expression of mouse Creld1 in C. elegans rescued crld-1a mutant phenotypes. Altogether these results identify a novel and evolutionarily-conserved maturational enhancer of AChR biogenesis, which controls the abundance of functional receptors at the cell surface.

Inhibition of histone acetylation by curcumin reduces alcohol-induced fetal cardiac apoptosis

BackgroundPrenatal alcohol exposure may cause cardiac development defects, however, the underlying mechanisms are not yet clear. In the present study we have investigated the roles of histone modification by curcumin on alcohol induced fetal cardiac abnormalities during the development.Methods and resultsQ-PCR and Western blot results showed that alcohol exposure increased gene and active forms of caspase-3 and caspase-8, while decreased gene and protein of bcl-2. ChIP assay results showed that, alcohol exposure increased the acetylation of histone H3K9 near the promoter region of caspase-3 and caspase-8, and decreased the acetylation of histone H3K9 near the promoter region of bcl-2. TUNEL assay data revealed that alcohol exposure increased the apoptosis levels in the embryonic hearts. In vitro experiments demonstrated that curcumin treatment could reverse the up-regulation of active forms of caspase-3 and caspase-8, and down-regulation of bcl-2 induced by alcohol treatment. In addition, curcumin also corrected the high level of histone H3K9 acetylation induced by alcohol. Moreover, the high apoptosis level induced by alcohol was reversed after curcumin treatment in cardiac cells.ConclusionsThese findings indicate that histone modification may play an important role in mediating alcohol induced fetal cardiac apoptosis, possibly through the up-regulation of H3K9 acetylation near the promoter regions of apoptotic genes. Curcumin treatment may correct alcohol-mediated fetal cardiac apoptosis, suggesting that curcumin may play a protective role against alcohol abuse caused cardiac damage during pregnancy.

Essential role of Cdc42 in cardiomyocyte proliferation and cell-cell adhesion during heart development

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/β-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

TMEM2 Is a SOX4-Regulated Gene That Mediates Metastatic Migration and Invasion in Breast Cancer.

The developmental transcription factor SOX4 contributes to the metastatic spread of multiple solid cancer types, but its direct target genes that mediate cancer progression are not well defined. Using a systematic molecular and genomic approach, we identified the TMEM2 transmembrane protein gene as a direct transcriptional target of SOX4. TMEM2 was transcriptionally activated by SOX4 in breast cancer cells where, like SOX4, TMEM2 was found to mediate proinvasive and promigratory effects. Similarly, TMEM2 was sufficient to promote metastatic colonization of breast cancer cells and its expression in primary breast tumors associated with a higher likelihood of metastatic relapse. Given earlier evidence that genetic inactivation of SOX4 or TMEM2 yield similar defects in cardiac development, our findings lead us to propose that TMEM2 may not only mediate the pathologic effects of SOX4 on cancer progression but also potentially its contributions to embryonic development. Cancer Res; 76(17); 4994-5005. ©2016 AACR.

Abstract 65: Latrophilin-2 is a Specific Cell-surface Marker for Cardiac Progenitor Cells and Specifies Cardiac Lineage Commitment and Development

Backgrounds: The identification of a lineage-specific marker plays a pivotal role in understanding developmental process and is utilized to isolate a certain cell type with high purity for the therapeutic purpose. We here report a new cardiac-specific marker, and demonstrate its functional significance in the cardiac development. Methods and Results: When mouse pluripotent stem cells (ES and iPS cells) were stimulated with BMP4, Activin A, bFGF and VEGF, they differentiated into cardiac cells. To screen cell-surface expressing molecules on cardiac progenitor cells compared to undifferentiated mouse iPS and ES cells, we isolated Flk1+/PDGFRa+ cells at differentiation day 4 and performed microarray analysis. Among candidates, we identified a new G protein-coupled receptor, Latrophilin-2 (LPHN2) whose signaling pathway and its effect on cardiac differentiation is unknown. In sorting experiments under cardiac differentiation condition, LPHN2+ cells derived from pluripotent stem cells strongly expressed cardiac-related genes (Mesp1, Nkx2.5, aMHC and cTnT) and exclusively gave rise to beating cardiomyocytes, as compared with LPHN2- cells. LPHN2-/- mice revealed embryonically lethal and huge defects in cardiac development. Interestingly, LPHN2+/- heterozygotes were alive and fertile. For the purpose of cardiac regeneration, we transplanted iPS-derived LPHN2+ cells into the infarcted heart of adult mice. LPHN2+ cells differentiated into cardiomyocytes, and systolic function of left ventricle was improved and infarct size was reduced. We confirmed LPHN2 expression on human iPS and ES cell-derived cardiac progenitor cells and human heart. Conclusions: We demonstrate that LPHN2 is a functionally significant and cell-surface expressing marker for both mouse and human cardiac progenitor and cardiomyocytes. Our findings provide a valuable tool for isolating cardiac lineage cells from pluripotent stem cells and an insight into cardiac development and regeneration.

Prokineticin receptor-1 signaling promotes Epicardial to Mesenchymal Transition during heart development.

The epicardium plays an essential role in coronary artery formation and myocardial development. However, signals controlling the developing epicardium and epicardial-mesenchymal transition (EMT) in the normal and diseased adult heart are studied less rigorously. Here we investigated the role of angiogenic hormone, prokineticin-2 and its receptor PKR1 in the epicardium of developing and adult heart. Genetic ablation of PKR1 in epicardium leads to partial embryonic and postnatal lethality with abnormal heart development. Cardiac developmental defects are manifested in the adult stage as ischemic cardiomyopathy with systolic dysfunction. We discovered that PKR1 regulates epicardial-mesenchymal transition (EMT) for epicardial-derived progenitor cell (EPDC), formation. This event affects at least three consequential steps during heart development: (i) EPDC and cardiomyocyte proliferation involved in thickening of an outer compact ventricular chamber wall, (ii) rhythmicity, (iii) formation of coronary circulation. In isolated embryonic EPDCs, overexpression or activation of PKR1 alters cell morphology and EMT markers via activating Akt signaling. Lack of PKR1 signal in epicardium leads to defective heart development and underlies the origin of congenital heart disease in adult mice. Our mice provide genetic models for congenital dysfunction of the heart and should facilitate studies of both pathogenesis and therapy of cardiac disorders in humans.

Abstract 16137: Essential Role of Cdc42 in Cardiomyocyte Proliferation and Cell-cell Adhesion During Heart Development

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/β-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

Nos3−/− iPSCs model concordant signatures of in utero cardiac pathogenesis

BackgroundThrough genome-wide transcriptional comparisons, this study interrogates the capacity of in vitro differentiation of induced pluripotent stem cells (iPSCs) to accurately model pathogenic signatures of developmental cardiac defects. Methods and resultsHerein, we studied the molecular etiology of cardiac defects in Nos3−/− mice via transcriptional analysis of stage-matched embryonic tissues and iPSC-derived cells. In vitro comparisons of differentiated cells were calibrated to in utero benchmarks of health and disease. Integrated systems biology analysis of WT and Nos3−/− transcriptional profiles revealed 50% concordant expression patterns between in utero embryonic tissues and ex vivo iPSC-derived cells. In particular, up-regulation of glucose metabolism (p-value=3.95×10−12) and down-regulation of fatty acid metabolism (p-value=6.71×10−12) highlight a bioenergetic signature of early Nos3 deficiency during cardiogenesis that can be recapitulated in iPSC-derived differentiated cells. ConclusionsThe in vitro concordance of early Nos3−/− disease signatures supports the utility of iPSCs as a cellular model of developmental heart defects. Moreover, this study supports the use of iPSCs as a platform to pinpoint initial stages of congenital cardiac pathogenesis.

Cardiac Development in Zebrafish and Human Embryonic Stem Cells Is Inhibited by Exposure to Tobacco Cigarettes and E-Cigarettes

BackgroundMaternal smoking is a risk factor for low birth weight and other adverse developmental outcomes.ObjectiveWe sought to determine the impact of standard tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo.MethodsZebrafish (Danio rerio) were used to assess developmental effects in vivo and cardiac differentiation of human embryonic stem cells (hESCs) was used as a model for in vitro cardiac development.ResultsIn zebrafish, exposure to both types of cigarettes results in broad, dose-dependent developmental defects coupled with severe heart malformation, pericardial edema and reduced heart function. Tobacco cigarettes are more toxic than e-cigarettes at comparable nicotine concentrations. During cardiac differentiation of hESCs, tobacco smoke exposure results in a delayed transition through mesoderm. Both types of cigarettes decrease expression of cardiac transcription factors in cardiac progenitor cells, suggesting a persistent delay in differentiation. In definitive human cardiomyocytes, both e-cigarette- and tobacco cigarette-treated samples showed reduced expression of sarcomeric genes such as MLC2v and MYL6. Furthermore, tobacco cigarette-treated samples had delayed onset of beating and showed low levels and aberrant localization of N-cadherin, reduced myofilament content with significantly reduced sarcomere length, and increased expression of the immature cardiac marker smooth muscle alpha-actin.ConclusionThese data indicate a negative effect of both tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Tobacco cigarettes are more toxic than E-cigarettes and exhibit a broader spectrum of cardiac developmental defects.

The Phosphorylation State of GSK3β Serine 9 Correlated to the Development of Valproic Acid-Associated Fetal Cardiac Teratogenicity, Fetal VPA Syndrome, Rescued by Folic Acid Administration.

The effects of the phosphorylation state of the glycogen synthase kinase 3β involved in the cardiac myocytes (jelly-like cells) epithelial-mesenchymal transition-associated migration during heart-valve formation were examined through the valproic acid-induced cardiac teratogenicity of transgenic line A34 of Tg in a the Brachydanio rerio embryo model. Valproic acid is an effective anti-epileptic drug; however, when taken by pregnant women to treat epilepsy, it can produce cardiac developmental defects in fetuses. In this study, the role of glycogen synthase kinase 3β in valproic acid-induced cardiac teratogenicity was investigated. Transgenic line A34 of zebrafish embryos was used at 3 days postfertilization. The results show that 78% (18/23) of the embryos treated with 0.10 mM valproic acid (group A) had incomplete chamber formation with normal looping and 22 % (5/23) had abnormal looping. Bradycardia was also found in comparison with control embryos (P < 0.001). For the embryos treated with 0.25 mM valproic acid (group B), 92% (22/24) demonstrated chamber formation failure and looping abnormality. Pericardial effusion, noncontracting ventricles, and enlarged, slowly beating atriums were observed at 6 days postfertilization. Valproic acid inhibited phosphorylation of serine 9 in glycogen synthase kinase 3β in a dose-dependent manner. According to immunochemical staining results, valproic acid was shown to inhibit the mass migration and proliferation of cardiomyocytes in the development of the heart-valve region through inhibition of the GSK3β Ser 9 phosphorylation. Folic acid rescued the GSK3β Ser 9 phosphorylation and reversed the valproic acid-induced cardiac morphological, functional, and biochemical defects.

Micro-RNA Expression in Hypoplastic Left Heart Syndrome

ObjectiveMicro-RNAs (miRNAs) are important regulators of gene expression through interaction with the 3′UTR of target messenger RNAs (mRNAs). The role of miRNAs has been extensively studied in adult human and nonhuman animal models of heart disease. Hypoplastic left heart syndrome (HLHS) is the most common form of severe congenital heart disease and is an important cause of morbidity and mortality in infants and children. The objective of this work was to analyze the miRNA profile in HLHS patients. Methods and ResultsmiRNA profile was determined in the right ventricle with the use of miRNA array, and expression was validated with the use of reverse-transcription polymerase chain reaction (RT-PCR). Based on bioinformatics analysis, targets were selected and their expression analyzed with the use of RT-PCR.We found that the miRNA profile of HLHS is novel, with few similarities between pediatric and adult idiopathic dilated cardiomyopathy. Moreover, our analysis identified putative targets for these miRNAs that are known to be important for cardiac development and disease, and that miRNAs and their putative targets are antithetically regulated. We also found that miRNA expression changes with stage of surgery, suggesting that volume unloading of the ventricle has important consequences for gene expression. ConclusionsOur data suggest a unique miRNA profile for HLHS that may be associated with defects in cardiac development and disease.