Exosomal let-7a-5p derived from human umbilical cord mesenchymal stem cells alleviates coxsackievirus B3-induced cardiomyocyte ferroptosis via the SMAD2/ZFP36 signal axis

Viral myocarditis(VMC)is one of the most common acquired heart diseases in children and teenagers.However,its pathogenesis is still unclear,and effective treatments are lacking.This study aimed to investigate the regulatory pathway by which exosomes alleviate ferroptosis in cardiomyocytes(CMCs)induced by coxsackievirus B3(CVB3).CVB3 was utilized for inducing the VMC mouse model and cellular model.Cardiac echocardiography,left ventricular ejection fraction(LVEF),and left ventricular fractional shortening(LVFS)were implemented to assess the cardiac function.In CVB3-induced VMC mice,cardiac insufficiency was observed,as well as the altered levels of ferroptosis-related indicators(glutathione) peroxidase 4(GPX4),glutathione(GSH),and malondialdehyde(MDA).However,exosomes derived from human umbilical cord mesenchymal stem cells(hucMSCs-exo)could restore the changes caused by CVB3 stimulation.Let-7a-5p was enriched in hucMSCs-exo,and the inhibitory ffect of hucMSCs-exoa-ie-pmimo on CVB3-induced ferroptosis was higher than that of hucMSCs-exommie N(NC:negative control).Mothers against decapentaplegic homolog 2(SMAD2)increased in the VMC group,while the expression of zinc-finger protein 36(ZFP36)decreased.Let-7a-5p was confirmed to interact with SMAD2 messenger RNA(mRNA),and the SMAD2 protein interacted directly with the ZFP36 protein.Silencing SMAD2 and overexpressing ZFP36 inhibited the expression of ferroptosis-related indicators.Meanwhile,the levels of GPX4,solute carrier family 7,member 11(SLC7A11),and GSH were lower in the SMAD2 overexpression plasmid(oe-SMAD2)+let-7a-5p mimic group than in the oe-NC+let-7a-5p mimic group,while those of MDA,reactive oxygen species(ROS),and Fe^(2+)increased.In conclusion,these data showed that ferroptosis could be regulated by mediating SMAD2 expression.Exo-let-7a-5p derived from hucMSCs could mediate SMAD2 to promote the expression of ZFP36,which further inhibited the ferroptosis of CMCs to alleviate CVB3-induced VMC.

Factors controlling fetal echocardiography determine the diagnostic accuracy of isolated ventricular septal defect

Background:Fetal echocardiography (FECG) is a key screening tool for prenatal cardiac abnormalities.Herein,we examined the ultrasonic factors determining prenatal ultrasonic diagnosis of isolated ventricular septal defect (IVSD).Methods:The diagnostic role of ultrasonic factors was investigated in patients in middle or late pregnancy,diagnosed with IVSD by FECG and confirmed using postnatal echocardiography.Results:One hundred and six patients with IVSD were enrolled;the majority had perimembranous VSD.The combined imaging mode of 2 dimentionalechocardiography (2DE) and color doppler flow imaging (CDFI) showed the highest rate (56.6%) of IVSD detection,while CDFI was more efficient than 2DE (32.1% vs.11.3%).The single-view mode was more efficient than multiple-view mode (75.5% vs.24.5%).The highest efficient mode to detect IVSD was achieved using combined imaging mode on the single view of the left ventricular outflow tract view (LVOTV) (28.3%).FECG correctly classified 71.7% of fetal IVSD.There was a significant difference of accuracy rate in classifying IVSD among the three different imaging modes (x2=7.141,P<0.05).The single imaging mode of CDFI and the mode of CDFI combined with 2DE correctly classified 75.9% and 75.0% of fetal IVSD,respectively.LVOTV was the most accurate view of fetal IVSD classification (85.2%;x2=15.782,P<0.05).There was no difference in accuracies of IVSD classification among multiple-view modes (x2=2.343,P>0.05) or between single-view mode and multiple-view mode (x2=0.32,P>0.05).Conclusion:Single LVOTV in CDFI or CDFI combined with 2DE of FECG were the most effective diagnostic modes for fetal IVSD diagnosis.

Disruption of Planar Cell Polarity Pathway Attributable to Valproic Acid-Induced Congenital Heart Disease through Hdac3 Participation in Mice

Background： Valproic acid （VPA） exposure during pregnancy has been proven to contribute to congenital heart disease （CHD）. Our previous findings implied that disruption of planar cell polarity （PCP） signaling pathway in cardiomyocytes might be a factor for the cardiac teratogenesis of VPA. In addition, the teratogenic ability of VPA is positively correlated to its histone deacetylase （HDAC） inhibition activity. This study aimed to investigate the effect of the VPA on cardiac morphogenesis, HDAC1/2/3, and PCP key genes （Vangl2/Scrib/Rac 1）, subsequently screening out the specific HDACs regulating PCP pathway. Methods： VPA was administered to pregnant C57BL mice at 700 mg/kg intraperitoneally on embryonic day ! 0.5. Dams were sacrificed on E15.5, and death/absorption rates of embryos were evaluated. Embryonic hearts were observed by hematoxylin-eosin staining to identify cardiac abnormalities. H9C2 cells （undifferentiated rat cardiomyoblasts） were transfected with Hdac1/2/3 specific small interfering RNA （siRNA）. Based on the results of siRNA transfection, cells were transfected with Hdac3 expression plasmid and subsequently mock-treated or treated with 8.0 mmol/L VPA. Hdac1/2/3 as well as Vangl2/Scrib/Racl mRNA and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. Total HDAC activity was detected by colorimetric assay.Results： VPA could induce CHD （P 〈 0.001） and inhibit mRNA or protein expression of Hdac1/2/3 as well as Vangl2/Scrib in fetal hearts, in association with total Hdac activity repression （all P 〈 0.05）. In vitro, Hdac3 inhibition could significantly decrease Vangl2/Scrib expression （P 〈 0.01 ）, while knockdown of Hdac 1/2 had no influence （P 〉 0.05）, VPA exposure dramatically decreased the expression of Vanlg2/Scrib together with Hdac activity （P 〈 0.01 ）, while overexpression of Hdac3 could rescue the VPA-induced inhibition （P 〉 0.05）. Conclusion： VPA could inhibit Hdac1/2/3, Vang12/Scrib, or total Hdac activity both in vitro and in vivo and Hdac3 might participate in the process of VPA-induced cardiac developmental anomalies.

Effect of Histone Deacetylase Inhibition on the Expression of Multidrug Resistance-associated Protein 2 in a Human Placental Trophoblast Cell Line

Background： Placental multidrug resistance-associated protein 2 （MRP2）, encoded by ABCC2 gene in human, plays a significant role in regulating drugs＇ transplacental transfer rates. Studies o11 placental MRP2 regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the roles of epigenetic mechanisms in regulating placental drug transporters are still unclear. This study aimed to investigate the effect of histone deacetylases （HDACs） inhibition on MRP2 expression in the placental trophoblast cell line and to explore whether HDAC 1/2/3 are preliminarily involved in this process. Methods： The human choriocarcinoma-derived trophoblast cell line （Bewo cells） was treated with the HDAC inhibitors-trichostatin A （TSA） at different concentration gradients of 0.5, 1.0, 3.0, and 5.0 μmol/L. Cells were harvested after 24 and 48 h treatment. Small interfering RNA （siRNA） specific for HDACI/HDAC2/HDAC3 or control siRNA was transfected into cells. Total HDAC activity was detected by colorimetric assay kits. HDAC 1/2/3/ABCC2 messenger RNA （mRNA） and protein expressions were determined by real-time quantitative polymerase chain reaction and Western-blot analysis, respectively. Immunofluorescence for MRP2 protein expression was visualized and assessed using an immunofluorescence microscopy and ImageJ software, respectively. Results： TSA could inhibit total HDAC activity and HDAC 1/2/3 expression in company with increase ofM RP2 expression in Bewo cells. Reduction of HDAC 1 protein level was noted after 24 h of TSA incubation at 1.0, 3.0, and 5.0 μmol/L （vs. vehicle group, all P 〈 0.001 ）, accompanied with dose-dependent induction of MRP2 expression （P = 0.045 for 1.0 μmol/L, P = 0.001 for 3.0 μmol/L, and P 〈 0.001 for 5.0 μmol/L）, whereas no significant diferences in MRP2 expression were noted after HDAC2/3 silencing. Fluorescent micrograph images of MRP2 protein were expressed on the cell membrane. The fluorescent intensities of MRP2 in the control, HDAC2, and HDAC3 siRNA-transfected cells weir week, and no significant differences were noticed among these three groups （all P 〉 0.05）. However, MRP2 expression was remarkably elevated in H DAC1 siRNA-transfected cells, which displayed an almost 3.19-fold changes in comparison with the control siRNA-transfected cells （P 〈 0.001 ）. Conclusions： HDACs inhibition could up-regulate placental MRP2 expression in ritzy, and HDAC 1 was probably to be involved in this process.