Construction Of Zebrafish Heg1 Mutant And Its Application In Cardiovascular Drug Screening

Cardiovascular disease is a general term for diseases involving abnormal development of the heart or blood vessels,including cardiomyopathy,coronary heart disease,stroke,etc.,accounting for more than 40%of the world’s death causes,bringing a huge burden to the quality of human life.The zebrafish genome is highly homologous to the human genome,the embryo is transparent,and the entire cardiac tissue structure and blood cell flow can be clearly observed during development,making it an important model animal for cardiovascular disease research.CRISPR/Cas9 technology has become an essential tool for gene function research because of its simplicity and efficiency.HEG1 is a heart development-related protein that plays an important role in the development of the cardiovascular system.This study intends to use CRISPR/Cas9 technology to knock out the heg1 gene in zebrafish,in order to obtain a stably inherited cardiovascular disease model for drug screening and research of cardiovascular disease.In this study,we used CRISPR/Cas9 technology to perform gene editing in the exon of the zebrafish heg1 gene,and successfully obtained a stably inherited heg1 gene mutant strain.The mutant has a deletion of 25 bases in exon 1,and only 12 amino acids are retained.Morphological observation showed that the mutant exhibited severe cardiovascular malformations,including enlarged atrium and ventricle,slow heart rate,increased distance between venous sinus and bulbar arteriosus,enlarged sac area,venous thrombosis and blood flow retardation.Molecular biological analysis detected abnormal expression of zebrafish heart and blood vessel marker genes（myh7,cmlc2,vegfc,flt4,scl）.The above features are similar to the clinical phenotype of human dilated cardiomyopathy.In this study,the wild-type zebrafish（wt）and heg1 mutant zebrafish(heg1^-/-)developed to72 h were subjected to transcriptome sequencing.The results showed that the number of differential genes in the two groups was 4814,of which 2442 genes were up-regulated and2372 genes were down-regulated in heg1^-/-.GO analysis found that the mutant up-regulated genes were mainly enriched in cardiac development,including biological processes such as muscle organ development,muscle cell development,and myofibril assembly;mutant down-regulated genes were enriched in cardiovascular development,including blood vascular morphogenesis,blood vascular development,heart development and other biological processes.KEGG analysis showed that up-regulated genes were mainly enriched in Cell adhesion molecules,ECM-receptor interaction,Focal adhesion and other signaling pathways;down-regulated genes were mainly enriched in cardiomyocyte adrenergic signaling,Cardiac muscle contraction,VEGF and other signaling pathways.Transcriptomic results confirmed that HEG1 gene plays an important role in cardiovascular and myocardial development.In order to explore the application of heg1 mutants in drug screening,ten traditional Chinese medicines with the effect of treating cardiomyopathy were selected in this study,including Panax ginseng and Aconitum carmichaeli Debx,which have strong heart and qi.Paeoniae Radix Rubra,Leonurus japonicus Houtt,Ligusticum chuanxiong hort,Panax pseudo ginseng,and Carthamus tinctorius L that promote blood circulation and remove blood stasis.the diuretic Poria cocos（Schw.）Wolf,Draba nemorosa L and Cassia Twig.the results showed that the pericardial edema of heg1 mutant zebrafish was alleviated and the accumulation of blood cells was relieved to varying degrees after treatment with some traditional Chinese medicines.Among them,Panax ginseng has the highest recovery rate and the best effect.Then,three monomers,Ginsenoside Rb1,Hydroxysafflor yellow A,and Ligustrazine hydrochloride,were selected for further verification.The results showed that after the monomer treatment,the cardiovascular malformations of the heg1 mutants were alleviated more or less.Among them,Ginsenoside Rb1 had the highest recovery rate and the best effect.This study suggests that heg1 mutant can be used for the screening of therapeutic drugs for cardiovascular diseases,especially cardiomyopathy.Next,we further explored the efficacy and molecular mechanism of Panax ginseng and Ginsenoside Rb1 on heg1 mutant,combined with Tg(heg1^-/-;cmlc2:e GFP)cardiac transgenic zebrafish and Tg(heg1^-/-;flk1:e GFP)vascular transgenic zebrafish observed the morphological changes of the heart and blood vessels of the mutants after drug treatment.The results showed that both Panax ginseng and Ginsenoside Rb1 could restore the morphological abnormalities of the heart and blood vessels of the heg1 mutant to varying degrees,and the indexes such as heart rate,SV-BA distance,and cardiac sac area were all recovered after drug treatment..Combined with the results of RNA-seq,this study detected the rescue effect of human-involved Panax ginseng and Ginsenoside Rb1 on the key genes of the mutant myocardial contractility signaling pathway and the key genes of the VEGF signaling pathway by q RT-PCR.It is concluded that Panax ginseng and Ginsenoside Rb1may affect HEG1 gene through myocardial contractility signaling pathway or VEGF signaling pathway,thereby improving the cardiovascular malformation state of heg1mutant.In this study,heg1 deletion mutant zebrafish was successfully constructed by CRISPR/Cas9 gene editing technology.Phenotypic studies confirmed that HEG1 plays an important role in cardiovascular and cardiac development.RNA-seq explored the molecular mechanism of HEG1 affecting cardiovascular development.Ten kinds of traditional Chinese medicines and three monomers were selected to treat heg1 mutants and evaluate the therapeutic effect of the drugs.Finally,it was determined that Panax ginseng and Ginsenoside Rb1 with the best curative effect might be able to rescue the heg1 mutant through myocardial contraction and VEGF signaling pathway.This study provides a new zebrafish disease model for human cardiovascular disease research,which provides an effective tool for the screening of clinical therapeutic drugs for cardiovascular disease,and also provides a certain theoretical basis for the research of HEG1 in the cardiovascular field,and the treatment of cardiomyopathy by human participation in Panax ginseng and Ginsenoside Rb1.