| Background:Cardiovascular disease is a range of conditions associated with the dysfunction of heart structure and blood vessels,including,but not limited to,heart attacks caused by inadequate blood supply to the heart muscle,heart malformations,vascular embolisms,and a range of symptoms associated with these conditions,such as coronary heart disease,acute myocardial infarction,etc.Cardiovascular disease has the highest mortality rate in the world.There is a U-shaped relationship between hemoglobin levels and cardiovascular disease,and increasing or decreasing hemoglobin levels may increase the incidence of cardiovascular disease.Hemoglobin is an important component of the erythrocytes,whose main physiological functions are performed by hemoglobin.Hemoglobin is closely related to the functional activities of the body.Abnormal hemoglobin can cause a variety of diseases.Impairments in hemoglobin synthesis alter cell function due to reduced oxygen supply and lead to anemia,increasing the incidence of cardiovascular disease.In addition,decreased hemoglobin is also a valid indicator of poor prognosis in heart failure.However,the current research on cardiovascular disease has mainly focused on the energy metabolism regulated by mitochondria,and there has been few studies on the mechanism of the influence of hemoglobin on cardiovascular disease.Therefore,the detection of hemoglobin can provide a simple basis for risk assessment of cardiovascular disease,and understanding the regulation of hemoglobin production is important for the development of new therapies for anemia and cardiovascular diseases.Objective:In order to investigate the changes and possible mechanism of cardiovascular development in zebrafish after hemoglobin levels reduction by knockout of the hemoglobin gene on chromosome 3.Content and Methodology:（1）Using CRISPR/Cas9 technology to knock out the fragment of the hemoglobin gene hbba2 to hbae3 of zebrafish chromosome 3,a heterozygous mutant zebrafish(Hb+/-)with the deletion of this fragment was constructed.The Hb+/-was self-crossed and its offspring was screened.To explore the reasons for the absence of homozygous mutant(Hb-/-),analysis of Hb+/-segregation and genetic transmission was carried out,and embryos produced after the Hb+/-self-crossing were collected for genotype identification.The Hb+/-and wild type zebrafish（WT）were reciprocal crossed,and the gene segregation ratio of their offspring was counted.（2）A heterozygous mutant was used as a research model to compare the cardiovascular development of Hb+/-,a heterozygous mutant with hemoglobin gene deletion on chromosome 3,WT zebrafish,and to observe and analyze the differences in cardiovascular development between the two types.（1）Embryos of Hb+/-and WT at different stages were stained with o-Dianisidine to observe the hemoglobin content and distribution;（2）Cross-section of Hb+/-with myl7:EGFP transgenic zebrafish and analysis of 72 hpf heart rates;（3）Cross-section of Hb+/-with kdrl:EGFP transgenic zebrafish was used to observe embryonic vessel development;（4）The Hb+/-and WT adult zebrafish erythrocyte numbers at 5 months of age were detected and the differences were analyzed;（5）Total RNA from the hearts of adult Hb+/-and WT zebrafish was extracted using the TRIzol method for transcriptional library construction sequencing,and gene expression differences between heterozygous mutant zebrafish and wild-type zebrafish were analyzed,and the expression of relevant genes were validated by quantitative real-time PCR（q RT-PCR）.Results:（1）No Hb-/-was found in zebrafish offspring of adult Hb+/-.In order to explore the cause,the genotype of the Hb+/-self-crossing embryo was first identified.The ratio of WT,Hb+/-,Hb-/-was 55:225:2,Hb-/-only accounted for 0.71%,WT:Hb+/-about 1:4.We further explore the fraction of gametes produced by heterozygous mutants,allowing reciprocal crosses between Hb+/-and WT.The results showed that the ratio of Hb+/-to WT in crossing embryos of female Hb+/-and male WT was 47:1,and it was about 2:1in crossing embryos of male Hb+/-and female WT.The statistical results indicate that Hb+/-exhibits twisted genetic segregation.（2）Observations and analytical results on cardiovascular development:（1）o-dianisidine staining showed a significant reduction in Hb+/-hemoglobin content,and ectopic distribution was observed in embryos 50 hpf;（2）The heart rate statistics of 72hpf zebrafish after hybridization with myl7:EGFP showed bradycardia in Hb+/-（p<0.0001）;（3）After crossing with kdrl:EGFP transgenic zebrafish,it was observed that,compared to WT,both abdominal major vessels and common cardinal veins in Hb+/-developed slowly while sub-intestinal veins developed more rapidly and intersegmental vessels showed enlargement;（4）The erythrocyte count results of Hb+/-and WT adult zebrafish at 5 months showed no significant difference in the number of erythrocytes（p>0.05）;（5）Transcription analysis of adult zebrafish showed that the expression of Hb+/-hemoglobin gene hbba2 was significantly down-regulated,and the hypoxia-inducing gene hif3a was also significantly down-regulated,while the inducible nitric oxide synthase encoding gene nos2b and NADPH oxidase 2 encoding gene cybb were significantly up-regulated in HIF1 signaling pathway.Troponin T2 encoding gene tnnt2c was significantly upregulated in the myocardial systolic pathway,and genes associated with complex I-IV（a component member of the electron transport chain）and complex V（ATP synthase）were significantly upregulated in the oxidative phosphorylation pathway.Conclusion:Hemoglobin knockout impacts zebrafish embryonic hemoglobin levels and vascular development,while maintaining normal physiological activity in adulthood via control of gene expression and mitochondrial compensatory mechanisms. |
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