Functional Verification Of ABCC9 Gene Mutation Associated With Sick Sinus Syndrome In Zebrafish

Background:Sick sinus syndrome(SSS)is a symptomatic arrhythmia characterized by abnormal sinus node function and conduction.On the electrocardiogram,SSS is usually manifested as sinus bradycardia,sinus arrest,sinus conduction block,and sometimes accompanied by supraventricular tachyarrhythmia(slow-fast syndrome).The pathogenesis of SSS is still not very clear.Sinus atrial node degeneration and fiber infiltration related to age,cardiomyopathy,cardiovascular disease and other diseases can not explain the pathogenesis of all clinical SSS,especially in young patients.In recent years,more and more studies have found that the occurrence of SSS is related to a variety of genetic abnormalities.Genetic research on the pathogenesis of SSS has increasingly become a hot topic in this field,which also helps to understand the pathogenesis of SSS at the molecular level,and is expected to provide a way to develop new treatment methods for SSS.The group has already used whole-exome sequencing and bioinformatics analysis to identify 2 out of 3 family members with the suspected pathogenic mutation ABCC9 gene in the SSS family,and then applied the same research idea to find 6 out of 48 cases with the suspected pathogenic mutation ABCC9 gene in the disseminated SSS population by gene sequencing and bioinformatics analysis.Further verifying whether this gene mutation is a potential SSS pathogenic gene is helpful in revealing the pathogenesis of abnormal ion channel function of this gene mutation in SSS,contributing to the early diagnosis of SSS patients and early screening of family members,and providing a theoretical basis for future research and development of biological pacemakers.As a new generation of gene editing technology,CRISPR/Cas9 gene editing technology can currently achieve precise point mutation,point deletion,and insertion and replacement of artificially synthesized genes,which can be used for gene function research.Zebrafish,as an ideal animal model for studying cardiac genetic diseases,has the advantages of in vitro fertilization,short reproductive cycle,and visualization of embryonic development,which can be used for the preparation of cardiovascular disease models.Objectives:1.To verify the relationship between ABCC9 gene loss-of-function mutation and SSS.2.To investigate the effect of ABCC9 gene loss-of-function mutation on zebrafish heart function.Methods:1.The selection and confirmation of Cas9 target sites,using the ZiFiT website to design the knocking out target sites of the CRISPR/Cas9 system of ABCC9 gene,screen the most significant target sites that lead to ABCC9 gene mutation according to the target selection principle,and amplify the target sites with PCR technology,and sequence the real sequence of the target sites.2.Use gRNA and Cas9 mRNA in vitro transcription template vector to prepare gRNA and Cas9 mRNA,use agarose gel electrophoresis to detect whether the amplification product is correct,and use ultramicro spectrophotometer to determine the concentration of two kinds of RNA.3.Inject a certain concentration of gRNA and Cas9 mRNA into the fertilized eggs of F0 generation zebrafish under the microscope with a microinjection needle,and culture them in a constant temperature incubator.Total RNA was extracted from 48hpf zebrafish embryos,reverse transcribed to obtain cDNA,PCR amplified ABCC9 gene,and gene sequencing confirmed the knockout result of gRNA.4.Zebrafish embryos were reared to sexual maturity in the F0 generation,and the mRNA expression of ABCC9 gene was measured before and after gene knockout by clipping the tail fin of zebrafish.5.Measuring the effect of ABCC9 gene knockout on the heart function parameters of zebrafish,including heart rate,ejection fraction,stroke volume,end-systolic ventricular volume,end-diastolic ventricular volume.Results:1.The ABCC9 gene was effectively knocked out by the CRISPR/Cas9 system.The peak plots of zebrafish embryo target gene sequencing results showed significant sets of peaks and double peaks,indicating the presence of gene mutations around the target.The mRNA expression of ABCC9 gene in F0 generation zebrafish was detected to be significantly lower than that in wild-type zebrafish,indicating that ABCC9 gene expression was effectively inhibited.2.The heart rate was significantly lower in the ABCC9 knockout zebrafish group compared to the wild-type zebrafish group(the heart rate of zebrafish in the control group and experimental group is 120.00±13.93 beats/min vs.203.36±10.00 beats/min,P<0.001).3.The knockout of ABCC9 gene significantly altered cardiac function in zebrafish compared to wild-type zebrafish.In addition to significantly higher ejection fraction than wild-type zebrafish(The ratio between the control group and the experimental group is 0.35±0.09 to 0.47±0.07,P=0.001),the cardiac output(the control group compared with the experimental group:5918648.04±2676713.98 μm3 vs.2338604.9±1316439.77 μm3,P=0.001),the end-diastolic ventricular volume(1.00±0.24 in the control group vs.0.77±0.22 in the experimental group,P=0.018),and endsystolic ventricular volume(1.00±0.20 in the control group vs.0.67±0.19 in the experimental group,P<0.001)were significantly lower than wild-type zebrafish.Conclusion:1.Mutations in ABCC9 gene can affect the sinus node function and cause a significant decrease in heart rate in zebrafish,indicating that the ABCC9 gene and its mutations obtained from our screening in disseminated SSS cases are new possible causative genes and pathogenic mutations of SSS.2.Mutations in the ABCC9 gene also have an effect on cardiac function in zebrafish,causing a significant increase in ejection fraction and a significant decrease in cardiac output,end-diastolic ventricular volume,and end-systolic ventricular volume.