Research On The Mechanism Of The Defects In Atrioventricular Valve Development Caused By The Tnni1b Knockout In Zebrafish

The proper development of atrioventricular(AV)valves is critical for heart morphogenesis,the formation of atrial and ventricular chamber septation and the cardiac conduction system.The AV valves can prevent the blood reflux in the heart to maintain the correct direction of blood flow and normal pumping function of the heart.Defects in AV valve development are the most common type of congenital heart defect.The mechanisms underlying AV valves development are complex but relative conserved in different vertebrates,and are involved in multiple signaling pathways.Myocardial contract function and blood flow are the major epigenetic factors in regulating AV valves development.The connection between regulating genes and the different signaling pathways or epigenetic factors,however,have not been clearly identified.Thus,identify and study novel regulators in AV valves development,explore their connection with known regulating factors,acquire a better understanding of the molecular details of valve development,are essential and meaningful in the clinical detection and treatment of AV valve defects.Cardiac trpponin i(ctnni),a structural and regulatory protein involved in cardiac muscle contraction,is a subunit of troponin complex and a highly sensitive and specific marker of myocardial injury.The slow skeletal muscle tnni(sstnni)is predominantly expressed in mouse embryo hearts as well as in fetal hearts and is then down-regulated and completely replaced by the cardiac-specific isoform ctnni after birth.This expression pattern of tnni regulated by an unknown mechanism in the heart causes some difficulties in studying the function and molecular mechanisms of ctnni in early embryo heart development.On the other hand,tnni1 b served as the main tnni isoform in zebrafish heart,it is expressed in the early heart and continues to be expressed in the heart until adulthood in zebrafish without the developmental switch from sstnni to ctnni found in higher vertebrates.Thus,it is feasible to study the cardiac-specific isoform of tnni in the early development of the heart in zebrafish.In this study,we created a knockout zebrafish model in which tnni1 b was deleted using the CRISPR / Cas9 system.In the homozygous mutant,the embryos showed severe pericardial edema,malformation of the heart tube,reduction of heart rate without contraction and with almost no blood flow,and lack of an endocardial ring or valve leaflet,resulting in 88.8 ± 6.0% lethality at 7 d post fertilization.Deletion of tnni1 b caused the abnormal expression of several markers involved in AV valve development,including bmp4,cspg2,has2,notch1 b,spp1,and Alcam.Myocardial re-expression of tnni1 b in mutants partially rescued the pericardial edema phenotype and AV canal(AVC)developmental defects,indicating the knockout of tnni1 b is the direct reason for the AV valve defects in mutant embryos.Moreover,we further showed that tnni1 b knockout in zebrafish and ctnni knockdown in rat h9c2 myocardial cells inhibited cardiac wnt signaling and that myocardial reactivation of wnt signaling partially rescued the abnormal expression of AVC markers caused by the tnni1 b deletion.These results confirm that tnni1 b knockout can inhibit myocardial Wnt signaling,which is independent from the cardiomyocyte contraction and blood flow,to induce the abnormal expression of AVC specific markers and then cause the defects of AV valve development.In summary,we construct and report the first time of the zebrafish tnnib gene knockout model,observe and describe the phenotype of the mutant model.The data in this report suggest a vital role of tnni1 b in zebrafish AV valve development,reveal a new regulatory axis,the ctnni and Wnt axis which is independent from the epigenetic factors like cardiomyocyte contraction and blood flow,in regulating the development of AV valve.This is an important breakthrough of the molecular mechanisms in AV valves development.This report contributes to the study of sarcomere-related,protein-associated heart diseases,provides a new viewpoint and insight into the detection and treatment of AV valve defects.