The Study Of Vrtn Functions In Cardiomyocyte Proliferation

Objective:The morbidity of cardiovascular disease is increasing annually,which is the main cause of death in China.After the adult heart is damaged,due to the massive loss of cardiomyocytes and the limited ability to proliferate and regenerate,the heart will undergo undesirable remodeling and fibrosis,which will inevitably lead to heart failure.In the previous study,our group found that the deletion of a transcription factor Vertnin（Vrtn）would affect the development of the mouse heart,which may be related to the early heart development.Therefore,this study used mice as the research model,and explored the effect and mechanism of Vrtn transcription factor on heart development through molecular biological experiments and pathological experiments.Methods:1.We constructed a Vrtn deletion(Vrtn^+/-)mouse using the TAL effect endonuclease technology,mated the Vrtn^+/-female and male mice,and observed and counted the number and morphology of Vrtn^+/+,Vrtn^+/-and Vrtn^-/-mouse embryos at six time points in the 8.5-12.5 days according to the Theiler stage standard of mouse embryo development.2.RNA sequencing technology was utilized to detect and analyze the differential gene expression in Vrtn^+/+and Vrtn^-/-mouse embryos at 9.0 days,and the biological processes and signal pathways in which the differentially expressed genes are mainly involved.The genes with higher differential multiple were screened and verified by q PCR and Western blot.3.The number of Vrtn^+/+and Vrtn^+/-mice in offspring from the embryonic stage to postnatal stage were counted and analyzed,and the cardiac structure of adult mice of these two genotypes was detected by transmission electron microscopy.4.The expression of Vrtn transcription factor in the embryo of mice（C57BL/6）at8 time points at 6.5-13.5 days after mating was quantified by PCR,and the main period of action of Vrtn transcription factor in cardiac development was assessed.5.Use CRISPR/Cas9 technology to build and breed cardiac specific conditional Vrtn gene knockout mice Vrtn^flox/flox,Nkx2-5-Cre⁺.This is the experimental group and Vrtn^flox/flox mice are the control group.Combined with cardiac ultrasound and HE staining,observe and analyze the cardiac function and structure of the two groups of mice.Results:1.Vrtn^-/-mouse embryonic heart development abnormality,late deathIn the study,we collected the number and morphology of Vrtn^+/+,Vrtn^+/-and Vrtn^-/-mouse embryos at six time points,namely 8.5,9.0,9.5,10.5,11.5 and 12.5 days,after mating Vrtn^+/-female and male mice.Through observation and statistics,it was found that compared with Vrtn^+/+and Vrtn^+/-,the actual number of Vrtn^-/-mouse embryos were smaller and the morphological development was abnormal.The specific manifestation was that the heart began to cyclic abnormally at 9.0 days,and some pericardial effusion occurred at 10.5 days;Vrtn^-/-mouse embryos died between 11.5-12.5 days.2.Vrtn^-/-down-regulation of specific transcription factors related to embryonic heart developmentIn order to explore the causes of the low number of Vrtn^-/-embryos and the late death,we sequence the RNA of 9.0 d Vrtn^+/+and Vrtn^-/-mouse embryos.By analyzing the differentially expressed genes,it was found that compared with Vrtn^+/+mice,the expression of Bmp10,Actc1,Gja5,Myh7,Myl2,Myl3 and Nppa,the specific transcription factors related to the heart development of Vrtn^-/-mouse embryos,were down-regulated.The biological processes involved in down-regulated genes include myocardial contraction,cardiac contraction,cardiac cavity morphogenesis,ventricular development and ventricular morphogenesis,which are all related to cardiac development.We also used Western blot technology and q PCR technology to quantify the down-regulated differential genes,and the results were in agreement with RNA sequencing.3.Partial deletion of Vrtn transcription factor affects the development of the mouse heart,resulting in a decline in the survival rate of offspringThe mouse embryonic heart had matured at 13.5 days.During the study,it was found that when Vrtn^-/-embryonic heart related transcription factors were down-regulated,Vrtn^-/-embryos died before 13.5 days;However,the proportion of Vrtn^+/-and Vrtn^+/+mice in the offspring of mice gradually decreased from 13.5 days in the embryonic period to 21 days after birth,from 1 to 0.76.At the same time,we detected the hearts of 3-month-old Vrtn^+/+and Vrtn^+/-mice by transmission electron microscopy,and found that the heart structure of Vrtn^+/+mice was normal,and the myocardium of Vrtn^+/-mice was disordered and fibrosis was serious.This indicates that partial deletion of Vrtn gene affects the development of the mouse heart,resulting in a decrease in the survival rate of offspring.4.Vrtn transcription factor is highly expressed between 7.5-9.5 days of embryonic developmentIn order to study the effect and mechanism of Vrtn on cardiac development,we first quantified the expression level of Vrtn transcription factors.We mated C57BL/6J female and male mice and collected the whole embryo RNA of the embryos at four time points of 6.5,7.5,8.5 and 9.5 days,the RNA of the first,second and third heart regions of the 9.5 day mouse embryos,and the RNA of the 10.5,11.5,12.5 and 13.5day embryonic hearts.After extracting and reverse transcription of distinct RNA,the expression level was detected by PCR technology.Through analysis,it was found that Vrtn transcription factor was highly expressed in the period of 7.5-9.5 days during embryonic development.This period belongs to the early stage of cardiac development,and the heart is taken from the differentiation and development of progenitor cells.Previous studies found that the lack of Vrtn transcription factor led to abnormal cardiac development,which may be caused by abnormal differentiation and proliferation of primary cardiac progenitor cells.5.The effect of Vrtn transcription factor deletion on the heart has nothing to do with the first cardiac regionThe heart is mainly derived from the differentiation and development of two progenitor cell groups,namely,the primary heart region and the secondary heart region.We utilized CRISPR/Cas9 technology to construct cardiac specific conditional Vrtn gene knockout mice Vrtn^flox/flox,Nkx2-5-Cre⁺,knock out Vrtn transcription factor in the heart,and retain Nkx2-5 transcription factor from the progenitor cell group of the first cardiac region.Cardiac ultrasound and HE staining were performed on Vrtn^flox/flox,Nkx2-5-Cre⁺and Vrtn^flox/flox mice.It was found that there was no difference in cardiac function and structure between the two groups,indicating that the effect area of Vrtn transcription factor deletion on cardiac development might be in the second cardiac region,not in the second cardiac region.Conclusion:Vrtn transcription factor began to regulate cardiac development at the early stage of embryonic development,mainly by regulating the proliferation of early cardiac progenitor cells to regulate cardiac development,and the regulatory region has nothing to do with the first cardiac region.Because most of the progenitor cells related to cardiac development originate from the second cardiac region,the study of the main regulatory factors of the second cardiac region can provide potential new targets for the treatment of cardiovascular diseases in the future.