Effects And Mechanisms Of Cardiac-specific Knockout Of TNNI3K Gene On Cardiac Function In Mice And The Effects Of Pitavastatin On Lipoprotein Particles In Patients With Atherosclerosi

Part 1 Effects and mechanism of cardiac-specific knockout of TNNI3K gene on cardiac function in miceBackground and objective:The chromosomal location of the cardiac troponin I-interacting kinase(TNNI3K)gene is close to the region where the atrioventricular septal defect-susceptible gene exists,suggesting that it may be involved in the normal development of the heart.Previous studies have shown that TNNI3K is closely related to cardiomyocyte differentiation,viral myocarditis,dilated cardiomyopathy,arrhythmia and other cardiovascular diseases,as well as obesity and metabolic diseases.Based on the research foundation of the specific high expression of TNNI3K gene in the heart,this project constructed a cardiac-specific knockout mouse model of TNNI3K gene.Theoretical and scientific rationale for potential therapeutic targets in cardiovascular disease.Methods:First,a cardiomyocyte-specific TNNI3K knockout mouse(Tnni3k-cKO)model was constructed by CRISPR-Cas9 technology.Male(WT)mice were used as the control group(n=24).and male Tnni3k-cKO were used as the experimental group(n=24).The above-mentioned two groups of mice were divided into four groups according to different months(1,2,6 and 10 months old),with 6 mice in each group,and the mice in each group were fed a normal diet.Secondly,we used echocardiography to evaluate cardiac function in mice at different months of age.Heart failure markers ANP and BNP were detected via ELISA.Myocardial tissue was collected for HE staining and wheat germ agglutinin staining(Wheat Germ Agglutinin staining.WGA)staining.Masson staining.Sirius red staining,TUNEL staining and immunofluorescence double staining were used to evaluate the pathological changes of mouse cardiomyocyte cross-sectional area,myocardial hypertrophy,myocardial fibrosis and cardiomyocyte apoptosis.Apoptosis-related proteins and signaling pathway protein were detected via Western blot.Finally,the TNNI3K inhibition model of neonatal rat ventricular myocytes was constructed by inhibiting TNNI3K by GSK114.The normal cultured cardiomyocytes were divided into the control group while the cells treated with GSK114 were divided into the TNNI3K inhibition group and the expression of p38 MAPK was tested by Western blot.Results:Compared with the male WT mice,male Tnni3k-cKO mice showed a significant decrease in cardiac function at different months(1,2,6 and 10),and gradually deteriorated with the increase of months,including contraction Functional indicators ejection fraction(EF),fractional shortening(FS),and indicators of diastolic function The ratio of peak early filling velocity to mitral annular early diastolic velocity,E/e’).In addition,10-month-old Tnni3k-cKO mice showed signs of heart faiure.ELISA assays showed that Tnni3k-cKO mice had significant levels of ANP and BNP.Pathological staining results showed that with the increase of age,Tnni3k-cKO mice gradually developed pathological changes such as increased heart size,progressive enlargement of the left ventricular cavity and thinning of the ventricular wall.At the same time,the degree of myocardial hypertrophy,myocardial fibrosis and myocardial cell apoptosis gradually increased in Tnni3k-cKO mice;p38 MAPK signaling pathway may be involved in cardiomyocyte apoptosis induced by cardiac-specific knockout of TNNI3K gene.Conclusions:The results of this study showed that cardiac-specific knockout of TNNI3K gene resulted in myocardial hypertrophy,myocardial fibrosis,increased myocardial apoptosis,and cardiac dysfunction in mice.The induced cardiomyocyte apoptosis may be related to the p38 MAPK signaling pathway.This study suggests that the TNNI3K gene is important for maintaining normal cardiac structure and function.Part 2 Effect of Pitavastatin on Lipoprotein Subfractions in Patients with AtherosclerosisBackground and objective:Atherosclerosis(AS)poses a serious threat to human health and is one of the leading causes of cardiovascular death worldwide.Epidemiological studies have shown that lowering low-density lipoprotein cholesterol(LDL-C)can reduce major adverse cardiovascular events(MACEs);at the same time increasing high-density lipoprotein cholesterol(high-density lipoprotein cholesterol)cholesterol,HDL-C)is also a new target for the treatment of atherosclerotic diseases.The traditional four items of blood lipids(total cholesterol,triglyceride,LDL-C and HDL-C)have certain value for predicting atherosclerosis,but recent studies have shown that the analysis of lipoprotein particle subfractions can better predict atherosclerosis sclerosing cardiovascular disease(ASCVD).Small dense low-density lipoprotein(sdLDL)produces stronger atherogenic effects than large buoyant low-density lipoprotein(1bLDL).At present,the first-line lipid-lowering drugs commonly used in clinical practice are statins.Pitavastatin has become a key drug for the treatment of atherosclerotic plaque due to its anti-inflammatory,anti-oxidative stress and endothelial function improvement.This study was designed to examine the effect of pitavastatin on lipoprotein particles in patients with AS.Methods:We consecutively enrolled 36 patients undergoing elective coronary angiography.Patients with AS who were treated with pitavastatin were divided into a treatment group(n=18)and healthy subjects as a control group(n=18).We collected clinical and laboratory data about baseline.The Lipoprint Lipoprotein Classifier classifies and detects LDL and HDL particles,respectively.The LDL particles were divided into 7 subtypes(LDL1-7)by the method of Lipoprint System;HDL particles are divided into 10 subtypes,of which subtypes 1-3 are defined as small particle HDL.subtypes 4-7 are defined as medium particle HDL,and subtypes 8-10 are referred to as large particle HDL.After 8 weeks of pitavastatin treatment,the changes of lipoprotein particles before and after treatment in the two groups were compared.Results:Compared with before treatment,the concentrations of LDL-C,total cholesterol(TC),triglyceride(TG)and apolipoprotein B(ApoB)were significantly increased after pitavastatin treatment.decreased(P<0.05),the concentrations of large,medium and small LDL-C and the percentages of medium and small LDL were also significantly decreased(P<0.05).At the same time,HDL-C was markedly increased(P<0.05),and the concentration of ox-LDL was significantly decreased(P<0.05).Conclusions:The results of this study show that pitavastatin 2 mg/d treatment for 8 weeks can effectively reduce the concentration of large,medium and small LDL-C and the percentage of medium and small LDL,and reduce plasma ox-LDL levels.The results of this study showed that pitavastatin exerted its anti-atherosclerotic effect by improving the distribution of lipoprotein particle subtypes,revealing a new mechanism of its lipid-lowering effect.