KLK11 Targets AKT-mTOR Signaling Pathway To Facilitate Cardiac Hypertrophy

Objective: The purpose of this study was to explore the regulatory pathway and activation mode of the tissue bradykinin protein KLK11 involved in cardiac hypertrophy,and to reveal the regulatory effect of KLK11 on AKT-m TOR signaling pathway and downstream proteins in cardiac hypertrophy.To clarify the mechanism of action of bradykininase in the development of cardiac hypertrophy,it will provide new ideas and methods for preventing and treating cardiac hypertrophy.Methods:1.Using animal models(preparation of mouse cardiac hypertrophy by TAC method)and clinical human donor heart samples,real-time quantitative PCR(q RT-PCR)and Western blot(WB)were used to detect KLK11 in human and mouse hypertrophic heart tissues The expression of KLK11 and myocardial hypertrophy were analyzed according to the data of experimental animals and clinical cases;2.In vitro experiments: With the help of a cell model,angiotensin II(Ang II)was used to induce cardiomyocyte hypertrophy in vitro,and by up-regulating(overexpression mediated by adeno-associated virus AAV9)or silencing(si RNA knockdown)KLK11,systematically studied the role of KLK11 in cardiomyocyte hypertrophy.The role in: comparing the size of cardiomyocytes after the expression of KLK11 was increased or decreased by cell staining and the detection of cardiac hypertrophy markers atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and β-myosin heavy chain by q RT-PCR(MYH7)m RNA changes;3.In vivo experiment: Adeno-associated virus AAV9 was used to mediate the low expression of sh KLK11.C57BL/6 mice were injected with normal saline or AAV9 vector through the jugular vein.After 8 weeks,they underwent TAC or sham surgery to verify that the knockout rate was satisfactory.Fractional shortening(FS)and ejection fraction(EF)of cardiac function in four groups of mice were detected respectively,and then heart weight/body weight(HW/BW),tibia length/tibia length/(HW/TL)was used to evaluate myocardial hypertrophy,and then myocardial tissue staining was performed to observe the difference in myocardial cell size and the expression of myocardial hypertrophy markers,and to evaluate whether knockdown of KLK11 in vivo had an effect on TAC-induced myocardial hypertrophy;4.After KLK11 was overexpressed and knocked down under the same experimental conditions,the phosphorylation of S6K1 and 4EBP1 and the changes in protein synthesis(3H-leucine incorporation assay)were detected by WB assay,and the relationship between KLK11 and protein synthesis was analyzed;5.In vitro cell experiments,using rapamycin to inhibit the AKT-m TOR pathway,infect mouse cardiomyocytes with adenovirus for 24 hours,and then add Ang II(1 μM)under the culture condition of rapamycin(100 n M)for 24 hours.For hours,WB was used to verify the effect of KLK11 on Akt,m TOR,S6K1,4EBP phosphorylation and protein synthesis,Image J to detect the size of cardiomyocytes and q RT-PCR to detect whether the effect of the expression of hypertrophic myocardial markers would be blocked.Results:1.ANP,BNP and MYH7 were overexpressed in the myocardial tissue of mouse myocardial hypertrophy model and clinical cardiac hypertrophy patients(P<0.001),and the m RNA level and protein expression of KLK11 were also significantly up-regulated compared with normal myocardial group(P<0.001);2.In vitro,knockdown of KLK11 reduced Ang II-induced hypertrophic growth of cardiomyocytes,overexpression of hypertrophic marker genes(P<0.001),on the contrary,overexpression of KLK11 promoted Ang II-induced hypertrophic growth of cardiomyocytes,and Overexpression of cardiomyocyte hypertrophy marker genes(P<0.001);3.In vivo,knockdown of KLK11 reversed TAC-induced cardiac function and myocardial hypertrophy in mice: echocardiography showed that the myocardial FS and EF of mice in the AAV9-sh KLK11+TAC group were significantly lower than those in the AAV9-sh Ctrl+TAC group.The control group was significantly improved,and the heart weight ratios of HW/BW and HW/TL were decreased in TAC-induced mice.Pathological section staining showed the size of cardiomyocytes,and the expressions of ANP,BNP and MYH7 were also significantly increased(P<0.001);4.Cell experiments confirmed that knockdown of KLK11 could inhibit Ang II-induced cardiomyocyte protein synthesis and inhibit the phosphorylation of S6K1 and 4EBP1(P<0.001).Phosphorylation of S6K1 and4EBP1(P<0.001);5.Both in vitro and in vivo experiments indicated that the down-regulation of KLK11 inhibited the phosphorylation of Akt-m TOR.In vitro experiments suggested that overexpression of KLK11 promoted the phosphorylation of Aktm TOR,and the addition of rapamycin inhibited the phosphorylation of S6K1 and 4EBP1 mediated by KLK11,hypertrophy of cardiomyocytes and the expression of hypertrophic marker genes(P<0.001).Conclusions:1.KLK11 is a regulator of cardiac hypertrophy,which promotes hypertrophy of cardiomyocytes and reduces cardiac function;2.The effect of KLK11 on promoting cardiomyocyte hypertrophy is accomplished by activating the AKT-m TOR signaling pathway,that is,KLK11 regulates m TOR signaling to target cardiac hypertrophy;3.After the activation of the AKT-m TOR signaling pathway,the protein synthesis of S6K1 and 4EBP1 is further promoted;4.S6K1 and 4EBP1 may be downstream targets of KLK11 in promoting cardiac hypertrophy.