Vaspin Alleviates Pathological Cardiac Hypertrophy By Regulating Autophagy-dependent Myocardial Senescence

BackgroundVisceral adipose tissue-derived serine protease inhibitor(vaspin),a member of the serine protease inhibitor family,is secreted by visceral adipose tissue and released into the blood circulation to play a biological role.Previous reports showed that vaspin could alleviate insulin resistance,obesity,metabolic disturbance,and hepatic steatosis through autocrine and paracrine mechanisms.In recent years,it has been found that the level of vaspin secreted into peripheral blood is closely related to the occurrence and development of cardiovascular diseases such as hypertension and atherosclerosis.The serum level of vaspin in coronary artery disease(CAD)patients significantly lower than that in healthy people of the same age and sex,and the incidence of adverse cardiovascular events in CAD patients with lower vaspin levels was higher,suggesting that vaspin is an important predictor of prognosis in CAD patients.In addition,vaspin also has a protective effect on myocardial injury caused by ischemia/reperfusion and diabetes,and the mechanism is related to the regulation of apoptotic,inflammation,and autophagy.However,whether vaspin can affect pathological cardiac hypertrophy remains unclear,which requires further investigation by researchers.Objectives1.To explore the effects of vaspin on pathological cardiac remodelling;2.To verify whether vaspin can regulate premature myocardial senescence in pathological cardiac hypertrophy animal models;3.To explore the specific molecular target of vaspin’s effects on pathological cardiac hypertrophy and to provide basic experimental support for inhibiting the process of malignant heart remodeling in clinical practice.MethodsIn vivo study1.The target exon DNA sequence of the Tec gene was cut by the CRISPR system,making a DSB,resulting in a frameshift mutation by the NHEJ DNA repair mechanism,resulting in vaspin knockout mice;2.Pathologic cardiac hypertrophy was induced in male C57BL/6J wild-type(WT)and vaspin-knockout(vaspin-KO)mice.An in vivo study was conducted using a cardiac hypertrophy model established by subcutaneous injection of ISO(5mg/kg/d,dissolved in saline)once daily for 9 days.Buparlisib(PI3K inhibitor,50 mg/kg)and rapamycin(mTOR inhibitor,20 mg/kg)were pre-and co-administered to vaspin-KO mice daily for a period of 15 days.Induction of pathological cardiac hypertrophy was performed by the subcutaneous administration of isoproterenol(ISO)(5 mg/kg)into mice from the 7th to the 15th day;3.Cardiac function in mice was evaluated with echocardiography after 9 days of ISO subcutaneous injection;4.The mice’s body weight and heart weight were measured after 9 days of ISO subcutaneous injection;5.Serial heart sections were stained with haematoxylin-eosin or wheat germ agglutinin to measure myocyte cross-sectional areas in each group of mice after 9 days of ISO subcutaneous injection;6.Sirius red staining and masson staining were used to determine myocardium fibrosis levels in each group of mice after 9 days of ISO subcutaneous injection;7.RT-PCR was used to detect the mRNA transcription levels of hypertrophic genes as well as fibrotic genes,such as:Collal,Col3al,ANP,BNP,Myh7,β-MHC and other genes in each group of mice;8.Immunohistochemical staining was performed to assess the expression of P16 in myocardial tissue in each group of mice after 9 days of ISO subcutaneous injection;9.WB was performed to determine the expressions of senescence and autophagy markers(P16,P53,P21,γ-H2AX,PI3K,AKT,P-AKT,mTOR,P-mTOR,LC3-Ⅱ/Ⅰ,P62)and GAPDH in in myocardial tissue.In vitro study1.Establish pathological cardiac hypertrophy models in H9C2 cells H9C2 cells were divided into groups as follows:1)con,vaspin,ISO,ISO+vaspin;2)con,ISO,ISO+vaspin;3)con,ISO,ISO+vaspin,ISO+vaspin+CQ;4)ISO,ISO+CQ,ISO+vaspin,ISO+vaspin+CQ;2.Cell senescence β-galactosidase staining kit was used to detect the senescence level of H9C2 cells in each group with different stimuli;3.Immunofluorescence staining was used to label the expression of senescent protein P21 in H9C2 cells of each group after adding different stimuli;4.Before adding different stimuli to H9C2 cells,mRFP-GFP-LC3 adenovirus was transfected to detect the formation of autophagosome and autolysosomes,and whether autophagy flow was patent;5.The expression levels of autophagy molecules and senescence related molecular proteins such as P16,P53,P21,y-H2AX,PI3K,AKT,P-AKT,P-mTOR,mTOR LC3-II,P62 and GAPDH in H9C2 cells were detected by Western Blot;6.Electron microscopy to detect autophagosomes and autolysosomes in H9C2 cells;7.The mRNA transcriptional levels of hypertrophy genes such as ANP,BNP,β-MHC,and 18S in H9C2 cells were detected by RT-PCR.Results1.Vaspin-KO aggravated ISO-induced cardiac hypertrophy in vivo;2.Vaspin-KO aggravated ISO-induced premature myocardial senescence in vivo;3.Vaspin-KO aggravated ISO-induced autophagy reduction in vivo,and recombinant human vaspin can rescue the ISO-induced autophagy decline in vitro;4.Recombinant human vaspin could reverse the cardiomyocyte senescence phenotype in the ISO-induced cardiomyocyte hypertrophy model,which was blocked by CQ;5.Vaspin restores the level of autophagy flow in myocardial tissue,improves pathological remodeling of cardiac tissue and premature myocardial stress senescence,and is achieved by inhibiting the PI3K-Akt-mTOR pathway.ConclusionsThe protective effect of vaspin on premature myocardial senescence and cardiac hypertrophy depends on the PI3K-AKT-mTOR pathway-dependent activation of autophagy;Vaspin alleviates pathological cardiac hypertrophy by regulating autophagy-dependent myocardial senescence.