Effect Of MiR-451 On Acute Heart Failure And The Mechanisms

Background: Heart failure(HF)is the final stage of cardiovascular disease,which can be divided into chronic heart failure(CHF)and acute heart failure(AHF)according to the time and speed of occurrence of HF.CHF has a slow development process and is the main key node for HF prevention,while AHF is the main battlefield for clinical diagnosis and treatment.At present,the pathophysiological mechanism of AHF has not been fully elucidated,and it is believed that the main causes are primary myocardial injury and dysfunction,such as ischemic heart disease and cardiotoxic damage(including antineoplastic drugs,antidepressants,antiarrhythmic drugs and so on).Among them,the cardiotoxic effect of doxorubicin(DOX)can cause the manifestation of AHF,and its pathophysiological changes include neurohormone activation,inflammatory reaction,oxidative stress and so on.Since the specific mechanism of AHF is still unclear,it is of great significance to find disease-related target genes and study their specific mechanism in AHF for prevention and treatment of AHF.The human miR-451 gene is located on the chromosome 17q11.2.The non-coding small molecule RNA,with a length of about 22 nucleotides can bind to the 3’UTR of a specific gene and negatively regulate the protein expression of the target gene to exert its biological function.miR-451 is highly conserved in vertebrates.It has been reported that miR-451 shows expression disorder in some tumors(including lung cancer,gastric cancer,colorectal cancer,glioma,pancreatic cancer and leukemia),suggesting that miR-451 may play a key role in tumorigenesis.miR-451 is highly expressed in the heart and plays an important regulatory role in some pathological processes.For example,miR-451 can regulate the process of autophagy of cardiomyocytes through TSC1 protein in patients with hypertrophic cardiomyopathy.miR-451 can also inhibit the transformation of endothelial cells into mesenchymal cells to reduce myocardial fibrosis in the heart of diabetic mice.Another study have shown that miR-451 is closely related to palmitic acid-induced myocardial inflammation and apoptosis.The above studies show that miR-451 is closely related to the occurrence and development of AHF from pathological mechanism level.Therefore,it is necessary to conduct in-depth study and research on miR-451.Objective: The main objective of this study is to observe the changes of miR-451 expression in serum of patients with AHF and to evaluate whether miR-451 expression is related to the severity of AHF.AHF model of mice induced by DOX was established to evaluate the effect of myocardial miR-451 expression on cardiac function and oxidative stress,and to further explore the effect of miR-451 gene inhibition on the above effect.To explore the mechanism of miR-451 on oxidative stress and apoptosis at the cellular level.Methods: This topic was divided into three parts: Part 1: the change of miR-451 expression in patients with AHF and DOX-induced acute cardiomyopathy in mice;Part 2: the role of mir-451 in DOX induced AHF;Part 3: the molecular mechanism of mir-451 regulating DOX induced AHF.The three parts will be described one by one as follows.Part Ⅰ: 55 patients with AHF and 24 patients without heart failure were enrolled in the cardiology department of Renmin Hospital of Wuhan University.Fasting blood samples were collected within 24 hours after admission,and miR-451 expression in serum was detected by RT-PCR.C57BL/6 wild-type healthy mice(male,8-week-old,weight of 20～23g)were randomly divided into normal saline(NS)group and DOX group.DOX group was intraperitoneally injected with DOX(15mg/kg)to establish the DOX-induced acute cardiomyopathy model,and NS group was intraperitoneally injected with the same volume of NS.Seven days after operation,the heart tissue was surgically separated and miR-451 expression in the heart of each group was detected by RT-PCR.H9c2 cardiomyocytes from rats were stimulated with 1 μmol/l DOX for12 hours,while the control group was treated with PBS.The cardiomyocytes were collected to detect the miR-451 expressionPart Ⅱ: C57BL/6 wild-type mice(male,8-week-old,weight of 20～23g)were selected and divided into four groups(n=10): blank control group(Saline+NC inhibitor),Saline+miR-451 inhibitor group,DOX+NC inhibitor group and DOX+miR-451 inhibitor group.The model was established by intraperitoneal injection of DOX(15mg/kg)or the same volume of NS according to the group,and NC/miR-451 inhibitor(5nmol/l/day)was injected through the tail vein of the mice every other day from the day before the model was established.5 days later,the blood was taken from the retroophthalmic vein,and the expression of c Tn I,CK and NT-pro BNP in serum were detected by ELISA.7 days later,the cardiac function of mice in each group was evaluated by hemodynamic test,and then these mice were weighed,recorded,sampled and examined by pathology and molecular biology.After extracting myocardial protein,the expression of apoptosis-related proteins(Bcl-2,Bax)was detected by Western blot,and the changes of oxidative stress indexes(4-HNE,MDA,GSH/GSSG,SOD)were detected by ELISA method.Cardiomyocytes damage was evaluated by HE staining and their apoptosis was evaluated by TUNEL staining.In addition,H9c2 cardiomyocytes were cultured,and the changes of oxidative stress index,cell activity and ROS concentration were also detected to verify the protection effect of miR-451 inhibitor in vitro.Part Ⅲ: the protein extracted from mice cardiomyocytes and H9c2 cells in Part 2was used to detect the expression of CAB39 protein and the phosphorylation of AMPKα/m TOR pathway by Western blot.After H9c2 cardiomyocytes were cultured,AMPK inhibitor Compound C(CPC)(1 μmol/l)was added,then MTT was used to detect the cell activity to verify whether miR-451 gene inhibition exerted the cytoprotective effect through AMPKα/m TOR pathway.Finally,the phosphorylation of AMPKα and m TOR after inhibition of AMPKα activity was detected by Western blot.Results:Part Ⅰ: LAD in the heart failure group was significantly higher than that in the non-heart failure group(44.4±1.1mm vs 34.8±1.1mm,P<0.05);LVEDD was also significantly higher(52.0±1.1mm vs 45.00±0.7 mm,P<0.05);while LVEF decreased significantly(59.3±0.4% vs 45.5±1.3%,P<0.05).The median NT-pro BNP(pg/ml)of heart failure group was significantly higher than that of non-heart failure group(3665(1709,4179)vs 61(48.5,77.8),P<0.05).In heart failure group,the median of NT-pro BNP(pg/ml)in HFr EF subgroup was significantly higher than that in HFmr EF subgroup and HFp EF subgroup(3832(2630,8783.8)vs 2032.5(1541.8,3264.8),2212(1355,4179),P<0.05);there was no significant difference in NT-pro BNP between HFmr EF subgroup and HFp EF subgroup(2032.5(1541.8,3264.8)vs 2212(1355,4179),P≥0.05).The relative miR-451 expression in heart failure group was significantly higher than that in non-heart failure group(3.44±0.4vs 1.0±0.32,P<0.05).The miR-451 expression in patients with IV grade of cardiac function was significantly higher than that in patients with III grade and II grade(5.5±1.0 vs 2.55±0.42,3.03±0.56,P < 0.05).However,there was no significant difference in miR-451 concentration between II and III subgroups(2.55±0.42 vs3.03±0.56,P≥0.05).In DOX-induced acute cardiomyopathy,miR-451 expression in cardiomyocytes was significantly up-regulated,and the miR-451 expression in H9c2 cardiomyocytes was significantly up-regulated after DOX inducement,and the difference was statistically significant(P<0.05).Part Ⅱ: according to myocardial injury markers(cTnI,CK,NT-proBNP)in serum,miR-451 gene inhibition could reverse acute DOX-induced myocardial injury(P<0.05),and miR-451 gene inhibition could significantly reduce the decrease of body weight,heart weight/tibia length induced by DOX(P<0.05).miR-451 gene inhibition could reverse the decrease of cardiac function indexes such as DOX-induced +dp/dt,-dp/dt and ejection fraction(EF)(P<0.05).miR-451 gene inhibition could reduce the severity of DOX-induced oxidative stress,which showed that miR-451 gene inhibition could decrease the concentrations of 4-HNE,MDA and ROS,while increase the concentrations of SOD and GSH/GSSG(P<0.05).Under HE staining,miR-451 gene inhibition could reduce the injury of cardiomyocytes induced by DOX.Western blot and TUNEL staining showed that miR-451 gene inhibition could reduce cardiomyocytes apoptosis induced by DOX,such as apoptosis-related proteins(up-regulated Bcl-2 and down-regulated Bax),and decreased Caspase-3activity and enhanced cell activity(P<0.05).Part Ⅲ: in vivo and in vitro experiments both showed that miR-451 gene inhibition could significantly up-regulate the expression of target protein CAB39,increase the phosphorylation of AMPKα,and inhibit the phosphorylation of m TOR and p70S6 K.When CPC was added to H9c2 cardiomyocytes to inhibit AMPK activity,the cytoprotective effect of miR-451 gene inhibition disappeared,cell activity decreased,AMPKα phosphorylation decreased,while phosphorylation of m TOR and p70S6 K increased.Conclusion:1.The miR-451 expression in serum of patients with AHF was up-regulated,which was related to the severity of cardiac function,and the miR-451 expression was up-regulated in DOX-induced acute cardiomyopathy and H9c2 cardiomyocytes.2.MiR-451 gene inhibition can reduce the DOX-induced acute myocardial injury and improve cardiac function.miR-451 gene inhibition can reduce the severity of DOX-induced oxidative stress and reduce apoptosis.3.MiR-451 gene inhibition can up-regulate the expression of CAB39,increase the phosphorylation of AMPKα,inhibit the phosphorylation of m TOR and p70S6 K,and protect the cell activity.The functional deficiency of AMPKα can reverse the phosphorylation of AMPKα inhibited by miR-451 gene and up-regulate the phosphorylation of downstream proteins.