The Effects And Mechanism Of Mm9_circ₀08009 On Cardiac Function And Impairment Of AGEs On Cardiomyocytes

Aims:In the mm9＿circ＿008009^+/-heterozygote mice model,this study aimed to observed the effect of mm9＿circ＿008009 on cardiac function.Methods:CRISPR/Cas9 gene editing technology was used to construct the mm9＿circ＿008009 knockdown animal model mouse(mm9＿circ＿008009^+/-).The above model mice（2 months old,3 months old,n=6）and wild type（WT）mice（2 months old,3 months old,n=6）were used for experiments.Doppler was used to evaluate the cardiac function.In addition,H&E and WGA staining were introduced for cardiac hypertrophy.Masson-trichrome staining method was used and myocardial collagen volume fraction（CVF）was calculated to observe the degree of myocardial fibrosis.Immunohistochemical staining was used to observe the expression of collagen type III（col-III）,and to evaluate myocardial fibrosis.Results:Mm9＿circ＿008009 knockdown mouse model was successfully constructed by CRISPR/Cas9 gene editing technology.Detected by Doppler echocardiography,the heart cavity of the 2-month-old mm9＿circ＿008009^+/-mice became larger than that of WT mice at the same week age,and the ejection fraction and fractional shortening of the mice were reduced to 67.6%and 36.6%,and the end diastolic volume and end systolic volume were increased;compared with WT mice of the same week age,the heart cavities of 3-month-old mm9＿circ＿008009^+/-mice was increased,and their ejection fraction and shortening fraction were reduced to 60.9%and 32.2%,and the end diastolic volume and end systolic volume were increased（p<0.05）.Mm9＿circ＿008009^+/-mouse heart function was impaired,manifested as dilated cardiac insufficiency.The morphology of cardiomyocyte hypertrophy was irregularly arranged in mm9＿circ＿008009^+/-mice compared with the WT group at the same week age detected by H&E staining.The data of WGA staining results The staining results also showed that the myocardial cells of mm9＿circ＿008009^+/-mice were significantly enlarged compared with the WT group,and they increased with time.Masson-trichrome staining results showed that compared with the WT group,mm9＿circ＿008009^+/-mice had increased CVF,increased extracellular matrix collagen fibers,and increased fibrosis with time.Immunohistochemical staining results showed that compared with the WT group,mm9＿circ＿008009^+/-mice showed increased expression of col-III and increased myocardial fibrosis with time.Conclusion:The down-regulation of mm9＿circ＿008009 in cardiac tissue promotes cardiac hypertrophy and causes the occurrence and development of congestive heart failure.Aims: To observe the effect of mm9_circ₀08009 on advanced glycaadvanced glycationend products（AGEs）-induced myocardial injury and explore its possible molecular mechanism.Methods: Neonatal mouse myocardial cells were cultured successfully.After adenovirus-mediated overexpression of mm9_circ₀08009,AGEs（200 μg/ml）were treated for 48 h.Western blot was used to detect changes in the expression of cell pyrolysis protein: NLRP3,Caspase-1,and IL-1β.Fluorescence in situhybridization（FISH）was used to detect the intracellular localization of mm9_circ₀08009 and mi R-471-3p.The luciferase gene reporting experiment was used to verify the binding between mm9_circ₀08009 and mi R-471-3p,mi R-471-3p and SIRT1.RT-q PCR was used to detect the expression of mm9_circ₀08009 and mi R-471-3p.Results: Western blot results showed that AGEs upregulate the expression of NLRP3,Caspase-1,and IL-1β in cultred neonatal mouse myocardial cells,and over-expression of mm9_circ₀08009 effectively inhibited the impairment of AGEs（200 μg/ml,24 h）on the cardiomyocytes.In mm9_circ₀08009+/-mouse heart tissue,mi R-471-3p expression is up-regulated.FISH detected that the expression of mm9_circ₀08009 and mi R-471-3p were colocalized in the cytoplasm.After adenovirus-mediated mm9_circ₀08009 knockdown /overexpression,mi R-471-3p was negatively correlated with its expression.The data of luciferase gene report experiment showed that mm9_circ₀08009 not bind mi R-471-3p,mm9_circ₀08009 might indirectly on mi R-471-3p,while mi R-471-3p could bind to SIRT1（p <0.001）.Conclusion: The down-regulation of mm9_circ₀08009 in cardiac tissue promotes cardiac hypertrophy and causes the occurrence and development of congestive heart failure.