MiR-155 Mediates Myocardial Metabolism Remodeling Induced By Chronic Hypoxia

Hypoxia refers to the pathological process of abnormal changes in the metabolism,function and morphological structure of tissues due to insufficient oxygen supply or oxygen barriers in tissues.Numerous studies have shown that hypoxia can lead to ventricular remodeling.Cardiomyocyte hypertrophy,cardiac fibroblast proliferation,interstitial fibrosis,and myocardial cell death are the main pathological features of ventricular remodeling.The systematical research of chronic hypoxia on cardiac remodeling has been carried out,partially revealing the pathophysiological mechanism of myocardial structural remodeling and electrical remodeling caused by hypoxia.Many researches indicate that metabolic remodeling（also known as metabolic reprogramming）plays an important role in cardiac remodeling induced by hypoxia,but the exact mechanism of metabolic remodeling caused by hypoxia is not well understood.microRNA（miRNA）is a non-coding small RNA that regulates post-transcriptional expression of gene.Studies have shown that a variety of miRNAs play an important role in metabolic remodeling.As a member of the miRNA family,miR-155 has been reported to be involved in the regulation of inflammatory responses to cardiovascular diseases and participated in the regulation of vascular remodeling.However,the molecular mechanism of miR-155 in myocardial metabolic remodeling caused by hypoxia has not been reported.Therefore,in this study,we focused on the role of miR-155 in ventricular metabolic remodeling and its molecular mechanism.Part one Establishment of a model of myocardial metabolic remodeling induced by chronic hypoxiaObjective:To investigate cardiac structure and function induced by chronic hypoxia.Methods:1.Echocardiographic assessment was used to detect changes in cardiac function in normal mice and hypoxia mice.2.Heart HE staining was used to observe the heart morphology of normal and hypoxia mice.3.Collagen content in the heart was detected by sirius red staining andα-actin and Col1a2 immunofluorescence assay.4.The expression of glucose transfer protein-1（GLUT1）mRNA and protein was detected respectively by qRT-PCR and Western Blot.5.The content of lactic acid and pyruvic acid was detected by chromatography-mass assay.Results:1.Chronic hypoxia induced cardiac hypertrophy in miceCompared with normal mice,hypoxia induced the increase of the heart rate of mice after hypoxia.Meantime,left ventricular systolic and diastolic inner diameter decreased in the hypoxia group.2.Chronic hypoxia led to cardiac injuryFormation of cardiac myocytes was observed with Hematoxylin and Eosin staining.Swelling of cardiac myocytes occurs during chronic hypoxia.Cardiac myocytes appeared vacuolar degeneration and the intercellular space expanded after hypoxia.This indicated that hypoxia led to cardiac injury.3.Chronic hypoxia led to myocardial fibrosisMyocardial fibrosis is abnormal hyperplasia of collagen in cardiac interstitial,which attenuates severly cardiac function.Collagen of the heart was stained by Sirius red to observe the effects of the hypoxia on myocardial fibrosis.Sirius red staining and collagen type Iα2 chain immunofluorescence assay showed that the collagen content in the heart tissue increased after hypoxia,which indicated that hypoxia induced myocardial fibrosis.4.Chronic hypoxia promoted glucose uptake in myocardial tissueGlucose transporter 1（GLUT1）can indirectly represent glucose uptake.The expression and translocation of GLUT1 expression were detected by qRT-PCR,WB and immunofluorescence assay,respectively.The results showed that was hypoxia increased expression and translocation of GLUT1.5.Chronic hypoxia promoted glycolysis in myocardial tissueThe content of pyruvate and lactic acid in the heart tissue of normal and hypoxia mice was detected by liquid chromatography-mass spectrometry.The results showed that the content of pyruvate and lactic acid in the heart tissue of mice after hypoxia increased,indicating that the utilization of glucose was changed from aerobic oxidation to glycolysis.Summary:Chronic hypoxia induces ventricular structural changes,promotes glycolysis in heart tissue.Part two miR-155 mediates cardiac fibroblast metabolism reprogra- mming induced by hypoxiaObjective:To investigate the role and molecular mechanism of miR-155on metabolism reprogramming induced by chronic hypoxia in cardiac fibroblast.Methods:1.The effect of hypoxia on the proliferation of cardiac fibroblasts was detected by MTT assay.2.Glucose uptake capacity was detected by glucose uptake assay after hypoxia in cardiac fibroblast.3.Western Blot was used to detect the expression of GLUT1 in the normoxia group and hypoxia group.4.LC-MS method was used to detect the concentration of pyruvic acid and lactic acid in normal and hypoxic cell culture medium.5.Real-time PCR was used to detect the expression of miR-155 in cells after hypoxia.6.Cardiac fibroblasts in miR-155^WT and miR-155^-/-mice were cultured in hypoxia or normoxia,and qRT-PCR was used to detect the expression of GLUT1,PDK1,PKM2 and HK.7.Glycogen staining was used to detect the effect of hypoxia on the accumulation of glycogen in miR-155^WT and miR-155^-/-mice.Results:1.Chronic hypoxia promoted proliferation of mouse cardiac fibroblasts.Primary mouse cardiac fibroblasts were treated with normoxia and hypoxia,respectively.MTT results showed that hypoxia promoted the proliferation of mouse cardiac fibroblasts.2.Chronic hypoxia promoted glucose uptake in cardiac fibroblasts.Glucose uptake experiments have shown that chronic hypoxia promotes uptake of glucose by cardiac fibroblasts.In addition,the expression of mRNA and protein of GLUT1 was also significantly increased in the hypoxia group,further suggesting that hypoxia promotes glucose uptake by cells.3.Chronic hypoxia promotes glycolysis in cardiac fibroblasts.The concentration of pyruvic acid and lactic acid in the hypoxic cardiac fibroblast medium was increased using LC-MS assay,which suggesting that the utilization of glucose induced by hypoxia is converted from aerobic oxidation to glycolysis.To further verify the above results,we examined the expression of the key enzymes of glycolysis metabolism,such as,PDK1,PKM2,and hexokinase.The results showed that hypoxia significantly induced the expression of PDK1,PKM2 and hexokinase in cardiac fibroblasts.4.Hypoxia down-regulated the expression of miR-155 in cardiac fibroblasts.To further study the molecular mechanism of hypoxia-induced metabolic reprogramming of cardiac fibroblasts,primary mouse cardiac fibroblasts were cultured under normoxia or hypoxia,and the expression of miR-155 was detected by Real-time.The results showed that hypoxia significantly reduced the expression of miR-155 in cardiac fibroblasts.5.Knockout the expression of miR-155 promoted the proliferation of cardiac fibroblasts.To further clarify the role of miR-155 in hypoxia-induced cardiac fibroblast metabolism reprogramming,cardiac fibroblasts of miR-155^WT and miR-155^-/-mice were cultured in hypoxia or normoxia condition.MTT assay was used to detect the proliferation of miR-155^WT and miR-155^-/-cardiac fibroblasts after hypoxia.The results showed that knockout the expression of miR-155 promoted cell proliferation in the absence of oxygen compared to the control group.6.Knock out the expression of miR-155 promoted uptake of glucose in cardiac fibroblasts.We further investigated relationship between cardiac fibroblast prolif-eration and glucose uptake induced by miR-155.The results showed that knockout the expression of miR-155 significantly increased glucose uptake and GLUT1 expression after hypoxia in cardiac fibroblast.7.Knock out the expression of miR-155 reduced glycogen synthesis and enhanced glycolysis in cardiac fibroblasts.Glycogen accumulation in the miR-155^-/-mice was reduced after hypoxia compared with the control group;qRT-PCR results showed that knockout the expression of miR-155 promoted the expression of PDK1,PKM2,HK,etc.after hypoxia.Summary:Chronic hypoxia promoted the proliferation of cardiac fibroblasts and down-regulated the expression of miR-155 in cardiac fibroblasts.Knockout the expression of miR-155 enhanced myocardial fibroblast proliferation and glycolysis after chronic hypoxia.Conclusion:1.Chronic hypoxia down-regulated the expression of miR-155 in cardiac fibroblasts.2.Knock out the expression of miR-155 promoted metabolism reprogramming induced by hypoxia in cardiac fibroblast.