The Role And Regulation Mechanism Of MicroRNA-210 On The Energy Metabolism During H₂O₂ Induced Oxidative Stress Damage Of Cardiomyocytes

BackgroundIschemic heart disease?IHD?is a common health problem and one of the leading causes of cardiac dysfunction in the world.IHD is a common chronic disease that leads to myocardial ischemia,hypoxia,and even necrosis,posing a serious threat to human health.IHD manifests as oxidative stress in hypoxia which leads to apoptosis,autophagy and the energy metabolism shift.The energy metabolism shift,characterized by the repression of the tricarboxylic acid?TCA?cycle,mitochondrial electron transport,and oxidative phosphorylation?OXPHOS?and the enhancement of glycolysis,is a typical adaptive response under hypoxia-induced oxidative stress.The energy metabolism shift effectively improves the energy supply under oxidative stress.Therefore,an in-depth study of the myocardial energy metabolism shift under the conditions of oxidative stress is essential for the improved treatment and prognosis of IHD.The activities of proteins which regulate the mitochondrial energy metabolism are closely related to acetylation of the family of Sirtuins.SIRT3 locates in the mitochondrial matrix.SIRT3 is a deacetylase that regulates the majority process of the energy metabolism,such as IDH,SDH and MDH which are the enzymes of TCA cycle.Nicotinamide adenine dinucleotide?NAD⁺?has roles as cofactors during SIRT3-mediated deacetylation and it is a major factor affecting SIRT3 activity.The significance of ISC is that it is a functional cofactor for many enzymes and proteins.The iron-sulfur cluster assembly protein?ISCU?is a key chaperone for the assembly of cellular ISCs and their transportation to enzymes,such as the mitochondrial respiratory complexes?complexes I,II,and III?,which are responsible for mitochondrial respiration and energy production.Complex I is the key regulator to maintain the dynamic balance of NAD⁺/NADH.ISCU also transfers the mature ISC to the target proteins.Studies have found that the knockdown of ISCU affects the activity of complex I and causes the dysfunction of oxidative respiratory chain.MicroRNAs?miRNAs?are single-stranded,non-coding small molecule RNAs that inhibit translation or induce target molecule degradation.MiR-210 is a hypoxia-induced miR that plays an important role in the processes of apoptosis,autophagy,and angiogenesis.In our previous study,we found that the expression of mi R-210 in H9c2cardiomyocytes was up-regulated under H₂O₂ treatment.MiR-210 plays a role in protecting cardiomyocytes by inhibiting apoptosis and autophagy.However,the relationship between mi R-210 and cardiomyocyte energy metabolism shift during oxidative stress remains unclear.ObjectsIn this study,H9c2 cells were treated with H₂O₂ to establish a model of oxidative stress injury and observe the energy metabolism shift in cardiomyocytes.We take SIRT3 and ISCU as the study points aiming to investigate the effects of miR-210 on the energy metabolism shift of cardiomyocytes under oxidative stress injury and its mechanism,in order to provide new clues for the application of miR-210 in the diagnosis and treatment of cardiovascular diseases.Methods1.Cells were treated with H₂O₂?1 mM?or left untreated?controls?.Cell viability was detected by CCK.Cellular ATP and lactate were detected by assay kits to evaluate cellular energy metabolism.The expression of glucose metabolism genes was detected by PCR array.2.H9c2 cells were transduced with lentiviral vector containing miR-Scr and Pre-210.The expression of mi R-210 in cells was detected by qPCR.Cellular lactate of Pre- 210?miR-210 overexpression?group and control group was detected by assay kits. The effect of miR-210 on glucose metabolism genes was detected by PCR array.3.The expression of SIRT3 protein in Pre-210 group and control group were detected by Western Blot.The expression of SIRT3 mRNA in Pre-210 group and control group were detected by qPCR.The relative NAD⁺/NADH ratios and the activity of SIRT3 were detected by assay kits.4.H9c2 cells were transduced with lentiviral vector containing miR-Scr and Anti-210. The expression of mi R-210 in cells was detected by qPCR.The expression of ISCU and NDUFA9 protein in Pre-210 group,Anti-210?miR-210 inhibition?group and control group were detected by Western Blot.The expression of ISCU mRNA in Pre-210 group,Anti-210 group and control group were detected by qPCR.The activity of Complex?were detected by assay kits.Results1.H₂O₂ inhibited cell viability in a time-dependent manner.The expression of ATP showed a downward trend and lactate increased under H₂O₂ treatment,with the highest expression of lactate after 1 h of treatment.Cell viability and ATP levels decreased significantly in the H₂O₂?1mM,1 h?+2-DG?10 mM,1 h?group compared to those in the H₂O₂?1mM,1 h?group.Similar results were obtained for the lactate assay.We examined the expression of glucose metabolism genes using the RT2 profiler glucose metabolism PCR array.We found that four genes were significantly reduced following H₂O₂?1mM,1 h?treatment.Among these, glucokinase?GCK?,hexokinase 3?HK3?,and pyruvate kinase,liver and RBC ?PKLR?are glycolysis genes.2.We examined the relative expression of miR-210 by qPCR and found that H₂O₂ ?1mM,1 h?treatment up-regulated the expression of miR-210.Lactate levels were higher in the Pre-210 group,whether treated with H₂O₂?1mM,1 h?or not,than in the miR-Scr group.Four genes were found to be differentially expressed between the Pre-210 control group and the miR-Scr control group.At the same time,many genes were found to be differentially expressed between the Pre-210+H₂O₂?1mM, 1 h?group and the miR-Scr+H₂O₂?1mM,1 h?group.This included the upregulation of the glycolysis genes HK3 and phosphoglycerate kinase 2?PGK2?.While some glycogen metabolism genes were also upregulated,there were many more TCA cycle genes that were downregulated,such as isocitrate dehydrogenase 2?IDH2?, succinate dehydrogenase complex subunit A?SDHA?and malate dehydrogenase 1 ?MDH1?.Gluconeogenesis genes,such as Pck1?Pc were also were downregulated.3.Protein levels of SIRT3 decreased in the miR-Scr group with H₂O₂?1mM,1 h? treatment.They were also lower in the Pre-210 group,with or without H₂O₂?1mM, 1 h?treatment,than in the mi R-Scr group.Analysis of the mRNA expression of SIRT3 showed similar results.Furthermore,the relative levels of NAD⁺and its reduced form NADH served as an additional index of the expression of SIRT3.The relative NAD⁺/NADH ratios were consistent with the above results.The relative activity of SIRT3 decreased in the miR-Scr group under H₂O₂?1mM,1 h? conditions.Transduction with Pre-210 suppressed the activity of SIRT3 under H₂O₂ ?1mM,1 h?conditions,whereas there was no observed difference between the Pre- 210 control group and the miR-Scr control group.4.The mRNA expression of ISCU decreased in the Pre-210 group with or without H₂O₂?1mM,1 h?treatment and increased in the Anti-210 group under the same conditions compared with levels in the miR-Scr group.The protein levels of ISCU exhibited similar results.To further confirm the specificity of miR-210's regulation of ISCU,we tested the effect of miR-210 on an additional protein,NDUFA9. Neither transduction nor H₂O₂?1mM,1 h?treatment affected the protein levels of NDUFA9.Finally,in order to determine that miR-210 regulates mitochondrial respiration by inhibiting ISCU,we measured the activity of complex I.This was reduced in the Pre-210 group with or without H₂O₂?1mM,1 h?treatment and elevated in the Anti-210 group under the same conditions compared with levels in the miR-Scr group.ConclusionThe energy metabolism shift was occurring during H₂O₂-induced oxidative stress injury.MiR-210 promotes the energy metabolism shift under H₂O₂-induced oxidative stress injury:?1?MiR-210 promotes the expression of glycolytic genes and inhibits the expression of gluconeogenesis and TCA cycle-related genes;?2?MiR-210 indirectly reduced the expression and activity of SIRT3 and inhibited the function of TCA cycle;?3?MiR-210 inhibited the function of mitochondrial oxidative respiratory chain by inhibiting the expression of ISCU.Innovation and significanceIn this study,we investigated the effect of miR-210 on myocardial energy metabolism shift under oxidative stress injury.We also screened the glucose metabolism genes which may be regulated by miR-210 under oxidative stress injury by PCR array.This study provides new experimental evidences for myocardial oxidative stress injury.And it provides new theoretical basis for the occurrence and development of cardiovascular diseases from the point of myocardial energy metabolism shift.It also provides new ideas for promoting the application of miR-210 in clinical diagnosis and treatment.