| Background:Myocardial infarction is one of the cardiovascular diseases with highest morbidity and mortality,which seriously affects human health and increases social economic burden.In China,the prevalence of myocardial infarction is as high as 27% in people over 60 years.Myocardial infarction is resulted from myocardial ischemia and hypoxia caused by atherosclerotic plaque,thrombus and embolism,resulting in the death of a large number of myocardial cells.Adult myocardium cannot be repaired by effective self-proliferation,so most heart functions continue to deteriorate and eventually develop into heart failure.Epidemiological studies show that about 26 million people worldwide suffer from heart failure,with a high mortality similar to that of malignant cancers.Current treatments for myocardial infarction can only delay the pathological progress,but cannot effectively repair the damaged myocardium and prevent the occurrence of terminal events.Classically,the mammalian hearts are terminal differentiated organ and have no regenerative capacity.With the development of research methods in recent years,myocardial regeneration has been proved,but the low regeneration efficiency is still an urgent problem to be solved.A representative study of the human heart has shown that cardiomyocytes renew at a very low rate of approximately 0.3% to 1% per year,suggesting the presence of endogenous cardiac regeneration,but this inefficient renewal is insufficient to produce clinically significant cardiac regeneration after cardiac injury.Therefore,to explore the mechanism of endogenous myocardial regeneration and to regulate it is the current research focus.Mammals still maintain strong myocardial proliferative ability in the early stage of life,but this ability declines sharply in the 7th day after birth.Simultaneously,the myocardial energy metabolism was transformed from glycolysis to fatty acid oxidation.The metabolic reprogramming time point was synchronized with the close of the myocardial proliferation window,indicating that the metabolic reprogramming may be a key factor affecting the myocardial proliferation ability.Recent studies have found that inhibiting fatty acid metabolism can promote glycolysis and cardiomyocyte proliferation,suggesting that metabolic mode plays an important role in regulating myocardial proliferation.However,how metabolic changes affect cardiac proliferation is unclear.Therefore,we focused on the spectral changes of metabolites caused by the metabolic conversion from glycolysis to fatty acid oxidation.Recent non-targeted metabolomics analysis found that the hearts of 1 day and7 days postnatal mice exist significant difference in metabolites,among them,α-ketoglutarate,α-KG)has attracted our attention due to its large variation and high content,which has been showed is important on promoting proliferation,for example: Exogenous α-KG can significantly promote the proliferation of embryonic stem cells.In addition,the application ofα-KG has been reported to improve the prognosis of cardiac dysfunction caused by stress load.However,it has not been reported whether α-KG affects the proliferation of cardiomyocytes.Therefore,the purpose of this study is to investigate whether α-KG acts as the main metabolite for interfering with energy metabolism to promote adult cardiomyocyte proliferation,to determine whether α-KG can promote post-infarction myocardial repair and regeneration,and to clarify its downstream mechanism.Methods:Part Ⅰ:1.To explore the role of α-KG in cardiomyocyte proliferation,we first treated cardiomyocytes with different final concentrations of α-KG(0.1m M,1m M,5m M),and then detected the cardiomyocyte proliferation with the proliferation marker Ki67 immunofluorescence staining 24 hours later.2.In cardiomyocytes,the Oxoglutarate Dehydrogenase(OGDH)catalyzes the generation of α-KG to Succinyl-Co A and carbon dioxide(CO2).In order to further explore the effect of endogenous α-KG on cardiomyocytes proliferation,we used si RNA to interfere with OGDH in cardiomyocytes,block the passage of α-KG,and increase its content in cardiomyocytes.Then the proliferation markers Ki67 and PH3 were detected by immunofluorescence staining.3.To determine the effect of α-KG on cardiomyocyte proliferation during cardiac development,we intraperitoneally injected α-KG(30mg/ KG)into 7-day old C57/BL6 J mice for two consecutive weeks.After two weeks,the heart-weight ratio of the mice was measured to compare the size of their hearts.The size of cardiomyocytes was compared by Wheat germ agglutinin(WGA)staining.Proliferation of cardiomyocytes was detected by incorporation of5-ethynyl-2’-deoxyuridine(Ed U).Part Ⅱ:1.α-KG as the coenzyme of Jumonji domain containing protein 3(JMJD3)can activate the activity of JMJD3 and specifically demethylase the trimethylation level of the 27 th lysine(H3K27)of H3 histone(H3K27me3).H3K27me3 is a marker of gene transcriptional inhibition,and its demethylation can activate gene transcription,while JMJD3 demethylase can specifically remove the methylation state of H3K27me3 in the promoter region of the gene,thus releasing the inhibitory effect.To investigate whether α-KG can also regulate the activity of JMJD3 in cardiomyocytes,we compared the expression level of H3K27me3 after treating cardiomyocytes with α-KG.To further clarify whether JMJD3 is a target for α-KG regulation of myocardial proliferation,we used si RNA and Succinate to interfere with and inhibit JMJD3 in cardiomyocytes respectively,and then observed the PH3 positive rate of cardiomyocytes with immunofluorescence staining.2.To clarify the association between the JMJD3 modified H3K27me3 and the cardiomyocyte proliferation,we targeted C57/BL6 J mice aged 1 day,7 days and 28 days,which indicated that the myocardial proliferation ability was gradually weakened.The protein expression of H3K27me3 in mouse heart was compared.3.After treating cardiomyocytes with α-KG,the effect of H3K27me3 on cell cycle regulation genes was detected by Chromatin immunoprecipitation-quantitative Polymerase Chain Reaction(Ch IP-q PCR),for further clarifying the effect of α-KG on the inhibition of H3K27me3 on cell cycle regulation genes.Part Ⅲ:1.To further explore whether α-KG can promote the proliferation in adult cardiomyocytes and promote myocardial repair after infarction,we established MI model in C57/BL6 J mice and injected-kg intraperitoneally every day for two weeks.After two weeks,heart functions were observed.The hearts were harvested and sectioned,and the degree of cardiac fibrosis after infarction was detected by Masson staining.The ability of myocardial proliferation was detected by Ed U incorporation.Angiogenesis was detected by immunofluorescence staining of CD31,a marker of vascular endothelial cells.Result:Part Ⅰ:1.After the treatment of cardiomyocytes with different final concentrations of α-KG(0.1mm,1m M,5m M),the ratio of ki67-positive cardiomyocytes increased significantly compared with the control group,especially when the concentration was 5m M.2.After knockdown of OGDH with si RNA in cardiomyocytes to increase endogenous levels of α-KG,the number of Ki67 and PH3 positive cardiomyocytes was significantly higher than that of the control group.3.Compared with the control group,the mice in the α-KG injection group had larger heart volume and increased heart-weight ratio,while the WGA staining showed no increase in cardiomyocyte size.These results suggest that α-KG injection can promote the cardiomyocyte proliferation in vivo without affecting cardiac hypertrophy.The ratio of Ed U-positive cardiomyocytes was increased by α-KG injection,which further indicated that α-KG could promote the proliferation of cardiomyocytes.Part Ⅱ:1.To investigate whether JMJD3 is the target in α-KG induced cardiomyocyte proliferation,we used si RNA to knock down JMJD3 in cardiomyocytes.After knocking down JMJD3,the number of PH3 positive cardiomyocytes decreased significantly compared with the control group.Moreover,after knocking down JMJD3,the pro-proliferative effect ofα-KG on cardiomyocytes was blocked.Similarly,the results of Succinate inhibition of JMJD3 were consistent with that of si RNA interference.It indicates that JMJD3 is a key molecule in the regulation of cardiac proliferation and a target molecule for promoting cardiac proliferation of α-KG.2.The cardiomyocyte proliferation of 1-day old mice was the strongest,and the cardiomyocyte proliferation of 7-day old mice was significantly decreased,and the cardiomyocyte proliferation of 28-day old mice was almost lost.The expression of H3K27me3 was the lowest in the myocardium of 1-day old mice,the relatively higher in the hearts of 7-day old mice,and the highest in the hearts of 28-day old mice,indicating that H3K27me3 may inhibit the proliferation of cardiomyocytes.3.Ch IP-q PCR results further demonstrated that H3K27me3 could be enriched in Cell cyclin B1(CCNB1),Cell cyclin D1(CCND1),Cell cycle dependent kinase 1(CDK1)and Cell cycle dependent kinase 4(CDK4).These cell cycle genes are important for cardiomyocyte proliferation.The enrichment of H3K27me3 on the promoter of cell cycle regulating gene was significantly reduced by the treatment with α-KG,thus the inhibition of H3K27me3 on the transcription expression of cell cycle gene was relieved.Part Ⅲ:1.Two weeks after myocardial infarction,the cardiac functions of α-KG treated group were significantly improved compared with that of the control group.Masson staining showed that the scar area of the α-KG group was reduced,CD31 immunofluorescence staining showed that the injection of α-KG promoted angiogenesis,and Ed U immunofluorescence staining showed that the injection of α-KG promoted the proliferation of cardiomyocytes.It suggested that α-KG could promote the proliferation of adult cardiomyocytes and promote post-infarction repair.Conclusion:α-KG as the main medium to intervene energy metabolism can promote adult cardiomyocyte proliferation.α-KG activates JMJD3 activity,enhances its demethylation of H3K27me3,and thus removes the inhibition of H3K27me3 on cell cycle regulating genes,and ultimately promotes the proliferation of adult cardiac cells,so as to repair myocardial infarction and prevent heart failure. |
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