| Part Ⅰ: Effects and mechanism of LKB1 in cardiomyocyte proliferationBackground: Morbidity and mortality of heart failure are increasing worldwide,with significant impacts on individual,social and health care expenditures.Heart failure occurs while the heart cannot supply oxygen-rich blood to the body for energy consumption.Cardiac hypertrophy and myocardial infarction are two important reasons for heart failure.Cardiomyocytes death after myocardial infarction laid the foundation of heart failure.The low proliferation ability of cardiomyocytes is not enough to make up for the reduction of myocardial contractility caused by a large number of cardiomyocytes death.How to enhance the proliferation ability of cardiomyocytes is the focus of current research on heart regeneration.Liver kinase B1(LKB1)is a serine/ threonine kinase that plays an important role in biological processes such as cell proliferation,cell polarity and tumor inhibition.Previous studies have shown that the proliferation ability of cardiomyocytes gradually decreases over time,while the expression of LKB1 in the hearts gradually increases,and LKB1 is involved in the regulation of various types of cell proliferation,so we speculated that LKB1 may also be able to participate in the regulation of cell cycle of cardiomyocytes.Methods: 1.To study the effect of LKB1 expression on the proliferation of adult cardiomyocytes,we subcutaneously injected AAV9-LKB1-shRNA virus into C57/BL6 J neonatal mice on the second day after birth to reduce LKB1 expression in cardiomyocytes.We ligated the left anterior descending coronary artery to establish acute myocardial infarction(MI)models at 8 weeks old.The positive rates of Edu,Ki67 and pH3 in cardiomyocytes were detected by immunofluorescence staining,and the expression of cell cycle related genes was detected by qPCR.2.NRVMs were used as the research subjects to observe the AMPK and AMPK phosphorylation levels after LKB1 siRNA treatments.At the same time,AMPK phosphorylation inactivation plasmid was constructed.Under the condition of LKB1 overexpression in cardiomyocytes,AMPK WT plasmid and AMPK-T172 A mutant plasmid were transferred into NRVMs,respectively.Western blot(WB)was used to observe the expression of AMPK,and immunofluorescence staining was used to detect the expression of Ki67 and pH3 in cardiomyocytes.Results: 1.By constructing MI models and detecting cardiomyocyte proliferation 7 days after MI,we found that LKB1 knockdown promoted the positive rates of Edu,Ki67 and pH3 in cardiomyocytes,and promoted the expression of cell cycle related genes in cardiomyocytes.2.When LKB1 expression is decreased in cardiomyocytes,AMPK phosphorylation level is significantly decreased.Meanwhile,transfection of AMPK-T172 A mutant plasmid partially reversed LKB1 overexpression mediated Ki67 and pH3 down-regulation.Conclusion: 1.The expression of LKB1 is negatively correlated with the proliferation ability of cardiomyocytes.2.LKB1 regulates the proliferation of cardiomyocytes mainly through AMPK phosphorylation,AMPK phosphorylation inactivated can reverse LKB1 OE caused the reduction of cardiomyocyte proliferation.Part Ⅱ: Effects and mechanism of methyltransferase PCIF1 in cardiac hypertrophyBackground: When heart was exposed to chronic stress stimuli,heart initially reacts compensatory by increasing its volume,thickening ventricular wall,normalizing the wall stress and maintaining the normal ejection function.Over time,chronic stimuli eventually developed into ventricular dilatation and decreased systolic function,and lead to heart failure.Current studies hold the view that during the process of heart failure,cardiac hypertrophy plays a fundamental role in the pathological features of heart failure.Lengthening of single cardiomyocyte progresses to ventricular dilatation and wall thinning,ultimately leading to heart failure with preserved or reduced ejection fraction(HFpEF,HFrEF).Understanding the mechanism of occurrence and development of cardiac hypertrophy will help us to intervene the process of cardiac hypertrophy in the early stage and find new therapeutic methods to reverse the process of cardiac hypertrophy and prevent the occurrence of bad endings.Epigenetic modification of RNA is an important research field that has received extensive attention in recent years,and it is involved in various biological processes of the occurrence and development of diseases.At present,it has been reported that RNA modification m6A is involved in the occurrence and development of cardiac hypertrophy,but whether m6Am is involved in the regulation of cardiac hypertrophy has not been studied.Our early studies found that m6Am was significantly changed after cardiac hypertrophy.PCIF1,the only enzyme found to catalyze m6Am formation in mammals,also changed significantly after cardiac hypertrophy.Therefore,we proposed that PCIF1 may involve in the regulation of cardiac hypertrophy through the function of catalyzing m6Am formation.This study took PCIF1 as our starting point to figure out the correlation between cardiac hypertrophy and RNA modification of m6Am,and explore the possibility of new therapeutic intervention targets for cardiac hypertrophy.Methods: Chapter 11.1.We first constructed mice models of cardiac hypertrophy,taking C57/BL6 mice as our research objects,and performed transverse aortic coarctation(TAC)surgeries at 8 weeks.4 weeks after operations,mRNAs of hearts were extracted and purified to obtain m7G-enriched RNA fragments,and then dot blots were performed to measure the level of m6Am.1.2.C57/BL6 mice were selected as the research objects,TAC surgeries were conducted at 8 weeks,and the expression of PCIF1 in mice with cardiac hypertrophy was detected by extracting total RNA and protein at 4 weeks after operation.Neonatal rat ventricular myocytes(NRVMs)were taken as the research object and treated with PE serum-free for 48 hours to induce cardiomyocyte hypertrophy.Then,the expression of PCIF1 in NRVMs after hypertrophy was detected by extracting cell RNA and protein.1.3.NRVMs were used as the research objects,using PCIF1 siRNA to reduce the expression of PCIF1 and detected the levels of ANP,BNP and β-MHC,and measured the areas of cardiomyocytes by immunofluorescence staining.At the same time,the levels of ANP,BNP in cardiomyocytes were observed after PE treated on the premise of decreased PCIF1 expression,and the areas of cardiomyocytes were measured by immunofluorescence staining as well.1.4.NRVMs were used as the research objects,using PCIF1 overexpression vector to increase the expression of PCIF1 and detected the levels of ANP,BNP and β-MHC,and measured the areas of cardiomyocytes by immunofluorescence staining.At the same time,the levels of ANP,BNP in cardiomyocytes were observed after PE treated on the premise of increased PCIF1 expression,and the areas of cardiomyocytes were measured by immunofluorescence staining as well.Chapter 2 2.1.In order to clarify the effect of PCIF1 on cardiac hypertrophy in vivo,we treated C57/BL6 J newborn mice on the second day of life with AAV9-cTNT-PCIF1-shRNA virus subcutaneously to reduce PCIF1 expression in mice hearts.When mice were 8 weeks old,TAC surgeries were performed,4 weeks post-operation,cardiac function was observed and the areas of cardiomyocytes were measured by WGA staining.2.2.We increased the expression of PCIF1 in hearts by subcutaneous AAV9-cTnT-PCIF1-OE virus in C57/BL6 J newborn mice on the second day of life.When mice were 8 weeks old,TAC surgeries were performed to induce cardiac hypertrophy.The cardiac function of mice was observed and the areas of cardiomyocytes were measured by WGA staining at 4 weeks postoperations.Chapter 33.1.The adenovirus which inactivated by mutation of PCIF1 methylase active site constructed,and NRVMs were used as the research objects,then NRVMs were treated with PE.After 24 hours,the PCIF1 overexpression plasmid and the mutant adenovirus were transfected into NRVMs.The levels of ANP,BNP and β-MHC in cardiomyocytes were detected and the areas of cardiomyocytes were observed by immunofluorescence staining.3.2.In order to clarify the regulation of PCIF1 on cardiac hypertrophy and screen out the key genes mediating PCIF1 regulation on cardiac hypertrophy,we used adenovirus knockdown of PCIF1 expression on NRVMs,and then performed transcriptome sequencing.3.3.To clarify the changes in m6Am modification of downstream genes caused by PCIF1 knockdown,we treated NRVMs with adenoviruses that knocked down PCIF1 expression,isolated and purified cap-rich mRNA fragments,and performed m6Am-seq.3.4.The downstream target molecule Adora1 was screened by RNA-seq and m6Am-seq,and the changes in the areas of cardiomyocytes were observed by intervening the expression of Adora1 in cardiomyocytesResults: Chapter 1:1.1.After cardiac hypertrophy,the level of m6Am in the heart was significantly reduced,suggesting that cardiac hypertrophy causes a reduction in m6Am modification.1.2.In the C57/BL6 mice cardiac hypertrophy models,mRNA and protein levels of PCIF1 in the heart tissue were significantly decreased.1.3.In NRVMs hypertrophy models induced by PE,mRNA and protein levels of PCIF1 in cardiomyocytes were significantly decreased.1.4.After PCIF1 siRNA treatments,the expression of PCIF1 in NRVMs was decreased,which led to an increase in ANP,BNP and β-MHC,and the immunofluorescence showed that the areas of NRVMs were increased.Under the precondition of reduced expression of PCIF1,the expression of ANP,BNP in NRVMs induced by PE increased more significantly,and the areas of cardiomyocytes were further increased.1.5.After treatments of NRVMs with PCIF1 OE plasmid,the expression of PCIF1 in cardiomyocytes was increased,resulting in a certain degree of decrease in ANP,BNP and β-MHC in cardiomyocytes,but the immunofluorescence showed that the areas of cardiomyocytes did not change significantly.However,under the precondition of increased expression of PCIF1,the expression of ANP,BNP decreased significantly and the areas of cardiomyocytes were significantly decreased after PE induced.Chapter 2:2.1.The expression of PCIF1 in the hearts of AAV9-cTnT-PCIF1 shRNA virus treated mice was decreased.After TAC surgeries,the cardiac function of AAV9-cTNT-PCIF1 shRNA virus group was further deteriorated compared with the control group,accompanied by the increase of cardiomyocyte cell areas.2.2.The expression of PCIF1 in the hearts of AAV9-cTnT-PCIF1 OE virus treated mice was increased.After TAC operations,the cardiac function of AAV9-cTNT-PCIF1 OE virus group was restored to a certain extent,accompanied by the reduction of cardiomyocyte cell areas compared with the control group.Chapter 3:3.1.After PE induced cardiac hypertrophy,if the activity of PCIF1 methylase disappeared,overexpression of PCIF1 could not reverse cardiac hypertrophy,suggesting that PCIF1-mediated cardiac hypertrophy is mainly through the action of PCIF1 methylase,that is,catalytic formation of m6Am is a key factor of PCIF1-regulated cardiac hypertrophy.3.2.Transcriptome sequencing were performed after down-regulation of PCIF1 expression in NRVMs,a large number of up-regulated genes were enriched in molecular functions and signaling pathways related to cardiac hypertrophy present in Gene Ontology(GO)analysis.3.3.m6Am-seq results showed that the m6Am level of mRNA in cardiomyocytes decreased after the reduction of PCIF1 expression.KEGG results showed that the differentially expressed genes were enriched in hypertrophy-related signaling pathways.3.4.The downstream target molecule Adora1 was screened by RNA-seq and m6Am-seq.By interfering with the expression of Adora1 in NRVMs,it was found that activation of Adora1 expression could reverse PCIF1 siRNA-induced cardiac hypertrophy.Conclusion:1.RNA m6Am modification was significantly reduced after cardiac hypertrophy,and PCIF1,as a key enzyme catalyzing m6Am formation,was significantly reduced after cardiac hypertrophy.It referred to that PCIF1 was involved in the regulation of cardiomyocyte hypertrophy.When PCIF1 expression decreased,cardiomyocyte hypertrophy is obvious.When PCIF1 overexpressed,PE induced cardiomyocyte hypertrophy can be reversed.2.In vivo,decreased expression of PCIF1 aggravates TAC-induced cardiac hypertrophy and leads to further deterioration of cardiac function,while increased expression of PCIF1 can reverse TAC-induced cardiac hypertrophy and restore cardiac function to a certain extent.3.Adora1,as a key influencing factor,participates in the process of PCIF1-mediated cardiac hypertrophy. |
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