The Effect Of Ten-Eleven Translocation-2(TET2) In Cardiac Hypertrophy

Part I Dynamic changes of DNA demethylation during postnatal cardiac developmentObjective:DNA demethylation is a process in which 5-methylcytosine(5mC)is catalyzed by the ten-eleven translocation(TET)protein to form 5-hydroxymethylcytosine(5hmC).5hmC is considered to be an epigenetic modification with biological function.However,the dynamic changes of 5hmC and TET in cardiac development are not yet clear,so we detect the 5hmC and TET level at different time points to know the dynamic changes of DNA demethylation during cardiac development.Methods:Cardiac tissues were obtained on postnatal day 1(P1),day 14(P14)and 8weeks(adult).DNA dot blot and immunofluorescence were used to detect the level of 5hmC at different point.The expression of TETs were detected by qRT-PCR and western blot.The level of 5hmC was detected by DNA dot blot after Tet2 knock out.Results:DNA dot blot and immunofluorescence showed that the level of 5hmC was increased from P1 to adult.The level of TET2 was the most highest among TET protein in cardiac tissues at P1 and adult,and the expression level of Tet2 was decreased from P1 to adult.The level of 5hmC was significantly decreased after Tet2 KO in P1 and adult cardiac tissues.Conclusion:DNA demethylation was dynamic during postnatal cardiac development.The level of 5hmC was increased with the development after birth.TET2 was the main enzyme in the formation of 5hmC in heart,and its level was decreased with development.Part ? Loss of Tet2 promotes cardiac hypertrophyObjective:Cardiac hypertrophy is the compensatory response caused by long-term pressure overload.It manifests as cardiomyocytes hypertrophy and cardiac fibrosis.The molecular mechanism of cardiac hypertrophy is still unclear.DNA demethylation is dynamic during postnatal cardiac development.It has been reported that the pattern of 5hmC modification changes in hypertrophic cardiomyocyte,suggesting that 5hmC is associated with cardiac hypertrophy.TET2 is the main enzyme responsible for the formation of 5hmC in the heart may participate in the occurrence of cardiac hypertrophy.This study investigated the effect of TET2 on cardiac hypertrophy.Methods:We used adult wild type(WT)and Tet2 knock out(KO)mice for experiment,treated with isoproterenol(ISO)to induce cardiac hypertrophy.The ratio of heart weight to body weight was compared in different groups.We used cardiac tissue paraffin sections for HE staining,Masson staining and WGA staining.The level of 5hmC was detected by DNA dot blot.The mRNA level of Myh7,Nppa,Nppb and Col1a2 were detected by qRT-PR.The cardiac function in each group was detected by echocardiographic.Neonatal mouse cardiomyocytes were isolated and used lentivirus to knonck down Tet2 combined with phenylephrine treated.Immunofluorescence was used to compare the cell area of each group.Results:The ratio of heart weight to body weight was increase in Tet2 KO mice when compared with the WT mice.Masson staining suggested cardia fibrosis in Tet2 KO mice.WGA staining suggested the cardiomyocytes area was increased in Tet2 KO mice.After ISO treated the ratio of heart weight to body weight,the area of fibrosis and cardiomyocytes was increased when compared with the WT control.The mRNA level of Myh7,Nppa,Nppb and Col1a2 was increased after Tet2 KO.The ejection fraction and fraction shortening was decreased after Tet2 KO.The decrease was obvious after ISO treated.The level of 5hmC was decreased in ISO-induced cardiac hypertrophic mice.After Tet2 knock down and treated with phenylephrine in vitro,the cardiomyocyte cell area was obvious when compared with control group.Conclusion:The level of 5hmC changes in cardiac hypertrophy.The loss of Tet2 promote cardiac hypertrophy.Part ? Mechanism of Tet2 Deletion Promote Cardiac HypertrophyObjective:The study mainly invested the mechanism of cardiac hypertrophy after Tet2 KO.Methods:Genomic sequencing of 5hmC was used to obtain the differential hydroxymethylated regions(DhMRs)of WT and Tet2 KO mice heart.GO and KEGG analysis were performed on DhMRs associated genes.RNA sequencing was performed on cardiac tissues of WT and Tet2 KO mice.Differentially expressed genes were analyzed by GO analysis.Combined with Genomic sequencing of 5hmC and RNA sequencing to obtain the common genes for GO analysis.The protein level of p-ERK was detect by western blot after Tet2 KO.The mRNA level and protein level of Hspalb were detected after Tet2 KO.The protein level of p-ERK was detect by western blot after overexpression of Tet2 or Hspalb in H9C2 cells.We used lentivirus to knock down Tet2 in neonatal mouse cardiomyocytes to detect the level of HSPA1B and p-ERK.Results:2957 WT DhMRs associated genes and 10554 Tet2 KO DhMRs associated genes was detected by genomic sequencing of 5hmC,of which 2556 genes overlapped.RNA sequencing showed that 226 genes were up-regulated and 292 genes were down-regulated after Tet2 KO.Among the genes related to Tet2 KO DhMRs associated genes,337 genes were altered,173 were up-regulated and 164 were down-regulated.The expression of cardiac hypertrophy related-genes is altered by 5hmC modification changes.The level of Tet2 could affect the activation of ERK1/2.The level of 5hmC of Hspalb was decreases after Tet2 KO,and the level of p-ERK decreases when Hspalb was overexpressed.After Tet2 knock down,the level of HSPA1B was decreased while p-ERK was increased.Conclusion:The changes of 5hmC is related to the occurrence of cardiac hypertrophy.After Tet2 deletion,the level of 5hmC in Hspalb was decreased which led to the decrease of its expression and ERK1/2 pathway activation.