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Cardiac Myocyte De Novo DNA Methyltransferases 3a/3b Are Dispensable for Cardiac Function and Remodeling after Chronic Pressure Overload in Mice.

Nührenberg TG, Hammann N, Schnick T, Preißl S, Witten A, Stoll M, Gilsbach R, Neumann FJ, Hein L - PLoS ONE (2015)

Bottom Line: Here, we tested whether cardiomyocyte-specific loss of de novo DNA methyltransferases Dnmt3a and Dnmt3b altered cardiac function and remodeling after chronic left ventricular pressure overload.DKO cardiomyocytes showed virtual absence of targeted Dnmt3a and Dnmt3b mRNA transcripts.The absence of cardiac pathology in the presence of the predicted molecular phenotype suggests that de novo DNA methylation in cardiomyocytes is dispensable for adaptive mechanisms after chronic cardiac pressure overload.

View Article: PubMed Central - PubMed

Affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg, Germany; Universitäts-Herzzentrum Freiburg • Bad Krozingen, Klinik für Kardiologie und Angiologie II, Bad Krozingen, Germany.

ABSTRACT

Background: Recent studies reported altered DNA methylation in failing human hearts. This may suggest a role for de novo DNA methylation in the development of heart failure. Here, we tested whether cardiomyocyte-specific loss of de novo DNA methyltransferases Dnmt3a and Dnmt3b altered cardiac function and remodeling after chronic left ventricular pressure overload.

Methods: Mice with specific ablation of Dnmt3a and Dnmt3b expression in cardiomyocytes were generated by crossing floxed Dnmt3afl and Dnmt3bfl alleles with mice expressing Cre recombinase under control of the atrial myosin light chain gene promoter. The efficacy of combined Dnmt3a/3b ablation (DKO) was characterized on cardiomyocyte-specific genomic DNA and mRNA levels. Cardiac phenotyping was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (TAC). Under similar conditions, cardiac genome-wide transcriptional profiling was performed and DNA methylation levels of promoters of differentially regulated genes were assessed by pyrosequencing.

Results: DKO cardiomyocytes showed virtual absence of targeted Dnmt3a and Dnmt3b mRNA transcripts. Cardiac phenotyping revealed no significant differences between DKO and control mice under sham and TAC conditions. Transcriptome analyses identified upregulation of 44 and downregulation of 9 genes in DKO as compared with control sham mice. TAC mice showed similar changes with substantial overlap of regulated genes compared to sham. Promoters of upregulated genes were largely unmethylated in DKO compared to control mice.

Conclusion: The absence of cardiac pathology in the presence of the predicted molecular phenotype suggests that de novo DNA methylation in cardiomyocytes is dispensable for adaptive mechanisms after chronic cardiac pressure overload.

No MeSH data available.


Related in: MedlinePlus

Reduced promoter CpG methylation and enhanced RNA expression.Left panels display the methylation levels of single CpGs in the promoter regions (TSS ± 500 bp) of indicated genes under sham conditions. The black bars below the genomic loci indicate the location of the respective CpGs analyzed by pyrosequencing. Right panels depict mRNA levels for the respective genes under sham and TAC conditions assessed by quantitative real-time PCR. (A) Aldehyde dehydrogenase 1 family, member L1 (Aldh1l1), (B) keratin 8 (Krt8) and (C) solute carrier family 9, subfamily A (Slc9a3). * p < 0.05, ** p < 0.01, *** p < 0.001.
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pone.0131019.g007: Reduced promoter CpG methylation and enhanced RNA expression.Left panels display the methylation levels of single CpGs in the promoter regions (TSS ± 500 bp) of indicated genes under sham conditions. The black bars below the genomic loci indicate the location of the respective CpGs analyzed by pyrosequencing. Right panels depict mRNA levels for the respective genes under sham and TAC conditions assessed by quantitative real-time PCR. (A) Aldehyde dehydrogenase 1 family, member L1 (Aldh1l1), (B) keratin 8 (Krt8) and (C) solute carrier family 9, subfamily A (Slc9a3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Mentions: In order to analyze the effect of Dnmt3a/3b ablation on DNA methylation, three genes that showed increased expression in DKO mice and had the highest additional increase after TAC, were selected as candidate genes for pyrosequencing. In analyses of sorted cardiomyocyte nuclei from sham-operated hearts, 9 out of 11 single CpG sites were significantly hypomethylated in DKO compared to control mice confirming the suppression of de novo DNA methylation (Fig 7, left panels). In line with the array analysis of sham mice, gene expression of Aldh1l1, Slc9a3 and Krt8 was significantly higher in DKO mice. Under TAC conditions, changes in gene expression were again in line with the array results (Fig 7, right panels).


Cardiac Myocyte De Novo DNA Methyltransferases 3a/3b Are Dispensable for Cardiac Function and Remodeling after Chronic Pressure Overload in Mice.

Nührenberg TG, Hammann N, Schnick T, Preißl S, Witten A, Stoll M, Gilsbach R, Neumann FJ, Hein L - PLoS ONE (2015)

Reduced promoter CpG methylation and enhanced RNA expression.Left panels display the methylation levels of single CpGs in the promoter regions (TSS ± 500 bp) of indicated genes under sham conditions. The black bars below the genomic loci indicate the location of the respective CpGs analyzed by pyrosequencing. Right panels depict mRNA levels for the respective genes under sham and TAC conditions assessed by quantitative real-time PCR. (A) Aldehyde dehydrogenase 1 family, member L1 (Aldh1l1), (B) keratin 8 (Krt8) and (C) solute carrier family 9, subfamily A (Slc9a3). * p < 0.05, ** p < 0.01, *** p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476733&req=5

pone.0131019.g007: Reduced promoter CpG methylation and enhanced RNA expression.Left panels display the methylation levels of single CpGs in the promoter regions (TSS ± 500 bp) of indicated genes under sham conditions. The black bars below the genomic loci indicate the location of the respective CpGs analyzed by pyrosequencing. Right panels depict mRNA levels for the respective genes under sham and TAC conditions assessed by quantitative real-time PCR. (A) Aldehyde dehydrogenase 1 family, member L1 (Aldh1l1), (B) keratin 8 (Krt8) and (C) solute carrier family 9, subfamily A (Slc9a3). * p < 0.05, ** p < 0.01, *** p < 0.001.
Mentions: In order to analyze the effect of Dnmt3a/3b ablation on DNA methylation, three genes that showed increased expression in DKO mice and had the highest additional increase after TAC, were selected as candidate genes for pyrosequencing. In analyses of sorted cardiomyocyte nuclei from sham-operated hearts, 9 out of 11 single CpG sites were significantly hypomethylated in DKO compared to control mice confirming the suppression of de novo DNA methylation (Fig 7, left panels). In line with the array analysis of sham mice, gene expression of Aldh1l1, Slc9a3 and Krt8 was significantly higher in DKO mice. Under TAC conditions, changes in gene expression were again in line with the array results (Fig 7, right panels).

Bottom Line: Here, we tested whether cardiomyocyte-specific loss of de novo DNA methyltransferases Dnmt3a and Dnmt3b altered cardiac function and remodeling after chronic left ventricular pressure overload.DKO cardiomyocytes showed virtual absence of targeted Dnmt3a and Dnmt3b mRNA transcripts.The absence of cardiac pathology in the presence of the predicted molecular phenotype suggests that de novo DNA methylation in cardiomyocytes is dispensable for adaptive mechanisms after chronic cardiac pressure overload.

View Article: PubMed Central - PubMed

Affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg, Germany; Universitäts-Herzzentrum Freiburg • Bad Krozingen, Klinik für Kardiologie und Angiologie II, Bad Krozingen, Germany.

ABSTRACT

Background: Recent studies reported altered DNA methylation in failing human hearts. This may suggest a role for de novo DNA methylation in the development of heart failure. Here, we tested whether cardiomyocyte-specific loss of de novo DNA methyltransferases Dnmt3a and Dnmt3b altered cardiac function and remodeling after chronic left ventricular pressure overload.

Methods: Mice with specific ablation of Dnmt3a and Dnmt3b expression in cardiomyocytes were generated by crossing floxed Dnmt3afl and Dnmt3bfl alleles with mice expressing Cre recombinase under control of the atrial myosin light chain gene promoter. The efficacy of combined Dnmt3a/3b ablation (DKO) was characterized on cardiomyocyte-specific genomic DNA and mRNA levels. Cardiac phenotyping was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (TAC). Under similar conditions, cardiac genome-wide transcriptional profiling was performed and DNA methylation levels of promoters of differentially regulated genes were assessed by pyrosequencing.

Results: DKO cardiomyocytes showed virtual absence of targeted Dnmt3a and Dnmt3b mRNA transcripts. Cardiac phenotyping revealed no significant differences between DKO and control mice under sham and TAC conditions. Transcriptome analyses identified upregulation of 44 and downregulation of 9 genes in DKO as compared with control sham mice. TAC mice showed similar changes with substantial overlap of regulated genes compared to sham. Promoters of upregulated genes were largely unmethylated in DKO compared to control mice.

Conclusion: The absence of cardiac pathology in the presence of the predicted molecular phenotype suggests that de novo DNA methylation in cardiomyocytes is dispensable for adaptive mechanisms after chronic cardiac pressure overload.

No MeSH data available.


Related in: MedlinePlus