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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

Gene expression analysis.(A) Assessment of ventricular mRNA expression by quantitative real-time PCR of atrial natriuretic peptide (Nppa, upper panel) and β-myosin heavy chain (Myh7, lower panel). n ≥ 6 for control (CTL, filled bars), n ≥ 6 for DKO (open bars) mice, **p < 0.01, *p < 0.05. (B, C) Genome-wide assessment of ventricular mRNA expression by Illumina bead chip array analysis in sham- and TAC-operated CTL and DKO mice. n = 4 for each genotype under sham conditions, n = 6 for each genotype under TAC conditions. (B) Pie charts indicate the number of up- and downregulated genes between genotypes under sham conditions (left pie chart) and under TAC conditions (right pie chart). The Venn diagram displays the number of genes regulated both under sham- and TAC-conditions and the overlapping proportion. (C, D) Heatmaps of the differentially regulated genes. Individual expression values are displayed after unsupervised complete linkage clustering. (C) Genes differentially regulated between CTL and DKO under sham conditions. (D) Genes with differential regulation between CTL and DKO genotypes only under TAC conditions.
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pone.0131019.g006: Gene expression analysis.(A) Assessment of ventricular mRNA expression by quantitative real-time PCR of atrial natriuretic peptide (Nppa, upper panel) and β-myosin heavy chain (Myh7, lower panel). n ≥ 6 for control (CTL, filled bars), n ≥ 6 for DKO (open bars) mice, **p < 0.01, *p < 0.05. (B, C) Genome-wide assessment of ventricular mRNA expression by Illumina bead chip array analysis in sham- and TAC-operated CTL and DKO mice. n = 4 for each genotype under sham conditions, n = 6 for each genotype under TAC conditions. (B) Pie charts indicate the number of up- and downregulated genes between genotypes under sham conditions (left pie chart) and under TAC conditions (right pie chart). The Venn diagram displays the number of genes regulated both under sham- and TAC-conditions and the overlapping proportion. (C, D) Heatmaps of the differentially regulated genes. Individual expression values are displayed after unsupervised complete linkage clustering. (C) Genes differentially regulated between CTL and DKO under sham conditions. (D) Genes with differential regulation between CTL and DKO genotypes only under TAC conditions.

Mentions: Expression of genes with known induction in heart hypertrophy and failure such as Nppa and Myh7 was assessed by qPCR (Fig 6A). These genes were analyzed as general markers of heart failure. Especially the Myh7 locus is completely demethylated in adult cardiomyocytes [7]. Thus, we did not expect differences in expression between genotypes. Both genes were strongly upregulated after pressure overload, but again no significant differences were noted between the genotypes. To gain a more detailed insight into genome-wide transcriptional changes, we performed mRNA array analysis for DKO and control mice, both for sham and TAC conditions. In the sham group, 44 genes were upregulated in DKO mice while 9 genes were downregulated compared to control mice (Fig 6B, left pie chart). Results after TAC operation were largely similar with 37 genes showing increased and 6 genes showing decreased expression in DKO mice (Fig 6B, right pie chart). Of these 43 genes, 26 (60%) were similarly regulated in both sham and TAC-operated mice (Fig 6B, Venn diagram). Next, genes that were regulated between genotypes under sham conditions were clustered together with respective gene expression values after TAC (Fig 6C). The resulting heatmap demonstrated that the genotype rather than the disease state had a strong influence on clustering. For those 17 genes that exhibited differential expression exclusively after TAC, a separate heatmap was generated (Fig 6C). All genes showed similar direction of regulation in sham mice.


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)

Gene expression analysis.(A) Assessment of ventricular mRNA expression by quantitative real-time PCR of atrial natriuretic peptide (Nppa, upper panel) and β-myosin heavy chain (Myh7, lower panel). n ≥ 6 for control (CTL, filled bars), n ≥ 6 for DKO (open bars) mice, **p < 0.01, *p < 0.05. (B, C) Genome-wide assessment of ventricular mRNA expression by Illumina bead chip array analysis in sham- and TAC-operated CTL and DKO mice. n = 4 for each genotype under sham conditions, n = 6 for each genotype under TAC conditions. (B) Pie charts indicate the number of up- and downregulated genes between genotypes under sham conditions (left pie chart) and under TAC conditions (right pie chart). The Venn diagram displays the number of genes regulated both under sham- and TAC-conditions and the overlapping proportion. (C, D) Heatmaps of the differentially regulated genes. Individual expression values are displayed after unsupervised complete linkage clustering. (C) Genes differentially regulated between CTL and DKO under sham conditions. (D) Genes with differential regulation between CTL and DKO genotypes only under TAC conditions.
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pone.0131019.g006: Gene expression analysis.(A) Assessment of ventricular mRNA expression by quantitative real-time PCR of atrial natriuretic peptide (Nppa, upper panel) and β-myosin heavy chain (Myh7, lower panel). n ≥ 6 for control (CTL, filled bars), n ≥ 6 for DKO (open bars) mice, **p < 0.01, *p < 0.05. (B, C) Genome-wide assessment of ventricular mRNA expression by Illumina bead chip array analysis in sham- and TAC-operated CTL and DKO mice. n = 4 for each genotype under sham conditions, n = 6 for each genotype under TAC conditions. (B) Pie charts indicate the number of up- and downregulated genes between genotypes under sham conditions (left pie chart) and under TAC conditions (right pie chart). The Venn diagram displays the number of genes regulated both under sham- and TAC-conditions and the overlapping proportion. (C, D) Heatmaps of the differentially regulated genes. Individual expression values are displayed after unsupervised complete linkage clustering. (C) Genes differentially regulated between CTL and DKO under sham conditions. (D) Genes with differential regulation between CTL and DKO genotypes only under TAC conditions.
Mentions: Expression of genes with known induction in heart hypertrophy and failure such as Nppa and Myh7 was assessed by qPCR (Fig 6A). These genes were analyzed as general markers of heart failure. Especially the Myh7 locus is completely demethylated in adult cardiomyocytes [7]. Thus, we did not expect differences in expression between genotypes. Both genes were strongly upregulated after pressure overload, but again no significant differences were noted between the genotypes. To gain a more detailed insight into genome-wide transcriptional changes, we performed mRNA array analysis for DKO and control mice, both for sham and TAC conditions. In the sham group, 44 genes were upregulated in DKO mice while 9 genes were downregulated compared to control mice (Fig 6B, left pie chart). Results after TAC operation were largely similar with 37 genes showing increased and 6 genes showing decreased expression in DKO mice (Fig 6B, right pie chart). Of these 43 genes, 26 (60%) were similarly regulated in both sham and TAC-operated mice (Fig 6B, Venn diagram). Next, genes that were regulated between genotypes under sham conditions were clustered together with respective gene expression values after TAC (Fig 6C). The resulting heatmap demonstrated that the genotype rather than the disease state had a strong influence on clustering. For those 17 genes that exhibited differential expression exclusively after TAC, a separate heatmap was generated (Fig 6C). All genes showed similar direction of regulation in sham mice.

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