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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

Cardiomyocyte-specific deletion of Dnmt3a and Dnmt3b.(A, D) Visualization of the Dnmt3a (A) and Dnmt3b (D) gene loci and protein domain structure. The prolylcysteinyl (PC) dipeptide providing thiolate at the active catalytic site is encoded in exon 18 of Dnmt3a (A) and in exon 19 of Dnmt3b (D). LoxP sites are represented by red triangles. Green (A) and blue (D) bars indicate intron-spanning primers for three different amplicons for determination of Dnmt3a (A) and Dnmt3b (D) transcripts. (B, E)Dnmt3a (B) and Dnmt3b (E) mRNA levels in embryonic stem (ES) cells (n = 3) and in cardiac tissue at the indicated time points (n = 4–5). (C, F)Dnmt3a (C) and Dnmt3b (F) mRNA levels in sorted cardiomyocytes. Dnmt3 copies/104Rps29 copies are visualized for the intron-spanning amplicons as indicated. n = 5 for control (CTL, filled bars), n = 4 for DKO (open bars) cardiomyocytes, *p < 0.05, ***p < 0.001.
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pone.0131019.g001: Cardiomyocyte-specific deletion of Dnmt3a and Dnmt3b.(A, D) Visualization of the Dnmt3a (A) and Dnmt3b (D) gene loci and protein domain structure. The prolylcysteinyl (PC) dipeptide providing thiolate at the active catalytic site is encoded in exon 18 of Dnmt3a (A) and in exon 19 of Dnmt3b (D). LoxP sites are represented by red triangles. Green (A) and blue (D) bars indicate intron-spanning primers for three different amplicons for determination of Dnmt3a (A) and Dnmt3b (D) transcripts. (B, E)Dnmt3a (B) and Dnmt3b (E) mRNA levels in embryonic stem (ES) cells (n = 3) and in cardiac tissue at the indicated time points (n = 4–5). (C, F)Dnmt3a (C) and Dnmt3b (F) mRNA levels in sorted cardiomyocytes. Dnmt3 copies/104Rps29 copies are visualized for the intron-spanning amplicons as indicated. n = 5 for control (CTL, filled bars), n = 4 for DKO (open bars) cardiomyocytes, *p < 0.05, ***p < 0.001.

Mentions: In a breeding of homozygous floxed Dnmt3afl and Dnmt3bfl mice with one paternal MLC2aCre allele, litters contained DKO mice that developed normally and were fertile. The loxP sites in the Dnmt3a and Dnmt3b genes were located up- and downstream of exon 18 or exon 19, respectively (Fig 1A and 1D). Expression of Dnmt3a and Dnmt3b showed distinct time courses in embryonic stem cells and in the developing heart (Fig 1B and 1E). Cardiac Dnmt3a expression peaked during embryonic development but remained strongly expressed in adult mouse hearts (Fig 1B), whereas Dnmt3b was almost exclusively expressed in embryonic stem cells (Fig 1E). The effectiveness of cardiomyocyte-specific ablation of Dnmt3a and Dnmt3b was determined at multiple levels. In order to validate the deletion of Dnmt3a and Dnmt3b on the transcriptional level, RNA was extracted from FACS-sorted cardiomyocytes of DKO and control mice. In cardiomyocytes, Dnmt3a mRNA levels at the deleted exon 18 were almost completely absent (-94%, p = 0.0004, Fig 1C). In addition, mRNA levels at exons upstream or downstream of the floxed exon 18 were significantly lower as well (Fig 1C). Dnmt3b expression in cardiomyocytes was at the lower limit of detection and thus further reduction by cardiomyocyte-specific knockout could not be detected (Fig 1F). In order to assess the frequency of recombination, cardiomyocyte nuclei were isolated and purified by FACS sorting (Fig 2A). After identification by 7-AAD staining (left panel), nuclei were classified into PCM-1- non-myocyte and PCM-1+ cardiomyocyte nuclei (middle panel). Purity of sorted cardiomyocyte nuclei was 94.0% ± 0.7%. The recombination frequency in sorted cardiomyocyte nuclei was 78.1% ± 3.9% at exon 18 of Dnmt3a and 76.5% ± 1.9% at exon 19 of Dnmt3b (Fig 2B).


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)

Cardiomyocyte-specific deletion of Dnmt3a and Dnmt3b.(A, D) Visualization of the Dnmt3a (A) and Dnmt3b (D) gene loci and protein domain structure. The prolylcysteinyl (PC) dipeptide providing thiolate at the active catalytic site is encoded in exon 18 of Dnmt3a (A) and in exon 19 of Dnmt3b (D). LoxP sites are represented by red triangles. Green (A) and blue (D) bars indicate intron-spanning primers for three different amplicons for determination of Dnmt3a (A) and Dnmt3b (D) transcripts. (B, E)Dnmt3a (B) and Dnmt3b (E) mRNA levels in embryonic stem (ES) cells (n = 3) and in cardiac tissue at the indicated time points (n = 4–5). (C, F)Dnmt3a (C) and Dnmt3b (F) mRNA levels in sorted cardiomyocytes. Dnmt3 copies/104Rps29 copies are visualized for the intron-spanning amplicons as indicated. n = 5 for control (CTL, filled bars), n = 4 for DKO (open bars) cardiomyocytes, *p < 0.05, ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131019.g001: Cardiomyocyte-specific deletion of Dnmt3a and Dnmt3b.(A, D) Visualization of the Dnmt3a (A) and Dnmt3b (D) gene loci and protein domain structure. The prolylcysteinyl (PC) dipeptide providing thiolate at the active catalytic site is encoded in exon 18 of Dnmt3a (A) and in exon 19 of Dnmt3b (D). LoxP sites are represented by red triangles. Green (A) and blue (D) bars indicate intron-spanning primers for three different amplicons for determination of Dnmt3a (A) and Dnmt3b (D) transcripts. (B, E)Dnmt3a (B) and Dnmt3b (E) mRNA levels in embryonic stem (ES) cells (n = 3) and in cardiac tissue at the indicated time points (n = 4–5). (C, F)Dnmt3a (C) and Dnmt3b (F) mRNA levels in sorted cardiomyocytes. Dnmt3 copies/104Rps29 copies are visualized for the intron-spanning amplicons as indicated. n = 5 for control (CTL, filled bars), n = 4 for DKO (open bars) cardiomyocytes, *p < 0.05, ***p < 0.001.
Mentions: In a breeding of homozygous floxed Dnmt3afl and Dnmt3bfl mice with one paternal MLC2aCre allele, litters contained DKO mice that developed normally and were fertile. The loxP sites in the Dnmt3a and Dnmt3b genes were located up- and downstream of exon 18 or exon 19, respectively (Fig 1A and 1D). Expression of Dnmt3a and Dnmt3b showed distinct time courses in embryonic stem cells and in the developing heart (Fig 1B and 1E). Cardiac Dnmt3a expression peaked during embryonic development but remained strongly expressed in adult mouse hearts (Fig 1B), whereas Dnmt3b was almost exclusively expressed in embryonic stem cells (Fig 1E). The effectiveness of cardiomyocyte-specific ablation of Dnmt3a and Dnmt3b was determined at multiple levels. In order to validate the deletion of Dnmt3a and Dnmt3b on the transcriptional level, RNA was extracted from FACS-sorted cardiomyocytes of DKO and control mice. In cardiomyocytes, Dnmt3a mRNA levels at the deleted exon 18 were almost completely absent (-94%, p = 0.0004, Fig 1C). In addition, mRNA levels at exons upstream or downstream of the floxed exon 18 were significantly lower as well (Fig 1C). Dnmt3b expression in cardiomyocytes was at the lower limit of detection and thus further reduction by cardiomyocyte-specific knockout could not be detected (Fig 1F). In order to assess the frequency of recombination, cardiomyocyte nuclei were isolated and purified by FACS sorting (Fig 2A). After identification by 7-AAD staining (left panel), nuclei were classified into PCM-1- non-myocyte and PCM-1+ cardiomyocyte nuclei (middle panel). Purity of sorted cardiomyocyte nuclei was 94.0% ± 0.7%. The recombination frequency in sorted cardiomyocyte nuclei was 78.1% ± 3.9% at exon 18 of Dnmt3a and 76.5% ± 1.9% at exon 19 of Dnmt3b (Fig 2B).

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