7DNA methylation patterns at promoters of hypertrophy-associated genes in an engineered heart tissue model of cardiac hypertrophy

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Objectives: Engineered Heart Tissue (EHT) can be used to investigate cardiomyocyte hypertrophy in vitro. In this model coherently beating rat cardiomyocytes in 3D tissue culture are acutely forced to work against an increased afterload. They subsequently display hypertrophy and functional and transcriptional alterations stereotypically associated with heart failure. In the present study we used the model to investigate DNA methylation patterns of cardiomyocyte hypertrophy. We assessed two different approaches: An unbiased microarray and bisulfite sequencing of promoter regions of genes associated with pathological hypertrophy.

Methods: EHTs were cultured on hollow silicone posts in a 24-well format for 2 weeks until maturation. Afterload was then acutely increased (“afterload enhanced”, AE) by inserting metal braces into the hollow silicone posts for one week. Additional EHTs were treated with phenylephrine to induce hypertrophy (PE), control EHTs were left untreated. Methylated DNA fragments were enriched by MBD2 pull-down and quantified by microarray. Data was evaluated and mapped using DEVA, R and DAVID software. Additionally, DNA from EHTs was sodium bisulfite treated to convert all non-methylated cytosines to thymidines. Promoter region fragments of Nppa, Nppb, Acta-1 and Atp2a2 (ANP, BNP, alpha skeletal muscle actin and Serca2a) and methylation positive and negativ controls Oxt and Ube2b were PCR amplified and subjected to next generation sequencing (NGS). Single-base resolution methylation data was extracted by Bismark software.

Results: In the microarray analysis PE and AE clustered together. Methylation changes among AE or PE and controls respectively mapped to pathways relevant for pathological hypertrophy. Further analyses identified previously unidentified differentially methylated regions and confirmed differential methylation in well-studied genes such as Serca2a, supporting the validity of the approach. Targeted NGS confirmed subtle but reproducible DNA methylation alterations in promoter regions of hypertrophy associated genes. A region at -3000 bp of the Serca2a transcriptional start site was identified as differentially methylated using the microarray approach and confirmed by NGS.

Conclusion: Exposing EHTs to enhanced afterload rapidly and specifically alters DNA methylation in cardiac myocytes. In vitro hypertrophy is accompanied by a common DNA methylation signature irrespective of the cause of hypertrophy. Its probable regulatory function and high plasticity suggest DNA methylation as a possible drug target in cardiac hypertrophy.

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