Background:
DNA methylation patterns influence transcription factor binding and chromatin remodelling and thereby transcriptional regulation. During pre- and perinatal development, cardiomyocytes (CMs) are able to proliferate. Thus, DNA methylation needs to be preserved during DNA replication. DNA methyltransferase 1 (Dnmt1) recognizes hemi-methylated DNA in cooperation with other proteins and maintains symmetrical methylation of DNA at CpG sites. Consequently, the aim of this study was to unravel the impact of Dnmt1 during heart development.

Methods:
Dnmt1 deletion in cardiomyocytes was achieved using the Cre-loxP system. Breeding mice inheriting the Cre recombinase under the control of the Myl7 promotor (Myl7-Cre). Myl7 is expressed in atrial and ventricular CMs. Embryonic hearts were collected at different timepoints during development (E9.5-P0). Morphological alterations were determined by electron microscopy and immunofluorescent staining. Functional properties were characterised by nanoindentation and sharp electrode approaches. Analyses of cardiomyocyte-specific features were succeeded with fluorescence activated nuclei sorting (FANS) and single nuclei RNA sequencing (snRNA-seq). Epigenetic signatures, like transcriptome and DNA methylation analyses were done with bulk RNA- (RNA-seq) and Whole genome bisulfite sequencing, respectively.

Results:
Deletion of the Dnmt1 gene in CMs is not compatible with life and led to a complete lethality around embryonic day E13.5. Global DNA methylation changed (WT 68 % vs KO 49 %) in CMs as well as the frequencies of fully and unmethylated DNA. Loss of DNA methylation in CMs is accompanying the derepression of endogenous repetitive elements especially intracisternal A particles (IAPs). IAPs could also detected in electron microscopy and qPCR. Due to the expression of these virus-like particles, yH2A.X-positive DNA double-strand breaks per CM increased in Dnmt1 deleted CMs (WT 13.9 % vs KO 26.9 %, p<0.0001). DNA damage response pathways like the p53 pathway activation could be detected resulting in cell cycle arrest and proliferation stop leading to a differentiation delay based on bulk and single nuclei transcriptome analysis. The loss of CMs, as a fraction of total nuclei, led to a compositional change of the ventricles (FANS: WT 45.2 % vs KO 24.2 % p<0.05 n=6; snRNAseq WT 48.3 % vs KO 32.0 %). Ventricles at E13.5 showed a thinner wall (control 64.9 ± 3.3 µm vs KO 40.2 ± 2.1 µm, p<0.001, n=8) and reductions of the compact area of the total heart area (WT 28.4 % vs KO 19.9 %, p<0.01, n=6/8). These anatomical changes led to functional alterations in terms of contraction and electrical behaviour. The amplitude of contraction is reduced in KO hearts compared to WT ones (WT 550 nm vs KO 200 nm n=10/7) and the action potential durations are shorter in KO hearts compared to WT ones (APD20: WT 68.8 ms vs KO 49.7 ms p<0.01, APD50: WT 96.3 ms vs KO 73.0 ms p<0.01, APD90: WT 117.0 ms vs KO 93.2 ms p<0.05, n=8/4).

Conclusion:
DNA methylation preservation in cardiomyocytes during embryogenesis is necessary for a complete heart development. Dnmt1 deletion is essential for a proper cell division, differentiation and functional behaviour of the hearts.