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Background: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes., Methods: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward., Results: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding., Conclusion: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

Background and Objective: The progesterone receptor (PR) is a ligand-activated transcription factor existing in two isoforms, A (PRA) and B (PRB), resulting from alternative promoter usage. It has long been speculated that genetic variants of PR are associated with the risk for various benign and malignant diseases, but data from clinical trials and in-vitro studies remain contradictory. The most extensively studied variant is termed PROGINS and consists of an intronic 320-bp Alu insertion and two coding (Ser344Thr, Val660Leu) and one silent single nucleotide polymorphism in complete linkage disequilibrium (allele frequency in Caucasians 9-19%). Our study aimed at elucidating the functional consequences of the PROGINS-associated single nucleotide polymorphisms of PRA and PRB (i.e. Thr344 and Leu660) as compared with wild-type PR (Ser344, Val660)., Methods: The two PRA and two PRB full-length receptor variants were expressed by adenovirus in the PR-negative human breast cancer cell line T47D-Y and assayed with respect to transactivational properties, c-src activation, combined net mRNA and protein stability and hormone-binding characteristics., Results: In all experiments the wild-type PR and the PROGINS variant were undistinguishable., Conclusion: Though there still might be tissue specific effects of the variants, our data indicate that these common PR variants do not functionally differ, which may provide a basis to explain the heterogeneous outcome of association studies.