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 (hiPSC). Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue (EHT) from knockout hiPSC-derived cardiomyocytes. Methods: DNMT3A was knocked out in hiPSCs by CRISPR/Cas9 gene editing. Fibrin-based EHTs were generated from knockout (KO) and control hiPSC-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. EHTs were subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological and ultrastructural analyses were performed afterwards. 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 vs. wild-type. (2) Aberrant activation of the glucose/lipid metabolism regulator PPARγ was associated with accumulation of lipid vacuoles within KO cardiomyocytes. (3) HIF-1 protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering KO EHTs sensitive to metabolic stress such as serum withdrawal and restrictive feeding. Conclusions: 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.

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.