Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.

CS glycosaminoglycans accumulate during cardiac pathological remodeling and mediate myocardial inflammation and fibrosis. rhASB targets CS effectively as a novel therapeutic approach for the treatment of heart failure.

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.