Background Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modelling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) towards an adult phenotype under defined conditions. Methods We systematically investigated cell composition, matrix and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We employed morphological, functional, and transcriptome analyses to benchmark maturation of EHM. Results EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M-bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency-response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β1- and β2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and NT-proBNP release; all are classical hallmarks of heart failure. Additionally, we demonstrate scalability of EHM according to anticipated clinical demands for cardiac repair. Conclusions We provide proof-of-concept for a universally applicable technology for the engineering of macro-scale human myocardium for disease modelling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.

Background Hemodynamic load regulates myocardial function and gene expression. We tested the hypothesis that afterload and preload despite similar average load result in different phenotypes. Methods and Results Afterload and preload were compared in mice with transversal aortic constriction (TAC) and aorto-caval shunt (Shunt). When compared to sham mice, six hours after surgery, systolic wall stress (afterload) was increased in TAC (+40%, P<0.05), diastolic wall stress (preload) was increased in Shunt (+277%, P<0.05) and TAC (+74%, P<0.05) and mean total wall stress was similarly increased in TAC (69%) and Shunt (67%) (TAC vs. Shunt: not significant (n.s.), each P<0.05 vs. Sham). At 1 week, left ventricular weight/tibia length was significantly increased by 22% in TAC and 29% in Shunt (n.s. TAC vs. Shunt). After 24 hours and 1 week, calcium/calmodulin dependent protein kinase II (CaMKII) signaling was increased in TAC. This resulted in altered calcium cycling, including increased L-type calcium current, calcium transients, fractional SR release and calcium spark frequency. In Shunt, Akt phosphorylation was increased. TAC was associated with inflammation, fibrosis and cardiomyocyte apoptosis. The latter was significantly reduced in CaMKIIδ-KO TAC mice. 157 mRNAs and 13 microRNAs were differentially regulated in TAC vs. Shunt. After 8 weeks, fractional shortening was lower and mortality higher in TAC Conclusions Afterload results in maladaptive fibrotic hypertrophy with CaMKII-dependent altered calcium cycling and apoptosis. Preload is associated with Akt activation without fibrosis, little apoptosis, better function and lower mortality. This indicates that different loads result in distinct phenotype differences which may require specific pharmacological interventions.

Aims Deregulation of epigenetic processes and aberrant gene expression are important mechanisms in heart failure. Here we studied the potential relevance of m6A RNA methylation in heart failure development. Methods and results We analysed m6A RNA methylation via next‐generation sequencing. We found that approximately one quarter of the transcripts in the healthy mouse and human heart exhibit m6A RNA methylation. During progression to heart failure we observed that changes in m6A RNA methylation exceed changes in gene expression both in mouse and human. RNAs with altered m6A RNA methylation were mainly linked to metabolic and regulatory pathways, while changes in RNA expression level mainly represented changes in structural plasticity. Mechanistically, we could link m6A RNA methylation to altered RNA translation and protein production. Interestingly, differentially methylated but not differentially expressed RNAs showed differential polysomal occupancy, indicating transcription‐independent modulation of translation. Furthermore, mice with a cardiomyocyte restricted knockout of the RNA demethylase Fto exhibited an impaired cardiac function compared to control mice. Conclusions We could show that m6A landscape is altered in heart hypertrophy and heart failure. m6A RNA methylation changes lead to changes in protein abundance, unconnected to mRNA levels. This uncovers a new transcription‐independent mechanisms of translation regulation. Therefore, our data suggest that modulation of epitranscriptomic processes such as m6A methylation might be an interesting target for therapeutic interventions.

Barth syndrome (BTHS) is a cardiomyopathy caused by the loss of tafazzin, a mitochondrial acyltransferase involved in the maturation of the glycerophospholipid cardiolipin. It has remained enigmatic as to why a systemic loss of cardiolipin leads to cardiomyopathy. Using a genetic ablation of tafazzin function in the BTHS mouse model, we identified severe structural changes in respiratory chain supercomplexes at a pre‐onset stage of the disease. This reorganization of supercomplexes was specific to cardiac tissue and could be recapitulated in cardiomyocytes derived from BTHS patients. Moreover, our analyses demonstrate a cardiac‐specific loss of succinate dehydrogenase (SDH), an enzyme linking the respiratory chain with the tricarboxylic acid cycle. As a similar defect of SDH is apparent in patient cell‐derived cardiomyocytes, we conclude that these defects represent a molecular basis for the cardiac pathology in Barth syndrome.