Highlights d Cardiac fibroblasts and endothelial cells induce hiPSCcardiomyocyte maturation d CX43 gap junctions form between cardiac fibroblasts and cardiomyocytes d cAMP-pathway activation contributes to hiPSCcardiomyocyte maturation d Patient-derived hiPSC-cardiac fibroblasts cause arrhythmia in microtissues

FOXO transcription factors have important roles in metabolism, cellular proliferation, stress tolerance, and aging. FOXOs are negatively regulated by protein kinase B/c-Akt-mediated phosphorylation. Here we show that FOXO factors are also subject to regulation by reversible acetylation. We provide evidence that the acetyltransferase CREB-binding protein (CBP) binds FOXO resulting in acetylation of FOXO. This acetylation inhibits FOXO transcriptional activity. Binding of CBP and acetylation are induced after treatment of cells with peroxide stress. Deacetylation of FOXOs involves binding of the NAD-dependent deacetylase hSir2 SIRT1 . Accordingly, hSir2 SIRT1 -mediated deacetylation precludes FOXO inhibition through acetylation and thereby prolongs FOXO-dependent transcription of stress-regulating genes. These data demonstrate that acetylation functions in a second pathway of negative control for FOXO factors and provides a novel mechanism whereby hSir2 SIRT1 can promote cellular survival and increase lifespan.The Forkhead box, class O subfamily of forkhead transcription factors (FOXO) 1 consists of the functionally related proteins FOXO1, FOXO3a, and FOXO4 (also known as FKHR, FKHRL1, and AFX, respectively; Ref. 1). The growth factorstimulated phosphatidylinositol 3-kinase-protein kinase B (PKB)/c-Akt pathway negatively regulates FOXO factors by phosphorylation-mediated nuclear exclusion (2-4). This pathway is evolutionarily conserved between Caenorhabditis elegans and humans. DAF-16, the C. elegans homologue of mammalian FOXO, is also controlled by phosphatidylinositol 3-kinase/PKB signaling. DAF-16 regulates daver formation in larvae, and responses to various environmental stresses and longevity in adult worms (5-8). In parallel, mammalian FOXO transcription factors have been implicated in regulating metabolism, cell cycle progression, and stress tolerance (9, 10; reviewed in Ref. 11).In C. elegans, overexpression of the NAD-dependent deacetylase Sir2 (silent information regulator 2) increases lifespan, which requires DAF-16 (12). The Sir2 family of genes is a highly conserved group of genes with seven human homologues, of which the SIRT1 gene encodes the closest homologue of yeast and C. elegans Sir2, hence named hSir2 SIRT1 (13). Recently, deacetylation of p53 by hSir2 SIRT1 has been demonstrated, and it has been suggested that this functions in increasing cellular resistance against stress. However, subsequent studies (14) showed that in HEK293T cells, which lack functional p53, activation of hSir2 SIRT1 by resveratrol treatment still increases cellular resistance against gamma-radiation, thus suggesting alternative pathways. As DAF-16 is necessary for lifespan extension by Sir2, FOXOs may well function in such an alternative pathway. EXPERIMENTAL PROCEDURESCell Culture, Transfection, and Treatment-HEK293T, DL23 (DLD-1 human colon carcinoma cells expressing a conditionally active version of FOXO3a; Ref. 10), and A14 cells (human insulin receptor overexpressing mouse NIH3T3 cells (15) and C2C12 mouse myoblast...

Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of the human Ether-a-go go Related Gene (hERG) ion channel in particular is associated with life-threatening arrhythmias, such as Torsade de Pointes (TdP). Here we investigated human cardiomyocytes derived from pluripotent (embryonic) stem cells (hESC) as a renewable, scalable, and reproducible system on which to base cardiac safety pharmacology assays. Analyses of extracellular field potentials in hESC-derived cardiomyocytes (hESC-CM) and generation of derivative field potential duration (FPD) values showed dose-dependent responses for 12 cardiac and noncardiac drugs. Serum levels in patients of drugs with known effects on QT interval overlapped with prolonged FPD values derived from hESC-CM, as predicted. We thus propose hESC-CM FPD prolongation as a safety criterion for preclinical evaluation of new drugs in development. This is the first study in which dose responses of such a wide range of compounds on hESC-CM have been generated and shown to be predictive of clinical effects. We propose that assays based on hESC-CM could complement or potentially replace some of the preclinical cardiac toxicity screening tests currently used for lead optimization and further development of new drugs.

The polymer polydimethylsiloxane (PDMS) is widely used to build microfluidic devices compatible with cell culture. Whilst convenient in manufacture, PDMS has the disadvantage that it can absorb small molecules such as drugs. In microfluidic devices like “Organs-on-Chip”, designed to examine cell behavior and test the effects of drugs, this might impact drug bioavailability. Here we developed an assay to compare the absorption of a test set of four cardiac drugs by PDMS based on measuring the residual non-absorbed compound by High Pressure Liquid Chromatography (HPLC). We showed that absorption was variable and time dependent and not determined exclusively by hydrophobicity as claimed previously. We demonstrated that two commercially available lipophilic coatings and the presence of cells affected absorption. The use of lipophilic coatings may be useful in preventing small molecule absorption by PDMS.

Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we have generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers that were also present in primary cardiac microvasculature, suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducible in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs.