Single cardiomyocytes contain myofibrils that harbor the sarcomerebased contractile machinery of the myocardium. Cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) have potential as an in vitro model of heart activity. However, their fetallike misalignment of myofibrils limits their usefulness for modeling contractile activity. We analyzed the effects of cell shape and substrate stiffness on the shortening and movement of labeled sarcomeres and the translation of sarcomere activity to mechanical output (contractility) in live engineered hPSC-CMs. Single hPSC-CMs were cultured on polyacrylamide substrates of physiological stiffness (10 kPa), and Matrigel micropatterns were used to generate physiological shapes (2,000-μm 2 rectangles with length:width aspect ratios of 5:1-7:1) and a mature alignment of myofibrils. Translation of sarcomere shortening to mechanical output was highest in 7:1 hPSC-CMs. Increased substrate stiffness and applied overstretch induced myofibril defects in 7:1 hPSC-CMs and decreased mechanical output. Inhibitors of nonmuscle myosin activity repressed the assembly of myofibrils, showing that subcellular tension drives the improved contractile activity in these engineered hPSC-CMs. Other factors associated with improved contractility were axially directed calcium flow, systematic mitochondrial distribution, more mature electrophysiology, and evidence of transverse-tubule formation. These findings support the potential of these engineered hPSC-CMs as powerful models for studying myocardial contractility at the cellular level.contractility | sarcomeres | cardiomyocyte | stem cell | single cell

Genomic multiplication of the locus-encoding human ␣-synuclein (␣-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of ␣-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age-and dose-dependent ␣-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting Ϸ2% of the population over 65 years of age.Caenorhabditis elegans ͉ neuroprotection ͉ synuclein

SUMMARY Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.

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Introduction: Heart function relies on the contractility of its muscle cells (cardiomyocytes). Human pluripotent stem cells (hPSC) can be differentiated into cardiomyocytes (hPSC-CMs). However, while their myogenic maturity increases with time, they do not resemble adult cardiomyocytes in their morphology, structural organization, mechanical output and electrophysiology. The low maturity of hPSC-CMs limits their potential to model, study and treat heart diseases. Hypothesis: Since the organization of sarcomeres regulates the mechanical output of cardiomyocytes, we hypothesized that enhanced sarcomere maturity correlates with the mechanical output of these cells and the physiology of matured cardiomyocytes. Methods: We cultured single hPSC-CMs on 2000 μm2 rectangular protein patterns on polyacrylamide substrates with a modulus of 10 kPa to resemble the morphology of ventricular cardiomyocytes and match healthy adult ventricular stiffness. We infected seeded cells with rAV CAG-LifeAct-Tag RFP to label actin and image sarcomeres in contracting live cells. We estimated forces generated during single-cell contractile events with traction force microscopy and calculated power (force x cell shortening velocity). We varied cell elongation by changing the cell aspect ratio (1:1 to 7:1) and maintaining constant area. We used substrates having stiffness of 6 kPa (embryonic myocardium), 10 kPa (adult) and 35 kPa (infarcted myocardium). Results: Optimized power output was observed in cells with a 7:1 aspect ratio. The size, organization and dynamics of sarcomeres in these elongated hPSC-CMs are more mature. Optimized power output was observed in cells with a 7:1 aspect ratio. The power generated by hPSC-CMs on 35 kPa substrates was significantly reduced, while reduced variations in the power output across different aspect ratios was observed in 6 kPa. Forces generated by patterned hPSC-CMs were related to the organization, dynamics and size of sarcomeres. Electrophysiological parameters closer to that of mature cardiomyocytes were also observed with patch-clamp measurements in these hPSC-CMs with mature sarcomere organization. Conclusions: Stiffness and shape contribute to the structural and electrophysiological maturation of hPSC-CMs.