Rationale: Human Pluripotent Stem Cell-Derived Cardiomyocytes (hPSC-CMs) are a readily available, robustly reproducible and physiologically appropriate human cell source for cardiac disease modeling, drug discovery, and toxicity screenings in vitro. However, unlike adult myocardial cells in vivo, hPSC-CMs cultured in vitro maintain an immature metabolic phenotype where majority of ATP is produced through aerobic glycolysis instead of oxidative phosphorylation in the mitochondria. Little is known about the underlying signaling pathways controlling hPSC-CMs’ metabolic and functional maturation. Objective: To define the molecular pathways controlling CMs’ metabolic pathway selections and improve CM metabolic and functional maturation. Methods and Results: We cultured hPSC-CMs in different media compositions including glucose-containing media, glucose-containing media supplemented with fatty acids, and glucose-free media with fatty acids as the primary carbon source. We found that CMs cultured in the presence of glucose utilized primarily aerobic glycolysis and aberrantly upregulated hypoxia-inducible factor 1-alpha (HIF1α) and its downstream target lactate dehydrogenase A (LDHA). Conversely, glucose deprivation promoted oxidative phosphorylation and repressed HIF1α. Small molecule inhibition of HIF1α or LDHA resulted in a switch from aerobic glycolysis to oxidative phosphorylation. Likewise, siRNA inhibition of HIF1α stimulated oxidative phosphorylation while inhibiting aerobic glycolysis. This metabolic shift was accompanied by an increase in mitochondrial content and cellular ATP levels. Furthermore, functional gene expressions, sarcomere length and contractility were improved by HIF1α/LDHA inhibition. Conclusions: We show that under standard culture conditions, the HIF1α-LDHA axis is aberrantly upregulated in hPSC-CMs, preventing their metabolic maturation. Chemical or siRNA inhibition of this pathway results in an appropriate metabolic shift from aerobic glycolysis to oxidative phosphorylation. This in turn improves metabolic and functional maturation of hPSC-CMs. These findings provide key insight into molecular control of hPSC-CMs’ metabolism and may be used to generate more physiologically mature CMs for drug screening, disease modeling and therapeutic purposes.

Summary Many nociceptors detect mechanical cues, but the ion channels responsible for mechanotransduction in these sensory neurons remain obscure. Using in vivo recordings and genetic dissection, we identified the DEG/ENaC protein, DEG-1, as the major mechanotransduction channel in ASH, a polymodal nociceptor in Caenorhabditis elegans. But, DEG-1 is not the only mechanotransduction channel in ASH: loss of deg-1 revealed a minor current whose properties differ from those expected of DEG/ENaC channels. This current was independent of two TRPV channels expressed in ASH. Although loss of these TRPV channels inhibits behavioral responses to noxious stimuli, we found that both mechanoreceptor currents and potentials were essentially wild-type in TRPV mutants. We propose that ASH nociceptors rely on two genetically-distinct mechanotransduction channels and that TRPV channels contribute to encoding and transmitting information. Because mammalian and insect nociceptors also co-express DEG/ENaCs and TRPVs, the cellular functions elaborated here for these ion channels may be conserved.

SummaryThe quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening.

Background: Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC) at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated.

Cellular shape change and movement are central to biologic processes that range from normal embryonic development to inflammatory diseases and cancer. Quantitative visual phenotyping of dynamic cellular behaviors creates unique challenges for image capture, analysis and storage. Despite substantial technological advances in molecular biology, biochemistry, genomics and proteomics, investigating cellular processes remains tremendously challenging and labor-intensive. We have developed algorithms and software implementations that allow for fully-automated analysis of experiments designed to investigate a range of cellular and organismal behaviors. By enabling cellular phenotyping, this automated approach creates a unique opportunity for investigators to perform large-scale experiments designed to determine gene function or to screen for small molecule modulators of important cellular behaviors.