Metabolic Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes by Inhibition of HIF1α and LDHA.

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 cardiomyocytes' metabolic pathway selections and improve cardiomyocyte 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 cardiomyocytes cultured in the presence of glucose used primarily aerobic glycolysis and aberrantly upregulated HIF1α (hypoxia-inducible factor 1α) and its downstream target lactate dehydrogenase A. Conversely, glucose deprivation promoted oxidative phosphorylation and repressed HIF1α. Small molecule inhibition of HIF1α or lactate dehydrogenase A 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/lactate dehydrogenase A inhibition. CONCLUSIONS: We show that under standard culture conditions, the HIF1α-lactate dehydrogenase A 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 cardiomyocytes for drug screening, disease modeling, and therapeutic purposes.

Characteristics and opinions of MD-PhD students and graduates from different European countries: a study from the European MD-PhD Association.

BACKGROUND: MD-PhD programmes throughout the world provide a platform for medical trainees to commit to a physician-scientist career, qualifying with both a medical degree (MD or equivalent) and Doctor of Philosophy (PhD). However, there are limited studies assessing the characteristics of MD-PhD programmes in Europe and the outcomes of MD-PhD students and graduates. PURPOSE: This study aims at a first country-wise exploration of characteristics, opinions, and academic outcomes of MD-PhD students and graduates in Europe. METHODS: Two questionnaires were developed to assess the demographics, MD-PhD programme characteristics, opinions, future career paths and academic outcomes of European MD-PhD students and graduates. An online survey of 278 MD-PhD students and 121 MD-PhD graduates from nine and six European countries, respectively, was completed between April 2016 and December 2017. The country-wise categorical responses were then compared through chi-square analysis followed by multiple logistic regression. RESULTS: Responses from 266 MD-PhD students and 117 MD-PhD graduates were considered valid. Significant country-wise differences (p <0.05) were observed for age group, resident status, clinical time allocation, duration of studies, sources of funding, publications, average impact factor of the journals in which the research was published, satisfaction with the duration of MD-PhD studies and future career choices of MD-PhD students. Responses related to self-perception about clinical and research competence and challenges faced during MD-PhD training did not show a significant country-wise difference. CONCLUSION: The MD-PhD workforce in Europe is highly diverse in their demographics, programme characteristics and career paths but does not differ in opinions related to the challenges faced. The results of this study may be helpful for implementation and improvement of MD-PhD programmes.

Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates.

Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC-CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single-cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc.

Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes.

The 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.