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

Abstract

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.

Maturation of human induced pluripotent stem cell-derived cardiomyocytes for modeling hypertrophic cardiomyopathy

“…To investigate whether hiPSC-CMs could be shifted towards more adult-like morphology and behavior, a combinatorial approach was employed. First, instead of using media where glucose is the main energy source (GLUC) our cells were cultured in media where glucose was substituted with a combination of galactose, oleic acid, and palmitic acid (maturity medium, MM) based on previous reports of similar methods that improve cellular morphology, contractility, ATP content, and metabolic behavior 7,8 . Although both GLUC and MM each contain the B-27™ supplement, which contains several fatty acids, total fatty acid content in MM is ~150µmol versus ~144nM in B-27™-supplemented RPMI 9 .…”

Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques

“…We confirm that, in the case of lactate purification, the enriched cell population shifts towards a higher dependence on oxidative phosphorylation and show that oxygen consumption rate per cardiomyocyte does not vary. Within one post-natal week, human fetal cardiomyocytes shift from mostly glycolysis to oxidative phosphorylation due to increased metabolic demand [27], and hPSC-cardiomyocytes have been shown (similar to fetal cardiomyocytes) to utilize mostly glycolysis for energy production [28,29]. The high…”

Barth Syndrome: Exploring Cardiac Metabolism with Induced Pluripotent Stem Cell-Derived Cardiomyocytes

“…The Krebs cycle is primary fueled by acetyl-CoA produced from glucose and fatty acids oxidation. In general, in comparison to the adult cardiomyocytes, iPS-CM cells are highly glycolytic due to their non-mature phenotype [21]. To encourage iPS-CMs to use both glucose and palmitic acid as a carbon sources, we performed all our experiments in 10 mM glucose conditions.To determine the effect of the TAZ 517delG on the production of Krebs cycle intermediates from glucose, we supplemented cell culture media with 10 mM labeled 13 C 6 -glucose and 0.4 mM unlabeled palmitate (conjugated to BSA) as a carbon sources.…”