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Metabolic Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes by Inhibition of HIF1? 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 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 HIF1? (hypoxia-inducible factor 1?) and its downstream target lactate dehydrogenase A. Conversely, glucose deprivation promoted oxidative phosphorylation and repressed HIF1?. Small molecule inhibition of HIF1? or lactate dehydrogenase A resulted in a switch from aerobic glycolysis to oxidative phosphorylation. Likewise, siRNA inhibition of HIF1? 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 HIF1?/lactate dehydrogenase A inhibition. CONCLUSIONS:We show that under standard culture conditions, the HIF1?-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.

SUBMITTER: Hu D

PROVIDER: S-EPMC6208155 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Hu Dongjian D Linders Annet A Yamak Abir A Correia Cláudia C Kijlstra Jan David JD Garakani Arman A Xiao Ling L Milan David J DJ van der Meer Peter P Serra Margarida M Alves Paula M PM Domian Ibrahim J IJ

Circulation research 20181001 9

<h4>Rationale</h4>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 ...[more]

PMID: 30355156

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Project description:Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including ?-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.

Dataset Information

Metabolic Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes by Inhibition of HIF1? and LDHA.

Publications

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

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