AimsIron deficiency is common in patients with heart failure and associated with a poor cardiac function and higher mortality. How iron deficiency impairs cardiac function on a cellular level in the human setting is unknown. This study aims to determine the direct effects of iron deficiency and iron repletion on human cardiomyocytes.Methods and resultsHuman embryonic stem cell‐derived cardiomyocytes were depleted of iron by incubation with the iron chelator deferoxamine (DFO). Mitochondrial respiration was determined by Seahorse Mito Stress test, and contractility was directly quantified using video analyses according to the BASiC method. The activity of the mitochondrial respiratory chain complexes was examined using spectrophotometric enzyme assays. Four days of iron depletion resulted in an 84% decrease in ferritin (P < 0.0001) and significantly increased gene expression of transferrin receptor 1 and divalent metal transporter 1 (both P < 0.001). Mitochondrial function was reduced in iron‐deficient cardiomyocytes, in particular ATP‐linked respiration and respiratory reserve were impaired (both P < 0.0001). Iron depletion affected mitochondrial function through reduced activity of the iron–sulfur cluster containing complexes I, II and III, but not complexes IV and V. Iron deficiency reduced cellular ATP levels by 74% (P < 0.0001) and reduced contractile force by 43% (P < 0.05). The maximum velocities during both systole and diastole were reduced by 64% and 85%, respectively (both P < 0.001). Supplementation of transferrin‐bound iron recovered functional and morphological abnormalities within 3 days.ConclusionIron deficiency directly affects human cardiomyocyte function, impairing mitochondrial respiration, and reducing contractility and relaxation. Restoration of intracellular iron levels can reverse these effects.
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.
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.
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