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      Fatty Acid-Treated Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes Exhibit Adult Cardiomyocyte-Like Energy Metabolism Phenotypes

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          Abstract

          Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) (iPSC-CMs) are a promising cell source for myocardial regeneration, disease modeling and drug assessment. However, iPSC-CMs exhibit immature fetal CM-like characteristics that are different from adult CMs in several aspects, including cellular structure and metabolism. As an example, glycolysis is a major energy source for immature CMs. As CMs mature, the mitochondrial oxidative capacity increases, with fatty acid β-oxidation becoming a key energy source to meet the heart’s high energy demand. The immaturity of iPSC-CMs thereby limits their applications. The aim of this study was to investigate whether the energy substrate fatty acid-treated iPSC-CMs exhibit adult CM-like metabolic properties. After 20 days of differentiation from human iPSCs, iPSC-CMs were sequentially cultured with CM purification medium (lactate+/glucose-) for 7 days and maturation medium (fatty acids+/glucose-) for 3–7 days by mimicking the adult CM’s preference of utilizing fatty acids as a major metabolic substrate. The purity and maturity of iPSC-CMs were characterized via the analysis of: (1) Expression of CM-specific markers (e.g., troponin T, and sodium and potassium channels) using RT-qPCR, Western blot or immunofluorescence staining and electron microscopy imaging; and (2) cell energy metabolic profiles using the XF96 Extracellular Flux Analyzer. iPSCs-CMs (98% purity) cultured in maturation medium exhibited enhanced elongation, increased mitochondrial numbers with more aligned Z-lines, and increased expression of matured CM-related genes, suggesting that fatty acid-contained medium promotes iPSC-CMs to undergo maturation. In addition, the oxygen consumption rate (OCR) linked to basal respiration, ATP production, and maximal respiration and spare respiratory capacity (representing mitochondrial function) was increased in matured iPSC-CMs. Mature iPSC-CMs also displayed a larger change in basal and maximum respirations due to the utilization of exogenous fatty acids (palmitate) compared with non-matured control iPSC-CMs. Etomoxir (a carnitine palmitoyltransferase 1 inhibitor) but not 2-deoxyglucose (an inhibitor of glycolysis) abolished the palmitate pretreatment-mediated OCR increases in mature iPSC-CMs. Collectively, our data demonstrate for the first time that fatty acid treatment promotes metabolic maturation of iPSC-CMs (as evidenced by enhanced mitochondrial oxidative function and strong capacity of utilizing fatty acids as energy source). These matured iPSC-CMs might be a promising human CM source for broad biomedical application.

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          Energy metabolic phenotype of the cardiomyocyte during development, differentiation, and postnatal maturation.

          Dramatic maturational changes occur in cardiac energy metabolism during cardiac development, differentiation, and postnatal growth. These changes in energy metabolism have important impacts on the ability of the cardiomyocyte to proliferate during early cardiac development, as well as when cardiomyocytes terminally differentiate during later development. During early cardiac development, glycolysis is a major source of energy for proliferating cardiomyocytes. As cardiomyocytes mature and become terminally differentiated, mitochondrial oxidative capacity increases, with fatty acid beta-oxidation becoming a major source of energy for the heart. The increase in mitochondrial oxidative capacity seems to coincide with a decrease in the proliferative ability of the cardiomyocyte. The switch from glycolysis to mitochondrial oxidative metabolism during cardiac development includes both alterations in the transcriptional control and acute alterations in the control of each pathway. Interestingly, if a hypertrophic stress is placed on the adult heart, cardiac energy metabolism switches to a more fetal phenotype, which includes an increase in glycolysis and decrease in mitochondrial fatty acid beta-oxidation. In this article, we review the impact of alterations in energy substrate metabolism on cardiomyocyte proliferation, differentiation, and postnatal maturation.
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            The permeability transition pore controls cardiac mitochondrial maturation and myocyte differentiation.

            Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find that closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less-polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox-signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation. Copyright © 2011 Elsevier Inc. All rights reserved.
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              Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays.

              Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities. Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls. We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.
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                Author and article information

                Journal
                Cells
                Cells
                cells
                Cells
                MDPI
                2073-4409
                17 September 2019
                September 2019
                : 8
                : 9
                : 1095
                Affiliations
                [1 ]Department of Emergency Medicine, Asahikawa Medical University, Asahikawa, Hokkaido 078-8510, Japan; yhorikoshi-11@ 123456outlook.com (Y.H.); sfujita@ 123456asahikawa-med.ac.jp (S.F.)
                [2 ]Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA; hishiha@ 123456juntendo.ac.jp
                [3 ]Department of Cell Biology, Neuroscience & Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA; yashengyan@ 123456mcw.edu
                [4 ]Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA; mterashv@ 123456mcw.edu (M.T.); zbosnjak@ 123456mcw.edu (Z.J.B.)
                [5 ]Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
                [6 ]Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, Tokyo 113-8421, Japan
                [7 ]Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA
                Author notes
                [* ]Correspondence: xibai@ 123456mcw.edu ; Tel.: +1-414-955-5755; Fax: +1-414-955-6517
                Author information
                https://orcid.org/0000-0001-9598-7423
                https://orcid.org/0000-0002-9342-4480
                Article
                cells-08-01095
                10.3390/cells8091095
                6769886
                31533262
                462a1a99-ff49-4a74-bcd0-ac853717821d
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 05 April 2019
                : 14 September 2019
                Categories
                Article

                induced pluripotent stem cells,cardiomyocytes,maturation,metabolism

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