Additional file 1 of Characterization of cardiac metabolism in iPSC-derived cardiomyocytes: lessons from maturation and disease modeling
Creators: name: Vučković, Sofija
name: Dinani, Rafeeh
name: Nollet, Edgar E.
name: Kuster, Diederik W. D.
name: Buikema, Jan Willem
name: Houtkooper, Riekelt H.
name: Nabben, Miranda
name: van der Velden, Jolanda
name: Goversen, Birgit
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Cancer
subject: Computational Biology

description: Additional file 1: Figure S1: Seahorse analyses to measure cardiomyocyte metabolism. A: OCR can be used to determine the role of oxygen consumption in cellular respiration. Basal respiration: oxygen consumption of the cell at baseline conditions. ATP-linked respiration (or ATP turnover) is the proportion of basal respiration that is used for ATP production. Proton leak are protons that return to the mitochondrial matrix independent of the ATP synthase, caused by incomplete coupling of substrate oxidation and ADP phosphorylation. Proton leak can be considered as the basal respiration that is not coupled to ATP production, and it can be calculated as the difference between basal and ATP-linked respiration. Maximal respiration is the maximum oxygen consumption by the ETC. The spare respiratory capacity indicates the cardiomyocyte’s ability to respond to increasing energy demand. It can be calculated as the difference between maximal and basal respiration. Non-mitochondrial oxygen consumption are processes outside of the mitochondria that consume oxygen. B: The ETC in the mitochondrial membrane comprising of five transmembrane proteins (Complex I–IV &amp; Complex V (ATP synthase). Oligomycin: inhibits Complex V by decreasing electron flow causing a reduction in OCR. It can be used to isolate ATP production. FCCP: uncoupling agent. Electron flow through the ETC is unlimited allowing maximal oxygen consumption. It can be applied to measure maximal respiration. Rotenone: inhibits Complex I and Antimycin A inhibits Complex III. Together mitochondrial respiration is decreased to a minimum, which allows determination of non-mitochondrial respiration. C: ECAR can be used to isolate the glycolytic processes in the cardiomyocyte and is determined by measuring proton excretion. Glucose injection stimulates the cardiomyocyte to catabolize glucose. The resulting increase in ECAR indicates glycolysis (or glycolytic rate). ECAR before glucose injection is a measure for non-glycolytic acidification, attributed to processes other than glycolysis that excrete protons. Oligomycin inhibits mitochondrial ATP production, reducing energy production through oxidative phosphorylation and increasing energy production through glycolysis to a maximum, providing a measure for the maximal glycolytic capacity. Glycolytic reserve is the ability of the cardiomyocyte to respond to increased energy demand and is the difference between maximal glycolytic capacity and glycolysis. Adding 2-DG, a glucose analog, completely inhibits glycolysis. The resulting decrease in ECAR confirms that the proton excretion is indeed caused by glycolysis in the cardiomyocyte. D: Assay to determine the proportion of cellular respiration linked to FAO. Four conditions are measured: BSA as a control, fatty acids such as palmitate, both with and without the FAO-inhibitor Eto. Basal, maximal and non-mitochondrial oxygen consumption are similar to (A) but then specific for fatty acid oxidation. Figures are based on the figures from the user guides of Agilent Technologies, Santa Clara, California, and the USA. FCCP = Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; Eto = etomoxir. Figure S2: Flux analysis to measure metabolites in the cardiomyocyte. Schematic overview of metabolic flux analysis using 13C glucose as an example (Long &amp; Antoniewicz, 2019). A: The first step is isotope tracer design to determine the most optimal isotope for further experiments. Then cells are cultured in presence of the isotope after which external rate measurements are performed. Labeled metabolites are measured using mass spectrometry, for example by GC–MS or LC–MS. Results from the labeling experiments are fitted into a metabolic network model, which can then be used to estimate the flux. Statistical analysis is applied to determine how well the data fit into the model. B: Result of the flux analysis, exemplified for glucose that enters glycolysis and the TCA cycle. Each reaction then yields a number for the flux present in that pathway. Flows are reported as mmol/gDW/h. GC–MS = Gas chromatography–mass Spectrometry; LC–MS = Liquid chromatography–mass spectrometry; G6P = Glucose 6-phosphate; P5P = Pentose 5-phosphate; F6P = Fructose 6-phosphate; DHAP = Dihydroxyacetone phosphate; FBP = Fructose 1,6-biphosphate; G3P = Glyceraldehyde 3-phosphate; PGA = 3-Phosphoglyceric acid; PEP = Phosphoenolpyruvic acid; Pyr = Pyruvate; AcCoA = Acetyl CoA; Cit = Citrate; AKG = α-Ketoglutarate; SucCoA = Succinyl CoA; Suc = Succinate; Fum = Fumarate; Mal = Malate; OAC = Oxaloacetate. Table S1: Metabolic characteristics of matured iPSC-CMs. Overview of all studies on maturation in iPSC-CMs that measure quantitative metabolic properties. The values that are depicted as ‘fold’ increase or decrease, are calculated as ratios between matured and control iPSC-CMs. Values were retrieved from the text of the papers or requested from the authors of the respective studies. When no response was obtained from the authors, the values were estimated from the bar graphs in the figures of the papers by two independent observers. Values were not allowed to vary more than 10% between observers. Values were averaged and the average is depicted by ~ . Values that do not change more than 1.04-fold are depicted by ± . n.s. = non-significant findings. ACAA2 = Acetyl CoA acyltransferase 2; ACACA/B = Acetyl-CoA carboxylase A/B; ACAD = Acyl-CoA dehydrogenase; ACADM = medium-chain acyl-CoA dehydrogenase; ACADVL = Acyl-CoA Dehydrogenase Very Long Chain; ACAT1 = Acetyl-CoA acetyltransferase 1; ACC = Acetyl-CoA carboxylase; ACFs = adult cardiac fibroblasts; ACL(Y) = ATP citrate lyase; ACO = Acyl-CoA oxidase; ACOT = Acyl-Coa thioesterase 1; ACSL1/3/4 = Long-chain acyl-CoA synthetase 1/3/4; ACSS2 = Acyl-CoA synthetase short chain family member 2; AKAP1 = A-kinase anchoring protein 1; Akt = Protein kinase B; ALDH = Aldehyde dehydrogenase; ALDOA/C = Fructose-biphosphate aldolase A/C; AMPK = AMP-activated protein kinase; ATP5a/b = ATP Synthase; ATP5G3 = ATP synthase H + transporting, mitochondrial F0 complex, subunit C3; ATP5J = ATP synthase-coupling factor 6;ATPAF1 = ATP synthase mitochondrial F1 complex assembly factor 1; ATPG = ATP synthase gamma chain; BABP2 = Bile acid-binding protein 2; BECN1 = Beclin I; BPGM = Biphosphoglycerate mutase; CD36 = Cluster of Differentiation 36 – also known as fatty acid translocase CD36 (FAT-CD36); COX (3/5B/10/17) = Cardiac cytochrome C oxidase (subunit 3/5B/10/17); CPT1A/B, CPT2 = Carnitine Palmitoyltransferase 1 alpha/beta, 2; CS = Citrate synthase; CYP27A1/CYP4F12 = Cytochrome P450 Family 27/4 Subfamily A/F Member 1/12; Cyt-C = Cytochrome C;DGAT1 = DiGlyceride Acyltransferase 1/2; DNM1L = Dynamin 1-like; ECH1 = Enoyl-CoA hydratase 1; ECHS1 = Enoyl-CoA hydratase, short chain 1; ECI1 = Enoyl-CoA delta isomerase 1; ENO1/2 = Enolase 1/2; ESCH = Enoyl-CoA hydratase; ESRRA/G = Estrogen related receptor alpha/gamma; ETFDH = Electron transfer flavoprotein dehydrogenase FABP3 = Fatty acid-binding protein 3; FADS2 = Fatty acid desaturase 2; FASN = Fatty acid synthase; FH = Fumarate hydratase; FIS1 = Mitochondrial fission 1 protein; GABPA = GA binding protein transcription factor subunit alpha; GAPDH = Glyceraldehyde-3-phosphate dehydrogenase; GFAT2 = glutamine-fructose-6-phosphate aminotransferase 2; GLUT1/4 = Glucose transporter type 1/4; GP6PD = glucose-6-phosphate dehydrogenase; HADHA/B = Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit A/B; HBP = Hexosamine Biosynthesis Pathway; HIF-1 alpha = Hypoxia-inducable factor 1-alpha; HK2 = Hexokinase 2; IDH1/3A = Isocitrate dehydrogenase 1/3A; iPSC-ECs = induced pluripotent stem cell-derived endothelial cells; iPSC-CFs = induced pluripotent stem cell-derived cardiac fibroblasts; LCAD = Long-chain acyl-CoA dehydrogenase; LDHA = Lactate dehydrogenase A; LPL = Lipoprotein lipase; MAP1LC3A = Microtubule Associated Protein 1 Light Chain 3 Alpha; MCAD = Medium-chain acyl-CoA dehydrogenase; MCT4 = monocarboxylate transporter 4; MFF = Mitochondrial Fission Factor; MFN1/2 = Mitofusion 1/2; MLYCD = Malonyl-CoA decarboxylase, mitochondrial; MPC1 = Mitochondrial pyruvate carrier; mtND1/2 = Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1/2; NDUB9 = NAD dehydrogenase (ubiquinone) 1 beta subcomplex subunit 9NDUF = NADH dehydrogenase ubiquinone flavoprotein; NDUFA1/C2/S3 = NADH:Ubiquinone oxidoreductase subunit A1/C2/core subunit S3;NFE2L2 = Nuclear factor (erythroid-derived 2)-like 2; NRF1 = Nuclear respiratory factor 1; OGDHL = Oxyglutarate dehydrogenase; OPA1 = Optic Atrophy 1; OXCT1 (SCOT) = 3-Oxoacid CoA-transferase 1; PPP = Pentose Phosphate Pathway; P(PAR)GC-1A/B = Peroxisome proliferator-activated receptor Gamma Coactivator-1 alpha/beta; PARK2 = Parkin; PDH = pyruvate dehydrogenase; PDK1/4 = Pyruvate Dehydrogenase Kinase 1/4; PDP1 = pyruvate dehydrogenase phosphatase-1; PFKm/l = Phosphofructokinase, muscle/liver; PGAM1/4 = Phosphoglycerate mutase 1/4; PGD = 6-phosphogluconate dehydrogenase; PGK1 = Phosphoglycerate kinase 1; PKM2 = Pyruvate kinase M2; PPARA/D/G = Peroxisome Proliferator-Activated Receptor alpha/delta/gamma; PRKAA2 = Protein kinase AMP-activated catalytic subunit alpha 2;SCD = Stearol-CoA desaturase; SCOT1 = Sunnicyl-CoA:3-ketoacid coenzyme A transferase 1; SCP = Sterol carrier protein; SDH = Succinate dehydrogenase; SDHC = Succinate dehydrogenase complex subunit C; SFs = skin fibroblasts; SLC2A1/A3/A4/A6/A12/A6, SLC24A6, SLC25A1/A20/A29, SLC27A6 = Solute Carrier (Family) (Member); SREBF1 = Sterol regulatory element-binding factor 1; STC1 = Stanniocalcin-1;SUCLG = Succinyl-CoA ligase [GDP-forming]; TBF2M = Mitochondrial transcription factor B2; TFAM = Transcription factor A mitochondrial; THIM = 3-ketoacyl-CoA thiolase; TPI1 = Triosephosphate isomerase; UCP2 = Mitochondrial uncoupling protein 2. *See “Appendix” for additional information. Table S2: Metabolic characteristics of HCM iPSC-CMs. Overview of all studies in iPSC-CMs on HCM and metabolic syndromes with a cardiomyopathy phenotype that measure quantitative metabolic properties. Information on the type of iPSC-CMs that are used as control and hypertrophic properties are also included. The values that are depicted as ‘fold’ increase or decrease, are calculated as ratios between HCM and control iPSC-CMs. Values were retrieved from the text of the papers or requested from the authors of the respective studies. When no response was obtained from the authors, the values were estimated from the bar graphs in the figures of the papers by two independent observers. Values were not allowed to vary more than 10% between observers. Values were averaged and the average is depicted by ~ . Values that do not change more than 1.04-fold are depicted by ± . n.s. = non-significant findings. ACTC1 = Actin Alpha Cardiac Muscle 1; BNP = Brain Natriuretic Peptide; CD36 = Cluster of Differentiation 36; CMTs = Cardiac microtissues; CPT1A/B, CPT2 = Carnitine Palmitoyltransferase 1 alpha/beta, 2, ESRRA = Estrogen related receptor alpha; FABP3 = Fatty acid-binding protein 3; F6P = Fructose-6-phosphate; GLA = α-Galactosidase A; GLUT4 = Glucose transporter type 4; GP = glycogen phosphorylase; GYS1 = Glycogen Synthase 1; G1P = Glucose-1-Phosphate; G6P = Glucose-6-Phosphate; hEHTs = Human engineered heart tissues; HNF4 = Hepatocyte Nuclear Factor 4; LAMP-2 = Lysosomal Associated Membrane Protein type-2; MPO = Myeloperoxidase; MT-ATP6P1 = Mitochondrially encoded ATP synthase 6 pseudogene 1; MT-ATP8 = Mitochondrially encoded ATP synthase membrane subunit 8; MT-CO2 = Mitochondrially encoded cytochrome C oxidase II; MT-CYB = Mitochondrially encoded cytochrome B; MT-ND4L/5/6 = Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4L/5/6; MT-RNR1 = Mitochondrially encoded 12 s RRNA; MT-TP = Mitochondrially encoded TRNA-Pro; MYBPC3 = Myosin-binding protein C3; MYH6 = Myosin heavy chain alpha isoform; MYH7 = Myosin heavy chain beta isoform; NFAT = Nuclear factor of activated T cells; NRF1 = Nuclear respiratory factor 1; PDK4 = Pyruvate Dehydrogenase Kinase 4; PFK-1 = Phosphofructokinase 1; PPARGC1A = Peroxisome proliferator-activated receptor Gamma Coactivator-1 alpha; PPARA/G = Peroxisome Proliferator-Activated Receptor alpha/gamma; PRKAG2 = Protein Kinase AMP-Activated Non-Catalytic Subunit Gamma-2; SLC2A1/A4 = = Solute Carrier (Family) (Member); TAZ = Tafazzin; TFAM = Transcription factor A mitochondrial; TNNT2 = Troponin T2, Cardiac Type; 1,3BPG = 1,3-bisphosphoglycerate; 3PG = 3-phosphoglycerate