Bioenergetic and metabolic aberrations in induced pluripotent stem cell-derived cardiomyocytes generated from a patient with Wolff-Parkinson-White syndrome caused a PRKAG2 mutation

Figures

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  Figure 1. Glycolytic profile of PRKAG2-mutant and healthy iPSC-CMs (CMs) measured by XFe96 Seahorse metabolic flux analyzer. (a) Representative profile of glycolytic function in healthy CMs. Extracellular acidification rate (ECAR) is recorded during sequential, in-port additions of the glycolysis modulators: (1) Glucose, fuels glycolysis and thus leads to basal glycolysis and elevation of ECAR levels; (2) Oligomycin, an ATP Synthase inhibitor used to increase glycolysis dependency, leads to maximal ECAR values; (3) 2-DG, glucose-competitive inhibitor, reduces glycolysis-dependent ECAR levels to a minimum. (b) ECAR in PRKAG2-mutant (red) compared to healthy (black) CMs recorded during sequential additions of the glycolysis modulators: glucose, oligomycin and 2-DG. (c) Non-glycolytic acidification, glycolysis and glycolytic capacity rates calculated based on panel b. A significant decrease in glycolysis and glycolytic capacity rates was measured in PRKAG2-mutant compared to healthy CMs, while the non-glycolytic acidification rate was similar in these groups. ECAR values are normalized to μg protein measured using a modified Lowry protein assay. Sample sizes (n) refer to the number of wells…

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  Figure 2. Respiration profile of healthy, PRKAG2-mutant, and isogenic control iPSC-CMs (CMs) by the XFe96 Seahorse metabolic flux analyzer. (a) Representative respirometer profile of healthy control iPSC-CMs. Oxygen consumption rate is recorded during sequential, in-port additions of mitochondrial modifiers: (1) Oligomycin, an ATP synthase inhibitor used to measure the oxygen consumption coupled to ATP synthesis; (2) FCCP, dissipates the mitochondrial proton gradient and stimulates maximal mitochondrial respiration capacity; (3) Rotenone and antimycin A, inhibitors of complex III and I, hence blockers of mitochondrial respiration. (b) Oxygen consumption rate (OCR) in mutant (red), isogenic control (blue) and healthy (black) CMs recorded during sequential additions of mitochondrial respiration modulators: Oligomycin, FCCP, and rotenone plus antimycin A. (c–e) Non-mitochondrial oxygen consumption, basal respiration, ATP production, proton leak, maximal respiration and spare respiratory capacity rates calculated based on plot B. A significant increase in non-mitochondrial oxygen consumption, maximal respiration and spare respiratory capacity measured in mutant, isogenic control and healthy…

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  Figure 3. Mitochondrial content and activity in beating iPSC-CMs (CMs). (a) Representative confocal images of mitochondria in healthy (left), PRKAG2-mutant (middle), and isogenic control (right) CMs, simultaneously stained with 2 fluorescent dyes: MTG (green; ΔΨ independent), and TMRE (red; ΔΨ dependent). (b) Quantification of total mitochondrial content by MTG intensity. (c) Quantification of total mitochondrial activity calculated by the ratio of TMRE over MTG fluorescence (TMRE/MTG), of each image. The summary shows a significant increase in both mitochondrial activity and content in the mutant cells, compared to healthy and isogenic control. Cell Discoverer 7, Objective X20. Sample sizes (n) refer to image segments (defined areas within confocal images containing mitochondria). Multiple images were analyzed per well, and each image contained several segments. Healthy, n = 94; Mutant, n = 57; Isogenic control, n = 59. One-way ANOVA, ns = non-significant, *p < 0.05, **** p < 0.0001.

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  Figure 4. Principal component analysis and heatmap of metabolomic profiles in PRKAG2 mutant and isogenic control iPSC-CMs (CMs), and in PRKAG2 mutated and WT mice. (a,b) PCA of the complete metabolomic dataset comparing PRKAG2 mutant and controls. (a) PRKAG2-mutant (red) vs. isogenic control (blue) CMs. (b) PRKAG2-mutated (red) vs. WT (black) mice hearts. (c,d) Heatmap of the top 50 differentially expressed metabolites in (c) PRKAG2-mutant vs. isogenic control CMs, and (d) PRKAG2-mutated vs. WT mice hearts. Metabolite abundance is represented by a color gradient (red = high, blue = low). The figure illustrates that the mutant CMs/mutated mice hearts exhibit distinct metabolic alterations compared to the control groups, especially in the top 50 metabolites visualized in the heatmaps. mutant CMs, n = 5; isogenic control CMs, n = 5; mutated mice hearts, n = 7; WT mice hearts, n = 6.

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  Figure 5. Metabolic pathways related to stress response and energy regulation in PRKAG2-mutant iPSC-CMs (CMs) and mice hearts, analyzed by ingenuity pathway analysis (IPA). (a) Increased O-GlcNAc Transferase (OGT) activity in mutant CMs and mice hearts is indicated by elevated levels of key metabolites, including D-glucose, L-glutamic acid and glutathione (red). These findings suggest a heightened cellular stress response. (b) Elevated reactive oxygen species (ROS) formation is observed in mutant CMs and mice hearts. Increased levels of L-carnitine, L-methionine, and glutathione (red), along with orange predictions, indicate increased oxidative stress. (c) Disruptions in the mTOR pathway are shown, with elevated levels of 2-oxoglutaric acid and glucose-6-phosphate (red) in both mutant CMs and mice hearts. Orange predictions indicate increased mTOR pathway activity, suggesting heightened energy regulation and biosynthetic processes. The upper numbers refer to log2(fold change), and the lower numbers represent the False Discovery Rate (FDR). In these visualizations, “E” represents experimentally observed changes in metabolite levels, while “C” denotes computational predictions, including…

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  Figure 6. Metabolic pathways related to amino acid metabolism in PRKAG2-mutant iPSC-CMs (CMs) and mice hearts, as analyzed by ingenuity pathway analysis (IPA). (a) Altered efflux of amino acids in mutant CMs and mice hearts is indicated by higher levels of L-methionine, L-leucine, and beta-alanine (red), suggesting disruptions in amino acid transport. (b) Disruptions in the L2HGDH (L-2-hydroxyglutarate dehydrogenase) pathway are observed, with elevated levels of L-phenylalanine, L-histidine, and glutathione (red). These changes suggest enhanced amino acid degradation and shifts in energy metabolism. (c) Alterations in the GATA4 pathway are shown, with higher levels of D-glucose, L-methionine, and betaine (red), indicating the pathway's role in regulating amino acid metabolism and the cardiac stress response. The upper numbers refer to log2(fold change); the lower numbers represent the FDR. Color codes: Red = higher levels of metabolites; Green = lower levels of metabolites; Orange = predicted increased function; Blue = predicted decreased function. The curved arrows represent feedback loops or alternative pathway fluxes, while the gradient blue shades correspond to varying levels of…

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  Figure 7. Metabolic pathways related to glucose and lipid metabolism in PRKAG2-mutant iPSC-CMs (CMs) and mice hearts, as analyzed by the ingenuity pathway analysis (IPA). (a) Altered glucose transport in mutant CMs and mice hearts is indicated by higher levels of D-glucose, L-histidine, and L-methionine (red), suggesting disruptions in glucose handling and energy homeostasis. (b) Changes in triacylglycerol (TAG) metabolism are observed, with elevated levels of D-glucose, L-glutamic acid, and L-carnitine (red), indicating increased lipid storage and metabolic dysfunction in both PRKAG2-mutant models. The upper numbers refer to log2(fold change), and the lower numbers represent the FDR. Color codes: Red = higher levels of metabolites; Green = lower levels of metabolites; Orange = predicted increased function; Blue = predicted decreased function.

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  Figure 8. Lipidomic analysis in PRKAG2-mutant and isogenic control iPSC-CMs (CMs). (a) Scores plot from PCA showing separation between PRKAG2-mutant (red, n = 5) and isogenic control (blue, n = 5) CMs, based on lipidomic data. The PCA demonstrates distinct lipid profiles between the two cell groups, with PC1 accounting for 58.4% of the variation and PC2 accounting for 8.1%. (b) Heatmap representing the lipidomic profiles of all 1,051 lipid species detected in PRKAG2-mutant and isogenic control CMs. Lipid abundance is represented by a color gradient, with red indicating higher values and blue indicating lower values. The heatmap shows clear clustering of the samples, highlighting the differences in lipid profiles between the two groups. (c) A bar plot showing fold changes of lipid species with at least a 2-fold difference between mutant and isogenic control CMs. Positive and negative fold changes represent both upregulation and downregulation of lipid species in the mutant cells relative to controls.

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  Figure 9. Differentially expressed genes (DEGs) in PRKAG2-mutant vs. isogenic control iPSC-CMs (CMs). Volcano plot representing differentially expressed genes (DEGs) in mutant CMs compared to isogenic control CMs. Each dot represents a single gene, with green dots representing upregulated genes (541 DEGs) and red dots representing downregulated genes (273 DEGs). The x-axis shows the log2 fold gene expression change (mutant vs. isogenic control CMs), while the y-axis represents the −log10 of the adjusted p-value, indicating the statistical significance of gene expression changes. Genes with p-adjusted < 0.05 are considered significantly differentially expressed.

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  Figure 10. The effect of metformin treatment on respiration and mitochondria. (a,b) Oxygen consumption rate (OCR) in (a) untreated PRKAG2-mutant (red), isogenic control (blue) and healthy (black) CMs, and (b) mutant (deep red), isogenic control (dark blue) and healthy (grey) CMs treated with 1 mM metformin for 24 h. (c–e) Basal respiration, non-mitochondrial oxygen consumption, and maximal respiration rates are calculated based on plot b. (f) Quantification of total mitochondrial content by MTG intensity. (g) Quantification of total mitochondrial activity calculated by the ratio of TMRE over MTG fluorescence (TMRE/MTG), of each image. The summary demonstrates that 24-hour metformin treatment decreased maximal respiration in the mutant CMs to the level of isogenic control and healthy CMs, while having no effect on non-mitochondrial OCR. In addition, metformin treatment caused a significant decrease in mitochondrial content and activity in mutant CMs, compared to isogenic control and healthy CMs. The statistical significance is as follows: (a–e) Healthy, n = 21; PRKAG2-mutant, n = 18; isogenic control, n = 20. The Shapiro–Wilk test for normality was performed on the data. One-way ANOVA was…

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  Figure 11. Metformin blocks the arrhythmias in PRKAG2-mutant iPSC-CMs. (a) Action potentials of untreated mutant CMs at Day 0 (upper row) and after 24-hour (Day 1) incubation in fresh medium (lower row). As shown, arrhythmogenic activity persisted at both time points. (b) Action potentials of mutant CMs at Day 0 (upper row) and 24 h (Day 1) after treatment with 1 mM metformin (lower row). In Experiments 1, 2 and 4 metformin treatment prevented the arrhythmias, whereas in Experiment 3, the arrhythmogenic phenotype was markedly attenuated.

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  Figure 12. Action potential characteristics of PRKAG2-mutant iPSC-CMs (CMs) in the absence and presence of metformin. Characteristics of the action potentials shown in Figure 11b at day 0 and after 24-hour (Day 1) incubation with metformin. APA: action potential amplitude; Peak: action potential peak; MDP: maximal diastolic potential; dV/dtmax: maximal upstroke velocity of phase zero depolarization; APD90: action potential duration at 90% repolarization; Rate: Beats per minute. Comparisons between the untreated and treated CMs was performed by means of unpaired t-test. *p < 0.05. n = 6 CMs in each group.