Multi-Omics Profiling of Hypertrophic Cardiomyopathy Reveals Altered Mechanisms in Mitochondrial Dynamics and Excitation–Contraction Coupling

Figures

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  Figure 1. Study design and mass spectrometry workflows. (A) Schematic of wild-type and MYH7 mutant human induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) and clinical (myectomy) specimens. (B) hiPSC-CMs differentiation protocol; cultures were differentiated over 20 days from CRISPR/Cas9-edited MYH7R403Q+/− hiPSCs via targeted small-molecule, monolayer manipulation of the Wnt signaling pathway. (C) Sample processing followed by parallel mass spectrometry-based phosphoproteomics and metabolomics and subsequent bioinformatic analysis; LLE = liquid–liquid extraction, LC/MS = liquid chromatography/mass spectrometry, TMT = tandem mass tag.

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  Figure 2. Global phosphoproteomic and proteomic expression profiles. (A) Venn diagrams depicting total feature coverage (high confidence proteins and phosphoproteins) quantified in hiPSC-CM (left) versus patient (hypertrophic cardiomyopathy specimens and healthy control) myectomy specimens (right). (B) Volcano plots of differentially expressed features. Plots show negative log-transformed p-values (moderated t-test) against log2-transformed fold-change difference measured between the mutant and wild-type specimens. Non-grey values highlight significant difference between groups (moderated t-test; FDR adjusted p < 0.05).

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  Figure 3. Mitochondrial dynamics across early and advanced specimens. (A) Top differential pathways from gene set enrichment analysis of mutant versus control hiPSC-CM proteome (left) with select heatmaps (right) showing hierarchically clustered (Euclidean distance) profiles of corresponding annotated protein components. (B) Depiction of base excision repair and glutathione-based redox proteins quantified in hiPSC-derived cardiomyocytes. (C) Mitochondrial fission-related phosphosites quantified in myectomy specimens. (D) Thiol-based antioxidant response enriched according to the affected patient proteome samples.

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  Figure 4. Integrated metabolomic analysis of patient myectomies and hiPSC-CMs. (A) Top differential pathways enriched in mutant versus control samples based on metabolic enrichment network analysis of the hiPSC-CM proteomic datasets (left), volcano plot of differential metabolite features from global (positive ion mode) metabolomic analysis of CM metabolites (middle), and enriched biochemical pathways revealed by metabolic enrichment network analysis of the patient myectomy samples (right). (B) Targeted mass-spectrometry-based metabolomics of hiPSC-CMs using isotopically heavy labeled 13C glucose, including after treatment with BPTES over a 90 min period. (C) Targeted mass-spectrometry-based metabolomics using isotopically labeled 13C glutamine. Individual data plots across all experiments are shown with means ± SD (N = 8). F1,6BP = fructose 1,6-bisphosphate, GA3P = glyceraldehyde 3-phosphate, 3-PG = 3-phosphoglycerate, PYR = pyruvate, Ac-CoA = acetyl-CoA, CIT = citrate, ISOCIT = isocitrate, 2OG = 2-oxoglutarate, SUC = succinate, FUM = fumarate, MAL = malate, OAA = oxaloacetate.

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  Figure 5. Phosphoproteomic landscape of excitation contraction coupling proteins. (A) Hierarchical clustering (Euclidean distance) showing changes in phosphosite abundance of significantly differentially phosphorylated proteins linked to excitation contraction coupling in hiPSC-CMs and (B) patient myectomy samples. (C) Schematic depiction of select SR phosphoproteins in hiPSC-CMs and (D) clinical specimens. (E) Plots showing average Ca2+ transients (N = 3; n > 75 cells) normalized to WT transient, with error bands representing SEM (left), relative time for Ca2+ release into and reuptake from the cytoplasm compared to average WT cell (N = 3; n > 83 cells) (middle), and relative intensity of Ca2+ release into cytoplasm from SR following caffeine bolus relative to the average WT cell (right). Individual data points (n) across all experiments (N) are shown with means ± SEM.