Variable Penetrance in Hypertrophic Cardiomyopathy

Figures

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  Fig. 1:. Distinct Phenotype Penetrance & Variability of Different MYH7 Mutations is Reflected in hiPSC-CM Model. (A) Schematic of experimental set-up (B) I. UMAP representation of MYH7 H251N CMs (MYH7 WT/H251N) and the isogenic counterpart (MYH7 WT/WT) clustered by the presence of H251N mutation. II. Unbiased clustering of H251N and WT hiPSC-CMs. III. Relative contribution to each cluster by WT and H251N hiPSC-CMs. IV. Comparison of MYH7 gene expression in WT and H251N hiPSC-CMs. Fc = log2 fold change. (C) I. UMAP representation of MYH7 G256E CMs (MYH7 WT/G256E) and the isogenic counterpart (MYH7 WT/WT) clustered by the presence of G256E mutation. II. Unbiased clustering of G256E and WT hiPSC-CMs. III. Relative contribution to each cluster by WT and G256E hiPSC-CMs. IV. Comparison of MYH7 gene expression in WT and G256E hiPSC-CMs. Fc = log2 fold change. (D) Quantification of run-to-run expression variability of WT vs MUT DEGs between three independent scRNAseq runs. DEGs were defined as log fold change >0.5 & adjusted p-value < 0.05. Each dot represents the average run-to-run expression difference of one DEG for a specific clone. N(H251N) = 495 (3 clones, 165 DEGs); n(G256E) = 140 (5…

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  Fig. 2:. Beta-Adrenergic Stimulation Reveals a Metabolic Stress Response Phenotype in Variably Penetrant MYH7 G256E Mutation. (A) Schematic of experimental set-up. (B) Quantification of beating rate and contraction velocity of WT (Basal&Iso: n=30, 2 isogenic clones) and MYH7 G256E HET (Basal&Iso: n=45, 3 isogenic clones) CMs with and without isoproterenol (Iso) treatment. (C) I. UMAP representation of isoproterenol treated G256E CMs and the isogenic WT counterpart clustered by the presence of the G256E mutation. II. Unbiased clustering of G256E and WT hiPSC-CMs. III. Relative contribution to each cluster of WT and G256E hiPSC-CMs. IV. Comparison of MYH7 gene expression in WT and G256E hiPSC-CMs. Fc = log2 fold change. (D) Venn diagram comparing WT vs. G256E DEGs of unstressed (No iso treatment) and stressed (Iso treatment) CMs. (E) Gene expression of established hypertrophy response genes in WT and G256E CMs with and without isoproterenol treatment (WT n=1332, HET n=1106, WT+Iso n=1604, HET+Iso n=5958). (F) Gene expression of oxidative and cellular stress response genes in WT and G256E CMs with and without isoproterenol treatment. Fc = log2 fold change. (G) Quantification of mitochondrial…

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  Fig. 3:. Increase in Mutant Gene Dosage Results in Consistent Change in Transcriptional Signature. (A) Schematic of experimental set-up. (B) Sanger Sequencing of MYH7 locus at G256E mutation site of HET clone 141 and HOM clone 15. (C) I. UMAP representation of HOM MYH7 G256E mutant cells (MYH7 G256E/G256E) and the isogenic WT counterpart (MYH7 WT/WT) clustered by the presence of G256E mutation. II. Unbiased clustering of G256E and WT hiPSC-CMs. III. Relative contribution to each cluster by WT and G256E HOM hiPSC-CMs. IV. Comparison of MYH7 gene expression in WT and G256E HOM hiPSC-CMs. Fc = log2 fold change. (D) Venn diagram comparing WT vs G256E HOM DEGs with WT vs. G256E HET DEGs. (E) Quantification of run-to-run expression variability of DEGs between independent scRNAseq runs. DEGs were defined as log fold change >0.5 & adjusted p-value < 0.05. Each dot represents the average run-to-run expression difference of one DEG for a specific clone. N(G256E HET) = 140 (5 clones, 28 DEGs); n(G256E HOM) = 1040 (4 clones, 260 DEGs). (F) Quantification of clone-to-clone expression variability of DEGs between two isogenic G256E HOM CM clones, three isogenic G256E HET CM clones and two isogenic WT CM…

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  Fig. 4:. Proteomic Profiling Confirms Gene Dose Dependent Transcriptional Changes at the Protein Level. (A) Schematic of experimental set-up. Three biological replicates were quantified per clone (WT Cl.113 & Cl.174, HET Cl.141 & Cl.157, HOM Cl.15 & Cl.17) (B) Number of features quantified in the TMT experiment. (C) Quantification of MYH7 proteoforms in isogenic G256E HET and HOM CM clones. I. Schematic of the myosin heavy chain 7 sequence. Wild-type and G256E MYH7 proteoforms are differentiated based on the tryptic peptide spanning amino acids 250–257. II. Quantification of the mutant MYH7 G256E peptide in HET and HOM CMs. Data are presented as mean ± SE. (D+E) Volcano plot of differently abundant proteins in G256E HET vs. WT CMs (D) and G256E HOM vs. WT CMs (E). Salmon color indicating statistical significance defined as p-value < 0.05 and false discovery rate (FDR) < 0.1. Green color indicating both statistical significance and effect size of log2-fold change > 0.58 (F) Scatterplot comparing HET vs WT and HOM vs WT fold change regulation of previously identified HOM vs WT DEPs (n=58). Grey area indicates gene-dose dependent regulation of DEPs, red line represents linear regression fit.…

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  Fig. 5:. Homozygosity Reveals a Hypercontractile Phenotype in Variably Penetrant HCM Mutation. (A) Representative images of WT and G256E HOM cardiomyocytes showing, from left to right, α-actinin–GFP fluorescence, sarcomere orientation (color indicates local sarcomere orientation relative to the horizontal axis [cyan = horizontal, red = ±90°, vertical]), and sarcomere tracking (color-coded by Z-to-Z distance from longest [pink] to shortest [blue]). Scale bars: 50 μm (field of view, sarcomere orientation), 10 μm (sarcomere tracking). (B) Quantification of contraction velocity (CV), fractional sarcomere shortening (FSS), sarcomere orientation (SO), and relaxed Z-to-Z distance (RZ) in G256E HOM (CV n = 30; FSS, SO, RZ n = 20; 2 isogenic clones) and WT cardiomyocytes (CV n = 15; FSS, SO, RZ n = 10; 3 isogenic clones). Data are presented as mean ± SEM; functional parameters were analyzed using linear mixed-effects models (lmerTest) with genotype as a fixed effect and clone as a random effect (Satterthwaite’s approximation) (B). *P < 0.05, **P < 0.01, ****P < 0.0001.

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  Fig. 6:. Identification of Common vs Mutation-specific HCM Markers (A) Venn diagram of DEGs across all four experimental conditions. (B) Dotplot expression of identified markers consistently upregulated in all G256E & H251N transcriptomic experiments. (C-E) Top GO-term enrichment clusters for identified DEGs specific to only G256E HOM and H251N HET CMs (C), only H251N HET CMs (D) and only G256E HOM CMs (E).