Myosin Heavy Chain Converter Domain Mutations Drive Early-Stage Changes in Extracellular Matrix Dynamics in Hypertrophic Cardiomyopathy

Figures

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  FIGURE 1. Base editing and characterization of the MYH7/MYH6 mutant hiPSCs. (A) Schematic of MYH7 locus as shown in the red box on chromosome 14 and c.2167 location as pointed by the red arrow for base editing. (B) Chromatogram of MYH7 mutant locus in hiPSCs showing c.C2167T (p.Arg723Cys, p.R723C) mutation and its isogenic parental genotype. (C) Bright field images and immunostaining of Oct-4 using the isogenic control and MYH7/MYH6 mutant hiPSCs. DAPI labels the nuclei (blue). (D) qPCR analysis for Oct-4 expression level using RNA isolated from the isogenic control and MYH7/MYH6 mutant hiPSCs. (E) Cardiac differentiation timeline and analysis regime. (F) Quantitative analysis of the differentiation efficiency based on the cTnT staining in the control and MYH7/MYH6 mutant cardiomyocytes. Note that mutation did not result in any significant difference in the cardiomyocyte differentiation efficiency. (G) Cardiac Troponin T (cTnT) and DAPI-stained hiPSC-CMs on Day 15, 30, 45, and 60 after the start of differentiation. Data represents mean ± SEM. (Scale Bar = 100 μm) (N.S.: not significant; Student t test).

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  FIGURE 2. MYH7/MYH6 mutation recapitulates the hypertrophic cardiomyopathy phenotype in the hiPSC-CMs. (A,B) Bright-field images (A) and quantification (longest axis) (B) of the re-plated control and mutant cardiomyocytes prior to their attachment to the substrate to analyze their size at Day 15–60 of differentiation. Notice the hypertrophy of the mutant cells from Day 15–60. (Scale Bar = 100 μm). Data represents mean ± SEM. (**p < 0.01; ***p < 0.005; Student t test; n = 24 for six randomly picked particles with round and intact boundary from four bright field images per condition) (C) MYH7:MYH6 ratio obtained from qPCR analysis. Data represents mean ± SEM. (**p < 0.01; N.S.: not significant; Student t test; three wells for each condition and two experimental replicates for Day 15, Day 30, and Day 60, three experimental replicates for Day 45; n = 6 for Day 15, Day 30, and Day 60, respectively, and n = 9 for Day 45 conditions) (D) Representative images of cTnT-immunostained and DAPI-stained hiPSC-CMs to visualize nucleation in the differentiated cardiomyocytes using the isogenic control and MYH7/MYH6 mutant lines. Red arrows indicate multi-nucleated cells. Left images show Day 30 re-plated…

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  FIGURE 3. Functional assessment of the MYH7/MYH6 mutant cardiomyocytes. (A) Calcium transient trends for isogenic control and MYH7/MYH6 mutant hiPSC-CMs at Day 15–60 of differentiation. (B) Beat rate measured from calcium transient analysis using the isogenic control and MYH7/MYH6 mutant cardiomyocytes at Day 15–60 of differentiation. Note the significant reduction in beats per minute in the mutant cardiomyocytes relative to control. Data represents mean ± SEM; ***p < 0.005; Student t test; three wells of differentiated cardiomyocytes and four videos from separated locations in each well, and n = 12 for each condition. (C) Beat rate in frequency domain for both isogenic control and MYH7/MYH6 mutant cardiomyocytes at Day 15–60 of differentiation. (D) Quantitative result for irregular beating in the isogenic control and MYH7/MYH6 mutant cardiomyocytes at Day 15–60 of differentiation. The level of beating irregularity was defined as the average standard deviation for all beat intervals over twelve calcium transient measurements for each cell line at each time point. Data represents mean ± SEM; *p < 0.05; Student t test; three wells of differentiated cardiomyocytes and four videos from…

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  FIGURE 4. Bulk RNAseq analysis of MYH7/MYH6 dual mutation-induced hypertrophic cardiomyopathy in Day 15 hiPSC-CMs to evaluate the molecular mechanism. (A) Heatmap for comparing HCM mutant and control hiPSC-CMs showing the dysregulated transcripts in the mutant cardiomyocytes relative to control. (B) Volcano plot showed a total of 3,317 differentially expressed genes (DEGs) with the mutation. (p < 0.05, |log2fold change| > 0) (C) fpkm distribution showing similar log counts between the control and mutant lines. (D) Top 20 upregulated gene-disease associations by DisGeNet analysis. (E) Top 20 upregulated diseases annotated by disease ontology (DO) analysis. Size of the dot indicate the number of gene associated with the given disease and red color indicate the significance of this association. (F) Heatmap for 10 up-regulated DEGs that associated with hypertrophic cardiomyopathy. (G) Heatmap for 10 up-regulated DEGs that associated with calcium signal pathway. (H) Heatmap for 10 up-regulated DEGs and 1 downregulated DEG associated with cardiac muscle contraction.

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  FIGURE 5. MYH7/MYH6 mutation results in dysregulation of the metabolic pathways and ADP/ATP dynamics. (A) Bar graph showing top 20 significantly altered metabolic pathways in the mutant cardiomyocytes relative to control cardiomyocytes; *p < 0.05. (B) Heatmap of the DEGs mitochondrial transcripts in the mutant cardiomyocytes relative to control cardiomyocytes. (C) ADP:ATP ratio using the lysate from control and mutant cardiomyocytes. Notice the sharp peak at Day 45 of differentiation in the mutant cells relative to control cells. Data represents mean ± SEM; ***p < 0.005; N.S.: not significant; Student t test; n = 3 wells of differentiated cardiomyocytes for each condition. (D) qPCR analysis for ATP2B4 transcripts using the RNA isolated from control and mutant cardiomyocytes at Day 15 and Day 45 of differentiation. Data represents mean ± SEM; *p < 0.05; **p < 0.01; Student t test; n = 9 cell lysate samples for Day 15 conditions, n = 7 cell lysate samples for Day 30 conditions, n = 10 cell lysate samples for Day 45 conditions, n = 6 cell lysate samples for Day 60 conditions.

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  FIGURE 6. MYH7/MYH6 mutation drives altered ECM dynamics and ECM-receptor interaction. (A) Reactome analysis showing the top 20 annotated down-regulated pathways in the mutant as compared to control lines. Note that the most significantly dysregulated were the pathways associated with ECM organization. (B) Heatmap showing the dysregulated transcripts involving in ECM. (C) Top 20 annotated pathways determined by KEGG analysis with down-regulated DEG list. Note that ECM-receptor interaction transcripts were most significantly altered in the mutant cells relative to control cells. (D) Heatmap for up-regulated DEGs that associated with ECM-receptor interaction. (E) Representative images of cTnT and pFAK-immunostained hiPSC-CMs using isogenic control and mutant cells. White stars point out nucleus location and white arrows indicate activated FAK signal at the vicinity of the cell-substrate attachment site. DAPI labels the nuclei (blue). (Scale Bar = 100 μm) (F) qPCR analysis for ITGA7, ITGAV, and ITGB1 transcripts using RNA isolated from control and mutant cardiomyocytes at Day 15 and Day 45 of differentiation. Data represents mean ± SEM; *p < 0.05; **p < 0.01; Student t test; n = 4 cell lysate…

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  FIGURE 7. Schematic depicting the impact of MYH7/MYH6 mutation in mediating hypertrophic cardiomyopathy. Our data indicate that MYH7/MYH6 mutation results in hypertrophy, dysregulation of metabolic as well as ECM dynamics. Solid black arrows and bold text represent HCM phenotypes that have been reported for other MYH7 mutations, grey text represents previously reported features of MYH7 mutations. Solid blue arrows represent the mutation associated ECM dynamics which is novel to the field and could provide targets to develop drug therapies potent in the early stages of HCM.