Mitochondrial DNA: Hotspot for Potential Gene Modifiers Regulating Hypertrophic Cardiomyopathy

Figures

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  Figure 1. Harnessing isogenic hPSC-CM models to investigate varying HCM mitochondrial phenotypes. (a) Study workflow: hPSC were generated from three different healthy cell sources, followed by the introduction of the p.R453C-βMHC mutation by CRISPR/Cas9. Isogenic healthy and diseased (heterozygote or homozygote) mutant hPSCs were differentiated into cardiomyocytes and mtDNA content and sequence was evaluated. (b) Ratiometric qPCR analysis of mitochondrial: nuclear DNA highlighted variations in mitochondrial content across healthy (WT) lines, with AT1 lines showing 50–60% lower mtDNA content relative to HUES7 and REBL-PAT hPSC-CMs. Isogenic (c) AT1 and (d) REBL-PAT hPSC-CMs did not display striking changes in mtDNA content, with the exception of the AT1 MYH7-knockout line. Data are presented as box and whiskers plots, n = 5–8 biological replicates, * p < 0.05, ** p < 0.01 (Student’s t-test in (b) and one-way ANOVA + Dunnett’s correction relative to WT in (c,d)).

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  Figure 2. mtDNA NGS of three separate isogenic sets bearing the p.R453C-βMHC mutation. Analysis of mtDNA sequence of isogenic sets of fibroblasts, hPSCs and hPSC-CMs of AT1 (top), REBL-PAT (middle) and HUES7 (bottom) identified variants specific to the starting cell sources. Legend map on the right side indicates type of mtDNA mutation (DEL, deletion; INS, insertion; SNV, single nucleotide variant) and percent heteroplasmy is correlated with the size of the symbol (WT, wild-type; het, heterozygous; HOM, homozygous; KO, knock-out).

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  Figure 3. mtDNA NGS of three separate isogenic sets bearing the p.E99K-ACTC1 mutation. Analysis of mtDNA sequence of isogenic sets of fibroblasts, hPSCs and hPSC-CMs of E99K1 (top), E99K2 (middle) and NC-Edit-E99K (bottom) identified variants specific to the starting cell sources. Legend map on the right side indicates type of mtDNA mutation (DEL, deletion; INS, insertion; SNV, single nucleotide variant) and percent heteroplasmy is correlated with the size of the symbol. (WT, wild-type; het, heterozygous).

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  Figure 4. Mito-modulating effects of mtDNA variants identified in hPSC-CMs. (a) Mitochondrial respiration was evaluated by Seahorse analysis of healthy hPSC-CM lines used to model the p.R453C-βMHC sarcomeric mutation, showing (b) higher oxidative phosphorylation activity in REBL-PAT cardiomyocytes relative to the AT1 line, presenting different mtDNA variants. (c,d) The healthy (gene-corrected) E99K1-WT hPSC-CM line showed lower mitochondrial respiration activity relative to NC hPSC-CMs displaying diverse mtDNA variants. (e,f) Evaluation of mitochondrial ROS production by flow cytometry of MitoSOX-labelled hPSC-CMs showed no difference between healthy AT1 vs REBL-PAT hPSC-CMs, indicating that the mtDNA variants identified in these lines do not contribute to mito-dysfunction. Data are presented as mean ± SEM, N = 5-7 biological replicates, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., non-significant (Student’s t-test); OCR, oxygen consumption rate.

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  Figure 5. Analysis of mtDNA variants in unrelated HCM patients. (a–c) Pedigree chart of three unrelated families where the p.E99K-ACTC1 mutation is common (red numbers indicate more severe phenotypes and green numbers milder ones). (d) NGS sequencing analysis of mtDNA of fibroblasts derived from patient skin biopsies identified variants shared between patients or specific to individual families. Legend map on the right side indicates type of mtDNA mutation (DEL, deletion; INS, insertion; SNV, single nucleotide variant) and percent heteroplasmy is correlated with the size of the symbol.