Disturbed mitochondrial maturation in cardiolipin remodeling-deficient cardiomyocytes

Figures

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  Figure 1. Compromised mitochondrial adaptation to fatty acid exposure in TAZ-deficient cardiomyocytes Where indicated, WT and TAZ KO cardiomyocytes were cultured under Pal/car for 8 days (A) Immunoblot analysis of TAZ expression (mean ± SEM, n = 5–6 biological replicates). (B) Monolyso-cardiolipin (MLCL) to cardiolipin (CL) ratio (mean ± SEM, n = 4 biological replicates). (C) Oxygen consumption rate (OCR) measured by Seahorse XF96e FluxAnalyzer with glucose, glutamine, pyruvate, and palmitic acid as substrates (100K/well; mean ± SEM, n = 12–16 with 4 biological replicates and 3–4 technical replicates). Dashed lines: untreated WT. (D–F) Cristae morphology analyzed by electron microscopy. (D) Representative images. Scale bar length: 500 nm. See also Figure S6. (E) Quantification of cristae detachment from the inner boundary membrane (IBM) in WT cardiomyocytes. Data are from 3 biological replicates, with a total of 411 (untreated) and 259 (Pal/car-treated) mitochondria. Median and quartiles are shown. (F) Cristae morphology was categorized as follows: defined (>50% cristae with clear and aligned membranes), intermediate (25–50% cristae with clear and aligned membranes), irregular (<25%…

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  Figure 2. Cardiomyocyte maturation increases mitochondrial respiration and cristae density WT and TAZ KO cardiomyocytes were cultured with Pal/car + Dex/T3 for 2 weeks as indicated. (A) Sarcomere gene expression (qPCR, normalized to EEF1A1; mean ± SEM, n = 5 biological replicates). Dashed lines: untreated WT (Figure S8A). (B) Immunoblot analysis of cardiac Troponin I (cTnI) and slow skeletal muscle Troponin I (ssTnI) (mean ± SEM, n = 4–6 biological replicates). (C) Electrophysiological gene expression (qPCR, normalized to EEF1A1; mean ± SEM, n = 5 biological replicates). Dashed lines: untreated WT (Figure S8B). (D) OCR measured by Seahorse XF96e FluxAnalyzer with glucose, glutamine, pyruvate, and palmitic acid as substrates (80K/well; mean ± SEM, n = 9–12 with 3 biological replicates and 3–4 technical replicates). Dashed lines: untreated WT. (E) Representative electron microscopy images (n = 3–4 biological replicates). Scale bar length: 500 nm. (F) Quantitative analysis of cristae density (sum of cristae length per mitochondrion) in WT cardiomyocytes. Data are from 4 biological replicates, with a total of 282 (untreated) and 260 (+Pal/car, +Dex/T3) mitochondria. Median and quartiles are…

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  Figure 3. Cardiomyocyte maturation develops the inner mitochondrial membrane protein machinery WT and TAZ KO cardiomyocytes were cultured with the indicated treatments for 2 weeks (A) Immunoblot analysis of OXPHOS subunits (Complexes I, II, III, IV, and V). Representative images from n = 5–6 biological replicates are shown. (B and C) Individual respiratory complexes. Triton X-100 or n-dodecyl-β-D-maltoside (DDM)-solubilized cell extracts (protein:detergent = 1:4) were analyzed by blue native-PAGE and immunoblot. (D) Immunoblot analysis of adenine nucleotide translocators. Representative images from n = 5–6 biological replicates are shown. (E and F) Respiratory supercomplex assembly. Digitonin-solubilized cell extracts (protein:digitonin = 1:4) were analyzed by blue native-PAGE and immunoblot. Numerical subscripts specify the number of individual complex units comprising the indicated assembly; “n” denotes a variable number. Quantifications were performed on immunoblots against NDUFB6 (mean ± SEM, n = 3 biological replicates). (G) Mitochondrial DNA copy number analyzed by qPCR (mean ± SEM, n = 3–5 biological replicates). Statistical significance: one-way ANOVA with Tukey’s test; ∗p < 0.05,…

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  Figure 4. Cardiomyocyte maturation drives cardiolipin remodeling WT and TAZ KO cardiomyocytes were cultured for 2 weeks under the indicated conditions. (A) Principal component analysis (PCA) of cardiolipin and monolyso-cardiolipin species. (B) Monolyso-cardiolipin (MLCL) to cardiolipin (CL) ratio (mean ± SEM, n = 3–4 biological replicates). (C) Immunoblot analysis of TAZ (mean ± SEM, n = 6 biological replicates). (D) Heatmap of cardiolipin biogenesis gene expression (qPCR, normalized to EEF1A1; mean, n = 3–6 biological replicates). Statistical significance: one-way ANOVA with Tukey’s test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. ∗ WT vs. TAZ KO within treatment; # vs. untreated within genotype (in D).

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  Figure 5. TAZ-mediated cardiolipin remodeling is essential for proper mitochondrial respiration The indicated iPSC lines were differentiated into cardiomyocytes and treated with Pal/car + Dex/T3 for 2 weeks (A) Immunoblot analysis of TAZ (mean ± SEM, n = 3–4 biological replicates). (B) Monolyso-cardiolipin (MLCL) to cardiolipin (CL) ratio (mean ± SEM, n = 3–4 biological replicates). (C) OCR measured by Seahorse XF96e FluxAnalyzer with glucose, glutamine, pyruvate, and palmitic acid as substrates (80K/well; mean ± SEM, n = 17–34 with 3–5 biological replicates and 2–6 technical replicates). Statistical significance: one-way ANOVA with Tukey’s test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

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  Figure 6. TAZ deficiency causes cardiomyocyte dysfunction (A) NPPA and NPPB gene expression (qPCR, normalized to YWHAZ) in WT and TAZ KO cardiomyocytes cultured for 8 days (mean ± SEM, n = 5 biological replicates). (B) NPPA and NPPB gene expression (qPCR, normalized to EEF1A1) after 2 weeks of treatment (mean ± SEM, n = 5 biological replicates). Dashed lines: untreated WT. (C–H) Cardiomyocytes derived from the indicated iPSC strains were cultured under Pal/car + Dex/T3 for 2 weeks with or without 10 nM endothelin-1 (ET-1). (C) Secreted proBNP levels in the culture supernatant were quantified by ELISA (mean ± SEM, n = 3–6 biological replicates). (D–H) Cardiomyocyte contractions were analyzed by video recordings. Monolayered cardiomyocyte sheets were stimulated with increasing concentrations of isoproterenol (ISO) (mean ± SEM, n = 5–8 biological replicates). (D) Contraction frequency. (E) Contraction amplitude. (F) Amplitude at baseline. (G, H) Systolic and diastolic peak times at 20% (G) and 50% (H) thresholds at baseline. Limited response in ET-1-treated TAZ KO cells precluded data display (n.d., not detected). Statistical significance: one-way ANOVA with Tukey’s test; ∗p < 0.05, ∗∗p <…

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  Figure 7. Phenotypic spectrum of TAZ-deficient cardiomyocytes C11 WT and TAZ KO cardiomyocytes were cultured under Pal/car +Dex/T3 for 2 weeks (A) Immunoblot analysis of TAZ (mean ± SEM, n = 4 biological replicates). (B) Monolyso-cardiolipin (MLCL) to cardiolipin (CL) ratio (mean ± SEM, n = 3 biological replicates). (C) Immunoblot analysis of cardiac Troponin I (cTnI) and slow skeletal muscle Troponin I (ssTnI) (mean ± SEM, n = 4 biological replicates). (D) OCR measured by Seahorse XF96e FluxAnalyzer with glucose, glutamine, pyruvate, and palmitic acid as substrates (40K/well; mean ± SEM, n = 21–29 with 4–5 biological replicates and 5–6 technical replicates). (E–I) Indicated cardiomyocytes were cultured under Pal/car + Dex/T3 with or without 10 nM ET-1. Cardiomyocyte contractions were analyzed by video recordings with ISO stimulation (mean ± SEM, n = 4–9 biological replicates). (E) Contraction frequency. (F) Contraction amplitude. (G) Baseline amplitude. (H and I) Systolic and diastolic peak times at 20% (H) and 50% (I) thresholds at baseline. Limited response in ET-1-treated TAZ KO.10 cells (n = 2/5) precluded error bar and statistical significance display. (J) Secreted proBNP levels in…