Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies

Abstract

Study of monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combined patient-derived and genetically engineered induced pluripotent stem cells (iPSCs) with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene encoding tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS 'heart-on-chip' tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole-cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies and advances iPSC-based in vitro modeling of cardiomyopathy.

Fig. 1. Mitochondrial abnormalities in BTHS iCMs

a. Mass spectrum of control and BTH-H phospholipids showing mature cardiolipin (CL) and immature cardiolipin (monolysocardiolipin; MLCL) content of BTHS vs control iPSC-CMs. b. Comparison of MLCL/CL ratio in BTH-H, BTH-C, and control iPSC-CMs. The dashed line indicates the clinical diagnostic threshold for BTHS. c. ATP levels in BTHS and control iPSC-CMs cultured in galactose. n=3. NS, not significant. d–e. Assessment of BTH-H and control iPSC-CM mitochondrial function in galactose culture. Function was measured using cellular oxygen consumption rate (OCR), normalized to total protein. Oligo, oligomycin. FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. A/R, antimycin plus rotenone. Measures of mitochondrial function (defined in Supplementary Fig. 5b,c) were quantitatively compared between control and BTH-H iCMs. n=3. *, P<0.05. f. ATP levels in BTHS and control iPSC-CMs cultured in glucose. n=3.

Fig. 2. TAZ deficiency is necessary to cause the iPSC-CM metabolic phenotype

a. Subcellular localization of FLAG-tagged TAZ, delivered to iPSC-CMs by modified RNA (modRNA) transfection. Localization to mitochondria was assessed by co-localization with virally delivered RFP targeted to mitochondria. FLAG co-localized with RFP. Bar = 10 µm. b. TAZ modRNA restored cardiolipin biogenesis. BTH-H or control iPSC-CMs were transfected with the indicated modRNA and cardiolipin composition was measured by mass spectroscopy. MLCL/CL below 0.3 (dotted line) is considered normal. c. Effect of TAZ modRNA on mitochondrial function in control and BTHS iPSC-CMs. Mitochondrial function was measured by extracellular flux analysis as detailed in Supplementary Fig. 5b,c. d. Quantitation of mitochondrial functional parameters from c. n=3. e. F1F0 ATP synthase specific activity, measured by selective complex immunocapture. n=6. *, P<0.05 compared to each other group.

Fig. 3. Construction and characterization of TAZ mutant and isogenic control iPSCs by Cas9-mediated genome editing

a. Schematic of genome editing strategy. b. Strategy for modification of TAZ exon 6 using Cas9, the indicated guide RNA (gRNA), and the indicated repair oligo. c. Sequence of a region of TAZ exon 6 in selected genome-edited cell lines. Purple boxes indicate deletion (Δ) or insertion. PGP1-TAZ^WT was handled in parallel but was not modified and had wild-type TAZ sequence. d. Cardiolipin maturation in TAZ and isogenic control iPSC-CMs, as determined by phospholipid mass spectroscopy. e. Basal ATP level of TAZ mutant and isogenic control iPSC-CMs cultured in galactose media. **, p<0.01. n=3. f. F1F0 ATPase specific activity, measured from selectively immunocaptured proteins, in TAZ mutant and isogenic control iPSC-CMs. **, p<0.01. n=3. g. Abnormalities of mitochondrial function in TAZ mutant iPSC-CMs, as measured by extracellular flux mitochondrial function assay. iPSC-CMs were transfected with the indicated modified RNA for 5 days. **, p<0.01 by 1-way ANOVA with Dunnett’s post-hoc test compared to PGP1-TAZ^WT+nGFP. n=3.

Figure 4. Impaired sarcomere organization in BTHH mutant iPSC-CMs

iPSC-CMs were seeded on micropatterned fibronectin rectangles with length:width ratios of 7:1 (95 µm × 13 µm), fixed, and stained for α-actinin. Sarcomere organization was quantitated by an unbiased 2D Fourier transform-based algorithm (see Supplemental Methods). Bar = 10 µm. a. Sarcomere organization of patient-derived BTH-H and control iPSC-CMs. Sarcomere organization was tested in the indicated culture medium and after transfection with the indicated modified RNA. P < 0.05 vs: *, BTH-H + nGFP, gal; #, BTH-H + nGFP, glu. b. Sarcomere organization of genome-edited, isogenic PGP1 iPSC-CMs containing the indicated TAZ variant. iPSC-CMs were transfected with the indicated modified RNA. P < 0.05 vs: *, PGP1-TAZ-517delG + nGFP; #, PGP1-TAZ-517ins. c. Sarcomere organization of patient-derived BTH-C and control iPSC-CMs. Statistical comparisons were made by ANOVA with Fisher LSD post-hoc test. NS, not significant. Sample number is displayed within each bar.

Figure 5. Depressed contractile stress generation by BTHS myocardial tissue constructs

a. iPSC-CMs seeded onto thin elastomers with patterned lines of fibronectin self-organized into anisotropic myocardial tissues referred to muscular thin films (MTFs). Cardiomyocyte stress generation reduces the radius of curvature of the construct as it contracts from diastole to peak systole. Red lines indicate automated MTF tracking projected onto the horizontal plane. Blue lines indicate lengths of MTFs prior to peeling from substrate. Bar, 100 µm. b–d. Twitch stress and peak systolic stress generated by MTFs from patient-derived BTH-H and control iPSC-CMs (b), genome-edited TAZ frameshift and control iPSC-CMs (c), and patient-derived BTH-C and control iPSCs (d) at 2 Hz pacing. (b) P<0.05 vs: *, BTHH+nGFP, gal; #, BTH-H+nGFP+glu. (c) P<0.05 vs: *, PGP1-TAZ^c.517delG+nGFP; #, PGP1-TAZ^c.517ins+nGFP. (d) *, P<0.05 vs: BTH-C+nGFP. Statistical comparisons were made by Kruskal-Wallis One Way ANOVA on ranks and Dunn’s post-hoc test. Sample size is indicated by number inside each bar.

Fig. 6. Effect of small molecules on BTHS iCM ATP levels and mitochondrial function

a–c. BTHS iCMs were treated with linoleic acid (LA), bromoenol lactone (BEL), or arginine plus cysteine (A/C) for 5 days in galactose-containing media, and the effect on cardiolipin maturation (a), cellular ATP levels (b, n=3), and mitochondrial function (c, n=3) were measured. *, P<0.05 vs. control. V, vehicle. d. Effect of LA treatment on sarcomeric organization compared to vehicle (V). LA increased BTHH sarcomeric organization but did not have a significant effect on BTHC sarcomeres. One-way ANOVA. P < 0.05 vs: *, BTHH + V; #, BTHC + V. e. Effect of LA treatment on muscular thin film contractile stress generation. Twitch strength of BTHH and BTHC constructs were dramatically enhanced by LA treatment compared to vehicle (V). Kruskal-Wallis with Dunn’s post-hoc test. Numbers inside bars indicate sample size. P < 0.05 vs: *, BTHH + V; #, BTHC + V f. Lipid peroxidation in BTHS and control iPSC-CMs. Cells were cultured in glucose and treated with LA and the indicated modRNA for 5 days. The lipid peroxidation product 4-hydroxynonenal was measured by ELISA. One-way ANOVA. n=6. *, P<0.05 vs *, WT1 + nGFP; #, BTHH + nGFP. g. Mitochondrial ROS in BTHS and control iPSC-CMs and its suppression by MitoTEMPO. Cells were treated with MitoTEMPO (MT) or vehicle (V). Mitochondrial ROS was assessed by FACS measurement of Mitosox fluorescence of isolated mitochondria. Representative histograms are shown to the right, with dotted line indicating the peak MitoSox signal intensity from WT1+V. One-way ANOVA. n=3. P<0.05 vs *, BTHH + V; †, PGP1-TAZ^c.517delG + V. h. Effect of MitoTEMPO on sarcomeric organization of iPSC-CMs plated on fibronectin rectangles. Numbers inside bars indicate cells analyzed. One-way ANOVA. P<0.05 vs *, BTHH + V; †, PGP1-TAZ^c.517delG + V. i. Effect of MitoTEMPO on force generation by iPSC-CM myocardial tissue constructs. Numbers inside bars indicate thin films analyzed. Kruskal-Wallis with Dunn’s post-hoc test. P<0.05 vs *, BTHH + V; †, PGP1-TAZ^c.517delG + V.