Metabolic crisis and TRPM4 activation cause QT prolongation in TANGO2 deficiency disorder

Figures

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  Figure 1. Continuous telemetry over 12 min showing sinus rhythm with macroscopic T-wave alternans and episodes of torsades de pointes (TdP). After IV verapamil, there is marked normalization of repolarization and sinus rhythm with no ventricular ectopy or arrhythmias. Red boxes are magnified images of macroscopic T-wave alternans, TdP, and sinus rhythm with normal QT immediately after verapamil.

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  Figure 2. Palmitate, but not glucose, induces bioenergetic dysfunction in TANGO2−/− hiPSC-CMs. (A) Intracellular ATP/ADP ratio was reduced by palmitate, and further by fasting in TANGO2−/− L1 and L2 hiPSC-CMs (n = 32–48). a, P = 2.7e−6; b, P = 2.1e−5; c, P < 2.2e−16; d, P < 2.2e−16. (B) OCR traces measured via Seahorse XF analyzer in control and TANGO2−/− L1 and L2 hiPSC-CMs under 0.1 mmol/L palmitate conditions with/without 24-h fasting. Quantification of palmitate-dependent basal OCR (C), ATP-linked OCR (D), and intracellular ATP production rate (E) in control and TANGO2−/− L1 and L2 hiPSC-CMs (n = 51–62). (F and G) Substrate preference assay using 5.6 mmol/L glucose and 0.1 mmol/L palmitate shows that TANGO2−/− L1 and L2 hiPSC-CMs have impaired palmitate oxidation [fatty acid oxidation (FAO)] but preserved glucose oxidation (Gox) (n = 52–60). FAO-dependent OCR was calculated as the differences between baseline OCR and OCR after 0.1 mmol/L etomoxir treatment. Gox-dependent OCR represents the remaining OCR post-etomoxir treatment. Data points represent individual replicates from n = 3 independent cardiac differentiation batches. Data are presented as mean ± SD. Statistical analyses were…

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  Figure 3. Extracellular field potentials reveal metabolic substrate-dependent electrical abnormalities in TANGO2−/− hiPSC-CMs. Representative field potential traces recorded at 1 Hz pacing and 37°C under conditions with (A) 5.6 mmol/L glucose, (B) 0.1 mmol/L palmitate, or (C) 0.1 mmol/L palmitate following 24-h fasting. (D) Field potential upstroke duration (QRS duration) remained unchanged across all conditions, confirming preserved depolarization (n = 20–82). Asterisks (*) indicate TampA alternans and FPD alternans in TANGO2−/− hiPSC-CMs. (E and F) T-wave amplitude (TampA) and beat-to-beat variation in TampA (TampA alternans) increased significantly in TANGO2−/− hiPSC-CMs exclusively under post-fasting palmitate conditions. (G and H) FPD and beat-to-beat variation in FPD (FPD alternans) significantly increased in TANGO2−/− hiPSC-CMs upon palmitate exposure, with further exacerbation after fasting. Data points represent individual monolayers from n = 3 independent cardiac differentiation. Data in (D) are presented as mean ± SD. Data in (E–H) are presented as median ± interquartile range. For (E)–(H), data were log-transformed (E, F, and H) and cube root-transformed (G) to achieve normal…

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  Figure 4. Palmitate induces action potential dysregulation in TANGO2−/− hiPSC-CMs. (A) Representative AP traces recorded under 0.5 Hz pacing in three different conditions: 5.6 mmol/L glucose, 0.1 mmol/L palmitate, and 0.1 mmol/L palmitate after a 24-h fasting period. (B) Representative AP traces demonstrating alternans in TANGO2−/− L1 hiPSC-CMs under 0.5 Hz pacing with 0.1 mmol/L palmitate following 24-h fasting. Palmitate exposure (0.1 mmol/L) induces APD90 prolongation (C) and beat-to-beat AP alternans (D) in TANGO2−/− L1 hiPSC-CMs, with both parameters further exacerbated following a 24-h fasting. In contrast, glucose exposure (5.6 mmol/L) produced no differences in APD90 (C) and beat-to-beat AP alternans (D) between control and TANGO2−/− L1 hiPSC-CMs. Sample sizes: glucose, n = 8–9; palmitate, n = 10–12; palmitate post-fasting, n = 32–41. Data points indicate individual cells from n = 3 independent cardiac differentiation batches. Data in (C) are presented as mean ± SD. Data in (D) are presented as median ± interquartile range. For panels (D), data were square root-transformed to achieve normal distribution prior to statistical analysis. Statistical analyses were performed using the…

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  Figure 5. ATP restoration or LTCC blockade prevents palmitate-induced action potential prolongation in TANGO2−/− hiPSC-CMs post-24-h fasting. (A) Representative AP traces recorded in control and TANGO2−/− L1 hiPSC-CMs following 24-h fasting and 0.1 mmol/L palmitate exposure. Intracellular dialysis of 2.5-unit creatine kinase (CK) and 2 mmol/L PCr via patch pipette abolished the differences in APD90 (B) and AP alternans (C) between control and TANGO2−/− L1 hiPSC-CMs (n = 10–13). (D) Representative APs recorded in control and TANGO2−/− L1 hiPSC-CMs before (baseline) and after treated with 400 nmol/L verapamil under 0.5 Hz pacing, following exposure to 0.1 mmol/L palmitate after 24-h fasting. 400 nmol/L verapamil treatment abolishes palmitate-induced APD90 prolongation (E) and AP alternans (F) in TANGO2−/− L1 hiPSC-CMs (n = 9–14). Data points indicate individual cells from n = 3 independent cardiac differentiation batches. Data in (B) and (E) are presented as mean ± SD. Data in (C) and (F) are presented as median ± interquartile range. For (C) and (E), data were square root-transformed to achieve normal distribution prior to statistical analysis. Statistical analyses were performed using the…

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  Figure 6. Following a 24-h fasting, palmitate induces intracellular Ca abnormalities in TANGO2−/− hiPSC-CMs, which are prevented by verapamil. (A) Representative intracellular Ca transients and caffeine-induced transients in fura-2-loaded control and TANGO2−/− L1 hiPSC-CMs pretreated with vehicle (H2O) or 100 nmol/L verapamil for 1 h, upon exposure to 0.1 mmol/L palmitate following 24-h fasting. Comparison of diastolic Ca (B), Ca decay kinetics (τ) (C), fractional SR Ca release (D), and Ca transient alternans (E) in the presence of vehicle or 100 nmol/L verapamil (n = 110–113 cells in vehicle-treated groups; n = 45–54 cells in verapamil-treated groups). Data points indicate individual cells from n = 2 independent cardiac differentiation batches. Data in (B) and (D) are presented as mean ± SD. Data in (C) and (E) are presented as median ± interquartile range. For (C) and (E), data were square root-transformed to achieve normal distribution prior to statistical analysis. Statistical analyses were performed using the hierarchical statistical model with Bonferroni correction to account for batch-to-batch variation in cardiac differentiation. a, P = 1.7e−5; b, P = 1.3e−3; c, P = 1.5e−2; d, P =…

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  Figure 7. TRPM4 mediates the palmitate-induced action potential prolongation in TANGO2−/− hiPSC-CMs following 24-h fasting. (A) Relative mRNA expression of TRPM4 in TANGO2−/− L1 hiPSC-CMs upon exposure to 0.1 mmol/L palmitate following 24-h fasting, normalized to ACTN2 and expressed as fold change relative to control (n = 9). (B) Relative TRPM4 mRNA expression in control and TANGO2−/− L1 hiPSC-CMs treated with negative siRNA or TRPM4-targeting siRNA. Fold change was calculated relative to corresponding untreated group (n = 5). (C) Representative APs recorded in control and TANGO2−/− L1 hiPSC-CMs under 0.5 Hz pacing, transfected with negative or TRPM4 siRNA, following exposure to 0.1 mmol/L palmitate after 24-h fasting. (D) Effect of siRNA-mediated TRPM4 knockdown on APD90 in control and TANGO2−/− L1 hiPSC-CMs (n = 14–20). Data points represent biological replicates from independent cardiac differentiation batches (A and B) or individual cells from n = 3 independent cardiac differentiation batches (D). Statistical significance was assessed using unpaired two-tailed Student’s t test (A), two-way analysis of variance with Šídák's multiple comparison test (B), or hierarchical statistical model…