Cellular and electrophysiological characterization of triadin knockout syndrome using induced pluripotent stem cell-derived cardiomyocytes

Figures

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  Figure 1. Phenotype of patients with triadin knockout syndrome (TKOS) and TKOS patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) (A) Representative 12-lead electrocardiograms from TKOS patients 1 and 2 showing QT prolongation and T-wave inversions in precordial leads V1–V3. (B) Family pedigrees for TKOS patients 1 (red) and 2 (blue). Circles denote female, and squares denote male. (C) Protein topology map of TRDN-encoded cardiac triadin 1. Each white dot represents the location of a corresponding pathogenic variant. The KEKE domain is the region known to be responsible for the interaction and binding of both CASQ2 and RYR2 (amino acids 210–224). (D) Western blot detecting cardiac triadin 1 in unrelated control (lane 1) and patient-derived (lanes 2–3) iPSC-CMs. (E and F) Representative action potential tracings and (F) action potential duration at 90% repolarization (APD90) in iPSC-CMs derived from unrelated control (one differentiation), TKOS patient 1 (one differentiation), TKOS patient 1 transfected with either empty vector (EV; one differentiation) or human cardiac triadin 1 cDNA-containing plasmid (hTRDN; one differentiation), and TKOS patient 2 (one…

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  Figure 2. Generation of a variant-inserted TRDN−/− iPSC-CM model of TKOS (A) A schematic showing the insertion of a homozygous c.53_56delACAG variant (p. D18fs∗13) into a healthy donor iPSC line using CRISPR-Cas9 and western blot detecting cardiac triadin 1 in isogenic control (lane 1) and in TRDN−/− iPSC-CMs (lane 2). (B and C) Representative action potential traces and (C) action potential duration at 20% (APD20), 50% (APD50), and 90% (APD90) repolarization at baseline and after treatment with 1 μM isoproterenol (ISO) in isogenic control (two differentiations) and TRDN−/− (four differentiations) iPSC-CMs. Graphs are presented as mean ± SEM. Two-way ANOVAs were conducted to determine significance. Ctrl, control.

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  Figure 3. TRDN−/− iPSC-CMs display cellular arrhythmias (A) Representative action potential traces derived from isogenic control (black) and TRDN−/− (red) iPSC-CMs recording during 1-Hz pacing or spontaneous beating. Black arrows indicate individual delayed afterdepolarizations (DADs) or early afterdepolarization (EAD)-like events. Black boxes contain zoomed-in representative action potential traces. (B) The two left panels depict the number of cells showing DADs from either paced at 1 Hz or spontaneous action potential (gap-free) measurements and the number of DADs per action potential (DADs/episode) from spontaneous action potential measurements only in isogenic control (two differentiations) and TRDN−/− (four differentiations) iPSC-CMs at baseline and after treatment with 1 μM ISO. Fischer’s exact tests were used to determine significance between two groups (first panel). Two-way ANOVAs with Tukey’s multiple comparisons tests were used to determine statistical significance for more than three groups (second panel). The two right panels depict the number of cells showing either EAD-like events or alternans from either paced at 1 Hz or spontaneous action potential measurements in isogenic…

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  Figure 4. TRDN−/− iPSC-CMs display slow CaV1.2 inactivation and increased persistent current at low EGTA conditions (A) Whole-cell CaV1.2 representative traces from isogenic control and TRDN−/− iPSC-CMs determined from a holding potential of −90 mV to testing potential of +70 mV in 10-mV increments with 500-ms duration. (B and C) CaV1.2 current-voltage relationship and (C) CaV1.2 inactivation-activation curves in isogenic control (three differentiations) and TRDN−/− (three differentiations) iPSC-CMs. Steady-state inactivation was determined from a holding potential of −90 mV to prepulse of 20 mV in 10-mV increments with 5-s duration followed by a test pulse of 30 mV with 500-ms duration. (D) Inactivation time constants (τ) for the fast phase of CaV1.2 decay time in isogenic control (three differentiations) and TRDN−/− (three differentiations) iPSC-CMs at 20 mV. Time constants were determined by fitting a biexponential function to current decay. (E) Representative CaV1.2 traces at 20 mV showing slow inactivation in TRDN−/− iPSC-CMs compared with isogenic control. (F) Inactivation time constants (τ) for the slow phase of CaV1.2 decay time in isogenic control (three differentiations) and…

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  Figure 5. Ba2+ replacement eliminates differences in CaV1.2 inactivation and persistent current at low EGTA conditions in TRDN−/− iPSC-CMs (A) Whole-cell CaV1.2 representative traces from isogenic control and TRDN−/− iPSC-CMs determined from a holding potential of −90 mV to testing potential of +70 mV in 10-mV increments with 500-ms duration. (B) Inactivation time constants (τ) for the fast phase of CaV1.2 decay time in isogenic control (two differentiations) and TRDN−/− (two differentiations) iPSC-CMs at 0 mV. Time constants were determined by fitting a biexponential function to current decay. (C) Representative CaV1.2 traces at 0 mV showing no change in inactivation between isogenic control and TRDN−/− iPSC-CMs. (D) Inactivation time constants (τ) for the slow phase of CaV1.2 decay time in isogenic control (two differentiations) and TRDN−/− (two differentiations) iPSC-CMs at 0 mV. (E) Persistent current normalized to peak current at −20 mV shown as percentages for isogenic control (two differentiations) and TRDN−/− (two differentiations) iPSC-CMs. Graphs are presented as mean ± SEM. Unpaired Student’s t tests were conducted to determine significance.

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  Figure 6. TRDN−/− iPSC-CMs display impaired calcium release (A) Representative Fluo-4 calcium imaging traces derived from isogenic control and TRDN−/− iPSC-CMs at baseline (top) and after treatment with 1 μM ISO (bottom). (B) Calcium transient duration at 50% (CTD50; left) and 90% (CTD90; right) decay at baseline and after treatment with ISO. (C) Calcium transient peak to 50% (left) and 90% (right) decay time at baseline and after treatment with ISO. (D) Calcium transient upstroke time (left) and upstroke velocity (right) at baseline and after treatment with ISO. (E) Calcium transient amplitude at baseline and after treatment with ISO (baseline: isogenic control, n = 115, two differentiations; TRDN−/−, n = 115, four differentiations; ISO: isogenic control, n = 80, two differentiations; TRDN−/−, n = 85, four differentiations). Graphs are presented as mean ± SEM. Two-way ANOVAs were used to determine significance.

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  Figure 7. TRDN−/− iPSC-CMs display decreased expression and co-localization of key calcium handling proteins (A) Representative immunofluorescence images and quantification of RYR2 and CASQ2 protein expression in isogenic control (two differentiations) and TRDN−/− (four differentiations) iPSC-CMs. (B and C) Western blots detecting CASQ2 and RYR2 and subsequent quantification of relative expression (normalized to α-Actinin) in isogenic control (three differentiations) and TRDN−/− iPSC-CMs (six differentiations). (D) Representative immunofluorescence images showing co-localization of RYR2 and CASQ2 (yellow) and subsequent quantification of Pearson’s coefficient for RYR2 and CASQ2 co-localization in isogenic control or TRDN−/− iPSC-CMs were analyzed using ImageJ with JACoP plug-in. Scale bars, 50 μm. Graphs represent mean ± SEM. Unpaired Student’s t tests (immunofluorescence) or Mann-Whitney tests (western blot) were used to determine significance.