Disease modeling of a mutation in α‐actinin 2 guides clinical therapy in hypertrophic cardiomyopathy

Figures

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  Figure 1. HCM‐affected family and molecular characterization of human tissue and hiPSC‐derived CMs from 2D and 3D models APedigree of HCM‐affected family carrying a novel ACTN2 mutation (c.740C>T; T247M). Individuals who were genotyped are marked with an apostrophe, and HCM patients are indicated by filled symbols and carriers of this ACTN2 mutation with a (+) sign. Encircled in red with the arrow is the index patient (II.4) from whom the HCM hiPSC line was created.BRepresentative Sanger sequencing results of the ACTN2 locus in the Ctrl, HCM, and HCMrep hiPSCs (higher magnification of the locus shown on the right, red rectangle). Depicted is the mutation on position g.54,208 (c.740C>T), the repair template from g.54,148 to 54,270 (‐strand), the according silent mutations encoded on the repair template on position g.54,200 (G>C; ‐strand) and g.54,245 (C>G; ‐strand), and sgRNAs A and B.C, DWestern blot analysis of hiPSC‐CMs (C) and ‐EHTs (D) stained with antibodies directed against α‐actinin 2 and cTnT. Quantification of α‐actinin 2 protein levels in single samples, normalized to cTnT and related to Ctrl, is depicted [n = number of samples/differentiations; 2D: Ctrl (n = 4/3); HCM (n = 4/3);…

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  Figure 2. Disease modeling with 2D‐ and 3D‐cultured hiPSC‐derived CMs ARepresentative immunofluorescence images of the Ctrl, the HCM, and isogenic control HCMrep. HiPSC‐CMs were stained after 30 days in 2D culture with an antibody against α‐actinin 2 and Hoechst for nuclei staining (scale: 50 μm) and with higher magnification (scale: 10 μm).BQuantification of cell area analyzed with Fiji software [n = number of analyzed cells/wells/differentiations: Ctrl (n = 112/11/3); HCM (n = 424/7/4); HCMrep (n = 443/5/3)].CBlinded analysis of myofibrillar disarray using high‐resolution pictures [n = number of analyzed sarcomeres/cells/differentiations: Ctrl (n = 90/30/3); HCM (n = 132/44/2); HCMrep (n = 86/29/2)].DRepresentative EHTs from each cell line.ERepresentative single traces of EHTs in 1.8 mM Ca2+ Tyrode's solution under 1‐Hz pacing at 37°C.F–HFunctional parameters of force (F), time to peak 80% (T180%, G), and time from peak to baseline 80% (T280%, H) were measured under paced conditions at 1 Hz in 1.8 mM Ca2+ Tyrode's solution at 37°C [n = number of measurements/EHTs/differentiations: Ctrl (n = 36/25/4); HCM (n = 64/60/6); HCMrep (n = 41/29/4)].IForce–pCa relationships were performed with…

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  Figure 3. Electrophysiological characterization with 3D‐ and 2D‐cultured hiPSC‐derived CMs ARepresentative action potentials (APs) measured with sharp microelectrode in EHTs at 1‐Hz pacing.BQuantification of AP duration at 90% repolarization (APD90) of Ctrl, HCM, and HCMrep [n = number of EHTs/differentiations; Ctrl (20/8), HCM (13/6), HCMrep (14/5)].CCycle length dependence of APD90 of Ctrl (n = 7/3), HCM (n = 5/2), and HCMrep (n = 3/1).D, ERepresentative traces (D) and quantification (E) of single‐cell patch clamp recordings of L‐type calcium currents [n = number of isolated cardiomyocytes/differentiations; Ctrl (10/3), HCM (20/3), HCMrep (13/3)].FSimulated effect of 1.5‐fold ICa,L density in in silico model of human hiPSC‐CM AP and Force.Data information: Data are expressed as mean ± SEM, one‐way ANOVA with Bonferroni's post‐test.

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  Figure 4. Diltiazem intervention in 3D‐cultured hiPSC‐derived EHT A, BRepresentative force traces at 1‐Hz pacing under baseline condition and with 3 μM diltiazem in HCM‐ (A) and HCMrep‐EHTs (B).C, DQuantification of the diltiazem effect in percent to baseline (BL) levels for force (C) and time to baseline 80% [T280%, D; n = number of EHTs/differentiations; HCM (n = 23/2); HCMrep (n = 18/2)].E, FRepresentative action potentials (AP) paced at 1 Hz under BL condition (red for HCM, gray for HCMrep) and with 3 μM diltiazem (green) in HCM‐ (E) and HCMrep‐EHTs (F).G, HQuantification of AP duration at 50% (G) and 90% (H) of repolarization in HCM (n = 6/1) and HCMrep (n = 6/1).I–KRepresentative 12‐lead surface ECG with averaged signal and overlay of all leads (in gray, apart from V5 in black) of patient III.4 with different antihypertensive medication: (I) ramipril 5 mg, (J) ramipril 10 mg + 5 mg amlodipine, (K) ramipril 5 mg + diltiazem 180 mg for 1 month (QTcB: QT interval with Bazett correction; QTcF: with Fredericia correction).Data information: Data are expressed as mean ± SEM, unpaired Student's t‐test.

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  Figure 5. Monophasic action potential from HCM‐affected family member II.4 A, BFluoroscopy during the recording of monophasic action potentials in right anterior oblique projection (A) and left anterior oblique projection (B) at 30°. Atrial stimulation was performed by a catheter in coronary sinus (CS) and a second catheter for the recording of monophasic action potentials (MAPs) at the septum of the right ventricle, and the ablation catheter (Abl) was not used for the recording.CRepresentative MAPs traces.DComparison of MAPD90 and QTc (Bazett) in the index patients II.4 to a control and long QT (LQT) cohort taken from Shimizu et al (1991).