The Junctophilin-2 Mutation p.(Thr161Lys) Is Associated with Hypertrophic Cardiomyopathy Using Patient-Specific iPS Cardiomyocytes and Demonstrates Prolonged Action Potential and Increased Arrhythmogenicity

Figures

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  Figure 1. Localization of JPH2 in cardiomyocytes; (a) Overview of human JPH2 localized in the junctional membrane complex (JMC) of a cardiomyocyte. The highlighted region is the joining region that contains the mutation JPH2-Thr161Lys; (b) Schematic in the right-side panel represents the JPH2 protein topology (1–696 amino acids), which contains the N terminus, an N-terminal membrane occupation, and recognition nexus (MORN) region repeating eight times along the cell membrane phospholipids (1–143 and 284–322). Between the MORN-motifs I and II, the joining region (143–284) resides in direct interaction with the ryanodine-2 receptor (RyR2) [16]. The zoom-in on the joining region shows the JPH2-Thr161Lys mutation. The following structure consists of an alpha helix (322–423), divergent region (423–676), and a transmembrane domain (676–696) in the C-terminal that anchors the JPH2 to the ER/SR to maintain proximity to the PM. SR, Sarcoplasmic Reticulum; PLB, Phospholamban; SERCA, Sarco/Endoplasmic Reticulum Ca2+-ATPase; SPEG, Striated Muscle Enriched Protein Kinase; RyR2, Ryanodine Receptor 2; JMC, Junctional Membrane Complex; JPH2, Junctophilin-2; PM, Plasma Membrane; VGCC, Voltage-Gated Calcium…

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  Figure 2. Characterization of the 09703.HCMJp hiPSC line. (a) The hiPSCs formed colonies expressing Nanog, Sox2, Oct3/4, SSEA-4, TRA-60, and TRA-81. Scale bars 200 µm. (b) hiPSCs expressed endogenous SOX2 (151 bp), Nanog (287 bp), OCT3/4 (144 bp), cMYC (328 bp), and REX1 (306 bp). Virally transferred Sendai exogenes exo-OCT4 (483 bp), exo-KLF4 (410 bp), exo-SOX2 (451 bp), and exo-cMYC (532 bp) were absent in hiPSCs. (c) As a proof of pluripotency, hiPSCs were differentiated into EBs, which expressed markers from all germ layers: endoderm (SOX17 (120 bp), AFP (209 bp)), mesoderm (α-cardiac actin (486 bp), KDR (218 bp)), and ectoderm (SOX1 (166 bp), PAX6 (274 bp)). GAPDH (302 bp) was used as a housekeeping control in each PCR experiment. (d) The hiPSC line was karyotypically normal (46, XY) in the KaryoLite BoBs Assay (PerkinElmer). Karyotype analyses of the 09703.HCMJp cell line. Red and blue dots indicate chromosomal signal ratios of sample DNA against female (red) and male (blue) reference normal karyotype DNA. Signal from normal chromosomes against both male and female references should lie inside the reference area around value 1, whereas with an abnormal karyotype both signals lie…

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  Figure 3. Generation of the isogenic control cell line and localization of JPH2 in cardiomyocytes; (a) The heterozygous mutation JPH2 c.482C > A,p. (Thr161Lys) in the mutated CMs was corrected using CRISPR-CAS9 genome editing. Validation of the corrected isogenic line by sanger sequencing is shown. The mutated amino acid codon is boxed and M represents adenosine or cytosine in the patient line; (b) The sequence alignment of the reference sequence, patient line 09703.HCMJp, and the isogenic line is shown.

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  Figure 4. Phenotypic characterization of T161K and isogenic hiPSC-CMs; (a) The cell size of hiPSC-derived CMs after 37 days of culture as single cells. Representative immunofluorescent images of JPH2 hiPSC-CMs (left) and isogenic hiPSC-CMs (right) stained with antibodies for cTnT (green) and DAPI (magenta). Scale bars 100 µm. JPH2 hiPSC-CMs present with significantly larger (b) cell bodies and (c) nuclei sizes than isogenic hiPSC-CMs; (d) Representative immunofluorescent image stained with antibodies for cTnT (green) and DAPI (magenta), showing multinucleation (white frame) in hiPSC-CMs. Scale bars 100 µm; (e) T161K hiPSC-CMs have a larger fraction of multinucleated (blue) hiPSC-CMs than the other the analyzed cells; (f) Representative immunofluorescent images of hiPSC-CMs with misaligned (left) and aligned sarcomeres stained with antibodies for cTnT (green) and DAPI (magenta). Scale bars 100 um; (g) T161K hiPSC-CMs have increased sarcomeric disarray. Data represent mean ± SE. ** p < 0.01, *** p < 0.001; Mann–Whitney U-test. Numbers in parentheses represent the number of cells used.

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  Figure 5. Baseline action potential properties in T161K and isogenic hiPSC-CMs; (a) Representative spontaneous AP traces from T161K hiPSC-CMs and isogenic hiPSC-CMs. (b,c) Averaged values for action potential duration (APD) at 50% (APD50) and 90% (APD90) repolarization. (d) Average values for upstroke velocity (dVdt). (e) The hiPSC-CMs were stimulated at 1 Hz, representative traces of stimulated APs are shown. (f,g) Average values for APD at −20 mV (APD −20 mV) and at −60 mV (APD −60 mV). (h) Average values for upstroke velocity (Vmax). (i) Representative AP trace exhibiting phase 3 EAD and (j) phase-3-EAD-induced triggered arrhythmias indicated by red arrows. (k) Percentage of occurrence of phase 3 EAD during spontaneous beating. (l) Percentage of occurrence of phase 3 EAD during stimulation at 1 Hz. Data are presented as mean ± S.E:M. * p < 0.05; unpaired t-test. Numbers in parentheses represent the number of cells used.

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  Figure 6. Calcium current (ICa) in T161K and isogenic hiPSC-CMs. (a) Current density–voltage relationship of ICas recorded from T161K and isogenic hiPSC-CMs. (b) Voltage-dependent inactivation curve of ICas. (c) Average time course of recovery from inactivation of ICa. Peak calcium currents elicited by P2 were normalized (I2/I1) and plotted as a function of the recovery interval. (d) ICa responses to step voltage protocol. (e) Comparison of cell capacitances (* p < 0.05, unpaired t-test). (f) Comparison of ICa current densities at 10 mV. (g) Comparison of decay time constant of ICa obtained from monoexponential fitting. (* p < 0.05, unpaired t-test). (h,i) Correlation test (Pearson correlation) between ICa current densities and APD (−20 mV) and APD (−60 mV) (Pearson Correlation). (j,k) Correlation test between time constant and APD (−20 mV) and APD (−60 mV). Numbers in parentheses represent the number of cells used. For fitting procedures, see text. Inset: voltage-clamp protocol used.

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  Figure 7. Action potential clamp (APC) study in T161K and isogenic hiPSC-CMs. (a) The modified protocol for the APC technique to study the calcium current elicited at 10 mV from −40 mV. (b) Under action potential clamping conditions (above panel), the ICa elicited by P1 and P2 originated at −40 mV (below panel). (c) The modified protocol for the APC technique to study the calcium current elicited at 10 mV from −60 mV. (d) Under action potential clamping conditions (above panel), the ICa elicited by P1 and P2 originated at −60 mV (below panel). (e) Comparison of ICa current densities in response to protocol A (e1) P1, (e2) P2, (e3) I1/I2, and (e4) time constant (* p < 0.05, unpaired t-test). (f) Comparison of ICa current densities in response to protocol C (f1) P1, (f2) P2, (f3) I1/I2, and (f4) time constant (* p < 0.05, unpaired t-test). Correlation test (Pearson correlation) between the ICa current densities obtained from protocol A and (g) APD (−20 mV) and (h) APD (−60 mV). Correlation test between the time constant of ICa inactivation obtained from protocol A and (i) APD (−20 mV) and (j) APD (−60 mV).

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  Figure 8. Phase 3 EAD in the T161K hiPSC-CMs. Representative action potential (AP) traces of T161K hiPSC-CMs (a) with and (b) without the presence of phase 3 EAD indicated by red arrows. Comparison of (c) APD (−20 mV) and (d) APD (−60 mV) between T161K hiPSC-CMs with and without presence of phase 3 EAD. Comparison of (e) I1, (f) I2, and (g) I2/I1 elicited by using the protocol 1 action potential clamp between T161K hiPSC-CMs with and without presence of phase 3 EAD. (h) Comparison of the time constant of calcium currents elicited by using the protocol 1 action potential clamp between T161K hiPSC-CMs with and without the presence of phase 3 EAD. ** p < 0.01; paired t-test.

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  Figure 9. Functional phenotyping of contractility; (a) Illustration of a beating signal. 1: Time of contraction (phase 1), 2: Time contracted (phase 2), and 3: Time of relaxation (phase 3); (b) Representative beating traces of T161K and isogenic hiPSC-CMs; (c) Durations of beating phases 1–3; (d) Beating rate; (e) Absolute time of contraction against BPM; (f) Absolute time of relaxation against BPM. Data are presented as mean ± SE. Numbers in parentheses represent the number of cells used. *** p < 0.001.