Intracellular calcium current disorder and disease phenotype in OBSCN mutant iPSC-based cardiomyocytes in arrhythmogenic right ventricular cardiomyopathy

Figures

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  Figure 1. Information on the ARVC patient and establishment of induced pluripotent stem cell. (A) Squares indicate males; circles, females; filled-in symbols, clinically affected individuals; blank symbols, clinically unaffected. (B)Twelve-lead electrocardiography of patient with ARVC. T-wave inversion in leads V1 to V3, and epsilon (red arrow) wave appears after the QRS complex. (C) Schematic diagram representing the deletion mutation in obscurin (OBSCN), resulting in frameshift mutation at position 5218. (D) Morphology of peripheral blood mononuclear cells (PBMC) from a patient and establishment of ARVC-iPSCs on number 1 passage, clone 1. (E) iPSC lines express similar RNA levels of the POU5F1 and NODAL pluripotency-related genes as those observed in hESCs, as revealed by qRT-PCR analysis. (F) iPSCs are stained with pluripotency markers (SSEA-4 and TRA-1-60) as indicated by flow cytometry. (G) iPSCs retained long telomeres and a normal karyotype. (H, i) iPSCs contributed to typical teratomas, made up of distinct structures representing the 3-germ layers (neural rosettes and retinal pigmented epithelium [ectoderm], intestinal epithelium [endoderm] and cartilage, bone and fatness, as well…

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  Figure 2. Identification of induced pluripotent stem cell based cardiomyocytes (A) Immunofluorescence staining of Oct4 (a)(d), TRA-1-60 (a)(d), SOX2 (b)(e) and SSEA4 (b)(e) in iPSC of ARVC or control groups. Positive staining of Oct4 (red) and SOX2 (red) within the cell nuclei, and positive staining of TRA-1-60 (green) and SSEA4 (green) on the cell membrane. Immunofluorescence staining of TNNT-2 (c)(f) and NKX2.5 (c)(f) in iPSC-CMs of ARVC or control group. Positive staining of NKX2.5 (red) within cell nuclei, and positive staining of TNNT-2 (green) on the cell membrane. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bars of 50μm (images on the upper left); and 20μm (images on the upper right). (B) Images of Oil Red O staining images of CMs from ARVC and the control groups (a)(d). Most ARVC-iPSC-CMs were positivity stained for lipid droplets (red) within cells, whereas only a small number of control cells were stained positive for lipids. Scale bar: 100 μm. TEM images of iPSC-CMs containing lipid droplets in cell cytoplasm (b and e, 10000×) and the ultrastructure of CMs (c and f, 30000×). Small-scale lipid droplets (L) observed close to the nucleus…

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  Figure 3. Electrophysiological characterization of ARVC-iPSC-CMs (A) Action potential profile of a typical ventricular-like cell and its corresponding reaction to 1μΜ nifedipine and 0.1μM of isoproterenol (ISO). (B) Representative L-type calcium current trace and I-V curve in voltage-clamp pattern resulting from a holding potential of -60 mV and test pulses ranging from -60 to +60 mV.

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  Figure 4. Identification of differentially expressed genes (DEGs) (A) Heatmap showing DEGs of ARVC and control groups. Samples are classified in columns, and genes are classified in rows. (B) Volcano plot of all DEGs. Purple dots stand for genes with no significant differences in their expression between the 2 groups. Green dots represent downregulated genes, and red dots represent upregulated genes. (C) Trajectory plots of PCA scores displaying differences arising in the ARVC and control groups. Blue point, control group; yellow point, ARVC group.

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  Figure 5. Candidate driver genes and pathways in ARVC-iPSC-CMs (A) GO and pathway summary of DEGs in the ARVC group. GO and pathway categories divided according to the functional theme. (B) GOChord plot shows the genes related to over 3 pathways and in association with the ARVC state, contributing to the enrichment, organized according to the order of their expression level. OBSCN is at the forefront. (C) GOCluster plot explaining the relevance between DEGs of high association with ARVC and their related GO terms. For all genes, their high/low logFC values are interpreted by brown (turquoise) rectangles. These genes are mainly focused on calcium ion binding (green), heart development (red) and focal adhesion (cyan). (D) Chart revealing KEGG enrichment of DEGs in signaling pathways. The Y-axis label stands for the pathway, and the X-axis label represents the gene ratio (gene ratio = number of DEGs improved in the pathway/number of all genes within the background gene set). Squares, triangles, and circles represent pathways, cellular processes, and cardiovascular diseases, respectively. Size and color stands for the number of enriched DEGs of bad prognosis sets in the pathway and the…

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  Figure 6. Mapping of junctional protein N-cadherin and obscurin-binding ankyrin 1 (Ank1.5) for exploring the influence of OBSCN mutations on subcellular structures (A) There is no significant difference in the localization of N-cadherin (green) between the two groups, but the expression level of N-cadherin is increased in the disease group: obscurin (red) is located at the M-line level, cross distributed at approximately equal intervals (60×; oil mirror). The distribution of obscurin markers in the ARVC group is disordered (white box). (B) Observation under an ultra-high-resolution microscope reveals that in the control group actin (green) is located at the Z-disk level, whereas obscurin (red) is located at the M-line level, cross distributed at approximately equal intervals, with a spacing of about 200 nm (60×; oil mirror). The distribution of obscurin markers in the ARVC group is disordered (white arrow). (C) Ank1.5 binds to the ankyrin binding domain of obscurin in the sarcoplasmic reticulum. In fully developed striated muscle cells, ank1.5 is mainly located at the M-line level (red). In the control group, actinin (green) and ank1.5 (red) show regular interval distribution. In contrast,…

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  Figure 7. Expression analysis of specific genes and proteins in iPSC-CMs (A) qRT-PCR was performed to survey the expression of plakoglobin (JUP), desmoplakin (DSP), plakophilin2 (PKP2), connexin43 (GJA1), and α-actinin (ACTN1) genes in iPSC-CMs of both groups. (B, C) The enriched scores of adipocytokine and PPAR signaling pathways were higher in the ARVC group by the ssGSEA method. (D) Comparison of mRNA results of target and adipogenesis pathway-related genes (OBSCN, N-Cadherin, Ank1.5, PPARγ, C/EBPα and FABP4) in two groups. (E, F) Detection of target and adipogenesis pathway-related proteins in iPSC-CMs by Western blots. β-actin was used as a loading control. Bands were quantified with Image J software. One asterisk indicates P <0.05, and three asterisks are shown as P <0.001 compared with the control group.