Dysfunction of PDE4DIP contributes to LVNC development by regulating cell polarity, skeleton, and energy metabolism via Rho-ROCK pathway

Figures

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  Figure 1. LVNC-hiPSC-CMs have abnormal skeleton, polarity, and mitochondria compared with the NC-hiPSC-CMs. (A) Representative transmission electron microscope images of hiPSC-CMs between the NC group and LVNC group. Myofibrils, yellow arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B, C) The analysis of the proportion of the vacuolated mitochondria (n = 3 samples per group) and ATP content (nmol per mg protein) in hiPSC-CMs among the NC-hiPSC-CMs and LVNC-hiPSC-CMs groups (n = 3 samples per group). (D–H) Immunostaining of hiPSC-CMs for F-actin, vinculin, and Par3, and the analysis of the fluorescence intensity level (n = 30–80 cells per group). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, and ∗p < 0.05 versus the NC group.

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  Figure 2. Changes of PDE4DIP expression after differentiation of hiPSCs into hiPSC-CMs. (A) Quantitative reverse transcription PCR analysis of PDE4DIP relative mRNA expression on days 0, 5, 10, 15, 20, 25, and 30 during hiPSC-CM differentiation. (B, C) Protein expression of PDE4DIP on the 30th day of inducement, and the analysis of the PDE4DIP protein expression (n = 3 samples per group). (D, E) Immunostaining of hiPSC-CMs for PDE4DIP (scale bar = 10 μm) among the NC-hiPSC-CMs and LVNC-hiPSC-CMs on the 30th day of inducement, and the analysis of PDE4DIP protein expression in 30th hiPS-CMs (n = 80–120 cells per group). (F–H) The schematic diagram shows the location of PDE4DIP in the Golgi apparatus (F), the supposed 3D structure map of PDE4DIP protein (G), and the interaction of PDE4DIP with other cytoskeleton-related genes in red boxes (H). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 versus the NC group.

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  Figure 3. P-PDE4DIP has abnormal skeleton, polarity, and mitochondria in H9C2 cells compared with the P-NC. (A) Representative transmission electron microscope images of H9C2 cells between the P-NC group and P-PDE4DIP group. Myofibrils, yellow arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B) The analysis of the proportion of the vacuolated mitochondria (n = 3 samples per group). (C, D) Representative confocal microscope images of mitochondrial morphology stained by MitoTracker-Green (scale bar = 10 μm) in H9C2 cells transfected with plasmids, and the analysis of the fluorescence intensity level (n = 80–120 cells per group). (E) Skeletonization of the mitochondrial network from MitoTracker staining by NIS analysis software, and ATP content (F) (nmol per mg protein) in H9C2 among the P-NC and P-PDE4DIP groups (n = 6). (G–M) Immunostaining of H9C2 transfected with plasmids for vinculin, F-actin (G–I), Par6 (J, K), and α/β-tubulin (L, M), and the analysis of the fluorescence intensity level (n = 80–120 cells per group). (N, O) Protein expression of H9C2 transfected with plasmids of Scribble, Crb2, α/β-tubulin, and vinculin, and the analysis of the protein expression (n…

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  Figure 4. P-PDE4DIP has abnormal skeleton, polarity, and mitochondria in primary cardiomyocytes of neonatal Sprague–Dawley rats within 3 days of birth (PC), compared with the P-NC. (A) Representative transmission electron microscope images of primary cardiomyocytes between the P-NC group and P-PDE4DIP group. Myofibrils, yellow arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B) The analysis of the proportion of the vacuolated mitochondria (n = 3 samples per group). (C, D) Representative confocal microscope images of mitochondrial morphology stained by MitoTracker-Green (scale bar = 10 μm) in PC transfected with plasmids, and the analysis of the fluorescence intensity level (n = 80–120 cells per group). (E, F) Skeletonization of the mitochondrial network from MitoTracker staining by NIS analysis software (E), and ATP content (nmol per mg protein) in PC among the P-NC and P-PDE4DIP groups (n = 6) (F). (G–M) Immunostaining of PC transfected with plasmids of vinculin, F-actin (G–I), Par6 (J, K), and α/β-tubulin (L, M), and the analysis of the fluorescence intensity level (n = 80–120 cells/group). (N, O) Protein expression of Scribble, vinculin, and α/β-tubulin, and the…

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  Figure 5. Changes in skeleton, polarity, and mitochondria after transfection of siRNA-PDE4DIP in H9C2 cells. (A) Representative transmission electron microscope images of H9C2 cells between the si-NC group and si-PDE4DIP group. Myofibrils, yellow arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B) The analysis of the proportion of the vacuolated mitochondrial (n = 3 samples per group). (C, D) Representative confocal microscope images of mitochondrial morphology stained by MitoTracker-Green (scale bar = 10 μm) in H9C2 cells transfected with siRNA, and the analysis of the fluorescence intensity level (n = 80–120 cells per group). (E, F) Skeletonization of the mitochondrial network from MitoTracker staining by NIS analysis software (F), and ATP content (nmol per mg protein) in H9C2 among the si-NC and si-PDE4DIP groups (n = 6) (F). (G–M) Immunostaining of H9C2 transfected with plasmids of vinculin, F-actin (G–I), Par6 (J, K), and α/β-tubulin (L, M), and the analysis of the fluorescence intensity level (n = 80–120 cells per group). (N, O) Protein expression of PC transfected with siRNA of Scribble, Crb2, vinculin, and α/β-tubulin, and the analysis of the protein expression…

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  Figure 6. Changes in skeleton, polarity, and mitochondria after transfection of siRNA-PDE4DIP in the primary cardiomyocyte (PC) of neonatal Sprague–Dawley rats within 3 days of birth. (A) Representative transmission electron microscope images of primary cardiomyocytes between the si-NC group and si-PDE4DIP group. Myofibrils, yellow arrow; Z-disks, red arrow; endoplasmic reticulum, blue arrow; mitochondria, green arrow. (B) The analysis of the proportion of the vacuolated mitochondria (n = 3 samples per group). (C, D) Representative confocal microscope images of mitochondrial morphology stained by MitoTracker-Green (scale bar = 10 μm) in PC transfected with siRNA, and the analysis of the fluorescence intensity level (n = 80–120 cells per group). (E, F) Skeletonization of the mitochondrial network from MitoTracker staining by NIS analysis software (E), and ATP content (nmol per mg protein) in PC among the si-NC and si-PDE4DIP groups (n = 6) (F). (G–M) Immunostaining of PC transfected with siRNA for vinculin, F-actin (G–I), Par6 (J, K), and α/β-tubulin (L, M), and the analysis of the fluorescence intensity level (n = 80–120 cells per group). (N, O) Protein expression of PC transfected with…

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  Figure 7. Changes of H9C2 cell migration and proliferation after transfection of plasmid-PDE4DIP and siRNA-PDE4DIP. (A) H9C2 cell migration in scratch wound experiment. (B) The analysis of the proportion of migration area (%) (n = 5 samples per group). (C, D) The H9C2 cells transfected with plasmid-NC, plasmid-PDE4DIP, siRNA-NC, and siRNA-PDE4DIP were investigated by the CCK-8 assay (n = 5). ∗∗∗∗p < 0.0001, ∗∗p < 0.01, and ∗p < 0.05 versus the NC group.

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  Figure 8. PDE4DIP plays a role in cell polarity and skeleton through the RhoA-ROCK pathway. (A–C) Protein expression in H9C2 after transfection of plasmid-PDE4DIP, including CDC42 and RhoA, analysis of the protein expression (n = 3 samples per group), and quantitative reverse transcription PCR analysis was employed to assess the relative mRNA expression levels of RhoA-ROCK pathway genes, including CDC42, RhoA, and Rac1, between the P-NC group and P-PDE4DIP group (n = 4 samples per group). (D–F) Protein expression in PC after transfection of plasmid-PDE4DIP, including CDC42 and RhoA, analysis of the protein expression (n = 3 samples per group), and quantitative reverse transcription PCR analysis was employed to assess the relative mRNA expression levels of RhoA-ROCK pathway genes, including CDC42, RhoA, and Rac1, between the P-NC group and P-PDE4DIP group (n = 4 samples per group). (G–I) Protein expression in H9C2 after transfection of siRNA-PDE4DIP, including CDC42 and RhoA, analysis of the protein expression (n = 3 samples/group), and quantitative reverse transcription PCR analysis of was employed to assess the relative mRNA expression levels of RhoA-ROCK pathway genes, including CDC42,…

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  Figure 9. Possible mechanism of PDE4DIP causing LVNC through Rho-ROCK pathway. (A) PDE4DIP is located in the Golgi apparatus and regulates a series of physiological activities of cells through the Rho-ROCK pathway. (B) Simplified mechanism of PDE4DIP possibly causing LVNC.

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