Apremilast improves cardiomyocyte cohesion and arrhythmia in different models for arrhythmogenic cardiomyopathy

Figures

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  Fig. 1. Apremilast (Apr) enhances cardiomyocyte cohesion in HL-1 cells and wild-type murine cardiac slices. a HL-1 cells were treated with apremilast (Apr, 1 and 10 µM) for 30 min at 37 °C, and cAMP was measured; FR (5 µM/10 µM; 1 h, 37 °C) was used as a positive control in all assays. Treatments were compared to non-treated Ctrl conditions. N = 4–6. b Dissociation assays were performed in HL-1 cells treated with 1 or 10 µM Apr for 1 h at 37 °C. N = 7. The cartoon represents the dissociation assay method, and the panel below displays representative wells for each condition. MTT was used to show the viability of cells. c Dissociation assays were performed in wild-type murine cardiac slices treated with 1 µM Apr for 1 h at 37 °C. N = 4. The cartoon represents the dissociation assay method. d Immunostaining and confocal microscopy were performed for DP (green) and DSG2 (red) in HL-1 cells treated with FR (5 µM/10 µM) and 1 µM Apr for 1 h at 37 °C. Scale bar: 10 μm. e Quantification of DSG2-DP colocalization area. Each data point in the graph represents one confocal image performed across 3–4 experimental repeats. f Representative Western blots for PG-pS665, PG, pERK1/2, and ERK1/2 in HL-1…

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  Fig. 2. Apremilast requires plakoglobin to enhance cardiomyocyte cohesion. a Dissociation assays were performed with cardiac slices of PG-S665 phosphorylation deficient mice (JUPS665A), N = 5–6, and b cardiomyocyte-specific deletion of PG (Jup−/−) mice treated with apremilast (Apr, 1 µM) for 1 h at 37 °C, N = 4. The cartoons represent the dissociation assay method. c Representative Western blots for PG-pS665 and PG, in cardiac slices from JUPS665 (WT) and JupS665A mice treated with Apr (1 µM) and FR (5 µM/10 µM) for 1 h at 37 °C. The quantification of Western blots is displayed in the panel below. N = 6–7. d Immunostaining and confocal microscopy were performed for DSG2 (Cyan), PG (red) and WGA (green, shown in the merge images) in cardiac slices from wild-type, JUPS665A and Jup−/− treated with Apr (1 µM) for 1 h at 37 °C. Scale bar 10 μm. e The thickness of DSG2 and PG staining in the ICD was measured and represented in µm. Each data point in the graph represents the mean of multiple ICDs in one confocal image and measured across cardiac slices from 4–5 mice for each condition. Adjacent slices from a single mouse heart were used as a control for the respective treatments. All bar graphs…

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  Fig. 3. Apremilast-enhanced cardiomyocyte cohesion depends on ERK1/2 phosphorylation. a Dissociation assays in HL-1 cells treated with Apr (1 µM) with or without the MEK inhibitor U0126 (1 µM) for 1 h at 37 °C. U0126 was added 30 min prior to Apr. N = 7. The panel above displays representative wells for each condition b Representative Western blots for PG-pS665, PG, pERK1/2, and ERK1/2 in HL-1 cells treated with Apr (1 µM) with or without U0126 (1 µM) for 1 h at 37 °C. α-tubulin was used as a loading control. c Quantification of Western blots from b. N = 5–8. d Immunostaining and confocal microscopy were performed for DP (green) and DSG2 (red) in HL-1 cells treated with Apr (1 µM) with or without U0126 (1 µM) for 1 h at 37 °C. Scale bar 10 μm. e Quantification of DS2-DP colocalization area. Each data point in the graph represents one confocal image performed across 3–4 experimental repeats. α-tubulin (α-TUB) was used as a loading control. All bar graphs in the figure represent mean ± SEM. Two-way ANOVA with Holm-Šidák post-hoc analysis was performed to determine the statistical significance. *p<0.05, **p<0.005 and ***p < 0.0005

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  Fig. 4. Characterization of hiPSC line NP0151-11 F and hiPSC-CMs from ACM patient carrying the heterozygous mutation c.2854G > T in the DSP gene. a Post-mortem cardiac tissue (left ventricle) of ACM patient NP0151 showing large areas of cardiomyocyte loss, adipogenesis and fibrosis. Scale bar 1 mm. b Immunostaining for desmosomal proteins (DP, PG and DSG2) and gap junction protein Cx43 in the post-mortem cardiac tissue of ACM index patient revealed the absence of DSP at the ICD. Scale bar 10 μm. c Colony morphology of NP0151-11 F (DSPp.Glu952*/WT) hiPSC line. Scale bar 100 μm. d Expression of indicated pluripotency-associated transcripts in ACM-hiPSC line NP0151-11 F as determined by semiquantitative RT-PCR. GAPDH was amplified as a positive control. e Confirmation of the absence of the reprogramming Sendai virus vector in NP0151-11 F hiPSC master banks (passage 20) using RT-PCR. Early passage NP0151-11 hiPSCs (passage 11), which still had the reprogramming vector, were used as a positive control for the SeV presence. f DNA sequencing revealed the presence of the DSP mutation c.2854G > T in a patient-specific hiPSC line NP0151-11 F. In addition, silent heterozygous variant c.2862 C > T…

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  Fig. 5. Apremilast enhances cohesion of ACM-hiPSC-CMs. a Dissociation assays were performed in non-relative healthy (Non-rel), healthy mother (Healthy-rel) hiPSC- and ACM-hiPSC-derived cardiomyocytes for 1, 2, and 5 min to compare the cohesive strengths between healthy and ACM patient-derived cardiomyocytes. N = 3–6. b Dissociation assays were performed in ACM-hiPSC-CMs treated with FR (5 µM/10 µM) and Apr (1 µM) for 1 and 24 h at 37 °C. N = 4–5. c Representative Western blots for desmosomal proteins DP, DSG2, PKP2, PG-pS665, PG, the adherens junction protein N-CAD, and the signaling molecule pERK1/2 and ERK1/2 in ACM-hiPSC-CMs treated with FR (5 µM/10 µM) and Apr (1 µM) for 1 and 24 h at 37 °C. α-tubulin was used as a loading control. d Quantification of Western blots from c. N = 5–7. e and f Immunostaining and representative confocal microscopy images of PG-pS665 (cyan) and DSG2 (magenta) in ACM-hiPSC-CMs treated with FR (5 µM/10 µM) and Apr (1 µM) for 1 and 24 h at 37 °C. Merge images show actin staining using Phalloidin Alexa Fluor 488 (yellow) and nuclei staining using DAPI (blue). All bar graphs in the figure represent mean ± SEM. Scale bar 10 μm. Two-way ANOVA (a) and one-way…

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  Fig. 6. Apremilast reduces arrhythmia in a murine ACM model. Heart rate variability was measured as the standard deviation of R-R intervals (SDNN), which was used to measure arrhythmia. For this purpose, a MEA was performed in the ventricular cardiac slices from Jup+/+ and Jup-/- mice with or without Apr (1 µM). Each data point represents one cardiac slice measured from a heart. Graph shows before-after effects of Apr, which is depicted with a connecting line between 2 data points measured on the same cardiac slice. N = 3 mice per genotype. b Langendorff heart perfusion experiments were performed in isolated hearts from Jup+/+ and Jup-/- mice. Hearts were perfused for 20 min and then treated with Apr (1 µM) for 20 min. The graph shows the average heart rate measured across the whole experimental time with its corresponding SEM. N = 4 mice per genotype. c The graph shows the average heart rate measured in b for 1 min before adding Apr (Ctrl) and after 10 min of Apr treatment. d Representative ECG curves (for 5 s) obtained from the Langendorff experiments in Jup-/-mice before (upper panel) and after 10 min of Apr (lower panel). N = 4. e SDNN changes measured using the Langendorff perfusion…