Cardiac-targeted PIASy gene silencing mediates deSUMOylation of caveolin-3 and prevents ischemia/reperfusion-induced Nav1.5 downregulation and ventricular arrhythmias

Figures

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  Fig. 1. Efficiency and safety of AAV9-mediated PIASy silencing in the rat heart. a Distribution of Evans blue dye (2% v/w in 50 μl) in the rat heart at 10 min after intraventricular injection. b Green fluorescent protein (ZsGreen) expression mediated by the AAV9 vector in liver, lung, and heart samples at 15 d after intraventricular injection of AAV9-ZsGreen-shRNA (50 μl, 1.3 × 1011 vg). Arrows indicate ZsGreen expressed in the heart. c–d AAV9 PIASy shRNA-mediated PIASy silencing in the rat heart. RNA and protein samples from the left ventricle were generated for qRT-PCR and Western blotting, respectively, at 15 d after AAV9-ZsGreen-shRNA administration (n = 5). GAPDH was used as an internal control. Values are the mean ± standard error of the mean, *P < 0.05 vs. Scramble-shRNA and determined by Student’s t-test. e The effect of AAV9 PIASy shRNA on rat survival over the 15 d following AAV9 vector treatment (n = 20, P > 0.05, Kaplan–Meier method). f Histological analysis after H&E staining of rat tissue samples from the left ventricle free wall at 15 d after intraventricular AAV9-ZsGreen-shRNA injection. AAV9 adeno-associated virus subtype 9, H&E hematoxylin and eosin, PIASy protein…

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  Fig. 2. Effects of I/R and PIASy shRNA on PIASy, Nav1.5, and Cav-3 expression. a H/R-induced changes in PIASy, Nav1.5, Cav-3, and SUMO2/3 expression levels in isolated cardiomyocytes from the left ventricle of adult rats (arrows). PIASy (red) and SUMO2/3 (green) fluorescence were detected at high levels, while Nav1.5 (green) and Cav-3 (red) showed decreased localization, especially on membrane. b I/R and PIASy shRNA altered PIASy, Nav1.5 and Cav-3 mRNA levels in rat left ventricle tissues by qRT-PCR. c–e PIASy, Nav1.5, and Cav-3 protein expression in whole cardiomyocytes (c), membrane (d) and cytoplasmic (e) fractions after I/R in Scramble-shRNA and PIASy-shRNA groups. The left panel represents Western blot bands while the right represents their densitometric analysis. Values are expressed as the mean ± standard error of the mean (n = 10). *P < 0.05, **P < 0.01, one-way ANOVA. ANOVA analysis of variance, Cav-3 caveolin-3, H/R hypoxia/reoxygenation, I/R ischemia/reperfusion, Nav1.5 voltage-gated sodium channel 1.5, PIASy protein inhibitor of activated STAT Y, qRT-PCR quantitative real-time polymerase chain reaction, shRNA short hairpin RNA

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  Fig. 3. Effects of PIASy silencing on Cav-3 binding to SUMO2/3 and Nav1.5, and Nav1.5 distribution. a SUMO-modified Cav-3 in the rat heart. Rat myocardial lysates were collected and immunoprecipitated with an anti-Cav-3 antibody, then immunoblotting with anti-SUMO2/3 (left panel) or anti-SUMO1 antibodies (right panel) after I/R injury. b Cav-3 binding to SUMO2/3 in HEK293 cells co-transfected with Cav-3, Nav1.5, ubc9, SUMO2/3, and incremental doses of PIASy plasmid (left panel) or identical dose of PIASy plasmid and subjected to hypoxia or H/R (right panel). c Physical binding of Cav-3 and Nav1.5 in the rat heart, and Cav-3 and SUMO machinery in co-transfected HEK293 cells. Protein samples from normal left ventricle or HEK293 cells 48 h after transfection were immunoprecipitated with an anti-Cav-3 antibody and immunoblotted with an anti-Nav1.5 antibody. d Effects of PIASy shRNA on binding between Cav-3 and Nav1.5 by Co-IP in the I/R rat heart. e Cav-3 and Nav1.5 co-localization in rat myocardium. Immunofluorescence staining of the peri-infarct zone in the rat left ventricle showing the localization and expression levels of Nav1.5 (red) and Cav-3 (green), mainly on lateral membrane…

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  Fig. 4. The effects of Cav-3 mutations at SUMO consensus sites on Nav1.5 abundance in transfected HEK293 cells. a Schematic showing Cav-3 SUMO consensus sites. Cav-3 has a SUMO consensus motif (ΨKxD/E) near the caveolin scaffolding domain at the N-terminus. The SUMO consensus sites K38 and K144 at the C-terminus are highly conserved across different species. b-c Changes in Nav1.5 abundance in transfected HEK293 cells between the WT and mutants. HEK293 cells were co-transfected with SUMO machinery, including HA-SUMO2/3, ubc9, Flag-hPIASy, Nav1.5, and Flag-tagged Cav-3 (WT, K38R mutant or K144R mutant). Membrane protein was assessed by Western blotting at 48 h after transfection. Immunofluorescence staining was performed with an anti-Nav1.5 antibody (red, Nav1.5, arrows) and DAPI (blue, nucleus). d–e Effects of Cav-3 mutations on Nav1.5 abundance after H/R. HEK293 cells transfected with SUMO machinery, Nav1.5, and Cav-3 (WT, K38R mutant or K144R mutant) were exposed to 3 h hypoxia followed by 2 h reoxygenation. Membrane and cytoplasmic proteins were immunoblotted using anti-Nav1.5 and anti-flag (Cav-3) antibodies, respectively. Na+/K+ ATPase and β-actin were used as internal controls for…

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  Fig. 5. Effects of PIASy silencing on Nav1.5 phosphorylation in the I/R rat model. Rat cardiomyocyte lysates were immunoprecipitated with an anti-Nav1.5 antibody, followed by immunoblotting with anti-phospho-tyrosine and anti-phospho-threonine antibodies. I/R ischemia/reperfusion, Nav1.5 voltage-gated sodium channel 1.5, PIASy protein inhibitor of activated STAT Y, shRNA short hairpin RNA

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  Fig. 6. Effects of PIASy silencing on P duration, PR interval, QRS duration, and QTc values during I/R. a Changes in P duration, PR interval, QRS duration, and QTc values during I/R. b Representative changes in QRS waves and QT intervals (upper panel) in ECG, and statistical analyses of QRS durations and QTc values (bottom panel) at 20 min after ischemia and 1 h after reperfusion. Values are expressed as the mean ± standard error of the mean (n = 10). *P < 0.05, **P < 0.01, two-way ANOVA. ANOVA analysis of variance, ECG electrocardiography, isch ischemia, I/R ischemia/reperfusion, QTc corrected QT interval, PIASy protein inhibitor of activated STAT Y, rep reperfusion, shRNA short hairpin RNA

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  Fig. 7. Effects of PIASy silencing on lethal arrythmias during myocardial I/R. a Schematic diagram showing VT and VF during I/R. b-c Duration values of sustained VT (b) and VF (c) after I/R. d Arrhythmia scores in I/R-injured rats with PIASy-shRNA or Scramble-shRNA treatments. e–g Episodes of VT (e), sustained VT (f), and VF (g) within consecutive 5 min blocks after ligation of the left anterior descending coronary artery. Values are expressed as the mean ± standard error of the mean (n = 10). *P < 0.05, **P < 0.01. Kruskal–Wallis tests were performed in b, c, and e–g; one-way ANOVA was performed in d. AAV9 adeno-associated virus subtype 9, ANOVA analysis of variance, ECG electrocardiography, I/R ischemia/reperfusion, PIASy protein inhibitor of activated STAT Y, shRNA short hairpin RNA, VF ventricular fibrillation, VT ventricular tachycardia

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  Fig. 8. Schematic showing proposed signaling involving PIASy-mediated Cav-3 SUMOylation effects on reperfusion arrhythmias. PIASy-mediated Cav-3 SUMOylation by SUMO2/3 controls the magnitude of Cav-3/Nav1.5 interaction, which affects Nav1.5 abundance on cardiomyocyte membrane and modulate lethal reperfusion arrhythmias. Cav-3 caveolin-3, Nav1.5 voltage-gated sodium channel 1.5, PIASy protein inhibitor of activated STAT Y, shRNA short hairpin RNA