Cardiac-targeted PIASy gene silencing mediates deSUMOylation of caveolin-3 and prevents ischemia/reperfusion-induced Na_(v)1.5 downregulation and ventricular arrhythmias

Background:Abnormal myocardial voltage-gated sodium channel 1.5(Nav1.5)expression and function cause lethal ventricular arrhythmias during myocardial ischemia–reperfusion(I/R).Protein inhibitor of activated STAT Y(PIASy)-mediated caveolin-3(Cav-3)small ubiquitin-related modifier(SUMO)modification affects Cav-3 binding to the Nav1.5.PIASy activity is increased after myocardial I/R,but it is unclear whether this is attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias.Methods:Using recombinant adeno-associated virus subtype 9(AAV9),rat cardiac PIASy was silenced using intraventricular injection of PIASy short hairpin RNA(shRNA).After two weeks,rat hearts were subjected to I/R and electrocardiography was performed to assess malignant arrhythmias.Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurements.Results:PIASy was upregulated by I/R(P<0.01),with increased SUMO2/3 modification of Cav-3 and reduced membrane Nav1.5 density(P<0.01).AAV9-PIASy shRNA intraventricular injection into the rat heart down-regulated PIASy after I/R,at both mRNA and protein levels(P<0.05 vs.Scramble-shRNA+I/R group),decreased SUMO-modified Cav-3 levels,enhanced Cav-3 binding to Nav1.5,and prevented I/R-induced decrease of Nav1.5 and Cav-3co-localization in the intercalated disc and lateral membrane.PIASy silencing in rat hearts reduced I/R-induced fatal arrhythmias,which was reflected by a modest decrease in the duration of ventricular fibrillation(VF;P<0.05 vs.Scramble-shRNA+I/R group)and a significantly reduced arrhythmia score(P<0.01 vs.Scramble-shRNA+I/R group).The anti-arrhythmic effects of PIASy silencing were also evidenced by decreased episodes of ventricular tachycardia(VT),sustained VT and VF,especially at the time 5–10 min after ischemia(P<0.05 vs.Scramble-shRNA+IR group).Using in vitro human embryonic kidney 293 T(HEK293T)cells and isolated adult rat cardiomyocyte models exposed to hypoxia/reoxygenation(H/R),we confirmed that increased PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R.Mutation of SUMO consensus lysine sites in Cav-3(K38R or K144R)altered the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells.Conclusions:I/R-induced cardiac PIASy activation increased Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5-related ventricular arrhythmias.Cardiac-targeted PIASy silencing mediated Cav-3 deSUMOylation and partially prevented I/R-induced Nav1.5 downregulation in the plasma membrane of cardiomyocytes,and subsequent ventricular arrhythmias in rats.PIASy was identified as a potential therapeutic target for life-threatening arrhythmias in patients with ischemic heart diseases.