Department of Anesthesiology, Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Department of Cardiology, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, Jiangsu, China.
Mil Med Res. 2022 Oct 14;9(1):58. doi: 10.1186/s40779-022-00415-x.
Abnormal myocardial Na1.5 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) SUMO modification affects Cav-3 binding to the voltage-gated sodium channel 1.5 (Na1.5). PIASy activity is increased after myocardial I/R, but it is unclear whether this is attributable to plasma membrane Na1.5 downregulation and ventricular arrhythmias.
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.
PIASy was upregulated by I/R (P < 0.01), with increased SUMO2/3 modification of Cav-3 and reduced membrane Na1.5 density (P < 0.01). AAV9-PIASy shRNA intraventricular injection into the rat heart downregulated 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 Na1.5, and prevented I/R-induced decrease of Na1.5 and Cav-3 co-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 Na1.5/Cav-3 dissociation after H/R. Mutation of SUMO consensus lysine sites in Cav-3 (K38R or K144R) altered the membrane expression levels of Na1.5 and Cav-3 before and after H/R in HEK293T cells.
I/R-induced cardiac PIASy activation increased Cav-3 SUMOylation by SUMO2/3 and dysregulated Na1.5-related ventricular arrhythmias. Cardiac-targeted PIASy silencing mediated Cav-3 deSUMOylation and partially prevented I/R-induced Na1.5 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.
异常的心肌 Na1.5 表达和功能导致心肌缺血再灌注（I/R）期间致命性室性心律失常。蛋白激活 STAT Y 抑制剂（PIASy）介导的小窝蛋白-3（Cav-3）SUMO 修饰影响 Cav-3 与电压门控钠通道 1.5（Na1.5）的结合。心肌 I/R 后 PIASy 活性增加，但尚不清楚这是否归因于质膜 Na1.5 下调和室性心律失常。
使用重组腺相关病毒 9 型（AAV9），通过心室内注射 PIASy 短发夹 RNA（shRNA）沉默大鼠心脏的 PIASy。两周后，对大鼠心脏进行 I/R，并进行心电图评估恶性心律失常。收集左心室梗死区周围组织进行分子生物学测量。
PIASy 在 I/R 后被上调（P<0.01），Cav-3 的 SUMO2/3 修饰增加，质膜 Na1.5 密度降低（P<0.01）。AAV9-PIASy shRNA 心室内注射到大鼠心脏中，在 I/R 后下调 PIASy 的 mRNA 和蛋白水平（P<0.05 与 Scramble-shRNA+I/R 组相比），降低 SUMO 修饰的 Cav-3 水平，增强 Cav-3 与 Na1.5 的结合，并防止 I/R 诱导的 Na1.5 和 Cav-3 在闰盘和侧膜中的共定位减少。大鼠心脏中的 PIASy 沉默减少了 I/R 诱导的致命性心律失常，这反映在心室颤动（VF）持续时间略有减少（P<0.05 与 Scramble-shRNA+I/R 组相比）和心律失常评分显著降低（P<0.01 与 Scramble-shRNA+I/R 组相比）。PIASy 沉默的抗心律失常作用也通过减少室性心动过速（VT）、持续 VT 和 VF 的发作得到证实，尤其是在缺血后 5-10 分钟（P<0.05 与 Scramble-shRNA+IR 组相比）。通过体外人胚肾 293T（HEK293T）细胞和分离的成年大鼠心肌细胞缺氧/复氧（H/R）模型，我们证实，增加的 PIASy 通过 SUMO2/3 促进 Cav-3 修饰，并在 H/R 后促进 Na1.5/Cav-3 解离。Cav-3 的 SUMO 保守赖氨酸位点（K38R 或 K144R）的突变改变了 H/R 前后 HEK293T 细胞中 Na1.5 和 Cav-3 的膜表达水平。
I/R 诱导的心脏 PIASy 激活增加了 Cav-3 的 SUMO 化，并通过 SUMO2/3 调节了与 Na1.5 相关的室性心律失常。心脏靶向的 PIASy 沉默介导了 Cav-3 的去 SUMO 化，并部分预防了 I/R 诱导的质膜中 Na1.5 的下调，随后在大鼠中出现室性心律失常。PIASy 被确定为缺血性心脏病患者危及生命的心律失常的潜在治疗靶点。
