Department of Critical Care Medicine, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 200233, Shanghai, China.
Cell Death Dis. 2021 Jun 15;12(6):614. doi: 10.1038/s41419-021-03876-5.
Among several leading cardiovascular disorders, ischemia-reperfusion (I/R) injury causes severe manifestations including acute heart failure and systemic dysfunction. Recently, there has been increasing evidence suggesting that alterations in mitochondrial morphology and dysfunction also play an important role in the prognosis of cardiac disorders. Long non-coding RNAs (lncRNAs) form major regulatory networks altering gene transcription and translation. While the role of lncRNAs has been extensively studied in cancer and tumor biology, their implications on mitochondrial morphology and functions remain to be elucidated. In this study, the functional roles of Zinc finger protein 36-like 2 (ZFP36L2) and lncRNA PVT1 were determined in cardiomyocytes under hypoxia/reoxygenation (H/R) injury in vitro and myocardial I/R injury in vivo. Western blot and qRT-PCR analysis were used to assess the levels of ZFP36L2, mitochondrial fission and fusion markers in the myocardial tissues and cardiomyocytes. Cardiac function was determined by immunohistochemistry, H&E staining, and echocardiogram. Ultrastructural analysis of mitochondrial fission was performed using transmission electron microscopy. The mechanistic model consisting of PVT1 with ZFP36L2 and microRNA miR-21-5p with E3 ubiquitin ligase MARCH5 was assessed by subcellular fraction, RNA pull down, FISH, and luciferase reporter assays. These results identified a novel regulatory axis involving PVT1, miR-21-5p, and MARCH5 that alters mitochondrial morphology and function during myocardial I/R injury. Using an in vivo I/R injury mouse model and in vitro cardiomyocytes H/R model, we demonstrated that ZFP36L2 directly associates with PVT1 and alters mitochondrial fission and fusion. PVT1 also interactes with miR-21-5p and suppresses its expression and activity. Furthermore, we identified MARCH5 as a modifier of miR-21-5p, and its effect on mitochondrial fission and fusion are directly proportional to PVT1 expression during H/R injury. Our findings show that manipulation of PVT1-miR-21-5p-MARCH5-mediated mitochondrial fission and fusion via ZFP36L2 may be a novel therapeutic approach to regulate myocardial I/R injury.
在几种主要的心血管疾病中，缺血再灌注（I/R）损伤会导致严重的临床表现，包括急性心力衰竭和全身功能障碍。最近，越来越多的证据表明，线粒体形态和功能的改变也在心脏疾病的预后中起着重要作用。长链非编码 RNA（lncRNA）形成主要的调节网络，改变基因转录和翻译。虽然 lncRNA 的作用已在癌症和肿瘤生物学中得到广泛研究，但它们对线粒体形态和功能的影响仍有待阐明。在这项研究中，我们在体外缺氧/复氧（H/R）损伤和体内心肌 I/R 损伤的心肌细胞中确定了锌指蛋白 36 样 2（ZFP36L2）和 lncRNA PVT1 的功能作用。Western blot 和 qRT-PCR 分析用于评估心肌组织和心肌细胞中 ZFP36L2、线粒体分裂和融合标志物的水平。通过免疫组织化学、H&E 染色和超声心动图来确定心脏功能。使用透射电子显微镜对线粒体分裂的超微结构进行分析。通过亚细胞部分、RNA 下拉、FISH 和荧光素酶报告基因测定评估包含 PVT1、miR-21-5p 和 E3 泛素连接酶 MARCH5 的 PVT1-ZFP36L2-miR-21-5p-MARCH5 机制模型。这些结果确定了一种新的调节轴，涉及 PVT1、miR-21-5p 和 MARCH5，它们在心肌 I/R 损伤期间改变线粒体形态和功能。使用体内 I/R 损伤小鼠模型和体外心肌细胞 H/R 模型，我们证明 ZFP36L2 直接与 PVT1 结合并改变线粒体分裂和融合。PVT1 还与 miR-21-5p 相互作用并抑制其表达和活性。此外，我们确定 MARCH5 是 miR-21-5p 的调节剂，其对线粒体分裂和融合的影响与 H/R 损伤期间 PVT1 的表达成正比。我们的研究结果表明，通过 ZFP36L2 操纵 PVT1-miR-21-5p-MARCH5 介导的线粒体分裂和融合可能是调节心肌 I/R 损伤的一种新的治疗方法。
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