Huang Xiaozhi, Zhang Jiayin, Xu Chen, Cao Ranran, Jiang Peijun, Ji Xue, Wang Wenyi, Huang Zhishan, Han Peidong
Center for Genetic Medicine, the Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, China (X.H., J.Z., C.X., R.C., P.J., X.J., P.H.).
International School of Medicine, International Institute of Medicine, Zhejiang University, Yiwu, China (X.H., J.Z., C.X., R.C., P.J., X.J., P.H.).
Circ Res. 2025 Jan 31;136(3):279-296. doi: 10.1161/CIRCRESAHA.124.325290. Epub 2025 Jan 7.
Cardiac ischemia/reperfusion disrupts plasma membrane integrity and induces various types of programmed cell death. The ESCRT (endosomal sorting complex required for transport) proteins, particularly AAA-ATPase Vps4a (vacuolar protein sorting 4a), play an essential role in the surveillance of membrane integrity. However, the role of ESCRT proteins in the context of cardiac injury remains unclear.
We simultaneously visualized the formation of membrane blebs and the subcellular translocation of Vps4a during a variety of cell death programs in primary cardiomyocytes. cardiomyocyte-specific knockout and overexpression mice were generated and characterized. In vivo and ex vivo surgeries were performed to determine the effects of altered Vps4a expression levels on plasma membrane repair and cell survival. Given the role of Ripk3 (receptor-interacting kinase 3)-mediated pore formation in regulating cell membrane integrity, hearts from and double-knockout mice were examined. The sequential recruitment of upstream ESCRT components that promote the translocation of Vps4a to injured sites was also assessed using genetic gain- and loss-of-function approaches. Finally, we overexpressed a mutated form of Vps4a with defective ATPase activity and investigated its function during cardiomyocyte membrane repair.
Ischemia/reperfusion stimulation or forced induction of apoptosis, necroptosis, and pyroptosis in primary cardiomyocytes leads to membrane blebbing and the exposure of phosphatidylserine to the extracellular space. In response to injury, Vps4a promptly translocates to injured sites to reseal damaged membranes. gain- and loss-of-function in the postnatal stage minimally affects cardiac structure formation and function. However, in the context of ischemia/reperfusion stimulation, overexpression of protects cardiomyocytes against injury, whereas -deficient hearts are more susceptible to cell damage. Additionally, deletion abrogates the detrimental effects of deficiency during ischemia/reperfusion injury, and the Ca-Alix-Ist1 axis plays an essential role in recruiting Vps4a to the injured site. Mechanistically, Vps4a promotes the shedding of plasma membrane blebs to restrict permeability to the extracellular environment, and the surveillance of membrane integrity requires the ATPase activity of Vps4a.
These results demonstrate that Vps4a-mediated plasma membrane repair is an intrinsic cell protection machinery that antagonizes cardiac ischemia/reperfusion injury, and our findings may contribute to the development of therapeutic strategies towards attenuating cardiac injury.
心脏缺血/再灌注会破坏质膜完整性，并诱导多种类型的程序性细胞死亡。转运所需内体分选复合物（ESCRT）蛋白，尤其是AAA - ATP酶Vps4a（液泡蛋白分选4a），在监测膜完整性方面发挥着重要作用。然而，ESCRT蛋白在心脏损伤背景下的作用仍不清楚。
我们在原代心肌细胞的各种细胞死亡程序中同时观察了膜泡的形成和Vps4a的亚细胞转位。构建并鉴定了心肌细胞特异性敲除和过表达小鼠。进行体内和体外手术以确定Vps4a表达水平改变对质膜修复和细胞存活的影响。鉴于受体相互作用激酶3（Ripk3）介导的孔形成在调节细胞膜完整性中的作用，检测了Ripk3和Vps4a双敲除小鼠的心脏。还使用基因功能获得和缺失方法评估了促进Vps4a转位至损伤部位的上游ESCRT组分的顺序募集。最后，我们过表达了一种具有缺陷ATP酶活性的Vps4a突变形式，并研究了其在心肌细胞膜修复过程中的功能。
原代心肌细胞中的缺血/再灌注刺激或凋亡、坏死性凋亡和炎性小体介导的细胞死亡的强制诱导导致膜泡形成以及磷脂酰丝氨酸暴露于细胞外空间。响应损伤时，Vps4a迅速转位至损伤部位以重新封闭受损膜。出生后阶段的功能获得和缺失对心脏结构形成和功能的影响最小。然而，在缺血/再灌注刺激的背景下，Vps4a的过表达可保护心肌细胞免受损伤，而Vps4a缺陷的心脏对细胞损伤更敏感。此外，Vps4a的缺失消除了缺血/再灌注损伤期间Ripk3缺陷的有害影响，并且Ca - Alix - Ist1轴在将Vps4a募集至损伤部位中起重要作用。从机制上讲，Vps4a促进质膜泡的脱落以限制对细胞外环境的通透性，并且膜完整性的监测需要Vps4a的ATP酶活性。
这些结果表明，Vps4a介导的质膜修复是一种内在的细胞保护机制，可对抗心脏缺血/再灌注损伤，我们的发现可能有助于开发减轻心脏损伤的治疗策略。
