Background: 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.
Methods: We simultaneously visualized the formation of membrane blebs and the subcellular translocation of Vps4a during a variety of cell death programs in primary cardiomyocytes. Vps4a 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 Ripk3 and Vps4a 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.
Results: 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. Vps4a 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 Vps4a protects cardiomyocytes against injury, whereas Vps4a-deficient hearts are more susceptible to cell damage. Additionally, Ripk3 deletion abrogates the detrimental effects of Vps4a deficiency during ischemia/reperfusion injury, and the Ca2+-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.
Conclusions: 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.
Keywords: cell death; cell membrane; myocytes, cardiac; protein transport; reperfusion injury.