MG53 participates in ischaemic postconditioning through the RISK signalling pathway

Abstract

Aims: Recent studies show that ischaemic postconditioning (PostC), similar to the well-established ischaemic preconditioning (IPC), confers cardioprotection against ischaemia/reperfusion (IR) injury, and both IPC and PostC can activate the reperfusion injury salvage kinase (RISK) pathway and the survivor activating factor enhancement (SAFE) pathway. PostC is clinically more attractive because of its therapeutic application at the predictable onset of reperfusion. Our previous studies have demonstrated that MG53 is a primary component of the IPC machinery. Here, we investigated the potential role of MG53 in PostC-mediated myocardial protection and explored the underlying mechanism.

Methods and results: Using Langendorff perfusion, we investigated IR injury in wild-type (wt) and MG53-deficient (mg53(-/-)) mouse hearts with or without PostC. IR-induced myocardial damage was markedly exacerbated in mg53(-/-) hearts compared with wt controls. PostC protected wt hearts against IR-induced myocardial infarction, myocyte necrosis, and apoptosis, but failed to protect mg53(-/-) hearts. The loss of PostC protection in mg53(-/-) hearts was attributed to selectively impaired PostC-activated RISK signalling. Mechanistically, MG53 is required for the interaction between caveolin 3 (CaV3) and the p85 subunit of phosphoinositide 3-kinase (p85-PI3K) and PostC-mediated activation of the RISK pathway. Importantly, a structure-function study revealed that the MG53 tripartite motif (TRIM) domain (aa1-284) physically interacted with CaV3 but not p85-PI3K, whereas the MG53 SPRY domain (aa285-477) interacted with p85-PI3K but not CaV3, indicating that MG53 binds to CaV3 and p85 at its N- and C-terminus, respectively.

Conclusions: We conclude that MG53 participates in PostC-mediated cardioprotection largely through tethering CaV3 and PI3K and subsequent activation of the RISK pathway.

Figure 1

MG53 knockout hearts are vulnerable to IR injury and resistant to ischaemic PostC protection. (A) Schematic illustration of the protocol used for mouse global ex vivo IR (30 min ischaemia followed by reperfusion) with or without PostC (6 episodes of 10 s ischaemia followed by 10 s reperfusion). (B) Representative photographs and statistical data of infarct size in perfused wt and mg53^−/− mouse hearts subjected to IR with or without PostC (n = 8, scale bar denotes 1 mm). (C) Change of LDH concentration in the efflux of perfused hearts from wt and mg53^−/− mice subjected to 30 min ischaemia and 10 min of reperfusion with or without PostC (n = 8). (D) Representative photographs and statistical data of TUNEL staining of myocardial sections from perfused hearts of wt and mg53^−/− mice subjected to IR with or without PostC (n = 8, scale bar denotes 30 µm). For (B–D), data are presented as mean ± s.e.m. (*P < 0.05 vs. all three other groups; †P < 0.05 vs. wt IR).

Figure 2

MG53 is essential for PostC-induced activation of RISK pathway. (A–C): Representative immunoblots and statistical data of phosphorylated and total Akt (A), GSK3β (B), and ERK 1/2 (C) in lysates from perfused wt and mg53^−/− mouse hearts with or without PostC (n = 8, *P < 0.05 vs. all three other groups; †P < 0.05 vs. both wt groups). Note that MG53 ablation impaired PostC-induced phosphorylation of Akt, GSK3β, and ERK 1/2. (D) Average data of the infarct size in perfused mg53^−/− hearts subjected to 30 min ischaemia followed by 120 min reperfusion without or with PostC or SB216763 pretreatment (3 µM for 10 min before 30 min ischaemia. n = 8, *P < 0.05 as indicated).

Figure 3

SAFE pathway is independent of MG53. (A and B) Representative immunoblots and average data of phosphorylated and total STAT3 protein from total (A) and nuclear fraction (B) in hearts from wt and mg53^−/− mice with or without PostC (n = 6 for each group, *P < 0.05 vs. control). (C) Representative immunoblots and average data of STAT3 protein levels in myocardial tissue from wt and mg53^−/− mice (n = 8).

Figure 4

MG53 interacts with p85 subunit of PI3K as well as CaV3. (A) Schematic diagram of structure of the plasmids expressing flag-MG53, MG53-NT, flag-MG53-TRIM and flag-MG53-SPRY, p85-myc, flag-p85, or CaV3-myc. (B) Co-IP of flag-MG53 and p85-myc in lysates of HEK293 cells (n = 4). (C) Co-IP of p85-PI3K and CaV3 in the lysates of the perfused mouse hearts subjected to IR with or without PostC treatment (n = 4). (D) Confocal immunofluorescence co-staining to visualize the colocalization of flag-MG53 (green) and p85-myc (red) in HEK293 cells (Scale bar denotes 5 µm). (E and F) Representative blots of pull-downed MG53-myc recombinant protein with CaV3-myc or p85-myc protein, and CaV3-myc with flag-p85 in the presence or absence of MG53-NT recombinant protein, respectively. (G) Co-IP of MG53-TRIM or MG53-SPRY with p85-PI3K-myc or CaV3-myc in lysates of HEK293 cells (n = 4). (H) Schematic presentation to show the role of MG53 in tethering CaV3 and p85-PI3K and subsequent activation of the RISK pathway.

Figure 5

Schematic shows that MG53 forms a complex with CaV3 and p85-PI3K, which is involved in PostC-induced activation of the RISK but not the SAFE pathway.