GenCefe Client Article | IF: 33.5 (Cell Discovery)丨MARCH2 protects myocardial ischemia-reperfusion injury by inhibiting pyroptosis

Myocardial infarction (MI) remains one of the leading causes of disability and death worldwide [1]. Timely restoration of blood flow to occluded coronary arteries is the best processing for patients with myocardial infarction [2]. However, this reperfusion may trigger a series of harmful injuries to the myocardium, called ischemia-reperfusion (I/R) injury [2-3]. Studies have shown that activation of the inflammasome is one of the causes of inflammation-driven myocardial ischemia-reperfusion injury [4-5]. However, its underlying regulatory mechanisms remains to be discovered.

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In February 2024, Zhang Yingmei and Ge Junbo of Fudan University and Hu Ronggui of the CAS jointly published an article in Cell Discovery titled "The E3 ubiquitin ligase MARCH2 protects against myocardial ischemia-reperfusion injury through inhibiting pyroptosis via negative regulation of PGAM5/MAVS/NLRP3 axis" [6] revealed the molecular mechanism by which E3 ubiquitin ligase alleviates myocardial ischemia-reperfusion injury.

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In this study, PGAM5-mcherry, MAVS-EGFP, selected siRNA and negative controls were designed and synthesized by GENCEFE BIOTECH.

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Result

1. Through a series of experiments such as sequencing and qPCR, the research team found that MARCH2 protein levels increased in the hearts of ICM (ischemic cardiomyopathy) patients and mice during myocardial I/R, and further verified that MARCH2 deficiency can aggravate I /R cardiac dysfunction during injury.

2. MARCH2 deletion induces NLRP3 inflammasome activation after myocardial I/R; MARCH2 directly interacts with PGAM5 to regulate myocardial I/R injury (Figure 1)

3. MARCH2 mediates the ubiquitination and proteasomal degradation of PGAM5 by catalyzing K48-linked polyubiquitination.

To simulate H/R injury and pro-inflammatory environment in vitro, cardiomyocytes were treated with macrophage-conditioned medium (MCM) and H/R (hypoxia and reoxygenation, 6h/3h). Before processing, HL-1 cardiomyocytes were transfected with plasmids expressing MARCH2-Flag and PGAM5-HA, or with an empty plasmid vector (as a control). Transfection of a catalytically inactive mutant of MARCH2 (C64/67S) eliminated MARCH2-accelerated PGAM5 degradation (Fig. 2a–c), indicating that the catalytic activity of MARCH2 plays an important role in reducing PGAM5 protein levels; MARCH2 promotes the proteasome of PGAM5 dependent degradation (Fig. 2d–g).

After MCM+H/R processing, WTMARCH2 (but not the E3 ligase inactive mutant) mediates PGAM5 polyubiquitination (polyUb), and it is known that different Lys(K)-linked PolyUb chains can confer different signaling conduction outcomes. [7]. Experimental results demonstrated the critical role of the TM and PDZ domains of MARCH2 in direct interaction with PGAM5 (Fig. 2h,i). Subsequently, the researchers generated the PGAM5KOHL-1 cardiomyocyte cell line through CRISPR/Cas9-mediated genome editing technology to identify the ubiquitination site of PGAM5. A series of experiments demonstrated that MARCH2 promotes the ubiquitination and degradation of PGAM5 by binding K48-linked polyubiquitin to K88 and K141 sites (Figure 2j-l).

4. MARCH2 inhibits PGAM5-dependent NLRP3 inflammasome activation after myocardial I/R

The protein level of PGAM5 in ICM patients was negatively correlated with the protein level of MARCH2 (Fig. 3a); in MARCH2-overexpressing NMCM treated with MCM+H/R, the protein level of PGAM5 was reduced (Fig. 3b, c), PGAM5K48 in MARCH2KO hearts There was an associated decrease in ubiquitination (Fig. 5d). PGAM5 was also elevated in mouse hearts after I/R surgery, and this effect was further enhanced by genetic ablation of MARCH2 (Fig. 3e–g). The increase in ASC caused by MCM+H/R was abolished by MARCH2 overexpression, which in turn was counteracted by coexpression of PGAM5 (Fig. 3h, i).

5. PGAM5-MAVS co-condensate is formed under MCM+H/R processing, and PGAM5 promotes MAVS-dependent NLRP3 activation.

Database analysis and experiments have proven that endogenous MAVS interacts with PGAM5 protein; a series of experimental results show that MAVS exhibits a dynamic liquid state under MCM+H/R processing (Figure 4a-f); PGAM5-MAVS interaction is possible promotes MAVS gel-like transformation (Figure 4g-j); After MCM+H/R processing, PGAM5 overexpression leads to NLRP3 inflammasome activation, and this response is counteracted by MAVS knockdown (Figure 4k-q); MCM+ H/R-induced inflammasome activation was blocked by MARCH2 overexpression, while PGAM5 or MAVS overexpression eliminated the cardioprotective effect of MARCH2 (Fig. 4r-u).

6. MARCH2 overexpression can prevent myocardial I/R injury

The researchers constructed viral vectors to provide high MARCH2 expression in cardiomyocytes. Injecting mice with AAV9-cTnT-MARCH2 3 weeks before I/R surgery significantly increased MARCH2 protein levels in cardiomyocytes. Compared with the control group, mice injected with AAV9-cTnT-MARCH2 had significantly improved responses to I/R injury, including improvements in cardiac function (Fig. 5a–f). After I/R injury, PGAM5K48-linked ubiquitination increased (Fig. 5i) and PGAM5 protein levels decreased (Fig. 5j, k) in AAV9-cTnT-MARCH2 hearts. Injection of AAV9-cTnT-MARCH2 reversed mitochondrial localized NLRP3 inflammasome activation after I/R (Fig. 5l-n).

7. Cardiac PGAM5-MAVS signaling is critical for the protective effect of MARCH2 against myocardial I/R injury

By knocking down endogenous PGAM5 in cardiomyocytes via AAV9 viral vector-based RNA interference, we found that PGAM5/MAVS signaling is indispensable for the protective function of MARCH2 against cardiac I/R injury. AAV9 viral vector was used to stably express MARCH2, PGAM5 and MAVS respectively. Mice were delivered with AAV9-cTnT-MARCH2 with or without AAV9-cTnT-PGAM5 and AAV9-cTnT-MAVS for 3 weeks prior to I/R. Experimental results showed that MARCH2 prevented myocardial I/R injury by inhibiting NLRP3 inflammatory assembly and pyroptosis in a PGAM5/MAVS-dependent manner (Figure 6).

Findings from this study not only established the E3 ligase MARCH2 as a novel cardioprotective molecule against myocardial I/R injury, but also identified a previously unknown mechanism that negatively regulates PGAM5/MAVS/NLRP3 inflammasome signaling and pyroptosis to maintain homeostasis and normal function of mammalian hearts. Targeting MARCH2 may thus represent a novel opportunity to battle against myocardial injuries.

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【References】

[1] Reed, G. W., Rossi, J. E. & Cannon, C. P. Acute myocardial infarction. Lancet 389, 197–210 (2017).

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[2] Yellon, D. M. & Hausenloy, D. J. Myocardial reperfusion injury. N. Engl. J. Med. 357, 1121–1135 (2007).

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[3] Hausenloy, D. J. & Yellon, D. M. Myocardial ischemia-reperfusion injury: a neglected therapeutic target. J. Clin. Invest. 123, 92–100 (2013).

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[4] Toldo, S. & Abbate, A. The NLRP3 inflammasome in acute myocardial infarction. Nat. Rev. Cardiol. 15, 203–214 (2018).

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[5] Sandanger, ?. et al. The NLRP3 inflammasome is up-regulated in cardiac fibroblasts and mediates myocardial ischaemia-reperfusion injury. Cardiovasc. Res. 99, 164–174 (2013).

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[6] Liu, S., Bi, Y., Han, T. et al. The E3 ubiquitin ligase MARCH2 protects against myocardial ischemia-reperfusion injury through inhibiting pyroptosis via negative regulation of PGAM5/MAVS/NLRP3 axis. Cell Discov 10, 24 (2024).

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[7] Sheng, X., Xia, Z., Yang, H. & Hu, R. The ubiquitin codes in cellular stress responses. Protein Cell https://doi.org/10.1093/procel/pwad045 (2023).

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