The COP9 Signalosome Suppresses Cardiomyocyte Necroptosis

Background Loss of cardiomyocyte (CMs) due to apoptosis and regulated necrosis contributes to heart failure. However, the molecular mechanisms governing regulated CM necrosis remain obscure. The COP9 signalosome (CSN) formed by 8 unique protein subunits (COPS1 through COPS8) functions to deneddylate Cullin-RING ligases (CRLs), thereby regulating the functioning of the CRLs. Mice with CM-restricted knockout of Cops8 (Cops8-cko) die prematurely, following reduced myocardial performance of autophagy and the ubiquitin-proteasome system (UPS) as well as massive CM necrosis. This study was aimed to determine the nature and underlying mechanisms of the CM necrosis in Cops8-cko mice.Methods We examined myocardial expression and activities of key proteins that reflect the status of the RIPK1-RIPK3 pathway, redox, and caspase 8 in Cops8-cko mice. Moreover, we used in vivo CM uptake of Evan’s blue dye (EBD) as an indicator of necrosis and performed Kaplan-Meier survival analyses to test whether treatment with a RIPK1 kinase inhibitor (necrostatin-1) or an antioxidant (N-acetyl-L-cysteine), global knockout of the RIPK3 or the Ppif gene, CM-restricted knockout of the Nrf2 gene, or cardiac HMOX1 overexpression could rescue the Cops8-cko phenotype.Results Compared with littermate control mice, myocardial protein levels of RIPK1, RIPK3, MLKL, the RIPK1-bound RIPK3, protein carbonyls, full-length caspase 8, Nrf2, Ser40-phosphorylated Nrf2 and BCL2, as well as histochemical staining of superoxide anions were significantly increased but the cleaved caspase 8 and the overall caspase 8 activity were markedly decreased in Cops8-cko mice, indicating that the RIPK1-RIPK3 and the Nrf2 pathways are activated and caspase 8 activation is suppressed by Cops8-cko. Continuous necrostatin-1 infusion initiated at 2 weeks of age nearly completely blocked CM necrosis at 3 weeks and markedly delayed premature death of Cops8-cko mice. RIPK3 haploinsufficiency or cardiac-specific Nrf2 heterozygous knockout discernably attenuated CM necrosis and/or delayed mouse premature death; conversely, Ppif knockout, N-acetyl-L-cysteine treatment, and cardiac overexpression of HMOX1 exacerbated CM necrosis and mouse premature death.Conclusions Cardiac Cops8/CSN malfunction causes RIPK1-RIPK3 mediated CM necroptosis in mice; sustained Nrf2 activation and reductive stress pivot cardiomyocytes to necroptosis when autophagy and the UPS are impaired; and the CSN plays an indispensable role in suppressing CM necroptosis.