Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

PANoptosis integrates features of pyroptosis, apoptosis, and necroptosis. The roles of ZBP1, ADAR1, RIPK1, and RIPK3 in PANoptosis are not fully understood, particularly in the context of heart transplantation. Our studies revealed that Z-DNA can induce PANoptosis in human microvascular endothelial cells via ZBP1. ADAR1 and RIPK1 collaboratively regulate the activation of the ZBP1-RIPK3 complex for PANoptosis in a time-dependent manner. The interaction of ADAR1 with ZBP1 protected against Z-DNA-induced cell death by limiting the activation of ZBP1 and RIPK3 at an early stage of cell death. As well, RIPK1 promoted the phosphorylation of RIPK3 and furthered ZBP1-RIPK3 interaction and cell death. Our in vivo studies showed that ZBP1 and its ligand, Z-DNA, were significantly upregulated in the grafts after mouse heart transplantation. Importantly, ZBP1 deficiency in the mouse heart graft inhibited cardiac PANoptosis, attenuated acute and chronic graft injury, anti-donor immune responses, and induced long-term graft survival. In conclusion, this study elucidates the roles of ADAR1 and RIPK1 in regulating the ZBP1-RIPK3 complex and PANoptosis. Inhibition of ZBP1 can prevent heart graft injury and rejection. Our studies may inform the development of novel therapeutic strategies targeting multiple programmed cell death pathways to improve graft survival.