LUBAC-mediated linear ubiquitination drives proinflammatory activation of macrophage in cardiac ischemia-reperfusion injury

Background Cardiac ischemia-reperfusion (I/R) injury, triggered by blood flow restoration after acute myocardial infarction, is characterized by inflammation that severely aggravates tissue damage. The linear ubiquitin chain assembly complex (LUBAC), with HOIL-1-interacting protein (HOIP, encoded by Rnf31) as its critical catalytic subunit, is the sole E3 ligase that generates M1-linked linear ubiquitin chains. However, the cell-type-specific functions and substrate spectrum of LUBAC-mediated linear ubiquitination in macrophage-driven inflammation during cardiac I/R injury remain unclear. Purpose To investigate the role of LUBAC-mediated linear ubiquitination in cardiac I/R injury, and further explore the downstream inflammatory signal. Methods Western blot (WB), quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence staining (IF) were applied to examine expression of linear ubiquitination and LUBAC subunits in heart tissue and isolated macrophages following cardiac I/R injury, with validation in peripheral blood mononuclear cells (PBMCs) from patients. Myeloid-specific HOIP knockout mice were used to determine effects on inflammatory phenotypes. Key substrates and signaling axes were identified by co-immunoprecipitation (co-IP) and mass spectrometry (MS), followed by evaluation of drug efficacy in mice and cardiac organoid systems. Results During cardiac I/R injury, we found the level of linear ubiquitination in cardiac macrophages significantly increased, accompanied by elevated expression of HOIP, whereas the other LUBAC subunits showed minor changes. Notably, in PBMCs from patients, increased linear ubiquitination and HOIP expression correlated with cTnT, CK-MB and NT-proBNP. Myeloid-specific HOIP knockout mice demonstrated reduced infiltration of CCR2+/CD64+ macrophages and improved cardiac function. Mechanistically, proteomics and IP-MS identified Acyl-coenzyme A synthetase short-chain family member 2 (ACSS2) as the key downstream substrate. HOIP interacted with ACSS2 and raised its enzymatic activity, thereby increasing acetyl-CoA production. This process enhanced histone H3 lysine 27 acetylation (H3K27ac) at the promoters of proinflammatory genes, including Il1b, Ccl2, and the NF-κB-related transcription factor Irf1, driving inflammation activation. To intervene, we developed macrophage-targeted lipid nanoparticles encapsulating HOIPIN8, which showed safety and efficacy in both mice and cardiac organoid systems. Conclusion LUBAC-mediated linear ubiquitination represents a critical driver of macrophage-mediated inflammation following cardiac I/R injury through the HOIP-ACSS2 axis, which can be alleviated by targeted LNP-HOIPIN8 intervention.