Adam8 aggravates myocardial infarction injury by modulating macrophage mitochondrial homeostasis and ROS generation

Abstract Aims Myocardial infarction (MI) is one of the leading causes of cardiovascular mortality worldwide. Macrophages play critical roles in post-MI inflammation and repair, while excessive mitochondrial reactive oxygen species (ROS) production aggravates cardiac injury. Our previous study demonstrated that ADAM8 expression is upregulated in cardiac macrophages after MI and promotes inflammatory responses and adverse cardiac remodeling. This study aimed to elucidate the mechanism by which ADAM8 disrupts macrophage mitochondrial metabolic homeostasis and promotes ROS generation. Methods Macrophage-specific ADAM8 conditional knockout (CKO) mice were generated by crossing Adam8flox/flox mice with Lyz2-Cre mice. Male C57BL/6 mice aged 6–8 weeks were subjected to permanent ligation of the left anterior descending coronary artery to establish the MI model. Cardiac macrophages were isolated and purified using F4/80+ magnetic-activated cell sorting. Intracellular and mitochondrial ROS levels were measured by flow cytometry using CM-H2DCFDA and MitoSOX probes. Single-cell RNA sequencing was performed to identify ADAM8-dependent signaling pathways on cardiac macrophages. Mitochondrial function was evaluated by measuring ATP levels and oxygen consumption rate (OCR). Co-immunoprecipitation (Co-IP) and Western blotting were used to verify the interactions among ADAM8, integrin α9β1, and focal adhesion kinase (FAK). Results (1) Single-cell RNA sequencing revealed that MI significantly increased the number of cardiac macrophages and upregulated ROS-related pathways. (2) Intracellular ROS, mitochondrial ROS, and malondialdehyde (MDA) levels were markedly elevated in cardiac macrophages 3 days after MI, accompanied by reduced glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities. (3) Macrophage-specific ADAM8 deficiency after MI significantly suppressed ROS production in the infarcted myocardium. Single-cell transcriptomic analysis showed that ADAM8 CKO downregulated macrophage oxidoreductase activity and restored mitochondrial ATP synthesis-coupled proton transport. (4) ADAM8-deficient macrophages exhibited higher ATP levels, improved mitochondrial OCR, and alleviated mitochondrial damage. (5) Mechanistically, ADAM8 directly interacted with integrin α9β1 via its disintegrin domain. ADAM8 deletion reduced integrin β1 and FAK Y397 phosphorylation and restored the expression of mitochondrial ATP synthase subunits. Inhibition of ATP synthase reversed the mitochondrial protective effects of ADAM8 deficiency. Conclusions ADAM8 disrupts mitochondrial metabolic homeostasis and promotes mitochondrial ROS generation in macrophages by binding integrin α9β1 and activating FAK Y397 signaling, thereby exacerbating post-MI cardiac injury.GraphicAbstract