Copper exposure disrupts the stability of SAM50-MICOS-ATAD3-mtDNA axis to activate the cGAS-STING and NLRP3 pathway in pig liver.

Copper (Cu) is an essential mineral nutrient in animal, however, excess Cu causes mitochondrial homeostasis imbalance and multiple regulated cell death. This study explored how Cu overload disrupts the SAM50-MICOS-ATAD3-mtDNA axis and further activates cGAS-STING-NLRP3-mediated pyroptosis in pig liver via in vivo feeding model and in vitro hepatocyte with Cu chelator TTM, SAM50 overexpression and H₂O₂ positive control. We verified that excess dietary Cu triggers hepatic lipid metabolic disorder and impairs mitochondrial respiratory activity. Mechanistically, Cu weakens bidirectional protein interactions within the SAM50-MICOS-ATAD3 complex without altering total protein abundance of these subunits, thus breaking mtDNA retention homeostasis. Therefore, Cu exposure significantly increased the mtDNA (D-LOOP, mtATP6, mtCO1, mtND1, mtCO2 and mtND2) released to cytoplasm and plasma compared with that in the control group and raised the level of IFN-β (P < 0.05). Additionally, Cu exposure significantly increased the mRNA and protein expression of cGAS-STING pathway and NLRP3 dependent pyroptosis compared with the control group, while obviously decreased the mRNA and protein expressions of TFAM. Immunohistochemistry and immunofluorescence validated elevated GSDMD and NLRP3 as well as reduced TFAM in Cu stressed liver tissue. Notably, SAM50 overexpression effectively restored the disrupted complex, curtailed mtDNA egress, and attenuated downstream inflammatory activation, phenocopying the protective effects of TTM chelation. Collectively, our findings demonstrated Cu overload destroyed the stability of SAM50-MICOS-ATAD3-mtDNA axis, promotes mtDNA released and triggered the cGAS-STING and NLRP3 pathways in pig hepatocytes.