RIPK3 promotes ASIC1a-mediated fibroblast-like synoviocyte migration and invasion via malate shuttle-driven mitochondrial respiration in rheumatoid arthritis

Objective: Synovial fibroblast migration and invasion are critical contributors to the progression of rheumatoid arthritis (RA). Acidification of local joint tissue exacerbates RA progression, but the underlying mechanisms remain unclear. This study aimed to investigate the role of acid-sensitive ion channel ASIC1a and its mediator, the RIPK3-MDH1 axis, in regulating the migration and invasion of RA fibroblast-like synoviocytes (RA-FLSs). Methods: The expression of ASIC1a, RIPK3, and MDH1 in synovial tissue from RA patients and arthritic mice was analyzed using immunofluorescence and Western blotting. RA-FLSs were stimulated with extracellular acidification (pH 6.8, mimicking local tissue conditions), and their migration and invasion were assessed via Transwell assays. The interaction between ASIC1a and RIPK3 was predicted using molecular docking and confirmed by co-immunoprecipitation (CO-IP). RIPK3^-/- mice were used to establish a collagen antibody-induced arthritis (CAIA) model. Pharmacological inhibitors of ASIC1a (PcTX1) and RIPK3 (GSK-872) were employed to evaluate their therapeutic effects on migration and invasion in vitro and arthritis progression in vivo using the collagen-induced arthritis (CIA) model. Bioinformatics analyses, along with glucose, ATP, NAD⁺ and NADH assays, and oxygen consumption rate (OCR) measurements, were conducted to investigate the regulation of mitochondrial respiration by the RIPK3-MDH1 axis. Results: Extracellular acidification (pH 6.8) significantly enhanced the migration and invasion of RA-FLSs, effects that were abrogated by ASIC1a knockdown or pharmacological inhibition. ASIC1a activated RIPK3 through its kinase function, independent of its ion channel activity. RIPK3 activation promoted mitochondrial respiration and ATP production via MDH1-mediated malate shuttle activation. Furthermore, inhibition of the malate shuttle using Aminooxyacetic acid (Carboxymethoxylamine) hemihydrochloride (AOA) suppressed ASIC1a-mediated RA-FLSs migration and invasion. The RIPK3-MDH1 axis also maintained malate shuttle activity by enhancing glycolysis and glutamate metabolism through GLS1. Mechanistically, ASIC1a activated RIPK3, which in turn promoted MDH1-mediated malate shuttle activation, enhancing mitochondrial respiration and ATP synthesis, thereby driving RA-FLSs migration and invasion. In vivo, pharmacological inhibition of ASIC1a or RIPK3, as well as RIPK3 knockdown, significantly alleviated arthritis progression in CIA and CAIA mouse models. Conclusion: The RIPK3-MDH1 malate shuttle drives RA-FLSs migration and invasion in RA. Activation of the ASIC1a-RIPK3-MDH1 axis enhances mitochondrial respiration and ATP synthesis in RA-FLSs, highlighting this pathway as a potential therapeutic target for RA.