Complement C3 inhibition restores myasthenia gravis AChR antibody-mediated muscle pathophysiology

Abstract

In the autoimmune disorder myasthenia gravis (MG), the nicotinic acetylcholine receptors (nAChRs) are the primary targets of pathogenic antibodies. While MG mechanisms have been extensively studied in animal models, functional insights into how antibody binding disrupts nAChR-dependent calcium signaling and the effects of complement inhibition in human muscle cells remain limited. Methods We used real-time live-cell calcium imaging to assess the effects of cholinergic stimulation or inhibition on human muscle cells with pharmacological agents, sera from patients with acetylcholine receptor antibody seropositive (AChR+) MG, purified recombinant antibodies targeting α- and β-subunits, and a complement C3 inhibitor. Transcriptional changes in nAChR subunits, muscle markers, voltage-gated calcium channels (VGCCs), and complement-related genes were analysed by RT-qPCR. Immunocytochemistry and quantitative image analysis were performed to assess nAChR distribution and membrane attack complex (MAC) deposition. Findings Cholinergic stimulation of human muscle cells activated nAChRs, resulting in membrane depolarisation, which in turn led to VGCC opening and calcium transients. MG-associated pathogenic antibodies, particularly those present in sera from patients with AChR + MG and pure recombinant AChR α-subunit-specific monoclonal antibody (mAb), but not β-subunit-specific, abolished choline-induced calcium responses in myotubes. α-subunit-specific mAb also induced transcriptional upregulation of nAChR subunits, muscle structural proteins, and complement components, and were associated with nAChR loss and MAC formation. Importantly, pharmacological inhibition of C3 activation restored calcium signaling, preserved nAChR distribution, and reduced MAC formation induced by α-subunit-specific mAb, implicating complement activation as a key driver of pathogenic effects. Interpretation These findings indicate that targeting the α-subunit impairs nAChR-dependent calcium signaling and can induce complement activation. Disrupted signaling and reduced nAChR levels were effectively restored by C3 inhibition, which blocks multiple downstream pathways; thus, terminal complement activation leading to MAC formation is a suggested, but it may not be the only mechanism. Overall, the results highlight C3 as a promising upstream therapeutic target and support combining subunit-specific interventions with proximal complement blockade in MG.