Palmatine blocks GAPDH K263 lactylation, thereby inhibiting glycolysis and osteogenic differentiation in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a prevalent cardiovascular disorder for which no effective pharmacotherapy has been approved. This study aims to investigate the therapeutic potential of palmatine (PAL) in CAVD and to elucidate the underlying molecular mechanism, with a focus on its role in metabolic-epigenetic crosstalk. The anti-calcification effect of PAL was evaluated in human aortic valve interstitial cells (hVICs) using qRT-PCR, Western blotting, and Alizarin Red S staining. Integrated multi-omics profiling (transcriptomics and metabolomics) together with lactylomics revealed that PAL primarily interferes with glycolytic metabolism and global lysine lactylation levels. The interaction between PAL and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was assessed via molecular docking and surface plasmon resonance (SPR). The functional significance of GAPDH K263 lactylation (K263lac) was investigated using site-specific mutagenesis. PAL treatment significantly inhibited osteogenic differentiation and calcification of hVICs. Mechanistically, PAL directly bound to GAPDH, selectively reducing its lactylation at the K263 residue. Mutating K263 to arginine (K263R) mimicked the effect of PAL, abrogating the PAL-mediated suppression of glycolysis, osteogenic gene expression, and calcium deposition. Immunofluorescence analysis further confirmed that PAL treatment markedly reduced cytoplasmic K263lac levels, and the K263R mutation completely abolished this modification. In vivo, PAL administration in a high-fat diet-fed ApoE-/- mouse model significantly alleviated aortic valve calcification, as evidenced by echocardiography and von Kossa staining. In conclusion, GAPDH K263lac represents a novel druggable target in CAVD. By directly targeting this modification, palmatine disrupts glycolysis and osteogenic differentiation, highlighting its therapeutic potential for CAVD treatment.