The Senokine Gas6 Promotes Aortic Valve Calcification Through CaMKII–DRP1–Mediated Terminal Scission of Mitochondrial-Derived Vesicles

Abstract

Calcific aortic valve disease (CAVD) is an age-associated valvular disorder in which calcification expands from discrete foci into adjacent tissue. We investigated whether mitochondrial-derived vesicles (MDVs) released by human valvular interstitial cells (hVICs) exert pro-calcific effects on recipient cells and how MDV release is regulated. Using ex vivo hemodynamic perfusion of human CAVD tissue, multi-omics profiling, live-cell single-organelle tracking, MDV transfer assays, and in vivo Gas6 deletion, we found that calcifying hVICs hypersecreted MDVs that exerted pro-calcific effects in recipient hVICs. High-resolution imaging showed that mitochondria remodeled into donut-shaped structures under pro-osteogenic conditions; these structures served as physical platforms for MDV budding. Human CAVD valve tissues and hVICs treated with pro-osteogenic medium also exhibited senescence-associated features. Multi-omics profiling identified growth arrest-specific 6 (Gas6), a senokine, among 36 concordantly upregulated candidates. Functional analyzes showed that Gas6 promoted terminal MDV scission while leaving donut-shaped mitochondrial precursor formation intact. Mechanistically, Gas6 promoted intracellular Ca²⁺ accumulation, calcium/calmodulin-dependent protein kinase II (CaMKII) activation, and dynamin-related protein 1 (DRP1) phosphorylation at Ser616, supporting a Ca²⁺–CaMKII–DRP1 axis that facilitates terminal MDV scission. Pharmacological inhibition of CaMKII or DRP1 reduced MDV hypersecretion and subsequent recipient-cell calcification, and Gas6 deletion in vivo attenuated valvular stenosis, calcification, and fibrotic remodeling. These findings identify Gas6-dependent terminal MDV scission as a mechanism contributing to the intercellular propagation of valvular calcification in a CAVD context characterized by senescence-associated features.