Inadequate Camk2a and Excess Camk2b: The Key to Vitamin D Irresponsiveness in an In Vitro Model of Sarcopenia

Abstract

Vitamin D plays a crucial role in sarcopenia, yet supplementation often proves ineffective in treating the condition. We hypothesized that calcium signaling regulated by vitamin D may be involved in sarcopenia and could influence the effectiveness of vitamin D treatment. Calcium signaling pathway activity was analyzed through bioinformatics, and its regulation was validated in an in vitro model of sarcopenia using TNF-α-treated C2C12 cells. Analysis of transcriptomic data (GSE225576) revealed inhibition of the calcium signaling pathway, while proteomic data (PXD040722) showed extensive dysregulation. Notably, Camk2a expression was reduced and Camk2b increased at both mRNA and protein levels in aged mouse muscle tissue. Silencing Camk2a in C2C12 cells led to NF-κB activation and elevated expression of senescence-associated secretory phenotype (SASP) markers, including IL-6, IL-1β, IL-8, and MCP-1. Treatment with 1α,25(OH)₂D₃ suppressed NF-κB activation and SASP marker expression in shNC control cells, but this effect was lost in Camk2a-silenced cells and enhanced in Camk2b-silenced cells. Additionally, the reduction of NF-κB activity and SASP markers by 1α,25(OH)₂D₃ in Camk2b-silenced cells was reversed by co-transfection with siPdia3, but not siVDR, suggesting that 1α,25(OH)₂D₃ acts through the Pdia3–PLAA–Camk2 pathway to alleviate sarcopenia. Finally, co-immunoprecipitation confirmed the binding of calmodulin to both Camk2a and Camk2b both with and without 1α,25(OH)₂D₃ treatment. Collectively, these findings suggest that the downregulation of Camk2a and upregulation of Camk2b may contribute to vitamin D treatment resistance in sarcopenia-related muscle atrophy, potentially via NF-κB activation and SASP expression driven by Camk2b.