Canonical Wnt signaling affects calcium homeostasis in serum-treated AC16 cells through MLN-mediated SERCA2a regulation

The canonical Wnt/β-catenin pathway critically regulates cardiac calcium homeostasis, yet its interplay with microenvironmental factors remains unclear. This study reveals that fetal bovine serum (FBS) treatment alters Wnt-mediated calcium dynamics in AC16 cardiomyocytes. While Wnt activation elevates cytosol calcium in serum-free conditions, FBS supplementation reverses this response: Wnt inhibitors (SFRP2, XAV939, and LF3) induce cytosol calcium accumulation, while the activators (LiCl and Wnt3a) lose efficacy. Mechanistically, FBS ablates RyR2 expression, uncoupling calcium-induced calcium release. Consequently, calcium handling shifts to SERCA2a-dependent regulation. We identify myoregulin (MLN) as a pivotal effector of the Wnt/β-catenin signaling with Wnt inhibition upregulating MLN to suppress SERCA2a activity. MLN knockdown (90% suppression) abolishes the effects of Wnt inhibitors on SERCA2a function and calcium distribution patterns. RyR2 reconstitution in FBS-treated cells restores calcium release but not Wnt activation responses, confirming the dominant role of MLN. Crucially, a combination of RyR2 overexpression and MLN depletion fully restores Wnt-calcium responses, phenocopying serum-free conditions. Our work establishes a serum-dependent regulatory axis where Wnt/β-catenin signaling maintains calcium homeostasis by repressing MLN, thereby preserving SERCA2a function. This FBS-induced shift mirrors pathological adaptations in heart failure, positioning MLN as a therapeutic target for calcium-handling disorders.