Mitochondrial Calcium Uniporter-Mediated Regulation of the SIRT3/GSK3β/β-Catenin Signaling Pathway in Vascular...

Calcium homeostasis plays a crucial role in regulating the phenotype of vascular smooth muscle cells (VSMCs) and vascular remodeling. This study aims to investigate the role of the mitochondrial calcium uniporter (MCU), which facilitates the uptake of Ca²⁺ into the mitochondria, in vascular remodeling and its underlying regulatory mechanisms. Vascular remodeling in rats was induced through either 21-day hindlimb unloading (HU) or 21-day angiotensin II (Ang II) infusion (0.7 mg/kg/day). Phenotypic switching of VSMCs and vascular remodeling were assessed. To induce phenotypic switching and clarify the underlying regulatory mechanisms, VSMCs were treated with Ang II (100 μmol/L). Gene manipulation was performed using plasmids, lentivirus, and adeno-associated virus serotype 9 (AAV9). Mitochondrial oxidative stress, Ca²⁺ distribution, and the expression of MCU, SIRT3, GSK3β, and β-catenin, along with GSK3β activity, SIRT3 ubiquitination, and GSK3β acetylation, were evaluated. The expression of MCU and SIRT3 in rat cerebral arteries was downregulated following HU and Ang II administration, which resulted in an increase in cytoplasmic Ca²⁺, a decrease in mitochondrial Ca²⁺, and a shift toward a synthetic phenotype in VSMCs. In vitro, Ang II treatment of VSMCs led to reduced expression of MCU, SIRT3, and GSK3β, and increased nuclear translocation of β-catenin. Knockdown of MCU caused an increase in cytoplasmic Ca²⁺ and a reduction in mitochondrial Ca²⁺, while MCU overexpression had the opposite effect, decreasing cytoplasmic Ca²⁺ and increasing mitochondrial Ca²⁺. Additionally, MCU overexpression decreased SIRT3 ubiquitination, mitochondrial oxidative stress, GSK3β acetylation, nuclear translocation of β-catenin, and VSMC phenotypic switching-these effects were blocked by SIRT3 knockdown. Moreover, MCU overexpression partially mitigated vascular remodeling in HU and hypertensive rats by inhibiting the GSK3β/β-catenin pathway and preserving SIRT3. Ang II regulates MCU protein expression, which is reduced in the HU and Ang II-induced hypertensive rat cerebral arteries. This reduction impairs cellular Ca²⁺ buffering and promotes mitochondrial oxidative stress. The stress response triggers the downstream degradation of SIRT3, which subsequently inhibits the activity of GSK3β via acetylation and promotes the nuclear translocation of β-catenin, thereby facilitating phenotypic switching and vascular remodeling.