Serpina3c deficiency in perivascular adipocytes impairs MAM-dependent mitochondrial calcium homeostasis and accelerates atherosclerosis

Background Perivascular adipose tissue (PVAT) is a metabolically active organ surrounding vessels, and its dysfunction leads to local inflammation reaction and aggravates atherosclerosis. Dysregulated mitochondria-associated endoplasmic reticulum membranes (MAMs) in adipocytes drives excessive mitochondrial Ca²⁺ influx from ER, inducing oxidative stress and pro-inflammatory signaling. However, whether Serpina3c modulates MAM-dependent mitochondrial dysfunction in adipocytes and contributes to atherosclerosis remains unclear. Purpose This study aimed to determine whether Serpina3c affects PVAT functions and mediates atherosclerosis through regulating MAM-dependent mitochondrial calcium homeostasis. Methods Differentiated 3T3-L1 adipocytes and human adipose-derived stem cells (ADSCs) were exposed to palmitic acid (PA) to establish a high-fat microenvironment. In vitro, Serpina3c and Vdac1 were knocked down using siRNA. MAM formation was assessed, and mitochondrial calcium, oxidative stress and apoptosis levels were detected. Bioinformatic analysis and luciferase assay were used to identify potential transcriptional regulators of Vdac1. Male ApoE-/- mice were fed a high-fat diet for 16 weeks to establish the model of atherosclerosis, and an adipocyte-specific Serpina3c knockdown model in PVAT was constructed using local adenovirus-associated shRNA delivery. Results PA exposure inhibited Serpina3c protein expression, whereas it increased the expression of inflammation-related genes and mitochondrial ROS levels, enhanced ER-mitochondria coupling, and promoted apoptosis (Fig. 1A-E). These changes were significantly reversed by recombinant Serpina3c (Fig. 1B-E). Serpina3c knockdown promoted MAM formation and IP3R1-VDAC1 complex assembly in PA-exposed adipocytes, thereby triggering mitochondrial calcium overload and enhancing inflammation reaction (Fig. 1F-J). RNA sequencing analysis found enrichment of MAM-related pathways and Vdac1 upregulation following Serpina3c knockdown in 3T3-L1 cells (Fig. 2A-D). Vdac1 knockdown rescued mitochondrial dysfunction induced by Serpina3c deficiency (Fig. 2E-G). Bioinformatic analysis identified YY1 as a potential upstream regulator of Vdac1, and luciferase assays demonstrated its direct activation to the Vdac1 promoter (Fig. 2H-I). The above effects were also observed in ADSCs. In vivo, adipocyte-specific Serpina3c knockdown in PVAT similarly promoted MAM formation, mitochondrial calcium overload, and increased ROS levels, aggravating tissue inflammation and atherosclerosis. Conclusion This work suggests Serpina3c is a critical mediator of MAM-dependent mitochondrial calcium signaling in PVAT, thereby modulating atherosclerosis. This may represent a potential therapeutic strategy for atherosclerosis.