OR18-01 Autocrine Wnt16 Signals Sustain Mitochondrial Respiratory Capacity in Arterial Smooth Muscle and Osteoblasts, Regulating Mitochondrial Unfolded Protein Response and Cell Death Programs

Abstract D. Zhong: None. A. Behrmann: None. S. Xie: None. A. Lemoff: None. J. Kozlitina: None. D.A. Towler: None. We recently demonstrated expression of Wnt16 mRNA and protein in both human and mouse arterial smooth muscle (VSM). Hypomorphic Wnt16 alleles that reduce bone mass significantly increase cardiovascular risk in humans by 25% (Dallas Heart Study) and worsen abnormal arterial remodeling in mice (angiotensin II - induced aneurysm). Wnt16 augmented VSM contraction and contractile phenotype, mediated in part via Taz (Wwtr1) signaling relays. By electron microscopy, the aortic VSM mitochondria of Wnt16-null mice appear swollen, exhibiting an electron-dense mitochondrial matrix with indistinct cristae as compared to controls. Given the high energy demands of VSM contraction — and the concomitant cardiovascular and skeletal fragility with Wnt16 insufficiency — we examined the impact of Wnt16 deficiency on mitochondrial respiratory function and protein composition in both VSM and osteoblasts. In Seahorse Oximetry Mito Stress Test assays (25 mM glucose:1 mM pyruvate: 6 mM glutamine), arterial VSM from Wnt16-null mice exhibited significantly reduced basal (-26%), maximal (-23% , and spare reserve (-19%) respiratory capacity (all p < 0.001) - the latter characteristic of advanced age. Conversely treatment with recombinant Wnt16 augmented spare respiratory capacity 3-fold (p< 0.01), reversing the consequence of Wnt16 deficiency. RNAi targeting Taz (Wwtr1) reduced oxygen consumption. Conversely, siRNA targeting the upstream Yap/Taz inhibitor Ccm2 increased maximal respiration. Compared to arterial VSM, calvarial osteoblasts exhibit lower respiratory rates with very little spare respiratory capacity. However, basal (-41% ) and maximal (-48%) respiration were markedly and significantly reduced in osteoblasts of Wnt16-null mice. Label-free quantitative analyses by LC-MS/MS of mitochondrial protein extracts from both Wnt16-deficient VSM and calvarial osteoblasts revealed significant upregulation of mitochondrial unfolded protein response constituents (Lonp1, Clpp, Hspa9; confirmed by western blot), increased mitochondrial hexokinase Hk2, upregulation of mitophagy regulator Bcl2L13, and downregulation of the apoptosis inhibitor Bag3. TiterTACS assay demonstrated increased apoptosis in Wnt16-null VSM. Moreover, concentrations of Wnt16 (8 nM) that increase contractile phenotype, support contraction, and suppress the myomitokine Gdf15 significantly reduced VSM apoptosis on type I collagen - coated stiff (>= 100 kPa; - 22% change, p = 0.03) but not soft (=<50 kPa; 0% change) matrices. Wnt16 exerts cell-autonomous actions on mitochondrial composition and physiology in both arterial VSM and bone. Reduced autocrine Wnt16 signaling may increase cardiovascular and skeletal frailty in part via age-related accrual of mitochondrial dysfunction. Saturday, June 1, 2024