Abstract
Backgroud: TMEM16A is an intrinsic constituent of the Ca2+-activated chloride channel in vascular smooth muscle cells (VSMCs) and other cell types. It is involved in VSMCs proliferation and hypertension-induced vascular remodeling. However, the functional significance of TMEM16A in the process of apoptosis in basilar artery smooth muscle cells (BASMCs) remains elusive. Here, we investigated whether and how TMEM16A contribute to the development of apoptosis in BASMCs.
Methods: Cell viability assay, flow cytometry, western blotting, mitochondrial membrane potential test, immunogold labeling, and co-immunoprecipitation (co-IP) were performed in this study.
Results: we found that H2O2 induced BASMCs apoptosis via mitochondria-dependent pathway, including increase the apopstosis rate, upregulate the expression of pro-apoptotic protein of Bax, potentiate the loss of mitochondial membrane potential and the release of cytochrome c from mitochondria to cytoplasm. These effects were reversed by silence of TMEM16A, but further potentiated by overexpression of TMEM16A. The endogenous TMEM16A was detected in mitochondrial fraction and co-IP revealed an interaction between TMEM16A and Cyclophilin D, which was an component of the mitochondrial permeability transition pore (mPTP), and this interaction was upregulated by H2O2, but restricted by cyclosporin A, an inhibitor of Cyclophilin D. TMEM16A increased mPTP opening, resulting in the release of cytochrome c and the subsequent activation of caspase-9 and caspase-3, and finally developed to apoptosis. The results from aortas of TMEM16A smooth muscle-specific knockin mice were consistent with the results from BASMCs.
Conclusions: This study suggests that TMEM16A promotes H2O2-induced apoptosis via modulation of mitochondrial membrane permeability in VSMCs. This study establishes TMEM16A as a target for therapeutic intervention in several remodeling prevalent diseases, such as hypertension, stroke, aortic aneurysm, and arteriosclerosis.
