Genetic silencing of K Ca 3.1 inhibits atherosclerosis in ApoE null mice

Abstract Increased expression of K Ca 3.1 has been found in vascular smooth muscle (SMC), macrophages, and T cells in atherosclerotic lesions from humans and mice. Proliferating SMC cells increase the expression of K Ca 3.1, such that it becomes a dominant K + channel and contributes to SMC cell migration. The efficacy of pharmacological inhibition of K Ca 3.1 in limiting atherosclerosis progression has been demonstrated in mice and pigs, however direct, loss-of-function, i.e. gene silencing, studies are absent. To investigate the role of K Ca 3.1, we used CRISPR/Cas9 to generate K Ca 3.1 -/- Apoe -/- (DKO) mice and assessed lesion development in the brachiocephalic artery (BCA) of DKO versus Apoe -/- mice on a Western diet for 3 months. Notably, the loss of K Ca 3.1 did not affect serum total cholesterol or body weight. In BCAs of DKO mice, lesion size (0.036 mm² vs. 0.118 mm², p<0.05) and relative stenosis (13.9% vs. 43.0%, p<0.05) were reduced by 70% compared to Apoe -/- mice, with no effect on medial or lumen area. Additionally, DKO mice exhibited a significant reduction in macrophage content within atherosclerotic plaques compared to Apoe -/- mice, independent of sex. In vitro migration assays further showed a significant reduction in migration of bone marrow-derived macrophages (BMDMs) from DKO mice compared to those from Apoe -/- mice. Furthermore, in vitro experiments using rat aortic smooth muscle cells (RAOSMCs) revealed significant inhibition of PDGF-BB-induced MCP1/Ccl21 expression upon K Ca 3.1 inhibition, while activation of K Ca 3.1 further enhanced MCP1/Ccl21 expression. Both in vivo and in vitro analyses showed that silencing K Ca 3.1 and sex had no significant effect on the collagen content of plaque. RNAseq analysis of BCA samples from DKO and Apoe -/- mice revealed PPAR-dependent signaling as a potential key mediator of the reduction in atherosclerosis due to K Ca 3.1 silencing. Overall, this study provides the first genetic evidence that K Ca 3.1 is a critical regulator of atherosclerotic lesion development and composition and provides novel mechanistic insight into the link between K Ca 3.1 and atherosclerosis.