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bioRxiv · 10.1101/2024.12.11.628017

Genetic silencing of KCa3.1 inhibits atherosclerosis in ApoE null mice

Alam, P.·Tharp, D. L.·Bowles, H. J.·Grisanti, L.·Bui, H. S.·Bender, S. B.·Bowles, D. K.
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Abstract

Increased expression of KCa3.1 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 KCa3.1, such that it becomes a dominant K+ channel and contributes to SMC cell migration. The efficacy of pharmacological inhibition of KCa3.1 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 KCa3.1, we used CRISPR/Cas9 to generate KCa3.1-/-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 KCa3.1 did not affect serum total cholesterol or body weight. In BCAs of DKO mice, lesion size (0.036 mm{superscript 2} vs. 0.118 mm{superscript 2}, 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 KCa3.1 inhibition, while activation of KCa3.1 further enhanced MCP1/Ccl21 expression. Both in vivo and in vitro analyses showed that silencing KCa3.1 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 KCa3.1 silencing. Overall, this study provides the first genetic evidence that KCa3.1 is a critical regulator of atherosclerotic lesion development and composition and provides novel mechanistic insight into the link between KCa3.1 and atherosclerosis.

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Alam, P., Tharp, D. L., Bowles, H. J., Grisanti, L., Bui, H. S., Bender, S. B., Bowles, D. K.. 2024-12-16. Genetic silencing of KCa3.1 inhibits atherosclerosis in ApoE null mice. https://doi.org/10.1101/2024.12.11.628017

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