481-P: KCNMA1 Transcriptional Regulation by Nrf2 in Diabetic Vessels

Abstract

KCNMA1 encodes the large conductance Ca2+-activated K+ channel pore-forming subunits (BK-α), a major ionic mechanism that regulates vascular smooth muscle physiology and is impaired in high fat diet-induced (HFD) diabetic mice. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor that regulates a wide variety of cellular proteins by binding to the antioxidant response elements (AREs). We found that the promoter region of KCNMA1 contains multiple AREs, but whether Nrf2 regulates KCNMA1 expression is unknown. In this study, we hypothesize that Nrf2 is a transcriptional regulator of KCNMA1 and the reduced Nrf2 activity in diabetes contributes to BK channelopathy. Using videomicroscopy, patch clamp recordings, molecular and cellular approaches, we found that BK channel activity and BK channel-mediated vasodilation were diminished in the coronary arteries of mice after 4 months on HFD. This was accompanied by significant reductions in BK-α and Nrf2 protein expression. In cultured coronary artery smooth muscle cells (CASMCs), adenoviral transduction with Nrf2 (50 MOI) resulted in an increase of BK-α mRNA by 10-fold and BK-α protein expression by 2-fold, while a 70% knockdown of Nrf2 by shRNA produced a 30% downregulation of BK-α protein levels. Luciferase reporter assays showed robust luciferase signals in cells 48 h after co-transfection with Nrf2 vectors and reporter vectors containing a segment of the KCNMA1 promoter region with a predicted ARE-Nrf2 binding site, but not those with empty vectors or KCNMA1 reporter vectors with the ARE-Nrf2 binding site deleted. Most importantly, treatment with dimethyl fumarate (a Nrf2 activator) increased BK-α mRNA and protein expression in cultured CASMCs, and rescued BK channel function and BK channel-mediated vasodilation in coronary arteries of HFD mice. Our results indicate that Nrf2 transcriptionally regulates KCNMA1 expression in CASMCs and has a therapeutic potential in the treatment of BK channelopathy and vasculopathy in diabetes. Disclosure H. Lee: None. X. Sun: None. X. Wang: None. T. Lu: None. Funding American Diabetes Association (1-18-IBS-210 to H-C.L.); National Institutes of Health (R01HL74180)