Abstract P3002: Endoplasmic Reticulum Stress Plays a Role in Nox5 Mediated Vascular Contraction

Abstract

We previously reported that Nox5 regulates contraction through mechanisms involving ROS and Ca 2+ channels in the endoplasmic reticulum (ER). In young mice expressing human Nox5 in VSMCs (Nox5+SM22+), we observed hypercontractility, without changes in blood pressure. Here we tested the hypothesis that Nox5 influences ER Ca 2+ homeostasis and vascular function and that Nox5 amplifies aging-associated vascular dysfunction through processes involving ER stress. Female WT and Nox5+SM22+ mice aged 20 and 35weeks were studied. Blood pressure (BP) was assessed by tail-cuff and vascular function/structure by myography. BP was similar in all groups. Vascular contraction to U46619, a TXA2 analogue, was increased in aged Nox5+SM22+ (EMax - %KCl: 114±2.8 vs WT 95±2.4, p<0.05). Hypercontractility was reversed by NAC (antioxidant - 0.01 mM, EMax: 92±5%), melittin (Nox5 inhibitor - 0.1 μM, EMax 92±3.2%) and dantrolene (RyR Ca 2+ channel blocker - 0.01 mM, EMax: 67±4.2%) (p<0.05). VSMCs isolated from 20 and 35 wk WT and Nox5+SM22+ mice were used to study molecular mechanisms whereby Nox5 influences the contractile machinery, focusing on the ER. Expression of BIP, a marker of ER stress, was increased only in VSMCs from aged Nox5+SM22+ mice (AU: 0.13±0.01 vs WT 0.05±0.002, p<0.05). 4-PBA, an inhibitor of ER stress (1 mM), reversed the hypercontractile responses in 35 wk Nox5+SM22+ mice (EMax: 87±3%, p<0.05). We identified calreticulin, important in ER Ca 2+ homeostasis and channel function, as a molecular target of Nox5. As Nox regulates signalling by oxidation, we assessed calreticulin oxidation by pulldown using dimedone based probe (DCP-Bio). Calreticulin oxidation was increased in mesenteric arteries, aorta and VSMCs from 35 week Nox5+SM22+. Moreover, expression of calreticulin (23.5±2%) and BIP (27.6±9%) was increased by U46619 in VSMCs from Nox5+SM22+ (p<0.05); an effect inhibited by melittin and 4-PBA. Our study highlights molecular mechanisms whereby Nox5 regulates contraction, through oxidation of calcium regulatory proteins, such as calreticulin, and ER stress in aged Nox5 mice. These age related changes may predispose Nox5 mice to cardiovascular damage when challenged with factors associated with hypertension.