Activation of PERK branch of ER stress mediates homocysteine-induced BKCa channel dysfunction in coronary artery via FoxO3a-dependent regulation of atrogin-1

Abstract

The molecular mechanism of endoplasmic reticulum (ER) stress in vascular pathophysiology remains inadequately understood. We studied the role of ER stress in homocysteine-induced impairment of coronary dilator function, with uncovering the molecular basis of the effect of ER stress on smooth muscle large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels. The vasodilatory function of BK_Ca channels was studied in a myograph using endothelium-denuded porcine small coronary arteries. Primary cultured porcine coronary artery smooth muscle cells were used for mRNA and protein measurements and current recording of BK_Ca channels. Homocysteine inhibited vasorelaxant response to the BK_Cachannel opener NS1619, lowered BK_Ca β1 subunit protein level and suppressed BK_Ca current. Inhibition of ER stress restored BK_Ca β1 protein level and NS1619-evoked vasorelaxation. Selective blockade of the PKR-like ER kinase (PERK) yielded similarly efficient restoration of BK_Ca β1, preserving BK_Ca current and BK_Ca-mediated vasorelaxation. The restoration of BK_Ca β1 by PERK inhibition was associated with reduced atrogin-1 expression and decreased nuclear localization of forkhead box O transcription factor 3a (FoxO3a). Silencing of atrogin-1 prevented homocysteine-induced BK_Ca β1 loss and silencing of FoxO3a prevented atrogin-1 upregulation induced by homocysteine, accompanied by preservation of BK_Ca β1 protein level and BK_Ca current. ER stress mediates homocysteine-induced BK_Ca channel inhibition in coronary arteries. Activation of FoxO3a by PERK branch underlies the ER stress-mediated BK_Ca inhibition through a mechanism involving ubiquitin ligase-enhanced degradation of the channel β1 subunit.