科研之友
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摘要
Aquaporin-1 (AQP1) is expressed in human and mouse hearts, but little is known about its cellular and subcellular localization and regulation. The aim of this study was to investigate the localization of AQP1 in the mouse heart and to determine the effects of ischemia and hypoxia on its expression. Mouse myocardial cells were freshly isolated and split into cardiomyocyte and non-cardiomyocyte fractions. Isolated, Langendorff-perfused C57B16 mouse hearts (n=46) were harvested with no intervention, subjected to 35 min of ischemia or ischemia followed by 60 min of reperfusion. Eleven mouse hearts were perfusion-fixed for electron microscopy. Forty C57B16 mice were exposed to normobaric hypoxia for one or two weeks (n = 12). Needle biopsies of human left ventricular myocardium were sampled (n = 30) during coronary artery bypass surgery before cardioplegia and after 30 min of reperfusion. Human umbilical vein endothelial cells (HUVECs) were subjected to 4 h of hypoxia with reoxygenation for either 4 or 24 h. AQP1 expression was studied by electron microscopy with imrnunogold labeling, Western blot, and qPCR Expression of miR-214 and miR-320 in HUVECs with hypoxia was studied with qPCR HUVECs were then transfected with precursors and inhibitors of miR-214. AQP1 expression was confined to cardiac endothelial cells, with no signal in cardiomyocytes or cardiac fibroblasts. Immunogold electron microscopy showed AQP1 expression in endothelial caveolae with equal distribution along the basal and apical membranes. Ischemia and reperfusion tended to decrease AQP1 mRNA expression in mouse hearts by 37 +/- 9% (p = 0.06), while glycosylated AQP1 protein was reduced by 16 +/- 9% (p = 0.03). No difference in expression was found between ischemia alone and ischemia-reperfusion. In human left ventricles AQP1 mRNA expression was reduced following cardioplegia and reperfusion (p = 0.008). Hypoxia in mice reduced AQP1 mRNA expression by 20 +/- 7% (p<0.0001), as well as both glycosylated (-47 +/- 10%, p = 0.03) and glycan-free protein (-34 +/- 16%, p = 0.05). Hypoxia and reoxygenation in HUVECs downregulated glycan-free AQP1 protein (-34 +/- 24%, p = 0.04) and upregulated miR-214 (+287 +/- 52%, p<0.05). HUVECs transfected with anti-miR-214 had increased glycosylated (1.5 fold) and glycan-free (2 fold) AQP1. AQP1 in mouse hearts is localized to endothelial cell membranes and caveolae. Cardioplegia, ischemia and hypoxia decrease AQP1 mRNA as well as total protein expression and glycosylation, possibly regulated by miR-214.
- 出版日期2013-3
