高盐上调TMEM16A致小鼠冠状动脉重构的机制

Mechanism of high salt-induced coronary artery remodeling in mice by upregulating TMEM16A

摘要:

目的]探讨高盐摄入上调跨膜蛋白16A(TMEM16A)致小鼠冠状动脉重构的机制。 [方法]36只8周龄雄性C57BL/6J小鼠随机分为3组:对照组(饮食中未额外添加NaCl)、10 g/L NaCl组(1 L纯水中含10 g NaCl)和20 g/L NaCl组(1 L纯水中含20 g NaCl),喂养8周。每周测量小鼠体重和收缩压(SBP)；8周后,收集血清测量小鼠血清Na＋浓度；测量小鼠左心室质量指数(LVMI)以评估高盐对小鼠心脏的影响；Langendorff逆行恒压灌流法测定小鼠冠状动脉流量(CF)；HE染色评估小鼠冠状动脉形态学改变,并计算管壁厚度百分比及管壁面积百分比；微血管张力记录仪检测小鼠离体冠状动脉收缩性变化,以冠状动脉血管环面积标化其张力；Western blot检测小鼠冠状动脉中TMEM16A蛋白表达；免疫荧光双染检测小鼠冠状动脉中TMEM16A和α-平滑肌肌动蛋白(α-SMA)表达。 [结果]8周后,10 g/L NaCl组和20 g/L NaCl组小鼠体重变化率均低于对照组,SBP均显著高于对照组(P＜0.05或P＜0.01)。与对照组相比,10 g/L NaCl组和20 g/L NaCl组小鼠血清Na＋浓度均增加(P＜0.05或P＜0.01)；高盐摄入可增加小鼠LVMI,降低小鼠CF,且均以20 g/L NaCl组小鼠最显著(P＜0.01)；10 g/L NaCl组和20 g/L NaCl组小鼠冠状动脉的管壁厚度百分比和管壁面积百分比均显著增加(均P＜0.01)；10 g/L NaCl组和20 g/L NaCl组小鼠冠状动脉对60 mmol/L KCl和0.03 μmol/L内皮素1的收缩反应均显著增强(P＜0.01)；20 g/L NaCl组小鼠冠状动脉中TMEM16A蛋白表达显著增加(P＜0.01),10 g/L NaCl组和20 g/L NaCl组小鼠冠状动脉中TMEM16A和α-SMA荧光表达均显著增加(均P＜0.01),且TMEM16A与α-SMA在冠状动脉中存在显著共定位。 [结论]高盐摄入可致小鼠冠状动脉重构,其机制可能与上调冠状动脉TMEM16A的表达有关。

Abstract:

Aim To investigate the mechanism by which high-salt intake upregulates transmembrane protein 16A (TMEM16A) and induces coronary artery remodeling in mice. Methods Thirty-six 8-week-old male C57BL/6J mice were randomly divided into three groups:control group (no additional NaCl in the diet), 10 g/L NaCl group (1 L of pure water contains 10 g of NaCl), and 20 g/L NaCl group (1 L of pure water contains 20 g of NaCl). Mice were fed for 8 weeks. Mouse body weight and systolic blood pressure (SBP) were measured weekly. After 8 weeks, serum was collected to measure serum Na＋ concentrations in mice; left ventricular mass index (LVMI) was measured to assess the effects of high-salt intake on the mouse heart; coronary flow (CF) was determined using the Langendorff retrograde constant-pressure perfusion method; HE staining was used to assess morphological changes in the coronary arteries, and calculate wall thickness (WT) percentage and wall area (WA) percentage. Microvascular tension recording instruments were employed to measure contractile changes in isolated coronary arteries from each group, standardizing coronary artery tension based on the area of the vascular ring. Western blot was used to detect TMEM16A protein expression in mouse coronary arteries. Dual immunofluorescence staining was used to detect TMEM16A and α-SMA expression in mouse coronary arteries. Results After 8 weeks, mice in the 10 g/L NaCl group and 20 g/L NaCl group showed a lower body weight change rate than control group, and SBP was significantly higher in both salt groups (P＜0.05 or P＜0.01). Compared with control mice, serum Na＋ concentration was increased in the 10 g/L NaCl group and 20 g/L NaCl group (P＜0.05 or P＜0.01). High-salt intake elevated LVMI and reduced CF, with the 20 g/L NaCl group exhibiting the most pronounced changes (P＜0.01). Percent wall thickness and percent wall area of coronary arteries were significantly greater in both salt groups (all P＜0.01). Contractile responses to 60 mmol/L KCl and 0.03 μmol/L endothelin-1 in coronary arteries were significantly enhanced in the 10 g/L and 20 g/L NaCl groups (P＜0.01). TMEM16A protein expression in coronary arteries was markedly up-regulated in the 20 g/L NaCl group (P＜0.01), and immunofluorescence revealed significantly increased TMEM16A and α-SMA signals in both salt groups (all P＜0.01). In addition, there was an obvious co-localization between TMEM16A and α-SMA. Conclusion High-salt intake causes coronary artery remodeling in mice, and its mechanism may be related to increasing TMEM16A expression.