Ca^(2+)/CaM依赖的蛋白激酶Ⅱ对神经肽γ诱导心肌细胞内钙重分布的作用

The Effect of CaMK Ⅱ on NPY-lnduced Redistribution of Ca^(2+)in Cardiomyocytes

背景神经肽 Y(NPY)是中枢神经及末梢神经重要的调节剂,作用广泛,包括调节心脏及血管正常的生理活动,并参与许多心血管疾病的发生发展。研究表明,急性的 NPY 刺激可促进心肌细胞钙活动,对心肌产生正性肌力作用。而持续 NPY 刺激对心肌钙活动的影响及其机制,目前还未见报道。目的观察 NPY 刺激对大鼠心肌细胞胞浆钙和肌浆网(SR)内钙分布的影响,以及 Ca^(2+)/CaM 依赖的蛋白激酶Ⅱ(CaMKⅡ)在其中的作用。方法用100 nmol/L NPY 刺激 Sprague Dawley 乳鼠心肌细胞24 h,CaMKⅡ特异性抑制剂 KN-93干预。应用荧光染料 Fluo-4 AM 负载胞浆钙;Fluo-5N AM 负载肌浆网内游离钙离子,所有钙影像均由激光共聚焦显微镜记录。应用Western-blot 法和免疫荧光法检测 Ca^(2+)-ATP 酶(SERCA2a)和 ryanodine 受体(RyR_2)两种蛋白的分布及蛋白量的变化。结果经100 nmol/L NPY 刺激24 h 后,与对照组相比,心肌细胞胞浆游离钙浓度明显升高(65.3±6.2 vs50.7±4.1,P<0.05),心肌细胞肌浆网内游离钙含量明显低于对照组(67.6±8.3 vs 85.5±6.0,P<0.05),而KN-93可抑制上述效应;NPY 可增加 SERCA2a 和 RyR_2的蛋白表达(SERCA2a:2.4±0.7 vs 对照组:1.4±0.3;RyR_2:2.3±0.4 vs 对照组:1.2±0.4),KN-93可抑制上述作用。结论 CaMKⅡ通过影响 SERCA2a 和 RyR_2,调控 SR 的钙转运,进而介导 NPY 刺激下的细胞内钙重分布效应。

Background and Objective Neuropeptide Y （NPY） is an important modulator factor in central and peripharal nervous system, involving in regulating cardiac or vascular physiological functions, as well as the development of cardiovascular diseases. Previous study showed acute NPY stimulated promotes Ca^2＋ activation in cardiomyocytes, and resulted in positive inotrophic effect. However, it still remains unknown the effect of sustained NPY stimulation on Ca^2＋ mobilization in cardiomyocytes. Methods Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY 100 nmol/L or NPY in addition with CaMK Ⅱ specific inhibitor KN-93 for 24 h. Fluorescent indicator Fluo-4 AM was used to detect Ca^2＋ in cell plasma. Fluo-5N AM was used to show Ca^2＋ in sarcoplasmic reticulum（SR）. Calcium images were recorded by laser scanning confocal microscope. The alternation of SERCA2a and RyR2 expression were assessed by Western blot and immunofluorescence method. Results Twenty four hours after incubation with NPY, compared with control group, plasma（Ca^2＋ i） was significantly elevated （ NPY. 65.3 ± 6.2 vs control： 50.7 ± 4.1, P〈0.05 ）, concomitantly with an decreases in free Ca^2＋ in SR （Ca^2＋SR） （NPY： 67.65±8.3 vs control： 85.5±6.0, P〈0. 01 ）. Protein expressions of SERCA2a and RyR2 were increased （SERCA2a, NPY： 2.45±0. 7 vs control： 1.4±0.3; RyR2, NPY：2.3±0.4 vs control： 1.2±0.4, P〈 0. 05）, which were attenuated by KN-93. Conclusion CaMK Ⅱ modulated Ca^2＋ mobilization in SR by affecting SERCA2a and RyR2, which is responsible critically for Ca^2＋ redistribution induced by NPY.