β肾上腺素能受体活化蛋白激酶C_ε诱导心肌细胞肥大及其机制探讨

发布时间：2018-09-18 21:29

【摘要】：背景：心肌肥大是高血压、冠心病、瓣膜病及先心病等多种心血管疾病共同的病理改变,是心肌细胞对多种不良刺激的病理性代偿过程。心肌肥大是导致心血管疾病发病率和病死病残率显著升高的独立危险因素。心肌肥大的发生发展涉及多种促心肌肥大信号(如理化刺激、激素水平、组织因子等),这些信号单独或共同激活多条信号转导通路,最终促成和/或加重心肌肥大。阐明心肌肥大的病因、发病机理,对改进防治方法、开发有效药物和降低病死病残率意义重大。尤其对心肌肥大信号转导机制的研究近年来已成为国内外研究的热点。心脏的肾上腺素能信号转导通路包括α肾上腺素能受体(αAR)和β肾上腺素能受体(βAR),当αAR和βAR被激活时,分别引起磷脂酶C(PLC)/蛋白激酶C(PKC)和腺苷酸环化酶/蛋白激酶A (PKA)信号通路的活化。体外心肌细胞培养和转基因鼠活体研究均证实心脏的肾上腺素能信号转导通路对心肌肥大起重要作用。自1948年Ahlquist等将肾上腺素受体分为αAR和βAR以来,一直认为PKC信号转导通路是由αAR介导的,但Schmidt等的研究表明,环磷酸腺苷(cAMP)可活化一种新近发现的鸟嘌呤交换因子：直接被cAMP激活的交换蛋白(Epac),从而激活PLCs和PKC,提示βAR与PKC之间也可能存在由Epac和PLC介导的信号转导通路。目的：探讨pAR激动剂异丙肾上腺素(Iso)刺激心肌细胞后是否激活PKCs并阐明其机制。探讨Epac和磷脂酶C在其中发挥的作用,及其与细胞外信号调节激酶(ERK)信号转导通路的关系和对心肌肥大的影响。方法：以原代培养的Wistar乳鼠心肌细胞为实验细胞,研究组分别用βAR激动剂Iso (lμmol/L, lmin~30min、Epac激动剂8-CPT (lμmol/L,1Omin)、磷脂酶C抑制剂U73122(2μmo/L,30min)处理细胞。分别用携带具有显性抑制效应(DN)的突变型Epac (Epac R279K, Epac抑制剂)腺病毒、编码兔肌肉cAMP依赖的蛋白激酶抑制剂(Ad.PKI)腺病毒和标记绿色荧光蛋白(GFP)的腺病毒感染心肌细胞,确认感染成功后,给予Iso (lμmol/L, lmin)处理。采用Western blot方法半定量检测心肌细胞颗粒部分的PKCs蛋白,激光共聚焦显微镜观察心肌细胞内PKCs的转位情况。用特异性PKCs转位抑制肽(PKCε inhibitor peptide)和阴性对照肽(PKCε scramble peptide)转染心肌细胞,分别测定经Iso (10μmol/L)孵育48h后心肌细胞表面积和蛋白含量,以及经Iso(lμmol/L,1Omin)处理后磷酸化ERK1/2(pERK1/2)蛋白表达的变化。研究中每项处理均设有严格对照。采用SPSS11.0统计软件进行分析,以P0.05为差异有统计学意义。结果：①刺激pAR引起PKCs活化转位,心肌细胞颗粒部分PKCs在加入Iso孵育1min后开始增加,30min时恢复至基线水平。观察到活化转位的PKCs在心肌细胞内分布于核膜周围,以Iso孵育后1min~15min明显。②加入Iso (lμmol/L, lmin)和Epac激动剂8-CPT孵育细胞后,均引起细胞颗粒部分PKCε增加(P0.05): Iso和8-CPT均引起PKCε转位于细胞核周围,两组PKCs核周染色评分均高于对照组(P均0.05)。③预先用Ad. PKI腺病毒感染心肌细胞,抑制PKA活性后,Iso所致的细胞颗粒部分PKCε增加未受抑制(P0.01),定性和定量观测Ad.PKI也未能抑制Iso引起的PKCε核周转位(P0.01)。④用编码Epac R279K的腺病毒感染心肌细胞,抑制或阻断Epac的作用后再给Iso处理,细胞颗粒部分PKCε没有增加,Iso所致的PKCε活化被阻断。⑤磷脂酶C抑制剂U73122(2μmol/L,30min)与心肌细胞预孵育后,Iso刺激引起的PKCs活化转位消失,细胞颗粒部分PKCε无增加(P0.05), PKCε也未发生核周转位。⑥Iso引起PKCε活化可导致pERK1/2表达增加并诱导心肌细胞肥大。分别用PKCs转位抑制肽和阴性对照肽转染心肌细胞后,再给Iso处理(lμmol/L,1Omin),阴性对照肽组中Iso增加pERK1/2的表达(P0.05): PKCε抑制肽组中Iso未增加pERK1/2表达(P0.05),提示抑制PKCε活化后,Iso所致的pERKl/2表达增加受到抑制。用PKCs阴性对照肽转染心肌细胞后,经Iso处理组心肌细胞表面积和蛋白含量均较对照组增加,对照组和Iso组心肌细胞表面积分别为1319.79±460.00μm2和1874.36±479.52μm2(P0.05),蛋白含量分别为0.64±0.06和0.92±0.11(P0.01)。用PKCε转位抑制肽转染细胞后,经Iso处理组心肌细胞表面积和蛋白含量与对照组比较无增加,对照组和Iso组细胞表面积分别为1268.78±501.63μm2和1604.85±489.88μm2,蛋白含量分别为0.73±0.12和0.62±0.07(P均0.05),提示PKCε抑制肽阻断了Iso诱导的心肌细胞肥大。结论：①刺激心肌细胞βAR引起PKCε活化转位,提示心肌细胞中pAR与PKCε可能存在相互作用。②刺激βAR引起的PKCε活化转位可能不依赖于PKA.③Epac口PLC可能介导了刺激βAR引起的PKCε激活。pAR活化PKCε的途径可能是：激活βAR引起Epac活化,活化的EpaC激活PLC,从而介导PKCε活化转位至胞核周围.④pERK1/2表达增加和心肌细胞肥大是Iso激活PKCε信号转导通路的不良效应之一。βAR活化PKCε可激活ERK信号转导通路,可能参与了诱导心肌细胞肥大。
[Abstract]:BACKGROUND: Myocardial hypertrophy is a common pathological change in many cardiovascular diseases, such as hypertension, coronary heart disease, valvular disease and congenital heart disease. It is a pathological compensatory process of myocardial cells to a variety of adverse stimuli. These signals alone or together activate multiple signal transduction pathways that ultimately contribute to and/or increase myocardial hypertrophy. To clarify the etiology and pathogenesis of myocardial hypertrophy is of great significance for improving prevention and treatment methods, developing effective drugs and reducing mortality and disability. The study of signal transduction mechanism of cardiac hypertrophy has become a hot spot in recent years. Adrenergic signal transduction pathways in the heart include alpha-adrenergic receptor (alpha-AR) and beta-adrenergic receptor (beta-AR). When alpha-AR and beta-AR are activated, phospholipase C (PLC)/protein kinase C (PKC) and adenylate cyclase/protein excitation, respectively. Activation of the enzyme A (PKA) signaling pathway. Cardiac adrenergic signaling pathway plays an important role in myocardial hypertrophy in vitro and in vivo in transgenic mice.
Since Ahlquist et al classified adrenergic receptors into alpha-AR and beta-AR in 1948, it has been thought that the PKC signal transduction pathway is mediated by alpha-AR. However, studies by Schmidt et al have shown that cyclic adenosine monophosphate (cAMP) can activate a recently discovered guanine-exchange factor: the exchange protein (Epac) directly activated by cAMP, thereby activating PLCs and PKC, suggesting that beta-AR signal transduction pathway is mediated by alpha-AR. There may also be signal transduction pathways mediated by Epac and PLC between PKC.
AIM: To investigate whether isoproterenol (Iso), a pAR agonist, activates PKCs after stimulation of cardiomyocytes and elucidate its mechanism.
METHODS: The primary cultured Wistar neonatal rat cardiomyocytes were treated with beta-AR agonist Iso (lUMol/L, lmin~30min), Epac agonist 8-CPT (lUMol/L, 1Omin) and phospholipase C inhibitor U73122 (2UMO/L, 30min), respectively. The cells were treated with mutant Epac (Epac R279K, Epac inhibitor) carrying dominant inhibitory effect (DN) respectively. Adenovirus, adenovirus encoding cAMP-dependent protein kinase inhibitor (Ad.PKI) and adenovirus labelled green fluorescent protein (GFP) were used to infect cardiomyocytes. Iso (l_ micromol/L, lmin) was used to treat the infected cells. The PKCs protein in the granular part of cardiomyocytes was semi-quantitatively detected by Western blot and observed by confocal laser microscopy. To investigate the translocation of PKCs in cardiomyocytes, the myocardial cells were transfected with specific PKCs epsilon inhibitor peptide (PKC epsilon inhibitor peptide) and negative control peptide (PKC epsilon scramble peptide). The surface area and protein content of cardiomyocytes were measured 48 hours after incubation with Iso (10 micromol/L), and the phosphorylated ERK1/2 (pERK1/2) eggs were treated with Iso (1 micromol/L, 1 Omin). The changes of white expression were analyzed with SPSS11.0 statistical software, and the difference was statistically significant with P 0.05.
RESULTS: Activation and translocation of PKCs were induced by stimulation of pAR. Some PKCs in myocardial granules began to increase after incubation with Iso for 1 minute, and returned to baseline level at 30 minutes. Activation and translocation of PKCs were observed in myocardial cells around the nuclear membrane, which was evident at 1 to 15 minutes after incubation with Iso. 2. Incubation with Iso (lUMol/L, lmin) and Epac agonist 8-CPT was fine. Both Iso and 8-CPT induced PKC epsilon translocation around the nucleus, and the perinuclear staining scores of PKCs in both groups were higher than those in the control group (P 0.05). 3. After adenovirus Ad. PKI was used to infect cardiomyocytes in advance, the increase of PKC epsilon induced by Iso was not inhibited (P 0.01). Quantitative observation of Ad.PKI also failed to inhibit PKC epsilon turnover induced by Iso (P 0.01). 4. Cardiac myocytes were infected with adenovirus encoding Epac R279K and treated with Iso after inhibiting or blocking the effect of Epac. Partial PKC epsilon of cell granules did not increase and PKC epsilon activation induced by Iso was blocked. After pre-incubation, the activation and translocation of PKCs induced by Iso stimulation disappeared, the partial PKC epsilon did not increase (P 0.05), and PKC epsilon did not occur._PKC epsilon activation induced by Iso could increase the expression of pERK1/2 and induce cardiomyocyte hypertrophy.After transfected with PKCs inhibitory peptide and negative control peptide respectively, the myocardial cells were treated with Iso (lmicromol/L, 1O control peptide). Iso increased the expression of pERK1/2 in negative control peptide group (P 0.05): Iso did not increase the expression of pERK1/2 in PKC epsilon inhibitory peptide group (P 0.05), suggesting that the increase of pERKl/2 expression induced by Iso was inhibited after inhibiting the activation of PKC epsilon. In addition, the surface area and protein content of myocardial cells in control group and Iso group were 1319.79, 1874.36 6550 The area was 1268.78 [501.63] micron 2 and 1604.85 [489.88] micron 2, respectively, and the protein content was 0.73 [0.12] and 0.62 [0.07], respectively (P 0.05), suggesting that PKC epsilon inhibitory peptide blocked Iso-induced cardiomyocyte hypertrophy.
CONCLUSION: The activation and translocation of PKC epsilon induced by stimulation of beta-AR in cardiomyocytes suggest that there may be interaction between pAR and PKC epsilon in cardiomyocytes. The activation and translocation of PKC epsilon induced by stimulation of beta-AR may not depend on PKA. Activated EpaC activates PLC and thus mediates PKC epsilon activation and translocation to the perinucleus. Increased expression of pERK1/2 and cardiomyocyte hypertrophy are one of the adverse effects of Iso-activated PKC epsilon signal transduction pathway. Activated PKC epsilon by beta-AR may activate ERK signal transduction pathway and may be involved in inducing cardiomyocyte hypertrophy.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

【共引文献】