【摘要】：背景：心肌肥大是高血壓、冠心病、瓣膜病及先心病等多種心血管疾病共同的病理改變,是心肌細胞對多種不良刺激的病理性代償過程。心肌肥大是導致心血管疾病發(fā)病率和病死病殘率顯著升高的獨立危險因素。心肌肥大的發(fā)生發(fā)展涉及多種促心肌肥大信號(如理化刺激、激素水平、組織因子等),這些信號單獨或共同激活多條信號轉導通路,最終促成和/或加重心肌肥大。闡明心肌肥大的病因、發(fā)病機理,對改進防治方法、開發(fā)有效藥物和降低病死病殘率意義重大。尤其對心肌肥大信號轉導機制的研究近年來已成為國內外研究的熱點。心臟的腎上腺素能信號轉導通路包括α腎上腺素能受體(αAR)和β腎上腺素能受體(βAR),當αAR和βAR被激活時,分別引起磷脂酶C(PLC)/蛋白激酶C(PKC)和腺苷酸環(huán)化酶/蛋白激酶A (PKA)信號通路的活化。體外心肌細胞培養(yǎng)和轉基因鼠活體研究均證實心臟的腎上腺素能信號轉導通路對心肌肥大起重要作用。 自1948年Ahlquist等將腎上腺素受體分為αAR和βAR以來,一直認為PKC信號轉導通路是由αAR介導的,但Schmidt等的研究表明,環(huán)磷酸腺苷(cAMP)可活化一種新近發(fā)現(xiàn)的鳥嘌呤交換因子：直接被cAMP激活的交換蛋白(Epac),從而激活PLCs和PKC,提示βAR與PKC之間也可能存在由Epac和PLC介導的信號轉導通路。 目的：探討pAR激動劑異丙腎上腺素(Iso)刺激心肌細胞后是否激活PKCs并闡明其機制。探討Epac和磷脂酶C在其中發(fā)揮的作用,及其與細胞外信號調節(jié)激酶(ERK)信號轉導通路的關系和對心肌肥大的影響。 方法：以原代培養(yǎng)的Wistar乳鼠心肌細胞為實驗細胞,研究組分別用βAR激動劑Iso (lμmol/L, lmin~30min、Epac激動劑8-CPT (lμmol/L,1Omin)、磷脂酶C抑制劑U73122(2μmo/L,30min)處理細胞。分別用攜帶具有顯性抑制效應(DN)的突變型Epac (Epac R279K, Epac抑制劑)腺病毒、編碼兔肌肉cAMP依賴的蛋白激酶抑制劑(Ad.PKI)腺病毒和標記綠色熒光蛋白(GFP)的腺病毒感染心肌細胞,確認感染成功后,給予Iso (lμmol/L, lmin)處理。采用Western blot方法半定量檢測心肌細胞顆粒部分的PKCs蛋白,激光共聚焦顯微鏡觀察心肌細胞內PKCs的轉位情況。用特異性PKCs轉位抑制肽(PKCε inhibitor peptide)和陰性對照肽(PKCε scramble peptide)轉染心肌細胞,分別測定經(jīng)Iso (10μmol/L)孵育48h后心肌細胞表面積和蛋白含量,以及經(jīng)Iso(lμmol/L,1Omin)處理后磷酸化ERK1/2(pERK1/2)蛋白表達的變化。研究中每項處理均設有嚴格對照。采用SPSS11.0統(tǒng)計軟件進行分析,以P0.05為差異有統(tǒng)計學意義。 結果：①刺激pAR引起PKCs活化轉位,心肌細胞顆粒部分PKCs在加入Iso孵育1min后開始增加,30min時恢復至基線水平。觀察到活化轉位的PKCs在心肌細胞內分布于核膜周圍,以Iso孵育后1min~15min明顯。②加入Iso (lμmol/L, lmin)和Epac激動劑8-CPT孵育細胞后,均引起細胞顆粒部分PKCε增加(P0.05): Iso和8-CPT均引起PKCε轉位于細胞核周圍,兩組PKCs核周染色評分均高于對照組(P均0.05)。③預先用Ad. PKI腺病毒感染心肌細胞,抑制PKA活性后,Iso所致的細胞顆粒部分PKCε增加未受抑制(P0.01),定性和定量觀測Ad.PKI也未能抑制Iso引起的PKCε核周轉位(P0.01)。④用編碼Epac R279K的腺病毒感染心肌細胞,抑制或阻斷Epac的作用后再給Iso處理,細胞顆粒部分PKCε沒有增加,Iso所致的PKCε活化被阻斷。⑤磷脂酶C抑制劑U73122(2μmol/L,30min)與心肌細胞預孵育后,Iso刺激引起的PKCs活化轉位消失,細胞顆粒部分PKCε無增加(P0.05), PKCε也未發(fā)生核周轉位。⑥Iso引起PKCε活化可導致pERK1/2表達增加并誘導心肌細胞肥大。分別用PKCs轉位抑制肽和陰性對照肽轉染心肌細胞后,再給Iso處理(lμmol/L,1Omin),陰性對照肽組中Iso增加pERK1/2的表達(P0.05): PKCε抑制肽組中Iso未增加pERK1/2表達(P0.05),提示抑制PKCε活化后,Iso所致的pERKl/2表達增加受到抑制。用PKCs陰性對照肽轉染心肌細胞后,經(jīng)Iso處理組心肌細胞表面積和蛋白含量均較對照組增加,對照組和Iso組心肌細胞表面積分別為1319.79±460.00μm2和1874.36±479.52μm2(P0.05),蛋白含量分別為0.64±0.06和0.92±0.11(P0.01)。用PKCε轉位抑制肽轉染細胞后,經(jīng)Iso處理組心肌細胞表面積和蛋白含量與對照組比較無增加,對照組和Iso組細胞表面積分別為1268.78±501.63μm2和1604.85±489.88μm2,蛋白含量分別為0.73±0.12和0.62±0.07(P均0.05),提示PKCε抑制肽阻斷了Iso誘導的心肌細胞肥大。 結論：①刺激心肌細胞βAR引起PKCε活化轉位,提示心肌細胞中pAR與PKCε可能存在相互作用。②刺激βAR引起的PKCε活化轉位可能不依賴于PKA.③Epac口PLC可能介導了刺激βAR引起的PKCε激活。pAR活化PKCε的途徑可能是：激活βAR引起Epac活化,活化的EpaC激活PLC,從而介導PKCε活化轉位至胞核周圍.④pERK1/2表達增加和心肌細胞肥大是Iso激活PKCε信號轉導通路的不良效應之一。βAR活化PKCε可激活ERK信號轉導通路,可能參與了誘導心肌細胞肥大。
[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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

【共引文獻】

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