过表达肌浆网钙ATP酶2a对心肌细胞PI3K/Akt信号通路短期作用的研究
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摘要
心力衰竭是各种心脏疾病发展的最终归宿,是导致患者死亡的主要原因。目前针对慢性心衰的治疗已有长足发展,但其五年死亡率仍居高不下。分子生物学研究提示心力衰竭心肌细胞中存在某些相关基因表达和调控异常,因此心衰被认为是一种多基因突变疾病。基因治疗为心力衰竭提供了新的治疗途径。肌浆网Ca2+转运障碍是各种心力衰竭的共同通路,肌浆网钙ATP酶2a (sarcoplasmic reticulum calcium ATPase 2a, SERCA2a)是调节细胞内Ca2+稳态的决定因素。既往研究表明,转基因过表达SERCA2a可以改善心力衰竭心肌细胞的功能、纠正Ca2+稳态失衡、延长存活时间和逆转心室肥厚等。为了更好的评价这种治疗方法,就必须探讨其保护心肌的分子机制。本实验室前期的研究工作显示转基因过表达SERCA2a可使心肌细胞内与提供能量有关的酶类表达增加,并纠正MHC亚型的异常表达。其他研究小组发现转基因过表达SERCA2a可以纠正心力衰竭时蛋白激酶B(Akt1)的异常表达。
蒽环类抗生素广泛应用于各种恶性肿瘤的治疗,但其存在严重的心脏毒性。心力衰竭是此类药物心脏毒性的最终表现。研究显示,蒽环类药物影响心肌细胞SERCA2a蛋白的表达和功能,SERCA2a功能改变是此类药物导致心脏毒性的关键因素。另外蒽环类药物诱发心肌毒性与心肌细胞内Akt磷酸化水平降低有关。大量事实证明,多种心肌保护因子通过激活Akt通路抑制蒽环类药物的心肌毒性。
Akt是一种蛋白质丝／苏氨酸激酶,心肌细胞主要表达Akt1亚型。胰岛素等各种生长因子通过磷酯酰肌醇3-激酶(Phosphatidylinositol 3-kinase, PI3K)激活Akt,Akt被激活后广泛参与调节心肌细胞生长、能量代谢和抗调亡等过程。Akt是细胞内信号传导通路网络的中心分子,短期适度激活Akt对心肌细胞有保护作用,而大量长期激活会促进心力衰竭的发生。转基因过表达SERCA2a可以改善心力衰竭动物的长期预后,而过表达SERCA2a可以纠正心衰心肌细胞Akt1的异常表达,因此我们推测过表达SERCA2a的长期有益作用可能与纠正Akt1通路的异常有关。
本研究在体外培养的H9c2心肌细胞系和原代心肌细胞,采用重组腺病毒转基因瞬时过表达SERCA2a,观察过表达SERCA2a对正常心肌细胞PI3K/Akt信号通路的作用,并探讨此通路下游分子GSK3β和GLUT4活性改变情况,明确在正常心肌细胞过表达SERCA2a是否可以激活PI3K/Akt通路。首先,本研究采用HEK-293T细胞扩增重组腺病毒Ad-GFP和Ad-SERCA2a,并验证扩增后的腺病毒在体外可以有效感染目的细胞。利用Ad-GFP感染H9c2心肌细胞和MTT实验观察病毒感染对心肌细胞损伤程度,确定最佳感染复数(MOI)值,为后期实验奠定基础。其次,通过在体外培养的H9c2心肌细胞系和原代心肌细胞内转基因过表达SERCA2a,观察对Akt通路的影响。结果显示,在体外培养的H9c2心肌细胞过表达SERCA2a可以激活Akt,Akt磷酸化水平升高,是对照组的2.7倍(P<0.05)；其下游底物GSK3β磷酸化水平升高,是对照组的3.1倍(P<0.05); GLUT4蛋白表达无明显变化,但通过免疫荧光染色发现有GLUT4由细胞浆移位至细胞膜的现象。进一步细胞膜蛋白分析显示,过表达SERCA2a组细胞膜GLUT4蛋白量增加,是对照组的2.1倍(P<0.05)。在原代心肌细胞过表达SERCA2a也可以激活Akt通路,Akt磷酸化水平升高,是对照组的2.1倍(P<0.05)；其下游底物GSK3β磷酸化水平升高,是对照组的1.6倍(P<0.05)；采用PI3K抑制剂LY294002可以阻断以上蛋白磷酸化水平改变,提示过表达SERCA2a通过PI3K激活Akt通路。最后,采用阿霉素诱导心肌细胞坏死,观察过表达SERCA2a对阿霉素诱导心肌损伤的影响。结果显示,阿霉素诱导心肌细胞坏死时,细胞LDH漏出量增加,细胞内Ca2+离子水平升高,SERCA2a蛋白表达降低。过表达SERCA2a可以抑制阿霉素诱导的心肌损伤,表现为心肌细胞死亡率降低,心肌细胞活性增加(P<0.05),心肌细胞内Ca2+离子水平降低(P<0.05),同时伴有Akt信号通路的激活。LY294002仅能部分阻断SERCA2a对阿霉素诱导心肌损伤的保护作用,提示过表达SERCA2a抑制阿霉素诱导的心肌损伤除激活PI3K/Akt信号通路发挥作用外,可能还存在其他的保护机制。
Heart failure is the end stage of various cardiac diseases and is a major cause of mortality. Although progress in conventional treatment has improved survival, its 5-year mortality rate is still high. Molecular biology studies have shown that heart failure is a multi-gene disease. Gene therapy for heart failure provides a new therapeutic approach. Sarcoplasmic reticulum Ca2+ transport impairment is a common pathway for varieties of heart failure. Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) plays a key role in controlling intracellular Ca2+ handling. Previous studies showed that transgenic overexpression of SERCA2a was able to improve the function of myocardial cells of heart failure, correct the abnormal of Ca2+ homeostasis, extend survival time and reverse ventricular hypertrophy and so on. In order to better evaluate the treatment, to explore the molecular mechanisms of the treatment is necessary. Preliminary work in our laboratory shows that transgenic overexpression of SERCA2a can not only increase the expression of related enzymes that provide energy for the myocardial cells, but also correct the abnormal expression of MHC isoforms. In addition, other studies indicate transgenic overexpression of SERCA2a can correct myocardial protein kinase B (Akt1) abnormal expression in heart failure.
Anthracycline antibiotics are widely used in the treatment of various malignancies, but they have serious cardiac toxicity. Heart failure is the final performance cardiac toxicity. It was investigated that the drugs impacted on the expression and function of myocardial SERCA2a protein and the change of SERCA2a function of these drugs is a key factor in cardiac toxicity. Anthracycline-induced cardiotoxicity is related to the decrease of Akt phosphorylation. Many facts have proven that a variety of myocardial protection factors through activating Akt pathway restrain the anthracycline cardiac toxicity.
Akt is a protein serine/threonine kinase and Aktl is mainly in myocardial cell. Insulin and other growth factors activate Akt via PI3K. Akt activated extensively involved in the regulation of myocardial cell growth, the energy metabolism and resistance to apoptosis and other processes. Akt is the central elements within the cellular signal transduction networks, and the short-term moderate activated Akt has a protective effect on myocardial cells, while long-term activated one may promote heart failure. Transgenic overexpression of SERCA2a can improve the long-term prognosis in animal models of heart failure, while overexpression of SERCA2a can correct the abnormal expression of Aktl in myocardial cell of heart failure, we speculated that long-term beneficial effects of overexpression of SERCA2a may correct the abnormalities of Aktl pathway. In this study, the growth of cardiac cells is in vitro culture, and observed in normal cardiomyocytes PI3K/Akt signaling pathway effects by using recombinant adenovirus gene transient overexpression of SERCA2a. Morever, whether the downstream of GSK3βand GLUT4 molecular being activated is also investigated. First, HEK-293T cells were used to amplification of recombinant adenovirus Ad-GFP and Ad-SERCA2a, and verified that the amplified adenovirus can infect target cells effectively in vitro. Myocardial cell injury is observed by using Ad-GFP infected cardiomyocytes and MTT expremental to determine the optimal MOI in order to provide the basic material for the later experiments. Second, affected Akt pathway is observed througn overexpressing SERCA2a in cultured H9c2 myocardial cell lines and primary myocardial cells. The results showed that overexpression of SERCA2a in vitro can active Akt signaling pathway and result in the elevation of Akt phosphorylation level, which is 2.7 times as high as control group (P<0.05). Then the phosphorylation of downstream substrate GSK3βis also higher and it is 3.1 times as high as control (P<0.05). Although GLUT4 protein expression has on significant changes, the shift from the cytoplasm translocation to the plasm membrane was observed. Further analysis indicated membrane GLUT4 proteins increased in the groups of SERCA2a overexpression, which is 2.1 times as high as control (P<0.05). Overexpression of SERCA2a in primary myocardial cell also can active Akt signaling pathway and result in the elevation of Akt phosphorylation level, which is 2.1 times as high as control group (P<0.05). Then the phosphorylation of downstream substrate GSK3βis also higher and it is 1.6 times as high as control group (P<0.05). PI3K inhibitor (LY294002) blocked the phosphorylation level of the above changes, suggesting that SERCA2a activated Akt through PI3K signaling pathway. Finally, the use of adriamycin induced myocardial injury, the overexpression of SERCA2a observed decreased on the cardiac toxicity of doxorubicin. The results showed that Adriamycin induced myocardial necrosis, increase of cells LDH leakage and intracellular Ca2+ level and decrease of SERCA2a protein expression. Overexpression of SERCA2a could inhibit adriamycin-induced myocardial injury, manifested as myocardial cell death and increased cell survival of myocardial cells (P<0.05), myocardial cells reduce the level of Ca2+ level (P<0.05), accompanied by the activation of Akt signaling pathway. LY294002 only partially blocked by SERCA2a overexpression on adriamycin induced myocardial injury, suggesting that overexpression of SERCA2a inhibition of adriamycin induced myocardial injury via activation of PI3K/Akt signaling pathway, might have other protection mechanisms.
蒽环类抗生素广泛应用于各种恶性肿瘤的治疗,但其存在严重的心脏毒性。心力衰竭是此类药物心脏毒性的最终表现。研究显示,蒽环类药物影响心肌细胞SERCA2a蛋白的表达和功能,SERCA2a功能改变是此类药物导致心脏毒性的关键因素。另外蒽环类药物诱发心肌毒性与心肌细胞内Akt磷酸化水平降低有关。大量事实证明,多种心肌保护因子通过激活Akt通路抑制蒽环类药物的心肌毒性。
Akt是一种蛋白质丝／苏氨酸激酶,心肌细胞主要表达Akt1亚型。胰岛素等各种生长因子通过磷酯酰肌醇3-激酶(Phosphatidylinositol 3-kinase, PI3K)激活Akt,Akt被激活后广泛参与调节心肌细胞生长、能量代谢和抗调亡等过程。Akt是细胞内信号传导通路网络的中心分子,短期适度激活Akt对心肌细胞有保护作用,而大量长期激活会促进心力衰竭的发生。转基因过表达SERCA2a可以改善心力衰竭动物的长期预后,而过表达SERCA2a可以纠正心衰心肌细胞Akt1的异常表达,因此我们推测过表达SERCA2a的长期有益作用可能与纠正Akt1通路的异常有关。
本研究在体外培养的H9c2心肌细胞系和原代心肌细胞,采用重组腺病毒转基因瞬时过表达SERCA2a,观察过表达SERCA2a对正常心肌细胞PI3K/Akt信号通路的作用,并探讨此通路下游分子GSK3β和GLUT4活性改变情况,明确在正常心肌细胞过表达SERCA2a是否可以激活PI3K/Akt通路。首先,本研究采用HEK-293T细胞扩增重组腺病毒Ad-GFP和Ad-SERCA2a,并验证扩增后的腺病毒在体外可以有效感染目的细胞。利用Ad-GFP感染H9c2心肌细胞和MTT实验观察病毒感染对心肌细胞损伤程度,确定最佳感染复数(MOI)值,为后期实验奠定基础。其次,通过在体外培养的H9c2心肌细胞系和原代心肌细胞内转基因过表达SERCA2a,观察对Akt通路的影响。结果显示,在体外培养的H9c2心肌细胞过表达SERCA2a可以激活Akt,Akt磷酸化水平升高,是对照组的2.7倍(P<0.05)；其下游底物GSK3β磷酸化水平升高,是对照组的3.1倍(P<0.05); GLUT4蛋白表达无明显变化,但通过免疫荧光染色发现有GLUT4由细胞浆移位至细胞膜的现象。进一步细胞膜蛋白分析显示,过表达SERCA2a组细胞膜GLUT4蛋白量增加,是对照组的2.1倍(P<0.05)。在原代心肌细胞过表达SERCA2a也可以激活Akt通路,Akt磷酸化水平升高,是对照组的2.1倍(P<0.05)；其下游底物GSK3β磷酸化水平升高,是对照组的1.6倍(P<0.05)；采用PI3K抑制剂LY294002可以阻断以上蛋白磷酸化水平改变,提示过表达SERCA2a通过PI3K激活Akt通路。最后,采用阿霉素诱导心肌细胞坏死,观察过表达SERCA2a对阿霉素诱导心肌损伤的影响。结果显示,阿霉素诱导心肌细胞坏死时,细胞LDH漏出量增加,细胞内Ca2+离子水平升高,SERCA2a蛋白表达降低。过表达SERCA2a可以抑制阿霉素诱导的心肌损伤,表现为心肌细胞死亡率降低,心肌细胞活性增加(P<0.05),心肌细胞内Ca2+离子水平降低(P<0.05),同时伴有Akt信号通路的激活。LY294002仅能部分阻断SERCA2a对阿霉素诱导心肌损伤的保护作用,提示过表达SERCA2a抑制阿霉素诱导的心肌损伤除激活PI3K/Akt信号通路发挥作用外,可能还存在其他的保护机制。
Heart failure is the end stage of various cardiac diseases and is a major cause of mortality. Although progress in conventional treatment has improved survival, its 5-year mortality rate is still high. Molecular biology studies have shown that heart failure is a multi-gene disease. Gene therapy for heart failure provides a new therapeutic approach. Sarcoplasmic reticulum Ca2+ transport impairment is a common pathway for varieties of heart failure. Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) plays a key role in controlling intracellular Ca2+ handling. Previous studies showed that transgenic overexpression of SERCA2a was able to improve the function of myocardial cells of heart failure, correct the abnormal of Ca2+ homeostasis, extend survival time and reverse ventricular hypertrophy and so on. In order to better evaluate the treatment, to explore the molecular mechanisms of the treatment is necessary. Preliminary work in our laboratory shows that transgenic overexpression of SERCA2a can not only increase the expression of related enzymes that provide energy for the myocardial cells, but also correct the abnormal expression of MHC isoforms. In addition, other studies indicate transgenic overexpression of SERCA2a can correct myocardial protein kinase B (Akt1) abnormal expression in heart failure.
Anthracycline antibiotics are widely used in the treatment of various malignancies, but they have serious cardiac toxicity. Heart failure is the final performance cardiac toxicity. It was investigated that the drugs impacted on the expression and function of myocardial SERCA2a protein and the change of SERCA2a function of these drugs is a key factor in cardiac toxicity. Anthracycline-induced cardiotoxicity is related to the decrease of Akt phosphorylation. Many facts have proven that a variety of myocardial protection factors through activating Akt pathway restrain the anthracycline cardiac toxicity.
Akt is a protein serine/threonine kinase and Aktl is mainly in myocardial cell. Insulin and other growth factors activate Akt via PI3K. Akt activated extensively involved in the regulation of myocardial cell growth, the energy metabolism and resistance to apoptosis and other processes. Akt is the central elements within the cellular signal transduction networks, and the short-term moderate activated Akt has a protective effect on myocardial cells, while long-term activated one may promote heart failure. Transgenic overexpression of SERCA2a can improve the long-term prognosis in animal models of heart failure, while overexpression of SERCA2a can correct the abnormal expression of Aktl in myocardial cell of heart failure, we speculated that long-term beneficial effects of overexpression of SERCA2a may correct the abnormalities of Aktl pathway. In this study, the growth of cardiac cells is in vitro culture, and observed in normal cardiomyocytes PI3K/Akt signaling pathway effects by using recombinant adenovirus gene transient overexpression of SERCA2a. Morever, whether the downstream of GSK3βand GLUT4 molecular being activated is also investigated. First, HEK-293T cells were used to amplification of recombinant adenovirus Ad-GFP and Ad-SERCA2a, and verified that the amplified adenovirus can infect target cells effectively in vitro. Myocardial cell injury is observed by using Ad-GFP infected cardiomyocytes and MTT expremental to determine the optimal MOI in order to provide the basic material for the later experiments. Second, affected Akt pathway is observed througn overexpressing SERCA2a in cultured H9c2 myocardial cell lines and primary myocardial cells. The results showed that overexpression of SERCA2a in vitro can active Akt signaling pathway and result in the elevation of Akt phosphorylation level, which is 2.7 times as high as control group (P<0.05). Then the phosphorylation of downstream substrate GSK3βis also higher and it is 3.1 times as high as control (P<0.05). Although GLUT4 protein expression has on significant changes, the shift from the cytoplasm translocation to the plasm membrane was observed. Further analysis indicated membrane GLUT4 proteins increased in the groups of SERCA2a overexpression, which is 2.1 times as high as control (P<0.05). Overexpression of SERCA2a in primary myocardial cell also can active Akt signaling pathway and result in the elevation of Akt phosphorylation level, which is 2.1 times as high as control group (P<0.05). Then the phosphorylation of downstream substrate GSK3βis also higher and it is 1.6 times as high as control group (P<0.05). PI3K inhibitor (LY294002) blocked the phosphorylation level of the above changes, suggesting that SERCA2a activated Akt through PI3K signaling pathway. Finally, the use of adriamycin induced myocardial injury, the overexpression of SERCA2a observed decreased on the cardiac toxicity of doxorubicin. The results showed that Adriamycin induced myocardial necrosis, increase of cells LDH leakage and intracellular Ca2+ level and decrease of SERCA2a protein expression. Overexpression of SERCA2a could inhibit adriamycin-induced myocardial injury, manifested as myocardial cell death and increased cell survival of myocardial cells (P<0.05), myocardial cells reduce the level of Ca2+ level (P<0.05), accompanied by the activation of Akt signaling pathway. LY294002 only partially blocked by SERCA2a overexpression on adriamycin induced myocardial injury, suggesting that overexpression of SERCA2a inhibition of adriamycin induced myocardial injury via activation of PI3K/Akt signaling pathway, might have other protection mechanisms.
引文
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