Gsα基因干扰对心力衰竭幼鼠心肌钙调控蛋白的作用研究
|
摘要
研究背景正常情况下,交感神经递质激活β-肾上腺素能受体(β-adrenoreceptor,β-AR)后,后者与Gs蛋白结合,Gs蛋白异源三聚体α、βγ解离,Gsα活化,β-AR- Gs蛋白-cAMP信号通路激活,引起L型Ca2+离子通道(L-type Ca2+ channel , LTCC)、受磷蛋白(phospholamban,PLB)、兰尼碱受体(ryanodine receptor2,RyR2)、肌钙蛋白(cTnI)等磷酸化,并通过与其附属蛋白相互作用,如FK506结合蛋白(FK506 binding protein,FKBP12.6 )、肌浆网Ca2+-ATP酶(sarcoplasmic reticulum Ca2+ ATPase,SERCA2α)等,调控钙的摄取与释放,诱发心肌兴奋收缩耦联。可见, Gsα作为该信号通路上游的关键偶联因子,其量和活性在维持正常心功能状态中具有重要意义。
慢性心力衰竭(heart failure, HF)时,交感神经系统、肾素-血管紧张素系统等活性增高,Gsα蛋白活性和(或)含量下降,β-AR信号通路下调,这些改变究竟是引起心功能恶化的始动因素,还是一种适应性的保护机制,目前仍未完全明了。因心衰时其下游的钙调控蛋白的量、活性与结构亦出现相应变化,鉴于钙离子稳态对维持心肌细胞正常功能至关重要,钙离子的转运调控异常,可导致心肌细胞兴奋收缩耦联功能障碍,进而影响收缩-舒张功能,并可诱发严重心律失常,因此探索心衰时Gsα变化对钙调控蛋白和心功能的影响具有重要理论和临床价值。其次目前对心衰时这一信号通路的研究所用实验动物多为成年动物,至于在幼年动物体内是否具有不同的表现目前仍不清楚。本实验拟通过构建Gsα蛋白基因干扰的慢性心衰幼鼠模型,探讨Gsα蛋白变化对心衰幼鼠心肌钙调控蛋白RyR2、FKBP12.6、PLB、SERCA2α的影响,探讨Gsα变化在心衰发生发展中的意义。实验共分三部分:
第一部分靶向大鼠gnas基因shRNA真核表达载体的构建与鉴定
目的构建并鉴定靶向大鼠gnas基因的短发夹RNA(short hairpin RNA ,shRNA)真核表达载体。
方法根据大鼠gnas基因mRNA序列设计并合成3条shRNA,退火形成双链后克隆进入线性化pGenesil-1.1载体,并进行酶切鉴定和测序,鉴定构建成功后进行后续的体外和体内实验。
结果经酶切和测序鉴定分析,构建的shRNA已成功插入载体,并且与设计序列完全相符。
结论成功构建了shRNA表达质粒载体pgnas-shRNA1、pgnas-shRNA2和pgnas-shRNA3,扩增和纯化获得理想的质粒产品,为后续研究Gsα蛋白在心衰中的作用奠定了实验基础。
第二部分RNAi选择性下调大鼠心肌细胞Gsα蛋白的体外实验
目的用RNA干扰技术(RNA interference, RNAi)选择性下调大鼠心肌细胞上Gsα蛋白的表达,筛选出抑制Gsα蛋白效果最明显的shRNA表达质粒。
方法将上述三种构建成功的RNA干扰质粒或对照质粒,通过脂质体Lipofectamine? LTX & PLUS转染原代培养的乳鼠心肌细胞,并设空白对照组,转染后通过半定量RT-PCR和Western-blot法检测GsαmRNA和蛋白的表达情况,以内参照三磷酸甘油醛脱氢酶(GAPDH)进行标化。
结果通过Lipofectamine? LTX & PLUS进行质粒转染,细胞毒性作用相对较小。shRNA1-3均使Gsα的mRNA和蛋白表达明显降低,差异有统计学意义(P<0.01),且shRNA3的抑制效果最显著,阴性对照质粒组与空白对照组Gsα表达差异无统计学意义。
结论利用RNAi技术成功下调了原代培养的乳鼠心肌细胞中Gsα的表达,筛选出沉默效果最明显的shRNA3真核表达质粒,为下一步体内实验奠定了基础。
第三部分Gsα基因干扰对心力衰竭幼鼠心肌钙调节蛋白的作用
目的构建Gsα蛋白基因干扰的慢性心衰幼鼠模型,阐明Gsα蛋白变化对心衰幼鼠心肌钙调控蛋白RyR2、FKBP12.6、PLB、SERCA2α的影响,探讨Gsα变化在心衰发生发展中的意义。
方法健康雄性4周龄Wistar幼鼠,随机分三组,彩色超声心动图测定后,(1)Gsa基因干扰心衰组( n=13),通过经腹心包腔内注射术将上述体外实验所筛选出来的基因沉默效果较好的质粒pgnas-shRNA3混合液,注入心包腔,并进一步通过腹主动脉和下腔静脉造瘘法构建慢性心衰模型,(2)正常鼠构建的心衰模型组(n=11),心包腔注射等量无菌PBS,并进一步通过腹主动脉和下腔静脉造瘘法构建慢性心衰模型,3)假手术组(n=9),心包腔注射等量的无菌PBS,只穿刺腹主动脉不与下腔静脉间造瘘。8周后复查超声心动图,然后,麻醉取心脏标本,进行组织病理学检查,RT-PCR、Western-blot检测心肌钙调控蛋白表达。
结果8周后彩色多普勒超声心动图对三组进行相关测定,与假手术组相比,两心衰组的多项心功能指标均有不同程度的下降,RyR2、FKBP12.6、SERCA2α表达降低,SERCA2a活性降低。Gsα基因干扰心衰组与正常鼠构建的心衰组相比,基因干扰心衰组的左室收缩末期容积(Left ventricular end-systolic volume, LVESV)下降,差异有统计学意义(P<0.05),左室射血分数(Left ventricular ejection fraction,LVEF)、左室短轴缩短率(Left ventricular fraction shortening,LVFS)升高,差异有统计学意义(P<0.05),左室舒张末期内径(Left ventricular internal dimension diastole,LVIDd )、左室收缩末期内径(Left ventricular internal dimension systole, LVIDs)、左室舒张末期容积( Left ventricular end-diastolic volume,LVEDV)略有降低,但差异无统计学意义(P>0.05);心肌钙调控蛋白RyR2、FKBP12.6表达升高,且有统计学意义(P<0.01),两者比值也升高;PLB基因与蛋白表达在各组间无显著差异(P>0.05),Gsα基因干扰心衰组与正常鼠构建的心衰组比,SERCA2a含量、活性增高,并具有统计学意义(P<0.01、P<0.05),SERCA2a与PLB比值亦升高。
结论体内试验表明Gsα基因干扰心衰模型,与正常鼠构建的心衰模型相比,心肌钙调控蛋白表达升高,心功能的多项参数有改善趋势,推测心衰早期Gsα的低表达可能是一种保护机制。
Background Under normal physiological conditions, activation ofβ-adrenoreceptor(β-AR) by the sympathetic nervous system stimulate Gs proteins, and then Gsαdissociation from Gβγ, the classic Gs/adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) cascade activate , which leads to phosphorylation of various calcium regulatory proteins within the myocyte such as L-type Ca2+ channel(LTCC)、ryanodine receptor 2(RyR2)、phospholamban(PLB)、and regulate the expression of FK506 binding protein(FKBP12.6), sarcoplasmic reticulum Ca2+ ATPase(SERCA2a) et al. Theβ-AR/Gs/AC pathway is the most powerful physiological mechanism to accurately augment cardiac contractility, So Gsαproteins may be play an important roles in this system signaling.
Heart failure is a state characterized by enhanced sympathetic tone, theβ-AR/Gs/AC pathway exhibits marked alterations: down regulation or desensitization inβ-AR, and an impairment of the coupling ofβ-AR to Gs, the content and (or) activation of Gsαhas changed too, downstream mechanisms (calcium regulatory proteins) are altered. What is role of the changes of Gsαin heart failure is still unknown, is that a main factor causing further deterioration of heart function or adaptive mechanism? Moreover, previous studies in this signaling pathway mainly focused on adult animals, whether there are different manifestations in junior animals is still unclear.
So this study is to knockdown of Gsαgene by RNAi in junior rat and to investigate the effects of changes of Gsαin heart failure,, and the influence of Gsαon downstream mechanisms(calcium regulatory proteins, such as RyR2、FKBP12.6、PLB、SERCA2a ) .
Part 1 Construction and identification of eukaryotic expression plasmids containing short hairpin RNA targeting to gnas gene
Objective To construct eukaryotic expression plasmids containing short hairpin RNA (shRNA) that target to rat gnas gene, which encodes the a-subunit of the Gs protein (Gsα).
Methods Three pairs of shRNAs that target to gnas gene were designed. After annealed, the inserts were ligated into the linearized pGenesil-1.1 plasmids. The eukaryotic expression plasmids were constructed and identified using restriction enzyme analysis and sequencing analysis. And targeting no-isogeny gene was served as a negative control.
Results PGenesil-1.1 plasmids were confirmed by restriction enzyme analysis and sequencing analysis in accordance with design requiring.
Conclusion We successfully constructed 3 recombinant plasmids pGenesil-1.1-shRNA targeted to gnas gene, and it’s helpful for further research on the role of Gsαin heart failure by RNA interference (RNAi) technique.
Part 2 Selective knockdown of Gsαin primary cultured rat myocytes by RNAi in vitro
Objective To selectively knockdown the expression of Gsαprotein in rat myocytes by RNA interference (RNAi),and to select the plasmids having the best effect on inhibiting Gsα.
Method Eukaryotic expression plasmids containing shRNA targeting to gnas gene were constructed, then plasmids were transfected into primary cultured rat myocytes via liposome Lipofectamine? LTX & PLUS , there are plasmids carrying a nonspecific shRNA coding sequence(HK plasmids) as the negative control group and the blank control group involved. The mRNA and protein expressions of Gsαwere analyzed by RT-PCR and Western blot respectively, and were normalized to internal control gene GAPDH.
Results The expressions of mRNA and protein of Gsαof test groups were markedly decreased than that of control groups (P<0.01), and the shRNA3 has the mostly inhibitory effect. While no significant difference was found in control groups.
Conclusion RNAi can selectively knockdown Gsαexpression in primary cultured myocytes , and laid a foundation for future studies on Gsαin vivo.
Part 3 Effect of Gsαgene knockdown by RNAi on calcium regulatory proteins in junior rat with heart failure
Objective To knockdown of Gsαgene by RNAi in junior rats and investigate the effects of Gsαgene on on calcium regulatory proteins in rats with heart failure.
Methods Male Wistar rats, four weeks old, were randomly divided into 3 groups, measured by the high frequency ultrasound. (1)HF group which Gsαknockdown (n=13), a mixture test plasmids shRNA3 (diluted in sterile PBS) was injected transdiaphragmatically into the hearts, and then established the animal model of chronic heart failure by fistulation of abdominal aorta and inferior vena cave. (2) HF group (n=11), Equivalent of sterile PBS was injected into the hearts, and then established the animal model of chronic heart failure by the same approach. (3)Sham group (n=9). 8 weeks after the operation, the test rats were used to test the following procedures: measurement of cardiac dimensions and ejection fraction with echocardiogram, determination of proteins expression level of RyR2、FKBP12.6、PLB、SERCA2αin cardiac tissue with Western-blot and RT-PCR .
Results The echocardiogram showed that, compared with those of Sham group, many parameters of cardiac function decreased,and the expression level of RyR2, FKBP12.6 and SERCA2αalso decreased in the two HF groups, which have significant differences(P<0.01).While the expression level of PLB had no significant difference in these 3 groups (P>0.05). Compare with HF group, the Left ventricular end-systolic volume (LVESV) decreased(P<0.05), Left ventricular fraction shortening (LVFS)、Left ventricular ejection fraction(LVEF)increased (P<0.05), and Left ventricular internal dimension diastole (LVIDd )、Left ventricular internal dimension systole(LVIDs)、left ventricular end-diastolic volume (LVEDV) decreased, while had no statistically significant difference(P>0.05), the expression of RyR2、FKBP12.6、SERCA2αincreased(P<0.01), and FKBP12.6/ RyR2、SERCA2α/PLB increased in HF group which Gsa knockdown.
Conclusion Compare with HF group, the HF group which Gsa knockdown has improvement trends in cardiac function index and relatively high-level expression of calcium regulatory proteins. Our study suggests that the alterations of Gsαmay play an adaptive reaction to the increased sympathetic nervous system activity in heart failure.
慢性心力衰竭(heart failure, HF)时,交感神经系统、肾素-血管紧张素系统等活性增高,Gsα蛋白活性和(或)含量下降,β-AR信号通路下调,这些改变究竟是引起心功能恶化的始动因素,还是一种适应性的保护机制,目前仍未完全明了。因心衰时其下游的钙调控蛋白的量、活性与结构亦出现相应变化,鉴于钙离子稳态对维持心肌细胞正常功能至关重要,钙离子的转运调控异常,可导致心肌细胞兴奋收缩耦联功能障碍,进而影响收缩-舒张功能,并可诱发严重心律失常,因此探索心衰时Gsα变化对钙调控蛋白和心功能的影响具有重要理论和临床价值。其次目前对心衰时这一信号通路的研究所用实验动物多为成年动物,至于在幼年动物体内是否具有不同的表现目前仍不清楚。本实验拟通过构建Gsα蛋白基因干扰的慢性心衰幼鼠模型,探讨Gsα蛋白变化对心衰幼鼠心肌钙调控蛋白RyR2、FKBP12.6、PLB、SERCA2α的影响,探讨Gsα变化在心衰发生发展中的意义。实验共分三部分:
第一部分靶向大鼠gnas基因shRNA真核表达载体的构建与鉴定
目的构建并鉴定靶向大鼠gnas基因的短发夹RNA(short hairpin RNA ,shRNA)真核表达载体。
方法根据大鼠gnas基因mRNA序列设计并合成3条shRNA,退火形成双链后克隆进入线性化pGenesil-1.1载体,并进行酶切鉴定和测序,鉴定构建成功后进行后续的体外和体内实验。
结果经酶切和测序鉴定分析,构建的shRNA已成功插入载体,并且与设计序列完全相符。
结论成功构建了shRNA表达质粒载体pgnas-shRNA1、pgnas-shRNA2和pgnas-shRNA3,扩增和纯化获得理想的质粒产品,为后续研究Gsα蛋白在心衰中的作用奠定了实验基础。
第二部分RNAi选择性下调大鼠心肌细胞Gsα蛋白的体外实验
目的用RNA干扰技术(RNA interference, RNAi)选择性下调大鼠心肌细胞上Gsα蛋白的表达,筛选出抑制Gsα蛋白效果最明显的shRNA表达质粒。
方法将上述三种构建成功的RNA干扰质粒或对照质粒,通过脂质体Lipofectamine? LTX & PLUS转染原代培养的乳鼠心肌细胞,并设空白对照组,转染后通过半定量RT-PCR和Western-blot法检测GsαmRNA和蛋白的表达情况,以内参照三磷酸甘油醛脱氢酶(GAPDH)进行标化。
结果通过Lipofectamine? LTX & PLUS进行质粒转染,细胞毒性作用相对较小。shRNA1-3均使Gsα的mRNA和蛋白表达明显降低,差异有统计学意义(P<0.01),且shRNA3的抑制效果最显著,阴性对照质粒组与空白对照组Gsα表达差异无统计学意义。
结论利用RNAi技术成功下调了原代培养的乳鼠心肌细胞中Gsα的表达,筛选出沉默效果最明显的shRNA3真核表达质粒,为下一步体内实验奠定了基础。
第三部分Gsα基因干扰对心力衰竭幼鼠心肌钙调节蛋白的作用
目的构建Gsα蛋白基因干扰的慢性心衰幼鼠模型,阐明Gsα蛋白变化对心衰幼鼠心肌钙调控蛋白RyR2、FKBP12.6、PLB、SERCA2α的影响,探讨Gsα变化在心衰发生发展中的意义。
方法健康雄性4周龄Wistar幼鼠,随机分三组,彩色超声心动图测定后,(1)Gsa基因干扰心衰组( n=13),通过经腹心包腔内注射术将上述体外实验所筛选出来的基因沉默效果较好的质粒pgnas-shRNA3混合液,注入心包腔,并进一步通过腹主动脉和下腔静脉造瘘法构建慢性心衰模型,(2)正常鼠构建的心衰模型组(n=11),心包腔注射等量无菌PBS,并进一步通过腹主动脉和下腔静脉造瘘法构建慢性心衰模型,3)假手术组(n=9),心包腔注射等量的无菌PBS,只穿刺腹主动脉不与下腔静脉间造瘘。8周后复查超声心动图,然后,麻醉取心脏标本,进行组织病理学检查,RT-PCR、Western-blot检测心肌钙调控蛋白表达。
结果8周后彩色多普勒超声心动图对三组进行相关测定,与假手术组相比,两心衰组的多项心功能指标均有不同程度的下降,RyR2、FKBP12.6、SERCA2α表达降低,SERCA2a活性降低。Gsα基因干扰心衰组与正常鼠构建的心衰组相比,基因干扰心衰组的左室收缩末期容积(Left ventricular end-systolic volume, LVESV)下降,差异有统计学意义(P<0.05),左室射血分数(Left ventricular ejection fraction,LVEF)、左室短轴缩短率(Left ventricular fraction shortening,LVFS)升高,差异有统计学意义(P<0.05),左室舒张末期内径(Left ventricular internal dimension diastole,LVIDd )、左室收缩末期内径(Left ventricular internal dimension systole, LVIDs)、左室舒张末期容积( Left ventricular end-diastolic volume,LVEDV)略有降低,但差异无统计学意义(P>0.05);心肌钙调控蛋白RyR2、FKBP12.6表达升高,且有统计学意义(P<0.01),两者比值也升高;PLB基因与蛋白表达在各组间无显著差异(P>0.05),Gsα基因干扰心衰组与正常鼠构建的心衰组比,SERCA2a含量、活性增高,并具有统计学意义(P<0.01、P<0.05),SERCA2a与PLB比值亦升高。
结论体内试验表明Gsα基因干扰心衰模型,与正常鼠构建的心衰模型相比,心肌钙调控蛋白表达升高,心功能的多项参数有改善趋势,推测心衰早期Gsα的低表达可能是一种保护机制。
Background Under normal physiological conditions, activation ofβ-adrenoreceptor(β-AR) by the sympathetic nervous system stimulate Gs proteins, and then Gsαdissociation from Gβγ, the classic Gs/adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) cascade activate , which leads to phosphorylation of various calcium regulatory proteins within the myocyte such as L-type Ca2+ channel(LTCC)、ryanodine receptor 2(RyR2)、phospholamban(PLB)、and regulate the expression of FK506 binding protein(FKBP12.6), sarcoplasmic reticulum Ca2+ ATPase(SERCA2a) et al. Theβ-AR/Gs/AC pathway is the most powerful physiological mechanism to accurately augment cardiac contractility, So Gsαproteins may be play an important roles in this system signaling.
Heart failure is a state characterized by enhanced sympathetic tone, theβ-AR/Gs/AC pathway exhibits marked alterations: down regulation or desensitization inβ-AR, and an impairment of the coupling ofβ-AR to Gs, the content and (or) activation of Gsαhas changed too, downstream mechanisms (calcium regulatory proteins) are altered. What is role of the changes of Gsαin heart failure is still unknown, is that a main factor causing further deterioration of heart function or adaptive mechanism? Moreover, previous studies in this signaling pathway mainly focused on adult animals, whether there are different manifestations in junior animals is still unclear.
So this study is to knockdown of Gsαgene by RNAi in junior rat and to investigate the effects of changes of Gsαin heart failure,, and the influence of Gsαon downstream mechanisms(calcium regulatory proteins, such as RyR2、FKBP12.6、PLB、SERCA2a ) .
Part 1 Construction and identification of eukaryotic expression plasmids containing short hairpin RNA targeting to gnas gene
Objective To construct eukaryotic expression plasmids containing short hairpin RNA (shRNA) that target to rat gnas gene, which encodes the a-subunit of the Gs protein (Gsα).
Methods Three pairs of shRNAs that target to gnas gene were designed. After annealed, the inserts were ligated into the linearized pGenesil-1.1 plasmids. The eukaryotic expression plasmids were constructed and identified using restriction enzyme analysis and sequencing analysis. And targeting no-isogeny gene was served as a negative control.
Results PGenesil-1.1 plasmids were confirmed by restriction enzyme analysis and sequencing analysis in accordance with design requiring.
Conclusion We successfully constructed 3 recombinant plasmids pGenesil-1.1-shRNA targeted to gnas gene, and it’s helpful for further research on the role of Gsαin heart failure by RNA interference (RNAi) technique.
Part 2 Selective knockdown of Gsαin primary cultured rat myocytes by RNAi in vitro
Objective To selectively knockdown the expression of Gsαprotein in rat myocytes by RNA interference (RNAi),and to select the plasmids having the best effect on inhibiting Gsα.
Method Eukaryotic expression plasmids containing shRNA targeting to gnas gene were constructed, then plasmids were transfected into primary cultured rat myocytes via liposome Lipofectamine? LTX & PLUS , there are plasmids carrying a nonspecific shRNA coding sequence(HK plasmids) as the negative control group and the blank control group involved. The mRNA and protein expressions of Gsαwere analyzed by RT-PCR and Western blot respectively, and were normalized to internal control gene GAPDH.
Results The expressions of mRNA and protein of Gsαof test groups were markedly decreased than that of control groups (P<0.01), and the shRNA3 has the mostly inhibitory effect. While no significant difference was found in control groups.
Conclusion RNAi can selectively knockdown Gsαexpression in primary cultured myocytes , and laid a foundation for future studies on Gsαin vivo.
Part 3 Effect of Gsαgene knockdown by RNAi on calcium regulatory proteins in junior rat with heart failure
Objective To knockdown of Gsαgene by RNAi in junior rats and investigate the effects of Gsαgene on on calcium regulatory proteins in rats with heart failure.
Methods Male Wistar rats, four weeks old, were randomly divided into 3 groups, measured by the high frequency ultrasound. (1)HF group which Gsαknockdown (n=13), a mixture test plasmids shRNA3 (diluted in sterile PBS) was injected transdiaphragmatically into the hearts, and then established the animal model of chronic heart failure by fistulation of abdominal aorta and inferior vena cave. (2) HF group (n=11), Equivalent of sterile PBS was injected into the hearts, and then established the animal model of chronic heart failure by the same approach. (3)Sham group (n=9). 8 weeks after the operation, the test rats were used to test the following procedures: measurement of cardiac dimensions and ejection fraction with echocardiogram, determination of proteins expression level of RyR2、FKBP12.6、PLB、SERCA2αin cardiac tissue with Western-blot and RT-PCR .
Results The echocardiogram showed that, compared with those of Sham group, many parameters of cardiac function decreased,and the expression level of RyR2, FKBP12.6 and SERCA2αalso decreased in the two HF groups, which have significant differences(P<0.01).While the expression level of PLB had no significant difference in these 3 groups (P>0.05). Compare with HF group, the Left ventricular end-systolic volume (LVESV) decreased(P<0.05), Left ventricular fraction shortening (LVFS)、Left ventricular ejection fraction(LVEF)increased (P<0.05), and Left ventricular internal dimension diastole (LVIDd )、Left ventricular internal dimension systole(LVIDs)、left ventricular end-diastolic volume (LVEDV) decreased, while had no statistically significant difference(P>0.05), the expression of RyR2、FKBP12.6、SERCA2αincreased(P<0.01), and FKBP12.6/ RyR2、SERCA2α/PLB increased in HF group which Gsa knockdown.
Conclusion Compare with HF group, the HF group which Gsa knockdown has improvement trends in cardiac function index and relatively high-level expression of calcium regulatory proteins. Our study suggests that the alterations of Gsαmay play an adaptive reaction to the increased sympathetic nervous system activity in heart failure.
引文
[1] Wehrens XH, Marks AR. Altered function and regulation of cardiac ryanodine receptors in cardiac disease [J] .Trends Biochem Sci, Dec 2003; 28(12): 671-8.
[2] Zolk O, Kouchi I, Schnabel P, et al. Heterotrimeric G proteins in heart disease [J] .Can J Physiol Pharmacol, 2000, 78 187～198.
[3] Martin J. Lohse, Stefan Engelhardt, Thomas Eschenhagen .What is the Role of ?-Adrenergic Signaling in Heart Failure? [J] .Circ. Res, Nov 2003; 93: 896 - 906.
[4] Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference [J]. Nat Biotechnol, Mar 2004; 22(3): 326-30.
[5] Naito YK, Tomoyuki Yamada, Kumiko Ui-Tei,et al. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference[J].Nucleic Acids Res., Jul 2004; 32: W124 - W129.
[6] Geng Y-J, Ishikawa Y, Vatner DE, et al. Apoptosis of cardiac myocytes in Gs transgenic mice[J ] .Circ Res.1999;84:34–42.
[7] Vatner DE, Yang G-P, Geng Y-J, et al Determinants of the Cardiomyopathic Phenotype in Chimeric Mice Overexpressing Cardiac Gs{alpha}[J].Circ. Res, April 14, 2000; 86(7): 802 - 806.
[8] Hardt SE, Geng Y-J, Montagne O, et al. Accelerated Cardiomyopathy in Mice With Overexpression of Cardiac Gs{alpha} and a Missense Mutation in the {alpha}-Myosin Heavy Chain [J]. Circulation, February 5, 2002; 105(5):614 - 620.
[9] Vatner SF, Vatner DE, Homcy CJ. ?-Adrenergic Receptor Signaling: An Acute Compensatory Adjustment—Inappropriate for the Chronic Stress of Heart Failure? : Insights from Gs Overexpression and Other Genetically Engineered Animal Models [J].Circ. Res., Mar 2000; 86: 502 - 506.
[10] M?kinen PI, Koponen JK, K?rkk?inen AM, et al. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain [J]. Gene Med 2006; 8: 433-441
[11] Luquan Wang , Mu FY. A Web-based design center for vector-based siRNA and siRNA cassette[J]. Bioinformatics, Jul 2004; 20: 1818 - 1820.
[1]梁丽,刘春峰.SIRS大鼠心肌刺激性G蛋白的变化及地塞米松对其影响的实验观察.中国医科大学学报,2004,33(6):495 - 498.
[2] Gregory J,Hannon.RNA interference.Nature,2002;418:244～251.
[3] Ohnishi Y, Tokunaga K, Hohjoh H. Influence of assembly of siRNA elements into RNA-induced silencing complex by fork-siRNA duplex carrying nucleotide mismatches at the 3'- or 5'-end of the sense-stranded siRNA element. Biochem Biophys . Res Commun 2005; 329: 516-521
[4] Zamore PD RNA interference.listening to the sound of silence. Nature Structural Biology 2001; (8)746-750
[5] Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference [J]. Nat Biotechnol, 2004; 22(3): 326-330.
[6] Naito Y, Yamada T, Kumiko U T, et al. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference[J]. Nucleic Acids Res,2004,32:124-129.
[7] M?kinen PI, Koponen JK, K?rkk?inen AM, et al. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain [J]. Gene Med 2006; 8: 433-441
[8] Luquan Wang , Mu FY. A Web-based design center for vector-based siRNA and siRNA cassette[J]. Bioinformatics, Jul 2004; 20: 1818 - 1820.
[9] Datiles M J, Johnson E A, McCarty R E. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles[J].Biochim Biophys Acta. 2008,1777(4):362-368.
[10] Kongkaneramit L, Sarisuta N, Azad N,et al. Dependence of Reactive Oxygen Species and FLICE Inhibitory Protein onLipofectamine-Induced Apoptosis in Human Lung Epithelial Cells[J]. J Pharmacol Exp Ther.2008,325: 969 - 977.
[11]李凡东.质粒介导RNAi抑制大鼠心肌细胞KCNJ2基因表达的实验研究[D].山东:山东大学.2006.
[1] Wehrens XH, Marks AR. Altered function and regulation of cardiac ryanodine receptors in cardiac disease [J] .Trends Biochem Sci, Dec 2003; 28(12): 671-8.
[2] Martin J. Lohse, Stefan Engelhardt, Thomas Eschenhagen .What is the Role of ?-Adrenergic Signaling in Heart Failure? [J] .Circ. Res, Nov 2003; 93: 896 - 906.
[3] Jang DW,Xiao BL,Yang DM,et al.RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced.Ca2+ release[J].PNAS.204;101(35):13062-13067
[4] Gergs U,Berndt T,Buskase J,et al.On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure[J]. Am J Physiol Heart Circ Physiol.2007;293(1):H728-734
[5] Wehrens XH, Lehnart SE, Huang F, et.al.FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death[J]. Cell. 2003 Jun 27;113(7):829-40.
[6] Fromes Y, Salmon A, Wang X, et.al.Gene delivery to the myocardium by intrapericardial injection[J]. Gene Ther. 1999 6(4):683-8.
[7]惠海鹏李小鹰彭江.大鼠经腹心包腔内注射术的研究[J].医学研究生学报. 2005 18(10):879-881
[8]朱彤莹,黄国英,顾勇等.两种心衰模型大鼠心功能的比较[J].中国实验动物学报.2002,10(1):51-53
[9]米青.缬沙坦对心力衰竭幼鼠心肌钙调节蛋白作用的研究[D].重庆:重庆医科大学,2008.
[10] Zolk O, Kouchi I, Schnabel P, et al. Heterotrimeric G proteins in heart disease [J] .Can J Physiol Pharmacol, 2000, 78 187～198.
[11] Wehrens XH, Lehnart SE, Reiken S, et al. Ryanodine receptor/calcium release channel PKA phosphorylation: A critical mediator of heart failure progression[J].Proc Natl Acad Sci USA 2006;103:511-8.
[12] Wehrens XH, Lehnart SE, Reiken S, et al. Enhancing calstabin binding to ryanodine receptors improves cardiac and skeletal muscle function in heart failure[J].Proc Natl Acad Sci U S A 2005;102:9607-12.
[13] 1wase M ,Uechi M ,Vatner DE,et a1.Cardiomyopathy in—duced by cardiac Gs alpha overexpression.Am J Physiol,1997,272:H585～H589.
[14] Yin F, Wang YY, Du JH, et al . Noncanonical cAMP pathway and p38 MAPK mediate beta2-adrenergic receptor-induced IL-6 production in neonatal mouse cardiac fibroblasts.[J]J Mol Cell Cardiol. 2006 Mar;40(3):384-93. .
[15] Jaclyn W. McAlees and Virginia M. SandersHematopoietic Protein Tyrosine Phosphatase Mediatesβ2- Adrenergic Receptor-Induced Regulation of p38 Mitogen-Activated Protein Kinase in B Lymphocytes.[J] Mol. Cell. Biol.Feb 2009; 29: 675 - 686.
[16] Wehrens XH, Marks AR. Molecular determinants of altered contractility in heart failure[J]. Ann Med 2004;36. 1:70-80.
[17] Marks AR.Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death[J] JMoI Cell Cardiol.2001;33:615-24.
[18] Marx SO, Reiken S, Hisamatsu Y, et.al. Phosphorylation-dependent Regulation of Ryanodine Receptors:A Novel Role for Leucine/Isoleucine Zippers [J]. JCell Bio1 2001;153:699-708.
[19] Hoshijima M. Gene therapy targeted at calcium handling as an approach to the treatment of heart failure[J]. Phhearmacol Ther 2005;105:211-28.
[20] Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of beta-adrenergic signaling in heart failure?[J] Circ Res 2003:93:896-906.
[21] Leineweber K, Rohe P, Beilfuss A,. G-protein-coupled receptor kinase activity in human heart failure: effects of beta-adrenoceptor blockade[J]. Cardiovasc Res 2005;66:512-9.
[1] Murat Bastepe. et al.The GNAS Locus: Quintessential Complex Gene Encoding Gsα, XLαs, and other Imprinted Transcripts .Curr Genomics. 2007 September; 8(6): 398–414.
[2] lwase M,Uechi M,Vatner DE,et a1.Cardiomyopathy induced by cardiac Gs alpha overexpression[J]. Am J Physiol,1997,272: H585-H589.
[3] Zolk 0,Kouchi I, Schnabel P, et a1 . Heterotrimeric G proteins in heart disease[J].Can J Physiol Pharmacol,2000,78:187-198.
[4] Janet D Robishaw and Catherine H Berlot. Translating G protein subunit diversity into functional specificity[J] . 2004 ,16, (I2): 206-209.
[5] Irene Garcia-Higuera, Jessica Fenoglio, Ying Li, et a1.Folding of Proteins with WD-Repeats: Comparison of Six Members of the WD-Repeat Superfamily to the G ProteinβSubunit. Biochemistry, 1996, 35 (44), 13985–13994.
[6]钟前进,汪曾炜,肖颖彬.心肌缺血再灌注时Gsα和Giα蛋白含量mRNA水平变化及意义[J].《第四军医大学学报》2005(26)20:1851-1854
[7] Sara A. Epperson, Laurence L. Brunton, Israel Ramirez-Sanchez, et.al Adenosine receptors and second messenger signaling pathways in rat cardiac fibroblasts.[J]Am J Physiol Cell Physiol, May 2009; 296: C1171–1177
[8] J.C. Braz, O.F. Bueno, Q. Liang, B.J. Wilkins, Y.S. Dai, S. Parsons, J.Braunwart, B.J. Glascock, R. Klevitsky, T.F. Kimball, T.E. Hewett, J.D.Molkentin, Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling, J. Clin. Invest. 111 (2003) 1475–1486.
[9] Yin F, Wang YY, Du JH, et al . Noncanonical cAMP pathway and p38 MAPKmediate beta2-adrenergic receptor-induced IL-6 production in neonatal mouse cardiac fibroblasts.[J]J Mol Cell Cardiol. 2006;40(3):384-93.
[10] Clark CJ ,M illigan G, Gonnell JM. G prtein alter at ions in young Milan hyperten- sive strain rats. Biochim Biophys Acta, 1994, 1225: 149—157.
[11] Regitz-Zagrosek V, Fleck E. Heart failure: clinical aspects from basic research. Introduction. Eur Heart J. 1994;(15) D:2-155.
[12] ChengTH,ShihNL,ChenCH,et al. Role of mitogen-activated protein kinase path -way in reactive oxygen species-mediated endothelin-1-inducedβ-myosin heavy chain gene expression and cardiomyocyte hypertrophy. J Biomed sci 2005(12)123.
[13] Jie Ren , Shaosong Zhang , Attila Kovacs ,YibinWang , Anthony J. Muslin, Role of p38MAPK in cardiac apoptosis and remodeling after myocardial infarction, Journal of Molecular and Cellular Cardiology 2005(38)617–623
[14] Shen W, Vatner DE, Vatner SF ,et al. Progressive Loss of Creatine Maintains a Near Normal DeltaG( approximately ATP) in Transgenic Mouse Hearts with Cardiomyopathy Caused by Over Expressing Gsalpha[J]. J Mol Cell Cardiol. 2010 Apr;48(4):591-9.、
[15] Takao Nishizawaa, Stephen F. Vatnera, Chull Honga,et.al. Overexpressed cardiac Gsαin rabbits[J]. Journal of Molecular and Cellular Cardiology.2006;41(1): 44-50
[2] Zolk O, Kouchi I, Schnabel P, et al. Heterotrimeric G proteins in heart disease [J] .Can J Physiol Pharmacol, 2000, 78 187～198.
[3] Martin J. Lohse, Stefan Engelhardt, Thomas Eschenhagen .What is the Role of ?-Adrenergic Signaling in Heart Failure? [J] .Circ. Res, Nov 2003; 93: 896 - 906.
[4] Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference [J]. Nat Biotechnol, Mar 2004; 22(3): 326-30.
[5] Naito YK, Tomoyuki Yamada, Kumiko Ui-Tei,et al. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference[J].Nucleic Acids Res., Jul 2004; 32: W124 - W129.
[6] Geng Y-J, Ishikawa Y, Vatner DE, et al. Apoptosis of cardiac myocytes in Gs transgenic mice[J ] .Circ Res.1999;84:34–42.
[7] Vatner DE, Yang G-P, Geng Y-J, et al Determinants of the Cardiomyopathic Phenotype in Chimeric Mice Overexpressing Cardiac Gs{alpha}[J].Circ. Res, April 14, 2000; 86(7): 802 - 806.
[8] Hardt SE, Geng Y-J, Montagne O, et al. Accelerated Cardiomyopathy in Mice With Overexpression of Cardiac Gs{alpha} and a Missense Mutation in the {alpha}-Myosin Heavy Chain [J]. Circulation, February 5, 2002; 105(5):614 - 620.
[9] Vatner SF, Vatner DE, Homcy CJ. ?-Adrenergic Receptor Signaling: An Acute Compensatory Adjustment—Inappropriate for the Chronic Stress of Heart Failure? : Insights from Gs Overexpression and Other Genetically Engineered Animal Models [J].Circ. Res., Mar 2000; 86: 502 - 506.
[10] M?kinen PI, Koponen JK, K?rkk?inen AM, et al. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain [J]. Gene Med 2006; 8: 433-441
[11] Luquan Wang , Mu FY. A Web-based design center for vector-based siRNA and siRNA cassette[J]. Bioinformatics, Jul 2004; 20: 1818 - 1820.
[1]梁丽,刘春峰.SIRS大鼠心肌刺激性G蛋白的变化及地塞米松对其影响的实验观察.中国医科大学学报,2004,33(6):495 - 498.
[2] Gregory J,Hannon.RNA interference.Nature,2002;418:244～251.
[3] Ohnishi Y, Tokunaga K, Hohjoh H. Influence of assembly of siRNA elements into RNA-induced silencing complex by fork-siRNA duplex carrying nucleotide mismatches at the 3'- or 5'-end of the sense-stranded siRNA element. Biochem Biophys . Res Commun 2005; 329: 516-521
[4] Zamore PD RNA interference.listening to the sound of silence. Nature Structural Biology 2001; (8)746-750
[5] Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference [J]. Nat Biotechnol, 2004; 22(3): 326-330.
[6] Naito Y, Yamada T, Kumiko U T, et al. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference[J]. Nucleic Acids Res,2004,32:124-129.
[7] M?kinen PI, Koponen JK, K?rkk?inen AM, et al. Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain [J]. Gene Med 2006; 8: 433-441
[8] Luquan Wang , Mu FY. A Web-based design center for vector-based siRNA and siRNA cassette[J]. Bioinformatics, Jul 2004; 20: 1818 - 1820.
[9] Datiles M J, Johnson E A, McCarty R E. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles[J].Biochim Biophys Acta. 2008,1777(4):362-368.
[10] Kongkaneramit L, Sarisuta N, Azad N,et al. Dependence of Reactive Oxygen Species and FLICE Inhibitory Protein onLipofectamine-Induced Apoptosis in Human Lung Epithelial Cells[J]. J Pharmacol Exp Ther.2008,325: 969 - 977.
[11]李凡东.质粒介导RNAi抑制大鼠心肌细胞KCNJ2基因表达的实验研究[D].山东:山东大学.2006.
[1] Wehrens XH, Marks AR. Altered function and regulation of cardiac ryanodine receptors in cardiac disease [J] .Trends Biochem Sci, Dec 2003; 28(12): 671-8.
[2] Martin J. Lohse, Stefan Engelhardt, Thomas Eschenhagen .What is the Role of ?-Adrenergic Signaling in Heart Failure? [J] .Circ. Res, Nov 2003; 93: 896 - 906.
[3] Jang DW,Xiao BL,Yang DM,et al.RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced.Ca2+ release[J].PNAS.204;101(35):13062-13067
[4] Gergs U,Berndt T,Buskase J,et al.On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure[J]. Am J Physiol Heart Circ Physiol.2007;293(1):H728-734
[5] Wehrens XH, Lehnart SE, Huang F, et.al.FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death[J]. Cell. 2003 Jun 27;113(7):829-40.
[6] Fromes Y, Salmon A, Wang X, et.al.Gene delivery to the myocardium by intrapericardial injection[J]. Gene Ther. 1999 6(4):683-8.
[7]惠海鹏李小鹰彭江.大鼠经腹心包腔内注射术的研究[J].医学研究生学报. 2005 18(10):879-881
[8]朱彤莹,黄国英,顾勇等.两种心衰模型大鼠心功能的比较[J].中国实验动物学报.2002,10(1):51-53
[9]米青.缬沙坦对心力衰竭幼鼠心肌钙调节蛋白作用的研究[D].重庆:重庆医科大学,2008.
[10] Zolk O, Kouchi I, Schnabel P, et al. Heterotrimeric G proteins in heart disease [J] .Can J Physiol Pharmacol, 2000, 78 187～198.
[11] Wehrens XH, Lehnart SE, Reiken S, et al. Ryanodine receptor/calcium release channel PKA phosphorylation: A critical mediator of heart failure progression[J].Proc Natl Acad Sci USA 2006;103:511-8.
[12] Wehrens XH, Lehnart SE, Reiken S, et al. Enhancing calstabin binding to ryanodine receptors improves cardiac and skeletal muscle function in heart failure[J].Proc Natl Acad Sci U S A 2005;102:9607-12.
[13] 1wase M ,Uechi M ,Vatner DE,et a1.Cardiomyopathy in—duced by cardiac Gs alpha overexpression.Am J Physiol,1997,272:H585～H589.
[14] Yin F, Wang YY, Du JH, et al . Noncanonical cAMP pathway and p38 MAPK mediate beta2-adrenergic receptor-induced IL-6 production in neonatal mouse cardiac fibroblasts.[J]J Mol Cell Cardiol. 2006 Mar;40(3):384-93. .
[15] Jaclyn W. McAlees and Virginia M. SandersHematopoietic Protein Tyrosine Phosphatase Mediatesβ2- Adrenergic Receptor-Induced Regulation of p38 Mitogen-Activated Protein Kinase in B Lymphocytes.[J] Mol. Cell. Biol.Feb 2009; 29: 675 - 686.
[16] Wehrens XH, Marks AR. Molecular determinants of altered contractility in heart failure[J]. Ann Med 2004;36. 1:70-80.
[17] Marks AR.Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death[J] JMoI Cell Cardiol.2001;33:615-24.
[18] Marx SO, Reiken S, Hisamatsu Y, et.al. Phosphorylation-dependent Regulation of Ryanodine Receptors:A Novel Role for Leucine/Isoleucine Zippers [J]. JCell Bio1 2001;153:699-708.
[19] Hoshijima M. Gene therapy targeted at calcium handling as an approach to the treatment of heart failure[J]. Phhearmacol Ther 2005;105:211-28.
[20] Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of beta-adrenergic signaling in heart failure?[J] Circ Res 2003:93:896-906.
[21] Leineweber K, Rohe P, Beilfuss A,. G-protein-coupled receptor kinase activity in human heart failure: effects of beta-adrenoceptor blockade[J]. Cardiovasc Res 2005;66:512-9.
[1] Murat Bastepe. et al.The GNAS Locus: Quintessential Complex Gene Encoding Gsα, XLαs, and other Imprinted Transcripts .Curr Genomics. 2007 September; 8(6): 398–414.
[2] lwase M,Uechi M,Vatner DE,et a1.Cardiomyopathy induced by cardiac Gs alpha overexpression[J]. Am J Physiol,1997,272: H585-H589.
[3] Zolk 0,Kouchi I, Schnabel P, et a1 . Heterotrimeric G proteins in heart disease[J].Can J Physiol Pharmacol,2000,78:187-198.
[4] Janet D Robishaw and Catherine H Berlot. Translating G protein subunit diversity into functional specificity[J] . 2004 ,16, (I2): 206-209.
[5] Irene Garcia-Higuera, Jessica Fenoglio, Ying Li, et a1.Folding of Proteins with WD-Repeats: Comparison of Six Members of the WD-Repeat Superfamily to the G ProteinβSubunit. Biochemistry, 1996, 35 (44), 13985–13994.
[6]钟前进,汪曾炜,肖颖彬.心肌缺血再灌注时Gsα和Giα蛋白含量mRNA水平变化及意义[J].《第四军医大学学报》2005(26)20:1851-1854
[7] Sara A. Epperson, Laurence L. Brunton, Israel Ramirez-Sanchez, et.al Adenosine receptors and second messenger signaling pathways in rat cardiac fibroblasts.[J]Am J Physiol Cell Physiol, May 2009; 296: C1171–1177
[8] J.C. Braz, O.F. Bueno, Q. Liang, B.J. Wilkins, Y.S. Dai, S. Parsons, J.Braunwart, B.J. Glascock, R. Klevitsky, T.F. Kimball, T.E. Hewett, J.D.Molkentin, Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling, J. Clin. Invest. 111 (2003) 1475–1486.
[9] Yin F, Wang YY, Du JH, et al . Noncanonical cAMP pathway and p38 MAPKmediate beta2-adrenergic receptor-induced IL-6 production in neonatal mouse cardiac fibroblasts.[J]J Mol Cell Cardiol. 2006;40(3):384-93.
[10] Clark CJ ,M illigan G, Gonnell JM. G prtein alter at ions in young Milan hyperten- sive strain rats. Biochim Biophys Acta, 1994, 1225: 149—157.
[11] Regitz-Zagrosek V, Fleck E. Heart failure: clinical aspects from basic research. Introduction. Eur Heart J. 1994;(15) D:2-155.
[12] ChengTH,ShihNL,ChenCH,et al. Role of mitogen-activated protein kinase path -way in reactive oxygen species-mediated endothelin-1-inducedβ-myosin heavy chain gene expression and cardiomyocyte hypertrophy. J Biomed sci 2005(12)123.
[13] Jie Ren , Shaosong Zhang , Attila Kovacs ,YibinWang , Anthony J. Muslin, Role of p38MAPK in cardiac apoptosis and remodeling after myocardial infarction, Journal of Molecular and Cellular Cardiology 2005(38)617–623
[14] Shen W, Vatner DE, Vatner SF ,et al. Progressive Loss of Creatine Maintains a Near Normal DeltaG( approximately ATP) in Transgenic Mouse Hearts with Cardiomyopathy Caused by Over Expressing Gsalpha[J]. J Mol Cell Cardiol. 2010 Apr;48(4):591-9.、
[15] Takao Nishizawaa, Stephen F. Vatnera, Chull Honga,et.al. Overexpressed cardiac Gsαin rabbits[J]. Journal of Molecular and Cellular Cardiology.2006;41(1): 44-50