肌纤生成调节因子-1致心肌细胞肥大的分子机制研究
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摘要
研究背景：
人肌纤生成调节因子-1(human myofibrillogenesis regulator 1, hMR-1)是本课题组克隆的一个人类新功能基因,它定位于人类染色体2q35,全长755bp,编码一段142个氨基酸组成的蛋白质。前期工作证实,MR-1高表达于心肌和骨骼肌,免疫组化证实人类心肌肌原纤维上有MR-1分布,血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)诱导肥大心肌细胞中MR-1表达显著升高,以RNA干扰(RNA interference, RNAi)下调MR-1表达后则降低AngⅡ诱导的心肌肥大效果,提示MR-1与心肌肥大有关。既往工作通过酵母双杂交和GST pull-down实验发现,MR-1与肌球蛋白调节型轻链(myosin light chain-2, MRLC)、myomesin-1和β-烯醇酶(β-enolase)等与肌肉收缩装置相关的蛋白间存在相互作用,提示MR-1可能通过调节心肌肌原纤维引起心肌肥大。不过,MR-1是否可以直接引起心肌肥大以及MR-1致心肌肥大的分子机制尚不清楚。研究目的：
制备有效的抗MR-1抗体,构建MR-1真核表达质粒,建立MR-1沉默方法,在体外培养的乳大鼠心肌细胞模型上验证MR-1过表达直接引起肥大以及MR-1对心肌细胞肌原纤维生成与肌原纤维排列产生影响,阐明MR-1调节心肌肌原纤维改变的分子机制,旨在证实“MR-1通过促进肌原纤维生成引起心肌细胞肥大”。
研究方法：
1.抗体制备：生物信息学分析人类MR-1 (hMR-1)优势抗原肽,合成并混合后免疫家兔,抗血清以亲和层析法纯化后检测抗体的特异性及适用范围；质粒构建：根据NCBI GenBank数据库的hMR-1 mRNA信息克隆全长序列至pcDNA3.1-HisB(-)-Myc载体的多克隆位点；RNA干扰实验：根据大鼠源MR-1 (rMR-1)基因序列设计靶点并合成稳定修饰型stealth siRNA,筛选高沉默效率的MR-1-stealth siRNA.
2.乳大鼠心肌细胞培养与心肌细胞肥大模型的建立：培养的乳大鼠心肌细胞以终浓度1×10-7mol／L的血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)孵育48h,以复制AngⅡ致心肌细胞肥大模型；专业图像软件分析细胞平均表面积、[3H]-亮氨酸掺入法检测细胞蛋白合成速率,以及RT-PCR法检测心房利钠因子(atrial natriuretic factor, ANF)、脑钠肽(brain natriuretic peptide, BNP)的转录水平,以证实心肌肥大的形成。
3.肌原纤维生成(myofibrillogenesis)与排列观察：以FITC-鬼笔环肽特异性标记F-actin,观察横纹样F-actin的形成。
4.细胞内信号分子检测：以细胞免疫荧光技术检测分子的亚定位、共定位及转位现象,以RT-PCR和Western Blot方法检测分子在mRNA和蛋白水平的表达。
5.[Ca2+]i和钙调神经磷酸酶(calcineurin, CaN)活性检测：Fluo-3AM荧光探针标记胞浆Ca2+并半定量分析平均荧光强度,比色法检测CaN比活力。
6.图像分析、数据处理及统计学分析：细胞平均表面积、条带积分光密度、[Ca2+]i平均荧光强度以专业图像软件Image Pro-Plus (version 4.11)进行半定量分析；各组实验数据以均数±标准差(x±s)表示,以统计学软件SPSS13.0分析,多组间两两比较用Bonferroni's test,P<0.05认为具有显著性差异。
实验结果
1.制备了兔抗hMR-1多克隆抗体,可识别人源性及鼠源性MR-1抗原,适用于Western Blot和细胞免疫荧光实验。构建了MR-1真核表达质粒并在乳大鼠心肌细胞成功过表达外源hMR-1蛋白。根据沉默效率筛选stealth siRNA并转染乳大鼠心肌细胞,有效沉默了内源rMR-1表达。
2.MR-1过表达引起心肌细胞肥大：细胞表面积、[3H]-亮氨酸掺入和ANF和BNP转录明显增加(P<0.05)；MR-1-RNAi对ANF和BNP的转录水平有所降低而对细胞表面积和蛋白合成速率无显著影响；此外,RNAi对AngⅡ诱导的三项肥大指标增加均明显逆转。
3.MR-1过表达对心肌肌原纤维的影响：正常对照组心肌细胞F-actin呈“非横纹样(nonstriated-like)”和“非肌肉类型(nonmuscule-like pattern) ",主要沿胞膜内侧面分布；过表达MR-1组F-actin呈节段状,为典型的“横纹样”类型,且有25.0±6.7%的细胞肌原纤维呈紊乱排列,提示MR-1过表达促进肌原纤维生成(myofibrillogenesis)及其排列紊乱；MR-1-RNAi组F-actin呈“应力纤维样(stress fiber-like) ",与随机RNA对照组类似,并且MR-1-RNAi明显逆转了AngⅡ诱导的横纹样F-actin形成。
4.对MR-1调节肌原纤维改变的分子机制研究：
1)MR-1与MRLC共定位明显,均分布于胞浆并浓集于核周。在正常培养的乳大鼠心肌细胞中,MR-1与myomesin-1无共定位,但MR-1过表达引起myomesin-1转位至胞浆后,myomesin-1与胞浆的MR-1发生部分共定位。
2)MR-1过表达明显上调myomesin-1和MRLC mRNA及蛋白的表达(P<0.05),MR-1-RNAi对myomesin-1和MRLC表达无显著影响,但逆转了AngⅡ引起的myomesin-1和上调。
3)MR-1过表达促进myomesin-1的核-浆转位,MR-1-RNAi对转位无显著影响,但可在一定程度上抑制AngⅡ引起的myomesin-1转位。
4)过表达SUMO-1引起myomesin-1核-浆转位,促进肌原纤维生成；抑制MR-1明显逆转SUMO-1过表达引起的myomesin-1转位及肌原纤维生成,提示MR-1可能在myomesin-1的SUMO化修饰及SUMO化介导的肌纤生成过程中发挥关键作用。
5)MR-1过表达引起[Ca2+]i、CaN活性、肌细胞增强因子-2C(myocytes enhancer factor 2C, MEF-2C)和磷酸化-MEF-2C水平显著升高(P<0.05),MR-1-RNAi对这些无显著影响(P>0.05),但可逆转AngⅡ引起的升高。
研究结论：
1 MR-1过表达直接诱导乳大鼠心肌细胞肥大。
2 MR-1通过促进肌原纤维生成及排列紊乱致乳大鼠心肌细胞肥大。
3 MR-1通过调节myomesin-1与MRLC影响肌原纤维生成,从而引起心肌细胞肥大。(主要结论)
4 MR-1可能在“Ca2+→CaN→MEF-2C”这一致心肌肥大的经典信号途径中发挥重要作用。
Background
Homologous myofibrillogenesis regulator 1(hMR-1) is a novel characterized human functional gene cloned from human skeletal muscle cDNA library by our research group. It was found that hMR-1 gene locates on the human chromosome 2q35, with a full length of 755 bps and encodes a 142 amino acid-protein. Previous studies suggested that MR-1 found in human myofibrils by immunohistochemistry was highly expressed in myocardium and skeletal muscle, MR-1 is significantly up-regulated in hypertrophied cardiomyocytes induced by angiotensinⅡ(AngⅡ) whereas its silencing using RNAi would abolish the AngⅡ-induced hypertrophy, indicating that MR-1 is involved in hypertrophy. However, whether MR-1 induces hypertrophy directly or not and the mechanism is still unclear. The results from yeast two-hybrid screen and in vitro GST pull-down assay indicated that MR-1 could interact with the contractile apparatus associated proteins, i.e. myosin light chain-2 (MRLC), myomesin-1 andβ-enolase etc. These findings suggested that MR-1 induces cardiac hypertrophy by regulating the myofibrillogenesis.
Objective
In order to validate the hypothesis that MR-1 induces cardiac hypertrophy by promoting myofibrillogenesis, we aimed to verify the direct contribution of MR-1 to hypertrophy in vitro and influence of MR-1 on myofibrillogenesis and myofibrillar orgnization, as well as the regulatory mechnisms. Thus, we were to prepare the anti-MR-1 antibody, to construct the MR-1 eucaryotic expressing vector and to establish the MR-1 silencing method at first.
Methods
1. Antibody preparation; superior epitopes were selected based on the bioinformatics analysis, synthesized and mixed for immunization; anti-serum was purified by immunoaffinity chromatography and the specificity and application were measured as well. Plasmid construction; the full length of hMR-1 mRNA from NCBI GenBank database was cloned to the MCS of pcDNA3.1-HisB(-)-Myc vector. RNA interference (RNAi) assay; The rat original MR-1 (rMR-1) stably-modified stealth siRNA was designed, synthesized and then selected according to the silencing effect.
2. Neonatal rat cardiomyocytes culture and hypertrophy model in vitro; cardiomyocytes were cultured and incubated with angiotensin II (Ang II) 1x10-7 mol/L for 48h to develop hypertrophy, to validate the effect, hallmarks i.e. mean area of cell surface analyzed by professional software, protein synthesis velocity assessed by [3H]-Leucine incorporation and mRNA levels of atrial natriuretic factor and brain natriuretic peptide measured by RT-PCR were all employed.
3. Investigation on myofibrillogenesis and myofibril-alignment; striated-like F-actin pattern which indicated the myofibrillogenesis was characterized by specific staining with FITC-phalloidin.
4. Stuadies on interesting proteins; the sublocation, collocation and translocation of thosed molecules were detected using immunocytofluorescent assay, whereas their expression at mRNA and protein levels were measured using RT-PCR and Western Blot, respectively.
5. Measurement of [Ca2+]i and activity of calcineurin (CaN); cytoplasmic free Ca2+ was probed by fluo-3AM whereafter its fluorescent density was semi-quantitatively analyzed; enzymatic activity of CaN was measured by chromatometry.
6. Image analysis, data management and statistics analysis; semi-quantitative analysis on mean cell surface area, integrated optical density of bands and fluorescent density of [Ca2+]i were performed with software Image Pro-Plus (version 4.11), experimental data were represented by mean±standard deviation ((?)±s). SPSS 13.0 statistics software was introduced. Bonferroni's test was employed to analyze statistical differences between every two-sample among several groups. P<0.05 was considered statistically significant.
Results
1. The rabbit anti-hMR-1 polyclonal antibody detects either human original or rat original epitopes, available for both Western Blot and immunocytofluorescent assays were successfully prepared; the hMR-1 eukaryotic expression plasmid pcDNA3.1-Myc/HisB(-)-hMRl for exogenous hMR-1 overexpression in neonatal rat cardiomyocytes was constructed; the stealth siRNA selected according to its silencing effect was transfected to cardiomyocytes and successfully inhibited the expression of endogenous rMR-1.
2. The mean surface area, incorporation of [3H]-Leucine and transcriptional levels of ANF and BNP were all significantly increased with MR-1-overexpression (P<0.05); MR-1-RNAi decreased the transcription of ANF and BNP (P<0.05) instead of the mean surface area and protein synthesis. (P>0.05) In particular, it would reverse the Ang II-induced increases on those three hypertrophic hallmarks.
3. Cardiomyocytes in the normal culture exhibited a nonstriated and nonmuscle-like F-actin, which mainly distributed along the internal surface of the cellular membrane, whereas a quite different organization that periodic and characteristic striated-like F-actin was observed in MR-1-overexpressed myocytes,25.0±6.7% of which, however, exhibited an irregularly organized F-actin, suggesting a promoted but derangement myofibrillogenesis. MR-1-silenced myocytes showed a stress fiber-like F-actin, which is similar to the random-RNAi control, otherwise, MR-1-RNAi would abolish the striated-like F-actin induced by AngⅡ.
4. MR-1-involved regulation in myofibrillogenesis has been recognized detailed as follows;
1) Whereas MRLC collocates with MR-1 extensively in cytoplasm, especially the peri-nucleus area where they both enriched, the nucleus-located myomesin-1 does not, unless its translocation is initiated that myomesin-1 shifts to cytoplasm where its partial collocation with MR-1 was found.
2) Myomesin-1 and MRLC were significantly increased at mRNA and protein levels in MR-1-overexpressed culture; MR-1-RNAi does not affect their expression instead of the AngⅡ-induced upregulation.
3) MR-1-overexpression promotes myomesin-1 shuttling from nucleus to cytoplasm, this phenomenon would not be affected by MR-1-RNAi, but RNAi may reverse the AngⅡ-induced translocation of myomesin-1.
4) Overexpression of SUMO-1 promotes myomesin-1 translocation and the translocation involved myofibrillogenesis, which provide evidence that MR-1 may serve a crucial role in the SUMOylated modification of myomesin-1 and SUMOylated myomesin-1 mediated myofibrillogenesis.
5) [Ca2+]i, CaN-activity, myocytes enhancer factor 2C (MEF-2C) and phospho-MEF-2C levels were found significantly increased in MR-1-overexpressed myocytes (P<0.05) but not any significant disparity in MR-1-RNAi culture (P>0.05); nevertheless, RNAi applied myocytes would reverse the AngⅡ-induced upregulation of those molecules.
Conclusion
1. Overexpression of MR-1 directly induces hypertrophy in neonatal rat cardiomyocytes.
2. MR-1 induces hypertrophy in neonatal rat cardiomyocytes by promoting myofibrillogenesis and the myofibril-alignment.
3. MR-1 induces hypertrophy in cardiomyocytes through regulating myomesin-1 and MRLC mediated myofibrillogenesis. (major conclusion)
4. MR-1 may play an important role in the classic cardiac hypertrophy "Ca2+→CaN→MEF-2C" signaling pathway.
人肌纤生成调节因子-1(human myofibrillogenesis regulator 1, hMR-1)是本课题组克隆的一个人类新功能基因,它定位于人类染色体2q35,全长755bp,编码一段142个氨基酸组成的蛋白质。前期工作证实,MR-1高表达于心肌和骨骼肌,免疫组化证实人类心肌肌原纤维上有MR-1分布,血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)诱导肥大心肌细胞中MR-1表达显著升高,以RNA干扰(RNA interference, RNAi)下调MR-1表达后则降低AngⅡ诱导的心肌肥大效果,提示MR-1与心肌肥大有关。既往工作通过酵母双杂交和GST pull-down实验发现,MR-1与肌球蛋白调节型轻链(myosin light chain-2, MRLC)、myomesin-1和β-烯醇酶(β-enolase)等与肌肉收缩装置相关的蛋白间存在相互作用,提示MR-1可能通过调节心肌肌原纤维引起心肌肥大。不过,MR-1是否可以直接引起心肌肥大以及MR-1致心肌肥大的分子机制尚不清楚。研究目的：
制备有效的抗MR-1抗体,构建MR-1真核表达质粒,建立MR-1沉默方法,在体外培养的乳大鼠心肌细胞模型上验证MR-1过表达直接引起肥大以及MR-1对心肌细胞肌原纤维生成与肌原纤维排列产生影响,阐明MR-1调节心肌肌原纤维改变的分子机制,旨在证实“MR-1通过促进肌原纤维生成引起心肌细胞肥大”。
研究方法：
1.抗体制备：生物信息学分析人类MR-1 (hMR-1)优势抗原肽,合成并混合后免疫家兔,抗血清以亲和层析法纯化后检测抗体的特异性及适用范围；质粒构建：根据NCBI GenBank数据库的hMR-1 mRNA信息克隆全长序列至pcDNA3.1-HisB(-)-Myc载体的多克隆位点；RNA干扰实验：根据大鼠源MR-1 (rMR-1)基因序列设计靶点并合成稳定修饰型stealth siRNA,筛选高沉默效率的MR-1-stealth siRNA.
2.乳大鼠心肌细胞培养与心肌细胞肥大模型的建立：培养的乳大鼠心肌细胞以终浓度1×10-7mol／L的血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)孵育48h,以复制AngⅡ致心肌细胞肥大模型；专业图像软件分析细胞平均表面积、[3H]-亮氨酸掺入法检测细胞蛋白合成速率,以及RT-PCR法检测心房利钠因子(atrial natriuretic factor, ANF)、脑钠肽(brain natriuretic peptide, BNP)的转录水平,以证实心肌肥大的形成。
3.肌原纤维生成(myofibrillogenesis)与排列观察：以FITC-鬼笔环肽特异性标记F-actin,观察横纹样F-actin的形成。
4.细胞内信号分子检测：以细胞免疫荧光技术检测分子的亚定位、共定位及转位现象,以RT-PCR和Western Blot方法检测分子在mRNA和蛋白水平的表达。
5.[Ca2+]i和钙调神经磷酸酶(calcineurin, CaN)活性检测：Fluo-3AM荧光探针标记胞浆Ca2+并半定量分析平均荧光强度,比色法检测CaN比活力。
6.图像分析、数据处理及统计学分析：细胞平均表面积、条带积分光密度、[Ca2+]i平均荧光强度以专业图像软件Image Pro-Plus (version 4.11)进行半定量分析；各组实验数据以均数±标准差(x±s)表示,以统计学软件SPSS13.0分析,多组间两两比较用Bonferroni's test,P<0.05认为具有显著性差异。
实验结果
1.制备了兔抗hMR-1多克隆抗体,可识别人源性及鼠源性MR-1抗原,适用于Western Blot和细胞免疫荧光实验。构建了MR-1真核表达质粒并在乳大鼠心肌细胞成功过表达外源hMR-1蛋白。根据沉默效率筛选stealth siRNA并转染乳大鼠心肌细胞,有效沉默了内源rMR-1表达。
2.MR-1过表达引起心肌细胞肥大：细胞表面积、[3H]-亮氨酸掺入和ANF和BNP转录明显增加(P<0.05)；MR-1-RNAi对ANF和BNP的转录水平有所降低而对细胞表面积和蛋白合成速率无显著影响；此外,RNAi对AngⅡ诱导的三项肥大指标增加均明显逆转。
3.MR-1过表达对心肌肌原纤维的影响：正常对照组心肌细胞F-actin呈“非横纹样(nonstriated-like)”和“非肌肉类型(nonmuscule-like pattern) ",主要沿胞膜内侧面分布；过表达MR-1组F-actin呈节段状,为典型的“横纹样”类型,且有25.0±6.7%的细胞肌原纤维呈紊乱排列,提示MR-1过表达促进肌原纤维生成(myofibrillogenesis)及其排列紊乱；MR-1-RNAi组F-actin呈“应力纤维样(stress fiber-like) ",与随机RNA对照组类似,并且MR-1-RNAi明显逆转了AngⅡ诱导的横纹样F-actin形成。
4.对MR-1调节肌原纤维改变的分子机制研究：
1)MR-1与MRLC共定位明显,均分布于胞浆并浓集于核周。在正常培养的乳大鼠心肌细胞中,MR-1与myomesin-1无共定位,但MR-1过表达引起myomesin-1转位至胞浆后,myomesin-1与胞浆的MR-1发生部分共定位。
2)MR-1过表达明显上调myomesin-1和MRLC mRNA及蛋白的表达(P<0.05),MR-1-RNAi对myomesin-1和MRLC表达无显著影响,但逆转了AngⅡ引起的myomesin-1和上调。
3)MR-1过表达促进myomesin-1的核-浆转位,MR-1-RNAi对转位无显著影响,但可在一定程度上抑制AngⅡ引起的myomesin-1转位。
4)过表达SUMO-1引起myomesin-1核-浆转位,促进肌原纤维生成；抑制MR-1明显逆转SUMO-1过表达引起的myomesin-1转位及肌原纤维生成,提示MR-1可能在myomesin-1的SUMO化修饰及SUMO化介导的肌纤生成过程中发挥关键作用。
5)MR-1过表达引起[Ca2+]i、CaN活性、肌细胞增强因子-2C(myocytes enhancer factor 2C, MEF-2C)和磷酸化-MEF-2C水平显著升高(P<0.05),MR-1-RNAi对这些无显著影响(P>0.05),但可逆转AngⅡ引起的升高。
研究结论：
1 MR-1过表达直接诱导乳大鼠心肌细胞肥大。
2 MR-1通过促进肌原纤维生成及排列紊乱致乳大鼠心肌细胞肥大。
3 MR-1通过调节myomesin-1与MRLC影响肌原纤维生成,从而引起心肌细胞肥大。(主要结论)
4 MR-1可能在“Ca2+→CaN→MEF-2C”这一致心肌肥大的经典信号途径中发挥重要作用。
Background
Homologous myofibrillogenesis regulator 1(hMR-1) is a novel characterized human functional gene cloned from human skeletal muscle cDNA library by our research group. It was found that hMR-1 gene locates on the human chromosome 2q35, with a full length of 755 bps and encodes a 142 amino acid-protein. Previous studies suggested that MR-1 found in human myofibrils by immunohistochemistry was highly expressed in myocardium and skeletal muscle, MR-1 is significantly up-regulated in hypertrophied cardiomyocytes induced by angiotensinⅡ(AngⅡ) whereas its silencing using RNAi would abolish the AngⅡ-induced hypertrophy, indicating that MR-1 is involved in hypertrophy. However, whether MR-1 induces hypertrophy directly or not and the mechanism is still unclear. The results from yeast two-hybrid screen and in vitro GST pull-down assay indicated that MR-1 could interact with the contractile apparatus associated proteins, i.e. myosin light chain-2 (MRLC), myomesin-1 andβ-enolase etc. These findings suggested that MR-1 induces cardiac hypertrophy by regulating the myofibrillogenesis.
Objective
In order to validate the hypothesis that MR-1 induces cardiac hypertrophy by promoting myofibrillogenesis, we aimed to verify the direct contribution of MR-1 to hypertrophy in vitro and influence of MR-1 on myofibrillogenesis and myofibrillar orgnization, as well as the regulatory mechnisms. Thus, we were to prepare the anti-MR-1 antibody, to construct the MR-1 eucaryotic expressing vector and to establish the MR-1 silencing method at first.
Methods
1. Antibody preparation; superior epitopes were selected based on the bioinformatics analysis, synthesized and mixed for immunization; anti-serum was purified by immunoaffinity chromatography and the specificity and application were measured as well. Plasmid construction; the full length of hMR-1 mRNA from NCBI GenBank database was cloned to the MCS of pcDNA3.1-HisB(-)-Myc vector. RNA interference (RNAi) assay; The rat original MR-1 (rMR-1) stably-modified stealth siRNA was designed, synthesized and then selected according to the silencing effect.
2. Neonatal rat cardiomyocytes culture and hypertrophy model in vitro; cardiomyocytes were cultured and incubated with angiotensin II (Ang II) 1x10-7 mol/L for 48h to develop hypertrophy, to validate the effect, hallmarks i.e. mean area of cell surface analyzed by professional software, protein synthesis velocity assessed by [3H]-Leucine incorporation and mRNA levels of atrial natriuretic factor and brain natriuretic peptide measured by RT-PCR were all employed.
3. Investigation on myofibrillogenesis and myofibril-alignment; striated-like F-actin pattern which indicated the myofibrillogenesis was characterized by specific staining with FITC-phalloidin.
4. Stuadies on interesting proteins; the sublocation, collocation and translocation of thosed molecules were detected using immunocytofluorescent assay, whereas their expression at mRNA and protein levels were measured using RT-PCR and Western Blot, respectively.
5. Measurement of [Ca2+]i and activity of calcineurin (CaN); cytoplasmic free Ca2+ was probed by fluo-3AM whereafter its fluorescent density was semi-quantitatively analyzed; enzymatic activity of CaN was measured by chromatometry.
6. Image analysis, data management and statistics analysis; semi-quantitative analysis on mean cell surface area, integrated optical density of bands and fluorescent density of [Ca2+]i were performed with software Image Pro-Plus (version 4.11), experimental data were represented by mean±standard deviation ((?)±s). SPSS 13.0 statistics software was introduced. Bonferroni's test was employed to analyze statistical differences between every two-sample among several groups. P<0.05 was considered statistically significant.
Results
1. The rabbit anti-hMR-1 polyclonal antibody detects either human original or rat original epitopes, available for both Western Blot and immunocytofluorescent assays were successfully prepared; the hMR-1 eukaryotic expression plasmid pcDNA3.1-Myc/HisB(-)-hMRl for exogenous hMR-1 overexpression in neonatal rat cardiomyocytes was constructed; the stealth siRNA selected according to its silencing effect was transfected to cardiomyocytes and successfully inhibited the expression of endogenous rMR-1.
2. The mean surface area, incorporation of [3H]-Leucine and transcriptional levels of ANF and BNP were all significantly increased with MR-1-overexpression (P<0.05); MR-1-RNAi decreased the transcription of ANF and BNP (P<0.05) instead of the mean surface area and protein synthesis. (P>0.05) In particular, it would reverse the Ang II-induced increases on those three hypertrophic hallmarks.
3. Cardiomyocytes in the normal culture exhibited a nonstriated and nonmuscle-like F-actin, which mainly distributed along the internal surface of the cellular membrane, whereas a quite different organization that periodic and characteristic striated-like F-actin was observed in MR-1-overexpressed myocytes,25.0±6.7% of which, however, exhibited an irregularly organized F-actin, suggesting a promoted but derangement myofibrillogenesis. MR-1-silenced myocytes showed a stress fiber-like F-actin, which is similar to the random-RNAi control, otherwise, MR-1-RNAi would abolish the striated-like F-actin induced by AngⅡ.
4. MR-1-involved regulation in myofibrillogenesis has been recognized detailed as follows;
1) Whereas MRLC collocates with MR-1 extensively in cytoplasm, especially the peri-nucleus area where they both enriched, the nucleus-located myomesin-1 does not, unless its translocation is initiated that myomesin-1 shifts to cytoplasm where its partial collocation with MR-1 was found.
2) Myomesin-1 and MRLC were significantly increased at mRNA and protein levels in MR-1-overexpressed culture; MR-1-RNAi does not affect their expression instead of the AngⅡ-induced upregulation.
3) MR-1-overexpression promotes myomesin-1 shuttling from nucleus to cytoplasm, this phenomenon would not be affected by MR-1-RNAi, but RNAi may reverse the AngⅡ-induced translocation of myomesin-1.
4) Overexpression of SUMO-1 promotes myomesin-1 translocation and the translocation involved myofibrillogenesis, which provide evidence that MR-1 may serve a crucial role in the SUMOylated modification of myomesin-1 and SUMOylated myomesin-1 mediated myofibrillogenesis.
5) [Ca2+]i, CaN-activity, myocytes enhancer factor 2C (MEF-2C) and phospho-MEF-2C levels were found significantly increased in MR-1-overexpressed myocytes (P<0.05) but not any significant disparity in MR-1-RNAi culture (P>0.05); nevertheless, RNAi applied myocytes would reverse the AngⅡ-induced upregulation of those molecules.
Conclusion
1. Overexpression of MR-1 directly induces hypertrophy in neonatal rat cardiomyocytes.
2. MR-1 induces hypertrophy in neonatal rat cardiomyocytes by promoting myofibrillogenesis and the myofibril-alignment.
3. MR-1 induces hypertrophy in cardiomyocytes through regulating myomesin-1 and MRLC mediated myofibrillogenesis. (major conclusion)
4. MR-1 may play an important role in the classic cardiac hypertrophy "Ca2+→CaN→MEF-2C" signaling pathway.
引文
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[2]Hempelmann A, Kumar S, Muralitharan S et al. Myofibrillogenesis regulator 1 gene (MR-1) mutation in an Omani family with paroxysmal nonkinesigenic dyskinesia [J]. Neurosci Lett,2006,402:118-120.
[3]王晓礽,刘秀华.人肌纤生成调节因子1研究进展[J].生理科学进展．2009；40(3)：245-248.
[4]Lee HY, Xu Y, Huang Y et al. The gene for paroxysmal non-kinesigenic dyskinesia encodes an enzyme in a stress response pathway [J]. Human Molecular Genetics,2004,13 (24):3161-3170.
[5]Li TB, Hu Y, Feng S, Zuo ZY, Wang YG. Expression of a human myofibrillogenesis regulator 1 gene in E. coli and its immunogenicity [J]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao,2005; 27(1):42-47.
[6]冀瑞平,王迪浔.心肌细胞肥大的病理生理研究进展[J].心肺血管病杂志，2000第2期.
[7]Liu XH, Li TB, Sun S, Xu FF, Wang YG. Role of myofibrillogenesis regulator-1 in myocardial hypertrophy [J]. Am J Physol Heart Circ Physiol,2006; 290:279-285.
[8]刘海英,杨微,武正华等.细胞色素P450 3A4抗原表位的分析及其抗体的制备与鉴定[J].细胞与分子免疫学杂志，2007,23(1)：79-81.
[9]Sharma A, Trivedi NR, Zimerman MA, Tuveson DA, Smith CD, Robertson GP. Mutant V599E B-Raf regulates growth and vascular development of malignant melanoma tumors [J]. Cancer Res,2005; 65(6):2412-2421.
[10]Liu XH, Wu XD, Cai LR et al. Hypoxic preconditioning of cardiomyocytes and cardioprotection:phosphorylation of HIF-la induced by p42/p44 mitogen-activated protein kinases is involved [J]. Pathophysiology,2003; 9 (4):201-205.
[11]Kodama H, Fukuda K, Pan J, Makino S, Baba A, Hori S, and Ogawa S. Leukemia inhibitory factor, a potent cardiac hypertrophic cytokine, activates the JAK/STAT pathway in rat cardiomyocytes [J]. Circ Res,1997; 81(5):656-663.
[12]Bradford MM. A rapid and sensitive method for the quantitation of microgram of protein utilizing the principle of protein-dye binding [J]. Anal Biochem,1976; 72:248-254.
[13]王晓礽,刘秀华,李婷,宋泉声,韩文玲.人肌纤生成调节因子-1抗体的制备、鉴定及其在乳鼠心肌细胞中的应用.中国病理生理杂志,2010,26(1)：42-47.
[14]Hayashi D, Kudoh S, Shiojima I et al. Atrial natriuretic peptide inhibits cardiomyocyte
hypertrophy through mitogen-activated protein kinase phosphatase-1 [J]. Biochem Biophys Res Commun.2004,322:310-319.
[15]Barry SP, Davidson SM, Townsend PA. Molecular regulation of cardiac hypertrophy [J]. IntJBiochem Cell Biol,2008,40:2023-2039.
[16]Lloyd CM, Berendse M, Lloyd DG et al. A novel role for non-muscle g-actin in skeletal muscle sarcomere assembly [J]. Exp Cell Res,2004,29782-29796.
[17]Du A, Sanger JM, Linask KK et al. Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm [J]. Dev Biol,2003; 257(2):382-394.
[18]Reddy KB, Fox JE, Price MG et al. Nuclear localization of Myomesin-1:possible functions [J]. J Muscle Res Cell Motil,2008; 29 (1):1-8.
[19]Potthoff MJ, Arnold MA, McAnally J et al. Regulation of skeletal muscle sarcomere integrity and postnatal muscle function by Mef2c [J]. Mol Cel Bio,2007,8143-8151.
[20]吴秀山主编.信号调控与心脏发育[M].化学工业出版社／现代生物技术与医药科技出版中心.2006.238-248.
[21]Li J, Puceat M, Perez-Terzic C et al. Calreticulin reveals a critical Ca2+ checkpoint in cardiac myofibrillogenesis [J]. J Cel Bio.2002,158 (1):103-113.
[22]Li HL, She ZG, Li TB et al. Overexpression of myofibrillogenesis regulator-1 aggravates cardiac hypertrophy induced by angiotensin Ⅱ in mice [J]. Hypertension.2007; 49(6): 1399-1408.
[23]Nicholls MG. Hypertension, hypertrophy, heart failure [J]. Heart,1996,76 (3 Suppl 3): 92-97.
[24]Gradman AH, Alfayoumi F. From left ventricular hypertrophy to congestive heart failure: management of hypertensive heart disease [J]. Prog Cardiovasc Dis,2006,48 (5): 326-341.
[25]Sanger JW, Chowrashi P, Shaner NC et al. Myofibrillogenesis in Skeletal Muscle Cells [J]. Clinical Orthopaedics and related research,2002,403S:S153-S162.
[26]Auerbach D, Bantle S, Keller et al. Different domains of the M-Band protein myomesin are involved in myosin binding and M-band targeting [J]. Mol Biol Cell,1999; 10(5): 1297-1308.
[27]Fukuzawa A, Lange S, Holt M. Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band-Implications for hereditary myopathies [J]. J Cell Sci, 2008; 121 (Pt 11):1841-1851.
[28]Grove BK, Kurer V, Lehner C et al. A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies [J]. J Cell Biol,1984; 98 (2):518-524.
[29]Vinkemeier U, Obermann W, Weber K et al. The globular head domain of titin extends into the center of the sarcomeric M band. cDNA cloning, epitope apping and immunoelectron microscopy of two titin-associated proteins [J]. J Cell Sci,1993; 106(Pt
1):319-330.
[30]Price MG, Gomer RH. Skelemin, a cytoskeletal M-disc periphery protein, contains motifs of adhesion/recognition and intermediate filament proteins [J].J Biol Chem,1993; 268 (29):21800-21810.
[31]Bantle S, Keller S, Haussmann I et al. Tissue-specific isoforms of chicken myomesin are generated by alternative splicing [J]. J Biol Chem,1996; 271(32):19042-19052.
[32]Bertoncini P, Schoenauer R, Agarkova I et al. Study of the mechanical properties of myomesin proteins using dynamic force spectroscopy [J], J Mol. Biol,2005; 348 (5): 1127-1137.
[33]Obermann WMJ, van der Ven PFM, Steiner F, Weber K and Furst DO. Mapping of a myosin-binding domain and a regulatory phosphorylation site in M-protein, a structural protein of the sarcomeric M band [J]. Mol Biol Cell,1998; 9(4):829-840.
[34]Deshmukh L, Tyukhtenko S, Liu J et al. Structural insight into the interaction between platelet integrin {alpha} IIbbeta3 and cytoskeletal protein skelemin [J]. J Biol Chem, 2007; 282(44):32349-32356.
[35]Wang SM, Lo MC, Shang C et al. Role of M-Line proteins in sarcomeric titin assembly during cardiac myofibrillogenesis [J]. J Cell Biochem,1998; 71(1):82-95.
[36]Young P, Ehler E, Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly [J]. J Cell Biol,2001; 154 (1): 123-136.
[37]Borisov AB, Kontrogianni-Konstantopoulos A, Bloch RJ et al. Dynamics of obscurin localization during differentiation and remodeling of cardiac myocytes:obscurin as an Integrator of myofibrillar structure [J]. JHistochem Cytochem,2004; 52(9):1117-1127.
[38]McElhinny AS, Perry CN, Witt CC, Labeit S, Gregorio CC. Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development [J]. J Cell Sci,2004; 117 (15): 3175-3188.
[39]Lange S, Agarkova I, Perriard JC, Ehler E. The sarcomeric M-band during development and in disease [J]. J Muscle Res Cell Motil,2005; 26(6-8):375-379.
[40]韦玮,张浩,毛建平,叶棋浓。蛋白质SUMO化修饰研究进展[J]。中国生物工程杂志(China Biotechnology),2008,28 (7):122-126.
[41]Duda DM, Waardenburg RCAM, Borg LA et al. Structure of a SUMO-binding-motif Mimic Bound to Smt3p-Ubc9p:Conservation of a Non-covalent Ubiquitin-like Protein-E2 Complex as a Platform for Selective Interactions within a SUMO Pathway [J]. J Mol. Biol.2007,369:619-630.
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