心脏特异性新激酶TNNI3K在心肌细胞缺氧/缺血损伤时的作用及机制
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摘要
本协作研究小组成员在构建正常成人主动脉和心脏cDNA文库并进行大规模ESTs(Expressed Sequence Tags)测序的基础上,在1号染色体短臂3区(lp31.1)发现了一个可能与细胞骨架调节信号传导通路相关的新基因。在前期的研究中1、酵母双杂交结果表明它能与心肌肌钙蛋白I(cardiac troponin I,cTnI)、心肌肌动蛋白(α-actin)等一系列肌小节收缩调节蛋白发生作用,故而正式命名为TNNI3K(cTnI-interacting kinase);2、Northern Blot和多组织点阵杂交表明它只在心脏部位特异性表达;3、体外激酶活性分析显示它是具有功能活性的有丝分裂原激活蛋白激酶激酶激酶(MAPKKK);4、被Manning编录的人类蛋白激酶基因组收录,在其绘制的系统进化树(www.kinase.com/human/ kinome)中,TNNI3K(原称HH498)的序列号为SK494,体现了对这一基因鉴定工作的充分肯定。作为一个新认识的心脏特异性表达的新激酶家族成员,其调节底物、作用机制、以及对病理条件下心血管系统产生的生物学功能及其有关信号传导通路都还不清楚。
为深化上述研究思路,本课题设计通过细胞分子学(腺病毒载体基因转导原代培养心肌细胞)、动物实验(基因修饰干细胞治疗)及临床患者(缺血性心脏病血清学检查)三个层面,从分子生物学的微观改变和病理生理、组织结构功能及生化指标的宏观变化,多角度探讨TNNI3K在心肌缺氧/缺血损伤时的意义,以深入了解这一新的激酶的功能作用及可能机制。该课题所做工作为源头创新,国内外未有报道。
第一部分:TNNI3K对缺氧所致心肌损伤的影响及可能机制
目的:
利用体外培养心肌细胞并结合缺氧造成损伤模型,观察不同缺氧时间段(0h、3h、6h、12h、24h)TNNI3K水平的变化;进而利用腺病毒转导心肌细胞使得TNNI3K基因高表达,从而研究该基因对缺氧造成心肌损伤的影响,并探讨产生作用的有关信号转导通路。
方法:
新生乳鼠原代心室肌细胞由胰酶消化法获得,缺氧环境在密闭的AnaeroPack方盒(Mitsubishi Gas Chemical Co, Inc)中经一次性缺氧催化袋GENbox anaer (BioMérieux?sa, France)(它能吸收O2 ,产生CO2)产生。实验分三个部分1、观察本实验设计的缺氧模型/无血清培养0h、3h、6h、12h、24h五个时间点心肌细胞损伤的情况:倒置显微镜下观测各组心肌细胞搏动的频率和节律,全自动生化分析仪测定心肌细胞培养液乳酸脱氢酶(LDH)活性,以确立损伤模型的建立;Western blot法半定量TNNI3K基因的表达,明确缺氧损伤对TNNI3K基因的表达是否具有影响,这是确立整篇研究的基础。2、腺病毒载体TNNI3K基因转导在缺氧/无血清干预造成原代大鼠心肌细胞损伤中的作用。用本组前期包装和扩增的绿色荧光蛋白(GFP)标记Ad-TNNI3K(携带TNNI3K基因的腺病毒)、Ad- TNNI3K-AS(携带反义TNNI3K基因腺病毒)和Ad-C(Ad-LacZ,即空载体腺病毒)瞬时转导原代培养心肌细胞,观察TNNI3K基因对缺氧导致的细胞病理损伤包括LDH活性、心肌细胞搏动的频率和节律、细胞存活率(MTT法)等的影响,并用Hoechst 33258染色和DeadEndTM Colorimetric TUNEL System检测各组细胞凋亡及凋亡指数。3、探讨TNNI3K基因高表达抗缺氧/无血清诱导心肌细胞损伤或凋亡的机制。用Western blot法半定量磷酸化细胞外信号调节激酶C(phosphorylated extracellular signal-related kinases, p-ERK)、c-jun氨基末端激酶(phosphorylated c-Jun N- terminal kinases,p-JNK)和p38(p- p38)激酶的表达,观察MAPK信号通路中三条主要途径的参与作用;同时,选择缺氧12h为观察时间点,用酶标仪法测Caspase-3活性,免疫细胞化学和激光共聚焦显微镜检测不同组别缺氧前后细胞色素C在细胞胞浆和线粒体中的定位改变,Rhodamine 123染色流式细胞仪测定线粒体膜电位,以及Western blot法半定量致凋亡蛋白Bax的表达,共同阐述TNNI3K基因对线粒体信号通路的作用。
结果:
1、利用AnaeroPack缺氧盒,配合无血清DMEM培养成功建立缺氧模型,缺氧0.5h后,细胞培养液中的氧分压达40mmHg以下,氧饱和度20%左右,达到缺氧实验所要求的标准。缺氧6h可见心肌细胞搏动频率减慢,节律出现不规整,至缺氧24h绝大多数细胞已无搏动,极少数浅慢搏动;培养液LDH水平在缺氧6h以后即有显著性升高(P<0.05),24h更甚(P<0.01),提示细胞出现损伤性改变;同时TNNI3K蛋白表达在缺氧早期(3h)即上调,12h达到高峰,24h后渐回落,提示TNNI3K在心肌细胞缺氧应激时可能发挥作用。
2、利用腺病毒作为载体,成功转导原代心肌细胞,缺氧可使心肌细胞存活率下降,但在12h和24h时Ad-TNNI3K、Ad-TNNI3K-AS两组的细胞生存率优于Ad-C组,前两组之间无显著性差异;缺氧12h后,Ad-C、Ad-TNNI3K、Ad-TNNI3K-AS三组均见LDH升高,而Ad-TNNI3K组要明显低于Ad-C、Ad-TNNI3K-AS两组,后两组之间比较,Ad-TNNI3K-AS组又明显低于Ad- C,表明TNNI3K高表达对细胞的保护作用;同时,Hoechst 33258染色和TUNEL检测显示,Ad- TNNI3K基因高表达能使缺氧12和24h的凋亡指数均低于Ad-C组,提示TNNI3K基因具有抗细胞凋亡的作用。由于反义TNNI3K组的作用介于TNNI3K和Ad-C组之间,有时和TNNI3K组无明显差异,推测反义TNNI3K未能抑制大鼠心室肌细胞内TNNI3K同源基因的表达,一方面可能是因为种属差异,另一方面,可能是由于反义核酸技术本身存在作用的不可预知性的问题,故在有关机制的研究中只对Ad-C组和Ad- TNNI3K两组进行了分析。
3、对TNNI3K产生作用的机制研究表明,(1)MAPK信号通路中,缺氧前Ad-C组和Ad- TNNI3K两组心肌细胞即有较低水平的ERK1/2和JNK磷酸化,两组之间无显著差异。缺氧3h后,Ad-C组p-ERK1/2表达水平明显升高,持续到6h,而Ad-TNNI3K组改变不明显,两组间比较显见不同(p<0.05);但到12h、24h则两组均趋于下降,组间差异不明显。p-JNK与上述不一样,Ad-C组在缺氧3h时一度轻度上升,但随即处于极低表达状态,Ad- TNNI3K组则升高更加明显,且高峰持续至缺氧12h,于6h和12h两个时间点两组差别极显著(P<0.01),至24h时两组均处于很低水平。而反复行western blot实验,在38KD附近检测到的杂交信号非常弱,无法判别所需的目的条带,推测在本研究设计的缺氧干预腺病毒转导心肌细胞中,p38蛋白基本没有被磷酸化,或磷酸化的水平很低。(2)高表达TNNI3K基因能够抑制缺氧各时间点Caspase-3的激活程度,至缺氧12h、24h差异显著;能明显减轻由缺氧12小时导致的细胞色素C从线粒体到胞浆的释放水平,抑制凋亡蛋白Bax的表达;提示TNNI3K基因的抗凋亡作用通过线粒体信号途径。
结论:
在体外培养的原代心肌细胞实验中,首次证明心脏特异性表达的新激酶基因TNNI3K具有抗缺氧造成的细胞损伤和凋亡作用。抑制MAPK级联信号通路中ERK1/2的早期、瞬间磷酸化激活以及提高JNK1/2的磷酸化水平;同时,抑制线粒体信号途径中Caspase-3的激活和细胞色素C的释放,以及凋亡蛋白Bax的表达是其发挥抗凋亡作用的机制。
第二部分:心外膜心肌注射TNNI3K对心肌梗死后兔心功能的影响
目的:
观察整体动物急性心肌梗死后,通过使局部高表达TNNI3K,能否改善心肌重塑而达到减轻心功能损害。
方法:
P19CL6细胞是鼠科胚胎瘤(embryonal carcinoma,EC)P19细胞的衍生物,P19细胞是一种来源于鼠科畸胎瘤的未分化干细胞,P19CL6细胞形态上与之相似,但不表达特异性的EC标记物SSEA-1抗原,已经证实在加入DMSO作为诱导剂的培养基中,它能有效分化成表达心脏特异基因的搏动的心肌细胞,同时具有心肌细胞的电生理特性。利用本协作研究组成员在前期工作中分别获得的质粒载体高表达TNNI3K转染细胞株(P19CL6/TNNI3K)和FLAG多肽标签质粒转染P19CL6细胞株(P19CL6/FLAG),作为本实验待用的基因修饰干细胞。实验共分四组(i)假手术对照组(Control组)(ii)单纯心肌梗死+生理盐水注射组(NaCl组)(iii)心肌梗死+ P19CL6/ pCDNA6-FLAG注射组(FLAG组)(iv)心肌梗死+ P19CL6/ pCDNA6-FLAG -TNNI3K注射组(TNNI3K组)。1、结扎兔左冠状动脉回旋支的左室支造成心肌梗死模型;2、体外用1%DMSO诱导P19CL6/TNNI3K或P19CL6/FLAG两株细胞5天后,重新计数细胞,分别收集4x105个细胞,生理盐水200μl重悬,置于皮试用1ml注射器中,在兔梗死与正常心肌交界部位暗红色的充血带四周分6点注射,每点30μl;3、彩色多普勒超声心动图观测上述四个实验组第四周末心脏左室舒张末径(LVEDD)、左室收缩末径(LVESD)、左室壁和室间隔厚度,计算左室射血分数(LVEF);4、4道生理记录仪经颈动脉插管检测上述四个实验组第四周末血流动力学指标,包括左室舒张末压(LVEDP)、左室收缩末压(LVESP)、左室内压最大上升速率(+dp/dtmax)和左室内压最大下降速率(-dp/ dtmax);5、第四周末取四组含梗死区心肌组织,切片,HE(苏木精-伊红)染色,观察病理形态学差别。
结果:
P19CL6-TNNI3K和P19CL6-FLAG两株细胞在1%DMSO诱导后,均在大约10-12天,局部开始出现收缩,到约12-16天,几乎所有的片层出现同步的搏动。成功造成兔心肌梗死,按分组观察,术前、术后各组兔心率之间无明显差异。术后四周超声心动图显示,心梗各组心功能均较假手术组显著降低,但TNNI3K组心功能下降的程度显然轻于NaCl及FLAG两组,FLAG组和NaCl两组间无显著差异。比较左室壁和室间隔厚度,NaCl和FLAG两组可见有不同程度变薄,而TNNI3K组则与对照组相仿,组间差异显著。提示TNNI3K基因治疗能减轻心肌重构,改善心功能。进一步通过4道生理记录仪经颈动脉插管检测血流动力学指标,可见心肌梗死各组均有LVEDP明显升高,而LVESP和±dp/dtmax则均明显下降,提示左室收缩和舒张功能的减退,TNNI3K组的变化处于control和FLAG/ NaCl两者之间,但优于FLAG或NaCl组,后两组间无显著差异。心脏组织切片HE染色显示TNNI3K组心肌纤维化程度明显轻于NaCl和FLAG两组。
结论:
急性心肌梗死后,经开胸直接心外膜心肌局部注射使高表达TNNI3K基因,可有效改善梗死后的心脏重塑,减轻心肌纤维化,减少心脏舒缩功能的损害。
第三部分:缺血性心脏病TNNI3K的血清学变化及临床意义
目的:
建立一个准确性高、可信度大、重复性好的ELISA实验条件,在此基础上检测正常人、急性心肌梗死、心肌梗死后心脏重构或缺血性心肌病出现心力衰竭、以及扩张性心肌病心力衰竭患者的TNNI3K血清水平并比较其差异,从临床角度探讨TNNI3K在缺血性心脏病中的作用及其临床意义。
方法:
1、从三株纯化的单克隆抗体中选定最合适的一株,并确定使用浓度,同时确定合适的血清样本与二抗的稀释倍数;建立纯化的TNNI3K蛋白的标准曲线;通过批内和批间重复性实验建立一个准确性高、可信度大、重复性好的ELISA实验条件;2、选择既往无心血管疾病的正常献血员为正常对照;冠状动脉粥样硬化性心脏病(CHD),包括急性心肌梗死(AMI)和缺血性心肌病心力衰竭(CHD/HF)(EF≤45%),扩张型心肌病(DCM)心力衰竭(EF≤45%)患者,均根据病史、症状、体征、客观检查(包括实验室检查、X线胸片、心电图、超声心动图、核素、核磁、冠状动脉造影等)综合作出确诊。抽取受试者清晨空腹肘静脉血2ml,不抗凝,2500 rpm/min离心10min,取血清分装,-20℃冻存备用。TNNI3K水平用上述建立的直接ELISA法测定;3、比较入选不同患者组间血清TNNI3K水平的差异及其临床意义。
结果:
1、正常对照和入选患者血清中均可检测到TNNI3K抗原。但无论CHD或DCM组,TNNI3K血清水平均高于正常对照(P<0.01),两类患组之间差异无统计学意义。2、在CHD患者中,比较不同发病时期AMI和CHD/HF两组血清TNNI3K浓度无明显不同;3、尽管缺血性心肌病和扩张型心肌病引起的心力衰竭发病机制不同,两组血清TNNI3K浓度亦无统计学差异。
结论:
TNNI3K基因与冠状动脉粥样硬化性心脏病密切相关。但以急性心肌梗死和以心力衰竭为临床发病的冠心病不同类型之间其血清水平并无差别;冠心病导致的心力衰竭和扩张型心肌病心力衰竭对TNNI3K血清水平的影响相似。
While constructing human adult heart and aorta cDNA libraries and making large-scale ESTs(Expressed Sequence Tags) sequencing, our research group members identified a novel gene localized on 1p31.1 based on bioinformatics analyses which might interact with cytoskeleton regulating signal pathway. A yeast two-hybrid screen proved it could interect with a series of sarcomeric regulating protein among which the most frequent candidate was cTnI and then was named TNNI3K (cTnI-interacting kinase) (GenBank accession no. AF116826), results that were further confirmed by co-immunoprecipitation in vivo. Multiple fetal and adult northern blot and 76-tissue array suggest TNNI3K is cardiac-specific gene and was undetectable in other tissues. In vitro kinase assays using immunoprecipitates of the series of FLAG-tagged TNNI3K mutants which found that TNNI3K not only autophosphorylated but also phosphorylated tyrosine substrate myelin basic protein (MBP) demonstrated that TNNI3K is a functional protein kinase. Phylogenetic parsimony analysis further clearly indicates TNNI3K belong to MAPKKKs superfamily in the evolutionary tree.
A great deal pre-work indicated that TNNI3K, as a novel cardiac-specific protein kinase, has functional activities. But its downstream targets, acting mechanism and biological signal pathway under pathological stress still remain poorly understood. So further study in this work will be carried out from molecular cytological level (primary cultured neonatal rat cardiomyocytes transducted with adenovirus carrying/ non-carrying TNNI3K),animal model and histological level (treating with TNNI3K modified stem cells),and then clinical serological level to clarify its biological significance under hypoxia/ischemia intervention.
All researches in this work are original novel and never been reported.
Part I: Effects and mechanism of TNNI3K on hypoxia induced cardiocyte injury
Objective:
First time to prove whether the TNNI3K levels could be changed with different hypoxia time periods(0h,3h,6h,12h and 24h)in vitro cultured primary cardiomyocytes. Then the effects of TNNI3K over-expression as a result of adenovirally mediated gene transfer on neonatal rat cardiac myocyte injury induced by hypoxia were investigated and its relative signal transduction pathway was elucidated.
Methods:
Primary ventricular cardiomyocytes were prepared from neonatal rats by collagenase digest method. Hypoxia was achieved by using an anaerobic jar (AnaeroPack Series, Mitsubishi Gas Chemical Co, Inc) equipped with an AnaeroPack, disposable O2-absorbing and CO2-generating agent, and an indicator to monitor oxygen depletion. Whole experiment contains three parts:1.Cell injury induced by hypoxia/serum free culture was observed at different time periods (0h,3h,6h,12h,24h). The cardiomyocytes beating rate and rhythm in each group were documented under inverted microscope and lactate dehydrogenase(LDH) in cultured cell medium was assayed using automatic biochemical analysis instrument(Beckman,USA) in order to confirm the successfully established cell injury model. Then western blot was used to semi-quantitate TNNI3K gene expression whose alteration is the base for all next work.2.Effect of TNNI3K on neonatal rat cardiac myocyte injury induced by hypoxia/serum-free. Primary cultured neonatal rat cardiomyocytes were transiently transducted with Ad-C (adenovirus-LacZ as control), Ad-TNNI3K(adenovirus carrying sense of TNNI3K) and Ad-TNNI3K-AS (adenovirus carrying anti-sense of TNNI3K) in which all were labelled with green fluorescent protein(GFP).The effects of TNNI3K gene overexpression on neonatal rat cardiac myocytes injury induced by hypoxia/serum-free were probed by evaluating LDH release,cells beating rate and rhythm and viability(assessed by MTT assay). Cell apoptosis was determined by both Hoechst 33258 staining and DeadEndTM Colorimetric TUNEL System.3.The action mechanism of TNNI3K overexpression on cardiomyocytes apoptosis induced by hypoxia/serum-free. Evaluation of the participative roles of three main classic MAPK pathways activation using semi-quantitating phosphorylated extracellular signal-related kinase( p-ERK), phosphorylated c-Jun N- terminal kinase(p-JNK) and phosphorylated p38(p-p38) by western blot. Furthermore,to explore the mitochondrial apoptotic signal pathway effects of TNNI3K at 12h exposing to hypoxia through measuring Caspase-3 activity by Microplate Reader, the release of cytochrome C from mitochondrial to cytoplasm using immunochemical method combining laser scanning confocal microscope(LSCM),change of cell mitochondrial membrane potential( m) which staining with Rhodamine 123 assessing by FACS(flow cytometry) and proapoptotic protein Bax expression by western blot.
Results:
1.The AnaeroPack jar is capable of depleting the concentration of O2 down to PO2<30mmHg or SO2 about 20% in 1 hour which successfully meets our experimental requirement in this study.After 6hs of hypoxia,cardiomyocytes beated slower than before exposing to hypoxia and the beating rhythm became irregular which still worsen and almost no-beating for majority of the cells after 24hs hypoxia.LDH level in medium increased significantly at 6hs post hypoxia(P<0.05)and much dramatically at the 24h which suggest the injury of the cells. The elevation of TNNI3K protein expression occurred at early hypoxia time(3h) and peaked at 12h then went down slowly at 24h post hypoxia.
2.All Adenovirus-mediated transduction cells divided into Ad-C、Ad-TNNI3K and Ad-TNNI3K-AS groups. After exposing to hypoxia 12 h and 24h, cell viability decreased in three groups but this was much better in Ad-TNNI3K and Ad-TNNI3K- AS groups than that in Ad-C group while there was no significant difference between the former two. Furthermore,12h- and 24h- hypoxia increased medium LDH levels in all three groups but those were relatively much lower in group Ad-TNNI3K than those in groups Ad-C and Ad-TNNI3K-AS even though when compared between them those in Ad- TNNI3K-AS group showd significant lower than in Ad-C group. Overexpress- ion of TNNI3K in Ad-TNNI3K group also decreased apoptosis at 12h and 24h by Hoechst 33258 staining which showed less chromatin condensation and aggregation under the nuclear membrane and less nuclear fragmentation and apoptosis body formation. Apoptosis index calculation by DeadEndTM Colorimetric TUNEL System assay was also in accordance with this result. All above revealed the very important cytoprotective and anti-apoptotic effect of TNNI3K gene on hypoxia induced injury. The paradoxical results of Ad-TNNI3K-AS group whose activity was sometimes between group Ad-TNNI3K and Ad-C whereas sometimes no significant difference compared with Ad- TNNI3K group possibly due to the species difference becouse the anti-sense TNNI3K deriving from human sequence might not inhibit the homologous gene of TNNI3K in rat ventricular myocyte or probably due to the unpredictable problems of anti-sense nucleotide technique per se.So,in all next studies on mechanism, we only focused in Ad-C and Ad- TNNI3K groups.
3. Based on western blot analysis,we delineate the MAPKs signaling pathway roles in hypoxia induced apoptosis. The results indicted that (1) only ERK1/2 and JNK signal pathways involve in TNNI3K functional effect responding to hypoxia treatment. There were low expression levels of p-JNK and p-ERK1/2 in both Ad-C and Ad- TNNI3K groups at 0h hypoxia. After 3h hypoxia, p-ERK1/2 in Ad-C group signifiantly increased and still higher at 6h of hypoxia while which couldnot be seen in Ad-TNNI3K group(p<0.05),then followed by a decline in both groups and made no difference between them at 12 or 24h.For p-JNK, the pattern of change was considerably different to that seen for p- ERK1/2.Levels in Ad-C group initially slightly increased at 3h and then maintained very low throughout the time course while that in Ad- TNNI3K group increased at 3h and 6h and peaked at 12h making significant difference between two groups at 6 and 12h(P<0.01) and then kept quite low levels in both groups at 24h.(2)High expression of TNNI3K could inhibit activation of caspase-3 and alleviate the release of cytochrom C from mitochondria to cytoplasm and reduce the expression of proapoptotic protein Bax.All above results suggest that the antiapoptotic effect of TNNI3K might be through mitochondria dependent signal pathway.
Conclusion:
In vitro primary cultured neonatal rat cardiomyocytes experiment,we first approved that cardiac-specific gene TNNI3K has the protective effect on cell injury and apoptosis induced by hypoxia.This function might be through inhibiting the early stage and trensient activation of p- ERK1/2 and increasing the expression of p- JNK in MAPK cascade signal pathway.The anti-apoptotic mechanism was motochondria dependent through inhibiting the activation of caspase-3 and reducing the release of cytochrom C from mitochondria to cytoplasm and the expression of proapoptotic protein Bax.
Part II: Effect of Intramyocardial Administration of TNNI3K on Rabbit Post- myocardial infarction Cardiac Function
Objective:
To observe the effect of intramyocardial injection of TNNI3K on cardiac function in a rabbit model of infarction.
Methods:
P19CL6,a derivation of mouse embryonal carcinoma(EC) P19 cells which is an undifferentiated stem cell derived from murine teratocarcinoma, is morphologically similar to P19 cells but does not express SSEA-1 (stage specific embryonic antigen-1) antigen, a specific EC marker.It is known that P19CL6 cells efficiently differentiate into beating cardiomyocytes expressing cardiac- specific genes after culture in the presence of an inducer, dimethylsulfoxide (DMSO). The vector pcDNA6-FLAG /TNNI3K was obtained from our prophase work and the stable transfected P19CL6 cells with pcDNA6-FLAG(FLAG only) or pcDNA6-TNNI3K were endowed by our Japanese cooperator.Briefly, the vectors pcDNA6-FLAG (FLAG only) and pcDNA6- FLAG/TNNI3K were transfected into P19CL6 cells using electropoie 2000 (Invitro- gen) and maintained in medium containing 500μg/ml blasticidin (Invitrogen). Acute myocardial infarction (AMI) was produced in rabbits using ligation of left ventricular branch of left circumflex coronary.After 5 days being induced by 1%DMSO, early differentiated P19CL6 cells with or without high expression of TNNI3K were collected respectively and resuspended with normal saline. 24 experimental rabbits were randomly divided into four groups(i) Sham operation control group,n=6(C) ; (ii)AMI+ intramyocardial normal saline injection group,n=6 (NaCl);(iii) AMI+ intramyocardial pcDNA6-FLAG injection group, n=6 (FLAG); (iiii) AMI+ intramyocardial pcDNA6-FLAG/ P19CL6 injection group,n=6 (P19CL6). 4 weeks after operation ,left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular wall thickness and interventricular septum thickness were measured by Color Doppler Eechocardiography and then left ventricular ejection (LVEF) was calculated. Haemodynamics parameters including left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP),maximum LV pressure rising velocity(+dp/dtmax) and maximum LV pressure decreasing velocity(-dp/dtmax) were recorded with an electrophysiolograph and then the hearts were excised. Specimens were coded and fixed in 10% buffered formalin for subsequent histopathological analyses using hematoxylin and eosin stain (HE stain).
Results:
Around 10 to 12 days differentiation induced by 1%DMSO, P19CL6-TNNI3K and P19CL6-FLAG cells which seeded at 4 x105 per 60mm dish contracted first at a restricted area then on days 12 to 16 almost all of the sheet beated synchronously. Myocardial infarction was successfully produced in rabbits using coronary artery ligation. NaCl group received an intramyocardial injection of totally 200μl of normal saline at the border zone between infarcted and normal myocardial,and the same amount volume injection containing 4x105 P19CL6-TNNI3K and P19CL6-FLAG cells designated as TNNI3K and FLAG group. Sham operation group was set as control. Ventricular function was evaluated by echocardiography 4 weeks later.There were no significant difference in heart rate among all groups before and 4 weeks after AMI. LVESD or LVEDD were less expanded and cardiac function was less impaired in TNNI3K group than those in FLAG and NaCl groups even though for all post- MI groups ,including TNNI3K, FLAG and NaCl ,there were more deteriorative cardiac function than those in control. TNNI3K group still had thicker (p<0.05)ventricular which similar to control compared with the NaCl or FLAG groups at 4 weeks. Hemodynamics study using four-channel physiologic recorder also confirmed even there were higher LVEDP and lower LVESP or±dp/dtmax in all MI groups while which were better in TNNI3K than in FLAG and NaCl groups.We could not find significant difference for all above parameters between FLAG and NaCl groups. Further histological analyses revealed less myocardial fibrosis in TNNI3K group than that in FLAG and NaCl groups.
Conclusion:
Intramyocardial injection of high-expression TNNI3K gene afterAMI can effectively improve ventricular function by means of attenuating cardiac remodelling and myocardial fibrosis.
Part III. Clinical Observation of TNNI3K Serum Levels in Ischemic Heart Disease
Objective:
To establish an ELISA (Enzyme-linked immunosorbent assay) with high veracity, reliability and repeatability system and detect serum TNNI3K levels in normal humans, acute myocardial infarction (AMI),heart failure caused by post-myocardial infarction remodelling or ischemic cardiomyopathy(CHD/HF)(EF≤45%)and the heart failure patients with dilated cardiomyopathy (DCM).Then discrepancies were compared among groups so as to explore the significance of TNNI3K on ischemic heart disease from clinical point of view. Methods:
Set up a standard curve of purified TNNI3K protein first and establish a high veracity, reliability and repeatability ELISA system by repeatitive intra- or inter- batch experiment. The healthy blood donators were enrolled as the Control and the definite diagnosis of patients with AMI, CHD/HF(EF≤45%)and DCM were maken through patients’history of chief complaint, symptoms, signs, lab examination, radiography,electrocardiogram(ECG),echocardiography,coronary angiography,et al. Serum TNNI3K levels in controls or different group patients were detected using above direct ELISA system. Results:
TNNI3K can be detected in the serum of both normal control and patients groups but the levels in CHD(including AMI and CHD/HF) and DCM were significantly higher than those in control(P<0.01)even though there was no significant difference between them.We couldnot find any significant difference either between AMI and CHD/HF groups as to different clinical stage or between the heart failure caused by CHD and DCM based on two-type mechanism.
Conclusion:
Serum level of TNNI3K elevated in coronary heart disease which suggests that it may play an important role in the occurrence and development of this disease.
为深化上述研究思路,本课题设计通过细胞分子学(腺病毒载体基因转导原代培养心肌细胞)、动物实验(基因修饰干细胞治疗)及临床患者(缺血性心脏病血清学检查)三个层面,从分子生物学的微观改变和病理生理、组织结构功能及生化指标的宏观变化,多角度探讨TNNI3K在心肌缺氧/缺血损伤时的意义,以深入了解这一新的激酶的功能作用及可能机制。该课题所做工作为源头创新,国内外未有报道。
第一部分:TNNI3K对缺氧所致心肌损伤的影响及可能机制
目的:
利用体外培养心肌细胞并结合缺氧造成损伤模型,观察不同缺氧时间段(0h、3h、6h、12h、24h)TNNI3K水平的变化;进而利用腺病毒转导心肌细胞使得TNNI3K基因高表达,从而研究该基因对缺氧造成心肌损伤的影响,并探讨产生作用的有关信号转导通路。
方法:
新生乳鼠原代心室肌细胞由胰酶消化法获得,缺氧环境在密闭的AnaeroPack方盒(Mitsubishi Gas Chemical Co, Inc)中经一次性缺氧催化袋GENbox anaer (BioMérieux?sa, France)(它能吸收O2 ,产生CO2)产生。实验分三个部分1、观察本实验设计的缺氧模型/无血清培养0h、3h、6h、12h、24h五个时间点心肌细胞损伤的情况:倒置显微镜下观测各组心肌细胞搏动的频率和节律,全自动生化分析仪测定心肌细胞培养液乳酸脱氢酶(LDH)活性,以确立损伤模型的建立;Western blot法半定量TNNI3K基因的表达,明确缺氧损伤对TNNI3K基因的表达是否具有影响,这是确立整篇研究的基础。2、腺病毒载体TNNI3K基因转导在缺氧/无血清干预造成原代大鼠心肌细胞损伤中的作用。用本组前期包装和扩增的绿色荧光蛋白(GFP)标记Ad-TNNI3K(携带TNNI3K基因的腺病毒)、Ad- TNNI3K-AS(携带反义TNNI3K基因腺病毒)和Ad-C(Ad-LacZ,即空载体腺病毒)瞬时转导原代培养心肌细胞,观察TNNI3K基因对缺氧导致的细胞病理损伤包括LDH活性、心肌细胞搏动的频率和节律、细胞存活率(MTT法)等的影响,并用Hoechst 33258染色和DeadEndTM Colorimetric TUNEL System检测各组细胞凋亡及凋亡指数。3、探讨TNNI3K基因高表达抗缺氧/无血清诱导心肌细胞损伤或凋亡的机制。用Western blot法半定量磷酸化细胞外信号调节激酶C(phosphorylated extracellular signal-related kinases, p-ERK)、c-jun氨基末端激酶(phosphorylated c-Jun N- terminal kinases,p-JNK)和p38(p- p38)激酶的表达,观察MAPK信号通路中三条主要途径的参与作用;同时,选择缺氧12h为观察时间点,用酶标仪法测Caspase-3活性,免疫细胞化学和激光共聚焦显微镜检测不同组别缺氧前后细胞色素C在细胞胞浆和线粒体中的定位改变,Rhodamine 123染色流式细胞仪测定线粒体膜电位,以及Western blot法半定量致凋亡蛋白Bax的表达,共同阐述TNNI3K基因对线粒体信号通路的作用。
结果:
1、利用AnaeroPack缺氧盒,配合无血清DMEM培养成功建立缺氧模型,缺氧0.5h后,细胞培养液中的氧分压达40mmHg以下,氧饱和度20%左右,达到缺氧实验所要求的标准。缺氧6h可见心肌细胞搏动频率减慢,节律出现不规整,至缺氧24h绝大多数细胞已无搏动,极少数浅慢搏动;培养液LDH水平在缺氧6h以后即有显著性升高(P<0.05),24h更甚(P<0.01),提示细胞出现损伤性改变;同时TNNI3K蛋白表达在缺氧早期(3h)即上调,12h达到高峰,24h后渐回落,提示TNNI3K在心肌细胞缺氧应激时可能发挥作用。
2、利用腺病毒作为载体,成功转导原代心肌细胞,缺氧可使心肌细胞存活率下降,但在12h和24h时Ad-TNNI3K、Ad-TNNI3K-AS两组的细胞生存率优于Ad-C组,前两组之间无显著性差异;缺氧12h后,Ad-C、Ad-TNNI3K、Ad-TNNI3K-AS三组均见LDH升高,而Ad-TNNI3K组要明显低于Ad-C、Ad-TNNI3K-AS两组,后两组之间比较,Ad-TNNI3K-AS组又明显低于Ad- C,表明TNNI3K高表达对细胞的保护作用;同时,Hoechst 33258染色和TUNEL检测显示,Ad- TNNI3K基因高表达能使缺氧12和24h的凋亡指数均低于Ad-C组,提示TNNI3K基因具有抗细胞凋亡的作用。由于反义TNNI3K组的作用介于TNNI3K和Ad-C组之间,有时和TNNI3K组无明显差异,推测反义TNNI3K未能抑制大鼠心室肌细胞内TNNI3K同源基因的表达,一方面可能是因为种属差异,另一方面,可能是由于反义核酸技术本身存在作用的不可预知性的问题,故在有关机制的研究中只对Ad-C组和Ad- TNNI3K两组进行了分析。
3、对TNNI3K产生作用的机制研究表明,(1)MAPK信号通路中,缺氧前Ad-C组和Ad- TNNI3K两组心肌细胞即有较低水平的ERK1/2和JNK磷酸化,两组之间无显著差异。缺氧3h后,Ad-C组p-ERK1/2表达水平明显升高,持续到6h,而Ad-TNNI3K组改变不明显,两组间比较显见不同(p<0.05);但到12h、24h则两组均趋于下降,组间差异不明显。p-JNK与上述不一样,Ad-C组在缺氧3h时一度轻度上升,但随即处于极低表达状态,Ad- TNNI3K组则升高更加明显,且高峰持续至缺氧12h,于6h和12h两个时间点两组差别极显著(P<0.01),至24h时两组均处于很低水平。而反复行western blot实验,在38KD附近检测到的杂交信号非常弱,无法判别所需的目的条带,推测在本研究设计的缺氧干预腺病毒转导心肌细胞中,p38蛋白基本没有被磷酸化,或磷酸化的水平很低。(2)高表达TNNI3K基因能够抑制缺氧各时间点Caspase-3的激活程度,至缺氧12h、24h差异显著;能明显减轻由缺氧12小时导致的细胞色素C从线粒体到胞浆的释放水平,抑制凋亡蛋白Bax的表达;提示TNNI3K基因的抗凋亡作用通过线粒体信号途径。
结论:
在体外培养的原代心肌细胞实验中,首次证明心脏特异性表达的新激酶基因TNNI3K具有抗缺氧造成的细胞损伤和凋亡作用。抑制MAPK级联信号通路中ERK1/2的早期、瞬间磷酸化激活以及提高JNK1/2的磷酸化水平;同时,抑制线粒体信号途径中Caspase-3的激活和细胞色素C的释放,以及凋亡蛋白Bax的表达是其发挥抗凋亡作用的机制。
第二部分:心外膜心肌注射TNNI3K对心肌梗死后兔心功能的影响
目的:
观察整体动物急性心肌梗死后,通过使局部高表达TNNI3K,能否改善心肌重塑而达到减轻心功能损害。
方法:
P19CL6细胞是鼠科胚胎瘤(embryonal carcinoma,EC)P19细胞的衍生物,P19细胞是一种来源于鼠科畸胎瘤的未分化干细胞,P19CL6细胞形态上与之相似,但不表达特异性的EC标记物SSEA-1抗原,已经证实在加入DMSO作为诱导剂的培养基中,它能有效分化成表达心脏特异基因的搏动的心肌细胞,同时具有心肌细胞的电生理特性。利用本协作研究组成员在前期工作中分别获得的质粒载体高表达TNNI3K转染细胞株(P19CL6/TNNI3K)和FLAG多肽标签质粒转染P19CL6细胞株(P19CL6/FLAG),作为本实验待用的基因修饰干细胞。实验共分四组(i)假手术对照组(Control组)(ii)单纯心肌梗死+生理盐水注射组(NaCl组)(iii)心肌梗死+ P19CL6/ pCDNA6-FLAG注射组(FLAG组)(iv)心肌梗死+ P19CL6/ pCDNA6-FLAG -TNNI3K注射组(TNNI3K组)。1、结扎兔左冠状动脉回旋支的左室支造成心肌梗死模型;2、体外用1%DMSO诱导P19CL6/TNNI3K或P19CL6/FLAG两株细胞5天后,重新计数细胞,分别收集4x105个细胞,生理盐水200μl重悬,置于皮试用1ml注射器中,在兔梗死与正常心肌交界部位暗红色的充血带四周分6点注射,每点30μl;3、彩色多普勒超声心动图观测上述四个实验组第四周末心脏左室舒张末径(LVEDD)、左室收缩末径(LVESD)、左室壁和室间隔厚度,计算左室射血分数(LVEF);4、4道生理记录仪经颈动脉插管检测上述四个实验组第四周末血流动力学指标,包括左室舒张末压(LVEDP)、左室收缩末压(LVESP)、左室内压最大上升速率(+dp/dtmax)和左室内压最大下降速率(-dp/ dtmax);5、第四周末取四组含梗死区心肌组织,切片,HE(苏木精-伊红)染色,观察病理形态学差别。
结果:
P19CL6-TNNI3K和P19CL6-FLAG两株细胞在1%DMSO诱导后,均在大约10-12天,局部开始出现收缩,到约12-16天,几乎所有的片层出现同步的搏动。成功造成兔心肌梗死,按分组观察,术前、术后各组兔心率之间无明显差异。术后四周超声心动图显示,心梗各组心功能均较假手术组显著降低,但TNNI3K组心功能下降的程度显然轻于NaCl及FLAG两组,FLAG组和NaCl两组间无显著差异。比较左室壁和室间隔厚度,NaCl和FLAG两组可见有不同程度变薄,而TNNI3K组则与对照组相仿,组间差异显著。提示TNNI3K基因治疗能减轻心肌重构,改善心功能。进一步通过4道生理记录仪经颈动脉插管检测血流动力学指标,可见心肌梗死各组均有LVEDP明显升高,而LVESP和±dp/dtmax则均明显下降,提示左室收缩和舒张功能的减退,TNNI3K组的变化处于control和FLAG/ NaCl两者之间,但优于FLAG或NaCl组,后两组间无显著差异。心脏组织切片HE染色显示TNNI3K组心肌纤维化程度明显轻于NaCl和FLAG两组。
结论:
急性心肌梗死后,经开胸直接心外膜心肌局部注射使高表达TNNI3K基因,可有效改善梗死后的心脏重塑,减轻心肌纤维化,减少心脏舒缩功能的损害。
第三部分:缺血性心脏病TNNI3K的血清学变化及临床意义
目的:
建立一个准确性高、可信度大、重复性好的ELISA实验条件,在此基础上检测正常人、急性心肌梗死、心肌梗死后心脏重构或缺血性心肌病出现心力衰竭、以及扩张性心肌病心力衰竭患者的TNNI3K血清水平并比较其差异,从临床角度探讨TNNI3K在缺血性心脏病中的作用及其临床意义。
方法:
1、从三株纯化的单克隆抗体中选定最合适的一株,并确定使用浓度,同时确定合适的血清样本与二抗的稀释倍数;建立纯化的TNNI3K蛋白的标准曲线;通过批内和批间重复性实验建立一个准确性高、可信度大、重复性好的ELISA实验条件;2、选择既往无心血管疾病的正常献血员为正常对照;冠状动脉粥样硬化性心脏病(CHD),包括急性心肌梗死(AMI)和缺血性心肌病心力衰竭(CHD/HF)(EF≤45%),扩张型心肌病(DCM)心力衰竭(EF≤45%)患者,均根据病史、症状、体征、客观检查(包括实验室检查、X线胸片、心电图、超声心动图、核素、核磁、冠状动脉造影等)综合作出确诊。抽取受试者清晨空腹肘静脉血2ml,不抗凝,2500 rpm/min离心10min,取血清分装,-20℃冻存备用。TNNI3K水平用上述建立的直接ELISA法测定;3、比较入选不同患者组间血清TNNI3K水平的差异及其临床意义。
结果:
1、正常对照和入选患者血清中均可检测到TNNI3K抗原。但无论CHD或DCM组,TNNI3K血清水平均高于正常对照(P<0.01),两类患组之间差异无统计学意义。2、在CHD患者中,比较不同发病时期AMI和CHD/HF两组血清TNNI3K浓度无明显不同;3、尽管缺血性心肌病和扩张型心肌病引起的心力衰竭发病机制不同,两组血清TNNI3K浓度亦无统计学差异。
结论:
TNNI3K基因与冠状动脉粥样硬化性心脏病密切相关。但以急性心肌梗死和以心力衰竭为临床发病的冠心病不同类型之间其血清水平并无差别;冠心病导致的心力衰竭和扩张型心肌病心力衰竭对TNNI3K血清水平的影响相似。
While constructing human adult heart and aorta cDNA libraries and making large-scale ESTs(Expressed Sequence Tags) sequencing, our research group members identified a novel gene localized on 1p31.1 based on bioinformatics analyses which might interact with cytoskeleton regulating signal pathway. A yeast two-hybrid screen proved it could interect with a series of sarcomeric regulating protein among which the most frequent candidate was cTnI and then was named TNNI3K (cTnI-interacting kinase) (GenBank accession no. AF116826), results that were further confirmed by co-immunoprecipitation in vivo. Multiple fetal and adult northern blot and 76-tissue array suggest TNNI3K is cardiac-specific gene and was undetectable in other tissues. In vitro kinase assays using immunoprecipitates of the series of FLAG-tagged TNNI3K mutants which found that TNNI3K not only autophosphorylated but also phosphorylated tyrosine substrate myelin basic protein (MBP) demonstrated that TNNI3K is a functional protein kinase. Phylogenetic parsimony analysis further clearly indicates TNNI3K belong to MAPKKKs superfamily in the evolutionary tree.
A great deal pre-work indicated that TNNI3K, as a novel cardiac-specific protein kinase, has functional activities. But its downstream targets, acting mechanism and biological signal pathway under pathological stress still remain poorly understood. So further study in this work will be carried out from molecular cytological level (primary cultured neonatal rat cardiomyocytes transducted with adenovirus carrying/ non-carrying TNNI3K),animal model and histological level (treating with TNNI3K modified stem cells),and then clinical serological level to clarify its biological significance under hypoxia/ischemia intervention.
All researches in this work are original novel and never been reported.
Part I: Effects and mechanism of TNNI3K on hypoxia induced cardiocyte injury
Objective:
First time to prove whether the TNNI3K levels could be changed with different hypoxia time periods(0h,3h,6h,12h and 24h)in vitro cultured primary cardiomyocytes. Then the effects of TNNI3K over-expression as a result of adenovirally mediated gene transfer on neonatal rat cardiac myocyte injury induced by hypoxia were investigated and its relative signal transduction pathway was elucidated.
Methods:
Primary ventricular cardiomyocytes were prepared from neonatal rats by collagenase digest method. Hypoxia was achieved by using an anaerobic jar (AnaeroPack Series, Mitsubishi Gas Chemical Co, Inc) equipped with an AnaeroPack, disposable O2-absorbing and CO2-generating agent, and an indicator to monitor oxygen depletion. Whole experiment contains three parts:1.Cell injury induced by hypoxia/serum free culture was observed at different time periods (0h,3h,6h,12h,24h). The cardiomyocytes beating rate and rhythm in each group were documented under inverted microscope and lactate dehydrogenase(LDH) in cultured cell medium was assayed using automatic biochemical analysis instrument(Beckman,USA) in order to confirm the successfully established cell injury model. Then western blot was used to semi-quantitate TNNI3K gene expression whose alteration is the base for all next work.2.Effect of TNNI3K on neonatal rat cardiac myocyte injury induced by hypoxia/serum-free. Primary cultured neonatal rat cardiomyocytes were transiently transducted with Ad-C (adenovirus-LacZ as control), Ad-TNNI3K(adenovirus carrying sense of TNNI3K) and Ad-TNNI3K-AS (adenovirus carrying anti-sense of TNNI3K) in which all were labelled with green fluorescent protein(GFP).The effects of TNNI3K gene overexpression on neonatal rat cardiac myocytes injury induced by hypoxia/serum-free were probed by evaluating LDH release,cells beating rate and rhythm and viability(assessed by MTT assay). Cell apoptosis was determined by both Hoechst 33258 staining and DeadEndTM Colorimetric TUNEL System.3.The action mechanism of TNNI3K overexpression on cardiomyocytes apoptosis induced by hypoxia/serum-free. Evaluation of the participative roles of three main classic MAPK pathways activation using semi-quantitating phosphorylated extracellular signal-related kinase( p-ERK), phosphorylated c-Jun N- terminal kinase(p-JNK) and phosphorylated p38(p-p38) by western blot. Furthermore,to explore the mitochondrial apoptotic signal pathway effects of TNNI3K at 12h exposing to hypoxia through measuring Caspase-3 activity by Microplate Reader, the release of cytochrome C from mitochondrial to cytoplasm using immunochemical method combining laser scanning confocal microscope(LSCM),change of cell mitochondrial membrane potential( m) which staining with Rhodamine 123 assessing by FACS(flow cytometry) and proapoptotic protein Bax expression by western blot.
Results:
1.The AnaeroPack jar is capable of depleting the concentration of O2 down to PO2<30mmHg or SO2 about 20% in 1 hour which successfully meets our experimental requirement in this study.After 6hs of hypoxia,cardiomyocytes beated slower than before exposing to hypoxia and the beating rhythm became irregular which still worsen and almost no-beating for majority of the cells after 24hs hypoxia.LDH level in medium increased significantly at 6hs post hypoxia(P<0.05)and much dramatically at the 24h which suggest the injury of the cells. The elevation of TNNI3K protein expression occurred at early hypoxia time(3h) and peaked at 12h then went down slowly at 24h post hypoxia.
2.All Adenovirus-mediated transduction cells divided into Ad-C、Ad-TNNI3K and Ad-TNNI3K-AS groups. After exposing to hypoxia 12 h and 24h, cell viability decreased in three groups but this was much better in Ad-TNNI3K and Ad-TNNI3K- AS groups than that in Ad-C group while there was no significant difference between the former two. Furthermore,12h- and 24h- hypoxia increased medium LDH levels in all three groups but those were relatively much lower in group Ad-TNNI3K than those in groups Ad-C and Ad-TNNI3K-AS even though when compared between them those in Ad- TNNI3K-AS group showd significant lower than in Ad-C group. Overexpress- ion of TNNI3K in Ad-TNNI3K group also decreased apoptosis at 12h and 24h by Hoechst 33258 staining which showed less chromatin condensation and aggregation under the nuclear membrane and less nuclear fragmentation and apoptosis body formation. Apoptosis index calculation by DeadEndTM Colorimetric TUNEL System assay was also in accordance with this result. All above revealed the very important cytoprotective and anti-apoptotic effect of TNNI3K gene on hypoxia induced injury. The paradoxical results of Ad-TNNI3K-AS group whose activity was sometimes between group Ad-TNNI3K and Ad-C whereas sometimes no significant difference compared with Ad- TNNI3K group possibly due to the species difference becouse the anti-sense TNNI3K deriving from human sequence might not inhibit the homologous gene of TNNI3K in rat ventricular myocyte or probably due to the unpredictable problems of anti-sense nucleotide technique per se.So,in all next studies on mechanism, we only focused in Ad-C and Ad- TNNI3K groups.
3. Based on western blot analysis,we delineate the MAPKs signaling pathway roles in hypoxia induced apoptosis. The results indicted that (1) only ERK1/2 and JNK signal pathways involve in TNNI3K functional effect responding to hypoxia treatment. There were low expression levels of p-JNK and p-ERK1/2 in both Ad-C and Ad- TNNI3K groups at 0h hypoxia. After 3h hypoxia, p-ERK1/2 in Ad-C group signifiantly increased and still higher at 6h of hypoxia while which couldnot be seen in Ad-TNNI3K group(p<0.05),then followed by a decline in both groups and made no difference between them at 12 or 24h.For p-JNK, the pattern of change was considerably different to that seen for p- ERK1/2.Levels in Ad-C group initially slightly increased at 3h and then maintained very low throughout the time course while that in Ad- TNNI3K group increased at 3h and 6h and peaked at 12h making significant difference between two groups at 6 and 12h(P<0.01) and then kept quite low levels in both groups at 24h.(2)High expression of TNNI3K could inhibit activation of caspase-3 and alleviate the release of cytochrom C from mitochondria to cytoplasm and reduce the expression of proapoptotic protein Bax.All above results suggest that the antiapoptotic effect of TNNI3K might be through mitochondria dependent signal pathway.
Conclusion:
In vitro primary cultured neonatal rat cardiomyocytes experiment,we first approved that cardiac-specific gene TNNI3K has the protective effect on cell injury and apoptosis induced by hypoxia.This function might be through inhibiting the early stage and trensient activation of p- ERK1/2 and increasing the expression of p- JNK in MAPK cascade signal pathway.The anti-apoptotic mechanism was motochondria dependent through inhibiting the activation of caspase-3 and reducing the release of cytochrom C from mitochondria to cytoplasm and the expression of proapoptotic protein Bax.
Part II: Effect of Intramyocardial Administration of TNNI3K on Rabbit Post- myocardial infarction Cardiac Function
Objective:
To observe the effect of intramyocardial injection of TNNI3K on cardiac function in a rabbit model of infarction.
Methods:
P19CL6,a derivation of mouse embryonal carcinoma(EC) P19 cells which is an undifferentiated stem cell derived from murine teratocarcinoma, is morphologically similar to P19 cells but does not express SSEA-1 (stage specific embryonic antigen-1) antigen, a specific EC marker.It is known that P19CL6 cells efficiently differentiate into beating cardiomyocytes expressing cardiac- specific genes after culture in the presence of an inducer, dimethylsulfoxide (DMSO). The vector pcDNA6-FLAG /TNNI3K was obtained from our prophase work and the stable transfected P19CL6 cells with pcDNA6-FLAG(FLAG only) or pcDNA6-TNNI3K were endowed by our Japanese cooperator.Briefly, the vectors pcDNA6-FLAG (FLAG only) and pcDNA6- FLAG/TNNI3K were transfected into P19CL6 cells using electropoie 2000 (Invitro- gen) and maintained in medium containing 500μg/ml blasticidin (Invitrogen). Acute myocardial infarction (AMI) was produced in rabbits using ligation of left ventricular branch of left circumflex coronary.After 5 days being induced by 1%DMSO, early differentiated P19CL6 cells with or without high expression of TNNI3K were collected respectively and resuspended with normal saline. 24 experimental rabbits were randomly divided into four groups(i) Sham operation control group,n=6(C) ; (ii)AMI+ intramyocardial normal saline injection group,n=6 (NaCl);(iii) AMI+ intramyocardial pcDNA6-FLAG injection group, n=6 (FLAG); (iiii) AMI+ intramyocardial pcDNA6-FLAG/ P19CL6 injection group,n=6 (P19CL6). 4 weeks after operation ,left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular wall thickness and interventricular septum thickness were measured by Color Doppler Eechocardiography and then left ventricular ejection (LVEF) was calculated. Haemodynamics parameters including left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP),maximum LV pressure rising velocity(+dp/dtmax) and maximum LV pressure decreasing velocity(-dp/dtmax) were recorded with an electrophysiolograph and then the hearts were excised. Specimens were coded and fixed in 10% buffered formalin for subsequent histopathological analyses using hematoxylin and eosin stain (HE stain).
Results:
Around 10 to 12 days differentiation induced by 1%DMSO, P19CL6-TNNI3K and P19CL6-FLAG cells which seeded at 4 x105 per 60mm dish contracted first at a restricted area then on days 12 to 16 almost all of the sheet beated synchronously. Myocardial infarction was successfully produced in rabbits using coronary artery ligation. NaCl group received an intramyocardial injection of totally 200μl of normal saline at the border zone between infarcted and normal myocardial,and the same amount volume injection containing 4x105 P19CL6-TNNI3K and P19CL6-FLAG cells designated as TNNI3K and FLAG group. Sham operation group was set as control. Ventricular function was evaluated by echocardiography 4 weeks later.There were no significant difference in heart rate among all groups before and 4 weeks after AMI. LVESD or LVEDD were less expanded and cardiac function was less impaired in TNNI3K group than those in FLAG and NaCl groups even though for all post- MI groups ,including TNNI3K, FLAG and NaCl ,there were more deteriorative cardiac function than those in control. TNNI3K group still had thicker (p<0.05)ventricular which similar to control compared with the NaCl or FLAG groups at 4 weeks. Hemodynamics study using four-channel physiologic recorder also confirmed even there were higher LVEDP and lower LVESP or±dp/dtmax in all MI groups while which were better in TNNI3K than in FLAG and NaCl groups.We could not find significant difference for all above parameters between FLAG and NaCl groups. Further histological analyses revealed less myocardial fibrosis in TNNI3K group than that in FLAG and NaCl groups.
Conclusion:
Intramyocardial injection of high-expression TNNI3K gene afterAMI can effectively improve ventricular function by means of attenuating cardiac remodelling and myocardial fibrosis.
Part III. Clinical Observation of TNNI3K Serum Levels in Ischemic Heart Disease
Objective:
To establish an ELISA (Enzyme-linked immunosorbent assay) with high veracity, reliability and repeatability system and detect serum TNNI3K levels in normal humans, acute myocardial infarction (AMI),heart failure caused by post-myocardial infarction remodelling or ischemic cardiomyopathy(CHD/HF)(EF≤45%)and the heart failure patients with dilated cardiomyopathy (DCM).Then discrepancies were compared among groups so as to explore the significance of TNNI3K on ischemic heart disease from clinical point of view. Methods:
Set up a standard curve of purified TNNI3K protein first and establish a high veracity, reliability and repeatability ELISA system by repeatitive intra- or inter- batch experiment. The healthy blood donators were enrolled as the Control and the definite diagnosis of patients with AMI, CHD/HF(EF≤45%)and DCM were maken through patients’history of chief complaint, symptoms, signs, lab examination, radiography,electrocardiogram(ECG),echocardiography,coronary angiography,et al. Serum TNNI3K levels in controls or different group patients were detected using above direct ELISA system. Results:
TNNI3K can be detected in the serum of both normal control and patients groups but the levels in CHD(including AMI and CHD/HF) and DCM were significantly higher than those in control(P<0.01)even though there was no significant difference between them.We couldnot find any significant difference either between AMI and CHD/HF groups as to different clinical stage or between the heart failure caused by CHD and DCM based on two-type mechanism.
Conclusion:
Serum level of TNNI3K elevated in coronary heart disease which suggests that it may play an important role in the occurrence and development of this disease.
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