病理性心肌肥大向心衰转变的相关分子机制研究
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摘要
病理性心肌肥大是心脏对抗各种损伤因素的共同反应,持续的心肌肥大最终将导致心肌重构、心室扩张和心功能障碍。越来越多的研究旨在确立心肌肥大发展及向心衰转变的分子机制。
目前许多研究通过药理性抑制剂显示,ROCK在心肌肥大和心肌重构中发挥作用。本论文第一章将利用基因敲除技术,在动物水平深入探讨ROCK家族的ROCK1亚型在心肌病理性重构中的作用。
心肌细胞凋亡一直被认为是病理性心肌肥大向心衰转变的关键因素。然而即使在病理性刺激下,成年心脏心肌细胞凋亡率仍然很低,因此,心肌细胞凋亡在心肌病理性重构中的作用越来越受到质疑。基于此,本论文第二章将通过对比生理和病理情况下心肌细胞凋亡的年龄相关性差异,探讨心肌细胞凋亡在不同年龄心肌病变中的重要性。
第一章ROCK1缺失抑制病理性心肌肥大模型心肌扩张并改善收缩功能
研究背景
病理性心肌细胞肥大是心脏对抗血流动力学增加、肌损伤和神经激素应激等的共同反应,持续的心肌肥大最终将导致失代偿性充血性心衰,表现为心室扩张和收缩功能障碍。越来越多的研究旨在确立心肌肥大发展以及向心衰转变的分子机制。
Gq为一类异三聚体GTP结合蛋白。由α、p和γ亚基组成。大量研究表明,Gq信号通路是心肌肥大发展和心脏失代偿的关键调节因素。心肌细胞特异性转基因过表达Gα亚单位(Gαq)小鼠是一个已经定性的病理性心肌肥大模型,在分子水平、细胞水平和整体水平模拟了许多压力超负荷诱导的心肌肥大的特征,如：左心室扩张、基础水平和p-肾上腺素受体刺激后的心室收缩功能减退、基础水平和激动剂刺激的腺苷环化酶(adenylyl cyclase, AC)活性下降、以及心肌基因表达向胎儿模式转变。许多研究阐述了Gαq过表达所致心肌细胞肥大、心室扩张和心功能障碍的可能机制,包括：PKCα表达和活性增加、AC表达和活性下降、MEKK1/JNK信号途径的异常等。
ROCK是RhoA GTPase的下游信号分子,在RhoA调控压力纤维形成、平滑肌收缩、细胞粘附等多种事件中扮演关键角色。最近有在体实验使用ROCK抑制剂Y27632和法舒地尔提示,ROCK在心肌肥大和心肌重构中发挥作用。但这些药理性抑制剂不能区分ROCK家族两个亚型ROCK1和ROCK2。我们最近的研究显示,ROCK1基因缺失纯合子小鼠(ROCK-/-)心肌在对压力超负荷的反应中,心肌间质纤维和心肌细胞凋亡较WT小鼠显著减少,但仍然发展了心肌细胞肥大。另一个研究也显示,在血管紧张素2刺激后,ROCK+/-杂合子小鼠发展了心肌肥大,但血管外周纤维化较WT明显减少。
许多研究通过体外实验表明,RhoA/ROCK协同Gαq信号通路,参与了G蛋白偶联受体激动剂诱导的心肌细胞肥大。然而,至今没有在体实验表明两条通路之间的相互作用。本研究联合心肌细胞特异性Gαq过表达心肌肥大模型和ROCK1基因敲除小鼠,检验了ROCK1和Gαq通路间的相互作用,探讨了ROCK1缺失对Gαq过表达小鼠心肌内部变化所致扩张型心肌病变的影响,也为进一步明确ROCK1和ROCK2亚型的独立作用奠定了基础。
第一节ROCK1缺失阻止Gαq小鼠心肌扩张并改善心收缩功能
方法
(1)生存实验：
FVB雄性小鼠,分设4组(n=5)：野生型组(WT)、心肌细胞特异性Gaq过表达组(Gαq)、Gαq过表达与ROCK1基因敲除混合组(Gαq/ROCK1-/-)、ROCK1基因敲除组(ROCK1-/-)。所有动物正常饲养至12月龄,采用SPSS软件绘制Caplan-Meier生存曲线。在10月龄行超声心动分析。
(2)其他蛋白分析、组织学和功能实验：
FVB雄性小鼠,分设4组(n=4)：WT、Gαq、Gαq/ROCK1-/-、 ROCK1-/-。所有动物至12周龄,行心功能分析后,断颈处死并分离心脏。心脏保存于液氮,或行冰冻或石蜡包埋。
结果
1.Gαq、ROCK表达水平分析：
(1)在Gαq和Gαq/ROCK1-/-小鼠心肌组织内,Gαq表达水平较WT组和ROCK1-/-组显著增高,且在各转基因组小鼠表达一致,ROCK缺失没有影响心肌组织内Gαq的过表达水平；
(2)ROCK1-/-和Gαq/ROCK1-/-小鼠心肌组织内不表达ROCK1; ROCk2的表达在各组小鼠间没有差异；
(3)ROCK活性水平没有随ROCK1缺失和Gαq过表达而改变；
2.ROCK1缺失显著延长了Gaq小鼠的存活时间。
3.ROCK1缺失没有阻止Gαq小鼠心肌细胞肥大性发展：
(1)形态学分析显示,ROCK1缺失没有明显抑制Gαq过表达诱导的心脏重量增大；
(2)超声扫描分析显示,ROCK1缺失没有明显降低Gαq过表达诱导的小鼠左心室质量增大；
(3)组织学切片染色显示,ROCK1没有明显降低Gαq过表达诱导的左心室心肌细胞肥大；
4.ROCK1缺失部分抑制了Gαq过表达诱导的心肌肥大标志物mRNA水平上调；
5.ROCK1缺失阻止了心肌细胞特异性Gαq过表达诱导的左心室扩张和左心室收缩功能障碍：
(1)超声扫描分析显示,ROCK1缺失明显抑制了Gαq小鼠左室内径增大及室壁变薄；
(2)组织学切片染色显示,Gαq小鼠为左室离心性肥大,而Gαq/ROCK1-/-混合小鼠为心室向心性肥大；
(3)超声扫描分析显示,ROCK1缺失明显阻止了Gαq小鼠左室短轴缩短率(fractional shortening, FS)和射血分数(ejection fraction,EF)降低；
(4)形态学分析显示,ROCK1缺失阻止了Gαq小鼠的肺水肿。
结论
ROCK1基因缺失抑制了心肌细胞特异性过表达Gαq转基因小鼠的心室扩张,改善了其心功能,并显著延长了Gαq小鼠的寿命。
刍二节ROCK1缺失阻止Gαq小鼠心肌AC5/6下调并恢复心肌β-AR功能
方法
FVB雄性小鼠,分设4组：WT、Gαq、Gαq/ROCK1-/-、ROCK1-/-。所有动物至12周龄,行心功能分析后,断颈处死并分离心脏。心脏保存于液氮,或行冰冻或石蜡包埋。部分实验动物给予腹腔注射异丙肾上腺素(ISO,100ng/g体重),在注射前和注射后5分钟分别进行超声心动检测。
结果
1.Gaq和Gαq/ROCK1-/-小鼠心肌组织没有发展明显的心肌纤维化。
2.Gαq和Gαq/ROCK1-/-小鼠没有明显的心肌凋亡。
3.异丙肾上腺素刺激后,ROCK1基因缺失显著改善了Gαq心肌对p-肾上腺素受体刺激的低反应性。
4.ROCK1缺失恢复了心肌特异性Gαq过表达诱导的AC5/6下调。
5.ROCK1缺失没有抑制心肌细胞特异性Gαq过表达诱导的MAPK活化和PKCα活化。
结论
ROCK1缺失明显抑制了心肌细胞特异性Gαq过表达诱导的AC5/6蛋白水平下调和p-肾上腺素受体功能障碍,从而改善了Gαq小鼠的心功能不全。
ROCK1基因缺失明显抑制了心肌细胞特异性Gαq过表达诱导的心室扩张、AC5/6的蛋白水平下调和β-肾上腺素受体功能障碍,从而改善了Gαq小鼠的心功能,并显著延长了Gαq小鼠的存活时间。
第一节Caspases依赖性心肌细胞凋亡的年龄差异性比较研究背景
介导Caspases依赖性细胞凋亡的信号通路包括外部凋亡途径和内部调亡途径,caspase8和caspase9分别是外部和内部通路的凋亡起始者,两条通路最终汇合于凋亡执行者caspase3的活化。在内部凋亡途径,Apafl作为接头蛋白,连接细胞色素C和pro-caspase9,为pro-caspase9活化所必需。Bcl-2家族是内部凋亡途径最主要的调节因子,包括：促凋亡蛋白Bax、Bak、BH3、蛋白如Bad、Bid、Nix和BNip3,抑凋亡蛋白Bcl-2,Bcl-XL和Mcl-1。
心肌细胞凋亡在心脏胚胎发育期扮演关键角色,而在出生后成熟的心肌非常罕见。无数研究表明心肌细胞凋亡在许多心肌病变中成百倍上调,然而即使在这些病理情况下,成年心肌细胞凋亡率仍然很低。研究也显示,幼年心脏比成年心脏对各种刺激更加敏感。我们推测心肌细胞死亡和增殖相关基因表达模式或蛋白活性的年龄依赖性变化,是幼年和成年心脏对刺激敏感性不同的根本原因之一。
我们首先系统比较了正常小鼠从出生到成年心脏发育过程中,生理条件下心肌细胞凋亡及其关键信号分子包括：caspase3、caspse8、 caspase9、Apafl、Bax等的表达或活性的变化；并将TUNEL原位染色法与特异性标记心肌细胞核的方法结合,较准确地定量了生理条件下不同年龄阶段的心肌细胞凋亡。
方法
实验使用FVB品系野生型小鼠(WT)或心肌细胞核特异性过表达lacZ的FVB小鼠(αMHC-nlacZ)。有关caspase3活性检测和caspases蛋白表达分析的实验部分,WT小鼠分为6组：雄或雌性出生后1天、出生后3天、1周龄、2周龄、雄性3周龄、雄性5周龄。有关活化caspase3免疫组化染色、TUNEL染色及其他凋亡相关蛋白分析的实验部分,WT或nlacZ小鼠分设3组：雄或雌性1周龄、雄性3周龄、雄性5周龄；在活化caspase3免疫组化染色和TUNEL染色实验中,先行p-半乳糖苷(β-gal)染色,以特异性标记心肌细胞核。
结果
1. aMHC-nlacZ小鼠心脏冰冻切片行β-gal染色后,可见心肌细胞核呈淡蓝色,而胞浆和其它类型细胞核未染。
2. Caspase3活性实验显示,小鼠心肌组织caspase3活性随年龄增大而显著降低。出生1周内的心肌组织具有相对高的caspase3活性,2周龄时仍可以检测到少量caspase3活性,3周以后已基本检测不到活化的caspase3。
3. β-gal染色结合免疫组化染色、以及Western blot结果显示,小鼠心肌细胞内活化caspase3的表达水平随年龄增大而显著减少,3周龄以后的心肌细胞不表达活化caspase3。
4. p-gal染色结合TUNEL染色显示,小鼠心室肌细胞凋亡随年龄增大而显著减少。1周龄时心室肌细胞仍有一定比率的凋亡发生,而3周以后罕见心肌细胞凋亡。
5. Western blot结果显示,出生1周内pro-caspase3的表达水平较高,2周后明显降低,3周后极其微弱；pro-caspase9和pro-caspase8的表达水平同样随年龄增大而明显降低。
6. Western blot结果显示,心肌组织内部凋亡途径信号分子Bax、Apafl的表达随年龄增大而显著下降。
7.心肌组织内其他细胞增殖和生存相关蛋白如Akt/p-Akt。p-P53、p-p38和p-p42/44的表达水平均没有呈现年龄相关性变化。
结论
出生后心肌生理性发育的早期,细胞凋亡通路及心肌细胞凋亡较成年心肌尤为活跃,这可能是幼年心脏对病理性刺激高敏感性的分子基础之一。
研究背景
阿霉素(Doxorubicin, DOX)是目前应用最广泛和有效的抗肿瘤药物之一。但是严重的心脏毒性限制了DOX的使用剂量和疗程。研究显示心肌细胞凋亡在DOX诱导的心肌毒性反应中扮演重要角色。但目前有关DOX心肌毒性的在体研究绝大部分是采用成年动物模型,其病理情况下的心肌细胞凋亡率仍然很低；而且目前有关原位检测心肌细胞凋亡的方法如TUNEL染色,均不能准确区分心肌细胞和非心肌细胞。因此心肌细胞凋亡在病理性因素比如DOX刺激诱导的心肌病理性发展、尤其是成年后心肌重构中的重要性越来越受到质疑。
临床研究也表明,接受DOX治疗的儿童和青少年对其心脏毒性影响较成年尤为敏感,大部分儿童在完成疗程一年或多年以后发生心肌病变。可能的原因为心肌细胞丢失、心肌生长损害,导致左心室发育不全和心肌病变。然而其最根本的分子机制至今不明。我们在前一节所述的动物实验以及其他少数研究提示,幼年心肌细胞内部凋亡途径的相对高水平、尤其是凋亡执行者caspase3的高表达,可能是儿童对DOX心脏毒性较成年更为敏感的原因之一。
因此本实验中,我们使用在体给予DOX的心肌毒性动物模型,比较了在DOX刺激的早期,幼年和成年心肌细胞凋亡及其相关信号通路的差异。并结合特异性标记心肌细胞核的方法,准确定量DOX诱导的心肌细胞凋亡,衡量了心肌细胞凋亡在不同年龄阶段DOX所至心肌病变中的重要性。
方法
实验使用FVB品系WT或αMHC-nlacZ小鼠：
(1)观察不同浓度DOX对1周龄小鼠心肌组织caspase3表达的影响：
分为5组(n=4)：对照组(1wk-ctrl); DOX5mg/kg处理组(1wk+5);DOX10mg/kg处理组(1wk+10)；DOX15mg/kg处理组(1wk+15)；DOX20mg/kg处理组(1wk+20)。所有实验动物单次腹腔注射不同浓度DOX或等量生理盐水,24小时后断颈处死动物,迅速收集心脏,存于液氮。
(2) DOX10mg/kg对1周龄小鼠心肌组织caspase3表达影响的时间曲线分析：
分为7组(n=4)：DOX处理0小时组(对照组),DOX处理2,4,8,12,16,24小时组。DOX注射方式及标本采集方式同上。
(3)观察DOX(低浓度和高浓度)对不同年龄小鼠心肌细胞凋亡及凋亡信号通路的影响：
分为9组(n=4)：1周龄对照组(生理盐水组,1wk-ctrl);1周龄DOX10mg/kg处理组(1wk+10)；1周龄DOX20mg/kg处理组(1wk+20)；3周龄对照组(3wk-ctrl);3周龄DOX10mg/kg处理组(3wk+10)；3周龄DOX20mg/kg处理组(3wk+20)；5周龄对照组(5wk-ctrl);5周龄DOX10mg/kg处理组(5wk+10)；5周龄DOX20mg/kg处理组(5wk+20)。DOX注射方式及标本采集方式同上。
结果
1.1周龄小鼠心肌组织活化caspase3的表达水平及PARP剪切产物呈DOX浓度依赖性增加,在DOX10mg/kg处理后增加具统计显著性。
2. DOX10mg/kg处理后,1周龄小鼠心肌组织活化caspase3的表达水平逐渐增高,并在处理24小时后达峰值。
3. DOX10mg/kg处理24小时后,1周龄小鼠心肌组织caspase3活性显著增高,DOX20mg/kg处理后进一步增高。
4.1周龄小鼠心肌细胞凋亡率呈DOX浓度依赖性显著增高。
5.1周龄小鼠心肌组织活化caspase9的表达水平呈DOX浓度依赖
性显著增加。
6.DOX处理后,Bax的表达水平在初生和成年小鼠心肌组织均显著增高。
7.DOX10或20mg/kg处理24h后,3周龄和5周龄小鼠心肌组织均没有呈现caspase3活性。
8.DOX10或20mg/kg处理24h后,在3周龄和5周龄小鼠心肌组织没有检测到活化caspase3表达。
9.DOX10或20mg/kg处理没有诱导3和5周龄小鼠心肌细胞凋亡发生。
10.DOX10或20mg/kg处理没有诱导3和5周龄小鼠心肌组织活化caspase9表达。
11.各年龄心肌组织pro-caspase3、pro-caspase9、Bax、Apaf、XIAP、 pro-caspase8、活化caspase8的表达没有呈现DOX相关性变化。
12.各年龄心肌组织细胞增殖或生存相关蛋白如Akt/p-Akt、p-P53、p-P38、p-P44/42等的表达水平均没有呈现DOX相关性改变。
结论
心肌细胞凋亡是幼年时期接受DOX治疗后心肌病变的早期和根本原因之一,也是幼年心脏对DOX毒性较成年心脏更为敏感的根本原因之一。
本研究首次通过动物模型证实,幼年心脏心肌凋亡能力明显高于成年心脏,心肌细胞凋亡是幼年时期接受阿霉素治疗后心肌病变的早期和根本原因之一,也是幼年心脏对阿霉素毒性较成年心脏更为敏感的根本原因之一。
Pathological cardiomyocyte hypertrophy is the common response when the heart endures damage factors. Persistent hypertrophy eventually leads to cardiac remodeling, chamber dilation and cardiac dysfunction. There is a growing interest in identifying the signaling mechanisms underlying the development of cardiac hypertrophy and the transition to heart failure.
Numerous studies have suggested a role of ROCK in mediating cardiac hypertrophy and remodeling by using pharmacological inhibitors of ROCK. The first chapter of this thesis will focus on the potential role of ROCK1, one of the two isoforms of ROCK family, on the cardiac remodeling in pathological hypertrophy model via in vivo loss-of-function of ROCK1.
Cardiomyocyte apoptosis has been considered as an essential factor during the transition of cardiac hypertrophy to heart failure. However, even under pathological conditions, the absolute prevalence of cardiomyocyte apoptosis is still low. Thus, the significance of apoptosis to human ventricular remodeling continues to be debated. Based on this consideration, in the second chapter we will investigate the significance of cardiomyocyte apoptosis in cardiomyopathy at different ages, through elucidating the age-related difference of cardiomyocyte apoptosis under physiological and pathological conditions.
Chapter Ⅰ Disruption of ROCK1gene attenuates cardiac dilation and improves cardiac function in pathological cardiac hypertrophy
BACKGROUND
Pathological cardiomyocyte hypertrophy is the common response when the heart endures increased hemodynamic load, myocardial injury, and neurohormonal stress. Persistent stress eventually leads to a decompensate congestive heart failure, which is characterized by chamber dilation and myocardial dysfunction. There is a growing interest in identifying the signaling mechanisms underlying the development of cardiac hypertrophy and the transition to heart failure.
The Gq class of heterotrimeric G proteins is one important signal transducer that is responsible for the development of cardiac hypertrophy and subsequent cardiac decompensation. Transgenic overexpression of the α subunit of Gq in the myocardium is a well-characterized in vivo pathological hypertrophy model which recapitulates many cellular, molecular, and functional characteristics of pressure overload induced hypertrophy including left ventricular dilation, depressed ventricular contractility at baseline and in response to β-adrenergic receptor stimulation, diminished basal and agonist stimulated adenylyl cyclase (AC) activity, and activation of a fetal cardiac gene program. A number of studies have delineated several potential mechanisms by which Gocq overexpression leads to cardiomyocyte hypertrophy, cardiac dilation, and dysfunction. These mechanisms include increased expression and activity of protein kinase C (PKC)α, decreased AC expression and catalytic activity, and increased activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase kinase1(MEKK1)/c-Jun N-terminal kinase (JNK) signaling pathway.
Rho-associated coiled-coil containing protein kinase (ROCK) is a downstream mediator of RhoA GTPase, and is believed to play a critical role in mediating the effects of RhoA on stress fiber formation, smooth muscle contraction, cell adhesion, membrane ruffling, and cell motility. Recent in vivo studies using ROCK inhibitors, Y27632and fasudil, suggest a role of ROCK in mediating cardiac hypertrophy and remodeling. However these pharmacological inhibitors are not able to distinguish between the two isoforms of the family, ROCK1and ROCK2. We have recently shown that ROCK1homozygous deficient mice developed cardiac myocyte hypertrophy in response to pressure overload induced by transverse aortic constriction, indicating that ROCK1is not critical for the development of cardiac hypertrophy. However these mice exhibited significantly reduced interstitial fibrosis and cardiomyocyte apoptosis. This observation is consistent with another recent study using ROCK1haploinsufficient mice which did not show decreased hypertrophy but decreased perivascular fibrosis induced by angiotensin Ⅱ. A number of the previous in vitro studies performed in cultured rat neonatal cardiomyocytes have shown that RhoA/ROCK pathway cooperated with Gαq pathway for the induction of cardiomyocyte hypertrophy by G protein-coupled receptor agonists. The combination of the well-characterized transgenic Gαq cardiac hypertrophy model with the ROCK1deficient mice provides an opportunity to examine the potential in vivo interaction between ROCK1and Gαq pathways and to test effects of ROCK1deletion on the development of dilated cardiomyopathy induced by intrinsic cardiac effects in Gαq overexpression transgenic mice.
Part One ROCK1deletion prevents cardiac dilation and improves contractile function in Gαq mice
METHODS
(1) Survival study:
Sex (male)-matched FVB mice were divided into4groups (n=5): wild type group (WT), cardiac specific Gαq overexpressing group (Gαq), Gαq-oevrexpressing and ROCK1-knockout compound group (Gαq/ROCK1-/-), and ROCK1-knockout group (ROCK1-/-). All these mice underwent echocardiography at the age of10months, and were followed for12months.
(2) Other protein analyses, histological and functional analyses:
Male FVB mice were divided into4groups:wild type (WT)、Gαq、 Gαq/ROCK1-/-and ROCK1-/-. All these mice underwent echo-cardiography and were sacrificed at the age of12weeks, and the hearts were saved for western blot analysis and histological analysis.
RESULTS
1. Expression levels of Gαq and ROCK:
(1)Gαq expression level was increased significantly and invariant in Gαq and Gαq/ROCK1-/-mice. ROCK1deficiency did not change the expression level of Gαq transgene in ventricular tissue.
(2)ROCK1expression was absent in ROCK1-/-and Gαq/ROCK1-/-mice.
(3)ROCK2expression remained similar in all four groups.
(4)No significant difference was detected for ROCK activity assessed by Western blot analysis among these groups.
2. ROCK1deletion prolonged the survival time of Gαq mice significantly.
3. Disruption of ROCK1did not prevent development of cardiomyocyte hypertrophy induced by cardiac-specific overexpression of Gαq.
4. ROCK1deletion partially prevented the induction of embryonic marker genes by cardiac-specific overexpression of Gαq.
5. ROCK1deletion prevented left ventricular dilation and dysfunction induced by cardiac-specific overexpression of Gαq.
CONCLUSION
ROCK1deficiency prevents cardiac dilation, improves cardiac function, and prolongs survival time of cardiac-specific Gαq overex-pression transgenic mice.
METHODS
4groups of FVB mice were enrolled in this study:WT、Gαq、Gαq/ROCK1-/-and ROCK1-/-. All these mice were sacrificed right after undergoing echocardiography under baseline or Isoproteronol stimulation at the age of12weeks. Ventricular tissue homogenate was used for western blot analysis, and whole heart was used for histological analysis.
RESULTS
1. Fibrosis was not consistently observed in the Gαq and Gαq/ROCK1-/cardiac tissue.
2. There was no significant increase in cardiac apoptosis in Gαq and Gαq/ROCK1-/-groups compared with WT or ROCK1-/-groups.
3. Gαq/ROCK1-/-mice exhibited improved contractility in response to β-adrenergic receptor stimulation when compared with Gαq mice.
4. ROCK1deletion did not inhibit MAPK activation and PKCα activation induced by cardiac-specific overexpression of Gαq.
5. ROCK1deletion rescued down-regulation of adenylyl cyclase type Ⅴ/Ⅵ induced by cardiac-specific overexpression of Gαq.
CONCLUSION
ROCK1deletion prevents down-regulation of adenylyl cyclase type Ⅴ/Ⅵ and β-adrenergic dysfunction induced by cardiac-specific overexpression of Gαq, which is consistent with improved contractile function by ROCK1deletion.
CONCLUSIONS OF CHAPTER Ⅰ
ROCK1deletion prevents cardiac dilation, down-regulation of adenylyl cyclase type Ⅴ/Ⅵ and β-adrenergic dysfunction induced by cardiac-specific overexpression of Gαq, which is consistent with the improved contractile function and prolonged survival time by ROCK1deletion.
Chapter Ⅱ The age-related difference of cardiomyocyte apoptosis and the relationship between cardiomyocyte apoptosis and cardiomyopathy at different ages
Part One The age-related difference of caspases-dependent cardiomyocyte apoptosis
BACKGROUND
Two canonical pathways involved in caspases-dependent apoptosis include the extrinsic pathway and the intrinsic pathway, which are initiated through activation of caspase8and caspse9respectively, and converge on the cleavage of apoptotic executioner caspases3,6,7. In the intric pathway, Apaf1serves as adaptor protein to link cytochrome C and pro-caspase9, and is necessary to the activation of pro-caspase9. Bcl-2family, which is believed to be a major regulator, contains pro-apoptotic proteins, such as Bax, Bak, BH3-only proteins, and anti-apoptotic proteins, such as Bcl-2, Bcl-XL and Mcl-1.
Cardiomyocyte apoptosis is essential to the embryonic cardiac development, and becomes very rare after birth. Numerous studies have demonstrated the significance of cardiomyocyte apoptosis in cardiomyopathy, however even under the pathological conditions, the absolute prevalence of cardiomyocyte apoptosis is still very low. Another question which has been attracting much attention is that the young hearts present more susceptibility to the cardiac damages than the adult hearts. We speculate that the age-dependent changes of the cell death and proliferation-related genes and proteins may be linked to the difference of the sensitivity to stimuli between young and adult hearts.
At first, the changes of the cardiomyocyte apoptosis and the apoptotic molecules, including caspase3, caspase8, caspase9, Apaf1, Bax, etc, were determined from the neonate to the adult under physiological conditions; Furthermore, the combination of the specific labeling of cardiomyocyte nuclei in which the lacZ gene is overexpressed and in situ TUNEL staining gave us an access to precisely quantify the cardiomyocyte apoptosis at different ages.
METHODS
FVB wild type mice (WT) and transgenic FVB mice overexpressing lacZ in the nuclei of cardiomyocytes (αMHC-nlacZ) were enrolled in this study. About the caspase3activity measurement and the caspases protein analysis,6groups of WT mice were included:1-day-old group,3-day-old group,1-week-old group,2-week-old group,3-week-old group and5-week-old group. Regarding the immunostaining of active-caspase3, TUNEL staining and other protein analysis,3groups of WT or nlacZ mice were included:1-week-old group,3-week-old group and5week-old group. The β-gal staining to specifically label the cardiomyocytes was performed before immuno staining and TUNEL staining.
RESULTS
1. The nuclei of cardiomyocytes in αMHC-nlacZ presented blue color after β-Gal staining compared with the cytosol and other types of cells.
2. Caspase3activity assay showed that the caspase3activity in myocardium was decreased significantly with the age. The caspase3 activity was much higher in1-week-old cardiac tissue, decreasing and going to be undetectable after3weeks.
3. Immunostaining assay and western blot showed that the expression level of active caspase3in cardiomyocytes was decreased significantly with the age, and no active caspase3was detected in the cardiomyocytes after3weeks.
4. Combination of β-Gal staining and TUNEL staining showed that the cardiomyocyte apoptosis was decreased significantly with the age. Some apoptotic cardiomyocytes could be observed at the age of1week, however rare cardiomyocyte apoptosis occurred3weeks after birth.
5. From the results of western blot, the expression level of pro-caspase3was much higher in1-week-old cardiac tissue, and decreased significantly after2weeks, and became very weak at the age of3weeks; also, the expression levels of pro-caspase9and pro-caspase8were decreased significantly with the age; Active-caspase9, but no active-caspase8, was detected in1-week-old myocardium, but not in3-and5-week-old myocardium.
6. From the results of western blot, the expression levels of other proteins which are essential for the intrinsic apoptotic pathway, including Bax and Apafl, were significantly down-regulated with the age.
7. The expression of other proliferation-and survival-related proteins, such as Akt, p-Akt, p-P53, p-p38and p-p42/44, did not present the age-dependent change.
CONCLUSION
In the earlier stage of the postnatal cardiac development, the cardiac apoptotic potential is much higher than that in the adult hearts, which may render a greater tendency towards cardiac apoptosis in the neonatal heart with damage, and may serve as one of the pivotal mechanisms for the higher susceptibility of the neonatal heart to the cardiac damage.
BACKGROUND
Doxorubicin (DOX) is one of the most widely used and successful antitumor drugs. Cardiotoxicity is a major limiting factor in anticancer therapy. Numerous studies indicate that cardiomyocyte apoptosis plays a key role in the DOX-induced cardiotoxicity. However, most of the in vivo studies about DOX-induced cardiotoxicity only focus on the adult hearts, in which the rate of cardiomyocyte apoptosis is very low, even under the pathological conditions. In addition, all the methods to measure the cardiomyocyte apoptosis in situ are unable to precisely distinguish cardiomyocytes and non-cardiomyocytes. Thus, the significance of apoptosis to cardiomyopathy and ventricular remodelling induced by DOX or other stimuli continues to be debated.
As demonstrated by clinical studies, children and adolescents are particularly susceptible to the cardiotoxic effects of DOX. The possible rationale was due to the loss of myocytes and impaired cardiac growth resulting in inadequate left ventricular mass and cardiomyopathy a year or more after cessation of chemotherapy. The results from the preceding section and other studies suggest that the more active intrinsic apoptotic pathway, particularly the higher expression level of pro-caspase3, may contribute to the more severe DOX cardiotoxicity in the neonatal hearts.
In the present study, we further observed the difference of the cardiomyocyte apoptosis and the apoptotic pathways between neonatal and adult hearts at the earlier stage of DOX-induced cardiotoxicity, precisely quantified the DOX-induced cardiomyocyte apoptosis via combining the β-gal staining and TUNEL staining, and measured the significance of cardiomyocyte apoptosis to cardiomyopathy at different ages.
METHODS
FVB-WT and aMHC-nlacZ mice were recruited in this study.
(1)The effects of DOX with different concentrations on the expression of caspase3in1-week-old mouse hearts: Mice were divided into5groups (n=4):Saline-treated group (1wk-ctrl), DOX5mg/kg-treated group (1wk+5), DOX10mg/kg-treated group (1wk+10), DOX15mg/kg-treated group (1wk+15), DOX20mg/kg-treated group (1wk+20). DOX was given by i.p injection. Mice were sacrificed to harvest hearts24h after injection.
(2) The time-course analysis of effects of DOX10mg/kg on the caspase3expression in1-week-old hearts: Mice were divided into7groups:treated with DOX for0hour,2,4,8,12,16,24hours (n=4).
(3) The effects of DOX with low (10mg/kg) and high (20mg/kg) concentrations on cardiomyocyte apoptosis and apoptotic pathways in mouse hearts with different ages: Mice were divided into9groups:1wk-ctrl,1wk+10,1wk+20;3wk-ctrl,3wk+10,3wk+20;5wk-ctrl,5wk+10,5wk+20.
RESULTS
1. The expression levels of active caspase3and the production of cleaved PARP were increased in a DOX concentration-dependent manner in the1-week-old mouse hearts, and became significantly higher with the dose increasing to10mg/kg.
2. The expression levels of active caspase3were induced gradually by DOX, and peaked at24hours after DOX administration in1-week-old myocardium.
3. The caspase3activity was increased significantly in the1-week-old myocardium after treatment with low-dose DOX (10mg/kg), and further higher in the high-dose group (20mg/kg).
4. The rate of cardiomyocyte apoptosis was increased significantly in the1-week-old mouse hearts in a DOX concentration-dependent manner.
5. The expression level of active caspase9was increased significantly in a concentration-dependent manner after treatment with DOX in1-week-old hearts.
6. The expression of Bax was induced significantly by DOX treatment in both neonatal and adult hearts.
7. The caspase3activity was undetectable in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.
8. The expression of active caspase3was undetectable in3-and5-week-old hearts after treatment with DOX10or20mg/kg for24hours.
9. The cardiomyocyte apoptosis was not present in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.
10.The expression of active caspase9was undetectable in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.
11.DOX exerted no effects on the expression of pro-caspase3, pro-caspase9, Apaf1, XIAP, pro-caspase8and active-caspase8in both neonata1and adult hearts.
12.DOX exerted no effects on the expression of Akt/p-Akt, p-P53, p-p38, p-p42/44in both neonatal and adult hearts.
CONCLUSION
The higher activity of apoptotic pathways leads to a higher occurrence of apoptosis in the neonatal heart after DOX treatment, suggesting that excessive cardiomyocyte apoptosis is one of the primary and major factors involved in the earlier cardiotoxicity induced by DOX in the juvenile hearts, and is one of the underlying mechanisms by which the neonatal hearts are exposed to a high susceptibility to the DOX-induced cardiotoxicity.
Our present in vivo study suggests for the first time that the cardiac apoptotic potential is much higher in the neonatal hearts than that in the adult hearts, which is one of the primary and major factors involved in the earlier cardiotoxicity induced by DOX in the juvenile hearts, and is one of the underlying mechanisms by which the juvenile patients are exposed to a higher susceptibility to the DOX-mediated cardiotoxicity.
目前许多研究通过药理性抑制剂显示,ROCK在心肌肥大和心肌重构中发挥作用。本论文第一章将利用基因敲除技术,在动物水平深入探讨ROCK家族的ROCK1亚型在心肌病理性重构中的作用。
心肌细胞凋亡一直被认为是病理性心肌肥大向心衰转变的关键因素。然而即使在病理性刺激下,成年心脏心肌细胞凋亡率仍然很低,因此,心肌细胞凋亡在心肌病理性重构中的作用越来越受到质疑。基于此,本论文第二章将通过对比生理和病理情况下心肌细胞凋亡的年龄相关性差异,探讨心肌细胞凋亡在不同年龄心肌病变中的重要性。
第一章ROCK1缺失抑制病理性心肌肥大模型心肌扩张并改善收缩功能
研究背景
病理性心肌细胞肥大是心脏对抗血流动力学增加、肌损伤和神经激素应激等的共同反应,持续的心肌肥大最终将导致失代偿性充血性心衰,表现为心室扩张和收缩功能障碍。越来越多的研究旨在确立心肌肥大发展以及向心衰转变的分子机制。
Gq为一类异三聚体GTP结合蛋白。由α、p和γ亚基组成。大量研究表明,Gq信号通路是心肌肥大发展和心脏失代偿的关键调节因素。心肌细胞特异性转基因过表达Gα亚单位(Gαq)小鼠是一个已经定性的病理性心肌肥大模型,在分子水平、细胞水平和整体水平模拟了许多压力超负荷诱导的心肌肥大的特征,如：左心室扩张、基础水平和p-肾上腺素受体刺激后的心室收缩功能减退、基础水平和激动剂刺激的腺苷环化酶(adenylyl cyclase, AC)活性下降、以及心肌基因表达向胎儿模式转变。许多研究阐述了Gαq过表达所致心肌细胞肥大、心室扩张和心功能障碍的可能机制,包括：PKCα表达和活性增加、AC表达和活性下降、MEKK1/JNK信号途径的异常等。
ROCK是RhoA GTPase的下游信号分子,在RhoA调控压力纤维形成、平滑肌收缩、细胞粘附等多种事件中扮演关键角色。最近有在体实验使用ROCK抑制剂Y27632和法舒地尔提示,ROCK在心肌肥大和心肌重构中发挥作用。但这些药理性抑制剂不能区分ROCK家族两个亚型ROCK1和ROCK2。我们最近的研究显示,ROCK1基因缺失纯合子小鼠(ROCK-/-)心肌在对压力超负荷的反应中,心肌间质纤维和心肌细胞凋亡较WT小鼠显著减少,但仍然发展了心肌细胞肥大。另一个研究也显示,在血管紧张素2刺激后,ROCK+/-杂合子小鼠发展了心肌肥大,但血管外周纤维化较WT明显减少。
许多研究通过体外实验表明,RhoA/ROCK协同Gαq信号通路,参与了G蛋白偶联受体激动剂诱导的心肌细胞肥大。然而,至今没有在体实验表明两条通路之间的相互作用。本研究联合心肌细胞特异性Gαq过表达心肌肥大模型和ROCK1基因敲除小鼠,检验了ROCK1和Gαq通路间的相互作用,探讨了ROCK1缺失对Gαq过表达小鼠心肌内部变化所致扩张型心肌病变的影响,也为进一步明确ROCK1和ROCK2亚型的独立作用奠定了基础。
第一节ROCK1缺失阻止Gαq小鼠心肌扩张并改善心收缩功能
方法
(1)生存实验：
FVB雄性小鼠,分设4组(n=5)：野生型组(WT)、心肌细胞特异性Gaq过表达组(Gαq)、Gαq过表达与ROCK1基因敲除混合组(Gαq/ROCK1-/-)、ROCK1基因敲除组(ROCK1-/-)。所有动物正常饲养至12月龄,采用SPSS软件绘制Caplan-Meier生存曲线。在10月龄行超声心动分析。
(2)其他蛋白分析、组织学和功能实验：
FVB雄性小鼠,分设4组(n=4)：WT、Gαq、Gαq/ROCK1-/-、 ROCK1-/-。所有动物至12周龄,行心功能分析后,断颈处死并分离心脏。心脏保存于液氮,或行冰冻或石蜡包埋。
结果
1.Gαq、ROCK表达水平分析：
(1)在Gαq和Gαq/ROCK1-/-小鼠心肌组织内,Gαq表达水平较WT组和ROCK1-/-组显著增高,且在各转基因组小鼠表达一致,ROCK缺失没有影响心肌组织内Gαq的过表达水平；
(2)ROCK1-/-和Gαq/ROCK1-/-小鼠心肌组织内不表达ROCK1; ROCk2的表达在各组小鼠间没有差异；
(3)ROCK活性水平没有随ROCK1缺失和Gαq过表达而改变；
2.ROCK1缺失显著延长了Gaq小鼠的存活时间。
3.ROCK1缺失没有阻止Gαq小鼠心肌细胞肥大性发展：
(1)形态学分析显示,ROCK1缺失没有明显抑制Gαq过表达诱导的心脏重量增大；
(2)超声扫描分析显示,ROCK1缺失没有明显降低Gαq过表达诱导的小鼠左心室质量增大；
(3)组织学切片染色显示,ROCK1没有明显降低Gαq过表达诱导的左心室心肌细胞肥大；
4.ROCK1缺失部分抑制了Gαq过表达诱导的心肌肥大标志物mRNA水平上调；
5.ROCK1缺失阻止了心肌细胞特异性Gαq过表达诱导的左心室扩张和左心室收缩功能障碍：
(1)超声扫描分析显示,ROCK1缺失明显抑制了Gαq小鼠左室内径增大及室壁变薄；
(2)组织学切片染色显示,Gαq小鼠为左室离心性肥大,而Gαq/ROCK1-/-混合小鼠为心室向心性肥大；
(3)超声扫描分析显示,ROCK1缺失明显阻止了Gαq小鼠左室短轴缩短率(fractional shortening, FS)和射血分数(ejection fraction,EF)降低；
(4)形态学分析显示,ROCK1缺失阻止了Gαq小鼠的肺水肿。
结论
ROCK1基因缺失抑制了心肌细胞特异性过表达Gαq转基因小鼠的心室扩张,改善了其心功能,并显著延长了Gαq小鼠的寿命。
刍二节ROCK1缺失阻止Gαq小鼠心肌AC5/6下调并恢复心肌β-AR功能
方法
FVB雄性小鼠,分设4组：WT、Gαq、Gαq/ROCK1-/-、ROCK1-/-。所有动物至12周龄,行心功能分析后,断颈处死并分离心脏。心脏保存于液氮,或行冰冻或石蜡包埋。部分实验动物给予腹腔注射异丙肾上腺素(ISO,100ng/g体重),在注射前和注射后5分钟分别进行超声心动检测。
结果
1.Gaq和Gαq/ROCK1-/-小鼠心肌组织没有发展明显的心肌纤维化。
2.Gαq和Gαq/ROCK1-/-小鼠没有明显的心肌凋亡。
3.异丙肾上腺素刺激后,ROCK1基因缺失显著改善了Gαq心肌对p-肾上腺素受体刺激的低反应性。
4.ROCK1缺失恢复了心肌特异性Gαq过表达诱导的AC5/6下调。
5.ROCK1缺失没有抑制心肌细胞特异性Gαq过表达诱导的MAPK活化和PKCα活化。
结论
ROCK1缺失明显抑制了心肌细胞特异性Gαq过表达诱导的AC5/6蛋白水平下调和p-肾上腺素受体功能障碍,从而改善了Gαq小鼠的心功能不全。
ROCK1基因缺失明显抑制了心肌细胞特异性Gαq过表达诱导的心室扩张、AC5/6的蛋白水平下调和β-肾上腺素受体功能障碍,从而改善了Gαq小鼠的心功能,并显著延长了Gαq小鼠的存活时间。
第一节Caspases依赖性心肌细胞凋亡的年龄差异性比较研究背景
介导Caspases依赖性细胞凋亡的信号通路包括外部凋亡途径和内部调亡途径,caspase8和caspase9分别是外部和内部通路的凋亡起始者,两条通路最终汇合于凋亡执行者caspase3的活化。在内部凋亡途径,Apafl作为接头蛋白,连接细胞色素C和pro-caspase9,为pro-caspase9活化所必需。Bcl-2家族是内部凋亡途径最主要的调节因子,包括：促凋亡蛋白Bax、Bak、BH3、蛋白如Bad、Bid、Nix和BNip3,抑凋亡蛋白Bcl-2,Bcl-XL和Mcl-1。
心肌细胞凋亡在心脏胚胎发育期扮演关键角色,而在出生后成熟的心肌非常罕见。无数研究表明心肌细胞凋亡在许多心肌病变中成百倍上调,然而即使在这些病理情况下,成年心肌细胞凋亡率仍然很低。研究也显示,幼年心脏比成年心脏对各种刺激更加敏感。我们推测心肌细胞死亡和增殖相关基因表达模式或蛋白活性的年龄依赖性变化,是幼年和成年心脏对刺激敏感性不同的根本原因之一。
我们首先系统比较了正常小鼠从出生到成年心脏发育过程中,生理条件下心肌细胞凋亡及其关键信号分子包括：caspase3、caspse8、 caspase9、Apafl、Bax等的表达或活性的变化；并将TUNEL原位染色法与特异性标记心肌细胞核的方法结合,较准确地定量了生理条件下不同年龄阶段的心肌细胞凋亡。
方法
实验使用FVB品系野生型小鼠(WT)或心肌细胞核特异性过表达lacZ的FVB小鼠(αMHC-nlacZ)。有关caspase3活性检测和caspases蛋白表达分析的实验部分,WT小鼠分为6组：雄或雌性出生后1天、出生后3天、1周龄、2周龄、雄性3周龄、雄性5周龄。有关活化caspase3免疫组化染色、TUNEL染色及其他凋亡相关蛋白分析的实验部分,WT或nlacZ小鼠分设3组：雄或雌性1周龄、雄性3周龄、雄性5周龄；在活化caspase3免疫组化染色和TUNEL染色实验中,先行p-半乳糖苷(β-gal)染色,以特异性标记心肌细胞核。
结果
1. aMHC-nlacZ小鼠心脏冰冻切片行β-gal染色后,可见心肌细胞核呈淡蓝色,而胞浆和其它类型细胞核未染。
2. Caspase3活性实验显示,小鼠心肌组织caspase3活性随年龄增大而显著降低。出生1周内的心肌组织具有相对高的caspase3活性,2周龄时仍可以检测到少量caspase3活性,3周以后已基本检测不到活化的caspase3。
3. β-gal染色结合免疫组化染色、以及Western blot结果显示,小鼠心肌细胞内活化caspase3的表达水平随年龄增大而显著减少,3周龄以后的心肌细胞不表达活化caspase3。
4. p-gal染色结合TUNEL染色显示,小鼠心室肌细胞凋亡随年龄增大而显著减少。1周龄时心室肌细胞仍有一定比率的凋亡发生,而3周以后罕见心肌细胞凋亡。
5. Western blot结果显示,出生1周内pro-caspase3的表达水平较高,2周后明显降低,3周后极其微弱；pro-caspase9和pro-caspase8的表达水平同样随年龄增大而明显降低。
6. Western blot结果显示,心肌组织内部凋亡途径信号分子Bax、Apafl的表达随年龄增大而显著下降。
7.心肌组织内其他细胞增殖和生存相关蛋白如Akt/p-Akt。p-P53、p-p38和p-p42/44的表达水平均没有呈现年龄相关性变化。
结论
出生后心肌生理性发育的早期,细胞凋亡通路及心肌细胞凋亡较成年心肌尤为活跃,这可能是幼年心脏对病理性刺激高敏感性的分子基础之一。
研究背景
阿霉素(Doxorubicin, DOX)是目前应用最广泛和有效的抗肿瘤药物之一。但是严重的心脏毒性限制了DOX的使用剂量和疗程。研究显示心肌细胞凋亡在DOX诱导的心肌毒性反应中扮演重要角色。但目前有关DOX心肌毒性的在体研究绝大部分是采用成年动物模型,其病理情况下的心肌细胞凋亡率仍然很低；而且目前有关原位检测心肌细胞凋亡的方法如TUNEL染色,均不能准确区分心肌细胞和非心肌细胞。因此心肌细胞凋亡在病理性因素比如DOX刺激诱导的心肌病理性发展、尤其是成年后心肌重构中的重要性越来越受到质疑。
临床研究也表明,接受DOX治疗的儿童和青少年对其心脏毒性影响较成年尤为敏感,大部分儿童在完成疗程一年或多年以后发生心肌病变。可能的原因为心肌细胞丢失、心肌生长损害,导致左心室发育不全和心肌病变。然而其最根本的分子机制至今不明。我们在前一节所述的动物实验以及其他少数研究提示,幼年心肌细胞内部凋亡途径的相对高水平、尤其是凋亡执行者caspase3的高表达,可能是儿童对DOX心脏毒性较成年更为敏感的原因之一。
因此本实验中,我们使用在体给予DOX的心肌毒性动物模型,比较了在DOX刺激的早期,幼年和成年心肌细胞凋亡及其相关信号通路的差异。并结合特异性标记心肌细胞核的方法,准确定量DOX诱导的心肌细胞凋亡,衡量了心肌细胞凋亡在不同年龄阶段DOX所至心肌病变中的重要性。
方法
实验使用FVB品系WT或αMHC-nlacZ小鼠：
(1)观察不同浓度DOX对1周龄小鼠心肌组织caspase3表达的影响：
分为5组(n=4)：对照组(1wk-ctrl); DOX5mg/kg处理组(1wk+5);DOX10mg/kg处理组(1wk+10)；DOX15mg/kg处理组(1wk+15)；DOX20mg/kg处理组(1wk+20)。所有实验动物单次腹腔注射不同浓度DOX或等量生理盐水,24小时后断颈处死动物,迅速收集心脏,存于液氮。
(2) DOX10mg/kg对1周龄小鼠心肌组织caspase3表达影响的时间曲线分析：
分为7组(n=4)：DOX处理0小时组(对照组),DOX处理2,4,8,12,16,24小时组。DOX注射方式及标本采集方式同上。
(3)观察DOX(低浓度和高浓度)对不同年龄小鼠心肌细胞凋亡及凋亡信号通路的影响：
分为9组(n=4)：1周龄对照组(生理盐水组,1wk-ctrl);1周龄DOX10mg/kg处理组(1wk+10)；1周龄DOX20mg/kg处理组(1wk+20)；3周龄对照组(3wk-ctrl);3周龄DOX10mg/kg处理组(3wk+10)；3周龄DOX20mg/kg处理组(3wk+20)；5周龄对照组(5wk-ctrl);5周龄DOX10mg/kg处理组(5wk+10)；5周龄DOX20mg/kg处理组(5wk+20)。DOX注射方式及标本采集方式同上。
结果
1.1周龄小鼠心肌组织活化caspase3的表达水平及PARP剪切产物呈DOX浓度依赖性增加,在DOX10mg/kg处理后增加具统计显著性。
2. DOX10mg/kg处理后,1周龄小鼠心肌组织活化caspase3的表达水平逐渐增高,并在处理24小时后达峰值。
3. DOX10mg/kg处理24小时后,1周龄小鼠心肌组织caspase3活性显著增高,DOX20mg/kg处理后进一步增高。
4.1周龄小鼠心肌细胞凋亡率呈DOX浓度依赖性显著增高。
5.1周龄小鼠心肌组织活化caspase9的表达水平呈DOX浓度依赖
性显著增加。
6.DOX处理后,Bax的表达水平在初生和成年小鼠心肌组织均显著增高。
7.DOX10或20mg/kg处理24h后,3周龄和5周龄小鼠心肌组织均没有呈现caspase3活性。
8.DOX10或20mg/kg处理24h后,在3周龄和5周龄小鼠心肌组织没有检测到活化caspase3表达。
9.DOX10或20mg/kg处理没有诱导3和5周龄小鼠心肌细胞凋亡发生。
10.DOX10或20mg/kg处理没有诱导3和5周龄小鼠心肌组织活化caspase9表达。
11.各年龄心肌组织pro-caspase3、pro-caspase9、Bax、Apaf、XIAP、 pro-caspase8、活化caspase8的表达没有呈现DOX相关性变化。
12.各年龄心肌组织细胞增殖或生存相关蛋白如Akt/p-Akt、p-P53、p-P38、p-P44/42等的表达水平均没有呈现DOX相关性改变。
结论
心肌细胞凋亡是幼年时期接受DOX治疗后心肌病变的早期和根本原因之一,也是幼年心脏对DOX毒性较成年心脏更为敏感的根本原因之一。
本研究首次通过动物模型证实,幼年心脏心肌凋亡能力明显高于成年心脏,心肌细胞凋亡是幼年时期接受阿霉素治疗后心肌病变的早期和根本原因之一,也是幼年心脏对阿霉素毒性较成年心脏更为敏感的根本原因之一。
Pathological cardiomyocyte hypertrophy is the common response when the heart endures damage factors. Persistent hypertrophy eventually leads to cardiac remodeling, chamber dilation and cardiac dysfunction. There is a growing interest in identifying the signaling mechanisms underlying the development of cardiac hypertrophy and the transition to heart failure.
Numerous studies have suggested a role of ROCK in mediating cardiac hypertrophy and remodeling by using pharmacological inhibitors of ROCK. The first chapter of this thesis will focus on the potential role of ROCK1, one of the two isoforms of ROCK family, on the cardiac remodeling in pathological hypertrophy model via in vivo loss-of-function of ROCK1.
Cardiomyocyte apoptosis has been considered as an essential factor during the transition of cardiac hypertrophy to heart failure. However, even under pathological conditions, the absolute prevalence of cardiomyocyte apoptosis is still low. Thus, the significance of apoptosis to human ventricular remodeling continues to be debated. Based on this consideration, in the second chapter we will investigate the significance of cardiomyocyte apoptosis in cardiomyopathy at different ages, through elucidating the age-related difference of cardiomyocyte apoptosis under physiological and pathological conditions.
Chapter Ⅰ Disruption of ROCK1gene attenuates cardiac dilation and improves cardiac function in pathological cardiac hypertrophy
BACKGROUND
Pathological cardiomyocyte hypertrophy is the common response when the heart endures increased hemodynamic load, myocardial injury, and neurohormonal stress. Persistent stress eventually leads to a decompensate congestive heart failure, which is characterized by chamber dilation and myocardial dysfunction. There is a growing interest in identifying the signaling mechanisms underlying the development of cardiac hypertrophy and the transition to heart failure.
The Gq class of heterotrimeric G proteins is one important signal transducer that is responsible for the development of cardiac hypertrophy and subsequent cardiac decompensation. Transgenic overexpression of the α subunit of Gq in the myocardium is a well-characterized in vivo pathological hypertrophy model which recapitulates many cellular, molecular, and functional characteristics of pressure overload induced hypertrophy including left ventricular dilation, depressed ventricular contractility at baseline and in response to β-adrenergic receptor stimulation, diminished basal and agonist stimulated adenylyl cyclase (AC) activity, and activation of a fetal cardiac gene program. A number of studies have delineated several potential mechanisms by which Gocq overexpression leads to cardiomyocyte hypertrophy, cardiac dilation, and dysfunction. These mechanisms include increased expression and activity of protein kinase C (PKC)α, decreased AC expression and catalytic activity, and increased activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase kinase1(MEKK1)/c-Jun N-terminal kinase (JNK) signaling pathway.
Rho-associated coiled-coil containing protein kinase (ROCK) is a downstream mediator of RhoA GTPase, and is believed to play a critical role in mediating the effects of RhoA on stress fiber formation, smooth muscle contraction, cell adhesion, membrane ruffling, and cell motility. Recent in vivo studies using ROCK inhibitors, Y27632and fasudil, suggest a role of ROCK in mediating cardiac hypertrophy and remodeling. However these pharmacological inhibitors are not able to distinguish between the two isoforms of the family, ROCK1and ROCK2. We have recently shown that ROCK1homozygous deficient mice developed cardiac myocyte hypertrophy in response to pressure overload induced by transverse aortic constriction, indicating that ROCK1is not critical for the development of cardiac hypertrophy. However these mice exhibited significantly reduced interstitial fibrosis and cardiomyocyte apoptosis. This observation is consistent with another recent study using ROCK1haploinsufficient mice which did not show decreased hypertrophy but decreased perivascular fibrosis induced by angiotensin Ⅱ. A number of the previous in vitro studies performed in cultured rat neonatal cardiomyocytes have shown that RhoA/ROCK pathway cooperated with Gαq pathway for the induction of cardiomyocyte hypertrophy by G protein-coupled receptor agonists. The combination of the well-characterized transgenic Gαq cardiac hypertrophy model with the ROCK1deficient mice provides an opportunity to examine the potential in vivo interaction between ROCK1and Gαq pathways and to test effects of ROCK1deletion on the development of dilated cardiomyopathy induced by intrinsic cardiac effects in Gαq overexpression transgenic mice.
Part One ROCK1deletion prevents cardiac dilation and improves contractile function in Gαq mice
METHODS
(1) Survival study:
Sex (male)-matched FVB mice were divided into4groups (n=5): wild type group (WT), cardiac specific Gαq overexpressing group (Gαq), Gαq-oevrexpressing and ROCK1-knockout compound group (Gαq/ROCK1-/-), and ROCK1-knockout group (ROCK1-/-). All these mice underwent echocardiography at the age of10months, and were followed for12months.
(2) Other protein analyses, histological and functional analyses:
Male FVB mice were divided into4groups:wild type (WT)、Gαq、 Gαq/ROCK1-/-and ROCK1-/-. All these mice underwent echo-cardiography and were sacrificed at the age of12weeks, and the hearts were saved for western blot analysis and histological analysis.
RESULTS
1. Expression levels of Gαq and ROCK:
(1)Gαq expression level was increased significantly and invariant in Gαq and Gαq/ROCK1-/-mice. ROCK1deficiency did not change the expression level of Gαq transgene in ventricular tissue.
(2)ROCK1expression was absent in ROCK1-/-and Gαq/ROCK1-/-mice.
(3)ROCK2expression remained similar in all four groups.
(4)No significant difference was detected for ROCK activity assessed by Western blot analysis among these groups.
2. ROCK1deletion prolonged the survival time of Gαq mice significantly.
3. Disruption of ROCK1did not prevent development of cardiomyocyte hypertrophy induced by cardiac-specific overexpression of Gαq.
4. ROCK1deletion partially prevented the induction of embryonic marker genes by cardiac-specific overexpression of Gαq.
5. ROCK1deletion prevented left ventricular dilation and dysfunction induced by cardiac-specific overexpression of Gαq.
CONCLUSION
ROCK1deficiency prevents cardiac dilation, improves cardiac function, and prolongs survival time of cardiac-specific Gαq overex-pression transgenic mice.
METHODS
4groups of FVB mice were enrolled in this study:WT、Gαq、Gαq/ROCK1-/-and ROCK1-/-. All these mice were sacrificed right after undergoing echocardiography under baseline or Isoproteronol stimulation at the age of12weeks. Ventricular tissue homogenate was used for western blot analysis, and whole heart was used for histological analysis.
RESULTS
1. Fibrosis was not consistently observed in the Gαq and Gαq/ROCK1-/cardiac tissue.
2. There was no significant increase in cardiac apoptosis in Gαq and Gαq/ROCK1-/-groups compared with WT or ROCK1-/-groups.
3. Gαq/ROCK1-/-mice exhibited improved contractility in response to β-adrenergic receptor stimulation when compared with Gαq mice.
4. ROCK1deletion did not inhibit MAPK activation and PKCα activation induced by cardiac-specific overexpression of Gαq.
5. ROCK1deletion rescued down-regulation of adenylyl cyclase type Ⅴ/Ⅵ induced by cardiac-specific overexpression of Gαq.
CONCLUSION
ROCK1deletion prevents down-regulation of adenylyl cyclase type Ⅴ/Ⅵ and β-adrenergic dysfunction induced by cardiac-specific overexpression of Gαq, which is consistent with improved contractile function by ROCK1deletion.
CONCLUSIONS OF CHAPTER Ⅰ
ROCK1deletion prevents cardiac dilation, down-regulation of adenylyl cyclase type Ⅴ/Ⅵ and β-adrenergic dysfunction induced by cardiac-specific overexpression of Gαq, which is consistent with the improved contractile function and prolonged survival time by ROCK1deletion.
Chapter Ⅱ The age-related difference of cardiomyocyte apoptosis and the relationship between cardiomyocyte apoptosis and cardiomyopathy at different ages
Part One The age-related difference of caspases-dependent cardiomyocyte apoptosis
BACKGROUND
Two canonical pathways involved in caspases-dependent apoptosis include the extrinsic pathway and the intrinsic pathway, which are initiated through activation of caspase8and caspse9respectively, and converge on the cleavage of apoptotic executioner caspases3,6,7. In the intric pathway, Apaf1serves as adaptor protein to link cytochrome C and pro-caspase9, and is necessary to the activation of pro-caspase9. Bcl-2family, which is believed to be a major regulator, contains pro-apoptotic proteins, such as Bax, Bak, BH3-only proteins, and anti-apoptotic proteins, such as Bcl-2, Bcl-XL and Mcl-1.
Cardiomyocyte apoptosis is essential to the embryonic cardiac development, and becomes very rare after birth. Numerous studies have demonstrated the significance of cardiomyocyte apoptosis in cardiomyopathy, however even under the pathological conditions, the absolute prevalence of cardiomyocyte apoptosis is still very low. Another question which has been attracting much attention is that the young hearts present more susceptibility to the cardiac damages than the adult hearts. We speculate that the age-dependent changes of the cell death and proliferation-related genes and proteins may be linked to the difference of the sensitivity to stimuli between young and adult hearts.
At first, the changes of the cardiomyocyte apoptosis and the apoptotic molecules, including caspase3, caspase8, caspase9, Apaf1, Bax, etc, were determined from the neonate to the adult under physiological conditions; Furthermore, the combination of the specific labeling of cardiomyocyte nuclei in which the lacZ gene is overexpressed and in situ TUNEL staining gave us an access to precisely quantify the cardiomyocyte apoptosis at different ages.
METHODS
FVB wild type mice (WT) and transgenic FVB mice overexpressing lacZ in the nuclei of cardiomyocytes (αMHC-nlacZ) were enrolled in this study. About the caspase3activity measurement and the caspases protein analysis,6groups of WT mice were included:1-day-old group,3-day-old group,1-week-old group,2-week-old group,3-week-old group and5-week-old group. Regarding the immunostaining of active-caspase3, TUNEL staining and other protein analysis,3groups of WT or nlacZ mice were included:1-week-old group,3-week-old group and5week-old group. The β-gal staining to specifically label the cardiomyocytes was performed before immuno staining and TUNEL staining.
RESULTS
1. The nuclei of cardiomyocytes in αMHC-nlacZ presented blue color after β-Gal staining compared with the cytosol and other types of cells.
2. Caspase3activity assay showed that the caspase3activity in myocardium was decreased significantly with the age. The caspase3 activity was much higher in1-week-old cardiac tissue, decreasing and going to be undetectable after3weeks.
3. Immunostaining assay and western blot showed that the expression level of active caspase3in cardiomyocytes was decreased significantly with the age, and no active caspase3was detected in the cardiomyocytes after3weeks.
4. Combination of β-Gal staining and TUNEL staining showed that the cardiomyocyte apoptosis was decreased significantly with the age. Some apoptotic cardiomyocytes could be observed at the age of1week, however rare cardiomyocyte apoptosis occurred3weeks after birth.
5. From the results of western blot, the expression level of pro-caspase3was much higher in1-week-old cardiac tissue, and decreased significantly after2weeks, and became very weak at the age of3weeks; also, the expression levels of pro-caspase9and pro-caspase8were decreased significantly with the age; Active-caspase9, but no active-caspase8, was detected in1-week-old myocardium, but not in3-and5-week-old myocardium.
6. From the results of western blot, the expression levels of other proteins which are essential for the intrinsic apoptotic pathway, including Bax and Apafl, were significantly down-regulated with the age.
7. The expression of other proliferation-and survival-related proteins, such as Akt, p-Akt, p-P53, p-p38and p-p42/44, did not present the age-dependent change.
CONCLUSION
In the earlier stage of the postnatal cardiac development, the cardiac apoptotic potential is much higher than that in the adult hearts, which may render a greater tendency towards cardiac apoptosis in the neonatal heart with damage, and may serve as one of the pivotal mechanisms for the higher susceptibility of the neonatal heart to the cardiac damage.
BACKGROUND
Doxorubicin (DOX) is one of the most widely used and successful antitumor drugs. Cardiotoxicity is a major limiting factor in anticancer therapy. Numerous studies indicate that cardiomyocyte apoptosis plays a key role in the DOX-induced cardiotoxicity. However, most of the in vivo studies about DOX-induced cardiotoxicity only focus on the adult hearts, in which the rate of cardiomyocyte apoptosis is very low, even under the pathological conditions. In addition, all the methods to measure the cardiomyocyte apoptosis in situ are unable to precisely distinguish cardiomyocytes and non-cardiomyocytes. Thus, the significance of apoptosis to cardiomyopathy and ventricular remodelling induced by DOX or other stimuli continues to be debated.
As demonstrated by clinical studies, children and adolescents are particularly susceptible to the cardiotoxic effects of DOX. The possible rationale was due to the loss of myocytes and impaired cardiac growth resulting in inadequate left ventricular mass and cardiomyopathy a year or more after cessation of chemotherapy. The results from the preceding section and other studies suggest that the more active intrinsic apoptotic pathway, particularly the higher expression level of pro-caspase3, may contribute to the more severe DOX cardiotoxicity in the neonatal hearts.
In the present study, we further observed the difference of the cardiomyocyte apoptosis and the apoptotic pathways between neonatal and adult hearts at the earlier stage of DOX-induced cardiotoxicity, precisely quantified the DOX-induced cardiomyocyte apoptosis via combining the β-gal staining and TUNEL staining, and measured the significance of cardiomyocyte apoptosis to cardiomyopathy at different ages.
METHODS
FVB-WT and aMHC-nlacZ mice were recruited in this study.
(1)The effects of DOX with different concentrations on the expression of caspase3in1-week-old mouse hearts: Mice were divided into5groups (n=4):Saline-treated group (1wk-ctrl), DOX5mg/kg-treated group (1wk+5), DOX10mg/kg-treated group (1wk+10), DOX15mg/kg-treated group (1wk+15), DOX20mg/kg-treated group (1wk+20). DOX was given by i.p injection. Mice were sacrificed to harvest hearts24h after injection.
(2) The time-course analysis of effects of DOX10mg/kg on the caspase3expression in1-week-old hearts: Mice were divided into7groups:treated with DOX for0hour,2,4,8,12,16,24hours (n=4).
(3) The effects of DOX with low (10mg/kg) and high (20mg/kg) concentrations on cardiomyocyte apoptosis and apoptotic pathways in mouse hearts with different ages: Mice were divided into9groups:1wk-ctrl,1wk+10,1wk+20;3wk-ctrl,3wk+10,3wk+20;5wk-ctrl,5wk+10,5wk+20.
RESULTS
1. The expression levels of active caspase3and the production of cleaved PARP were increased in a DOX concentration-dependent manner in the1-week-old mouse hearts, and became significantly higher with the dose increasing to10mg/kg.
2. The expression levels of active caspase3were induced gradually by DOX, and peaked at24hours after DOX administration in1-week-old myocardium.
3. The caspase3activity was increased significantly in the1-week-old myocardium after treatment with low-dose DOX (10mg/kg), and further higher in the high-dose group (20mg/kg).
4. The rate of cardiomyocyte apoptosis was increased significantly in the1-week-old mouse hearts in a DOX concentration-dependent manner.
5. The expression level of active caspase9was increased significantly in a concentration-dependent manner after treatment with DOX in1-week-old hearts.
6. The expression of Bax was induced significantly by DOX treatment in both neonatal and adult hearts.
7. The caspase3activity was undetectable in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.
8. The expression of active caspase3was undetectable in3-and5-week-old hearts after treatment with DOX10or20mg/kg for24hours.
9. The cardiomyocyte apoptosis was not present in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.
10.The expression of active caspase9was undetectable in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.
11.DOX exerted no effects on the expression of pro-caspase3, pro-caspase9, Apaf1, XIAP, pro-caspase8and active-caspase8in both neonata1and adult hearts.
12.DOX exerted no effects on the expression of Akt/p-Akt, p-P53, p-p38, p-p42/44in both neonatal and adult hearts.
CONCLUSION
The higher activity of apoptotic pathways leads to a higher occurrence of apoptosis in the neonatal heart after DOX treatment, suggesting that excessive cardiomyocyte apoptosis is one of the primary and major factors involved in the earlier cardiotoxicity induced by DOX in the juvenile hearts, and is one of the underlying mechanisms by which the neonatal hearts are exposed to a high susceptibility to the DOX-induced cardiotoxicity.
Our present in vivo study suggests for the first time that the cardiac apoptotic potential is much higher in the neonatal hearts than that in the adult hearts, which is one of the primary and major factors involved in the earlier cardiotoxicity induced by DOX in the juvenile hearts, and is one of the underlying mechanisms by which the juvenile patients are exposed to a higher susceptibility to the DOX-mediated cardiotoxicity.
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