RhoA/ROCK信号通路在心肌肥厚和心力衰竭中的作用及法舒地尔的干预效应
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摘要
RhoA作为小分子GTP结合蛋白,是各种细胞功能的分子开关包括细胞迁移、胞质分裂和基因表达等。Rho激酶(Rho kinase, ROCK)是第一个被发现的RhoA的效应子,属于丝氨酸/苏氨酸蛋白激酶家族成员,具有广泛的生物学功能。ROCK有两种亚型,分别命名为ROCKⅠ和ROCKⅡ。ROCKⅠ主要在非神经组织中高表达,而ROCKⅡ是主要在脑组织中表达。细胞和分子生物学最新研究进展表明RhoA/ROCK信号通路广泛参与心血管系统的各种生理病理进程,例如心肌细胞凋亡,心肌肥厚,心肌梗死后心室重塑等。因此,抑制RhoA/ROCK信号通路可能是治疗包括高血压,动脉粥样硬化,糖尿病和肥厚性心衰等许多心血管疾病的有效办法。
法舒地尔是临床最常用的特异性ROCK抑制剂,能有效阻断RhoA/ROCK信号通路。根据动物实验研究,法舒地尔可以降低缺血再灌注的损伤程度。对于慢性高血压导致的充血性心衰大鼠,法舒地尔可降低心肌梗死后梗死面积和心肌纤维化程度。此外,根据临床观察,法舒地尔能显著改善心绞痛患者心肌缺血程度,并且不影响心率和血压,具有良好的耐受性。这表明RhoA/ROCK信号通路参与了人类心血管疾病的发病机制。在此基础上,我们使用法舒地尔作为工具药,研究了不同动物模型心肌病的发病机制,及RhoA/ROCK信号通路在这些机制中的调控作用,并探讨了在心血管疾病治疗方面,法舒地尔新的应用潜力。
本研究主要内容包括：(1) RhoA/ROCK信号通路在阿霉素诱导的大鼠心肌病中的作用及法舒地尔的干预效应；(2) RhoA/ROCK与JNK和ERK1/2信号通路的相互对话,参与异丙肾上腺素诱导的大鼠心力衰竭；(3) RhoA/ROCK信号通路干预大鼠甲亢性心肌病的发病机制。
目的：研究RhoA/ROCK信号通路在ADR诱导的大鼠心肌肥厚的发病机制中的作用及法舒地尔的干预效应
方法：清洁级雄性SD大鼠60只,体重200～240g,购自河北医科大学动物中心,在室温20℃～22℃,空气湿度45%-55%的条件下饲养一周后,随机分成5组：空白对照组(CONT)、ADR模型组(ADR)、卡托普利组(CAP+ADR)、法舒地尔低剂量组(FASL+ADR)、法舒地尔高剂量组(FASH+ADR),每组12只。FASL+ADR组和FASH+ADR组分别给予法舒地尔2mg·kg-1·d-1和10mg·kg-1·d-1,分两次腹腔注射给药,连续6 d; CAP+ADR组给予卡托普利30 mg·kg-1·d-1灌胃,连续6 d; ADR组、空白对照组给予等容积生理盐水。除空白对照组外,其余各组大鼠在实验第四天腹腔注射ADR 10mg·kg-1·d-1。各组大鼠注射ADR后,全部存活,死亡率为0。停药24 h后检测下列指标：(1)血流动力学：包括心率(heart rate, HR)、左心室收缩压(left ventricular systolic pressure, LVSP)、左心室舒张末期压力(left ventricular end diastolic pressure, LVEDP)、左心室室内压最大上升速率(maximum ascending rate of left ventricular pressure,+dp/dtmax)、左心室室内压最大下降速率(maximum descending rate of left ventricular pressure,-dp/dtmax)等；(2)组织学检测：包括HE染色观察心肌的组织病理学变化和透射电镜观察心肌超微结构的病理学变化；(3)血液生化指标：血清中乳酸脱氢酶(LDH)和肌酸磷酸激酶(CPK)的活性测定；(4)RT-PCR测定ROCKⅠ, c-fos, c-jun, c-FLIPL mRNA的表达；(5) Western Blotting测定bax, bcl-2蛋白的表达；(6) ELISA试剂盒检测NF-κB的活性。
结果：
1法舒地尔对左心室血流动力学参数的影响
与CONT比较,ADR模型组HR显著升高(从327.5±30.4升至408.7±34.8bpm,p<0.05); LVEDP也显著升高(从6.7±1.64升至18.6±5.15 mmHg,p<0.05)。此外,ADR模型组的LVSP显著降低(从144.6±26.4降至99.5±21.7 mmHg,p<0.05);±dp/dtmax也显著降低(+dp/dtmax从6865±1451降至4509±1524mmHg/s, p<0.05;-dp/dtmax从6024±1297降至3890±813mmHg/s,p<0.05)。LVSP和：±dp/dtmax对左心室前负荷和后负荷都敏感,血流动力学参数改变表明ADR模型组左心室功能紊乱,造模成功。与ADR模型组相比,CAP+ADR组HR显著下降(从408.7±34.8降至332.4±57.1 bpm,p<0.05); LVEDP显著降低(从18.6±5.15降至12.7±2.55mmHg,p<0.05);而LVSP显著升高(从99.5±21.7升至129.2±28.1mmHg, p<0.05);±dp/dtmax显著升高(+dp/dtmax从4509±1524升至6257±1402 mmHg/s,p<0.01;-dp/dtmax从3890±813升至5334±1372 mmHg/s,p<0.05)。与ADR模型组相比,FASL+ADR组HR显著降低(从408.7±34.8降至338.4±45.3 bpm,p<0.05); LVEDP显著降低(从18.6±5.15降至14.8±3.21 mmHg,p<0.05);但是LVSP显著升高(从99.5±21.7升至114.8±16.5 mmHg,p<0.05);±dp/dtmax显著升高(+dp/dtmax从4509±1524升至5937±1195 mmHg/s, p<0.01; -dp/dtmax从3890±813升至5104±1380 mmHg/s,p<0.05)。与ADR模型组相比,FASH+ADR组HR显著降低(从408.7±±34.8降至336.2±33.2bpm,p<0.05); LVEDP显著降低(从18.6±5.15降至12.1±2.82 mmHg,p<0.05),而LVSP显著升高(从99.5±-21.7升至122.4±21.3 mmHg, p<0.05);±dp/dtmax显著升高(+dp/dtmax从4509±1524升至6158±1421 mmHg/s, p<0.05;-dp/dtmax从3890±±813升至5545±1050 mmHg/s, p<0.05)。
2法舒地尔对左心室组织结构的影响
在光镜和透射电子显微镜下观察心肌细胞结构,显示正常心肌细胞肌原纤维排列整齐,明暗带清晰。椭圆形细胞核,核膜完整,含有丰富的线粒体。ADR模型组肌纤维呈波浪状,局部断裂溶解,余者心肌细胞萎缩明显,心肌线粒体肿胀,Z线扭曲变窄,部分消失,肌原纤维溶解断裂。与ADR模型组相比,CAP+ADR, FASL+ADR和FASH+ADR组大鼠心肌形态特征有明显好转,并且法舒地尔对ADR诱导的大鼠心肌肥厚的抑制呈剂量依赖性。
3法舒地尔对血清中乳酸脱氢酶(LDH)和肌酸磷酸激酶(CPK)的活性影响
与正常大鼠比较,模型组大鼠血清中LDH和CPK活性明显升高。卡托普利或法舒地尔处理后,活性显著降低(p<0.05或p<0.01),但不能达到正常组水平。
4法舒地尔对左心室ROCKⅠ, c-fos, c-jun和c-FLIPL mRNA表达的影响
与CONT比较,ADR模型组ROCKⅠmRNA表达显著升高(p<0.01)。与ADR模型组比较,CAP+ADR, FASL+ADR和FASH+ADR组ROCKⅠmRNA表达显著降低(p<0.0])。与CONT比较,ADR模型组的c-fos/GAPDH和c-jun/GAPDH mRNA水平显著升高(p<0.01),而c-FLIPL/GAPDH mRNA水平显著降低(p<0.01)。与ADR模型组比较,CAP+ADR组c-fos mRNA水平显著降低(p<0.01),而FASL+ADR和FASH+ADR组的c-fos mRNA变化没有统计学意义。与ADR模型组比较,CAP+ADR, FASL+ADR和FASH+ADR组c-jun mRNA水平显著降低(p<0.01)。与ADR模型组比较,CAP+ADR组c-FLIPL mRNA表达略有升高,但无统计学意义;而FASL+ADR和FASH+ADR组c-FLIPL mRNA表达显著升高(p<0.05)。
5法舒地尔对左心室bax和bcl-2蛋白表达的影响
与CONT比较,ADR模型组bcl-2蛋白表达显著降低(p<0.01),而bax蛋白表达显著升高(p<0.01)。给药后,与ADR模型组比较,CAP+ADR, FASL+ADR和FASH+ADR组bax蛋白表达显著降低(p<0.01),而bcl-2蛋白表达显著升高(p<0.05或p<0.01)。bax/bcl-2的比值是诱导凋亡的关键因子,我们的结果显示阿霉素处理大鼠后,bax/bcl-2的比值升高到743%。法舒地尔可以降低bax/bcl-2的比值,并呈剂量依赖性
6法舒地尔对左心室NF-κB活性的影响
用ELISA试剂盒检测ADR诱导的大鼠心肌肥厚中左心室NF-κB的活性,结果显示,ADR模型组NF-κB活性是CONT组的4倍数(417%,p<0.01)。卡托普利和法舒地尔可以显著减弱NF-κB的活性。
小结：在ADR诱导的大鼠心肌肥厚的发病过程中,RhoA/ROCK信号通路通过调控c-jun, c-fos, bax, bcl-2和NF-κB的活性而参与其病理过程。法舒地尔通过抑制RhoA/ROCK信号通路的激活而改善ADR诱导的心力衰竭,缓解超负荷心脏的心肌肥厚。
目的：研究RhoA/ROCK信号通路与JNK和ERK1/2信号通路的相互对话对ISO诱导的大鼠心力衰竭的发病机制的影响,以及法舒地尔的抗心力衰竭作用。
方法：清洁级雄性SD大鼠48只,体重200-240g,购自河北医科大学动物中心,在室温20℃-22℃,空气湿度45%-55%的条件下饲养一周后,随机分成4组：空白对照组(CONT)、ISO模型组(ISO)、法舒地尔低剂量组(FASL+ISO)、法舒地尔高剂量组(FASH+ISO),每组12只。FASL+ISO组和FASH+ISO组分别给予法舒地尔2mg·kg-1·d-1、10mg·kg-1·d-1,分两次腹腔注射给药,连续7d；ISO模型组、空白对照组给予等容积生理盐水；除空白对照组外,其余各组大鼠腹腔注射ISO,每天一次5 mg/kg/d,连续7 d,造成大鼠心力衰竭模型。各组大鼠注射ISO后全部存活,死亡率为0。停药24 h后,开始检测下列指标：(1)心重量指数：包括体重(body weight, BW);心脏重量(heart weight, HW);心脏重量／体重(HW/BW);左心室湿重(left ventricle wet weight, LV WW)／体重(LV WW/BW);左心室干重(left ventricle dry weight, LV DW)体重(LV DW/BW);(2)组织学检测：包括HE染色观察心肌的组织病理学变化和马松染色观察心肌纤维化变化；(3)血流动力学：包括心率(HR)、左室收缩压(LVSP)、左室舒张末压(LVEDP)、左室内压最大上升速率(+dp/dtmax)、左室内压最大下降速率(-dp/dtmax)等；(4)心电图：标准Ⅱ导联体表心电图,用RM6240C多通道生理信号采集与处理系统采图；(5)Western Blotting测定MYPT-1, p-MYPT-1, ERK1/2, p-ERK1/2, JNK, p-JNK和c-FLIPL蛋白的表达；(6) RT-PCR测定ROCKⅠ, c-fos, c-jun, c-FLIPL mRNA的表达。
结果：
1法舒地尔对心重量指数的影响
与CONT组比较,ISO模型组HW/BW升高141.53%(ISO:4.26±0.14 vs. CONT:3.01±0.04,p<0.01); LV WW/BW升高127.60%(ISO:3.19±0.06 vs. CONT:2.50±0.28,p<0.05); LV DW/BW升高138.18%(ISO:0.76±0.01 vs. CONT:0.55±0.07, p<0.05)。注射法舒地尔干预,HW/BW显著降低FASL+ISO组(FASL+ISO:3.77±0.10 vs. ISO:4.26±0.14,p<0.05)和FASH+ISO组(FASH+ISO:3.44±0.08 vs. ISO:4.26±0.14,p<0.01)。FASL+ISO组LV WW/BW显著降低15.05%(FASL+ISO:2.71±0.16 vs. ISO:3.19±0.06, p<0.05); FASH+ISO组显著降低28.84%(FASH+ISO:2.27±0.02 vs. ISO: 3.19±0.06, p<0.01)。LV DW/BW与LV WW/BW有相似的趋势,也显著降低(FASL+ISO:0.63±0.06 vs. ISO:0.76±0.01,p<0.05; FASH+ISO: 0.60±0.01 vs. ISO:0.76±0.01,p<0.01)。
2法舒地尔对左心室形态学变化的影响
心肌组织HE染色和马松染色后分别进行左心室形态学和左心室纤维化研究。ISO导致心肌劳损和纤维化(p<0.01),好发部位在心内膜。用Image-Pro Plus软件统计胶原纤维化面积。ISO模型组与CONT组比较,心肌纤维化升高约84.56%,法舒地尔可以减轻纤维化程度并呈剂量依赖性(FASL+ISO组43.79%,FASH+ISO组20.01%)。
3法舒地尔对血流动力学参数的影响
与CONT比较,ISO模型组HR显著升高(从343±9.6增至424±8.2bpm,p<0.01)；LVEDP也显著升高(从4.77±0.38增至15.37±0.49 mmHg,p<0.01)。此外,ISO模型组的LVSP显著降低(从143±5.8降至98±8.2 mmHg,P<0.05)；±dp/dtmax也显著降低(+dp/dtmax从8311±437降至4339±232 mmHg/s,p<0.05；-dp/dtmax从7116±272降至3623±±598mmHg/s,p<0.05)。LVSP和：±dp/dtmax对前负荷和后负荷都敏感,血流动力学参数改变表明ISO模型组左心室功能紊乱,造模成功。与ISO模型组相比,法舒地尔处理后,FASL+ISO组可明显改善心肌功能,收缩压和舒张压显著升高,LVSP从98±8.2升高到126±6.7 mmHg(p<0.05),±dp/dtmax显著升高(+dp/dtmax从4339±232增至5787±246 mmHg/s,p<0.01；-dp/dtmax从3623±598增至5130±1344 mmHg/s,p<0.05),LVEDP(从15.37±0.49降至11.73±1.31 mmHg,p<0.05).FASH+ISO组LVSP从98±8.2增至140±8.0 mmHg(p<0.05),±dp/dtmax显著升高(+dp/dtmax从4339±232增至7861±324 mmHg/s,p<0.01；-dp/dtmax从3623±598增至6660±334 mmHg/s,p<0.05),LVEDP(从15.37±0.49降至7.60±0.33 mmHg,p<0.05).
4法舒地尔对体表心电图(ECG)的影响
ISO可引起大鼠ECG标准Ⅱ导联ST段下移,T波反向,典型的心率失常,法舒地尔治疗一周后可以显著改善心肌功能紊乱症状。
5法舒地尔对ROCK活性的影响
从左心室提总RNA,进行RT-PCR检测。与CONT比较,ISO模型组ROCKⅠmRNA活性显著升高(p<0.01),法舒地尔处理后ROCKⅠmRNA活性显著降低(p<0.01)。我们还检测了RhoA/ROCK信号通路下游的靶蛋白MYPT-1活性。ISO使MYPT-1磷酸化增加(p<0.01),但是法舒地尔可以抑制MYPT-1磷酸化增加,并呈剂量依赖性(p<0.05或p<0.01)。
6法舒地尔对细胞核内ERK活性的影响
Western blotting检测结果显示,与CONT比较,ISO可明显提高ERK转运进入细胞核的能力(p<0.01),而法舒地尔减少ERK转运进入细胞核的量(p<0.01)。
7法舒地尔对JNK活性的影响
Western blotting检测结果显示,与CONT相比,ISO使JNK磷酸化提高到189.24%(p<0.01),而法舒地尔减少JNK磷酸化约24.71%(p<0.01)和43.37%(p<0.01)。
8法舒地尔对c-fos和c-jun mRNA表达的影响
RT-PCR检测结果显示,与CONT比较,ISO模型组c-fos和c-jun mRNA表达显著升高到252.92%(p<0.01)或247.45%(p<0.01)。高剂量法舒地尔减少c-fos和c-jun mRNA表达30.84%(p<0.01)和41.25%(p<0.01),而低剂量的法舒地尔效果明显。
9法舒地尔对c-FLIPL mRNA和蛋白表达的影响
与CONT比较,ISO模型组c-FLIPL mRNA表达降低,而法舒地尔使c-FLIPL mRNA表达有所恢复(p<0.01)。c-FLIPL蛋白表达与c-FLIPL mRNA表达一致。
小结：RhoA/ROCK信号通路与JNK和ERK1/2信号通路的相互对话,介导了ISO诱导大鼠心力衰竭的发病机制。法舒地尔通过抑制RhoA/ROCK信号通路,调控ISO诱导的心肌肥厚,改善心肌纤维化,缓解超负荷心脏的心力衰竭。
目的：研究RhoA/ROCK信号通路对大鼠甲亢性心肌病的影响以及法舒地尔的干预效应。
方法：清洁级雄性SD大鼠48只,体重200-240 g,购自河北医科大学动物中心,在室温20℃-22℃,空气湿度45%-55%的条件下饲养一周后,随机分成4组：空白对照组(CONT)、左旋甲状腺素模型组(T4模型组)、法舒地尔低剂量组(FASL+T4)、法舒地尔高剂量组(FASH+T4),每组12只。FASL+T4组和FASH+T4组分别给予法舒地尔2 mg·kg-1·d-1和10mg·kg-1·d-1,分两次腹腔注射给药,连续14d；T4模型组、空白对照组给予等容积生理盐水；除空白对照组外,其余各组大鼠腹腔注射左旋甲状腺素(L-thyroxine,T4)每天一次0.25 mg/kg/d,连续14 d,造成大鼠甲亢模型。大鼠注射T4后,全部存活,死亡率为O。停药24 h后,开始检测下列指标：(1)心重量指数：包括BW、HW、HW/BW;(2)甲状腺素检测：取血4ml,3500g离心收集血清,采用放射性免疫鉴定法检测血清中T4含量；(3)血流动力学测定：包括HR、LVSP、LVEDP、±dp/dtmax；(4)组织学检测：HE染色观察心肌的组织病理学变化；(5)TUNEL染色测定心肌细胞的凋亡情况；(6)RT-PCR测定ROCKⅠ,c-fos,c-jun, c-FLIPL mRNA的表达；(7)Western Blotting测定bax,bcl-2和p-MYPT-1蛋白的表达。
.结果：
1法舒地尔对心重量指数的影响
与CONT组比较,.T4模型组HW/BW显著升高(4.33±0.13 mg/g vs. 3.12±0.15 mg/g,n=6；p<0.01),体重却显著降低(191.2±3 g vs. 229.4±5 g, n=6；p<0.01)。注射法舒地尔干预,与T4模型组比较,FASL+T4组和FASH+T4组HW/BW显著降低(4.07±0.15 mg/g vs. 4.33±0.13 mg/g,n=6；p<0.05；3.92±0.09 mg/g vs. 4.33±0.13 mg/g,n=6;p<0.01)
2法舒地尔对血清T4含量的影响
大鼠连续14天注射T4后,T4模型组血清中T4含量约为CONT的4倍(120±13 nmol/l vs. 3.8±3nmol/l,n=6；p<0.01),法舒地尔对大鼠血清中T4含量没有明显的影响,与T4模型组相比无显著差异(FASL+T4组：118±14 nmol/l vs. 120±13 nmol/l,n=6；p>0.05；FASH+T4组：124±17 nmol/l vs. 120±13 nmol/l,n=6；p>0.05)。
3法舒地尔对血流动力学参数的影响
与CONT比较,T4模型组±dp/dtmax降低约1/3(+dp/dtmax从6856±524降至4489±371；-dp/dtmax从6019±357降至3890±312 mmHg/s)；LVSP降低约28%(从142.2±22.4降至102.8±21.4 mmHg)；HR升高约14%(从357.5±10.4降至408.7±34.8 bpm)；LVEDP升高约3倍(从6.7±0.58降至17.6±1.15).FASL+T4和FASH+T4两组大鼠HR,LVEDP,LVSP,+dp/dtmax和-dp/dtmax与T4模型组相比明显好转,与T4模型组相比有显著性差异(FASL+T4组：HR降至338.4±43.8 bp, LVEDP降至14.7±2.01 mmHg, LVSP升至109.4±10.3mmHg,+dp/dtmax升至5887±393 mmHg/s,-dp/dtmax升至5104±380 mmHg; FASH+T4组：HR降至330.2±32.2 bpm, LVEDP降至12.1±1.68 mmHg, LVSP升至127.2±19.7 mmHg,+dp/dtmax升至6162±553 mmHg/s,-dp/dtmax升至5545±437 mmHg)。
4法舒地尔对心脏形态学的影响
HE染色用来进行形态学观察。T4模型组细胞核肿大,心肌肥厚及间质纤维化,肌纤维重度紊乱。法舒地尔可以缓解T4导致的心肌肥厚,并呈剂量依赖性。
5法舒地尔对左心室凋亡的影响
TUNEL染色检测左心室的凋亡。T4模型组TUNEL染色阳性细胞数目显著提高。法舒地尔可以引起凋亡细胞的减少,并呈剂量依赖性。
6法舒地尔对ROCK活性的影响
从左心室提总RNA,进行RT-PCR检测。与CONT比较,T4模型组ROCKⅠmRNA活性显著升高近2倍(p<0.01),法舒地尔处理后ROCKⅠmRNA活性显著降低(p<0.01)。RhoA/ROCK信号通路下游的靶蛋白MYPT-1活性检测结果表明,T4使MYPT-1磷酸化增加(p<0.01),而法舒地尔可以抑制MYPT-1磷酸化增加(p<0.01)。
7法舒地尔对c-fos, c-jun和c-FLIPL mRNA表达的影响
RT-PCR检测结果显示,与CONT比较,T4模型组c-fos和c-jun mRNA表达显著升高(p<0.01),而c-FLIPL mRNA表达显著降低(p<0.01)。在FASL+T4和FASH+T4组,法舒地尔显著减少c-fos和c-jun mRNA表达(p<0.01)。显著升高c-FLIPL mRNA表达(p<0.01)。
8法舒地尔对bax和bcl-2表达的影响
与CONT比较,T4模型组bax表达显著升高是CONT组近5倍,而bcl-2显著降低(p<0.01)。注射法舒地尔干预,bax表达显著降低(p<0.01),并呈剂量依赖性。高剂量法舒地尔可以引起bcl-2表达显著升高(p<0.01),但是低剂量法舒地尔虽可使bcl-2表达有所升高,但无统计学意义。
小结：大鼠甲亢性心肌病发病过程中心肌肥厚明显,负荷增加,这些病理过程伴随着RhoA/ROCK信号通路的激活,进而使AP1 (c-fos和c-jun)活性增加,而使c-FLIPL表达下调。法舒地尔通过抑制RhoA/ROCK信号
1 RhoA/ROCK信号通路通过提高c-jun, c-fos, bax和NF-κB的活性而参与ADR诱导的心肌病的发病过程。法舒地尔干预可抑制RhoA/ROCK介导的心肌细胞凋亡和左心室功能紊乱。
2 ISO导致的心衰与激活RhoA/ROCK信号通路有关。法舒地尔抑制RhoA/ROCK信号通路,从而抑制JNK活性,ERK转运入核,AP-1 (c-fos和c-jun)的表达和引起c-FLIPL的上调,改善ISO所致心衰的各种病理变化。
3 T4提高ROCK和AP1的活性,降低c-FLIPL的表达,引起甲亢性心肌病。法舒地尔可以抑制这一通路而对抗T4所致的各种心肌病变。
本研究结果显示,不同原因所致心肌肥厚、心力衰竭的病理发展过程与RhoA/ROCK信号通路的激活有关,ROCK抑制剂法舒地尔干预可获得有益的治疗效果。
Recent cellular and molecular biology studies have indicated an important role of the RhoA/Rho kinase (ROCK) cascade in many aspects of cardiovascular function such as cardiomyocyte apoptosis, cardiac hypertrophy and ventricular remodeling following myocardial infarction. ROCK, the first Rho effector to be described, is a serine/threonine kinase that is important in fundamental processes of cell migration, cell proliferation and cell survival. There are two isoforms of ROCK, known as ROCKⅠandⅡ. ROCKⅠshows the highest expression level in non-neuronal tissues, whereas ROCKⅡis preferentially expressed in the brain. Inhibition of the RhoA/ROCK signaling pathway may be a suitable target for a number of cardiovascular diseases including hypertension, atherosclerosis, diabetes and hypertrophic heart failure.
The knowledge of the involvement of ROCKs in the cardiovascular system came mostly from studies utilizing pharmacological inhibitors. Fasudil, a ROCK inhibitor, was shown to decrease ischemia-reperfusion injury, infarct size and myocardial fibrosis in response to experimental myocardial infarction and in a rat model of chronic hypertension induced congestive heart failure. Moreover, in clinical practice, fasudil treatment significantly ameliorated pacing-induced myocardial ischemia in patients with effort angina, indicating involvement of the ROCK pathway in the pathogenesis of cardiovascular disease in humans. Fasudil did not affect heart rate or blood pressure, and was well tolerated. Based on the above information, we investigated the effect of fasudil on cardiovascular disease and the mechanisms involved, to explore the new potential application of fasudil in the treatment of cardiovascular disease. In this study, we use fasudil investigated:(1) RhoA/ROCK may play an important role in adriamycin (ADR)-induced cardiomyopathy; (2) RhoA/ROCK mediates isoproterenol-induced heart failure in rats via JNK and ERK1/2 pathways; (3) RhoA/ROCK may involve in cardiac hypertrophy induced by experimental hyperthyroidism.
Part 1 RhoA/ROCK signaling pathway may play an important role in adriamycin-induced cardiomyopathy
Objective:To investigate the role of RhoA/ROCK signaling pathway in cardiomyopathy induced by ADR in rats and the inhibitory effect of fasudil on this pathway.
Methods:60 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. The rats were divided into five groups of twelve animals in each group as follows:control (CONT), ADR alone- (ADR), captopril+ADR (CAP+ADR), low-dose fasudil+ADR (FASL+ADR), high-dose fasudil+ADR (FASH+ADR). All Drugs were given as follows: low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously. Captopril (30 mg/kg/day) were orally administered. Fasudil and captopril were administered over a period of 6 days. ADR (10 mg/kg) was injected intraperitoneally on the 4th day. The CONT animals received an injection of the equivalent volume of saline. The rats given ADR administration were all alive. All evaluations were performed 24 h after the last fasudil and captopril administration.
The indices as follow:
1 Haemodynamic parameters, including heart rate(HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and the maximum ascending and descending rate of left ventricular pressure (maximum rate of pressure development+dp/dtmax and maximum rate of pressure decrease-dp/dtmax)
2 Histopathological examinations, including hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM)
3 Measurement of plasma LDH and CPK levels
4 Reverse transcription polymerase chain reaction (RT-PCR):Total RNA was extracted from fresh-frozen myocardium using the Trizol reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.
5 Western Blotting, The expressions of bax, bcl-2 andβ-actin protein were determined by Western Blotting.
6 A colorimetric non-radioactive NF-κB p65 transcription factor ELISA Assay, was to determine NF-κB activation.
Results:
1 The effects of fasudil on hemodynamic variables of ADR-induced heart failure
Treatments of rats with a single dose of ADR (10 mg/kg) caused significant increases (p<0.05) in HR (from 327.5±30.4 to 408.7±34.8bpm, p<0.05) and LVEDP (from 6.7±1.64 to 18.6±5.15 mmHg, p<0.05) compared to the control values. On the other hand, ADR caused significant decreases in LVSP (from 144.6±26.4 to 99.5±21.7 mmHg,p<0.05) and±dp/dtmax compared to the control values (+dp/dtmax from 6865±1451 to 4509±1524 mmHg/s,p<0.05;-dp/dtmax from 6024±1297 to 3890±813 mmHg/s, p<0.05). LVSP and±dp/dtmax are sensitive to changes in preload and afterload. Haemodynamic parameters changes indicated left ventricular dysfunction in ADR rats. Treatment of ADR-injected rats with fasudil for 6 days markedly normalized the heart function. Both systolic and diastolic function were significantly enhanced, and HR (from 408.7±34.8 to 338.4±45.3 bpm, p<0.05), LVSP (from 99.5±21.7 to 114.8±16.5 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4509±1524 to 5937±1195 mmHg/s, p<0.01;-dp/dtmax from 3890±813 to 5104±1380 mmHg/s, p<0.05) and LVEDP (from 18.6±5.15 to 14.8±3.21 mmHg, p<0.05) were all markedly improved in the FASL+ ADR group compared with the ADR group. HR (from 408.7±34.8 to 336.2±33.2 bpm,p<0.05), LVSP (from 99.5±21.7 to 122.4±21.3 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4509±1524 to 6158±1421 mmHg/s, p<0.05;-dp/dtmax from 3890±813 to 5545±1050 mmHg/s,p<0.05) and LVEDP (from 18.6±5.15 to 12.1±2.82 mmHg, p<0.05) were all markedly improved in the FASH+ADR group compared with the ADR group. 2 The effects of fasudil on heart histopathology
Normal cardiac myocytes were regularly shaped cylinders, comprised the Z lines with diads, sarcomeres, elliptical nucleus, numerous mitochondria and prominent myofilaments as shown in the CONT group. The early manifestations of myocardial necrosis, such as wavy fibers, myocyte vacuolization, mitochondrial swelling, thinning of the Z lines, and loss of myofibrils were detected for rat hearts in the ADR group. The vacuolization of the cytoplasm of the LV was significantly higher in ADR-treated rats (about 31.09%). However, the progressive LV vacuolization was attenuated by fasudil and the effect was in a dose dependent manner (about 24.95% in FASL +ADR and 18.51% in FASH+ADR).
3 The effects of fasudil on the serum LDH and CPK
The assessment of serum LDH and CPK activities showed that the administration of ADR significantly increased its activity levels in rat serum compared with control (p<0.01). Treatment of rats with fasudil or captopril, compared to ADR group, markedly lowered the serum LDH and CPK (p< 0.05 or p<0.01). The protective effects obtained by fasudil administration, however, were not complete and did not reach those of the control group
4 The effects of fasudil on LV mRNA expressions of ROCKⅠ, c-fos, c-jun, and c-FLIPL
Agarose gels with representative PCR products for ROCKⅠ, c-fos, c-jun, and c-FLIPL are depicted. The level of ROCKⅠmRNA showed a significant increase in ADR group compared with CONT group (p<0.01), whereas the mRNA level of ROCKⅠremarkably decreased in CAP+ADR, FASL+ADR and FASH+ADR groups (p<0.01). The c-fos/GAPDH and c-jun/GAPDH mRNA levels in the LV significantly increased in ADR group compared with CONT group (p<0.01), whereas the c-FLIPL/GAPDH mRNA levels were significantly reduced. The level of c-fos mRNA expression in CAP+ADR group was significantly decreased (p<0.01) relative to ADR group whereas there was no statistical difference in both FASL+ADR and FASH+ADR groups. In CAP+ADR group, c-jun mRNA expression was significantly reduced as well as in both FASL+ADR and FASH+ADR groups (p<0.01). But c-FLIPL mRNA expression in CAP+ADR group was higher than in ADR group. In FASL+ADR and FASH+ADR groups, c-FLIPL mRNA expressions were significantly increased (p<0.05).
5 The effects of fasudil on cardiac muscle tissue protein expressions of bax and bcl-2
In the LV from ADR group, a dramatic decrease of bcl-2 compared with the CONT group was detected (p<0.01). On the other hand, bax expression in ADR group significantly increased compared with the CONT group (p<0.01). After captopril was administrated, bax expression decreased (p<0.01), but bcl-2 expression showed a significant increase (p<0.05). Meanwhile, fasudil significantly enhanced bcl-2 expressions and reduced bax expressions both in FASL+ADR group (p<0.01) and FASH+ADR group (p<0.01). Because the ratio of bax and bcl-2 expression is the determining factor for the induction of apoptosis, we found that the ratio of bax/bcl-2 protein expression was 7 fold increase in the ADR group (743% of the control, p<0.01), and the ratio of bax/bcl-2 protein expression was dose-dependently decrease after fasudil treatment (p<0.01).
6 The effects of fasudil on cardiac muscle tissue NF-κB activity
The CONT group showed a constitutive activation of NF-κB. Treatment with ADR induced a 4 fold increase in NF-κB activation (417% of the CONT group, p<0.01), which was significantly attenuated by captopril, low-dose fasudil or high-dose fasudil (p<0.01).
Conclusions:In summary, the present study demonstrated that the RhoA/ROCK signaling pathway plays an important role in the progression of heart failure induced by adriamycin (Fig.6). Furthermore, fasudil is capable of suppressing ADR-induced cardiomyopathy, which is associated with inhibition of bax and NF-κB activation, AP-1 expression and up-regulation of c-FLIP (L) and bcl-2 expressions. These results suggested that fasudil might have therapeutic potential through suppressing RhoA/ROCK dependent cardiocyte apoptosis and preserving LV function in the stressed heart. The study will provide further insight into the molecular mechanisms underlying the beneficial effects of fasudil on heart diseases.
Part 2 RhoA/ROCK mediates isoproterenol-induced heart failure in rats via JNK and ERK1/2 pathways
Objective:To investigate the roles of crosstalk between the ROCK, JNK and ERK1/2 signaling pathways signaling pathway in heart failure induced by ISO in rats, and clarify the possible molecular mechanisms underlying the beneficial effects of fasudil against heart failure.
Methods:48 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. The rats were divided into four groups of twelve animals in each group as follows:control (CONT), ISO alone- (ISO), low-dose fasudil treatment+ISO (FASL+ISO) or high-dose fasudil treatment +ISO (FASH+ISO) groups. All Drugs were given as follows:low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously. Fasudil was administered over a period of 7 days. ISO was administered once daily by intraperitoneal injection at 5 mg/kg/day for 7 days. The CONT animals received an injection of the equivalent volume of saline. The rats given ISO administration were all alive after 7 days. All evaluations were performed 24 h after the last ISO administration.
The indices as follow:
1 Heart weight indexs, including body weight (BW), heart weight (HW), Heart weight to-body weight ratio (HW/BW), left ventricular wet weight (LV WW)-to-body weight ratio (LV WW/BW), left ventricular dry weight (LV DW)-to-body weight ratio (LV DW/BW);
2 Histological analysis, including HE staining and masson's trichrome staining protocol;
3 Haemodynamic parameters, including HR, LVSP, LVEDP and±dp/dt;
4 Electrocardiography:the leadⅡof a standard body surface electrocardiogram (ECG) is easily reproducible approach by capturing the whole cardiac cycle using a RM6240C Multi-channel physiological signal acquisition and processing system;
5 Western blotting, the expressions of myosin phosphatase target subunit-1 (MYPT-1), p-MYPT-1, The extracellular signal-regulated kinases 1/2 (ERK1/2), p-ERK1/2, the the c-jun NH 2-terminal kinase (JNK), p-JNK, c-FLIPL protein were determined by Western Blotting;
6 RT-PCR:Total RNA was extracted from fresh-frozen myocardium using the Trizol Reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.
Results:
1 Effect of fasudil on the heart weight parameters'alterations of ISO-induced heart failure
HW/BW was increased by about 41.53% in ISO group as compared to CONT group (ISO:4.26±0.14 versus CONT:3.01±0.04,p<0.01). Moreover, LV WW/BW was increased to 127.60%(ISO:3.19±0.06 versus CONT:2.50±0.28,p<0.05) and LV DW/BW was increased to 138.18%(ISO:0.76±0.01 versus CONT:0.55±0.07,p<0.05). After being administrated with fasudil, HW/BW was significantly reduced in FASL+ISO group (FASL+ISO:3.77±0.10 versus ISO:4.26±0.14, p<0.05) and FASH+ISO group (FASH+ISO: 3.44±0.08 versus ISO:4.26±0.14, p<0.01). WW/BW of LV was significantly reduced by 15.05% in FASL group (FASL+ISO:2.7±0.16 versus ISO:3.19±0.06,p<0.05) and 28.84% in FASH group (FASH+ISO: 2.27±0.02 versus ISO:3.19±0.06, p<0.01) as compared to ISO group. DW/BW of LV showed a similar tendency as LV WW/BW (FASL+ISO:0.63±0.06 versus ISO:0.76±0.01,p<0.05; FASH+ISO:0.60±0.01 versus ISO: 0.76±0.01,p<0.01).
2 Effect of fasudil on the morphological alterations of ISO-induced heart failure
HE was for morphological analysis and Masson's stain was for fibrosis
HE was for morphological analysis and Masson's stain was for fibrosis analysis. Treatment with ISO resulted in marked myocyte loss and increased fibrosis (p<0.01), primarily limited to the subendocardium of the LV free wall and septum. The collagen fraction was determined by measuring the area of blue-stained tissue within a given field with Image-Pro Plus software. The stained area was calculated as a percentage of the total area within an image. The blue-appearing collagen of the LV endocardium was significantly higher in ISO-treated rats (about 84.56%). However, the progressive LV fibrosis was attenuated by fasudil and the effect was in a dose dependent manner (about 43.79% in FASL+ISO and 20.01% in FASH+ISO).
3 Effect of fasudil on hemodynamic variables of ISO-induced heart failure
Treatments of rats with a single dose of ISO (5 mg/kg/day) caused significant increases in HR (from 343±9.6 to 424±8.2bpm, p<0.01) and LVEDP compared to the control value (from 4.77±0.38 to 15.37±0.49 mmHg, p<0.01), indicating LV haemodynamic overload. On the other hand, ISO caused significant decreases in LVSP (from 143±5.8 to 98±8.2 mmHg, p<0.01) and±dp/dtmax compared to the control values (+dp/dtmax from 8311±437 to 4339±232 mmHg/s,p<0.01;-dp/dtmax from 7116±272 to 3623±598 mmHg/s, p<0.01). LVSP and±dp/dtmax are sensitive to changes in preload and afterload. Haemodynamic parameters changes indicated left ventricular dysfunction in ISO rats. Treatment of ISO-injected rats with fasudil for 7 days markedly normalized the heart function. Both systolic and diastolic function were significantly enhanced, and LVSP (from 98±8.2 to 126±6.7 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4339±232 to 5787±246 mmHg/s,p<0.01;-dp/dtmax from 3623±598 to 5130±1344 mmHg/s, p<0.05) and LVEDP (from 15.37±0.49 to 11.73±1.31 mmHg,p<0.05) were all markedly improved in the FASL+ISO group compared with the ISO group. LVSP (from 98±8.2 to 140±8.0 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4339±232 to 7861±324 mmHg/s,p<0.01;-dp/dtmax from 3623±598 to 6660±334 mmHg/s,p<0.01) and LVEDP (from 15.37±0.49 to 7.60±0.33 mmHg, p<0.01) were all markedly improved in the FASH+ISO group compared with the ISO group.
4 Effect of fasudil on the ECG alterations of ISO-induced heart failure
The result shows the standard ECGs traces of leadⅡfrom the CONT rats during baseline, ST segment depression with a negative T-wave and typical arrhythmias in the ISO group. Treatment of ISO-injected rats with fasudil for 7 days ameliorated the heart dysfunction.
5 Effect of fasudil on ISO-mediated ROCK activation
Total RNA was extracted from the LVs of the CONT, ISO, FASL+ISO and FASH+ISO groups. ISO caused a significant up-regulation of ROCK I mRNA expression compared with the CONT group (p<0.01), and treatment with fasudil resulted in a significant reduction in ROCKⅠmRNA expression (p<0.01). We tested the activation of RhoA/ROCK pathway by the amount of phosphorylated MYPT-1. ISO caused phosphorylation of MYPT-1(p<0.01), which was inhibited by fasudil in a dose-dependent manner (p<0.05 or p<0.01).
6 Fasudil reduces ISO-induced activation of ERK
We determined the effect of fasudil on the phosphorylation status of ERK in the nucleus as it may participate in ISO-induced heart failure. ISO increased nuclear translocation of ERK (p<0.01), while fasudil reduced this ISO-induced p-ERK to various degrees (p<0.01).
7 Fasudil attenuates ISO-induced JNK activation
The effect of fasudil on ISO-induced JNK activation was observed. ISO increased the activities of JNK phosphorylation to 189.24%(p<0.01). Compared with ISO group, fasudil reduced JNK phosphorylation by about 24.71%(p<0.01) at low dose and 43.37%(p<0.01) at high dose.
8 Effect of fasudil on c-fos and c-jun mRNA expressions
When the rats were administrated by ISO, the expressions of c-fos and c-jun mRNA quickly increased, reaching about 252.92%(p<0.01) and 247.45%(p<0.01) respectively compared with CONT group. The increases of c-fos and c-jun mRNA expressions by ISO were inhibited by high-dose fasudil by approximately 30.84%(p<0.01) and 41.25%(p<0.01), respectively. There were no significant decreases in the c-fos and c-jun mRNA expressions in low-dose fasudil administrated rats.
9 Effect of fasudil on c-FLIPL mRNA and protein expressions
Both the mRNA and protein expressions of c-FLIPL were determined. The c-FLIPL mRNA expression decreased in the ISO group. By contrast, treatment with fasudil resulted in a significant increase (p<0.01). Simultaneously, the c-FLIPL protein levels changed similarly to that of the c-FLIPL mRNA.
Conclusions:RhoA/ROCK is activated by ISO and its activation is essential for heart failure induced by ISO. Furthermore, fasudil is capable of suppressing ISO-induced heart failure, which is associated with inhibition of JNK activation, ERK translocation to the nucleus, subsequent AP-1 (c-fos and c-jun) expression and up-regulation of c-FLIPL expression.
Part 3 RhoA/ROCK may involve in cardiac hypertrophy induced by experimental hyperthyroidism.
Objective:To further investigate the mechanism of cardiovascular dysfunction in the hyperthyroid condition, the role of RhoA/ROCK signaling pathway and effects of fasudil were examined in rats treated with L-thyroxine (T4).
Methods:48 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. Rats were randomly assigned to one of four groups: control (CONT), thyroxine (T4), low-dose fasudil+T4 (FASL+T4), and high-dose fasudil+T4 (FASH+T4). All Drugs were given as follows: L-Thyroxine (T4) was dissolved in 99% ethanol by adding a small volume (20μl) of 25% NaOH and diluted 33 times by adding 0.9% NaCl to obtain a stock solution of 1 mg/ml. The solution was made in 0.9% NaCl to obtain a concentration of 50μg T4/ml. T4 in daily doses of 0.25mg/kg body weight was given subcutaneously once daily for 14 days. This treatment results in a long-term moderate hyperthyroidism. The control rats were treated with subcutaneous injections of normal saline given once daily for 14 days. Low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously at the same time. The rats given T4 administration were all alive. All evaluations were performed 24 h after the last fasudil and T4 administration.
The indices as follow:
1 Measurement of cardiac hypertrophy
Cardiac hypertrophy was assessed by the measurement of HW (in mg), BW (in g) and HW/BW (in mg/g).
2 Hormone concentrations
Rats were anaesthetized, and then the blood samples (4 ml) were collected for the analysis of plasma T4 levels. Following centrifugation (3500 g), plasma was separated and stored at -70℃. Total plasma T4 levels were determined by radioimmunoassay according to procedures of the kits.
3 Measurement of mechanical function
A miniature pressure transducer (Millar Micro-Tip) was inserted into the LV via the right carotid artery. The HR, LVEDP, LVSP, and±dp/dtmax were monitored by using MS4000U-1C biological signal quantitative recording and analyzing system.
4 Histology analysis
The LV was removed and fixed, and routinely embedded. Paraffin sections were cut at 5μm thickness and mounted on glass slides. Slides were routinely stained with hematoxylin and eosin for histopathologic review.
5 TUNEL assays
TUNEL assays were performed with the use of 3,3'-diaminobenzidine (DAB). Photographs were taken with the use of a microscope. The numbers of TUNEL-positive cardiac myocyte nuclei were quantified using Image Pro Plus software (Media Cybernetics, Silver Spring, MD). The chosen fields of view represented areas of heaviest TUNEL staining.
6 Western blotting
The expressions of bax, bcl-2 and p-MYPT-1 protein were determined by Western Blotting.
7 RNA extraction and RT-PCR analysis
Total RNA was extracted from fresh-frozen myocardium using the Trizol Reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.
Results:
1 Heart weights
T4 induced a significantly increased HW/BW ratio (T4:4.33±0.13 mg/g vs. CONT:3.12±0.15 mg/g, n=6 each; p<0.01) accompanied by a significantly decreased body weight than that seen in the CONT (T4:191.2±3 g vs. CONT:229.4±5 g, n= 6 each; p<0.01). Combined treatment by T4 and low-dose fasudil (4.07±0.15 mg/g vs.4.33±0.13 mg/g, n=6;p<0.05), T4 and high-dose fasudil (3.92±0.09 mg/g vs. 4.33±0.13 mg/g, n=6;p<0.01) showed significantly lower HW/BW ratios than that of T4。
2 Thyroid state of T4-treated animals
Rats treated with T4 for 14 days had a serum T4 level 4 times higher than that seen in the control (euthyroid) rats (120±13 nmol/l vs. 38±3 nmol/l, n=6 each; p<0.01). Combined treatment by T4 and low-dose fasudil (118±14 nmol/l vs. 120±13 nmol/l, n= 6; p<0.05), T4 and high-dose fasudil (124±17 nmol/l vs. 120±13 nmol/l, n= 6; p<0.01) resulted in T4 concentrations not differing from T4 treated group values.
3 Measurement of mechanical function
T4 depressed left ventricular±dp/dtmax by～1/3 (from 6856±524 to 4489±371 and 6019±357 to 3890±312 mmHg/s for +dp/dtmax and -dp/dtmax, respectively) and LVSP by 28%(from 142.2±22.4 to 102.8±21.4 mmHg). T4 increased HR by 14%(from 357.5±10.4 to 408.7±34.8 bpm) and LVEDP by nearly 3 times (from 6.7±0.58 to 17.6±1.15). FASL+T4 and FASH+T4 groups demonstrated return toward normal HR, LVEDP, LVSP,+dp/dtmax and-dp/dtmax (for FASL+T4 and FASH+T4 groups, HR decreased to 338.4±43.8 and 330.2±32.2 bpm, LVEDP decreased to 14.7±2.01 and 12.1±1.68 mmHg, LVSP increased to 109.4±10.3 and 127.2±19.7 mmHg,+dp/dtmax increased to 5887±393 and 6162±553 mmHg/s,-dp/dtmax increased to 5104±380 and 5545±437 mmHg, respectively).
4 Myocardial histology analysis
Myocardial hypertrophy with large nuclei, severe myofiber disorganization, and interstitial fibrosis was found in T4 group. T4 induced cardiomyocyte hypertrophy was significantly attenuated by fasudil even though cardiomyocyte areas from the FASL+T4 group remained significantly greater that those of CONT. Cardiomyocyte hypertrophy was significantly attenuated in FASH+T4 group.
5 Apoptotic changes in the LVs
Because HW/BWs were observed to significantly increase in the T4 group compared to the CONT group, the possible involvement of apoptosis was examined. Apoptosis was evaluated within the LV tissue by the TUNEL method. The number of TUNEL-positive cells per microscopic field was significantly increased in the T4 group compared in the CONT group. Fasudil caused a decrease in apoptosis in the cardiac tissue, more significantly at the higher dose, compared to the T4 group
6 ROCK activation
In left ventricle mRNAs of ROCKⅠand GAPDH were detected by RT-PCR analysis. The expression of ROCKⅠmRNA was significantly up-regulated by nearly 2 times in T4 group compared with CONT group (p<0.01). Compared with T4 group, the mRNA level of ROCKⅠremarkably decreased in FASL+T4 and FASH+T4 groups (p<0.01). Consistent with RT-PCR observations, p-MYPT-1 expression was significantly higher in T4-treated rats and remarkably decreased in FASL+T4 and FASH+T4 groups.
7 The effects of fasudil on LV mRNA expressions of c-fos, c-jun, and c-FLIPL
Agarose gels with representative PCR products for c-fos, c-jun, and c-FLIPL are depicted. The c-fos/GAPDH and c-jun/GAPDH mRNA levels in the LV significantly increased in T4 group compared with CONT (p<0.01), whereas the c-FLIPL/GAPDH mRNA levels significantly reduced (p<0.01). The level of c-fos/c-jun mRNA expressions in FASL+T4 and FASH+T4 groups significantly decreased (p<0.01) relative to T4 group. c-FLIPL mRNA expressions in FASL+T4 and FASH+T4 groups significantly increased (p<0.01).
8 Bax and bcl-2 expressions
The left ventricle bax expression of T4 group was 5 times higher than the CONT group. Bcl-2 expression in T4 group significantly decreased compared with the CONT group (p<0.01). Accompany with fasudil administrated, bax expression significantly decreased (p<0.01) on dose-dependent manner. Meanwhile, high-dose fasudil increased the expression of bcl-2 (p<0.01). Low-dose fasudil treated group showed no significant vary on bcl-2 expression.
Conclusions:this study demonstrated that T4 enhanced cardiac hypertrophy by concomitant enhancement of ROCK and AP1 activity and reduction of c-FLIPL expression. It also confirmed that this response can be inhibited by fasudil in dose dependent manner.
1 RhoA/ROCK is activated by ADR and its activation is essential for ADR-induced cardiomyopathy. Furthermore, fasudil is capable of suppressing ADR-induced cardiomyopathy, which is associated with inhibition of bax and NF-κB activations, subsequent AP-1(c-fos and c-jun) expression and up-regulation of c-FLIPL expression.
2 RhoA/ROCK is activated by ISO and its activation is essential for heart failure induced by ISO. Furthermore, fasudil is capable of suppressing ISO-induced heart failure, which is associated with inhibition of JNK activation, ERK translocation to the nucleus, subsequent AP-1(c-fos and c-jun) expression and up-regulation of c-FLIPL expression.
3 T4 enhanced cardiac hypertrophy by concomitant enhancement of ROCK and API activity and reduction of c-FLIPL expression. It also confirmed that this response can be inhibited by fasudil.
In summary, the present studies demonstrated that RhoA/ROCK is activated by heart failure and cardiac hypertrophy in different experimental model of rats, fasudil has beneficial effects on hypertrophic heart failure by suppressing the pathological processes.
法舒地尔是临床最常用的特异性ROCK抑制剂,能有效阻断RhoA/ROCK信号通路。根据动物实验研究,法舒地尔可以降低缺血再灌注的损伤程度。对于慢性高血压导致的充血性心衰大鼠,法舒地尔可降低心肌梗死后梗死面积和心肌纤维化程度。此外,根据临床观察,法舒地尔能显著改善心绞痛患者心肌缺血程度,并且不影响心率和血压,具有良好的耐受性。这表明RhoA/ROCK信号通路参与了人类心血管疾病的发病机制。在此基础上,我们使用法舒地尔作为工具药,研究了不同动物模型心肌病的发病机制,及RhoA/ROCK信号通路在这些机制中的调控作用,并探讨了在心血管疾病治疗方面,法舒地尔新的应用潜力。
本研究主要内容包括：(1) RhoA/ROCK信号通路在阿霉素诱导的大鼠心肌病中的作用及法舒地尔的干预效应；(2) RhoA/ROCK与JNK和ERK1/2信号通路的相互对话,参与异丙肾上腺素诱导的大鼠心力衰竭；(3) RhoA/ROCK信号通路干预大鼠甲亢性心肌病的发病机制。
目的：研究RhoA/ROCK信号通路在ADR诱导的大鼠心肌肥厚的发病机制中的作用及法舒地尔的干预效应
方法：清洁级雄性SD大鼠60只,体重200～240g,购自河北医科大学动物中心,在室温20℃～22℃,空气湿度45%-55%的条件下饲养一周后,随机分成5组：空白对照组(CONT)、ADR模型组(ADR)、卡托普利组(CAP+ADR)、法舒地尔低剂量组(FASL+ADR)、法舒地尔高剂量组(FASH+ADR),每组12只。FASL+ADR组和FASH+ADR组分别给予法舒地尔2mg·kg-1·d-1和10mg·kg-1·d-1,分两次腹腔注射给药,连续6 d; CAP+ADR组给予卡托普利30 mg·kg-1·d-1灌胃,连续6 d; ADR组、空白对照组给予等容积生理盐水。除空白对照组外,其余各组大鼠在实验第四天腹腔注射ADR 10mg·kg-1·d-1。各组大鼠注射ADR后,全部存活,死亡率为0。停药24 h后检测下列指标：(1)血流动力学：包括心率(heart rate, HR)、左心室收缩压(left ventricular systolic pressure, LVSP)、左心室舒张末期压力(left ventricular end diastolic pressure, LVEDP)、左心室室内压最大上升速率(maximum ascending rate of left ventricular pressure,+dp/dtmax)、左心室室内压最大下降速率(maximum descending rate of left ventricular pressure,-dp/dtmax)等；(2)组织学检测：包括HE染色观察心肌的组织病理学变化和透射电镜观察心肌超微结构的病理学变化；(3)血液生化指标：血清中乳酸脱氢酶(LDH)和肌酸磷酸激酶(CPK)的活性测定；(4)RT-PCR测定ROCKⅠ, c-fos, c-jun, c-FLIPL mRNA的表达；(5) Western Blotting测定bax, bcl-2蛋白的表达；(6) ELISA试剂盒检测NF-κB的活性。
结果：
1法舒地尔对左心室血流动力学参数的影响
与CONT比较,ADR模型组HR显著升高(从327.5±30.4升至408.7±34.8bpm,p<0.05); LVEDP也显著升高(从6.7±1.64升至18.6±5.15 mmHg,p<0.05)。此外,ADR模型组的LVSP显著降低(从144.6±26.4降至99.5±21.7 mmHg,p<0.05);±dp/dtmax也显著降低(+dp/dtmax从6865±1451降至4509±1524mmHg/s, p<0.05;-dp/dtmax从6024±1297降至3890±813mmHg/s,p<0.05)。LVSP和：±dp/dtmax对左心室前负荷和后负荷都敏感,血流动力学参数改变表明ADR模型组左心室功能紊乱,造模成功。与ADR模型组相比,CAP+ADR组HR显著下降(从408.7±34.8降至332.4±57.1 bpm,p<0.05); LVEDP显著降低(从18.6±5.15降至12.7±2.55mmHg,p<0.05);而LVSP显著升高(从99.5±21.7升至129.2±28.1mmHg, p<0.05);±dp/dtmax显著升高(+dp/dtmax从4509±1524升至6257±1402 mmHg/s,p<0.01;-dp/dtmax从3890±813升至5334±1372 mmHg/s,p<0.05)。与ADR模型组相比,FASL+ADR组HR显著降低(从408.7±34.8降至338.4±45.3 bpm,p<0.05); LVEDP显著降低(从18.6±5.15降至14.8±3.21 mmHg,p<0.05);但是LVSP显著升高(从99.5±21.7升至114.8±16.5 mmHg,p<0.05);±dp/dtmax显著升高(+dp/dtmax从4509±1524升至5937±1195 mmHg/s, p<0.01; -dp/dtmax从3890±813升至5104±1380 mmHg/s,p<0.05)。与ADR模型组相比,FASH+ADR组HR显著降低(从408.7±±34.8降至336.2±33.2bpm,p<0.05); LVEDP显著降低(从18.6±5.15降至12.1±2.82 mmHg,p<0.05),而LVSP显著升高(从99.5±-21.7升至122.4±21.3 mmHg, p<0.05);±dp/dtmax显著升高(+dp/dtmax从4509±1524升至6158±1421 mmHg/s, p<0.05;-dp/dtmax从3890±±813升至5545±1050 mmHg/s, p<0.05)。
2法舒地尔对左心室组织结构的影响
在光镜和透射电子显微镜下观察心肌细胞结构,显示正常心肌细胞肌原纤维排列整齐,明暗带清晰。椭圆形细胞核,核膜完整,含有丰富的线粒体。ADR模型组肌纤维呈波浪状,局部断裂溶解,余者心肌细胞萎缩明显,心肌线粒体肿胀,Z线扭曲变窄,部分消失,肌原纤维溶解断裂。与ADR模型组相比,CAP+ADR, FASL+ADR和FASH+ADR组大鼠心肌形态特征有明显好转,并且法舒地尔对ADR诱导的大鼠心肌肥厚的抑制呈剂量依赖性。
3法舒地尔对血清中乳酸脱氢酶(LDH)和肌酸磷酸激酶(CPK)的活性影响
与正常大鼠比较,模型组大鼠血清中LDH和CPK活性明显升高。卡托普利或法舒地尔处理后,活性显著降低(p<0.05或p<0.01),但不能达到正常组水平。
4法舒地尔对左心室ROCKⅠ, c-fos, c-jun和c-FLIPL mRNA表达的影响
与CONT比较,ADR模型组ROCKⅠmRNA表达显著升高(p<0.01)。与ADR模型组比较,CAP+ADR, FASL+ADR和FASH+ADR组ROCKⅠmRNA表达显著降低(p<0.0])。与CONT比较,ADR模型组的c-fos/GAPDH和c-jun/GAPDH mRNA水平显著升高(p<0.01),而c-FLIPL/GAPDH mRNA水平显著降低(p<0.01)。与ADR模型组比较,CAP+ADR组c-fos mRNA水平显著降低(p<0.01),而FASL+ADR和FASH+ADR组的c-fos mRNA变化没有统计学意义。与ADR模型组比较,CAP+ADR, FASL+ADR和FASH+ADR组c-jun mRNA水平显著降低(p<0.01)。与ADR模型组比较,CAP+ADR组c-FLIPL mRNA表达略有升高,但无统计学意义;而FASL+ADR和FASH+ADR组c-FLIPL mRNA表达显著升高(p<0.05)。
5法舒地尔对左心室bax和bcl-2蛋白表达的影响
与CONT比较,ADR模型组bcl-2蛋白表达显著降低(p<0.01),而bax蛋白表达显著升高(p<0.01)。给药后,与ADR模型组比较,CAP+ADR, FASL+ADR和FASH+ADR组bax蛋白表达显著降低(p<0.01),而bcl-2蛋白表达显著升高(p<0.05或p<0.01)。bax/bcl-2的比值是诱导凋亡的关键因子,我们的结果显示阿霉素处理大鼠后,bax/bcl-2的比值升高到743%。法舒地尔可以降低bax/bcl-2的比值,并呈剂量依赖性
6法舒地尔对左心室NF-κB活性的影响
用ELISA试剂盒检测ADR诱导的大鼠心肌肥厚中左心室NF-κB的活性,结果显示,ADR模型组NF-κB活性是CONT组的4倍数(417%,p<0.01)。卡托普利和法舒地尔可以显著减弱NF-κB的活性。
小结：在ADR诱导的大鼠心肌肥厚的发病过程中,RhoA/ROCK信号通路通过调控c-jun, c-fos, bax, bcl-2和NF-κB的活性而参与其病理过程。法舒地尔通过抑制RhoA/ROCK信号通路的激活而改善ADR诱导的心力衰竭,缓解超负荷心脏的心肌肥厚。
目的：研究RhoA/ROCK信号通路与JNK和ERK1/2信号通路的相互对话对ISO诱导的大鼠心力衰竭的发病机制的影响,以及法舒地尔的抗心力衰竭作用。
方法：清洁级雄性SD大鼠48只,体重200-240g,购自河北医科大学动物中心,在室温20℃-22℃,空气湿度45%-55%的条件下饲养一周后,随机分成4组：空白对照组(CONT)、ISO模型组(ISO)、法舒地尔低剂量组(FASL+ISO)、法舒地尔高剂量组(FASH+ISO),每组12只。FASL+ISO组和FASH+ISO组分别给予法舒地尔2mg·kg-1·d-1、10mg·kg-1·d-1,分两次腹腔注射给药,连续7d；ISO模型组、空白对照组给予等容积生理盐水；除空白对照组外,其余各组大鼠腹腔注射ISO,每天一次5 mg/kg/d,连续7 d,造成大鼠心力衰竭模型。各组大鼠注射ISO后全部存活,死亡率为0。停药24 h后,开始检测下列指标：(1)心重量指数：包括体重(body weight, BW);心脏重量(heart weight, HW);心脏重量／体重(HW/BW);左心室湿重(left ventricle wet weight, LV WW)／体重(LV WW/BW);左心室干重(left ventricle dry weight, LV DW)体重(LV DW/BW);(2)组织学检测：包括HE染色观察心肌的组织病理学变化和马松染色观察心肌纤维化变化；(3)血流动力学：包括心率(HR)、左室收缩压(LVSP)、左室舒张末压(LVEDP)、左室内压最大上升速率(+dp/dtmax)、左室内压最大下降速率(-dp/dtmax)等；(4)心电图：标准Ⅱ导联体表心电图,用RM6240C多通道生理信号采集与处理系统采图；(5)Western Blotting测定MYPT-1, p-MYPT-1, ERK1/2, p-ERK1/2, JNK, p-JNK和c-FLIPL蛋白的表达；(6) RT-PCR测定ROCKⅠ, c-fos, c-jun, c-FLIPL mRNA的表达。
结果：
1法舒地尔对心重量指数的影响
与CONT组比较,ISO模型组HW/BW升高141.53%(ISO:4.26±0.14 vs. CONT:3.01±0.04,p<0.01); LV WW/BW升高127.60%(ISO:3.19±0.06 vs. CONT:2.50±0.28,p<0.05); LV DW/BW升高138.18%(ISO:0.76±0.01 vs. CONT:0.55±0.07, p<0.05)。注射法舒地尔干预,HW/BW显著降低FASL+ISO组(FASL+ISO:3.77±0.10 vs. ISO:4.26±0.14,p<0.05)和FASH+ISO组(FASH+ISO:3.44±0.08 vs. ISO:4.26±0.14,p<0.01)。FASL+ISO组LV WW/BW显著降低15.05%(FASL+ISO:2.71±0.16 vs. ISO:3.19±0.06, p<0.05); FASH+ISO组显著降低28.84%(FASH+ISO:2.27±0.02 vs. ISO: 3.19±0.06, p<0.01)。LV DW/BW与LV WW/BW有相似的趋势,也显著降低(FASL+ISO:0.63±0.06 vs. ISO:0.76±0.01,p<0.05; FASH+ISO: 0.60±0.01 vs. ISO:0.76±0.01,p<0.01)。
2法舒地尔对左心室形态学变化的影响
心肌组织HE染色和马松染色后分别进行左心室形态学和左心室纤维化研究。ISO导致心肌劳损和纤维化(p<0.01),好发部位在心内膜。用Image-Pro Plus软件统计胶原纤维化面积。ISO模型组与CONT组比较,心肌纤维化升高约84.56%,法舒地尔可以减轻纤维化程度并呈剂量依赖性(FASL+ISO组43.79%,FASH+ISO组20.01%)。
3法舒地尔对血流动力学参数的影响
与CONT比较,ISO模型组HR显著升高(从343±9.6增至424±8.2bpm,p<0.01)；LVEDP也显著升高(从4.77±0.38增至15.37±0.49 mmHg,p<0.01)。此外,ISO模型组的LVSP显著降低(从143±5.8降至98±8.2 mmHg,P<0.05)；±dp/dtmax也显著降低(+dp/dtmax从8311±437降至4339±232 mmHg/s,p<0.05；-dp/dtmax从7116±272降至3623±±598mmHg/s,p<0.05)。LVSP和：±dp/dtmax对前负荷和后负荷都敏感,血流动力学参数改变表明ISO模型组左心室功能紊乱,造模成功。与ISO模型组相比,法舒地尔处理后,FASL+ISO组可明显改善心肌功能,收缩压和舒张压显著升高,LVSP从98±8.2升高到126±6.7 mmHg(p<0.05),±dp/dtmax显著升高(+dp/dtmax从4339±232增至5787±246 mmHg/s,p<0.01；-dp/dtmax从3623±598增至5130±1344 mmHg/s,p<0.05),LVEDP(从15.37±0.49降至11.73±1.31 mmHg,p<0.05).FASH+ISO组LVSP从98±8.2增至140±8.0 mmHg(p<0.05),±dp/dtmax显著升高(+dp/dtmax从4339±232增至7861±324 mmHg/s,p<0.01；-dp/dtmax从3623±598增至6660±334 mmHg/s,p<0.05),LVEDP(从15.37±0.49降至7.60±0.33 mmHg,p<0.05).
4法舒地尔对体表心电图(ECG)的影响
ISO可引起大鼠ECG标准Ⅱ导联ST段下移,T波反向,典型的心率失常,法舒地尔治疗一周后可以显著改善心肌功能紊乱症状。
5法舒地尔对ROCK活性的影响
从左心室提总RNA,进行RT-PCR检测。与CONT比较,ISO模型组ROCKⅠmRNA活性显著升高(p<0.01),法舒地尔处理后ROCKⅠmRNA活性显著降低(p<0.01)。我们还检测了RhoA/ROCK信号通路下游的靶蛋白MYPT-1活性。ISO使MYPT-1磷酸化增加(p<0.01),但是法舒地尔可以抑制MYPT-1磷酸化增加,并呈剂量依赖性(p<0.05或p<0.01)。
6法舒地尔对细胞核内ERK活性的影响
Western blotting检测结果显示,与CONT比较,ISO可明显提高ERK转运进入细胞核的能力(p<0.01),而法舒地尔减少ERK转运进入细胞核的量(p<0.01)。
7法舒地尔对JNK活性的影响
Western blotting检测结果显示,与CONT相比,ISO使JNK磷酸化提高到189.24%(p<0.01),而法舒地尔减少JNK磷酸化约24.71%(p<0.01)和43.37%(p<0.01)。
8法舒地尔对c-fos和c-jun mRNA表达的影响
RT-PCR检测结果显示,与CONT比较,ISO模型组c-fos和c-jun mRNA表达显著升高到252.92%(p<0.01)或247.45%(p<0.01)。高剂量法舒地尔减少c-fos和c-jun mRNA表达30.84%(p<0.01)和41.25%(p<0.01),而低剂量的法舒地尔效果明显。
9法舒地尔对c-FLIPL mRNA和蛋白表达的影响
与CONT比较,ISO模型组c-FLIPL mRNA表达降低,而法舒地尔使c-FLIPL mRNA表达有所恢复(p<0.01)。c-FLIPL蛋白表达与c-FLIPL mRNA表达一致。
小结：RhoA/ROCK信号通路与JNK和ERK1/2信号通路的相互对话,介导了ISO诱导大鼠心力衰竭的发病机制。法舒地尔通过抑制RhoA/ROCK信号通路,调控ISO诱导的心肌肥厚,改善心肌纤维化,缓解超负荷心脏的心力衰竭。
目的：研究RhoA/ROCK信号通路对大鼠甲亢性心肌病的影响以及法舒地尔的干预效应。
方法：清洁级雄性SD大鼠48只,体重200-240 g,购自河北医科大学动物中心,在室温20℃-22℃,空气湿度45%-55%的条件下饲养一周后,随机分成4组：空白对照组(CONT)、左旋甲状腺素模型组(T4模型组)、法舒地尔低剂量组(FASL+T4)、法舒地尔高剂量组(FASH+T4),每组12只。FASL+T4组和FASH+T4组分别给予法舒地尔2 mg·kg-1·d-1和10mg·kg-1·d-1,分两次腹腔注射给药,连续14d；T4模型组、空白对照组给予等容积生理盐水；除空白对照组外,其余各组大鼠腹腔注射左旋甲状腺素(L-thyroxine,T4)每天一次0.25 mg/kg/d,连续14 d,造成大鼠甲亢模型。大鼠注射T4后,全部存活,死亡率为O。停药24 h后,开始检测下列指标：(1)心重量指数：包括BW、HW、HW/BW;(2)甲状腺素检测：取血4ml,3500g离心收集血清,采用放射性免疫鉴定法检测血清中T4含量；(3)血流动力学测定：包括HR、LVSP、LVEDP、±dp/dtmax；(4)组织学检测：HE染色观察心肌的组织病理学变化；(5)TUNEL染色测定心肌细胞的凋亡情况；(6)RT-PCR测定ROCKⅠ,c-fos,c-jun, c-FLIPL mRNA的表达；(7)Western Blotting测定bax,bcl-2和p-MYPT-1蛋白的表达。
.结果：
1法舒地尔对心重量指数的影响
与CONT组比较,.T4模型组HW/BW显著升高(4.33±0.13 mg/g vs. 3.12±0.15 mg/g,n=6；p<0.01),体重却显著降低(191.2±3 g vs. 229.4±5 g, n=6；p<0.01)。注射法舒地尔干预,与T4模型组比较,FASL+T4组和FASH+T4组HW/BW显著降低(4.07±0.15 mg/g vs. 4.33±0.13 mg/g,n=6；p<0.05；3.92±0.09 mg/g vs. 4.33±0.13 mg/g,n=6;p<0.01)
2法舒地尔对血清T4含量的影响
大鼠连续14天注射T4后,T4模型组血清中T4含量约为CONT的4倍(120±13 nmol/l vs. 3.8±3nmol/l,n=6；p<0.01),法舒地尔对大鼠血清中T4含量没有明显的影响,与T4模型组相比无显著差异(FASL+T4组：118±14 nmol/l vs. 120±13 nmol/l,n=6；p>0.05；FASH+T4组：124±17 nmol/l vs. 120±13 nmol/l,n=6；p>0.05)。
3法舒地尔对血流动力学参数的影响
与CONT比较,T4模型组±dp/dtmax降低约1/3(+dp/dtmax从6856±524降至4489±371；-dp/dtmax从6019±357降至3890±312 mmHg/s)；LVSP降低约28%(从142.2±22.4降至102.8±21.4 mmHg)；HR升高约14%(从357.5±10.4降至408.7±34.8 bpm)；LVEDP升高约3倍(从6.7±0.58降至17.6±1.15).FASL+T4和FASH+T4两组大鼠HR,LVEDP,LVSP,+dp/dtmax和-dp/dtmax与T4模型组相比明显好转,与T4模型组相比有显著性差异(FASL+T4组：HR降至338.4±43.8 bp, LVEDP降至14.7±2.01 mmHg, LVSP升至109.4±10.3mmHg,+dp/dtmax升至5887±393 mmHg/s,-dp/dtmax升至5104±380 mmHg; FASH+T4组：HR降至330.2±32.2 bpm, LVEDP降至12.1±1.68 mmHg, LVSP升至127.2±19.7 mmHg,+dp/dtmax升至6162±553 mmHg/s,-dp/dtmax升至5545±437 mmHg)。
4法舒地尔对心脏形态学的影响
HE染色用来进行形态学观察。T4模型组细胞核肿大,心肌肥厚及间质纤维化,肌纤维重度紊乱。法舒地尔可以缓解T4导致的心肌肥厚,并呈剂量依赖性。
5法舒地尔对左心室凋亡的影响
TUNEL染色检测左心室的凋亡。T4模型组TUNEL染色阳性细胞数目显著提高。法舒地尔可以引起凋亡细胞的减少,并呈剂量依赖性。
6法舒地尔对ROCK活性的影响
从左心室提总RNA,进行RT-PCR检测。与CONT比较,T4模型组ROCKⅠmRNA活性显著升高近2倍(p<0.01),法舒地尔处理后ROCKⅠmRNA活性显著降低(p<0.01)。RhoA/ROCK信号通路下游的靶蛋白MYPT-1活性检测结果表明,T4使MYPT-1磷酸化增加(p<0.01),而法舒地尔可以抑制MYPT-1磷酸化增加(p<0.01)。
7法舒地尔对c-fos, c-jun和c-FLIPL mRNA表达的影响
RT-PCR检测结果显示,与CONT比较,T4模型组c-fos和c-jun mRNA表达显著升高(p<0.01),而c-FLIPL mRNA表达显著降低(p<0.01)。在FASL+T4和FASH+T4组,法舒地尔显著减少c-fos和c-jun mRNA表达(p<0.01)。显著升高c-FLIPL mRNA表达(p<0.01)。
8法舒地尔对bax和bcl-2表达的影响
与CONT比较,T4模型组bax表达显著升高是CONT组近5倍,而bcl-2显著降低(p<0.01)。注射法舒地尔干预,bax表达显著降低(p<0.01),并呈剂量依赖性。高剂量法舒地尔可以引起bcl-2表达显著升高(p<0.01),但是低剂量法舒地尔虽可使bcl-2表达有所升高,但无统计学意义。
小结：大鼠甲亢性心肌病发病过程中心肌肥厚明显,负荷增加,这些病理过程伴随着RhoA/ROCK信号通路的激活,进而使AP1 (c-fos和c-jun)活性增加,而使c-FLIPL表达下调。法舒地尔通过抑制RhoA/ROCK信号
1 RhoA/ROCK信号通路通过提高c-jun, c-fos, bax和NF-κB的活性而参与ADR诱导的心肌病的发病过程。法舒地尔干预可抑制RhoA/ROCK介导的心肌细胞凋亡和左心室功能紊乱。
2 ISO导致的心衰与激活RhoA/ROCK信号通路有关。法舒地尔抑制RhoA/ROCK信号通路,从而抑制JNK活性,ERK转运入核,AP-1 (c-fos和c-jun)的表达和引起c-FLIPL的上调,改善ISO所致心衰的各种病理变化。
3 T4提高ROCK和AP1的活性,降低c-FLIPL的表达,引起甲亢性心肌病。法舒地尔可以抑制这一通路而对抗T4所致的各种心肌病变。
本研究结果显示,不同原因所致心肌肥厚、心力衰竭的病理发展过程与RhoA/ROCK信号通路的激活有关,ROCK抑制剂法舒地尔干预可获得有益的治疗效果。
Recent cellular and molecular biology studies have indicated an important role of the RhoA/Rho kinase (ROCK) cascade in many aspects of cardiovascular function such as cardiomyocyte apoptosis, cardiac hypertrophy and ventricular remodeling following myocardial infarction. ROCK, the first Rho effector to be described, is a serine/threonine kinase that is important in fundamental processes of cell migration, cell proliferation and cell survival. There are two isoforms of ROCK, known as ROCKⅠandⅡ. ROCKⅠshows the highest expression level in non-neuronal tissues, whereas ROCKⅡis preferentially expressed in the brain. Inhibition of the RhoA/ROCK signaling pathway may be a suitable target for a number of cardiovascular diseases including hypertension, atherosclerosis, diabetes and hypertrophic heart failure.
The knowledge of the involvement of ROCKs in the cardiovascular system came mostly from studies utilizing pharmacological inhibitors. Fasudil, a ROCK inhibitor, was shown to decrease ischemia-reperfusion injury, infarct size and myocardial fibrosis in response to experimental myocardial infarction and in a rat model of chronic hypertension induced congestive heart failure. Moreover, in clinical practice, fasudil treatment significantly ameliorated pacing-induced myocardial ischemia in patients with effort angina, indicating involvement of the ROCK pathway in the pathogenesis of cardiovascular disease in humans. Fasudil did not affect heart rate or blood pressure, and was well tolerated. Based on the above information, we investigated the effect of fasudil on cardiovascular disease and the mechanisms involved, to explore the new potential application of fasudil in the treatment of cardiovascular disease. In this study, we use fasudil investigated:(1) RhoA/ROCK may play an important role in adriamycin (ADR)-induced cardiomyopathy; (2) RhoA/ROCK mediates isoproterenol-induced heart failure in rats via JNK and ERK1/2 pathways; (3) RhoA/ROCK may involve in cardiac hypertrophy induced by experimental hyperthyroidism.
Part 1 RhoA/ROCK signaling pathway may play an important role in adriamycin-induced cardiomyopathy
Objective:To investigate the role of RhoA/ROCK signaling pathway in cardiomyopathy induced by ADR in rats and the inhibitory effect of fasudil on this pathway.
Methods:60 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. The rats were divided into five groups of twelve animals in each group as follows:control (CONT), ADR alone- (ADR), captopril+ADR (CAP+ADR), low-dose fasudil+ADR (FASL+ADR), high-dose fasudil+ADR (FASH+ADR). All Drugs were given as follows: low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously. Captopril (30 mg/kg/day) were orally administered. Fasudil and captopril were administered over a period of 6 days. ADR (10 mg/kg) was injected intraperitoneally on the 4th day. The CONT animals received an injection of the equivalent volume of saline. The rats given ADR administration were all alive. All evaluations were performed 24 h after the last fasudil and captopril administration.
The indices as follow:
1 Haemodynamic parameters, including heart rate(HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and the maximum ascending and descending rate of left ventricular pressure (maximum rate of pressure development+dp/dtmax and maximum rate of pressure decrease-dp/dtmax)
2 Histopathological examinations, including hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM)
3 Measurement of plasma LDH and CPK levels
4 Reverse transcription polymerase chain reaction (RT-PCR):Total RNA was extracted from fresh-frozen myocardium using the Trizol reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.
5 Western Blotting, The expressions of bax, bcl-2 andβ-actin protein were determined by Western Blotting.
6 A colorimetric non-radioactive NF-κB p65 transcription factor ELISA Assay, was to determine NF-κB activation.
Results:
1 The effects of fasudil on hemodynamic variables of ADR-induced heart failure
Treatments of rats with a single dose of ADR (10 mg/kg) caused significant increases (p<0.05) in HR (from 327.5±30.4 to 408.7±34.8bpm, p<0.05) and LVEDP (from 6.7±1.64 to 18.6±5.15 mmHg, p<0.05) compared to the control values. On the other hand, ADR caused significant decreases in LVSP (from 144.6±26.4 to 99.5±21.7 mmHg,p<0.05) and±dp/dtmax compared to the control values (+dp/dtmax from 6865±1451 to 4509±1524 mmHg/s,p<0.05;-dp/dtmax from 6024±1297 to 3890±813 mmHg/s, p<0.05). LVSP and±dp/dtmax are sensitive to changes in preload and afterload. Haemodynamic parameters changes indicated left ventricular dysfunction in ADR rats. Treatment of ADR-injected rats with fasudil for 6 days markedly normalized the heart function. Both systolic and diastolic function were significantly enhanced, and HR (from 408.7±34.8 to 338.4±45.3 bpm, p<0.05), LVSP (from 99.5±21.7 to 114.8±16.5 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4509±1524 to 5937±1195 mmHg/s, p<0.01;-dp/dtmax from 3890±813 to 5104±1380 mmHg/s, p<0.05) and LVEDP (from 18.6±5.15 to 14.8±3.21 mmHg, p<0.05) were all markedly improved in the FASL+ ADR group compared with the ADR group. HR (from 408.7±34.8 to 336.2±33.2 bpm,p<0.05), LVSP (from 99.5±21.7 to 122.4±21.3 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4509±1524 to 6158±1421 mmHg/s, p<0.05;-dp/dtmax from 3890±813 to 5545±1050 mmHg/s,p<0.05) and LVEDP (from 18.6±5.15 to 12.1±2.82 mmHg, p<0.05) were all markedly improved in the FASH+ADR group compared with the ADR group. 2 The effects of fasudil on heart histopathology
Normal cardiac myocytes were regularly shaped cylinders, comprised the Z lines with diads, sarcomeres, elliptical nucleus, numerous mitochondria and prominent myofilaments as shown in the CONT group. The early manifestations of myocardial necrosis, such as wavy fibers, myocyte vacuolization, mitochondrial swelling, thinning of the Z lines, and loss of myofibrils were detected for rat hearts in the ADR group. The vacuolization of the cytoplasm of the LV was significantly higher in ADR-treated rats (about 31.09%). However, the progressive LV vacuolization was attenuated by fasudil and the effect was in a dose dependent manner (about 24.95% in FASL +ADR and 18.51% in FASH+ADR).
3 The effects of fasudil on the serum LDH and CPK
The assessment of serum LDH and CPK activities showed that the administration of ADR significantly increased its activity levels in rat serum compared with control (p<0.01). Treatment of rats with fasudil or captopril, compared to ADR group, markedly lowered the serum LDH and CPK (p< 0.05 or p<0.01). The protective effects obtained by fasudil administration, however, were not complete and did not reach those of the control group
4 The effects of fasudil on LV mRNA expressions of ROCKⅠ, c-fos, c-jun, and c-FLIPL
Agarose gels with representative PCR products for ROCKⅠ, c-fos, c-jun, and c-FLIPL are depicted. The level of ROCKⅠmRNA showed a significant increase in ADR group compared with CONT group (p<0.01), whereas the mRNA level of ROCKⅠremarkably decreased in CAP+ADR, FASL+ADR and FASH+ADR groups (p<0.01). The c-fos/GAPDH and c-jun/GAPDH mRNA levels in the LV significantly increased in ADR group compared with CONT group (p<0.01), whereas the c-FLIPL/GAPDH mRNA levels were significantly reduced. The level of c-fos mRNA expression in CAP+ADR group was significantly decreased (p<0.01) relative to ADR group whereas there was no statistical difference in both FASL+ADR and FASH+ADR groups. In CAP+ADR group, c-jun mRNA expression was significantly reduced as well as in both FASL+ADR and FASH+ADR groups (p<0.01). But c-FLIPL mRNA expression in CAP+ADR group was higher than in ADR group. In FASL+ADR and FASH+ADR groups, c-FLIPL mRNA expressions were significantly increased (p<0.05).
5 The effects of fasudil on cardiac muscle tissue protein expressions of bax and bcl-2
In the LV from ADR group, a dramatic decrease of bcl-2 compared with the CONT group was detected (p<0.01). On the other hand, bax expression in ADR group significantly increased compared with the CONT group (p<0.01). After captopril was administrated, bax expression decreased (p<0.01), but bcl-2 expression showed a significant increase (p<0.05). Meanwhile, fasudil significantly enhanced bcl-2 expressions and reduced bax expressions both in FASL+ADR group (p<0.01) and FASH+ADR group (p<0.01). Because the ratio of bax and bcl-2 expression is the determining factor for the induction of apoptosis, we found that the ratio of bax/bcl-2 protein expression was 7 fold increase in the ADR group (743% of the control, p<0.01), and the ratio of bax/bcl-2 protein expression was dose-dependently decrease after fasudil treatment (p<0.01).
6 The effects of fasudil on cardiac muscle tissue NF-κB activity
The CONT group showed a constitutive activation of NF-κB. Treatment with ADR induced a 4 fold increase in NF-κB activation (417% of the CONT group, p<0.01), which was significantly attenuated by captopril, low-dose fasudil or high-dose fasudil (p<0.01).
Conclusions:In summary, the present study demonstrated that the RhoA/ROCK signaling pathway plays an important role in the progression of heart failure induced by adriamycin (Fig.6). Furthermore, fasudil is capable of suppressing ADR-induced cardiomyopathy, which is associated with inhibition of bax and NF-κB activation, AP-1 expression and up-regulation of c-FLIP (L) and bcl-2 expressions. These results suggested that fasudil might have therapeutic potential through suppressing RhoA/ROCK dependent cardiocyte apoptosis and preserving LV function in the stressed heart. The study will provide further insight into the molecular mechanisms underlying the beneficial effects of fasudil on heart diseases.
Part 2 RhoA/ROCK mediates isoproterenol-induced heart failure in rats via JNK and ERK1/2 pathways
Objective:To investigate the roles of crosstalk between the ROCK, JNK and ERK1/2 signaling pathways signaling pathway in heart failure induced by ISO in rats, and clarify the possible molecular mechanisms underlying the beneficial effects of fasudil against heart failure.
Methods:48 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. The rats were divided into four groups of twelve animals in each group as follows:control (CONT), ISO alone- (ISO), low-dose fasudil treatment+ISO (FASL+ISO) or high-dose fasudil treatment +ISO (FASH+ISO) groups. All Drugs were given as follows:low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously. Fasudil was administered over a period of 7 days. ISO was administered once daily by intraperitoneal injection at 5 mg/kg/day for 7 days. The CONT animals received an injection of the equivalent volume of saline. The rats given ISO administration were all alive after 7 days. All evaluations were performed 24 h after the last ISO administration.
The indices as follow:
1 Heart weight indexs, including body weight (BW), heart weight (HW), Heart weight to-body weight ratio (HW/BW), left ventricular wet weight (LV WW)-to-body weight ratio (LV WW/BW), left ventricular dry weight (LV DW)-to-body weight ratio (LV DW/BW);
2 Histological analysis, including HE staining and masson's trichrome staining protocol;
3 Haemodynamic parameters, including HR, LVSP, LVEDP and±dp/dt;
4 Electrocardiography:the leadⅡof a standard body surface electrocardiogram (ECG) is easily reproducible approach by capturing the whole cardiac cycle using a RM6240C Multi-channel physiological signal acquisition and processing system;
5 Western blotting, the expressions of myosin phosphatase target subunit-1 (MYPT-1), p-MYPT-1, The extracellular signal-regulated kinases 1/2 (ERK1/2), p-ERK1/2, the the c-jun NH 2-terminal kinase (JNK), p-JNK, c-FLIPL protein were determined by Western Blotting;
6 RT-PCR:Total RNA was extracted from fresh-frozen myocardium using the Trizol Reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.
Results:
1 Effect of fasudil on the heart weight parameters'alterations of ISO-induced heart failure
HW/BW was increased by about 41.53% in ISO group as compared to CONT group (ISO:4.26±0.14 versus CONT:3.01±0.04,p<0.01). Moreover, LV WW/BW was increased to 127.60%(ISO:3.19±0.06 versus CONT:2.50±0.28,p<0.05) and LV DW/BW was increased to 138.18%(ISO:0.76±0.01 versus CONT:0.55±0.07,p<0.05). After being administrated with fasudil, HW/BW was significantly reduced in FASL+ISO group (FASL+ISO:3.77±0.10 versus ISO:4.26±0.14, p<0.05) and FASH+ISO group (FASH+ISO: 3.44±0.08 versus ISO:4.26±0.14, p<0.01). WW/BW of LV was significantly reduced by 15.05% in FASL group (FASL+ISO:2.7±0.16 versus ISO:3.19±0.06,p<0.05) and 28.84% in FASH group (FASH+ISO: 2.27±0.02 versus ISO:3.19±0.06, p<0.01) as compared to ISO group. DW/BW of LV showed a similar tendency as LV WW/BW (FASL+ISO:0.63±0.06 versus ISO:0.76±0.01,p<0.05; FASH+ISO:0.60±0.01 versus ISO: 0.76±0.01,p<0.01).
2 Effect of fasudil on the morphological alterations of ISO-induced heart failure
HE was for morphological analysis and Masson's stain was for fibrosis
HE was for morphological analysis and Masson's stain was for fibrosis analysis. Treatment with ISO resulted in marked myocyte loss and increased fibrosis (p<0.01), primarily limited to the subendocardium of the LV free wall and septum. The collagen fraction was determined by measuring the area of blue-stained tissue within a given field with Image-Pro Plus software. The stained area was calculated as a percentage of the total area within an image. The blue-appearing collagen of the LV endocardium was significantly higher in ISO-treated rats (about 84.56%). However, the progressive LV fibrosis was attenuated by fasudil and the effect was in a dose dependent manner (about 43.79% in FASL+ISO and 20.01% in FASH+ISO).
3 Effect of fasudil on hemodynamic variables of ISO-induced heart failure
Treatments of rats with a single dose of ISO (5 mg/kg/day) caused significant increases in HR (from 343±9.6 to 424±8.2bpm, p<0.01) and LVEDP compared to the control value (from 4.77±0.38 to 15.37±0.49 mmHg, p<0.01), indicating LV haemodynamic overload. On the other hand, ISO caused significant decreases in LVSP (from 143±5.8 to 98±8.2 mmHg, p<0.01) and±dp/dtmax compared to the control values (+dp/dtmax from 8311±437 to 4339±232 mmHg/s,p<0.01;-dp/dtmax from 7116±272 to 3623±598 mmHg/s, p<0.01). LVSP and±dp/dtmax are sensitive to changes in preload and afterload. Haemodynamic parameters changes indicated left ventricular dysfunction in ISO rats. Treatment of ISO-injected rats with fasudil for 7 days markedly normalized the heart function. Both systolic and diastolic function were significantly enhanced, and LVSP (from 98±8.2 to 126±6.7 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4339±232 to 5787±246 mmHg/s,p<0.01;-dp/dtmax from 3623±598 to 5130±1344 mmHg/s, p<0.05) and LVEDP (from 15.37±0.49 to 11.73±1.31 mmHg,p<0.05) were all markedly improved in the FASL+ISO group compared with the ISO group. LVSP (from 98±8.2 to 140±8.0 mmHg, p<0.05),±dp/dtmax (+dp/dtmax from 4339±232 to 7861±324 mmHg/s,p<0.01;-dp/dtmax from 3623±598 to 6660±334 mmHg/s,p<0.01) and LVEDP (from 15.37±0.49 to 7.60±0.33 mmHg, p<0.01) were all markedly improved in the FASH+ISO group compared with the ISO group.
4 Effect of fasudil on the ECG alterations of ISO-induced heart failure
The result shows the standard ECGs traces of leadⅡfrom the CONT rats during baseline, ST segment depression with a negative T-wave and typical arrhythmias in the ISO group. Treatment of ISO-injected rats with fasudil for 7 days ameliorated the heart dysfunction.
5 Effect of fasudil on ISO-mediated ROCK activation
Total RNA was extracted from the LVs of the CONT, ISO, FASL+ISO and FASH+ISO groups. ISO caused a significant up-regulation of ROCK I mRNA expression compared with the CONT group (p<0.01), and treatment with fasudil resulted in a significant reduction in ROCKⅠmRNA expression (p<0.01). We tested the activation of RhoA/ROCK pathway by the amount of phosphorylated MYPT-1. ISO caused phosphorylation of MYPT-1(p<0.01), which was inhibited by fasudil in a dose-dependent manner (p<0.05 or p<0.01).
6 Fasudil reduces ISO-induced activation of ERK
We determined the effect of fasudil on the phosphorylation status of ERK in the nucleus as it may participate in ISO-induced heart failure. ISO increased nuclear translocation of ERK (p<0.01), while fasudil reduced this ISO-induced p-ERK to various degrees (p<0.01).
7 Fasudil attenuates ISO-induced JNK activation
The effect of fasudil on ISO-induced JNK activation was observed. ISO increased the activities of JNK phosphorylation to 189.24%(p<0.01). Compared with ISO group, fasudil reduced JNK phosphorylation by about 24.71%(p<0.01) at low dose and 43.37%(p<0.01) at high dose.
8 Effect of fasudil on c-fos and c-jun mRNA expressions
When the rats were administrated by ISO, the expressions of c-fos and c-jun mRNA quickly increased, reaching about 252.92%(p<0.01) and 247.45%(p<0.01) respectively compared with CONT group. The increases of c-fos and c-jun mRNA expressions by ISO were inhibited by high-dose fasudil by approximately 30.84%(p<0.01) and 41.25%(p<0.01), respectively. There were no significant decreases in the c-fos and c-jun mRNA expressions in low-dose fasudil administrated rats.
9 Effect of fasudil on c-FLIPL mRNA and protein expressions
Both the mRNA and protein expressions of c-FLIPL were determined. The c-FLIPL mRNA expression decreased in the ISO group. By contrast, treatment with fasudil resulted in a significant increase (p<0.01). Simultaneously, the c-FLIPL protein levels changed similarly to that of the c-FLIPL mRNA.
Conclusions:RhoA/ROCK is activated by ISO and its activation is essential for heart failure induced by ISO. Furthermore, fasudil is capable of suppressing ISO-induced heart failure, which is associated with inhibition of JNK activation, ERK translocation to the nucleus, subsequent AP-1 (c-fos and c-jun) expression and up-regulation of c-FLIPL expression.
Part 3 RhoA/ROCK may involve in cardiac hypertrophy induced by experimental hyperthyroidism.
Objective:To further investigate the mechanism of cardiovascular dysfunction in the hyperthyroid condition, the role of RhoA/ROCK signaling pathway and effects of fasudil were examined in rats treated with L-thyroxine (T4).
Methods:48 Male SD rats (200-240 g) were purchased from the Laboratory Animal Center (Hebei Medical University, China) and housed in rust-free cages at 20℃～22℃temperature,45%～55% relative humidity on a 12 h light-dark cycle. Rats were randomly assigned to one of four groups: control (CONT), thyroxine (T4), low-dose fasudil+T4 (FASL+T4), and high-dose fasudil+T4 (FASH+T4). All Drugs were given as follows: L-Thyroxine (T4) was dissolved in 99% ethanol by adding a small volume (20μl) of 25% NaOH and diluted 33 times by adding 0.9% NaCl to obtain a stock solution of 1 mg/ml. The solution was made in 0.9% NaCl to obtain a concentration of 50μg T4/ml. T4 in daily doses of 0.25mg/kg body weight was given subcutaneously once daily for 14 days. This treatment results in a long-term moderate hyperthyroidism. The control rats were treated with subcutaneous injections of normal saline given once daily for 14 days. Low-dose fasudil (2 mg/kg/day) and high-dose fasudil (10 mg/kg/day) were injected subcutaneously at the same time. The rats given T4 administration were all alive. All evaluations were performed 24 h after the last fasudil and T4 administration.
The indices as follow:
1 Measurement of cardiac hypertrophy
Cardiac hypertrophy was assessed by the measurement of HW (in mg), BW (in g) and HW/BW (in mg/g).
2 Hormone concentrations
Rats were anaesthetized, and then the blood samples (4 ml) were collected for the analysis of plasma T4 levels. Following centrifugation (3500 g), plasma was separated and stored at -70℃. Total plasma T4 levels were determined by radioimmunoassay according to procedures of the kits.
3 Measurement of mechanical function
A miniature pressure transducer (Millar Micro-Tip) was inserted into the LV via the right carotid artery. The HR, LVEDP, LVSP, and±dp/dtmax were monitored by using MS4000U-1C biological signal quantitative recording and analyzing system.
4 Histology analysis
The LV was removed and fixed, and routinely embedded. Paraffin sections were cut at 5μm thickness and mounted on glass slides. Slides were routinely stained with hematoxylin and eosin for histopathologic review.
5 TUNEL assays
TUNEL assays were performed with the use of 3,3'-diaminobenzidine (DAB). Photographs were taken with the use of a microscope. The numbers of TUNEL-positive cardiac myocyte nuclei were quantified using Image Pro Plus software (Media Cybernetics, Silver Spring, MD). The chosen fields of view represented areas of heaviest TUNEL staining.
6 Western blotting
The expressions of bax, bcl-2 and p-MYPT-1 protein were determined by Western Blotting.
7 RNA extraction and RT-PCR analysis
Total RNA was extracted from fresh-frozen myocardium using the Trizol Reagent. Expressions of ROCKⅠ, c-fos, c-jun, c-FLIPL and GAPDH in cardiomyocytes were examined by RT-PCR.
Results:
1 Heart weights
T4 induced a significantly increased HW/BW ratio (T4:4.33±0.13 mg/g vs. CONT:3.12±0.15 mg/g, n=6 each; p<0.01) accompanied by a significantly decreased body weight than that seen in the CONT (T4:191.2±3 g vs. CONT:229.4±5 g, n= 6 each; p<0.01). Combined treatment by T4 and low-dose fasudil (4.07±0.15 mg/g vs.4.33±0.13 mg/g, n=6;p<0.05), T4 and high-dose fasudil (3.92±0.09 mg/g vs. 4.33±0.13 mg/g, n=6;p<0.01) showed significantly lower HW/BW ratios than that of T4。
2 Thyroid state of T4-treated animals
Rats treated with T4 for 14 days had a serum T4 level 4 times higher than that seen in the control (euthyroid) rats (120±13 nmol/l vs. 38±3 nmol/l, n=6 each; p<0.01). Combined treatment by T4 and low-dose fasudil (118±14 nmol/l vs. 120±13 nmol/l, n= 6; p<0.05), T4 and high-dose fasudil (124±17 nmol/l vs. 120±13 nmol/l, n= 6; p<0.01) resulted in T4 concentrations not differing from T4 treated group values.
3 Measurement of mechanical function
T4 depressed left ventricular±dp/dtmax by～1/3 (from 6856±524 to 4489±371 and 6019±357 to 3890±312 mmHg/s for +dp/dtmax and -dp/dtmax, respectively) and LVSP by 28%(from 142.2±22.4 to 102.8±21.4 mmHg). T4 increased HR by 14%(from 357.5±10.4 to 408.7±34.8 bpm) and LVEDP by nearly 3 times (from 6.7±0.58 to 17.6±1.15). FASL+T4 and FASH+T4 groups demonstrated return toward normal HR, LVEDP, LVSP,+dp/dtmax and-dp/dtmax (for FASL+T4 and FASH+T4 groups, HR decreased to 338.4±43.8 and 330.2±32.2 bpm, LVEDP decreased to 14.7±2.01 and 12.1±1.68 mmHg, LVSP increased to 109.4±10.3 and 127.2±19.7 mmHg,+dp/dtmax increased to 5887±393 and 6162±553 mmHg/s,-dp/dtmax increased to 5104±380 and 5545±437 mmHg, respectively).
4 Myocardial histology analysis
Myocardial hypertrophy with large nuclei, severe myofiber disorganization, and interstitial fibrosis was found in T4 group. T4 induced cardiomyocyte hypertrophy was significantly attenuated by fasudil even though cardiomyocyte areas from the FASL+T4 group remained significantly greater that those of CONT. Cardiomyocyte hypertrophy was significantly attenuated in FASH+T4 group.
5 Apoptotic changes in the LVs
Because HW/BWs were observed to significantly increase in the T4 group compared to the CONT group, the possible involvement of apoptosis was examined. Apoptosis was evaluated within the LV tissue by the TUNEL method. The number of TUNEL-positive cells per microscopic field was significantly increased in the T4 group compared in the CONT group. Fasudil caused a decrease in apoptosis in the cardiac tissue, more significantly at the higher dose, compared to the T4 group
6 ROCK activation
In left ventricle mRNAs of ROCKⅠand GAPDH were detected by RT-PCR analysis. The expression of ROCKⅠmRNA was significantly up-regulated by nearly 2 times in T4 group compared with CONT group (p<0.01). Compared with T4 group, the mRNA level of ROCKⅠremarkably decreased in FASL+T4 and FASH+T4 groups (p<0.01). Consistent with RT-PCR observations, p-MYPT-1 expression was significantly higher in T4-treated rats and remarkably decreased in FASL+T4 and FASH+T4 groups.
7 The effects of fasudil on LV mRNA expressions of c-fos, c-jun, and c-FLIPL
Agarose gels with representative PCR products for c-fos, c-jun, and c-FLIPL are depicted. The c-fos/GAPDH and c-jun/GAPDH mRNA levels in the LV significantly increased in T4 group compared with CONT (p<0.01), whereas the c-FLIPL/GAPDH mRNA levels significantly reduced (p<0.01). The level of c-fos/c-jun mRNA expressions in FASL+T4 and FASH+T4 groups significantly decreased (p<0.01) relative to T4 group. c-FLIPL mRNA expressions in FASL+T4 and FASH+T4 groups significantly increased (p<0.01).
8 Bax and bcl-2 expressions
The left ventricle bax expression of T4 group was 5 times higher than the CONT group. Bcl-2 expression in T4 group significantly decreased compared with the CONT group (p<0.01). Accompany with fasudil administrated, bax expression significantly decreased (p<0.01) on dose-dependent manner. Meanwhile, high-dose fasudil increased the expression of bcl-2 (p<0.01). Low-dose fasudil treated group showed no significant vary on bcl-2 expression.
Conclusions:this study demonstrated that T4 enhanced cardiac hypertrophy by concomitant enhancement of ROCK and AP1 activity and reduction of c-FLIPL expression. It also confirmed that this response can be inhibited by fasudil in dose dependent manner.
1 RhoA/ROCK is activated by ADR and its activation is essential for ADR-induced cardiomyopathy. Furthermore, fasudil is capable of suppressing ADR-induced cardiomyopathy, which is associated with inhibition of bax and NF-κB activations, subsequent AP-1(c-fos and c-jun) expression and up-regulation of c-FLIPL expression.
2 RhoA/ROCK is activated by ISO and its activation is essential for heart failure induced by ISO. Furthermore, fasudil is capable of suppressing ISO-induced heart failure, which is associated with inhibition of JNK activation, ERK translocation to the nucleus, subsequent AP-1(c-fos and c-jun) expression and up-regulation of c-FLIPL expression.
3 T4 enhanced cardiac hypertrophy by concomitant enhancement of ROCK and API activity and reduction of c-FLIPL expression. It also confirmed that this response can be inhibited by fasudil.
In summary, the present studies demonstrated that RhoA/ROCK is activated by heart failure and cardiac hypertrophy in different experimental model of rats, fasudil has beneficial effects on hypertrophic heart failure by suppressing the pathological processes.
引文
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