HSP70上调对快速心房起搏致兔房颤离子通道重构的影响
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摘要
目的：探讨预先诱导心肌HSP70表达上调,对兔快速心房起搏致AF电重构以及Cav1.2,α1c、KCa 3.1的mRNA和蛋白的影响。方法：将32只健康成年新西兰大白兔随机分成热应激±起搏组(n=8)；阿托伐他汀±起搏组(n=8)；起搏组(n=8)和假手术组(n=8)。热应激：将新西兰大白兔放入恒温箱中加热,测缸内温度达43℃后持续15min,放入室温恢复24h。给药组于起搏前一周按2.0mg-Kg-1·d-1进行灌胃,每日一次。以600次/分行右心房起搏,测量0h、2h、4h、6h的右房ERP (AERP200、AERP150), AERP频率适应性,AF诱发率。假手术组只测量不起搏。用RT-PCR和免疫组织化学检测各组心肌HSP70mRNA、HSP70, Cav1.2,α1c,mRNA、Cav1.2,α1c蛋白、KCa3.1 mRNA和Kca3.1蛋白的含量。结果：(1)快速心房起搏后,起搏组AERP200和AERP150立即缩短,起搏2h达最小值[AERP200 (79.38±6.23) ms, AERP150 (71.25±6.94) ms, P<0.01];执应激组、阿托伐他汀组和假手术组起搏前后无显著变化。(2)快速心房起搏前,起搏组RA处(AERP2oo-AERP150)/50ms为0.21±0.10,起搏2h、4h、6h后分别为0.16±0.07,0.14±0.05,0.13±0.05,P<0.01,提示AERP频率适应性逐渐降低；热应激组、阿托伐他汀组和假手术组起搏前后该值差异无显著变化。(3)予以程序性刺激和burst刺激,假手术组各时间点均为25%,起搏后Oh、2h、4h、6h,起搏组AF诱发率分别为37.5%、75%、100%、100%；热应激组起搏后各时间点AF诱发率分别为25%、37.5%、37.5%、50%；阿托伐他汀组AF诱发率分别为37.5%、50%、50%、62.5%,假手术组起搏前后无显著变化。(4)热应激组和阿托伐他汀组心脏各部位HSP70和HSP70mRNA表达较起搏组和假手术组明显增高(P<0.05),起搏组和假手术组间无显著变化；起搏组Cav1.2,α1c,mRNA、Cav1.2,alc蛋白和Kca3.1mRNA、KCa3.1蛋白表达均较热应激±起搏组和假手术组明显减少(P<0.05),热应激组、阿托伐他汀组和假手术组无显著差异。结论(1)热应激使兔肛温达41℃持续15min可诱导心房肌HSP70高表达,HSP70高,表达有显著预防心房肌电重构和AF诱发作用；(2)阿托伐他汀可增加心肌HSP70mRNA及其蛋白的表达,显著预防心肌电重构和AF诱发作用；(3)HSP70表达量是影响心房肌ER和AF诱发的重要因素；(4)心肌HSP70表达上调可抑制快速心房起搏对Cav1.2,α1c和Kca3.1的重构。(5)阿托伐他汀可通过提高HSP70表达抑制快速心房起搏对Cav1.2,α1c和Kca3.1的重构。(6)HSP70高表达预防心房肌ER和AF的作用可维持6h。
Objective:To investigate effects of upregulation expression of myocardium heat shock protein 70 due to heat stress on electrical remodeling and expression of the Cav1.2,α1c、Kca 3.1mRNA and protein induced by rapid atrial pacing in in rabbits. Methods:Thirty-two healthy adult New Zealand rabbits were randomly divided into heat stress groups (n=8), Pacing groups (n=8),atorvastatin groups(n=8) and sham groups (n=8). Heat stress is that New Zealand rabbits were administered ketamine with intraperitoneal in 30mg/kg, muscles injection, then placed them into the heated incubator, used ice packs to protect their heads, measured the cylinder temperatures to 41℃, continued for 15 minuets, then placed them in room temperature for 24h. Atorvastatin pretreatment group was given atorvastatin by 2.0mg-Kg-1·-d-1by gavage one week before pacing.After rapid right atrial pacing at 600 bpm 6h, the parameters reflecting the change of atrial electrophysiology properties were measurred, during AERP, adaptation of AERP, the inducibility of atrial fibrillation at 0h,2h,4h,6h. sham groups received electrophysiological testing only without pacing. HSP70mRNA,HSP70, LVDCCα1c, mRNA, LVDCCalc protein, KCa3.1 mRNA and Kca3.1protein of the myocardial content was detected by RT-PCR. Results:(1) after pacing, AERP200 and AERP150 were immediately shortened AERP200 (79.38±6.23) ms, AERP,150 (71.25±6.94) ms, P< 0.01]; but there were no significant changes in heat stress group, atorvastatin group and the sham group before and after pacing. (2) Before rapid atrial pacing, at RA in pacing group was (AERP2oo-AERP150)/50ms 0.21±0.10, and it was 0.16±0.07,0.14±0.05,0.13±0.05, P<0.01, after pacing for 2h,4h,6h, this showed that tips AERP rate adaptation decreased gradually; there was no significant difference in other groups before and after pacing. (3) the inducibility of atrial fibrillation in each group were by programmed and burst stimulation.The the inducibility of atrial fibrillation of sham groups were 25% at each time point;The the inducibility of atrial fibrillation of pacing groups respectively were 37.5%,75%,100%,100%at pacing 0h,2h,4h,6h.Them were respective 25%,37.5%,37.5%,50% in heat stress groups;Them were respective 37.5%,50%,50%,62.5% inatorvastatin group, It did not significant change during the 6h of pacing in sham groups. (4)The expression of HSP70 and HSP70mRNA of all parts of the heart in Heat stress group and the atorvastatin group was significantly higher (P< 0;05). There was no significant changes between pacing group and the sham group. The expression of Cav1.2,α1c,mRNA,Cav1.2,α1c and Kca3.1mRNA,Kca3.1 protein in pacing group was decreased compared with heat stress group and the sham group.But it had no statistical difference among heat stress group, A asked atorvastatin group and the sham group. Conclusions:(1) heat stress to rabbit maintain rectal temperature at 41℃for 15min induced upregulation of atrial expression of HSP70, high expression of HSP70 was able to reduce of inducement of atrial electrical remodeling and AF by atrial pacing significantly; (2) Atorvastatin increased the expression of HSP70mRNA in myocardial, and it had significant preventive role to myocardial electrical remodeling and AF induced by atrial pacing; (3) the Expression of HSP70 is an important factor which can influence atrial ER and AF; (4) the upregulation of HSP70 in Myocardial inhibited rapid atrial pacing induced reconstruction of Cav1.2,α1c and Kca3.1; (5) Atorvastatin suppressed rapid atrial pacing inducd Reconstruction of Cav1.2,α1c and Kca3.1 via increasing HSP70 expression; (6) Preventative effect of HSP70 high expression to atrial ER and AF maintained 6 h.
Objective:To investigate effects of upregulation expression of myocardium heat shock protein 70 due to heat stress on electrical remodeling and expression of the Cav1.2,α1c、Kca 3.1mRNA and protein induced by rapid atrial pacing in in rabbits. Methods:Thirty-two healthy adult New Zealand rabbits were randomly divided into heat stress groups (n=8), Pacing groups (n=8),atorvastatin groups(n=8) and sham groups (n=8). Heat stress is that New Zealand rabbits were administered ketamine with intraperitoneal in 30mg/kg, muscles injection, then placed them into the heated incubator, used ice packs to protect their heads, measured the cylinder temperatures to 41℃, continued for 15 minuets, then placed them in room temperature for 24h. Atorvastatin pretreatment group was given atorvastatin by 2.0mg-Kg-1·-d-1by gavage one week before pacing.After rapid right atrial pacing at 600 bpm 6h, the parameters reflecting the change of atrial electrophysiology properties were measurred, during AERP, adaptation of AERP, the inducibility of atrial fibrillation at 0h,2h,4h,6h. sham groups received electrophysiological testing only without pacing. HSP70mRNA,HSP70, LVDCCα1c, mRNA, LVDCCalc protein, KCa3.1 mRNA and Kca3.1protein of the myocardial content was detected by RT-PCR. Results:(1) after pacing, AERP200 and AERP150 were immediately shortened AERP200 (79.38±6.23) ms, AERP,150 (71.25±6.94) ms, P< 0.01]; but there were no significant changes in heat stress group, atorvastatin group and the sham group before and after pacing. (2) Before rapid atrial pacing, at RA in pacing group was (AERP2oo-AERP150)/50ms 0.21±0.10, and it was 0.16±0.07,0.14±0.05,0.13±0.05, P<0.01, after pacing for 2h,4h,6h, this showed that tips AERP rate adaptation decreased gradually; there was no significant difference in other groups before and after pacing. (3) the inducibility of atrial fibrillation in each group were by programmed and burst stimulation.The the inducibility of atrial fibrillation of sham groups were 25% at each time point;The the inducibility of atrial fibrillation of pacing groups respectively were 37.5%,75%,100%,100%at pacing 0h,2h,4h,6h.Them were respective 25%,37.5%,37.5%,50% in heat stress groups;Them were respective 37.5%,50%,50%,62.5% inatorvastatin group, It did not significant change during the 6h of pacing in sham groups. (4)The expression of HSP70 and HSP70mRNA of all parts of the heart in Heat stress group and the atorvastatin group was significantly higher (P< 0;05). There was no significant changes between pacing group and the sham group. The expression of Cav1.2,α1c,mRNA,Cav1.2,α1c and Kca3.1mRNA,Kca3.1 protein in pacing group was decreased compared with heat stress group and the sham group.But it had no statistical difference among heat stress group, A asked atorvastatin group and the sham group. Conclusions:(1) heat stress to rabbit maintain rectal temperature at 41℃for 15min induced upregulation of atrial expression of HSP70, high expression of HSP70 was able to reduce of inducement of atrial electrical remodeling and AF by atrial pacing significantly; (2) Atorvastatin increased the expression of HSP70mRNA in myocardial, and it had significant preventive role to myocardial electrical remodeling and AF induced by atrial pacing; (3) the Expression of HSP70 is an important factor which can influence atrial ER and AF; (4) the upregulation of HSP70 in Myocardial inhibited rapid atrial pacing induced reconstruction of Cav1.2,α1c and Kca3.1; (5) Atorvastatin suppressed rapid atrial pacing inducd Reconstruction of Cav1.2,α1c and Kca3.1 via increasing HSP70 expression; (6) Preventative effect of HSP70 high expression to atrial ER and AF maintained 6 h.
引文
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[11]Parcellier A, Gurbuxani S, Schmitt E, et al. Heat shock proteins, cellular chaperones that modulate mitochondrial cell death pathways[J]. Biochem Biophys Res Commun, 2003,304(3):505-512.
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[14]庄梅,方颖,吴立荣,等.地塞米松诱导热休克蛋白72减轻大鼠心肌缺血再灌注损伤[J].中国现代医学杂志,2008,18(15)：2117-2120.
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[16]潘宴青,陈鑫.慢性心房颤动患者血清及心肌热休克蛋白26/60/70的表达及其意义[J].现代医学,2008,36(4)：260-263.
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[18]李萍,富青,熊凡,等.SHR和WKY大鼠心肌细胞凋亡、HSP70、iNOS水平的比较研究[J].微循环学杂志,2005,15(2)：15-17.
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[20]Julie YH, Chen CO, Ling LW, et al. Heat shock protein 70 confers cardiovascular protection during endotoxemia via inhibition of nuclear factor-β activation and inducible nitric oxide synthase expression in the rostra 1 ventro lateral medulla [J]. Circulation,2004,110(3):3560-3566.
[21]许丽丽,石武祥,邬堂春.热休克蛋白70与心肌炎的关系研究进展[J].现代预防医学,2008,35(3)：406-408.
[22]Das M. Apoptpsis as a therapeutic target in heart failure[J]. Am J Physiol Heart Cir Physiol,2007,293:1322-1323.
[23]Shiomi T, Tsutsui H, Matsuska H, et al. Overexpression of glutathione peroxidase prevents left ventricular remodeling and failure after myocardial infarction in mice[J]. Circulation,2004,109:544-549.
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[26]Rafiee P, Shi Y, Pritchard KA, et al. Cellular redistribution of inducible Hsp70 protein in the human and rabbit heart in response to the stress of chronic hypoxia[J]. Biol Chem,2003,278:43636-43644.
[27]Brundel BJ, Shiroshita-Takashita A, Qi X, et al. Induction of heat shock response protects the heart against atrial fibrillation[J]. Circ Res,2006,99(12):1394-1402.
[28]刘广忠,李悦,李为明,等.热休克蛋白与心房颤动时的心房颤动[J].中国心脏起搏与电生理杂志,2009,23(6)：548-551.