调节底物代谢对衰竭心肌代谢重构的干预作用及相关机制研究
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摘要
研究背景
心脏是高耗能器官,心肌细胞需要持续而巨大的能量供应来保证其收缩功能和自身的需要。研究发现,慢性心力衰竭(Chronic Heart Hailure,CHF)时,心肌代谢将会发生很大改变,ATP生成不足,处于“能量饥饿”状态。线粒体是能量代谢的细胞器,学者们逐渐认识到CHF可能伴随着线粒体功能障碍,线粒体功能障碍才是CHF心肌代谢紊乱的核心,研究衰竭心肌线粒体改变对揭示CHF能量代谢改变的机制具有重要意义。解耦联蛋白2(uncoupling protein 2,UCP2),是线粒体内膜的一种质子转运蛋白,当它被激活时,可产生质子的渗漏,使氧化磷酸化解偶联,ATP合成降低。由于UCP2的生理作用,于衰竭心肌能量缺乏的情况是不利的。研究表明,CHF时UCP2表达增高,且与心肌作功减低相关,其增高原因及在CHF能量代谢障碍中意义有待研究。
CHF时,交感系统活性和儿茶酚胺水平增加,血浆游离脂肪酸(FFA)水平升高,FFA代谢增加对心力衰竭的心肌具有损害作用,过多的FFA在线粒体内参加β氧化易引起线粒体氧化应激;同时有研究表明血浆中高浓度的FFA可促进大鼠心肌、骨骼肌UCP2的表达上调,导致产能减少。大型流行病学调查显示,高FFA水平与心源性猝死显著相关,而限制游离FFA和加强葡萄糖的利用,如使用胰岛素和/或葡萄糖降低血FFA水平,抗交感活性治疗,抑制FFA进入线粒体,或者使用曲美他嗪(TMZ)抑制FFA氧化将改善心肌缺血状态。在这种理念的指导下,临床上使用曲美他嗪治疗缺血性心肌病取得了良好的效果,有研究提示长期使用曲美他嗪将改善心衰患者的心功能分级,提高左室功能,我们推测这一作用可能与抑制FFA的利用有关,但其潜在的机制仍不明了,需要进一步研究。
本课题从动物模型和心肌细胞培养两个层面进行研究,并给予相应的药物干预,探讨CHF时线粒体能量生成、氧化呼吸功能的改变及FFA对其影响和UCP2表达变化在其中的作用及意义,为心衰代谢治疗提供新的理论依据及治疗靶点。
研究方法
1.建立腹主动脉缩窄后心力衰竭大鼠模型,分为心力衰竭组(HF20w组),假手术组(SH20w组)和正常对照组(N组)。术后20周后检测:(1)超声心动图:测定舒张末期内径(LVEDd)及室间隔厚度(IVSTd)和左室后壁厚度(LVPWd),计算左心室射血分数(EF%);(2)血流动力学指标:测量心率(HR)、左心室舒张末压(LVEDP)、左心室压力最大上升和下降速率(±dp/dt max),计算平均主动脉压(MAP);(3)病理苏木素-伊红(HE)染色和透射电镜观察衰竭心肌病理变化;(4)分离大鼠心肌线粒体,Clark氧电极法测定线粒体氧化活性及电子传递链活性,HPLC法分析心肌组织内高能磷酸盐含量,罗丹明123法测线粒体膜电位(MMP);(5)测定血FFA含量,RT-PCR、Western-blot检测心肌UCP2表达变化,并对FFA含量与UCP2表达水平、ATP含量和UCP2表达水平进行相关分析。
2.分离培养大鼠心肌细胞,然后观察向培养基中添加不同浓度的FFA(1mmol/l、2mmol/l和4mmol/l)后不同时相点(6h、12h、24h)对细胞凋亡和UCP2表达的影响。其中,用定量RT-PCR和Western-blot检测UCP2表达情况,Tunnel法检测细胞凋亡率,Western-blot检测凋亡相关蛋白bax、bcl-2的表达情况。
3.构建UCP2 RNA干扰腺病毒质粒载体并转染心肌细胞,分为UCP2干扰质粒转染组(siUCP2组)、空白干扰质粒转染组(siCon组),2组培养条件为2mmol/l FFAs,空白对照组(Con组)正常条件培养,设12h、24h两个不同时像点,检测:(1)心肌细胞内ATP、ADP、AMP含量;(2)细胞凋亡率;(3)Wsetern-blot检测UCP2、PPARα、PPARγ及凋亡相关蛋白bax、bcl-2表达。
4.PPARα阻断剂MK88、PPARγ阻断剂GW9662预处理大鼠心肌细胞,后以2mmol/l FFAs培养,相应分组为:FFAs+MK886组、FFAs+GW9662组及FFAs组,同时设空白对照组,Wsetern-blot检测各组UCP2表达变化。
5.20周存活的心衰组大鼠再随机分为:曲美他嗪治疗组(T组,曲美他嗪10 mg·kg -1·d -1)、阿西莫司治疗组(A组,5 mg·kg -1·d -1)、心衰对照组(HF组)和假手术组(SH组),给药8周后检测:(1)超声心动图(指标同方法1) (2)透射电镜观察衰竭心肌超微结构变化;(3)Clark氧电极法测定线粒体氧化活性及电子传递链活性,HPLC法分析心肌组织内高能磷酸盐含量; (4)测定血腹血葡萄糖及FFA水平;(5)RT-PCR、Western-blot检测UCP2表达变化。
结果
1.腹主动脉缩窄术后20周,HF20w组大鼠心脏超声提示:心腔轻度扩大、LVEDd明显增加,EF值显著降低;血流动力学提示:LVEDP、MAP增高,±dp/ dtmax下降;与SH20w组和N组相差显著,符合心力衰竭表现。
2.与同时间N组和SH20W组比较,HF20W组心肌组织线粒体内ATP、ADP、AMP及Pcr含量均降低;线粒体MMP、ST3、PCR水平降低,差异显著;HF20W组ST4略有增高,但各组间无显著差异。
3.HF20W组血FFA水平较N组与SH20W组增高约1.7倍;HF20W组大鼠心肌UCP2 mRNA、蛋白水平较对照组表达显著增加;相关性分析显示,心肌组织ATP含量与UCP2蛋白表达呈显著负相关(r = -0.929);心肌UCP2蛋白表达与血清FFA呈显著正相关(r = 0.89)。
4.2 mmol/l FFAs培养大鼠心肌细胞6h后,UCP2 mRNA及蛋白表达水平较对照组有所增高,至24h时表达量达顶峰;1, 2和4 mmol/l三种浓度FFAs培养心肌细胞12h后UCP2 mRNA及蛋白表达水平较对照组均增高。
5.通过RNAi技术构建siUCP2腺病毒表达载体,转染大鼠心肌细胞24h后,siUCP2组UCP2蛋白表达下降了78%,对照组无明显改变。2mmol/l FFAs培养大鼠心肌细胞12h后:细胞内ATP、ADP较对照组均降低,AMP含量略有增高;至24h,ATP、ADP含量进一步降低;siUCP2组ATP含量明显高于对照组。
6.2mmol/l FFAs培养心肌细胞6h细胞凋亡无明显增加,至12h、24h细胞凋亡数量则明显增加,同时1, 2及4 mmol/l FFAs培养心肌细胞24h,细胞凋亡数量均明显增加;与对照组相比,2mmol/lFFAs培养心肌细胞12 h、24h,凋亡蛋白bax表达明显增高,抗凋亡蛋白bcl-2降低,bcl-2/bax比值降低亦( 2.041降至0.480);抑制UCP2表达后,心肌细胞凋亡率明显降低;WB结果提示:siUCP2组细胞bax表达下降同时bcl-2增高,bcl-2/bax增高(0.48至1.63)。
7.2 mmol/l FFAs培养心肌细胞12、24 h后,Western结果提示:PPARα、PPARγ表达水平无明显改变;使用PPARα阻断剂MK886和PPARγ阻断剂GW9662 30 min预处理心肌细胞,2 mmol/l FFAs培养24h,MK886组UCP2 mRNA及蛋白表达水平的增高受到抑制, GW9662组无明显改变。
8.药物干预8周后对各组动物进行心脏超声检测:T组大鼠EF较HF组、A组有所改善;与HF组相比,A组、T组心肌线粒体ST3及PCR水平明显改善,T组ATP、ADP、PCr含量亦有所提高,各组间ST4水平无显著改变。
9.药物干预后,A组血清FFA明显降低,HF组和T组FFA水平较SH组仍增高;与SH组相比HF组葡萄萄水平增高,A组与HF组则明显降低;与HF组相比,T组、A组UCP2mRNA、蛋白表达明显降低。
结论
1.衰竭心肌发生代谢重构:心肌组织腺苷酸含量和PCr含量降低,线粒体呼吸功能发生改变,MMP降低。细胞能量供应不足、消耗增多可能是心功能减退的直接原因。
2.衰竭心肌UCP2表达增高,并与线粒体ATP含量减少有关,抑制大鼠心肌细胞UCP2表达可减弱高浓度FFA诱导的ATP下降,UCP2表达的增高可能是衰竭心肌ATP生成不足的分子机制之一。
3.UCP2参与了FFA诱导成年大鼠心肌细胞凋亡,抑制UCP2表达后可以减弱FFA诱导的细胞凋亡,同时FFA诱导大鼠心肌细胞UCP2表达是通过PPARα而不是PPARγ实现的。
4.曲美他嗪通过改善线粒体呼吸功能、抑制UCP2表达,减少质子漏,促进线粒体ATP生成,从而改善心肌能量代谢。将心肌底物利用由脂肪酸转向葡萄糖,优化底物代谢,改善线粒体功能,可能是曲美他嗪保护心脏功能的机制。
5.阿西莫司可改善线粒体氧化呼吸功能,降低UCP2水平,但没有改善ATP生成,对心功能无明显改善,推测与其降低FFA的同时降低了血葡萄糖水平有关,我们认为优化衰竭心肌底物代谢需要在降低FFA的同时促进葡萄糖利用。
Background and Objectives
Heart is a high energy-consuming organ, and myocardial cells need enormous and sustained energy to keep its normal function. Recent study revealed that myocardial metabolism will alter greatly after chronic heart failure (CHF), resulting insufficient of adenosine triphosphate adenosine (ATP)and leaving the heart in a state of“energy hungry”. Mitochondria are organelles of energy metabolism, and it is gradually revealed that CHF might accompanied by mitochondria dysfunction. Mitochondria dysfunction maybe play a pivotal role in myocardial metabolism disorder in CHF, so it is important to investigate the biological change of mitochondria in CHF. Uncoupling protein 2 (UCP2) is a proton transporter protein located in the inner membrane of mitochondria, and when it is activated, can leads to proton leakage, uncoupling of oxidative phosphorylation, and decrease of ATP production. Thus the activation of UCP2 is unfavorable to the energy metabolism of heart in CHF. It has been found that the expression of UCP2 increases in CHF and is related to the decreased myocardial performance. However, the reason and the significance of increased UCP2 expression in CHF need further investigation.
The sympathetic system activity and the level of catecholamine increase in CHF, which in turn can lead to increment of plasma free fatty acid (FFA). Excess FFA is detrimental to failed myocardium by causing mitochondrial oxidative stress by participating inβoxidation. Also, the expression of UCP2 is closed related to the concentration of plasma FFA, and it has been found that high plasma FFA can lead to increasing expression of UCP2 in skeletal muscle. Large-scale epidemiological investigation showed that high FFA levels were significantly related to sudden cardiac death. Restriction of the utilization of FFA and enhancement of the utilization of glucose, such as by reducing plasma level of FFA through application of insulin and/or glucose, measures of anti-sympathetic activity, and application of trimetazindine, an inhibitor of FFA oxidation, will improve the ischemia state of myocardium. Under the direction of such concept, trimetazidine have been used clinically for treatment of ischemic cardiomyopathy and achieved good results. Clinical studies show that long-term use of trimetazidine can improve cardiac function in patients with heart failure, improve the left ventricular function, and can improve energy metabolism of myocardium. We speculate this effect may relate to the restriction of use of the FFA, but the underlying mechanisms remain unclear and need further study.
To provide a new theoretical basis and treatment targets for the prevention and treatment of CHF, research work were developed in two levels, namely cell culture in vitro and animal models, to investigate the alteration of metabolism and oxidation respiration function of mitochondria, and the role of FFA treatment targets in CHF development as well as after medicine intervention.
Methods
1. CHF model were developed in rats by constriction of abdominal aorta, and sham operation and normal rats served as control. Twenty weeks later, the rats were sacrificed and the following items were examined. (1) The Left ventricular end diastolic diameter (LVEDd), end-diastolic interventricular septum dimensions(IVSTd) and left ventricular posterior wall dimensions(LVPWd) were measured by echocardiography, and then ejection fraction were calculated. (2) Hemodynamics and CHF parameters including heart rate (HR), LVEDP and±dp/ dtmax were measured, and then mean aortic pressure were calculated. (3) To observe the pathological changes of failed myocardium by Hematoxylin-eosin staining and transmission electron microscopy. (4) Mitochondria were isolated from ventricle of failed heart by density gradient centrifugation. Then mitochondrial oxidative respiratory function was measured by Clark oxygen electrode, the content of adenine nucleotide pools were measured by high performance liquid chromatography (HPLC), and the mitochondrial membrane potential (MMP) was detected by rhodamine 123method. (5) The concentration of FFA and the expression level of UCP2 were measured, and then the correlation analysis were made between FFA concentration and UCP2 expression level, and between ATP content and UCP2 expression level.
2. Cardiomyocytes were isolated and cultured, and then different concentrations of FFA were added to the culture medium to observe the effect of FFA on the expression of FFA and the apoptosis of cardiomyocytes at different time points (6h, 12h and 24h). The expression of UCP2 was examined by RT-PCR and Western-blot, and the apoptosis-related protein bax and bcl-2 were monitored by Tunnel method and Western-blot.
3. MicroRNA interference adenovirus vector of UCP2 was constructed and used to transfect cultured cardiomyocytes to observe the effect of down-regulation of UCP2 expression on apoptosis rates of cardiomyocytes, the content of ATP, ADP and AMP in cardiomyocytes, and the expression of UCP2, PPARα, PPARγ, bax and bcl-2. Empty vector and saline served as control and all culture medium containing 2mmol/l FFA. 4. To observe the effect of PPAR on the expression of UCP2, 5 mM MK886 (a specific
antagonist of PPAR-a) and 10 mM GW9662 (a specific antagonist of PPAR-γ) was added to the culture medium of cardiomyocytes and then 2mmol/l FFAs were added. The expression of UCP2 in cardiomyocytes was then examined by Western blot.
5. Twenty weeks after the operation when CHF were successfully introduced, the rats of CHF were randomly divided into three groups: trimetazidine treated group (group T), acipimox treated group (group A), untreated group (group HF) and sham operation group (group SH). Then the following parameters were examined: (1) LVEDd、IVSTd、LVPWd and EF evaluated by echocardiography.(2) The hemodynamics and heart failure parameters including HR, LVEDP,±dp/dtmax and MAP were measured. (3) The mitochondrial oxidative respiratory function was measured by Clark oxygen electrode and the content of adenine nucleotide pools were measured by high performance liquid chromatography (HPLC). (4) Plasma FFA and blood glucose concentrations were measured. (5) The expression of UCP2 in mitochondria was detected by PT-PCR and Western blot analysis.
Results
1. Twenty weeks after the ligation, echocardiography examination showed that the heart were enlarged, and left ventricular ejection fraction and±dp/dtmax of group SH20w decreased in group SH20w when compared with control groups. Also, the results of hemodynamics examination showed that LVEDP and MAP increased, and±dp/ dtmax decreased in group SH20w when compared with control groups. Together, these data indicating that CHF model could be successfully introduced in rats by abdominal aorta constriction method.
2. The content of ATP, ADP, AMP, Pcr, the level of MMP and ST3 were lower in HF20w rats than in control rats (P<0.05). The level of ST4 were higher in HF20w rats than in control rats, but no significant difference exist among groups (P>0.05).
3. The concentration of plasma FFA were 1.7 times higher in in HF20w rats than in control rats (P<0.05), and the expression of UCP2 at mRNA and protein levels were significant higher in HF20w rats than in control rats (P<0.05). Correlation analysis showed myocardial ATP content and UCP2 protein expression was significantly negatively correlated (r = -0.929), and cardiac UCP2 expression and serum FFA was significantly positvely correlated (r = 0.89).
4. Six hours after treatment of rat cardiomyocytes with FFAs (2 mmol/L), the mRNA and protein levels of UCP2 increased significantly when compared with the control group, reaching a peak at 24 hours after treatment. Incubating cells with FFAs for 12 hours at concentrations of 1, 2, or 4 mmol/L significantly increased the expression of UCP2 compared with the control group.
5. suppressed the expression of UCP2 using RNAi. The inhibitory effect of siRNA on UCP2 expression was evident 24 hours after transfection, when the level of UCP2 protein had decreased by 78%. No apparent effect on UCP2expression in control group. When culture in the medium containing 2mmol/l FFAs for 12h,the content of ATP and ADP in cardiomyocytes were lower than control cells, and decreased further 24 hours after the adding of FFAs. The content of ATP increased significantly in cardiomyocytes cultured in 2mmol/l FFAs and SiRNA treatment than in cardiomyocytes cultured in 2mmol/l FFAs only.
6. When cells were treated with FFAs for 6 hours, the number of apoptotic cells did not increase significantly when compared with the untreated control group. However, at 12 and 24 hours after treatment, the number of apoptotic cells significantly increased significantly when compared with the control group. Furthermore, incubating cells with FFAs for 24 hours at concentrations of 1, 2, or 4 mmol/L resulted in a significant increase of apoptosis cells when compared with the control group. After 12- and 24-hour culture in medium containing 2mM FFAs, an increase in bax and a reduction in bcl-2 were observed when compared with control cells. Also, the bcl-2/bax ratio was clearly reduced from 2.041 to 0.48. RNAi-mediated knockdown of UCP2 attenuated FFA-induced apoptosis in cardiomyocyte. Western blot analysis showed that UCP2 knockdown prevented bax increase and bcl-2 reduction. The bcl-2/bax ratio went from 0.48 to 1.63 after treatment with FFAs plus UCP2 knockdown.
7. Incubating cells with 2 mmol/l FFAs for 12 or 24 hours did not change the level of both PPAR-a and PPAR-γexpression. Pretreated adult rat cardiomyocytes with MK886 (5 mM) blocked FFA-induced UCP2 expression at both the mRNA and protein levels, whereas GW9662 had no effect.
8.Echocardiography were performed eight weeks after medicine intervention, and the results showed that EF improved in rats of group T than those in group HF and group A. When compared with group HF and group A, the ST3 and Pcr level in group T ameliorated significantly (P<0.05), and the content of ATP and ADP increased too in group F. There was no significant change in ST4 level in each group.
9. Eight weeks after medicine intervention, the concentration of plasma FFA in group A reduced significantly, while the concentration in group HF and gourp T were still higher than that in group SH. The level of glucose in group HF were higher than that in group SH, while were significant lower than that in group A. When the mRNA and protein expression level of UCP2 were concerned, group T and group A were significantly lower than group HF (P<0.05); and there were no significant difference among other groups (P>0.05).
Conclusions
1. Metabolic remolding such as decrease of the content of ATP and Pcr, alteration of respiratory function and reduction of MMP in mitochondria take place in chronic failed myocardium. Insufficient energy supply and increasing consume is the possible cause of reduction of cardiac function.
2. The expression of UCP2 in chronic failed myocardium increase and is correlated with the reduction of ATP content in mitochondria, and inhibition of UCP2 expression in cardiomyocytes can reduce the extent of ATP reduction inducing by FFA. The increased expression of UCP2 is one of the mechanisms of insufficient ATP production in CHF.
3. UCP2 plays a role in FFA-induced apoptosis of adult rat cardiomyocytes, and knockdown of UCP2 expression attenuates FFA-induced apoptosis in these cells. Also, FFA induction of UCP2 expression involved PPAR-a, but not PPAR-γ.
4. Trimetazidine can promote energy production of mitochondria by ameliorating respiratory function of mitochondria, inhibiting the expression UCP2 and the lekeage of proton in mitochondria, which can in turn improve the energy metabolism of myocardium. To transfer the metabolic substrate of myocardium from FFA to glucose, improve the function of mitochondria and optimize the substrate metabolism is the mechanism of protection effect of trimetazidine on cardiac function.
5. Acipimox can ameliorate oxidation respiration function of mitochondria, decrease UCP2 expression level, but can not increase ATP production, thus showing no obvious effect to improve cardiac function. This may lie in the reduction of glucose level in myocardiocyte at the same time. These data indicating increment of glucose consumption that and reduction of FFA are needed simultaneously to optimize the metabolism in failed myocardium.
心脏是高耗能器官,心肌细胞需要持续而巨大的能量供应来保证其收缩功能和自身的需要。研究发现,慢性心力衰竭(Chronic Heart Hailure,CHF)时,心肌代谢将会发生很大改变,ATP生成不足,处于“能量饥饿”状态。线粒体是能量代谢的细胞器,学者们逐渐认识到CHF可能伴随着线粒体功能障碍,线粒体功能障碍才是CHF心肌代谢紊乱的核心,研究衰竭心肌线粒体改变对揭示CHF能量代谢改变的机制具有重要意义。解耦联蛋白2(uncoupling protein 2,UCP2),是线粒体内膜的一种质子转运蛋白,当它被激活时,可产生质子的渗漏,使氧化磷酸化解偶联,ATP合成降低。由于UCP2的生理作用,于衰竭心肌能量缺乏的情况是不利的。研究表明,CHF时UCP2表达增高,且与心肌作功减低相关,其增高原因及在CHF能量代谢障碍中意义有待研究。
CHF时,交感系统活性和儿茶酚胺水平增加,血浆游离脂肪酸(FFA)水平升高,FFA代谢增加对心力衰竭的心肌具有损害作用,过多的FFA在线粒体内参加β氧化易引起线粒体氧化应激;同时有研究表明血浆中高浓度的FFA可促进大鼠心肌、骨骼肌UCP2的表达上调,导致产能减少。大型流行病学调查显示,高FFA水平与心源性猝死显著相关,而限制游离FFA和加强葡萄糖的利用,如使用胰岛素和/或葡萄糖降低血FFA水平,抗交感活性治疗,抑制FFA进入线粒体,或者使用曲美他嗪(TMZ)抑制FFA氧化将改善心肌缺血状态。在这种理念的指导下,临床上使用曲美他嗪治疗缺血性心肌病取得了良好的效果,有研究提示长期使用曲美他嗪将改善心衰患者的心功能分级,提高左室功能,我们推测这一作用可能与抑制FFA的利用有关,但其潜在的机制仍不明了,需要进一步研究。
本课题从动物模型和心肌细胞培养两个层面进行研究,并给予相应的药物干预,探讨CHF时线粒体能量生成、氧化呼吸功能的改变及FFA对其影响和UCP2表达变化在其中的作用及意义,为心衰代谢治疗提供新的理论依据及治疗靶点。
研究方法
1.建立腹主动脉缩窄后心力衰竭大鼠模型,分为心力衰竭组(HF20w组),假手术组(SH20w组)和正常对照组(N组)。术后20周后检测:(1)超声心动图:测定舒张末期内径(LVEDd)及室间隔厚度(IVSTd)和左室后壁厚度(LVPWd),计算左心室射血分数(EF%);(2)血流动力学指标:测量心率(HR)、左心室舒张末压(LVEDP)、左心室压力最大上升和下降速率(±dp/dt max),计算平均主动脉压(MAP);(3)病理苏木素-伊红(HE)染色和透射电镜观察衰竭心肌病理变化;(4)分离大鼠心肌线粒体,Clark氧电极法测定线粒体氧化活性及电子传递链活性,HPLC法分析心肌组织内高能磷酸盐含量,罗丹明123法测线粒体膜电位(MMP);(5)测定血FFA含量,RT-PCR、Western-blot检测心肌UCP2表达变化,并对FFA含量与UCP2表达水平、ATP含量和UCP2表达水平进行相关分析。
2.分离培养大鼠心肌细胞,然后观察向培养基中添加不同浓度的FFA(1mmol/l、2mmol/l和4mmol/l)后不同时相点(6h、12h、24h)对细胞凋亡和UCP2表达的影响。其中,用定量RT-PCR和Western-blot检测UCP2表达情况,Tunnel法检测细胞凋亡率,Western-blot检测凋亡相关蛋白bax、bcl-2的表达情况。
3.构建UCP2 RNA干扰腺病毒质粒载体并转染心肌细胞,分为UCP2干扰质粒转染组(siUCP2组)、空白干扰质粒转染组(siCon组),2组培养条件为2mmol/l FFAs,空白对照组(Con组)正常条件培养,设12h、24h两个不同时像点,检测:(1)心肌细胞内ATP、ADP、AMP含量;(2)细胞凋亡率;(3)Wsetern-blot检测UCP2、PPARα、PPARγ及凋亡相关蛋白bax、bcl-2表达。
4.PPARα阻断剂MK88、PPARγ阻断剂GW9662预处理大鼠心肌细胞,后以2mmol/l FFAs培养,相应分组为:FFAs+MK886组、FFAs+GW9662组及FFAs组,同时设空白对照组,Wsetern-blot检测各组UCP2表达变化。
5.20周存活的心衰组大鼠再随机分为:曲美他嗪治疗组(T组,曲美他嗪10 mg·kg -1·d -1)、阿西莫司治疗组(A组,5 mg·kg -1·d -1)、心衰对照组(HF组)和假手术组(SH组),给药8周后检测:(1)超声心动图(指标同方法1) (2)透射电镜观察衰竭心肌超微结构变化;(3)Clark氧电极法测定线粒体氧化活性及电子传递链活性,HPLC法分析心肌组织内高能磷酸盐含量; (4)测定血腹血葡萄糖及FFA水平;(5)RT-PCR、Western-blot检测UCP2表达变化。
结果
1.腹主动脉缩窄术后20周,HF20w组大鼠心脏超声提示:心腔轻度扩大、LVEDd明显增加,EF值显著降低;血流动力学提示:LVEDP、MAP增高,±dp/ dtmax下降;与SH20w组和N组相差显著,符合心力衰竭表现。
2.与同时间N组和SH20W组比较,HF20W组心肌组织线粒体内ATP、ADP、AMP及Pcr含量均降低;线粒体MMP、ST3、PCR水平降低,差异显著;HF20W组ST4略有增高,但各组间无显著差异。
3.HF20W组血FFA水平较N组与SH20W组增高约1.7倍;HF20W组大鼠心肌UCP2 mRNA、蛋白水平较对照组表达显著增加;相关性分析显示,心肌组织ATP含量与UCP2蛋白表达呈显著负相关(r = -0.929);心肌UCP2蛋白表达与血清FFA呈显著正相关(r = 0.89)。
4.2 mmol/l FFAs培养大鼠心肌细胞6h后,UCP2 mRNA及蛋白表达水平较对照组有所增高,至24h时表达量达顶峰;1, 2和4 mmol/l三种浓度FFAs培养心肌细胞12h后UCP2 mRNA及蛋白表达水平较对照组均增高。
5.通过RNAi技术构建siUCP2腺病毒表达载体,转染大鼠心肌细胞24h后,siUCP2组UCP2蛋白表达下降了78%,对照组无明显改变。2mmol/l FFAs培养大鼠心肌细胞12h后:细胞内ATP、ADP较对照组均降低,AMP含量略有增高;至24h,ATP、ADP含量进一步降低;siUCP2组ATP含量明显高于对照组。
6.2mmol/l FFAs培养心肌细胞6h细胞凋亡无明显增加,至12h、24h细胞凋亡数量则明显增加,同时1, 2及4 mmol/l FFAs培养心肌细胞24h,细胞凋亡数量均明显增加;与对照组相比,2mmol/lFFAs培养心肌细胞12 h、24h,凋亡蛋白bax表达明显增高,抗凋亡蛋白bcl-2降低,bcl-2/bax比值降低亦( 2.041降至0.480);抑制UCP2表达后,心肌细胞凋亡率明显降低;WB结果提示:siUCP2组细胞bax表达下降同时bcl-2增高,bcl-2/bax增高(0.48至1.63)。
7.2 mmol/l FFAs培养心肌细胞12、24 h后,Western结果提示:PPARα、PPARγ表达水平无明显改变;使用PPARα阻断剂MK886和PPARγ阻断剂GW9662 30 min预处理心肌细胞,2 mmol/l FFAs培养24h,MK886组UCP2 mRNA及蛋白表达水平的增高受到抑制, GW9662组无明显改变。
8.药物干预8周后对各组动物进行心脏超声检测:T组大鼠EF较HF组、A组有所改善;与HF组相比,A组、T组心肌线粒体ST3及PCR水平明显改善,T组ATP、ADP、PCr含量亦有所提高,各组间ST4水平无显著改变。
9.药物干预后,A组血清FFA明显降低,HF组和T组FFA水平较SH组仍增高;与SH组相比HF组葡萄萄水平增高,A组与HF组则明显降低;与HF组相比,T组、A组UCP2mRNA、蛋白表达明显降低。
结论
1.衰竭心肌发生代谢重构:心肌组织腺苷酸含量和PCr含量降低,线粒体呼吸功能发生改变,MMP降低。细胞能量供应不足、消耗增多可能是心功能减退的直接原因。
2.衰竭心肌UCP2表达增高,并与线粒体ATP含量减少有关,抑制大鼠心肌细胞UCP2表达可减弱高浓度FFA诱导的ATP下降,UCP2表达的增高可能是衰竭心肌ATP生成不足的分子机制之一。
3.UCP2参与了FFA诱导成年大鼠心肌细胞凋亡,抑制UCP2表达后可以减弱FFA诱导的细胞凋亡,同时FFA诱导大鼠心肌细胞UCP2表达是通过PPARα而不是PPARγ实现的。
4.曲美他嗪通过改善线粒体呼吸功能、抑制UCP2表达,减少质子漏,促进线粒体ATP生成,从而改善心肌能量代谢。将心肌底物利用由脂肪酸转向葡萄糖,优化底物代谢,改善线粒体功能,可能是曲美他嗪保护心脏功能的机制。
5.阿西莫司可改善线粒体氧化呼吸功能,降低UCP2水平,但没有改善ATP生成,对心功能无明显改善,推测与其降低FFA的同时降低了血葡萄糖水平有关,我们认为优化衰竭心肌底物代谢需要在降低FFA的同时促进葡萄糖利用。
Background and Objectives
Heart is a high energy-consuming organ, and myocardial cells need enormous and sustained energy to keep its normal function. Recent study revealed that myocardial metabolism will alter greatly after chronic heart failure (CHF), resulting insufficient of adenosine triphosphate adenosine (ATP)and leaving the heart in a state of“energy hungry”. Mitochondria are organelles of energy metabolism, and it is gradually revealed that CHF might accompanied by mitochondria dysfunction. Mitochondria dysfunction maybe play a pivotal role in myocardial metabolism disorder in CHF, so it is important to investigate the biological change of mitochondria in CHF. Uncoupling protein 2 (UCP2) is a proton transporter protein located in the inner membrane of mitochondria, and when it is activated, can leads to proton leakage, uncoupling of oxidative phosphorylation, and decrease of ATP production. Thus the activation of UCP2 is unfavorable to the energy metabolism of heart in CHF. It has been found that the expression of UCP2 increases in CHF and is related to the decreased myocardial performance. However, the reason and the significance of increased UCP2 expression in CHF need further investigation.
The sympathetic system activity and the level of catecholamine increase in CHF, which in turn can lead to increment of plasma free fatty acid (FFA). Excess FFA is detrimental to failed myocardium by causing mitochondrial oxidative stress by participating inβoxidation. Also, the expression of UCP2 is closed related to the concentration of plasma FFA, and it has been found that high plasma FFA can lead to increasing expression of UCP2 in skeletal muscle. Large-scale epidemiological investigation showed that high FFA levels were significantly related to sudden cardiac death. Restriction of the utilization of FFA and enhancement of the utilization of glucose, such as by reducing plasma level of FFA through application of insulin and/or glucose, measures of anti-sympathetic activity, and application of trimetazindine, an inhibitor of FFA oxidation, will improve the ischemia state of myocardium. Under the direction of such concept, trimetazidine have been used clinically for treatment of ischemic cardiomyopathy and achieved good results. Clinical studies show that long-term use of trimetazidine can improve cardiac function in patients with heart failure, improve the left ventricular function, and can improve energy metabolism of myocardium. We speculate this effect may relate to the restriction of use of the FFA, but the underlying mechanisms remain unclear and need further study.
To provide a new theoretical basis and treatment targets for the prevention and treatment of CHF, research work were developed in two levels, namely cell culture in vitro and animal models, to investigate the alteration of metabolism and oxidation respiration function of mitochondria, and the role of FFA treatment targets in CHF development as well as after medicine intervention.
Methods
1. CHF model were developed in rats by constriction of abdominal aorta, and sham operation and normal rats served as control. Twenty weeks later, the rats were sacrificed and the following items were examined. (1) The Left ventricular end diastolic diameter (LVEDd), end-diastolic interventricular septum dimensions(IVSTd) and left ventricular posterior wall dimensions(LVPWd) were measured by echocardiography, and then ejection fraction were calculated. (2) Hemodynamics and CHF parameters including heart rate (HR), LVEDP and±dp/ dtmax were measured, and then mean aortic pressure were calculated. (3) To observe the pathological changes of failed myocardium by Hematoxylin-eosin staining and transmission electron microscopy. (4) Mitochondria were isolated from ventricle of failed heart by density gradient centrifugation. Then mitochondrial oxidative respiratory function was measured by Clark oxygen electrode, the content of adenine nucleotide pools were measured by high performance liquid chromatography (HPLC), and the mitochondrial membrane potential (MMP) was detected by rhodamine 123method. (5) The concentration of FFA and the expression level of UCP2 were measured, and then the correlation analysis were made between FFA concentration and UCP2 expression level, and between ATP content and UCP2 expression level.
2. Cardiomyocytes were isolated and cultured, and then different concentrations of FFA were added to the culture medium to observe the effect of FFA on the expression of FFA and the apoptosis of cardiomyocytes at different time points (6h, 12h and 24h). The expression of UCP2 was examined by RT-PCR and Western-blot, and the apoptosis-related protein bax and bcl-2 were monitored by Tunnel method and Western-blot.
3. MicroRNA interference adenovirus vector of UCP2 was constructed and used to transfect cultured cardiomyocytes to observe the effect of down-regulation of UCP2 expression on apoptosis rates of cardiomyocytes, the content of ATP, ADP and AMP in cardiomyocytes, and the expression of UCP2, PPARα, PPARγ, bax and bcl-2. Empty vector and saline served as control and all culture medium containing 2mmol/l FFA. 4. To observe the effect of PPAR on the expression of UCP2, 5 mM MK886 (a specific
antagonist of PPAR-a) and 10 mM GW9662 (a specific antagonist of PPAR-γ) was added to the culture medium of cardiomyocytes and then 2mmol/l FFAs were added. The expression of UCP2 in cardiomyocytes was then examined by Western blot.
5. Twenty weeks after the operation when CHF were successfully introduced, the rats of CHF were randomly divided into three groups: trimetazidine treated group (group T), acipimox treated group (group A), untreated group (group HF) and sham operation group (group SH). Then the following parameters were examined: (1) LVEDd、IVSTd、LVPWd and EF evaluated by echocardiography.(2) The hemodynamics and heart failure parameters including HR, LVEDP,±dp/dtmax and MAP were measured. (3) The mitochondrial oxidative respiratory function was measured by Clark oxygen electrode and the content of adenine nucleotide pools were measured by high performance liquid chromatography (HPLC). (4) Plasma FFA and blood glucose concentrations were measured. (5) The expression of UCP2 in mitochondria was detected by PT-PCR and Western blot analysis.
Results
1. Twenty weeks after the ligation, echocardiography examination showed that the heart were enlarged, and left ventricular ejection fraction and±dp/dtmax of group SH20w decreased in group SH20w when compared with control groups. Also, the results of hemodynamics examination showed that LVEDP and MAP increased, and±dp/ dtmax decreased in group SH20w when compared with control groups. Together, these data indicating that CHF model could be successfully introduced in rats by abdominal aorta constriction method.
2. The content of ATP, ADP, AMP, Pcr, the level of MMP and ST3 were lower in HF20w rats than in control rats (P<0.05). The level of ST4 were higher in HF20w rats than in control rats, but no significant difference exist among groups (P>0.05).
3. The concentration of plasma FFA were 1.7 times higher in in HF20w rats than in control rats (P<0.05), and the expression of UCP2 at mRNA and protein levels were significant higher in HF20w rats than in control rats (P<0.05). Correlation analysis showed myocardial ATP content and UCP2 protein expression was significantly negatively correlated (r = -0.929), and cardiac UCP2 expression and serum FFA was significantly positvely correlated (r = 0.89).
4. Six hours after treatment of rat cardiomyocytes with FFAs (2 mmol/L), the mRNA and protein levels of UCP2 increased significantly when compared with the control group, reaching a peak at 24 hours after treatment. Incubating cells with FFAs for 12 hours at concentrations of 1, 2, or 4 mmol/L significantly increased the expression of UCP2 compared with the control group.
5. suppressed the expression of UCP2 using RNAi. The inhibitory effect of siRNA on UCP2 expression was evident 24 hours after transfection, when the level of UCP2 protein had decreased by 78%. No apparent effect on UCP2expression in control group. When culture in the medium containing 2mmol/l FFAs for 12h,the content of ATP and ADP in cardiomyocytes were lower than control cells, and decreased further 24 hours after the adding of FFAs. The content of ATP increased significantly in cardiomyocytes cultured in 2mmol/l FFAs and SiRNA treatment than in cardiomyocytes cultured in 2mmol/l FFAs only.
6. When cells were treated with FFAs for 6 hours, the number of apoptotic cells did not increase significantly when compared with the untreated control group. However, at 12 and 24 hours after treatment, the number of apoptotic cells significantly increased significantly when compared with the control group. Furthermore, incubating cells with FFAs for 24 hours at concentrations of 1, 2, or 4 mmol/L resulted in a significant increase of apoptosis cells when compared with the control group. After 12- and 24-hour culture in medium containing 2mM FFAs, an increase in bax and a reduction in bcl-2 were observed when compared with control cells. Also, the bcl-2/bax ratio was clearly reduced from 2.041 to 0.48. RNAi-mediated knockdown of UCP2 attenuated FFA-induced apoptosis in cardiomyocyte. Western blot analysis showed that UCP2 knockdown prevented bax increase and bcl-2 reduction. The bcl-2/bax ratio went from 0.48 to 1.63 after treatment with FFAs plus UCP2 knockdown.
7. Incubating cells with 2 mmol/l FFAs for 12 or 24 hours did not change the level of both PPAR-a and PPAR-γexpression. Pretreated adult rat cardiomyocytes with MK886 (5 mM) blocked FFA-induced UCP2 expression at both the mRNA and protein levels, whereas GW9662 had no effect.
8.Echocardiography were performed eight weeks after medicine intervention, and the results showed that EF improved in rats of group T than those in group HF and group A. When compared with group HF and group A, the ST3 and Pcr level in group T ameliorated significantly (P<0.05), and the content of ATP and ADP increased too in group F. There was no significant change in ST4 level in each group.
9. Eight weeks after medicine intervention, the concentration of plasma FFA in group A reduced significantly, while the concentration in group HF and gourp T were still higher than that in group SH. The level of glucose in group HF were higher than that in group SH, while were significant lower than that in group A. When the mRNA and protein expression level of UCP2 were concerned, group T and group A were significantly lower than group HF (P<0.05); and there were no significant difference among other groups (P>0.05).
Conclusions
1. Metabolic remolding such as decrease of the content of ATP and Pcr, alteration of respiratory function and reduction of MMP in mitochondria take place in chronic failed myocardium. Insufficient energy supply and increasing consume is the possible cause of reduction of cardiac function.
2. The expression of UCP2 in chronic failed myocardium increase and is correlated with the reduction of ATP content in mitochondria, and inhibition of UCP2 expression in cardiomyocytes can reduce the extent of ATP reduction inducing by FFA. The increased expression of UCP2 is one of the mechanisms of insufficient ATP production in CHF.
3. UCP2 plays a role in FFA-induced apoptosis of adult rat cardiomyocytes, and knockdown of UCP2 expression attenuates FFA-induced apoptosis in these cells. Also, FFA induction of UCP2 expression involved PPAR-a, but not PPAR-γ.
4. Trimetazidine can promote energy production of mitochondria by ameliorating respiratory function of mitochondria, inhibiting the expression UCP2 and the lekeage of proton in mitochondria, which can in turn improve the energy metabolism of myocardium. To transfer the metabolic substrate of myocardium from FFA to glucose, improve the function of mitochondria and optimize the substrate metabolism is the mechanism of protection effect of trimetazidine on cardiac function.
5. Acipimox can ameliorate oxidation respiration function of mitochondria, decrease UCP2 expression level, but can not increase ATP production, thus showing no obvious effect to improve cardiac function. This may lie in the reduction of glucose level in myocardiocyte at the same time. These data indicating increment of glucose consumption that and reduction of FFA are needed simultaneously to optimize the metabolism in failed myocardium.
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