心衰大鼠心肌Na+-K+泵电流及其相应α亚基的改变
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摘要
心力衰竭(heart failure, HF)是全世界流行的一种心血管疾病,其发病具有复杂性和多因素性,其中室性心率失常和心肌收缩减弱是引发心衰的主要原因,而细胞内Ca2+调节的改变则是心脏收缩障碍和心律失常发生的共同通路[1]。
无论最初何种起因(高血压,心肌缺血,心肌病等),一旦心肌受到损伤,结果都会导致心力衰竭。最初阶段,可以通过激活交感神经和肾素-血管紧张素系统,对初始心肌损害造成的心肌收缩功能减弱进行代偿,并逐渐导致左心室扩大和/或肥厚。但久而久之,若心脏功能持续减弱,并伴随神经内分泌的过度激活,那么心脏的损伤就会累积和不可逆,而且不能满足机体的代谢需要,导致心衰[2]。
在过去50年里,有以下机制解释心脏的功能异常:(1)能量产生和利用的损伤(2)前负荷和后负荷的增加(3)神经激素和信号转导的改变(4)细胞内Ca2+超载和Ca2+调节的异常。由于心衰常常伴有心脏形状和体积的改变,这提示心肌损伤可能导致心肌改型。压力超负荷和容量过度负荷造成的心衰是心肌改型的结果[3]。
在心肌肥厚和心衰期间,细胞内Na+增加可以通过Na+/Ca2+交换体增加Ca2+内流,从而有助于增加收缩力或者维持收缩力。在心衰这种病理生理学状态下,一些Na+转运体的表达和功能受到影响,都可以导致细胞内Na+增加。如有文献报道Na+-K+泵的活性下降和Na+/H+交换体的表达上调都会导致细胞内Na+增加[4]。
Na+-K+泵通过调节细胞内Na+来调节细胞内Ca2+浓度[5]。Na+/Ca2+交换体与Na+-K+泵是紧密相连的,抑制Na+-K+泵后可通过Na+/Ca2+交换体来增加细胞内Ca2+,从而加强心肌收缩力[6-9]。所以Na+-K+泵活性的改变对于心肌收缩力影响十分重要[10,11]。
Na+-K+泵系由α亚基(~110 kDa)和β亚基(~55 kDa)组成的异源二聚体。α亚基调节酶的催化活性以及包括和Na+,K+,ATP以及强心苷的结合位点[12-14]。α亚基有四种不同的亚型(α1,α2,α3,α4),其分布具有组织特异性和种属差异[15]。α1和α2主要分布在大鼠、豚鼠和小鼠的心脏,α1、α2和α3主要分布在人的心脏[16,17]。
人心衰后,心肌细胞Na+-K+泵的α1、α3和β1的表达均有显著性下降,而α2没有改变,Na+-K+泵的活性下降[18]。在心衰家兔的心肌细胞上,Na+-K+泵的活性没有改变[19]。免疫印迹结果表明,心衰大鼠α1和α2的表达较正常大鼠分别下降了~19%和~74%;而Svein等报道,心衰大鼠的α1和β1亚基在mRNA和蛋白表达水平均无显著改变,仅α2亚基在mRNA和蛋白表达水平分别下降了25%和55% [20,21]。
鉴于文献关于心衰后Na+-K+泵的活性及其亚基改变的报道并不一致,本实验拟在来自阿霉素致大鼠心衰模型的心肌细胞,以全细胞膜片钳技术测定Na+-K+泵电流(Ip)和分子生物学技术测定Na+-K+泵α亚基表达,旨在研究心衰大鼠Na/K泵活性的改变和相应亚基的改变,并分析Na+-K+泵在心衰发病机制中的作用。
目的:检测心衰大鼠Na+-K+泵活性的变化,以及Na+-K+泵α亚基的在蛋白水平的改变。
方法:(1)采用阿霉素(ADR)致大鼠心衰的方法建立大鼠心衰模型:雄性SD大鼠,体重200-250 g,由河北医科大学实验动物中心提供。将36只SD大鼠随机分为对照组(NOR)和心衰组(HF)。心衰组第2、4天腹腔注射ADR 1 mg/kg,第6、8天注射ADR 2 mg/kg,第10、12天注射ADR 3 mg/kg,第14、16天注射ADR 3 mg/kg。对照组每次腹腔注射等容积的生理盐水。17-20天后,通过行为体征观察,体重(BW)、肺重比体重(LW/BW)、左心室重比体重(LVW/BW)及心功能如心率(HR)、左心室收缩压(LVSP)、左心室舒张末期压(LVEDP)、左室压力最大上升速度(+dp/dtmax)、左室压力最大下降速度(-dp/dtmax)的测定进行心衰评价。
(2)酶解法急性分离大鼠心室肌细胞:取实验用SD大鼠,腹腔注射肝素2500 U·kg-1和12 mg·L-1的戊巴比妥钠50 mg·kg-1,待动物麻醉后,使用Langendorff装置,行主动脉插管逆行灌流(温度,压力恒定),先灌流无Ca2+台式液约10 ml,再灌流含胶原酶Ⅱ(0.6 mg·ml-1)及CaCl2(30μM·L-1)的台式液30 ml消化心脏,反复循环灌流20-25 min,急性分离大鼠心室肌细胞。
(3)Na+-K+泵电流测定:以全细胞膜片钳技术(whole-cell patch-clamp technique)记录Na+-K+泵电流(Ip),Na+-K+泵一次主动转运可将3个Na+泵出细胞外,同时泵进2个K+,因而可产生一个纯净的向外电流。在特定的细胞外液和电极内液阻断细胞膜上其他离子通道和交换体电流的条件下(主要是K电流,Ca电流,Na+/Ca2+交换体电流),灌流Na+-K+泵的抑制剂哇巴因(OUA),使膜电流产生内向移动,此差别电流反应Ip大小。1 mmol·L-1的哇巴因可以完全阻断Na+-K+泵,产生的电流即Ip。电流密度即是电流大小与电容之比。以哇巴因浓度([OUA])对数值为横坐标,以哇巴因对Na+-K+泵电流抑制的百分率(ΔIp)为纵坐标,绘制ΔIp-[OUA]量效关系曲线,并采用两点结合模型公式进行曲线拟合。
(4)采用Western Blot方法测定Na+-K+泵α1和α2亚基蛋白表达的变化:提取NOR和HF组大鼠心室肌细胞膜蛋白,用BCA蛋白定量法进行蛋白定量,10%SDS-聚丙烯酰胺电泳,转膜,5%脱脂奶粉振荡封闭1小时,加α亚基特异性的抗体,4℃孵育过夜,TBST洗三次,加二抗37℃孵育1小时,TBS洗三次,DAB显色。
结果:(1)心衰模型相关指标评价:HF组大鼠10天后出现中毒现象,表现为精神状态差,BW、HR、LVSP以及+dp/dtmax、-dp/dtmax都明显下降(p<0.05),而LVW/BW、LW/BW、LVEDP有显著升高(p<0.05)。给药前正常组BW、LVW/BW、LW/BW、HR、LVSP、LVEDP、+dp/dtmax、-dp/dtmax分别为238±15 g、2.14±0.11 mg·g-1、4.11±0.27 mg·g-1、483±23次/min、129±4 mmHg、5.9±0.9 mmHg、3357±101 mmHg/s、3314±127 mmHg/s。给药后心衰组BW、LVW/BW、LW/BW、HR、LVSP、LVEDP、+dp/dtmax、-dp/dtmax分别为207±13 g、2.61±0.04 mg·g-1、5.51±0.31 mg·g-1,388±34次/min、80±4 mmHg、11.3±1.2 mmHg、1727±55 mmHg/s、1550±45 mmHg/s。
(2)心衰大鼠Ip的改变:与正常大鼠Na+-K+泵电流密度相比,心衰大鼠Na+-K+泵电流密度显著下降( 0.5070+0.06297 pA/pF vs 0.2106+0.04890 pA/pF,p<0.05)。绘制ΔIp-[OUA]量效关系曲线,对ΔIp-[OUA]量效关系曲线以两个以上α亚基两位点公式进行拟合,结果表明正常和心衰大鼠均存在高低亲和力两种Na+-K+泵。不同的是,正常大鼠低亲和力钠泵K值为2.85×10-5 M,高亲和力钠泵K值为6.74×10-8 M,阿霉素致大鼠心衰后,其低亲和力钠泵K值为3.55×10-5 M,而高亲和力钠泵K值为2.97×10-7 M。由以上结果可知,大鼠心衰后高亲和力钠泵发生了变化。
(3)心衰大鼠Na+-K+泵α1和α2亚基蛋白表达的变化:我们使用Western blot检测技术在正常及心衰大鼠的心肌均检测到α1和α2亚基蛋白(112 KD)。与正常组相比,心衰组Na+-K+泵低亲和力α1亚基蛋白表达无明显改变,而高亲和力α2亚基蛋白表达则明显下降,约下降了50%(p<0.05)。这进一步提示阿霉素致大鼠心衰的机制是Na+-K+泵的α2亚基发生改变,与电生理结果一致。
结论:大鼠心衰后电流密度显著下降。ΔIp-[OUA]量效关系曲线表明心衰大鼠Na+-K+泵的高亲和力泵发生改变,而且心衰大鼠心肌Na+-K+泵α2亚基蛋白表达明显下降,但α1亚基蛋白的表达没有改变。提示心衰的发病机制可能与Na+-K+泵的α2亚基密切相关。
Heart failure (HF) is the main cardiovascular disease in the world. The genesis of HF syndromes is complex and multifactorial, while ventricular arrhythmias and degression of the myocardial contractility are the main causes of human heart failure, but the altered cellular Ca2+ regulation may be a final common pathway in both contractile dysfunction and arrhythmogenesis [1].
In a heart that has suffered from myocardial damage, regardless of the initial cause of the damage (hypertension, myocardial ischemia, cardiomyopathy, etc), HF eventually occurs if such damage persists for a prolonged period. In the initial stages, compensation for the depressed myocardial contractility due to myocardial damage can occur via activation of both the sympathetic nervous system and the renin-angiotensin system, resulting in left ventricle dilatation and/or hypertrophy. However, if the depressed cardiac function persists, with a parallel activation of neurohumoral factors, the myocardial damage becomes progressive and irreversible, and the heart can no longer meet the metabolic demand of the body, resulting in HF [2].
Over the past 50 years, various mechanisms, including: (1) defects in energy production and utilization, (2) increased preload and afterload, (3) altered neurohormonal profile and signal transduction, and (4) occurrence of intracellular Ca2+-overload and Ca2+-handling abnormalities, have been indicated to explain cardiac dysfunction in HF. Since HF is invariably associated with changes in the shape and size of the heart, it has been suggested that the myocardial damage may be lead to cardiac remodelling. HF induced by pressure overload, or volume overload is a consequence of cardiac remodelling [3].
During cardiac hypertrophy and HF, a rise in intracellular Na+ concentration ([Na+]i) could potentially contribute to increasing or maintaining contractility, as it would increase Ca2+ influx via the Na+/Ca2+ exchanger. During the pathophysiological states of HF, expression and function of several of the Na transporters are affected, which can lead to an increase in [Na+]i. Several studies have reported a decrease in activity of the Na+/K+-pump and the up-regulation of the Na+/H+ exchanger would result in an increase in [Na+]i [4].
The cardiac Na+/K+-pump regulates intracellular Ca2+ concentration ([Ca2+]i) in the heart by regulating [Na+]i [5]. The Na+/Ca2+ exchanger is closely associated with the Na+/K+-pump, which suggests that inhibition of the Na+/K+-pump in the heart, through effects on the Na+/Ca2+ exchanger, raises the [Ca2+]i and strengthens cardiac contraction [6-9]. Hence, the alterations of Na+/K+-pump activity can have profound effects on cardiac myocytes contractility [10,11].
The active Na+/K+-pump contains a catalyticαsubunit (~110 kDa) and a smallβsubunit (~55 kDa). Theαsubunit mediates the catalytic activity of the enzyme and contains the specific binding sites for Na+, K+, ATP and cardiac glycosides [12-14]. Four isoforms of theαsubunit (α1,α2,α3,α4) have been identified and posssess tissue specific distribution and species differences [15].α1 andα2 isoforms are expressed in rat, guinea pig and mouse heart, while three isoforms (α1,α2, andα3) are present in human heart [16,17].
In human with heart failure, the Na+/K+-pump subunitsα1,α3, andβ1 were significantly reduced in ventricular myocardium and the Na+/K+-pump activity decreased [18]. The Na+/K+-pump function is unchanged in rabbit with heart failure[19]. Immunoblots showed a ~19% lower expression ofα1 isoform and a ~74% lower expression ofα2 isoform in HF rat [20]. Svein reported the expressions of theα1 andβ1 subunits (mRNA and protein) of the Na+/K+-pump were not significantly different in CHF, and the mRNA and protein levels of theα2 isoform were lower by 25 and 55%, respectively [21].
Because the reports about the activity and the protein expression of the Na+/K+-pump in HF models are not coincident, we established rat model HF induced by Adriamycin (ADR), measured the Na+/K+-pump current (Ip) of the single myocardial cell by the whole-cell patch-clamp technique, and detected the protein expression of Na+/K+-pumpα1 andα2 isoform by Western Blot analysis. The aim of this study was to investigate the changes of Na+/K+-pump activity and the protein expressions in HF rats. We also analyze the role of the Na+/K+-pump in the development of HF.
Objective: The purpose of this study was to investigate the changes of the activity of Na+/K+-pump and the expression of Na+/K+-pumpαisoforms in HF rats.
Methods: (1) The establishment of the HF rats: The SD rats were randomly divided into two groups: control group (NOR) and HF group (HF). The model of HF was established by the ip of ADR once every two days, at a dose of 1 mg/kg for the first two times, 2 mg/kg for the third and forth time, 3 mg/kg for the last four times. The NOR received an equivalent volume of the saline solution. During about twenty days after ADR administration, the indexes of HF, behavior,body weight (BW), ratio of left ventricular weigh and body weight (LVW/BW), ratio of lung weight and body weight (LW/BW) were evaluated. The parameters of heart function such as heart rate (HR), left ventricular systolic pressure (LVSP),left ventricular end-diastolic pressure (LVEDP),±dP/dtmax were measured.
(2)Enzymatic isolation of rats ventricular myocytes: The rats were anaesthetized by intraperitoneal injection of sodium pentobarbitone (50 mg/kg) and heparin (2500 U·kg-1) solutions. Hearts were excised, perfused in a retrograde fashion through Langendorff apparatus and ventricular myocytes were isolated by enzymatic dispersion (0.6 mg/mL Type-2 collagenase) .
(3)The record of the Na+/K+-pump current: Whole-cell patch-clamp technique was performed to record the Na+/K+-pump current (Ip), the Na+/K+-pump exchanges three intracellular Na+ for two external K+ aross the cell membrane during each active transport process and generates a net outward current (Ip). With selected external and pipette solutions, membrane current through K+ channel, Ca2+ channel, Na+/Ca2+ exchanger were minimized. Under the above experimental conditions, Ip was measured as the ouabain-blocked current. 1 mmol·L-1 ouabain (OUA) can completely block the activity of Na+/K+-pump, so the current generated by 1 mmol·L-1 OUA is defined as Ip. The Ip density is the ratio of Ip and capacitance. TheΔIp–OUA relation curve was plotted by normalized Na+/K+-pump current vs log OUA concentration and fitted by a two-site binding model formula.
(4)Western Blot analysis was used to detect the protein expression of Na+/K+-pumpα1 andα2 isoforms: Membrane proteins were extrcted from the left ventricles of NOR and HF rats and the concentration of the protein was measured by BCA protein assay. Membrane proteins were electrophoresed into 10% SDS-PAGE gels and blotted onto PVDF membrane. Membrane were blocked for 1 h in 5% nonfat dry milk in TBST, then incubated overnight with Na+/K+-pumpα1 andα2 isoforms antibody at 4℃, washed three times with TBST, and incubated with secondary antibody at 37℃for 1 h, washed with TBS for three times. The resulting bands were finally visualized using DAB.
Results: (1)The evaluation of HF indexes: The HF rats appeared poisoned symptom on 10th day after ADR injection, showed inanimate behavior, and BW, HR, LVSP, +dp/dtmax, and -dp/dtmax were decreased (238±15 g vs 207±13 g, 483±23次/min vs 388±34次/min, 129±4 mmHg vs 80±4 mmHg, 3357±101 mmHg/s vs 1727±55 mmHg/s, 3314±127 mmHg/s vs 1550±45 mmHg/s, p<0.05), but the LVW/BW, LW/BW and LVEDP were increased significantly (2.14±0.11 mg·g-1 vs 2.61±0.04 mg·g-1, 4.11±0.27 mg·g-1 vs 5.51±0.31 mg·g-1, 5.9±0.9 mmHg vs 11.3±1.2 mmHg, p<0.05).
(2)The change of Ip of venticular myocytes from HF rats: Compared with those from NOR rats, the Na+/K+-pump current density of venticular myocytes from HF rats was decreased (0.5070+0.06297 pA/pF vs 0.2106+0.04890 pA/pF, p<0.05). TheΔIp–OUA relation curve was plotted by normalized Na+/K+-pump current vs log OUA concentration and fitted by a two-site binding model formula. The results indicates that, there are two kinds ofαisoforms in both NOR rats and HF rats, which are with high affinity (α2 isoform) and low affinity (α1 isoform) for OUA binding. But in NOR rats, the K ofα1 isoform are 2.85×10-5 M andα2 isoform are 6.74×10-8 M; and in HF rats the K ofα1 isoform are 3.55×10-5 M andα2 isoform are 2.97×10-7 M, indicating that HF can mainly reduce the high affinity Na+/K+-pump of hearts.
(3)The change of protein expression ofα1 andα2 isoform of Na+/K+-pump in the HF rats left ventricles: In the NOR and HF rats left ventricles, we used Western blot to detect theα1 andα2 isoforms (112 KD). Compared with NOR rats, the protein expression ofα1 isoform of HF rats had no change, but the protein expression ofα2 isoform showed a significant decrease, about 50% (p<0.05). The results imply the mechanism of HF is closely related to the reduction of Na+/K+-pumpα2 isoform, which is in accordance with the results of electrophysiology.
Conclusion: In HF rats, the density of Na+/K+-pump current was significant decreased, the high affinity Na+/K+-pump was changed, and theα2 isoforms showed a significant decrease. These results imply the pathogenesis of HF is closely related to the reduction expression of Na+/K+-pumpα2 isoforms.
无论最初何种起因(高血压,心肌缺血,心肌病等),一旦心肌受到损伤,结果都会导致心力衰竭。最初阶段,可以通过激活交感神经和肾素-血管紧张素系统,对初始心肌损害造成的心肌收缩功能减弱进行代偿,并逐渐导致左心室扩大和/或肥厚。但久而久之,若心脏功能持续减弱,并伴随神经内分泌的过度激活,那么心脏的损伤就会累积和不可逆,而且不能满足机体的代谢需要,导致心衰[2]。
在过去50年里,有以下机制解释心脏的功能异常:(1)能量产生和利用的损伤(2)前负荷和后负荷的增加(3)神经激素和信号转导的改变(4)细胞内Ca2+超载和Ca2+调节的异常。由于心衰常常伴有心脏形状和体积的改变,这提示心肌损伤可能导致心肌改型。压力超负荷和容量过度负荷造成的心衰是心肌改型的结果[3]。
在心肌肥厚和心衰期间,细胞内Na+增加可以通过Na+/Ca2+交换体增加Ca2+内流,从而有助于增加收缩力或者维持收缩力。在心衰这种病理生理学状态下,一些Na+转运体的表达和功能受到影响,都可以导致细胞内Na+增加。如有文献报道Na+-K+泵的活性下降和Na+/H+交换体的表达上调都会导致细胞内Na+增加[4]。
Na+-K+泵通过调节细胞内Na+来调节细胞内Ca2+浓度[5]。Na+/Ca2+交换体与Na+-K+泵是紧密相连的,抑制Na+-K+泵后可通过Na+/Ca2+交换体来增加细胞内Ca2+,从而加强心肌收缩力[6-9]。所以Na+-K+泵活性的改变对于心肌收缩力影响十分重要[10,11]。
Na+-K+泵系由α亚基(~110 kDa)和β亚基(~55 kDa)组成的异源二聚体。α亚基调节酶的催化活性以及包括和Na+,K+,ATP以及强心苷的结合位点[12-14]。α亚基有四种不同的亚型(α1,α2,α3,α4),其分布具有组织特异性和种属差异[15]。α1和α2主要分布在大鼠、豚鼠和小鼠的心脏,α1、α2和α3主要分布在人的心脏[16,17]。
人心衰后,心肌细胞Na+-K+泵的α1、α3和β1的表达均有显著性下降,而α2没有改变,Na+-K+泵的活性下降[18]。在心衰家兔的心肌细胞上,Na+-K+泵的活性没有改变[19]。免疫印迹结果表明,心衰大鼠α1和α2的表达较正常大鼠分别下降了~19%和~74%;而Svein等报道,心衰大鼠的α1和β1亚基在mRNA和蛋白表达水平均无显著改变,仅α2亚基在mRNA和蛋白表达水平分别下降了25%和55% [20,21]。
鉴于文献关于心衰后Na+-K+泵的活性及其亚基改变的报道并不一致,本实验拟在来自阿霉素致大鼠心衰模型的心肌细胞,以全细胞膜片钳技术测定Na+-K+泵电流(Ip)和分子生物学技术测定Na+-K+泵α亚基表达,旨在研究心衰大鼠Na/K泵活性的改变和相应亚基的改变,并分析Na+-K+泵在心衰发病机制中的作用。
目的:检测心衰大鼠Na+-K+泵活性的变化,以及Na+-K+泵α亚基的在蛋白水平的改变。
方法:(1)采用阿霉素(ADR)致大鼠心衰的方法建立大鼠心衰模型:雄性SD大鼠,体重200-250 g,由河北医科大学实验动物中心提供。将36只SD大鼠随机分为对照组(NOR)和心衰组(HF)。心衰组第2、4天腹腔注射ADR 1 mg/kg,第6、8天注射ADR 2 mg/kg,第10、12天注射ADR 3 mg/kg,第14、16天注射ADR 3 mg/kg。对照组每次腹腔注射等容积的生理盐水。17-20天后,通过行为体征观察,体重(BW)、肺重比体重(LW/BW)、左心室重比体重(LVW/BW)及心功能如心率(HR)、左心室收缩压(LVSP)、左心室舒张末期压(LVEDP)、左室压力最大上升速度(+dp/dtmax)、左室压力最大下降速度(-dp/dtmax)的测定进行心衰评价。
(2)酶解法急性分离大鼠心室肌细胞:取实验用SD大鼠,腹腔注射肝素2500 U·kg-1和12 mg·L-1的戊巴比妥钠50 mg·kg-1,待动物麻醉后,使用Langendorff装置,行主动脉插管逆行灌流(温度,压力恒定),先灌流无Ca2+台式液约10 ml,再灌流含胶原酶Ⅱ(0.6 mg·ml-1)及CaCl2(30μM·L-1)的台式液30 ml消化心脏,反复循环灌流20-25 min,急性分离大鼠心室肌细胞。
(3)Na+-K+泵电流测定:以全细胞膜片钳技术(whole-cell patch-clamp technique)记录Na+-K+泵电流(Ip),Na+-K+泵一次主动转运可将3个Na+泵出细胞外,同时泵进2个K+,因而可产生一个纯净的向外电流。在特定的细胞外液和电极内液阻断细胞膜上其他离子通道和交换体电流的条件下(主要是K电流,Ca电流,Na+/Ca2+交换体电流),灌流Na+-K+泵的抑制剂哇巴因(OUA),使膜电流产生内向移动,此差别电流反应Ip大小。1 mmol·L-1的哇巴因可以完全阻断Na+-K+泵,产生的电流即Ip。电流密度即是电流大小与电容之比。以哇巴因浓度([OUA])对数值为横坐标,以哇巴因对Na+-K+泵电流抑制的百分率(ΔIp)为纵坐标,绘制ΔIp-[OUA]量效关系曲线,并采用两点结合模型公式进行曲线拟合。
(4)采用Western Blot方法测定Na+-K+泵α1和α2亚基蛋白表达的变化:提取NOR和HF组大鼠心室肌细胞膜蛋白,用BCA蛋白定量法进行蛋白定量,10%SDS-聚丙烯酰胺电泳,转膜,5%脱脂奶粉振荡封闭1小时,加α亚基特异性的抗体,4℃孵育过夜,TBST洗三次,加二抗37℃孵育1小时,TBS洗三次,DAB显色。
结果:(1)心衰模型相关指标评价:HF组大鼠10天后出现中毒现象,表现为精神状态差,BW、HR、LVSP以及+dp/dtmax、-dp/dtmax都明显下降(p<0.05),而LVW/BW、LW/BW、LVEDP有显著升高(p<0.05)。给药前正常组BW、LVW/BW、LW/BW、HR、LVSP、LVEDP、+dp/dtmax、-dp/dtmax分别为238±15 g、2.14±0.11 mg·g-1、4.11±0.27 mg·g-1、483±23次/min、129±4 mmHg、5.9±0.9 mmHg、3357±101 mmHg/s、3314±127 mmHg/s。给药后心衰组BW、LVW/BW、LW/BW、HR、LVSP、LVEDP、+dp/dtmax、-dp/dtmax分别为207±13 g、2.61±0.04 mg·g-1、5.51±0.31 mg·g-1,388±34次/min、80±4 mmHg、11.3±1.2 mmHg、1727±55 mmHg/s、1550±45 mmHg/s。
(2)心衰大鼠Ip的改变:与正常大鼠Na+-K+泵电流密度相比,心衰大鼠Na+-K+泵电流密度显著下降( 0.5070+0.06297 pA/pF vs 0.2106+0.04890 pA/pF,p<0.05)。绘制ΔIp-[OUA]量效关系曲线,对ΔIp-[OUA]量效关系曲线以两个以上α亚基两位点公式进行拟合,结果表明正常和心衰大鼠均存在高低亲和力两种Na+-K+泵。不同的是,正常大鼠低亲和力钠泵K值为2.85×10-5 M,高亲和力钠泵K值为6.74×10-8 M,阿霉素致大鼠心衰后,其低亲和力钠泵K值为3.55×10-5 M,而高亲和力钠泵K值为2.97×10-7 M。由以上结果可知,大鼠心衰后高亲和力钠泵发生了变化。
(3)心衰大鼠Na+-K+泵α1和α2亚基蛋白表达的变化:我们使用Western blot检测技术在正常及心衰大鼠的心肌均检测到α1和α2亚基蛋白(112 KD)。与正常组相比,心衰组Na+-K+泵低亲和力α1亚基蛋白表达无明显改变,而高亲和力α2亚基蛋白表达则明显下降,约下降了50%(p<0.05)。这进一步提示阿霉素致大鼠心衰的机制是Na+-K+泵的α2亚基发生改变,与电生理结果一致。
结论:大鼠心衰后电流密度显著下降。ΔIp-[OUA]量效关系曲线表明心衰大鼠Na+-K+泵的高亲和力泵发生改变,而且心衰大鼠心肌Na+-K+泵α2亚基蛋白表达明显下降,但α1亚基蛋白的表达没有改变。提示心衰的发病机制可能与Na+-K+泵的α2亚基密切相关。
Heart failure (HF) is the main cardiovascular disease in the world. The genesis of HF syndromes is complex and multifactorial, while ventricular arrhythmias and degression of the myocardial contractility are the main causes of human heart failure, but the altered cellular Ca2+ regulation may be a final common pathway in both contractile dysfunction and arrhythmogenesis [1].
In a heart that has suffered from myocardial damage, regardless of the initial cause of the damage (hypertension, myocardial ischemia, cardiomyopathy, etc), HF eventually occurs if such damage persists for a prolonged period. In the initial stages, compensation for the depressed myocardial contractility due to myocardial damage can occur via activation of both the sympathetic nervous system and the renin-angiotensin system, resulting in left ventricle dilatation and/or hypertrophy. However, if the depressed cardiac function persists, with a parallel activation of neurohumoral factors, the myocardial damage becomes progressive and irreversible, and the heart can no longer meet the metabolic demand of the body, resulting in HF [2].
Over the past 50 years, various mechanisms, including: (1) defects in energy production and utilization, (2) increased preload and afterload, (3) altered neurohormonal profile and signal transduction, and (4) occurrence of intracellular Ca2+-overload and Ca2+-handling abnormalities, have been indicated to explain cardiac dysfunction in HF. Since HF is invariably associated with changes in the shape and size of the heart, it has been suggested that the myocardial damage may be lead to cardiac remodelling. HF induced by pressure overload, or volume overload is a consequence of cardiac remodelling [3].
During cardiac hypertrophy and HF, a rise in intracellular Na+ concentration ([Na+]i) could potentially contribute to increasing or maintaining contractility, as it would increase Ca2+ influx via the Na+/Ca2+ exchanger. During the pathophysiological states of HF, expression and function of several of the Na transporters are affected, which can lead to an increase in [Na+]i. Several studies have reported a decrease in activity of the Na+/K+-pump and the up-regulation of the Na+/H+ exchanger would result in an increase in [Na+]i [4].
The cardiac Na+/K+-pump regulates intracellular Ca2+ concentration ([Ca2+]i) in the heart by regulating [Na+]i [5]. The Na+/Ca2+ exchanger is closely associated with the Na+/K+-pump, which suggests that inhibition of the Na+/K+-pump in the heart, through effects on the Na+/Ca2+ exchanger, raises the [Ca2+]i and strengthens cardiac contraction [6-9]. Hence, the alterations of Na+/K+-pump activity can have profound effects on cardiac myocytes contractility [10,11].
The active Na+/K+-pump contains a catalyticαsubunit (~110 kDa) and a smallβsubunit (~55 kDa). Theαsubunit mediates the catalytic activity of the enzyme and contains the specific binding sites for Na+, K+, ATP and cardiac glycosides [12-14]. Four isoforms of theαsubunit (α1,α2,α3,α4) have been identified and posssess tissue specific distribution and species differences [15].α1 andα2 isoforms are expressed in rat, guinea pig and mouse heart, while three isoforms (α1,α2, andα3) are present in human heart [16,17].
In human with heart failure, the Na+/K+-pump subunitsα1,α3, andβ1 were significantly reduced in ventricular myocardium and the Na+/K+-pump activity decreased [18]. The Na+/K+-pump function is unchanged in rabbit with heart failure[19]. Immunoblots showed a ~19% lower expression ofα1 isoform and a ~74% lower expression ofα2 isoform in HF rat [20]. Svein reported the expressions of theα1 andβ1 subunits (mRNA and protein) of the Na+/K+-pump were not significantly different in CHF, and the mRNA and protein levels of theα2 isoform were lower by 25 and 55%, respectively [21].
Because the reports about the activity and the protein expression of the Na+/K+-pump in HF models are not coincident, we established rat model HF induced by Adriamycin (ADR), measured the Na+/K+-pump current (Ip) of the single myocardial cell by the whole-cell patch-clamp technique, and detected the protein expression of Na+/K+-pumpα1 andα2 isoform by Western Blot analysis. The aim of this study was to investigate the changes of Na+/K+-pump activity and the protein expressions in HF rats. We also analyze the role of the Na+/K+-pump in the development of HF.
Objective: The purpose of this study was to investigate the changes of the activity of Na+/K+-pump and the expression of Na+/K+-pumpαisoforms in HF rats.
Methods: (1) The establishment of the HF rats: The SD rats were randomly divided into two groups: control group (NOR) and HF group (HF). The model of HF was established by the ip of ADR once every two days, at a dose of 1 mg/kg for the first two times, 2 mg/kg for the third and forth time, 3 mg/kg for the last four times. The NOR received an equivalent volume of the saline solution. During about twenty days after ADR administration, the indexes of HF, behavior,body weight (BW), ratio of left ventricular weigh and body weight (LVW/BW), ratio of lung weight and body weight (LW/BW) were evaluated. The parameters of heart function such as heart rate (HR), left ventricular systolic pressure (LVSP),left ventricular end-diastolic pressure (LVEDP),±dP/dtmax were measured.
(2)Enzymatic isolation of rats ventricular myocytes: The rats were anaesthetized by intraperitoneal injection of sodium pentobarbitone (50 mg/kg) and heparin (2500 U·kg-1) solutions. Hearts were excised, perfused in a retrograde fashion through Langendorff apparatus and ventricular myocytes were isolated by enzymatic dispersion (0.6 mg/mL Type-2 collagenase) .
(3)The record of the Na+/K+-pump current: Whole-cell patch-clamp technique was performed to record the Na+/K+-pump current (Ip), the Na+/K+-pump exchanges three intracellular Na+ for two external K+ aross the cell membrane during each active transport process and generates a net outward current (Ip). With selected external and pipette solutions, membrane current through K+ channel, Ca2+ channel, Na+/Ca2+ exchanger were minimized. Under the above experimental conditions, Ip was measured as the ouabain-blocked current. 1 mmol·L-1 ouabain (OUA) can completely block the activity of Na+/K+-pump, so the current generated by 1 mmol·L-1 OUA is defined as Ip. The Ip density is the ratio of Ip and capacitance. TheΔIp–OUA relation curve was plotted by normalized Na+/K+-pump current vs log OUA concentration and fitted by a two-site binding model formula.
(4)Western Blot analysis was used to detect the protein expression of Na+/K+-pumpα1 andα2 isoforms: Membrane proteins were extrcted from the left ventricles of NOR and HF rats and the concentration of the protein was measured by BCA protein assay. Membrane proteins were electrophoresed into 10% SDS-PAGE gels and blotted onto PVDF membrane. Membrane were blocked for 1 h in 5% nonfat dry milk in TBST, then incubated overnight with Na+/K+-pumpα1 andα2 isoforms antibody at 4℃, washed three times with TBST, and incubated with secondary antibody at 37℃for 1 h, washed with TBS for three times. The resulting bands were finally visualized using DAB.
Results: (1)The evaluation of HF indexes: The HF rats appeared poisoned symptom on 10th day after ADR injection, showed inanimate behavior, and BW, HR, LVSP, +dp/dtmax, and -dp/dtmax were decreased (238±15 g vs 207±13 g, 483±23次/min vs 388±34次/min, 129±4 mmHg vs 80±4 mmHg, 3357±101 mmHg/s vs 1727±55 mmHg/s, 3314±127 mmHg/s vs 1550±45 mmHg/s, p<0.05), but the LVW/BW, LW/BW and LVEDP were increased significantly (2.14±0.11 mg·g-1 vs 2.61±0.04 mg·g-1, 4.11±0.27 mg·g-1 vs 5.51±0.31 mg·g-1, 5.9±0.9 mmHg vs 11.3±1.2 mmHg, p<0.05).
(2)The change of Ip of venticular myocytes from HF rats: Compared with those from NOR rats, the Na+/K+-pump current density of venticular myocytes from HF rats was decreased (0.5070+0.06297 pA/pF vs 0.2106+0.04890 pA/pF, p<0.05). TheΔIp–OUA relation curve was plotted by normalized Na+/K+-pump current vs log OUA concentration and fitted by a two-site binding model formula. The results indicates that, there are two kinds ofαisoforms in both NOR rats and HF rats, which are with high affinity (α2 isoform) and low affinity (α1 isoform) for OUA binding. But in NOR rats, the K ofα1 isoform are 2.85×10-5 M andα2 isoform are 6.74×10-8 M; and in HF rats the K ofα1 isoform are 3.55×10-5 M andα2 isoform are 2.97×10-7 M, indicating that HF can mainly reduce the high affinity Na+/K+-pump of hearts.
(3)The change of protein expression ofα1 andα2 isoform of Na+/K+-pump in the HF rats left ventricles: In the NOR and HF rats left ventricles, we used Western blot to detect theα1 andα2 isoforms (112 KD). Compared with NOR rats, the protein expression ofα1 isoform of HF rats had no change, but the protein expression ofα2 isoform showed a significant decrease, about 50% (p<0.05). The results imply the mechanism of HF is closely related to the reduction of Na+/K+-pumpα2 isoform, which is in accordance with the results of electrophysiology.
Conclusion: In HF rats, the density of Na+/K+-pump current was significant decreased, the high affinity Na+/K+-pump was changed, and theα2 isoforms showed a significant decrease. These results imply the pathogenesis of HF is closely related to the reduction expression of Na+/K+-pumpα2 isoforms.
引文
1 Pogwizd SM, Schlotthauer K, Li L, et al. Arrhythmogenesis and contractile dysfunction in heart failure [J]. Circulation Research, 2001, 88(11): 1159-1167
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5 Berry RG, Despa S, Fuller W, et al. Differential distribution and regulation of mouse cardiac Na+/K+-ATPaseα1 andα2 subunits in T-tubule and surface sarcolemmal membranes [J]. Cardiovascular Research, 2007, 73(1): 92-100
6 Matchkov VV, Gustafsson H, Rahman A, et al. Interaction between Na+/K+-Pump and Na+/Ca2+-Exchanger modulates intercellular communication [J]. 2007, 100(7): 1026-1035
7 James PF, Grupp IL, Grupp G, et al. Identification of a specific role for the Na,K-ATPaseα2 isoform as a regulator of calcium in the heart [J]. Molecular Cell, 1999, 39(5): 555-563
8 Juhaszova M, Blaustein MP. Na+ pump low and high ouabain affinity a subunit isoforms are differently distributed in cells [J]. Cell Biology, 1997, 94(5): 1800-1805
9 Arnon A, Hamlyn JM, Blaustein MP. Ouabain augments Ca2+ transients in arterial smooth muscle without raising cytosolic Na+ [J]. Am J Physiol Heart Circ Physiol, 2000, 279(5): 679-691
10 Despa S, Bers DM. Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes [J]. Am J Physiol Cell Physiol, 2007, 293(1): 321-327
11 Swift F, Tovsrud N, Enger UH, et al. The Na+/K+-ATPaseα2-isoform regulates cardiac contractility in rat cardiomyocytes [J]. Cardiovascular Research, 2007, 75(1): 109-117
12 Feng J, Orlowski J, Lingre JB. Identification of a functional thyroid hormone response element in the upstream flanking region of the humanNa,K-ATPaseβ1 gene [J]. Nucleic Acids Research, 1993, 21(11): 2619-2626
13 Pacholczyk T, Sweadner KJ. Epitope and mimotope for an antibody to the Na,K-ATPase [J]. Pmrein Science, 1997, 6(7): 1537-1548
14 Pathak BG, Neumann JC, Croyle ML, et al. The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPaseα3 subunit gene are required for brain expression in transgenic mice [J]. Nucleic Acids Research, 1994, 22(22): 4748-4755
15 Shamraj OI, Lingrel JB. A putative fourth Na+/K+-ATPaseα-subunit gene is expressed [J]. Proc Natl Acad Sci USA, 1994, 91(26): 12952-12956
16 Wang J, Velotta JB, Mcdonough AA, et al. All human Na+-K+-ATPaseα-subunit isoforms have a similar affinity for cardiac glycosides [J]. Am J Physiol Cell Physiol, 2001, 281(4): 1336-1343
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4 Despa S, Islam MA, Weber CR, et al. Intracellular Na+ concentration is elevated in heart failure but Na/K pump function is unchanged [J]. Circulation, 2002, 105(21): 2543-2548
5 Farkas AS, Acsai K, Nagy N, et al. Na+/Ca2+ exchanger inhibition exerts a positive inotropic effect in the rat heart, but fails to influence the contractility of the rabbit heart [J]. British Journal of Pharmacology, 2008, 154(1): 93-104
6 Pieske B, Maier LS, Bers DM, et al. Ca2+ handling and sarcoplasmicreticulum Ca2+ content in isolated failing and nonfailing human myocardium [J]. Circulation Research, 1999, 85(1): 38-46
7 Feng J, Orlowski J, Lingre JB. Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPaseβ1 gene [J]. Nucleic Acids Research, 1993, 21(11): 2619-2626
8 Pacholczyk T, Sweadner KJ. Epitope and mimotope for an antibody to the Na,K-ATPase [J]. Pmrein Science, 1997, 6(7): 1537-1548
9 Pathak BG, Neumann JC, Croyle ML, et al. The presence of both negative and positive elements in the 5'-flanking expression in transgenic mice [J]. Nucleic Acids Research, 1994, 22(22): 4748-4755
10 Antoloviv R, Bruller HJ, Bunk S, et al. Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+,K+-ATPase are located on the cytoplasmic side of the membrane [J]. Eur J Biochem, 1991, 199(1): 195-202
11 Shamraj OI, Lingrel JB. A putative fourth Na+/K+-ATPaseα-subunit gene is expressed [J]. Proc Natl Acad Sci USA, 1994, 91(26): 12952-12956
12 Gao J, Wymore R, Wymore RT, et al. Isoform-specific regulation of the sodium pump byαandβ-adrenergic agonists in the guinea-pig ventricle [J]. Journal of Physiology, 1999, 516(2): 377-383
13 Wang J, Velotta JB, Mcdonough AA, et al. All human Na+-K+-ATPaseα-subunit isoforms have a similar affinity for cardiac glycosides [J]. Am J Physiol Cell Physiol, 2001, 281(4): 1336-1343
14 Schwinger RH, Bundgaard H, Muller Ehmsen J, et al. The Na, K-ATPase in the failing human heart [J]. Cardiovascular Research, 2003, 57(4): 913- 920
15 Dostanic-Larson I, Lorenz JN, Van Huysses JW, et al. Physiological role of theα1-andα2-isoform of the Na+/K+-ATPase, and biological significant of their cardiac glycoside binding site [J]. Am J Physiol Regul Integr Comp Physiol, 2006, 290:524-528
16 Lines GT, Sande JB, Louch WE, et al. Contribution of the Na+/Ca2+exchanger to rapid Ca2+ release in cardiomyocytes [J]. Biophysical Journal, 2006, 91(3): 779-792
17 Shigekawa M, Iwamoto T. Cardiac Na+-Ca2+ exchange molecular and pharmacological aspects [J]. Circulation Research, 2001, 88(9): 864-876
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20 Berry RG, Despa S, Fuller W, et al. Differential distribution and regulation of mouse cardiac Na+/K+-ATPaseα1 andα2 subunits in T-tubule and surface sarcolemmal membranes [J]. Cardiovascular Research, 2007, 73(1): 92-100
21 Matchkov VV, Gustafsson H, Rahman A, et al. Interaction between Na+/K+-Pump and Na+/Ca2+-Exchanger modulates intercellular communication [J]. 2007, 100(7): 1026-1035
22 Juhaszova M, Blaustein MP. Na+ pump low and high ouabain affinity a subunit isoforms are differently distributed in cells [J]. Cell Biology, 1997, 94(5): 1800-1805
23 Arnon A, Hamlyn JM, Blaustein MP. Ouabain augments Ca2+ transients in arterial smooth muscle without raising cytosolic Na+ [J]. Am J Physiol Heart Circ Physiol, 2000, 279(2): 679-691
24 Despa S, Bers DM. Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes [J]. Am J Physiol Cell Physiol, 2007, 293(1): 321-327
25 Swift F, Tovsrud N, Enger UH, et al. The Na+/K+-ATPaseα2-isoform regulates cardiac contractility in rat cardiomyocytes [J]. Cardiovascular Research, 2007, 75(1): 109-117
26 James PF, Grupp IL, Grupp G, et al. Identification of a specific role for the Na,K-ATPaseα2 isoform as a regulator of calcium in the heart [J]. Molecular Cell, 1999, 39(5): 555-563
27 Sweadner KJ, Herrera VL, Amato S, et al. Immunologic identification ofNa+,K+-ATPase isoforms in myocardium [J]. Circulation Research, 1994, 74(4): 669-678
28 Mcdonough AA, Zhang Y, Shin V, et al. Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes [J]. Am J Physiol Cell Physiol, 1996, 270(4): 1221-1227
29 Fransen P, Hendrickx J, Brutsaert DL. Distribution and role of Na+,K+-ATPase in endocardial endothelium [J]. Cardiovasc Res, 2001, 52(3): 487-499
30 Silverman BZ, Fuller W, Eaton P, et al. Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na+,K+-ATPase [J]. Cardiovasc Res, 2005, 65(1): 93-103
31 Harada K, Lin H, Endo Y, et al. Subunit composition and role of Na+,K+-ATPases in ventricular myocytes [J]. Phys Sci, 2006, 56(1): 113-121
32 Schwinger RH, Wang J, Frank K, et al. Reduced sodium pumpα1,α3 andβ1-iosform protein levels and Na+/K+-ATPase activity but unchanged Na+/Ca2+-exchanger protein levels in human heart failure [J]. Circulation, 1999, 99(16): 2105-2112
33 Studer R, Reinecke H, Bilger J, et al. Gene expression of the cardiac Na+-Ca2+ exchanger in end-stage human heart failure [J]. Circ Res, 1994, 75(3): 443-453
34 Pogwizda SM, Sipidob KR, Verdonckc F, et al. Intracellular Na+ in animal models of hypertrophy and heart failure: contractile function and arrhythmogenesis [J]. Cardiovascular Research, 2003, 57(4): 887-896
35 Ove Semb S, Lunde PK, Holt E, et al. Reduce myocardial Na,K-pump capacity in congestive heart failure following myocardial infarction in rats [J] Journal of Molecular and Cellular Cardiology, 1998, 30(7): 1311-1328
36 Muller-Ehmsen J, Wang J, Schwinger RH, et al. Region specific regulation of sodium pupm isoform and Na+-Ca2+ exchanger expression in the failing human heart-right atrium vs left ventricle [J]. Cell-Mol-Biol-(Noisy-le- grand), 2001, 47(2): 373-381
37 Gray RP, McIntyre H, Sheridan DS,et al. Intracellular sodium and contractile function in hypertrophied human and guinea-pig myocardium [J]. Pfluger’s Arch, 2001, 442(1): 117-123
38 Meszaros J, Khananshvili D, Hart G. Mechanisms underlying delayed afterdepolarizations in hypertrophied left ventricular myocytes of rats [J]. Am J Physiol, 2001, 281(2): 903-914
39 Verdonck F, Volders PG, Vos MA, et al. Increased Na+ concentration and altered Na/K pump activity in hypertrophied canine ventricular cells [J]. Cardiovascular Research, 2003, 57(4): 1035-1043
40 Baudet S, Noireaud J, Leoty C. Intracellular Na activity measurements
41 in the control and hypertrophied heart of the ferret: an ion-sensitive micro-electrode study [J]. Pfluger’s Arch, 1991, 418(4): 313-318
42 Baartscheer A, Borren MV, Schumacher C, et al. Increased Na+/H+- exchanger activity in heart failure causes disturbed calcium hand-ling [J]. Biophys J, 2002, 81: 598
43 Pieskea B, Houser SR. [Na+]i handling in the failing human heart [J]. Cardiovascular Research, 2003, 57(4): 874-886
44 Dostanic I, Lorenz JN, Schultz JE, et al. Theα2 Isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice [J]. The Journal of biological chemistry, 2003, 278(52): 53026-53034
45 Dostanic I, Schultz JE, Lorenz JN, et al. Theα1 Isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart [J]. The Journal of biological chemistry, 2004, 279(52): 54053-54061
46那开宪,余平,田娟.慢性充血性心力衰竭的药物治疗(上) [J].中国临床医生, 2005, 33(4): 48-49
47朱文青.慢性充血性心力衰竭的药物治疗现状[A].中国临床医学, 2005, 12(1): 1-5
2 Masafumi Y, Yasuhiro I, Masunori M. Altered intracellular Ca2+ handling in heart failure [J]. The Journal of Clinical Investigation, 2005, 115(3):556-564
3 Dhalla NS, Saini-Chohan HK, Rodriguez-Leyva D, et al. Subcellular remodelling may induce cardiac dysfunction in congestive heart failure [J]. Cardiovascular Research, 2009, 81(3): 429-438
4 Verdonck F, Volders PG, Vos MA, et al. Increased Na+ concentration and altered Na/K pump activity in hypertrophied canine ventricular cells [J]. Cardiovascular Research, 2003, 57(4): 1035-1043
5 Berry RG, Despa S, Fuller W, et al. Differential distribution and regulation of mouse cardiac Na+/K+-ATPaseα1 andα2 subunits in T-tubule and surface sarcolemmal membranes [J]. Cardiovascular Research, 2007, 73(1): 92-100
6 Matchkov VV, Gustafsson H, Rahman A, et al. Interaction between Na+/K+-Pump and Na+/Ca2+-Exchanger modulates intercellular communication [J]. 2007, 100(7): 1026-1035
7 James PF, Grupp IL, Grupp G, et al. Identification of a specific role for the Na,K-ATPaseα2 isoform as a regulator of calcium in the heart [J]. Molecular Cell, 1999, 39(5): 555-563
8 Juhaszova M, Blaustein MP. Na+ pump low and high ouabain affinity a subunit isoforms are differently distributed in cells [J]. Cell Biology, 1997, 94(5): 1800-1805
9 Arnon A, Hamlyn JM, Blaustein MP. Ouabain augments Ca2+ transients in arterial smooth muscle without raising cytosolic Na+ [J]. Am J Physiol Heart Circ Physiol, 2000, 279(5): 679-691
10 Despa S, Bers DM. Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes [J]. Am J Physiol Cell Physiol, 2007, 293(1): 321-327
11 Swift F, Tovsrud N, Enger UH, et al. The Na+/K+-ATPaseα2-isoform regulates cardiac contractility in rat cardiomyocytes [J]. Cardiovascular Research, 2007, 75(1): 109-117
12 Feng J, Orlowski J, Lingre JB. Identification of a functional thyroid hormone response element in the upstream flanking region of the humanNa,K-ATPaseβ1 gene [J]. Nucleic Acids Research, 1993, 21(11): 2619-2626
13 Pacholczyk T, Sweadner KJ. Epitope and mimotope for an antibody to the Na,K-ATPase [J]. Pmrein Science, 1997, 6(7): 1537-1548
14 Pathak BG, Neumann JC, Croyle ML, et al. The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPaseα3 subunit gene are required for brain expression in transgenic mice [J]. Nucleic Acids Research, 1994, 22(22): 4748-4755
15 Shamraj OI, Lingrel JB. A putative fourth Na+/K+-ATPaseα-subunit gene is expressed [J]. Proc Natl Acad Sci USA, 1994, 91(26): 12952-12956
16 Wang J, Velotta JB, Mcdonough AA, et al. All human Na+-K+-ATPaseα-subunit isoforms have a similar affinity for cardiac glycosides [J]. Am J Physiol Cell Physiol, 2001, 281(4): 1336-1343
17 Schwinger RH, Bundgaard H, Muller Ehmsen J, et al. The Na, K-ATPase in the failing human heart [J]. Cardiovascular Research, 2003, 57(4): 913-920
18 Schwinger RH, Wang J, Frank K, et al. Reduced sodium pumpα1,α3 andβ1-iosform protein levels and Na+/K+-ATPase activity but unchanged Na+/Ca2+-exchanger protein levels in human heart failure [J]. Circulation, 1999, 99(16): 2105-2112
19 Despa S, Islam MA, Weber CR, et al. Intracellular Na+ concentration is elevated in heart failure but Na/K pump function is unchanged [J]. Circulation, 2002, 105(21): 2543-2548
20 Swift F, Birkeland JA, Tovsrud N, et al. Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPaseα2-isoform in heart failure [J]. Cardiovascular Research, 2008, 78(1): 71-78
21 Ove Semb S, Lunde PK, Holt E, et al. Reduce myocardial Na,K-pump capacity in congestive heart failure following myocardial infarction in rats [J] Journal of Molecular and Cellular Cardiology, 1998, 30(7): 1311-1328
22 Dostanic I, Lorenz JN, Schultz JE, et al. Theα2 Isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice [J]. The Journal ofbiological chemistry, 2003, 278(52): 53026-53034
23 Dostanic-Larson I , Lorenz JN, Van Huysses JW, et al. Physiological role of theα1-andα2-isoform of the Na+/K+-ATPase, and biological significant of their cardiac glycoside binding site [J]. Am J Physiol Regul Integr Comp Physiol, 2006, 290:524-528
24 Wang Y, Gao J, Mathias RT, et al.α-Adrenergic effects on Na+-K+-pump current in guinea-pig ventricular myocytes [J]. Journal of Physiology, 1998, 509(1): 117-128
25 Ishizuka N, Fieldding AJ, Berlin JR, et al. Na pump current can separated into ouabain-sensitive and- insensitive components in single rat ventricular myocytes [J]. Japanese Journal of Physiolopy, 1996, 46(3): 215-223
26 Dostanic I, Schultz JE, Lorenz JN, et al. Theα1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in Heart [J]. The Journal of biological chemistry, 2004, 279(52): 54053-54061
27 Pogwizda SM, Sipidob KR, Verdonckc F, et al. Intracellular Na+ in animal models of hypertrophy and heart failure: ontractile function and arrhythmogenesis [J]. Cardiovascular Research , 2003, 57(4): 887-896
28杨建业,张迎春,唐俊明,等.阿霉素诱导大鼠心衰模型的建立[J].郧阳医学院学报, 2005,24(5): 269-271
29李梅秀,田国忠,欧叶涛,等.大鼠阿霉素慢性心衰模型的制备与心衰指标的判定[J].解剖学研究, 2005,27(3): 176-178
30 Farkas AS, Acsai K, Nagy N, et al. Na+/Ca2+ exchanger inhibition exerts a positive inotropic effect in the rat heart, but fails to influence the contractility of the rabbit heart [J]. British Journal of Pharmacology, 2008, 154(1): 93-104
1薛如庄.慢性充血性心力衰竭的现代治疗[J].广西民族学院学报, 2004, 1(4): 123-126
2 Kawai M, Hussain M, Orchard CH. Excitation-contraction coupling in rat ventricular myocytes after formamide-induced detubulation [J]. Am J Physiol Heart Circ Physiol, 1999, 277(2): 603-609
3 Swift F, Birkeland JA, Tovsrud N, et al. Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPaseα2-isoform in heart failure [J]. Cardiovascular Research, 2008, 78(1): 71-78
4 Despa S, Islam MA, Weber CR, et al. Intracellular Na+ concentration is elevated in heart failure but Na/K pump function is unchanged [J]. Circulation, 2002, 105(21): 2543-2548
5 Farkas AS, Acsai K, Nagy N, et al. Na+/Ca2+ exchanger inhibition exerts a positive inotropic effect in the rat heart, but fails to influence the contractility of the rabbit heart [J]. British Journal of Pharmacology, 2008, 154(1): 93-104
6 Pieske B, Maier LS, Bers DM, et al. Ca2+ handling and sarcoplasmicreticulum Ca2+ content in isolated failing and nonfailing human myocardium [J]. Circulation Research, 1999, 85(1): 38-46
7 Feng J, Orlowski J, Lingre JB. Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPaseβ1 gene [J]. Nucleic Acids Research, 1993, 21(11): 2619-2626
8 Pacholczyk T, Sweadner KJ. Epitope and mimotope for an antibody to the Na,K-ATPase [J]. Pmrein Science, 1997, 6(7): 1537-1548
9 Pathak BG, Neumann JC, Croyle ML, et al. The presence of both negative and positive elements in the 5'-flanking expression in transgenic mice [J]. Nucleic Acids Research, 1994, 22(22): 4748-4755
10 Antoloviv R, Bruller HJ, Bunk S, et al. Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+,K+-ATPase are located on the cytoplasmic side of the membrane [J]. Eur J Biochem, 1991, 199(1): 195-202
11 Shamraj OI, Lingrel JB. A putative fourth Na+/K+-ATPaseα-subunit gene is expressed [J]. Proc Natl Acad Sci USA, 1994, 91(26): 12952-12956
12 Gao J, Wymore R, Wymore RT, et al. Isoform-specific regulation of the sodium pump byαandβ-adrenergic agonists in the guinea-pig ventricle [J]. Journal of Physiology, 1999, 516(2): 377-383
13 Wang J, Velotta JB, Mcdonough AA, et al. All human Na+-K+-ATPaseα-subunit isoforms have a similar affinity for cardiac glycosides [J]. Am J Physiol Cell Physiol, 2001, 281(4): 1336-1343
14 Schwinger RH, Bundgaard H, Muller Ehmsen J, et al. The Na, K-ATPase in the failing human heart [J]. Cardiovascular Research, 2003, 57(4): 913- 920
15 Dostanic-Larson I, Lorenz JN, Van Huysses JW, et al. Physiological role of theα1-andα2-isoform of the Na+/K+-ATPase, and biological significant of their cardiac glycoside binding site [J]. Am J Physiol Regul Integr Comp Physiol, 2006, 290:524-528
16 Lines GT, Sande JB, Louch WE, et al. Contribution of the Na+/Ca2+exchanger to rapid Ca2+ release in cardiomyocytes [J]. Biophysical Journal, 2006, 91(3): 779-792
17 Shigekawa M, Iwamoto T. Cardiac Na+-Ca2+ exchange molecular and pharmacological aspects [J]. Circulation Research, 2001, 88(9): 864-876
18 Barry WH. Na+-Ca2+ exchange in failing myocardium: friend or foe? [J]. Circulation Research, 2000, 87(7): 529-531
19 Blaustein MP, Jonathan LW. Sodium/Calcium exchange: its physiological implications [J]. Physiological Reviews, 1999, 79(3): 763-854
20 Berry RG, Despa S, Fuller W, et al. Differential distribution and regulation of mouse cardiac Na+/K+-ATPaseα1 andα2 subunits in T-tubule and surface sarcolemmal membranes [J]. Cardiovascular Research, 2007, 73(1): 92-100
21 Matchkov VV, Gustafsson H, Rahman A, et al. Interaction between Na+/K+-Pump and Na+/Ca2+-Exchanger modulates intercellular communication [J]. 2007, 100(7): 1026-1035
22 Juhaszova M, Blaustein MP. Na+ pump low and high ouabain affinity a subunit isoforms are differently distributed in cells [J]. Cell Biology, 1997, 94(5): 1800-1805
23 Arnon A, Hamlyn JM, Blaustein MP. Ouabain augments Ca2+ transients in arterial smooth muscle without raising cytosolic Na+ [J]. Am J Physiol Heart Circ Physiol, 2000, 279(2): 679-691
24 Despa S, Bers DM. Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes [J]. Am J Physiol Cell Physiol, 2007, 293(1): 321-327
25 Swift F, Tovsrud N, Enger UH, et al. The Na+/K+-ATPaseα2-isoform regulates cardiac contractility in rat cardiomyocytes [J]. Cardiovascular Research, 2007, 75(1): 109-117
26 James PF, Grupp IL, Grupp G, et al. Identification of a specific role for the Na,K-ATPaseα2 isoform as a regulator of calcium in the heart [J]. Molecular Cell, 1999, 39(5): 555-563
27 Sweadner KJ, Herrera VL, Amato S, et al. Immunologic identification ofNa+,K+-ATPase isoforms in myocardium [J]. Circulation Research, 1994, 74(4): 669-678
28 Mcdonough AA, Zhang Y, Shin V, et al. Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes [J]. Am J Physiol Cell Physiol, 1996, 270(4): 1221-1227
29 Fransen P, Hendrickx J, Brutsaert DL. Distribution and role of Na+,K+-ATPase in endocardial endothelium [J]. Cardiovasc Res, 2001, 52(3): 487-499
30 Silverman BZ, Fuller W, Eaton P, et al. Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na+,K+-ATPase [J]. Cardiovasc Res, 2005, 65(1): 93-103
31 Harada K, Lin H, Endo Y, et al. Subunit composition and role of Na+,K+-ATPases in ventricular myocytes [J]. Phys Sci, 2006, 56(1): 113-121
32 Schwinger RH, Wang J, Frank K, et al. Reduced sodium pumpα1,α3 andβ1-iosform protein levels and Na+/K+-ATPase activity but unchanged Na+/Ca2+-exchanger protein levels in human heart failure [J]. Circulation, 1999, 99(16): 2105-2112
33 Studer R, Reinecke H, Bilger J, et al. Gene expression of the cardiac Na+-Ca2+ exchanger in end-stage human heart failure [J]. Circ Res, 1994, 75(3): 443-453
34 Pogwizda SM, Sipidob KR, Verdonckc F, et al. Intracellular Na+ in animal models of hypertrophy and heart failure: contractile function and arrhythmogenesis [J]. Cardiovascular Research, 2003, 57(4): 887-896
35 Ove Semb S, Lunde PK, Holt E, et al. Reduce myocardial Na,K-pump capacity in congestive heart failure following myocardial infarction in rats [J] Journal of Molecular and Cellular Cardiology, 1998, 30(7): 1311-1328
36 Muller-Ehmsen J, Wang J, Schwinger RH, et al. Region specific regulation of sodium pupm isoform and Na+-Ca2+ exchanger expression in the failing human heart-right atrium vs left ventricle [J]. Cell-Mol-Biol-(Noisy-le- grand), 2001, 47(2): 373-381
37 Gray RP, McIntyre H, Sheridan DS,et al. Intracellular sodium and contractile function in hypertrophied human and guinea-pig myocardium [J]. Pfluger’s Arch, 2001, 442(1): 117-123
38 Meszaros J, Khananshvili D, Hart G. Mechanisms underlying delayed afterdepolarizations in hypertrophied left ventricular myocytes of rats [J]. Am J Physiol, 2001, 281(2): 903-914
39 Verdonck F, Volders PG, Vos MA, et al. Increased Na+ concentration and altered Na/K pump activity in hypertrophied canine ventricular cells [J]. Cardiovascular Research, 2003, 57(4): 1035-1043
40 Baudet S, Noireaud J, Leoty C. Intracellular Na activity measurements
41 in the control and hypertrophied heart of the ferret: an ion-sensitive micro-electrode study [J]. Pfluger’s Arch, 1991, 418(4): 313-318
42 Baartscheer A, Borren MV, Schumacher C, et al. Increased Na+/H+- exchanger activity in heart failure causes disturbed calcium hand-ling [J]. Biophys J, 2002, 81: 598
43 Pieskea B, Houser SR. [Na+]i handling in the failing human heart [J]. Cardiovascular Research, 2003, 57(4): 874-886
44 Dostanic I, Lorenz JN, Schultz JE, et al. Theα2 Isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice [J]. The Journal of biological chemistry, 2003, 278(52): 53026-53034
45 Dostanic I, Schultz JE, Lorenz JN, et al. Theα1 Isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart [J]. The Journal of biological chemistry, 2004, 279(52): 54053-54061
46那开宪,余平,田娟.慢性充血性心力衰竭的药物治疗(上) [J].中国临床医生, 2005, 33(4): 48-49
47朱文青.慢性充血性心力衰竭的药物治疗现状[A].中国临床医学, 2005, 12(1): 1-5