摘要
心力衰竭是多数心血管病的最终归宿和主要死因。其症状复杂，预后严峻，是当今心血管疾病治疗中的一大难题，严重危害公共健康。
强心苷（Cardiotonic steroid，Cs）是治疗心衰的传统经典药物，研究证明其治疗心衰的作用机制与钠钾泵（Na+/K+-ATPase，Na/K pump，NKA，钠泵）活性有关。钠钾泵是普遍存在于人类和动物细胞膜上的一种酶，其α亚基上存在强心苷的结合位点。目前认为强心苷治疗心衰的机制在于抑制心肌细胞膜上的钠钾泵，使细胞内Na+增加，通过Na+/Ca2+交换，引起胞浆Ca2+的升高，并促进肌浆网对钙的摄取及随后的钙释放（CICR），从而使心肌细胞的收缩力增强。
但研究也发现，临床上强心苷有效治疗心衰的血清游离药物浓度（10-9-10-8M）比其抑制钠钾泵发挥离子转运功能的浓度要低得多，而且Gao等指出，低浓度强心苷（10-9-10-8M的双氢哇巴因）不但不抑制反而会兴奋钠钾泵。我室以往的实验也证实低浓度强心苷（10-10-10-8M的双氢哇巴因）能够产生兴奋性钠钾泵电流。这使得传统的钠钾泵抑制理论难以圆满解释治疗浓度强心苷的强心作用。因而，我们提出了低浓度强心苷可能通过直接兴奋钠钾泵而强心治疗心衰的假设，并首次在离体灌流豚鼠心脏发现nM为强心苷的治疗浓度，具有浓度依赖性持续强心作用和钠钾泵兴奋作用，且其强心作用与其兴奋钠钾泵作用呈显著正相关，首次为“强心苷兴奋钠钾泵可能为其强心机制”的假说提供了证据。但在分离的左心室内膜心肌细胞上虽也曾偶尔观察到此浓度强心苷的强心作用，却始终未得到稳定的测定结果。
心肌细胞收缩舒张功能的测定对于心力衰竭的发生发展极其药物治疗机制的研究起着至关重要的作用。此类实验对细胞质量要求极高，不仅需要细胞能耐受持续刺激，而且要求细胞在一定时间内能维持稳定的收缩舒张功能。有文献报道，温度变化影响心肌细胞的收缩特性。本室以往实验中也发现，细胞的收缩力随着季节交替和温度变化而变化。因此，本实验拟首先以心肌细胞收缩功能为指标，通过连续升温和恒温孵育两种方法确定一个能使心肌细胞产生最大且最稳定收缩的最佳温度。并在此温度下测定低浓度(10-8M)OUA对大鼠心室肌细胞的正性肌力作用，观察钠钾泵各信号转导通路抑制剂对低浓度OUA正性肌力作用的影响。
一、温度对大鼠心肌细胞收缩力的影响
目的：确定一个能使心肌细胞产生最大且最稳定收缩的最佳温度。
方法：用酶解法急性分离大鼠左室心肌细胞并覆钙，然后采用可视化单细胞动缘探测系统（Video-based motion edge-detection system）同步检测比较连续升温和固定恒温对单个大鼠心肌细胞舒张、收缩功能的影响。
结果：
1连续升温对心肌细胞收缩力的影响
25℃—37℃逐步升温过程中，每个温度刺激下心肌细胞收缩力各不相同。若将25℃时（room temperature）大鼠心室肌细胞收缩幅度标准化为100%,则27℃、29℃、30℃、31℃使细胞收缩幅度分别增大至（107.694±2.042）%（p<0.01）、（127.0±6.3）%（p<0.01）、（142.5±17.0）%（p<0.05）、（139.0±17.7）%（p<0.05）；33℃、35℃、37℃时心室肌细胞收缩幅度分别为（130.4±18.3）%（p>0.05）、（132.5±25.6）%（p>0.05）、（129.8±33.3）%（p>0.05），与25℃时相比没有显著性差异。而且，30℃后随着温度的继续上升，细胞会出现不规律收缩，某些细胞还会出现形态改变，甚至挛缩死亡。以上结果提示，在25℃—37℃逐步升温过程中，温度为30℃时心肌细胞可达最佳收缩。
2固定恒温对心肌细胞收缩力的影响
25℃、30℃、35℃孵育1h后大鼠心室肌细胞收缩幅度有差异。25℃孵育的细胞收缩幅度为4.54±0.13，30℃孵育的细胞收缩幅度为6.92±0.33，35℃孵育的细胞收缩幅度为2.58±0.22，三组数据两两比较p<0.01。该结果表明：25℃、30℃、35℃三个温度恒温孵育后的细胞收缩幅度存在显著差异，其中30℃孵育的细胞收缩幅度最大且稳定，为25℃时收缩幅度的152%，此结果与连续升温时测定的结果一致，但增加比例较连续升温至30℃时的增加比例（142%）为大。与连续升温测定结果所不同的是，35℃时细胞收缩幅度最小，仅为25℃时收缩幅度的57%。
结论：温度变化影响大鼠心室肌细胞的收缩舒张功能，30℃时细胞收缩力强且稳定，可能与低温升高细胞内钙和提高肌丝对钙的敏感性有关。
二、低浓度OUA对大鼠心室肌细胞收缩力的影响及机制研究
目的：观察低浓度(10-8M)OUA是否能增加大鼠心肌细胞收缩力，并研究与治疗浓度OUA发挥的正性肌力作用相关的钠钾泵信号转导通路。
方法：（1）检测并比较10-9-10-4M浓度OUA增强大鼠心室肌细胞收缩力的作用。（2）先分别给予PP2（1μM）、NAC（10μM、100μM）、PD98059（10μM、50μM）、U73122（1μM）或Xestospongin C（1μM）5min，分别检测5种信号转导抑制剂对大鼠心室肌细胞收缩力的影响；之后继续分别用PP2（1μM）、NAC（10μM、100μM）、PD98059（10μM、50μM）、U73122（1μM）或Xestospongin C（1μM）与10-8M OUA共灌流，记录5种信号转导抑制剂对10-8M OUA增强大鼠心室肌细胞收缩力的影响。
结果：
1不同浓度OUA对大鼠心室肌细胞收缩力的作用
10-9M，10-8M，10-7M，10-6M，10-5M，10-4M的OUA使大鼠心室肌细胞收缩幅度分别增大至（111.973±3.885）%、（112.092±0.524）%、（123.045±2.199）%、（139.27±7.58）%、（179.46±36.323）%、(180.35±29.191)%，（p<0.01）。
2信号转导抑制剂对10-8M OUA正性肌力作用的影响
(1)1μM PP2（Src抑制剂）部分抑制10-8M OUA的正性肌力作用
本组10-8M OUA使大鼠心室肌细胞收缩幅度增大至（114.16±1.776）%，（p<0.01）；灌流1μM PP2+10-8M OUA的收缩幅度为（102.527±6.415）%，（p>0.05）；而1μM PP2+10-8M OUA组与10-8M OUA组相比收缩幅度减小（p<0.05）。提示1μM PP2部分抑制了10-8M OUA的增加心肌细胞收缩力的效应。
(2)100μM NAC（ROS抑制剂）部分抑制10-8M OUA的正性肌力作用
本组10-8M OUA使大鼠心室肌细胞收缩幅度增大至（114.246±2.16）%，（p<0.01）；灌流100μM NAC+10-8M OUA的收缩幅度变为（101.846±5.614）%，（p>0.05）;而100μM NAC+10-8M OUA组与10-8M OUA组相比收缩幅度减小（p<0.05）。提示100μM NAC部分抑制了10-8M OUA的增加心肌细胞收缩力的效应。
(3)1μM U73122（PLC抑制剂）部分抑制10-8M OUA的正性肌力作用
本组10-8M OUA使大鼠心室肌细胞收缩幅度增大至（114.487±4.074）%，（p<0.01）；灌流1μM U73122+10-8M OUA的收缩幅度为（102.337±1.938）%，（p>0.05）。1μM U73122+10-8M OUA组与10-8M OUA组相比收缩幅度减小（p<0.05）。提示1μM U73122部分抑制了10-8M OUA的增加心肌细胞收缩力的效应。
(4)1μM Xestospongin C（IP3抑制剂）部分抑制10-8M OUA的正性肌力作用
本组10-8M OUA使大鼠心室肌细胞收缩幅度增大至（111.516±7.201）%，（p<0.01）；灌流1μM Xestospongin C+10-8M OUA的收缩幅度为（99.07±10.404）%，（p>0.05）。1μM Xestospongin C+10-8M OUA组与10-8M OUA组相比收缩幅度减小（p<0.01）。提示1μM Xestospongin C部分抑制了10-8M OUA的增加心肌细胞收缩力的效应。(5)10μM和50μM PD98059（MEK抑制剂）没有抑制10-8M OUA的正性肌力作用
本组10-8M OUA使大鼠心室肌细胞收缩幅度增大至（113.366±2.128）%，（p<0.01）；灌流10μM PD98059+10-8M OUA使收缩幅度增大至（119.186±5.134）%，（p<0.01）；10μM PD98059+10-8M OUA与10-8MOUA组相比收缩幅度没有显著性差异（p>0.05）。提示10μM PD98059没有抑制10-8M OUA的增加心肌细胞收缩力的效应。
本组10-8M OUA使大鼠心室肌细胞收缩幅度增大至（114.246±2.167）%，（p<0.01）；50μM PD98059+10-8M OUA使收缩幅度增大至（121.707±5.762）%，（p<0.01）;50μM PD98059+10-8M OUA与10-8MOUA组相比收缩幅度没有显著性差异（p>0.05）。提示50μM PD98059没有抑制10-8M OUA的增加心肌细胞收缩力的效应。
结论：10-9-10-4M的OUA均能增加大鼠心室肌细胞收缩性，且具有浓度依赖性。低浓度OUA的正性肌力作用与钠钾泵的信号转导功能有关，其中Src/EGFR/Ras/ROS信号途径和Src/EGFR/PLC/PIP2/IP3信号途径参与调节了10-8M的OUA的正性肌力作用，而Ras/Raf/MEK/ERK1/2cascade通路没有参与。
Congestive heart failure (CHF) is the ultimate and main death cause ofmost cardiovascular diseases (CVD), being one of the difficult problems inCVD treatment area. It is harmful to public health severely since complexsymptom and tense prognosis.
Cardiotonic steroid (Cs) is the traditional and classical medicine to curecongestive heart failure. The therapeutic mechanism of Cs might associatewith the activity of Na+/K+-ATPase (Na/K pump，NKA) that is widely foundin the membrane of human and animals. The Na/K pump plays an importantrole in transporting three Na+out of the cell and two K+in of cell against theirconcentration gradients by utilizing ATP.
There is a binding site of cardiac glycosides in α subunit of Na/K pump.The traditional cardiotonic mechanism considered that：The binding of cardiacglycosides to Na/K pump located in the membrane of failed cardiocytes leadsto the intracellular Na+increase ([Na+] i), and in turn causing intracellular Ca2+increase ([Ca2+]i) by Na+/of Ca2+exchange, thus promoting the calciumuptake and subsequent release of calcium of sarcoplasmic reticulum (CICR),causing an increase in the contraction of cardiocytes finally. However, thestudy found that the serum concentration （10-9-10-8M） of Cs for effectivetreatment of heart failureis much lower than the concentration of inhibiting theNa/K pump. Further more, Gao proposed that low concentration of cardiacglycoside（10-9-10-8M dihydroouabain）had activated rather than inhibitedNa+/K+-ATPase. Our previous experiment proved that low concentration ofcardiac glycosides（10-10-10-8M dihydroouabain）had induced stimulatoryNa/K pump current. It is difficult to satisfactorily explain the cardiotoniceffects of therapeutic concentrations of cardiac glycosides with the traditionalNa/K pumpinhibitory theory. Therefore, we hypothesize that low concentration of cardiac glycoside can treat CHF by direct stimulating Na/Kpump and try to prove by experiment. The nMconcentration of cardiacglycosides is the therapeutic concentration, which was found in the isolatedheart of guinea pig in vitro for the first time, and has the effects of enhancingthe heart contraction continuously and the stimulating Na/K pump inconcentration-dependent manner, and the two effects have significantpositively correlation, which prompt that “Na/K pump stimulation” might bethe cardiotonic mechanism of low concentration OUA. Although lowconcentration of cardiac glycosides could occasionally increase the contractileforce of left ventricular endocardial cell, stable contraction induced by lowconcentration of OUA had not been observed due to the quality ofcardiomyocytes.
The measurement of cardiocytes systolic and diastolic function plays animportant role in researching the development and treatment of CHF. Thismeasurement has much more requirement on the quality of cells, which notonly must be able to resist the repeatable stimulation, but also should possessthe stable function of the contraction and relaxation. However, some studiesreported that temperature changes affected the contraction of cardiocytes. Wealso found that the extent of contraction changed with the changes of theseasons and temperature. Thus, the purpose of the present experiment was todetermine an optimum temperature firstly, which enables the cardiocytes toproduce the largest and the most stable contraction, by observing the effects ofthe two methods on contraction, which were the continuous rising temperatureand the constant temperature incubation, and then detect the effect of lowconcentration (10-8M) OUA on the contraction of rat cardiocytes at thistemperature and explore signal transduction pathways related topositive inotropic action using different inhibitor.
Part1Effects of temperature on contraction of rat cardiocytes
Objective: To determine the optimum temperature that enables ratcardiocytes to produce the maximal and most stable contraction.
Methods：Left ventricular myocytes were enzymatically isolated. Then the contraction of rat cardiocytes was measured by a video-based motionedge-detection system and the difference of the contractile amplitude wascompared under the conditions of the continuous rising temperature and theconstant temperature incubation.
Results:
1Effects of continuous rising temperature on contraction in rat cardiocytes
When temperature raising from25℃to37℃step by step， thecontractility of rat cardiocytes was different at each temperature. Theamplitude of contraction was normalized using the contractile value at25℃as100%. The amplitude of the contraction at27℃,29℃,30℃, and31℃increased to （107.694±2.042）%（p<0.01）,（127.0±6.3）%（p<0.01）,（142.5±17.0）%（p<0.05）, and（139.0±17.7）%（p<0.05）, respectively.Those at33℃,35℃, and37℃were（130.4±18.3）%（p>0.05）（,132.5±25.6）%（p>0.05）, and （129.8±33.3）%（p>0.05），respectively, which were similarto that at25℃. Moreover, with increasing temperature to above30℃, cellcrispation and morphological changes occured, and even cell death. Theseresults suggested that30℃wasthe optimum temperature, at which thecardiocytes could produce the largest and the most stable contraction.
2Effects of constant temperature incubation on contraction in rat cardiocytes
When the cardiocytes were pre-incubated at25℃,30℃, or35℃for1hrespectively, there were significant differences (p<0.01) in contractionamplitude of rat cardiocytes. The contraction amplitude of cardiocytes at25℃,30℃, and35℃were4.54±0.13，6.92±0.33, and2.58±0.22, respectively. Theresults showed that after the incubation at30℃, the cardiocytes could producethe largest and the most stable contraction. This result was consistent with theresult from continuous rising temperature above.
Conclusion：Temperature can affect on systolic and diastolic function ofrat cardiocytes. The cardiocytes reveal the strongest and most stablecontraction at30℃, this may be related to hypothermia increased intracullularcalcium and myfilament calcium sensitivity.
Part2Effect and mechanism of low concentration OUA on contraction inrat cardiocytes
Objective: To observe wether low concentration (10-8M) of OUA canincrease the contractility of rat cardiocytes and investigate the Na/K pumpsignal transduction pathways related to positive inotropic action following thetherapeutic concentration of OUA.
Methods:(1) Detected and compared the potentiations of10-9-10-8MOUA on the contractility of rat cardiocytes.(2) The cardiocytes werepre-treated with PP2（1μM）, NAC（10μM,100μM）, PD98059（10μM,50μM）,U73122（1μM）or xestospongin C（1μM）for5min, and then detected theeffects of the five signal transduction inhibitors on the contractility ofcardiocytes, eventually, the cells were perfused with PP2（1μM）, NAC（10μM,100μM）, PD98059（10μM,50μM）, U73122（1μM）, or xestospongin C（1μM）, and10-8M OUA, and recorded the effects of the5kinds of signalstransduction inhibitors on the positive inotropic effect of10-8M OUA.
Results:
1Effects of10-9-10-8M OUA on the contractility of cardiocytes in ratsOUA10-9M，10-8M，10-7M，10-6M，10-5M，and10-4M increased thecontractility of rat cardiocytes to（110.0±3.9）%,（112.0±0.5）%,（123.0±2.2）%,（139.3±7.6）%,（179.5±36.3）%, and (180.4±29.2)%（p<0.01）.
2Effects of signal transduction inhibitors on the positive inotropic effect of10-8M OUA
(1)1μM PP2（Src inhibitor）partially inhibited the positive inotropic effect of10-8M OUAThe contraction amplitude of rat cardiocytes increased to（114.2±1.8）%（p<0.01）with10-8M OUA and became（102.5±6.4）%（p>0.05）with1μMPP2+10-8M OUA. Compared1μM PP2+10-8M OUA group with10-8M OUAgroup, the contraction amplitude decreased, which indicated1μM PP2partially inhibited the contraction effect of cardiocytes enhanced by10-8MOUA.
(2)100μM NAC（ROS inhibitor）partially inhibited the positive inotropiceffect of10-8M OUA
The contraction amplitude of rat cardiocytes increased to（114.2±2.2）%（p<0.01）with10-8M OUA and became（101.8±5.6）%（p>0.05）with100μMNAC+10-8M OUA. Compared100μM NAC+10-8M OUA group with10-8MOUA group, the contraction amplitude decreased, which indicated100μMNAC partially inhibited the contraction effect of cardiocytes enhanced by10-8M OUA.
(3)1μM U73122（PLC inhibitor）partially inhibited the positive inotropiceffect of10-8M OUA
The contraction amplitude of rat cardiocytes increased to （114.5±4.1）%（p<0.01）with10-8M OUA and became（102.3±1.9）%（p>0.05）with1μM U73122+10-8M OUA. Compared1μM U73122+10-8M OUA group with10-8M OUA group, the contraction amplitude decreased, which indicated1μMU73122partially inhibited the contraction effect of cardiocytes enhanced by10-8M OUA.
(4)1μM xestospongin C（IP3inhibitor）partially inhibited the positiveinotropic effect of10-8M OUA
The contraction amplitude of rats' cardiocytes increased to （111.5±7.2）%（p<0.01）with10-8M OUA and became（99.1±10.4）%（p>0.05）with1μMxestospongin C+10-8M OUA. Compared1μM xestospongin C+10-8M OUAgroup with10-8M OUA group, the contraction amplitude decreased, whichindicated1μM xestospongin C partially inhibited the contraction effect ofcardiocytes enhanced by10-8M OUA.
(5)10μM and50μM PD98059（MEK inhibitor）didn't inhibit the positiveinotropic effect of10-8M OUA
The contraction amplitude of rats' cardiocytes increased to （113.4±2.1）%（p<0.01）with10-8M OUA and grew to （119.2±5.1）%（p<0.01）with10μM PD98059+10-8M OUA. There was no significant difference incontraxtile amplitudes between10μM PD98059+10-8M OUA group and10-8M OUA group, which indicated10μM PD98059didn't inhibit the contractioneffect of cardiocytes enhanced by10-8M OUA.
The contraction amplitude of rat cardiocytes increased to （114.2±2.2） %（p<0.01）with10-8M OUA and grew to （121.7±5.7）%（p<0.01）with50μM PD98059+10-8M OUA. There was no significant difference incontraxtile amplitudes between50μM PD98059+10-8M OUA group and10-8M OUA group, which indicated50μM PD98059didn't inhibit the contractioneffect of cardiocytes enhanced by10-8M OUA.
Conclusion：10-9-10-4M OUA could increase the contraction amplitudeof cardiocytes in rats in concentration-dependent manner. Positive inotropiceffect of therapeutic concentrations of OUA is related to Na/K pump signaltransduction. Multiple signal pathways regulate the positive inotropic effect of10-8M of OUA, including the Src/EGFR/the Ras/ROS signal pathway andSrc/EGFR/PLC/PIP2/IP3signal pathway, while Ras/Raf/MEK/ERK1/2cascade pathway do not participate.
引文
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