Effect And Mechanism Of Low Concentration OUA On Contraction In Rat Cardiocytes

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 cardiocytesObjective: 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 cardiocytesWhen 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 cardiocytesWhen 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 cardiocytesObjective: 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 OUAThe 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 OUAThe 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 OUAThe 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.