The Effect Of Urantide, An UT Receptor Antagonist, Against Myocardial Ischemia-Reperfusion Injury In Rats And Its Underlying Mechanism

Background:Cardiovascular diseases are the commonly and frequently encountered diseaseswhich are severely harmful to health. It is well known that cardiovascular diseases havehigh mortality. Ischemic heart disease is one of the high incidence diseases, which ischaracterized by the myocardium lack blood and oxygen supply rapidly. For the pastfew years, Coronary Artery Bypass, thrombolytic therapy, percutaneous transluminalcoronary angioplasty (PTCA), external ciculation of cardiac surgical procedures andcardiopulmonary cerebral resuscitation were established and extensively applied toclinics. All the methods mentioned above can make the ischemic heart regain bloodsupply rapidly. On the contrary, the damage of myocardium was observed to becomeaggratated even proceed irreversibility injury after reperfusion. This phenomemon wasnamed as the ischemical reperfusion injury. Therefore, more and more attention waspaid to develop the effective medicines for prevention or therapy toischemia-reperfusion injury.Urotensin Ⅱ (UⅡ) is a cyclic peptide initially isolated from the caudalneurosecretory system of teleost fish. The cyclic region of UⅡ, which is responsible forthe biological activity of the peptide, has been fully conserved from fish to human. Thematching of the11amino acid peptide human urotensin-Ⅱ (hU-Ⅱ) with an orphanhuman G-protein coupled receptor with homology to rat GPR14, which was named UTreceptor by Ames in1999. hU-Ⅱ was considered to be the most potent mammalianvasoconstrictor identified so far, which vasoconstrictor potency is higher than ET-1.Many clinical analysis show that the hUⅡ in plasma level is elevated in differentcardiovascular diseases including hypertension, heart failure and so on. No doubt hUⅡ and UT receptors play an important role in cardiovascular diseases.Urantide, is the most potent UT receptor antagonist compound so far reported inthe rat isolated aorta, and is also endowed with high affinity at human UT receptors. Ourprevious studies indicated that urantide had a protective effect against myocardialischemia reperfusion injury in rats and mice. The mechanism may related toantioxidation, reducing calcium overload in cardiomyocytes and augmenting thesynthesis of NO. The present study was going to investigate the effects of urantide usingthe ischemia reperfusion rat model in vivo and hypoxia reoxygenation cellular model invitro respectively. The study was to observe the effects and mechanism of urantide oncardiomyocyte protection. In this study, chelerythrine which is an inhibitor of the PKCsignaling pathway and LY294002which is an inhibitor of the PI3K-Akt signalingpathway were used to assess the relationship between the effects of urantide and relativesignal transduction pathway. On the other hand, by using RNA interference to knockdown the expression of UT receptor it was to be investigated if the UT receptor playsrole in protection of urantide in hypoxia reoxygenation cardiomyocytes.Methods and ResultsPart I Effects of urantide on myocardial ischemia-reperfusion injury in rats andits underlying mechanismsMethods:1. The ischemical reperfusion model was set up by ligating and untying left anteriordescending coronary artery in rats. The electrocardiogram was recorded during theexperiment, the serum was collected to measure the content of NO, MDA and cTnI aswell as the activities of CK, LDH and NOS. The heart was harvested to determinemyocardial ischemic size and infarct size by dual staining with Evans blue andtriphenyl-tetrazolium-chloride.2. The rats were randomized into8groups: All hearts were subjected to theischemia/reperfusion (I/R) protocol consisting of30min of regional ischemia and90 min of reperfusion except for the sham group.(1) the sham group: the animals' chest were operated, but the coronary ligatures werenot tied.(2) the model group: the hearts were subjected to the ischemia/reperfusion (I/R)protocol consisting of30min of regional ischemia and90min of reperfusion(3) urantide3μg·kg^-1group: Urantide (3μg·kg^-1)was given through intravenous drugperfusion ten minutes before ischemia.(4) urantide10μg·kg^-1group: Urantide(10μg·kg^-1) was given through intravenous drugperfusion ten minutes before ischemia.(5) urantide30μg·kg^-1group: Urantide(30μg·kg^-1) was given through intravenous drugperfusion ten minutes before ischemia.(6) verapamil1.6mg·kg^-1group: ver (1.6mg·kg^-1) was given through intravenous drugperfusion ten minutes before ischemia as the positive control(7) urantide30μg·kg^-1+CHE1mg·kg^-1(chelerythrine, the inhibitor of PCK signaling)group: the chelerythrine was given through intravenous drug perfusion afterstabilization, urantide(30μg·kg^-1) was given through intravenous drug perfusion fiveminutes after the previous administration, the ischemia reperfusion operation wascarried out ten minutes after the urantide afford.(8)urantide30μg·kg^-1+LY2940020.3mg·kg^-1(the inhibitor of PI3K-Akt signaling)group: LY294002was given through intravenous drug perfusion after stabilization,urantide(30μg·kg^-1) was given through intravenous drug perfusion five minutesafter the previous administration, the ischemia reperfusion operation was carried outten minutes after the Urantide afford.3. The hearts were fixed at the end of each experiment. The histopathologic examinationof myocardium in rat was observed by hematoxylin and eosin stain. Theultrastructural morphology in rats cardiomyocyte was measured by transmissionelectron microscope. 4.TUNEL labeling was used for the measurement of apoptosis in myocardium.5. The expressions of Bcl-2and Bax protein in cardiomyocytes of rats were measuredby immunohistochemistry technology.6. The expressions of Akt and p-Akt protein in cardiomyocytes of rats were measuredby western blot analysis.Results:1. The ST segment of ECG markedly elevated during the process of ischemiareperfusion, while urantide (10,30μg·kg^-1) could markedly inhibit the elevation ofST segment of ECG. Urantide (10,30μg·kg^-1) could also significantly reduce infarctsize as compared to model hearts, while pharmacological inhibition of PKC andPI3K/Akt signaling completely abrogated urantide-induced inhibition in elevation ofST segment and reduction of infarct size.2. The cTnI content and CK,LDH activity elevated in model group. Urantide (10,30μg·kg^-1) could markedly inhibit the elevation of cTnI content and CK, LDH activitiesin serum. However, those decreased injury indexes increased evidently in urantide30μg·kg^-1+CHE group and urantide30μg·kg^-1+LY294002group.3. Morphological observation by HE staining and transmission electron microscopeshowed that the ischemic reperfusion injury was improved by urantide (10,30μg·kg^-1)administration. But myocardial injury of the urantide30μg·kg^-1+CHEgroup and urantide30μg·kg^-1+LY294002group showed to become more seriouscompared to that of urantide30μg·kg^-1group.4. Urantide (10,30μg·kg^-1) could markedly inhibit the MDA content, and increase theSOD, NOS activities and NO content in serum. However, pharmacological inhibitionof PKC and PI3K/Akt signaling completely abolished changes of these indexesinduced by urantide administration.5. Compared with those in the sham group, the number of TUNEL-positive cells in I/R group was significantly increased. Urantide (3,10,30μg·kg^-1) markedly decreasedthe number of TUNEL-positive cells. On the contrary, pharmacological inhibition ofPKC and PI3K/Akt signaling markedly inhibited urantide-induced decrease ofTUNEL-positive cells.6. Expression level of Bax protein was significantly higher in model group than that inurantide administration group, whereas the level of Bcl-2protein and Bcl-2/Bax ratiowere markedly higher in30μg·kg^-1urantide group than those in model group,respectively. Two proteins expression in urantide30μg·kg^-1+CHE group and urantide30μg·kg^-1+LY294002group were no statistical difference in comparison with modelgroup.7. Levels of Akt (total-Akt) and its phosphorylation at Ser473(p-Akt) were determinedby western blotting and phosphorylation of Akt was expressed as the ratio betweenp-Akt and total-Akt. The results showed that Urantide could rise the ratio at threedifferent dose. Furthermore, both CHE and LY294002significantly reduced Aktphosphorylation and decrease the ratio between p-Akt and total-Akt.Part Ⅱ Effects of knockdown of UT receptor on protection of urantide oncultured neonatal rat cardiomyocytes subjected to hypoxia reoxygenation injuryMethods:1.Cardiomyocytes of neonatal rat were isolated and cultured in vitro. Morphocytologychanges and cell vitality were observed at different period during cell culture. Cellpurity was evaluated through troponin I expression by immunohistochemistrytechnology.2.Cardiomyocytes were transfected with three different UT siRNA which were designedby Shanghai Gene Pharma Co. Ltd. The UT mRNA and protein expression weredetected by real-time PCR and Western blotting respectively. Thus choosing the mosteffective UT siRNA to knock down the UT receptor. 3. Cardiomyocytes were transfected with the most effective UT siRNA prior to hypoxiathree hours and reoxygenation three hours. Urantide10-6mol·L^-1was incubated beforehypoxia reoxygenation. At the end of experiment, the cell culture medium wascollected and proteins were harvested to detect different index.4. Cell viability was measured by trypan blue staining and MTT staining respectively.Hoechst33258assay and flow cytometric techniques were used to detect apoptoticcells. The content of MDA,NO and cTnI and activities of SOD, CK, LDH, NOS incell culture medium were measured by commercial kits.Results:1. Cardiocytes began to adhere and grow after cell culture. It can be observed thatCardiocytes beat spontaneously and rhythmically on the third day, and the frequencebecome more and more after that. The frequence reduced gradually from the seventhday of cell culture. Cardiocytes proliferate obviously on the third and the fourth dayof cell culture. Immunohistochemistry staining confirmed the cell with purity higherthan95％.2. The levels of UT mRNA and protein expression showed that three differentUTsiRNA could all inhibit UT mRNA and protein express distinctively. The siRNA3was the most effective one among those siRNA, so siRNA3was adopted in latterexperiments.3. It was found that10-6mol·L^-1urantide increased the viability of cardiocytes subjectedto hypoxia/reoxygenation by using both trypan blue staining and MTT staining assay.However, down-regulation of UT receptor in cardiocytes by using UTsiRNA3led toa decrease to increase of cell viability produced by urantide.4. On the H/R model of myocardial cells, urantide (10-6mol·L^-1) could evidently inhibitthe increase of cTn I content, reduce the rise of CK and LDH activities in the cellculture medium. Whereas down-regulation of UT receptor in cardiocytes could also abolish aforementioned effects of urantide.5. During H/R, Urantide (10-6mol·L^-1) could evidently inhibit the increase of MDA andthe decreases of activities of SOD and NOS and content of NO in the cell culturemedium. UT siRNA3-mediated down-regulation of UT receptor could abolish theinhibitory effect of urantide on increase of MDA and decrease of SOD, but had noeffect on urantide-induced alteration of NO content and NOS activity.6. By using Hoechst33258assay and flow cytometric techniques, it was observed thatthe apoptosis rate in model group was much higher than that in sham group.Pretreatment of10-6mol·L^-1urantide significantly inhibited apoptosis induced by H/R.While knock-down of UT receptor by using UT siRNA3could abolish the inhibitioneffects of urantide on H/R-induced apoptosis.Conclusions:1. Urantide has a significant protective effect against myocardial ischemic/reperfusioninjury in rats, which may be related to anti-lipidperoxidation, elevating the NOsynthesis and inhibiting apoptosis via modulating the expression o f Bc l-2and Bax.2. The protective effect of urantide on myocardial ischemic/reperfusion injury in ratsmay be mediated via activation of PKC-TK-MAPK signal transduction pathway andPI3K-Akt signal transduction pathway.3. UT receptor involves in the protective of urantide on hypoxia reperfusion injury in ratcardiomyocyte.4. UT receptor mediates the mechanisms of anti-lipidperoxidation and anti-apoptoticeffect of urantide, however UT receptor did not involve urantide-induced synthesisand release of NO.