| BackgroundIschemic heart disease is expected to become the leading worldwide cause of death, and acute myocardial infarction is a major cause of such mortality. It is well known that heart is an organ consumed lots of oxygen and absent of anaerobic glycolysis. Therefore, when myocardium underwent ischemia, it would induce serious myocardial injury and lead to contraction dysfunction due to the oxygen disequilibrium between supplement and requirement. In patients with acute myocardial infarction, rapid restoration of blood flow (reperfusion) is the most effective treatment for myocardial salvage. However, it is now clear that reperfusion has the potential to induce additional lethal injuries, which is reperfusion injury. For this reason, it is very critical to develop strategies to limit the reperfusion injury.In 1986, Murry et al. firstly introduced the concept of ischemic preconditioning (PreC) in which repetitive brief periods of ischemia protected the myocardium from a subsequent longer ischemic insult. As a potent endogenous mechanism of cardioprotection against ischemia/reperfusion (I/R) injury, PreC has been extensively investigated in the hope of identifying new rational approaches to therapeutic protection of the ischemic myocardium.However, the use of PreC as a clinical cardioprotective strategy is limited by the inability to predict the onset of ischemia. In 2003, Zhao et al. reported another concept of ischemic postconditioning (PostC) as a novel strategy to harness nature's protection against myocardial I/R injury. PostC is induced by a short series of repetitive cycles of brief reperfusion and re-occlusion of the coronary artery applied immediately at the onset of reperfusion. Since its introduction, PostC has been consistently reproduced in several experimental preparations and different animal species. Although the protocol of PostC varies among multiple experimental models and conditions, its effects were comparable to that of PreC. Whereas PostC, in contrast to PreC, is more clinically feasible because the onset of reperfusion is more predictable and is under the clinician's control. As such, it has provoked renewed interest in the reperfusion phase as a target for cardioprotection. However, the mechanisms responsible for the PostC-induced cardioprotection are so complicated and still remain to be elucidated. To clarify the mechanisms of PostC would help to induce the protective effects like PostC in clinic, which will result in the recovery of heart function.A critical component of myocardial ischemia is hypoxia, while hypoxia inducible factor-1 (HIF-1) is the principal transcription factor involved in the regulation of adaptive responses to hypoxia. HIF-1 is a dimeric transcriptional complex composed of HIF-1αand HIF-1? subunits that has been recognized primarily for its role in the maintenance of oxygen homoeostasis. Although HIF-1? subunit is constitutively expressed and maintained at constant level regardless of oxygen availability, the expression and function of HIF-1αare precisely regulated by cellular oxygen concentrations, and hence HIF-1αis the key determinant of HIF-1 activity. Under normoxic conditions, HIF-1αis rapidly degraded through the ubiquitin-proteasomal pathway which is triggered by prolyl hydroxylases (PHDs) in the presence of oxygen. Whereas HIF-1αis stabilized and accumulates instantaneously under hypoxic conditions by escaping from the degradation process due to diminished oxygen content. HIF-1 has been shown to promote transcription of multiple genes that are required for the cell to be able to survive the ischemic insult, thus showing its protective role against cellular ischemic injury.Over the past decade, it has been indicated that HIF-1 activation plays an essential role in triggering cellular protection and metabolic alterations from the consequences of oxygen deprivation during myocardial ischemia. This suggests that HIF-1 activation should confer protection against ischemia/reperfusion (I/R) injury. Moreover, it has been reported that an increase in the level of HIF-1αwas one of the first adaptive responses, at a molecular level, of human myocardium to ischemia or infarction. At present, it has been demonstrated that chronic hypoxia increased some vasoactive substances concluding inducible nitric oxide synthase (iNOS) expression via upregulated HIF-1α, leading to adaptive responses to hypoxic conditions. Recently, Eckle et al demonstrated that HIF-1 played a central role in the cardioprotection by preconditioning. Since PreC triggers cardioprotective responses before"index"ischemia while PostC alters events after prolonged index ischemia, the mechanisms and the timing of those mechanisms are likely quite different between the two maneuvers. Accordingly, whether HIF-1 activation also plays an important role in the cardioprotection elicited by PostC remain to be clarified. In recent years, there are evidences that increased expression of iNOS, NO production and followed cGMP formation may have a protective role as a mediator in ischemic postconditioning. However, it still remains unknown whether the activation of iNOS-NO-cGMP signaling by PostC is related to HIF-1.At present it is important to consider that the effectiveness of PostC was demonstrated mostly in normal experimental models, which is far from the clinical reality. In fact, hypercholesterolemia is a major risk factor for atherosclerosis and ischemic heart disease in clinic. But very little is known about the effect of PostC on hyperlipidemia and there is a controversy. Recently, Martin et al. have shown that PostC reduces infarct size in hypercholesterolemic animals through activation of A1 and K+ATP channels. But it is very interesting that Iliodromitis et al. have found that the infarct size-limiting effect of PostC is lost in rabbits with experimental hyperlipidemia and atherosclerosis. However, it must be considered that these authors evaluated an advanced stage of atherosclerosis which may influence the effects of PostC, and most of these studies are confirmed in isolated hearts. Accordingly, whether PostC protects the myocardium against I/R-injury in the hyperlipidemic rats is unknown.In summary, it has been reported that ischemia/reperfusion and PreC significantly increased HIF-1αprotein level. However, whether HIF-1αexpression is influenced by PostC; whether HIF-1αplays a role in the protective mechanisms of PostC; and whether the protective effects by PostC are afforded in the hyperlipidemic conditions. All these doubts remain unkown.SECTION ONE Effects of Ischemic Postconditioning on Myocardial Injury Induced by Ischemia/Reperfusion and the Expression of Hypoxia Inducible Factor-1αObjective1. To investigate the effects of postconditioning on the expression of HIF-1α;2. To analyze the potential relationship between HIF-1αlevels and the degree of myocardial injury.Methods1. Male Wistar rats were randomly assigned to one of these groups:(1) Sham (n=10): animals underwent the same surgical procedures except that the suture passed under the LAD was not tightened for 210 min;(2) Control (n=10): animals were subjected to 30 min of LAD occlusion followed by 180 min of reperfusion;(3) PostC (n=10): at the onset of reperfusion, 3 cycles of 10 s reperfusion and 10 s LAD re-occlusion preceded 180 min of reperfusion.2. Determination of area at risk (AAR) and infarct size using TTC staining, the AAR was expressed as a percentage of the left ventricular area (AAR/LV), and the An as a percentage of the AAR (An/AAR, infarct size);3. Determination of plasma creatine kinase (CK) activity to reflect the degree of myocardial injury;4. Myocardial apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase -mediated dUTP nick end labeling) staining and Caspase-3 activity assay;5. The protein expression level of HIF-1αand iNOS were determined by Western-blot;6. HIF-1αlocalization was detected by immunohistochemical staining;7. The gene expression level of HIF-1αand iNOS were determined by real time-PCR.Results1. Effects of Postconditioning on the myocardial injury induced by ischemia/ reperfusion.The AAR was comparable in all groups (Fig 1A), averaging between 28% and 35%, indicating that a comparable degree of ischemia was induced in these groups.1.1 Postconditioning attenuated the myocardial infarct size.Infarct size in the Control group was greatly increased than that in the Sham group (36±5.29% vs. 3.05±0.27%, P<0.01). The extension of infarct size was significantly reduced by PostC relative to the Control group (27.3±4.16% vs. 36±5.29%, P<0.01, Fig 1B).1.2 Plasma creatine kinase (CK) activity was decreased by postconditioning. Plasma CK activity, a surrogate measure of morphological injury, increased significantly in the Control group, confirmed the extension of infarct size measured by TTC staining. CK activity in the PostC group was significantly lower than that in the Control group (0.38±0.06U/ml vs. 0.48±0.04U/ml, P<0.05, Fig 2) which was consistent with those observed for infarct size. This suggested that cardiomyocyte membrane may be damaged by ischemia/reperfusion leading to the leakage of CK, while PostC appeared to protect the membrane from I/R-induced injury.1.3 Postconditioning attenuated myocardial apoptosis induced by I/R. Apoptosis plays a key role in the formation of infarct area. Therefore, in the present study, myocardial apoptosis was first determined by highly sensitive terminal dUTP nick end-labeling (TUNEL) assay. Cardiomyocytes exhibited significant TUNEL positive staining in the Control group. The myocardial apoptosis index in the Control group was significantly higher than that in the Sham group (17.68±2.44% vs. 1.45±0.41%, P<0.01). Postconditioning greatly reduced the apoptosis index relative to the Control group (11.34±1.54% vs. 17.68±2.44%, P<0.01, Fig 3). Caspase-3 has been identified as being a key protein in the final pathway of cell apoptosis. To further verify the I/R-induced myocardial cell apoptosis, we also performed Caspase-3 activity assay. As shown in Fig 4, the activity of Caspase-3 significantly increased in the Control group compared to the Sham group (3.79±0.64 vs. 1±0.29, P<0.01). Postconditioning reduced remarkably the Caspase-3 activity in comparison to that in the Control group (1.85±0.5 vs. 3.79±0.64, P<0.01). Coupled with the above-mentioned TUNEL assay, these results indicate that postconditioning is able to attenuate the I/R-induced apoptosis.These results suggested that postconditioning attenuated the myocardial injury induced by I/R.2. Effects of postconditioning on HIF-1αexpression2.1 HIF-1αprotein levels were further increased by postconditioning. As shown in Fig 5, the HIF-1αprotein level in Sham group was extremely low because of its rapid degradation under normoxic condition. When the heart was subjected to ischemia/reperfusion insult, HIF-1αprotein level was greatly increased to 2.85-fold as compared to that of Sham group. Whereas after postconditioning treatment, HIF-1αprotein level was further significantly increased attaining at 5.76-fold compared to that of Sham group. This indicates that after a prolonged"index"ischemia, the postconditioning procedure is still able to trigger the expression of HIF-1αprotein. The elevation of HIF-1αprotein levels by postconditioning was comparable to that induced by preconditioning.Consistent with these results, immunohistochemical staining showed enhanced HIF-1αexpression in area at risk myocardium in the Control animals (Fig 6). However, HIF-1αexpression was markedly increased in the PostC group.2.2 No changes in HIF-1αmRNA level were observed in all groups. To determine whether the change in HIF-1αprotein expression in both Control and PostC groups is regulated at a transcriptional level, steady-state mRNA level of HIF-1αwas analyzed by a real time-PCR assay. There were no significant changes in HIF-1αmRNA level among all groups (Fig7, Fig8).These results suggested that the changes of HIF-1αexpression by I/R and postconditioning treatment were regulated at its post-translational level but not at gene level.2.3 Effects of postconditioning on the expression of iNOSIt has been well documented that HIF-1αis a transcriptional activator of iNOS expression, but it is unknown whether iNOS expression could be modulated through PostC-enhanced HIF-1α. Moreover, in order to illustrate whether upregulated HIF-1 by postconditioning was activated, in this study, we measured the levels of iNOS mRNA and protein by real time-PCR and Western blot. As shown in Fig 9, Fig 10, expression level of iNOS mRNA was increased to 3.75-fold by I/R insult, and to 10.39-fold by postconditioning. Western blot analysis showed increases in the expression of iNOS protein with an increasing tendency for each corresponding group similar to that observed for iNOS mRNA (Fig 11). These changes in the expression of iNOS gene are consistent with those of HIF-1αas mentioned above. These results suggest that HIF-1αis remarkably upregulated and activated by postconditioning3. The relationship between HIF-1αprotein level and myocardial infarct sizeTo explore whether the up-regulation of HIF-1αlevel correlates to the infarct-sparing effect of PostC, a linear correlation analysis between the degree of cardiac injury as represented by infarct size and HIF-1αprotein level was conducted. As shown in Fig 12, there was a linear inverse relationship between myocardial infarct size and HIF-1αprotein level (r=-0.799, P<0.01). This result suggests a role of HIF-1αup-regulation in PostC-elicited cardioprotection.According to all these results, it could be presumed that the attenuation in the myocardial injury by postconditioning may be related to the upregulation of HIF-1α.Summary1. Postconditioning further upregulates the expression of HIF-1αin myocardial tissue and enhances its activation;2. The attenuation in the myocardial injury by postconditioning may be related to the upregulation of HIF-1α.SECTION TWO Changes of Hypoxia Inducible Factor-1αExpression with DMOG and siRNA influences the Cardioprotection Elicited by Ischemic PostconditioningObjective1. After upregulation of HIF-1αin myocardium with pharmacological agent (DMOG, a prolyl hydroxylase inhibitor), to investigate the effects of it on cardioprotection against ischemia/reperfusion-injury by postconditioning;2. After silence HIF-1αgene by siRNA method in the cultured cardiocyte in vitro, to investigate the effects of postconditioning on the insult of cardiocytes. Methods1. 40 male Wistar rats were randomly assigned to one of these groups:(1) Control (n=10): animals were subjected to 30 min of LAD occlusion followed by 180 min of reperfusion;(2) DMOG+Control (n=10): 24 hours before I/R treatment, rats were received 40mg/kg DMOG administrated intraperitoneally, and then followed by the same treatment as Control group;(3) Vehicle + PostC (n=10): 24 hours before I/R treatment, animals were randomized to receive a similar volume of sterile saline as vehicle, and at the onset of reperfusion, 3 cycles of 10 s reperfusion and 10 s LAD re-occlusion preceded the 180 min of reperfusion;(4) DMOG + PostC (n=10): 24 hours before I/R treatment, animals were randomized to receive 40mg/kg DMOG and then these animals underwent the same treatment as PostC group.2. Determination of area at risk (AAR) and infarct size using TTC staining, the AAR was expressed as a percentage of the left ventricular area (AAR/LV), and the An as a percentage of the AAR (An/AAR, infarct size);3. Determination of plasma creatine kinase (CK) activity to reflect the degree of myocardial injury;4. Myocardial apoptosis was analyzed by Caspase-3 activity assay;5. The protein expression level of HIF-1αand iNOS were determined by Western blot;6. The gene expression level of HIF-1αand iNOS were determined by real time-PCR;7. The content of myocardial cGMP was detected by the method of radioimmunology;8. The hypoxia/reoxygenation and hypoxic postconditioning model were prepared to simulate the myocardial ischemia/reperfusion in the cultured H9c2 cell line, which is a kind of embryonic rat heart-derived cardiac muscle.(1) Nomoxic Control: cultured in an incubator full of 5% CO2 and 95% air for 5 h;(2) Hypoxia/Reoxygenation: hypoxia with 95% N2 and 5% CO2 for 3 h, then followed by 2h reoxygenation with 5% CO2 and 95% air;(3) PostC: hypoxia for 3h, then received 3 cycles of 5min reoxygenation and 5min hypoxia preceded the 1.5 h reoxygenation;(4) DMOG + PostC: before hypoxia, 0.1mM, 0.5mM and 1mM DMOG was added into the medium respectively, then followed by similar postconditioning treatment.9. Determination of lactate dehydrogenase (LDH) activity, viability of cardiocytes and caspase-3 activity to reflect the effects of hypoxia/reoxygenation and hypoxic postconditioning on the cultured cardiocyte;10. Silence HIF-1αgene using RNAi method.ResultsIn normoxia, HIF-1 activation is controlled by HIF-1αprolyl hydroxylases, which target HIF-1αfor ubiquitination and proteasomal degradation. Thus, dimethyloxaloylglycine (DMOG), as a pan-hydroxylase inhibitor, could be able to promote upregulation and activation of HIF-1α.1. Enhancement of PostC-modulated HIF-1αby DMOG1.1 DMOG pretreated significantly increased the level of HIF-1αprotein. As shown in Fig 13, pretreatment with DMOG at 24 h before an index ischemia in the Control animals significantly elevated HIF-1αprotein level to a same extent as seen in the PostC group. There was significant difference between DMOG alone, PostC and the Control (2.1-fold and 2.0-fold vs. Control group, respectively, P<0.01). However, PostC-modulated HIF-1αprotein was further increased by 3.3-fold when DMOG was given 24h before application of PostC, which was significantly different compared to those in the Control and PostC alone groups.1.2 DMOG did not influence the expression of HIF-1αin mRNA level. The levels of HIF-1αmRNA were not changed by DMOG pretreatment (Fig 14, Fig 15).2. Enhancement of PostC-mediated reduction in myocardial injury by DMOG2.1 DMOG further reduced the myocardial infarct size.Consistent with modulation of HIF-1αprotein by DMOG, infarct size was also significantly reduced by pretreatment with DMOG. As shown in Fig16, the infarct size in the DMOG alone and PostC alone groups was reduced by 24.2% and 25.7%, respectively, compared to the Control group. However, a reduction in infarct size by PostC was further enhanced when DMOG was given 24 h before applying PostC, averaging 50% reduction compared to those in both DMOG alone and PostC alone groups. These results suggest a role of HIF-1αup-regulation in PostC-mediated protection after an index ischemia.2.2 DMOG further reduced the plasma CK activity.As shown in Fig 17, CK activity in the DMOG alone and PostC alone groups was reduced compared to the Control group. However, a reduction in CK activity by PostC was further enhanced by DMOG pretreatment compared to those in both DMOG alone and PostC alone groups. 2.3 DMOG further attenuated the activity of Caspase-3.Consistent with modulation of infarct size and CK activity by DMOG, Caspase-3 activity was also significantly reduced by pretreatment with DMOG. In addition, the effects of DMOG and PostC were additive (Fig 18).2.4 Modulation of iNOS expression by PostC and DMOGNO is enzymatically produced by inducible nitric oxide synthase (iNOS) through the oxidation of arginine, and is known to exert anti-inflammatory and vasodilatory properties against ischemia/reperfusion injury. It has been well documented that HIF-1αis a transcriptional activator of iNOS expression, but it is unknown whether iNOS expression could be modulated through PostC and DMOG-enhanced HIF-1α. In this study, we measured the levels of iNOS mRNA and protein by real time-PCR and Western blot. Ischemia/reperfusion caused significant increase in both iNOS mRNA and iNOS protein. Pretreatment with DMOG alone increased iNOS mRNA and iNOS protein to a compatible level as seen in the PostC group. However, their levels were markedly increased when DMOG treatment was given before applying PostC. Exact tendency in modulation of iNOS mRNA and iNOS protein by DMOG and PostC was consistent with that in DMOG and PostC-modulated change in HIF-1α, suggesting a role of HIF-1αin regulation of iNOS in these experimental setting (Fig 19, Fig 20, Fig 21).2.5 Modulation of cGMP content by PostC and DMOGPostC increased the content of cGMP in myocardial tissue compared with that in Control group (P<0.05). While DMOG with PostC further enhanced the increase in cGMP content by DMOG alone and PostC alone. This result suggested that consistent with modulation of HIF-1αby PostC and DMOG, the downstream pathway iNOS-NO-cGMP was also activated by PostC and DMOG, which may further confirmed the role of HIF-1αplayed in the mechanism of PostC (Fig 22)3. Attenuation of cardioprotective effects by postconditioning through HIF-1αgene silencingIn order to clarify whether HIF-1αwas involved in the mechanisms of postconditioning, we pursued another study in which HIF-1αgene was silenced using a siRNA strategy in cultured cardiocytes.3.1 Hypoxia/reoxygenation and hypoxic postconditioning models of H9c2 cell simulated myocardial ischemia/reperfusionThe H9c2 embryonic rat heart-derived (cardiac muscle) cell line has been widely selected to investigate signaling mechanisms after hypoxia and reoxygenation. After underwent 3h of hypoxia and 2h of reoxygenation, the cell viability reduced by 46% compared with that in the nomoxia Control in normoxic conditions (P<0.01, Fig 23). Consistent with the change of viability, the leakage of LDH was increased respectively by hypoxia and reoxygenation. However, hypoxic postconditioning attenuated the cardiomyocyte injury including viability and LDH release (Fig 24). Moreover, DMOG pretreatment in different concentration reduced the myocyte injury in doses-dependent. These results suggested that the models of hypoxia/reoxygenation and hypoxic postconditioning have been successful.3.2 The effects of HIF-1αgene silencing in H9c2 on the effectiveness of postconditioning HIF-1αmRNA expression was assessed in H9c2 transfected with siRNA-HIF-1αand compared cells transfected with the normal control. HIF-1αsiRNA produced significant reductions in HIF-1αmRNA compared with the control (Fig 25).When HIF-1αgene silencing using siRNA, the postconditioning-mediated reduction in LDH activity was attenuated (Fig 26), and caspase-3 activity was increased compared with hypoxic postconditioning itself (Fig 27). It suggests that the cardioprotection by postconditioning was remarkably inhibited after HIF-1αsilencing, and indicates that HIF-1αinvolves in the mechanisms of postconditioning.Summary1. DMOG upregulated the level of HIF-1αprotein, and the increase degree is similar to that by postconditioning. Moreover, DMOG with postconditioning further increases the level of HIF-1αprotein. This indicates an additive effect of DMOG and postconditioning. However, DMOG did not influence the level of HIF-1αmRNA.2. Upregulation of HIF-1αprotein with DMOG attenuated the myocardial injury, and further enhanced the cardioprotective effects of postconditioning which showed an additive result. The results suggested that there may be a relationship between upregulation of HIF-1αand the cardioprotection by postconditioning.3. Associating with upregulation of HIF-1αexpression and the increase of cardioprotective effects by DMOG and postconditioning, the expressions of iNOS and the contents of cGMP in myocardium increased. This result may suggest that HIF-1αmay involve in the protective mechanisms of postconditioning via the downstream iNOS-cGMP signaling.4. HIF-1αgene silencing may inhibit the attenuation of myocardial injury by postconditioning. SECTION THREE Postconditioning Attenuates the Myocardial Injury Induced by Ischemia/Reperfusion in the Hyperlipidemic Rats and the Involvement of Hypoxia Inducible Factor-1αUpregulationObjective1. To investigate the effects of hyperlipidemia on the myocardial injury induced by ischemia/reperfusion;2. To investigate the effects of postconditioning on the myocardial ischemia/reperfusion injury in the hyperlipidemic rats;3. To detect the effects of hyperlipidemia on myocardial HIF-1αexpression.Methods1. Sixty male Wistar rats weighing 120±10g were randomly assigned to two different dietary groups:(1) Normal (control) diet group (N=30): Animals were fed with normal diet for 8 weeks.①N-Sham (n=10): Animals underwent the same surgical procedures except that the suture passed under the LAD was not tightened;②N-Control ((n=10): Animals was subjected to 30 min of LAD occlusion followed by 180 min of reperfusion;③N-PostC (n=10): at the onset of reperfusion, 3 cycles of 10 s reperfusion and 10 s LAD re-occlusion preceded the 180 min of reperfusion.(2) Hyperlipidemia group (N=30): Animals were fed with high fat diet for 8 weeks.①HC-Sham (n=10): the treatment is similar to the N-Sham group;②HC-Control (n=10): the treatment is similar to the N-Control group;③HC-PostC (n=10): the treatment is similar to the N-PostC group.2. Assay of the levels of plasma lipid using chromatometry;3. Determination of area at risk (AAR) and infarct size using TTC staining, the AAR was expressed as a percentage of the left ventricular area (AAR/LV), and the An as a percentage of the AAR (An/AAR, infarct size);4. Determination of plasma creatine kinase (CK) activity to reflect the degree of myocardial injury;5. Myocardial apoptosis was analyzed by Caspase-3 activity assay; 6. The protein expression level of HIF-1αand iNOS were determined by Western blot;7. The gene expression level of HIF-1αand iNOS were determined by Real time-PCR.ResultsIn the present study, we selected rats to be exposed to dietary cholesterol for 8 weeks without development of atherosclerosis, because rats do not develop significant atherosclerosis from consumption of a high-cholesterol diet.1. The levels of plasma lipidThere were no significant differences in plasma lipid concentrations among the groups at the beginning of the 8-week feeding period (Table 1). After the 8-week feeding period, plasma total cholesterol (TC), total triglyceride (TG) and low density lipoprotein (LDL) levels were markedly increased in cholesterol-fed rats than in normally fed rats (P<0.01, Table 2).2. Hyperlipidemia enhanced the susceptibility of myocardium to I/R injury.2.1 Hyperlipidemia further increased the myocardial infarct size induced by I/R.The AAR was comparable in all groups, indicating that a comparable degree of ischemia was induced in these groups. Infarct size in the Control group was greatly increased than that in the Sham group in both normal diet group and in hyperlipidemia group (33.38±1.4% vs. 3.05±0.99% and 39.54±1.16% vs. 4.98±0.83%, P<0.01). Hyperlipidemia increased the extention of myocardial infarct size, but there was no significant difference. However, hyperlipidemia amplified the increase in infarct size induced by I/R than control normolipidemia (P<0.05, Fig 28).2.2 Hyperlipidemia reinforced the increase of plasma creatine kinase (CK) activity by I/R.I/R increased plasma CK activity, which was a surrogate measure of myocardial morphological injury, in both hyperlipidemic group and normolipidemic group. But hyperlipidemia further reinforced the increase of CK activity induced by I/R than normolipidemia (0.56±0.06 vs. 0.47±0.04,P<0.01, Fig 29A)2.3 Hyperlipidemia further augmented myocardial apoptosis induced by I/R.The activity of Caspase-3 significantly increased in the Control group compared to the Sham group in both hyperlipidemia and normolipidemia. However, hyperlipidemia further augmented the increase of Caspase-3 activity induced by I/R (4.63±0.42 vs. 2.31±0.27, P<0.01, Fig 29B) These results suggested that hyperlipidemia leaded to significant aggravation of myocardial I/R injury. 3. Effects of postconditioning on the myocardial injury in the hyperlipidemic rats3.1 Postconditioning attenuated the myocardial infarct size in hyperlipidemic and normolipidemic rats.In both hyperlipidemic and normolipidemic group, postconditioning attenuated the myocardial infarct size induced by I/R. There were no differences in the degree of the attenuation by postconditioning between the hyperlipidemic and normolipidemic group (Fig 30).3.2 Plasma CK activity was decreased by postconditioning in both hyperlipidemic and normolipidemic group.Postconditioning significantly reduced CK activity up to 0.38±0.06 in normolipiemic group and 0.43±0.05 in hyperlipidemic group, respectively (P<0.01, vs. normal-Control and hyperlipidemia-Control groups). This result confirmed the extension of infarct size measured by TTC staining (Fig 31A).3.3 Postconditioning attenuated myocardial Caspase-3 activity in both hyperlipidemic and normolipidemic rats.As the effects of postconditioning on the infarct size and plasma CK activity, postconditioning attenuated the increase of Caspase-3 activity induced by I/R in both normolipidemic and hyperlipidemic group (Fig 31B).All these results suggested that postconditioning significantly reduced the I/R-injury in the hyperlipidemic animals down to the same value in normolipidemic group. Therefore, we conclude that the reduction of the injury by PostC is greater in the hyperlipidemic animals than in the normolipidemic ones.4. Hyperlipidemia increased myocardial tissue HIF-1αexpression after I/R and postconditioning.4.1 Effects on HIF-1αprotein expressionIn normal diet rats, I/R extremely increased the HIF-1αprotein level, while postconditioning further enhanced the increase of HIF-1αprotein expression induced by I/R. But under the hyperlipidemic condition, HIF-1αprotein level was much higher in both Sham and Control groups than in the similar treated normal diet groups respectively. Postconditioning also markedly increased HIF-1αprotein level up to 4.05±0.18 compared to that of Control group in hyperlipidemic rats (P<0.01, Fig 32).4.2 Effects on HIF-1αmRNA expressionThe mRNA level of HIF-1αwas assayed by Real time-PCR. There were no significant changes in HIF-1αmRNA expression in all groups investigated. The results suggested that the changes of HIF-1αexpression by I/R, postconditioning and hyperlipidemia were regulated at its post-translational level but not at gene level (Fig 33, Fig 34).4.3 Effects on iNOS mRNA and protein expressionNO is enzymatically produced by inducible nitric oxide synthase (iNOS) through the oxidation of arginine, and is known to exert anti-inflammatory and vasodilatory properties against ischemia/reperfusion injury. It has been well documented that HIF-1αis a transcriptional activator of iNOS expression, but it is unknown whether iNOS expression could be modulated through PostC and hyperlipidemia-enhanced HIF-1α. In this study, we measured the levels of iNOS mRNA and protein by real time-PCR and Western blot. Consistent with the changes of HIF-1αby postconditioning and hyperlipidemia, iNOS (a downstream target gene of HIF-1α) expression both in mRNA level and in protein level was increased in hyperlipidemic rats (Fig 35, Fig 36, Fig 37) .These results indicated that HIF-1αmay be involved in the cardioprotection of postconditioning in hyperlipidemic rats.Summary1. Hyperlipidemia enhanced the susceptibility of myocardium to ischemia/reperfusion injury.2. Postconditioning reduced the myocardial injury in hyperlipidemic rats.3. Hyperlipidemia increased the expression of HIF-1α, and which may be related to the attenuation of myocardial ischemia/reperfusion injury by postconditioning.Conclusion1. Postconditioning attenuates the myocardial injury induced by ischemia/reperfusion in normal rats.2. Postconditioning increased the expression of HIF-1αin myocardium.3. HIF-1αmay be involved in the cardioprotective mechanisms of p...
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