| The Cardioprotection of Ischemic Preconditioning against Myocardial No-Reflow and Ischemia-Reperfusion Injury is Partially Mediated by the Protein Kinase A PathwayObjectives:To investigate whether the cardioprotective effects of ischemic preconditioning (IPC) against myocardial no-reflow and ischemia-reperfusion injury relates to the activation of protein kinase A (PKA) pathway.Methods:In a 90-minute ischemia and 180-minute reperfusion model,40 minipigs were randomized into sham, myocardial infarction (MI), IPC (3 cycles of 5 minutes ischemia and 5 minutes of reperfusion 30 minutes before the prolonged ischemia), IPC+H-89 (a PKA inhibitor) and H-89 groups (n=8 in each group). Hemodynamics was monitored during the procedure. The area of no-reflow (ANR), area of necrosis (AN), and water content in left ventricle and myocardium of ischemia and non-ischemia were determined by pathological studies. Microvascular permeability was determined by a methods of FITC-labeled dextran staining. Myocardial hemorrhage, infiltration of polymorphonuclear leukocytes (PMNs), and cardiomyocyte cross-sectional area (CSA) were evaluated by histological analysis. Under electron microscope, ultrastructure of microvascular endothelial cells and cardiomyocytes, and mitochondria cross-sectional area (MSA) were studied. Activities of creatine kinase (CK), PKA, myeloperoxidase (MPO), malondialdehyde (MDA), and nitric oxide synthase (NOS) were determined by biochemical methods. Myocardial apoptosis was detected with TUNEL assay. Myocardial expression of PKA, Thr198 phosphorated (p)-PKA, endothelial NOS (eNOS), p-eNOS (Ser1179 and Ser635), Ser133 phosphorated cAMP response element-binding protein (p-CREB), TNF-?, P-selectin, aquaporins (AQPs), Bcl-2, Bax, and caspase-3 were detected by Western blotting analysis.Results:During ischemia and reperfusion, hemodynamics was worsened in the MI group compared to baseline (P<0.05), IPC decreased heart rates after 90 minutes of ischemia (P<0.05), while H-89 increased dp/dtmin after 90 minutes of ischemia (P<0.05).Analysis of the areas of myocardial no-reflow and infarction revealed that IPC reduced ANR from 48.64%to 9.71%(P<0.01), and attenuated AN from 78.46%to 31.3%(P<0.01), and H-89 partially canceled the effects of IPC. After 90 minutes of ischemia and 180 minutes of reperfusion, plasma CK activities in the MI group increased by about 2.7 and 3.45 times relative to the sham group (P<0.05), IPC lowered CK activities by about 51.5%and 31.9%compared to the MI group (P<0.05), while H-89 partially canceled the IPC effects. The results indicated that the cardioprotection of IPC was partly mediated by the PKA pathway.Myocardial PKA activities in reflow and no-reflow areas of the MI group elevated by about 1.58 and 2.02 times compared to the sham group (P<0.001), IPC further increased PKA activities by about 1.27 and 1.28 times relative to the MI group (P<0.001), but decreased by about 40.8%and 32.4%in the IPC+H-89 group compared with the IPC group (P<0.05). In reflow area, the expression of Thr198 p-PKA and Ser133 p-CREB in the MI group were higher than that in the sham group (P<0.05), and the expression of Ser133 p-CREB was higher in the IPC group than that in the MI group (P<0.01); In no-reflow area, the expression of Ser133 p-CREB in the MI group was higher than that in the sham group (P<0.05), and the expression of PKA, Thr198 p-PKA and Ser133 p-CREB were higher in the IPC group than that in the MI group (P<0.05); However, the expression of Ser'33 p-CREB in the reflow and no-reflow areas of the IPC+H-89 group were lower than that in the IPC group (P<0.05). The data suggested that the PKA pathway was activated by myocardial ischemia and reperfusion and was further activated by IPC pretreatment.Histological examination revealed that IPC reduced local hemorrhage score from 1.73 to 0.75 in the reflow area (P<0.01), and from 1.8 to 1.23 in the no-reflow area (P<0.01), and H-89 partially inhibited the IPC effect (P<0.05). PMN infiltration in ischemic myocardium increased in the MI and IPC+H-89 groups, but decreased in the IPC group (P<0.01). Electron microscope revealed that microvascular endothelium and cardiomyocytes were severely damaged in the MI and IPC+H-89 groups, but greatly improved in the IPC group. Microvascular permeability in the reflow and no-reflow myocardium of the MI group increased by about 1.83 and 1.86 times than the sham group (P<0.05), but reduced by about 25.9%and 29.6%in the IPC group relative to the MI group (P<0.05), and H-89 partially inhibited the effect of IPC. The results revealed that the endothelial protection of IPC was partially mediated by the PKA pathway. Myocardial iNOS activities in the reflow and no-reflow areas of the MI group were higher by about 48.7%and 57.9%than the sham group (P<0.01), IPC increased myocardial activities of tNOS and cNOS by about 52.7%and 70.7%compared to the MI group (P<0.05), but H-89 canceled the IPC effect. The expression of Ser1179 p-eNOS and Ser635 p-eNOS in the MI group were higher than that in the sham group, and were further increased in the IPC group compared to the MI group (P<0.05), but decreased in the IPC+H-89 group relative to the IPC group (P<0.05).After ischemia and reperfusion, MPO activities in reflow and no-reflow areas of the MI group increased by about 2.5 and 1.92 times (P<0.05), and MDA content increased by about 4.74 and 4.63 times (P<0.05). Compared to the MI group, IPC inhibited MPO activities in reflow and no-reflow areas by about 6.25 and 1.63 times (P<0.05), and decreased MDA content in reflow and no-reflow areas by about 1.99 and 0.86 times, but H-89 blocked the IPC effect. The expression of TNF-a and P-selectin in ischemic myocardium increased in the MI group compared to the sham group (P<0.05), decreased in the IPC group relative to the MI group (P<0.05), but again elevated in the IPC+H-89 group when compared with the IPC group (P<0.05).Myocardial water content in left ventricle and the myocardium of reflow and no-reflow increased by about 1.43%,7.36%and 5.83%times in the MI group than that in the sham group (P<0.05). IPC reduced the water content in left ventricle and reflow myocardium by about 0.95%and 5.09%relative to the MI group (P<0.05), but H-89 eliminated the IPC effect. CSA in reflow and no-reflow areas of the MI group swelled by about 1.96 and 2.1 times than the sham group (P<0.01), IPC reduced CSA in reflow area by about 19.7%relative to the MI group (P<0.01), but again increased by about 12.8%in the IPC+H-89 compared to the IPC group (P<0.01). MSA in reflow and no-reflow areas of the MI group were higher by about 2.17 and 2.99 times than the sham group (P<0.001), but were smaller in the IPC group by about 34.7%and 28.5%than the MI group (P<0.05), but were larger by about 30.8%and 24.2%in the IPC+H-89 group than in the IPC group (P>0.05). Myocardial expression of AQP-1,-8 and-9 in reflow area increased in the MI group, but AQP-8 decreased in the IPC group (P<0.01).In no-reflow area, the expression of AQP-1 and-9 increased in the MI group (P<0.05); IPC decreased the expression of AQP-4,-8 and-9 (P<0.05), but H-89 diminished the effect of IPC. Our results demonstrated that IPC-reduced myocardial edema was related to the inhibition of AQPs via the PKA pathway.TUNNEL study shown that cardiomyocytes apoptosis in reflow and no-reflow areas increased by about 10.49 and 14.99 times in the MI group compared with the sham group (P<0.01). Compared with the MI group, IPC abated cardiomyocytes apoptosis in the reflow and no-reflow areas by about 2.29 and 1.34 times (P<0.001), while cardiomyocytes apoptosis in the IPC+H-89 group was higher by about 2.89 and 1.2 times than that in the IPC group (P<0.05). IPC increased the expression of Bcl-2 and the ratio of Bcl-2/Bax, and decreased the expression of Bax compared with the MI group (P<0.05), and H-89 partially canceled the effects of IPC.Conclusions:1. IPC pretreatment 30 minutes before AMI can protect the swine hearts against myocardial no-reflow and ischemia-reperfusion injury, by improving the structure and function of the microvascular endothelium, attenuating myocardial inflammation, reducing myocardial edema and apoptosis; 2. The cardioprotection of IPC is partially mediated by the PKA pathway. The Cardioprotection of Simvastatin against Myocardial No-Reflow and Ischemia-Reperfusion Injury is Partially Mediated by the Protein Kinase A PathwayObjectives:To investigate whether the cardioprotective effects of simvastatin (SIM) against myocardial no-reflow and ischemia-reperfusion injury relates to the activation of protein kinase A (PKA) pathway in reperfused swine hearts.Methods:In a 90-minute ischemia and 180-minute reperfusion model,40 Chinese minipigs were randomized into sham, myocardial infarction (MI), SIM (2 mg·kg-1, delivered 1 hour before myocardial ischemia), SIM+H-89 (1.0?g·kg-1-min-1, a PKA inhibitor) and H-89 groups (n=8 in each group). Other methods used during and after the procedure were as same as part 1.Results:During ischemia and reperfusion, hemodynamics was worsened in the MI group compared to baseline (P<0.05); SIM partly inhibited the deterioration of the hemodynamics after 180 minutes of reperfusion without statistical difference (P<0.05), while heart rates and dp/dtmin in the SIM+H-89 group were decreased after 90 minutes of ischemia (P<0.05).Analysis of the areas of myocardial no-reflow and infarction revealed that SIM reduced the ANR from 48.64%to 36.1%(P<0.01), and the AN from 78.46%to 64.1% (P<0.01), whereas addition of H-89 partially canceled the beneficial effects of SIM. After 90 minutes of ischemia and 180 minutes of reperfusion, plasma CK activity in the MI group increased by about 2.7 and 3.45 times relative to the sham group (P<0.05), SIM lowered CK activities by about 40.9%and 26.9%compared to the MI group (P<0.05), while H-89 partially canceled the SIM's effects, indicating that the cardioprotection of SIM was partly mediated by the PKA pathway.Myocardial tissue PKA activities in the reflow and no-reflow areas of the MI group elevated by about 1.58 and 2.02 times compared to the sham group (P<0.001), SIM pretreatment further increased PKA activities in the reflow and no-reflow myocardium by about 1.28 and 1.19 times relative to the MI group (P<0.001), and by about 1.3 and 1.26 times compared to the SIM+H-89 group (P<0.05). In reflow area, the expression of Thr198 p-PKA and Ser133 p-CREB in the MI group were higher than that in the sham group (P<0.05), and the expression of Ser133 p-CREB was higher in the SIM group than that in the MI group (P<0.01); in no-reflow area, the expression of Ser133 p-CREB in the MI group was higher than that in the sham group (P<O.05), and the expression of PKA, Thr198 p-PKA and Ser133 p-CREB were higher in the SIM group than that in the MI group (P<0.05); the expression of PKA and Ser133 p-CREB were decreased in the reflow and no-reflow areas of the SIM+H-89 group relative to the SIM group (P<0.01). The data suggested that the PKA pathway was activated by myocardial ischemia and reperfusion and was further activated by SIM pretreatment.Histological examination revealed that SIM significantly reduced the local hemorrhage score in the reflow and no-reflow myocardium from 1.73 and 1.8 both to 1.18 (P<0.01), and reduced the PMN infiltration score in the no-reflow area by about 31.9%relative to the MI group (P<0.01), but H-89 partially inhibited the SIM's effect (P<0.05). Ultrastructure examination by electron microscope revealed that SIM greatly mitigated the damage in microvascular endothelium and cardiomyocytes, and the SIM's effect was again diminished by H-89. Microvascular permeability in the reflow and no-reflow myocardium of the MI group increased by about 1.83 and 1.86 times than in the sham group (P<0.05), but reduced by about 25.97%in reflow area of the SIM group relative to the MI group (P<0.05), but again increased by about 1.29 times in the reflow area of the SIM+H-89 group compared to the SIM group (P<0.05). SIM increased myocardial cNOS activities by about 67.5%and 46.3%in reflow and no-reflow areas compared to the MI group (P<0.05), but H-89 canceled the SIM effect. The expression of Ser1179 p-eNOS and Ser635 p-eNOS in reflow area were higher in the SIM group than that in the MI group (P<0.05), but Ser635 p-eNOS were lower in the SIM+H-89 group relative to the SIM group (P<0.05); Ser635 p-eNOS in no-reflow area of the SIM group was higher than that in the MI group (P<0.05). The results revealed that the endothelial protection of SIM was partially mediated by the PKA pathway.MPO activities in the reflow and no-reflow areas of the MI group were higher by about 2.5 and 1.92 times than in the sham group (P<0.05), and MDA content were higher by about 4.74 and 4.63 times than the sham group (P<0.05). Compared to the MI group, SIM inhibited MPO activities in the reflow and no-reflow areas by about 74.2%and 59.8%(P<0.05), and decreased MDA content in the reflow and no-reflow areas by about 46.7%and 53.8%(P<0.05), but H-89 blocked the effect of SIM. The expression of TNF-a and P-selectin in the ischemic myocardium significantly increased in the MI group than in the sham group (P<0.05); SIM greatly inhibited the expression of TNF-a and P-selectin relative to the MI group (P<0.05), but the expression of P-selectin in reflow and no-reflow areas of the SIM+H-89 were higher than that in the SIM group (P<0.05).Myocardial water content in left ventricle and the myocardium of reflow and no-reflow increased by about 1.43%,7.36%and 5.83%in the MI group than in the sham group (P<0.05). SIM reduced the water content in reflow myocardium by about 5.6% relative to the MI group (P<0.05), but H-89 eliminated the reductive effect of SIM on water content. CSA in the reflow and no-reflow areas of the MI group swelled by about 1.96 and 2.1 times than the sham group (P<0.01), SIM reduced CSA in the reflow area by about 20.03%relative to the MI group (P<0.01), but CSA again increased by about 1.13 times in the reflow area of the SIM+H-89 group compared to the SIM group (P<0.01). MSA in the reflow and no-reflow areas of the MI group were higher by about 2.17 and 2.99 times than the sham group (P<0.00l); MSA in the reflow and no-reflow areas of the SIM group were smaller by about 39.1%and 33.0%than the MI group, and H-89 again counteracted the SIM's effect (P<0.05). In the reflow area, the expression of myocardial AQP-1,-8 and-9 were higher in the MI group than in the sham group (P<0.01); The expression of AQP-8 and-9 were decreased in the SIM group compared to the MI group (P<0.01), but were increased in the SIM+H-89 group relative to the SIM group (P<0.05). In the no-reflow area, the expression of AQP-1 and-9 were increased in the MI group relative to the sham group (P<0.05); SIM inhibited the expression of AQP-1,-4,-8 and-9 compared to the MI group (P<0.05), but H-89 diminished the reductive effect of SIM on the expression of AQP-4 (P<0.01). Our results demonstrated that SIM-reduced myocardial edema was related to the inhibition of AQPs via the PKA pathway.TUNNEL study shown that the apoptosis of cardiomyocytes in the reflow and no-reflow areas of the MI group increased by about 10.49 and 14.99 times compared with the sham group (P<0.01), SIM abated the apoptosis of cardiomyocytes in the reflow and no-reflow areas by about 57.9%and 27.4%relative to the MI group (P<0.001), but the apoptosis of cardiomyocytes in the reflow and no-reflow areas of the SIM+H-89 group again increased by about 1.76 and 1.24 times compared with the SIM group (P<0.01). Compared to the sham group, the expression of Bcl-2, the ratio of Bcl-2/Bax, and caspase-3 increased, while Bax decreased in the ischemic myocardium of the MI group (P<0.05). Compared with the MI group, SIM increased the expression of Bcl-2 and the ratio of Bcl-2/Bax, and decreased the expression of Bax (P<0.05), and the effects of SIM were partially canceled by H-89. Conclusion:1. Pretreatment with single loading dose of SIM just 1 hour before AMI can protect the swine hearts against myocardial no-reflow and ischemia-reperfusion injury, by improving the structure and function of the microvascular endothelium, attenuating myocardial inflammation, and reducing myocardial edema and apoptosis; 2. The cardioprotection of SIM is partially mediated by the PKA pathway. The Cardioprotection of Tongxinluo against Myocardial No-Reflow and Ischemia-Reperfusion Injury is Partially Mediated by the Protein Kinase A PathwayObjectives:To investigate whether the cardioprotective effects of tongxinluo (TXL) against myocardial no-reflow and ischemia-reperfusion injury relates to the activation of protein kinase A (PKA) pathway in reperfused swine hearts, and to reveal the mechanical difference of IPC, SIM and TXL in attenuating myocardial no-reflow and ischemia-reperfusion injury.Methods:In a 90-minute ischemia and 180-minute reperfusion model,40 minipigs were randomized into sham, myocardial infarction (MI), TXL (0.05 g-kg-1, delivered 1 hour before myocardial ischemia), TXL+H-89 (1.0?g·kg-1·min-1, a PKA inhibitor) and H-89 groups (n=8 in each group). Finally, compare the difference of IPC, SIM and TXL in attenuating myocardial no-reflow and ischemia-reperfusion injury. Other methods used during and after the procedure were as same as part 1.Results:During ischemia and reperfusion, hemodynamics was worsened in the MI group compared to baseline (P<0.05), TXL decreased heart rates after 90 minutes of ischemia (P<0.05), and there was no significant difference of hemodynamics between the IPC, SIM and TXL groups (P>0.05).Analysis of the areas of myocardial no-reflow and infarction revealed that TXL reduced the ANR from 48.64%to 9.7%(P<0.01), and the AN from 78.46%to 59.2% (P<0.01), whereas the no-reflow and infarct size of the TXL+H-89 group were larger by about 3.0 and 1.3 times than the TXL group (P<0.05). The effect of TXL reducing the no-reflow area was better than SIM (36.07%, P<O.01) and not inferior to IPC (9.71%, P>0.05), and the effect of TXL attenuating infarct size was inferior to IPC (31.3%, P<0.01) and no difference with SIM (64.14%, P>0.05). After 90 minutes of ischemia and 180 minutes of reperfusion, TXL lowered CK activities by about 35.6%and 25.1% compared to the MI group (P<0.05), while H-89 partially canceled the TXL effects. The results indicated that the cardioprotection of TXL was partly mediated by PKA pathway, and IPC was the best and SIM the worst in mitigating myocardial no-reflow and ischemia-reperfusion injury.TXL pretreatment increased PKA activities in the reflow myocardium by about 1.26 times relative to the MI group (P<0.05), but was lower by about 21.9%in the TXL+H-89 group than that in the TXL group (P<0.05). The expression of Ser133 p-CREB in reflow area and the expression of PKA, Thr198 p-PKA and Ser133 p-CREB in no-reflow area were higher in the TXL group than that in the MI group (P<0.05), and the expression of Ser133 p-CREB in no-reflow area of the TXL group was higher than that in the IPC and SIM groups (P<0.05). The data suggested that the PKA pathway was activated by TXL pretreatment, and TXL was better than IPC and SIM in activating the PKA pathway.Ultrastructure examination by electron microscope revealed that TXL greatly alleviated the damage in microvascular endothelium and cardiomyocytes relative to the MI group, and TXL's effect was similar to IPC and SIM, while H-89 blocked the effect of TXL. Microvascular permeability were reduced by about 37.8%and 32.8%in reflow and no-reflow areas of the TXL group relative to the MI group (P<0.05), and this effect was similar to IPC and better than SIM, but H-89 partially inhibited the effect of TXL. TXL increased the myocardial tNOS and cNOS activities in the no-reflow area by about 59.5%and 65.9%compared to the MI group (P<0.05), but H-89 canceled the TXL effect. The expression of Ser635 p-eNOS was higher in no-reflow area of the TXL group than that in the MI, TXL+H-89, IPC, and SIM groups (P<0.05), indicating that TXL was more effective in activating Ser635 p-eNOS. These results suggested that the endothelial protection of TXL was partially mediated by the PKA pathway, and the protective effect of TXL was not inferior to IPC and SIM.Compared to the MI group, TXL inhibited MPO activities in the reflow and no-reflow areas by about 63.5%and 30.5%(P<0.05), and decreased MDA content in the reflow and no-reflow areas by about 66.1%and 59.6%(P<0.05), but H-89 blocked the effect of TXL. TXL greatly inhibited the expression of TNF-a and P-selectin in no-reflow area relative to the MI group (P<0.05), and the effect of TXL reducing the expression of P-selectin was worse than IPC and SIM, but H-89 blocked the TXL effect.TXL reduced the water content in left ventricle and reflow myocardium by about 1.47%and 5.06%relative to the MI group (P<0.05), but H-89 eliminated the reductive effect of TXL on water content. TXL reduced CSA in reflow area by about 20.97% relative to the MI group (P<0.01), and decreased MSA in reflow and no-reflow areas by about 48.5%and 53.5%compared to the MI group (P<0.05); CSA in reflow area of the TXL+H-89 group was about 1.14 times higher and MSA in reflow and no-reflow areas were about 1.4 and 1.6 times higher compared to the TXL group (P<0.05). In the reflow area, the expression of AQP-8 was decreased in the TXL group compared to the MI group (P<0.01), and was as well decreased in the TXL+H-89 group relative to the TXL group (P<0.01). TXL inhibited the expression of AQP-1 and-9 in no-reflow area compared to the MI group (P<0.05), but H-89 diminished the effect of TXL. Our results demonstrated that TXL-reduced myocardial edema was related to the inhibition of AQPs via the PKA pathway. Although worse than IPC and SIM in inhibiting the expression of AQP-4,-8 and-9, TXL was not inferior to IPC and SIM in reducing myocardial edema.The apoptosis of cardiomyocytes were reduced by about 55.9%and 25.1%in reflow and no-reflow areas of the TXL group compared with the MI group (P<0.001), but was increased by about 1.2 times in no-reflow area of the TXL+H-89 group (P<0.05). Compared with the MI group, TXL increased the expression of Bcl-2 and the ratio of Bcl-2/Bax, and decreased the expression of Bax (P<0.05), and the effects of TXL were partially canceled by H-89.Conclusion:1. Pretreatment with single loading dose of TXL just 1 hour before AMI can protect the swine hearts against myocardial no-reflow and ischemia-reperfusion injury, by improving the structure and function of the microvascular endothelium, reducing myocardial inflammation, and attenuating myocardial edema and apoptosis; 2. The cardioprotection of TXL is partially mediated by the PKA pathway; 3. Among IPC, SIM and TXL, IPC is the best and SIM the worst in protecting the heart against myocardial no-reflow and ischemia-reperfusion injury. |
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