Myocardial Ischemia-reperfusion Injury In Mice Research Articles

Gaseous Hydrogen Sulfide Protects against Myocardial Ischemia-Reperfusion Injury in Mice Partially Independent from Hypometabolism

BackgroundIschemia-reperfusion injury (IRI) is a major cause of cardiac damage following various pathological processes. Gaseous hydrogen sulfide (H2S) is protective during IRI by inducing a hypometabolic state in mice which is associated with anti-apoptotic, anti-inflammatory and antioxidant properties. We investigated whether gaseous H2S administration is protective in cardiac IRI and whether non-hypometabolic concentrations of H2S have similar protective properties.MethodsMale C57BL/6 mice received a 0, 10, or 100 ppm H2S-N2 mixture starting 30 minutes prior to ischemia until 5 minutes pre-reperfusion. IRI was inflicted by temporary ligation of the left coronary artery for 30 minutes. High-resolution respirometry equipment was used to assess CO2-production and blood pressure was measured using internal transmitters. The effects of H2S were assessed by histological and molecular analysis.ResultsTreatment with 100 ppm H2S decreased CO2-production by 72%, blood pressure by 14% and heart rate by 25%, while treatment with 10 ppm H2S had no effects. At day 1 of reperfusion 10 ppm H2S showed no effect on necrosis, while treatment with 100 ppm H2S reduced necrosis by 62% (p<0.05). Seven days post-reperfusion, both 10 ppm (p<0.01) and 100 ppm (p<0.05) H2S showed a reduction in fibrosis compared to IRI animals. Both 10 ppm and 100 ppm H2S reduced granulocyte-influx by 43% (p<0.05) and 60% (p<0.001), respectively. At 7 days post-reperfusion both 10 and 100 ppm H2S reduced expression of fibronectin by 63% (p<0.05) and 67% (p<0.01) and ANP by 84% and 63% (p<0.05), respectively.ConclusionsGaseous administration of H2S is protective when administered during a cardiac ischemic insult. Although hypometabolism is restricted to small animals, we now showed that low non-hypometabolic concentrations of H2S also have protective properties in IRI. Since IRI is a frequent cause of myocardial damage during percutaneous coronary intervention and cardiac transplantation, H2S treatment might lead to novel therapeutical modalities.

Abstract 15: Remote Ischemic Preconditioning Confers Late Protection Against Myocardial Ischemia-Reperfusion Injury in Mice by Upregulating Interleukin-10

Remote ischemic preconditioning (RIPC) induces a prolonged late phase of multi-organ protection against ischemia-reperfusion (IR) injury. In the present study, we tested the hypothesis that RIPC confers late protection against myocardial IR injury by upregulating expression of IL-10. Mice were exposed to lower limb RIPC or sham leg ischemia. After 24 h, mice with RIPC demonstrated decreased myocardial infarct size and improved cardiac contractility following 30-min ischemia and 120-min reperfusion (I-30/R-120), accompanied by increased phosphorylation of Akt and eNOS in the heart. These effects of RIPC were completely blocked by anti-IL-10 receptor antibodies. Moreover, in IL-10 KO mice, RIPC was no longer able to provide cardioprotection against IR injury. IL-10 receptors were expressed in cardiomyocytes under basal conditions. Stimulation with exogenous IL-10 increased Akt phosphorylation and decreased myocardial infarct size after I-30/R-120 in isolated perfused hearts. Wildtype mice with RIPC demonstrated increased plasma IL-10 levels, while in the preconditioned gastrocnemius muscle, the phosphatase PTEN was inactivated and expression of IL-10 was increased through Stat3. Myocyte-specific PTEN inactivation led to increased Stat3 phosphorylation and IL-10 protein expression in the gastrocnemius muscle. Taken together, these results suggest that RIPC induces late protection against myocardial IR injury by increasing expression of IL-10 in the remote muscle, followed by release of IL-10 into the circulation, and activation of protective signaling pathways in the heart. This study provides a scientific basis for the use of RIPC to confer systemic protection against IR injury.

Genetically Induced Moderate Inhibition of the Proteasome in Cardiomyocytes Exacerbates Myocardial Ischemia-Reperfusion Injury in Mice

Both cardiomyocyte-restricted proteasome functional enhancement and pharmacological proteasome inhibition (PSMI) were shown to attenuate myocardial ischemia/reperfusion (I/R) injury. The role of cardiac proteasome dysfunction during I/R and the perspective to diminish I/R injury by manipulating proteasome function remain unclear. We sought to determine proteasome adequacy in I/R hearts, create a mouse model of cardiomyocyte-restricted PSMI (CR-PSMI), and test CR-PSMI impact on I/R injury. Myocardial I/R were modeled by ligation (30 minutes) and subsequent release of the left anterior descending artery in mice overexpressing GFPdgn, a validated surrogate proteasome substrate. At 24 hours of reperfusion, myocardial proteasome activities were significantly lower whereas total ubiquitin conjugates and GFPdgn protein levels were markedly higher in all regions of the I/R hearts than the sham controls, indicative of proteasome functional insufficiency. CR-PSMI in intact mice was achieved by transgenic (tg) overexpression of a peptidase-disabled mouse β5 subunit (T60A-β5) driven by an attenuated mouse mhc6 promoter. Overexpressed T60A-β5 can replace endogenous β5 and inhibits proteasome chymotrypsin-like activities in the heart. Mice with moderate CR-PSMI showed no abnormalities at the baseline but displayed markedly more pronounced structural and functional damage during I/R, compared with non-tg littermates. The exacerbation of I/R injury by moderate CR-PSMI was associated with significant increases in the protein level of PTEN and protein kinase Cδ (PKCδ), decreased Akt activation, and reduced PKCε. Myocardial I/R causes proteasome functional insufficiency in cardiomyocytes and moderate CR-PSMI augments PTEN and PKCδ, suppresses Akt and PKCε, increases cardiomyocyte apoptosis, and aggravates I/R injury in mice.

Abstract P271: Genetically Induced Moderate Inhibition of the Proteasome in Cardiomyocytes Exacerbates Myocardial Ischemia-Reperfusion Injury in Mice

Proteasome dysfunction is implicated human ischemic heart disease and observed in experimental myocardial ischemia-reperfusion (I/R) injury. Altered proteasome activities in I/R hearts were reported but it is unclear whether proteasome function in I/R hearts is adequate. Moreover, effects of pharmacological inhibition of the proteasome on I/R injury remain controversial. Hence we sought to determine the adequacy of proteasomal function in I/R hearts and the impact of moderate cardiomyocyte-restricted proteasome inhibition (CR-PSMI) on I/R injury in intact animals. First, myocardial I/R was created by ligation (30min) and subsequent release of the left anterior descending artery in the transgenic (tg) mice overexpressing GFPdgn, a previously validated surrogate proteasome substrate. Compared with the sham controls, myocardial GFPdgn protein levels in the remote area, the board zone, and the a rea a t r isk (AAR) of the ventricle 24hrs after reperfusion were significantly increased, indicative of proteasome functional insufficiency (PFI) in the I/R heart. The most important proteasome peptidase resides in β5 subunits of the 20S proteasome. The clinically used proteasome inhibitor bortezomib targets specially the β5 subunit. To achieve CR-PSMI in intact animals, we engineered and tested a catalytically inactive mouse β5 subunit mutant (T60A-β5) and created multiple tg mouse lines in which T60A-β5 was overexpressed under the control of an attenuated mouse mhc6 promoter. Baseline analyses of the tg lines confirm the ability of T60A-β5 to replace endogenous β5 and inhibit proteasome chymotrypsin-like activities in the heart in a dose-dependent manner. A stable tg line with ∼40% replacement was used in this study. The same I/R procedure caused a significantly greater infarct size in T60A-β5 tg mice (66.2% of AAR) than in the littermate Ntg mice (53.4% of AAR). Under either the sham surgery or the I/R condition, a decrease in phosphorylated AKT and an increase in PKCδ proteins were evident in the tg hearts, compared with the respective Ntg groups. These results show that I/R causes PFI in the heart and PFI plays a pathogenic role in I/R injury. A potential mechanism by which PFI contributes to I/R injury is to suppress AKT activation and exacerbate PKCδ signaling.

Sodium 4-phenylbutylate protects against myocardial ischemia-reperfusion injury by reducing unfolded protein response-mediated apoptosis in mice

Unfolded protein response (UPR)-mediated apoptosis plays a pivotal role in ischemia-reperfusion injury. Sodium 4-phenylbutyrate (PBA) has been reported to act as a chemical chaperone inhibiting UPR-mediated apoptosis triggered by ischemia in various organs other than the heart. Therefore we investigated whether PBA reduces UPR-mediated apoptosis and protects against myocardial ischemia-reperfusion injury in mice.

Abstract 5053: Deficiency of ASC (Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain) in Bone Marrow Cells Improved Myocardial Ischemia-Reperfusion Injury in Mice

Background: Inflammatory responses play a key role in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury. ASC is an adaptor protein that forms inflammasome whose activation leads to caspase-1-dependent interleukin (IL)-1 β generation and subsequent inflammatory responses; however, the role of ASC in myocardial I/R injury remains to be determined. Methods and Results: ASC deficient (ASC −/− ) and wild-type (WT) mice were subjected to 30 min LAD occlusion, followed by reperfusion. ASC −/− mice showed improved LV dysfunction (%FS: 34.0% vs. 25.7% at 14 days p&lt;0.01), reduced infarct area/area at risk (IA/AAR: 18.7% vs. 28.6% at 48 h, p&lt;0.01), and scar formation (scar/LV area: 9.7% vs. 14.6% at 14 days, p&lt;0.01) after myocardial I/R. Immunostaining revealed decreased infiltration of macrophages (Mac3) and neutrophils (Gr-1), but not neovascularization (CD31), in the injured myocardium of the ASC −/− mice. Real-time RT-PCR and ELISA analyses demonstrated that the myocardial mRNA and protein expression of inflammatory cytokines, such as IL-1 β , IL-6, and MCP-1, after I/R were significantly decreased in the ASC −/− mice. Further, improved myocardial I/R injury was also observed in the caspase-1 −/− mice, compared with WT mice (IA/AAR: 20.2% vs. 28.6% at 48 h, p&lt;0.05). Since double immunostaining revealed that ASC was expressed in the infiltrated macrophages and neutrophils, bone marrow-transplanted (BMT) mice were prepared. The IA/AAR was significantly decreased in the BMT ASC−/−→WT mice, compared with BMT WT→WT (IA/AAR: 17.8% vs. 27.9% at 48 h, p&lt;0.01). Further, in vitro experiments showed that LPS-induced production of inflammatory cytokines such as IL-1 β , IL-6, and MCP-1 in the ASC −/− -bone marrow cells was significantly decreased compared, with that in the WT-bone marrow cells. Conclusion: ASC deficiency improved myocardial I/R injury via inflammatory cell infiltration and cytokine expression. These results suggest that ASC is a novel therapeutic target for myocardial I/R injury.

F001 Prominent role for the endothelial gap junction protein connexin40 in myocardial ischemia-reperfusion injury in mice

Intercellular channels formed by connexins (Cx) have been shown to play a critical role in cardiovascular disease. Indeed, connexin-deficient mice showed modifications in cell signalling, that appeared crucial in atherosclerosis and restenosis. In the heart, Cx40 is expressed in atrial myocytes, the conduction system and in endothelial cells. Here, we study the implication of endothelial Cx40 during ischemia and reperfusion in mice. For this purpose, we used the Cre-loxP system to create a mouse line in which Cx40 is deleted from the endothelium only. Immunostainings on TIE2- Cre+Cx40fl/flApoE-/- mice confirmed the absence of Cx40 in the endothelium, whereas the protein was normally expressed in the atria and cardiac conduction system. Moreover, mean arterial pressure and heart rates wehat appeared crucial in atheroscTIE2-Cre-Cx40fl/flApoE-/- and TIE2-Cre+Cx40 wt/wtApoE-/-) and TIE2-Cre+Cx40fl/flApoE-/- animals. Sixteen-week-old mice with or without endothelial-specific deletion of Cx40 were subjected to in vivo left coronary artery occlusion for 30 minutes and sacrificed 24-hours after reperfusion for analysis of infarct size. Myocardial surfaces areas at risk and infracted areas were measured from computed images using NIH Image software. Areas at risk, normalized to total left ventricle surfaces areas, were similar between the experimental groups, i.e controls 29.17±1.19 % (N=11) and TIE2- Cre-Cx40fl/flApoE-/- 29.29±2.37 % (N=7, n.s.). Interestingly, the infarct area, normalized to areas at risk, was significantly increased in TIE2-Cre+Cx40fl/flApoE-/- mice as compared to controls (20.67±4.74 % and 8.47±1.45 %, respectively, P<0.01). We conclude that endothelial Cx40 is implicated in resistance of the heart to ischemia-reperfusion injury. These findings underline once more the importance of connexin-mediated intercellular communication in cardiovascular inflammation, and may point towards novel therapeutic strategies to limit the cardiac injury after coronary interventions.

Tissue Kallikrein Is Involved in the Cardioprotective Effect of AT1-Receptor Blockade in Acute Myocardial Ischemia

Angiotensin-converting enzyme inhibitors limit infarct size in animal models of myocardial ischemia reperfusion injury. This effect has been shown to be due to inhibition of bradykinin degradation rather than inhibition of angiotensin II formation. The purpose of this study was to determine whether angiotensin AT1 receptor blockade by losartan or its active metabolite EXP3174 protects against myocardial ischemia-reperfusion injury in mice and whether this protection is mediated by the kallikrein kinin system. We subjected anesthetized mice to 30 min of coronary artery occlusion followed by 3 h of reperfusion and evaluated infarct size immediately after reperfusion. Losartan (Los) or EXP3174 [2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yI)methyl]imidazole-5-carboxylic acid] were administered 5 min before starting reperfusion at dosages determined by preliminary studies of blood pressure effect and inhibition of angiotensin pressor response. Compared with saline, both drugs significantly reduced myocardial infarct size by roughly 40% (P < 0.001). Pretreatment of mice with the selective AT2 receptor antagonist PD123,319 [S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid] did not affect infarct size in the absence of losartan but abolished the reduction in infarct size provided by losartan. In tissue kallikrein gene-deficient mice (TK-/-), losartan no longer reduced infarct size. Pretreatment of wild-type mice with the B2 receptor antagonist icatibant reproduced the effect of TK deficiency. We conclude that AT1 receptor blockade provides cardioprotection against myocardial ischemia-reperfusion injury through stimulation of AT2 receptors. Kallikrein and B2 receptor are major determinants of this cardioprotective effect of losartan. Our results support the hypothesis of a coupling between AT2 receptors and kallikrein during AT1 receptor blockade, which plays a major role in cardioprotection.

Role of 5-lipoxygenase in myocardial ischemia-reperfusion injury in mice

Myocardial ischemia induces 5-lipoxygenase (LOX) translocation and leukotriene production in the heart. Leukotrienes increase inflammatory responses and could thereby aggravate ischemic injury. However, the role of lipoxygenase and leukotrienes in cardiac ischemia/reperfusion damage has not been well defined. Therefore, we tested the effect of ischemia reperfusion in mice with targeted deletion of 5-lipoxygenase, the enzyme converting arachidonic acid in leukotrienes. 5-LOX deficient (KO) and wild-type (WT) mice underwent 30 min of coronary artery ligation and 24 h of reperfusion in vivo. In mice with equivalent area at risk, infarct size was not significantly different between WT and KO mice (infarct/area at risk 61.7+/-3.9 vs. 55.8+/-6.6%, WT vs. KO, P=n.s.). However, neutrophil infiltration as well as tumor necrosis factor expression were increased in 5-lipoxygenase deficient mice. In summary, inhibition of 5-lipoxygenase does not affect cardiac ischemia-reperfusion injury but the post-ischemic inflammatory response.

CC Chemokine Receptor-2 Deficiency Attenuates Oxidative Stress and Infarct Size Caused by Myocardial Ischemia-Reperfusion in Mice

Monocyte chemoattractant protein-1 (MCP-1) and its major receptor, CC chemokine receptor 2 (CCR2), have been shown to contribute to left ventricular remodeling after myocardial infarction. However, it is unknown whether CCR2 deficiency protects the myocardium after myocardial ischemia-reperfusion. The purpose of the present study was to investigate the effects of CCR2 deficiency on myocardial ischemia-reperfusion injury in mice. Experiments were performed in CCR2(-/-) and wild-type mice subjected to 45 min of ischemia followed by reperfusion. Macrophage infiltration in ischemic lesions was markedly reduced in CCR2(-/-) mice compared with wild-type mice (p<0.01). The infarct size was significantly reduced in CCR2(-/-) mice compared with wild-type mice at 3 days after reperfusion (p<0.001). In situ zymography revealed augmented gelatinolytic activity at 3 days after reperfusion in wild-type mice, but significantly less activity in CCR2(-/-) mice. NADPH oxidase activity, the intensity of nitrotyrosine staining and expression of inducible nitric oxide synthase and thioredoxin-1 were significantly increased in ischemic myocardium in wild-type mice compared with CCR2(-/-) mice, indicating a role for CCR2 in oxidative stress after ischemia-reperfusion. Inhibition of the MCP-1/CCR2 pathway may be a useful strategy for attenuating myocardial ischemia-reperfusion injury.

Effect of Aurintricarboxylic acid on myocardial ischemia-reperfusion injury in mice

Effect of Aurintricarboxylic acid on myocardial ischemia-reperfusion injury in mice