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Recent studies have indicated that C-reactive protein (CRP) is a causal agent in atherogenesis rather than merely a marker of inflammatory processes. However, the effects of CRP on myocardial ischemia reperfusion injury, an urgent problem to be solved, and the mechanisms underlying have not been determined. Furthermore, whether statins have protective effects on CRP-mediated ischemia reperfusion injury is still unknown.
We simulated ischemia reperfusion using oxygen-glucose deprivation/ reoxygenation (OGD/R) in neonatal Sprague-dawley rat cardiomyocyte, which was induced by 3 hours of hypoxia plus glucose and serum deprivation, followed by 1 hour of reperfusion. Cell viability was tested with MTS assay and cardiomyocyte damage was evaluated by Lactate dehydrogenase (LDH) leakage. Mitochondrial membrane potential was determined by tetramethylrhodamine ethyl ester (TMRE) and mitochondrial permeability transition pore (mPTP) opening was measured by calcein/AM, using confocal laser scanning microscopy. In addition, we studied the signaling pathway underlying CRP-mediated ischemia reperfusion injury via western blot analysis.
Compared with the simple OGD/R group, after intervention with 10 μg/ml of CRP, the cell viability decreased markedly (82.36 %± 6.18% versus 64.84% ± 4.06%, P=0.0007) and the LDH leakage had a significant rise [(145.3±16.06) U/L versus (208.2±19.23) U/L, P=0.0122]. We also found that CRP directly activated mPTP opening and reduced mitochondrial membrane potential during ischemia reperfusion. Furthermore, pretreatment with 1 μM of atorvastatin (Ator) before CRP intervention protected cardiomyocytes from ischemia reperfusion injury. Interestingly, the mitochondrial KATP channel opener diazoxide and the mPTP inhibitor cyclosporin A can offset the effects of CRP on cell viability, LDH leakage and the fluorescence intensity values of calcein/AM and TMRE. Moreover, the expression of phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2 was significantly higher after pre-treatment with CRP compared with the value simply through OGD/R (170.4%±3.00% versus 93.53%±1.94%, P<0.0001). Western blot analysis also revealed that Akt expression was markedly activated after co-treatment with Ator and CRP, compared with the level after simply CRP pretreatment (184.2%±6.96% versus 122.7%±5.30%, P=0.0003). The difference was also significant in the change of phosphorylation of ERK between these two groups.
Our results suggested that CRP directly aggravates myocardial ischemia reperfusion injury in myocardial cells and may be mainly mediated by inhibiting mitoKATP Channels and promoting mPTP Opening, but Ator do the opposite. Ator can reduce CRP-induced ischemia reperfusion injury. Furthermore, one of the mechanisms in CRP-induced ischemia reperfusion injury may relate to the sustained activation of the ERK1/2 signaling pathway. Ator protected cardiomyocytes mainly through activating AKt signaling pathway and restraining phosphorylation of ERK signaling pathway.