Rosuvastatin Postconditioning Protects Isolated Hearts Against Ischemia-Reperfusion Injury

Background:With the development of social economy,arteriosclerotic cardiovascular disease has become the first rank of fatal diseases in China.The pathology of acute coronary syndrome is rupture or erosion of coronary atherosclerosis plaque which lead to complete or incomplete occlusive thrombosis and myocardial ischemia.Revascularization is the key to restore blood flow.But after restoring blood supply,ischemic tissues produce excess free radicals which cause more severe injury to the ischemia tissues.That is so called ischemia-reperfusion injury.A number of large clinical trials have shown statins are associated with decreased cardiovascular mortality in both primary and secondary prevention in coronary artery disease.Since statin cardioprotection is also observed in patients with normal cholesterol levels,it is supposed that statins may have a large spectrum of protective benefits in addition to their cholesterol-lowering effect.Both stains preconditioning and postconditioning are promising strategies of obtaining protection against myocardial ischemia/reperfusion?I/R?injury.Accumulating evidence suggests that opening of the mitochondrial permeability transition pore?mPTP?plays a crucial role in myocardial I/R injur.Glycogen synthase kinase-3??GSK-3??regulates a variety of cellular functions besides glycogen synthase and acts as an important component in the myocardial response to I/R injury.Several prosurvival kinases inhibit its activity by phosphorylation at Ser⁹.The inactivation of GSK-3?was reported to be involved in inhibition of mPTP openin.Previous study had shown that statins postconditioning reduced infarct size through activating phosphatidylinositol-3-kinase-protein kinase B?PI3K–Akt?signaling cascadeAims:GSK-3?and mPTP play an important role in myocardial ischemia reperfusion injury.The aim of this study is to investigate whether postconditioning with rosuvastatin is able to reduce myocardial ischemia-reperfusion injury and clarify the potential mechanisms.Methods:Two different protocols were performed.Protocol I aimed at evaluating infarct size analysis,biochemical determination and cardiac function measurement.Protocol II was used to assess western blot analysis and Ca²⁺-induced mitochondrial permeability transition.Sham hearts were not subjected to ischemia.Protocol ?:All hearts were stabilized for 15 min and subjected to 30 min of coronary occlusion followed by 60 min of reperfusion,the experimental groups receive one of the following treatments at the onset of reperfusion:1)I/R control?n=8?;2)rosuvastatin?n=8 each for 1,10,25 and 50 nmol/L,n=16 for 5 nmol/L?;and 3)PI3K inhibitor?LY294002?15?mol/L in the presence or absence of 5 nmol/L rosuvastatin?n=8?.Protocol ?:All hearts underwent 30 min of coronary occlusion followed by 10 min of reperfusion,the hearts were randomized to receive either 5 nmol/L rosuvastatin in the presence or absence of 15?mol/L LY294002 or vehicle?n=10 for each group?.At the end of the 10 min reperfusion,hearts were excised for further assessmentAt the end of reperfusion,Infarct size was stained using the TTC method.Infarct size was expressed as a percentage of the area at risk region.The cardiac parameters of left ventricular end-diastolic pressure?LVEDP?,left ventricular-developed pressure?LVDP??difference between left ventricular end systolic pressure and end diastolic pressure?,and maximal rise rate of left ventricular pressure?dP/dt max?were monitored at baseline,30 and 60 min after reperfusion.At the end of 60 min of reperfusion,both the activity of SOD and MDA in the supernatant,and the concentration of LDH and CK-MB in the coronary effluent liquid were determined by special kits.ROS generation was evaluated using fluorescence 2',7'-dichlorofluorescin diacetate?DCFH-DA?method.Isolation of mitochondria from the risk area was conducted using the Mitochondria Fractionation Kit.The mPTP opening was assessed following in vitro Ca²⁺overload.Western blot analysis was performed to determine the activity of signaling pathway.Results:1 Rosuvastatin showed no significant protection against I/R injury at 1 nM.Over the concentration range of 5 to 10 nM,rosuvastatin significantly attenuated infarct size compared to controls,peaking with 5 nM?25.69±3.75%vs 40.04±4.93%,P<0.05,?.However,this protection faded with the incremental dosage and even reversed to detrimental effect at 50 nM?47.75±5.27%vs 40.04±4.93%,P<0.05?.Parallel to these protective effects,in the 5 nM rosuvastatin-treated hearts,a significant reduction of CK-MB and LDH release was detected?P<0.05?.LY294002 abolished these protection produced by the beneficial effect of rosuvastatin.There were no statistically significant differences in hemodynamics between groups during baseline and coronary occlusion.During the reperfusion period,recovery of left ventricular systolic contractility and diastolic relaxation were significantly improved in rosuvastatin groups?P<0.05?.LY294002 significantly attenuated the increase in LVDP and dP/dt max and the decrease in LVEDP observed in rosuvastatin group2 MDA and ROS generation were remarkably increased while SOD activity was decreased in I/R control group compared with sham group?P<0.05?.Rosuvastatin postconditioning significantly reduced MDA and ROS level and increased SOD activity compared with I/R control group?P<0.05?.LY294002 abolished the effect of rosuvastatin on the oxidative stress.3 Rosuvastatin caused a significant increase in the phosphorylation of both Akt and GSK-3?compared with those in I/R control group?P<0.05?.The total amount of Akt and GSK-3?remained unchanged under different stimulations.Moreover,LY294002 did not modify the level of phosphorylated forms of Akt and GSK-3?detected in I/R hearts but attenuated the effects produced by rosuvastatin.The phosphorylation of Akt and GSK-3?were not activated in the sham group.4 In the sham group,the cumulative amount of Ca²⁺required to open the mPTP averaged 123±26?mol Ca²⁺/mg mitochondrial proteins.This concentration was reduced to 32±10?mol Ca²⁺/mg in mitochondria isolated from I/R control group?P<0.05 vs sham group?,suggesting that mPTP was more likely to open under the ischemia-reperfusion condition.Rosuvastatin significantly increased the amount of Ca²⁺overload required for mPTP opening to 90±18?mol Ca²⁺/mg?P<0.05 vs I/R control group?,indicating that rosuvastatin delayed mPTP opening.Importantly,rosuvastatin itself did not modify mPTP opening when administrated in sham group.Meanwhile,the inhibition of mPTP opening induced by rosuvastatin was attenuated by LY294002.Conclusion:Rosuvastatin administration upon reperfusion resulted in a concentration-dependent biphasic response,with cardioprotection exerted between a concentration range of 5 and 10 nM and potential detriment at a higher concentration of 50 nM.Rosuvastatin prevents myocardial ischemia-reperfusion injury by inducing phosphorylation of PI3K-Akt and GSK-3?.Rosuvastatin administration activates phosphorylation of Akt and GSK-3?,enhances the resistance of mPTP,and this is regulated by PI3K-Akt-GSK-3?pathway.In all,rosuvastatin reduced the I/R injure through the inhibition of oxidative stress regulated by PI3K-Akt pathwaypreventing oxidative stress and subsequent inhibition of mPTP opening.