Contribution Of Calpain To Myocardial Apoptosis Induced By Oxidative Stress In Mouse Ischemia/Reperfusion

Objective:In the present study, we aimed to explore the effects of calpain and itsinhibitor PD150606(PD) on oxidative stress induced myocardial apoptosis inmouse Ischemia/Reperfusion injury by using both in vivo animal and in vitrocell models.Method:Adult C57BL/6male mice were randomly divided into three groups.Control group, I/R group and PD plus I/R group. In the PD plus I/R group,animals were pretreated with PD150606(1mg/kg, i.v.) for30min, before theywere subjected to the ischemia for45min by ligating the left anteriordescending (LAD) coronary artery and subsequent reperfusion for3h. About3h after reperfusion, the area at the risk of the potential I/R injury andensuing infarct size per se were assessed by Evens Blue and TTC dye.Apoptosis in the heart tissue were assessed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay (TUNEL)、cytosolic cytochrome c levels determined by an ELISA kit and caspase-3activity in the cardiomyocytes assayed by a fluorimetric kit.The ventricularmyocytes of adult C57BL/6mice were isolated and cultured. Thecardiomyocytes were randomly divided into three groups, control group, H/Rgroup and PD plus H/R group. To achieve a suitable simulated H/R,cardiomyocytes were incubated in serum-free and glucose-free medium underconditions of hypoxia for20min, then they were returned to a normoxic environment with the normal culture medium at37°C for2hours. In PD plusH/R group, before the cardiomyocytes were subjected to H/R, they wereincubated in normal culture medium contains PD150606(25μM) for30min.The viability of cultured cardiomyocytes was examined by using trypan blueexclusion test. The cytosolic cytochrome c levels were determined by anELISA kit. The fluorimetric caspase-3kit was assayed to measure its activityin the cardiomyocytes. The Suc-LLVY-AMC was used as a fluorescencesubstrate to measure of calpain activity of cardiomyocytes. Formation of2-hydroxyethidium (2-EOH) in cardiomyocytes was measured by HPLC toquantify ROS production.Result：1in vivo1.1Myocardial infarction area: Although there is no obvious changes inthe regions at risk between these two distinct groups, PD150606pretreatmentcan significantly reduce the ratio of the infarct to risk regions (P<0.05).1.2TUNEL: The ratio of TUNEL-positive cardiomyocytes wassignificantly increased in I/R group (P<0.01), while the TUNEL-positiverate of PD+I/R group was significantly lower than the I/R group (P<0.05).1.3Cytoplasm Cyt C level：When compared with control group, thecytoplasm Cyt C in H/R group were increased (P <0.01); however，thePD+I/R group has a lower Cyt C level in the cytoplasm（P<0.05）.1.4Caspase-3activity: The caspase-3activity of I/R group wereincreased significantly (P<0.01), however, it was prevented in PD+I/R group.(P<0.05).2in vitro2.1Cell viability: Both viable cardiomyocytes (i.e. unstained and rod-shaped) and nonviable cardiomyocytes (i.e. blue stained and spherical)were visualized under microscopy, the viable rod-shaped cells in H/R groupwere decreased when compared with the control group, while the calpaininhibition by PD150606prevented the rod-shaped cells against H/R-induceddeath.2.2Calpain activity: After H/R, the Calpain activity of cardiomyocyteswere significantly increased (P<0.01), while PD150606pretreatment caneffectively inhibit Calpain activity increase caused by H/R.2.3Cardiomyocytes apoptosis: When compared with control group, thecell cytoplasm Cyt C level of H/R group were significantly increased(P<0.01), while PD150606pretreatment can significantly decreased thischange (P<0.05).2.4Caspase-3activity: The caspase-3activity of cardiomyocytes in H/Rgroup were significantly increased (P<0.01), however, PD150606preventedthe caspase-3activity increased (P<0.05).2.5Assessment of superoxide contents: The generation of superoxide inI/R group were significantly increased (P<0.01), while H/R induced ROSproduction can be inhibited by PD150606pretreatment.Conclusion：It is plausible that I/R-induced myocardial apoptosis is prevented by thePD150606inhibition of calpains, as a potent cytoprotective strategy of hearttranslational medicine.