Study On Mechanism Of Calpain Mediated Apoptosis In Cardiomyocytes Induced By Ischemia-reperfusion

Objective:Calpains have been compliciated in myocardial ischemia-reperfusion(I-R) injury. The two major members,μ- and m-calpain, are ubiquitousexpressed in mammalian. However, wheather both of them are avtivated inmyocardium during I-R remains unclear. The mitochondrial permeabilitytransition pore (mPTP) opens in response to calcium overload, andsubsequently triggers apoptotic cell death after reperfusion. However, themechanistic link between calpain activity, mPTP opening and apoptosis incardiomyocytes during I-R remains to be elucidated. This study wasundertaken to identify the activated members of calpain family, and toinvestigate whether the activation of calpain is associated with alterations inmPTP and subsequent apoptotic cell death in cardiomyocytes during I-R.Methods:Primary cultured neonatal C57BL/6 mouse cardiomyocytes weredeprived of oxygen and glucose to simulate ischemia and restored oxygenand sugar supply to simulate reperfusion (simulated I-R injury). Toinvestigate the time course of calpain activation during I-R, cells were randomly divided into five groups, including normal, ischemia for 6 hwithout reperfusion and ischemia for 6 h followed by 3 h, 6 h, 12 hreperfusion. Cell survival was assessed by trypan blue exclusion test. Thefodrin breakdown product (FBDP) and the N-terminal domains of thecatalytic subunit ofμ- and m-calpain in cardiomyocytes was detected byusing western blot analysis. To investigate the mechanistic link betweencalpain activity, mPTP opening and apoptosis during I-R, cells wererandomly divided into four groups, including control group, PD group inwhich cells were treatd with PD150606, a specific inhibitor of calpain, I-Rgroup and PD+I-R group in which cells were treated with PD150606 beforeI-R. Cell survival was assessed by trypan blue exclusion test. Apoptosis incardiomyocytes was determined by terminal deoxyribonucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay and Caspase-3activity analysis. Opened mPTP of cardiomyocytes were assessed by usingthe calcein–cobalt method and mitochondrial membrane potential (Δψm) byimaging cells loaded with JC-1.Results:Cell survival in group in which cells underwent ischemia for 6 hfollowed by 12 h reperfusion was lower than cells underwent ischemia for 6h without reperfusion (65.68±3.78% vs. 81.70±3.91%, p<0.05). Compared with normal or ischemia for 6 h without reperfusion groups, more FBDP wasdetected in groups in which cells underwent ischemia for 6 h followed by 3 h,6 h or 12 h reperfusion. The autolysis of the N-terminal domains of thecatalytic subunit ofμ-calpain was observed in groups in which cellsunderwent ischemia for 6 h followed by 3 h, 6 h or 12 h reperfusion.However, The autolysis of the N-terminal domains of the catalytic subunit ofm-calpain was not observed in any groups. I-R decreased cell survival(Compared with control or PD group, p<0.01), an effect was alleviated byPD150606 pretreatment (Compared with I-R group, p<0.01). Consistently,The ratio of TUNEL-positive cardiomyocytes and Caspase-3 activity wassignificantly less in PD+I-R compared with I-R group ( p<0.05). OpenedmPTP was detected in I-R group, however, it was prevented in PD+I-Rgroup. Compared with control or PD group,Δψm was lower in I-R group( p<0.01). However, the loss ofΔψm induced by I-R was prevented byPD150606 pretreatment (p<0.05).Conclusion:As one of the major members of calpain family,μ-calpain was activatedin cardiomyocytes during I-R. Inhibition of calpain prevented openging ofmPTP and declining ofψm, thus, alleviated apoptotic cell death.