Inhibiting Calpain Activity Attenuates Myocardial Ischemia/reperfusion Injury In Anesthetized Rat

Background: Ischemia/reperfusion injury often occurs in the treatment of acutemyocardial ischemia and heart surgery. However, the underlying mechanism is still notfully elucidated. Our previous studies demonstrated that inhibition of calpain reduces celldeath induced by ischemia/reperfusion in isolated rat heart, so it is necessary to determinewhether calpain is a key molecule mediating cell injury in the intact rat heart. In addition,as cell surviving molecule Akt is also activated by Ca2+in neurons, and calpain inendothelial cells, it is also necessary to obseve the possible role of Akt in calpain-mediatedheart injury.Methods: Male SD rats weighing250~300g were randomly divided into fourgroups as follows,(1) control group (Con), the rats were anesthetized and left thoracotomywas performed with no left coronary artery ligation;(2) ischemia/reperfusion group (IR),the rats underwent30min of coronary artery occlusion followed by120min ofreperfusion;(3) MDL28170group (MDL), the rats received an intravenous bolus injectionof10mg/kg MDL2810, which was given10min before ischemia/reperfusion.(4)MDL28170control group (MDL-C), the same experimental protocol as control group was performed, only a bolus of injection of MDL2810was given intravenously.At the end of experiments, the heart slices were stained with TTC and Evans-blue,and the infarct size was expressed as the ratio between infarct size and area at risk(IS/AAR). The concentration of LDH and cTnI in plasma were measured by ELISA kit.TUNEL staining was performed to evaluate the apoptotic index, and the activity ofcaspase3was measured by colormetric assay kit. The activity of Akt was measured bydetecting the phosphorylation on threonine308(Thr308) and serine473(Ser473) residue.The activity of GSK-3, a downstream target of Akt, was measured by detecting thephosphorylation on serine9(Ser9) residue.Results: Compared with control group, the hearts subjected to30min ischemia/2hreperfusion exhibited a marked increase in infarct size, plasma LDH and cTnIconcentration, caspase3activity and apoptotic index. MDL28170did not produce anyeffects in the control hearts; however, it significantly reduced the infarct size, plasma LDHconcentration, caspase3activity and apoptotic index, suggesting that activation of calpainresults in cell death in myocardial ischmemia/reperfusion. In addition, MDL28170did notsignificantly reduce the plasma cTnI concentration, but there is a certain trend.Compared with control group, the hearts in IR group had high levels of Aktphosphorylation on Ser308and Thr473and GSK-3phosphorylation on Ser9. MDL28170itself had no effect in control hearts; however, it significantly reduced the phosphorylationof both Akt and GSK-3in ischemia/reperfused hearts. These data suggest that the cellsurviving molecule Akt is the downstream target of calpain.Conclusions: Our data demonstrate that inhibiting calpain activity protects againstmyocardial ischemia/reperfusion injury in vivo. Our data also provide evidence suggestingthat calpain activates Akt. The pathophysiological implications need to be clarified.