Differential Expression Of Calpastatin In Isolated Rat Heart Of Ischemia-reperfusion Injury

IntroductionIn the myocardium, the evolution of ischemic damage is characterized by a wide array of functional and structural alterations. Cardiac contraction is blocked immediately at the onset of ischemia,and a prolonged restriction of coronary flow is associated with a progressive rise in diastolic pressure that likely reflects depletion of ATP and other metabolites.When coronary flow is re-established during reperfusion, mechanical activity is severely depressed and the degree of contracture is increased. Because Ca2+ ions play a crucial role in the regulation of cardiac muscle contraction, disturbances in Ca2+ homeostasis have been reported for myocardial ischemia-reperfusion (I/R),These observations have led to the suggestion that a Ca2+-activated, calpain and its endogenous inhibitor calpastatin may be playing a role in I/R.In present study, we observed the change of mRNA and protein expression levels of calpastatin domain L and doman 1-4 to know the pathophysiological roles of calpastatin in ischemia-reperfusion injury.Materials and Methods1. Reagents and equipmentsRT-PCR kit(TaKaRa), Trizol(Gibco,USA), antibody(from Saskatchewan university of England) NP-40, PMSF, APS, TEMED, SDS, methanol, biotin-labeled sheepanti-rabbit IgG, Tween-20, fat-free milk, were from Biosharp company, PCR machine(Bioer), imaging sysem(UVP, USA) etc。2. Animal model and groupsIn these models, myocardial injury was elicited with 30 min of regional ischemia and 120 min of reperfusion in vitro using rat models of myocardial ischemia-reperfusion injury. In these models were anesthetized by i.v. administration of sodium pentobarbital (30 mg/kg), followed by intubation and ventilation with 100%O2 using a positive pressure ventilator. A left thoracotomy was performed, the heart exposed, and a snare placed loosely around a prominent branch of the left coronary artery. The heart was rapidly removed from the chest, mounted on a Langendorff apparatus, and maintained by retrograde perfusion (nonrecirculating) with a modified Krebs'solution (118.5 mM NaCl,4.7 mM KCl,1.2mM MgSO4,1.2 mM KH2PO4,24.8 mM NaHCO3,2.5 mM CaCl2, and 10 mM glucose) at a constant pressure of 80 mm Hg and a temperature of 38.5℃. Perfusate pH was maintained at 7.4 to 7.5 by bubbling with 95% O2,5% CO2.3. RT-PCRTotal mRNA was extracted from rat left ventricle with the method of Trizol,RT-PCR reaction was carried out by RNA PCR Kit(AMV)Ve3.0 according to its instruction.The sequences of the primers were designed by Primer 5.0 software and synthesized by Shanghai Sangon Biological Engineering Technology Sequences of primer see chart 1. 4. Western blotProtein extracts of rat left ventricle were prepared with the similar method as previous reports.The primary antibodies against domain L, were used at 1:400 dilution.HRP-conjugated secondary antibody was diluted 1:6000.The proteins were detected using an ECL detection system.5. Statisitical AnanlysisMeasurements were expressed as mean±SD and statistical analysis was performed by one-way ANOVA test with SPSS 11.5 Software.P<0.05 was regarded as a significant difference.Each experiment was repeated six Animals and material.Results1. To analyze mRNA expression of calpastatin domain L in the condition of myocardial ischemia and reperfusion with RT-PCR. Compared with control group, the mRNA expression of ischemia group (both 45min and 60min) was down-regulated significantly (P<0.05). During reperfusion of 15min, domain L mRNA expression recovered.However, in the group of 60min-ischemia and 30min reperfusion, domain L mRNA was significantly decreased, compared with control.(P<0.05)2. To analyze mRNA expression of calpastatin domain 1-2 and domain 3-4 in the condition of myocardial ischemia and reperfusion with RT-PCR. Compared with control group, both domain 1-2 and domain 3-4 mRNA levels were significantly decreased in ischemia groups(P<0.05).3. To study protein expression of calpastatin domain L in the condition of myocardial ischemia and reperfusion with Western blot. The tendency was the similar with mRNA expression. In the group of 45min-ischemia, the Domain L protein expression was decreased significantly (P<0.05).Conclusion1. During caradiac ischemia and reperfusion, the mRNA expression of calpastatin domain L was down-regulated in ischemia group. During reperfusion of 15min, domain L mRNA expression recovered.2. During cardiac ischemia and reperfusion, the mRNA expression of calpastatin domain 1-2, domain 3-4 were both significantly decreased in ischemia group.3. The protein expression of calpastatin domain L was down-regulated in 45min-ischemia group.