Roles Of The Endoplasmic Reticulum Stress In Neca-Induced Cardioprotection Against Ischemia/Reperfusion Injury

Application of the thrombolytic therapy, percutaneous coronary in tervention, and coronary artery bypass graft surgery has initiated th e new era of reperfusion therapy for the treatment of acute myocardia1infarction. However, despite the fact that reperfusion therapy rest ores the blood supply to reduce or reverse cardiac damage, it also ca uses myocardial injury such as myocardial stunning, arrhythmias, and necrosis, namely myocardial reperfusion injury. Therefore, prevention of reperfusion injury is critical for the salvage of myocardium and improvement of the prognosis.Studies using various animal models have demonstrated that adenos ine and its agonists protects the heart form ischemia/reperfusion inj ury and the protective effects are inhibited by adenosine receptor b1ockers. Adenosine is a product of the energy metabolism, generated fr om ATP degradation. A short period of cardiac ischemia leads to ATP d egradation followed by accumulation of adenosine in myocardium. Adeno sine can trigger or mediate ischemic preconditioning and postconditio ning to prevent reperfusion injury. There are four adenosine receptor s including A1, A2a, A2b and A3receptors, among which A2receptors ma y play an important role in postconditioning to prevent reperfusion i njury. Recent studies have shown that some adenosine agonists can con fer cardioprotection by targeting A2receptors, but the exact mechani sm underlying the protection remains to be elucidated. Hausenloy et al. proposed that the ultimate target of cardioprote ctive signalings is the mitochondrial permeability transition pore (m PTP), and a number of drugs protect the heart from reperfusion injury by modulating the mPTP opening. Glycogen synthase kinase3β (GSK-3β) is-a multifunctional serine/threonine kinase, has a very importan t biological functions in the body. Studies have shown that GSK-3β i s a common target of many cardioprotective signal transduction pathwa ys, and inhibition of the activity of GSK-3β can prevent the mPTP op ening, thereby reducing reperfusion injury.Endoplasmic reticulum stress (ERS) is a pathological process of s ubcellular organs caused by the internal and external cellular envir onmental changes leading to aggregation of unfolded protein in the e ndoplasmic reticulum and destruction of calcium homeostasis, and dis order of physiological functions. ERS is the cellular initial reacti on to stress, which further mediates mitochondrial and nuclear stres ses. Recent studies have demonstrated that ERS plays a role in myoca rdial reperfusion injury. Glucose-regulated proteins (GRP78, GRP94) are the chaperones of the endoplasmic reticulum, and serve as the ma rkers of ERS. Martindale et al. found that ischemia followed by repe rfusion increased the levels of GRP78and GRP94in neonatal cardiomy ocytes, indicating that ischemia/reperfusion induces ERS. Zhang et a1. reported that Ghrelin can protect in vitro rat hearts subjected t o ischemia and reperfusion through inhibiting ERS However, whether a denosine protect the heart from reperfusion injury through modulatin g ERS is still nuclear.Objective:To determine whether NECA, an adenosine receptor agoni st, induces cardioprotection against reperfusion injury by inactivati ng GSK-3β and thereby inhibiting the mPTP opening through modulation of ERS.Methods:Study ⅠRat heart tissue-derived H9c2cells were exposed to H202(800μM) for20min to trigger mitochondrial oxidatives lesions. Mitochondrial membrane potential (△Ψm) was detected by staining cells with tetra methylrhodamine ethyl ester (TMRE) and the mPTP opening was examined with changes in△Ψm. Phosphorylion of GSK-3β at ser9, VASP at ser239and GRP94expression were determined with immunofluorescence stainin g and western blot.Study ⅡMale Wistar rats weighing250-300g were used to isolate heart s and hearts were mounted on Langendorff apparatus and the left coron ary artery was ligated to induce ischemia. Rats were randomly divided into3groups:control (ischemia/reperfusion, I/R), NECA (I/R+NECA) and ERS inhibitor TUDCA (I/R+TUDCA). NECA and TUDCA were dissolved in the perfusion solution, respectively. Cardiac tissue samples were collected from the risk zone10,30,60, and120min after the onset of reperfusion. Infarct size was measured with TTC staining. Western blot analysis was used to determine GRP94expression and phosphoryla tion of GSK-3β. Transmission electron microscope was used to evaluat e alterations of the myocardial ultrastructures in the setting of isc hemia/reperfusion.Results:Study I①Studies with laser confocal microscopy showed that the treatmen. t of cells with H2O2(800μM) for20min reduced the TMRE fluorescenc e intensity of cardiac H9c2cells (36.7±3.7%of control). In contr ast, NECA (0.01μM-10μM) at different concentrations suggested an obvious inhibition of TMRE fluorescence intensity weakening, with the peak at0.1μM (81.0±1.1%), indicating that NECA can inhibit the mPTP opening caused by oxidative stress.②Western blot analysis showed that compared with the control gro up (97.1±1.3%)(123.8±4.6%), NECA increased GSK-3β (Ser9) phos phorylation but decreased GRP94expression with the peak at0.1μM (138.8±5.2%)(55.7±1.5%), suggesting that NECA can inactivate GSK-3β and ERS may be involved in NECA-induced cardioprotection.③Western blot analysis revealed that the effect of NECA (0.1μM) on GSK-3β (Ser9) phosphorylation was blocked by the PKG inhibitor K T5823(0.1μM), suggesting that NECA may inactivate GSK-3β (Ser9) pho sphorylation and exert its cardioprotection through the cGMP/PKG sign aling pathway. Compared to the control group (102.1±2.1%) and (101.4±4.2%), cells treated with NECA (0.1μM) enhanced phosphorylatio n of VASP (Ser239)(156.9±1.3%) significantly but reduced GRP94expr ession (65.8±2.2%), an effect that was reversed by KT5823(0.1μM)(97.3±0.5%) and (85.9±2.1%). These results suggest that NECA ma y induce cardioprotection though inactivating GSK-3β via ERS and cGM P/PKG signaling pathway.④Western blot analysis also showed that NECA (0.1μM) can enh ance GSK-3β (Ser9) phosphorylation and this effect was prevented by the ERS inductor2-DG (20mM). Compared to the control group (101.6±3.9%) and (116.2±3.3%), giving cells doses of NECA (0.1μM) indicated a significant enhance in phosphorylation of GSK-30(Ser9)(159.7±15.0%), whereas the GRP94expression is significantly reduc ed (60.5±2.6%), which were inhibited by2-DG(20mM)(123.5±9.1%) and (101.2±3.4%), suggesting that NECA may trigger cardioprot ection by inactivating GSK-3β (Ser9) through prevention of ERS.⑤Western blot analysis further showed that H2O2reduced GSK-3β p hosphorylation but increased GRP94expression, an effect that was pre vented by NECA(0.1μM), implying that NECA induces cardioprotection by inactivating GSK-3β and preventing ERS.Study Ⅱ①Compare to the control, both NECA and TUDCA decreased myocardia1infarction (26.5±4.9%and36.0±5.1%respectively), indicati ng that ERS is involved in NECA-induced cardioprotection.②Western blot analysis showed that both NECA (0.1μM) and TUDCA reversed GRP94expression at reperfusion and increased GSK-3β phosp horylation, compared to the control (97.1±1.3%and123.8±4.6%respectively), suggesting that NECA protect the heart by inhibiting GSK-3β and ERS.③Transmission electron microscopy analysis showed that compared to the control, NECA and TUDCA reduced mitochondrial swelling and the endoplasmic reticulum expansion, suggesting that NECA can protect th e heart by preventing the mPTP opening through modulation of ERS.Conclusions:①NECA protects the heart from reperfusion injury by preventing t he mPTP opening through inactivation of GSK-3β via ERS inhibition.②The cGMP/PKG signaling is responsible for GSK-3β inactivatio n by NECA.