Mitochondrial Channels/Pore As The Targets For Cardioprotection

Mitochondria play a critical role in both the life and death of cells.When the organism is subjected to various stress stimuli,mitochondrial ATP decreases rapidly,glycolysis induces the accumulation of dangerous substances,activates many intrinsic mechanisms,then induces the events of cell apoptosis and necrosis.Mitochondrial dysfunction plays a central role in mediating both the necrotic and apoptotic components of reperfusion injury.How to protect the mitochondria and heart from injury arouses more and more attention.Mitoehondrial mechanisms have been emphasized as the key point for cardioprotection.There are many types of ion channels and one kind of pore on the mitochondrial inner and outer membranes which are involved in the mechanisms of cardioprotection.The mitochondrial permeability transition pore(abbreviated to mitoPTP)is a non-specific pore across the mitochondrial inner and outer membranes which plays an important role against ischemia and reperfusion injury.It may be the endpoint effector against ischemia and reperfusion injury.The activity of mitoPTP may be regulated by the upstream mitochondrial ion channels.The mitochondrial calcium uniporter(mito Ca²⁺ uniporter)is one of the important pathways by which mitochondria exchange calcium with the cytosol.The uniporter transports calcium from the cytosol into the intra-mitochondrial compartment and balances the calcium level between mitochondrial matrix and cytosol.The two potassium channels in mitochondria—mitochondrial ATP sensitive potassium(mitoK_ATP)channel and mitochondrial calcium activated potassium(mitoK_Ca)channel can maintain the potassium level between mitochondria and cytosol,keeping the mitochondrial volume.Meanwhile it can regulate mitochondrial Ca²⁺uptake and the release of mitochondrial reactive oxygen species(ROS).It is of concern whether these mitochondrial ion channels and the pore are the common pathway for cardioprotection,and whether these ion channels can modulate the activity of mitoPTP.In recent years,more and more interventions which affect cardioprotection have attracted attention.They mainly include ischemic preconditioning,pharmacological preconditioning and ischemic postconditioning.It has been verified that opening of mitoK_ATPchannel,inhibiting the opening of mito Ca²⁺uniporter and inhibiting the opening of mitoPTP are all involved in the mechanisms of ischemic preconditioning. Whether pharmacological preconditioning and ischemic postconditioning play their cardioprotective effects through similar mitochondrial mechanisms is unclear.In our study,we set up three layers of experimental models:the ischemia and reperfusion injury model in isolated heart,the anoxia and reoxygenation injury model in isolated cardiomyocytes and high concentration of calcium induced mitochondrial injury model to observe whether the three different types of interventions:an endogenous cytokine tumor necrosis factorαpreconditioning,an exogenous isoflavone puerarin preconditioning and a nonpharmacological intervention-ischemic postconditioning-can have cardioprotective effects through the common mitochondrial ion channels/pore and the downstream signal conduction pathway.We emphasized the role of mitoPTP,mitoK_Cachannel,mitoK_ATPand mitoCa²⁺uniporter in the cardioprotection of these three interventions,and further investigated the potential upstream or downstream mechanisms.In our experiments,the regional ischemia and reperfusion injury in isolated heart was set up by occluding the left coronary artery for 30 min to produce regional ischemia, releasing the snare for 120 min to produce reperfusion process,and hearts were subjected to global ischemia by stopping the K-H buffer perfusion,while reperfusion was achieved by restarting the perfusion.The left vertricular function was evaluated by recording the left ventricular developed pressure(LVDP)and rate-pressure product (RPP:LVDP×heart rate)and left ventricular end-diastolic pressure(LVEDP) continuously.The lactate dehydrogenase(LDH)activity in coronary flow was assessed using spectrophotometric method.The 2,3,5-triphenyltetrazolium chloride staining method was used to determine the infarct size and the production of formazan, which provides an index of myocardial viability,was measured by absorbance at 550 nm.The absorbance of mitochondria at 520 nm(A₅₂₀)which reflects the opening degree of mitochondrial permeability transition was measured by spectrophotometer.Meanwhile,we set up myocyte anoxia and reperfusion injury model and H₂O₂ induced injury model in cardiomyocytes.Trypan blue exclusion was used as an index of the viability of the ventricular myocytes.Mitochondrial membrane potential fluorescence and ROS fluorescence were measured by laser scanning confocol microscope and spectrofluorometer.We observed,when heart was subjected to ischemia and reperfusion in isolated heart model,pretreatment with endogenous cytokine TNF-αat 10 U/ml significantly raised RPP and CF,attenuated the increase of LVEDP,and reduced the infarct size and LDH release.Atractyloside(Atr),a mitoPTP opener,paxilline(Pax),the K_Cachannel inhibitor and the mito Ca²⁺uniporter opener spermine(Sper)partially canceled the effects of TNF-αon RPP,CF,LVEDP,infarct size and LDH release in I/R hearts.The mitoPTP inhibitor cyclosporin A(CsA)and the K_Cachannel activator NS 1619 significantly elevated RPP and CF,and reduced LVEDP,infarct size and LDH release in contrast to ischemia and reperfusion..The effects of NS 1619 were attenuated in the presence of Atr while the effects of CsA were not altered by pretreatment with Pax.The mitochondria were isolated after the hearts were treated with TNF-α.The decrease at A₅₂₀in the mitochondrial suspension was significantly attenuated compared with that of control mitochondria from untreated hearts.Incubation with Pax and Sper both attenuated the effect of TNF-αon the mitochondria.When heart was subjected to ischemia and reperfusion in isolated heart model,Pue significantly increased the myocardial content of formazan and reduced the release of LDH.Pue pretreatment also raised RPP,±dP/dtmax,and attenuated the increase of LVEDP during reperfusion.The mitoPTP opener Atr,K_Cachannel inhibitor Pax and mitoK_ATPchannel inhibitor 5-HD,all attenuated the effects of Pue.In the pelletting ischemia and H₂O₂ induced injury model,Pue significantly increased the proportion of surviving cells.Co-treatment with 5-HD,Pax,Atr and PKC inhibitor chelerytheline all attenuated the effect of Pue.Pue prevented the depolarization of mitochondrial membrane potential in pelletting ischemia process, and co-treatment with Atr or 5-HD attenuated the effect of Pue.Pretreatment with Pue prevented the enhancement of DCFH-DA fluorescence intensity,and co-treatment with Pax and chelerythrine attenuated the effect of puerarin.When mitochondria were isolated from hearts perfused with Pue for 5 min,the decrease at A₅₂₀was significantly attenuated compared with that of mitochondria from untreated hearts. Pretreatment with 5-HD or Pax both attenuated the effect of Pue on mitochondrial absorbance.Ischemic postconditioning significantly increased the myocardial content of formazan and reduced the release of LDH,raised RPP,±dP/dtmax and coronary flow, attenuated the increase of LVEDP during reperfusion.The K_Cachannel inhibitor Pax and mitoK_ATPchannel inhibitor 5-HD attenuated the effects of postconditioning.Theδ-opioid receptor inhibitor naltrindole andκ-opioid receptor inhibitor nor-binaltorphimine partially abolished the effects of postconditioning.The decrease at A₅₂₀in postconditioning was significantly attenuated compared with that of untreated hearts.In conclusion,the cardioprotection against ischemia and reperfusion injury by endogenous cytokine TNF-αis mediated by inhibiting the mitoPTP opening, inhibiting the mitoCa²⁺uniporter opening,and activating a K_Cachannel.The cardioprotective and antioxidant roles of exogenous drug puerarin are mediated by inhibiting opening of mitoPTP and activating the mitoK_ATPchannel.Meanwhile this is mediated by activating protein kinase C and opening the K_Cachannel.Ischemic postconditioning protects myocardium via inhibiting K_cachannel and mitoK_ATP channel,activatingδ-andκ-opioid receptors are also involved in the mechanisms of postconditioning.The mitoCa²⁺uniporter,K_Cachannel and mitoK_ATPchannel may be upstream from mitoPTP.It is suggested that the mitochondrial ion channels/pores are the common pathway for cardioprotection.The different types interventions including endogenous cytokine preconditioning,exogenous drug and ischemic postconditioning all revealed the mitochondrial ion channels/pores and the downstream signal transduction pathway as their common target.