Role Of Mitochodrial Calcium Uniporter In Cardioprotection By Ischemic Preconditioning And Its Mechanism

Ischemia/reperfusion injury of myocardium is a phenomenon often happened in clinical practice. In 1986, Murry et al. found that brief exposures of the myocardium to ischemia can protect potently the heart against subsequent severe ischemia, a phenomenon called ischemic preconditioning (IPC). Subsequent research found that not only local ischemic preconditioning but also ischemic preconditioning in a distant organ from heart can decrease the ischemia/reperfusion injury, this is called remote preconditioning (RPC). No matter what kind of way, ischemic preconditioning is an effective measure to protect against myocardial ischemia/reperfusion injury. Although there have been intensive studies on the mechanisms of IPC, the exact mechanism is still not fully elucidated.In recent years, it was found that mitochondrial Ca²⁺ plays important role both in myocardial ischemia/reperfusion injury and in cardioprotection by ischemic preconditioning. Not only mitochondrial Ca²⁺ overload is closely related to myocardial ischemia/reperfusion injury, but also mitochondrial Ca²⁺ homeostasis is closely related to cardioprotection by ischemic preconditioning. Mitochondrial calcium uniporter (MCU) is an important Ca²⁺ transporter located in the mitochondrial inner membrane, and can transport Ca²⁺ from cytosol to mitochondrial matrix, plays important role in modulating energy metabolism, mitochondrial Ca²⁺homeostasis and Ca2+signaling in cell, so we hypothesized that MCU may play important role in myocardial ischemia/reperfusion injury and cardioprotection by ischemic preconditioning.In order to test our hypothesis, in the present study, we investigated the role of MCU in cardioprotection of ischemic preconditioning, including classical ischemic preconditioning and remote preconditioning, by using in vitro and in vivo experiments.OBJECTIVES1. To observe the role of MCU in myocardial ischemia/reperfusion injury;2. To observe the role of MCU in cardioprotection by classical ischemic preconditioning and remote preconditioning;3. To investigate the modulation of mitochondrial permeability transition pore (MPTP) by MCU, and the role of this modulation in cardioprotection by ischemic preconditioning;4. To investigate the role of endogenous activation of opioid receptor in cardioprotection by remote preconditioning, and the role of MCU and its MPTP modulation in this process.METHODS1. Langendorff perfusion apparatus was used to record the contractile function and heart rate of isolated rat hearts.2. In vitro and in vivo ischemia/reperfusion models of heart were used to observe the role of MCU activity in ischemia/reperfusion injury.3. Local ischemic preconditioning model and limb remote preconditioning model were used to observe the role MCU activity in cardioprotection by ischemic preconditioning.4. The 2,3,5-triphenyltetrazolium chloride staining method was used to determine the infarct size of heart.5. The lactate dehydrogenase (LDH) activity and the marker enzyme activities of mitochondria were assessed by using spectrophotometric method.6. Electron microscopy was used to define the structure of isolated heart mitochondria.7. Atomic absorption spectrophotometry was used to measure the mitochondrial calcium content.8. Mitochondrial swelling method was used to measure the MPTP opening in isolated mitochondria.9. Laser scanning confocal microscope was used to measure MPTP opening in isolated cardiomyocytes.10. Radioimmunoassay method was used to detect the levels of dynorphin and met-enkephalin in plasma.RESULTSPart 1: Role of mitochondrial calcium uniporter in myocardialischemia/reperfusion injury1. In isolated perfused rat heart and intact rat, inhibition of MCU by using MCU inhibitor ruthenium red (RR) (5 umol/L in vitro and 2.5 mg/kg in vivo) enhanced the contractile recovery of ventricle during reperfusion, and decreased the infarct size and activities of lactate dehydrogenase (LDH) in coronary flow and plasma induced by ischemia/reperfusion.2. In isolated perfused rat heart and intact rat, activation of MCU by using spermine (Sper) (20 umol/L in vitro and 5 mg/kg in vivo) decreased the contractile recovery of ventricle during reperfusion, and increased activities of LDH in coronary flow and plasma induced by ischemia/reperfusion.3. In intact animal, the mean artery pressure (MAP) and heart rate decreased in all groups, and recovered to different levels in different groups, but there were no statistically significant difference between groups.Part 2: Role of mitochondrial calcium uniporter activity in ischemic preconditioning1. Ischemic preconditioning (IPC) greatly decreased the infarct size induced by ischemia/reperfusion and the activities of LDH in coronary effluent, and the contractile recovery of ventricle was greatly enhanced during reperfusion.2. The effect of IPC was abolished by MCU activator spermine (20 umol/L), and pretreatment with MCU specific inhibitor Ru360 (10 umol/L) or pretreatment with MCU inhibitor RR (5 jjmol/L) mimicked the cardioprotection by IPC.3. The limb remote preconditioning greatly decreased the infarct size and the activities of LDH in plasma induced by ischemia/reperfusion. Pretreatment with MCU activator Sper (5mg/kg) before RPC procedure abolished the cardioprotective effect of RPC.4. The MAP and heart rate decreased in all groups, and recovered to different levels in different groups, but there were no statistically significant difference between groups.Part 3: Modulation of mtiochondrial permeability transition pore by mitochondrial calcium uniporter1. Under electron microscope, isolated mitochondria showed intact membranes and dense matrix space before swelling, but disruption of the outer membrane and disappearance of the cristae were evident after swelling.2. The activities of succinate dehydrogenase, the inner membrane marker enzyme of mitochondria, and monoamine oxidase, the outer membrane marker enzyme ofmitochondria were higher in isolated mitochondria than that in the cytosol.3. In isolated mitochondria from heart, the treatment of MCU inhibitor RR (1 fxmol/L) or Ru360 (10 umol/L) inhibited the MPTP opening induced byCa2+ (200 umol/L). The treatment with MPTP activator Sper (20 umol/L) accelerated the opening of MPTP, and the latter effect was blocked by cyclosporine A (CsA, 0.2 umol/L), a specific MPTP inhibitor.4. In isolated calcein loaded cardiomyocytes, MCU inhibitor RR (5 umol/L) inhibited the decrease of calcein fluorescence intensity;however, MCU activator Sper (20 umol/L) increased the loss of calcein fluorescence intensity in mitochondria of cardiomyocytes.5. Ischemia/reperfusion and treatment with Sper greatly increased the mitochondrial Ca2+content, and RR (5 umol/L) and IPC significantly inhibited the mitochondrial Ca2+ increase. The effect of IPC was blocked by Sper.6. Inhibition of MCU by RR (5 umol/L) during reperfusion greatly decreased the ROS production induced by ischemia/reperfusion, and activation of MCU with Sper (20 umol/L) greatly increased the mitochondrial ROS production.Part 4: Role of mitochondrial calcium uniporter-mitochondrial permeability transition pore modulation in cardioprotection by ischemic preconditioning1. The effect of MCU activation with Sper on cardioprotection of IPC was abolished by CsA, a specific MPTP inhibitor.2. The decrease of absorbance in 520 nm in mitochondria isolated from preconditioned rat hearts was much less than the mitochondria from un-preconditioned rat hears.3. Pretreatment with kappa opioid receptor agonist U50,488H (10 mg/kg) or endogenous agonist of kappa opioid receptor dynorphin (300 pg/ml) significantly decreased the infarct size and lactate dehydrogenase level in plasma. The dynorphinlevels in plasma increased greatly after RPC procedure.4. The cardioprotection of RPC and U50,488H were blocked by pretreatment with nor-BNI (10 mg/kg), a specific kappa-opioid receptor antagonist.5. The 8 opioid receptor antagonist naltrindole (1 mg/kg) had no evident effect on the cardioprotection by RPC. The endogenous 8 opioid receptor agonist met-enkephalin in plasma showed no evident changes.6. Pretreatment with specific MPTP inhibitor CsA (10 mg/kg) abolished the effects of nor-BNI or Sper. Administration of atractyloside (5 mg/kg), a specific activator of the pore, attenuated the effects of RPC and U-50,488H.CONCLUSIONS1. Inhibition of mitochondrial calcium uniporter activity can protect myocardium against ischemic/reperfusion injury. The cardioprotection by ischemic preconditioning may be related with attenuation of mitochondrial calcium uniporter activity.2. The mitochondrial calcium uniporter-mitochondrial permeability transition pore modulation is one of the possible mechanisms of cardioprotection by ischemic preconditioning.3. Mechanism of cardioprotection by limb remote preconditioning involves release of endogenous kappa opioid receptor agonist and subsequent mitochondrial calcium uniporter-mitochondrial permeability transition pore modulation pathway.