Role Of Reverse Mode Na⁺/Ca²⁺ Exchanger In The Cardioprotection Of Ischemic Preconditioning And Its Underlying Mechanism

Ischemic preconditioning (IP) is a phenomenon in which brief exposures of myocardium to ischemia render it more resistant to a subsequent and more severse insult, termed index ischemia. IP protects the heart against infarction and incidence of arrhythmias caused by ischemia and reperfusion. But the underlying mechanisms of IP remain unidentified. It is now known that a transient increase in cytosolic Ca²⁺ during preconditioning contributes to all these protective effects. It has been demonstrated that the reverse mode of Na+/Ca²⁺ exchanger (NCX) is activated during ischemia, contributing to intracellular Ca²⁺ overload and thus causing cardiac injury. In essence, IP are several procedures of short ischemia/reperfusion (I/R) in pathophysiological situation. We therefore hypothesized that increased reverse mode NCX activity during preconditioning may induce cardioprotection.It has been found many end-effectors of IP. They play the central role in protecting structure and function of Mitochondrion. Many studies have been demonstrated that Mito KATP and Mito KCa act as trigger and/or mediator in IP and pharmacological preconditioning. Based on these finding, we hypothesized that both Mito KATP and Mito KCa channels are involved in the cardioprotective effects of prior stimulation of the reverse mode NCX.Objective1. The activity of reverse mode NCX was assessed by withdrawal extracellular Na+ and changes of [Ca²⁺]i were measured with Fura/AM.2. To observe the effcets of E4031 on the changes of [Ca²⁺]i and its specificity for reverse mode NCX.3. To evaluate the role of reverse mode NCX in the cardioprotection of MIP in rat ventricular myocytes.4. In perfused isolated rat heart, to observe the effect of prior stimulated reverse mode NCX on infarct size and arrhythmias caused by I/R.5. To evaluate the hypothesis that Mito KATP and Mito KCa channels are involved in the cardioprotective effects of prior stimulation of the reverse mode NCX.Methods1. Single ventricular myocytes were enzymatically isolated and loaded with Fura-2/AM. Withdrawal extracellular Na+ and changes of [Ca²⁺]i were measured.2. Cells were pretreated with ryanodine and thapsigargin to block the sarcoplasmic reticulum function. Administered nifedipine (inhibitor of L-type calcium channel) or Ni2+/KB-R7943 (inhibitor of reverse mode NCX) and the changes of [Ca²⁺]i were measured.3. pretreated with E4031, the activity of reverse mode NCX was assessed by withdrawal extracellular Na+ and changes of [Ca²⁺]i were measured with/without KB-R7943.4. Myocytes were subjected to metabolic inhibition preconditioning for 30 min. E4031 pretreatment was administered to the electrically stimulated ventricular myocytes for 10 min. After that, the cells were perfused with normal Tyrode solution with 0.2% bovine serum albumin for 10 min before they were subjected to severe metabolic inhibition for 8 min followed by reperfusion with normal solution for 10 min. KB-R7943 was given 5min before and during metabolic inhibition preconditioning or E4031 pretreatment.5. In perfused isolated rat heart, the ischemia or reperfusion by was occluding or loosing the coronary artery. IP was produced by 2 cycles of 5min regional ischemia followed by 5min reperfusion. Assessed the cardioprotection by infarct size and arrhythmia scores. E4031 was perfused with different concentration and for different time. KB-R7943 was perfused for a period of 5 min before the first ischemic episode to 5 min after the second ischemic episode, or E4031 pretreatment.6. 5-HD (a selective blocker of the mitoKATP), or paxilline (a selective blocker of the mitoKCa ) was either given for 30 min before 30 min of ischemia, or given from 5 min before 30 min of ischemia until 10 min after reperfusion. KB-R7943 was administered from 10 min before to 10 min after one cycle of 10 min treatment with diazoxide (a selective activator of the mitoKATP), or NS1619 (a selective activator of the mitoKCa ). Assessed the cardioprotection by infarct size and arrhythmia scores.Results1. [Ca²⁺]i was increased by the increasing of [Ca²⁺]o after ventricular myocytes exposed to Na+-free solution (NMDG solution), and returned to baseline following washout.2. Nifedipine did not affect NMDG solution-induced increases of [Ca²⁺]i , while both Ni2+ and KB-R7943 almost completely blocked the effect, an indication of the reverse mode NCX.3. E4031 significantly increased the change of [Ca²⁺]i upon NMDG, which was also blocked by KB-R7943.4. In isolated ventricular myocytes, MIP significantly increased the amplitude of twitch contraction. Pretreatment with E4031 also significantly increased the cell contraction, and the effect of MIP or E4031 was abolished by KB-R7943.5. Preconditioning with two cycles of 5 min ischemia each or pretreatment with E4031, significantly reduced the infarct size, and the effect was abolished by 5μM KB-R7943. Exposure to E4031 at 3 - 5μM for two cycles of 5 min each reduced the infarct size caused by index ischemia in a concentration-dependent manner. Exposure to E4031 at 5μM for one cycle of 10 min also significantly reduced the infarct size, but its effect was significantly weaker than that for twocycles. Similar to IP, preconditioning the heart with E4031 also significantly reduced the arrhythmia scores during index ischemia and reperfusion, and the anti-arrhythmic effect of IP and E-4301 pretreatment were abolished by KB-R7943, which itself had no effect.6. Treatment of the heart with 5-HD/paxilline, 30 min before ischemia, abolished the beneficial effects of E4031 on infarct size and arrhythmias, and administration of 5-HD/paxilline from 5 min before the 30-min ischemia until 10 min after reperfusion also abolished the cardioprotective effect of E4031. 5-HD/paxilline alone had no effect. Activation of the mito KATP/ KCa channel with Diazoxide/ NS1619 also reduced infarct size and arrhythmias and the protective effects were blocked by 5-HD/paxilline, These effects were not affected by administration of KB-R7943 before index ischemia.Conclusions1. The activity of reverse mode NCX was assessed by withdrawal extracellular Na+. It has been demonstrated that the special effcets of E4031 on activation/augmentation of reverse mode NCX.2. Reverse-mode NCX activation during preconditioning ischemia triggered cardioprotection by increased [Ca²⁺]i . Prior stimulation of the reverse-mode NCX ameliorated the contraction in rat ventricular myocytes and reduced the infarct size and arrhythmia in perfusion isolated rat heart.3. Both mitoKATP and mitoKCa channels mediate the protective effects of prior stimulation of the reverse-mode NCX against infarct size and arrhythmias caused by ischemia and reperfusion, and that the NCX is located upstream from both the mitoKATP and mitoKCa channels.