Role Of Reversed Sodium/Calcium Exchanger-mediated Calpainactivation In Rat Heart Subjected To Calcium Paradox

BackgroundMyocardial ischemia/reperfusion injury happens in the process of cardiacsurgery, which is one of the most important problems needed to be furtheraddressed. It has been demonstrated that intracellular calcium overload is animportant mechanism mediating myocardial ischemia/reperfusion injury.Therefore, it is of great significance to clarify the formation of calcium overloadand the signal transduction pathway of calcium-overload injury, which maypromote the development of new strategy of myocardial protection and theprogress of cardiac surgery.It was observed that an isolated heart rapidly deteriorates after a short periodof perfusion with a Ca2+-free, otherwise normal buffer （calcium depletion）followed by a buffer containing a physiological Ca2+concentration （calcium repletion）. This damage was called calcium paradox. A massive Ca2+influxduring the calcium repletion phase is thought to be responsible for the damageinduced by calcium paradox. Therefore, the route of the entry of Ca2+duringcalcium repletion becomes the focus of concerning research. Sodium/calciumexchanger （NCX） is an important modulator of intracellular calciumhomeostasis. In the model of isolated cardiomyocytes, SEA0400（an inhibitor ofreversed NCX） was observed to elicit cardioprotection agaist calcium paradox.However, the role of reversed NCX in calcium paradox remains to bedetermined due to the difference between the level of cell and the level of organ.The integrity of plasma membrane is a prerequisite for the physiologicalfunction of living cells. Alpha-fodrin forms the backbone of the membranecytoskeleton and contributes to the integrity of plasma membrane. It has beenrevealed that the loss of to the integrity of plasma membrane is a significantcause of calcium paradox-mediated myocardial injury. However, the effect ofcalcium paradox on the degradation of α-fodrin has not been elucidated. As isshown in many related study, α-fodrin is one of the substrates of calpain, theactivation of which depends on an inappropriate elevation of intracellularconcentration of Ca2+. Therefore, we speculate that calcium paradox leads to anactivation of reversed NCX, contributing to intracellular calcium overload,which in turn leads to an inappropriate activation of calpain. The overactivatedcalpain hydrolyses α-fodrin, which favors myocardial injury by causingsarcolemmal fragility.Based on the background above, this study will focus on the followings:Objectives1. To investigate the role of revere-mode NCX-mediated Ca2+influx in the myocardial damage of calcium paradox.2. To investigate whether the abnormal activation of calpain exists in heartsubjected to calcium paradox, and to explore the function and its mechanism ofthe activation of calpain in calcium paradox.3. To investigate whether the calcium influx through reversed NCX can producethe abnormal activation of calpain during calcium paradox.4. To tentatively explore the effect of the duration of the calcium depletion onthe mechanisms mediating calcium paradox.Methods1. Forty male adult SD rats were randomly divided into five groups, with eightrats per group. I. Normal control, perfusion with normal KH buffer for48minutes. II. Calcium paradox （Ca PD）,3min of calcium depletion followed by30min of calcium repletion. III. The treatment of a reversed NCX inhibitorKB-R7943in calcium paradox （KBR+Ca PD）. IV. The treatment of anotherinhibitor of reversed NCX SN-6in calcium paradox （SN+Ca PD）. V. Thetreatment of MDL-28170, an inhibitor of calpain, in calcium paradox （MDL+Ca PD）. Isolated perfusion of rat heart was prepared, and the hemodynamicindex during the process of perfusion was recorded and analysed. The coronaryeffluent was collected, and the content of lactate dehydrogenase （LDH） wasdetermined by enzyme-linked mmunosorbent assay. After the perfusion, theheart was taken down and cut into pieces for the determination of myocardialinfarct size with TTC staining. Samples for western blotting analysis werecollected in an additional series of hearts. The level of145/150KDcalpain-dependent α-fodrin fragments, which is closely related to the activationof calpain, was determined through western blot. The degradation of troponin I （TnI） and mitochondrial release of cytochrome c were also assayed.2. Male adult SD rats were randomly divided into nine groups, with eight ratsper group. I. Normal control, perfusion with normal KH buffer for48minutes. II.Calcium paradox induced by3min of calcium depletion followed by30min ofcalcium repletion （Ca PD₃）. III. The treatment of a reversed NCX inhibitorKB-R7943in Ca PD₃（KBR+Ca PD₃）. IV. The treatment of another inhibitorof reversed NCX SN-6in Ca PD₃（SN+Ca PD₃）. V. The treatment ofMDL-28170, an inhibitor of calpain, in Ca PD₃（MDL+Ca PD₃）. VI. Calciumparadox induced by5min of calcium depletion followed by30min of calciumrepletion （Ca PD₅）. VII. The treatment of KB-R7943in Ca PD₅（KBR+CaPD₅）. VIII The treatment of SN-6in Ca PD₅（SN+Ca PD₅）. IX The treatment ofMDL-28170in Ca PD₅（MDL+Ca PD₅）. Isolated perfusion of rat heart wasprepared, and the hemodynamic index during the process of perfusion wasrecorded and analysed. The coronary effluent was collected, and the content oflactate dehydrogenase （LDH） was determined by enzyme-linked mmunosorbentassay. After the perfusion, the heart was taken down and cut into pieces for thedetermination of myocardial infarct size with TTC staining.Results1. Calcium paradox was successfully induced by both3min of calciumdepletion followed by30min of calcium repletion. No obvious heart beat wasobserved in the hearts undergoing calcium paradox. In comparison with Controlgroup, the calcium paradoxic hearts showed a significant increase in leftventricular end-diastolic pressure （LVEDP） and marked decreases in leftventricular developed pressure （LVDP）, the highest rate of change of pressuredevelopment （+dp/dtmax） and the highest rate of change of pressure decay （-dp/dtmax）. The hearts also showed appreciable increases in LDH release,myocardial infarct size and TnI degradation as compared with control heats.2. Perfusion of Ca²⁺-paradoxic hearts with KB-R7943produced acardioprotection against calcium paradox, which was reflected by a significantimprovement in cardiac function and a marked decrease in LDH release,myocardial infarct size and TnI degradation as compared with Ca PD group. Thecardioprotection achieved by SN-6was similar to KB-R7943. All these resultsindicate that reversed NCX is involved in the heart injury induced by calciumparadox.3. An abnormal activation of calpain was observed in isolated rat heart subjectedto calcium paradox, which was reflected by a significant increase in145/150KDcalpain-dependent α-fodrin fragments in Ca PD group as compared with controlgroup. Treatment of reversed NCX inhibitor produced a marked reduction of thelevel of145/150KD fragmented α-fodrin, indicating that the activation ofcalpain is a downstream event following the activation of reversed NCX.4. The application of calpain inhibitor MDL-28170produced a cardioprotectionagainst calcium paradox, which was reflected by a significant improvement incardiac function and a marked decrease in LDH release and myocardial infarctsize as compared with Ca PD group, indicating that calpain activation isinvolved in the heart injury induced by calcium paradox.5. Calcium paradox produced a significant increase in LDH release, which was markedlyinhibited by the application of both reversed NCX inhibitor and calpain inhibitor. In view ofthe above findings that the activation of calpain is a downstream event followingthe activation of reversed NCX, we can conclude that the reversed NCXmediated-activation of calpain contributes to the necrotic cell death induced bycalcium paradox. 6. Calcium paradox induced a marked increase in the release of cytochrome c, which wassignificantly inhibited by the treatment of both reversed NCX inhibitor and calpain inhibitor.In consideration of the above findings that the activation of calpain is a downstreamevent following the activation of reversed NCX, we can conclude that thereversed NCX mediated-activation of calpain contributes to the apoptotic celldeath elicited by calcium paradox.7. The calcium paradox induced by3min of calcium depletion followed by30min of calcium repletion produced a similar heart injury to that induced by5min of calcium depletion followed by30min of calcium repletion, which wasreflected by that no significant difference in cardiac function, LDH release andmyocardial infarct size was observed between the two kinds of calcium paradox.8. Protection by reversed NCX inhibitor against Ca PD3was stronger than thatagainst Ca PD₅, which was reflected by a significant improvement of cardiacfunction and marked decreases in LDH release and myocardial infarct size wereachieved both in KBR+Ca PD3group as compared with KBR+Ca PD₅groupand in SN+Ca PD3group as compared with SN+Ca PD₅group. These findingsindicate that the weight of reversed NCX in the mechanisms mediating calciumparadox injury decreases with the extension of the duration of calciumdepletion.9. Protection by MDL-28170against Ca PD3was stronger than that against CaPD₅, which was reflected by a significant improvement of cardiac function andmarked decreases in LDH release and myocardial infarct size were achieved inMDL+Ca PD3group as compared with MDL+Ca PD₅group. These findingsindicate that the weight of calpain activation in the mechanisms mediatingcalcium paradox injury decreases with the extension of the duration of calciumdepletion. Conclusions1. Calcium paradox involves in its mechanisms mediating myocardial injury thesignaling pathway of reversed NCX–calcium overload–calpain–substrates（α-fodrin）.2. The signaling pathway of reversed NCX–calcium overload–calpaincontributes to the necrosis and apoptosis induced by calcium paradox.3. Both recersed NCX inhibitor KB-R7943/SN-6and calpain inhibitorMDL-28170elicit a significant cardioprotection against calcium paradox,indicating that either reversed NCX or calpain serves as an important target forprotection against calcium paradox.4. The duration of calcium depletion has no effect on the severity of myocardialinjury induced by calcium paradox, but has an effect on the mechanismmediating calcium paradox injury. The weight of reversed NCX mediatedactivation of calpain in the mechanisms mediating calcium paradox injurydecreases with the extension of the duration of calcium depletion.