Mitochondrial Mechanisms Of Urocortin I Postconditioning Against Myocardial Ischemia Reperfusion Injury

Objective: To investigate the mitochondrial mechanisms of cardioprotective effect of UrocortinⅠ postconditioning against myocardial ischemia/reperfusion injury.Methods:1.Ischemic reperfusion injury was prepared using the Langendorff isolated heart perfusion system. 48 healthy SD rats were randomly divided into four groups: normal group(Nor group), Ischemia group(I/R group), UrocortinⅠpostconditioning group(UcnⅠgroup), 5-HD antagonism group(5-HD+ UcnⅠgroup). Nor group: Kerbs-Henseleit(K-H) buffer was perfused continuously for 155 minutes. I/R group: K-H buffer was perfused for 20 min, and then cardiac ischemia was induced for 40 minutes before 95 min reperfusion was exerted. UcnⅠgroup: K-H containing UcnⅠwas administrated for 30 min followed by 65 min reperfusion. 5-HD+UcnⅠgroup: K-H buffer containing 5-HD before UcnⅠ postconditioning was used for 5 min, the rest managements were the same with UcnⅠ group. The following indexes of the four groups were evaluated at the end of equilibrium and at the termination of reperfusion respectively:(1) heart rate(HR), left ventricular end-diastolic pressure(LVDEP), left ventricular developed pressure(LVDP) and maximum dp/dt(dp/dtmax) were used to indicate heart function;(2) Mitochondria from ventricular tissues were extracted to analyze respiratory function and respiratory enzyme activity using oxygen electrode;(3) Microstructures of myocardial cell were scanned by transmission electron microscope. 2. Isolated myocardial cells of rats were obtained using the MPA isolated heart-perfusion system. The same batch of myocardial cells was divided into four groups after cultured for 24 hours: Nor group, I/R group, UcnⅠ group and 5-HD+ UcnⅠ group. Nor group was cultured continuously for 155 minutes in the 37℃ incubator while other groups experienced anoxia for 40 minutes and reoxygenation for 60 minutes. UcnⅠ was added into the dish in UcnⅠgroup for 30 min between anoxia and reoxygenation. 5-HD was added into the dish in 5-HD+UcnⅠ group before UcnⅠ was administrated, the rest managements were the same with UcnⅠ group. The following indexes at the end of reoxygenation were evaluated:(1) changes of mitochondrial membrane potential(MMP) were detected by laser scanning confocal microscope;(2) cell vitality were detected by cell counting kit. Results: 1.Indexes of heart function: before ischemia there was no significant difference among all the groups; at the end of reperfusion, HR, LVDP, LVEDP and dp/dt max in Nor group were superior to other groups(P<0.05); the indexes of heart function of UcnⅠ group were significantly greater than those in I/R group and 5-HD+UcnⅠgroup(P<0.05). There was difference between Group 5-HD+UcnⅠ and Group I/R in LVEDP and dp/dtmax(P<0.05), there was no significant difference between I/R group and 5-HD+Ucn I group in HR and LVDP(P>0.05). 2.Mitochondria respiratory function and respiratory enzyme activity: before ischemia there was no significant difference among all the groups; State3, RCR, NADH-OX, succinate oxidase and Cyt-OX in NOR group were significantly different from those in other groups(P<0.05). these indexes in UcnⅠ group were higher than those in I/R group and 5-HD+UcnⅠ group(P<0.05)while there was significant difference between I/R group and 5-HD+ UcnⅠgroup(P<0.05). 3.mitochondrial membrane potential measurement: The highest mitochondrial membrane potential was found in Nor group(P<0.01), while the potential of UcnⅠ group was higher than that in I/R group and 5-HD+UcnⅠ group(P<0.05). There was no significant difference between I/R group and 5-HD+Ucn Ⅰgroup(P>0.05). 4. the cell vitality: The greatest cell vitality was found in Nor group(P<0.05). the vitality of UcnⅠ group was higher than that in I/R group and 5-HD+UcnⅠ group(P<0.05)while there was no significant difference between I/R group and 5-HD+UcnⅠ group(P>0.05).Conclusion: UcnⅠpostconditioning can improve heart function by enhancing myocardial contractility and reducing end-diastolic pressure. UcnⅠpostconditioning can improve mitochondrial respiratory function and respiratory enzyme activity to maintain the normal process of oxidative phosphorylation and electron transference of respiratory chain. UcnⅠpostconditioning exerts protective effects on myocardium suffering from ischemia reperfusion by stabilizing mitochondrial membrane potential, protecting mitochondrial structure and function. It is found that mito-KATP is involved in the protective mechanism of UcnⅠpostconditioning.