The Effects Of MitoK_ATP Opening On Respiratory Chain In Myocardial Injury Induced By Hypercatecholamine

Objective: To investigate the effects of mitochondrial ATP-sensitive potassium(mitoK_ATP)channel opening on mitochondrial respiratory chain(cytochrome c, cytochrome c oxidase) in myocardial injury induced by hypercatecholamine, and explore the mechanism responsible for cardioprotective effects of mitoK_ATP openers.Methods: 1. Cardiac myocytes were dispersed from the ventricles of 1 -3-day-oldWistar rats by digestion with collagenaseⅡand pancreatin. Making use of different growth time between myocytes and fibroblast, and treatment with 5-Bromodeoxyuridine, cardiac myocytes were purified. The primary cultured cardiac myocytes were identified by means of phase contrast microscopy, HE staining, Trypan blue staining and immunohistochemistry.2. After being cultured for 72h, cardiac myocytes were randomly divided into five groups: CON group(control group): cardiac myocytes were incubated without any extra treatment for 48h; NIC group: cardiac myocytes were incubated with nicorandil(1mmol/L) for 48h; ISO group: cardiac myocytes were incubated for 48h with isoproterenol(10μmol/L); ISO+NIC group: nicorandil(lmmol/L) was administered 20min before treatment with ISO(10μmol/L), then incubated for another 48h; ISO+NIC+5-HD group: 5-hydroxydecanoate(500μmol/L) was administered 10min before treatment with NIC(1mmol/L), others treatment were as the same as the ISO+NIC group.3. The mitochondrial fraction was obtained by differential centrifugation. The cytochrome c release and the activity of the cytochrome c oxydase were examined by spectrophotometry. Cell survival was measured by Trypan blue staining.Results: 1. Activity of cytochrome c oxydase(COX): There was no significantdifference in activity of COX between CON group and NIC group(14.023 +++++2.604 vs 13.249±2.217μmolCyt-C·min^-1·mg^-1, P>0.05); Compared with CON group, the activity of COX was decreased significantly in ISO group (6.818±1.191 vs 14.023±2.604μmolCyt-C·min^-1·mg^-1, P<0.05); Compared with ISO group, the activity of COX was increased significantly in ISO+NIC group(11.965±1.641 vs 6.818±1.191μmolCyt-C·min^-1·mg^-1, P<0.05); Compared with ISO+NIC group, the activity of COX in ISO+NIC+5-HD group was decreased significantly( 11.965±1.641 vs 9.176±0.949μmolCyt-C·min^-1·mg^-1, P<0.05).2. Content of cytochrome c(Cyt-C) in extracellular fluid: There was no significant difference in extracellular Cyt-C content between CON group and NIC group (4.855±0.912vs5.421±1.329μmol·L^-1, P>0.05) ; Compared with CON group, the extracellular Cyt-C content was increased significantly in ISO group( 9.756±2.027 vs 4.855±0.912μmol·L^-1, P<0.05) ; Compared with ISO group, the extracellular Cyt-C content was decreased in ISO+NIC group (7.841±1.799 vs 9.756±2.027μmol·L^-1, P<0.05); Compared with ISO+NIC group, the extracellular Cyt-C content was increased in ISO+NIC+5-HD group (10.364±1.629 vs7.841±1.799μmol·L^-1, P<0.05) .3. Content of Cyt-C in the cytoplasm: Compared with CON group, the content of Cyt-C in the cytoplasm was increased significantly in ISO group (275.895 vs 164.173±21.644μmol·L^-1, P<0.05); Compared with ISO group, the cytoplasm Cyt-C content was decreased in ISO+NIC group (202.725±33.427 vs 275.895±25.391μmol·L^-1, P<0.05) .4. Content of Cyt-C in mitochondria: Compared with CON group, the content of Cyt-C in mitochondria was decreased significantly in ISO group (8.513±1.772 vs 16.558±2.051μmol·L^-1, P<0.05); Compared with ISO group, mitochondrial Cyt-C content was increased in ISO+NIC group (13.770±1.822 vs 8.513±1.772μmol·L^-1, P<0.05); Compared with ISO+NIC group, the mitochondrial Cyt-C content was decreased in ISO+NIC+5-HD group (11.142±1.088 vs 13.770±1.822 mol·L^-1, P<0.05)5. Viability of cardiac myocytes: The viability of cardiomyocytes in ISO group was lower than that in CON group (75.3 vs 96.2%, P< 0.005); Compared with ISO group, the viability in ISO+NIC group increased (83.9 vs 75.3%, P<0.005); There was no significant difference between CON group and NIC group(96.2 vs 93.2%, P >0.005). Conclusion 1. Under hypercatecholamine, the activity of cytochrome c oxydase ofcultured cardiomyocyte decrease, and the cytochrome c, one of the key element of mitochondrial respiratory chain, is not only released from mitochondrion into the cytoplasm, but leaves the cell.2. Hypercatecholamine can induce mitochondrial dysfunction, the viability of cardiaomyocytes decrease.3. Nicorandil, the K_ATP channel opener can protect the cardiomyocytes from death induced by hypercatecholamine, preserving the activity of COX and decreasing the release of cytochrome c may be part of the mechanism. The cardioprotection of nicorandil due to the opening of the mitoK_ATP channel, rather than sarcK_ATP channel.These observations may suggest new pharmaceutical strategies to diminish hypercatecholamine-mediated cardiomyocyte damage.