| The mechanism of ischemia-reperfusion (I/R) injury remain unknown at present, such intervention therapy could not solve I/R injury effectively. N-n-butyl haloperidol iodide (F2) was a novel compound which was screened from a series of quaternary ammonium salt derivatives of haloperidol synthesized by our drug research lab. Our previous studies showed that the cardioprotective mechanism of F2 might be associated with the inhibition of Ca2+ overload and suppressing the expression of early growth response-1 (Egr-1). PKC inhibitor could significantly reduce the levels of Egr-1 mRNA and protein inducedy hypoxia-reperfusion (H/R) in cultured cardiomyocytes.The protein kinase C (PKC) family of highly homologous enzymes is a group of closely related serine-threonine protein kinases, which is implicated in numerous cellular signal-transduction processes affecting a variety of functions. We aimed to observe the change of PKC activity, translocation of PKC isoforms in primary cultured cardiomyocytes induced by H/R. specifically, the PKC isoformsα,βⅡ,δandε, which predominate in myocardium. Further, we aimed to investigate the biological function of PKC isoforms on cardiomyocyte injury induced by H/R with PKC inhibitor and activator, which is helpful to elucidate the molecule mechanism of F2 in a cardiomyocyte H/R model.METHODS1. Primary culture of neonatal rat cardiomyocytes and preparation of H/R model:After replaced the initial culture medium with hypoxic buffer, the cardiomyocytes were incubated in an air-tight chamber gassed with pure N2 for 2 h of hypoxia. The buffer was then replaced with fresh oxygenated culture medium and the dishes were transferred into a normoxic incubator for 30 min of reoxygenation.2. PKC activity was measured by non-radioactive detection of PKC, and the translocation pattern of PKCα,βⅡ,δandεisoforms was assessed by fractionated Western-blot analysis.3. Measurements included cell damage evaluated by creatine kinase (CK) release, levels of cardiac troponin I (cTnI) in the medium were measured by two-site sandwich immunoassay to assess the degree of injury of cultured cardiomyocytes; Levels of TNF-αin the medium were mesure by sandwich enzyme-linked immunoabsorbent assay (ELISA) method to assess the degree of inflammation of cultured cardiomyocytes; apoptosis measured by annexin V-FITC assay. RESULTS1. In primary cultured cardiomyocytes exposed to H/R, PKC activity significantly increased after the onset of hypoxia, and peaked at 2 h of hypoxia, remained significantly activated at up to 60 min of reoxygenation, then returned to control values after 2 h of reoxygenation. However, the protein levels of PKC isoforms did not change after H/R, levels of PKCεprotein but PKCα,βⅡand 5 significantly decreased after 4 h of hypoxia. H/R caused an increase of CK and cTnI, elevation of TNF-αand apoptosis, upregulation of Egr-1 protein expression.2. PKCa,βⅡand s showed significant translocation from the soluble to the particulate fraction after H/R, but a decrease of PKCδin the particulate fraction. Pretreatment with F2 then H/R showed a significant increase in the translocation of PKCεfrom the soluble to particulate fraction, with a decrease in the translocation of PKCa from the soluble to particulate fraction; Pretreatment with F2 produced a significant increase of PKCεactivity in the particulate fraction and a decrease of PKCεactivity in the soluble fraction as compared with control cells. F2 had no effect on other isoforms of PKC (α,βⅡ,δ) in the particulate or soluble fractions in normal or H/R-induced cardiomyocytes. With the inhibitor of phospholipase C, U73122, the effect of F2 on activation of PKCεtranslocation to the particulate fraction was inhibited in normal or H/R-induced cardiomyocytes.3. Treatment with the conventional PKC inhibitor Go6976 reduced Egr-1 protein expression, decreased the levels of CK, cTnI and TNF-α, attenuated apoptosis. The PKCεinhibitor peptideεV1-2 increased CK leakage, cTnI release and apoptotic cells, without influencing TNF-a secretion from cardiomyocytes; pretreatment withεV1-2 peptide conferred no alteration in protein level of Egr-1 protein induced by H/R.4. Inhibition of Egr-1 protein level by F2 is blocked by the PKCa activator thymeleatoxin (TXA); pretreatment with F2 inhibited cardiomyocytes injury induced by H/R; the protection of F2 was blocked in part by TXA andεV1-2 peptide.CONCLUSIONS1. PKC activity changed with different styles in H/R-induced primary cardiomyocytes, activition of PKCa was associated with upregulation of Egr-1 protein expression.2. H/R caused injury by activiation and transcolation of PKC, which might be attributed to inhibition of PKCa that associated with alleviated cell damage, and activation of PKCε, that necessary for cardioprotection against cardiomyocyte apoptosis and cell damage.3. Myocardial protection of F2 depended on PKCa inhibition and PKCεactivation. |
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