The Protective Effect Of N-n-butyl Haloperidol Iodide And Ca～(2+) Channel Blockers On Hypoxia/Reoxygenation Injury Of Cardiomyocytes

Egr-1 is one of the immediate-early genes in response to stimulation. With Egr-1-null miceor antisense Egr-1 oligodeoxyribonucleotide, previous studies have showed that Egr-1 might be amaster switch in the pathogenesis of ischemia/reperfusion(I/R) injury due to its coordinatingupregulation of divergent gene families underlying pathophysiological event of I/R. Previousresearchs show that N-n-butyl haloperidol iodide (F₂) could reduce I/R-induced myocardialinjury through inhibiting Egr-1 RNA transcription and protein overexpression. However, we donot know whether there are protective effects elicited by F₂. We all know, calcium overload playsan important role in the pathogenesis of I/R injury, calcium antagonists are also used to preventI/R injury widely. We have previously shown that F₂ reduces I/R-induced myocardial injury viablocking intracellular Ca²⁺ overload, whether calcium antagonists could inhibit Egr-1 RNAtranscription and protein overexpression? The purpose of this study is to investigate the effectsof F₂ and calcium antagonist: Verapamil, Diltiazem, Nifedipine on Egr-1 mRNA transcriptionand protein expression in cultured cardiomyocytes after hypoxia/reoxygenation (H/R, I/R modelin vitro), which is helpful to elucidate the new molecular mechanisms of F₂.Methods(1) Preparation of H/R modelAfter replaced the initial culture medium with hypoxic buffer, the cardiomyocyteswere incubated in an air-tight chamber gassed with pure N₂ for 3 h of hypoxia. Thebuffer was then replaced with fresh oxygenated culture medium and the dishes weretransferred into a normoxic incubator for 1 h of reoxygenation. (2) Groups:the first part: The cardiomyocytes were randomly divided into one of five groups:Control group, H/R group, solvent [PEG+LIP (Lipfectemine)] group, antisensenucleotide(AS) group and AS+F₂ (1×10^-6mol/L) group.the second part: The cardiomyocytes were randomly divided into one of six groups:Control group, H/R group, DMSO group, Verapamil(Ver, 2×10^-6 mol/L)group,Diltiazem(Dil, 1×10^-5 mol/L) group and NifedipinefNif, 1×10^-5 mol/L) group andequal volumn of DMSO were added to the hypoxic buffer and reoxygenated medium.(3) The changes of morphology and spontaneous beat of cultured cardiomyocytes weredetected by the optic microscope.(4) Levels of LDH and CK in the medium and Levels of SOD and MDA culturedcardiomyocytes were measured by colorimetric method to assess the degree of injuryof cultured cardiomyocytes(5) Levels of cardiac troponin I (cTnI) in the medium were measured by two-site sandwichimmunoassay to assess the degree of injury of cultured cardiomyocytes.(6) Levels of TNF-αin the medium were mesure by sandwich enzyme-linkedimmunoabsorbent assay (ELISA) method to assess the degree of inflammation ofcultured cardiomyocytes.(7) The expression levels of Egr-1 mRNA in cultured cardiomyocytes were examined byRT-PCR.(8) The expression levels of Egr-1 protein in cultured cardiomyocytes were examined byWestern-blot.Results1. The effect of antisense oligodeoxynucleotides transfection in culturedcardiomyocytesFITC labeled antisense oligodeoxynucleotides emited green fluorescence. The location of fluorescence coincidented with the area of cardiomyocytes under theinverted phasecontrast microscope, which indicated that antisenseoligodeoxynucleotides had transfected cells successfully.2. The effect of F₂ on H/R injury of cultured cardiomyocytes2.1 The effect of F₂ on morphology and structure changes of cultured cardiomyocytesCompared with the Control group, H/R caused cultured cardiomyocytesparamorphia, arrhythmic beat and ultrastructural damage, which were not altered byPEG, but significantly attenuated by F₂ or VER.2.2 The effect of F₂ on Levels of CK, LDH, cTnI and TNF-αin culture mediumCompared with the Control group, H/R caused CK, LDH and cTnI leakage andTNF-αsecretion from cardiomyocytes. Treatment with AS or AS+F₂, not PEG+LIP, significantly reduced CK, LDH, cTnI release and TNF-αsecretion fromcardiomyocytes.2.3 The effect of F₂ on Levels of SOD and MDA in cultured cardiomyocytesCompared with the Control group, the levels of SOD in cultured cardiomyocyteswere significantly decreased and the levels of SOD in cultured cardiornyocytes weresignificantly increased in the H/R group. These changes in levels of SOD and MDAwere not altered by PEG+LIP, but significantly reduced when treated with AS orAS+F₂.3. The effect of F₂ on Egr-1 protein expression in cultured cardiomyocytesConsistent with changes in levels of Egr-1 mRNA in the H/R group, Egr-1protein in cultured cardiomyocytes measured by Western blot analysis weresignificantly increased relative to the Control group. This change in levels of Egr-1protein was not altered by PEG+LIP, but significantly reduced when treated with ASor AS+F₂.4. The effect of calcium antagonists on H/R injury of cultured cardiomyocytes4.1 The effect of calcium antagonists on Levels of CK and LDH in culture medium Compared with the Control group, H/R caused CK and LDH leakage fromcardiomyocytes. Treatment with Verapamil, Diltiazem and Nifedipine, not DMSO,significantly reduced CK and LDH release from cardiomyocytes.4.2 The effect of calcium antagonists on Levels of SOD and MDA in culturedcardiomyocytesCompared with the Control group, levels of SOD in cultured cardiomyocyteswere significantly decreased and the levels of SOD in cultured cardiomyocytes weresignificantly increased in the H/R group. These changes in levels of SOD and MDAwere not altered byDMSO, but significantly reduced when treated with Verapamil,Diltiazem and Nifedipine.5. The effect of calcium antagonists on Egr-1 mRNA expression in culturedcardiomyocytesRelative to the Control group, levels of ERr-1 mRNA in cultured cardiomyocytesmeasured by RT-PCR analysis in the H/R group were significantly increased at theend of experiment. These changes in levels of Egr-1 mRNA were not altered byDMSO, but significantly reduced when Verapamil, Diltiazem and Nifedipine wasadded into cultured cells.6. The effect of calcium antagonists on Egr-1 protein expression in culturedcardiomyocytesConsistent with changes in levels of Egr-1 mRNA in the H/R group, Egr-1protein in cultured cardiomyocytes measured by Western blot analysis weresignificantly increased relative to the Control group. This change in levels of Egr-1protein was not altered by DMSO, but significantly reduced when treated withVerapamil, Diltiazem and Nifedipine.Conclusions(1) Though inhibiting the overexpression of Egr-1 protein in cultured cardiomyocytescaused by H/R, F₂ protects cultured cardiomyoeytes from H/R injury, which mightbe one of the protective mechanisms of F₂ on myocardial I/R injury. (2) There are other protective mechanisms of F₂ on cultured cardiomyocytes H/Rinjury.(3) Verapamil, Diltiazem and Nifedipine inhibit the overexpression of Egr-1 mRNAand protein in cultured cardiomyocytes caused by H/R, which protects culturedcardiomyocytes from H/R injury.