Aims: Our previous studies have shown that N-n-butyl haloperidol iodide (F₂) can antagonize myocardial ischemia/reperfusion (I/R) injury by blocking intracellular Ca²⁺ overload. The present study is to test the hypothesis that the protective effects of F₂ on myocardial I/R injury is mediated by downregulating Egr-1 expression. Methods: The Sprague–Dawley rat myocardial I/R model and cardiomyocyte hypoxia/reoxygenation (H/R) model were established. With antisense Egr-1 oligodeoxyribonucleotide (ODN), the relationship between Egr-1 expression and myocardial I/R injury was investigated. Hemodynamic parameters, myeloperoxidase (MPO), cardiac troponin I (cTnI) and tumor necrosis factor-α (TNF-α) were measured to assess the degree of injury and inflammation of myocardial tissues and cells. Egr-1 mRNA and protein expressions were examined by Northern-blot and Western-blot analyses. Results: Treatment with antisense Egr-1 ODN significantly reduced Egr-1 protein expression and attenuated injury of myocardial tissues and cells. Meanwhile, treatment with F₂ significantly inhibited the overexpression of Egr-1 mRNA and protein in myocardial tissues and cells. Consistent with downregulation of Egr-1 expression by F₂, inflammation and other damages were significantly relieved evidenced by the amelioration of hemodynamics, the reduction in myocardial MPO activity as well as the decrease in leakage of cTnI and release of TNF-α from cardiomyocyte. Conclusions: These results suggested that the overexpression of Egr-1 was causative in myocardial I/R or H/R injury, and F₂ could protect myocardial tissues and cells from I/R or H/R injury, which was largely due to the inhibition of Egr-1 overexpresssion.

We have previously shown that N-n-butyl haloperidol iodide (F2) derived from haloperidol reduces ischemia/reperfusion-induced myocardial injury by blocking intracellular Ca2+ overload. This study tested the hypothesis that cardio-protection with F2 is associated with an attenuation in the expression of early growth response gene 1 (Egr-1). In an in vivo rat model of 60 min coronary occlusion followed by 180 min of reperfusion, treatment with F2 significantly reduced myocardial injury evidenced by the reduction in release of plasma creatine kinase, myocardial creatine kinase isoenzyme and lactate dehydrogenase. In cultured neonatal rat cardiomyocytes of hypoxia for 3 h and reoxygenation for 1 h, F2 treatment attenuated necrotic and apoptotic cell death, as demonstrated by electron microscopy. Concomitant with cardio-protection by F2, the increased expression levels of Egr-1 mRNA and protein were significantly reduced in myocardial tissue and cultured cardiomyocytes as detected by reverse transcription-polymerase chain reaction, immunohistochemistry and immunocytochemistry. In conclusion, these results suggest that the protective effect of F2 on ischemia/reperfusion- or hypoxia/reoxygenation-induced myocardial injury might be partly mediated by downregulating Egr-1 expression.

Aims: In this study, we tested whether Egr-1 is a potential target of calcium channel blockers in cardioprotection with I/R injury. Methods: We treated rats in vivo I/R and rat cultured cardiomyocytes in vitro hypoxia/reoxygenation (H/R) models with three types of classical calcium channel blockers (verapamil, diltiazem and nifedipine). Activity of creatine kinase (CK), lactate dehydrogenase (LDH), myeloperoxidase (MPO) superoxide dismutase (SOD) and level of malondialdehyde (MDA) in plasma and culture medium were measured to assess the degree of injury and inflammation of myocardial tissues and cells. Egr-1 mRNA and protein expressions were examined by RT-PCR and Western-blot analyses. Results: Calcium channel blockers (verapamil, diltiazem and nifedipine) significantly attenuated myocardial injury, as shown by reduced release of CK and LDH, preserved SOD activity and decreased MDA production and MPO activity. Concomitant with cardioprotection by calcium channel blockers, the mRNA and protein expression of Egr-1 increased with I/R and H/R injury was significantly reduced in myocardial tissue and cultured cardiomyocytes. Conclusions: These results suggested that the cardioprotective effects of calcium channel blockers with I/R or H/R injury might be mediated by downregulating Egr-1 expression. Egr-1 might be the potential target of calcium channel blockers in cardioprotection with ischemia/reperfusion injury.

Early growth response 1 (Egr-1) over-expression has been demonstrated in myocardial ischemia-reperfusion injury, which is closely associated with endothelial dysfunction. In the present study we investigated the expression of Egr-1 on cultured cardiac microvascular endothelial cells (CMECs) to help define the mechanism of myocardial ischemia-reperfusion injury. A model of cultured CMECs exposed to hypoxia-reoxygenation was developed in which synthesized Egr-1 sense and antisense oligodeoxyribonucleotide were transfected into the cells. The expression of Egr-1 was examined by Western blot analysis. Lactate dehydrogenase, malondialdehyde, superoxide dismutase, tumor necrosis factor alpha, and intercellular adhesion molecule 1 were measured after hypoxia-reoxygenation to assess cell function and injury. Cell morphology, cell viability, and neutrophil adhesion to the CMECs were measured to assess the degree of injury and inflammation. Only cells transfected with Egr-1 antisense oligodeoxyribonucleotide showed a significant reduction in Egr-1 protein expression following hypoxia-reoxygenation. Consistent with the down-regulation of Egr-1 expression, other forms of cell injury were significantly reduced in this group of cells, as evidenced by less alteration in cell morphology, a decrease in expression of tumor necrosis factor alpha and intercellular adhesion molecule 1, improved cell survival, and reduced neutrophil adhesion.