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The iron-sulfur cluster containing protein mitoNEET is known to modulate the oxidative capacity of cardiac mitochondria but its function during myocardial reperfusion injury after transient ischemia is unknown. The purpose of this study was to analyze the impact of mitoNEET on oxidative stress induced cell death and its relation to the glutathione-redox system in cardiomyocytes in an in vitro model of hypoxia and reoxygenation (H/R). Our results show that siRNA knockdown (KD) of mitoNEET caused an 1.9-fold increase in H/R induced apoptosis compared to H/R control while overexpression of mitoNEET caused a 53% decrease in apoptosis. Necrosis was not affected. Apoptosis of both, mitoNEET-KD and control cells was diminished to comparable levels by using the antioxidants Tiron and glutathione compound glutathione reduced ethyl ester (GSH-MEE), indicating that mitoNEET-dependent apoptosis is mediated by oxidative stress. The interplay between mitoNEET and glutathione redox system was assessed by treating cardiomyocytes with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio-carbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), known to effectively inhibit glutathione reductase (GSR) and to decrease the GSH/GSSG ratio. Surprisingly, inhibition of GSR-activity to 20% by 2-AAPA decreased apoptosis of control and mitoNEET-KD cells to 23% and 25% respectively, while at the same time mitoNEET-protein was increased 4-fold. This effect on mitoNEET-protein was not accessible by mitoNEET-KD but was reversed by GSH-MEE. In conclusion we show that mitoNEET protects cardiomyocytes from oxidative stress-induced apoptosis during H/R. Inhibition of GSH-recycling, GSR-activity by 2-AAPA increased mitoNEET-protein, accompanied by reduced apoptosis. Addition of GSH reversed these effects suggesting that mitoNEET can in part compensate for imbalances in the antioxidative glutathione-system and therefore could serve as a potential therapeutic approach for the oxidatively stressed myocardium.

Aims: Thrombomodulin (TM), via its lectin-like domain (LLD), exhibits anti-inflammatory properties partly by sequestering the pro-inflammatory cytokine, high-mobility group box 1 (HMGB1). Since myocardial damage after ischaemia and reperfusion is mediated by inflammation, we evaluated the cardioprotective effects of the LLD of TM. Using an in vivo mouse model of transient ischaemia and in vitro models of cardiomyocyte hypoxia, we assessed the ability of the LLD to suppress HMGB1-mediated activation of the receptors, receptor for advanced glycation endproducts (RAGEs) and Toll-like receptors (TLRs) 2 and 4., Methods and Results: Thirty-minute myocardial ischaemia was induced in isoflurane-anaesthetized mice followed by 24 h of reperfusion in wild-type (WT) mice, in mice lacking the LLD of TM (TM(LeD/LeD) mice), and in WT with systemic overexpression of the LLD of TM induced by hydrodynamic transfection. Infarct size, HMGB1 protein, and apoptotic cells were significantly increased in TM(LeD/LeD) mice when compared with WT. Neonatal rat cardiomyocytes transfected with TLR2-, TLR4-, and RAGE-siRNA were exposed to hypoxia (0.8% O2) and reoxygenation (21% O2). HMGB1 augmented hypoxia-induced apoptosis in TLR2- but not in RAGE- or TLR4-suppressed cells. Administration of HMGB1- and TLR2-blocking antibodies in TM(LeD/LeD) mice prior to myocardial ischaemia diminished apoptosis. Therapeutic systemic gene therapy using the LLD reduced the infarct size and HMGB1 protein levels 24 h after reperfusion., Conclusion: The LLD of TM suppresses HMGB1-induced and TLR2-mediated myocardial reperfusion injury and apoptosis in vitro and in vivo.

Aims: Myocardial infarction (MI) results in acute impairment of left ventricular (LV) function through the initial development of cardiomyocyte death and subsequent progression of LV remodelling. The expression of syndecan-4 (Sdc4), a transmembrane proteoglycan, is up-regulated after MI, but its function in the heart remains unknown. Here, we characterize the effects of Sdc4 deficiency in murine myocardial ischaemia and permanent infarction., Methods and Results: Targeted deletion of Sdc4 (Sdc4(-/-)) leads to increased myocardial damage after ischaemic-reperfusion injury due to enhanced cardiomyocyte apoptosis associated with reduced activation of extracellular signal-regulated kinase in cardiomyocytes in vitro and in vivo. After ischaemic-reperfusion injury and permanent infarction, we observed an increase in cardiomyocyte area, nuclear translocation of nuclear factor of activated T cells (NFAT), and transcription of the NFAT target rcan1.4 in wild-type mice. NFAT pathway activation was enhanced in Sdc4(-/-) mice. In line with the in vivo data, NFAT activation and hypertrophy occurs in isolated cardiomyocytes with reduced Sdc4 expression during phenylephrine stimulation in vitro. Despite the initially increased myocardial damage, echocardiography revealed improved LV geometry and function in Sdc4(-/-) mice 7 days after MI., Conclusion: Interception of the Sdc4 pathway enhances infarct expansion and hypertrophic remodelling during early infarct healing in ischaemic-reperfusion injury and permanent infarction mouse models and exerts net beneficial effects on LV function.

Background: Perioperative myocardial ischemia poses a vital threat to surgical patients. Means to protect postischemic myocardium are clinically not available. Lidocaine has been demonstrated to exert antiinflammatory pleiotropic effects. The authors set out to test if lidocaine protects ischemic myocardium from reperfusion injury., Method: A mouse model of transient coronary artery ligation (30 min) and reperfusion (24 h) was used with animal care committee approval. Infarct size and area-at-risk were determined. Leukocyte recruitment was quantified on immunohistochemical stainings. Apoptosis was assessed using enzyme-linked immunosorbent assay to detect histone modifications and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Lidocaine effects on leukocyte-endothelial interactions were assessed in vitro by using a parallel-plate flow chamber or static adhesion assays., Results: Infarct size per area-at-risk was reduced by 27% in mice treated with a lidocaine bolus (1 mg/kg) before a continuous infusion (0.6 mg . kg(-1) . h(-1)) during ischemia (P < 0.005). Neutrophil density in the infarct and periinfarct zone was not reduced by lidocaine, although the size of the infiltrated area was. Terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cardiomyocytes and endothelial cells were significantly reduced in the periinfarct zone by lidocaine. In vitro, no effect on leukocyte rolling or firm adhesion to resting or activated endothelium was demonstrable. In vitro, lidocaine reduced cardiomyocyte apoptosis induced by hypoxia and reoxygenation (3h/1h) significantly. Infarct size and in vitro cardiomyocyte apoptosis were likewise reduced when lidocaine bolus and infusion were administered after the ischemic insult., Conclusion: Lidocaine exerts cardioprotective effects when administered before or after the ischemic insult. This effect is mediated through an antiapoptotic and not through an antiinflammatory pathway and may be therapeutically exploitable.