Oxidative stress-dependent sphingosine kinase-1 inhibition mediates monoamine oxidase A-associated cardiac cell apoptosis.

The mitochondrial enzyme monoamine oxidase (MAO), its isoform MAO-A, plays a major role in reactive oxygen species-dependent cardiomyocyte apoptosis and postischemic cardiac damage. In the current study, we investigated whether sphingolipid metabolism can account for mediating MAO-A- and reactive oxygen species-dependent cardiomyocyte apoptosis. In H9c2 cardiomyoblasts, MAO-A-dependent reactive oxygen species generation led to mitochondria-mediated apoptosis, along with sphingosine kinase-1 (SphK1) inhibition. These phenomena were associated with generation of proapoptotic ceramide and decrease in prosurvival sphingosine 1-phosphate. These events were mimicked by inhibition of SphK1 with either pharmacological inhibitor or small interfering RNA, as well as by extracellular addition of C(2)-ceramide or H(2)O(2). In contrast, enforced expression of SphK1 protected H9c2 cells from serotonin- or H(2)O(2)-induced apoptosis. Analysis of cardiac tissues from wild-type mice subjected to ischemia/reperfusion revealed significant upregulation of ceramide and inhibition of SphK1. It is noteworthy that SphK1 inhibition, ceramide accumulation, and concomitantly infarct size and cardiomyocyte apoptosis were significantly decreased in MAO-A-deficient animals. In conclusion, we show for the first time that the upregulation of ceramide/sphingosine 1-phosphate ratio is a critical event in MAO-A-mediated cardiac cell apoptosis. In addition, we provide the first evidence linking generation of reactive oxygen species with SphK1 inhibition. Finally, we propose sphingolipid metabolites as key mediators of postischemic/reperfusion cardiac injury.

Oxidative stress by monoamine oxidase mediates receptor-independent cardiomyocyte apoptosis by serotonin and postischemic myocardial injury.

BACKGROUND: Serotonin (5-hydroxytryptamine [5-HT]), released by activated platelets during cardiac ischemia, is metabolized by the mitochondrial enzyme monoamine oxidase A (MAO-A). Because hydrogen peroxide is one of the byproducts of 5-HT degradation by MAO-A, we investigated the potential role of reactive oxygen species generated by MAOs in 5-HT-dependent cardiomyocyte death and post-ischemia-reperfusion cardiac damage. METHODS AND RESULTS: Treatment of isolated adult rat cardiomyocytes with 5-HT induced intracellular oxidative stress and cell apoptosis. The apoptotic cascade triggered by 5-HT involves release of cytochrome c, upregulation of proapoptotic Bax protein, and downregulation of antiapoptotic Bcl-2 protein. These effects were prevented by inhibition of amine transporter or MAO, antioxidants, or iron chelation. In contrast, cardiomyocyte apoptosis was only slightly affected by the 5-HT(2B) receptor antagonist SB 206553. In vivo, inhibition of MAO-A largely reduced myocardial ultrastructural damage induced by 30 minutes of ischemia followed by 60 minutes of reperfusion in the rat heart. Cardioprotective effects of MAO inhibitors were associated with the prevention of postischemic oxidative stress, neutrophil accumulation, and mitochondrial-dependent cell death and were not reverted by SB 206553. Administration of MAO-A inhibitors during ischemia was still effective in preventing cardiac damage. CONCLUSIONS: Our results supply the first direct evidence that oxidative stress induced by MAO is responsible for receptor-independent apoptotic effects of 5-HT in cardiomyocytes and postischemic myocardial injury. These findings provide new insight into the mechanisms of 5-HT action in the heart and may constitute the basis for novel therapies.

Involvement of peripheral benzodiazepine receptor in the oxidative stress, death-signaling pathways, and renal injury induced by ischemia-reperfusion.

The peripheral benzodiazepine receptor (PBR) is a critical component of the mitochondrial permeability transition pore, which is involved in the regulation of cell death. In the present study we investigated the role of PBR in the regulation of signaling pathways leading to apoptotic and necrotic damage and renal dysfunction in a rat model of ischemia-reperfusion. Renal ischemia-reperfusion led to extended tubular apoptosis and necrosis that were associated with peroxidative damage, high levels of proapoptotic Bax expression, and low levels of antiapoptotic Bcl-2 expression, cleavage of death substrate, poly(ADP-ribose) polymerase (PARP), and activation of a key effector of apoptosis, caspase-3. Rat pretreatment with a novel PBR antagonist, SSR180575, significantly decreased postreperfusion oxidative stress and tubular apoptosis and necrosis. This effect was associated with inhibition of caspase-3 activation and PARP cleavage, upregulation of Bcl-2, and downregulation of Bax. Furthermore, inhibition of PBR accelerated the recovery of normal renal function, as assessed by measurement of levels of plasma creatinine and blood urea nitrogen. These findings reveal a role for PBR as a modulator of necrotic and apoptotic cell death induced by ischemia-reperfusion and suggest that regulation of PBR may provide new therapeutic implications for the prevention of acute renal failure.

Role of peripheral benzodiazepine receptors in mitochondrial, cellular, and cardiac damage induced by oxidative stress and ischemia-reperfusion.

Mitochondrial dysfunction has been identified as a possible early event in ischemia-reperfusion damage. The peripheral benzodiazepine receptor, a mitochondrial inner membrane protein, has already been proposed to play a role in mitochondrial regulation, although its exact function remains unclear. The aim of this work was to determine the role of peripheral benzodiazepine receptor in ischemia-reperfusion injury and to test the potential beneficial effect of a novel potent peripheral benzodiazepine receptor ligand, 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (SSR180575). To characterize and link the mitochondrial, cellular, and cardiac consequences of ischemia-reperfusion, we examined the effects of SSR180575 in several in vitro and in vivo models of oxidative stress. Hydrogen peroxide decreased mitochondrial membrane potential, reduced oxidative phosphorylation capacities, and caused cytochrome c release, caspase 3 activation, and DNA fragmentation. SSR180575 (100 nM-1 microM) prevented all these effects. In perfused rat hearts, SSR180575 administered in vitro (100 nM-1 microM) or by oral pretreatment (3-30 mg/kg) greatly reduced the contractile dysfunction associated with ischemia-reperfusion. Furthermore, in anesthetized rats, SSR180575 (3-30 mg/kg p.o.) produced significant reductions in infarct size after coronary artery occlusion/reperfusion. In conclusion, we have demonstrated that peripheral benzodiazepine receptor play a major role in the regulation of cardiac ischemia-reperfusion injury and that SSR180575, a novel peripheral benzodiazepine receptor ligand, is of potential interest in these indications.

Top down analysis ceramide-induced mitochondrial dysfunctions: role of mitochondrial swelling.

Mitochondrial role in ceramide-induced apoptosis pathway remains unclear. Direct effects of ceramide on mitochondria (cytochrome c release, respiratory chain inhibition, oxygen radicals production...) have been reported [1, 2] and we previously showed that addition of ceramide to intact cells or isolated mitochondria triggers mitochondrial swelling which appeared to be insensitive to cyclosporin A (CsA) [3, 4]. The purpose of this work was to determine to which extent this CsA-insensitive mitochondrial swelling, therefore distinct from permeability transition, participates to ceramide-induced apoptosis. To achieve this, we applied Top-Down analysis of integrated mitochondrial function [5], in order to better understand ceramide-induced mitochondrial dysfunctions.

Deficiency of survivin in transgenic mice exacerbates Fas-induced apoptosis via mitochondrial pathways.

BACKGROUND & AIMS: Survivin is an inhibitor of apoptosis protein (IAP), which also is crucial for mitosis and cell cycle progression. IAPs participate in regulating Fas ligand-induced hepatic apoptosis. The aim was to study the contribution of survivin to hepatic apoptosis by generating transgenic mice lacking survivin. METHODS: The survivin gene was inactivated in mice by homologous recombination in embryonic stem cells. Survivin+/- and survivin+/+ mice were generated and injected with the Fas agonistic antibody Jo2. RESULTS: In 3 genetic backgrounds, survivin-/- embryos died before 4.5 days post coitum. Survivin+/- mice appeared normal, but liver lysates revealed baseline low-level activation of procaspase-8, Bid, procaspase-9, and procaspase-3, with accumulation of Bax, and release of cytochrome c, indicating a proapoptotic state. Intraperitoneal injection of low-dose Jo2 had no effect on survivin+/+ mice at 2 hours. However, in survivin+/- mice, Jo2 caused hemorrhagic necrosis of the liver, associated with prominent activation of the apoptotic pathway via the mitochondria, and up-regulation of hepatocellular expression of survivin in the cytosol, nuclei, and mitochondria. Isolated mitochondria from survivin+/- livers had more defects in oxidative phosphorylation after C(2)-ceramide exposure. CONCLUSIONS: Absence of survivin is incompatible with life. Although Jo2 induces expression of survivin, diminished baseline levels render the liver more sensitive to Fas, possibly due to functional effects on the mitochondria. This is the first in vivo documentation that survivin modulates caspase activation and that Fas-mediated hepatic apoptosis is regulated by survivin via mitochondrial pathways.