Background-Serotonin (5-hydroxytryptamine ), 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.

Abstract-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 speciesdependent 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-Adeficient 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. Key Words: sphingosine kinase-1 Ⅲ monoamine oxidases Ⅲ ischemia/reperfusion Ⅲ ROS Ⅲ serotonin T he ischemia/reperfusion (I/R) syndrome occurring during revascularization results in the progressive loss of cardiac myocytes, ultimately leading to heart failure. 1 A wealth of reports indicate a pivotal role for apoptosis in cardiac myocyte death after I/R. 2-4 Indeed, cardiomyocyte apoptosis was found during the early phase of reperfusion after myocardial ischemia in animal models 5,6 and after cardiac transplantation in humans. 7,8 In addition, prevention of apoptosis strongly reduced postischemic cardiac damage.Abundant evidence suggests that reactive oxygen species (ROS) play an important role in the development and progression of postischemic myocardial damage, leading to apoptosis. 9,10 The induction of oxidative stress is related to an activation of several enzymatic systems including monoamine oxidases (MAOs). 11-13 MAOs catalyze oxidative deamination of several monoamines (eg, serotonin [5-hydroxytryptamine{5-HT}], noradrenaline, dopamine), resulting in significant ROS production. 14 Based on their substrate preference and inhibitor specificity, 2 functional isoenzymes, MAO-A and MAO-B, have been identified. 15 In the heart, MAO-A is a predominant enzyme involved in the deamination of endogenous or exogenous amines. 16 Recently, we have shown that oxidative stress induced by MAO-A is responsible for receptor-independent, serotonin-mediated apoptosis during postischemic myocardial injury....