Serotonin 5-HT2A receptor-mediated hypertrophy is negatively regulated by caveolin-3 in cardiomyoblasts and neonatal cardiomyocytes.

The serotonin 5-HT(2A) receptor belongs to the G-protein-coupled receptors (GPCRs) superfamily and mediates the hypertrophic response to serotonin (5-HT) in cardiac myocytes. At present the regulatory mechanisms of 5-HT(2A) receptor-induced myocyte hypertrophy are not fully understood. The localization and the compartmentation of GPCRs within specialized membrane microdomains are known to modulate their signalling pathway. Therefore, we hypothesized that caveolae microdomains and caveolin-3, the predominant isoform of cardiac caveolae, might be regulators of 5-HT(2A) receptor signalling. We demonstrate that 5-HT(2A) receptors interact with caveolin-3 upon 5-HT stimulation and traffic into caveolae membrane microdomains. We provide evidence that caveolin-3 knockdown abolishes the redistribution of 5-HT(2A) receptors into caveolae and enhances 5-HT(2A) receptor-induced myocyte hypertrophic markers such as cell size, protein synthesis and ANF gene expression. Importantly, we demonstrate that caveolin-3 and caveolae structures are negative regulators of 5-HT(2A) receptor-induced nuclear factor of activated T cells (NFAT) transcriptional activation. Taken together, our data demonstrate that caveolin-3 and caveolae microdomains are important regulators of the hypertrophic response induced by 5-HT(2A) receptors. These findings thus open new insights to target heart hypertrophy under the enhanced serotonin system. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Mesenchymal stem cells promote matrix metalloproteinase secretion by cardiac fibroblasts and reduce cardiac ventricular fibrosis after myocardial infarction.

Recent studies showed that mesenchymal stem cells (MSCs) transplantation significantly decreased cardiac fibrosis; however, the mechanisms involved in these effects are still poorly understood. In this work, we investigated whether the antifibrotic properties of MSCs involve the regulation of matrix metalloproteinases (MMPs) and matrix metalloproteinase endogenous inhibitor (TIMP) production by cardiac fibroblasts. In vitro experiments showed that conditioned medium from MSCs decreased viability, alpha-smooth muscle actin expression, and collagen secretion of cardiac fibroblasts. These effects were concomitant with the stimulation of MMP-2/MMP-9 activities and membrane type 1 MMP expression. Experiments performed with fibroblasts from MMP2-knockout mice demonstrated that MMP-2 plays a preponderant role in preventing collagen accumulation upon incubation with conditioned medium from MSCs. We found that MSC-conditioned medium also decreased the expression of TIMP2 in cardiac fibroblasts. In vivo studies showed that intracardiac injection of MSCs in a rat model of postischemic heart failure induced a significant decrease in ventricular fibrosis. This effect was associated with the improvement of morphological and functional cardiac parameters. In conclusion, we showed that MSCs modulate the phenotype of cardiac fibroblasts and their ability to degrade extracellular matrix. These properties of MSCs open new perspectives for understanding the mechanisms of action of MSCs and anticipate their potential therapeutic or side effects.

Genetic deletion of MAO-A promotes serotonin-dependent ventricular hypertrophy by pressure overload.

The potential role of serotonin (5-HT) in cardiac function has generated much interest in recent years. In particular, the need for a tight regulation of 5-HT to maintain normal cardiovascular activity has been demonstrated in different experimental models. However, it remains unclear how increased levels of 5-HT could contribute to the development of cardiac hypertrophy. Availability of 5-HT depends on the mitochondrial enzyme monoamine oxidase A (MAO-A). Therefore, we investigated the consequences of MAO-A deletion on ventricular remodeling in the model of aortic banding in mice. At baseline, MAO-A deletion was associated with an increase in whole blood 5-HT (39.4+/-1.9 microM vs. 24.0+/-0.9 microM in KO and WT mice, respectively). Cardiac 5-HT(2A), but not 5-HT(2B) receptors were overexpressed in MAO-A KO mice, as demonstrated by real-time PCR and Western-blot experiments. After aortic banding, MAO-A KO mice demonstrated greater increase in heart wall thickness, heart to body weight ratios, cardiomyocyte cross-section areas, and myocardial fibrosis compared to WT. Exacerbation of hypertrophy in KO mice was associated with increased amounts of 5-HT in the heart. In order to determine the role of 5-HT and 5-HT(2A) receptors in ventricular remodeling in MAO-A KO mice, we administered the 5-HT(2A) receptor antagonists ketanserin (1 mg/kg/day) or M100907 (0.1 mg/kg/day) during 4 weeks of aortic banding. Chronic administration of these antagonists strongly prevented exacerbation of ventricular hypertrophy in MAO-A KO mice. These results show for the first time that regulation of peripheral 5-HT by MAO-A plays a role in ventricular remodeling via activation of 5-HT(2A) receptors.

3-[5-(4,5-dihydro-1H-imidazol-2-yl)-furan-2-yl]phenylamine (Amifuraline), a promising reversible and selective peripheral MAO-A inhibitor.

On the basis of the observation that the central side effects of MAO inhibitors may represent a major limit for their use in pathological processes involving peripheral MAOs, we investigated the possibility of generating novel inhibitors able to target specifically peripheral MAOs. To address this issue, we designed compounds 7-28. From biological results, the 2-(5-phenyl-furan-2-yl)-4,5-dihydro-1H-imidazole (Furaline, 17) proved to be a suitable lead. In fact, in enzyme assays on homogenate preparation from rat liver and HEK cells expressing MAO-A or MAO-B, compounds possessing the frame of 17 behaved as selective and reversible MAO-A inhibitors. Interestingly, in in vivo studies the amino derivative 21 (Amifuraline), endowed with good hydrophilic character, was able to significantly inhibit liver but not brain MAO-A.

Glucose handling in streptozotocin-induced diabetic rats is improved by tyramine but not by the amine oxidase inhibitor semicarbazide.

A soluble form of semicarbazide-sensitive amine oxidase (SSAO) circulating in plasma is known to increase in type 1 and 2 diabetes. This cuproenzyme generates hydrogen peroxide, ammonia, and aldehydes when oxidizing circulating biogenic or exogenous amines. Based on the angiotoxicity of these products, inhibition of SSAO has been proposed to prevent vascular complications of diabetes. However, substrates of SSAO and monoamine oxidase (MAO) have been recently evidenced to activate glucose utilisation in insulin-sensitive tissues and to exhibit antihyperglycemic actions. To determine whether amine oxidase blockade or activation could be beneficial for diabetes, we aimed at comparing the influence of prolonged treatments with semicarbazide (SSAO-inhibitor), pargyline (MAO-inhibitor), or tyramine (amine oxidase substrate) on amine oxidase activities and glycemic control in streptozotocin-induced diabetic rats. The increase in plasma SSAO was confirmed in diabetic rats, while MAO and SSAO were decreased in subcutaneous adipose tissue when compared with normoglycemic controls. Among the diabetic rats, only those receiving tyramine exhibited slightly decreased hyperglycemia and improved glucose tolerance. Adipocytes from untreated or treated diabetic rats shared similar sensitivity to insulin. However glucose uptake activation and lipolysis inhibition in response to amine oxidase substrates combined with vanadate were impaired in rats treated with amine oxidase inhibitors. Thus, amine oxidase inhibition does not improve metabolic control while prolonged administration of tyramine slightly improves glucose disposal. It is therefore concluded that amine oxidase activation by increased substrate supply elicits insulin-like actions that may be more beneficial in diabetes than SSAO inhibition formerly proposed to prevent vascular complications.

p53-PGC-1α pathway mediates oxidative mitochondrial damage and cardiomyocyte necrosis induced by monoamine oxidase-A upregulation: role in chronic left ventricular dysfunction in mice.

AIMS: Oxidative stress and mitochondrial dysfunction participate together in the development of heart failure (HF). mRNA levels of monoamine oxidase-A (MAO-A), a mitochondrial enzyme that produces hydrogen peroxide (H(2)O(2)), increase in several models of cardiomyopathies. Therefore, we hypothesized that an increase in cardiac MAO-A could cause oxidative stress and mitochondrial damage, leading to cardiac dysfunction. In the present study, we evaluated the consequences of cardiac MAO-A augmentation on chronic oxidative damage, cardiomyocyte survival, and heart function, and identified the intracellular pathways involved. RESULTS: We generated transgenic (Tg) mice with cardiac-specific MAO-A overexpression. Tg mice displayed cardiac MAO-A activity levels similar to those found in HF and aging. As expected, Tg mice showed a significant decrease in the cardiac amounts of the MAO-A substrates serotonin and norepinephrine. This was associated with enhanced H(2)O(2) generation in situ and mitochondrial DNA oxidation. As a consequence, MAO-A Tg mice demonstrated progressive loss of cardiomyocytes by necrosis and ventricular failure, which were prevented by chronic treatment with the MAO-A inhibitor clorgyline and the antioxidant N-acetyl-cystein. Interestingly, Tg hearts exhibited p53 accumulation and downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial function. This was concomitant with cardiac mitochondrial ultrastructural defects and ATP depletion. In vitro, MAO-A adenovirus transduction of neonatal cardiomyocytes mimicked the results in MAO-A Tg mice, triggering oxidative stress-dependent p53 activation, leading to PGC-1α downregulation, mitochondrial impairment, and cardiomyocyte necrosis. INNOVATION AND CONCLUSION: We provide the first evidence that MAO-A upregulation in the heart causes oxidative mitochondrial damage, p53-dependent repression of PGC-1α, cardiomyocyte necrosis, and chronic ventricular dysfunction.

Characterization of monoamine oxidases in mesenchymal stem cells: role in hydrogen peroxide generation and serotonin-dependent apoptosis.

Early death of grafted bone marrow mesenchymal stem cells (MSCs) represents a major limit to their use in cell therapy of solid organs. It is well known that oxidative stress plays a major role in cell death. We have recently shown that the serotonin-degrading enzyme monoamine oxidase A (MAO-A) generates large amount of hydrogen peroxide (H2O2) responsible for cell apoptosis. Hydrogen peroxide generation requires 5-HT internalization into the cell and its degradation by MAO-A. In the present study, we investigated whether MAO-A is expressed in MSCs and we defined its role in serotonin-dependent MSCs apoptosis. RT-PCR analysis and western blots showed that the serotonin transporter (SERT) and the 2 MAO isoenzymes, A and B, are expressed in MSCs. As shown by enzyme assays using [14C]serotonin or [14C]ß-phenylethylamine as selective MAO-A or MAO-B substrates, MAO-A is largely predominant in MSCs. Incubation of MSCs with the MAO substrate tyramine led to a time-dependent generation of H2O2 that was prevented by the MAO inhibitor pargyline. Finally, exposure of the cells to serotonin promoted an increase in MSCs apoptosis prevented by pargyline and the SERT inhibitor imipramine. The pro-apoptotic effect of serotonin was associated to a decrease in the expression of the anti-apoptotic factor Bcl-2. In conclusion, these results show for the first time that the 5-HT-degrading enzyme MAO-A is an important source of H2O2 in MSCs and plays a major role in 5-HT-dependent MSCs apoptosis.

Substrate-dependent regulation of MAO-A in rat mesangial cells: involvement of dopamine D2-like receptors.

In the present study, we investigated the existence of a back-regulation of the catecholamine-degrading enzyme monoamine oxidase (MAO)-A by dopamine in rat renal cells. In proximal tubule cells, MAO-A expression was not modified after dopamine receptor stimulation. In contrast, in mesangial cells, enzyme assay and Western blots showed that MAO activity and protein increased by approximately 80% after 48-h incubation with the D(2)-like receptor agonist bromocriptine and quinpirole but not with the D(1)-like receptor agonist SKF-38393. This effect was prevented by the D(2)-receptor antagonist sulpiride and domperidone. The increase in MAO-A protein was preceded by an augmentation of MAO-A mRNA that was prevented by the transcriptional inhibitor actinomycin D. Bromocriptine effect was mimicked by the PKA inhibitor H89 and inhibited by the PKA activator 8-bromo-cAMP. These results show for the first time the existence of a dopamine-dependent MAO-A regulation involving D(2)-like receptors, inhibition of the cAMP-PKA pathway, and an ex novo enzyme synthesis.