Platelet derived serotonin drives the activation of rat cardiac fibroblasts by 5-HT2A receptors.

International audiencePlatelet activation occurs in different acute and chronic heart diseases including myocardial infarction, obstructive hypertrophic cardiomyopathy and valve stenosis. Recent studies suggested that some factors secreted by activated platelets may participate in cardiac remodeling. In the present study, we investigated whether platelets and platelet-released serotonin (5-HT) are directly involved in the functional regulation of cardiac fibroblasts. Treatment of neonatal rat cardiac fibroblasts with platelet lysate, 5-HT and the 5-HT2A receptor agonist DOI increased the expression of alpha-SMA protein, a marker of fibroblast differentiation into myofibroblasts. Platelet lysate, 5-HT and DOI also induced a time-dependent stimulation of cardiac fibroblast migration that was inhibited by the 5-HT2A receptor antagonist ketanserin. Finally, incubation of cardiac fibroblasts with platelet lysate or 5-HT enhanced secretion of TGF-beta1 and expression of MMP-3 and MMP-13. As observed for fibroblast migration, these effects were prevented by ketanserin. These results demonstrated for the first time that factors released from platelet directly regulate cardiac fibroblasts by enhancing secretion of TGF-beta1 and MMPs and promoting their migration and differentiation. 5-HT released by platelets appears to be a major contributor of platelet effects which are mediated through 5-HT2A receptors

Role of endothelial AADC in cardiac synthesis of serotonin and nitrates accumulation.

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation

Inhibition of cardiac 5-HT synthesis <i>in vivo</i> in presence of the AADC inhibitor benserazide.

<p>(A) Mice were pretreated with benserazide (100 mg/kg ip) or saline 30 min before 5-HTP administration (20 mg/kg ip) and they were sacrified 30 min after the last injection. 5-HT and 5-HTP were measured in heart. (B) Mice were treated only with benserazide (100 mg/kg ip) or saline 30 min before euthanasia. 5-HT and 5-HTP were measured in heart and 5-HTP in plasma. Values are means ± SEM (<i>n</i> = 8–10 to each group). *<i>P</i><0.05; **<i>P</i><0.001 vs. saline.</p

Primer sets used in RT-PCR experiments.

<p>Primer sets used in RT-PCR experiments.</p

Expression and localization of AADC in cardiac tissue.

<p>(A) AADC mRNA expression in mouse cardiac tissue was defined by RT-PCR (left panel) and protein expression by Western blot (right panel) (MW: molecular weight). (B) Immunofluorescent double staining of heart frozen sections with antibodies directed against AADC (a,d green) and CD31 (b,e red). The merged pictures (c, f) show co-localization of AADC and CD31 proteins in microvascular (c) and coronary endothelium (d). Magnification 200X.</p

Expression and localization of TPH1 in heart.

<p>(A) TPH1 mRNA expression in cardiac tissue was defined by RT-PCR (MW: molecular weight) (left panel) and Western blot (right panel). Immunoblots showed a faint immunoreactive band in heart lysates of 129/SvJ mice but not in those from 129/SvJ TPH1 KO mice. (B) TPH1 immunostaining localized within vesicular granules in rat heart (right and upper panel). Metachromatic granules of mast cells stained with toluidine blue; magnification 400X (right panels). Few mast cells stained with toluidine blue were also present in heart of 129/SvJ mice, magnification 100X (left panel).</p

Cardiac and blood 5-HT level after 5-HTP administration.

<p>(A) 5-HT and 5-HTP were measured in heart of 129/SvJ mice treated with 5-HTP (5 to 20 mg/kg ip) or saline 30 min before euthanasia. (B) 5-HT concentration in blood of 5-HTP treated mice. Values are means ± SEM (<i>n</i> = 3–7 to each group). *<i>P</i><0.05; **<i>P</i><0.001; ***<i>P</i><0.0001 vs. saline.</p

AADC activity in cardiac tissue.

<p>(A) Enzyme activity was performed by measuring the conversion of different concentration of 5-hydroxytryptophan (5-HTP) (10–100 µM) into 5-HT in homogenates of mouse cardiac tissue. Values are means ± SEM of 5-HT concentration obtained from three different heart homogenates performed in triplicate. (B) Synthesis of 5-HT from 20 µM 5-HTP in presence or not of 50 µM of AADC inhibitor benserazide. Values are means ± SEM (<i>n</i> = 3 to each group) ***<i>P</i><0.0001.</p