Experimental Research On Role Of 5-HT 2B Receptor In Norepinephrine-induced Cardiac Hypertrophy In Rats

Serotonin or 5-hydroxytryptamine is a biogenic amine produced predominantly by intestinal enterochromaffin cells and stored in platelets in periphery. Its receptors are wide distributed in the cardiovascular system. 5-HT affects a wide variety of physiological and pathological functions on the cardiovascular system. Recent studies indicated that ventricular hypertrophy induced by 5-HT involves dual mechanisms of action through activation of 5-HT2 receptor, or receptor-independent mechanism. However, it is not well known that the ralationship between 5-HT and other tradational factors that could induce cardiac hypertrophy. Particularly, the machenism of 5-HT involves in cardiac hypertrophy induced by other factors is not known yet.In present study, we systemically investigated whether 5-HT2B receptors could be involved in the development of cardiac hypertrophy and the generation of apoptotic events associated with norepinephrine overload. 1. Attenuation of cardiac hypertrophy by 5-HT2B receptor antagonistRats treated with NE developed cardiac hypertrophy as indicated by increased ratios of left ventricular weight to body weight (LVW/BW) and left ventricular free wall thickness to body weight (LVWT/BW). In the meantime, the levels of 5-HT increased significantly in left ventricular tissues. Blockade of 5-HT2B receptor by SB204741 significantly attenuated the degree of cardiac hypertrophy and levels of 5-HT in a dose-dependent manner in the NE-injected rats; however, treatment of SB204741 alone had no effects on the degree of cardiac hypertrophy and levels of 5-HT in saline control rats. Another 5-HT2B receptor antagonist, SDZ SER 082, showed the similar effects and further confirmed the role of 5-HT2B receptor in formation of cardiac hypertrophy. The results suggest that 5-HT2B receptor involves in development of cardiac hypertrophy induced by NE over stimulation. There should be some synergic effects between 5-HT and NE through activation of 5-HT2B receptor.2. Anti-apoptotic activity of SB204741 in cardiac hypertrophy induced by NE over stimulationRemodeling cardiomyocytes upon exposure to increasing doses of NE are associated with increased cardiomyocyte apoptosis. Next, we detected whether treatment with SB204741 could prevent NE-induced cardiomyocyte apoptosis by TUNEL staining. Apoptotic cardiomyocytes were rarely seen in the heart of saline treated rats. However, TUNEL-positive nuclear staining increased significantly in the left ventricle from rats treated with NE. The number of TUNEL-positive nuclear staining was notably decreased by administration of SB204741, suggesting that activation of 5-HT2B receptors provoke the cardiac apoptosis induced by chronic NE overload.Anti-apoptotic proteins of the Bcl-2 family may exert cardiac protection by multiple mechanisms such as a direct anti-oxidant effect, inhibition of pro-apoptotic proteins, and mitochondrial membrane stabilization. However, caspases activated by NE play key roles in cardiomyocytes apoptosis. Cardiac Bcl-2 protein levels were significantly decreased, while Bax, caspase-3 and 5-HT2B receptor protein levels were increased in hearts of rats with chronic NE treatment. Blockade of 5-HT2B receptor with SB204741 showed an anti-apoptotic activity as shown by decreasing Bax/Bcl-2 ratio and caspase-3 expression. Moreover, expression of 5-HT2B receptor was significantly down-regulated.3. Effect of SB204741 on apoptosis and mitochondria injury of cardiomyocytes in cultured cardiomyocytes induced by NE over stimulationTo further provide direct evidence of 5-HT2B receptor actions on cardiomyocytes and exclude in vivo endogenous biochemical and compensatory effects, we performed Hoechst staining in the cultured neonatal cardiomyocytes to test the effects of SB204741 on apoptosis induced by NE. Nuclear fragmentation and chromatin clumping were shown in the cardiomyocytes after treatment with high dose of NE for 48 hours in the presence of 5μM serotonin. When counted the cells, we observed an increased percentage of apoptotic cardiomyocytes in NE treated group as compared with saline treated one. SB204741 notably attenuated apoptotic activities induced by NE treatment.Mitochondria are the important component of the apoptotic signaling, especially in the heart. We performed transmission electron microscopy detection in the cultured neonatal cardiomyocytes to test the effects of SB204741 on mitochondria injury induced by NE. Cultured cardiomyocytes treated with high dose of NE for 48 hours were severely damaged. Some of cardiomyocytes were floated in the culture media and contract of the adherent cardiomyocytes were weaker than that of the control cells. Ultrastructural studies of the cultured cardiomyocytes revealed mitochondria with swollen matrix, vague granules and cristae in the NE-treated cells as compared with the control ones. Administration of SB204741 blunted mitochondrial structure damage induced by NE incubation.4. Effect of SB204741 on calcium current of cardiomyocytes in development of cardiac hypertrophy induced by NE over stimulationTo further examine the activity of 5-HT2B receptors in the development of myocardial hypertrophy, we performed whole-cell patch-clamp recordings in the isolated single cardiomyocyte again. Size of left ventricular myocytes was increased significantly in NE treated rats as compared to the saline controls assayed in terms of cell membrane capacitance. SB204741 significantly attenuated membrane capacity of cardiomyocytes in NE treated rats.It is not known whether the electrophysiological and structural phenotypes of hypertrophy are causally linked with 5-HT, we examined whether they could be dissociated by 5-HT2B inhibition. Because intracellular Ca2+ is increasingly viewed as a central point of regulation in the pathogenesis of hypertrophy, apoptosis, and disease-related electrical remodeling, we measured the ICa-L in the presence and absence of hypertrophy. Current kinetics and current–voltage (I–V) relationships of ICa-L appeared similar in the three groups. Consistent with the previous report, ICa-L density (normalization to cell size capacitance) was increased markedly in the hypertrophic myocytes from the NE injected rats. Blockade of 5-HT2B receptors with SB204741 significantly reversed enhancement of ICa-L density in the single cardiomyocyte isolated from NE injected rats.It is not well known whether SB204741 directly inhibits the L-type calcium channels in vitro. So we performed whole-cell patch-clamp recordings in the isolated single cardiomyocyte. Consistent with the previous report, perfusion of 5-HT (1μM) could increase ICa-L current markedly in the isolated adult myocytes. This effect was abolished by perfusion of 2μM SB204741. Analysis of current kinetics of ICa-L revealed that SB204741 did not affect the current kinetics and current–voltage (I–V) relationships. It provided direct evidence that blockade of 5-HT2B receptors could prevent 5-HT-induced promotion of calcium influx through L-type calcium channels.Taken together, SB204741 could partially reverse cardiac hypertrophy induced by NE overload and decreased L-type calcium currents in the ventricular cardiomyocytes. In addition, SB204741 could notably attenuated myocardial apoptosis by down-regulation of Bax, caspase-3 activities, and up-regulation of anti-apoptotic Bcl-2 protein. In vitro, SB204741 notably attenuated apoptotic activities and blunted mitochondrial structure damage induced by NE treatment.Our data suggest an involvement of 5-HT2B receptors in development of cardiac hypertrophy and the generation of apoptotic events associated with cardiac remodeling during increased adrenergic stimulation.