Lycopene Inhibits Urotensin-II-Induced Cardiomyocyte Hypertrophy in Neonatal Rat Cardiomyocytes

Abstract

This study investigated how lycopene affected urotensin-II- (U-II-) induced cardiomyocyte hypertrophy and the possible implicated mechanisms. Neonatal rat cardiomyocytes were exposed to U-II (1 nM) either exclusively or following 6 h of lycopene pretreatment (1-10 μ M). The lycopene (3-10 μ M) pretreatment significantly inhibited the U-II-induced cardiomyocyte hypertrophy, decreased the production of U-II-induced reactive oxygen species (ROS), and reduced the level of NAD(P)H oxidase-4 expression. Lycopene further inhibited the U-II-induced phosphorylation of the redox-sensitive extracellular signal-regulated kinases. Moreover, lycopene treatment prevented the increase in the phosphorylation of serine-threonine kinase Akt and glycogen synthase kinase-3beta (GSK-3 β ) caused by U-II without affecting the protein levels of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). However, lycopene increased the PTEN activity level, suggesting that lycopene prevents ROS-induced PTEN inactivation. These findings imply that lycopene yields antihypertrophic effects that can prevent the activation of the Akt/GSK-3 β hypertrophic pathway by modulating PTEN inactivation through U-II treatment. Thus, the data indicate that lycopene prevented U-II-induced cardiomyocyte hypertrophy through a mechanism involving the inhibition of redox signaling. These findings provide novel data regarding the molecular mechanisms by which lycopene regulates cardiomyocyte hypertrophy.

Figure 1

Effect of  lycopene on U-II-induced cardiomyocyte hypertrophy. (a) Effect of  lycopene on U-II-induced morphologic changes in cardiomyocytes. Cultured cardiomyocytes were exposed to vehicle control (control: top left), to lycopene alone at 3 μM (lycopene: top right), or to U-II at 1 nM for 24 h in the absence (U-II: bottom left) or presence of lycopene (U-II + lycopene: bottom right) and then immunostained with an anti-α-actinin antibody (red), and the nucleus was stained with DAPI (blue). The figure shows a representative stain prepared based on 3 independent experiments. Scale bar shows 20 μm. (b) Relative cardiomyocyte size. The cultured cardiomyocytes were exposed to vehicle control or U-II at 1 nM or 24 h in the absence or presence of lycopene (1, 3, and 10 μM). The surface areas of the cardiomyocytes were measured using NIH image software in 60 randomly chosen cells from 3 dishes. The data are presented as mean ± SEM. (c) Measuring protein synthesis by using [³H]-leucine incorporation. The results are shown as mean ± SEM (n = 6). *P < 0.05 versus control; ^# P < 0.05 versus U-II alone.

Figure 2

Effects of lycopene on U-II-induced ROS production in cardiomyocytes. (a) ROS levels after adding lycopene or the NAD(P)H oxidase-4 (NOX-4) inhibitor apocynin in cells exposed to U-II for 2 min. The ROS levels of cells pretreated for 6 h with lycopene (3 μM) or 30 min with apocynin (0.3 mM) and exposed to U-II (1 nM) for 2 min. (b) Upper panels: representative western blot analysis of NOX-4 in cells pretreated for 6 h with lycopene (3 μM) then exposed to U-II. Lower panels: the indicated values represent the ratio of NOX-4 to GAPDH. The results are shown as mean ± SEM (n = 6).*P < 0.05 versus control.

Figure 3

Effects of lycopene on U-II-induced ERK phosphorylation in cardiomyocytes. Upper panels: representative western blot analyses of ERK in cells pretreated for 6 h with lycopene (3 μM) then exposed to U-II (1 nM) for 30 min. Lower panels: the indicated values represent the ratio of phosphorylated protein to total protein. The results are shown as mean ± SEM (n = 6). *P < 0.05 versus control; ^# P < 0.05 versus U-II alone.

Figure 4

Lycopene treatment prevents Akt/GSK-3β phosphorylation in U-II-stimulated neonatal rat cardiomyocytes. Protein extracts from cardiomyocytes were subjected to immunoblot analysis. (a) Effects of lycopene on U-II-induced Akt phosphorylation in cardiomyocytes. Upper panels: representative immunoblots using total and antiphospho-Akt (Ser473) antibodies. Lower panels: the indicated values represent the ratio of phosphorylated protein to total protein. (b) Effects of lycopene on U-II-induced GSK-3β phosphorylation in cardiomyocytes. Upper panels: representative immunoblots using total and antiphospho-GSK-3β (Ser9) antibodies. Lower panels: the indicated values represent the ratio of phosphorylated protein to total protein. Neonatal rat cardiomyocytes were nonstimulated (control) or stimulated with U-II (1 nM for 2 min). When indicated, lycopene (3 μM) was added 6 h before U-II stimulation. The results are shown as mean ± SEM (n = 6). *P < 0.05 versus control; ^# P < 0.05 versus U-II alone.

Figure 5

Lycopene treatment prevents PTEN oxidation in U-II-stimulated neonatal rat cardiomyocytes. (a) Lycopene does not affect PTEN protein levels in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to U-II (1 nM for 1 h) in the presence or absence of lycopene (3 μM) added 6 h prior to the stimuli. Representative immunoblots using anti-PTEN antibodies or anti-GAPDH antibodies are shown. The blot data are representative of 3 experiments. (b) Effect of lycopene on PTEN activity. Cardiomyocytes were treated with U-II (1 nM) in the absence or presence of lycopene (3 μM) as indicated. The oxidation of the endogenous PTEN in the cardiomyocytes exposed to 0.5 mM H₂O₂. The level of PTEN activity in each sample was detected using a PTEN Malachite Green Assay Kit. The relative level of PTEN activity is shown as mean ± SEM (n = 6). *P < 0.05 versus control; ^# P < 0.05 versus U-II alone.