PI3K/Akt signaling is involved in GSK-3β inhibition.

<p>Cardiac myocytes were pretreated for 30 min with Akt-inhibitor VIII (Akti-VIII, 10 μM), LY-294002 (10 μM), or PD98059 (50 μM) prior to testosterone stimulation (100 nM) by 30 min. The densitometry results show the ratio of phosphorylated protein to total protein (n = 4 for each condition). Values are presented as the mean ± SEM. *p < 0.05 <i>vs</i>. control.</p

GSK-3β inhibition activates NFAT.

<p>Cardiac myocytes were co-transfected with NFAT-Luc and <i>Renilla</i> luciferase plasmids. (A) Cells were pretreated with 1-Azk for 30 min (1 μM) prior to 100 nM testosterone stimulation for 24 h. (B) Cardiac myocytes were transfected with either siRNA-GSK-3β or a non-targeted siRNA control and then stimulated with 100 nM testosterone for 24 h. (C) Cardiac myocytes were transfected with either GSK-3βWT or GSK-3βS9A, and then stimulated with 100 nM testosterone for 24 h (n = 6 for each condition). Values are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 <i>vs</i>. control; # p < 0.05 <i>vs</i>. 1-Azk.</p

NFAT is involved in cardiac myocyte hypertrophy induced by testosterone.

<p>Cardiac myocytes were pretreated with FK506 (1 μM) or CsA (1 μM) or 11R-VIVIT (1 μM) and stimulated with 100 nM testosterone for 48 h. (A) For experiments involving cellular area measurement, the cardiac myocytes were incubated with CellTracker Green and visualized by confocal microscopy (n > 80 cells from 4 independent cell cultures). (B) Protein synthesis was determined based on [³H]-leucine incorporation. The data correspond to the ratio (basal counts/min)·μg^-1 protein for each experimental condition (n = 6 for each condition). Values are presented as the mean ± SEM. *p < 0.05, and ***p < 0.001 <i>vs</i>. control non-stimulated condition.</p

Testosterone activates NFAT in cardiac myocytes.

<p>NFAT activity was determined in cardiac myocytes transfected with a NFAT luciferase-reporter plasmid (NFAT-Luc) and normalized relative to <i>Renilla</i> luciferase activity in each sample. NFAT-Luc activity is expressed as fold induction relative to non-stimulated cells. (A) Cardiac myocytes were stimulated with 100 nM testosterone for 6, 12, 24, and 48 h. (B) Concentration-dependent effect of testosterone on NFAT-Luc activity. Cells were stimulated with 1, 10, 100, or 1000 nM testosterone for 24 h. Treatment with 100 and 1000 nM testosterone significantly increased NFAT-Luc activity as compared with non-stimulated cells. (C) Pretreatment of cardiac myocytes for 30 min with FK506 (1 μM), CsA (1 μM), or 11R-VIVIT (1 μM) prior testosterone stimulation (100 nM by 24 h) reduced NFAT-Luc activation. Values are presented as the mean ± SEM (n = 5 for each condition). *p < 0.05, **p < 0.01, and ***p < 0.001 <i>vs</i>. control non-stimulated condition.</p

Testosterone inhibits GSK-3β in cardiac myocytes.

<p>Cardiac myocytes were stimulated with 100 nM testosterone for 15, 30, 60, 90, and 120 min and subjected to Western blot analysis to determine (A) GSK-3β phosphorylation (p-GSK-3β, Ser9) and protein levels (n = 5) and (B) β-catenin phosphorylation and total β-catenin protein levels (n = 4). The densitometric analyses show the ratio of phosphorylated versus total protein. Testosterone increased GSK-3β phosphorylation at Ser9 and decreased β-catenin phosphorylation at Ser33, Ser37, and Thr41 after 30 min of stimulation. (C) Cardiac myocytes were stimulated with 100 nM testosterone for 3, 6, 9, 12, and 24 h and total β-catenin protein accumulation was determined through Western blotting (n = 5). Values are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 <i>vs</i>. control non-stimulated condition.</p

Testosterone-induced NFAT activation depends on androgen receptor.

<p>Cardiac myocytes expressing NFAT-Luc were pretreated with AR inhibitors. (A) Cells were pretreated for 30 min with bicalutamide (1 mM) or cyproterone (1 μM) before stimulation with testosterone (100 nM) for 24 h. (B) Cardiac myocytes were transfected with siRNA-AR (20 nM) or non-targeting siRNA as a control. AR downregulation abolished the increase in NFAT-Luc activity induced by testosterone. Values are presented as the mean ± SEM (n = 4 for each condition). *p < 0.05 and **p < 0.01 <i>vs</i>. control.</p