Testosterone activates glucose metabolism through AMPK and androgen signaling in cardiomyocyte hypertrophy

Background: Testosterone regulates nutrient and energy balance to maintain protein synthesis and metabolism in cardiomyocytes, but supraphysiological concentrations induce cardiac hypertrophy. Previously, we determined that testosterone increased glucose uptake—via AMP-activated protein kinase (AMPK)—after acute treatment in cardiomyocytes. However, whether elevated glucose uptake is involved in long-term changes of glucose metabolism or is required during cardiomyocyte growth remained unknown. In this study, we hypothesized that glucose uptake and glycolysis increase in testosterone-treated cardiomyocytes through AMPK and androgen receptor (AR). Methods: Cultured cardiomyocytes were stimulated with 100 nM testosterone for 24 h, and hypertrophy was verified by increased cell size and mRNA levels of β-myosin heavy chain (β-mhc). Glucose uptake was assessed by 2-NBDG. Glycolysis and glycolytic capacity were determined by measuring extracellular acidification rate (ECAR). Results: Testosterone induced cardiomyocyte hypertrophy that was accompanied by increased glucose uptake, glycolysis enhancement and upregulated mRNA expression of hexokinase 2. In addition, testosterone increased AMPK phosphorylation (Thr172), while inhibition of both AMPK and AR blocked glycolysis and cardiomyocyte hypertrophy induced by testosterone. Moreover, testosterone supplementation in adult male rats by 5 weeks induced cardiac hypertrophy and upregulated β-mhc, Hk2 and Pfk2 mRNA levels. Conclusion: These results indicate that testosterone stimulates glucose metabolism by activation of AMPK and AR signaling which are critical to induce cardiomyocyte hypertrophy.Fil: Troncoso, Mayarling Francisca. Universidad de Chile; ChileFil: Pavez, Mario. Universidad de Chile; ChileFil: Wilson Rodriguez, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Universidad de Chile; ChileFil: Lagos, Daniel. Universidad de Chile; ChileFil: Duran, Javier. Universidad de Chile; ChileFil: Ramos, Sebastián. Universidad de Chile; ChileFil: Barrientos, Genaro. Universidad de Chile; ChileFil: Silva, Patricio. Universidad Central de Chile; ChileFil: Llanos, Paola. Universidad de Chile; ChileFil: Basualto Alarcón, Carla. Universidad de Chile; Chile. Universidad de Aysén; ChileFil: Westenbrink, B. Daan. University of Groningen; Países BajosFil: Lavandero, Sergio. Universidad de Chile; Chile. Texas A&M University; Estados UnidosFil: Estrada, Manuel. Universidad de Chile; Chil

GDF11 Modulates Ca2+-Dependent Smad2/3 signaling to prevent cardiomyocyte hypertrophy

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor- family, has been shown to act as a negative regulator in cardiac hypertrophy. Ca2+ signaling modulates cardiomyocyte growth; however, the role of Ca2+-dependent mechanisms in mediating the effects of GDF11 remains elusive. Here, we found that GDF11 induced intracellular Ca2+ increases in neonatal rat cardiomyocytes and that this response was blocked by chelating the intracellular Ca2+ with BAPTA-AM or by pretreatment with inhibitors of the inositol 1,4,5-trisphosphate (IP3) pathway. Moreover, GDF11 increased the phosphorylation levels and luciferase activity of Smad2/3 in a concentration-dependent manner, and the inhibition of IP3-dependent Ca2+ release abolished GDF11-induced Smad2/3 activity. To assess whether GDF11 exerted antihypertrophic effects by modulating Ca2+ signaling, cardiomyocytes were exposed to hypertrophic agents (100 nM testosterone or 50 M phenylephrine) for 24 h. Both treatments increased cardiomyocyte size and [H-3]-leucine incorporation, and these responses were significantly blunted by pretreatment with GDF11 over 24 h. Moreover, downregulation of Smad2 and Smad3 with siRNA was accompanied by inhibition of the antihypertrophic effects of GDF11. These results suggest that GDF11 modulates Ca2+ signaling and the Smad2/3 pathway to prevent cardiomyocyte hypertrophy.Fondo Nacional de Ciencia y Tecnologia (FONDECYT) 1151118 CONICYT CONICYT 2115088

GDF-11 prevents cardiomyocyte hypertrophy by maintaining the sarcoplasmic reticulum-mitochondria communication

Growth differentiation factor 11 (GDF11) is a novel factor with controversial effects on cardiac hypertrophy both in vivo and in vitro. Although recent evidence has corroborated that GDF11 prevents the development of cardiac hypertrophy, its molecular mechanism remains unclear. In our previous work, we showed that norepinephrine (NE), a physiological pro-hypertrophic agent, increases cytoplasmic Ca2+ levels accompanied by a loss of physical and functional communication between sarcoplasmic reticulum (SR) and mitochondria, with a subsequent reduction in the mitochondrial Ca2+ uptake and mitochondrial metabolism. In order to study the anti-hypertrophic mechanism of GDF11, our aim was to investigate whether GDF11 prevents the loss of SR-mitochondria communication triggered by NE. Our results show that: a) GDF11 prevents hypertrophy in cultured neonatal rat ventricular myocytes treated with NE. b) GDF11 attenuates the NE-induced loss of contact sites between both organelles. c) GDF11 increases oxidative mitochondrial metabolism by stimulating mitochondrial Ca2+ uptake. In conclusion, the GDF11-dependent maintenance of physical and functional communication between SR and mitochondria is critical to allow Ca2+ transfer between both organelles and energy metabolism in the cardiomyocyte and to avoid the activation of Ca2+-dependent pro-hypertrophic signaling pathways

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

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