GSK-3β/NFAT Signaling Is Involved in Testosterone-Induced Cardiac Myocyte Hypertrophy

Area of Science:

• Cardiovascular Biology
• Molecular Endocrinology
• Cell Signaling

Background:

• Testosterone is known to induce cardiac hypertrophy, a process involving complex intracellular signaling.
• Nuclear Factor of Activated T-cells (NFAT) promotes cardiac hypertrophy, while Glycogen Synthase Kinase-3β (GSK-3β) acts as a negative regulator, primarily by modulating NFAT.
• The specific roles of calcineurin-NFAT and GSK-3β signaling in testosterone-induced cardiac hypertrophy were previously undefined.

Purpose of the Study:

• To investigate the molecular mechanisms by which testosterone stimulates cardiac myocyte hypertrophy.
• To determine the involvement of calcineurin-NFAT and GSK-3β signaling pathways in testosterone-mediated cardiac hypertrophy.
• To elucidate the crosstalk between androgen signaling and these key intracellular pathways.

Main Methods:

• Utilized NFAT-luciferase reporter assays to measure NFAT activity under testosterone stimulation.
• Employed calcineurin inhibitors (FK506, cyclosporine A) and a specific NFAT inhibitor (11R-VIVIT).
• Assessed GSK-3β activity via phosphorylation status and β-catenin levels, using inhibitors (1-azakenpaullone) and siRNA, and a constitutively active mutant (GSK-3βS9A).
• Quantified cardiac myocyte hypertrophy by measuring cell size and protein synthesis ([3H]-leucine incorporation).
• Investigated the role of the intracellular androgen receptor.

Main Results:

• Testosterone dose- and time-dependently increased NFAT activity, peaking at 24 hours with 100 nM testosterone.
• Testosterone inhibited GSK-3β activity, evidenced by increased GSK-3β phosphorylation at Ser9 and β-catenin accumulation.
• Calcineurin-NFAT inhibition abolished testosterone-induced hypertrophy, while GSK-3β inhibition promoted it.
• Inhibition of the intracellular androgen receptor blocked testosterone-induced NFAT-Luc activation.
• Testosterone-induced cardiac myocyte hypertrophy was confirmed by increased cell size and protein synthesis.

Conclusions:

• Testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3β inhibition.
• The findings reveal a cooperative mechanism linking androgen signaling to NFAT recruitment via calcineurin activation and GSK-3β inhibition.
• These pathways are critical mediators of testosterone's hypertrophic effects on cardiac myocytes.