Abstract
Testosterone is known to induce cardiac hypertrophy through androgen receptor (AR)-dependent and -independent pathways, but the molecular underpinnings of the androgen action remain poorly understood. Previous work has shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII) and myocyte-enhancer factor 2 (MEF2) play key roles in promoting cardiac myocyte growth. In order to gain mechanistic insights into the action of androgens on the heart, we investigated how testosterone affects CaMKII and MEF2 in cardiac myocyte hypertrophy by performing studies on cultured rat cardiac myocytes and hearts obtained from adult male orchiectomized (ORX) rats. In cardiac myocytes, MEF2 activity was monitored using a luciferase reporter plasmid, and the effects of CaMKII and AR signaling pathways on MEF2C were examined by using siRNAs and pharmacological inhibitors targeting these two pathways. In the in vivo studies, ORX rats were randomly assigned to groups that were administered vehicle or testosterone (125 mg⋅kg-1⋅week-1) for 5 weeks, and plasma testosterone concentrations were determined using ELISA. Cardiac hypertrophy was evaluated by measuring well-characterized hypertrophy markers. Moreover, western blotting was used to assess CaMKII and phospholamban (PLN) phosphorylation, and MEF2C and AR protein levels in extracts of left-ventricle tissue from control and testosterone-treated ORX rats. Whereas testosterone treatment increased the phosphorylation levels of CaMKII (Thr286) and phospholambam (PLN) (Thr17) in cardiac myocytes in a time- and concentration-dependent manner, testosterone-induced MEF2 activity and cardiac myocyte hypertrophy were prevented upon inhibition of CaMKII, MEF2C, and AR signaling pathways. Notably, in the hypertrophied hearts obtained from testosterone-administered ORX rats, both CaMKII and PLN phosphorylation levels and AR and MEF2 protein levels were increased. Thus, this study presents the first evidence indicating that testosterone activates MEF2 through CaMKII and AR signaling. Our findings suggest that an orchestrated mechanism of action involving signal transduction and transcription pathways underlies testosterone-induced cardiac myocyte hypertrophy.
Highlights
The biological effects of androgens on cardiac myocytes are tightly regulated by the circulating plasma levels of testosterone, the specific activation of intracellular signaling networks by this hormone, and, the functional integration of these signaling pathways
We examined the kinetics of calmodulindependent protein kinase II (CaMKII) phosphorylation over short (5–60 min) and long (3–24 h) periods of stimulation with 100 nM testosterone
CaMKII phosphorylation increased at 5 min and peaked at 15 min after testosterone stimulation (Figure 1A), whereas in the longer time-course, CaMKII phosphorylation peaked at 3 h, to a level lower than that at 15 min, and returned to its basal level within 6–9 h (Figure 1B)
Summary
The biological effects of androgens on cardiac myocytes are tightly regulated by the circulating plasma levels of testosterone, the specific activation of intracellular signaling networks by this hormone, and, the functional integration of these signaling pathways. An after-effect produced by elevated circulating levels of testosterone is cardiac hypertrophy; the initial growth of cardiac myocytes that is triggered here is an adaptive response, a sustained anabolic environment becomes detrimental and leads to an increase in cardiac morbidity and mortality (Thum and Borlak, 2002; Liu et al, 2003) Most of this evidence was gathered from studies on people who abused anabolic steroids or were administered multiple drugs, and this has precluded a clear understanding of the precise actions of androgens; the mechanisms underlying testosterone signaling in cardiac myocytes remain largely elusive
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