**6. Integrated model for androgen action**

Androgens diffuse across plasma membrane due to their lipophilic nature. Once inside the cell, they are recognized by the cytosolic androgen receptor, which forms a complex with its ligand. This complex works as a transcription factor, which translocates to cell nucleus, binds to DNA and promotes the activation of several genes (Beato, 1989). This is called the

Cardiovascular Effects of Androgens 73

depicted in Figure 2, testosterone activates a plasma membrane G-protein coupled receptor, a signaling pathway that results in a nongenomic mechanism. Activation of this pathway facilitates the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, thereby generating diacylglycerol and IP3. IP3 mediates opening of IP3 receptors in the sarcoplasmatic reticulum, which in turn produce rapid (1-5 min) Ca2+ release (Vicencio et al., 2006). Similar effects of testosterone have been identified in T-cells, skeletal muscle and neuroblastoma cells (Benten et al., 1999; Estrada et al., 2003, 2006). In cardiomyocytes, nongenomic signals activate the extracellular signal-regulated kinase (ERK) and the mTOR

pathways. Activation of Ca2+/ERK/mTOR axis is independent of androgen receptor.

regulates gene expression.

**7. Perspective** 

protocols.

**8. Acknowledgment** 

*Circ Res,* 84, 813-819.

**9. References** 

This work was supported by FONDECYT grant 1090276.

The anabolic properties of testosterone indicate that this hormone regulate multiple genes involved in growth and metabolism. Traditionally, ligand-dependent androgen receptors act on DNA within the nucleus and in this compartment there is cross-talk among the nuclear receptors and cytosolic signaling. We have determined that testosterone induce a hypertrophic pattern in cardiomyocytes, evaluated as increase in the expression of fetal proteins, cardiomyocyte size, and protein synthesis. Hypertrophy effects of testosterone were blocked by either inhibition of mTOR or androgen receptor (Altamirano et al., 2009). We propose that cell growth produced by androgens require both androgen receptor activity and translation control through mTOR signaling pathway. Thus, both mTOR pathway and androgen receptor could control protein synthesis by a coordinated mechanism, where mTOR regulates translation and the intracellular androgen receptor

The role of sex steroid hormones in modulating cardiovascular function is of the highest importance, given that androgen deficiency is strongly associated with common medical conditions including metabolic syndrome, obesity, diabetes, hypertension and atherosclerosis. However, cardiovascular side effects of high doses of anabolic/androgen steroid reduce its actual therapeutic use. Research in this field is necessary to know the real cardiovascular effects of androgens and to elaborate future optimal therapeutic replacement

Akkad, A. A.; Halligan, A. W.; Abrams, K.; & al-Azzawi, F. (1997). Differing responses in

Altamirano, F.; Oyarce, C.; Silva, P.; Toyos, M.; Wilson, C.; Lavandero, S.; Uhlen, P.; &

transdermal estrogen replacement therapy. *Obstet Gynecol,* 89, 97-103. Alexandersen, P.; Haarbo, J.; Byrjalsen, I.; Lawaetz, H.; & Christiansen, C. (1999). Natural

blood pressure over 24 hours in normotensive women receiving oral or

androgens inhibit male atherosclerosis: a study in castrated, cholesterol-fed rabbits.

Estrada, M. (2009). Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway. *J Endocrinol* 202, 299-307.

classic or genomic androgen response pathway. On the other hand, it has been reported that androgens, exert a rapid, non-genomic effect mediated by a putative plasma membrane receptor. Its activation implicates participation of kinases, intracellular calcium oscillations and transcription factor activation, among other fast events.

Cardiomyocytes are terminally differentiated cells. Cardiac hypertrophy is characterized by an increase in cell size and protein synthesis and reactivation of a fetal gene program. Emerging evidence indicate that androgen action involves a crosstalk between genomic and nongenomic mechanisms. These could imply a two-step process, where both non-genomic and genomic effects occur sequentially (**Figure 2**). First, fast effects would be generated at the plasma membrane level with second messenger participation. Second, there would be gene activation; the pathways used by each cell type could provide specificity to the signal through a cooperative mechanism, which can act in a concerted form.

Fig. 2. Integrated mechanism for testosterone effects in cardiomyocytes. In cardiomyocytes, testosterone activates plasma membrane androgen receptors coupled to heterotrimeric Gq protein. Gβγ dimers activate phospholipase C (PLC), which locally produces inositol IP3 thus activating IP3 receptors (IP3R). The consequent Ca2+ increase leads to the activation of several pathways, including the MEK/ERK/mTOR axis. These responses combined induce a series of genomic changes that lead to increased gene expression and increased protein synthesis, ultimately leading to cardiac hypertrophy.

While studying these hypertrophy effects on cardiomyocytes, our laboratory determined that testosterone uses a cooperative mechanism between the genomic and nongenomic pathways to produce the hypertrophic growth of cardiac cells. Thus, with normal levels of androgens the interactions among signaling pathways lead to the maintained health of cardiac cells, whereas high doses recruit additional signals which are hypertrophic. As

classic or genomic androgen response pathway. On the other hand, it has been reported that androgens, exert a rapid, non-genomic effect mediated by a putative plasma membrane receptor. Its activation implicates participation of kinases, intracellular calcium oscillations

Cardiomyocytes are terminally differentiated cells. Cardiac hypertrophy is characterized by an increase in cell size and protein synthesis and reactivation of a fetal gene program. Emerging evidence indicate that androgen action involves a crosstalk between genomic and nongenomic mechanisms. These could imply a two-step process, where both non-genomic and genomic effects occur sequentially (**Figure 2**). First, fast effects would be generated at the plasma membrane level with second messenger participation. Second, there would be gene activation; the pathways used by each cell type could provide specificity to the signal

Fig. 2. Integrated mechanism for testosterone effects in cardiomyocytes. In cardiomyocytes, testosterone activates plasma membrane androgen receptors coupled to heterotrimeric Gq protein. Gβγ dimers activate phospholipase C (PLC), which locally produces inositol IP3 thus activating IP3 receptors (IP3R). The consequent Ca2+ increase leads to the activation of several pathways, including the MEK/ERK/mTOR axis. These responses combined induce a series of genomic changes that lead to increased gene expression and increased protein

While studying these hypertrophy effects on cardiomyocytes, our laboratory determined that testosterone uses a cooperative mechanism between the genomic and nongenomic pathways to produce the hypertrophic growth of cardiac cells. Thus, with normal levels of androgens the interactions among signaling pathways lead to the maintained health of cardiac cells, whereas high doses recruit additional signals which are hypertrophic. As

synthesis, ultimately leading to cardiac hypertrophy.

and transcription factor activation, among other fast events.

through a cooperative mechanism, which can act in a concerted form.

depicted in Figure 2, testosterone activates a plasma membrane G-protein coupled receptor, a signaling pathway that results in a nongenomic mechanism. Activation of this pathway facilitates the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, thereby generating diacylglycerol and IP3. IP3 mediates opening of IP3 receptors in the sarcoplasmatic reticulum, which in turn produce rapid (1-5 min) Ca2+ release (Vicencio et al., 2006). Similar effects of testosterone have been identified in T-cells, skeletal muscle and neuroblastoma cells (Benten et al., 1999; Estrada et al., 2003, 2006). In cardiomyocytes, nongenomic signals activate the extracellular signal-regulated kinase (ERK) and the mTOR pathways. Activation of Ca2+/ERK/mTOR axis is independent of androgen receptor. The anabolic properties of testosterone indicate that this hormone regulate multiple genes involved in growth and metabolism. Traditionally, ligand-dependent androgen receptors act on DNA within the nucleus and in this compartment there is cross-talk among the nuclear receptors and cytosolic signaling. We have determined that testosterone induce a hypertrophic pattern in cardiomyocytes, evaluated as increase in the expression of fetal proteins, cardiomyocyte size, and protein synthesis. Hypertrophy effects of testosterone were blocked by either inhibition of mTOR or androgen receptor (Altamirano et al., 2009). We propose that cell growth produced by androgens require both androgen receptor activity and translation control through mTOR signaling pathway. Thus, both mTOR pathway and androgen receptor could control protein synthesis by a coordinated mechanism, where mTOR regulates translation and the intracellular androgen receptor
