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Endocrinology, doi:10.1210/en.2005-1139
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Endocrinology Vol. 147, No. 3 1386-1395
Copyright © 2006 by The Endocrine Society

Testosterone Induces an Intracellular Calcium Increase by a Nongenomic Mechanism in Cultured Rat Cardiac Myocytes

Jose Miguel Vicencio, Cristian Ibarra, Manuel Estrada, Mario Chiong, Dagoberto Soto, Valentina Parra, Guillermo Diaz-Araya, Enrique Jaimovich and Sergio Lavandero

Centro de Estudios Moleculares de la Célula (J.M.V., C.I., M.E., M.C., D.S., V.P., G.D.-A., E.J., S.L.), Instituto de Ciencias Biomedicas (M.E., E.J., S.L.), Facultad de Medicina, and Facultad de Ciencias Quimicas y Farmaceuticas (J.M.V., C.I., M.C., D.S., V.P., G.D.-A., S.L.), Universidad de Chile, Santiago 6640750, Chile

Address all correspondence and requests for reprints to: Drs. Sergio Lavandero or Enrique Jaimovich, Centro FONDAP (Fondo de Invesigación Avanzada en Areas Prioritarias) Estudios Moleculares de la Celula, Universidad de Chile, Olivos 1007, Santiago 6640750, Chile. E-mail: slavander{at}uchile.cl; or ejaimovi{at}med.uchile.cl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens are associated with important effects on the heart, such as hypertrophy or apoptosis. These responses involve the intracellular androgen receptor. However, the mechanisms of how androgens activate several membrane signaling pathways are not fully elucidated. We have investigated the effect of testosterone on intracellular calcium in cultured rat cardiac myocytes. Using fluo3-AM and epifluorescence microscopy, we found that exposure to testosterone rapidly (1–7 min) led to an increase of intracellular Ca2+, an effect that persisted in the absence of external Ca2+. Immunocytochemical analysis showed that these effects occurred before translocation of the intracellular androgen receptor to the perinuclear zone. Pretreatment of the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester and thapsigargin blocked this response, suggesting the involvement of internal Ca2+ stores. U-73122, an inhibitor of phospholipase C, and xestospongin C, an inhibitor of inositol 1,4,5-trisphosphate receptor, abolished the Ca2+ signal. The rise in intracellular Ca2+ was not inhibited by cyproterone, an antagonist of intracellular androgen receptor. Moreover, the cell impermeant testosterone-BSA complex also produced the Ca2+ signal, indicating its origin in the plasma membrane. This effect was observed in cultured neonatal and adult rat cardiac myocytes. Pertussis toxin and the adenoviral transduction of ß- adrenergic receptor kinase carboxy terminal peptide, a peptide inhibitor of ß{gamma}-subunits of G protein, abolished the testosterone-induced Ca2+ release. In summary, this is the first study of rapid, nongenomic intracellular Ca2+ signaling of testosterone in cardiac myocytes. Using various inhibitors and testosterone-BSA complex, the mechanism for the rapid, testosterone-induced increase in intracellular Ca2+ is through activation of a plasma membrane receptor associated with a Pertussis toxin-sensitive G protein-phospholipase C/inositol 1,4,5-trisphosphate signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIAC MUSCLE is a target for the action of endogenous anabolic androgenic steroids (AAS) (1) and synthetic derivatives of the hormone testosterone (2). AAS have been used by athletes to improve performance by increasing muscle mass and strength. Among the numerous documented toxic and hormonal effects of AAS, attention has been recently focused especially on the cardiovascular effects (3). It is known that there are increases in blood pressure and peripheral arterial resistance (4, 5), and there are also effects on the heart muscle, primarily left ventricular hypertrophy with restricted diastolic function (6, 7, 8). Severe cardiac complications (heart failure, ventricular fibrillation, ventricular thromboses, myocardial infarction, or sudden cardiac death) in individual strength athletes with acute AAS abuse have also been reported (3, 9, 10). These processes involve changes in gene expression controlled by intracellular androgen receptor-mediated pathways. Recent studies have, however, demonstrated alternative, rapid intracellular androgen receptor-independent mode of testosterone action. For example, administration of testosterone acutely induces vasodilatation in the systemic, coronary, and pulmonary vascular beds (11). Acting in this way, testosterone might increase myocardial tolerance to ischemia. Testosterone replacement in orchidectomized rats improved the recovery of myocardial function after ischemia/reperfusion injury (12). Er et al. (13) have recently shown that testosterone is directly cytoprotective in the myocardium through activation of ATP-sensitive K+ channels in the mitochondrial inner membrane.

Similar nongenomic effects are known in several cell types, including rat osteoblasts (14), macrophages (15), and skeletal muscle cells (16). It has been proposed that these rapid androgen actions may be exerted through membrane receptors that stimulate early intracellular signaling pathways through interaction with G proteins (17, 18). Common to these early effects are the fast intracellular Ca2+ increase, activation of Ca2+-dependent pathways and second-messenger cascades (19). In T cells, membrane androgen receptor mediates ligand-induced Ca2+ influx through nonvoltage-gated, Ni2+-blockable Ca2+ channels (18, 20). In osteoblasts, testosterone stimulates both the influx of extracellular Ca2+ via voltage-gated Ca2+ channels and Ca2+ release from intracellular stores through G protein-coupled receptors activating phospholipase C (PLC) via a Bordetella pertussis toxin (PTX)-sensitive G protein (14). Murine macrophages respond to testosterone by predominantly intracellular Ca2+ mobilization mediated through G protein-coupled receptors for testosterone (15, 21). We have previously shown that, in skeletal muscle cells, both testosterone and nandrolone produce rapid intracellular Ca2+ transients (16) involving Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores (22). This rapid Ca2+ transient was not modified by cyproterone, an inhibitor of steroid binding to its intracellular androgen receptor. Ca2+ signals elicited by steroid hormones through an increase in intracellular IP3 and associated with increased nucleoplasmic Ca2+ may have an important role in the regulation of different processes in the muscle cell (23).

In cardiac myocytes, intracellular Ca2+ regulates contraction, but an alternative role for Ca2+ in these cells as a regulator of gene expression has been proposed (17, 19). Cardiac myocytes contain intracellular androgen receptors (24), which regulate the expression of several genes (25, 26). In this work, we report the early effects of testosterone on intracellular Ca2+ in cultured cardiac myocytes. Our results show that, in cultured cardiac myocytes, testosterone induces a rapid and nongenomic intracellular Ca2+ release through activation of a plasma membrane androgen receptor associated with the PTX-sensitive G protein-PLC/IP3 signaling pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Testosterone, testosterone-BSA, BSA, cyproterone acetate (6-chloro-1ß,2ß-dihydro-17-hydroxy-3'H-cyclopropa[1, 2]pregna-1,4,6-triene-3,20-dione acetate), medium 199, DMEM, PTX, IP3, ryanodine, thapsigargin, and U-73122 were purchased from Sigma-Aldrich Corp. (St. Louis, MO). [3H]IP3 was from DuPont NEN Life Science Products (Boston, MA). Fluo3-acetoxymethylester (fluo3-AM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester (BAPTA-AM) were purchased from Molecular Probes (Eugene, OR). Genistein and xestospongin C were from Calbiochem (La Jolla, CA). Fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG was from Pierce Chemical Co. (Rockford, IL). Intracellular androgen receptor antibody (C-19) was from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents or molecular biology grade chemicals were obtained from Sigma Chemical Co.

Culture of cardiac myocytes
Neonatal rat cardiac myocytes were prepared from hearts of 1- to 3-d-old Sprague Dawley rats as described previously (27). For determination of IP3 and intracellular Ca2+, cardiac myocytes were plated at a final density of 0.7 x 103/mm2 on gelatin-precoated-60 mm dishes and 1.0 x 103/mm2 on gelatin-precoated coverslips, respectively. Serum was withdrawn 24 h before cells were used. To prevent the overgrowth of fibroblast and smooth muscle cells, 10 µM bromodeoxyuridine was used in our cell culture media. Cultured cardiac myocytes were identified using an anti-ß-myosin heavy-chain antibody. Cell cultures were at least 95% pure. Freshly dispersed adult rat cardiac myocytes were prepared from hearts of male adult Sprague Dawley rats (>250 g). Rats were anesthetized with ketamin and xylazin, hearts were removed, washed with Gerard buffer [0.19 mM NaH2PO4, 1.01 mM Na2HPO4, 10 mM HEPES, 128 mM NaCl, 4 mM KCl, 1.4 mM MgSO4, 5.5 mM glucose, 2 mM pyruvic acid (pH 7.4)] and retroperfused at 4 ml/min with 1 mM CaCl2 containing Gerard buffer during 5 min, followed by 1 mM EGTA containing Gerard buffer during 1 min and finally with 0.07% (wt/vol) collagenase and hyaluronidase containing Gerard buffer (digestion solution) for 30 min. Digested hearts were mechanically shattered in 5 ml Gerard buffer, mixed with 20 ml digestion solution, incubated at 37 C with constant agitation for 10 min, and supernatants centrifuged at 500 rpm for 30 sec. Remaining tissue was further digested with 20 ml digestion solutions. Pellets containing cardiac myocytes were resuspended in Gerard buffer and plated to obtain a final density of 1.0 x 103/mm2 on laminin-precoated coverslips. Rats were from the Animal Breeding Facility from the Faculty of Chemical and Pharmaceutical Sciences, University of Chile (Santiago, Chile). The Animal Investigation Committee approved all experimental procedures involving animals. This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (publication no. 85–23, revised 1985).

Adenovirus and inhibitors
Adenoviral vectors were propagated and purified as previously described (28). Two transgenes (a gift from Dr. W. J. Koch, Duke University Medical Center, Durham, NC) were used: adenovirus (Ad-) overexpressing ß-adrenergic receptor kinase carboxy terminal peptide (ßARKct) and an empty adenoviral construct (Ad-EV). ßARKct is a peptide inhibitor of Gß{gamma} signaling (29). Cardiac myocytes were transduced with adenoviral vectors at a multiplicity of infection (MOI) of 300 and incubated 24 h a 37 C in DMEM/medium 199. Cardiac myocytes were preincubated for 30 min before addition of the androgen with BAPTA-AM (100 µM), U-73122 (50 µM), U-73343 (50 µM), xestospongin C (100 µM), ryanodine (20 µM), cyproterone (1 µM), or genistein (50 µM) or for 6 h with PTX (1 µg/ml).

Intracellular calcium
Intracellular Ca2+ measurements were obtained from cardiac myocytes preloaded with the fluorescent Ca2+-sensitive dye fluo3-AM, using a fluorescence microscope (Olympus Diaphot-TMD, Nikon Corp., Tokyo, Japan) equipped with a cooled charge-coupled device camera and an image acquisition system (MCD 600 Spectra Source, Westlake Village, CA). Cardiac myocytes were washed three times with Ca2+-containing media (145 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1 mM MgCl2, 10 mM HEPES-Na, 5.6 mM glucose, adjusted to pH 7.4 with HCl) and loaded with 5.4 µM fluo3-AM (diluted from a 1.08 mM stock in 20% pluronic acid-dimethylsulfoxide) for 30 min at room temperature. After dye loading, cardiac myocytes were washed either with the Ca2+-containing medium or with a Ca2+-free medium (145 mM NaCl, 5 mM KCl, 1.0 mM EGTA, 3.6 mM MgCl2, 10 mM HEPES-Na, 5.6 mM glucose, adjusted to pH 7.4 with HCl) and used for experiments. Coverslips with adherent cells were mounted in a 1-ml-capacity plastic chamber and placed in the microscope for fluorescence measurements. Testosterone was either added directly or through rapid changing of medium (1 sec). The fluorescent images were collected every 2.0–5.0 sec and analyzed frame by frame with an image data acquisition program (Spectra Source). An objective lens PlanApo x60 (numerical aperture 1.4) was generally used. Most images were acquired at 512 x 120 pixels. Intracellular Ca2+ levels were expressed as relative total fluorescence [{Delta}F/F0: ratio of fluorescence difference, stimulated-basal (Fi-F0), to basal value (F0)] as a function of time. The fluorescence intensity increases proportionally with intracellular Ca2+ (30).

Digital image processing
Elimination of out-of-focus fluorescence was performed using both the no-neighbors deconvolution algorithm and Castleman’s (31) point spread function theoretical model, as described previously (16). For quantification of fluorescence, the summed pixel intensity was calculated from the section delimited by a contour. As a way of increasing efficiency of these data manipulations, action sequences were generated. To avoid interference from possible testosterone effects on the cellular volume, the area of each fluorescent cell was determined by image analysis using an adaptive contour and then creation of a binary mask, which was compared with its bright-field image.

Measurement of IP3 levels
Cardiac myocytes were rinsed and preincubated for 20 min at room temperature in 58 mM NaCl, 4.7 mM KCl, 3 mM CaCl2, 1.2 mM MgSO4, 0.5 mM EDTA, 60 mM LiCl, 10 mM glucose, and 20 mM HEPES, adjusted to pH 7.4 with HCl. Cells were stimulated by rapid addition of testosterone (final concentration 100 nM). At the indicated times, reactions were stopped by aspiration of the stimulating solution, addition of 0.8 M ice-cold perchloric acid and freezing with liquid nitrogen. Samples were allowed to thaw and cell debris was spun down for protein determination. Supernatants were neutralized with a solution of 2 M KOH, 0.1 M 2-(N-morpholine) ethane sulfonic acid, and 15 mM EDTA. The neutralized extracts were frozen at –80 C until required for IP3 determination. Measurements of IP3 mass were made by a validated radioreceptor assay (32). Briefly, a crude rat cerebellar membrane preparation was obtained after homogenization of tissue in 50 mM Tris-HCl (pH 7.7) containing 1 mM EDTA, 2 mM ß-mercaptoethanol, and centrifugation at 20,000 x g for 15 min. This procedure was repeated three times, suspending the final pellet in the same solution plus 0.3 M sucrose and freezing it at –80 C until required for use. The rat cerebellar membrane preparation was calibrated for IP3 binding with 1.6 nM [3H]IP3 and 2–120 nM cold IP3, with sample analysis performed in a similar way but replacing cold IP3 with a portion of the neutralized supernatant. [3H]IP3 radioactivity, which remained bound to membranes, was measured in an LS-6000TA liquid scintillation spectrometer (Beckman Instruments Corp., Fullerton, CA). Protein was determined by the Lowry method (33).

Immunocytochemistry
Intracellular androgen receptor was localized using indirect immunofluorescence. Testosterone-stimulated and nonstimulated cardiac myocytes were washed three times with PBS and then fixed with 100% methanol at –20 C for 20 min and treated with a blocking solution of 1% BSA in PBS for 30 min. Cells were incubated with the primary polyclonal antiintracellular androgen receptor antibody (1:100) overnight at 4 C. Later cardiac myocytes were washed in PBS and incubated with FITC-conjugated goat antirabbit IgG diluted 1:200 for 2 h at room temperature. Cells were washed and Vectashield (Vector Laboratories Inc., Burlingame, CA) was added to prevent bleaching. Cardiac myocytes were examined with a confocal microscope (135-M LSM Microsystems, Carl Zeiss AG, Oberkochen, Germany). Controls were performed as previously described (22).

Expression of results and statistical analysis
Data are means ± SE of the number of independent experiment indicated in the figure legends or are representative experiments performed on at least three separate occasions with similar outcomes. Data were analyzed by ANOVA, and comparisons between groups were performed using a protected Dunnett’s test. A value of P < 0.05 was set as the limit of statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of testosterone on intracellular Ca2+ levels in cultured cardiac myocytes
Cardiac myocytes were preloaded with fluo3-AM, and the relative fluorescence {Delta}F/F0 of single cells for each series of images was calculated. Cardiac myocytes maintained in Ca2+-containing medium showed basal oscillations of intracellular Ca2+ (Fig. 1AGo). Accordingly, Ca2+ oscillations were associated with Ca2+ mobilization due to spontaneous contraction of the myocytes. Testosterone stimulation (100 nM) did not modify the oscillation frequency of cardiac myocytes maintained in Ca2+-containing medium. The amplitude of oscillation varied from cell to cell, but the frequency was relatively constant at 0.12 ± 0.05 Hz (see Fig. 1Go, C and D). A sequence of fluorescence images showing intracellular Ca2+ oscillations in cardiac myocytes stimulated with testosterone (100 nM) is shown in Fig. 1BGo. Ryanodine (20 µM) completely abolished both spontaneous and testosterone-induced intracellular Ca2+ oscillations (Fig. 1Go, A and C), suggesting that ryanodine receptors were involved. This last result is consistent with the observation that Ca2+ oscillations are associated with contraction. It has been described that androgens inhibited Ca2+ influx by L-type Ca2+ channels in smooth muscle cells (34, 35, 36). To evaluate this possibility, cardiac myocytes were preincubated with L-type Ca2+ channel blockers (nifedipine or verapamil) and then stimulated with testosterone (100 nM) in Ca2+ containing medium. As shown in Fig. 1DGo, both L-type channel blockers have similar effects, eliminating fast influx of Ca2+ and Ca2+ oscillations in cultured cardiac myocytes treated with testosterone.


Figure 1
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  		FIG. 1. Testosterone increases intracellular Ca2+ levels in cultured rat cardiac myocytes maintained in Ca2+-containing medium. Cells were preloaded with fluo3-AM and maintained in Ca2+-containing medium at the moment of stimulation. A, Intracellular Ca2+ level oscillations in nonstimulated cells (black line) and pretreated with ryanodine (20 µM, gray line). B, Series of images showing intracellular Ca2+ level oscillations in cultured cardiac myocytes treated with testosterone (100 nM). C, {Delta}F/F0 was calculated from fluorescence images in B (black line) and those obtained from cardiac myocytes preincubated with ryanodine (20 µM, gray line) and stimulated with testosterone. D, {Delta}F/F0 calculated from fluorescence images of cardiac myocytes treated with testosterone (100 nM, black line) or preincubated with nifedipine (10 µM, gray line) or verapamil (10 µM, light gray line), and then stimulated with testosterone, respectively. Each graph is representative from triplicates of at least three independent experiments. Arrows indicate the time of addition of testosterone.

 
When experiments were performed in Ca2+-free medium, no Ca2+ oscillations were detected in both basal and testosterone-treated cardiac myocytes. Figure 2AGo shows a representative sequence of images depicting the effect of testosterone on intracellular Ca2+ levels in cardiac myocytes maintained in Ca2+-free medium. {Delta}F/F0 calculated from Fig. 2AGo showed that intracellular Ca2+ levels increased slower than in Ca2+-containing medium, reaching a maximum at approximately 3 min (Fig. 2BGo). The kinetics of the increases in nuclear and cytosolic Ca2+ was similar (Fig. 2CGo). A dose-response relationship was observed from 0 to 1 µM testosterone on the maximum values of relative fluorescence (RF max) (Fig. 2DGo). Maximal effect was obtained at 100 nM (Fig. 2DGo), and this concentration was chosen for all experiments.


Figure 2
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  		FIG. 2. Testosterone (T) effect on intracellular Ca2+ levels in cultured cardiac myocytes maintained in Ca2+-free medium. Cells were preloaded with fluo3-AM and maintained in Ca2+-free medium at the moment of stimulation. A, Serial of fluorescence images at the indicated times showing intracellular Ca2+ level changes in cardiac myocytes stimulated with 100 nM testosterone. B, {Delta}F/F0 calculated from the fluorescence images shown in A (black line), corresponding to cells treated with testosterone and from cells treated with saline solution (gray line). C, Nuclear (black line) and cytosolic (gray line) region of interest (ROI) analysis of the testosterone-induced Ca2+ signal from A. D, Concentration-dependent response. The graphic shows a statistical analysis of the RF max from all experiments performed with cardiac myocytes treated with testosterone or saline solution. The inset graphic is presented in logarithmic scale. Values are expressed as mean ± SE of triplicates from three independent experiments. **, P < 0.01 vs. 0 nM (saline). Arrows indicate the time of addition of testosterone.

 
Effect of testosterone on intracellular Ca2+ stores in cultured cardiac myocytes
The persistence of the Ca2+ signal in experiments with Ca2+-free medium was consistent with testosterone stimulating Ca2+ mobilization from intracellular stores. To determine the participation of internal Ca2+ stores in the testosterone-induced intracellular Ca2+ increase, cardiac myocytes were pretreated with BAPTA-AM before testosterone treatment. This compound eliminated the testosterone-induced signal observed in absence of external calcium (Fig. 3AGo). After depletion of intracellular Ca2+ stores with the specific sarcoendoplasmic reticulum Ca2+-ATPase pump inhibitor thapsigargin, testosterone no longer elicited the Ca2+ signal in cells incubated in Ca2+-free medium (Fig. 3BGo). Ryanodine did not abolish the Ca2+ signal induced by testosterone in cells maintained in Ca2+-free medium (Fig. 3CGo), indicating that ryanodine receptors do not participate in the slower component of the testosterone-induced calcium signal, which involves Ca2+ release from intracellular stores.


Figure 3
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  		FIG. 3. Effect of BAPTA-AM, thapsigargin, and ryanodine on the testosterone (T) effect on intracellular Ca2+ levels. Cardiac myocytes were preloaded with fluo3-AM and maintained in Ca2+-free medium at the moment of stimulation. A, Total {Delta}F/F0 calculated from fluorescence images of cardiac myocytes treated with testosterone (100 nM, black line) or preincubated with BAPTA-AM (100 µM for 30 min, gray line) and then stimulated with testosterone. B, Total {Delta}F/F0 calculated from fluorescence images of cardiac myocytes treated with thapsigargin (1 µM) for approximately 18 min to deplete intracellular calcium stores and then stimulated with testosterone (100 nM), as indicated by the respective arrows. C, Total {Delta}F/F0 calculated from images obtained from cells treated with testosterone 100 nM (black line) or preincubated with ryanodine (20 µM, gray line) and stimulated with testosterone. The inset graphics represent the statistical analysis of maximum relative fluorescence from the experiments performed with the respective inhibitors. Values are expressed as mean ± SE of duplicates from at least three independent experiments. **, P < 0.01 vs. control (testosterone). Arrows (A and C) indicate the time of addition of testosterone.

 
To investigate the role of the PLC-IP3-IP3 receptor (IP3R) signaling pathway on the increases in intracellular Ca2+ levels induced by testosterone, cardiac myocytes were pretreated with U-73122 (a general PLC inhibitor) or xestospongin C (an IP3R blocker). As shown in Fig. 4Go, A and C, both compounds abolished the testosterone effect on intracellular Ca2+ levels. There are some reports indicating that U-73122 may alter intracellular Ca2+ responses by PLC-independent effects (37, 38, 39, 40). U-73343 an isomer that is inactive as a PLC inhibitor was used as control. Figure 4BGo depicts that U-73343 did not modify the effect of testosterone on the intracellular Ca2+ levels. To further demonstrate that testosterone increases intracellular IP3 levels, cultured cardiac myocytes were exposed to this hormone at different times in Ca2+-free medium and IP3 contents were quantified by a radioreceptor assay. Figure 4DGo shows that testosterone significantly increased IP3 levels after 15 sec, reaching a maximum at 30 sec and then decreasing after 3 min. Collectively, these experiments suggest that the PLC-IP3-IP3R signaling pathway is involved in the testosterone induced Ca2+ release from intracellular stores.


Figure 4
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  		FIG. 4. Testosterone (T) activates the PLC/IP3 pathway in cultured cardiac myocytes. Cells were preloaded with fluo3-AM, treated for 30 min with U-73122 (50 µM, A), U-73343 (50 µM, B), or xestospongin C (100 µM, C) and stimulated with testosterone 100 nM in Ca2+-free resting medium. Total {Delta}F/F0 was calculated from fluorescence images. The inset graphics (A–C) correspond to the statistical analysis of RF max from experiments with the respective inhibitors and testosterone. Values are expressed as mean ± SE of triplicates from at least three independent experiments. **, P < 0.01 vs. controls. D, Cells were stimulated with 100 nM testosterone, and at the indicated times, IP3 mass was determined by a radioreceptor assay. Values are the mean ± SE of three independent experiments. **, P < 0.01; *, P < 0.05 vs. time 0.

 
Participation of a membrane receptor in the testosterone-induced Ca2+ transients in cultured cardiac myocytes
Androgens exert their genomic effects by binding to intracellular receptors and then translocate as a hormone-receptor complex to the nucleus (41). The intracellular androgen receptor antagonist cyproterone inhibits genomic activation in several cell types (42). Immunocytochemical experiments for intracellular androgen receptor were performed in cultured rat cardiac myocytes at different times of stimulation with testosterone. In control cells (Fig. 5AGo), intracellular androgen receptor was mainly found in the cytosol, a condition that was not modified after 5 min of testosterone treatment (Fig. 5BGo). Only after 60 min was there a decrease in the cytosolic distribution of the intracellular androgen receptor (Fig. 5CGo). The subcellular distribution of the androgen receptor was mainly perinuclear after 2 h of stimulation with testosterone (Fig. 5DGo). Cyproterone (1 µM for 30 min) had no effect on the distribution of the androgen receptor in nonstimulated cardiac cells (Fig. 5EGo) but completely blocked testosterone-induced translocation of the intracellular receptor (Fig. 5FGo), despite being unable to prevent the effect of on the Ca2+ signal (Fig. 6AGo). The time course for intracellular androgen receptor translocation induced by testosterone was also clearly longer than the time necessary for the testosterone-dependent increase in intracellular Ca2+ levels. It is unlikely, therefore, that the intracellular androgen receptor plays any role in the short-term testosterone effects.


Figure 5
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  		FIG. 5. Testosterone stimulates a slow intracellular androgen receptor translocation in cultured cardiac myocytes. Cells were stimulated with 100 nM testosterone for 0 min (A, control), 5 min (B), 1 h (C), or 2 h (D). Cells were pretreated with cyproterone and stimulated without (E) or with (F) testosterone for 2 h. Cells were stimulated with T-BSA for 2 h (G). After stimulation, the cardiac myocytes were fixed, incubated with an antibody against the intracellular androgen receptor, revealed with antirabbit IgG-FITC, and analyzed by confocal microscopy as described in Materials and Methods. Data are representative of three independent experiments.

 

Figure 6
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  		FIG. 6. Testosterone (T) increases intracellular Ca2+ levels by a mechanism involving a membrane androgen receptor, G protein, and ß{gamma}-dimmers in cultured cardiac myocytes. Cells were preloaded with fluo3-AM and maintained in a Ca2+-free medium at the moment of stimulation. A, Cardiac myocytes were stimulated with testosterone (100 nM, black line) or pretreated with cyproterone (1 µM for 30 min, gray line) and then stimulated with testosterone. B, Cells were stimulated with testosterone (100 nM, black line) or pretreated with genistein (Gen, 50 µM for 30 min, gray line) or PTX (1 µg/ml for 6 h, light gray line) and then stimulated with testosterone. Genistein did not modify testosterone-induced Ca2+ signal, whereas PTX completely abolished this response. C, Cells were transduced with Ad-ßARKct (a Gß{gamma} signaling peptide inhibitor, MOI = 300 for 24 h) or Ad-EV (control, MOI = 300 for 24 h) and stimulated with testosterone (100 nM). The inset graphics correspond to the statistical analysis of RF max from the respective experiments performed with inhibitors and controls with testosterone. Data in insets are mean ± SE of triplicates from three independent experiments. **, P < 0.01 vs. controls.

 
To further investigate the early signaling events triggered by testosterone on cardiac myocytes related to the effect on intracellular Ca2+ levels, cells were pretreated with genistein (50 µM for 30 min) or PTX (1 µg/ml for 2 h) and then stimulated with testosterone in absence of external Ca2+. Genistein did not modify the testosterone-induced Ca2+ signal, whereas with PTX, it was completely abolished (Fig. 6BGo). Ad-ßARKct, a peptide binding to and inactivating the ß{gamma}-subunits of G protein (28, 29), inhibited the testosterone-induced Ca2+ transients; control vector had no effect (Fig. 6CGo). These results indicated that a PTX-sensitive heterotrimeric G protein is a critical component of the testosterone-induced Ca2+ release in cardiac myocytes.

The participation of membrane steroid receptor on the different nongenomic steroid effects, including those on intracellular Ca2+ levels, has been proposed in many cell types (43, 44, 45). To further evaluate this possibility, testosterone covalently bound to albumin (T-BSA), which cannot enter the cell and exerts effects only at the cell membrane, was used. T-BSA did not change the distribution of intracellular androgen receptor after 2 h of stimulation as did the free hormone (Fig. 5GGo) but reproduced the Ca2+ increase seen with free testosterone. Moreover, control experiments with BSA alone were without effect (Fig. 7AGo). As shown in Fig. 7BGo, U-73122, xestospongin C, PTX, and Ad-ßARKct, but not cyproterone, blocked the effect of T-BSA on intracellular Ca2+ levels. These results suggest that testosterone affects intracellular Ca2+ levels through binding to a plasma membrane androgen receptor. To exclude any unspecific effect of testosterone on the cell membrane, which may lead to changes on intracellular Ca2+ levels, {Delta}4-androstene-3,7-dione (a metabolic precursor with reduced androgen activity) was used. Figure 7CGo shows that {Delta}4-androstene-3,7-dione also increased intracellular Ca2+ levels but with a weaker and not significant response in comparison with testosterone. To further evaluate the effect of testosterone in completely differentiated cardiac myocytes, we performed the same epifluorescence studies using adult rat cardiac myocytes. In the absence of extracellular calcium, testosterone (100 nM) elicited a similar response in intracellular calcium (Fig. 8Go); moreover, T-BSA at the same concentration mimicked the effect of the free hormone.


Figure 7
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  		FIG. 7. Testosterone covalently bound to albumin effect on intracellular Ca2+ levels in cultured rat cardiac myocytes. Cells were preloaded with fluo3-AM and maintained in a Ca2+-free medium at the moment of stimulation. A, Cells were stimulated with BSA (control, 100 nM, gray line) or T-BSA (100 nM, black line). B, Cells were pretreated with cyproterone (1 µM for 30 min), U-73122 (50 µM for 30 min), or xestospongin C (10 µM for 30 min), PTX (1 µg/ml for 6 h) or transduced with Ad-ßARKct (MOI = 300 for 24 h). Cells were then stimulated with BSA (100 nM) or T-BSA (100 nM) as indicated. Values are expressed as mean ± SE of at least three independent experiments. **, P < 0.01 vs. BSA; ++, P < 0.01 vs. T-BSA. C, Cells were stimulated with testosterone (T, 100 nM, black line) or {Delta}4-androstene-3,7-dione (100 nM, gray line), a metabolic precursor with less androgenic activity. The inset graphic is the statistical analysis of maximum relative fluorescence levels of cells treated with testosterone (black line) or {Delta}4-androstene-3,7-dione (gray line). Values are expressed as mean ± SE of duplicates from at least three independent experiments. **, P < 0.01 vs. testosterone.

 

Figure 8
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  		FIG. 8. Effect of testosterone (T) in adult rat cardiac myocytes. Cells were preloaded with fluo3-AM and maintained in Ca2+-free medium at the moment of stimulation. A, Fluorescence images at the indicated times showing intracellular Ca2+ level changes in cardiac myocytes stimulated with testosterone 100 nM. B, {Delta}F/F0 calculated from the fluorescence images shown in A (black line) and cells treated with T-BSA (100 nM, gray line). The inset graph (B) corresponds to the statistical analysis of Rf max from experiments with testosterone and T-BSA. Values are expressed as mean ± SE of triplicates from three independent experiments. The time of stimulation is indicated by the arrow.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first study describing an intracellular Ca2+ increase in response to testosterone in cardiac myocytes. This short-term, nongenomic effect of testosterone has been previously observed in other cell types (14, 20, 22, 44). Moreover, the increase in intracellular Ca2+ levels was dependent on Ca2+ release from intracellular stores by a PLC/IP3- dependent mechanism. This effect was not linked to the intracellular androgen receptor but to a putative plasma membrane androgen receptor associated with a PTX-sensitive G protein.

The different time course of testosterone-induced intracellular Ca2+ signals, observed in cardiac myocytes maintained with and without external Ca2+, suggests that at least two Ca2+ components were involved. Results showed that testosterone induces a fast Ca2+ influx, followed by a slower Ca2+ release from intracellular stores. The maximal effect on intracellular Ca2+ levels was obtained with 100 nM testosterone, a circulating concentration that mimics a pathophysiological state detected on individuals using the hormone therapeutically (46, 47).

Basal intracellular Ca2+ oscillations associated to external Ca2+ and ryanodine receptors are normally detected in cardiac myocytes. Testosterone did not change the frequency of these oscillations but increased their amplitude, possibly by increasing basal calcium levels. These oscillations may have an important regulatory function in the cardiac cells (48, 49, 50). The observation that intracellular Ca2+ oscillations induced by testosterone are seen only when there is a source of extracellular Ca2+ indicates that these oscillations are related to Ca2+ influx from the extracellular space. Ryanodine completely blocked the Ca2+ oscillations observed in testosterone-treated cardiac myocytes maintained in Ca2+-containing medium. Because ryanodine inhibits the oscillatory behavior, the source of this phenomenon must rely both on Ca2+ influx across the plasma membrane and on the process of calcium-induced calcium release. Consistently, acute inhibition of L-type calcium channels also blocked Ca2+ oscillations but did not alter the slow release of Ca2+ from internal stores triggered by testosterone. This effect of L-type calcium channel blockers was different from that reported by others (34, 36), in which testosterone inhibits the dihydropyridine-sensitive calcium influx. These differences may rely on different properties of alternatively spliced generated isoforms of L-type Ca2+ channels between smooth and cardiac muscle cells (51).

The question arises as to the origin of the Ca2+ signal when there is no extracellular Ca2+. Treatment with the Ca2+-chelator BAPTA-AM completely abolished this increase in the absence of external Ca2+, indicating that Ca2+ during this signal must be released from internal stores. Ryanodine-sensitive stores did not participate in the testosterone-induced slow Ca2+ release, and depletion of thapsigargin-sensitive Ca2+ stores from sarcoplasmic reticulum was in agreement with a role of intracellular Ca2+ in testosterone action. We and others have described that testosterone-induced Ca2+ release from IP3-sensitive stores in skeletal muscle cells and osteoblasts (14, 22). Our results using a PLC inhibitor and an IP3 receptor blocker indicated that testosterone acted with a similar mechanism in cultured cardiac myocytes. The possibility of unspecific effects of U-73122 was discarded using the inactive isomer U-73343 as control. The time courses of both IP3 mass and Ca2+ level increases support an IP3-dependent Ca2+ release by testosterone. The experiments with specific inhibitors (BAPTA-AM, ryanodine, thapsigargin, U-73122, and xestospongin C) in Ca2+-free medium are consistent with the sarcoplasmic reticulum being the source.

In many cell types, the actions of androgens involve binding to intracellular receptors leading to genomic responses (52, 53, 54). However, there is now evidence to suggest that androgens also trigger rapid and nongenomic effects leading to the activation of several signal pathways (19, 55). In cardiac myocytes, we found that testosterone stimulates both intracellular androgen receptor translocation from the cytosol to perinuclear regions and induces intracellular Ca2+ transients. The former process is inhibited by cyproterone, whereas the latter is not. In addition, the kinetics of the changes in intracellular Ca2+ is sufficiently fast, so this process is unlikely to be mediated at the genomic level. The experiments with T-BSA strongly suggest that such short-term effects of the androgen may involve an action of the hormone primarily at the cell surface through a plasma membrane androgen receptor. Nongenomic responses originated by plasma membrane receptors have been described for steroids such as sex hormones, glucocorticoids, mineralocorticoids, neurosteroids, or vitamin D (55, 56). Others and we (14, 22, 57) have shown that the putative membrane androgen receptor is associated with G protein. Our results shown here indicate that this G protein is sensitive to pertussis toxin and that the cascade also involves ß{gamma}-subunits.

Translocation of intracellular androgen receptor was inhibited by cyproterone, which has been previously reported to block genomic responses of androgens (42). The testosterone-induced Ca2+ signal was detected within 1–7 min and was not affected by cyproterone. Covalent linkage of testosterone to a large protein such as albumin impedes this hormone to cross the plasma membrane acting only at the plasma membrane level (18, 21, 58). Our results showed that T-BSA but not BSA triggered Ca2+ transients, suggesting the participation of a plasma membrane androgen receptor instead of an intracellular androgen receptor in the short-term effect of this hormone in cultured cardiac myocytes. To exclude any unspecific effect, additional experiments with {Delta}4-androstene-3,7-dione, a metabolic precursor with reduced androgenic activity, validated the results. However, it cannot completely rule out the possibility that testosterone may activate an unrelated membrane receptor coupled to this pathway.

To check the effect of testosterone in differentiated cells, we used adult rat cardiac myocytes. The increase on intracellular calcium levels persisted, but the kinetics of the signal was slower. The persistence of the signal suggests that this nongenomic effect is a conserved mechanism of androgen action during development.

Cardiac hypertrophy is a leading predictor of progressive heart disease that often leads to heart failure and a loss of cardiac contractile performance associated with profound alterations in intracellular calcium handling. Different hormones and growth factors stimulate cardiac myocyte hypertrophy through Ca2+-dependent signaling pathways (59). Calmodulin-activated phosphatase calcineurin, activated by increases in calcium, mediates the hypertrophic response through its downstream nuclear factor of activated T cells (60). Our results represent the first link between Ca2+ and AAS in cardiac myocytes, suggesting that this nongenomic effect of AAS can contribute to the documented androgen receptor mediated cardiotoxicity observed in AAS abuse. However, further work related with Ca2+ signaling pathways, such as calcineurin/nuclear factor of activated T cells, will be necessary to clarify the role of Ca2+ transients in the hypertrophic effects of androgens in the heart.

In conclusion, our results indicate that in rat neonatal cardiac myocytes, testosterone increases intracellular Ca2+ levels by a nongenomic mechanism, which involves a membrane androgen receptor, a PTX-sensitive G protein, PLC, IP3, and IP3R as signaling pathway.


   Acknowledgments
 
We thank Dr. W. J. Koch (Duke University Medical Center, Durham, NC) for the ßARKct and Ad-EV, Dr. J. L. Liberona for IP3 measurements, Professor C. I. Pogson for his critical reading of the manuscript, and Fidel Albornoz for his technical assistance.


   Footnotes
 
This work was supported by FONDAP (Fondo de Invesigación Avanzada en Areas Prioritarias) 15010006 (to S.L. and E.J.) and Fundación Andes C-14060-11 (to M.E.). J.M.V., C.I., and D.S. hold fellowships from Consejo Nacional de Ciencia y Tecnologia, Chile.

All authors have nothing to declare.

First Published Online December 8, 2005

Abbreviations: AAS, Anabolic androgenic steroids; Ad-ßARKct, adenovirus overexpressing ßARKct; Ad-EV, empty adenoviral construct; ßARKct, ß-adrenergic receptor kinase carboxy terminal peptide; BAPTA-AM, 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester; Ca2+ calcium; {Delta}F/F0, relative total fluorescence; FITC, fluorescein isothiocyanate; fluo3-AM, fluo3 acetoxymethylester; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; MOI, multiplicity of infection; PLC, phospholipase C; PTX, Bordetella pertussis toxin; RF max, maximum values of relative fluorescence; T-BSA, testosterone covalently bound to albumin.

Received September 6, 2005.

Accepted for publication November 30, 2005.


   References
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 Introduction
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 Results
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