Testosterone induces apoptosis in cardiomyocytes by increasing proapoptotic signaling involving tumor necrosis factor-α and renin angiotensin system

Abstract

Anabolic androgenic steroids lead to cardiac complications and have been shown to exhibit proapoptotic effects in cardiac cells; however, the mechanism involved in those effects is unclear. The aim of this study was to assess whether apoptosis and the activation of caspase-3 (Casp-3) induced by testosterone in high concentrations involves increments in tumor necrosis factor-α (TNF-α) concentrations and angiotensin-converting enzyme (ACE) activity in cardiomyocytes (H9c2) cell cultures. Cardiomyocytes were treated with testosterone (5 × 10⁻⁶ mol/L), doxorubicin (9.2 × 10⁻⁶ mol/L), testosterone + etanercept (Eta; 6.67 × 10⁻⁵ mol/L), testosterone + losartan (Los; 10⁻⁷ mol/L), and testosterone + AC-DEVD-CHO (10⁻⁵ mol/L; Casp-3 inhibitor). Apoptosis was determined by flow cytometry and by the proteolytic activity of Casp-3. We demonstrated that incubation of H9c2 cells for 48 h with testosterone causes the apoptotic death of 60–70% of the cells and co-treatments with Eta, Los, or AC-DEVD-CHO reduced this effect. Testosterone also induces apoptosis (concentration dependent) and increases the proteolytic activity of Casp-3, which were reduced by co-treatments. TNF-α and ACE activities were elevated by testosterone treatment, while co-treatment with Los and Eta reduced these effects. We concluded that an interaction between testosterone, angiotensin II, and TNF-α induced apoptosis and Casp-3 activity in cultured cardiomyocytes, which contributed to the reduced viability of these cells induced by testosterone in toxic concentrations.

Keywords

Anabolic agents apoptosis caspase-3 ACE TNF-α H9c2

Introduction

The abusive use of anabolic androgenic steroids (AAS) can cause serious health complications such as cardiac hypertrophy, cardiomyopathy, myocardial infarction, and sudden death.^1
–3 AAS exert a primary anabolic effect and promote the growth of cardiac tissue. However, AAS also have a direct toxic effect on cardiomyocytes, inducing ultrastructural changes similar to those observed in the early stages of congestive heart failure.^4,5 In addition, as apoptosis can be induced by AAS, which is a notable factor in the transition from compensatory cardiac hypertrophy to heart failure, apoptosis could account for the cardiotoxic effect of AAS.^6

–9

Actually, testosterone at physiological concentrations exhibits an antiapoptotic effect in cells exposed to hydrogen peroxide (H₂O₂) or isoproterenol.^10,11 However, in anabolic concentrations, this hormone has been implicated in the induction of apoptosis in cultured cardiac myocytes and the activation of apoptosis indicates a direct myocardial injury caused by testosterone.^6,7 Lopes et al. showed that testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway by reactive oxygen species increase.⁸

Two pathways are responsible for initiating apoptosis, intrinsic and extrinsic pathway, one mediated by mitochondria and another mediated by cell death receptors located on the membrane surface.¹² Several factors can activate the apoptotic process. The renin angiotensin aldosterone system and tumor necrosis factor α (TNF-α) are examples of activating factors of intrinsic and extrinsic pathway, respectively.^13,14

Among the factors that activate the intrinsic apoptosis pathway, angiotensin II (Ang II) is one of the most studied and their production depends on the activity of angiotensin-converting enzyme (ACE).¹⁵ ACE is a zinc-containing dipeptidase that converts Ang I to Ang II and has been shown to be dynamically regulated by hormones, such as testosterone. The use of AAS also promotes increased activity of this enzyme.¹⁶ ACE is increased in male mice compared to females, resulting in increased cardiac production of Ang II, which interacts with Ang II receptor type 1 (AT₁) or Ang II receptor type 2 (AT₂) initiating the apoptotic process.^13,17,18 We have demonstrated that cardiac cytokine imbalance and an increase in ACE activity mediates cardiac remodeling and injury in rats treated with nandrolone, which indicates a relationship between AAS, the renin angiotensin system (RAS) and cytokines in the promotion of the cardiac side effects of AAS.¹⁹ In fact, inhibition of ACE activity prevents the cardiovascular changes induced by nandrolone.²⁰ Wan et al.²¹ showed that the silencing of ACE by RNA interference prevented cardiomyocytes (H9c2) from apoptosis through regulation of the intracellular RAS.²¹

Testosterone also present effects in TNF-α production, which was demonstrated by Metcalfe et al.²², where it was observed that testosterone mediates increased production of TNF-α in renal tubular cells, and Wang et al.²³ observed that testosterone participates in the gender differences related to the deleterious effects of TNF-α on the myocardium.

However, no study has investigated the participation of RAS and TNF-α in the apoptosis of cardiac myocytes induced by AAS. Therefore, the aim of this study was to assess whether apoptosis and activation of caspase-3 (Casp-3) induced by testosterone in high concentrations involve increments in TNF-α concentrations and ACE activity in cell cultures of cardiac myocytes (H9c2).

Materials and methods

Cell culture

A cell line derived from embryonic rat heart (H9c2, ATCC CRL1446) was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St Louis, Missouri, USA) in a humidified stove at 37°C with 5% carbon dioxide (CO₂). The DMEM medium was supplemented with 10 mL of penicillin-G–glutamine–Streptomycin (Sigma-Aldrich) and 10% fetal bovine serum (FBS, Gibco, Invitrogen Corporation, Grand Island, New York, USA) with the pH adjusted to 7.2.

Cell treatment

All substances used in the treatment of the cell lines were obtained from Sigma and used at the following concentrations: doxorubicin (DOX): 9.2 × 10⁻⁶ mol/L, etanercept (Eta): 6.67 × 10⁻⁵ mol/L, losartan (Los): 10⁻⁷ mol/L, testosterone: 5 × 10⁻⁶ mol/L, and Ac-DEVD-CHO: 10⁻⁵ mol/L.^6,24

–27 The testosterone concentration used in this study was the concentration that showed cytotoxic effect above 50% in H9c2 cells and this concentration also corresponds to the concentration used by Zaugg et al.⁶ The time of exposure with the different substances in culture wells, alone or in combination, was 48 h.

Cytotoxicity assay

Cytotoxicity was determined by testing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich). The MTT salt is converted into formazan by living cells, forming a blueish precipitate.²⁸ H9c2 cells were seeded in 96-well plates to 5 × 10⁴ cells/mL and incubated with 190 μL of DMEM supplemented with 10% FBS and 10 μL of test substances for 48 h at 37°C in 5% CO₂. To determine the mean inhibitory concentration (IC₅₀), wells were incubated with different concentrations of testosterone (2.5 × 10⁻⁸, 2.5 × 10⁻⁷, 2.5 × 10⁻⁶, 5 × 10⁻⁶, and 10⁻⁵ mol/L). Furthermore, the testosterone (5 × 10⁻⁶ mol/L)⁶ also received co-treatment with Eta (6.67 × 10⁻⁵ mol/L), Los (10⁻⁷ mol/L), or the Casp-3 inhibitor (Ac-DEVD-CHO, 10⁻⁵ mol/L). DOX was used as positive control. After the incubation period, the supernatant was replaced with 200 µL of DMEM and 25 µL of MTT (5 mg/mL), and the cells were incubated for 4 h at 37°C in 5% CO₂. The insoluble formazan crystals were dissolved in 200 µL of isopropanol acidified with hydrochloric acid (4 × 10² M). Cell viability was evaluated in a manner proportional to the absorbance measured at 570 nm in an enzyme-linked immunosorbent assay (ELISA) reader (TP-Reader Thermoplate, China) and calculated according to the equation: $% i n h i b i t i o n = (N C - A) / N C \times 100$ , where NC is the negative control and A represents the absorbance of the well.

Determination of apoptosis by flow cytometry analysis

The amount of apoptosis in the cardiomyocytes was determined using the annexin V-fluorescein isothiocyanate (FITC) kit for the detection of apoptosis (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, 5 × 10⁵ cells/mL preincubated for 48 h in different treatments were harvested after the removal of cellular adhesion with trypsin, washed in phosphate-buffered saline, and stained with FITC-conjugated annexin V and propidium iodide (PI) for 15 min at room temperature under light. The percentage of positive cells determined over 10,000 acquired events was analyzed by a FACSCalibur system (BD BioSciences, San Jose, California, USA) equipped with a 488 nm argon laser and FCS Express 4 flow cytometry software (De Novo Software, Los Angeles, California, USA). Cells in the initial stage of apoptosis were defined as annexin V (+)/PI (−), while late apoptotic cells were defined as double positive. Three concentrations of testosterone (2.5 × 10⁻⁷, 2.5 × 10⁻⁶, and 5 × 10⁻⁶ mol/L) were used to assess whether the induction of apoptosis was concentration dependent. Testosterone at a concentration of 5 × 10⁻⁶ M/L also received co-treatment with Eta (6.67 × 10⁻⁵ mol/L), Los (10⁻⁷ mol/L), or inhibitor of Casp-3 (10⁻⁵ mol/L). H₂O₂ was used as a positive control (10⁻⁶ mol/L).

Determination of Casp-3 activity

All reagents used to evaluate the activity of Casp-3, including Ac-DEVD-CHO, were obtained from a Casp-3 colorimetric assay kit (Sigma-Aldrich). H9c2 cells were cultured in a 24-well plate, 10⁵ cells/mL. The cells were maintained for 48 h in the presence of test substances (testosterone (5 × 10⁻⁶ mol/L); testosterone + Eta (6.67 × 10⁻⁵ mol/L); testosterone + Los (10⁻⁷ mol/L); and testosterone + Casp-3 inhibitor (10⁻⁵ mol/L)). Procedures were performed following the manufacturer’s specifications. Reading the absorbance of the plate was performed on an ELISA reader at 405 nm (TP-Reader Thermoplate). Tests were performed in octuplicate, and the results were expressed as micromoles of p-nitroaniline (pNA) per minute per milliliter.²⁹

Quantification of TNF-α by ELISA assay

The levels of TNF-α were measured in wells with cells at a concentration of 1 × 10⁶ cells/well using a commercial kit (Invitrogen, San Jose, California, USA) according to the manufacturer’s instructions. After incubation for 48 h with different types of treatment (testosterone: 5 × 10⁻⁶ mol/L; testosterone + Eta; testosterone + Los; and DOX), the wells were read at 450 nm in an ELISA reader (TP-Reader Thermoplate).

Determination of ACE activity

ACE activity was determined by the quantification of the glycylglycine (Gly-Gly) cleavage product hippuryl-glycyl-glycine (Hip-Gly-Gly) by ACE.¹⁰ Briefly, H9c2 cells were cultured in a 24-well plate, 1 × 10⁵ cells/mL. After incubation for 48 h in the presence of test substances, the cell supernatant was diluted 1:1 with phosphate buffer (pH 8.3). For the enzyme reaction, an aliquot of supernatant or the positive control, rabbit lung solution (final concentration 9.1 mg/ml, Sigma), was combined with an assay buffer and substrate solution Hip-Gly-Gly (100 mmol, Sigma-Aldrich). After homogenization, the mixture was incubated for 35 min at 37°C. The reaction was stopped by the addition of sodium tungstate (300 mmol) and sulfuric acid (0.33 mmol). The system was mixed with trinitrobenzene sulfonate staining reagent (Sigma-Aldrich). After 20 min under light, the absorbance was read on a microtiter plate reader (TP-Reader Thermoplate) at 405 nm against a blank. The blank solution was prepared similarly except that sodium tungstate and sulfuric acid were added prior to the enzyme solution. The assay was also carried out in the presence of 10 μL of a solution of captopril (final concentration 64 nmol). Assays were performed in octuplicate. ACE activity was calculated according to the equation: $% a c t i v i t y = (A \times 100) / B$ , where A is the measured absorbance (at 405 nm) of the well of cells and B is the absorbance of rabbit lung.

Statistical analysis

Values are expressed as the mean ± standard error of the mean (SEM). The data were analyzed by one-way analysis of variance. The significance of the difference between the means was determined by a post hoc Tukey’s test adjusted for multiple comparisons. The level of significance was set at p < 0.05.

Results

Testosterone effect on H9c2 cell survival

Treatments with different concentrations of testosterone for 48 h decreased the cell viability of H9c2 cells in a concentration-dependent manner. Cell viability was 12.49 ± 1.54%, 21.36 ± 0.94%, 30.06 ± 1.38%, 64.74 ± 1.16%, and 74.85 ± 1.97%, at concentrations of 2.5 × 10⁻⁸, 2.5 × 10⁻⁷, 2.5 × 10⁻⁶, 5 × 10⁻⁶, and 10⁻⁵ mol/L, respectively (Figure 1). Concentrations of 5 × 10⁻⁶ and 10⁻⁵ mol/L exhibited the highest levels of cytotoxicity, which were greater than that induced by the positive control (DOX; 60.4 ± 1.7%; p < 0.01). The co-treatments with Eta or Los and testosterone (5 × 10⁻⁶ mol/L) prevented testosterone cytotoxicity (p < 0.01). Co-treatment with Ac-DEVD-CHO (a Casp-3 inhibitor) reduced the cell death induced by testosterone at the same concentration (p < 0.01; Table 1).

Figure 1.

Increased concentration-dependent inhibition of cell growth in cardiomyocytes (H9c2) treated with testosterone after 48 h exposure. Values represent mean ± SEM. **p < 0.01: in relation with the previous testosterone concentration.

Table 1.

Percentage of cell death induced by testosterone (5 × 10⁻ ⁶ mol/L) and co-treatment with inhibitors (Eta, Los, and Ac-DEVD-CHO) in H9c2 cardiomyocytes, with exposure time of 48 h.^a

Groups	T (5 × 10⁻⁶ mol/L)	T + Eta	T + Los	T + Ac-DEVD-CHO	DOX	H9c2
Growth inhibition (%)	64.7 ± 1.2^b	5.3 ± 1.7^c	6.4 ± 1.8^c	12.9 ± 3.5^b,c	60.4 ± 1.7^b	3.9 ± 0.8

T: testosterone; Eta: etanercept; Los: losartan; DOX: doxorubicin.

^aValues are expressed as the mean ± SEM.

^bp < 0.01: compared to H9c2.

^cp < 0.01: relative to T (5 × 10⁻⁶ mol/L).

Apoptosis and necrosis in H9c2 cells treated with testosterone

Figure 2 shows a concentration-dependent apoptosis and necrosis in H9c2 cells after treatment for 48 h with testosterone (Figure 2(a)). With increased concentration of testosterone, more H9c2 cells undergo apoptosis (annexin V (+)/PI (−), lower right quadrant; 2.5 × 10⁻⁷ mol/L: 2.84 ± 0.18%, 2.5 × 10⁻⁶ mol/L: 6.44 ± 0.14% and 5 × 10⁻⁶ mol/L: 13.15 ± 0.16%; p < 0.01; Figure 2(b)), and necrosis (annexin V (+)/PI (+), upper right quadrant; 2.5 × 10⁻⁷ mol/L: 1.96 ± 0.15%, 2.5 × 10⁻⁶ mol/L: 2.50 ± 0.13% and 5 × 10⁻⁶ mol/L: 6.13 ± 0.16%; p < 0.01; Figure 2(c)). A concentration of 5 × 10⁻⁶ mol/L was associated with the higher level of cellular apoptosis and necrosis, which was better than positive control to induce apoptosis (H₂O₂, 8.45 ± 0.16%; p < 0.01; Figure 2(b)) and lower than to induce necrosis (H₂O₂, 36.31 ± 0.20%; p < 0.01; Figure 2(c)). Co-treatment with Eta and Los reduced the apoptosis (Eta: 6.51 ± 0.62%; Los: 8.08 ± 0.54%; p < 0.01; Figure 2(b)) and necrosis (Eta: 1.89 ± 0.19%; Los: 2.29 ± 0.11%; p < 0.01; Figure 2(c)) induced by testosterone (5 × 10⁻⁶ mol/L).

Figure 2.

Concentration-dependent apoptosis and necrosis (2.5 × 10⁻⁷, 2.5 × 10⁻⁶, and 5 × 10⁻⁶ mol/L) induced by testosterone (T) in H9c2 cells. After the treatment period of 48 h, cells were stained with annexin V-FITC and PI followed by analysis in the flow cytometer. Cells in the initial stage of apoptosis were defined as annexin V (+)/PI (−), while necrotic cells were defined as double positive. Co-treatment with Eta H9c2 cells (Eta, 6.67 × 10⁻⁵ mol/L) or Los (Los, 10⁻⁷ mol/L) reduced apoptosis and necrosis induced by testosterone. (a) Data represent three independent experiments.(b) Percentage of apoptotic cells (sum of annexin V (+)/PI (−)]. (c) Percentage of necrotic cells (double positive). Values represent mean ± SEM. **p < 0.01: compared with positive control (H₂O₂); ++p < 0.01: relative concentrations previous testosterone; ##p < 0.01: compared to testosterone 5 × 10⁻⁶ mol/L. FITC: fluorescein isothiocyanate; PI: propidium iodide; Eta: etanercept; Los: losartan; H₂O₂: hydrogen peroxide.

Proteolytic activity of Casp-3 in H9c2 cells treated with testosterone

The proteolytic activity of Casp-3 on the substrate pNA was significantly increased after treatment with testosterone (0.320 ± 0.03 10⁻⁶ mol pNA/min/mL; p < 0.01 and p < 0.05 in relation to H9c2 and Casp-3, respectively; Figure 3). As expected, Ac-DEVD-CHO (INIB) inhibited the activity of Casp-3 (testosterone + INIB: 0.020 ± 0.003 10⁻⁶ mol pNA/min/mL; Casp-3 + INIB: 0.020 ± 0.002 10⁻⁶ mol pNA/min/mL; p < 0.01 in relation to testosterone and Casp-3), and a similar result was observed in treatment with Eta and Los (testosterone + Eta: 0.021 ± 0.001 10⁻⁶ mol pNA/min/mL; testosterone + Los: 0.016 ± 0.003 10⁻⁶ mol pNA/min/mL) that annulled the effect caused by testosterone on the proteolytic activity of Casp-3. The positive control assay of Casp-3 (0.413 ± 0.018 10⁻⁶ mol pNA/min/mL, p < 0.05) showed superior results to those found for testosterone.

Figure 3.

Increased activity of Casp-3 in cell line cardiomyocytes (H9c2) treated with testosterone (T) 5 × 10⁻⁶ mol/L. Co-treatment with Casp-3 inhibitor (Ac-DEVD-CHO, 10⁻⁵ M/L), Eta (6.67 × 10⁻⁵ mol/L), and Los (10⁻⁷ mol/L) prevented the increase of Casp-3 by testosterone after 48 h incubation. Values represent mean ± SEM. **p < 0.01: compared with the control (H9c2); ++p < 0.01: relative to Casp-3; ##p < 0.01: compared to testosterone 5 × 10⁻⁶ mol/L. Casp-3: caspase-3; Eta; etanercept; Los: losartan.

ACE activity and quantification of TNF-α in H9c2 cells treated with testosterone

In Figure 4(a), the effect of testosterone is shown on the levels of TNF-α. Cells treated with testosterone increased the amount of TNF-α (447.7 ± 8.7 pg/mL; p < 0.01) compared with the control (H9c2: 326.2 ± 8.3 pg/mL). ACE activity was also increased in cells treated with testosterone (171 ± 8%; p < 0.05, Figure 4(b)) compared with the control (H9c2: 118 ± 2%). Co-treatment with Eta and Los nullified the effects caused by testosterone in both measurements, TNF-α (testosterone + Eta: 369.4 ± 8.0 pg/mL; testosterone + Los: 372.6 ± 7.8 pg/mL) and ACE activity (testosterone + Eta: 104 ± 14%; testosterone + Los: 99 ± 8%) (p < 0.01). Treatment with the positive control (DOX) increased ACE activity (DOX: 131 ± 5%; p < 0.05) and the amount of TNF-α (DOX: 437.6 ± 13.8 pg/mL; p < 0.01).

Figure 4.

Testosterone induces increased TNF-α and ACE activity in cardiomyocytes. Cells (H9c2) were treated with testosterone (T) 5 × 10⁻⁶ mol/L with or without co-treatment with Eta (6.67 × 10⁻⁵ M/L) or Los (10^-7 mol/L) and DOX (9.2 × 10⁻⁶ mol/L; positive control) for 48 h. (a) Quantification of TNF-α. (b) Evaluation of ACE activity. Values represent mean ± SEM. *p < 0.05 or **p < 0.01: compared with control (H9c2); ##p < 0.01: compared to testosterone 5 × 10⁻⁶ mol/L. TNF-α: tumor necrosis factor-α; ACE: angiotensin-converting enzyme; Eta: etanercept; Los: losartan; DOX: doxorubicin; TNF-α: tumor necrosis factor-α.

Discussion

The results of this study indicate an interaction among TNF-α and ACE activity in the testosterone-induced cytotoxic effect in cultured cardiomyocytes due to apoptosis. Testosterone was able to promote apoptosis and necrosis in H9c2 cells, confirming the cytotoxic effect. The cytotoxicity induced by high concentrations of testosterone was concentration dependent and was reduced by co-treatment with inhibitor of Casp-3, confirming that apoptosis is involved in this process. Testosterone-induced apoptosis was confirmed by flow cytometry and the participation of Casp-3 was revealed by the proteolytic activity of Casp-3 under testosterone treatment. Zaugg et al. also reported the concentration-dependent induction of cytotoxicity and apoptosis by testosterone in adult cardiomyocytes in vitro. The cytotoxic effect was comparable to that determined by stanozolol, an AAS, and was similar to the cytotoxic effect elicited by testosterone at the IC₅₀ determined in this study (5 × 10⁻⁶ mol/L).⁶ In contrast, testosterone in physiological concentration or testosterone replacement therapy has a beneficial effect capable of reducing the apoptotic process. Kang et al. observed that replacement with testosterone reduced the apoptosis induced by isoproterenol in cardiac cells and increased the expression of Bcl-2.¹¹

Casp-3 has been well established as one of the mediators of apoptosis and treatment with testosterone increased the proteolytic activity of Casp-3. Co-treatment with Los and Eta prevented this increase, similar to the effect with Ac-DEVD-CHO, indicating that both TNF-α and Ang II (via AT₁ receptor activation) are involved in the increased activity of Casp-3 induced by testosterone. Actually, a relationship among androgens and RAS has been reported. The expression of the AT₁ receptor in the abdominal aorta is increased by androgens and elevated ACE activity in rat heart has been reported to influence AAS.^19,30
–32 In this study, testosterone induced an increase in ACE activity in cultured cardiomyocytes, indicating a possible increase of Ang II. Several mechanisms are proposed for Ang II-mediated apoptosis. Both AT₁ and AT₂ receptor can induce apoptosis in cardiovascular system.¹¹ Activation of AT₁ receptor signaling pathway involves the tumor suppressor protein p53 or the increase of cytosolic calcium, giving both apoptotic ability to Ang II.^18,33 Co-treatment with Los prevented this effect of testosterone, which can be explained by the fact that blockade of the AT₁ receptor leads to increased stimulation of AT₂ by Ang II, resulting in reduced ACE activity.^34
–36 Gokce et al. evidenced that ACE inhibition and AT₁ receptor blockade reduced the apoptotic changes in contralateral testis.³⁷

Treatment with testosterone also increased the content of TNF-α in the H9c2 cell culture and co-treatment with Eta prevented this increase. Du Toit et al. observed that high doses of AAS increase myocardial TNF-α concentrations in rat hearts.³⁸ Increased TNF-α by AAS was also observed in human peripheral blood lymphocytes in vitro and in the hearts of male CD1 mice.^39,40 Metcalfe et al. observed a testosterone-mediated increase in TNF-α production, Casp-8, -9, and -3 activation, apoptotic cell death, and fibrosis during renal obstruction and alterations in renal function.²²

A possible connection point between the action of testosterone and increased TNF-α and ACE activity in apoptosis could be an increased amount of p53, which has been implicated in the upregulation of both RAS (increasing AT₁ receptors, induction, and formation of Ang II) and of TNF-α receptor 1 in cardiomyocytes.^18,26,41,42 Therefore, the DNA damage caused by testosterone could increase the expression of p53, resulting in increased ACE activity and the amount of TNF-α. Interestingly, co-treatment with Eta or Los with testosterone reduced ACE activity and the content of TNF-α, respectively, indicating an interaction between Ang II and TNF-α. This interaction was recently documented by Chai et al. who showed that Los and Eta can block the cardiomyocyte apoptosis induced by AT₁ very effectively.⁴³ This suggests that AT₁ receptor blocker and TNF inhibitor therapy may prevent fetal cardiomyocyte apoptosis. Kalra et al. verified the influence of Ang II on TNF-α, suggesting that cardiomyocytes produce this cytokine after activation of AT₁ receptors by Ang II.⁴⁴ Additionally, cardiac fibroblasts treated with TNF-α increased the expression of messenger RNA for the Ang II-AT₁ receptor, which could promote upregulation of these receptors on the cell surface and thus increase the action of Ang II in cardiac fibroblasts.⁴⁵

To summarize, this study indicates an interaction between testosterone, Ang II, and TNF-α in the induction of apoptosis and increased activity of Casp-3 in cultured cardiomyocytes and that this process contributes to the reduced viability of these cells (cytotoxic effect) induced by testosterone at high concentrations and in a concentration-dependent manner.

Footnotes

Acknowledgment

We thank Professor Elenice Moreira Lemos for helping with ELISA.

Conflict of interest

The authors declared no conflicts of interest.

Funding

We thank the Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) and Fundação Nacional de Desenvolvimento do Ensino Superior Particular (Funadesp) for financial support.

References

Sullivan

Martinez

Gennis

. The cardiac toxicity of anabolic steroids. Prog Cardiovasc Dis 1998; 41: 1–15.

Nascimento

Medei

. Cardiac effects of anabolic steroids: hypertrophy, ischemia and electrical remodelling as potential triggers of sudden death. Mini Rev Med Chem 2011; 11: 425–429.

Montisci

El Mazloum

Cecchetto

. Anabolic androgenic steroids abuse and cardiac death in athletes: morphological and toxicological findings in four fatal cases. Forensic Sci Int 2012; 217 (1–3): 13–18.

Behrendt

Bofin

. Myocardial cell lesions caused by an anabolic hormone. Cell Tissue Res 1977; 181: 423–426.

Kindermann

. Cardiovascular side effects of anabolic-androgenic steroids. Herz 2006; 31 (6): 566–573.

Zaugg

Jamali

Lucchinetti

. Anabolic-androgenic steroids induce apoptotic cell death in adult rat ventricular myocytes. J Cell Physiol 2001; 187: 90–95.

Papamitsou

Barlagiannis

Papaliagkas

. Testosterone-induced hypertrophy, fibrosis and apoptosis of cardiac cells—an ultrastructural and immunohistochemical study. Med Sci Monit 2011; 17: 266–273.

Lopes

Neves

Pestana

. Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement. Am J Physiol Heart Circ Physiol 2014; 306 (11): H1485–H1494.

Hirota

Chen

Betz

. Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the onset of heart failure during biomechanical stress. Cell 1999; 97: 189–198.

10.

Vasconsuelo

Pronsato

Ronda

. Role of 17β-estradiol and testosterone in apoptosis. Steroids 2011; 76: 1223–1231.

11.

Kang

. Testosterone improves cardiac function and alters angiotensin II receptors in isoproterenol-induced heart failure. Arch Cardiovasc Dis 2012; 105: 68–76.

12.

Schmitz

Kirchhoff

Krammer

. Regulation of death receptor mediated apoptosis pathways. Int J Biochem Cell Biol 2000; 32: 1123–1136.

13.

Yamada

Akishita

Pollman

. Angiotensin II type 2 receptor mediates vascular smooth muscle cell apoptosis and antagonizes angiotensin II type 1 receptor action: an in vitro gene transfer study. Life Sci 1998; 63(19): PL289–PL295.

14.

Igney

Krammer

. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer 2002; 2 (4): 277–288.

15.

Buemi

Corica

Marino

. Cardiovascular remodeling, apoptosis, and drugs. Am J Hypertens 2000; 13 (4 Pt 1): 450–454.

16.

Ehlers

Fox

Strydom

. Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the C-terminal half of endothelial angiotensin-converting enzyme. Proc Natl Acad Sci USA 1989; 86 (20): 7741–7745.

17.

Freshour

Chase

Vikstrom

. Gender differences in cardiac ACE expression are normalized in androgen-deprived male mice. Am J Physiol Hearth Circ Physiol 2002; 283: 1997–2003.

18.

Liu

Wang

Zhou

. Angiotensin II-induced p53-dependent cardiac apoptotic cell death: its prevention by metallothionein. Toxicol Lett 2009; 191: 314–320.

19.

Franquni

do Nascimento

de Lima

. Nandrolone decanoate determines cardiac remodeling and injury by an imbalance in cardiac inflammatory cytokines and ACE activity, blunting of the Bezold-Jarisch reflex, resulting in the development of hypertension. Steroids 2013; 78 (3): 379–385.

20.

Andrade

Loiola

Alcure

. Role of the renin-angiotensin system in the nandrolone-decanoate-induced attenuation of the Bezold-Jarisch reflex. Can J Physiol Pharmacol 2011; 89: 891–897.

21.

Wan

Jiang

. Silencing of angiotensin converting enzyme by RNA interference prevents H9c2 cardiomyocytes from apoptosis induced by anoxia/reoxygenation through regulation of the intracellular renin-angiotensin system. Int J Mol Med 2013; 32 (6): 1380–1386.

22.

Metcalfe

Leslie

Campbell

. Testosterone exacerbates obstructive renal injury by stimulating TNF production and increasing proapoptotic and profibrotic signaling. Am J Physiol Endocrinol Metab 2008; 294: 435–443.

23.

Wang

Brewster

. Role of endogenous testosterone in TNF-induced myocardial injury in males. Int J Clin Exp Med 2012; 5: 96–104.

24.

Zheng

Liu

Yang

. Co-culture of apoptotic breast cancer cells with immature dendritic cells: a novel approach for DC-based vaccination in breast cancer. Braz J Med Biol Res 2012; 45: 510–515.

25.

Egberts

Cloosters

Noack

. Anti-tumor necrosis factor therapy inhibits pancreatic tumor growth and metastasis. Canc Res 2008; 68: 1443–1450.

26.

Pierzchalski

Reiss

Cheng

. p53 induces myocyte apoptosis via the activation of the renin–angiotensin system. Exp Cell Res 1997; 234: 57–65.

27.

Nicholson

Ali

Thornberry

. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995; 376: 37–43.

28.

Mosmann

. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 365: 55–63.

29.

Tada

Kagaya

Takeda

. Endogenous erythropoietin system in non-hematopoietic lineage cells plays a protective role in myocardial ischemia/reperfusion. Cardiovasc Res 2006; 71: 466–477.

30.

Henriques

Zhang

Yiannikouris

. Androgen increases AT1a receptor expression in abdominal aortas to promote angiotensin II-induced AAAs in apolipoprotein E deficient mice. Arterioscler Thromb Vasc Biol 2008; 28: 1251–1256.

31.

Do Carmo

Fernandes

Koike

. Anabolic steroid associated to physical training induces deleterious cardiac effects. Med Sci Sports Exerc 2011; 43: 1836–1848.

32.

Rocha

Carmo

Roque

. Anabolic steroids induce cardiac renin-angiotensin system and impair the beneficial effects of aerobic training in rats. Am J Physiol Heart Circ Physiol 2007; 293: 3575–3583.

33.

Goldenberg

Grossman

Jacobson

. Angiotensin II-induced apoptosis in rat cardiomyocyte culture: a possible role of AT₁ and AT₂ receptors. J Hypertension 2001; 19: 1681–1689.

34.

Abadir

Carey

Siragy

. Angiotensin AT2 receptors directly stimulate renal nitric oxide in bradykinin B2-receptor–null mice. Hypertension 2003; 42: 600–604.

35.

Carey

Howell

Jin

. Angiotensin type-2 receptor-mediated hypotension in angiotensin type-1 receptor-blocked rats. Hypertension 2001; 38: 1272–1277.

36.

Hunley

Tamura

Stoneking

. The angiotensin type II receptor tonically inhibits angiotensin-converting enzyme in AT2 null mutant mice. Kidney Inter 2000; 57: 570–577.

37.

Gokce

Karboga

Yildiz

. Effect of angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on apoptotic changes in contralateral testis following unilateral testicular torsion. Int Urol Nephrol 2008; 40 (4): 989–995.

38.

Du Toit

Rossouw

Van Rooyen

. Proposed mechanisms for the anabolic steroid-induced increase inmyocardial susceptibility to ischaemia/reperfusion injury. Cardiovasc J S Afr 2005; 16: 21–28.

39.

Hughes

Fulep

Juelich

. Modulation of immune responses by anabolic androgenic steroids. Int J Immunopharmacol 1995; 17: 857–863.

40.

Riezzo

De Carlo

Neri

. Heart disease induced by AAS abuse, using experimental mice/rats models and the role of exercise-induced cardiotoxicity. Mini Rev Med Chem 2011; 11: 409–424.

41.

Leri

Liu

. Up-regulation of AT1 and AT2 receptors in postinfarcted hypertrophied myocytes and stretch-mediated apoptotic cell death. Am J Pathol 2000; 156: 1663–1672.

42.

Reed

. Apoptosis-targeted therapies for cancer. Cancer Cell 2003; 3: 17–22.

43.

Chai

Zhang

Jin

. Angiotensin II type I receptor agonistic autoantibody-induced apoptosis in neonatal rat cardiomyocytes is dependent on the generation of tumor necrosis factor-α. Acta Biochim Biophys Sin 2012; 44: 984–990.

44.

Kalra

Sivasubramanian

Mann

. Angiotensin II induces tumor necrosis factor biosynthesis in the adult mammalian heart through a protein kinase C-dependent pathway. Circulation 2002; 105: 2198–2205.

45.

Guarantz

Cowling

Villarreal

. Tumor necrosis factor-alpha upregulates angiotensin II type 1 receptors on cardiac fibroblasts. Circ Res 1999; 85: 272–279.