International Journal of Cardiology 190 (2015) 11–14
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Testosterone regulates cardiac calcium homeostasis with enhanced ryanodine receptor 2 expression through activation of TGF-β Jung-Chieh Hsu a,1, Chen-Chuan Cheng b,c,1, Yu-Hsun Kao a,d, Yao-Chang Chen e, Cheng-Chih Chung a,f, Yi-Jen Chen a,f,⁎ a
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Division of Cardiology, Chi-Mei Medical Center, Tainan, Taiwan School of Medicine, Chung Shan Medical University, Taichung, Taiwan d Department of Medical Education and Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan e Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan f Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan b c
a r t i c l e
i n f o
Article history: Received 12 April 2015 Accepted 14 April 2015 Available online 15 April 2015 Keywords: Androgen Calcium transients Ryanodine receptor 2 Transforming growth factor β
Gender plays a critical role in the pathophysiology of cardiovascular diseases. Testosterone deficiency may contribute to the genesis of heart failure (HF). As to clinical needs, several reports showed that supplementation with testosterone improves exercise capacity and therapy in chronic HF patients [1]. Administration of testosterone increases cardiac output and regulates cardiac electrical activity [2]. Moreover, androgen receptor knock-out was associated with abnormal calcium regulation and testosterone replacement changes cardiac electrical activity [3]. Castrated animals exhibited an 80% decrease in Na+/Ca2+ exchanger gene expression, which led to reduced cardiac contractility; this was completely restored by testosterone supplementation [4]. Tsang et al. demonstrated that testosterone increased contraction and relaxation velocities that were associated with increased Ca2+ release and recovery through activities of the ryanodine receptor (RyR) and sarcoendoplasmic reticulum calcium transport ATPase (SERCA2) [5]. CD38(−/−) null mice exhibited markedly increased testosterone levels which were correlated with obvious increases in RyR2, and SERCA2 and α myosin heavy chain (α-MHC) gene expressions [6]. In contrast, ⁎ Corresponding author at: Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, 250 Wu-Xing Street, Taipei 11031, Taiwan. E-mail address: [email protected] (Y.-J. Chen). 1 Hsu JC, and Cheng CC contribute equally to this manuscript.
http://dx.doi.org/10.1016/j.ijcard.2015.04.116 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
hyperandrogenism may also produce hypertension, cardiac hypertrophy, and arrhythmias. Calcium regulation plays a critical role in cardiac electrical activity and arrhythmogenesis. Cardiac RyR2 determines calcium transients, which may contribute to cardiac contractility. Moreover, RyR2 dysfunction may result in cardiac arrhythmias. Abnormal RyR2 opening and closing (calcium leakage) impair cardiac electrical activity. Enhanced RyR2 opening can produce calcium overload and arrhythmogenesis. Aging and HF were shown to change the RyR2 expression. RyR2 messenger (m)RNA expression and calcium release are also regulated by transforming growth factor (TGF)-β in smooth muscles and many types of inflammatory cells [7]. However, the regulation of cardiac RyR expression remains unclear. It is not clear whether androgen or TGF-β can modulate RyR expression to regulate cardiac calcium homeostasis. Therefore, the purposes of this study were to investigate whether testosterone can change cardiac RyR2 expression and evaluate the underlying mechanisms in cell and animal models. Western blotting and PCR were used to measure the protein and RNA expressions of RyR2, TGF-β, and calcium transients in HL-1 cells with and without testosterone (10−8–10−6 M), nilutamide (5 μM), or LY2109761 (5 μM) for 24 h. Intracellular Ca2+ ([Ca2+]i) was measured from confocal microscopy with fluo-3 fluorescence during a 2-Hz field-stimulation with 10-ms twice-threshold strength square-wave pulses as described previously [8]. Male mice (8 weeks old with a C57BL/B6 background) were purchased from BioLASCO (Taipei, Taiwan). The experimental procedures were approved by the Institutional Animal Care and Use Committee of Taipei Medical University. Testosterone cypionate (80 mg/kg) was injected intramuscularly twice a week for 2 weeks. Atrial specimens were collected from control and testosterone-treated mice after euthanasia with an isoflurane overdose. All quantitative data are expressed as the mean ± SEM. A one-way repeated analysis of variance (ANOVA) test or unpaired T test was used to compare cells with and without drugs. A p value of b 0.05 was considered statistically significant. As shown in Fig. 1A, testosterone (10−8 M) increased the protein expression of RyR2 by 1.55-fold compared to control HL-1 cells. Similarly, testosterone treatment also increased the RNA expression of RyR2 compared to control HL-1 cells. Moreover, in the presence of nilutamide
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J.-C. Hsu et al. / International Journal of Cardiology 190 (2015) 11–14
Fig. 1. Testosterone (TES) increased ryanodine receptor 2 (RyR2) expressions and [Ca2+]i transients through enhanced transforming growth factor (TGF)-β1 protein expressions in HL-1 cells. (A) RyR2 expressions measured by Western blot in HL-1 cells treated with and without (control) testosterone (10−8–10−6 M) for 24 h (n = 4). (B) HL-1 cells were pretreated with nilutamide (Nilu; 5 μM) for 0.5 h, then incubated with testosterone (10−8–10−6 M) for further 24 h (n = 3). (C) TGF-β1 protein expression measured by Western blot in HL-1 cells treated with and without (control) testosterone (10−8–10−6 M) for 24 h (n = 3). (D) HL-1 cells were treated with LY2109761 (5 μM) for 0.5 h, then cells were changed to medium with or without (control) testosterone (10−8–10−6 M) and incubated for 24 h (n = 6). (E) TGF-β increased RyR2 protein expression in HL-1 cells. HL-1 cells were treated with TGF-β1 (10 μM) for 24 h (n = 6). (F) Testosterone-treated HL-1 cells (n = 12) had larger [Ca2+]i transients than control HL-1 cells (n = 11). In contrast, in the presence of nilutamide (5 μM), there were similar [Ca2+]i transients in HL-1 cells with (n = 18) and without (n = 13) nilutamide. *p b 0.05, **p b 0.01, ***p b 0.005.
(an androgen receptor blocker, 5 μM), testosterone (10−8 to 10−6 M) did not change the protein expression of RyR2 by HL-1 cells (Fig. 1B), which suggests that testosterone enhances RyR2 expression through androgen receptor signaling. We studied whether TGF-β plays a role in testosterone-mediated RyR2 expression and found that TGF-β1 protein levels were increased after testosterone treatment (Fig. 1C). However, in the presence of LY2109761 (a TGF-β receptor blocker,
5 μM), testosterone did not change RyR2 expression in HL1 cells (Fig. 1D). Similarly, TGF-β (10 μM) also increased RyR2 expression by 1.6-fold (Fig. 1E). These results suggest that testosterone induces RyR2 expression through TGF-β activation. We evaluated the effects of testosterone on calcium transients in HL-1 cells. As shown in Fig. 1F, testosterone-treated HL-1 cells had larger [Ca2+]i transients than did control cells. However, [Ca2+]i transients of
J.-C. Hsu et al. / International Journal of Cardiology 190 (2015) 11–14
13
Fig. 2. Effects of testosterone on ryanodine receptor 2 and transforming growth factor (TGF)-β expression in testosterone-treated mice. C57BL/B6 mice were treated with or without (control) testosterone cypionate. (A) RyR2 protein (n = 6) expressions of the atrium C57BL/B6 mice treated with or without testosterone were determined. GAPDH expression was used as an internal control. (B) RyR2 mRNA (n = 3) and (C) TGF-β (n = 3) expressions by the atrium of C57BL/B6 mice treated with or without testosterone was determined. *p b 0.05.
testosterone-treated HL-1 cells did not significantly change in the presence of nilutamide. Moreover, as compared to control atrium, the testosterone-treated atrium had a greater RyR2 protein expression by 1.2 fold (Fig. 2A). In addition, testosterone-treated atrium also had greater RNA expression of RyR2 by 1.26 fold (Fig. 2B) and TGF-β1 by 1.4 fold (Fig. 2C) than control atrium. In this study, we also demonstrated that testosterone can increase calcium transients in cardiomyocytes. Moreover, this study for the first time demonstrated that testosterone can upregulate RyR2 protein and RNA expressions in HL-1 cells. Although testosterone elicits Ca2+ release from internal stores and improves cardiovascular function with androgen insufficiency syndrome, abusing anabolic-androgen steroids (AAS) may increase sudden death due to cardiac arrhythmias [9,10]. Field studies of these athletes ingested a total AAS dose of as much as 3000–5000 mg of testosterone per week, which was similar to the testosterone used in the whole-animal experiments. Therefore, highly enhanced RyR expression with larger calcium transients may contribute to an increased arrhythmogenic risk due to calcium overload with hyperandrogenism. Since an androgen receptor blocker can attenuate the effects of testosterone on RyR2, our findings suggest that testosterone amplify RyR2 signaling via the androgen receptor pathway. TGF-β 1 was shown to regulate Ca2+ homeostasis and contractile responses. In this study, we found that TGF-β can increase RyR2 expression. Moreover, we found that a TGF-β receptor blocker can block the effects of testosterone, which suggests that TGF-β signaling may underlie the effects of testosterone. In addition, we also found a significant increase in cardiac TGF-β after mice were treated with testosterone. This effect may contribute to the higher RyR2 expression in testosteronetreated mice. In conclusion, testosterone can upregulate RyR2 expression through androgen receptor with activation of TGF-β signaling.
These effects may contribute to cardiac calcium dysregulation with hyperandrogenism. Conflict of interest None declared. Acknowledgments The current study was supported by grants from the National Science Council, Taiwan (NSC100-2628-B-038-001-MY4, NSC 1022811-B-038-024, NSC 102-2314-B-038-003-MY2 and 102-2628-B038-002-MY3), the Taipei Medical University (TMU101-AE1-B54), the Chi-Mei Medical Center (103CM-TMU-07, 104CM-TMU-07) and the Wan Fang Hospital, Taipei Medical University (102swf08, 104-wf-eva01). References [1] M. Toma, F.A. McAlister, E.E. Coglianese, V. Vidi, S. Vasaiwala, J.A. Bakal, et al., Testosterone supplementation in heart failure: a meta-analysis, Circ. Heart Fail. 5 (2012) 315–321. [2] W.C. Tsai, T.I. Lee, Y.C. Chen, Y.H. Kao, Y.Y. Lu, Y.K. Lin, et al., Testosterone replacement increases aged pulmonary vein and left atrium arrhythmogenesis with enhanced adrenergic activity, Int. J. Cardiol. 176 (1) (2014) 110–118. [3] W.C. Tsai, L.Y. Yang, Y.C. Chen, Y.H. Kao, Y.K. Lin, S.A. Chen, et al., Ablation of the androgen receptor gene modulates atrial electrophysiology and arrhythmogenesis with calcium protein dysregulation, Endocrinology 154 (2013) 2833–2842. [4] K.L. Golden, J.D. Marsh, Y. Jiang, T. Brown, J. Moulden, Gonadectomy of adult male rats reduces contractility of isolated cardiac myocytes, Am. J. Physiol. Endocrinol. Metab. 285 (2003) E449–E453. [5] S. Tsang, S.S. Wong, S. Wu, G.M. Kravtsov, T.M. Wong, Testosterone-augmented contractile responses to alpha1- and beta1-adrenoceptor stimulation are associated
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J.-C. Hsu et al. / International Journal of Cardiology 190 (2015) 11–14
with increased activities of RyR, SERCA, and NCX in the heart, Am. J. Physiol. Cell Physiol. 296 (2009) C766–C782. [6] L. Gan, W. Jiang, Y.F. Xiao, L. Deng, L.D. Gu, Z.Y. Guo, et al., Disruption of CD38 gene enhances cardiac functions by elevating serum testosterone in the male null mice, Life Sci. 89 (2011) 491–497. [7] G. Giannini, E. Clementi, R. Ceci, G. Marziali, V. Sorrentino, Expression of a ryanodine receptor-Ca 2 + channel that is regulated by TGF-beta, Science 257 (1992) 91–94.
[8] B. Lkhagva, S.L. Chang, Y.C. Chen, Y.H. Kao, Y.K. Lin, C.T. Chiu, et al., Histone deacetylase inhibition reduces pulmonary vein arrhythmogenesis through calcium regulation, Int. J. Cardiol. 177 (3) (2014) 982–989. [9] M.L. Sullivan, C.M. Martinez, P. Gennis, E.J. Gallagher, The cardiac toxicity of anabolic steroids, Prog. Cardiovasc. Dis. 41 (1998) 1–15. [10] G. Kanayama, J.I. Hudson, H.G. Pope Jr., Long-term psychiatric and medical consequences of anabolic-androgenic steroid abuse: a looming public health concern? Drug Alcohol Depend. 98 (2008) 1–12.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Testosterone regulates cardiac calcium homeostasis with enhanced ryanodine receptor 2 expression through activation of TGF-β Jung-Chieh Hsu a,1, Chen-Chuan Cheng b,c,1, Yu-Hsun Kao a,d, Yao-Chang Chen e, Cheng-Chih Chung a,f, Yi-Jen Chen a,f,⁎ a
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Division of Cardiology, Chi-Mei Medical Center, Tainan, Taiwan School of Medicine, Chung Shan Medical University, Taichung, Taiwan d Department of Medical Education and Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan e Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan f Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan b c
a r t i c l e
i n f o
Article history: Received 12 April 2015 Accepted 14 April 2015 Available online 15 April 2015 Keywords: Androgen Calcium transients Ryanodine receptor 2 Transforming growth factor β
Gender plays a critical role in the pathophysiology of cardiovascular diseases. Testosterone deficiency may contribute to the genesis of heart failure (HF). As to clinical needs, several reports showed that supplementation with testosterone improves exercise capacity and therapy in chronic HF patients [1]. Administration of testosterone increases cardiac output and regulates cardiac electrical activity [2]. Moreover, androgen receptor knock-out was associated with abnormal calcium regulation and testosterone replacement changes cardiac electrical activity [3]. Castrated animals exhibited an 80% decrease in Na+/Ca2+ exchanger gene expression, which led to reduced cardiac contractility; this was completely restored by testosterone supplementation [4]. Tsang et al. demonstrated that testosterone increased contraction and relaxation velocities that were associated with increased Ca2+ release and recovery through activities of the ryanodine receptor (RyR) and sarcoendoplasmic reticulum calcium transport ATPase (SERCA2) [5]. CD38(−/−) null mice exhibited markedly increased testosterone levels which were correlated with obvious increases in RyR2, and SERCA2 and α myosin heavy chain (α-MHC) gene expressions [6]. In contrast, ⁎ Corresponding author at: Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, 250 Wu-Xing Street, Taipei 11031, Taiwan. E-mail address: [email protected] (Y.-J. Chen). 1 Hsu JC, and Cheng CC contribute equally to this manuscript.
http://dx.doi.org/10.1016/j.ijcard.2015.04.116 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
hyperandrogenism may also produce hypertension, cardiac hypertrophy, and arrhythmias. Calcium regulation plays a critical role in cardiac electrical activity and arrhythmogenesis. Cardiac RyR2 determines calcium transients, which may contribute to cardiac contractility. Moreover, RyR2 dysfunction may result in cardiac arrhythmias. Abnormal RyR2 opening and closing (calcium leakage) impair cardiac electrical activity. Enhanced RyR2 opening can produce calcium overload and arrhythmogenesis. Aging and HF were shown to change the RyR2 expression. RyR2 messenger (m)RNA expression and calcium release are also regulated by transforming growth factor (TGF)-β in smooth muscles and many types of inflammatory cells [7]. However, the regulation of cardiac RyR expression remains unclear. It is not clear whether androgen or TGF-β can modulate RyR expression to regulate cardiac calcium homeostasis. Therefore, the purposes of this study were to investigate whether testosterone can change cardiac RyR2 expression and evaluate the underlying mechanisms in cell and animal models. Western blotting and PCR were used to measure the protein and RNA expressions of RyR2, TGF-β, and calcium transients in HL-1 cells with and without testosterone (10−8–10−6 M), nilutamide (5 μM), or LY2109761 (5 μM) for 24 h. Intracellular Ca2+ ([Ca2+]i) was measured from confocal microscopy with fluo-3 fluorescence during a 2-Hz field-stimulation with 10-ms twice-threshold strength square-wave pulses as described previously [8]. Male mice (8 weeks old with a C57BL/B6 background) were purchased from BioLASCO (Taipei, Taiwan). The experimental procedures were approved by the Institutional Animal Care and Use Committee of Taipei Medical University. Testosterone cypionate (80 mg/kg) was injected intramuscularly twice a week for 2 weeks. Atrial specimens were collected from control and testosterone-treated mice after euthanasia with an isoflurane overdose. All quantitative data are expressed as the mean ± SEM. A one-way repeated analysis of variance (ANOVA) test or unpaired T test was used to compare cells with and without drugs. A p value of b 0.05 was considered statistically significant. As shown in Fig. 1A, testosterone (10−8 M) increased the protein expression of RyR2 by 1.55-fold compared to control HL-1 cells. Similarly, testosterone treatment also increased the RNA expression of RyR2 compared to control HL-1 cells. Moreover, in the presence of nilutamide
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J.-C. Hsu et al. / International Journal of Cardiology 190 (2015) 11–14
Fig. 1. Testosterone (TES) increased ryanodine receptor 2 (RyR2) expressions and [Ca2+]i transients through enhanced transforming growth factor (TGF)-β1 protein expressions in HL-1 cells. (A) RyR2 expressions measured by Western blot in HL-1 cells treated with and without (control) testosterone (10−8–10−6 M) for 24 h (n = 4). (B) HL-1 cells were pretreated with nilutamide (Nilu; 5 μM) for 0.5 h, then incubated with testosterone (10−8–10−6 M) for further 24 h (n = 3). (C) TGF-β1 protein expression measured by Western blot in HL-1 cells treated with and without (control) testosterone (10−8–10−6 M) for 24 h (n = 3). (D) HL-1 cells were treated with LY2109761 (5 μM) for 0.5 h, then cells were changed to medium with or without (control) testosterone (10−8–10−6 M) and incubated for 24 h (n = 6). (E) TGF-β increased RyR2 protein expression in HL-1 cells. HL-1 cells were treated with TGF-β1 (10 μM) for 24 h (n = 6). (F) Testosterone-treated HL-1 cells (n = 12) had larger [Ca2+]i transients than control HL-1 cells (n = 11). In contrast, in the presence of nilutamide (5 μM), there were similar [Ca2+]i transients in HL-1 cells with (n = 18) and without (n = 13) nilutamide. *p b 0.05, **p b 0.01, ***p b 0.005.
(an androgen receptor blocker, 5 μM), testosterone (10−8 to 10−6 M) did not change the protein expression of RyR2 by HL-1 cells (Fig. 1B), which suggests that testosterone enhances RyR2 expression through androgen receptor signaling. We studied whether TGF-β plays a role in testosterone-mediated RyR2 expression and found that TGF-β1 protein levels were increased after testosterone treatment (Fig. 1C). However, in the presence of LY2109761 (a TGF-β receptor blocker,
5 μM), testosterone did not change RyR2 expression in HL1 cells (Fig. 1D). Similarly, TGF-β (10 μM) also increased RyR2 expression by 1.6-fold (Fig. 1E). These results suggest that testosterone induces RyR2 expression through TGF-β activation. We evaluated the effects of testosterone on calcium transients in HL-1 cells. As shown in Fig. 1F, testosterone-treated HL-1 cells had larger [Ca2+]i transients than did control cells. However, [Ca2+]i transients of
J.-C. Hsu et al. / International Journal of Cardiology 190 (2015) 11–14
13
Fig. 2. Effects of testosterone on ryanodine receptor 2 and transforming growth factor (TGF)-β expression in testosterone-treated mice. C57BL/B6 mice were treated with or without (control) testosterone cypionate. (A) RyR2 protein (n = 6) expressions of the atrium C57BL/B6 mice treated with or without testosterone were determined. GAPDH expression was used as an internal control. (B) RyR2 mRNA (n = 3) and (C) TGF-β (n = 3) expressions by the atrium of C57BL/B6 mice treated with or without testosterone was determined. *p b 0.05.
testosterone-treated HL-1 cells did not significantly change in the presence of nilutamide. Moreover, as compared to control atrium, the testosterone-treated atrium had a greater RyR2 protein expression by 1.2 fold (Fig. 2A). In addition, testosterone-treated atrium also had greater RNA expression of RyR2 by 1.26 fold (Fig. 2B) and TGF-β1 by 1.4 fold (Fig. 2C) than control atrium. In this study, we also demonstrated that testosterone can increase calcium transients in cardiomyocytes. Moreover, this study for the first time demonstrated that testosterone can upregulate RyR2 protein and RNA expressions in HL-1 cells. Although testosterone elicits Ca2+ release from internal stores and improves cardiovascular function with androgen insufficiency syndrome, abusing anabolic-androgen steroids (AAS) may increase sudden death due to cardiac arrhythmias [9,10]. Field studies of these athletes ingested a total AAS dose of as much as 3000–5000 mg of testosterone per week, which was similar to the testosterone used in the whole-animal experiments. Therefore, highly enhanced RyR expression with larger calcium transients may contribute to an increased arrhythmogenic risk due to calcium overload with hyperandrogenism. Since an androgen receptor blocker can attenuate the effects of testosterone on RyR2, our findings suggest that testosterone amplify RyR2 signaling via the androgen receptor pathway. TGF-β 1 was shown to regulate Ca2+ homeostasis and contractile responses. In this study, we found that TGF-β can increase RyR2 expression. Moreover, we found that a TGF-β receptor blocker can block the effects of testosterone, which suggests that TGF-β signaling may underlie the effects of testosterone. In addition, we also found a significant increase in cardiac TGF-β after mice were treated with testosterone. This effect may contribute to the higher RyR2 expression in testosteronetreated mice. In conclusion, testosterone can upregulate RyR2 expression through androgen receptor with activation of TGF-β signaling.
These effects may contribute to cardiac calcium dysregulation with hyperandrogenism. Conflict of interest None declared. Acknowledgments The current study was supported by grants from the National Science Council, Taiwan (NSC100-2628-B-038-001-MY4, NSC 1022811-B-038-024, NSC 102-2314-B-038-003-MY2 and 102-2628-B038-002-MY3), the Taipei Medical University (TMU101-AE1-B54), the Chi-Mei Medical Center (103CM-TMU-07, 104CM-TMU-07) and the Wan Fang Hospital, Taipei Medical University (102swf08, 104-wf-eva01). References [1] M. Toma, F.A. McAlister, E.E. Coglianese, V. Vidi, S. Vasaiwala, J.A. Bakal, et al., Testosterone supplementation in heart failure: a meta-analysis, Circ. Heart Fail. 5 (2012) 315–321. [2] W.C. Tsai, T.I. Lee, Y.C. Chen, Y.H. Kao, Y.Y. Lu, Y.K. Lin, et al., Testosterone replacement increases aged pulmonary vein and left atrium arrhythmogenesis with enhanced adrenergic activity, Int. J. Cardiol. 176 (1) (2014) 110–118. [3] W.C. Tsai, L.Y. Yang, Y.C. Chen, Y.H. Kao, Y.K. Lin, S.A. Chen, et al., Ablation of the androgen receptor gene modulates atrial electrophysiology and arrhythmogenesis with calcium protein dysregulation, Endocrinology 154 (2013) 2833–2842. [4] K.L. Golden, J.D. Marsh, Y. Jiang, T. Brown, J. Moulden, Gonadectomy of adult male rats reduces contractility of isolated cardiac myocytes, Am. J. Physiol. Endocrinol. Metab. 285 (2003) E449–E453. [5] S. Tsang, S.S. Wong, S. Wu, G.M. Kravtsov, T.M. Wong, Testosterone-augmented contractile responses to alpha1- and beta1-adrenoceptor stimulation are associated
14
J.-C. Hsu et al. / International Journal of Cardiology 190 (2015) 11–14
with increased activities of RyR, SERCA, and NCX in the heart, Am. J. Physiol. Cell Physiol. 296 (2009) C766–C782. [6] L. Gan, W. Jiang, Y.F. Xiao, L. Deng, L.D. Gu, Z.Y. Guo, et al., Disruption of CD38 gene enhances cardiac functions by elevating serum testosterone in the male null mice, Life Sci. 89 (2011) 491–497. [7] G. Giannini, E. Clementi, R. Ceci, G. Marziali, V. Sorrentino, Expression of a ryanodine receptor-Ca 2 + channel that is regulated by TGF-beta, Science 257 (1992) 91–94.
[8] B. Lkhagva, S.L. Chang, Y.C. Chen, Y.H. Kao, Y.K. Lin, C.T. Chiu, et al., Histone deacetylase inhibition reduces pulmonary vein arrhythmogenesis through calcium regulation, Int. J. Cardiol. 177 (3) (2014) 982–989. [9] M.L. Sullivan, C.M. Martinez, P. Gennis, E.J. Gallagher, The cardiac toxicity of anabolic steroids, Prog. Cardiovasc. Dis. 41 (1998) 1–15. [10] G. Kanayama, J.I. Hudson, H.G. Pope Jr., Long-term psychiatric and medical consequences of anabolic-androgenic steroid abuse: a looming public health concern? Drug Alcohol Depend. 98 (2008) 1–12.
