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Aldosterone induces myocyte apoptosis in the heart and skeletal muscles of rats in vivo
Journal of Molecular and Cellular Cardiology 39 (2005) 395–399 www.elsevier.com/locate/yjmcc
Rapid communication
Aldosterone induces myocyte apoptosis in the heart and skeletal muscles of rats in vivo Jatin G. Burniston a,*, Amarjit Saini a, Lip-Bun Tan b, David F. Goldspink a a
Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Webster Street, Liverpool L3 2ET, UK b Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds General Infirmary, Leeds LS2 9JT, UK Received 17 December 2004; received in revised form 30 March 2005; accepted 5 April 2005 Available online 23 May 2005
Abstract Over activation of the renin–angiotensin–aldosterone system is known to be cardiotoxic but the potential injurious effects on the skeletal musculature have not been investigated. Male Wistar rats were given subcutaneous injections of aldosterone (1 µg–10 mg kg–1) and killed 7 h later, or continuous infusion (1 mg kg–1 d–1) and killed 48 h later. The role of the mineralocorticoid receptor in mediating aldosterone-induced apoptosis in vivo was investigated using spironolactone (200 mg kg–1). The number of apoptotic (caspase 3 positive) myocytes was counted on cryosections of the heart, soleus and Tibialis Anterior muscles. Injections of aldosterone induced significant (P < 0.05) cardiomyocyte apoptosis (peak = 2.46 ± 0.6 per 104 viable myocytes) over the range of 100 µg–10 mg kg–1, whereas only administration of 1 mg kg–1 induced significant (P < 0.05) apoptosis (2.47 ± 0.8 per 104 viable myocytes) in the soleus muscle. In contrast, no apoptosis was detected in the striated muscles after administration of only the vehicle. Infusion of aldosterone induced less apoptosis than the same dose (1 mg kg–1) given as a single injection. Prior administration of spironolactone significantly (P < 0.05) protected the heart (90%) and soleus muscle (79%) against the apoptosis induced by a single injection of 1 mg kg–1 aldosterone. These data confirm a myotoxic effect of aldosterone on the heart and provide the first description of aldosterone-induced myocyte apoptosis in skeletal muscle. High circulating levels of aldosterone are clearly capable of damaging all types of striated muscle and this may lend support to the concept that heart failure is a generalised, rather than cardiac-specific, myopathy. © 2005 Elsevier Ltd. All rights reserved. Keywords: Aldosterone; Spironolactone; Caspase 3; Myocyte death; Cardiac and skeletal muscle
1. Introduction Experimental models have demonstrated that catecholamines [1], angiotensin II [2] and aldosterone [2,3] are all capable of inducing cardiomyocyte death. Recent work from our laboratory has shown that the myotoxicity of catecholamines also extends to skeletal muscles, with exposure to excess catecholamines causing both myocyte apoptosis and necrosis [4]. The loss of myocytes from the dysfunctional heart propagates a vicious cycle of events, which results in a reduced cardiac reserve capacity [1]. Myocyte apoptosis also occurs in the skeletal muscles of chronic heart failure patients [5,6] and this is associated with muscle wasting, weakness and exercise intolerance [5]. Furthermore, the loss of skeletal * Corresponding author. E-mail address: [email protected] (J.G. Burniston). 0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2005.04.001
muscle mass is an independent risk factor for mortality in patients with chronic heart failure [7]. The clinical and laboratory-based studies described above support the concept that heart failure involves a generalised, rather than cardiac specific, myopathic process. There is good evidence to suggest that chronically elevated levels of catecholamines [4] and angiotensin II [8] are at least partly responsible for mediating this myopathy. In comparison, relatively little is known about the myotoxicity of aldosterone. The cardiotoxic effects of aldosterone have recently been described in normotensive [2] and hypertensive [3] rats, and also on isolated cardiomyocytes in vitro [2,9]. Furthermore, aldosterone blockade, using spironolactone, significantly reduces morbidity and mortality of patients with severe heart failure, over and above that of standard therapies [10]. Skeletal myocytes also possess mineralocorticoid receptors and
396
J.G. Burniston et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 395–399
the current study tests the hypothesis that aldosterone is toxic to skeletal, as well as cardiac, myocytes.
2. Methods All experimental procedures were carried out under the auspices of the British Home Office Animal (Scientific Procedures) Act 1986. Wistar rats (310 ± 10 g; mean ± S.D.) were purchased from a commercial supplier (Bantin and Kingman, Hull, UK) and allowed a period of 4 d prior to the experimental procedures. Animals were housed in control conditions of 20 °C, 45% relative humidity, and a 12 h light (06:00– 18:00) and dark cycle, with water and food (containing 18.5% protein) available ad libitum. The dose dependency (from 1 µg to 10 mg kg–1) of aldosterone-induced apoptosis was investigated using independent groups of animals (n = 5, in each group). Aldosterone (Sigma Chemicals, IL, USA) was freshly prepared in a 5% ethanol, 154-mmol NaCl vehicle immediately prior to administration. Animals received a single subcutaneous injection of hormone or the vehicle only and were killed 7 h later. To investigate whether the aldosterone-induced apoptosis was mediated through the mineralocorticoid receptor in vivo, independent groups of animals (n = 8, in each group) were administered either 1 mg kg–1 aldosterone (positive control), 200 mg kg–1 spironolactone (negative control) or spironolactone and aldosterone. Spironolactone treated animals were given two doses (200 mg kg–1, via gavage) with a 24 h interval between each. Three hours after the last administration of spironolactone, animals were challenged with either a single injection (s.c.) of 1 mg kg–1 aldosterone or the vehicle only and were killed 7 h later. The potential myotoxic effects of continuous infusions of aldosterone were also investigated. Animals were implanted, under isoflurane anaesthesia, with preconditioned osmotic pumps (Alzet™ 1003D) dispensing (s.c.) either 1 mg kg– –1 1 d aldosterone or the vehicle only (n = 5, in each group), and were killed 48 h later. At the end of each experiment, rats were concussed and killed by cervical dislocation and the heart, slow-twitch soleus and fast-twitch Tibialis Anterior muscles rapidly isolated. The atria and great vessels were removed from the heart and the ventricles mounted apex uppermost on a piece of cork. A standardised segment of the mid-belly from each skeletal muscle (soleus and Tibialis Anterior) was mounted in transverse section and supported with liver. Tissues were snap-frozen in super-cooled isopentane and stored at –80 °C, prior to cryosectioning (5 µm) and storage at –20 °C. Apoptosis was detected by an indirect immunoperoxidase protocol using an anti-caspase-3 antibody (Ab; R&D Systems, Minneapolis, USA) on cryosections in vitro. Previous work from our laboratory [4] and others [2] has shown that caspase-3 activity co-localises with dUTP nick-end labelling on cryosections in vitro, and with annexin V-biotin detection of phosphatidylserine externalisation in vivo [11].
To quantify apoptosis in cardiac and skeletal muscle, the number of positively stained myocytes was counted relative to the total number of viable myocytes per muscle crosssection; these typically being 11,362 ± 442 for the left ventricle, 2436 ± 147 for the soleus and 4250 ± 211 for the Tibialis Anterior (mean ± S.D.). For clarity and ease of comparison, all data have been normalised to 104 viable myocytes. All experiment data are presented as mean ± S.E.M. Experiments were analysed by using one-way analysis of variance with multiple post hoc analyses. P values of < 0.05 were taken to indicate statistical significance. 3. Results Administration of aldosterone (from 1 µg to 10 mg kg–1) to normotensive rats induced clearly discernable myocyte apoptosis in the heart (Fig. 1A) and skeletal muscle (Fig. 1B) in vivo. Administration of the vehicle only did not induce any cardiac (Fig. 1C) or skeletal (Fig. 1D) myocyte death. Because of this zero baseline in the control muscles, all myocyte death induced by aldosterone may be reasonably regarded as being of biological significance. Apoptosis in the heart (Fig. 2A) reached statistically significant (P < 0.05) levels after the administration of aldosterone over the range of 100 µg–10 mg kg–1. While the dosedependent profile of apoptosis was very similar in the soleus and Tibialis Anterior muscles, only the administration of 1 mg kg–1 induced statistically significant (P < 0.05) levels of skeletal myocyte death in the soleus (Fig. 2B). Prior administration of the mineralocorticoid receptor antagonist, spironolactone, significantly (P < 0.05) prevented the myocyte apoptosis induced by a single injection of aldosterone in the heart (90%) and soleus muscle (79%) and completely abolished it in the Tibialis Anterior (Fig. 3). Continuous infusion of aldosterone (1 mg kg–1 d–1) was generally less myotoxic than the same dose given as a single injection. Myocyte apoptosis was detected in the heart (0.9 ± 0.4 per 104 viable cardiomyocytes) and soleus muscles (1.9 ± 0.6 per 104 viable myocytes; P < 0.05) of those animals infused with aldosterone for 48 h. In contrast, no myocyte apoptosis was detected in any of the striated muscles (heart, soleus and Tibialis Anterior) after infusion of only the vehicle. 4. Discussion To the best of our knowledge, this is the first description of aldosterone inducing myocyte apoptosis in skeletal muscle. These data also provide the first confirmation of the initial findings of De Angelis et al. [2] who recently described the apoptotic effects of aldosterone in the hearts of normotensive rats in vivo. In the current work, the amount of cardiomyocyte apoptosis (2.5 ± 0.6 per 104 viable cardiomyocytes) was greatest in
J.G. Burniston et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 395–399
397
Fig. 1. Immunohistochemical detection of myocyte apoptosis in the heart and soleus muscle. Myocyte apoptosis was detected, using an anti-caspase 3 Ab, on cryosections (5 µm) of the heart (A) and soleus muscle (B) 7 h after the administration of 1 mg kg–1 aldosterone. No myocyte apoptosis was detected on cryosections of the control heart (C) or skeletal muscle (D) from animals that received only the saline vehicle. Brown (DAB) staining represents antibody binding (arrows), contrasted against a blue (haematoxylin) background. All images are × 400 magnification.
response to a single injection of 1 mg kg–1 aldosterone. When measured after infusion of this dose for 48 h, the incidence of cardiomyocyte apoptosis was 0.9 ± 0.4 per 104 viable cardiomyocytes. The dose used in the current work (1 mg kg– –1 –1 1 d ) is approximately twice that (75 µg h ) used in the earlier studies of Brilla et al. [12], who investigated the fibrinogenic effects of long-term (8-week) hyperaldosteronism. However, the current work compares closely with that of De Angelis et al. [2] who counted 285 ± 18 apoptotic cardiomyocytes per 106 cells after infusion of 1 mg kg–1 d–1 aldosterone for 24 h. The mechanisms that mediate aldosterone-induced apoptosis are yet to be elucidated fully. Studies conducted using cultured cardiomyocytes in vitro have suggested both mineralocorticoid-dependent [2] and independent [9] pathways may be involved. Prior intervention with spironolactone in vivo significantly (P < 0.05) prevented aldosteroneinduced apoptosis in both the heart and skeletal muscles (Fig. 3). Suggesting that the myocyte apoptosis detected in the current studies was mediated through the mineralocorticoid receptor. Therefore, some of the positive outcomes observed in heart failure patients that have received spironolactone [10] may be related to its ability to protect the striated muscles from aldosterone-induced myocyte death. The plasma concentration of aldosterone in heart failure patients with cachexia is elevated approximately threefold, being 1 nmol l–1 compared with 0.5 nmol l–1 for noncachectic patients and 0.3 nmol l–1 for age-matched individuals without heart failure [13]. Although not measured in the
work, De Angelis et al. [2] reported that infusion of 1 mg kg–1 aldosterone to rats elevated their plasma aldosterone approximately 10-fold from 0.7 to 8 ng ml –1 , equivalent to 24 nmol l–1. Suggesting that some of the doses used in the current work may have resulted in plasma concentrations of aldosterone greater than those found in heart failure patients. Nevertheless, this work reveals the potential for aldosterone to induce apoptosis in striated muscles in vivo. Future work should determine whether long-term infusion of a lower dose of aldosterone is similarly myotoxic. Myocyte apoptosis has been detected in skeletal muscle biopsy samples from heart failure patients [6] and patients with cachexia and skeletal muscle wasting have a poorer prognosis than non-cachexic patients [7]. However, it is not known which of the hormonal perturbations concomitant with heart failure is responsible for mediating the skeletal muscle apoptosis. That is, plasma concentrations of aldosterone, norepinephrine, epinephrine and tumour necrosis factor-a (TNFa) are all significantly elevated in heart failure patients with cachexia [13]. Previous work from our laboratory [1] has revealed that catecholamines can induced myocyte apoptosis and necrosis in skeletal muscle, and Dalla Libera et al. [14] have described the induction of skeletal myocyte apoptosis by TNFa. Therefore, it may be postulated that the elevated levels of these hormones (aldosterone, catecholamines and TNFa) act in synergy to contribute to the muscle wasting and cachexia associated with heart failure, by inducing myocyte apoptosis.
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J.G. Burniston et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 395–399
Fig. 2. Dose dependent aldosterone-induced myocyte apoptosis. Myocyte apoptosis in the heart (A), soleus (B) and Tibialis Anterior muscle (C) in response to aldosterone. Independent groups (n = 5, in each group) of animals were administered either a single subcutaneous injection of aldosterone over the range 1 µg–10 mg kg–1 or the vehicle only and killed 7-h later. Apoptosis was detected on muscle cryosections by caspase-3 immunohistochemistry, this having been validated against other techniques [4,11]. Data are presented as mean ± S.E.M. *P < 0.05 denotes significant differences from vehicle controls.
While not a model of heart failure per se, the current findings from normal rats provide suggestive evidence that a key hormone known to be elevated in heart failure is capable of causing direct damage to skeletal myocytes and that this damage is mediated by the mineralocorticoid receptor. In light of the growing evidence in support of the concept that heart failure is a generalised, rather than cardiac specific, myopathy,
Fig. 3. Effect of mineralocorticoid receptor antagonism on aldosteroneinduced myocyte apoptosis. Myocyte apoptosis in the heart (A), soleus (B) and Tibialis Anterior muscle (C) after administration of 200 mg kg–1 spironolactone only, 1 mg kg–1 aldosterone only or spironolactone and aldosterone. Animals were killed 7 h after the administration of aldosterone and apoptosis was detected on muscle cryosections using an anti-caspase-3 Ab. Data are presented as mean ± S.E.M. (n = 8, in each group). *P < 0.05 denotes significant differences from administration of spironolactone only, †P < 0.05 denotes significant differences from administration of aldosterone only.
more studies should investigate skeletal muscle, as well as the heart, and pharmacological therapies be considered to prevent myocyte death in both types of striated muscle. Acknowledgements J.G.B. is a British Heart Foundation Junior Research Fellow (FS/04/028).
J.G. Burniston et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 395–399
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Goldspink DF, Burniston JG, Tan L-B. Cardiomyocyte death and the ageing and failing heart. Exp Physiol 2003;88:447–58. De Angelis N, Fiordaliso F, Latini R, Calvillo L, Funicello M, Gobbi M, et al. Appraisal of the role of angiotensin II and aldosterone in ventricular myocyte apoptosis in adult normotensive rat. J Mol Cell Cardiol 2002;34:1655–65. Rocha R, Stier CT, Kifor I, Ochoa-Maya MR, Rnnke HG, Williams GH, et al. Aldosterone: a mediator of myocardial necrosis and renal arteriopathy. Endocrinology 2000;141:3871–8. Goldspink DF, Burniston JG, Ellison GM, Clark WA, Tan LB. Catecholamine-induced apoptosis and necrosis in cardiac and skeletal myocytes of the rat in vivo: the same or separate death pathways? Exp Physiol 2004;89:407–16. Adams V, Jiang H, Yu J, Mobius-Winkler S, Fiehn E, Linke A, et al. Apoptosis in skeletal myocytes of patients with chronic heart failure is associated with exercise intolerance. JACC 1999;33:959–65. Vescovo G, Volterrani M, Zennaro R, Sandri M, Ceconi C, Lorusso R, et al. Apoptosis in the skeletal muscle of patients with heart failure: investigation of clinical and biochemical changes. Heart 2000;84: 431–7. Anker SD, Ponikowski P, Varney S, Chua TP, Clark AL, Webb-Peploe, et al. Wasting as an independent risk factor for mortality in chronic heart failure. Lancet 1997;349:1050–3.
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Dalla Libera L, Ravara B, Angelini A, Rossini K, Sandri M, Thiene G, et al. Beneficial effects on skeletal muscle of the angiotensin II type 1 receptor blocker irbesartan in experimental heart failure. Circulation 2001;103:2195–200. Mano A, Tatsumi T, Shiraishi J, Keira N, Nomura T, Takeda M, et al. Aldosterone directly induces myocyte apoptosis through calcineurindependent pathways. Circulation 2004;110:317–23. Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med 1999;341:709–17. Burniston JG, Tan L-B, Goldspink DF. b2-Adrenergic receptor stimulation in vivo induces apoptosis in the rat heart and soleus muscle. J Appl Physiol 2005;98:1379–86. Brilla CG, Matsubara LS, Weber KT. Anti-aldosterone treatment and the prevention of myocardial fibrosis in primary and secondary hyperaldosteronism. J Mol Cell Cardiol 1993;25:563–75. Anker SD, Chua TP, Ponikowski P, Harrington D, Swan JW, Kox WJ, et al. Hormonal changes and catabolic/anabolic imbalance in chronic heart failure and their importance for cardiac cachexia. Circulation 1997;96:526–34. Dalla Libera L, Sabbadini R, Renken C, Ravara B, Sandri M, Betto R, et al. Apoptosis in the skeletal muscle of rats with heart failure is associated with increased serum levels TNF-alpha and sphingosine. J Mol Cell Cardiol 2001;33:1871878.
Rapid communication
Aldosterone induces myocyte apoptosis in the heart and skeletal muscles of rats in vivo Jatin G. Burniston a,*, Amarjit Saini a, Lip-Bun Tan b, David F. Goldspink a a
Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Webster Street, Liverpool L3 2ET, UK b Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds General Infirmary, Leeds LS2 9JT, UK Received 17 December 2004; received in revised form 30 March 2005; accepted 5 April 2005 Available online 23 May 2005
Abstract Over activation of the renin–angiotensin–aldosterone system is known to be cardiotoxic but the potential injurious effects on the skeletal musculature have not been investigated. Male Wistar rats were given subcutaneous injections of aldosterone (1 µg–10 mg kg–1) and killed 7 h later, or continuous infusion (1 mg kg–1 d–1) and killed 48 h later. The role of the mineralocorticoid receptor in mediating aldosterone-induced apoptosis in vivo was investigated using spironolactone (200 mg kg–1). The number of apoptotic (caspase 3 positive) myocytes was counted on cryosections of the heart, soleus and Tibialis Anterior muscles. Injections of aldosterone induced significant (P < 0.05) cardiomyocyte apoptosis (peak = 2.46 ± 0.6 per 104 viable myocytes) over the range of 100 µg–10 mg kg–1, whereas only administration of 1 mg kg–1 induced significant (P < 0.05) apoptosis (2.47 ± 0.8 per 104 viable myocytes) in the soleus muscle. In contrast, no apoptosis was detected in the striated muscles after administration of only the vehicle. Infusion of aldosterone induced less apoptosis than the same dose (1 mg kg–1) given as a single injection. Prior administration of spironolactone significantly (P < 0.05) protected the heart (90%) and soleus muscle (79%) against the apoptosis induced by a single injection of 1 mg kg–1 aldosterone. These data confirm a myotoxic effect of aldosterone on the heart and provide the first description of aldosterone-induced myocyte apoptosis in skeletal muscle. High circulating levels of aldosterone are clearly capable of damaging all types of striated muscle and this may lend support to the concept that heart failure is a generalised, rather than cardiac-specific, myopathy. © 2005 Elsevier Ltd. All rights reserved. Keywords: Aldosterone; Spironolactone; Caspase 3; Myocyte death; Cardiac and skeletal muscle
1. Introduction Experimental models have demonstrated that catecholamines [1], angiotensin II [2] and aldosterone [2,3] are all capable of inducing cardiomyocyte death. Recent work from our laboratory has shown that the myotoxicity of catecholamines also extends to skeletal muscles, with exposure to excess catecholamines causing both myocyte apoptosis and necrosis [4]. The loss of myocytes from the dysfunctional heart propagates a vicious cycle of events, which results in a reduced cardiac reserve capacity [1]. Myocyte apoptosis also occurs in the skeletal muscles of chronic heart failure patients [5,6] and this is associated with muscle wasting, weakness and exercise intolerance [5]. Furthermore, the loss of skeletal * Corresponding author. E-mail address: [email protected] (J.G. Burniston). 0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2005.04.001
muscle mass is an independent risk factor for mortality in patients with chronic heart failure [7]. The clinical and laboratory-based studies described above support the concept that heart failure involves a generalised, rather than cardiac specific, myopathic process. There is good evidence to suggest that chronically elevated levels of catecholamines [4] and angiotensin II [8] are at least partly responsible for mediating this myopathy. In comparison, relatively little is known about the myotoxicity of aldosterone. The cardiotoxic effects of aldosterone have recently been described in normotensive [2] and hypertensive [3] rats, and also on isolated cardiomyocytes in vitro [2,9]. Furthermore, aldosterone blockade, using spironolactone, significantly reduces morbidity and mortality of patients with severe heart failure, over and above that of standard therapies [10]. Skeletal myocytes also possess mineralocorticoid receptors and
396
J.G. Burniston et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 395–399
the current study tests the hypothesis that aldosterone is toxic to skeletal, as well as cardiac, myocytes.
2. Methods All experimental procedures were carried out under the auspices of the British Home Office Animal (Scientific Procedures) Act 1986. Wistar rats (310 ± 10 g; mean ± S.D.) were purchased from a commercial supplier (Bantin and Kingman, Hull, UK) and allowed a period of 4 d prior to the experimental procedures. Animals were housed in control conditions of 20 °C, 45% relative humidity, and a 12 h light (06:00– 18:00) and dark cycle, with water and food (containing 18.5% protein) available ad libitum. The dose dependency (from 1 µg to 10 mg kg–1) of aldosterone-induced apoptosis was investigated using independent groups of animals (n = 5, in each group). Aldosterone (Sigma Chemicals, IL, USA) was freshly prepared in a 5% ethanol, 154-mmol NaCl vehicle immediately prior to administration. Animals received a single subcutaneous injection of hormone or the vehicle only and were killed 7 h later. To investigate whether the aldosterone-induced apoptosis was mediated through the mineralocorticoid receptor in vivo, independent groups of animals (n = 8, in each group) were administered either 1 mg kg–1 aldosterone (positive control), 200 mg kg–1 spironolactone (negative control) or spironolactone and aldosterone. Spironolactone treated animals were given two doses (200 mg kg–1, via gavage) with a 24 h interval between each. Three hours after the last administration of spironolactone, animals were challenged with either a single injection (s.c.) of 1 mg kg–1 aldosterone or the vehicle only and were killed 7 h later. The potential myotoxic effects of continuous infusions of aldosterone were also investigated. Animals were implanted, under isoflurane anaesthesia, with preconditioned osmotic pumps (Alzet™ 1003D) dispensing (s.c.) either 1 mg kg– –1 1 d aldosterone or the vehicle only (n = 5, in each group), and were killed 48 h later. At the end of each experiment, rats were concussed and killed by cervical dislocation and the heart, slow-twitch soleus and fast-twitch Tibialis Anterior muscles rapidly isolated. The atria and great vessels were removed from the heart and the ventricles mounted apex uppermost on a piece of cork. A standardised segment of the mid-belly from each skeletal muscle (soleus and Tibialis Anterior) was mounted in transverse section and supported with liver. Tissues were snap-frozen in super-cooled isopentane and stored at –80 °C, prior to cryosectioning (5 µm) and storage at –20 °C. Apoptosis was detected by an indirect immunoperoxidase protocol using an anti-caspase-3 antibody (Ab; R&D Systems, Minneapolis, USA) on cryosections in vitro. Previous work from our laboratory [4] and others [2] has shown that caspase-3 activity co-localises with dUTP nick-end labelling on cryosections in vitro, and with annexin V-biotin detection of phosphatidylserine externalisation in vivo [11].
To quantify apoptosis in cardiac and skeletal muscle, the number of positively stained myocytes was counted relative to the total number of viable myocytes per muscle crosssection; these typically being 11,362 ± 442 for the left ventricle, 2436 ± 147 for the soleus and 4250 ± 211 for the Tibialis Anterior (mean ± S.D.). For clarity and ease of comparison, all data have been normalised to 104 viable myocytes. All experiment data are presented as mean ± S.E.M. Experiments were analysed by using one-way analysis of variance with multiple post hoc analyses. P values of < 0.05 were taken to indicate statistical significance. 3. Results Administration of aldosterone (from 1 µg to 10 mg kg–1) to normotensive rats induced clearly discernable myocyte apoptosis in the heart (Fig. 1A) and skeletal muscle (Fig. 1B) in vivo. Administration of the vehicle only did not induce any cardiac (Fig. 1C) or skeletal (Fig. 1D) myocyte death. Because of this zero baseline in the control muscles, all myocyte death induced by aldosterone may be reasonably regarded as being of biological significance. Apoptosis in the heart (Fig. 2A) reached statistically significant (P < 0.05) levels after the administration of aldosterone over the range of 100 µg–10 mg kg–1. While the dosedependent profile of apoptosis was very similar in the soleus and Tibialis Anterior muscles, only the administration of 1 mg kg–1 induced statistically significant (P < 0.05) levels of skeletal myocyte death in the soleus (Fig. 2B). Prior administration of the mineralocorticoid receptor antagonist, spironolactone, significantly (P < 0.05) prevented the myocyte apoptosis induced by a single injection of aldosterone in the heart (90%) and soleus muscle (79%) and completely abolished it in the Tibialis Anterior (Fig. 3). Continuous infusion of aldosterone (1 mg kg–1 d–1) was generally less myotoxic than the same dose given as a single injection. Myocyte apoptosis was detected in the heart (0.9 ± 0.4 per 104 viable cardiomyocytes) and soleus muscles (1.9 ± 0.6 per 104 viable myocytes; P < 0.05) of those animals infused with aldosterone for 48 h. In contrast, no myocyte apoptosis was detected in any of the striated muscles (heart, soleus and Tibialis Anterior) after infusion of only the vehicle. 4. Discussion To the best of our knowledge, this is the first description of aldosterone inducing myocyte apoptosis in skeletal muscle. These data also provide the first confirmation of the initial findings of De Angelis et al. [2] who recently described the apoptotic effects of aldosterone in the hearts of normotensive rats in vivo. In the current work, the amount of cardiomyocyte apoptosis (2.5 ± 0.6 per 104 viable cardiomyocytes) was greatest in
J.G. Burniston et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 395–399
397
Fig. 1. Immunohistochemical detection of myocyte apoptosis in the heart and soleus muscle. Myocyte apoptosis was detected, using an anti-caspase 3 Ab, on cryosections (5 µm) of the heart (A) and soleus muscle (B) 7 h after the administration of 1 mg kg–1 aldosterone. No myocyte apoptosis was detected on cryosections of the control heart (C) or skeletal muscle (D) from animals that received only the saline vehicle. Brown (DAB) staining represents antibody binding (arrows), contrasted against a blue (haematoxylin) background. All images are × 400 magnification.
response to a single injection of 1 mg kg–1 aldosterone. When measured after infusion of this dose for 48 h, the incidence of cardiomyocyte apoptosis was 0.9 ± 0.4 per 104 viable cardiomyocytes. The dose used in the current work (1 mg kg– –1 –1 1 d ) is approximately twice that (75 µg h ) used in the earlier studies of Brilla et al. [12], who investigated the fibrinogenic effects of long-term (8-week) hyperaldosteronism. However, the current work compares closely with that of De Angelis et al. [2] who counted 285 ± 18 apoptotic cardiomyocytes per 106 cells after infusion of 1 mg kg–1 d–1 aldosterone for 24 h. The mechanisms that mediate aldosterone-induced apoptosis are yet to be elucidated fully. Studies conducted using cultured cardiomyocytes in vitro have suggested both mineralocorticoid-dependent [2] and independent [9] pathways may be involved. Prior intervention with spironolactone in vivo significantly (P < 0.05) prevented aldosteroneinduced apoptosis in both the heart and skeletal muscles (Fig. 3). Suggesting that the myocyte apoptosis detected in the current studies was mediated through the mineralocorticoid receptor. Therefore, some of the positive outcomes observed in heart failure patients that have received spironolactone [10] may be related to its ability to protect the striated muscles from aldosterone-induced myocyte death. The plasma concentration of aldosterone in heart failure patients with cachexia is elevated approximately threefold, being 1 nmol l–1 compared with 0.5 nmol l–1 for noncachectic patients and 0.3 nmol l–1 for age-matched individuals without heart failure [13]. Although not measured in the
work, De Angelis et al. [2] reported that infusion of 1 mg kg–1 aldosterone to rats elevated their plasma aldosterone approximately 10-fold from 0.7 to 8 ng ml –1 , equivalent to 24 nmol l–1. Suggesting that some of the doses used in the current work may have resulted in plasma concentrations of aldosterone greater than those found in heart failure patients. Nevertheless, this work reveals the potential for aldosterone to induce apoptosis in striated muscles in vivo. Future work should determine whether long-term infusion of a lower dose of aldosterone is similarly myotoxic. Myocyte apoptosis has been detected in skeletal muscle biopsy samples from heart failure patients [6] and patients with cachexia and skeletal muscle wasting have a poorer prognosis than non-cachexic patients [7]. However, it is not known which of the hormonal perturbations concomitant with heart failure is responsible for mediating the skeletal muscle apoptosis. That is, plasma concentrations of aldosterone, norepinephrine, epinephrine and tumour necrosis factor-a (TNFa) are all significantly elevated in heart failure patients with cachexia [13]. Previous work from our laboratory [1] has revealed that catecholamines can induced myocyte apoptosis and necrosis in skeletal muscle, and Dalla Libera et al. [14] have described the induction of skeletal myocyte apoptosis by TNFa. Therefore, it may be postulated that the elevated levels of these hormones (aldosterone, catecholamines and TNFa) act in synergy to contribute to the muscle wasting and cachexia associated with heart failure, by inducing myocyte apoptosis.
398
J.G. Burniston et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 395–399
Fig. 2. Dose dependent aldosterone-induced myocyte apoptosis. Myocyte apoptosis in the heart (A), soleus (B) and Tibialis Anterior muscle (C) in response to aldosterone. Independent groups (n = 5, in each group) of animals were administered either a single subcutaneous injection of aldosterone over the range 1 µg–10 mg kg–1 or the vehicle only and killed 7-h later. Apoptosis was detected on muscle cryosections by caspase-3 immunohistochemistry, this having been validated against other techniques [4,11]. Data are presented as mean ± S.E.M. *P < 0.05 denotes significant differences from vehicle controls.
While not a model of heart failure per se, the current findings from normal rats provide suggestive evidence that a key hormone known to be elevated in heart failure is capable of causing direct damage to skeletal myocytes and that this damage is mediated by the mineralocorticoid receptor. In light of the growing evidence in support of the concept that heart failure is a generalised, rather than cardiac specific, myopathy,
Fig. 3. Effect of mineralocorticoid receptor antagonism on aldosteroneinduced myocyte apoptosis. Myocyte apoptosis in the heart (A), soleus (B) and Tibialis Anterior muscle (C) after administration of 200 mg kg–1 spironolactone only, 1 mg kg–1 aldosterone only or spironolactone and aldosterone. Animals were killed 7 h after the administration of aldosterone and apoptosis was detected on muscle cryosections using an anti-caspase-3 Ab. Data are presented as mean ± S.E.M. (n = 8, in each group). *P < 0.05 denotes significant differences from administration of spironolactone only, †P < 0.05 denotes significant differences from administration of aldosterone only.
more studies should investigate skeletal muscle, as well as the heart, and pharmacological therapies be considered to prevent myocyte death in both types of striated muscle. Acknowledgements J.G.B. is a British Heart Foundation Junior Research Fellow (FS/04/028).
J.G. Burniston et al. / Journal of Molecular and Cellular Cardiology 39 (2005) 395–399
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