Characterisation of isoprenaline myotoxicity on slow-twitch skeletal versus cardiac muscle

International Journal of Cardiology 86 (2002) 299–309 www.elsevier.com / locate / ijcard

Characterisation of isoprenaline myotoxicity on slow-twitch skeletal versus cardiac muscle YeeLan Ng a , David F. Goldspink a , Jatin G. Burniston a , William A. Clark b , John Colyer c , d, Lip-Bun Tan * a

Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, 15 – 21 Webster Street, Liverpool L3 2 ET, UK b Michael Reese Hospital and Medical Center, 2929 S Ellis, Chicago, IL 60616 -3990, USA c Department of Biochemistry and Molecular Biology, The University of Leeds, Leeds LS2 9 JT, UK d Department of Medicine, The University of Leeds, Leeds LS2 9 JT, UK Received 7 December 2001; received in revised form 14 June 2002; accepted 16 July 2002

Abstract Background: Elevated catecholamines are known to be cardiotoxic, but their potential injurious effects on skeletal muscles are largely unknown. We have investigated whether isoprenaline induces in vivo myocyte necrosis in rat soleus muscle, and characterised the time-course, dose–response, spatial distribution and adrenoceptor involvement of its myotoxicity, in comparison with effects on cardiomyocytes in the same animals. Material and methods: Myocyte necrosis in response to subcutaneous isoprenaline was detected in vivo using a monoclonal anti-myosin antibody. Secondary immunoperoxidase staining (in vitro) facilitated the localisation of the damage and quantitative image analysis. Results: Using this sensitive technique we report a novel observation that isoprenaline induces significant myocyte necrosis (5–10%) in the soleus muscle. This toxic damage was initiated at lower doses of isoprenaline than in the myocardium (1 vs. 10 mg kg 21 s.c.), and peaked earlier (at 12 vs. 18 h post injection). Damage was distributed throughout the soleus muscle, whereas cardiomyocyte necrosis was most marked in left ventricular subendocardium where it was approximately 10 and three times greater than in the subepicardium and atria, respectively. Using selective adrenoceptor (AR) antagonism, we found that isoprenaline myotoxicity was mediated via b 2 -AR in the soleus and via b 1 -AR in the myocardium. Conclusion: The results show that the myopathic effects of isoprenaline are not confined to the heart. The involvement of skeletal muscle with different characteristics and mechanisms may have important implications in elucidating and treating the generalised myopathic processes seen in heart failure patients who have elevated levels of circulating catecholamines.  2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Catecholamines; Myocytes; Necrosis; Immunohistochemistry; Beta-adrenergic receptors; Beta-adrenergic antagonists

1. Introduction Activation of the sympathetic system and elevated circulating catecholamines are important parts of the ‘fight and flight’ survival responses. Normally these changes are short-lived, with transient increases *Corresponding author. Present address: G Floor, Martin Wing, Leeds General Infirmary, Great George St., Leeds LS1 3EX, UK. Tel.: 144113-392-5401; fax: 144-113-392-5395.

returning to baseline levels once the stress stimulus has been withdrawn. However, levels greatly in excess of these physiological responses are encountered in medical practice, such as pathophysiological levels seen in phaeochromocytoma and heart failure, and pharmacological levels in resuscitative and intensive care practice. The cardiotoxic effects of elevated levels of catecholamines have been known for many decades [1–3]. Little is known about whether these

0167-5273 / 02 / $ – see front matter  2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 02 )00369-8

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hormones have any damaging effects on other striated muscles. Detailed literature searches have failed to reveal any reports describing myocyte necrosis in skeletal muscles after exposure to excess catecholamines. Such information is of relevance in view of conditions with sympathetic over-activation, such as in heart failure, where peripheral muscle weakness [4] and the cachexia associated with a pronounced loss of muscle bulk and power in severe heart failure [5,6] have been reported to be associated with elevated serum adrenaline levels [7]. We have therefore tested the hypothesis that isoprenaline induces myocyte injury in skeletal muscle, and compared its effects on the soleus muscle to those observed in the heart. To complement standard histopathological methods, we have also employed monoclonal antimyosin antibody labelling of necrotic myocytes with disruption of cell membranes. Having found that isoprenaline does induce necrosis in both types of striated muscles, we proceeded to characterise its dose–responses, time courses and spatial distribution. We also explored which b-adrenergic receptor (AR) subtype(s) are involved in mediating these injurious effects of isoprenaline.

2. Methods The experimental model employed male, Wistar rats weighing 30162.4 g which were housed under controlled conditions of 25 8C, 50% relative humidity and a 12-h light (06:00–18:00 h) and 12-h dark cycle, with water and food (containing 18.5% protein) available ad libitum. All experimental procedures were carried out in accordance with the Home Office of Great Britain and Northern Ireland Animal (Scientific Procedures) Act 1986. The monoclonal antimyosin antibody used in this study to label necrotic myocytes has been derived as previously reported and shown to bind myosin protein specifically [8–10]. The technique employed in the labelling of in vivo myocyte necrosis has also been reported in previous publications [2,11–15]. This antibody has been shown not to gain access in vivo into intact normal myocytes, but is admitted through the disrupted sarcolemmal membranes of necrotic myocytes [2,11–15]. The rationale of employing the monoclonal antibody is that when the

plasma membrane disintegrates, it marks the irreversible stage of myocyte cell death [16]. The disrupted membrane releases macromolecules (e.g. CK, troponins) and admits the antibodies that bind avidly to the intracellular myosin, which can then be labelled immunochemically and identified [2]. That the monoclonal antibody specifically binds the myosin proteins in cardiac (Fig. 1a) and skeletal muscle (Fig. 1c) in vitro is further demonstrated in Fig. 1, while non-muscle tissues such as the kidney (Fig. 1e) and liver (not shown) failed to react when exposed to the anti-myosin antibody. Freshly made isoprenaline was injected subcutaneously in doses ranging from 1 mg kg 21 to 5.0 mg kg 21 for the dose–response studies. All animals received an intraperitoneal injection of 1 mg of antimyosin antibody kg 21 1 h before the subcutaneous administration of either isoprenaline (i.e. experimental group) or the saline vehicle (i.e. control group). For the time course study, rats were sacrificed by cervical dislocation at different intervals from 0 to 72 h following administration of isoprenaline. After sacrifice, the heart and soleus muscles were rapidly isolated. Small pieces of the soleus muscle were embedded in OCT (optimal cutting temperature) compound and snap frozen in super cooled isopentane. The atria were separated from the ventricles at the coronary sulcus, and embedded and frozen as above. The ventricles were mounted whole in OCT compound, frozen and subsequently sectioned transversely from the apex to the base (Fig. 2h). These provided information on the spatial distribution of cardiomyocyte damage in both ventricles. Transverse cryosections (5 mm thick) of all the above muscles were cut and stored at 220 8C. After development with a secondary anti-mouse horse radish peroxidase-conjugated antibody and diaminobenzidine, the damaged myocytes with the antibody bound to their sarcomeric myosin stained brown (as shown in Figs. 2d,f and 3d). Both positive and negative controls were run alongside sections from the experimental muscles. This method is very sensitive and can detect necrosis in single myocytes (e.g. Fig. 2f) in a way that would not be possible using a standard histological stains (e.g. H&E staining; Fig. 2e). Although this secondary procedure caused some swelling of the extracellular space (e.g. Fig. 3b,d), this artifact occurred in control and

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Fig. 1. The anti-myosin monoclonal antibody employed is specific for myocytes. Cryosections (5 mm) of the myocardium (A), soleus muscle (C) and kidney (E) were exposed in vitro to 5 mg ml 21 anti-myosin antibody, followed by the secondary anti-mouse, horse radish peroxidase-conjugated antibody and diaminobenzidine. The myocytes in both striated muscles (A–C) stained brown. Despite identical treatment there was no staining in the kidney (E). Exposure to a denatured form of the anti-myosin antibody served as negative controls, and identically treated myocardium (B), soleus muscle (D) and kidney (F) failed to react.

experimental tissues alike and therefore did not influence the overall assessment of myocyte damage. All sections were analysed blindly with respect to the investigator’s knowledge of the origin of the tissue specimens. The proportion of damaged myocytes in the skeletal muscle was calculated by counting a minimum of

500 myocytes in random fields from cross-sections of each soleus. Cardiomyocytes are much smaller and run in different planes throughout the ventricles. A different quantitative approach, combining light microscopy and image analysis, was therefore required for the heart. Within each field of view the area stained brown (i.e. labelled necrotic myocytes) was

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Fig. 2. Isoprenaline-induced damage in the subendocardium (c and d) and subepicardium (e and f) of the left ventricle. Twelve hours after administering 5 mg of isoprenaline kg 21 subcutaneously, 5-mm transverse sections (diagram g), including randomly selected subepicardial (crosses) and subendocardial (shaded) measurement sites, were cut from the apex to the base of the ventricles (diagram h). Serial sections were stained using either Haematoxylin and Eosin to show no damage in control tissue (a) or generalised tissue damage (i.e. c and e), or horse radish peroxidase conjugation (stained brown) to visualise the anti-myosin antibody located in necrotic myocytes (d and f). The anti-myosin in vivo is excluded from normal (control) cardiomyocytes (b), but enters damaged ones (d and f) through their disrupted sarcolemmal membranes [16]. Large areas of focal myocyte damage (identified by the arrows) were found in the subendocardium (c and d), compared with only isolated damage (see arrow) in the subepicardium (e and f) of the left ventricle. Scale bar530 mm.

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Fig. 3. Isoprenaline-induced damage of myocytes in the soleus muscle. Individual myocyte necrosis (stained brown through antimyosin antibody labelling and identified by arrows in d) and associated inflammatory cell infiltrates (c) were detected in serial sections of the soleus taken from the same rat as the heart in Fig. 2. (a) and (b) are control tissues from rats receiving the anti-myosin, but the saline vehicle instead of isoprenaline. Scale bar530 mm.

measured against the paler background of the normal myocardium. At least eight random fields from either the subepicardium (e.g. marked X in Fig. 2g) or subendocardium (i.e. shaded region, Fig. 2g) were analysed. The resultant data were averaged accordingly and expressed as a percent of the area of the whole field. In all muscles, serial sections were also cut and stained using H&E to study generalised tissue damage (Figs. 2c,e and 3c) alongside our myocyte-specific labelling method. Regions of inflammation, with accompanying infiltration of phagocytic cells, were apparent in areas of focal necrosis (e.g. Figs. 2c and 3c). In experiments designed to identify the adrenoceptors (AR) through which isoprenaline induces myocyte damage, specific AR-antagonists were ad-

ministered twice, 1 h before and 6 h after, the isoprenaline injection in an attempt to ensure complete inhibition of the receptors. By using a nonselective antagonist (e.g. propranolol, against b 1 - and b 2 -ARs, at 10 mg kg 21 ), a b 1 -AR selective antagonist (e.g. bisoprolol at 25 mg kg 21 ) or b 2 -AR selective antagonists (e.g. ICI-118551 at 25 mg kg 21 ), the involvement of specific b-AR subtype(s) was examined by a process of elimination. For completeness, a non-selective a-AR antagonist (e.g. phenoxybenzamine at 10 mg kg 21 ) was also used to rule out any involvement of a-AR in isoprenalineinduced damage. These animals were then sacrificed 12 h after administering the isoprenaline. Data are presented as means6S.E.M. and statistically significant differences determined using ANOVA with Tukey–Kramer post-test.

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3. Results Using our antimyosin antibody technique that enables us to detect sensitively and specifically myocyte cell death in vivo, we observed significant injury in the rat soleus muscle (Fig. 3d), as well as the heart (Fig. 2d,f), following acute exposure to isoprenaline. We proceeded to compare the dose– responses of these two muscles to the injurious effects of isoprenaline (Fig. 4), and defined the onset, time course (Fig. 5) and topographical distribution (Fig. 2g,h) of the necrosis in each muscle after an injection of isoprenaline. Interesting differences emerged between the responses of the myocytes in the skeletal and cardiac muscles. Myocyte necrosis in the soleus muscle was detected after administering 1 mg of subcutaneous isoprenaline per kg of body weight (Fig. 4a). Greater amounts of damage were observed at higher doses of the catecholamine, with necrosis reaching highest levels at between 0.1 and 5 mg kg 21 of isoprenaline (approximately 5–10% of the fibres; Fig. 4a). The earliest detection of damage was at 6 h after the isoprenaline injection, while maximal damage appeared around 12 h post injection (Fig. 5a). In contrast to these observations in the skeletal muscle, the minimum dose of isoprenaline required to induce necrosis in the cardiomyocytes of the subepicardium (Fig. 4b) and subendocardium (Fig. 4c) was an order of magnitude greater at 10 mg kg 21 . Thereafter, the amount of cardiomyocyte injury rose with increasing doses of isoprenaline reaching a peak at 5 mg kg 21 (Fig. 4b,c). Peak damage in both regions of the left ventricle was detected after 18 h (Fig. 5b,c), which was some 6 h later than in the soleus (Fig. 5a). Differences in the topographical distribution of myocyte necrosis were also apparent. Myocyte damage in the soleus was fairly uniformly distributed throughout the muscle, but considerable variations were found within the heart. More damage was consistently found in the left ventricle than in the right, and in the subendocardium (Figs. 2d and 4c) than in the subepicardium (Figs. 2f and 4b) within any transverse plane (Fig. 2g). The amount of myocyte necrosis also varied along the longitudinal axis, rising from low levels at the base to a peak occurring approximately one-third of the way from the apex of the ventricle (Fig. 2h).

Fig. 4. Dose responses of myocyte damage to isoprenaline challenge. Myocyte damage was measured in (a) the soleus, (b) the subepicardial and (c) the subendocardial regions of the left ventricle 18 h after giving a single injection of isoprenaline; the dose ranging from 0.001 to 5.0 mg kg 21 . Data are means6S.E.M. for a minimum of five rats per group. All isoprenaline-induced myocyte necrosis occurred against a background of zero damage in the heart and soleus muscles of control rats. To establish optimal doses, an ANOVA with Tukey–Kramer post-test was used to determine which, if any, values of necrosis were significantly different (*P,0.05; **P,0.01) from those induced by the largest dose of isoprenaline, i.e. 5 mg kg 21 .

As an initial step in elucidating the mechanisms underlying this new observation of skeletal myocyte damage, various adrenoceptor antagonists were used. This approach enabled us to establish the subtype(s) of ARs mediating the isoprenaline injuries, and whether this was the same for both types of striated

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Fig. 5. The onset and time course of the isoprenaline-induced myocyte damage. The amount of myocyte necrosis was measured in (a) the soleus, (b) subepicardium, and (c) subendocardium at different times after administering a single injection of 5 mg of isoprenaline kg 21 body weight. Data are means6S.E.M. (n5a minimum of three muscles) of the myocyte necrosis induced at each time point, against a background of nil necrosis in control (i.e. zero time) muscles. To establish the peaks of myocyte necrosis, an ANOVA with Tukey–Kramer post-test was employed to compare statistically significant differences (*P,0.05, **P, 0.01) between the maximum recorded values (i.e. at 12 h in the soleus and 18 h in the myocardium) and all other time points.

muscle. Inhibition of a-ARs by 10 mg kg 21 of a general antagonist (i.e. phenoxybenzamine; Fig. 6) showed no reduction of the isoprenaline-induced necrosis in either striated muscle. When the nonselective b-AR antagonist, propranolol (against b 1 and b 2 -ARs) was administered, the isoprenaline-in-

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Fig. 6. The effects of general a- and b-AR blockade on isoprenalineinduced damage in the soleus and cardiac muscles. The effectiveness of phenoxybenzamine (a 1 - and a 2 -), propranolol (b 1 - and b 2 -selective), or ICI 118551 (b 2 -selective) AR antagonists in preventing myocyte damage was investigated in (a) the soleus, (b) subepicardium and (c) subendocardium of the LV wall. Each AR antagonist was injected twice subcutaneously at the above doses 1 h before, and again 6 h after, the 5 mg of isoprenaline kg 21 . All rats were killed 12 h after the isoprenaline. Results are presented as means6S.E.M., with n53–9 muscles. An ANOVA with Tukey–Kramer post-test was employed (*P,0.05; **P,0.01) to determine the level of protection that each AR-antagonist provided relative to the isoprenaline damaged control (j).

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Fig. 7. Isoprenaline-induced damage to myocytes in the atria, in the presence and absence of b-AR antagonists. The right and left atria (subsequently pooled) were found to be equally damaged 12 h after exposure to a single injection of 5 mg of isoprenaline kg 21 . Significant (**P,0.01) protection against this myocyte necrosis was provided by b 1 - (bisoprolol), but not b 2 - (ICI-118551), AR antagonists.

duced damage was markedly reduced (Fig. 6). By a process of elimination using more selective b-AR antagonists, we were able to determine which subtypes of b-ARs were involved in mediating the isoprenaline-induced necrosis (Fig. 6). At a dose of 25 mg kg 21 , the b 2 -AR antagonist, ICI-118551, was effective in protecting the myocytes of the soleus, but not the cardiomyocytes (Fig. 6) from isoprenaline injury. When a selective b 1 -AR antagonist such as bisoprolol was used the reverse was true, as it was effective in preventing myocyte necrosis (Fig. 6) in the rat heart but not in the skeletal muscle. The myocytes of both atria were also found to be damaged by isoprenaline. As in the subepicardium this damage was diffuse in nature, but at 0.9460.4% was intermediate between the damage observed in the subepicardium and the focal damage found in the subendocardium (Fig. 7). Like the ventricular myocytes, the atria were protected by the b 1 -AR specific inhibitor bisoprolol (91%; P,0.001) but not by b 2 -AR blockade using ICI-118551. 4. Discussion As far as we know, this is the first time that

myocyte necrosis has been reported in skeletal muscle after acute exposure to isoprenaline in vivo. By carefully defining (in the same animals) the doses of isoprenaline that are required to initiate and cause maximal necrosis in both striated muscles, we found that skeletal myocytes are even more sensitive than cardiac myocytes to the injurious effects of this catecholamine (Figs. 4 and 5). We have previously shown that elevated levels of catecholamines are cardiotoxic [2], consistent with observations by others [1,3]. Our earlier work [2] has been extended here by defining the threshold dose of isoprenaline that is required to initiate cardiomyocyte necrosis (Fig. 4b,c), and by establishing more precisely the timing of peak myocardial injury (Fig. 5b,c) and its heterogeneous distribution throughout the atria and ventricles (Fig. 2g,h). This observation implies that whenever attempts are made to quantify myocardial damage, either in response to isoprenaline or other potentially toxic agents, such temporal and spatial distribution must be taken into account. We have also found that the myocyte necrosis in these two types of striated muscle is initiated through different subtypes of b-ARs: predominantly mediated by b 1 -ARs in the heart (Figs. 6 and 7) and by b 2 -ARs in the soleus (Fig. 6). These observations are

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of interest in the light of recent reports [17–19] indicating that the over-expression of b 1 -ARs, but not b 2 -ARs, in transgenic mice leads to heart failure. Despite the presence of pre-synaptic b-ARs that can enhance the release of noradrenaline, the extents of myocyte necrosis in the cardiac or skeletal muscle after blockade of a-ARs were similar to after isoprenaline alone, suggesting that a-ARs were not significantly involved. At this stage, we cannot explain why skeletal myocytes appear to be more responsive to the injurious effects of isoprenaline than the cardiomyocytes. This synthetic hormone is a more vasodilating analogue of adrenaline and both of these hormones demonstrate similar affinities for b 1 - and b 2 -ARs. The lower threshold for injury in the soleus does not appear to correlate with adrenoceptor availability as the overall density of b-ARs (i.e. b 1 1b 2 ) is approximately seven- to eightfold greater in the heart than in the soleus [20]. Further studies are required to elucidate the mechanisms involved. In heart failure both the sympathetic and renin– angiotensin–aldosterone systems are over-activated, resulting in sustained elevated levels of adrenaline, noradrenaline, angiotensin II and aldosterone [7]. Repeated injections of catecholamines in rats over several days have been shown to result in cardiac hypertrophy, with changes in cardiomyocyte size, electrophysiology and a prolongation of the cardiac action potential [21]. However, similar doses of isoprenaline have also been reported to cause irreversible myocyte necrosis and reactive fibrosis [2,3]. In clinical practice, raised circulating cardiac troponins are usually taken as biochemical markers of cardiomyocyte necrosis. The detection of elevated serum troponin levels in heart failure in the absence of any myocardial infarction implies that there may be a cumulative, on-going cardiomyocyte injury, with the extent of this damage relating to the severity of the heart failure [22–25]. The over-activated sympathetic system in heart failure may be causatively linked to enhanced levels of myocyte attrition. Comparison of the pharmacological doses of isoprenaline used in this animal model with levels of adrenoceptor stimulation seen clinically is often fraught with difficulties, but it may suffice to consider that the circulating levels of experimental isoprenaline are probably relatively dilute compared to the enhanced

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local post-synaptic concentrations of noradrenaline at sympathetic nerve endings stimulating the adrenoceptors of cardiomyocytes in heart failure. Cachexia, with a loss of muscle bulk, is also prevalent and prognostically significant in congestive heart failure [5,26]. Despite this, we currently have limited information about the factors and mechanisms that initiate and worsen skeletal myopathy. The observation that both skeletal and cardiac myocytes are severely damaged by isoprenaline (which mimicks the actions of adrenaline) provides a possible aetiological role for catecholamines in causing a generalised, rather than a cardiac specific, myopathy in heart failure. Opasich et al. [4] and others [27] have recently suggested that heart failure is a multiorgan phenomenon, with impairment of skeletal and respiratory muscles [28], as well as the heart. As skeletal muscle appears to possess a lower threshold for injury (Fig. 4), myocyte necrosis could become evident as a progressive loss of muscle bulk and power as the circulating levels of adrenaline rise (fivefold) with increasing severity of cachexic heart failure [4,7]. This would be consistent with both reported instances of peripheral weakness in mild heart failure [6] and dramatic muscle wasting in chronic heart failure [7,26,29]. Preventing such losses of muscle mass is important because the skeletal musculature plays a vital metabolic role in the body and its loss could significantly increase the risk of death [5,26]. However, to date, therapies are largely directed at the heart, and may need to be broadened to include treatment of generalised myopathy. Whether supplementary blockade of b 2 -ARs confers additional benefits over purely selective b 1 -AR inhibition has yet to be established. One supportive evidence is provided by recent data from the COPERNICUS trial which have shown that carvedilol, a non-selective b-blocker, was effective in ameliorating or reversing the progression of cachexia in patients with severe heart failure [30]. Future studies are warranted in view of the biological and clinical significance of these results. The fact that a neuro-endocrine system that is vital for survival of the organism can become injurious to the heart and skeletal musculature when excessively activated is consistent with the biological paradox of too much hormonal activation usually leads to harm (e.g. thyrotoxicosis). Clinically, recognised conditions

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fulfilling these criteria include phaeochromocytoma and heart failure. In both, adrenoceptor inhibition is of proven benefit [31–34]. Important future research will include, further investigations into the relative toxicity of various catecholamines, the mechanisms that are responsible for mediating the injurious processes downstream from the adrenoceptors, the parallel role (if any) of apoptosis in contributing to total myocyte death, and other agents capable of inhibiting these injurious processes for patients who cannot tolerate adrenoceptor blockade.

Acknowledgements We are grateful to the National Heart Research Fund and the British Heart Foundation (BHF) for their financial support. JGB is a PhD student (FS / 2000078), YN a Junior Research Fellow (FS / 98065), JC and LBT Senior Lecturers of the British Heart Foundation.

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