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1 The Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, 15–21 Webster Street, Liverpool L3 2ET, UK2 Michael Reese Hospital and Medical Center, 2929 S Ellis, Chicago, IL 60616-3990, USA3 Academic Unit of Molecular and Vascular Medicine, Martin Wing, Leeds General Infirmary, Leeds LS2 9JT, UK
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Abstract |
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(Received 18 February 2004;
accepted after revision 20 April 2004; first published online 6 May 2004)
Corresponding author D. F Goldspink: The Research Institute for Sport & Exercise Sciences, Liverpool John Moores University, 15–21 Webster Street, Liverpool L3 2ET, UK. Email: D.Goldspink{at}livjm.ac.uk
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Introduction |
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In recent years much greater emphasis has been placed on studying apoptosis, compared with other forms of cell death. Consequently, very few studies have attempted simultaneously to quantify the relative amounts of cellular apoptosis and necrosis in any tissue in vivo (Kajstura et al. 1996, 1998; Blom et al. 1999; Dumount et al. 2000; Sun et al. 2001). Even where attempted, in most cases insufficient preliminary studies were undertaken to establish the optimal conditions, with respect to the dose and temporal–spatial distributions of the injury, to ensure accurate and meaningful quantification of, and hence comparison between, different forms of cell death.
To address such issues we employed the isoprenaline challenge model that we, and others, have previously used to induce necrosis in cardiomyocytes of the heart (Benjamin et al. 1989; Teerlink et al. 1994; Ng et al. 2002; Tan et al. 2003). After optimizing the experimental conditions, myocyte-specific necrosis and apoptosis were detected and quantified in both the heart and a skeletal muscle using sensitive techniques of immunohistochemistry and image analysis. This approach has enabled us to question whether one form of cell death in vivo predominates over the other and whether apoptosis and necrosis in vivo represent two distinctive pathways or a continuum of events.
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Materials and methods |
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Myocyte death was induced in both cardiac and skeletal muscles by a single subcutaneous injection of isoprenaline. In dose–response studies this ranged from 0.01 µg to 5 mg (kg BW)–1. Myocyte-specific necrosis was detected by administering (via intraperitoneal injection) 1 mg of an anti-myosin monoclonal antibody (Ab) to each rat 1 h before either the isoprenaline (i.e. experimental groups) or saline vehicle (i.e. negative control group), as previously described (Clark et al. 1980; Kajstura et al. 1996, 1998). This Ab interacts with the ß-myosin found in all striated muscles (Clark et al. 1980; Ng et al. 2002). The Ab is too large to traverse the intact membranes of viable myocytes, but readily enters necrotic myocytes through their disrupted sarcolemmal membranes (Goldspink et al. 2003), enabling us to exploit this end-stage of cellular necrosis (Ng et al. 2002; Tan et al. 2003).
Rats were killed by cervical dislocation at different time intervals (0–24 h) after administration of the isoprenaline. Hearts were rapidly isolated and both atria removed. The intact ventricles were mounted whole, snap frozen in super cooled isopentane and transverse cryosections (5 µm) taken at 200 µm intervals along the longitudinal axis, from the apex to the base. Soleus muscles (predominantly slow-oxidative) were also isolated and samples taken from the mid-belly of each muscle. These were supported by blocks of liver and embedded in Optimal Cutting Temperature compound before being snap frozen in isopentane. Transverse and longitudinal cryosections (5 µm) were then cut.
In both striated muscles, necrotic myocytes, with the Ab bound to their myosin, stained brown after sections were developed with a secondary, horseradish-peroxidase-conjugated Ab and 3–3'diaminobenzedine (DAB) (Fig. 1C and F). In serial sections apoptotic cells stained brick red (Fig. 1B and E) when exposed to an Ab directed against the 17 kDa subunit of caspase-3 (R & D Systems, Minneapolis, USA) and Nova Red (Vector Laboratories, Burlingame, CA, USA). Using a combination of light microscopy and image analysis a minimum of 500 fibres in each soleus was counted and the proportions of apoptotic and necrotic myocytes determined. In the heart, cardiomyocyte damage was quantified in six randomly chosen fields of view of either the left or the right ventricular subendocardia or subepicardia. Areas staining either brown (i.e. necrotic myocytes) or red (apoptotic myocytes) were measured against the haematoxylin-counterstained background of normal cardiomyocytes. The values obtained in each of these areas were averaged and expressed as the percentage area of either apoptosis or necrosis. All sections were analysed blindly.
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To verify the apoptosis labelling using the caspase-3 Ab, a subset of animals (n= 3 in each group) were administered annexin V-biotin (Nexins Research, Netherlands) to detect the externalization of phosphatidylserine (PS) in vivo. Annexin V-biotin [25 mg (kg BW)–1] was administered intravenously 3.25 h after the administration of 5 mg (kg BW)–1 isoprenaline (experimental groups) or saline vehicle only (control group). Muscles were harvested at the optimized time point (3 h post-ß-agonist administration) and processed using standard immunohistochemical techniques, as previously described (Dumount et al. 2000). Annexin V-biotin is cleared through the kidney. Hence, renal tissue was used as the positive control (data not shown) to demonstrate both positive staining and to verify that the annexin V-biotin had reached, and passed through, the circulation. Tissues from the saline control animals were stained alongside experimental tissues to control for any post mortem changes and clearly demonstrated that apoptosis occurred in response to the experimental intervention.
Formamide-induced DNA denaturation detected using an anti-single strand DNA (ssDNA) Ab was used to confirm the nuclear changes concomitant with apoptosis.
Cryosections of muscles taken from control and experimental animals were heated in formamide as described by Frankfurt & Krishan (2001). The denatured DNA of apoptotic cells was then detected using an anti-ssDNA Ab (Chemicon International, Temecula, CA, USA) according to the manufacturer's instructions. Cryosections of mammary gland and small intestine were included as immunohistochemical controls alongside the experimental tissues; as a negative immunohistochemical control these sections were not heated in formamide.
Strand-breaks in the DNA of apoptotic cells were detected using a commercially available TUNEL kit (R & D Systems) according to the instructions provided. Positive control sections were exposed to DNase I and on separate sections the TdT enzyme was omitted to provide an immunohistochemical negative control.
With the exception of the topographical investigation, apoptosis in the heart was only quantified in the subendocardial region of the LV. For each section, six to eight fields of view (100 x magnification), encompassing the entire subendocardial region, were digitized. Positive staining (apoptosis) was differentiated from the haematoxylin background, quantified using image analysis and the incidence of apoptosis expressed as the percentage area relative to the field of view. The coefficient of variation using this technique was 4.7%.
To quantify apoptosis in the soleus, three random fields of view (100 x magnification) across each transverse section were digitized. Both apoptotic and viable myocytes were counted (> 700), and the number of apoptotic fibres expressed as a percentage of the total. The coefficient of variation for this technique was 3.5%.
Double immunofluorescence was used to investigate the co-localization of apoptosis and necrosis within the same myocytes. Primary detection of the anti-myosin (necrosis) and anti-caspase-3 (apoptosis) Ab was visualized with a Fluorescein-conjugated horse anti-mouse Ab (necrosis) or a Texas-Red-conjugated goat anti-rabbit Ab (apoptosis) and viewed as either separate or combined images on the image analyser.
To identify the adrenergic receptor pathway mediating the isoprenaline-induced myocyte necrosis and apoptosis, selective ß1- (bisoprolol) or ß2- (ICI-118551) adrenoceptor (AR) antagonists were injected at 25 and 10 mg (kg BW)–1, respectively, 1 h before the isoprenaline challenge. We have previously shown that these doses provide selective and near complete inhibition of these ß-AR subtypes (Tan et al. 2003).
Statistical analyses were carried out using one-way ANOVA, with multiple post hoc tests. P < 0.05 was considered to be significant.
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Results |
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By contrast, a single injection of isoprenaline [5 mg (kg BW)–1]in vivo induced significant (P < 0.05) apoptosis (Fig. 1B and E) and necrosis (Fig. 1C and F) in both striated muscles. However, skeletal myocyte death by either mode was induced by a much lower dose (about 100-fold lower; Fig. 2A and B). The extent of injury was dose dependent, with both apoptosis and necrosis occurring with a dose of 0.01 mg (kg BW)–1 in cardiac myocytes and with an even lower dose of 0.001 mg (kg BW)–1 in skeletal myocytes. Peak values were reached in response to a dose of isoprenaline of 5 mg (kg BW)–1. This optimal dose was employed in all subsequent studies.
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Quantification was more straightforward in the soleus muscle. Damaged myocytes (n= 350) in randomly selected fields of view from ten different muscles were examined 12 h after isoprenaline injection. All 350 fibres stained positively for the presence of the monoclonal anti-myosin Ab (Fig. 6D), whereas 249 (71%) were labelled as caspase-3 positive (Fig. 6B). On closer examination, we confirmed that all of the caspase-3-positive fibres were co-labelled as necrotic (Fig. 6F), leaving just 101 fibres (29%) staining as necrotic only with no detectable caspase-3-positive labelling. The probability that this observation was due to chance was P < 0.001, using a chi-squared association test (1, n= 350) = 62.58.
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Discussion |
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The lysis of apoptotic cells in vivo will undoubtedly instigate an inflammatory response that may result in the demise of adjacent cells that were not directly damaged by the original insult. In the current work, the very close similarities in dose–responses (Fig. 2), spatial distributions (Fig. 4) and receptor mediation (ß1-AR in the myocardium or ß2-AR in the soleus; Fig. 5) for myocyte apoptosis and necrosis suggest that either the two processes originate from common initial pathways or that they are one and the same process, i.e. a continuum of events. The earlier time course of cardiomyocyte apoptosis peaking at 3–6 h relative to necrosis peaking at 18 h (Fig. 3) and the greater number of necrotic compared with apoptotic myocytes (ranging from 3.5/1 to 10/1 in the heart, and 1.4/1 in the soleus) both suggest that some of the necrosis measured 18 h after isoprenaline administration may, in fact, be secondary to the apoptosis measured at the earlier time points, i.e. secondary necrosis. Confirmation of this is provided by the observation of caspase-3-positive myocytes with disrupted sarcolemmal membranes 12 h after the administration of isoprenaline (Fig. 6E and F). Intuition suggests that the greater size, particularly of skeletal myocytes, would impede their phagocytosis by tissue macrophages and so these cells would be more likely to progress to secondary necrosis. Indeed, a greater proportion (70%) of skeletal myocytes were co-labelled than the smaller myocytes of the myocardium (
30%). Despite this, the rate of removal of necrotic skeletal myocytes (Ng et al. 2002) appears to be similar, both to the smaller cardiomyocytes (Fig. 3) and to even smaller hepatocytes (Blom et al. 1999; Sun et al. 2001). If secondary necrosis does indeed occur in vivo, it would appear to do so before the formation of classical apoptotic bodies (Goldspink et al. 2003) as no such structures were seen in our studies. This further highlights the importance of recognizing differences in observations derived from studies in vitro and in vivo.
An alternative explanation to this ‘continuum of events’ hypothesis is provided by the growing body of research on the role of the mitochondria in cell death. This has provided some fascinating new insights into the relationship between apoptosis and necrosis. Apoptosis and necrosis have been shown to coexist in the heart following experimentally induced ischaemic damage (Kajstura et al. 1996) and myocardial infarctions (James, 1998). Permeablization of the outer membrane of mitochondria is a common feature of both apoptosis and necrosis (Lemasters et al. 1999, 2002). This process of simultaneous induction of apoptotic and necrotic death has been termed ‘necrapoptosis’ (Lemasters, 1999). After permeablization of the outer mitochondrial membrane, the subsequent phenotype of cell death (i.e. apoptotic or necrotic) may be determined by the availability of ATP (Eguchi et al. 1997; Leist & Jaattela, 2001). That is, cells depleted of ATP undergo necrosis rather than apoptosis. This dual induction of both cell death phenotypes following a common insult could equally well explain the current findings.
Regardless of the exact intracellular events leading to the observed myocyte death, the realisation that different cell death phenotypes can co-exist within the same cell or tissue in response to a single stimulus questions the validity of the findings of many publications that have focused only on one cell death pathway. Clearly, the data presented here support the proposal that both apoptosis and necrosis should be investigated simultaneously, with the distinctiveness of the different death pathways as yet unresolved. Indeed, the fact that we have not investigated the incidence of autophagic cell death represents a limitation to the current study, which we intend to rectify in the near future. The current findings also demonstrate that in order to attempt to evaluate the relative importance of the different cell death pathways, preliminary investigations of the dose dependency and time course of cell death are required.
Elevated catecholamines have long been known to induce necrotic damage in the heart (Benjamin et al. 1989; Teerlink et al. 1994; Ng et al. 2002; Tan et al. 2003). Recently, we added to knowledge in this area by showing that the slow-twitch soleus muscle can be similarly damaged by the synthetic catecholamine, isoprenaline (Ng et al. 2002). The current work reveals that the incidence of necrotic myocyte death in response to isoprenaline is three to ten times greater than that of apoptotic death, depending on the time point at which myocyte death is quantified. Clearly, not all myocytes responded equally to isoprenaline challenge, as myocyte death in the soleus was induced by a much lower dose than that required to induce damage in the heart (Fig. 2). In both studies the fast-twitch tibialis anterior muscle was not damaged by isoprenaline administration; this may be explained by the development-dependent disappearance of the caspase-3 protein in this muscle (Ruest et al. 2002).
Our data are supported by studies (Mann et al. 1992) on cardiomyocytes in vitro that have elegantly demonstrated that catecholamine-induced death is mediated by the ß1-AR and preceded by an elevation in cyclical AMP and Ca2+i overload. Using the current model, isoprenaline-induced myocyte death can be mimicked by the phosphodiesterase inhibitor, milrinone (our unpublished results), suggesting that the same mechanisms mediate myocyte death in vivo. Myocyte death observed in the soleus muscle was mediated through the ß2-AR. This is a novel observation and the involvement of different ß-AR subtypes, i.e. ß1-AR in the heart and ß2-AR in skeletal muscle, in catecholamine-induced myocyte death has potential clinical relevance (vide infra).
It is tempting to suggest that the disparate sensitivity of the two striated muscles is due to differences in ß-AR subtype density. However, by combining data on the density of ß-AR in the heart and the triceps surae (gastrocnemius, plantaris and soleus) muscles (Rothwell et al. 1987) and data on the ratio of the ß-AR subtypes in the heart (Van Veldhuisen et al. 1995) and skeletal muscles (Rothwell et al. 1987; Jenson et al. 1995), we estimate that the heart has approximately 3.5-fold more ß1-AR receptors than the soleus has ß2-AR. Therefore, ß-AR density per se cannot explain these findings. This suggests that stimulation of the ß2-AR pathway in the soleus may be a more potent mediator of cell death than the ß1-AR of cardiomyocytes. Alternatively, the Ca2+i handling capabilities of the two striated muscles may also account for the disparate sensitivities of these muscles to catecholamine challenge. Intracellular Ca2+ transients are inextricably linked to ß-AR signalling and deregulation of [Ca2+]1 is implicated in cell death. As mitochondria are involved in the regulation of [Ca2+]1, it is our hypothesis that the greater mitochondrial density of cardiomyocytes (Murakami et al. 1995) would afford these cells greater protection against hyper-stimulation of the ß-AR. This, however, has not been shown empirically.
Whether administered in vivo (Fig. 5A) or in vitro (Communal et al. 1999), the stimulation of ß1-AR by isoprenaline leads to cardiomyocyte apoptosis. Similarly, over-expression of the same AR subtype leads to heart failure (Engelhardt et al. 1999). The stimulation of cardiomyocyte ß1- or ß2-AR subtypes in vitro is known to regulate the apoptotic death of these cells differentially. That is, isoprenaline-induced apoptosis of cardiomyocytes is prevented by the ß1-AR antagonist, CGP 20712A (Communal et al. 1999), consistent with our findings using bisoprolol in vivo (Fig. 5A). However, Communal et al. (1999) also found that blockade of ß2-AR increased the incidence of apoptosis in response to isoprenaline. From this observation these authors concluded that ß2-AR agonism has an inhibitory affect on pro-apoptotic ß1-AR signalling. Clearly this is not the case when studied in vivo. In the current work prior blockade of ß2-AR did not increase, but rather decreased the incidence of cardiomyocyte death (Fig. 5A). Based on our knowledge of the cardiotoxic effects of the ß2-AR selective agonist, clenbuterol (Burniston et al. 2002), we propose that a component of the cardiomyocyte death induced by isoprenaline in vivo is due to its ß2-AR-mediated facilitation of noradrenaline release from the sympathetic varicosities.
Inevitably these observations raise a number of important questions. For example, when quantifying the amounts of apoptosis, either singularly or relative to other forms of non-apoptotic cell death, in a complex solid organ such as the heart highly systematic and reproducible sampling protocols are essential. Random sampling from the myocardium, either in time (Fig. 3) or in space (Fig. 4), is likely to give widely variable results, and such data could potentially give rise to misleading conclusions. For example, 9 h after isoprenaline administration cardiomyocyte death would appear to arise from equal contributions of apoptosis and necrosis (Fig. 3). However, if 3 h were chosen instead, the observation would consist entirely of apoptosis, whereas after 18 h the majority of the myocyte loss would appear to be due to necrosis. These findings serve as an important reminder that care should be exercised when interpreting results, paying particular attention to the sampling protocols used.
Our findings are also of clinical relevance because over-activation of the sympathetic system in patients with pheochromocytoma and heart failure leads to sustained pathophysiological levels of catecholamines (Anker et al. 1997). Pharmacological levels of these hormones and their analogues are also used in resuscitative and intensive care practice. Dramatic and widespread losses in muscle bulk (Murdoch & McMurray, 1999) and concomitant muscle weakness (Mancini et al. 1994), as well as progressive cardiac dysfunction, are known to occur in such conditions. Indeed, heart failure is now recognized as a generalized, rather than a mere cardio-specific myopathic process (Opasich et al. 1999). Hence, less selective ß-AR inhibitors (i.e. blocking both ß1-AR in the myocardium and ß2-AR in skeletal muscles) may provide a more effective overall treatment than cardio-specific ß1-AR antagonists.
Clearly, the processes associated with cell death in vivo are more complex than those in vitro, from where the bulk of our information is currently derived. There is a real need to acquire more information about all forms of cell death in vivo. In the case of catecholamine-induced injury to cardiac and skeletal myocytes, it is clear that measurements of apoptosis alone may substantially underestimate the total biological impact of the insult.
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References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Benjamin
IJ, Jalil
JE, Tan
LB, Cho
K, Weber
KT
&
Clark
WA (1989). Isoproterenol-induced myocardial fibrosis in relation to myocyte necrosis. Circ Res
65, 657–670.
Blom WM, De Bont HE, Meijerman I, Kuppen PJ, Mulder GJ & Nagelkerke JF (1999). Interleukin-2-activated natural killer cells can induce both apoptosis and necrosis in rat hepatocytes. Hepatology 29, 785–792.[CrossRef][Medline]
Burniston
JG, Ng
Y, Clark
WA, Colyer
J, Tan
L-B
&
Goldspink
DF (2002). Myotoxic effects of clenbuterol in the rat heart and soleus muscle. J Appl Physiol
93, 1824–1832.
Casey TM, Chen H, Plaut K & Chiu JF (1996). Involution of mouse mammary glands during whole organ culture via apoptosis of epithelial tissue. Cell Biol Int 20, 763–767.[CrossRef][Medline]
Clark WA, Everett A, Fitch FW, Frogner KS, Jakovcic S, Rabinowitz M, Warner AM & Zak R (1980). Characterization of monoclonal antibodies directed against determinants on cardiac myosin heavy chain. Biochem Biophys Res Commun 95, 1680–1686.[CrossRef][Medline]
Communal C, Singh K, Sawyer DB & Colucci WS (1999). Opposing effects of beta1- and beta2-adrenergic receptors on cardiac myocyte apoptosis. Circulation 100, 1210–1217.
Dumount
EAWJ, Hofstra
L, Van Heerde
WL, Vam Den Eijnde
S, Doevendans
PAF, DeMuinck
E, Daemen
MARC, Smits
JFM, Frederik
P, Wellens
HJJ, Daemen
MJAP
&
Reutelingsperger
CPM (2000). Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model. Circulation
102, 1564–1568.
Eguchi
Y, Shimizu
S
&
Tsujimoto
Y (1997). Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res
57, 1835–1840.
Engelhardt S, Hein L, Wiesmann F & Lohse MJ (1999). Progressive hypertrophy and heart failure in beta1-adrenergic receptor transgenic mice. Pharmacology 96, 7059–7064.
Frankfurt
OS
&
Krishan
A (2001). Identification of apoptotic cells by formamide-induced DNA denaturation in condensed chromatin. J Histochem Cytochem
49, 369–378.
Goldspink DF, Burniston JG & Tan L-B (2003). Cardiomyocyte death and the ageing and failing heart. Exp Physiol 88, 447–458.[Abstract]
Honda O, Kuroda M, Joja I, Asaumi J, Takeda Y, Akaki S, Togami I, Kanazawa S, Kawasaki S & Hiraki Y (2000). Assessment of secondary necrosis of Jurkat cells using a new microscopic system and double staining method with annexin V and propidium iodide. Int J Oncol 16, 283–288.[Medline]
James TN (1998). The variable morphological coexistence of apoptosis and necrosis in human myocardial infarction: significance for understanding its pathogenesis, clinical course, diagnosis and prognosis. Coronary Artery Dis 9, 291–307.[Medline]
Jenson J, Brors O & Dahl HA (1995). Different beta-adrenergic receptor density in different rat skeletal fibre types. Pharmacol Toxicol 76, 380–385.[Medline]
Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, Reed JC, Olivetti G & Anversa P (1996). Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 74, 86–107.[Medline]
Kajstura J, Liu Y, Baldini A, Li B, Olivetti G, Leri A & Anversa P (1998). Coronary artery constriction in rats: necrotic and apoptotic myocyte death. Am J Cardiol 82, 30k–41k.[CrossRef][Medline]
Knaapen
MWM, Davies
MJ, De Bie
MH, Aven
AJ, Martinet
W
&
Kockx
MM (2001). Apoptotic versus autophagic cell death in heart failure. Cardiovasc Res
51, 304–312.
Leist M & Jaattela M (2001). Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2, 589–598.[CrossRef][Medline]
Lemasters JJ (1999). Necrapoptosis and the mitochondrial permeability transition: shared pathways to apoptosis and necrosis. Am J Physiol 276, G1–G6.
Lemasters JJ, Qian T, Baradham CA, Brenner DA, Cascio WE, Trost LC, Nishimura Y, Nieminen A-L & Herman B (1999). Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. J Bioenerg Biomem 31, 305–319.[CrossRef][Medline]
Lemasters JJ, Qian T, He L, Kim J-S, Elmore SP, Cascio WE & Brenner DA (2002). Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis and autophagy. Antioxid Redox Signal 4, 769–781.[CrossRef][Medline]
Lockshin RA, Osborne BA & Zakeri Z (2000). Cell death in the third millennium. Cell Death Differ 7, 2–7.[CrossRef][Medline]
Majno G & Joris I (1995). Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol 146, 3–15.[Abstract]
Mancini DM, Henson D, LaManca J & Levine S (1994). Evidence of reduced respiratory muscle endurance in patients with heart failure. J Am Coll Cardiol 24, 972–981.[Abstract]
Mann
DL, Kent
RL, Parsons
B
&
Cooper
G (1992). Adrenergic effects on the biology of the adult mammalian cardiocyte. Circulation
85, 790–804.
Murakami T, Shimomura Y, Fujitsuka N & Sugiyama S (1995). Differential adaptation to endurance training between heart and gastrocnemius muscle mitochondria in rats. Biochem Mol Biol Int 36, 285–290.[Medline]
Murdoch
DR
&
McMurray
JJV (1999). Cardiac cachexia – lean and mean. Eur Heart J
20, 1609–1611.
Ng Y, Goldspink DF, Burniston JG, Clark WA, Colyer J & Tan L-B (2002). Characterisation of isoprenaline myotoxicity on slow-twitch verses cardiac muscle. Int J Cardiol 86, 299–309.[CrossRef][Medline]
Opasich
C, Ambroino
N, Felicetti
G, Aquilani
R, Pasini
E, Bergitto
D, Mazza
A, Cobelle
F
&
Tavazzi
L (1999). Heart failure-related myopathy. Eur Heart J
20, 1191–1200.
Rothwell NJ, Stock MJ & Sudera DK (1987). Changes in tissue blood flow and beta-receptor density of skeletal muscle in rats treated with the beta2-adrenoceptor agonist clenbuterol. Br J Pharmacol 90, 601–607.[Medline]
Ruest LB, Khalyfa A & Wang E (2002). Development-dependent disappearance of caspase-3 in skeletal muscle is post-transcriptionally regulated. J Cell Biochem 86, 21–28.[CrossRef][Medline]
Saraste
A
&
Pulkki
K (2000). Morphological and biochemical hallmarks of apoptosis. Cardiovasc Res
45, 528–537.
Sun F, Hamagawa E, Tsutsui C, Ono Y, Oriri K & Kojo S (2001). Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in the rat. Biochem Biophys 153, 186–191.
Tan L-B, Burniston JG, Clark WA, Ng Y & Goldspink DF (2003). Characterisation of adrenoceptor involvement in skeletal and cardiac myotoxicity induced by sympathomimetic agents: towards a new bioassay for beta-blockers. J Cardiovasc Pharmacol 41, 518–525.[CrossRef][Medline]
Teerlink
JR, Pfeffer
JM
&
Pfeffer
MA (1994). Progressive ventricular remodeling in response to diffuse isoproterenol-induced myocardial necrosis in rats. Circ Res
75, 105–113.
Trump BF, Berezesky IK, Chang SH & Phelps PC (1997). The pathways of cell death: oncosis, apoptosis and necrosis. Toxicol Pathol 25, 82–88.[Medline]
Van Veldhuisen DJ, Brodde OE, van Gilst WH, Schulze C, Hegeman H, Anthonio RL, Scholtens E, de Graeff PA, Wesseling H & Lie KI (1995). Relation between myocardial beta-adrenoceptor density and hemodynamic and neurohumoral changes in a rat model of chronic myocardial infarction: effects of ibopamine and captopril. Cardiovasc Res 30, 386–393.[CrossRef][Medline]
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) and necrosis (
) were measured in the heart (A) and soleus (B) 12 h after receiving a single subcutaneous injection of one of the above doses of isoprenaline. Values are means ±S.E.M., with n= 4–6. First, any detectable isoprenaline-induced apoptosis or necrosis must be of biological significance when compared with the absence of cell death in control muscles. Statistically significant differences (*P < 0.05) were also found between 5 mg kg–1 and other doses of isoprenaline, using one-way ANOVA, with Tukey post hoc test.
