© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Clenbuterol induces cardiac hypertrophy with normal functional, morphological and molecular features
aDivision of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, UK
bCentre for Cardiopulmonary Biochemistry, University College London, Rayne Institute, London, UK
* Corresponding author, at: Department of Cardiac Surgery, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. Tel.: +44 171 3518533; Fax: +44 171 3763442.
Received 31 January 1997; accepted 8 July 1997
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Abstract |
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Objective: Several pharmacological agents have been shown to produce ‘physiological’ or ‘pathological’ hypertrophy based on their functional characteristics. The aim of this study was to examine the features of cardiac hypertrophy induced by the selective β2-adrenergic agonist, clenbuterol. Methods: Cardiac hypertrophy was induced in 7-week-old Sprague-Dawley rats by daily injections of clenbuterol for 3 weeks. Thyroxine and isoproterenol were also used to produce cardiac hypertrophy to serve as positive controls for physiological and pathological hypertrophy, respectively. Left ventricular function was determined using an isolated rat heart preparation. Ventricular samples were used for morphological examination while interstitial collagen was measured using high-pressure liquid chromatography. Expression of sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a) and phospholamban (PLB) were measured by dot blot analysis. Results: Clenbuterol treatment induced 26% left ventricular hypertrophy. These hearts demonstrated normal systolic isovolumic parameters and diastolic (active relaxation and passive stiffness) function. In addition, left ventricular concentration of collagen and morphology was normal as were the expression of SERCA2a and PLB mRNA. Conclusion: These results suggest that clenbuterol-induced hypertrophy is ‘physiological’ in terms of its function, extracellular structure and gene expression.
KEYWORDS β2-adrenergic agonist; Hypertrophy; Rat, ventricle
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Previous studies have shown that different stimuli induce cardiac hypertrophy in animals with specific functional, molecular and morphological features [1–6]. Based on these characteristics, cardiac hypertrophy has been described as either ‘pathological’ or ‘physiological’. Pressure-overload left ventricular (LV) hypertrophy induces pathological hypertrophy with impairment in systolic function, myocardial relaxation and an increase in diastolic stiffness [1, 2]. Abnormalities in diastolic relaxation and systolic function are thought to be due to the down regulation of intracellular calcium handling proteins including sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a) and phospholamban (PLB) [7–9]. SERCA2a facilitates the transport of intracellular Ca2+ into the sarcoplasmic reticulum (SR) while phosphorylation of PLB increases the affinity of SERCA2a for Ca2+. In addition, increase in interstitial collagen has been shown to account for the increase in diastolic stiffness in pathological hypertrophy [2, 10]. In contrast, cardiac hypertrophy induced by thyroxine and swimming training has been shown to be physiological with enhanced systolic function [4, 11]and normal ventricular collagen content [12]. Furthermore, thyroxine treatment increases gene expression of SERCA2a while downregulating that of PLB [13]thereby, increasing Ca2+ transport into the SR.
There has been considerable interest in the effects of adrenergic agents in cardiac hypertrophy, as endogenous catecholamines are known to be involved in both cardiovascular conditioning with exercise [14]and hypertrophy [15]. The characteristics of cardiac hypertrophy by isoproterenol (β1- and β2-agonist) have been well described and been shown to be similar to that seen with pressure-overload [5, 6]. Clenbuterol, a selective β2-adrenergic agent, has also been shown to induce an increase in the mass of the rat heart, although to date there has been little known about the pathophysiology of such hypertrophy [16–19].
We have recently reported that clenbuterol induces cardiac hypertrophy in the rat with no changes in gene expression indicative of pathological hypertrophy (absence of skeletal actin and predominantly 
-cardiac myosin heavy chain isoform) [20]. However, LV function in clenbuterol-induced cardiac hypertrophy has not been previously investigated. The purpose of this study was to determine its LV function, collagen concentration and morphology. In addition, the mRNA expression of intracellular calcium handling proteins, SERCA2a and PLB, which are known to be important determinants of LV function in the rat, were measured.
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2 Methods |
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2.1 Animals and treatment regimen
The animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1985). Two sets of rats were prepared for: (a) measurement of LV function and extracellular collagen and histology, or (b) molecular analysis. Fifty-five 7-week-old male Sprague-Dawley (Harlan, UK) rats weighing 200–225 g were fed normal rat chow and water ad libitum over a 3-week experimental period. Eight experimental groups were studied: Groups I (n = 8) and V (n = 6) were used as controls and injected subcutaneously (s.c.) with 0.5 ml of saline (vehicle) once daily. Groups II (n = 8) and VI (n = 8) were injected s.c. with clenbuterol (a gift from Boehringer Ingelheim, UK) once daily at a dose of 2 µg/g body weight [20]. Groups III (n = 8) and VII (n = 8) were injected intraperitoneally with 3.5 mg/kg of DL-thyroxine (Sigma, UK) once daily. Groups IV (n = 6) and VIII (n = 5) received once daily s.c. injections of isoproterenol (1 mg/kg) for the last 2 weeks of the experimental period. Thyroxine and isoproterenol groups served as positive controls for physiological and pathological cardiac hypertrophy. The dose and mode of administration of these drugs were based on previous work and that reported in the literature [5, 20, 21].
2.2 Perfusion technique and evaluation of ventricular function
2.2.1 Perfusion technique
Groups I, II, III and IV were sacrificed for functional experiments. LV function was measured using an isolated rat heart preparation (Langendorff). At the end of the experimental period, animals were administered an intraperitoneal injection of 0.3–0.5 ml of 2.5% sodium thiopentone 2.5% (May and Baker, UK) and heparinised (5000 IU) via the femoral vein. The heart was rapidly excised and immediately arrested by placing in ice-cold perfusion buffer. The thoracic aorta was cannulated and the hearts retrogradely perfused with a constant perfusion pressure reservoir. Modified Krebs–Henseleit bicarbonate buffer used contained (mmol/l): NaCl 118.5, KCl 4.8, NaHCO3 25, KH2PO4 1.2, Mg O4·7H2O 1.48, CaCl2 1.25 and glucose 11. The buffer was gassed with a mix of 95% O2/5% CO2 and kept at 37°C (pH 7.40–7.45). The LV was vented through the apex to allow free passage of coronary effluent. LV pressure was measured using a compliant intraventricular balloon made from plastic film (Saranwrap) and attached via a short length of polypropylene tubing to a pressure transducer (SensorNor 840, Norway) and recorded on a chart recorder (Lectromed, UK). The heart was atrially paced at 5 Hz and coronary perfusion fixed at 100 cm H2O. The coronary flow/g LV tissue was similar in all groups (data not shown). Isovolumic LV pressure measurements were recorded with the balloon volume fixed at an end-diastolic pressure of 10 mmHg while pressure-volume measurements were recorded during increments of 0.02 ml of balloon volume. Two sets of pressure–volume measurements were recorded for each heart and reproducible results (±10%) used for analysis. Unstable preparations were also discarded and this resulted in the use of 30 of 38 preparations (79%).
2.2.2 LV function analysis
Systolic function was determined from the developed (systolic–diastolic) pressure normalised to the weight of the left ventricle and the ratio of the maximal rate of pressure increment to the instantaneous pressure (dP/dtmax/P) at an end-diastolic pressure (EDP) of 10 mmHg. Diastolic function was determined from dP/dtmin at an EDP of 10 mmHg, a measure of active relaxation and the passive stiffness of the intact ventricle (chamber) and myocardium (tissue). Chamber stiffness was analysed from the end-diastolic pressure–volume relationship (dP/dV). In order to correct for differences in LV mass and size of different hearts, the exponential stiffness coefficient was determined from the stress (
, g per cm2) and strain (
, %) for the midwall of the LV assuming a spherical geometry [2]:
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The diastolic pressure–volume and diastolic stress–strain curves were fitted to mono-exponential relations: EDP=A[eB(EDV–Vo)–1] and = C[eD()–1], respectively, where A, B, C and D are regression constants of the respective equations. By logarithmic transformation, each set of data was converted to a linear relationship and slope values used for comparison. The slope of the stress–strain relationship represents the exponential stiffness constant.
2.3 Collagen concentration and morphology
LV tissue from animals in the functional experiments (groups I, II, III and IV) were used for determination of collagen concentration quantified from the concentration of hydroxyproline by high pressure liquid chromatography (HPLC) [22]. Values were reported as nanograms of collagen assuming a hydroxyproline content of 12.2% (w/w) [23]. To identify any changes in morphology (myocyte necrosis, inflammatory cell infiltration), a coronal section was obtained at the equator of the LV from which 5-µm paraffin sections were prepared and stained with haematoxylin and eosin.
2.4 Analysis of mRNA
Animals were sacrificed at 3 weeks for molecular analysis. Samples were snap frozen in liquid nitrogen and stored at –80°C. RNA was extracted from tissue samples by the guanidium thiocyanate–phenol–chloroform technique [24].
Relative abundance of specific mRNAs was measured by dot blotting as described using 32P-labelled cDNA probe complementary to ANF (plasmid kindly provided by K. Khowlton, San Diego) [20], SERCA2a [25], PLB [26]and a 32P-endlabelled oligonucleotide complementary to 18S ribosomal RNA. The resultant dots were quantified by densitometry. Values for ANF, SERCA2a and PLB were standardised to the respective values for 18S. ‘Hot’ reverse transcriptase–polymerase chain reaction (RT–PCR) analysis of MHC Iso-mRNAs was also performed as previously described [20]by PCR amplification using a forward and reverse primer (identical to sequences for - and β-MHC) and an end-labelled [32P] forward primer, TaqI DNA Polymerase, 20 mM dNTPs. The MHC iso-RNAs were distinguished by digestion of 25 µl of PCR products with Tru9I digestion. The fragments (-MHC 309 base pairs, β-MHC 259 and 50 base pairs) were separated on an 8% SDS–polyacrylamide gel. The gel was dried and exposed to X-ray film at –70°C. The resultant bands on the autoradiogram were quantified by densitometry and the ratio of the β-MHC isoform to the respective quantity of total MHC isoforms.
2.5 Determination of mass data
Hearts were blot dried and the great vessels trimmed away before weighing. The atria were excised and the right and left ventricle (including interventricular septum) separated before weighing. LV mass index was calculated from the ratio of the left ventricular weight to left tibial length. As clenbuterol is known to cause skeletal muscle hypertrophy and catabolism of adipose tissue, the left gastrocnemius plantaris-soleus (GPS) and the left perinephric fat pad were removed and weighed as previously described [20].
2.6 Statistical analyses
All group data are presented as mean±s.e.m. and compared by analysis of variance (ANOVA) with Bonferroni bounds and two-tailed Student's t-test for unpaired data where appropriate. Results were considered significant when P<0.05.
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3 Results |
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3.1 Effects of clenbuterol on mass data
The mean body weight of clenbuterol treated rats were significantly greater than the control, isoproterenol and thyroxine groups by 15%, 13.9% and 13.1%, respectively (P<0.01) (Table 1). This increase was predominantly attributable to the growth of skeletal muscle mass as evidenced by an increase in the left GPS muscle mass which was significantly increased compared to control, isoproterenol and thyroxine groups by 33.5%, 26.0% and 31.7%, respectively (Table 1). These findings in addition to that of a significant decrease (41%) in perinephric fat in the clenbuterol group to the control group is consistent with clenbuterol's known repartitioning effect.
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The effects of pharmacological agents on the LV mass index are shown in Fig. 1. Clenbuterol significantly increased the LV mass index by 26.3±2.9% compared to controls. This confirms our previous findings and those of others of a significant increase in heart mass with clenbuterol treatment which in our study was similar to that induced by thyroxine (22.2±1.6%), although isoproterenol treatment produced the greatest degree of hypertrophy (42.8±3.8%).
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The effect of all three agents were not chamber-specific, and global hypertrophy was seen as demonstrated by the LV/RV ratios: control 5.19±0.18, clenbuterol 5.00±0.19, thyroxine 5.36±0.17 and isoproterenol 4.49±0.19 (all treated groups vs. control, P>0.05).
3.2 Left ventricular function
The normalised developed pressure and dP/dtmax/P were similar in the clenbuterol and thyroxine group compared to that of controls (Table 2). This is in contrast to the isoproterenol group, where dP/dtmax/P was significantly reduced although normalised developed pressure was normal.
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dP/dtmin in the clenbuterol group was also similar to that of the control and thyroxine groups (Table 2). Ventricular stiffness (dP/dV) as analysed from the EDP–volume relationship demonstrated normal stiffness in the clenbuterol and thyroxine groups in contrast to that of the isoproterenol group which was significantly increased (Fig. 2a,b). The exponential stiffness coefficient was also normal in the clenbuterol and thyroxine groups unlike that in the isoproterenol group (Fig. 2b).
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, n = 8), clenbuterol (
, n = 8), thyroxine (
, n = 8) and isoproterenol (
, n = 6) groups demonstrating an increased ‘stiffness’ in the intact ventricle in the isoproterenol group. (b) Bar graph of slope values of exponential end-diastolic pressure–volume and stress–strain data. Values represent mean±s.e.m. *P<0.05 vs. control.3.3 Collagen concentration and morphology
Fig. 3 demonstrates the LV collagen concentration between the groups. Clenbuterol-induced cardiac hypertrophy was associated with no significant differences in collagen concentration to the control group. This was similar to that with thyroxine treatment but was in contrast to that with isoproterenol which had a 65% increase in collagen. Histological examination of the clenbuterol and thyroxine groups further demonstrated normal morphology (Fig. 4b,c) unlike that seen in the isoproterenol samples which had patchy myocyte necrosis (inflammatory cell infiltrate, disruption of myocytes and fibrosis replacing myocardial tissue) within the subendocardium and interventricular septum (Fig. 4d). These findings are consistent with the normal diastolic passive properties found in clenbuterol-induced cardiac hypertrophy.
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3.4 RNA analyses in the heart
As a marker of myocardial hypertrophy, the LV expression of ANF was measured and found to be increased by 2-fold in clenbuterol-treated animals (Table 3). Thyroxine- and isoproterenol-treated rats had higher expressions of ventricular ANF (3- and 8-fold, respectively).
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The ratios of β-MHC to total MHC mRNA expression in clenbuterol and thyroxine groups were similar to that of the control group. The isoproterenol group showed a mild shift towards the β-MHC isoform compared to controls (P>0.05) (Table 3). The difference in proportion of β-MHC was, however, 2-fold between the clenbuterol and isoproterenol group (P<0.05).
Table 3 shows the expression of SERCA2a and PLB mRNA. Clenbuterol-treated hearts had normal expression of SERCA2a while thyroxine-treatment led to a 50% increase in SERCA2a expression (P = 0.19). There were no significant changes in SERCA2a expression in the isoproterenol group, although 2 of 5 hearts expressed only 50% of the mean control level. PLB mRNA expression was unchanged in all treated groups relative to controls.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Previous studies have shown that different adrenergic agonists induce specific types of hypertrophy through different mechanisms [15]. The adrenergic agents norepinephrine and isoproterenol have been shown to induce pathological changes [5, 6]. We hypothesised that the selective β2-agonist, clenbuterol, may induce physiological hypertrophy in the rat based on the gene expression of its ventricular MHC isoforms [20]. The present study has further demonstrated normal left ventricular function, morphology and collagen concentration, as well as mRNA expression of SERCA2a and PLB in clenbuterol-induced cardiac hypertrophy in rats.
The dose of 2 µg/g body weight used in this study was based on our preliminary work [20]. This dose is large in comparison to that which has been reported in human use [27], although no formal clinical dose–response trials have been performed. There have also been no scientific reports of cardiac hypertrophy following clenbuterol treatment in humans.
In rats, however, the modest increase in LV mass (26%) by clenbuterol as observed in this study is consistent with that previously reported by us [20]and similar to that seen with thyroxine. Isovolumic systolic parameters in clenbuterol-induced hypertrophy were normal, which is unlike the supraphysiological systolic function observed by others with thyroxine-induced hypertrophy [28]. However, this may be explained by differences in techniques used to measure contractile function, as thyroxine-treated hearts in our experiments also demonstrated equal performance to the controls. It was, however, important to find normal systolic function in clenbuterol-induced hypertrophy, as systolic function in cardiac hypertrophy induced by other adrenergic agents such as isoproterenol may be impaired as demonstrated in this study, and by others [29]. The reduced speed in contractility of isoproterenol-induced hypertrophy may be accounted for by the mild increase in proportion of β-MHC isoform. In small animals where the predominant MHC isoform is the -isoform, a switch to the β-MHC isoform is often seen in pathological hypertrophy and results in a reduced maximal shortening velocity [30]. As previously demonstrated [20], the proportion of -MHC isoform was found to be normal in clenbuterol-treated hearts.
Pathological cardiac hypertrophy is also associated with diastolic dysfunction detected either as an increase in relaxation times [1]or passive stiffness [2, 10]. The latter was demonstrated in isoproterenol-treated hearts in our study but, in contrast, clenbuterol-treated hearts demonstrated normal dP/dtmin and passive stiffness similar to that found with thyroxine. The finding of normal active relaxation is consistent with the normal expression of SERCA2a and PLB mRNA found in the clenbuterol- and thyroxine-treated hearts. The normal mRNA expression of SERCA2a and PLB in these ventricles is similar to that described in swimming rats [31]. In the case of thyroxine, however, our findings were disparate to other reports of thyroxine-induced hypertrophy which have shown an increase in SERCA2a and a decrease in PLB mRNA expression [13]. This may be accounted for by the use of different dose regimens or animal model. At the time points studied, expression of SERCA2a and PLB mRNA were not significantly reduced in the isoproterenol group, which is consistent with results found by Boluyt et al. when isoproterenol was administered for long periods [6]. This may be related to a downregulation of β-adrenergic receptors, and would also explain why a greater change in MHC isoform was not seen in the isoproterenol group.
Significantly, the increase in LV mass with clenbuterol was without a concomitant increase in extracellular collagen. This has important functional consequences, as increases in extracellular collagen are responsible for increases in passive diastolic stiffness seen in pathological hypertrophy such as that with isoproterenol [2, 10]. This increase in collagen may also account for the reduced speed of contraction observed with isoproterenol. Another important feature of clenbuterol-induced hypertrophy was the normal morphology observed which is in contradistinction to the characteristic myocyte death seen with isoproterenol [5].
The mechanism of myocardial growth with clenbuterol is poorly understood. We have previously suggested this to be a compensatory hypertrophy secondary to an increased haemodynamic demand from a greater skeletal muscle mass similar to that seen with sympathetically-mediated effects of cardiovascular conditioning [20]. Clenbuterol may act directly on β2-receptors which may be in the myocyte or even non-myocyte compartment, although our preliminary in vitro work has not demonstrated a change in cellular morphology with clenbuterol treatment [20]. There is, however, evidence that functional β2-receptors exist in the myocardium and are involved in ventricular function by a different pathway to that of β1-receptors [32]. However, antagonist studies by β-blockade have shown conflicting results on the hypertrophic response of clenbuterol [16–18]and other mechanisms may well be involved. Future studies should be directed towards defining the β1- and β2-receptor densities in hearts treated with clenbuterol and isoproterenol. It has also been suggested that clenbuterol's action on cardiac muscle may be independent of that on skeletal muscle by the fact that cyclo-oxygenase inhibitors have been shown to selectively inhibit clenbuterol's actions on the myocardium without antagonising skeletal muscle growth [19]. Further work to elucidate the mechanism of clenbuterol should involve investigating the transduction pathways including the measurement of the proto-oncogenes (c-fos, c-jun) and signalling pathways such as the G-proteins and mitogen-activated protein kinase cascade.
The finding that clenbuterol, a selective β2-adrenergic agonist, appears to induce physiological cardiac hypertrophy extends our understanding of the different mechanisms involved in the induction of hypertrophy and may have important clinical implications. Experimentally, growth hormone and its local effector, insulin-like growth factor, has been shown to enhance ventricular function in normal and failing hearts [33, 34]. Although one of the current aims of medical therapy in heart failure is the regression of cardiac hypertrophy, it has been suggested that the induction of physiological hypertrophy may decrease wall stress and improve ventricular function in patients with heart failure [35]. Our findings suggest that clenbuterol may also be a candidate for inducing physiological hypertrophy and could be used in such a setting. Furthermore, unlike growth hormone and thyroxine, it is devoid of major systemic side effects.
In conclusion, it would appear that unlike cardiac hypertrophy induced by other adrenergic agents, clenbuterol-induced cardiac hypertrophy is not associated with pathological changes and is physiological in terms of function, structure and gene expression. Further studies are needed to investigate its effects on experimental heart disease (cardiac hypertrophy and failure) and its possible role in clinical practice.
Time for primary review 31 days.
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Acknowledgements |
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We would like to thank Mr. P. Athanasspoulos for performing the HPLC analysis of the ventricular samples, Miss K. Morrison for the histological processing and staining of tissue samples and Dr. D. Robinson, Department of Mathematics and Statistics, University of Sussex, Brighton for his help in the statistical analysis.
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