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(Circulation. 1997;96:2239-2246.)
© 1997 American Heart Association, Inc.

Articles

Pharmacological Modulation of Pressure-Overload Cardiac Hypertrophy

Changes in Ventricular Function, Extracellular Matrix, and Gene Expression

Kit Wong, FRCS; Kenneth R. Boheler, PhD; Mario Petrou, FRCS; ; Magdi H. Yacoub, FRCS

From the Division of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, UK.

Correspondence to Professor Sir Magdi Yacoub, Department of Cardiac Surgery, Royal Brompton Hospital, Sydney St, London SW3 6NP, UK.

	Abstract

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Background Appropriate cardiac hypertrophy (CH) is necessary in several clinical settings, such as pulmonary artery banding in the two-stage arterial switch operation for transposition of the great arteries. Pressure-overload CH, however, produces ventricular dysfunction due to structural and molecular changes. The ß₂-adrenergic receptor agonist clenbuterol has been shown to induce CH without such adverse effects to the rat heart. This study was performed to determine its effects on left ventricular (LV) function, structure, and gene expression in pressure-overload CH.

Methods and Results Sprague-Dawley rats were assigned to one of four groups: 1, sham-operated (n=15); 2, banding of ascending aorta (n=22); 3, banding+clenbuterol (n=18); and 4, banding+thyroxine (n=17). At the end of 3 weeks, groups 2, 3, and 4 showed an increase in LV mass index of 49.7±5.1%, 66.1±3.8%, and 47.6±4.6%, respectively, relative to group 1. A subgroup with severe CH (>50%) in group 2 was found to have significantly impaired developed pressure and diastolic relaxation and an increase in passive stiffness, with significantly reduced LV expression of sarcoplasmic reticulum Ca²⁺-ATPase2a (SERCA2a) mRNA and increased LV collagen concentration. In comparison, similarly hypertrophied animals in groups 3 and 4 demonstrated improved developed pressure, normal relaxation and diastolic stiffness with normal collagen concentration, and a greater abundance of SERCA2a mRNA.

Conclusions Clenbuterol administration in conjunction with pressure overload produces a specific type of CH with preserved LV function. In addition, an increase in LV mass was associated with less fibrosis and greater expression of SERCA2a mRNA than banding alone.

Key Words: hypertrophy • pharmacology • collagen • sarcoplasmic reticulum

	Introduction

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Cardiac hypertrophy is a physiological response to an increase in hemodynamic load that initially normalizes wall stress and improves contractile function but eventually leads to ventricular dysfunction and heart failure.¹ In valvular disease, cardiomyopathy, hypertension, and ischemic heart disease, cardiac hypertrophy increases morbidity and mortality.^{2 3} The pathological characteristics of the hypertrophied ventricle have been well described and include early diastolic dysfunction followed by systolic impairment, interstitial fibrosis,^{4 5 6 7} and downregulation of the calcium-handling proteins SERCA2a and PLB. These proteins are important regulators of intracellular Ca²⁺ homeostasis and have been implicated in ventricular dysfunction; specifically, diastolic relaxation and reduction in these proteins are associated with increases in calcium transient times.^{8 9 10 11 12 13} Medical treatment is aimed at the regression of hypertrophy and reversing the features of pathological remodeling, for which ACE inhibitors have been shown to be beneficial.^{14 15} Paradoxically, a specific type of hypertrophy induced by growth hormone has been demonstrated to improve ventricular function in experimental heart failure and in patients with dilated cardiomyopathy.^{16 17} The induction of hypertrophy without adverse effects may also be desirable in cardiac surgery, namely, the two-stage ASO for transposition of the great arteries, in which the LV mass is increased by a preparatory first-stage operation (pulmonary artery banding with or without a systemic-to-pulmonary shunt) before ASO.¹⁸ However, in a proportion of these patients, LV systolic function after the ASO has been found to be impaired.^{19 20 21 22} The use of pharmacological agents to modulate the pathological features of pressure-overload hypertrophy has not been adequately explored.

Several agents have been used to induce physiological cardiac hypertrophy, including thyroxine, growth hormone, and its local effector (insulin-like growth factor-1).^{16 23 24 25 26} We have recently shown that clenbuterol, a selective ß₂-adrenergic agonist, induces a moderate degree of cardiac hypertrophy in the rat, with certain physiological features, including the absence of a shift to the ß-MHC isoform and skeletal actin at the mRNA level.²⁷ In addition, our studies have shown that systolic and diastolic (relaxation and passive stiffness) function in clenbuterol-induced cardiac hypertrophy is normal.²⁸ Unlike cardiac hypertrophy by other adrenergic agents (isoproterenol),²⁹ cardiac growth with clenbuterol treatment was not associated with an increase in interstitial collagen. The mRNA expression of SERCA2a and PLB and contractile proteins that influence ventricular function was also shown to be normal.

The aim of this study was to examine the effects of clenbuterol on pressure-overload cardiac hypertrophy by evaluating the changes in ventricular function, interstitial collagen, expression of calcium-handling genes (SERCA2a and PLB), and abundance of contractile (MHC isoforms) proteins. The pharmacological modulation of cardiac hypertrophy by such agents as clenbuterol may improve our understanding of the different mechanisms in cardiac hypertrophy and have important clinical implications.

	Methods

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Animals
Seven-week-old male Sprague-Dawley rats (Harlan, UK) weighing 200 to 225 g were used in all experiments. Feed and water were provided ad libitum, and rats were housed in a light- and temperature-controlled room over a 3-week experimental period. Rats were randomly grouped for the purpose of (1) measurement of ventricular function, collagen concentration, and histology or (2) molecular analysis.

Surgical Preparation of Animals
Pressure overload was achieved by ascending aortic banding. After induction of general anesthesia with enflurane, endotracheal intubation with a 16-gauge polyethylene cannula was performed. This cannula was connected to a rodent ventilator (Harvard), and anesthesia was maintained by a mixture of oxygen and 0.5% to 1.5% enflurane (Abbott). The ascending aorta was exposed via a left lateral thoracotomy (fourth intercostal space) and banded at its junction with the innominate artery using a 4/0 mersilk (Eticon) suture tied firmly around a hypodermic cannula (1.05 mm, based on pilot work showing this to produce 40% to 80% hypertrophy). The cannula was then withdrawn from under the ligature. Sham-operated animals had a similar operation apart from the banding procedure. The chest was closed under positive-pressure ventilation to reduce the potential of pneumothorax.

Transstenotic pressure gradients were determined from 4 animals in each group of banded animals. General anesthesia and mechanical ventilation were achieved as described above at the end of the experimental period. Left intraventricular systolic pressure and aortic pressure distal to the band were determined by direct puncture with an 18-gauge needle connected by a fluid-filled polyethylene tubing to a pressure transducer, with pressures recorded digitally on a monitor (Roche). This showed a mean gradient of 58.2 mm Hg (SEM, 5.9 mm Hg; range, 40 to 95 mm Hg).

Preparation of Experimental Groups
Group 1 (n=15) consisted of sham-operated rats injected once daily with 0.5 mL saline SC. Banded rats were allowed a 48-hour period of postoperative recovery and then randomly allocated to one of three groups: group 2 (n=22), banded rats injected daily with 0.5 mL saline SC; group 3 (n=18), banded rats injected with clenbuterol (a gift from Boehringer Ingelheim, UK) once daily at a dose of 2 µg/g body wt SC; and group 4 (n=17), banded rats injected with DL-thyroxine (Sigma) once daily at a dose of 3.5 mg/kg IP. The dose and mode of administration of these drugs were based on those used in previous work.^{27 29 30} Group 4 was studied as a positive control of a pharmacological agent known to modulate some features of pathological cardiac hypertrophy, including normalization of the ratio of - and ß-MHC isoforms in rats with pressure-overload hypertrophy³¹ and interstitial collagen concentration in rats given isoproterenol and thyroxine.²³

Isolated Heart Perfusion and Evaluation of LV Function
At the end of the experimental period, animals were anesthetized with an injection of 0.3 to 0.5 mL IP of 2.5% sodium thiopentone (May & Baker) and heparinized (5000 IU) through the femoral vein before the chest was entered. Each animal was examined for signs of cardiac failure (pericardial and pleural effusion and ascites). Hearts were rapidly excised and perfused by the Langendorff method with a constant-perfusion-pressure reservoir and modified Krebs-Henseleit buffer containing (in mmol/L) NaCl 118.5, KCl 4.8, NaHCO₃ 25, KH₂PO₄ 1.2, MgSO₄ · 7H₂O 1.48, CaCl₂ 1.25, and glucose 11. The buffer was gassed with 95% O₂/5% CO₂ (pH 7.4) at 37°C. The left ventricle was vented through the apex to allow free passage of coronary effluent. LV pressure was measured with a compliant intraventricular balloon³² attached via a short length of polypropylene tubing to a pressure transducer (SensorNor840) connected to a chart recorder (Lectromed). The heart was atrially paced at 5 Hz, and coronary perfusion was fixed at 100 cm H₂O for sham and 120 cm H₂O for banded animals. These differing perfusion pressures were used in view of the higher in vivo perfusion pressure in the banded animals and on the basis of pilot studies that showed this increment to produce comparable coronary flow per gram LV weight. LV pressures were recorded during increments of 0.02 mL of balloon volume for pressure-volume measurements⁵ and isovolumically at an end-diastolic pressure of 10 mm Hg.¹¹ All measurements were repeated twice, and only those with reproducible results (±10%) were included in the study. Preparations that were unstable were also excluded. Thirty-seven of 48 heart preparations (78%) were eventually used.

The isovolumic developed (systolic-diastolic) pressure normalized to the LV weight (grams) and the ratio of the maximal rate of pressure development to the instantaneous pressure [(dP/dt_max)/P] were used as indices of systolic function. The maximum rate of pressure decay (dP/dt_min) was used as a measure of active relaxation during diastole, and passive stiffness was determined from the diastolic pressure-volume measurements. Chamber stiffness was analyzed from the end-diastolic pressure-volume relationship (dP/dV). To correct for differences in LV mass and size of different hearts, the exponential stiffness coefficient, a measure of the myocardial (tissue) stiffness, was determined from the stress (, g/cm²)–strain (, %) relationship.³³ The diastolic pressure-volume and diastolic stress-strain curves were fitted to monoexponential relations: EDP=A[e^B(EDV-V°⁾-1] and =C[e^D()-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 were used for comparison. The slope of the stress-strain relationship represents the exponential stiffness constant.

Collagen Concentration and Morphology
Hearts from the functional experiments were used for measurement of collagen concentration and histological examination. A 15-mg piece of left ventricle was obtained from the free wall of the ventricle at the equator, and the concentration of hydroxyproline was determined by HPLC.³⁴ Collagen concentration was determined from this assuming a hydroxyproline content of 12.2% (wt/wt).³⁵ A coronal section at the equator of the LV was obtained, from which 5-µm paraffin coronal sections were prepared. These sections were stained with hematoxylin and eosin.

Molecular Analysis
Animals were euthanized at 3 weeks for molecular analysis. Samples were snap-frozen in liquid nitrogen and stored at -80°C. The guanidinium thiocyanate–phenol-chloroform technique was used for RNA extraction.³⁶

The relative abundance of specific mRNAs was measured by dot blotting as described, by use of a ³²P-labeled cDNA probe complementary to ANF,²⁷ SERCA2a,³⁷ PLB,⁹ and a ³²P-end-labeled oligonucleotide complementary to 18S ribosomal RNA. The resultant dots were quantified by densitometry, and the values for ANF, SERCA2a, and PLB were standardized to the respective values for 18S. MHC iso-mRNAs were analyzed by "hot" RT-PCR as previously described.²⁷ This PCR amplification used a forward and reverse primer (identical to sequences for - and ß-MHC) and an end-labeled (³²P) forward primer, Taq I DNA polymerase, 20 mmol/L dNTPs. The MHC iso-mRNAs were distinguished by digestion of 25 µL of PCR products with Tru9I digestion, and the fragments (-MHC, 309 base pairs; ß-MHC, 259 and 50 base pairs) were separated on an 8% SDS-polyacrylamide gel, which was then put to film. The resultant bands were quantified by densitometry.

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 ventricles (including interventricular septum) separated before weighing. LV mass index was calculated from the ratio of LV weight to left tibial length.²⁷

The left gastrocnemius-plantaris-soleus muscle and left perinephric fat pad were removed and weighed, because clenbuterol is known to cause skeletal muscle hypertrophy and catabolism of adipose tissue.²⁷ The ratio of wet to dry lung weights was determined to identify the presence of pulmonary congestion.

Statistical Analysis
All group data are presented as mean±SEM and compared by ANOVA with Bonferroni bounds and two-tailed Student's t test for unpaired data where appropriate. Results were considered significant when P<.05.

	Results

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Body, Muscle, and Lung Mass
The mean body weight of the banded+clenbuterol group was greater than those of the sham, banded, and banded+thyroxine groups by 7.1% (P<.05), 5.5% (P>.05), and 10.0% (P<.05), respectively (Table 1). The increase in left gastrocnemius-plantaris-soleus muscle mass in the banded+clenbuterol group relative to sham was 15.6% (P<.05), although the relative increase versus the banded (24.4%, P<.05) and banded+thyroxine (34%, P<.05) groups was much higher (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effect of Clenbuterol and Thyroxine Treatment on Body, Skeletal Muscle, and Perinephric Fat Weight in Banded Rats

The ratios of wet to dry lung weight as an index of pulmonary congestion and heart failure were not different between the groups (Table 1).

Banding alone produced an increase in the LV mass index by 49.73±5.06% compared with sham. Pharmacological intervention with clenbuterol and thyroxine produced increases of 66.08±3.83% and 47.55±4.59%, respectively (Fig 1). Clenbuterol therefore produced a trend toward increasing the weight of the banded hearts (P>.05). The effect of banding was chamber-specific, because the LV:RV values were increased in the banded (6.45±0.26), banded+clenbuterol (6.23±0.23), and banded+thyroxine (6.30±0.29) groups relative to sham (5.26±0.26) (P=.034). Our previous studies have shown that clenbuterol treatment alone induces a global hypertrophy (ie, no significant change in LV:RV relative to controls).

View larger version (25K): [in this window] [in a new window]   Figure 1. LV mass index (LV mass/tibial length ratio) of banded rats (n=22), banded rats given clenbuterol (n=18), and banded rats given thyroxine (n=17) expressed as ratio to mean ratio of sham group (n=15). Values are mean±SEM. *P<.05 vs sham.

LV Function
To examine the changes in LV function by pressure overload alone and that with concomitant administration of clenbuterol or thyroxine, hearts were perfused in a Langendorff apparatus. Table 2 shows the isovolumic systolic and diastolic parameters. With LV hypertrophy by pressure overload alone, ventricular impairment was found in a subgroup of rats with severe (>50%) hypertrophy. In this subgroup of rats, normalized developed pressure was 57.8% of the sham group (P<.01), and (dP/dt_max)/P was also found to be significantly reduced. In addition, the subgroup of rats with severe hypertrophy exhibited a reduction in dP/dt_min (P<.01) and an increase in chamber and myocardial stiffness versus the sham group (P<.01) (Fig 2A and 2B). No signs of heart failure were evident in these rats as identified by the above-mentioned parameters. When cardiac hypertrophy was <50%, ventricular function was found to be normal.

View this table: [in this window] [in a new window]   Table 2. Isovolumic Systolic and Diastolic Parameters at End-Diastolic Pressure of 10 mm Hg in Banded and Clenbuterol- or Thyroxine-Treated Banded Rats

View larger version (22K): [in this window] [in a new window]   Figure 2. A, LV end-diastolic pressure-volume relationship of groups: sham (, n=7), banded with <50% hypertrophy (, n=8), banded with >50% hypertrophy (, n=5), banded+clenbuterol with >50% hypertrophy (, n=8), and banded+thyroxine with >50% hypertrophy (, n=4). Unstressed balloon volumes (µL) for the groups were 45.7±7.2, 50±5.3, 62±8, 23.1±1.6, and 42.5±4.8, respectively. B, Slope values of exponential end-diastolic pressure-volume and stress-strain data. Values are mean±SEM. *P<.05 vs sham, +P<.05 banded >50% hypertrophy vs banded <50% hypertrophy or banded+clenbuterol or thyroxine >50% hypertrophy.

Banded rats with severe hypertrophy treated with clenbuterol or thyroxine produced a significantly greater developed pressure (both 75.4% of sham) than the banded subgroup with similar hypertrophy. In addition, (dP/dt_max)/P and dP/dt_min in both these groups were no different from those of the sham group. Furthermore, unlike that seen by banding alone, the development of this degree of hypertrophy by a combination of clenbuterol or thyroxine with pressure overload was not associated with an increase in chamber and myocardial stiffness (Fig 2A and 2B). No signs of heart failure were evident in these rats either.

LV Collagen Concentration and Morphology
To determine whether the observed changes in diastolic stiffness were associated with similar changes in collagen concentration, the concentration of collagen was measured by HPLC. There were no intergroup differences in collagen concentration between the banded groups and sham group. However, in the subgroup of banded animals with >50% hypertrophy, collagen concentration was significantly increased in comparison with the sham group (Fig 3). Histology also demonstrated a greater collagen deposition in the banded subgroup with severe hypertrophy (Fig 4). In contrast, banded animals treated with clenbuterol or thyroxine with similar hypertrophy had no significant difference in collagen concentration compared with the sham group (Fig 3). Histology also revealed less deposition of collagen in the interstitium than that of the banded-only group (Fig 4). These findings appear to be entirely consistent with the functional data.

View larger version (18K): [in this window] [in a new window]   Figure 3. Collagen concentration as measured by HPLC between groups: sham (n=7), banded with <50% hypertrophy (n=8), banded with >50% hypertrophy (n=5), banded+clenbuterol with >50% hypertrophy (n=8), and banded+thyroxine with >50% hypertrophy (n=4). Values are mean±SEM. *P<.05 vs sham, +P<.05 banded >50% hypertrophy vs banded <50% hypertrophy or banded+clenbuterol or thyroxine >50% hypertrophy.

View larger version (166K): [in this window] [in a new window]   Figure 4. Representative LV sections stained with hematoxylin-eosin demonstrating morphology in the sham group (a) and an increase in interstitial collagen in banded rats with severe hypertrophy (b). Although there was some deposition of collagen in banded rats treated with clenbuterol (c) or thyroxine (d) vs sham group, this was less marked than in banded animals. Scale bars=0.25 mm.

RNA Analyses of the Left Ventricle
As a marker of myocardial hypertrophy, the ventricular expression of ANF was measured and found to be increased in all banded groups (P<.05).

To determine whether the observed changes in systolic function and active relaxation were related to changes in MHC isoforms and expression of SERCA2a and PLB, the gene expressions of the respective mRNAs were measured. The ratio of ß-MHC to total MHC mRNA expression in the banded groups was significantly greater than in the sham group, with no intergroup differences even after the different subgroups of hypertrophy were accounted for.

SERCA2a mRNA expression in all banded groups was significantly reduced in comparison with that of the sham group, although there were no differences between the banded groups (Table 3). Because there were functional differences between rats with severe and moderate hypertrophy, a similar analysis was performed based on the degree of hypertrophy (Fig 5B). The cutoff point for demonstrable differences in SERCA2a mRNA expression was similar to that for functional changes (55% versus 50%, respectively). Banded rats with <55% hypertrophy had no significant difference in expression of SERCA2a mRNA from that of the sham group, whereas those with >55% hypertrophy had only 40.8% of normal expression of SERCA2a mRNA. Rats with >55% hypertrophy by banding and clenbuterol or thyroxine also had a significantly reduced expression of SERCA2a mRNA (59.1% and 64.5%, respectively) versus that of the sham group. However, they were significantly greater than that found in rats with similar hypertrophy by banding alone. No discernible differences were found in PLB mRNA expression between the groups.

View this table: [in this window] [in a new window]   Table 3. Molecular Analyses of LV Tissue From Banded and Clenbuterol- and Thyroxine-Treated Banded Animals

View larger version (23K): [in this window] [in a new window]   Figure 5. A, Representative dot blots of sham, comparably hypertrophied hearts (>55%) by banding (II), banding and clenbuterol (III), or thyroxine treatment (IV). This shows a decrease in ventricular SERCA2a mRNA in sample II and some preservation of SERCA2a expression in samples III and IV. B, Quantification of SERCA2a by densitometry showed significant reduction in banded samples with >55% hypertrophy compared with sham. Among the severely hypertrophied subgroups, the banded+clenbuterol or thyroxine subgroups had greater SERCA2a expression than the banded subgroup. SERCA2a is expressed in arbitrary units (normalized to 18S). *P<.05 vs sham, +P<.05 banded >55% hypertrophy vs banded+clenbuterol or thyroxine >55% hypertrophy.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that functional, collagen-concentration, and gene-expression characteristics of pressure-overload LV hypertrophy may be modulated by the concomitant administration of clenbuterol or thyroxine. Cardiac hypertrophy induced by this means resulted in an improvement in ventricular function, normalization of interstitial collagen, and increased expression of SERCA2a mRNA compared with similarly hypertrophied ventricles by banding alone.

We have previously shown that treatment with the selective ß₂-agonist clenbuterol induces 18% to 26% cardiac hypertrophy in normal rats.^{27 28} In addition, LV function, including normalized developed pressure, rate of contraction [(dP/dt_max)/P], diastolic relaxation (dP/dt_min), and passive stiffness were all found to be normal in these hearts.²⁸ This increase in LV mass was associated with normal collagen concentration and histology unlike that seen with the ß-agonist isoproterenol.^{29 38} Molecular analysis of LV samples revealed normal gene expression of MHC isoforms, SERCA2a, and PLB mRNA.²⁸

This study investigated the potential of clenbuterol to modulate pressure-overload cardiac hypertrophy in a rodent model. Chronic pressure overload is known to induce cardiac hypertrophy with ventricular dysfunction. Diastolic dysfunction occurs early and may manifest as an impairment in myocardial relaxation or an increase in passive stiffness. Impairment in myocardial relaxation has been shown to be secondary to a decrease in intracellular calcium-handling proteins, including SERCA2a and PLB.^{10 11 12 13} Experimentally, a reduced expression of SERCA2a mRNA has been correlated to the severity of hypertrophy¹⁰ and progression of compensated hypertrophy to heart failure.¹¹ Our findings with pressure-overload hypertrophy are consistent with those reported by de la Bastie et al,¹⁰ in which a decrease in the expression of SERCA2a mRNA was found only when cardiac hypertrophy was severe (without the presence of heart failure). This subgroup of banded rats also showed significant impairment in diastolic relaxation and developed pressure.

Furthermore, these banded rats with severe hypertrophy demonstrated an increase in diastolic stiffness and a higher LV collagen concentration. Unlike such models of cardiac hypertrophy as abdominal aortic or renal artery banding, in which interstitial fibrosis may be profound in the presence of moderate hypertrophy,^{5 39} our data suggest that an increase in LV collagen concentration is seen only when the pressure-overload stimulus is extreme enough to produce severe cardiac hypertrophy. This may be attributed to the raised circulating levels of angiotensin seen in the former³⁹ but not with the ascending aortic banding model.¹⁵

The concomitant administration of clenbuterol with pressure overload had a modest additive effect on cardiac mass (16.4%) whereas no change in mass was seen with banding and thyroxine. However, the important finding in this study was that severe hypertrophy induced by banding and clenbuterol or thyroxine was not detrimental to LV function and in fact improved the systolic and lusitropic function compared with the banded subgroup with similar hypertrophy. Thyroxine treatment similarly improved ventricular function, although there was no increase in LV mass. The improved functional changes observed in both these groups may be related to the upregulation seen in the expression of SERCA2a mRNA. Shifts in MHC isoforms are also known to alter contractile function,^{40 41} although such shifts would not appear to account for the improved function seen, because no differences in iso-MHC mRNAs were detected between the banded groups.

In addition, the subgroups of hearts severely hypertrophied by clenbuterol and pressure overload exhibited normal passive stiffness and interstitial collagen. This is a major finding of the study and demonstrates that clenbuterol may prevent interstitial fibrosis during the induction phase of pressure-overload hypertrophy. We can only speculate that this repartitioning agent exerts a differential effect on the rates of collagen synthesis and degradation in the extracellular matrix.

The mechanism of the myotrophic action of clenbuterol on the heart and skeletal muscle has not been elucidated and remains controversial.^{42 43 44} Metabolic studies have shown that clenbuterol increases protein synthesis, with a concomitant decrease in degradation.⁴⁵ The finding of increased LV mass with clenbuterol and pressure overload with significantly less interstitial collagen may be explained by a selectivity in its action on the myocyte compartment. The possible indirect role of clenbuterol in altering the local levels of peptide growth factors in the heart, such as basic fibroblast growth factor⁴⁶ or proto-oncogene expression,⁴⁷ as has been found in the brain and skeletal muscle, respectively, is plausible and needs to be investigated. It is unlikely that the effects of clenbuterol are due to an alteration in the hormonal milieu involving circulating levels of thyroxine and growth hormone, because no significant change in these hormones has been demonstrated in either small- or large-animal experiments.^{48 49}

The experimental findings in pressure-overload hypertrophy have important implications in clinical practice, particularly in the "training" of the LV in surgery for certain types of congenital heart disease. Pharmacological modulation of hypertrophy may improve surgical results, specifically that of the two-stage ASO, by reducing the degree of pressure overload needed to produce ventricular mass and preserving ventricular function. Previously investigated agents such as growth hormone and thyroxine may not be suitable for this purpose because of their extracardiac side effects. In this respect, clenbuterol has the advantage of being relatively free of long-term side effects, although its trophic effect on skeletal muscle has been reported to increase fatigability.⁵⁰

In conclusion, this study demonstrates that clenbuterol may be able to modulate pressure-overload hypertrophy, resulting in an increase in mass with preserved LV systolic and diastolic function. This was also associated with minimal fibrosis and a greater expression of SERCA2a mRNA compared with cardiac hypertrophy from pressure overload alone. These findings could have important clinical and physiological implications.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme ANF = atrial natriuretic factor ASO = arterial switch operation HPLC = high-pressure liquid chromatography LV = left ventricular MHC = myosin heavy chain PLB = phospholamban RT-PCR = reverse transcriptase–polymerase chain reaction SERCA2a = sarcoplasmic reticulum Ca2+-ATPase2a

	Acknowledgments

 
This work was supported in part by a BHF Project Grant (PG/94132) to Dr Boheler. We would like to thank P. Athanasspoulos for performing the HPLC analysis of the ventricular samples, 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.

Received January 2, 1997; revision received April 15, 1997; accepted April 21, 1997.

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