(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
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.
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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
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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.
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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/dtmax)/P] were used as indices of systolic
function. The maximum rate of pressure decay (dP/dtmin) 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[eB(EDV-V°)-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 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.
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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).
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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).
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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/dtmax)/P was also found to be
significantly reduced. In addition, the subgroup of rats with severe
hypertrophy exhibited a reduction in dP/dtmin
(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.
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, 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/dtmax)/P and
dP/dtmin 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.
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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.
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Discussion |
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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/dtmax)/P], diastolic relaxation (dP/dtmin), 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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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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