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-agonist administration affects cardiac function of adult but not old rats, independent of -adrenoceptor density
1Department of Physiology, The University of Melbourne, Melbourne, Victoria; and 2School of Agriculture, Charles Sturt University, Wagga Wagga, New South Wales, Australia
Submitted 13 December 2004 ; accepted in final form 18 February 2005
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ABSTRACT |
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-adrenoceptor agonists have clinical merit for attenuating the age-related loss of skeletal muscle mass and strength (sarcopenia), potential cardiac-related side effects may limit their clinical application. The aim of this study was to determine whether chronic -agonist administration impairs cardiac function in adult or aged rats. Adult (16 mo) and aged (28 mo) Fischer 344 rats were treated with fenoterol (1.4 mg·kg–1·day–1 ip) or vehicle for 4 wk. Heart function was assessed in vitro before analyses of cardiac structure and -adrenoceptor density. Heart mass increased 17% and 25% in fenoterol-treated adult and aged rats, respectively. The increased heart mass in aged, but not adult, rats was associated with a relative increase in collagen content. Cardiac hypertrophy in adult rats was associated with an increase in left ventricular developed pressure, a marked reduction in cardiac output, and a reduction in coronary flow per unit heart mass. In contrast, negligible differences in ventricular function were observed in fenoterol-treated aged rats. The differential effect on contractile function was not associated with age-related differences in -adrenoceptor density but, rather, an age-related increase in downregulation after treatment. Our results show that chronic -agonist treatment impairs cardiac function to a greater extent in adult than in aged rats. These results provide important information regarding the potential effects of chronic -agonist use on cardiac function and the future development of safe and effective treatments for sarcopenia.
adrenergic agonists; aging; hypertrophy; ventricular function; receptors
Whereas local administration of -adrenoceptor agonists (-agonists), by inhalation, is indicated for bronchodilation in the treatment of asthma, their systemic administration at higher doses can stimulate muscle hypertrophy (8). We have identified fenoterol as a potent -agonist that is capable of reversing age-associated muscle wasting and weakness in rats (20, 21). However, systemic administration of fenoterol in this manner can also stimulate cardiac hypertrophy (20). These data demonstrated that although -agonists have potential as an intervention for age-associated deterioration of skeletal muscle, they can have potentially deleterious effects on the heart, where 1- and 2-adrenoceptor subtypes influence myocardial contractility (16).
Some early clinical and preclinical observations have implicated -agonists in the development of hypertrophic myocardial pathology (2, 3, 7, 10, 12, 16, 24). Furthermore, recent reports suggest that chronic use of -agonists for prevention and relief of asthma symptoms may be associated with increased risk of adverse cardiovascular-related events (22). Therefore, if safe and effective treatments for sarcopenia are to be developed using -agonists, it is important to determine the effects of sustained medication on cardiac function and whether aging alters the response to these compounds.
There are few data that describe the effects of sustained -agonist administration on the contractile properties of the heart, and even less is known about cardiac adaptations to chronic -agonist administration in aged patients and animals. Experiments using left ventricular (LV) cardiomyocytes, isolated from rats, suggest that the contractile response to -agonists decreases with age (29). The reason for this decrease remains controversial but may result from decreased adrenoceptor density and/or function (14).
The aim of the present study was to characterize the effects of chronic -agonist administration on cardiac structure and function and to compare the responses of adult and aged rats. We tested the hypothesis that potent stimulation of myocardial -adrenoceptors by repeated administration of the 1- and 2-adrenoceptor agonist fenoterol would induce deleterious changes in cardiac structure and function of the hearts of adult and, to a lesser extent, aged rats. We observed that although fenoterol administration increased heart mass in adult and aged rats, only adult rats exhibited significant functional effects. These findings provide important preclinical information regarding the potential deleterious effects of chronic -agonist use on cardiac function and whether these effects are altered by advancing age.
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MATERIALS AND METHODS |
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Adult (16 mo old, n = 13) and aged (28 mo old, n = 11) male Fischer 344 rats (460–500 g; Harlan Sprague Dawley, Indianapolis, IN) were randomly allocated to a control or a treated group. Fenoterol (1.4 mg/kg ip; Sigma Chemical) in isotonic saline (
0.5 ml volume) was administered to the treated group once daily for 28 consecutive days. Untreated rats received an equivolume injection of isotonic saline. In previous studies, this dose and protocol of fenoterol administration have proved sufficient to restore skeletal muscle mass in aged rats with age-related muscle atrophy (21).
Perfusion of isolated hearts. Cardiac function was assessed in vitro on the basis of established techniques (18, 26). At 24 h after the last treatment, the rats were anesthetized with pentobarbital sodium (Nembutal, Rhone Merieux, Pinkenba, QLD, Australia; 60 mg/kg ip) and treated with heparin (2,000 U/kg). The chest cavity was opened, and the heart was rapidly excised and placed into ice-cold modified Krebs-Henseleit buffer (KHB; in mM: 118 NaCl, 24.9 NaHCO3, 11.1 D-glucose, 5 sodium pyruvate, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, and 0.5 EDTA, pH 7.4). A cannula (12 mm long, 1.8 mm ID) was placed in the aorta of the excised heart, tied with 4-0 silk suture, and attached to a column containing recirculating operating KHB (buffered with 95% O2-5% CO2 and maintained at 37°C), which provided the heart with retrograde perfusion of oxygen and substrate at 90 mmHg. The column was equipped with a compliance chamber containing 2 ml of air to provide the system with aortic compliance. The time from excision to commencement of reperfusion was usually <60 s.
Subsequent to establishment of "Langendorff" perfusion of the heart, the left atrium was located and cannulated (12 mm long, 1.8 mm ID) through the pulmonary vein and connected to a recirculating "preload" column containing buffered, thermoregulated KHB, which, when opened, provided the heart with antegrade perfusion of oxygen and substrate at 20 mmHg ("working heart" mode). Other apertures associated with the left atrium were sutured closed to eliminate leakage from the left atrium in the working-heart mode. A KHB-filled 25-gauge steel syringe needle connected to a pressure transducer (model MLT0698, ADInstruments, Castle Hill, NSW, Australia) was inserted into the LV through the apical portion of the heart for measurement of intraventricular pressure. Contractile and flow measurements were obtained under autonomous (basal) pacing conditions; then the ventricle was externally stimulated at 5 Hz [heart rate (HR) 300 beats/min; S88 stimulator, Grass Instruments, Quincy, MA]. Setup time for the heart preparation from excision to full working heart mode was typically <10 min. Contractile measurements were recorded using a four-channel PowerLab recorder (ADInstruments) run by a personal computer operating Chart data-acquisition software (version 4.1.2, ADInstruments). Data collection was completed within 20 min of establishment of the working heart mode. In-house stability tests of the preparation and apparatus have demonstrated maintenance of stable contractile parameters for >60 min, thereby verifying the reliability of short-term acquisition of data as described in the present study.
After functional testing, the heart was removed from the apparatus, trimmed of any adhering nonmuscle tissue, blotted quickly on filter paper, and weighed on an analytic balance. Hearts were subsequently bisected at the midventricle region, snap frozen in isopentane cooled in liquid nitrogen, and stored at –80°C for later histological examination and radioligand binding assays.
Morphological analyses.
The ventricles of each frozen heart were cryosectioned transversely from the apex to the midventricle region on a cryostat microtome at –20°C (CTI cryostat, IEC, Needham Heights, MA). Transverse sections (8 µm thick) of each heart were obtained from the apex and midway through the ventricles and placed onto uncoated glass microscope slides. Sections were stained with hematoxylin and eosin for examination of general tissue morphology. Collagen localization was determined by incubation of frozen sections in 1:3 acetic acid-alcohol for 10 min and then van Gieson stain (0.05 mM 1% acid fuchsin and 16.5 mM saturated picric acid). Stained sections were rinsed in ethanol, and coverslips were applied. A field of view covering the entire cross section was chosen for sections from the base and midway through the ventricles, and collagen was localized within the image. Images of all sections were acquired using a digital imaging camera (Spot model 1.3.0, Diagnostic Instruments, Sterling Heights, MI) attached to an upright microscope (model BX-51, Olympus, Tokyo, Japan). Image files were analyzed using an Analytical Imaging Station (version 6.0, Imaging Research Ontario) in a double-blind manner. The mean cross-sectional area (CSA) of individual myocytes was calculated by interactive determination of the circumference of 
150 adjacent longitudinal myocytes from the musculature of the LV.
Radioligand binding assays.
Ventricular tissue was assayed for 1- and 2-adrenoceptor density using methods described in detail previously (20, 21). Briefly, frozen tissues were homogenized and centrifuged to obtain the cell membrane fraction for analysis. Cell membrane samples were assayed for -adrenoceptor subtypes utilizing competitive radioligand binding with [125I]iodocyanpindolol in the presence and absence of selective adrenoceptor subtype antagonist (CGP-20712A). Results are expressed as receptor concentration per milligram of protein (20, 21). Protein concentration was measured using the Bradford technique.
Statistical analyses. Values are means ± SE. Data were compared between experimental groups using a two-factor ANOVA for influ-ences of treatment type and/or age, with Fisher's least-significant difference post hoc multiple-comparison procedure to identify differences between specific groups. In all cases, differences between groups were considered significant when P < 0.05.
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RESULTS |
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In aged rats, body mass increased 11% after fenoterol treatment; thereby, a mass similar to that in untreated adult rats was attained. Fenoterol-treated aged rats demonstrated a 25% greater heart mass than untreated rats, such that the heart mass-to-body mass ratio was 9% greater than in aged controls (P < 0.05). In contrast to fenoterol-treated adult rats, the increase in heart mass of treated aged rats was not associated with any change in LV myocyte CSA. However, there was a 6% increase in mid-LV collagen deposition (P < 0.05; Fig. 2).
Contractile properties.
All references to cardiac contractile performance are derived from recordings of the LV and volumetric output collected from the aorta and coronary vasculature. Once reanimated and equilibrated on the perfusion apparatus, the hearts of untreated adult and aged rats maintained an autonomous ex vivo contraction frequency (HR) of 233 ± 7 and 213 ± 16 beats/min, respectively (not statistically different; Table 1). There was no difference in the rate of relaxation (–dP/dtmin) between hearts of aged and untreated adult rats, although half-relaxation time (RT
) was prolonged in aged compared with adult rats (Table 1). No other differences in cardiac function were observed between adult and aged control rats as measured by contractile parameters and flow rates.
Fenoterol treatment reduced the HR of adult rats by 16% (P < 0.05) and increased LV systolic pressure by 18% compared with adult control rats. In association with the increase in LV systolic pressure, fenoterol increased LV developed pressure in adult rats by 18%. Fenoterol treatment did not alter LV diastolic pressure, peak rate of intraventricular pressure development (+dP/dtmax), or –dP/dtmin in adult rat hearts but prolonged RT 16 ms (28%) compared with hearts from untreated adult rats (P < 0.05; Table 1). The fenoterol intervention did not affect the mean cardiac power [i.e., product of mean arterial pressure and cardiac output (CO)] (5) of adult rat hearts (Table 1).
In contrast to the effects in adult rats treated with fenoterol, no difference in HR was detected between treated and untreated aged rats (Table 1). LV diastolic pressure and LV systolic pressure were comparable in the self-paced ex vivo hearts of aged control and aged treated rats, such that there was no difference in LV developed pressure between control and treated aged rats. As with adult rats, no differences in +dP/dtmax and –dP/dtmin or mean cardiac power were observed in aged rats after treatment with fenoterol, but fenoterol prolonged the RT of hearts from aged rats by 12.5% (Table 1).
Cardiac flow rates for adult and aged control and treated rats are presented in Table 2. Total CO [i.e., sum of aortic flow and coronary flow (CF)] was not different for the hearts of adult and aged control rats during autonomous pacing (Table 2). Because heart mass did not appear to change as a function of age, there was no change in aortic flow corrected for heart mass. The lack of change in CO and HR was mirrored by no change in calculated stroke volume (SV) between the hearts of adult and aged rats (Table 2).
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Fenoterol did not significantly reduce CO in aged rats. As such, fenoterol had no effect on the absolute or relative CF indexes in the hearts of aged rats (CF ÷ body mass; Table 2). SV was not altered by fenoterol treatment in aged rats.
Examination of cardiac function under external pacing conditions at 5 Hz (HR = 300 beats/min) did not show further impairments in cardiac function in any of the parameters measured under autonomous (basal) conditions (data not shown).
-Adrenoceptor density.
In hearts of untreated adult and aged rats, density and proportion of 1- (60%) to 2-(40%) adrenoceptors were similar (Fig. 3). In adult rats, fenoterol treatment reduced total -adrenoceptor density by 34%, which was accounted for entirely by a 58% reduction in 1-adrenoceptors (P < 0.05; Fig. 3). In aged rats, fenoterol treatment reduced total -adrenoceptor density by 59%, comprising a 71% reduction in 1-adrenoceptors and a 43% reduction in 2-adrenoceptors (Fig. 3). Although fenoterol elicited different changes in adrenoceptor density in the hearts of adult and aged rats, both treated cohorts experienced a shift in the ratio of 1- to 2-adrenoceptor density compared with untreated rats toward a majority of 2-receptors.
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DISCUSSION |
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-agonist fenoterol can stimulate significant cardiac hypertrophy in adult and aged rats, but only adult rats exhibited a severe impairment in cardiac function. Although the effects of acute -adrenoceptor agonist administration have been studied extensively in single myocytes and whole hearts (28), comparatively little is known about the effects of chronic agonist administration, and even less is known about the response in aged subjects. This is the first study to demonstrate that the effects of repeated administration of a -agonist on cardiac contractile properties vary as a function of a subject's age.
Cardiac morphology and function were comparable between untreated adult and aged rats, which suggests that age-related changes in normal cardiac function are largely completed during maturation from birth to adulthood (16 mo) or that significant effects do not commence in Fischer 344 rats before 28 mo of age. The hypertrophy induced by fenoterol in adult and aged rats is consistent with previously well-documented accounts of high-dose 2-agonist treatment (10, 20, 27). Our findings indicate that, despite equivalent changes in cardiac mass in adult and aged rats, an agonist-induced decrease in cardiac function is not observed in the latter. The mechanisms responsible for these different responses to -agonist administration warrant further attention.
Cardiac hypertrophy in fenoterol-treated aged rats was associated with an increase in midventricular collagen deposition, whereas adult rats exhibited no change in collagen with treatment. However, increased collagen in aged rats did not reduce cardiac function at rest. The increased collagen infiltration in aged rats was associated with prolonged relaxation. Such an effect could reduce diastolic filling time, which would become progressively more evident as HR increases, an effect that might impair cardiac function during exercise. Previous studies have found that -agonist treatment can increase collagen content in the heart (7), but the effect on function has not been fully characterized. We suggest that the fenoterol-mediated increase in collagen in the hearts of aged rats is not sufficient to impair function and that other mechanisms are responsible for the detrimental effects of fenoterol in adult rats. Furthermore, the 2-agonist clenbuterol at 5 mg/kg has been found (in rats) to cause a low level of myocyte apoptosis (4). Thus it is possible that similar damage contributed to the observed increase in collagen infiltration in aged rat hearts (present study) after fenoterol treatment.
In contrast to Wong and colleagues (27), who demonstrated that clenbuterol did not cause detrimental changes in cardiac function in adult rats, our results indicate that fenoterol is detrimental to cardiac function in adult rats. This discrepancy may be due to the limitations of the Langendorff technique employed by others, inasmuch as our results demonstrate that fenoterol impairs CF and, therefore, cardiac function, when assessed by a "working"-heart technique (3, 7, 24).
In contrast to acute administration of a -agonist, which causes a reduction in ventricular relaxation time, chronic fenoterol treatment prolonged RT. Acute stimulation of the 1- and 2-adrenoceptor-Gs pathways of the heart increases the activity of the cAMP-dependent protein kinase (PKA), which is known to phosphorylate phospholamban (23). The phosphorylation of phospholamban disinhibits sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), thus increasing SERCA activity and enhancing Ca2+ uptake potential (13). Our data demonstrating the prolongation of RT in hearts of adult rats treated chronically with fenoterol could be explained by the downregulation of 1-adrenoceptors, contributing to a reduction in phospholamban phosphorylation and a resultant increase in inhibition of SERCA activity. This hypothesis is supported by the observations of others considering alternative models of pathological cardiac hypertrophy, where there is a decreased response of the rate of relaxation after administration of the nonselective 1- and 2-adrenoceptor agonist isoproterenol, attributed to altered phosphorylation of phospholamban (25). Further investigation into the effect of chronic -agonist administration is required to confirm this hypothesis.
Our results show that, despite the absence of fenoterol-induced changes in cardiac function in the hearts of aged rats, there was no age-associated decrease in 1- or 2-adrenoceptor density. The exact mechanisms for the age-associated decline in cardiac 1- and 2-adrenoceptor signaling remain controversial but are likely due to a multitude of factors (28). Our results suggest that pathways involved in adrenoceptor downregulation and degradation after activation may play an important role.
The continued exposure of -adrenoceptors to an agonist initially results in a rapid attenuation of receptor responsiveness (desensitization) and, if the agonist is not removed, a decrease in -adrenoceptor number (downregulation) (9). Desensitization occurs when the -adrenoceptor becomes phosphorylated by PKA, PKC, or G protein-coupled receptor kinases. The phosphorylated adrenoceptor promotes the binding of cytosolic proteins called -arrestins. The interaction of -arrestins with adrenoceptors renders the adrenoceptor completely inactive or desensitized. Subsequent to receptor desensitization, the phosphorylated component is internalized in a clathrin-coated pit, where it is dephosphorylated and recycled to the cell surface for reactivation or degraded by lysosomes and, therefore, subtracted from the functional adrenoceptor population. The result of an increased downregulation in the hearts of aged rats suggests an increased signal for adrenoceptor degradation, rather than dephosphorylation and recycling. The increased susceptibility to downregulation is likely responsible for the absence of a fenoterol-mediated change in cardiac function of aged compared with adult rats. Additionally, the de novo synthesis of adrenoceptors as a mechanism of regulating receptor population in parallel with receptor degradation may differ between adult and aged subjects. Motifs identified in the 5'- and 3'-untranslated regions flanking the human -adrenergic receptor genes represent sites of interaction by signal transduction elements stimulated via -adrenoceptor activity. The net effects of these feedback mechanisms influence transcriptional activity and RNA stability during translation (11). It is conceivable that the activity of signal transduction elements utilized by -adrenoceptors varies with advancing age in a manner that not only stimulates receptor degradation but also suppresses receptor expression. The effect of advancing age on the interplay of factors influencing receptor degradation and synthesis is thus a point deserving of deeper investigation.
Some of the most serious consequences of aging relate to its effects on skeletal muscle, including a loss of muscle mass and strength (6, 19). As the number and proportion of older persons in the population continues to escalate, so does the need for therapeutics that aim to restore mass and strength. Previous experiments in rats indicate that fenoterol has a beneficial effect on skeletal muscle and is able to reverse the sarcopenia observed in limb muscles (21). The absence of detrimental effects on cardiac function in aged rats, at the same dose of fenoterol that prevents limb muscle atrophy, is encouraging. However, the increased heart mass associated with treatment of aged rats warrants attention, inasmuch as cardiac hypertrophy can be associated with increased cardiovascular events and mortality (1).
Although -agonists show considerable potential as an intervention for sarcopenia, much research is needed to test their efficacy and safety, especially the need to separate skeletal muscle and cardiac effects. Such issues need to be addressed before these agonists can be recommended for clinical application.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* P. Gregorevic and J. G. Ryall contributed equally to this work. 
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P < 0.05 vs. adult fenoterol.