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-adrenergic receptor function influences myocardial responses to isoproterenol stimulation in mice
1Division of Cardiology, Department of Medicine, Case Western Reserve University and University Hospitals of Cleveland, 2Department of Nutrition, Case Western Reserve University, Cleveland, Ohio; 3Cardiovascular Research Institute and Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey—New Jersey Medical School, Newark, New Jersey; 4Department of Pathology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio; and 5Department of Pathology, Emory University, Atlanta, Georgia
Submitted 25 June 2004 ; accepted in final form 24 February 2005
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ABSTRACT |
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-adrenergic receptor (-AR) density and/or coupling. A/J and B6 mice were randomized to receive daily isoproterenol (100 mg/kg sc) or isovolumic vehicle for 5 days. Animals were studied using echocardiography, tail-cuff blood pressure, histopathology, -AR density and percent high-affinity binding, and basal and stimulated adenylyl cyclase activities. One hundred twenty-eight mice (66 A/J and 62 B6) were studied. Isoproterenol-treated A/J mice demonstrated greater percent increases in echocardiographic LV mass/body weight (97 ± 11 vs. 20 ± 10%, P = 0.001) and in gravimetric heart mass/body weight versus same-strain controls than B6 mice. Histopathology scores (a composite of myocyte hypertrophy, nuclear changes, fibrosis, and calcification) were greater in isoproterenol-treated A/J vs. B6 mice (2.8 ± 0.2 vs.1.9 ± 0.3, P < 0.05), as was quantitation of myocyte damage (22.3 ± 11.5 vs. 4.3 ± 3.5%). Interstrain differences in basal -AR density, high-affinity binding, and adenylyl cyclase activity were not significant. However, whereas isoproterenol-treated A/J mice showed nonsignificant increases in all -AR activity measures, isoproterenol-treated B6 mice had lower -AR density (57 ± 6 vs. 83 ± 8 fmol/mg, P < 0.05), percent high-affinity binding (15 ± 2 vs. 26 ± 3%, P < 0.005), and GTP + isoproterenol-stimulated adenylyl cyclase activity (10 ± 1.1 vs. 5.8 ± 1.5 pmol cAMP·mg–1·min–1) compared with controls. High-dose, short-term isoproterenol produces greater macro- and microscopic cardiac hypertrophy and injury in A/J than B6 mice. A/J mice, unlike B6 mice, do not experience -AR downregulation or uncoupling in response to isoproterenol. Abnormalities in -adrenergic regulation may contribute to strain-related differences in the vulnerability to isoproterenol-induced cardiac changes.
echocardiography; myocyte injury
Inbred mouse strains provide a unique opportunity for the study of LVH. Recently, we showed that C57BL/6J (B6) mice, in contrast to A/J mice, have greater end-diastolic dimensions, increased LV mass, and unchanged relative wall thickness (i.e., eccentric LVH) (10). Although these phenotypic differences are well characterized in healthy mice, the effect of a hypertrophic stimulus on LV mass and cardiovascular performance in these two strains is unknown. We postulated that differences in the muscle gene program would induce a greater hypertrophic response in B6 than A/J mice. However, we show that in response to exogenous catecholamine, a well-established cause of myocyte hypertrophy and injury both in vitro and in vivo (19), isoproterenol (Iso) responses are more robust in A/J than B6 mice. We further tested the hypothesis that this disparate response can be explained partly by strain differences in -adrenergic receptor (-AR) density and/or coupling.
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METHODS |
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This investigation conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). All experiments were conducted in accordance with institutional guidelines, and the Institutional Animal Care and Use Committee at Case Western Reserve University approved the experimental protocol.
Study design.
All 128 animals received daily subcutaneous injections of either Iso or isovolumic vehicle (sterile water) for 5 days (days 1–5). Thirty-eight animals (19 A/J and 19 B6) underwent pre- and posttreatment echocardiography (on days 0 and 6, respectively) to assess cardiovascular performance; hearts from 20 of these animals (10 A/J and 10 B6) were subsequently examined histopathologically. Sixty-one animals (31 A/J and 30 B6) had pre- and posttreatment systolic blood pressure measurements. Forty-one of these animals (21 A/J and 20 B6) had blood pressure measured on days 0 and 6; these animals were killed, and their hearts were used for gravimetry. Hearts from 21 of these animals (11 A/J and 10 B6) were subsequently used for -AR binding assays; the remaining hearts were saved for additional studies not reported here. The acute hemodynamic effects of isoproterenol were studied in eight animals (4 A/J and 4 B6); tail-cuff measurements were made between 30 and 60 min and between 2 and 3 h after the subcutaneous injection of Iso. Twenty animals (10 A/J and 10 B6) had tail-cuff blood pressure measurements performed both before and 6 h after the injection on protocol days 0, 1, and 3 to determine the subacute hemodynamic effects of Iso administration. The remaining 21 animals (11 A/J and 10 B6) were killed, and their hearts were used for adenylyl cyclase assays.
Iso administration. Animals were randomly and blindly assigned to receive either Iso (0.01 ml/g body wt of a 10 mg/ml solution, dosed at 100 mg/kg) or isovolumic vehicle (sterile water) via daily subcutaneous injection for 5 days. Animals were injected at approximately the same time each day (12 noon).
Tissue preparation.
Animals were anesthetized on day 6 using a mixture of intraperitoneal ketamine (100 mg/kg), xylazine (20 mg/kg), and acepromazine (3 mg/kg). A median sternotomy was performed, and the heart was removed from the chest. The great vessels were trimmed away using microsurgical scissors; hearts used for gravimetric heart weight determinations were rinsed in iced sterile saline, blotted dry, and inspected for clots, and wet weights recorded on an analytic balance (Mettler; Columbus, OH). In another group, the atria and right ventricle, identified using x10 magnification, were trimmed away with microsurgical scissors. The remaining LV was rinsed in iced sterile saline, placed in 10% buffered formalin, and processed for histopathological examination. Hearts used for the -AR assays were rinsed in iced sterile saline, patted dry, frozen in liquid nitrogen, and stored at –80°C before study.
Blood pressure. Mice were acclimatized to the blood pressure apparatus to reduce stress-related blood pressure variability. The animals were placed in restraining units (2.5 x 10 cm) mounted on a warmed (27–28°C) surface, and their tails were passed through a cuff attached to a custom-built indirect mouse-tail pressure monitoring system (either Micro-Med; Louisville, KY; or Visitech Systems-2000; Apex, NC). Blood flow was detected photoelectrically (Harvard Apparatus; Holliston, MA) and digitally sampled at 200 Hz. The tail cuff was manually inflated to 200 mmHg and released; the first onset of the pulse was recorded as the systolic blood pressure. Blood pressure was taken as an average of three measurements within 5 mmHg; thus, if three consecutive measures varied by >5 mmHg, three additional measurements were obtained until this criterion was satisfied. Heart rate was determined as the average of the three pulse recordings from the corresponding tail-cuff blood pressure measurements for each animal.
Echocardiography. Mice were anesthetized with intraperitoneal 2.5% tribromoethanol (0.01 ml/g), and their chests were shaved and cleaned with alcohol pads. A warming pad (Deltaphase Isothermal Pad; Braintree Scientific, MA) was used to maintain body temperature. Warmed, centrifuged ultrasound transmission gel (Parker Laboratories) was applied to the precordium, and M-mode and two-dimensional echocardiographic studies were performed on each animal using a 15-MHz (15L8) transducer (Sequoia, Acuson; Mountainview, CA). Mice were imaged in the supine and shallow left lateral decubitus positions; short-axis and orthogonal long-axis views of the LV were obtained. Two-dimensional directed M-mode studies were taken from the short axis at the level of the largest LV diameter.
Calculated M-mode echocardiographic variables included LV fractional shortening [FS = (EDD – ESD)/EDD], LV mass [1.06 x (EDD + EDPWTh + EDAWTh)3 – (EDD)3], and LV wall thickness relative to radius [TH/R = (EDPWTh + EDAWTh/EDD)], where EDD is end-diastolic dimension, ESD is end-systolic dimension, EDPWTh is end-diastolic posterior wall thickness, and EDAWTh is end-diastolic anterior wall thickness. All measurements were made from digital images captured at the time of the study using the analysis program resident on the ultrasonograph. M-mode flows were made at a sweep speed of 200 mm/s. Three beats were averaged for each measurement.
Histopathology. Hearts were immersion fixed with 10% buffered formalin, and axial cross sections were made of the LV. Tissues were embedded in paraffin, and replicate 4-µm-thick sections were made at the basal, mid, and apical levels of the LV. These sections were stained with hematoxylin and eosin and Masson trichrome stains, producing a total of 6 sections/heart. Hearts were examined microscopically by a blinded observer. The observer graded each ventricle on a five-point scale (0–4); one point each was scored for increased myocyte size, nuclear changes (hyperchromasia and/or duplication), increased interstitial fibrosis, and dystrophic calcification.
To compliment the visual analysis, quantitation of the histopathological images was performed using a Nikon Axiophot 800 light microscope (Nikon; Garden City, NY) with a Nikon x10 apoplanachromatic primary lens group. The instrument was configured with Image-Pro Media Cybernetics (Image-Pro; Silver Spring, MD) morphometric analysis system. Images were analyzed morphometrically to define zones of damaged myocytes (basophilia, purple hue) versus healthy, viable myocytes (brick red hue) by selecting specific color ranges. Four zones were taken from each transverse section of the LV. The fraction of damaged myocardium was expressed as a percentage of the total tissue area in each frame and compared with ANOVA.
Cardiac -AR binding assays.
Mouse hearts were slowly thawed, minced, and homogenized in 20 volumes of ice-cold HEPES-buffered isotonic sucrose (pH brought to 7.4 with Tris base) containing protease inhibitor cocktail (Boerhinger-Mannheim; Indiapolis, IN) by using a polytron (Tekmar, setting 3 for 2 x 15 s). Homogenates were centrifuged at 1,000 g for 5 min at 4°C to remove nuclei and debris. The pellets (P1) were resuspended and recentrifuged. The combined supernatants were centrifuged at 48,000 g for 18 min at 4°C, and the resulting pellet (P2) was resuspended in 10–25 vol of 50 mM Tris·HCl buffer (pH 7.7) containing 5 mM EDTA. After recentrifugation at 48,000 g for 18 min, the resulting membrane pellet was flash frozen and stored at –70°C.
Radioligand binding assays with 125I-labeled pindolol (125I-pindolol) for determination of specific binding to -AR sites were performed as previously described (4) . Briefly, assays were conducted in a total volume of 250 µl consisting of 125 µl membrane suspension, 25 µl radioligand, and 100 µl of Iso or 0.1% ascorbic acid vehicle. Incubations were carried out for 60 min at 25°C. Nonspecific binding was defined in the presence of Iso (0.1 mM). Incubations were terminated by vacuum filtration over glass fiber filters using a cell harvester (Brandel; Gaithersburg, MD) and read in a gamma counter.
Adenylyl cyclase assays.
Adenylyl cyclase activity was assayed according to the method of Salomon et al. (18). Cardiac membranes were prepared from each individual mouse LV and septum (pooling of hearts was not necessary). The tissue was minced and homogenized in 1 ml of Tris buffer (50 mM Tris·HCl and 2 mM EDTA; pH 7.4) and then homogenized with a Polytron (Brinkman Instruments; Westbury, NY) at setting 6 for 15 s. The homogenate was centrifuged at 41,000 g for 20 min at 4°C. The pellet was resuspended in Tris buffer, homogenized, and centrifuged as above. The pellet was resuspended in Tris buffer. Protein measurements were made by the Bradford method (6). Cardiac membranes (25 µg) were added to a solution containing 1 mM ATP {2–3 x 104 counts/min (cpm) of [
-32P]ATP}, 20 mM creatine phosphate, creatine phosphokinase (1 unit), 1 mM cAMP (2,000–3,000 cpm of [3H]cAMP as an internal standard), 25 mM Tris·HCl, 5 mM MgCl2, 1 mM EDTA, and the test substance to measure adenylyl cyclase activity [GTP (0.1 mM), Iso (0.1 mM), and forskolin (0.1 mM)]. Ten microliters of stopping solution (20 mM ATP, 10 mM cAMP, and 2% SDS) were added to each tube to terminate the reaction, and the tubes were heated on a dry bath at 100°C for 3 min. cAMP was separated as described previously (18, 25). Recovery of added cAMP was 50–80%.
Statistics. All data are expressed as means ± SE. Radioligand binding data were analyzed by nonlinear curve fitting with GraphPad Prism using a rectangular hyperbola for saturation and a logistic equation for competition. Comparisons were made using two-factor (strain and treatment) ANOVA. Within-strain effects of treatment were assessed using paired t-tests. A P value of <0.05 was considered statistically significant.
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RESULTS |
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Microscopically, Iso-treated A/J mice received significantly higher histopathology scores than Iso-treated B6 mice (2.8 ± 0.2 vs. 1.9 ± 0.3, P < 0.05), whereas vehicle-treated B6 mice received higher histopathology scores than A/J mice (1.33 ± 0.33 vs. 0.25 ± 0.25, P = 0.114). Representative LV sections demonstrating myocyte damage and averaged quantitative data are shown in Fig. 2. The fraction of LVs with basophilia was 22.3 ± 11.5% in Iso-treated A/J mice versus 7.6 ± 2.6%, 4.3 ± 3.5%, and 5.0 ± 4.3% in vehicle-treated A/J mice, Iso-treated B6 mice, and vehicle-treated B6 mice, respectively (P < 0.01). These data support the histopathology scores indicating greater damage in the A/J than B6 strain in response to Iso.
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-AR regulation.
Among vehicle-treated animals, there were no significant strain-related differences in -AR density or apparent G protein coupling (percentage of binding sites in the high-affinity state). As shown in Fig. 3, -AR with high affinity for Iso made up about one-quarter of sites and were occupied by low nanomolar concentrations of Iso. The remaining three-quarters of -ARs required 100-fold higher concentrations of Iso. These data are comparable to those obtained in the rat lung (4). Iso treatment produced statistically significant -AR downregulation and uncoupling in B6 mice (Table 4 and Fig. 3). In contrast, Iso-treated A/J animals experienced nonsignificant increases in both -AR density and the degree of high-affinity binding. There were no significant differences in the affinity constants for either 125I-pindolol (Kd) or the high- and low-affinity binding sites for Iso (KiH and KiL, respectively).
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DISCUSSION |
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-AR downregulation (reduced the -AR density), uncoupling (reduced fraction of high-affinity agonist binding sites), or stimulated adenylyl cyclase activity. Importantly, the concentric cardiac hypertrophy that occurred in the A/J mice could not be attributed to a change in systolic arterial pressure. These findings suggest that strain-related differences in -AR signaling contribute to the observed strain-related changes in cardiac mass that are independent of hemodynamic load.
Acutely, high doses of Iso produce myocyte hypertrophy, necrosis, apoptosis, and interstitial fibrosis (3, 21, 22, 24), whereas chronic infusions at lower doses result in ventricular hypertrophy (12, 17) and interstitial fibrosis but fail to demonstrate either necrosis or chronic inflammation (11). Data suggest that -AR downregulation is a protective mechanism against these cellular responses. For example, the susceptibility of myocardium to acute Iso-induced cardiomyocyte injury was reduced in both homologous (9 days of pretreatment with Iso) and heterologous (propylthiouracil) models of -AR desensitization (22). Interestingly, transgenic mice overexpressing Gs fail to fully desensitize, and older animals develop a dilated cardiomyopathy (25); the lack of -AR desensitization in A/J mice is reminiscent of the response in these transgenic animals. Thus these data support the concept that -AR desensitization is potentially protective and that ineffective desensitization mechanisms are deleterious (26). Adenylyl cyclase activities confirm these data and are consistent with strain-dependent alterations in -AR signaling distal to the -AR.
The mechanisms through which exogenous -adrenergic stimulation promotes cardiac hypertrophy are incompletely understood. An in vivo animal study (19) has implicated catecholamine-induced increases in hemodynamic load as the primary contributor to hypertrophy. However, our findings are consistent with other studies employing cultured adult and neonatal myocytes that suggest that cell growth results from excessive -adrenergic stimulation in the absence of an abnormal hemodynamic load. Whereas -adrenergic stimulation promotes myocyte protein synthesis predominantly via the 1-receptor and its subsequent activation of phospholipase C through the Gq signaling pathway, -AR stimulation appears to promote hypertrophy through a more diverse array of interacting intra- and extracytoplasmic signaling cascades (14). For example, Iso produces modest activation of the MAPK cascades mediated by Gs (via PKA) and Gi (via Ras) (5, 27, 28). In addition, -agonists can promote phosphorylation and activation of Akt (with a resultant decrease in glycogen synthase kinase 3 expression) (15) and activation of IL-6 synthesis in cardiomyocytes and fibroblasts (7). Studies have also suggested roles for ornithine decarboxylase (20) and calcineurin (28) in -agonist-induced hypertrophy in mice. Irrespective of the mechanisms, these data indicate that strain-dependent differences in the -AR and signaling in response to short-term, high-dose Iso are associated with differential hypertrophic responses.
Limitations.
Several potential limitations and caveats should be addressed. First, rodent models of excessive -AR signaling, whether due to exogenous catecholamines or to overexpression of various -AR components, are usually studied in young animals in whom LV function is either preserved or enhanced (26), suggesting that -AR stimulation may be beneficial. Indeed, the LV fractional shortening increased a small but insignificant degree in our animals treated with Iso. However, older animals with transgenic forced overexpression of either Gs or 1-AR develop dilated cardiomyopathy (8, 25). In our study, there was histopathological evidence of myocyte necrosis in animals treated with Iso, predominantly among the A/J mice. Thus our model is best considered as one of myocardial hypertrophy and injury. Although one cannot exclude the possibility that baseline differences in LV mass and blood pressure between the two strains are responsible in part for altered vulnerability to catecholamines, we clearly demonstrate echocardiographic, biochemical, and histopathological strain-related differences in response to high-dose Iso in mouse hearts.
Second, the dose and duration of Iso infusion used to produce LVH were highly variable (11, 12, 21, 22, 24). In this regard, there are several advantages to our short-term, high-dose subcutaneous model, including greater ease of administration, shorter duration of treatment, lower cost, and (in the absence of an implanted minipump) greater animal mobility. However, extrapolating the results from this model to other hypertrophy models, such as hypertension and aortic banding, may not be reliable. Finally, the study describes a single "snapshot" in time; however, remodeling is a progressive, dynamic response to injury. Thus two subcutaneous injections of 85, 170, or 340 mg/kg Iso produced dose-dependent increases in LV end-diastolic pressure, volume indexes, and wall stress and impaired systolic function and cardiac output over16 wk (23, 24). Finally, Iso stimulates both 1- and 2-receptors, which were not separately measured in this study. However, we detected few 2-receptors in our preparations (data not shown), and it is the 1-subtype, not the 2-subtype, that primarily mediates the hypertrophic response to Iso in rodents (16).
In conclusion, short-term, high-dose Iso treatment produces significantly greater cardiac hypertrophy and histopathological changes in A/J than B6 mice. These strain-dependent differences may be partly due to observed variability in downregulation and uncoupling of -ARs and are consistent with the notion that receptor downregulation and uncoupling are cardioprotective, at least to the hypertrophic and histopathological effects of high-dose Iso administration. Finally, we describe a model for the toxic and hypertrophic effects of -AR stimulation.
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ACKNOWLEDGMENTS |
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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.
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REFERENCES |
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-adrenergic receptors in rat lung: upregulation by chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 265: L389–L394, 1993.-Adrenergic stimulation causes cardiocyte apoptosis: influence of tachycardia and hypertrophy. Am J Physiol Heart Circ Physiol 275: H961–H968, 1998.-Adrenergic receptor desensitisation may serve a cardioprotective role. Cardiovasc Res 26: 608–614, 1992.[Web of Science][Medline]
-Adrenergic receptor signaling: an acute compensatory adjustment inappropriate for the chronic stress of heart failure? Insights from Gsalpha overexpression and other genetically engineered animal models. Circ Res 86: 502–506, 2000.This article has been cited by other articles:
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 A. El-Armouche, K. Wittkopper, F. Degenhardt, F. Weinberger, M. Didie, I. Melnychenko, M. Grimm, M. Peeck, W. H. Zimmermann, B. Unsold, et al. Phosphatase inhibitor-1-deficient mice are protected from catecholamine-induced arrhythmias and myocardial hypertrophy Cardiovasc Res, December 1, 2008; 80(3): 396 - 406. [Abstract] [Full Text] [PDF]
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D. B. Walker Serum Chemical Biomarkers of Cardiac Injury for Nonclinical Safety Testing Toxicol Pathol, January 1, 2006; 34(1): 94 - 104. [Abstract] [Full Text] [PDF]
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P < 0.05 and 
P < 0.001, vehicle vs. Iso-treated A/J mice.
