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J Thorac Cardiovasc Surg 1995;109:236-241
© 1995 Mosby, Inc.
CARDIOPULMONARY BYPASS, |
Durham, N.C., La Jolla, Calif., and Houston, Tex.
Durham, N.C.
Supported in part by National Institutes of Health grants HL-16037 (R.J.l.), 5F32-CA09350 (C.A.M.), HL-18468 (P.C.D.), American Heart Association California Affiliate 92-300 (H.A.R.), and NIA-RO1-AG662 (R.A.B.)
Address for reprints: Robert J. Lefkowitz, MD, Duke University Medical Center, DUMC BOX 3821, Durham, NC 27710.
Abstract
Transgenic mice with intense cardiac expression of humanß-adrenergic receptor gene were engineered and shown to display market improvements in baseline myocardial and left ventricular function. Heart/body weight ratios and histologic appearance were not found to be significantly altered, suggesting that receptor gene expression did not induce pathologic changes. Given the substantial reduction inß-adrenergic receptor density and result reduction in inotropic responsiveness observed in chronic heart failure, these finding represent a novel approach for increasing myocardial function with important clinical implications. (J THORACCARDIOVASCSURG1995;109:236-41)
The primary physiologic mechanism for enhancing myocardial performance during stress involves stimulation of myocardial ß-adrenergic receptors (ß-ARs) by endogenous catecholamines. Furthermore, the main therapeutic approach for augmenting myocardial function involves administration of synthetic agonists, which stimulate myocardial ß-ARs (e.g., dopamine and dobutamine). However, in a number of cardiac disease states that result in chronic congestive heart failure, myocardial ß-AR function is impaired and ß1-AR density markedly reduced. 
1 This impairment in the myocardial ß-AR system significantly reduces inotropic responsiveness and may further limit the ability of the myocardium to compensate for the primary disease process. 1
We 2 have previously described the ability to target expression of an exogenous ß-AR gene to the myocardium of transgenic mice. Coupling the deoxyribonucleic acid (DNA) coding sequence of the human ß2-AR gene to an 
-myosin heavy chain promoter sequence can achieve intense myocardium-specific expression of the exogenous ß-AR. 2,3 In this report, enhancement of myocardial function induced by this ß-AR gene expression is described in detail. In addition, morphologic and histologic characterization of the transgenic hearts is presented. Finally in the context of the known reduction in ß-AR number in chronic heart failure, 1 potential therapeutic implications of this work are discussed.
METHODS
DNA construct and generation of transgenic animals
The DNA construct used for generation of the transgenic mice consisted of the human ß2 -AR coding sequence coupled to the -myosin heavy chain promoter. 2,3 This DNA was microinjected into the pronuclei of fertilized single cell mouse embryos, and the embryos were reimplanted into the oviduct of an adult female mouse. 4 Offspring were screened for the presence of the exogenous gene by Southern analysis of DNA extracted from tail cuts, and founder mice were identified. These founders were bred with normal mice to generate a line of transgenic mice; two lines of transgenic mice that contain the exogenous gene (TG4 and TG33) are described in this report. All animal manipulations were conducted after appropriate anesthesia in accordance with institutional review board and National Institutes of Health guidelines (NIH Publication No. 86-23, revised 1985). All data presented were obtained on either transgenic or control adult mice, approximately 2 months of age.
Ligand binding assay
Excised hearts were placed in ice cold lysis buffer (Tris HCl, 5 mmol/L [pH 7.4], and EDTA,* 5 mmol/L) and homogenized. After an initial 500 g centrifugation, the supernatant was removed and centrifuged at 40,000 g and the resultant pelleted membrane fraction was resuspended in binding buffer (Tris HCl, 75 mmol/L [pH 7.4], MgCl2, 12.5 mmol/L, and EDTA, 2 mmol/L.) Saturation binding assays were then performed on the membrane fractions in the presence of [125 I]-cyanopindolol, 500 pmol/L (a ß-AR-specific ligand); nonspecific binding was determined in the presence of alprenolol, 20 µmol/L. Reactions were conducted at 37°C for 1 hour and then terminated by vacuum suction through glass fiber filters. Filters were counted in a gamma counter and receptor density was expressed as mean membrane protein (picomoles per milligram) plus or minus standard error of the mean. 5
Immunohistochemical labeling
Frozen sections were cut at 7 µm for indirect immunofluorescence studies. Sections were rinsed three times for 3 minutes in phosphate-buffered saline solution (PBS) and 3 minutes in PBS with 0.05% Triton X-100 (Triton-PBS), blocked with serum diluent (10% goat serum in PBS with 0.1% bovine serum albumin and 0.1% sodium azide), and then rinsed for 15 minutes in Triton-PBS before overnight incubation at 4°C with a primary rabbit antihuman beta2-AR antiserum 6 (1:500 dilution in serum diluent). The sections were then washed four times for 10 minutes in Triton-PBS at room temperature and incubated for 1 hour in fluorescein isothiocyanate-conjugated goat antirabbit immunoglobulin G (1:50 dilution in serum diluent). After five 3-minute rinses in PBS, the sections were mounted with sodium iodide (25 gm/L) in 1:1 PBS/glycerol solution and photographed.
Isolated myocardial function
After excision of the heart, the atria were dissected and suspended at optimal resting tension in an organ bath in carbogenated modified Krebs bicarbonate solution (NaHCO3, 25 mmol/L [pH 7.4]; NaCl, 118 mmol/L; KCl, 4.8 mmol/L; MgSO4 7H2O, 1.2 mmol/L; KH2 PO4 , 1.2 mmol/L; CaCl2 2H2 O, 1.75 mmol/L; glucose, 10 mmol/L; NaS2O5 , 0.1 mmol/L; EDTA, 0.03 mmol/L), supplemented with ascorbic acid (1.1 x 10-4 mol/L), cocaine (1 x 10-5 mol/L), corticosterone (4 x 10-5 mol/L), and phentolamine (3 x 10-6 mol/L). Left atria were paced with a pulse duration of 3 msec and the voltage set at threshold plus 20%; rates were not different between the two groups. Isometric developed tension was measured at baseline and after administration of isoproterenol, 3 x 10-8 mol/L, the dose that effected a maximum response. Data are given as the means plus or minus standard error of the mean.
In vivo cardiac function
After anesthesia, a cervical incision was performed, the trachea intubated, and the animal connected to a volume-cycled ventilator. A carotid artery was then cannulated with a flame-stretched PE 50 catheter connected to a modified P50 Statham transducer (Viggo Spectramed Inc., Critical Care Div., Oxnard, Calif.). The chest and pericardium were then opened and a 2F high-fidelity micromanometer catheter (Millar Instruments, Inc., Houston, Tex.) was inserted into the left atrium, advanced across the mitral valve, and secured in the left ventricle (LV). Continuous aortic pressures, LV systolic and diastolic pressures, and the derivative of LV pressure (LV dP/dt) were recorded on an eight-channel chart recorder and in digitized form on computer disk for beat averaging (codas, Dataq Instruments, Akron, Ohio). 7 Ten sequential beats were averaged for each measurement (CORDAT, Essen, Germany) and the data were expressed as the mean plus or minus standard deviation. In separate experiments, these parameters were recorded both at baseline and 4 minutes after the systemic administration of 5 micrograms of ICI-118551, a ß2 -AR antagonist.
Heart/body weight ratios and histologic evaluation
Wet heart/body weight ratios were determined as previously described and expressed as the mean plus or minus standard deviation. 8 Hearts were then fixed in a 1% formaldehyde solution and processed for paraffin embedding. LV sagittal sections were then generated, labeled with wheat-germ agglutinin, and myocyte cross-sectional areas were determined. 8
Atrial natriuretic factor (ANF) Northern blots
Ventricular ribonucleic acid (RNA) was extracted, fractionated on a 1% agarose formaldehyde gel, and transferred to a nitrocellulose membrane as previously described. 8 Nitrocellulose membranes were prehybridized and then hybridized with a random-primer radiolabeled ANF cDNA probe 8; blots were stripped and reprobed with the rat glyceraldehyde 3-phosphate dehydrogenase cDNA probe (Ambion, Austin, Tex.).
RESULTS AND DISCUSSION
Immunohistochemistry performed on ventricular myocardial sections with an antiserum specific for the human ß2-AR demonstrated impressive labeling of the transgenic myocardium with virtually no labeling of control tissue (Fig. 1). ß-AR density was quantitated by radioligand binding and was found to be increased approximately 175-fold in the TG4 animals over control levels (Fig. 1, inset). This level of receptor overexpression results from the -myosin heavy chain promoter, which is a strong myocardial promoter. 2,3
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7 and baseline parameters were measured in TG4 transgenic mice and compared with control mice. Heart rates were significantly elevated in the transgenic mice relative to control mice (441 ± 44 versus 349 ± 21 beats/min, respectively; n = 7); mean aortic pressures and LV end-diastolic pressure, however, were not different. Under these conditions, LV maximal dP/dt (LV dP/dtmax ) is an accurate parameter of LV performance, and in the transgenic animals, significant increases in LV dP/dtmax were present relative to control values (Fig. 3, A).
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9*
Given the chronic, marked functional increases that were present in these transgenic mice, animals were evaluated for the development of cardiac disease. Heart/body weight ratios were minimally altered when a large group of transgenic animals were compared with controls (Table I). LV myocyte cross-sectional areas were not significantly increased in the transgenic hearts relative to controls, confirming the absence of hypertrophy (Table I). Furthermore, fibrosis or myocyte necrosis was not present in transgenic myocardial sections. Finally, ventricular ANF mRNA levels, a sensitive molecular marker associated with pressure-overload hypertrophy, 10 were assessed by Northern blot analysis (Fig. 4). A very weak ANF signal was present on Northern blots of normal control ventricular mRNA and mRNA from five TG4 animals. Conversely, mRNA from animals with genetically induced ventricular hypertrophy 8 characteristically had a very strong signal. Equivalent signals obtained with the glyceraldehyde 3-phosphate dehydrogenase probe excluded unequal mRNA loading or degradation. Together, these data suggest that the functional enhancement generated by ß-AR overexpression does not effect significant morphologic or histologic alteration. Furthermore, the normal myocyte gene expression appears unaltered.
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Recent studies have demonstrated the ability of recombinant, replication-deficient adenovirus to transfer genes to the myocardium of adult experimental animals and effect significant levels of expression. 11 Such an approach may be useful for myocardial ß-AR gene transfer and may achieve increased levels of receptor expression capable of enhancing cardiac function. Furthermore, given the specific defects in the ß-AR system in patients with chronic congestive heart failure, therapeutic efforts to transfer ß-AR genes to the myocardium in vivo represent a promising area for future clinical intervention.
Appendix: Discussion
Dr. Stanley K. C. Tam (Boston, Mass.).
In heart failure ß-adrenergic receptors are down-regulated, presumably to protect the myocardium from elevated levels of adrenergic stimuli. What do you think overexpression of this same receptor in a failing heart will do?
Is -myosin heavy chain up-regulated or down-regulated in heart failure as opposed to ß-myosin heavy chain?
Dr. Milano.
Reports have not demonstrated that down-regulation of ß-adrenergic receptors represents a protective effect, and we believe that this down-regulation may inhibit the ability of the myocardium to compensate for the primary disease that is causing the impairment of myocardial function.
The -myosin heavy chain may be down-regulated in heart failure, but this is a species-dependent process. However, other promoter sequences could be used to overexpress the ß-adrenergic receptor.
Mr. Thoralf M. Sundt (Harefield, England).
Two bands appeared to be expressed on your Northern blot. Is that correct, or was I misreading your blot?
Dr. Milano.
There is one predominant band of the appropriate size, which is roughly 2.4 kb. I think the lower labeling represents some degradation of the RNA on that blot.
Dr. Andrew S. Wechsler (Richmond, Va.).
I was surprised that in the isolated atria there appeared to be enhanced intrinsic contractile function, which almost looked greater than the increment that you got with a ß-adrenergic agonist. Were you surprised by that or can you explain that?
Dr. Milano.
Initially we thought we would find a greater response to isoproterenol rather than an increase in baseline function. However, what seems to be happening is that when the receptor is so greatly overexpressed, the percentage of the receptor that remains in the activated state even in the absence of agonist (for example, isoproterenol or catecholamine in the sense of the in vivo setting) becomes significant. Since there is such a large pool of receptors in these transgenic animals, the small fraction that remains activated becomes significant and results in a completely activated system in the absence of agonists.
Acknowledgments
We thank J. Robbins for clone 20 containing the murine myosin heavy chain promoter, B. K. Kobilka for the polyclonal antibody to the human ß2-AR, and K. R. Chien for the murine ANF cDNA.
Footnotes
From the Departments of Surgery,a Medicine,b Pediatrics,c and the Howard Hughes Medical Institute,d Duke University Medical Center, Durham, N.C.; the Department of Medicine,e University of California at San Diego, School of Medicine, La Jolla, Calif.; the Department of Pharmacology,f University of Houston, and the Department of Anesthesiology,g Baylor College of Medicine, Houston, Texas. 
Read at the Seventy-fourth Annual Meeting of The American Association for Thoracic Surgery, New York, N.Y., April 24-27, 1994.
*Ethylenediaminetetraacetic acid.
*Bond RA, Johnson TD, Milano CA, et al. Demonstration of inverse agaopnists in transgenic mice overexpressing the ß2-adrenoceptor. Unpublished data.
*Bond RA, Johnson TD, Milano CA, et al. Demonstration of inverse agaopnists in transgenic mice overexpressing the ß2-adrenoceptor. Unpublished data.
References
-myosin heavy chain gene promoter in transgenic mice. J Biol Chem 1991;266:24613-20.1 B-adrenergic receptor in transgenic mice induces cardiac hypertrophy. Proc Natl Acad Sci U S A [In press].This article has been cited by other articles:
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  Y. Wang, B. Lauffer, M. Von Zastrow, B. K. Kobilka, and Y. Xiang N-Ethylmaleimide-Sensitive Factor Regulates beta2 Adrenoceptor Trafficking and Signaling in Cardiomyocytes Mol. Pharmacol., August 1, 2007; 72(2): 429 - 439. [Abstract] [Full Text] [PDF]
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 L. Hein, M. E. Stevens, G. S. Barsh, R. E. Pratt, B. K. Kobilka, and V. J. Dzau Overexpression of angiotensin AT1 receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block PNAS, June 10, 1997; 94(12): 6391 - 6396. [Abstract] [Full Text] [PDF]
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 C. J. Magovern, C. A. Mack, J. Zhang, R. T. Hahn, W. Ko, O. W. Isom, R. G. Crystal, and T. K. Rosengart Direct In Vivo Gene Transfer to Canine Myocardium Using a Replication-Deficient Adenovirus Vector Ann. Thorac. Surg., August 1, 1996; 62(2): 425 - 433. [Abstract] [Full Text]
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 K. D. Becker, K. R. Gottshall, and K. R. Chien Strategies for Studying Cardiovascular Phenotypes in Genetically Manipulated Mice Hypertension, March 1, 1996; 27(3): 495 - 501. [Abstract] [Full Text]
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