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(Circulation. 2000;101:408.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Intracoronary Adenovirus-Mediated Delivery and Overexpression of the ß₂-Adrenergic Receptor in the Heart

Prospects for Molecular Ventricular Assistance

Ashish S. Shah, MD; R. Eric Lilly, MD; Alan P. Kypson, MD; Oliver Tai, BS; Jonathan A. Hata, BA; Anne Pippen, BS; Scott C. Silvestry, MD; Robert J. Lefkowitz, MD; Donald D. Glower, MD; Walter J. Koch, PhD

From the Departments of General and Thoracic Surgery (A.S.S., R.E.L., A.P.K., O.T., J.A.H., S.C.S., D.D.G, W.J.K.), Pharmacology and Cancer Biology (W.J.K.), Medicine and Biochemistry (A.P., R.J.L.), and The Howard Hughes Medical Institute (R.J.L.), Duke University Medical Center, Durham, NC.

Correspondence to Walter J. Koch, PhD, Laboratory of Molecular Cardiovascular Biology, Box 2606, MSRB Room 471, Duke University Medical Center, Durham, NC 27710. E-mail koch0002{at}mc.duke.edu

	Abstract

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Background—Genetic modulation of ventricular function may offer a novel therapeutic strategy for patients with congestive heart failure. Myocardial overexpression of ß₂-adrenergic receptors (ß₂ARs) has been shown to enhance contractility in transgenic mice and reverse signaling abnormalities found in failing cardiomyocytes in culture. In this study, we sought to determine the feasibility and in vivo consequences of delivering an adenovirus containing the human ß₂AR cDNA to ventricular myocardium via catheter-mediated subselective intracoronary delivery.

Methods and Results—Rabbits underwent percutaneous subselective catheterization of either the left or right coronary artery and infusion of adenoviral vectors containing either a marker transgene (Adeno-ßGal) or the ß₂AR (Adeno-ß₂AR). Ventricular function was assessed before catheterization and 3 to 6 days after gene delivery. Both left circumflex– and right coronary artery–mediated delivery of Adeno-ß₂AR resulted in 10-fold overexpression in a chamber-specific manner. Delivery of Adeno-ßGal did not alter in vivo left ventricular (LV) systolic function, whereas overexpression of ß₂ARs in the LV improved global LV contractility, as measured by dP/dt_max, at baseline and in response to isoproterenol at both 3 and 6 days after gene delivery.

Conclusions—Percutaneous adenovirus-mediated intracoronary delivery of a potentially therapeutic transgene is feasible, and acute global LV function can be enhanced by LV-specific overexpression of the ß₂AR. Thus, genetic modulation to enhance the function of the heart may represent a novel therapeutic strategy for congestive heart failure and can be viewed as molecular ventricular assistance.

Key Words: gene therapy • myocardium • receptors, adrenergic, ß • ventricles • heart failure • signal transduction

	Introduction

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Novel treatment strategies for ventricular dysfunction are of great importance, and the area of gene therapy is gaining increasing attention. Traditional approaches to the treatment and palliation of ventricular dysfunction and failure include medical management,¹ myocardial revascularization,² valve repair,³ and mechanical assist and transplantation.⁴ Although there have been advances in these therapies, congestive heart failure (CHF) remains a leading cause of death in the United States and worldwide.⁵ Thus, a molecular approach for treating the failing heart is attractive. Early work in cardiac gene therapy has concentrated on 2 distinct problems: reliable methods of delivery to working myocardium and identification of potential molecular targets. First, work by several groups demonstrated the feasibility of delivering transgenes via direct intramyocardial injection,^{6 7} ex vivo perfusion,^{8 9} and finally a transluminal intracoronary approach.¹⁰ Second, a spectrum of molecular targets has emerged through work done in genetically engineered mice.¹¹

One molecular target identified is the overexpression of the ß₂-adrenergic receptors (ß₂ARs). Transgenic mice with cardiac-specific overexpression of the human ß₂AR at either >100-fold over endogenous ß₂AR density¹² or significantly lower overexpression¹³ have enhanced contractility without overt pathological conditions. These models were developed to replace receptors that are lost during the development of CHF. In the failing heart, there is a 50% reduction of myocardial ß-adrenergic receptors (ß-ARs), with remaining receptors being functionally uncoupled.¹⁴ In addition to the positive phenotype of the ß₂AR-overexpressing mice, adenovirus-mediated overexpression of ß₂ARs in failing rabbit ventricular cardiomyocytes in culture has resulted in the functional rescue of the signaling abnormalities present in failing heart cells.¹⁵ Thus, genetically replacing lost ß-ARs in the failing heart represents a potentially novel therapeutic strategy to increase inotropy.

The primary hurdle to testing the feasibility of ß₂AR gene transfer in vivo is the development of a clinically relevant gene delivery system. Recent work by our laboratory¹⁶ and others¹⁷ has shown that it is possible to deliver transgenes globally to the myocardium by adenoviral vectors. In these 2 studies, adenoviruses were delivered via a surgically invasive approach in which the transgenes are injected into the left ventricular (LV) cavity while the aorta is cross-clamped, directing the adenoviral solution to perfuse the coronary arteries.^{16 17} Using this method, we showed that overexpression of ß₂ARs in the rabbit heart does enhance global in vivo LV function.¹⁶ Because this method of gene delivery has its limitations, we explored the feasibility of delivering the ß₂AR transgene to the rabbit heart in vivo via percutaneous subselective coronary catheterization and injection. A previous report showed that transluminal intracoronary artery delivery of marker transgenes in the rabbit is possible; however, that report did not include a study of myocardial function.¹⁰ The purpose of our study was to develop a reproducible percutaneous subselective intracoronary artery delivery method for efficient ventricle-targeted in vivo gene transfer of adenoviral transgenes to rabbit myocardium. Furthermore, we investigated whether ventricular overexpression of the ß₂AR in the rabbit heart could alter biochemical and in vivo cardiac function.

	Methods

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Adenoviral Constructs
Adenoviral constructs using a "first-generation" E1/E3-deleted replication-deficient adenovirus have been described previously.^{9 15} The ß₂AR construct (Adeno-ß₂AR) and the marker transgene ß-galactosidase (Adeno-ßGal) were driven by the CMV promoter. Large-scale preparations of these adenoviruses were purified from infected Epstein-Barr nuclear antigen–transfected 293 cells as described.^{9 15}

Percutaneous Intracoronary Adenovirus Delivery and Cardiac Functional Assessment
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the NIH. Forty-seven adult male New Zealand White rabbits (3 kg) were used in the present study. At the time of the initial study, animals were sedated with ketamine (50 mg/kg) and acepromazine (0.25 mg/kg), and an incision was made over the right neck to expose the right carotid and jugular vessels. A 2.5F micromanometer (Millar Inc) was placed into the LV cavity via the carotid artery under fluoroscopic guidance, and a 22-gauge angiocatheter was placed into the jugular vein. The micromanometer was coupled to a PC-based data acquisition system (Physiological Systems Inc, and LV pressure was obtained at baseline and after the infusion of isoproterenol at 0.5 µg · kg^-1 · min^-1 for 20 minutes as described.¹⁶ All hemodynamic studies were performed in a closed-chest model with an intact pericardium. After ventricular functional assessment of individual rabbits, a 4.5F radial arterial line sheath (Cook Inc) was placed into the carotid artery, and a 3F coronary catheter (Cook Inc) was placed into the left circumflex (LCx, n=37) or right coronary artery (RCA, n=10) under fluoroscopic guidance to deliver the adenoviral transgenes. The coronary catheter used to perform these subselective coronary artery adenovirus injections was a simple right-angle catheter with a single distal port (Cook Inc). Before virus infusion, adenosine (1.5 mg), lidocaine (0.33 mg), and heparin (500 U) were given via the jugular vein, and 5x10¹¹ total viral particles (TVP) of a replication-deficient adenovirus in 2.5 mL of PBS at 37°C was injected as a bolus into the respective coronary artery. In addition to adenovirus, a subset of animals (n=4) received only PBS as a control for the delivery technique. All animals were returned to their cages after they had recovered and were fully alert. In 3 days, cardiac hemodynamics of rabbits that received adenovirus or saline were studied as above. Thus, each rabbit served as its own control for cardiac function. All rabbits received methylprednisolone 5.0 mg · kg^-1 · d^-1 IM for 2 days after adenovirus delivery. After the final assessment, animals were euthanized and their hearts rapidly excised. Transmural samples of the LV free wall, right ventricle (RV), septum, left atrium, lung, brain, and liver were frozen in liquid nitrogen and stored for biochemical analysis.

To examine the effects of heart rate (HR) on maximal rate of pressure rise, rabbits (n=4) underwent transvenous atrial pacing via the right jugular vein, and LV pressure was measured as described above. HR was varied from baseline to 330 bpm.

Data Analysis of In vivo Cardiac Function
Analog data were digitized at 200 Hz and analyzed on a VAX workstation (Digital Equipment Corp) with custom software (Physiological Systems Inc). The maximal rate of pressure rise (dP/dt_max) was computed from the digital pressure waveform as a running 5-point polyorthogonal transformation. All hemodynamic data were derived from the average of 20 steady-state cardiac cycles.¹⁸

ß-AR Density
ß-AR binding was performed on myocardial sarcolemmal membrane preparations as we have previously described.^{12 16} Total myocardial ß-AR density was determined by incubating 25 µg of sarcolemmal membranes with a saturating concentration of ¹²⁵I-labeled cyanopindolol and 20 µmol/L alprenolol to define nonspecific binding.^{12 16} Assays were performed in triplicate, and ß-AR density was normalized to milligrams of membrane protein.

ß-Galactosidase Staining
After excision, transverse cross sections of myocardium at the midpapillary level were obtained for histological analysis and stored in 30% sucrose solution before paraffin embedding as described.¹⁶ Paraffin-embedded samples were mounted on a cryostat and sectioned into 5- to 10-µm sections, which were then transferred to a glass slide. ß-Gal staining was performed by standard procedures as described.^{9 16}

ß₂AR Immunohistochemistry
Frozen myocardial sections were cut at 10 µm for indirect immunofluorescence studies as we have described.^{9 12 16} Briefly, sections were rinsed in PBS and then in PBS with 0.05% Triton X-100 (Triton-PBS), blocked with serum diluent (10% goat serum in PBS with 0.1% BSA and 0.1% sodium azide), and then rinsed for 15 minutes in Triton-PBS before overnight incubation at 4°C with a primary rabbit anti-human ß₂AR antiserum (1:500 dilution in serum diluent). The sections were then washed, incubated for 1 hour in FITC-conjugated goat anti-rabbit immunoglobulin G (1:50 dilution in serum diluent), rinsed in PBS, mounted with sodium iodide (25 g/L) in 1:1 PBS/glycerol solutions, and photographed.^{9 12}

Statistical Analysis
All data are expressed as the mean±SEM. In vivo hemodynamic data were compared by a paired Student’s t test. Unpaired comparisons were made by use of a 1-way ANOVA. For all analyses, a value of P<0.05 was considered to be statistically significant.

	Results

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Adenovirus-Mediated Myocardial Overexpression of Transgenes After Intracoronary Delivery
We delivered adenoviral transgenes via either the LCx or RCA in a subselective manner. Transgene expression in the rabbit myocardium was assessed 3 days after delivery to allow time for adequate protein translation and before adenoviral immunological processes were evident. As further protection toward the latter, we treated rabbits (including all control groups) for 2 days with corticosteroids.

To assess the volume of myocardium expressing transgene, we delivered Adeno-ßGal (5x10¹¹ TVP) via either the LCx or RCA. Figure 1 shows representative X-Gal–stained cardiac cross sections at low and high power of magnification after Adeno-ßGal delivery. Chamber-specific expression of the ßGal gene that corresponded to staining in individual myocytes was evident (Figure 1). Thus, it appears that within the region served by the catheterized coronary artery, complete transmural expression is possible, especially in this adenoviral dose range.

View larger version (71K): [in this window] [in a new window]   Figure 1. Representative ß-Gal staining of (A) LV free wall and (B) RV free wall 3 days after catheterization and delivery of Adeno-ßGal (5x1011 TVP) via LCx and RCA, respectively. C, High magnification of LV section demonstrating transgene expression in individual myocytes.

We also studied the delivery and expression of the human ß₂AR, which represents a potentially therapeutic transgene. As with Adeno-ßGal, we delivered 5x10¹¹ TVP Adeno-ß₂AR via either the LCx or RCA and initially assessed ß-AR density. As shown in Figure 2A, Adeno-ß₂AR delivery in the LCx resulted in significant LV-specific overexpression, whereas catheterization and injection via the RCA results in RV-specific expression. ß₂AR overexpression ranged from 9- to 15-fold over endogenous myocardial ß-AR density. Expression of the ß₂AR transgene remained elevated at 6 days (n=4) after gene delivery (Figure 2B). Importantly, ß₂AR overexpression after in vivo coronary delivery was found to be localized to the sarcolemmal membranes of individual ventricular myocytes as visualized by immunohistochemical staining with an antibody specific for the human ß₂AR (Figure 3). As with the ßGal transgene distribution, ß₂AR overexpression found by immunohistochemistry was diffuse throughout the entire LV after LCx injection, and a representative section of an Adeno-ß₂AR–treated LV is shown in Figure 3.

View larger version (28K): [in this window] [in a new window]   Figure 2. A, ß2AR overexpression in LV, RV, or septum after delivery of Adeno-ß2AR (5x1011 TVP) via LCx (left, n=10) or RCA (right, n=10). Chamber-specific ß2AR overexpression was compared with ß-AR density in individual chambers from Adeno-ßGal–treated rabbit hearts (control). *P<0.05 vs density in controls. B, ß2AR overexpression in LV at 6 days compared with 3-day values.

View larger version (123K): [in this window] [in a new window]   Figure 3. Representative immunohistochemical detection of expressed human ß2ARs in rabbit LV 3 days after Adeno-ß2AR (5x1011 TVP) delivery via the LCx.

Functional Consequences of Subselective Intracoronary Adenovirus Delivery and Myocardial Overexpression of Adeno-ßGal and Adeno-ß₂AR Transgenes
The hemodynamic consequences of subselective intracoronary delivery of adenoviral transgenes were examined by use of dynamic intracavitary pressure measurements. The maximal first derivative of the LV intracavitary pressure (dP/dt_max) was used as a measure of global LV contractile performance. As described in the Methods, the hemodynamics of each rabbit in the study was measured before and then 3 to 6 days after gene delivery. Thus, each animal served as its own control, increasing the power of the analysis. Furthermore, animals that underwent catheterization and injection of saline served as controls for the delivery technique itself. ß-AR density determined for all animals in the study confirmed ß₂AR overexpression. Animals that received Adeno-ßGal via the LCx did not show a significant change in systolic function compared with both precatheterization values and animals injected with saline (Figure 4A). In animals that received Adeno-ß₂AR and subsequently showed ß₂AR overexpression in the LV, a significant increase in baseline dP/dt_max was seen compared with precatheterization values and Adeno-ßGal–treated animals (Figure 4B). LV systolic functional responses to isoproterenol were also significantly greater in ß₂AR-overexpressing animals compared with controls (Figure 4C).

View larger version (19K): [in this window] [in a new window]   Figure 4. A, Comparison of systolic function (LV dP/dtmax) 3 days after administration and overexpression of Adeno-ßGal (5x1011 TVP) via LCx. Post–gene delivery values (Post Cath) are compared with Pre Cath values or animals that received saline (PBS). B and C, Effect of ß2AR gene delivery on LV systolic performance: B, LV dP/dtmax 3 days after delivery (Post Cath) of Adeno-ß2AR (5x1011 TVP, n=10) via LCx vs Pre Cath values (P=0.007) and vs rabbits that received 5x1011 TVP Adeno-ßGal (n=10, P=0.01). C, LV contractile response to infusion of isoproterenol (0.5 µg · kg-1 · min-1) in these rabbits. *P<0.05 vs Pre Cath values; P<0.05 vs Post Cath values from Adeno-ßGal–treated rabbits.

Interestingly, in animals that received either Adeno-ß₂AR or Adeno-ßGal (5x10¹¹ TVP each), HRs and LV end-diastolic pressure (EDP) were significantly increased 3 days after gene delivery, whereas there was no change, with either treatment, in systolic blood pressure (Table 1). LV dP/dt_min in Adeno-ßGal–treated rabbits was significantly reduced, whereas no reduction was evident in Adeno-ß₂AR–treated rabbits (Table 1). Hemodynamic values were not altered 3 days after saline delivery including dP/dt_max (Figure 4A), dP/dt_min (before, -2387±148 mm Hg/s versus after, -2152±196 mm Hg/s, P=NS), and LV EDP (before, 0.7±0.15 mm Hg versus after, 0.9±0.4 mm Hg, P=NS).

View this table: [in this window] [in a new window]   Table 1. Summary of Hemodynamic Data Before (Pre-Cath) and 3 Days After (Post-Cath) Intracoronary LCx Delivery of Adeno-ß2AR (n=10) or Adeno-ßGal (n=10) (5x1011 TVP)

LV dP/dt_max measurements can be confounded by changes in HR and afterload. Accordingly, a subset of animals was studied to determine the effect of HR on LV dP/dt_max by use of transvenous atrial pacing. As shown in Figure 5, the HR effect in the range of 200 to 300 bpm did not significantly increase LV dP/dt_max in a normal rabbit, demonstrating that increased HR is not accountable for the significant increase in basal and isoproterenol-stimulated LV dP/dt_max found in Adeno-ß₂AR–treated rabbits.

View larger version (11K): [in this window] [in a new window]   Figure 5. Effect of HR on LV dP/dtmax after atrial transvenous pacing (n=4).

The functional benefit of ß₂AR overexpression in the LV was also studied 6 days (n=4) after gene delivery. Importantly, basal and isoproterenol-stimulated LV systolic performance remained elevated at 6 days compared with pre–gene delivery hemodynamic measurements (Table 2).

View this table: [in this window] [in a new window]   Table 2. Summary of In Vivo LV Performance Before (Pre-Cath) and 6 Days After (Post-Cath) Adeno-ß2AR Gene Delivery (n=4)

Finally, the effect of ß₂AR overexpression in the RV after Adeno-ß₂AR delivery via the RCA on LV function was also studied. No improvement in LV systolic performance was seen in animals that overexpressed the ß₂AR in the RV (n=6) from precatheterization baseline values (before catheterization, 2725.8±211 versus after catheterization, 2858.7±48 mm Hg/s, P=NS).

	Discussion

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The present study reports the novel findings that in vivo molecular manipulation of ventricular function by a percutaneous intracoronary gene delivery method is feasible. Accordingly, we have demonstrated that chamber-specific delivery of an adenovirus containing the human ß₂AR via subselective coronary catheterization and injection produces ventricular overexpression of the ß₂AR at a level that increases contractile function in vivo, demonstrating a therapeutic strategy to effectively produce molecular ventricular assistance in CHF.

The ß-AR signaling system is an appealing target for cardiac gene therapy, because specific molecular abnormalities have been well described in human CHF and in multiple animal models of failure and cardiac disease.^{11 19} A downregulation of ß-ARs, uncoupling from second messenger systems, and elevation of desensitizing G-protein receptor kinase activity are the fundamental alterations seen in heart failure.^{14 15 16 19} In myocytes isolated from hearts in CHF, these abnormalities can be reversed by ß₂AR overexpression or G-protein–coupled receptor kinase inhibition by use of transgenes delivered via adenoviral vectors.¹⁵ These studies, as well as studies in transgenic mice, form the basis of our ß₂AR gene therapy strategy.^{11 12 15}

The overexpression of ß₂ARs in the LV free wall appears to improve global LV systolic performance. dP/dt_max both at baseline and after isoproterenol administration was increased relative to the precatheterization values in each of the individual rabbits. This is consistent with our findings in a model of biventricular overexpression of ß₂ARs.¹⁶ It is extremely encouraging that the 20% improvement seen in this study occurred after only LV free wall transfection and a relatively modest 10-fold increase in LV ß-AR density. Furthermore, this functional benefit was sustained at 6 days after gene delivery. Previous reports in transgenic mice demonstrated greater improvement in LV contractile function with ß₂AR overexpression. However, these mice had considerably higher levels of ß₂AR overexpression, which was global in nature and not limited to a single chamber, as is the case in the rabbits of this study. This is the first animal model to examine the in vivo effects of chamber-specific overexpression of the ß₂AR in the intact circulation, where ventricular interactions limit the maximal improvement in performance. Rabbits treated with Adeno-ßGal did not show a significant difference in systolic function compared with saline-injected animals. This demonstrates that adenovirus delivery, and transgene expression in general, does not depress baseline dP/dt_max. Interestingly, ß₂AR overexpression directed to the RV did not enhance LV performance. Although this result is not surprising, RV delivery of ß₂ARs may offer benefit in conditions of isolated right-sided dysfunction such as pulmonary hypertension.

We did observe a degree of myocardial injury with this model. In Adeno-ßGal controls and ß₂AR-treated rabbits, EDP was elevated and Adeno-ßGal–treated rabbits had depressed dP/dt_min values. Saline-injected controls did not show alterations of EDP or dP/dt_min. Overall, these data suggest that bolus delivery of adenovirus caused an increase in LV stiffness. Interestingly, animals that overexpressed the ß₂AR had less depression of dP/dt_min at 3 and 6 days, suggesting a superimposed lusitropic effect. Nevertheless, this apparent negative effect of intracoronary adenovirus injection needs to be examined further.

The present study highlights a number of challenges to in vivo cardiac gene therapy. First, myocardial delivery of the adenoviral transgene has been achieved in this percutaneous model, but whether the methods used here will be applicable to other viral vectors is not certain. The kinetics of adenovirus-mediated transfer in the intact coronary vasculature favor high coronary perfusion flow and pressure as well as enhanced permeability,²⁰ most of which can induce myocardial injury. We use a relatively large injection volume for individual coronary arteries, which we have found leads to significant improvement in gene transfer and transgene expression, consistent with the ex vivo kinetic studies of intracoronary adenoviral gene delivery.²⁰ Furthermore, the present study uses higher concentrations of virus than previously reported; however, transmural myocardial expression is also greater than in previous reports.¹⁰ Accordingly, a significant percentage of ventricular myocytes must be transfected to achieve a global functional impact.

The duration of transgene expression was not examined in this study. Work in our laboratory has documented that the first-generation vectors used yield expression that lasts for 1 to 2 weeks in vivo in the myocardium.^{9 16} However, we have clearly shown that adenovirus-mediated transgene delivery can alter the in vivo function of the heart for 1 week, and the use of this method can only improve in the future as vector technology advances to allow for less inflammatory vectors that also support longer-term expression. For example, long-term myocardial expression has recently been reported with adeno-associated virus.²¹ It will be of particular future interest to determine whether our delivery method will effectively deliver adeno-associated virus transgenes.

There are several implications of our experimental findings. The present study is among the first to demonstrate the feasibility of cardiac gene transfer by clinically relevant and available methods. Previous studies have used open thoracotomy and aortic cross-clamping,^{16 17} which would not be as widely useful to the 4 million patients with CHF. We have also demonstrated the ability to enhance baseline myocardial function in the otherwise normal LV. This improvement of baseline systolic function is consistent with previous findings in ß₂AR-overexpressing transgenic mice,^{12 13} including transgenic mice that had myocardial-targeted ß₂AR overexpression in the range reached by our adenovirus-mediated gene transfer.¹³ Thus, the possibility of genetic manipulation via the coronary circulation offers potential therapy to patients not amenable to traditional approaches to cardiomyopathy. Recent work has documented polymorphisms of the human ß₂AR gene, which result in dysfunctional G-protein coupling and predict poor prognosis in affected patients with CHF.²² Therefore, gene delivery of a normal ß₂AR gene might be particularly critical in this patient population.

The recent clinical evidence that failing cardiomyocytes have the capacity to reverse their remodeling and regain normal contractile function supports the hypothesis that molecular ventricular assistance, as shown here with ß₂AR overexpression, could serve as a novel form of therapy.^{23 24 25} This hypothesis is further strengthened by the finding that overexpression of ß₂ARs and an inhibitor of ß-AR desensitization reverse abnormal signal transduction in cultured cardiomyocytes isolated from rabbits in CHF.¹⁵

A growing body of evidence supports the use of ß-blockade in the treatment of heart failure.²⁶ Although our strategy would seem contradictory to this clinical experience, it may in fact be supported. Recent experimental work has shown that ß-antagonist therapy with carvedilol is associated with a restoration of desensitized ß-AR signaling that may be the mechanism of benefit of this agent in CHF.²⁷ Therefore, overexpression of functional ß₂ARs serves as an alternative method to restore ß-AR signaling in failing myocardium.

In summary, the present study has demonstrated that subselective adenovirus-mediated delivery of a functional transgene is possible via the intact coronary circulation. Future studies will examine the consequences of acquired ß₂AR overexpression in models of CHF, and the present approach lends itself to that task. Furthermore, subselective gene delivery may allow the examination of new strategies for the management of hypertrophy, RV failure, and genetic cardiac maladaptation.

	Acknowledgments

 
This work was supported by NIH grants HL-59333 and HL-56205 (to Dr Koch). We also thank the Genzyme Corporation, Framingham, Mass, for purification of the Adeno-ß₂AR. Dr Lefkowitz is an investigator of the Howard Hughes Medical Institute. We would like to gratefully acknowledge the excellent technical assistance of Kyle Shotwell and Dimitri Baklanov, MD. We also thank Dr Paul C. Dolber for assistance with the ß₂AR immunohistochemistry.

Received April 30, 1999; revision received July 29, 1999; accepted August 11, 1999.

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