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Ann Thorac Surg 2002;73:1939-1946
© 2002 The Society of Thoracic Surgeons

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Original article: cardiovascular

Global cardiac-specific transgene expression using cardiopulmonary bypass with cardiac isolation

^a Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA
^b Department of Neurology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA

Accepted for publication February 7, 2002.

* Address reprint requests to Dr Bridges, Division of Cardiothoracic Surgery, Pennsylvania Hospital, 230 W Washington Sq, 3rd Flr, Philadelphia, PA 19106 USA
e-mail: cbridges{at}pahosp.com

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. The available techniques for intravascular gene delivery to the heart are inefficient and not organ-specific. Yet, effective treatment of heart failure will likely require transgene expression by the majority of cardiac myocytes. To address this problem, we developed a novel cannulation technique that achieves efficient isolation of the heart in situ using separate cardiopulmonary bypass (CPB) circuits for the heart and body in dogs.

Methods. The arterial inflow and venous effluent from the two circuits were physically isolated. The efficiency of separation was 98% to 99% in three preliminary experiments using Evans Blue dye-labeled albumin. In 6 dogs, the cardiac circuit was perfused with oxygenated crystalloid cardioplegia at 37°C containing 4 x 10¹¹ particles of an adenovirus encoding LacZ (AdCMVLacZ) with a perfusion pressure of 170 to 200 mm Hg for 15 minutes allowing virus to recirculate through the heart 15 times. Cross-clamp time was 26 ± 2 minutes and CPB time was 90 ± 3 minutes.

Results. Five animals survived and were euthanized at 7 days. ß-Galactosidase activities measured using a chemiluminescent assay were three orders of magnitude higher in all areas of the heart than in the liver. Histological analyses revealed heterogeneous X-Gal staining of myocytes in all areas of the myocardium.

Conclusions. Despite using a constitutive promoter, this technique yields relatively cardiac-specific transgene expression and is potentially translatable to clinical applications. Future studies will allow for further optimization of the conditions necessary for vector-mediated gene delivery to the heart.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Heart failure is a major public health problem with a prevalence of over 2,000,000 in the United States [1]. The incidence is more than 400,000 new cases per year [2] with more than 200,000 deaths per year [3]. The 5-year survival is less than 50% [3]. Nearly 60,000 patients per year could benefit from cardiac transplantation or long-term mechanical circulatory support. However, only approximately 2,500 donor hearts are available for transplantation each year in the United States [4]. Gene therapy is emerging as a potential new therapy for this vexing public health problem. Our hypothesis is that one important reason for the lack of effective gene-based therapy to date is the unique obstacle of obtaining efficient gene delivery to the majority of myocytes in situ. Furthermore, experimental validation requires that these difficulties be overcome in a clinically translatable large animal model. Experimental methods of delivering transgenes to the myocardium include intramuscular (IM) [5] or intracoronary [6–8] injection, or injection directly into the left ventricular (LV) cavity with cross-clamping of the aorta [9]. Intramural injection allows for local myocyte gene expression only [5]. The other two techniques result in a single-pass of vector through the heart with systemic release of the unabsorbed fraction typically resulting in undesirably high-level transduction of the liver [8]. Using adenovirus encoding LacZ (AdCMVLacZ) or adeno-associated virus encoding delta sarcoglycan (AAVCMVsarcoglycan) in rodents, our recent studies have shown that limb isolation with heterotopic groin transplantation of the heart results in gene expression by essentially 100% of cardiac and skeletal myocytes when vectors are coadministered with selected inflammatory mediators [10]. These observations suggest that overcoming the endothelial barrier is critical to achieving widespread myocyte gene expression with Ad or AAV vectors via the intravascular route.

As a first step toward achieving this end in the heart, we present an exciting new technique that allows for efficient isolation of the heart in vivo using separate cardiopulmonary bypass (CPB) circuits for the cardiac and systemic circulations in dogs. We [11], along with Davidson and colleagues [12], simultaneously published descriptions of the use of cardiopulmonary bypass for cardiac gene delivery. In contrast to their method which involves standard cold cardioplegic arrest [12, 13], our technique allows for isolation of the heart in situ. This novel configuration is utilized to increase the theoretical efficiency of transgene delivery using two separate, oxygenated circuits for the heart and systemic circulations, respectively, allowing for multiple passes of vector through the heart and control of perfusion temperature while minimizing transgene delivery to other organs.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
All animals were treated in compliance with NIH publication No. 82-23 as revised in 1985. Nine mongrel dogs (group 1, labeled albumin, n = 3; group 2, AdCMVLacZ, n = 6) were fasted overnight (12 hours). The animals were sedated and induced for anesthesia with ketamine (10 mg/kg, IM), diazepam (0.5 mg/kg, IV), atropine (0.05 mg/kg, IM), and the vocal cords sprayed with 2% lidocaine. They underwent endotracheal intubation and mechanical ventilation (Drager anesthesia monitor, North American Drager, Telford, PA) with 100% oxygen. Anesthesia was started with 2% to 3% isoflurane and maintained with 0.5% to 2% isoflurane. A rectal temperature probe was inserted, and a noninvasive pulse oximetry transcutaneous monitor placed on the tongue. The animals were prepared and draped using sterile technique. All animals received cefazolin (1 gram, IV) prior to anesthetic induction. The surface electrocardiogram was monitored throughout the procedure. A percutaneously placed right femoral catheter was used to monitor arterial blood pressure and obtain arterial blood gas samples. The right jugular vein was cannulated with an 8.5 Fr. introducer for volume infusion. The right common carotid artery was isolated, and the incision left open temporarily. The drapes were removed, and the animal was again prepped and draped using sterile technique including both the chest, the right jugular, and internal carotid arteries in the operative field. A sternotomy incision was performed using an oscillating saw. The pericardium was opened, and the heart was suspended in a pericardial cradle.

Cardiac isolation technique
Two separate cardiopulmonary bypass circuits were employed with separate pump-oxygenators and reservoirs for the systemic and cardiac circuits, respectively. Heparin (3 mg/kg) was given intravenously. Purse string sutures were placed in the proximal ascending aorta, proximal superior vena cava, and right atrium adjacent to the inferior vena cava. The left carotid artery was cannulated, and the cannula was connected to the arterial limb of the systemic cardiopulmonary bypass circuit. A cardioplegia cannula was passed through the purse string in the proximal ascending aorta and connected to the arterial limb of the cardiac circuit. Two 26 Fr. right angle venous cannulae were placed in the superior vena cava (SVC) and inferior vena cava (IVC), respectively. Their combined outflow was passed via a Y-connector to the venous limb of the systemic cardiopulmonary bypass circuit. Snares were placed around the SVC and IVC to allow diversion of all systemic venous return into the venous limb of the systemic cardiopulmonary bypass circuit during the cardiac isolation interval. Cardiopulmonary bypass was initiated at 37°C. Ventricular vent catheters were placed into the right and left ventricles, respectively. These vent catheters were connected via a Y-connector to become the venous limb of the cardiac circuit. The azygous vein was ligated. Systemic cooling to 30°C was performed. The ascending aorta was cross-clamped, and 300 cc of crystalloid cardioplegia (Plegisol, Abbott Laboratories, Chicago, IL; pH adjusted with sodium bicarbonate to pH = 7.4) at 4°C was administered via the aortic root. Diphenhydramine 50 mg and methyl prednisolone 100 mg were administered intravenously. The SVC and IVC snares were tightened. The pulmonary artery was cross-clamped proximally. In this way, all blood or crystalloid returning from the heart via the coronary sinus or the Thebesian veins was then routed via the right ventricular (RV) vent, and all blood that regurgitated across the aortic valve was routed via the left ventricular vent to the cardiac venous reservoir. The cardioplegia solution was oxygenated and returned to the coronary circulation via the cardioplegia cannula in the aortic root (cardiac arterial in-flow) and maintained at 37°C. All blood returning from the systemic circulation was routed via the SVC and IVC cannulae to the systemic venous reservoir. Arterial in-flow via the cardioplegia cannula perfused only the cardiac circulation via the left and right coronary arteries proximal to the aortic cross-clamp. Systemic arterial in-flow perfused only the systemic (noncardiac) circulation.

With the cardiac circulation effectively isolated from the systemic circulation, warm, oxygenated crystalloid cardioplegia (Plegisol), alone, (group 2) or containing Evans Blue dye-labeled albumin with histamine (10 mM) and papaverine (10 mg/L) (group 1) was infused into the cardiac circulation and re-circulated continuously for approximately 15 minutes. In either case, the solution pH was adjusted to 7.4 using sodium bicarbonate before starting the infusion. In group 2, 1 minute after cardiac isolation, a high-flow, four-way stopcock was used to rapidly inject AdCMVLacZ, 0.4 ml of 10¹² particles/ml directly into the cardioplegia stream without interruption. The cardioplegia/vector or cardioplegia/albumin solution was infused at a perfusion pressure of 170 to 200 mm Hg resulting in flows of approximately 250 to 400 cc/min. The high perfusion pressure was chosen empirically to maximize the gradient for transendothelial transport. This procedure allowed for passage of the entire volume of the cardiac circuit through the coronary circulation approximately 15 times. When the isolation interval was completed, 1 liter of Hespan (DuPont Pharmaceuticals, Wilmington, DE) with 100 mg of methyl prednisolone and 50 mg of diphenhydramine added was used to wash out the coronary circulation. The aortic cross-clamp was removed and snares loosened, merging the two circulations. Systemic rewarming was initiated. An additional dose of diphenhydramine 50 mg and methyl prednisolone 100 mg were administered IV. The LV and RV vent catheters were then used as systemic vents to decompress the heart. The animals were routinely weaned from bypass after 30 minutes of reperfusion with epinephrine (1 mcg/min) and lidocaine (1 mg/min) infusions. After 5 minutes of systemic reperfusion, the cross-clamp on the pulmonary artery was removed. Once the heat was contracting well, the LV and RV vent cannulae were removed, and inotropic support was discontinued. The cardiac isolation procedure is depicted in Figure 1.

View larger version (40K): [in this window] [in a new window]   Fig 1. Open chest canine preparation during cardiopulmonary bypass. Illustrated are all important components of the cardiac isolation scheme including the aortic cross-clamp, superior vena cava (SVC) and inferior vena cava (IVC) cannulae, SVC and IVC snares, pulmonary cross-clamp, cardiac arterial in-flow, and right ventricular (RV) and left ventricular (LV) vent catheters.

Isolated perfusion with AdCMVLacZ
For the 6 animals in group 2, after weaning from bypass, the sternum was rewired, the sternotomy wound was closed, and the animals were allowed to recover. Within approximately 4 hours after completion of the procedure, the animals were weaned from mechanical ventilation and inotropic support, and all chest tubes were removed. The animals were returned to an oxygen cage overnight and allowed access to food and water. The next morning they were transferred to cages without supplemental oxygen and sacrificed after 7 days. A total of 6 dogs underwent the procedure described. Tissues were procured at necropsy on day 7, following euthanasia of the dogs with an intravenous overdose of sodium pentobarbital. Cryostat sections of the heart, liver, and lung were incubated overnight at room temperature in X-Gal solution. ß-Galactosidase enzyme activity was quantified using a chemiluminescent reporter assay system (Galacto-Light Plus, Tropix, Inc, Bedford, MA).

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Labeled albumin studies
These preliminary experiments were used to develop the surgical technique utilized in the subsequent experiments. The concentrations of histamine (10 mM) and papaverine (0.3 mg/ml) used were based on the concentrations in previous isolated hindlimb experiments that resulted in efficient vector-mediated gene transfer to skeletal muscle and to the heterotopically transplanted heart [10]. All animals were successfully weaned from cardiopulmonary bypass. Evans Blue dye fluorescence histochemistry at 540 nm from specimens of the right and left ventricle and diaphragm obtained for histological studies are demonstrated in Figure 2. There is extensive and uniform exudation of labeled albumin into the interstitium of the myocardium of the left and right ventricles (Fig 2C and D, respectively), affecting the local environment of essentially 100% of the myocytes. The interstitial space is prominent with increased volume indicative of tissue edema. In contrast, there are only scant amounts of labeled albumin present in the interstitium of the diaphragm, and there is no interstitial edema (Fig 2A,B). In the diaphragm, the punctate "dots" of fluorescence (Fig 2A) represent small amounts of labeled albumin remaining in the systemic capillaries surrounding the muscle cells. The use of two separate CPB circuits with surgical isolation of the heart resulted in an average to systemic cardiac-labeled albumin concentration ratio of 66.5. This result is indicative of 98.3% efficiency of separation of the two circulations (Table 1). These findings are consistent with the hypothesis that histamine and papaverine result in a dramatic increase in endothelial permeability, an effect that remains localized to the myocardium.

View larger version (162K): [in this window] [in a new window]   Fig 2. Evans blue dye-labeled albumin fluorescence histochemistry: cross sections obtained from the diaphragm (A, 200x; B, 40x), left ventricle (C, 100x), and right ventricle (D, 100x).

View larger version (145K): [in this window] [in a new window]   Fig 3. Gene transfer into right ventricular (RV) cardiomyocytes after cardiopulmonary bypass with isolated perfusion of the cardiac circulation in vivo with AdCMVLacZ. (A) RV outflow tract, 100x; (B) RV free wall, 40x; (C) RV outflow tract, 200x; (D) RV free wall, 40x, after infusion of 4 x 1011 particles of AdCMVLacZ and staining with X-Gal 1 week postoperatively.

View larger version (140K): [in this window] [in a new window]   Fig 4. Gene transfer into left ventricular (LV) cardiomyocytes after cardiopulmonary bypass with isolated perfusion of the cardiac circulation in vivo with AdCMVLacZ. (A) LV apex, 100x; (B) LV anterior wall, 100x; (C) LV lateral wall, 100x; (D) intraventricular septum, 100x, after infusion of 4 x 1011 particles of AdCMVLacZ and staining with X-Gal 1 week postoperatively.

View larger version (133K): [in this window] [in a new window]   Fig 5. Gene transfer into left (LV) and right (RV) ventricular cardiomyocytes after cardiopulmonary bypass with isolated perfusion of the cardiac circulation in vivo with AdCMVLacZ. (A) LV endocardium, 40x; (B) RV, adjacent to a large artery and vein, 40x; (C) LV region remote from blood vessels, 40x; (D) RV region remote from blood vessels, 40x, after infusion of 4 x 1011 particles of AdCMVLacZ and staining with X-Gal 1 week postoperatively.

View larger version (44K): [in this window] [in a new window]   Fig 6. ß-Galactosidase activities in the heart, liver, and lung (relative light units [RLU]/µg/103, mean ± S.E.M.). (LV = left ventricle; RV = right ventricle.)

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
The results presented here differ in two important ways from most previously published studies. Despite the use of a constitutive promoter, relatively cardiac-specific gene expression was achieved. The ß-galactosidase activities in the heart were several orders of magnitude higher than those in the liver. In contrast, Lilly and coworkers found that intracoronary administration of AdCMVLuc in the adult rabbit left circumflex coronary artery resulted in a threefold to 40-fold higher luciferase activity in the liver compared to the particular region of the heart expected to have the greatest level of gene expression [8]. Davidson and associates also used cardiopulmonary bypass for delivery of adenovirus encoding the LacZ reporter gene and the ß-adrenoreceptor to the myocardium in neonatal pigs [13]. However, they used a conventional configuration for bypass with a single pump oxygenator. Their technique resulted in heterogenous gene expression throughout the myocardium. In contrast to the method described here, their approach did not include isolation of the cardiac circulation from the systemic circulation. As a result, cold (4°C) cardioplegia was required, and only a single pass of the vector through the cardiac circulation was achieved. After the cross-clamp interval, residual vector was washed out into the systemic circulation. It is, therefore, somewhat surprising that their technique did not result in more significant gene expression in the liver.

In the technique presented here, separate pump-oxygenators are used for the systemic and cardiac circuits. This novel configuration allows for optimization of a number of variables that have been shown to be important determinants of the efficiency of vector-mediated, intravascular cardiac gene delivery including temperature, pressure, ionic composition, flow rate, and exposure time [16]. Moreover, this system allows for recirculation of the vector through the cardiac circulation multiple times, theoretically increasing the efficiency of vector-mediated transduction of cardiac myocytes. At the end of the cross-clamp interval, this technique allows for removal of the vector from the cardiac circulation, minimizing the probability of transgene expression in other organs despite the use of a constitutive promoter.

In general, we found the highest ß-galactosidase activities in the right atrium and right ventricle (Fig 6). We speculate that the higher activities on the right side may be due to a combination of direct and transvascular transport of adenoviral vectors on the right side of the heart, while on the left side, primarily transvascular transport occurs. Note that we retrieve the venous return from the cardiac circuit from the right ventricular lumen via the right ventricular vent (Fig 1). Therefore, on the endocardial surface, right ventricular and right atrial myocytes are bathed directly with the cardiac circuit venous effluent containing adenoviral vector. Since the majority of cardiac venous return occurs via the coronary sinus, the highest concentrations of vector are likely to contact the endocardial surface of the right atrium. In contrast, the left ventricular endocardium has direct contact with adenovirus only in those areas bathed by any physiological aortic regurgitation and from Thebesian veins draining into the left ventricle or atrium. As expected, we found that the vast majority of the venous return was from the right ventricular vent and only a minimal amount from the left ventricular vent.

The available literature [5–8] and our own results [10] clearly indicate that the probability of successful transduction of any cardiac myocyte, P_t is much less than 1.0 when a vector such as AdCMVLacZ is delivered via a single pass through the cardiac circulation. Without isolation of the heart as accomplished here, injected Ad vectors are most likely to transduce hepatocytes rather than cardiac myocytes even when injected directly into the coronary arteries [8]. Therefore, in general, intracoronary injection [6–8] is likely to result in only a single pass of a given vector through the cardiac circulation. Based on these assumptions, the probability of myocyte transduction using the technique presented here should be P nP_t where n is the number of times the cardiac reservoir volume recirculates through the heart. Since, in the experiments presented, the reservoir plus tubing volume for the cardiac circuit is approximately 300 cc, at flow rates of 300 cc/minute for 15 minutes, n 15 resulting in a theoretical order of magnitude increase in transduction efficiency.

One possible limitation of our technique is the potential for inactivation of the adenovirus due to incompatibility with components of the bypass circuit including the tubing and pump-oxgenator surfaces. Marshall and colleagues showed elegantly that a variety of commonly used catheter constituents such as stainless steel, nitinol, and polycarbonate rapidly and efficiently inactivate adenovirus infectivity [17]. The primary component of tubing used for cardiopulmonary bypass circuits is polyvinyl chloride (PVC). PVC was not one of the polymers tested in this study. However, polycarbonate is commonly used in stopcocks and venous reservoirs, and the adenovirus was usually drawn up through a metal needle. If in aggregate, the components of the bypass circuit lead to significant viral inactivation, the theoretical advantages of virus recirculation would not be realized. Specific measures, such as the incorporation of human serum albumin into the cardioplegia solution then could be used to minimize adenovirus inactivation [17].

We chose to use crystalloid cardioplegia for perfusion of the cardiac circuit rather than blood cardioplegia since adenovirus-mediated transduction of isolated myocytes is significantly more efficient in crystalloid media than in media containing red blood cells [16]. An oxygenator was incorporated into the cardiac circuit, as well, to ensure adequate oxygen delivery to meet the metabolic demands of the normothermic heart. Oxygenated crystalloid cardioplegia solutions at 37°C have been shown to provide adequate myocardial protection for cross-clamp intervals greater than 40 minutes with results comparable to blood cardioplegia in humans [18]. Temperature is another important variable determining the efficiency of vector-mediated gene transfer. Donahue and colleagues found that transduction efficiency was approximately ten times higher at 37°C than at 4°C for isolated myocytes exposed to AdCMVLacZ for 10 to 15 minutes in vitro [16]. Therefore, we chose to perfuse the cardiac circuit with oxygenated crystalloid at 37°C to maximize the theoretical efficiency of vector-mediated gene transfer without compromising myocardial protection.

We were encouraged by the relative specificity of cardiac transgene expression achieved in this model, illustrating one clear advantage of this technique for cardiac gene delivery. Our failure to achieve successful transduction of the majority of cardiac myocytes in situ using this technique was somewhat disappointing yet not unexpected. Previous work in our laboratories has implicated the endothelial barrier as the critical obstacle to obtaining widespread Ad- or AAV-mediated gene transfer to skeletal myocytes in situ and to the heterotopically transplanted heart in the rat and sarcoglycan deficient hamster [10]. Overcoming this barrier by coinfusion of selected inflammatory mediators such as histamine and papaverine into the isolated hindlimb results in successful transduction of essentially 100% of skeletal myocytes in situ and 100% of cardiac myocytes in the heart heterotopically transplanted into the groin [10]. The results presented here support our "restrictive-endothelium" hypothesis that the blood vessel wall is rate limiting for intravascular Ad or AAV-mediated gene transfer to skeletal or cardiac myocytes in situ.

In summary, a new cardiac surgical technique has been presented that represents a novel approach to the goal of achieving highly efficient vector-mediated gene transfer throughout the myocardium in situ in a large animal. This technique allows for control of temperature, perfusion pressure, exposure time, and the ionic composition of the myocardial perfusion solution, all of which have been shown to be important determinants of the efficiency of vector-mediated gene transfer to the heart. Using two separate oxygenated bypass circuits for the cardiac and systemic circulations, 98% to 99% efficiency of separation of the two circulations was achieved. We found that gene expression appeared to be most marked in endothelial cells and in myocytes adjacent to blood vessels or the endocardial surface. Based on these and our own previously published results, we believe that the inclusion of strategies designed to increase endothelial permeability will be necessary to achieve successful transduction of the majority of cardiac myocytes in situ via the intravascular route. The technique presented allows for unique opportunities to investigate systematically a variety of approaches to manipulate the cardiac circulation to optimize the efficiency of vector-mediated gene transfer to the myocardium. This method is clinically translatable and could be used as an adjunct to other cardiac surgical procedures where cardiopulmonary bypass is utilized. The approach presented may also have applications to problems in drug delivery where isolation of the heart is desirable such as organ-specific chemotherapy for neoplastic disorders.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This study was supported by a grant from the NIH (5-P01-AR/NS43648); grants from the Muscular Dystrophy Association of America, the Association Francaise Contre les Myopahthies, and the U.S. Veterans Administration; and by funds from the Harrison Department of Surgical Research, University of Pennsylvania Health System. Dr Bridges was supported by an NIH Research Supplement for Minority Investigators (supplement to 5-P01-AR/NS43648).

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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