EJCTS Click here for details of sales representative
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Author home page(s):
Carmelo A. Milano
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jones, J. M.
Right arrow Articles by Milano, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jones, J. M.
Right arrow Articles by Milano, C. A.
Related Collections
Right arrow Cardiac - pharmacology
Right arrow Cardiac - physiology
Right arrow Molecular biology

Eur J Cardiothorac Surg 2004;26:1161-1168
© 2004 Elsevier Science NL

ß2 Adrenoceptor gene therapy ameliorates left ventricular dysfunction following cardiac surgery

J. Mark Jonesa,*, Jason A. Petrofskia, Katrina H. Wilsonb, Charles Steenbergenc, Walter J. Kocha, Carmelo A. Milanoa

a Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
b Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
c Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA

Received 21 June 2004; received in revised form 2 August 2004; accepted 10 August 2004.

* Corresponding author. Present address: Department of Cardiac Surgery, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA, UK. Tel.: +44 289 0633 500; fax: +44 289 0312 907. (E-mail: mark.jones{at}royalhospitals.n-i.nhs.uk).


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objective: Heart surgery is associated with impairment of the myocardial ß-adrenoceptor (ßAR) system. Effective therapies for post-operative ventricular dysfunction are limited. Prolonged inotrope exposure is associated with further ßAR down-regulation. Left ventricular (LV) dysfunction and myocardial ßAR impairment were assessed following cardiopulmonary bypass (CPB) and cardioplegic arrest in a pig model. Transfer of the human ß2-adrenoceptor transgene (Adeno-ß2AR) during cardioplegic arrest was then tested as a potential therapy. Methods: Five groups of six neonatal piglets were studied. One group did not undergo surgery (Group A). Adeno-ß2AR or phosphate buffered saline (PBS) were delivered via the aortic root during cardioplegic arrest. Groups B (PBS) and C (Adeno-ß2AR) were assessed at 2 days while Groups D (PBS) and E (Adeno-ß2AR) were assessed at 2 weeks from the time of surgery. An LV micromanometer was inserted under sedation to obtain pressure recordings following surgery. ßAR density was measured subsequently. Results: Following cardiac surgery LV ßAR density was reduced (104±5.7 vs 135±6.1fmol/mg membrane protein; P=0.007), and, in response to ß agonist stimulation, LV dP/dtmax was reduced (4337±405 vs 5328±194mmHg/s; P<0.05) compared to animals which did not undergo surgery. Adeno-ß2AR therapy during cardiac surgery resulted in elevated LV ßAR density (520±250.9fmol/mg) 2 days post-operatively compared to PBS (104±5.7fmol/mg; P=0.002) and compared to the no surgery group (135±6.1fmol/mg; P=0.002). Elevated LV ßAR density was also present at 2 weeks (315±74.1 vs 119±7.1fmol/mg; P=0.002). In addition, Adeno-ß2AR therapy enhanced ß agonist stimulated LV dP/dtmax (5348±121 vs 4337±405mmHg/s; P<0.05) and heart rate (209±6.9 vs 173±11.0bpm; P<0.05), and reduced LVEDP (2.1±0.4 vs 6.4±1.8mmHg; P<0.05) compared to PBS treatment. Interestingly, gene delivery was cardiac-selective and beneficial effects on function persisted for 2 weeks. Moreover, ß2AR gene transfer ameliorated LV dysfunction following surgery such that there were no significant differences between non-operated controls and animals treated with Adeno-ß2AR during CPB and cardioplegic arrest. Conclusions: Reduced ßAR density and impaired LV function were present following CPB and cardioplegic arrest. Cardiac-selective ß2AR gene transfer during CPB resulted in amelioration of LV dysfunction after cardiac surgery. Such a technique may offer a new approach to post-operative ventricular support.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
Heart surgery has been associated with impairment of the myocardial ßAR system. Cardiopulmonary bypass (CPB) is a potent stimulant to the release of endogenous catecholamines. Release of noradrenaline has been demonstrated from anoxic, isolated hearts and cooling maintains elevated catecholamines in ischaemic myocardium. Upon termination of CPB increased catecholamine levels may have been experienced locally within the myocardium and the rewarmed uncross-clamped heart is exposed to blood containing elevated circulating catecholamines. ßAR desensitization has been demonstrated to occur during CPB with down-regulation of ßAR 30min after termination of CPB [1]. Furthermore, ventricular dysfunction is a significant complication contributing to death after heart surgery [2]. Prolonged inotrope exposure is also associated with further down-regulation of ßARs which may reduce responsiveness to agonist in the post-operative period [3,4].

Myocardial gene transfer to modify ß adrenergic receptor signalling has been proposed as a novel treatment to enhance cardiac function [5]. Transgenic mouse models with myocardial specific overexpression of the human ß2 adrenergic receptor (ß2AR) or overexpression of the carboxyl terminus of ß adrenergic receptor kinase (ßARKct), an inhibitor of ßARK, have resulted in enhanced left ventricular (LV) function without detrimental effect, unlike overexpression of the human ß1 adrenergic receptor (ß1AR) [6,7]. Indeed, recent studies have found that signalling through ß1ARs differs significantly from signalling through ß2ARs. While both activate adenylyl cyclase via the stimulatory G protein, Gs, ß2ARs can also stimulate Gi. While small increases in ß1AR density can lead to cardiomyopathy, much greater overexpression of ß2AR was required before cardiomyopathy became apparent. Apoptosis has been implicated in the development of cardiomyopathy and heart failure. Although, chronic catecholamine stimulation does induce apoptosis this response seems to be mediated by ß1AR, while stimulation of the ß2AR does not yield such an effect, and may even protect against ß1AR mediated apoptosis [5].

Various experimental models have confirmed myocyte transgene expression following intracoronary vector delivery [8–11]. However, with these methods the vector is injected under high pressure, which could result in endothelial or myocardial injury. Furthermore, extracardiac transgene expression may be hazardous, resulting in adverse metabolic effects or causing activation of the inflammatory and immune responses in multiple organs [9,10]. We have, therefore, developed a surgical method of gene delivery and demonstrated that adenoviral-based gene transfer during CPB and cardioplegic arrest achieves efficient selective myocardial transgene expression [12,13]. Minimal coronary blood flow occurs during the period of aortic cross-clamping, which may enhance gene expression, since according to in vitro studies uptake of adenoviral vector is related to duration of contact. A range of myocardial protection techniques, including warm fibrillatory arrest, and warm and cold cardioplegia, were shown to be compatible with gene transfer during CPB. A neonatal pig model was used for these studies to limit the quantity of adenoviral vector required. However, neonates may have a reduced immune response to the vector. In addition, the pattern of heart growth by myocyte hyperplasia changes to hypertrophy during the perinatal period which may also have implications for the uptake and distribution of the adenoviral vector [14].

Further to earlier experiments, in this study, we characterize the association of post-operative LV dysfunction and myocardial ßAR impairment. Moreover, we tested the hypothesis that Adeno-ß2AR delivery during such cardiac surgery can ameliorate early post-operative LV dysfunction thus providing a novel, and potentially clinically applicable, method of ventricular support.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
2.1. Adenoviral transgenes
The adenoviral backbone used was a replication-deficient first generation type V adenovirus with deletions of the E1 and E3 genes. A vector was constructed containing the transgene for the human ß2AR (Adeno-ß2AR) as previously described [15]. Immediately prior to use the adenoviral vector was thawed from –80°C and reconstituted in 8ml of phosphate buffered saline (PBS).

2.2. Animals
Thirty neonatal piglets (1–2 weeks old) weighing 2.8±0.08kg were used. There was no significant difference in weight between groups. Six-month old pigs were sacrificed to provide fresh blood for priming of the CPB circuit. All animals received humane care in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised in 1985). The experimental protocol was approved by Duke University Animal Care and Use Committee.

Six animals did not undergo CPB and cardioplegic arrest and served as a control for normal neonatal function (Group A). All other animals underwent CPB and cardioplegic arrest and treatment with either 1x1012 total viral particles (tvp) Adeno-ß2AR in 8ml PBS or PBS alone. Vector or PBS was delivered via the aortic root following cardioplegic arrest. Two independent sets of animals were sacrificed following assessment of LV function at 2 days (n=6 per group; Group B: PBS; Group C: Adeno-ß2AR), or 2 weeks (n=6 per group; Group D: PBS; Group E: Adeno-ß2AR) from the time of surgery.

2.3. Surgery, CPB and gene delivery
Animals were anaesthetized with 10mg ketamine (Abbott Laboratories, North Chicago, IL) intramuscularly and inhaled 1–2% isoflurane (Abbott Laboratories, North Chicago, IL). Dexamethasone (American Regent Laboratories Inc., Shirley, NY) was administered pre-operatively (12.5mg IM) and on induction of anaesthesia (25mg IV). Ampicillin (Bristol-Myers Squibb Co., Princton, NJ) (1g IV) was used prophylactically.

Median sternotomy was performed, the thymus gland was removed, and the pericardium was opened in the midline according to standard technique. The piglet was given 300IU/kg heparin (Elkins-Sinn Inc., Cherry Hill, NJ). A Medtronic Minimax paediatric membrane oxygenator (Medtronic Cardiopulmonary, Anaheim, CA) was used for CPB. The circuit was primed with 450ml fresh heparinized blood from an adult donor pig. Non-pulsatile CPB was established at 100ml/kg per min [12,13]. Shed blood was returned to the venous reservoir by a second roller pump. The left ventricle (LV) was vented through the left atrium using a 19 guage butterfly cannula (Abbott Laboratories, North Chicago, IL) which was connected to the pump suction.

After establishment of CPB, a cross-clamp was applied to the aorta just proximal to the aortic cannula. Cold crystalloid cardioplegia was composed of lactated Ringer's solution containing 40mmol/l KCl (Abbott Laboratories, North Chicago, IL). A volume of 30ml/kg was injected at a rate of approximately 50ml/min, through a 21 guage butterfly cannula (Abbott Laboratories, North Chicago, IL) inserted into the proximal aorta. Preliminary studies demonstrated an aortic root pressure of 80mmHg under this infusion rate, which is within physiological limits. Simultaneously, the adenoviral vector was thawed from –80°C and reconstituted in 8ml of PBS (Dulbecco Phosphate Buffered Saline, Gibco BRL, Life Technologies, Grand Island, NY). Immediately following delivery of cardioplegia, the adenoviral vector was injected through the cardioplegia cannula. The catheter and proximal aorta were flushed with a further 2ml of PBS. The vector or PBS was allowed to dwell in the coronary circulation for 30min. The end of the butterfly cannula was then attached to the barrel of a 50ml syringe (Becton Dickinson & Co., NJ) in order to passively vent the aortic root. Saline slush was placed within the pericardial cavity to provide topical cooling and aid myocardial protection.

Frusemide, 5mg (American Regent Laboratories Inc., Shirley, NY) and lignocaine, 10mg (Abbott Laboratories, IL) were added to the venous reservoir, 5 and 2min, respectively, before releasing the aortic cross-clamp. The heart was defibrillated as necessary with a 5J shock (Life Pak 6S Cardiac Monitor, Hewlett Packard). The heart was allowed to stabilize for 10min prior to weaning from CPB. Sodium bicarbonate was administered as required. The venous and arterial cannulae were removed and heparin was reversed with 10mg protamine sulphate (American Pharmaceutical Partners Inc., Los Angeles, CA). An 8Fr anterior mediastinal chest drain was inserted through the subxiphoid region. Routine haemostasis was performed prior to sternal closure with 2/0 polydioxanone (Ethicon Inc., Somerville, NJ). The subcutaneous tissues and skin were closed in two layers with 2/0 and 3/0 Vicryl (Ethicon Inc., Somerville, NJ). After haemodynamic and metabolic stability were confirmed, the femoral arterial cannula used for pressure monitoring and blood gas sampling was removed, and the femoral artery ligated. The groin wound was also closed in two layers. Both the sternal and groin wounds were infiltrated with 10ml 0.25% bupivicaine (Abbott Laboratories, North Chicago, IL). Following the cessation of isoflurane anaesthesia, the piglet was disconnected from the ventilator, and oxygen was administered via a face mask when it was breathing satisfactorily. ECG monitoring was continued until the piglet made attempts to gain sternal recumbency. At this stage the chest drain and peripheral venous cannula were also removed. The piglet was then transferred to an infant incubator. The endotracheal tube was removed when the piglet was deemed to be able to maintain a patent airway. Post-operative opioid analgesia was administered as required (Butorphenol 0.5mg IM 4 hourly).

2.4. Haemodynamic assessment
Piglets were sedated with 100mg/kg ketamine and 100µg/kg acepromazine (Boehringer Ingelheim Vetmedica, Inc., MO). After local infiltration with 10mg lignocaine (Abbott Laboratories, North Chicago, IL) a longitudinal cervical incision was made over the strap muscles and the carotid sheath was incised. A 22 guage teflon venous cannula (Quik-Cath®, Baxter Healthcare Corporation, IL) was inserted into the left internal jugular vein. A 3 French Millar micromanometer (Millar Instruments Inc., Houston, TX) was inserted into the left common carotid artery and manipulated into the left ventricular cavity to record LV pressure. Data were acquired in a blinded fashion at baseline and after infusion of isoprenaline (Abbott Laboratories, North Chicago, IL) at 0.1µg/kg per min and analysed as previously described [9]. Group A underwent haemodynamic assessment without having undergone CPB and cardioplegic arrest and served as a control for normal neonatal function. Groups B and C which received PBS alone or Adeno-ß2AR in PBS respectively, underwent haemodynamic assessment 2 days following CPB and cardioplegic arrest. Groups D and E which received PBS alone or Adeno-ß2AR in PBS respectively, underwent haemodynamic assessment 2 weeks following CPB and cardioplegic arrest. Each animal underwent only one haemodynamic assessement after which they were euthanized.

2.5. Tissue analysis
One hour after completing haemodynamic assessment euthanasia was performed using 16mEq KCl intravenously. The heart was rapidly excised and samples of lung and liver were also collected.

Transverse cross-sections of myocardium (5mm) at the level of the base of the papillary muscle were obtained for histological analysis. Samples were placed immediately in 10% neutral buffered formalin (Surgipath, IL) and subsequently embedded in paraffin. Standard staining techniques with haematoxylin and eosin, CD3 immunological stain (rabbit anti-human antibody which cross-reacts with porcine antigens; Dako Corporation, Carpinteria, CA) with secondary and tertiary antibodies (Innovex Biosciences, Richmond, CA) and Masson trichrome were performed in triplicate on 5µm sections and reviewed by a blinded cardiac pathologist.

Tissue samples from the anterior LV, liver and lung were homogenized in lysis buffer (5mM Tris–HCl (pH 7.4), 5mM EDTA). Membrane fractions were resuspended in binding buffer (75mM Tris–HCl (pH 7.4), 12.5mM MgCl2, and 2mM EDTA) at a concentration of approximately 1mg/ml of membrane protein as determined by the Bradford method [16].

Ligand binding assays were performed in triplicate on membranes with saturating concentrations of the radiolabelled ß-adrenoceptor ligand, [125I] cyanopindolol. Non-specific binding was determined in the presence of alprenolol (20µM). Assays were performed at 37°C for 60min [6]. The resulting specific binding (estimated Bmax) was reported as the receptor density and was normalized to milligrams of membrane protein.

2.6. Statistics
Values are reported as mean±standard error of the mean (SEM), but in addition the range of values for receptor density are also quoted. Receptor density was compared using the Mann–Whitney U-test. Two-tailed unpaired Student's t-test were used to compare the effects of cardiac surgery and Adeno-ß2AR gene therapy on LV function under basal conditions and following isoprenaline stimulation. Two way analysis of variance (ANOVA) was used to assess the effect of Adeno-ß2AR on basal or maximal isoprenaline stimulated responses at both time-points.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
LV ßAR density was reduced 2 days following CPB and cardioplegic arrest (Group B) compared to animals that had not undergone surgery (Group A) (104±5.7 vs 135±6.1fmol/mg membrane protein; P=0.007). Following treatment with Adeno-ß2AR (Group C) total LV ßAR density was increased approximately 4-fold (520±250.9fmol/mg membrane protein; range 158–1731fmol/mg) compared to PBS (Group B) (104±5.7fmol/mg; range 90–125fmol/mg; P=0.002) and compared to the no surgery group (Group A) (135±6.1fmol/mg; range 119–148fmol/mg; P=0.002) (Fig. 1).



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1. LV ßAR density in normal animals (Group A) or 2 days following delivery of PBS (Group B) or Adeno-ß2AR (Group C) during CPB and cardioplegic arrest. (n=6 animals per group). *P<0.05 vs Group B, **P<0.005 vs Groups A and B.

 
Complete comparison of the recorded parameters is shown in Table 1. Basal LV function among the three groups was similar. Following CPB and cardioplegic arrest (Group B) isoprenaline stimulated LV dP/dtmax was reduced by over 25% compared to normal animals (Group A) (4337±405 vs 5328±194mmHg/s; P<0.05), and LV dP/dtmin was less negative (–1537±175 vs –1879±69mmHg/s; P=0.10). ß2AR gene transfer during CPB and cardioplegic arrest (Group C) resulted in elevated isoprenaline stimulated LV dP/dtmax (5348±121 vs 4337±405mmHg/s; P<0.05), heart rate (209±6.9 vs 173±11.0bpm; P<0.05), and reduced LVEDP (2.1±0.4 vs 6.4±1.8mmHg; P<0.05) compared to PBS delivery during CPB and cardioplegic arrest (Group B). The response of basal and isoprenaline stimulated dP/dtmax among the three groups is shown in Fig. 2.


View this table:
[in this window]
[in a new window]
  		Table 1. In vivo haemodynamic measurements at baseline and after infusion of isoprenaline at 0.01, 0.05 and 0.1µg/kg per min, in 18 neonatal piglets
 


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2. In vivo assessment of LV dP/dtmax in normal neonatal piglets (Group A), and also 2 days following delivery of PBS (Group B) or Adeno-ß2AR (Group C) during CPB and cardioplegic arrest (n=6 animals per group). *P<0.05 Group B vs A, **P<0.05 Groups B and C vs A.

 
These changes demonstrate a reduction in ßAR dependent contractility and relaxation following CPB and cardioplegic arrest. Importantly, the impaired LV function following CPB and cardioplegic arrest was restored towards normal by ß2AR gene therapy. Thus, to summarize, there were no significant differences between the group that received Adeno-ß2AR during CPB and cardioplegic arrest (Group C) and the group that did not undergo surgery (Group A).

In addition to the early time-point of post-operative day 2, comparison of animals 2 weeks after treatment showed persistent LV ßAR overexpression (315±74.1 (range 174–653) vs 119±7.1 (range 101–142) fmol/mg membrane protein; P=0.002) in the Adeno-ß2AR group (Group E) compared to the PBS group (Group D) (Fig. 3). There was no significant difference in ßAR overexpression at 2 weeks compared to 2 days (315±74.1 (range 174–653) vs 520±250.9 (range 158–1731) fmol/mg; P=0.94).



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. LV ßAR density 2 weeks following delivery of PBS (Group D) or Adeno-ß2AR (Group E) during CPB and cardioplegic arrest (n=6 animals per group). *P<0.05 vs Group D.

 
Liver and lung ßAR density was similar between animals treated with Adeno-ß2AR and PBS at 2 days and 2 weeks following surgery (Fig. 4). However, both liver and lung ßAR density were lower 2 days following surgery compared to the 2 week time-point, which suggests that surgery causes a reduction in both cardiac and extracardiac ßAR density, which was recovering by 2 weeks.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 4. Liver and lung ßAR density 2 and 14 days (n=6 animals per group) following delivery of Adeno-ß2AR or PBS during cardiac surgery. There were no significant differences between Adeno-ß2AR and PBS treatment at any time-point for either liver or lung ßAR density but there were differences between 2-day and 2-week values as indicated. (A) *P<0.05 Group B vs D, i.e. 2 day value compared to 2 week value. (B) *P<0.05 Group C vs E, i.e. 2-day value compared to 2-week value.

 
There was no obvious myocardial inflammatory response at 2 days in either Adeno-ß2AR, Adeno-ßgal or PBS treatment groups (data not shown). Two weeks following surgery there was very mild inflammation, predominantly lymphocytes (haematoxylin and eosin), with an associated T cell response (CD3) and greater staining of collagen (Masson trichrome) in Adeno-ß2AR treated animals (Group E) compared to PBS (Group D) (Fig. 5).



View larger version (130K):
[in this window]
[in a new window]
  		Fig. 5. Representative histological sections from hearts excised 2 weeks following delivery of either 1x1012 tvp Adeno-ß2AR or PBS alone during CPB and cardioplegic arrest. Haematoxylin and eosin stained sections show increased inflammation following Adeno-ß2AR (A) vs PBS (B); CD3 stained sections show greater infiltration of brown staining T cells following Adeno-ß2AR (C) vs PBS (D); Masson trichrome stained sections show greater blue staining collagen deposition following Adeno-ß2AR (E) vs PBS (F). All sections are shown at 10x magnification.

 
The global enhancement of LV function following Adeno-ß2AR delivery evident at 2 days was present at the later time-point of 2 weeks. Peak isoprenaline stimulated LV dP/dtmax was enhanced following Adeno-ß2AR treatment (Group E) compared to those treated with PBS alone (Group D) (4658±319 vs 3882±128mmHg/s; P<0.05; Fig. 6). In the absence of different heart rates (182±13.6 vs 161±7.6bpm; P>0.20) or LVEDP (65±2.3 vs 64±2.2mmHg; P>0.20), this is an indicator of enhanced contractility.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6. In vivo assessment of LV dP/dtmax 2 weeks following delivery of PBS (Group D) or Adeno-ß2AR (Group E) during CPB and cardioplegic arrest (n=6 animals per group). *P<0.05 vs Group D.

 
A comparison of all animals assessed at either 2 days or 2 weeks following CPB and cardioplegic arrest (Groups B–E) by two way ANOVA identified that while basal responses were similar, there was a significant effect of Adeno-ß2AR compared to PBS on heart rate (P=0.008), LV dP/dtmax (P=0.002) and LVEDP (P=0.02) after isoprenaline stimulation.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
This study demonstrates reduced ßAR density and LV function 2 days following CPB and cardioplegic arrest in pigs. Cardiac-selective overexpression of the human ß2AR was achieved within 2 days of Adeno-ß2AR delivery during surgery and persisted for at least 2 weeks. This ß2AR overexpression was associated with restoration of LV function. The data suggests that ß2AR gene transfer may represent a novel approach to improve post-operative LV dysfunction.

This model demonstrates that cardiac ßAR density is reduced during the early post-operative period following CPB and cardioplegic arrest. A canine model of cardioplegic arrest and CPB demonstrated reduced LV ßAR density 30min following the termination of CPB, although there was no reduction in LV ßAR density during CPB itself [1]. Since, post-operative myocardial samples cannot be obtained from patients, human studies of post-operative myocardial ßAR signalling have not been performed. However, lymphocytes, which have been used as a model to study ßARs on the less accessible myocardium, demonstrate a reduction in ßAR density in adults one day after CPB [17]. This is in agreement with the current study, in which reduced ventricular ßAR density was present 2 days following CPB and cardioplegic arrest.

LV dP/dtmax, an indicator of contractility, was reduced 2 days following CPB and cardioplegic arrest. There was also increased LVEDP and impaired LV dP/dtmin, an indicator of relaxation. The alteration in these haemodynamic parameters is consistent with the reduced ßAR density. Thus, this study has demonstrated down-regulation of ßAR and reduced responsiveness to ßAR agonist following CPB and cardioplegic arrest. Prolonged exposure to ßAR agonist, as occurs in the perioperative period, is known to be associated with desensitization and down-regulation [3]. Furthermore, there is evidence to suggest that the human heart has only a small receptor reserve for ßAR mediated inotropic effects which is consistent with these observations [4]. Patients with post-operative LV failure are often treated with ßAR agonists, which may result in progressive receptor down-regulation and further reduced responsiveness to agonist. Theoretically, ß2AR gene therapy may be more effective than agonist, since it would overcome the problem of down-regulation of the endogenous ßAR. Furthermore, given the presence of regulatable therapeutic gene expression cassettes, it may be possible to switch on therapeutic genes when required in the post-operative period [18]. Additionally, other studies have demonstrated proof of principle in the utilization of novel adrenergic receptors which respond only to synthetic agonist but not to endogenous adrenergic agonists [19].

Cardiac transgene overexpression was demonstrated by elevated ßAR density 2 days following delivery of Adeno-ß2AR which confirmed earlier findings with the marker gene ß-galactosidase [12]. The major goal of this study was, however, to assess if ß2AR overexpression following cardiac surgery could result in a positive change in LV function, restoring post-operative dysfunction towards normal. Importantly, Adeno-ß2AR treated animals had enhanced isoprenaline stimulated LV dP/dtmax 2 days post-CPB and cardioplegic arrest. Lower LVEDP after Adeno-ß2AR suggests that increased pre-load does not account for the increased LV dP/dtmax. This restored the impaired LV function following CPB and cardioplegic arrest towards normal such that there were no significant differences between the group that received Adeno-ß2AR during CPB and cardioplegic arrest and the group that did not undergo surgery. LV dP/dtmax was still enhanced at the later time-point of 2 weeks, although the functional changes at this time were less marked in keeping with other models of gene transfer using first generation adenoviral vectors [9].

Animals treated with the adenoviral vector showed very mild myocardial inflammation, collagen formation and T cell response relative to PBS at 2 weeks. Newer vectors may minimize the immune response as Adeno-ß2AR represents a first generation vector that has previously been shown to induce an inflammatory reaction in the heart [9,11]. Encouraging results have been reported using second or third generation adenoviral vectors, ‘gutted’ adenoviral vectors, or adeno-associated virus [20–22]. Finally, it should be emphasized that despite this mild myocardial inflammation at 2 weeks, LV function remained enhanced in Adeno-ß2AR treated animals relative to PBS controls demonstrating that the histologic change had minimal effects on function.

Neonatal piglets were chosen to limit the quantity of adenoviral vectors required. However, neonates may have a reduced immune response to the vector, and it is also known that the pattern of heart growth by myocyte hyperplasia changes to hypertrophy during the perinatal period [14]. Therefore, further studies must be performed in adult animals before extrapolating the current results to adults. Moreover, the enhanced functional effects of ß2AR gene therapy were limited such that there was, not unsurprisingly, no enhancement of function beyond that of normal animals which did not undergo surgery. Therefore, studies which introduce a model of myocardial dysfunction such as pacing induced heart failure would be interesting to assess the potential of ß2AR gene therapy to significantly improve heart function via this method of delivery.

This study has demonstrated the novel finding that gene transfer of the ß2AR can reverse the decreased ßAR density following cardiac surgery. Moreover, ß2AR gene therapy restores post-operative LV dysfunction towards normal. Unlike a recently described experimental method of gene delivery which involves two separate CPB circuits, this method of gene delivery could be utilized during standard cardiac surgical practice [23]. The method described here also appears to be cardiac selective unlike some other models of cardiac gene delivery which have shown evidence of extracardiac transgene expression [8–10]. In addition, tissue-selective promotors could be combined with these methods to further limit detrimental extracardiac transgene expression [18,24]. The further refinement of vectors and the addition of regulated gene expression raises the prospect of switching genes on and off as required during the post-operative period according to individual patient requirements for such molecular ventricular support [18]. However, it must be remembered that alteration of any cardiac cellular signalling pathway may ultimately turn out to have cardiotoxic effects, including arrhythmias. Thus, any genes which appear to cause enhanced cardiac function, must undergo extensive toxicity studies in animals prior to similar experiments being performed in human subjects [25].


   Acknowledgments
 
The authors thank Robert J. Lefkowitz for helpful discussions throughout these studies. George Quick, Ronnie Johnston and Kurt Campbell provided technical assistance. This work was supported in part by National Institutes of Health grants HL 56205 (W.J.K.) and HL 59533 (W.J.K.), and the American Heart Association Grant-in-aid (C.A.M.).


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 

  1. Schwinn DA, Leone BJ, Spahn DR, Chesnut LC, Page SO, McRae RL, Liggett SB. Desensitization of myocardial ß-adrenergic receptors during cardiopulmonary bypass: evidence for early uncoupling and late downregulation. Circulation 1991;84:2559-2567.[Abstract/Free Full Text]
  2. Jones JM, O'Kane H, Gladstone DJ, Sarsam MA, Campalani G, MacGowan SW, Cleland J, Cran GW. Repeat heart valve surgery: risk factors for operative mortality. J Thorac Cardiovasc Surg 2001;122:913-918.[Abstract/Free Full Text]
  3. Hausdorff WP, Caron MG, Lefkowitz RJ. Turning off the signal: desensitization of the ß-adrenergic receptor function. Fed Am Soc Exp Biol J 1990;4:2881-2889.
  4. Brown L, Deighton NM, Bals S, Söhlmann W, Zerkowski H-R, Michel MC, Brodde O-E. Spare receptors for ß-adrenoceptor-mediated positive inotropic effects of catecholamines in the human heart. J Cardiovasc Pharmacol 1992;19:222-232.[Medline]
  5. Lefkowitz RJ, Rockman HA, Koch WJ. Catecholamines, cardiac ß-adrenergic receptors, and heart failure. Circulation 2000;101:1634-1637.[Free Full Text]
  6. Milano CA, Allen LF, Rockman HA, Dolber PC, McMinn TR, Chien KR, Johnson TD, Bond RA, Lefkowitz RJ. Markedly enhanced myocardial function in transgenic mice with cardiac overexpression of the human ß2-adrenergic receptor. Science 1994;264:582-586.[Abstract/Free Full Text]
  7. Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing the ß-adrenergic receptor kinase or a ßARK inhibitor. Science 1995;268:1350-1353.[Abstract/Free Full Text]
  8. Hajjar RJ, Schmidt U, Matsui T, Guerrero JL, Lee K-H, Gwathmey JK, Dec GW, Semigran MJ, Rosenzweig A. Modulation of ventricular function through gene transfer in vivo. Proc Natl Acad Sci USA 1998;95:5251-5256.[Abstract/Free Full Text]
  9. Maurice JP, Hata JA, Shah AS, White DC, McDonald PH, Dolber PC, Wilson KH, Lefkowitz RJ, Glower DD, Koch WJ. Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary ß2-adrenergic receptor gene delivery. J Clin Invest 1999;104:21-29.[Medline]
  10. Barr E, Carroll J, Kalynych AM, Tripathy SK, Kozarsky K, Wilson JM, Leiden JM. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus. Gene Ther 1994;1:51-58.[Medline]
  11. Shah AS, Lilly E, Kypson AP, Tai O, Hata JA, Pippen A, Silvestry SC, Lefkowitz RJ, Glower DD, Koch WJ. Intracoronary adenovirus-mediated delivery and overexpression of the ß2-adrenergic receptor in the heart—prospects for molecular ventricular assistance. Circulation 2000;101:408-414.[Abstract/Free Full Text]
  12. Davidson MJ, Jones JM, Emani S, Wilson KH, Jaggers J, Koch WJ, Milano CA. Cardiac gene delivery with cardiopulmonary bypass. Circulation 2001;104:131-133.[Abstract/Free Full Text]
  13. Jones JM, Wilson KH, Koch WJ, Milano CA. Adenoviral gene transfer to the heart during cardiopulmonary bypass: effect of myocardial protection technique on transgene expression. Eur J Cardiothorac Surg 2002;21:847-852.[Abstract/Free Full Text]
  14. Rudolph AM. Myocardial growth before and after birth: clinical implications. Acta Paediatr 2000;89:129-133.[CrossRef][Medline]
  15. Drazner MH, Peppel KC, Dyer S, Grant AO, Koch WJ, Lefkowitz RJ. Potentiation of ß-adrenergic signaling by adenoviral mediated gene transfer in adult rabbit ventricular myocytes. J Clin Invest 1997;99:288-296.[Medline]
  16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248-254.[CrossRef][Medline]
  17. Smiley RM, Pantuck CB, Chadburn A, Knowles DM. Down-regulation and desensitization of the ß-adrenergic receptor system of human lymphocytes after cardiac surgery. Anesth Analg 1993;61:653-661.
  18. Phillips MI, Tang Y, Schmidt-Ott K, Qian K, Kagiyama S. Vigilant vector: heart-specific promoter in an adeno-associated virus vector for cardioprotection. Hypertension 2002;39(2 Pt 2):651-655.[Abstract/Free Full Text]
  19. Small KM, Brown KM, Forbes SL, Liggett SB. Modification of the ß2-adrenergic receptor to engineer a receptor–effector complex for gene therapy. J Biol Chem 2001;276:31596-31601.[Abstract/Free Full Text]
  20. Amalfitano A, Hauser MA, Hu H, Serra D, Begy CR, Chamberlain JS. Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted. J Virol 1998;72:926-933.[Abstract/Free Full Text]
  21. Gilbert R, Nalbantoglu J, Howell JM, Davies L, Fletcher S, Amalfitano A, Petrof BJ, Kamen A, Massie B, Karpati G. Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products. Hum Gene Ther 2001;12:1741-1755.[CrossRef][Medline]
  22. Svensson EC, Marshall DJ, Woodard K, Lin H, Jiang F, Chu L, Leiden JM. Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors. Circulation 1999;99:201-205.[Abstract/Free Full Text]
  23. Bridges CR, Burkman JM, Malekan R, Konig SM, Chen H, Yarnall CB, Gardner TJ, Stewart AS, Stecker MM, Patterson T, Stedman HH. Global cardiac-specific transgene expression using cardiopulmonary bypass with cardiac isolation. Ann Thorac Surg 2002;73:1939-1946.[Abstract/Free Full Text]
  24. Franz WM, Rothman T, Frey N, Katus HA. Analysis of tissue-specific gene delivery by recombinant adenoviruses containing cardiac-specific promoters. Cardiovasc Res 1997;35:560-566.[Abstract/Free Full Text]
  25. Isner JM. Myocardial gene therapy. Nature 2002;415:234-239.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger
Designing Heart Performance by Gene Transfer
Physiol Rev, October 1, 2008; 88(4): 1567 - 1651.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Author home page(s):
Carmelo A. Milano
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jones, J. M.
Right arrow Articles by Milano, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jones, J. M.
Right arrow Articles by Milano, C. A.
Related Collections
Right arrow Cardiac - pharmacology
Right arrow Cardiac - physiology
Right arrow Molecular biology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ANN THORAC SURG ASIAN CARDIOVASC THORAC ANN EUR J CARDIOTHORAC SURG
J THORAC CARDIOVASC SURG ICVTS ALL CTSNet JOURNALS