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Ann Thorac Surg 2005;80:1812-1820
© 2005 The Society of Thoracic Surgeons

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Original article: Cardiovascular

Attenuation of DNA Damage in Canine Hearts Preserved by Continuous Hypothermic Perfusion

^a Division of Cardiac Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland
^b Department of Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland
^c Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Accepted for publication April 25, 2005.

^_* Address correspondence to Dr Conte, Heart and Heart/Lung Transplantation, The Johns Hopkins Medical Institutions, 600 North Wolfe St, Blalock 618, Baltimore, MD 21287 (Email: jconte{at}csurg.jhmi.jhu.edu).

	Abstract

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
BACKGROUND: Continuous hypothermic perfusion is a novel cardiac preservation technique. Reactive oxygen species play a role in ischemia reperfusion injury and limit organ preservation. Oxidative stress mediates a DNA mismatch lesion (7, 8-dihydro-8-oxoguanine [8-oxo-G]), which is repaired by the enzymes MutY homologue (MYH), 8-oxo-G glycosylase (OGG1), and MutS homologue 2 (MSH2). We hypothesized that continuous hypothermic perfusion would allow for maintenance of cardiac function while attenuating myocardial DNA damage with respect to the current clinical practice of static preservation at 4°C.

METHODS: In our canine orthotopic transplant model, donor hearts were harvested after echocardiograms, and hemodynamic studies were obtained and served as controls. The hearts were transplanted after 24 hours of continuous hypothermic perfusion or 4 hours of static preservation, and were studied for 6 hours. Quantification of 8-oxo-G lesions, MYH, OGG1, and MSH2 concentrations were performed on biopsies using immunohistochemistry.

RESULTS: Postimplant echocardiograms, completed in 7 continuously perfused and 8 statically preserved hearts, demonstrated good function and normal wall motion. Positive staining for 8-oxoG was markedly increased in the static preservation group. Staining density for MYH, OGG1, and MSH2 were significantly decreased in statically preserved hearts and equivalent between continuously perfused and control hearts.

CONCLUSIONS: The DNA damage assayed by 8-oxoG was significantly increased in statically preserved versus continuously perfused hearts. The DNA repair enzymes MYH, OGG1, and MSH2 were also markedly decreased in the static preservation versus continuous hypothermic perfusion groups. Continuous hypothermic perfusion reduces oxidative damage and extends preservation without compromising function.

	Introduction

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

Doctor Gage discloses a financial relationship with Organ Recovery Systems, Baltimore, Maryland.

 

Successful organ preservation requires metabolic arrest, conservation of organ architecture, and minimization of ischemia-reperfusion injury. Numerous investigations of operative techniques, additives, and solutions have improved storage duration of abdominal organs. However, static preservation (SP) of the heart beyond 6 hours does not provide reliable allograft function [1, 2]. Preservation using continuous hypothermic perfusion (CHP) offers a number of potential advantages over SP including (1) continuous myocardial cooling through the native coronary circulation; (2) continuous substrate supplementation limiting anaerobic metabolism; and (3) toxic by-product washout [3].

Cardiovascular tissues are rich sources of reactive oxygen species that are increasingly recognized as having a major role mediating ischemia-reperfusion injury. Extended hypothermic preservation induces excessive reactive oxygen species production (oxidative stress) and causes myriad cellular injuries, including DNA damage, which results in cellular death if unrepaired [4, 5]. Oxidation of guanine produces a stable, deleterious adduct (7, 8-dihydro-8-oxoguanine [8-oxo-G]) that causes an adenine/cytosine base transversion error during replication (Fig 1) [6–11]. The DNA repair enzymes 8-oxo-G glycosylase (OGG1), MutY homologue (MYH), and MutS homologue 2 (MSH2) recognize misincorporated base pairs and repair the oxidative-damaged DNA [12, 13].

View larger version (27K): [in this window] [in a new window]   Fig 1. System of 7, 8-dihydro-8-oxoguanine (8-oxo-G) repair. (A = adenine; C = cytosine; G = guanine; MSH2 = MutS homologue 2; MYH = MutY homologue; OG = oxidatively-damaged guanine; OGG1 = 8-oxo-G glycosylase; T = thymine.)

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Canines used were 30 to 40 kg, heartworm-free, 6-month-old mongrels who receive humane care in compliance with the "Guide for the Care and Use of Laboratory Animals," published by the National Research Council (National Academy Press, 1996).

Study Group
Dogs were divided into two groups based on the donor heart preservation strategy employed. Donor hearts in the first group underwent SP for 4 hours at a temperature of 5°C, and were monitored with an intramyocardial temperature probe. The hearts in the second group underwent CHP for 24 hours at a pressure of 15 mm Hg and a temperature of 5°C. The same preservation solution was used for static storage in the SP group and for perfusion in the CHP group. The circulating perfusate was a proprietary modified Belzer solution supplemented with hydroxyethyl starch, glutathione, adenine, and fructose-1,6-bisphosphate (KPS-1; Organ Recovery Systems, Des Plains, Illinois) [15].

Operative Technique
Canines were intubated after induction with sodium pentobarbital and anesthesized using inhalational isofluorane. A midline sternotomy was performed and the heart arrested with Stanford cardioplegia after injection of 10 mg Regitine. The heart was topically cooled and excised, preserving the aortic arch. Baseline weights were obtained for all hearts. For the CHP group, a perfusion cannula was placed into the aortic root and the hearts were suspended on a Heart Transporter (Organ Recovery Systems). The Heart Transporter is a modified kidney perfusion device that is a lithium-powered, ultralightweight apparatus with easily controlled perfusion pressure, flow, and temperature (Fig 2). A retrograde cardioplegia catheter was inserted into the coronary sinus for effluent sampling, and CHP was initiated and maintained at a 15 mm Hg perfusion pressure for 24 hours. Myocardial temperature was maintained at 5°C. The heart was monitored by a perfusion technologist experienced in organ perfusion at the local Organ Procurement Organization (Transplant Resource Center of Maryland). It was maintained within the following temperature-corrected parameters: (1) pH 7.55-7.70; (2) P_aO₂150 to 250 mm Hg; and (3) 380 to 400 milliosmoles.

View larger version (153K): [in this window] [in a new window]   Fig 2. The Heart Transporter (Organ Recovery Systems, Des Plains, Illinois) used for continuous hypothermic perfusion.

Functional Assessment
Each canine heart was utilized as its own control to limit the number of animals required for experimentation. Frequent arterial blood gas analysis was used to adjust ventilator mechanics, inotropic support, and fluid administration. Epicardial echocardiography was performed at two time points, preharvest and at 1 hour post-CPB. These echocardiograms were assessed for ejection fraction and regional wall motion abnormalities. Chamber pressures and cardiac output, using a Millar catheter (Millar, Houston, Texas), were measured at baseline and compared against measurements at 1, 3, and 5 hours post-CPB. Cardiac enzymes, including creatine kinase, creatine kinase-MB, and troponin, were measured at baseline and at 1, 3, and 5 hours post-CPB.

Immunohistochemical Staining (IHCS): 8-Oxo-G
Tissues were dehydrated, deparaffined, and incubated with proteinase K in PBS for 40 minutes at 37°C. The DNA was denatured using HCL for 7 minutes and then neutralized with Tris-Base. The sections were immersed in 10% fetal bovine serum for 1 hour at room temperature to block nonspecific staining sites before overnight incubation at 4°C with anti–8-oxo-G monoclonal antibody. Endogenous peroxidase was neutralized using 3% H₂O₂ in methanol solution before incubating with a horseradish-peroxidase conjugated with anti-mouse IgG antibody. The sections were stained with diaminobenzamide tetrahydrochloride (DAB) and counterstained with hematoxylin. Positive staining was assessed by microscopic examination and changes expressed as the percentage of positive staining area over the entire section examined.

DNA Repair Enzymes
Tissue specimens were dehydrated, paraffin-embedded, and cut into 5 µm sections. Samples were then deparaffined and blocked with 10% donor goat serum in PBS before incubation at 4°C with a primary polyclonal antibody diluted in 10% donor goat serum and 0.3% Triton-X for 20 hours. Endogenous peroxidase activity was neutralized using 0.5% H₂O₂ before incubating with a second horseradish-peroxidase conjugated with anti-rabbit IgG antibody. The sections were then stained with DAB, counterstained with hematoxylin, and mounted. Significant differences were assessed and quantified by two independent observers using microscopic examination and densitometry.

Western Blot Analysis
Specimens were homogenized in a lysis buffer and centrifuged at 7,000g for 15 minutes; then sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed using a 10% polyacrylamide gel under nonreducing conditions. The supernatant was transferred to a nitrocellulose membrane using electroblotting and immersed for 1 hour in 10% nonfat dry milk to block nonspecific binding sites. The membrane was then incubated with primary (1:500) and secondary (1:1000) polyclonal antibodies against OGG1, MYH, and MSH2. Detection was performed using an Enhanced Chemilluminescence kit (Amersham Biosciences, Piscataway, New Jersey). Monoclonal antibodies against actin were used as a control.

In-Situ Detection of DNA Fragmentation (TUNEL Staining)
To detect DNA fragmentation in situ, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) was performed using an ApopTag in situ detection kit (Oncogene, Cambridge, Massachusetts). The sections were deparaffined, incubated with 20 µg/mL proteinase K for 10 minutes, and then treated with 3% hydrogen peroxide for 5 minutes to inactivate endogenous peroxidase. The sections were then processed for TUNEL staining. A masked examiner reviewed the specimens to determine TUNEL positivity. Apoptosis was quantified and expressed as a percentage of positive TUNEL nuclear positivity over total nuclei examined.

Microarray Analysis
One hundred milligrams of ventricular myocardium from SP and CHP hearts were used to extract total RNA using RNAssay Qiagen (Qiagen, Valencia, CA) columns. For in vitro transcription, 5 µg total RNA were used according to Affimetrix protocols, and analysis performed using the Affimetrix platform. After in vitro transcription, the samples were processed separately for hybridization to an HG-U133Plus2A microarray chip. The HG-U133Plus2A chip contains more than 47,000 probes. The resultant microarray profile was compared against baseline canine myocardium using GeneSpring software.

Statistical Analysis
All statistics are reported as mean ± SD and were calculated and compared using ² and analysis of variance (ANOVA).

	Results

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Myocardial Weight Gain
All hearts gained weight during preservation but the CHP hearts percent weight gain was significantly greater than the SP hearts (Table 1). We also compared weight gain in the CHP hearts that functioned well versus those that did not and found no significant differences, although the percent change tended to be greater in the poorly functioning hearts.

View larger version (118K): [in this window] [in a new window]   Fig 3. Staining density for 7, 8-dihydro-8-oxoguanine (8-oxo-G): arrows denote increased staining density in static preservation (SP) versus continuous hypothermic perfusion (CHP) canine hearts.

View larger version (114K): [in this window] [in a new window]   Fig 4. The DNA repair enzyme 8-oxo-G glycosylase: decreased staining is seen in static preservation (SP) versus continuous hypothermic perfusion (CHP) hearts after storage but is equivalent after implant.

View larger version (106K): [in this window] [in a new window]   Fig 5. The MutY homologue (MYH): decreased staining is seen in static preservation (SP) versus continuous hypothermic perfusion (CHP) hearts after storage but is equivalent after implant.

View larger version (107K): [in this window] [in a new window]   Fig 6. MutS homologue 2 (MSH2): decreased staining is seen in static preservation (SP) versus continuous hypothermic perfusion (CHP) hearts after storage but is equivalent after implant.

View larger version (59K): [in this window] [in a new window]   Fig 7. Western Blot analysis: 8-oxo-G glycosylase (OGG) and MutY homologue (MYH) protein levels were decreased in static preservation hearts (SP) versus continuous hypothermic perfusion hearts (CHP) after storage but were equivalent after implant. (Pre = before implant; Post = after implant.)

View larger version (100K): [in this window] [in a new window]   Fig 8. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining: static preservation (SP) hearts had increased positivity versus continuous hypothermic perfusion (CHP) both after storage and after implant. Arrows point out TUNEL positive stained cells representing the increased amount of apoptosis in the SP versus CHP hearts.

View larger version (23K): [in this window] [in a new window]   Fig 9. Gene microarray analysis: marked upregulation of metallothionein gene expression in static preservation hearts (gray bars) and continuous hypothermic perfusion hearts (black bars) after implant.

	Comment

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

Numerous studies have demonstrated that mechanical methods of CHP successfully extended storage duration of abdominal organs without compromising function. However, equal success has not been realized with the heart [16–18]. Perfused cadaveric renal allografts have a decreased incidence of graft dysfunction versus those statically preserved and CHP is now the preservation standard [19]. Wicomb and colleagues [20] compared 24-hour CHP versus 4-hour SP hearts on a Langendorf apparatus and found that, although markedly edematous, the CHP hearts had adequate function to support systemic circulation. Nuclear magnetic resonance spectroscopy of CHP hearts demonstrated increased ATP and high-energy phosphate concentrations with concomitant decreases in lactate versus SP hearts [21]. Clinical pursuit of perfusion therapy in thoracic transplantation has been delayed by device complexity and begrudging acceptance of current procurement methods. The Heart Transporter represents the next generation of transporters. Early use of this device by experienced organ perfusion technicians confirmed the user-friendly interface, simplicity and durability of the Heart Transporter. This device offers hope that CHP could easily be implemented if further studies confirm the efficacy of this technology.

Myocardial edema is a consequence of any technique of hypothermic preservation. It increases coronary resistance, impairing subendocardial perfusion and compromises ventricular function. Perfusion preservation can exacerbate edema formation and modifications including hyperosmolar solutions, low perfusion pressures, and intermittent, pulsatile flows have failed to mitigate weight gain [22]. Previous studies suggested that weight gain more than 25% of baseline independently predicted poor outcome. However, weight gain did not clearly correlate with function or outcome in our study. Edema formation is an unavoidable consequence of perfusion-based preservation therapy, and strategies to reduce its formation need to be explored. The optimal duration of perfusion and conditions under which it is performed are unknown. Factors that need to be studied include temperature, perfusion pressure, solution composition, and pharmacologic manipulations such as antioxidants and synthetic oxygen carriers.

The low oxidation potential of guanine results in preferential attack during oxidative DNA damage, forming 7, 8-dihydro-8-oxoguanine (8-oxo-G). The 8-oxo-G is a stable biomarker of oxidative damage as well as an "electron sink" that propagates further oxidative injury if unrepaired [11]. Quantification of 8-oxo-G is a reliable measurement to compare oxidative stress and DNA damage between the SP and CHP preservation strategies. The significantly increased expression of 8-oxo-G in the SP versus CHP hearts at all time points after preservation confirms that perfusion is more protective against oxidative DNA damage. As a family of DNA repair enzymes, OGG1, MYH, and MSH2 recognize oxidized guanine residues or the resultant adenine transversion injury and restore normal DNA configuration. Failure to repair the injury promotes cardiomyocyte dysfunction and apoptosis. Previous studies of MYH, using an ischemic rabbit spinal cord model, demonstrated that MYH was increased as early as 1 hour after reperfusion. Inadequate expression of MYH in the setting of increasing oxidative stress correlated with the severity of paraplegia [10, 12]. Each of the three DNA repair enzymes studied in this experiment were significantly reduced immediately after storage in the SP hearts but were equivalent with the CHP hearts by the postimplant stage. The decreased expression of the DNA repair enzymes in the SP group may reflect a temperature-dependent delay in enzyme synthesis, transcription downregulation, or interference with posttranscriptional processing. Perfusion may preserve the ability of the cardiomyocyte to respond to and initiate oxidative damage repair processes earlier, even in hypothermic conditions, than static preservation, potentially improving early and late allograft performance.

Heart transplantation is associated with activation of apoptotic pathways, but the impact on allograft function is not clearly known [23]. Apoptosis is a consistent feature of end-stage heart failure. Percentages of apoptosis in endomyocardial biopsies directly correlate with grades of rejection, clinical disease progression, and survival [24–26]. Imbalances between oxidative damage and DNA repair capability promote apoptosis, characterized by increased TUNEL positivity in the SP hearts [10]. Although TUNEL assays have been criticized for limited specificity, careful processing of fresh tissue, produces reproducible and reliable results [27]. Reduction of oxidative stress and preserved repair processes using CHP reduces cardiomyocyte loss and may significantly impact both the short- and long-term functional outcome of cardiac allografts.

Reactive oxygen species are generated during ischemia-reperfusion and mediate myocardial injury. Metallothioneins are thiol-rich proteins that inhibit the cytochrome-c mediated caspase-3 activation pathway and suppress hypoxia/reoxygenation-induced cardiomyocyte apoptosis [28, 29]. Although normally expressed at low basal levels, significant increases in metallothioneins are induced by stress conditions including heat, inflammation, and ischemia. Transgenic, metallothionein-overexpressing mice have significantly improved contractile function versus controls in response to ischemia-induced myocardial injury [30]. Metallothionein proteins were upregulated to a greater degree in the CHP versus SP group, suggesting that perfusion better preserves allograft response to ongoing injury during hypothermic preservation.

Our animal experiment was purposefully designed to recreate the clinical situation and compare a short, standard period of SP with an extreme, extended CHP period. It was not done to directly compare the two preservation strategies. It was done to demonstrate the effectiveness of CHP in an extreme situation using a standard clinically relevant period of static preservation as a reference. Using this rationale, equivalent outcomes would portend a positive outlook for this technology. Less DNA damage and equivalent hemodynamic performance after 24 hours of CHP achieves this. It is clinically unlikely to be necessary to store a heart for 24 hours. The use of a simple, portable apparatus to lengthen preservation without compromising function offers several potential advantages. In addition to improving outcomes, this technique can potentially expand the donor pool in several ways: (1) by allowing HLA typing; (2) by resuscitating nonbeating heart donor organs; (3) by facilitating ex vivo interventions improving graft quality; and (4) by increasing organ sharing. Use of continuous hypothermic perfusion in thoracic transplantation is extremely promising with far-reaching clinical applications; however, more animal studies need to be completed, confirming reliability and reproducibility, before it can be utilized for human transplantation.

Limitations
This study evaluated a new device to perfuse hearts for a prolonged period of time. Adequate hemodynamic performance was assessed by separation from CPB and left ventricular function on echocardiogram. In the hearts that failed to wean from CPB, technical problems and human errors were identified and resulted in complications unrelated to CHP. These animals were excluded from analysis to prevent prejudicing opinion against CHP but were included in the results for full disclosure.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We would like to thank Melissa Jones, Jeffrey Brawn, and Tamara Treat for technical assistance with this project. Financial funding for this project was made available by Organ Recovery Systems, Baltimore, Maryland.

	References

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Torin P. Fitton Christopher J. Barreiro Pramod N. Bonde John V. Conte Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fitton, T. P. Articles by Conte, J. V. Search for Related Content PubMed PubMed Citation Articles by Fitton, T. P. Articles by Conte, J. V. Related Collections Transplantation - heart