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Ann Thorac Surg 1999;68:903-907
© 1999 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Prolonged preservation of the blood-perfused canine heart with glycolysis-promoting solution

^a Department of Cardiac Surgery, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
^b Department of Anesthesiology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
^c Division of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Address reprint requests to Dr del Nido, Department of Cardiac Surgery, Children’s Hospital, 300 Longwood Ave, Boston, MA 02115;
e-mail: delnido{at}a1.tch.harvard.edu

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Prolonged ischemia and inadequate myocardial preservation remain significant perioperative risk factors in cardiac transplantation. Long-term preservation techniques that have been effective in small rodent hearts have not been as effective in larger animal models or in clinical studies. We developed a cardioplegia solution formulated to promote high-energy phosphate production from glycolysis and determined its efficacy in a blood perfused canine heart model subjected to 24 hours of ischemia.

Methods. Hearts harvested from adult dogs (n = 6 per group) were flushed with a histidine-buffered cardioplegia solution containing glucose or University of Wisconsin solution. The hearts were maintained at 4°C for 24 hours then reperfused with autologous blood. After reperfusion, left ventricular pressures were measured with an intracavitary balloon at varying balloon volumes and compared with control nonischemic hearts. Predicted stroke volume and ejection fraction were calculated at an end-systolic pressure of 70 mm Hg and end-diastolic pressure of 15 mm Hg.

Results. Developed pressure was better preserved in the hearts that received histidine-buffered solution (93 ± 9 versus 38 ± 7 mm Hg, p < 0.05), along with a higher end-diastolic volume at 15 mm Hg (31 ± 3 versus 22 ± 2 mL histidine-buffered versus University of Wisconsin solutions, respectively, p < 0.05). Stroke volume and ejection fraction were also higher in the histidine group (17 ± 2.5 versus 2.3 ± 1.2 mL and 50% ± 3.5% versus 9% ± 4.5%, respectively) in the presence of dobutamine.

Conclusions. The highly buffered glycolysis-promoting cardioplegia solution provided effective preservation of the blood perfused canine heart with superior recovery of pump performance after 24 hours of hypothermic ischemia compared with University of Wisconsin solution in this model.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
Extending the period of donor graft preservation could expand the available donor pool and provide a wider safety margin for long-distance organ procurement. Inadequate myocardial protection during prolonged ischemia has been shown to contribute to early posttransplant heart failure and death unrelated to rejection or infection.

During prolonged ischemia, myocardial stores of high-energy phosphates are depleted, eventually leading to irreversible contracture. To slow these events, myocardial preservation techniques for prolonged ischemia have centered around deep hypothermia for metabolic arrest and cardioplegia to prevent contractile activity. Even at very low temperatures, however, adenosine triphosphate (ATP) hydrolysis still occurs resulting in hydrogen ion (H⁺) production and accumulation [1], which leads to intracellular acidosis. Glucose metabolism for energy production via the glycolytic pathway can proceed at low temperatures but, in the absence of oxidative metabolism, leads to lactate generation. Hydrogen ion and lactate accumulation from ATP breakdown and anaerobic glycolysis can suppress glycolytic enzymes preventing further ATP regeneration from glycolysis [2]. However, removing proton ions and lactate generated by the ischemic myocardium has been shown to accelerate glycolytic flux during ischemia and lead to enhanced high-energy phosphate preservation as well as recovery of cardiac function after ischemia [3].

We previously showed that by removing lactate and H⁺ by buffering the protons with the amino acid histidine (100 mM), anaerobic glycolysis can be promoted, leading to improved ATP preservation and postischemic contractile function in rabbit heart [4, 5]. We also showed that high concentrations of glucose, insulin, and adenosine further promote anaerobic glycolysis even when the heart is at very low temperatures (< 10°C) [6]. The purpose of the present study was to evaluate the efficacy of this cardioplegic solution formulated to promote anaerobic energy production from glucose metabolism, in a blood perfused canine heart subjected to 24 hours of ischemia. We compared this formulation with the University of Wisconsin solution, which has been used clinically in cardiac transplantation [7].

	Material and methods

Top Abstract Introduction Material and methods Results Comment References

 
Animal preparation
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the Institute of Laboratory Animal Resources and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 85-23, revised 1985).

Twelve mongrel dogs weighing 17.5 to 27.6 kg were anesthetized with sodium pentobarbital (30 mg/kg) and placed on a respirator after orotracheal intubation. After a median sternotomy exposing the aortic arch, heparin (5 mg/kg) was administered intravenously followed by insertion of a 12-F perfusion catheter (Sherwood, St. Louis, MO) into the ascending aorta through the innominate artery. After 1,000 mL of blood was collected by way of the catheter, the aorta was clamped and the heart arrested with 30 mL/kg of either University of Wisconsin solution (UWS) or histidine-buffered solution (HBS). The composition of both solutions is shown in Table 1. The inferior vena cava and left atrial appendage were incised during infusion to prevent ventricular distension. Cardiectomy was then completed, and the heart was stored in a plastic bag containing either 1 L of UWS or HBS solution at 4°C. After 24 hours of storage, the heart was then attached to an isolated heart perfusion apparatus. The heart was perfused with oxygenated autologous blood using a centrifugal pump (Bio-pump; Bio-Medicus Inc, Eden Prairie, MN) and a membrane oxygenator (ECMO 0600; SciMed Life System Inc, Minneapolis, MN) gassed with 95% oxygen and 5% carbon dioxide to maintain partial pressure of oxygen or partial pressure of carbon dioxide within the physiologic range. One hundred milliliters of 20% human serum albumin was added, and 1,800 mg of glucose and 20 IU of regular insulin were added to the perfusate system. Potassium and calcium levels were maintained within the physiological range by addition of calcium chloride or potassium chloride. A perfusion pressure of 70 to 90 mm Hg was maintained throughout the experiment. Another four hearts were arrested with high-potassium cardioplegia (30 mL/kg), then immediately mounted on the perfusion circuit. These hearts served as the control nonischemic perfusion group. A latex fluid-filled balloon connected to a catheter-tipped micromanometer (MPC-350; Miller, Houston, TX) was inserted into the left ventricle. The balloon volume was varied by a syringe. Cardiac function measurements were made after 1 and 2 hours of perfusion.

The ventricular function variables (systolic and diastolic pressure, stroke volume, ejection fraction, end-diastolic volume, and developed pressure) were obtained under two sets of conditions. A set of baseline measurements was taken 1 and 2 hours after reperfusion, and repeat measurements were made in the presence of low-dose (0.2 mg/L) or high-dose (1 mg/L) dobutamine at the end of the 2-hour reperfusion period.

Statistical analysis
Data were analyzed using the computer program Stat View (Abacus Concepts, Inc, Berkeley, CA). Nonparametric tests were used for data analysis because of the small sample size. The Kruskal-Wallis rank test was used in all cases among three groups. Results were considered significant if p value was less than 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
After 24 hours of preservation and 2 hours of reperfusion, postischemic contractile function was reduced in all groups compared with control nonischemic hearts. However, recovery of developed pressure was substantially better in the hearts preserved in HBS (93 ± 9 mm Hg) compared with the hearts preserved in UWS (38 ± 7 mm Hg, p < 0.05; Fig 1). Peak systolic pressure for different balloon volumes after 1 and 2 hours of reperfusion and the response to dobutamine are shown in Figure 2. The HBS-treated hearts generated consistently higher systolic pressures than the UWS group at all balloon volumes, and this difference was even more significant in the presence of dobutamine. There was no significant improvement in systolic function in the UWS group with low- or high-dose dobutamine in contrast to the HBS group, which had a substantial response to dobutamine.

View larger version (15K): [in this window] [in a new window]   Fig 1. Peak developed pressure at a diastolic pressure of 15 mm Hg in control hearts (nonischemic) and after 2 hours of reperfusion in hearts subjected to 24 hours of 4°C ischemia receiving histidine-buffered solution (HBS) or University of Wisconsin solution (UWS). *p < 0.05 between HBS and UWS groups.

View larger version (34K): [in this window] [in a new window]   Fig 2. Peak systolic pressure at different balloon volumes in hearts subjected to 24 hours of 4°C ischemia receiving histidine-buffered solution (closed circles) or University of Wisconsin solution (open circles). Top, After 1 hour (left) and 2 hours (right) of reperfusion. Bottom, In the presence of low-dose (0.2 mg/L, left) or high-dose (1 mg/L, right) dobutamine in the perfusate. *p < 0.05 between groups.

View larger version (33K): [in this window] [in a new window]   Fig 3. End-diastolic pressure at different balloon volumes in hearts subjected to 24 hours of 4°C ischemia receiving histidine-buffered solution (closed circles) or University of Wisconsin solution (open circles). Top, After 1 hour (left) and 2 hours (right) of reperfusion. Bottom, In the presence of low-dose (0.2 mg/L, left) or high-dose (1 mg/L, right) dobutamine in the perfusate. *p < 0.05 between groups.

View larger version (16K): [in this window] [in a new window]   Fig 4. End-diastolic volume at a diastolic pressure of 15 mm Hg in control hearts (nonischemic) and hearts subjected to 24 hours of 4°C ischemia receiving histidine-buffered solution (HBS) or University of Wisconsin solution (UWS). There were no significant differences between control and HBS. *p < 0.05 between groups.

	Comment

Top Abstract Introduction Material and methods Results Comment References

We hypothesized that the limited supply of high-energy stores and substrates for energy metabolism in the nonperfused heart is the main cause of graft deterioration with prolonged ischemia. The inevitable loss of high-energy stores even at low temperatures results in an inability of the cells to maintain ionic homeostasis leading to sodium and calcium overload and necrosis. Cardioplegia solutions with ionic compositions similar to intracellular ion concentrations, such as University of Wisconsin solution, prevent cation accumulation in most cells. However, excitable cells such as myocytes contain large intracellular stores of calcium in the sarcoplasmic reticulum. These large stores of calcium must be maintained by energy consuming calcium-adenosine triphosphatases (Ca-ATPases) to prevent cation leakage into the cytosol leading to the onset of contracture during prolonged ischemia [8]. Thus, maintaining ATP levels above the minimum required by the calcium-adenosine triphosphatases in excitable cells is necessary to prevent calcium overload.

After the onset of ischemia, oxygen tension in the tissue rapidly decreases to nearly zero, and substrate metabolism for ATP production immediately switches to anaerobic glycolysis. Intracellular lactate then starts to accumulate in association with intracellular acidosis from ATP breakdown [1, 2]. Accumulation of these two end-products then inhibits key glycolytic enzymes, further limiting ATP production. The central concept of the cardioplegia solution containing substrate and a proton-buffering agent is that, by providing glucose and removing or buffering the protons accumulated in the myocytes during ischemia, anaerobic glycolysis and ATP production can continue during ischemia. Continued anaerobic metabolism can then provide more high-energy phosphates for the ion pumps and thus delay the onset of contracture. Removing H⁺ from the cell, however, requires either cotransport with an anionic compound such as lactate, or most commonly, H⁺ exchange for extracellular sodium ions (Na⁺) by the electroneutral Na⁺/H⁺ exchanger on the cell membrane. In the myocyte, the Na⁺/H⁺ exchanger is considered the major mechanism responsible for maintaining intracellular pH within the physiologic range [9]. Intracellular Na⁺ accumulation during ischemia can lead to calcium ion (Ca²⁺) accumulation through the Na⁺/Ca²⁺ antiporter on the cell membrane, if the sodium-potassium ATPase is inactive. To prevent this ionic shift, the ideal method of maintaining intracellular pH within the normal range during ischemia is to buffer or bind H⁺ inside the cell and remove it with an anionic carrier such as histidine.

Bretschneider and colleagues [10] previously described the use of histidine, in high concentration (180 to 195 mmol) in cardioplegia solutions without the presence of glucose. In that technique, they relied almost entirely on breakdown of intracellular stores of glycogen for anaerobic glycolysis. In a previous study, however, we were unable to demonstrate any augmentation of anaerobic glycolysis with histidine alone in ischemic canine hearts [4]. With the addition of glucose and insulin to the histidine-containing solution, anaerobic glycolysis was significantly enhanced in association with improved ATP preservation and postischemic contractile function [5, 6].

In the components of our solution, glucose provides the substrate for anaerobic glycolysis, with insulin added to facilitate glucose transport into the cells. To further enhance glucose transport into myocytes, adenosine was added to the cardioplegia solution. We previously showed that high-dose adenosine significantly improves glucose transport and metabolism during hypothermic ischemia in the heart [6].

University of Wisconsin solution is an intracellular type solution with high potassium (125 mmol/L) and a relatively low sodium concentration (20 mmol/L). Conceptually, UWS was designed to prevent the expansion of extracellular and intracellular space and to prevent sodium and calcium entry into the cell by eliminating the transmembrane gradient of these cations [11]. University of Wisconsin solution contains impermeants in the form of raffinose, a nonmetabolizable trisaccharide, and lactobionate, an anion that substitutes for the freely permeable chloride anion. These components provide an extracellular osmotic force that has been shown to limit hypothermic cell swelling in the kidney, pancreas, and liver [12]. In addition, pentastarch, a type of hydroxyethyl starch, provides a colloidal oncotic pressure that will limit the expansion of the interstitial space. In our solution, sodium and calcium concentrations are also low, although higher than intracellular levels. The sodium channel blocker lidocaine was also added to further prevent Na⁺ and, in turn, Ca²⁺ accumulation in the myocytes during ischemia.

Although direct comparisons between the two solutions with respect to individual ingredients cannot be made, an important difference between the two preservation solutions is the presence of substrate for metabolism. The University of Wisconsin solution contains no glucose and, in fact, high-dose insulin (100 U/L) is added primarily to inhibit glycogen breakdown. On the basis of previous studies, we propose that substrate metabolism during ischemia, with removal of waste products, promotes ATP regeneration [3, 4]. The glycolytically derived ATP is used by the various ATPases in the myocytes to maintain cation homeostasis and improve tolerance to prolonged ischemia, particularly in large mammal and human hearts.

Another potential mechanism of protection with HBS solution is the effect of glucose and insulin on glycogen stores. Glucose and insulin at the concentrations present in the HBS solution might prevent glycogen breakdown during ischemia, resulting in higher glycogen content on reperfusion. Because we did not measure glycogen content or the rate of anaerobic glycolysis during ischemia in this study, we cannot state conclusively the contribution of either mechanism toward improved postischemic recovery.

In heart preservation studies with UWS, Mankad and colleagues [13] stored pig heart for 8 hours and evaluated recovery of cardiac function in an isovolumically contracting heart model. They found that developed pressure recovered to only 53% of control after 8 hours of ischemia followed by 2 hours of reperfusion. Jeevanandam and associates [7] reported that UWS was able to achieve adequate hypothermic preservation of heart for only up to 14.2 ± 1.6 hours during orthotopic baboon allotransplantation. These results are in contrast to experiments using small rodent hearts, where UWS was shown to be effective for 24 hours [14]. In larger mammal heart models, however, the results reported by the other studies are comparable to our findings that UWS is inadequate for protection from a prolonged (24-hour) ischemic insult. Superior prolonged preservation (24 hours) was accomplished with glycolysis-promoting cardioplegia in a blood perfused canine heart model.

	References

Top Abstract Introduction Material and methods Results Comment References

 

1. Gevers W. Generation of protons by metabolic processes in heart cells. J Mol Cell Cardiol 1979;9:867-874.
2. Wilkie D.R. Generation of protons by metabolic process than other glycolysis I muscle cells. J Moll Cell Cardiol 1979;11:325-330.[Medline]
3. Del Nido P.J., Wilson G.J., Mickle D.A.G., et al. The role of cardioplegia solution buffering in myocardial protection. J Thorac Cardiovasc Surg 1985;89:689-699.[Abstract]
4. Ohkado A., Cao-Danh H., Sommers K.E., del Nido P.J. Evaluation of a highly buffered low-calcium solution for long term preservation of the heart. Comparison with University of Wisconsin solution. J Thorac Cardiovasc Surg 1994;108:262-271.
5. McGowan F., Cao-Danh H., Takeuchi K., del Nido P.J. Neonatal myocardial preservation using a highly buffered low calcium solution. J Thorac Cardiovasc Surg 1994;108:772-779.[Abstract/Free Full Text]
6. Friehs I., Cao-Danh H., Takahashi S., Buenaventura P., Glynn P., McGowan F.X., del Nido P.J. Adenosine prevents protein kinase C activation during hypothermic ischemia. Circulation 1997;96:II221-II225.
7. Jeevanandam V., Auteri J.S., Sanchez J.A., et al. Cardiac transplantation after prolonged graft preservation with the University of Wisconsin solution. J Thorac Cardiovasc Surg 1992;104:224-228.[Abstract]
8. Jimenez E., del Nido P.J., Sarin M., Nakamura H., Feinberg H., Levitsky S. Effects of low extracellular calcium on cytosolic calcium and ischemic contracture. J Surg Res 1990;50:293-295.
9. Grace A.A., Kirschenlohr H.L., Metcalfe J.C., et al. Regulation of intracellular pH in the perfused heart by external HCO₃^-- and Na⁺–H⁺ exchange. Am J Physiol 1993;265:H289-H298.[Abstract/Free Full Text]
10. Bretschneider H.J., Hubuer G., Knoll D., Lohr B., Norbeck H., Spieckermann P.G. Myocardial resistance and tolerance to ischemia. J Caridovasc Surg 1975;16:241-260.
11. Belzer F.O., Southard J.H. Principles of solid-organ preservation by cold storage. Transplantation 1988;45:673-676.[Medline]
12. Wahlberg J.A., Southard J.H., Belzer F.O. Development of a cold storage solution for pancreas preservation. Cryobiology 1986;23:477-482.[Medline]
13. Mankad P.S., Severs N.J., Lachno D.R., Rothery S., Yacoub M.H. Superior qualities of University of Wisconsin solution for ex vivo preservation of the pig heart. J Thorac Cardiovasc Surg 1992;104:229-240.[Abstract]
14. Galinanes M., Murashita T., Hearse D.J. Long-term hypothermic storage of the mammalian heart for transplantation. J Heart Lung Transplant 1992;11:624-635.[Medline]

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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): Koh Takeuchi Pedro J. del Nido Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Takeuchi, K. Articles by del Nido, P. J. Search for Related Content PubMed PubMed Citation Articles by Takeuchi, K. Articles by del Nido, P. J.