Ann Thorac Surg 1997;64:73-80
© 1997 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Zinc-bis-Histidinate Preserves Cardiac Function in a Porcine Model of Cardioplegic Arrest
Saul R. Powell, PhD,
Roy L. Nelson, MD,
JeanMarie Finnerty, BSc,
Daniel Alexander, BSc,
George Pottanat, BSc,
Karlene Kooker, BSc,
Russell J. Schiff, MD,
Jeffrey Moyse, BSc,
Saul Teichberg, PhD,
Anthony J. Tortolani, MD
Departments of Surgery, Pediatrics, Laboratories, and Research, North Shore University Hospital, Manhasset, New York
Accepted for publication January 9, 1997.
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Abstract
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Background. We examined the ability of zinc-bis-histidinate to preserve postarrest myocardial function when added to a standard crystalloid cardioplegic solution.
Methods. Domestic pigs (35 to 50 kg) on left-sided cardiopulmonary bypass were subjected to 90 minutes of regional ischemia followed by 60 minutes of hypothermic cardioplegic arrest induced by antegrade infusion of 20 mL/kg cold St. Thomas' #2 cardioplegic solution with or without 100 µmol/L of zinc-bis-histidinate and maintained by infusion of 10 mL/kg of the same every 20 minutes. During reperfusion function was assessed at 1 and 3 hours over increasing preloads using the right-sided bypass method.
Results. At roller pump flows up to 2,000 mL/min, stroke work index–end-diastolic pressure curves were significantly (p < 0.05) higher and shifted to the left in treated hearts. In a series of pigs, echocardiography was used to determine end-diastolic and end-systolic volumes. At roller pump flows up to 3,500 mL/min, end-systolic pressure–end-systolic volume curves were significantly higher and shifted to the left in treated hearts. Left ventricular ejection fraction, fractional shortening, stroke volume, and cardiac output were significantly (p < 0.05) higher in treated hearts. Electron microscopy revealed that mitochondria in tissue not at risk appeared more swollen in control hearts.
Conclusions. The results of this study support the conclusion that zinc-bis-histidinate is effective as a myocardial preservative when added to a crystalloid cardioplegic solution.
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Introduction
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The iatrogenic ischemia and reperfusion associated with open heart operation is a serious concern. Despite the many improvements in surgical techniques and advances in myocardial preservation, postoperative morbidity and mortality continue to be a major problem. The possible involvement of reactive oxygen intermediates in postischemic injury has received considerable attention. It is now generally accepted that reactive oxygen intermediates play a role in reperfusion injury after short periods of ischemia that do not result in cell death but rather in arrhythmogenesis or reversible cell injury [1]. Over the last 15 years there have been innumerable studies evaluating potential interventions, based on the ability of various agents to scavenge reactive oxygen intermediates. This approach may be of limited value because it is kinetically impractical to remove these reactive species after they are formed. An alternate approach is to affect the basic processes that promote formation of reactive oxygen intermediates.
Formation of the highly reactive hydroxyl radical (•OH) from less reactive species, such as superoxide, is thought to require trace amounts of redox-active transition metals, such as iron and copper, as catalysts. Over the past several years, we have been examining the relationship between transition metals and postischemic reperfusion injury and have identified a different approach to this problem. Evidence from our laboratory and others suggests that zinc, a non–redox-active transition metal, can interfere with the production of the more destructive secondary reactive oxygen intermediates, such as •OH [2, 3]. In vitro isolated heart studies from our laboratory have shown that zinc can exert a powerful protective effect on the ischemic myocardium, as demonstrated by its antiarrhythmic effect [4, 5] and preservation of postischemic function and cardiac morphology [2]. We believe that zinc may be useful as an additive to cardioplegic solutions to prevent reperfusion injury. In an in vitro isolated perfused heart model of hypothermic cardioplegic arrest, we observed that recovery of postarrest function was significantly greater in zinc-treated hearts [6]. In the current study, we examine the effect of zinc-bis-histidinate (Zn-His2) on postarrest myocardial function in an in vivo porcine model of hypothermic cardioplegic arrest and demonstrate its possible utility as a myocardial preservative additive to cardioplegic solutions.
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Material and Methods
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Animals
All studies were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985) and were approved by the Institutional Animal Care and Use Committee of North Shore University Hospital. Male domestic pigs (35 to 55 kg) were obtained from Biomedical Associates (Friedensburg, PA). The animals fasted for 24 hours before the experimental procedure. A total of 59 pigs were used in this study; 15 were excluded for various reasons (surgical difficulties, 5; development of malignant arrhythmias, 4; pericarditis, 3; preischemic development of pulmonary edema, 1; adverse reaction to donor blood, 1; and inadvertent introduction of air, 1).
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Surgical Procedures and Experimental Protocol
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The protocol (Fig 1
) used in these experiments was based on the model described by Lazar and colleagues [7, 8]. Briefly, fasting adult pigs (35 to 55 kg) were premedicated with ketamine, 25 mg/kg intramuscularly, anesthetized with 0.1 mg/kg midazolam (Roche Pharmaceuticals, Manati, Puerto Rico) and 7 µg/kg sufentanil citrate (Janssen Pharmaceuticals, Titusville, NJ) intravenously (IV), and then placed on positive-pressure tracheal ventilation. After insertion of a femoral IV line, 
-chloralose, 65 mg/kg IV, was administered as the maintenance anesthetic with booster doses of 25% of the initial dose administered every 2 hours. Vencuronium bromide (Organon, Inc., West Orange, NJ), 0.1 mg/kg, was administered as needed for muscle relaxation. Central arterial pressure was monitored via a fluid-filled catheter inserted into the abdominal aorta through the femoral artery. Blood gases (model M170 blood gas analyzer; CIBA-Corning, Medfield, MA) and hematocrit were determined approximately every 30 minutes and as needed. Hematocrit was maintained at or above 24% through the infusion of donor blood obtained from a local slaughterhouse (Dealaman Enterprises, Warren, NJ).
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Fig 1. . Protocol for in vivo testing of zinc-bis-histidinate in swine. (CPB = cardiopulmonary bypass; EF = ejection fraction.)
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A median sternotomy was performed; heparin, 500 units/kg IV, administered; and a fluid-filled catheter inserted into the left ventricle through the apex to monitor left ventricular pressures. A 24F venous return catheter (DLP, Inc, Grand Rapids, MI) was placed into the right atrial appendage and an 18F catheter (DLP, Inc) was placed into the left atrial appendage for volume infusion. A 16F pediatric arterial catheter (DLP, Inc) was placed into the left femoral artery. Lidocaine (Abbott Laboratories, North Chicago, IL), 100 mg, was administered by IV push and a lidocaine drip, 0.5 mg/min, was started. The second and third diagonal branches of the left anterior descending coronary artery were occluded by snares placed just distal to their takeoffs. If severe ventricular arrhythmias (ventricular fibrillation or tachycardia) developed during the period of ischemia that were refractory to additional pharmacologic intervention (bretylium tosylate; Elkins-Sinn, Inc, Cherry Hill, NJ) or defibrillation, left-sided bypass was initiated and the animal then defibrillated (necessary in 9 control and 8 treated hearts). Otherwise, 20 minutes before the start of aortic cross-clamping, the animal was placed on left-sided cardiopulmonary bypass. Roller pump (American Optical Corp) head speed was adjusted to maintain central mean arterial pressure in the range of 45 to 60 mm Hg. Metaraminol bitartrate (Merck & Co, West Point, PA) was administered to effect for pressure control. Ten minutes later, the animal was cooled to approximately 28°C. Ten minutes after that, the aorta was cross-clamped and cold (4° to 6°C) cardioplegic solution (Plegisol; Abbott Laboratories) with or without 100 µmol of Zn-His2, 20 mL/kg, was administered in an antegrade fashion through a 14-gauge aortic root cannula (DLP, Inc). The Zn-His2 used in these experiments was prepared fresh daily as previously described [2]. Cardiac temperature was maintained at approximately 10° to 14°C through infusion of cold cardioplegic solution (with or without 100 µmol of Zn-His2), 10 mL/kg, every 20 minutes and the heart was bathed with a cold saline slush. Just before the last infusion of cardioplegic solution, the snares were released to allow for perfusion of these vascular beds with cardioplegic solution. Ten minutes before release of the cross-clamp, rewarming was initiated and the animal was gradually warmed to 37°C. After a total of 60 minutes of cross-clamp time, the aorta was released and reperfusion with warm blood commenced. The heart was defibrillated if necessary and reperfusion continued for up to 3 hours, during which time the animal remained on left-sided bypass. Results from the first 20 experiments indicated that there were no differences between control and Zn-His2-treated animals with respect to hematocrit or blood pH and gases (data not shown).
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Assessment of Cardiac Function
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At 1 and 3 hours of reperfusion, cardiac function was tested using the right-sided bypass method. Initial experiments examining the ventricular function curve and directional changes in contractility varied preload using roller pump speeds up to 2,000 mL/min. Left ventricular stroke work index (LVSWI) was calculated [9, 10] using the following formula: LVSWI (g•m/kg) = ((MAP - LVEDP)/HR) x CO/kg x 1.36/100, where MAP = mean arterial pressure, LVEDP = left ventricular end-diastolic pressure, HR = heart rate, CO = cardiac output delivered by the calibrated roller pump, and kg is the weight of the animal. In this series of experiments, 17 animals were assigned to the control group and 15 animals to the treated group.
In a second series of experiments, intraoperative echocardiography (Ultramark 7 ultrasound system; ATL, Inc, Bothell, WA) was performed using a 5-MHz biplane transesophageal probe as a flexible epicardial transducer to examine ejection phase indices of the left ventricle. The area and length of the ventricle were obtained from both the apical four-chamber and two-chamber views of the left ventricle. Left ventricular end-diastolic and end-systolic volumes were then calculated using the ellipsoid method (Dodge's approximation) [11]. Ejection fractions were calculated as (left ventricular end-diastolic volume - left ventricular end systolic volume)/left ventricular end-diastolic volume. Stroke volume was calculated as the difference between left ventricular end-diastolic and end-systolic volume, and cardiac output was calculated as the stroke volume times the heart rate. Left ventricular (LV) fractional shortening was determined from two dimensional guided M-mode echocardiography in the transverse plane at the level of the mid-papillary muscles from the following relationship: LVinternal dimension diastole - LVinternal dimension systole/LVinternal dimension diastole.
In this series of experiments 6 animals were assigned to the control group and 6 to the treated group.
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Infarct Size and Area at Risk
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Infarct size and area at risk were determined using standard histochemical techniques [12, 13]. After 3 hours of reperfusion, phthalocyanine blue dye was injected into the aortic root to outline the area at risk. Triphenyltetrazolium chloride was then used to distinguish between viable and nonviable tissue. Areas were calculated using computer-assisted planimetry (SigmaScan Image Analysis software package; Jandel Corp, Davis, CA).
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Light and Electron Microscopy
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FIXATION AND EMBEDDING.
After the experiments, hearts from a subset of 3 control or 3 Zn-His2–treated animals were fixed in situ by perfusion of 100 mL of isotonic (300 mosm/kg), 2% glutaraldehyde in 0.05 mol/L cacodylate (pH 7.3) at room temperature through the aortic root cannula after aortic cross-clamping. Hearts were stored in buffered fixative for one to several days and then dissected at specific sites established to be outside of the area at risk: the apex and the ventricular septum. At each site three sequential levels were cut and labeled to make comparisons between control and Zn-His2–treated hearts at equivalent loci. After dissection, slabs of cardiac tissue, less than 1 mm in diameter, were prepared for light and electron microscopy as previously described [2].
QUANTITATIVE MORPHOLOGIC ANALYSIS.
For quantitative morphometric studies the most peripheral level of the ventricular apex was selected for detailed analysis. One-micrometer plastic sections of tissue were initially cut, stained with toluidine blue, and examined by light microscopy. Areas of cardiac tissue proximal to well-perfused vasculature were thinly sectioned, stained with uranyl acetate and lead citrate, and examined on a JEOL JEM 100 CXII transmission electron microscope. Micrographs were taken at an initial magnification of x4,000 and photographically enlarged to x11,400. For quantitative analysis there were four electron micrographs per heart apex per animal from a total of 6 pigs, 3 controls and 3 Zn-His2–treated animals. The sample evaluated in our study consisted of a total of 300 to 500 mitochondria per ventricular apex per pig. The electron micrographs were analyzed using computer-assisted planimetry (SigmaScan Image Analysis software package; Jandel Corp).
The following morphometric parameters were subsequently calculated: volume density of mitochondria, number of mitochondria/µm2 cardiac tissue reference area, outer surface density of mitochondria, mean mitochondrial area, and mean mitochondrial volume, as previously described in detail [2, 14]. Mitochondrial surface: volume ratio was calculated as 4/
x 
of mitochondrial perimeters/ of mitochondrial areas in the reference area, or as described by Schmiedl and associates [15].
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Statistical Analysis and Treatment of Data
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Analysis of differences of cardiac functional recovery were analyzed with either a repeated-measures analysis of variance or a regression analysis followed by t test for slope analysis. Differences between two individual groups was analyzed with an independent Student's t test. In all cases, results were considered to significant at the p less than 0.05 level. All statistics were performed with the SPSS/PC+ (SPSS Inc, Chicago, IL) statistical analysis software package. In some experiments, drop-out of data resulted because end-diastolic pressure exceeded 15 to 20 mm Hg before maximal roller pump flow (2,000 or 3,500 mL/min) was achieved. After statistical consultation, we decided to use the "principle of last value carried forward" to prevent the groups from becoming uneven. As this maneuver was more likely to be applied to control groups, a negative bias was inserted into the results.
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Results
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Ventricular Function Curves and Contractility
In a series of studies we determined that Zn-His2 preserved postischemic ventricular function and had a positive effect on contractility. Initial experiments examined the effect of Zn-His2 on ventricular function curves and directional changes in contractility at roller pump flows up to 2,000 mL/min or until the end-diastolic pressure increased to greater than 20 mm Hg. As illustrated in Figure 2, ventricular function was significantly (p < 0.05, t test for slopes) better in the Zn-His2–treated group at both 1 and 3 hours. Ventricular function appeared to decrease by the 3-hour time point in the control group, but not significantly.
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Fig 2. . Effect of zinc on stroke work index at increasing preloads during right-sided bypass hemodynamic function testing. Swine were subjected to 90 minutes of regional ischemia followed by 60 minutes of hypothermic cardioplegic arrest while on left-sided bypass. At 1 and 3 hours of reperfusion, bypass was changed to the right side to alter left ventricular preload. Statistical analysis is presented in the Results section. Values are expressed as the mean (± standard error of the mean) of 13 to 17 separate experiments in each group.
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In an additional experiment, the relationship between developed systolic pressure and end-systolic volume was used as an indicator of directional changes in contractility. As illustrated in Figure 3, although pressure development appears to be equal among the groups, the developed systolic pressure•end-systolic volume curves for the Zn-His2–treated animals are shifted to the left indicating pressure development at a lower systolic volume denoting a positive effect on left ventricular contractility. Furthermore, the curve that is depicted for the control group at 3 hours is somewhat misleading, as the "principle of last value carried forward" was applied. In actuality, beyond the fourth point on this curve, developed systolic pressure goes to zero, as 0/5 control hearts were able to perform beyond roller pump flows of 2,500 mL/min, whereas 3/5 Zn-His2–treated hearts attained this level (Table 1).
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Fig 3. . Effect of zinc on developed systolic pressure at increasing end-systolic volumes. Swine were subjected to 90 minutes of regional ischemia followed by 60 minutes of hypothermic cardioplegic arrest while on left-sided bypass. At 1 and 3 hours of reperfusion, bypass was changed to the right side to alter left ventricular preload. Echocardiographic imaging was employed to determine ventricular volumes at increasing preloads. Statistical analysis is presented in the Results section. Values are expressed as the mean (± standard error of the mean) of 5 separate experiments in each group. The preischemic value is the mean (± standard error of the mean) of all experiments.
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Ejection Phase Parameters
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We also determined that several ejection phase parameters were improved in zinc-treated hearts. These parameters were determined at roller pump flows up to 3,500 mL/min through measurement of left ventricular volumes using intraoperative echocardiography. At both 1 and 3 hours, ejection fractions were significantly (p < 0.05, repeated-measures analysis of variance) higher in the Zn-His2–treated group (see Table 1
). The stroke volume•end-systolic volume and cardiac output•end systolic volume curves are presented in Figure 4. Both stroke volume and cardiac output were significantly higher at both 1 and 3 hours of reperfusion (p < 0.05, t test for slopes) in the Zn-His2–treated animals. What is apparent from these results is that ejection phase performance degrades significantly between 1 and 3 hours in control hearts. We observed that the calculated cardiac output of Zn-His2–treated hearts shows a high degree of correlation with actual roller pump flow at both 1 and 3 hours of reperfusion. The last ejection phase parameter that was examined was left ventricular fractional shortening. As demonstrated in Table 2, preischemic fractional shortening was 0.62. At both 1 and 3 hours, the fractional shortening decreased in the range of 0.35 in control hearts, again illustrating the severity of the injury to these hearts. At the 1-hour time point, fractional shortening of Zn-His2–treated hearts was significantly greater (p < 0.05, repeated-measures analysis of variance) and not different from preischemic values. However, by 3 hours performance as measured by this parameter had degraded to the point that the only value that was significantly higher was at a flow of 2,000 mL/min (p < 0.05, t test). Nonetheless, even at this time point fractional shortening appears to be greater in the Zn-His2–treated hearts.
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Fig 4. . Effect of zinc on left ventricular stroke volume and cardiac output at increasing end-systolic volumes. Figure and symbol legends are in Figure 3. Statistical analysis is presented in the Results section. Values are expressed as the mean (± standard error of the mean) of 5 separate experiments in each group. The preischemic value is the mean (± standard error of the mean) of all experiments.
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Histologic Studies
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AREA AT RISK AND INFARCT SIZE.
To determine if Zn-His2 affected the area of regional ischemia, we assessed the area at risk and infarct size. Regional ischemia of a total of 130 minutes resulted in 22.7% ± 3.2% of the left ventricle at risk, which was unaffected by inclusion of Zn-His2 in the cardioplegic solution (22.1% ± 4.7%). Of this "at risk" area, 29.5% ± 6.8% was infarcted. Treatment with Zn-His2 had no effect on the size of the infarct (32.8% ± 5.6%).
LIGHT AND ELECTRON MICROSCOPY.
Histologic and electron microscopic studies were performed on cardiac tissue from outside the area at risk. Detailed electron microscopic analysis focused on areas adjacent to well perfused blood vessels that were evidently well preserved in both control and Zn-His2–perfused cardiac tissue.
Light microscopic cross-sectional profiles of cardiac fibers from Zn-His2–treated hearts contained numerous oval to rod-shaped cytoplasmic structures that stained darkly with toluidine blue. These corresponded to mitochondria that were normal in size with a dense inner matrix when evaluated by electron microscopy. In control hearts, light microscopy showed fewer cardiac fibers with rod-shaped toluidine blue staining granules, whereas many fibers showed paler structures. Electron microscopic study revealed that the lack of staining in control preparations was due to swelling of the mitochondria and a marked loss of inner matrix density (Fig 5).
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Fig 5. . Representative morphology of the postischemic, postarrest myocardium from (A) control and (B) zinc-bis-histidinate–treated hearts: electron micrographs of representative cardiac fibers from hearts of swine subjected to 90 minutes regional ischemia followed by 60 minutes of hypothermic cardioplegic arrest while on left-sided bypass. At the end of 3 hours of reperfusion, hearts were perfusion-fixed in situ and then processed for electron microscopic analysis as described in the Material and Methods section. (x20,000 before 52% reduction; bar equals 1 µm.)
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MORPHOMETRIC STUDIES.
To confirm qualitative observations and quantify differences in mitochondria between control and Zn-His2–treated hearts, we carried out a morphometric analysis. As shown in Table 3, mitochondrial volume density, mean mitochondrial area, mean mitochondrial volume (p < 0.05, t test), and the mitochondrial surface density were all greater in control untreated preparations than in the Zn-His2–treated hearts, whereas mitochondrial surface/volume ratio (p < 0.05, t test) was decreased in controls as compared with Zn-His2–treated preparations. On the other hand, the number of mitochondria/cardiac tissue area and mitochondrial numeric density were indistinguishable in control and Zn-His2–treated hearts. Overall, these quantitative morphometric data support our qualitative observations of mitochondrial swelling in hearts perfused under control conditions and the prevention of this process by perfusion with the cardioplegic solution containing Zn-His2.
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Comment
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This study clearly demonstrates the cardioprotective effect of Zn-His2 in an in vivo porcine model of acute infarct followed by hypothermic cardioplegic arrest. In these experiments, 100 µmol/L Zn-His2 was added to St. Thomas' #2 cardioplegic solution (Plegisol; Abbott Laboratories), which is a commonly used hyperkalemic, hypermagnesemic crystalloid cardioplegic solution. Postischemic left ventricular contractile function and ejection phase indices were preserved in swine that had been treated with Zn-His2–supplemented cardioplegic solution. The ischemic model that we used represents a global stun injury superimposed over an acute regional infarct. This acute model, although clearly not the best, is used to simulate the clinical situation, ie, the vast majority of patients who undergo open heart operations generally have multiartery disease with long-standing regional ischemia and may have had one or more myocardial infarcts. On top of this, the surgeon induces global ischemia when the aorta is cross-clamped. A situation is created that involves superimposition of global ischemia over regional ischemia and possibly infarct.
In this study, we examined primarily global indices of cardiac function, despite the presence of regional dysfunction. Because calculation of fractional shortening and ejection fraction in only one plane would have been subject to error with discrete wall motion abnormalities that result from the regional dysfunction, biplanar ejection fractions that included the area of regional dysfunction were calculated to minimize this error. The higher ejection fractions in the treated hearts indicates that Zn-His2 improved postischemic ejection phase parameters. Moreover, the higher and leftward shift of the Starling curve suggests that postischemic contractility was also improved by this complex. Although it appears that the values for stroke work index that we report are low, at similar end-diastolic pressures they are comparable with those reported by Lazar and Rivers [10] in a similar swine model of approximately the same size. In fact, in this experiment the highest roller pump flow was only 2,000 mL/min, when in fact as we found out in later experiments, we may have been able to increase the roller pump flow to 3,500 mL/min in treated hearts, which would have yielded a much higher stroke work index. We chose to express this ventricular function curve as stroke work index versus end-diastolic pressure, in as much as Lazar and Rivers [10] have previously shown in a similar model excellent correlation between end-diastolic pressure and end-diastolic volume. Furthermore, end-diastolic pressure can be measured directly, whereas end-diastolic volume is estimated. Despite this potential shortcoming, the differences between the treatment groups are unequivocal and the zinc complex clearly improved ventricular function.
Further evidence for myocardial preservation can be derived from the histologic, morphologic, and morphometric studies. We initially examined the possibility that zinc preserves ventricular function through an effect on the regional area of ischemia by determining infarct size within the area at risk. However, infarct size was not different, which would have been disturbing were it not that the regional area at risk was not exposed to the zinc complex until 20 minutes before the end of cross-clamping. By this time, a total of 130 minutes of ischemia had elapsed and much of this tissue has already become irreversibly damaged. Thus, to make the conclusion that zinc does not have an effect on infarct size would be inappropriate based on this model. Nonetheless, this led us to suspect that the major effect of zinc was not on the regional component, but perhaps on the global component.
Morphologic and morphometric studies conducted on cardiac tissue from outside the area at risk demonstrate a protective effect of Zn-His2 on cardiac morphology. The morphometric analysis quantifying the changes in mitochondrial size confirmed qualitative electron microscopic findings indicating the presence of swollen mitochondria in control hearts. The data show the same number of mitochondria with increased volume density and mitochondrial volume and decreased mitochondrial surface: volume ratio in controls. Although it is unclear whether the disturbances in the mitochondria are cause or effect, these changes are consistent with several recent studies suggesting that this organelle may be a primary site of radical production in ischemic tissue [16, 17]. Clearly, mitochondria are highly susceptible to membrane damage and typically one may expect loss of their ability to osmoregulate volume with consequent swelling. Further, these observations in swine heart were entirely consistent with what we have published previously regarding the isolated heart [6] and are suggestive that Zn-His2 exerts a global "antistun" effect. This does not preclude the possibility that zinc may also effect hibernating myocardium that may be present within the infarcted area [18]. Furthermore, although previous in vitro studies [2, 5] suggest that Zn-His2 acts through inhibition of oxidative stress and thus form the rationale for this study, this aspect was not the focus of the present series of experiments. Thus to invoke a similar mechanism at this time would be pure speculation.
Whether the protective effect is due to the zinc or the histidine is an important question. Histidine is an excellent buffer and has in addition been shown to scavenge various reactive oxygen species [19]. Yet these effects apparently require the presence of millimolar concentrations (Bretschneider's histidine-tryptophan-ketoglutarate cardioplegic solution contains 180 mmol/L histidine) and have not been observed at the micromolar concentrations used in this study [20].
Finally, an important issue raised by these studies is the rather poor protection afforded by St. Thomas' #2 crystalloid cardioplegic solution. Other investigators using animal models have reported similar problems. Lazar and coworkers, in several studies [7, 8, 10], have reported incomplete protection with this crystalloid solution. Moreover, Schmeidl and associates [15] have previously reported morphologic and morphometric parameters to be less favorable in dog hearts treated with this crystalloid alone. This raises the issue of whether the zinc complex would have been less effectual as an additive to cardioplegic solutions that already contain trace amounts of zinc, such as blood cardioplegia. Plasma contains only 10 to 15 µmol/L zinc, and a large portion of this metal is bound to proteins and hence unavailable to the heart [21]. We have repeatedly shown in crystalloid-perfused Langendorf models, in which it must be assumed that all of the metal is available to the heart, that at least 30 µmol/L Zn-His2 is necessary for optimal protection [2, 4–6]. Thus it is likely that the protective effect represents a pharmacologic effect of zinc that may very well represent an extension of one of the physiologic functions of this metal.
Since our initial report [4], zinc has been shown to improve postischemic kidney function in rabbits [22] and decrease lipid peroxidation and area of erosion in the postischemic stomach and reduce infarct size and edema after temporary focal cerebral ischemia in rats [23, 24]. Based on these studies and the results of our studies, we believe that zinc merits further investigation of its protective effects on ischemic tissue. The observation of myocardial protection in a porcine model would suggest that a possible clinical use for this complex might be as an additive to at least crystalloid cardioplegic solutions.
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Acknowledgments
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We acknowledge Ms Ellen Gurzenda, Mr Joshua Dines, and Mr Jared Sender for their assistance in the performance of the swine experiments. We are grateful to Martin Lesser, PhD, Director of the Division of Biostatistics, for his guidance in statistical analysis of these data. Finally, we are indebted to Mr Curtis Caldwell and Bentley Laboratories, Inc (Baxter Healthcare Corp) for donation of many of the oxygenators and cardiotomy reservoirs, and to Mr Larry McTierney and DLP, Inc (Grand Rapids, MI) for donation of many of the arterial and venous cannulas used in the performance of these studies.
Funded in part by grant HL45534 from the National Institutes of Health.
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Footnotes
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Address reprint requests to Dr Powell, Boas-Marks Biomedical Science Research Bldg, North Shore University Hospital, 350 Community Dr, Manhasset, NY 11030.
Doctors Powell and Tortolani hold the patent for the application of this zinc complex as an additive to cardioplegic solutions, but no commercial licenses have been issued.
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Abstract
Introduction
