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Ann Thorac Surg 1996;62:1765-1772
© 1996 The Society of Thoracic Surgeons
Cardiac Surgical Research Unit, Baker Medical Research Institute, Prahran, Institute of Reproduction and Development, Monash Medical Centre, Clayton, and Department of Biochemistry, Alfred Hospital, Prahran, Victoria, Australia
Accepted for publication June 21, 1996.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. Acute myocardial infarction was produced in dogs. They then received either orotic acid or placebo (control) orally (n = 12 per group). Fractional radial shortening and systolic wall thickening were measured by two-dimensional echocardiography before and 1 and 3 days after infarction with and without ß-adrenergic blockade, and in 6 dogs up to 9 days after infarction. Global function was measured under anesthesia 4 days after infarction.
Results. In control animals, fractional radial shortening in the infarct decreased from 20.6% ± 5.1% before infarction to 3.0% ± 2.2% at day 1 and to 1.9% ± 1.9% at day 3 (p < 0.01). In the border zone radial shortening declined from 21.9% ± 3.7% to 11.0% ± 2.3% at day 1 and 9.3% ± 2.8% at day 3 (p < 0.05). In the noninfarcted myocardium radial shortening also declined from 27.1% ± 1.9% before infarction to 18.3% ± 2.3% on day 1 (p < 0.05) and to 16.0% ± 2.8% on day 3 after infarction (p < 0.05) with recovery to preinfarct levels by 9 days after infarction. These findings were confirmed by measurements of systolic thickening. Before infarction ß-receptor blockade decreased fractional shortening in all regions of the left ventricle, but this effect was absent on day 3 after infarction, implying that the myocardium had become less responsive to ß-adrenergic stimulation. Measurements of global function 4 days after infarction showed marked depression of stroke work. There was no effect of orotic acid treatment on regional or global function.
Conclusions. Myocardial infarction causes reversible depression of resting function and ß-adrenergic responsiveness in the remote and border zone areas, which is not prevented by metabolic therapy with orotic acid. This finding may explain the adverse response of the infarcted heart to cardioplegic arrest.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We have also shown that recent infarction reduces high-energy phosphates in the remaining, noninfarcted myocardium and that orotic acid therapy prevents this reduction [6], providing an explanation both for the sensitivity of the recently infarcted heart to cardioplegic arrest and for the beneficial effects of orotic acid. These findings encouraged us to believe that metabolic therapy with orotic acid might improve cardiac function after myocardial infarction, independent of any effect of cardioplegia, which would have important implications not only in the setting of postinfarct operation but also for the management of myocardial infarction in general. Indeed, several clinical reports from the Soviet Union in the 1970s showed dramatic improvements in cardiac function with orotic acid therapy after myocardial infarction [7, 8]. However, the true magnitude of the orotic acid effect and the site of action in the infarcted heart are essentially unknown.
Therefore, in this study we aimed to investigate the effect of acute myocardial infarction on regional and global function and the effect of orotic acid therapy in the early postinfarct period. We used ß-adrenoreceptor blockade to attenuate the effect of sympathetic influences on the heart to measure intrinsic cardiac performance.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Immediately after infarction, dogs were randomized to receive either oral orotic acid (50 mg/kg per day) or placebo (lactose), given three times a day in blinded fashion, commencing 2 hours after infarction (Fig 1
). To confirm absorption of the orotic acid, urinary orotic acid levels were measured using a spectrophotometric assay [9]. The urine specimens were frozen after collection, and analyzed after completion of the study. Elevated urinary levels of orotic acid were demonstrated in all orotic acid-treated dogs.
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All dogs received humane treatment according to the Code of Practice of the National Health and Medical Research Council of Australia.
Assessment of Regional Left Ventricular Function
Regional left ventricular (LV) function was assessed by measuring fractional radial shortening and percentage systolic wall thickening, from cross-sectional two-dimensional echocardiographic (2-D echo) images. To assess the effect of orotic acid therapy on regional function, studies were performed in the conscious, nonsedated state at three time points: 2 days before infarction, and 1 and 3 days after infarction. As intrinsic changes in myocardial performance may be confounded by the increased circulating catecholamines and increased sympathetic activity that accompany myocardial infarction [10], the preinfarction and day 3 postinfarction 2-D echo studies were repeated after ß-adrenoreceptor blockade with propranolol. ß-Blockade was not attempted on the first day after infarction because of the potential problems of hypotension and modification of infarct size [11].
To assess the longer term effects of myocardial infarction and orotic acid therapy on regional function, a subset of 6 dogs, 3 in the orotic acid group and 3 in the placebo group, were studied echocardiographically until day 9 after infarction.
During the 2-D echo studies, the dogs lay recumbent on a padded table, right side down in a quiet and darkened room. During the study before infarction, lead II of the electrocardiogram and the heart rate were recorded, and after infarction the arterial blood pressure and left atrial pressure were also recorded on a chart recorder. Hearts were imaged with a 77500 beta scanner (Hewlett-Packard, Andover, MA) coupled to a 3.5-MHz transducer positioned beneath the right side of the thorax. Short-axis views of the left ventricle were obtained at the midpapillary level [12]. Images were recorded on a video recorder (Panasonic NV-8200, Matsushita Electric, Japan) and gated with the electrocardiogram to display the end-diastolic and end-systolic frames.
All 2-D echo studies were performed at a constant rate of 150 beats/min. Preliminary studies indicated that this rate was the slowest that would regularly prevent breakthrough arrhythmias after infarction. Before infarction, heart rate was increased to this target rate by administration of intravenous atropine, 0.6 to 1.0 mg; after infarction atrial pacing was used. Baseline before and after infarction heart rates could not be matched in 9 dogs due to postinfarction tachycardia or persistent arrhythmias (5 in the placebo and 4 in the orotic acid group); therefore, these measurements were discarded. However, after ß-adrenoreceptor blockade, rates were matched before and after infarction in all dogs. To control ventricular arrhythmias during the echocardiographic study, intravenous lignocaine was administered (2 mg/kg followed by an infusion of 4 mg/min). Measurements of fractional shortening made before (18.0% ± 3.1%) and during the lignocaine administration (17.5% ± 1.5%) in 5 dogs showed no significant effect on contractility (p = 0.9).
For the ß-adrenoreceptor blockade studies, 2-D echo images were also recorded 10 minutes after the administration of intravenous propranolol, 0.5 mg/kg. The completeness of the ß-adrenoreceptor blockade achieved was confirmed in 6 dogs before infarction using an isoprenaline dose-response test.
To standardize the loading conditions under which the 2-D echo studies were performed after myocardial infarction, the mean left atrial pressure was maintained at 5 mm Hg above the level of the thoracic spine, by intravenous infusion of isotonic saline solution and 3.5% polygeline (Haemaccel).
Echocardiographic Analysis
The 2-D echo images were analyzed with a computerized analysis system (Cardio 500; Kontron Bildanalyse, Munich, Germany). After manual definition of the endocardial contours of the end-diastolic and end-systolic frames on the computer screen, the outlines were automatically digitized by the analysis program to provide appropriate regional measurements. To assess regional contractile function, the fractional radial shortening (as a percentage) of the ventricular cavity was calculated over 36 radiants at 10-degree intervals as 100 x [(EDR - ESR)/EDR], where EDR = end-diastolic radius (in millimeters) and ESR = end-systolic radius (in millimeters) [13, 14]. The zero reference of the radiants was defined as the mid-point between the anterior and posterior papillary muscles [14]. A fixed rather than a floating centroid was used to avoid spurious increases in shortening within the infarct zone [13]. The radiants corresponding to the infarct, border zone, and remote (noninfarcted) regions of the echocardiographic image were defined by comparison of the 2-D echo images with the postmortem mid-papillary slice of the formalin-fixed heart. To evaluate the contractile function of the infarcted area, five radiants encompassing the center of the infarct were selected, and the mean fractional radial shortening calculated (Fig 2). For the border zone, three radiants on either side of the infarct in the adjacent noninfarcted myocardium were identified, and an average fractional radial shortening calculated. The choice of three radiants adjacent to the infarct zone was based on the observation that the functional border zone extends circumferentially for approximately 30 degrees beyond the interface of infarcted/noninfarcted myocardium [15]. Mean fractional radial shortening for the remote myocardium was obtained using the five radiants directly opposite the infarct. Three to five beats were analyzed from each recording. Repeated analysis of the same recordings indicated that the measurement technique was highly reproducible (coefficient of variation, 8%).
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Assessment of Global Left Ventricular Function
On the fourth day after infarction, global LV function was measured in 8 placebo and 8 orotic acid-treated dogs. The animals were anesthetized with intravenous ketamine (3 mg/kg) and xylazine (0.3 mg/kg), intubated, and mechanically ventilated. Anesthesia was maintained with an infusion of alpha-chloralose (25 mg • kg-1 • h-1) and ketamine (1 mg • kg-1 • h-1). Electrocardiogram, femoral artery pressure, left atrial pressure, arterial blood gases, end-tidal partial pressure of carbon dioxide, and rectal temperature were monitored. The chest incision was reopened. A thermodilution cardiac output catheter (Edwards Laboratories, Irving, CA) was inserted into the pulmonary artery through a jugular vein. To minimize reflex autonomic stimulation of the heart during changes in loading conditions, the ansa subclavia and cervical vagosympathetic trunks were sectioned bilaterally [17] and ß-adrenoreceptors were blocked with propranolol (loading dose of 0.5 mg/kg, followed by an infusion of 40 µ • kg-1 • min-1). Heart rate was maintained at 150 beats/min with atrial pacing. The dogs were heparinized (3 mg/kg) and a cannula, connected to a reservoir containing a mixture of Haemaccel and donor dog blood, was inserted into the left atrial appendage to control left atrial filling pressure. For comparison with normal values, the same procedure was carried out in 6 noninfarcted dogs.
To assess global LV function, cardiac output was measured during stepwise increases in left atrial pressure to achieve a maximum left atrial pressure of 15 mm Hg. The LV function curves were generated for the relation between the stroke work index (SWI) and mean left atrial pressure. SWI (g-m/kg) was calculated as [CO x (MAP - LAP) x 1.36]/[HR x BW x 100], where CO = cardiac output (in milliliters per minute), MAP = mean arterial pressure (in mm Hg), LAP = mean left atrial pressure (in mm Hg), HR = heart rate (in beats per minute), and BW = body weight (kilograms).
Infarct Delineation
At the end of the experiment, 250 mL of 2% triphenyltetrazolium chloride was infused into the left atrium to define the infarcted area [18]. The dogs were then killed with phenobarbitone sodium. The heart was excised and fixed in formalin for 7 days. The atria and right ventricular free wall were removed, and the left ventricle was cut perpendicular to its long axis into slices 7 mm in thickness. The infarcted area on each slice, defined as the area not stained by triphenyltetrazolium chloride [18], was traced onto acetate transparencies and planimetered on a digitizing tablet. The proportion of infarcted myocardium in each slice was calculated, each slice was weighed, and the infarcted areas of all slices summed to yield the percentage of infarct for the entire left ventricle.
Statistical Analysis
Longitudinal changes in regional function before and after infarction and the effect of orotic acid were analyzed with repeated measures analysis of variance. The effect of ß-adrenoreceptor blockade on fractional radial shortening was compared before infarction and on day 3 after infarction using paired t tests. Global function in the placebo and orotic acid-treated groups was compared using analysis of variance and the Newman-Keuls multiple comparison procedure. Values are reported as mean ± standard error, and a p value less than 0.05 was considered significant.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Hemodynamics
The resting heart rate in the control dogs increased from 98 ± 6 beats/min before infarction to 157 ± 9 beats/min on day 1 after infarction (p < 0.01), and was similar on day 3 after infarction at 139 ± 5 beats/min. In the treated animals heart rate was 89 ± 5 beats/min before infarction, 149 ± 5 beats/min on day 1, and 123 ± 8 beats/min at day 3, which was not significantly different from the control animals.
After infarction, there was no difference in the mean arterial pressure or left atrial pressures at day 1 and day 3 between the control and treated animals.
After control of the heart rate at 150 beats/min, elevation of the left atrial pressure to 5 mm Hg, and administration of lignocaine (on day 1 only), the mean arterial pressure on day 1 was 107 ± 6 mm Hg in the control and 97 ± 3 mm Hg in the treated dogs, and on day 3 was 108 ± 3 mm Hg in the control and 100 ± 5 mm Hg in the treated dogs (p = not significant).
Effect of Infarction on Regional Function
In the control animals, in the infarct zone the fractional radial shortening decreased markedly on day 1 and on day 3 (p < 0.01) (Table 1 and Fig 3). In both the border zone and the remote myocardium the fractional radial shortening in the control animals declined on day 1 and on day 3 (p < 0.05 in both areas).
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Effect of ß-Blockade on Radial Shortening
In the control group before infarction (n = 12), ß-blockade produced a significant decline in fractional radial shortening in all three zones (Fig 4). In the future infarct zone the radial shortening declined from 20.6% ± 5.1% to 10.5% ± 2.8% (p < 0.001), in the prospective border zone from 21.9% ± 3.7% to 14.5% ± 2.0% (p < 0.02), and in the remote myocardium the radial shortening declined from 27.1% ± 1.9% to 21.7% ± 1.6% (p < 0.01). In the orotic acid-treated group before infarction, ß-blockade produced similar declines in fractional shortening in all zones.
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), the border zone (9.3% ± 2.8% before versus 8.2% ± 2.8% after blockade, p = 0.77), or in the remote myocardium (16.0% ± 2.8% before versus 15.1% ± 1.8% after blockade, p = 0.77). Comparing the preinfarction and day 3 postinfarction values after ß-blockade, radial shortening was reduced in the remote myocardium (p < 0.05) and in the infarct zone (p < 0.001), and not statistically different in the border zone. In the orotic acid-treated group (n = 12) the effects of blockade were similar to the control group in all instances.
Effect of Orotic Acid on Regional Function After Infarction
In the treated animals, in both the infarct zone and the border zone, there were significant reductions in fractional shortening on both day 1 and day 3 compared with measurements obtained before infarction, but these were not different from the control group (Table 1, Fig 3). In the remote myocardium the fractional radial shortening decreased on day 1 (p < 0.05) and on day 3, and was not significantly different from the control group (p = 0.8). After ß-blockade (day 3 only) there was no significant difference between the control and treated groups in fractional shortening in any of the three regions.
Regional Function After Prolonged Recovery
The 6 dogs studied up to 9 days after infarction (3 control and 3 orotic acid treated) showed a similar pattern of reduced radial shortening at day 1 and 3 followed by progressive recovery. Because there was no effect of orotic acid discernible in either the early (days 1 and 3) or late (days 5 and 9) measurements, the results of both groups were combined to study the delayed recovery of regional function (Fig 5). In these 6 dogs, the radial shortening in the infarct declined significantly from 19.9% ± 7.2% to 1.8% ± 4.7% on day 1 (p < 0.001), remained stable at day 3 and 5, and then showed a small, nonsignificant increase by day 9. In the border zone the radial shortening declined significantly from 22.1% ± 1.9% before infarction to 8.7% ± 2.30% at day 1 and 8.7% ± 1.73% at day 3, and then improved significantly to 17.5% ± 2.3% at day 9 (p < 0.01 for day 9 versus day 3). In the remote myocardium the radial shortening declined from 26.4% ± 1.8% before infarction to 19.0% ± 1.8% at day 1 and 21.4% ± 2.3% at day 3, before recovering to 29.4% ± 1.8% at day 9 (p < 0.01 for day 1 versus day 9 and p = 0.05 for day 3 versus day 9).
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). There was no significant difference between the maximum stroke work attainable in the control infarcted dogs before ß-blockade 2.2 ± 0.17 g-m/kg and the treated group 1.73 ± 0.24 g-m/kg (p = 0.25) or after ß-blockade, 1.8 ± 0.33 g-m/kg in the control dogs and 1.6 ± 0.37 g-m/kg in the treated animals (p = 0.7).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Depressed Function in Remote Noninfarcted Myocardium
Previous studies of the remote, noninfarcted myocardium after myocardial infarction have reported that the contractile function may be augmented, unaltered [19–21], or reduced [22]. Reduced function accords with the morphologic and biochemical abnormalities that have been demonstrated in this region, including reduced levels of high-energy phosphates [23, 6], reduced glycogen [24, 2], and decreased activity of mitochondrial enzymes [25], as well as intracellular edema and mitochondrial structural degeneration [24]. Areas of focal necrosis (microinfarction) associated with abnormal lactate metabolism have been observed in the remote myocardium [26]. The decrease in high-energy phosphate levels demonstrated in dogs by Gubdjarnason and colleagues [23] was present between 1 and 4 days after infarction, consistent with the time-related changes in function that we observed.
The functional and metabolic changes in the remote zone suggest an imbalance between energy utilization and production, and that the remote noninfarcted myocardium is under stress, resulting in a reduced functional reserve. The remaining myocardium incurs an obvious functional stress as the reduced mass of viable muscle must work harder to maintain a satisfactory cardiac output. It also suffers a mechanical disadvantage attributable to the presence of the akinetic or dyskinetic infarct, because energy expended in the motion of this area is wasted. Furthermore, the remaining myocardium is under neurohumoral stress from the increased sympathetic drive and elevated levels of circulating catecholamines [10, 27].
The fact that the remaining myocardium possesses a reduced functional reserve helps explain (1) why the recently infarcted heart responds poorly to the additional insult of global ischemia associated with cardioplegic arrest [2–4] and (2) why the recently infarcted heart responds favorably to myocardial protective methods during operation that minimize global ischemia [22].
Changes in ß-Adrenergic Responsiveness
Before infarction we found, as would be expected, that ß-receptor blockade produced a decrease in fractional shortening in all regions of the left ventricle. However, on day 3 after infarction, ß-receptor blockade failed to produce a significant change in fractional shortening in any region. This implies that before infarction removal of the effects of circulating catecholamines reduced contractility, whereas after infarction removal of these effects produced no change in function. The most likely explanation is that as a result of persistent catecholamine stimulation after infarction the myocardium had become less responsive to ß-adrenergic activity. This is consistent with previous studies of the remote myocardium [27] that demonstrated loss of response to isoproterenol stimulation at 3 days after infarction, with a reduced number of ß-receptor binding sites and reduced affinity of the remaining ß-receptors.
Orotic Acid Effect
Orotic acid is an important intermediate in the synthetic pathway for the pyrimidine nucleotides uridine and cytidine. Uridine nucleotides are important components of glycogen and RNA, whereas cytidine is an important constituent of the phospholipid component of cell membranes. After myocardial infarction there is a reduction in high-energy phosphates and glycogen in the remaining myocardium [23], and we have shown that orotic acid can prevent this [2, 6]. However, in the present study we were unable to demonstrate a significant effect of orotic acid on function in any zone, up to 9 days after infarction. This finding of a lack of orotic acid effect after infarction alone is consistent with our previous findings in rats [3, 4, 6] of no major effect of orotic acid administration on global LV function after infarction alone, but a significant and powerful effect of orotic acid after the added stress of cardioplegic arrest. Increased energy stores may improve tolerance to cardioplegic arrest but may not necessarily improve resting cardiac function. The previous clinical studies from Russia in the 1960s and 1970s [7, 8], showing an impressive benefit of orotic acid therapy after myocardial infarction, were not blinded or placebo controlled; however, it is possible that the hearts in these studies were more stressed than in the present study and hence could show an effect of orotic acid.
It is probable that the role of orotic acid is to provide a form of "metabolic preconditioning" to improve the metabolism of the stressed heart. Thus, treatment with orotic acid might provide no detectable improvement in baseline function of the heart after infarction but, after the additional ischemic stress of cardioplegic arrest, the treated myocardial cells might be better able to maintain cellular function.
Limitations of the Study
The relatively small size of the study population is a problem in that a negative result may not reflect the true absence of a beneficial effect of treatment, but reflect the small sample size. This is clearly a problem with studies involving animals that are a scarce resource and in particular in this study because there was considerable attrition from fatal ventricular arrhythmias.
For the comparison of radial shortening at days 1 and 3, full and satisfactory data were collected in 7 control and 8 treated animals. On the basis of the standard errors of the data presented, and assuming a p value of less than 0.05 (two-tailed) and power of 0.8, to detect a significant difference in the infarct zone between the two groups would require an absolute difference in the radial shortening of 4%. Similarly, in the border zone the same criteria would require an absolute difference of 6%. Although it is possible that a small benefit of orotic acid therapy on regional wall motion has not been detected due to the small sample size, there is little evidence of a clinically important trend in the data.
Clinical Significance
It is generally accepted that after a large myocardial infarction there is a vulnerable period beginning at 6 hours and lasting up to a week when mortality and morbidity from operation is significantly increased. Despite recent improvements in operation and myocardial preservation, patients with recent infarcts or mechanical complications of infarction still have a higher mortality from surgical revascularization [28, 29]. Previously, we have postulated that this is due to a reduced tolerance of the noninfarcted myocardium to ischemia, because of the mechanical and metabolic stress placed on it by the infarct [2]. The present study, by demonstrating both impairment of global function and impairment of regional function in all zones of the left ventricle, confirms that the reduced function after infarction is attributable to the combined effect of both the noncontractile infarct and the impaired function in the remote zone. Furthermore, the time course of depressed function in the remote myocardium that we have observed is similar to the period of higher surgical risk in clinical practice. Although in this study we did not determine the precise reasons for this functional impairment, these observations are in accord with studies in our own laboratory [6, 2] and elsewhere [23–25] that describe major biochemical derangements in the noninfarcted zone. In turn, these disturbances are likely to be exacerbated by chronic ß-adrenoreceptor overstimulation.
We conclude that in the recently infarcted heart, the remote and border zone myocardium may exhibit depressed function. Consequently, in patients with recent infarction undergoing operation, techniques of myocardial preservation should be used that minimize ischemic damage to the already stressed myocardium. Metabolic therapy with orotic acid would be expected to produce no major benefit in the patient with an uncomplicated infarct. Nevertheless, on the basis of our previous work [2–4] we believe that orotic acid therapy is beneficial in the period after infarction in patients with complications that may necessitate cardiac operation within 1 week of infarction and use it routinely in these patients in our clinical practice.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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