PET Imaging of Oxidative Metabolism Abnormalities in ...

PET Imaging of Oxidative Metabolism Abnormalities in Sympathetically Denervated Canine Myocardium Gary D. Hutchins, Timothy Chen, Kathy A. Carlson, Richard L. Fain, Wendy Winkle, Triad Vavrek, Bruce H. Mock and Douglas P. Zipes Division of Imaging Science, Department of Radiology and Krannert Institute of Cardiology, Department of Medicine, Indiana University Medical Center, Indianapolis, Indiana

This study was designed to test the hypothesis that regional sympathetic denervation produces perfusion and metabolic alter ations in myocardial tissue under resting conditions. Methods: PET studies of myocardial sympathetic innervation, myocardial perfusion and oxygen utilization using ["CJhydroxyephedrine (HED), [13N]ammonia and 1-[11C]acetate, respectively, were per formed before and approximately 2 and 8 wk after surgical left thoracotomy and regional chemical sympathetic denervation (n = 5). A second group of animals underwent the same surgical procedure but, so that they could serve as a sham control group, were not sympathetically denervated (n = 5). The second group of animals was imaged before and 2 wk after surgery. Images of the retention of [1'C]HED taken from 50 to 60 min postinjection were used to differentiate sympathetically innervated and dener vated regions of the left ventricle. Regions of interest were defined on polar plots of the [11C]HED retention, including the sympathetically denervated territory and normally innervated regions. Regions defined on the HED polar plots were then transferred to the [13N]ammonia and 1-[11C]acetate image data, and tracer kinetic models were fit to the regional time-activity curves to generate estimates of myocardial perfusion and oxidative metabolism. Results: The average percentage of the left ventricle denervated in the group I animals was 13.1% ±7.3%. Significant reductions in oxidative metabolism were observed in the sympathectomized tissue both at 2 and 8 wk after surgery (22% and 15% reductions, respectively). Significant alterations in regional perfusion were not observed. No significant changes in oxidative metabolism or perfusion were observed in the sham control group. Conclusion: Regional sympathetic denervation alters oxidative metabolism but not perfusion in the denervated region of the heart. Key Words: PET; myocardial oxidative metabolism;myocardial blood flow; autonomie nervous system; sympathetic denervation J NucÃ-Med 1999; 40:846-853

he autonomie nervous system of the heart regulates physiologic response to external conditions through sympa-

Received Apr. 27,1998; revision accepted Sep. 25,1998. For correspondence or reprints contact: Gary D. Hutchins, PhD, Director, Division of Imaging Science, Department of Radiology, Indiana University Medical Center CL 120, 541 Clinical Dr., Indianapolis, IN 46202-5111.

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thetic and vagai control of heart rate, conduction and cardiac muscle contractility (/). These systems modulate cardiac function through receptors on myocytes coupled to guanosine triphosphate-binding proteins that, in turn, are coupled to adenylate cyclase. The binding of neurotransmitters to muscarinic or ß-adrenergicreceptors located on the surface of myocytes regulates the activity of adenylate cyclase, resulting in modulation of cell-to-cell communications. The autonomie nervous system also plays a role in the regulation of the coronary microvasculature through direct innervation as well as endothelial-mediated mechanisms (2-6). Remod eling of the heart in cardiac disease disrupts cellular communication and autonomie innervation, producing a substrate that can lead to electrophysiologic disturbances and produce fatal ventricular tachyarrhythmias (7). Numerous studies have shown that denervation of other wise normal myocardial tissue leads to an exaggerated electrophysiologic response of the tissue to adrenergic agonists (8-13). This exaggerated response has been termed denervation supersensitivity, and both presynaptic and postsynaptic mechanisms have been shown to be involved in this altered response to agonist administration (8-13). Several scintigraphic imaging studies using [I23l]metaiodobenzylguanidine (MIBG) and [HC]hydroxyephedrine (HED) have shown that regional sympathetic denervation occurs in otherwise apparently normal tissue in several myocardial disease processes (14-21). Although numerous studies have investigated the electrophysiologic properties of sympatheti cally denervated myocardium, the effect of regional denerva tion on myocardial perfusion and oxidative metabolism is poorly understood. In a recent study of cardiac transplant patients using PET imaging, the response of coronary blood flow to sympathetic stimulation was shown to be related to the norepinephrine content of the cardiac sympathetic nerve terminals (22). In addition, it has been shown that the blockade of myocardial ßlreceptors reduces myocardial oxygen requirements and myocardial blood flow (23) and that regional sympathetic denervation results in the absence of normal metabolic responses during direct left stellate nerve stimulation in dogs (24). The objective of this study was to test the hypothesis that regional sympathetic denerva-

THE JOURNALOF NUCLEARMEDICINE• Vol. 40 • No. 5 • May 1999

the phenol application are restricted to a depth of only 0.25 mm (26). The LAD region was selected in an attempt to disrupt major trunks of the sympathetic nervous system to produce regions of denervation larger than the area of phenol application. This approach was used to minimize any potential direct effects of phenol on cardiac myocytes. In the first two animals, only a single application of phenol was used. These animals did not show any evidence of denervation on the basis of HED imaging and were subsequently included in our sham control group. Kaye et al. (26) have demonstrated the potential ineffectiveness of this technique in regions with epicardial fat. Therefore, in all subsequent animals, phenol was applied three times at approximately 10-min intervals

tion of the left ventricle produces an altered physiologic substrate, resulting in regional disruption of myocardial blood flow and metabolism in the resting state. To test this hypothesis, a series of PET imaging studies of myocardial sympathetic innervation, perfusion and oxidative metabo lism was performed in dogs before and after regional chemical sympathetic denervation using epicardial applica tion of phenol. MATERIALS AND METHODS

Study Groups

to ensure regional denervation. The chest in each animal was closed and negative intrathoracic pressure was re-established. The dogs were treated with cephazolium sodium (500 mg twice a day for 3 d), and their recovery was monitored by a veterinarian in the university's animal care facility. Dogs were maintained in accor

Two groups of mongrel dogs were studied. The study protocol for each group is shown in Figure 1. The five dogs in group I underwent surgery, and a region of denervation was created by the application of phenol to the surface of the left ventricle. Group II included three dogs that underwent surgery, with a similar region of the heart painted with saline, and two dogs in which the application of phenol did not create any evidence of denervation on the basis of the regional HED uptake. In each group, PET studies of sympa thetic innervation, myocardial perfusion and oxidative metabolism were performed both before and approximately 2 wk after surgery. A third series of PET imaging studies was performed on four of the group I animals at approximately 8 wk after surgery to examine the reproducibility of the oxidative metabolism and innervation results. The fifth animal in group I died unexpectedly before the third imaging session.

dance with guidelines specified by the National Institutes of Health in its Guide for the Care and Use of Laboratory Animals (27). Each animal was allowed to recover for approximately 2 wk before imaging.

Radiopharmaceuticals Studies of sympathetic innervation, oxidative metabolism and perfusion were performed using ["C]HED, l-["C]acetate and [13N]ammonia. HED was synthesized using ["C]methyltriflate to label the metaraminol precursor in a method reported by Rosenspire et al. (28), l-["C]acetate was prepared by reacting ["C]CO2 with CH3MgBr (29) and [13N]ammonia was generated in target

Denervation Model

through the addition of a small amount of ethanol (2 mmol/L) to an [16O]water target irradiated with 11-MeV protons. Each radiophar-

The animals were premedicated with morphine sulfate (2 mg/kg, injected intramuscularly) and anesthetized with sodium thiopental (25 mg/kg, intravenously). Anesthesia was maintained with alpha chloralose (60 mg/kg, intravenously, followed by 20 mg/kg/h, intravenously). Each animal was intubated with a cuffed endotracheal tube, and the lungs were ventilated with positive pressure. Each dog was kept on a circulating hot water pad to prevent hypothermia. The chest was opened with a left thoracotomy, the pericardial sac was sectioned and a circular region (approximately 1.5 cm in diameter) of phenol (88% carbolic acid) was painted in the territory of the left anterior descending coronary artery (LAD) at a midventricular level (25). Histologie changes associated with

j/PET

maceutical was prepared for sterile administration to the animals for imaging.

PET Imaging Dynamic imaging protocols were performed using a Siemens 951/31R PET tomograph (Siemens/CTI, Inc., Knoxville, TN) in the Indiana University Hospital. Anesthesia was initiated for the imaging studies using thiopental (18-22 mg/kg, intravenously) and was maintained throughout the imaging procedures using propofol infusion (0.2-0.4 mg/kg/min). Each animal was placed in a supine position in the PET scanner, and transmission scans were obtained

1 Group I / Denervation (N = 5 Dogs)

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FIGURE 1. PET imaging study protocols. Animals in groups I and II underwent PET imaging studies of myocardial perfusion, oxidative metabolism and sympathetic inner vation on entering protocol. After initial imag ing session, surgery was performed and regional sympathetic denervation was cre ated in group I animals but not in group II animals. Two weeks after surgery, PET imaging studies were repeated. Group I animals were also imaged again approxi mately 8 wk after surgery.

PET IMAGING OFDENERVATED MYOCARDIUM • Hutchins et al.

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to measure the attenuation of the torso. After the transmission scan, emission studies were initiated consisting of a slow intravenous bolus injection (spread over approximately 30 s) of 20 mCi HED, acetate or ammonia. The slow bolus was used to enable adequate sampling of the arterial blood curve shape from the image data for tracer kinetic analysis of the data (30).

database, and the extent of the phenol-induced defect was character ized as the percentage of the left ventricular wall with HED retention fractions 3 SD or greater below the control database values. Pre- and postsurgery results in both groups were compared using a paired r test. RESULTS

PET Data Analysis The emission images acquired with each radiopharmaceutical were reconstructed with a conventional filtered backprojection algorithm using a Manning window that produces images with a spatial resolution of approximately 1.0 cm full width at half maximum. The transverse section images were then combined to form a three-dimensional volume of the heart and resliced into 12 short-axis 1.0-cm-thick planes extending from the base to the apex of the left ventricle. Each short-axis plane, except for the most apical plane, was divided into 16 sectors, and myocardial profiles were generated across the heart wall. The centroid of each profile was then calculated and a region of interest (ROI) shaped like the sector cut out of an anulus was placed at the centroid location. This process was repeated for each image plane, and a single circular region was defined on the apex, creating a total of 177 ROIs. The 177 ROIs were then propagated through the entire dynamic series of images, creating time-activity curves for each region of the left ventricle. The HED data were analyzed by creating estimates of tracer retention fractions for a time window extending from 50 to 60 min postinjection:

RF =

ri=6

Eq.

JL The kinetic data for [13N]ammonia were analyzed by fitting a three-compartment tracer kinetic model to the regional timeactivity curves, producing estimates of myocardial perfusion (31,32). Estimates of myocardial oxidative metabolism were obtained by fitting a two-compartment model to the regional time-activity curves using an approach previously reported by Raylman et al. (33). Polar Map Analysis Retention fractions for HED and kinetic model parameters for [l3N]ammonia and l-["C]acetate were reformatted and displayed as polar maps for image analysis. In group I, ROIs were derived from the postsurgery HED retention fraction polar map image. Three regions, consisting of a sympathetically denervated zone, a septal wall region and an inferior wall region, were defined. In the studies for group II, ROIs were generated at a mid-ventricle level for each of the four major territories of the left ventricle (septal, anterior, lateral and inferior walls). The septal and inferior wall ROIs were defined in an analogous fashion for both groups. The regions defined on the HED polar maps were subsequently applied to the ammonia and acetate polar maps for calculation of regional perfusion and oxidative metabolism. All regions were normalized by the average of the septal and inferior wall ROI values to account for global variability across studies. The presurgery HED retention fraction polar maps from both groups were also combined to form a polar map database, consisting of mean and SD values for each of the 177 ROIs in the polar plots. Postsurgery HED retention fraction polar plots were compared with the HED retention fraction

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A comparison of retention fraction images for [BN]ammonia and ["C]HED pre- and postsurgery for one of the group I animals is shown in Figure 2. The postsurgery sympathetic denervation is easily identifiable in the anterior-lateral wall region of the short-axis image. In contrast, the ammonia retention fraction image visually appears normal in the region of denervation. A comparison of the mean HED retention fraction polar plots both pre- and postsurgery is shown in Figure 3. The sympathetic zone of denervation can be identified clearly in the postsurgery mean image for group I. The percentage of the left ventricular wall with retention fractions equal to or less than 3 SD below the mean retention fraction from control studies at approximately 2 wk after surgery was 13.1% ±7.3% (Table 1). Quantitative analysis of the [13N]ammonia image data did not reveal any signifi cant differences in myocardial blood flow in any region of the heart when compared with the presurgery scans (Tables 2 and 3). The dog with the largest sympathetic defect (24.8%) died unexpectedly, presumably as a result of sudden cardiac death, between the first and second postsurgery studies in the group I animals. The average HED retention fraction defect size decreased from 10.2% ±3.8% (2 wk after surgery) to 7.9% ±6.5% (8 wk after surgery) for the four animals studied (Table 1). Figure 4 shows a comparison of the mean polar map images for the group I studies pre- and postsurgical denervation. The arrows in Figure 4 demonstrate defects in the average polar maps of oxidative metabolism that

Control Regional Denervation (Phenol)

Perfusion (|!\-13|

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Innervation ((C-11JHED)

FIGURE 2. Mid-ventricular short-axis images of myocardial perfusion and sympathetic innervation. Shown are example images from animal in group I. Images on left show retention of [13N]ammonia in myocardium both without and after sympathetic denervation. Images on right show corresponding HED retention ¡mages.Regional denervation is clearly evident in image in lower right panel (arrow).

THE JOURNALOF NUCLEARMEDICINE• Vol. 40 • No. 5 • May 1999

Group I (N=5)

Group n

(N=5)

Pre Surgery HED

Post Surgery HED

correspond nicely with the zone of sympathetic denervation on the HED retention fraction maps. The mean polar plot maps for the group II studies are also shown in Figure 4 to demonstrate the absence of HED retention fraction and metabolism defects. ROI data are shown in the bar graphs of Figure 5 and are given in Tables 4 and 5. Statistical analysis of the ROI data from the l-["C]acetate studies revealed significant reductions in the k2 and normalized k2 values used as indices of oxidative metabolism (P < 0.05) in the denervated region in group I at 2 wk postsurgery. In the repeat studies performed at approximately 8 wk postsurgery, a significant reduction in oxidative metabolism as assessed using normalized k2 values persisted in the denervated region (P < 0.05). Acetate K, (myocardial blood flow X extraction fraction) and k2 values are plotted as a function of the heart rate and systolic blood pressure product (RPP) for group I in Figure 6. The slopes for linear regres sion fit to the K, data were 1.98E - 05 and 1.87E - 05 for

FIGURE 3. Mean polar maps of HED retention fractions. Average HED retention is shown for group I and group II animals. Sympathetic ¿¡enervation created in group I animals is clearly visible in image in upper right panel (arrow).

the control and denervated regions, respectively (P = NS). The regression slopes for k2 were 9.35E - 06 and 4.48E - 06 in the control and denervated regions, respectively (P = 0.10). The regression slope for k2 in the group II animals was similar to the group I control studies (slope = LUE —05; data not shown in Figure 6). Comparison of the ammonia myo cardial blood flow estimates and K! estimates for acetate ob tained from control and 2 wk postsurgery studies revealed a strong correlation (slope = 1.07, intercept = —0.12,r2 = 0.93). DISCUSSION New Findings

In this study, we examined changes in baseline metabo lism and blood flow that occur as a result of regional sympathetic denervation of the heart in dogs. The results of TABLE 2 Myocardial Perfusion Estimates Derived from Ammonia Kinetics*

TABLE 1 Extent of HED Retention Fraction Defects in Group I Animals

StudyDogiDog

2Dog 3Dog 4DogoAverage (1-4)Average (1-5)%

(control)0.58 StudyGroup (2wk)0.62 Defect Defect (2wk)13.86.813.37.124.810.2 (8wk)17.53.54.06.7—7.91DefectInferiorSeptalGroup 0.100.64 ± 0.130.66 ± 0.100.73 ± 0.140.81 ± 0.060.93 ± 0.170.97 ± IIAnteriorInferiorLateralSeptalAmmonia 0.300.79 ± 0.340.91 ± 0.190.79 ± 0.270.94 ± 0.180.94 ± 0.301.01 ± 3.813.1 ± 6.5— ± ±0.31Ammonia ±0.31P0.330.200.370.360.120.110.28 ±7.3%

HED = [11C]hydroxyephedrine.

"Estimates in mL/g/min.

PET IMAGINGOF DENERVATED MYOCARDIUM • Hutchins et al.

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TABLE 3 Myocardial Perfusion Estimates Normalized by Average of Septal and Inferior Wall Estimates*

StudyGroup

(control)0.85

(2wk)0.84

indicated a trend with P = 0.10). A reduction in the mean size of the denervated region was observed between 2 and 8 wk after chemical denervation. Although this change was not statistically significant, the observation is consistent with previously reported reinnervation times of approximately 14 wk (34).

1DefectInferiorSeptalGroup 0.090.93 ± 0.020.90 ± Other Studies 0.051 ± 0.061.10 ± 0.051.06 .07 ± 0.061 ± These observations are consistent with previous studies IIAnteriorInferiorLateralSeptalAmmonia suggesting that resting coronary flow is not substantially 0.070.92 ± 0.070.95 .00 ± affected by either humoral or neural adrenergic influences 0.060.93 ± 0.030.98 ± (2,22,24,35). However, our observation of changes in myo0.061 ± 0.141 ± cardial oxidative metabolism in regionally denervated myo.08 ±0.06Ammonia .05 ±0.03P0.440.080.080.080.170.200.16 'Estimates

in mL/g/min.

this study demonstrate a 22% reduction in the k2 values and a 19% reduction in the normalized k2 values estimated from the measured kinetics of l-["C]acetate in sympathetically denervated myocardium at 2 wk after regional chemical denervation. In studies repeated in four of the five original animals at 8 wk after denervation (one animal died of sudden cardiac death before the repeat study), this reduction in oxidative metabolism persisted with 15% and 14% reduc tions observed relative to presurgical study k2 values and normalized k2 values (only the differences in the normalized values were statistically significant). Estimates of myocardial perfusion using [l3N]ammonia at 2 wk postsurgery did not show any significant changes in the relative perfusion of the denervated myocardium when compared with presurgery perfusion studies. Apparent differences in the perfusion levels between the group I and group II animals were the result of differences in the RPP between the animals that made up each group. The slope from the linear regression fit to the acetate k2 data as a function of RPP was reduced by 52% in the defect region of the group I animals relative to the innervated regions (a t test comparing these slopes

FIGURE 4. Mean polar maps of HED retention fraction (RF) values and acetate k2 estimates in group I and II animals. Upper and lower polar maps display mean values for HED retention and acetate k2estimates, respectively, for control studies from both groups I and II on the left, for group I at 2 wk postsurgery (middle) and for group II (right). Note reduction in k2 that corresponds with region of sympathetic denervation observed in group I.

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cardial tissue under baseline conditions differs from the results of a study of láclate and oxygen extraction ratios performed by Chilian et al. (24), who found no differences between innervated and sympathectomized regions. How ever, in the study by Chilian et al., the oxygen extraction ratio increased in the normally innervated region with left stellate stimulation but not in the sympathectomized region. Our results suggest that baseline sympathetic tone plays a role in the regulation of regional myocardial oxygen metabo lism under resting conditions in the dog. In a related study, Mori et al. (36) demonstrated that characteristic regional myocardial contractility and coronary vascular resistance responses to sympathetic stimulation were abolished in phenol denervated regions. This study also provided evi dence that coronary resistance in a denervated region may be influenced more by coronary autoregulation than by direct sympathetic vasoconstriction. These observations are consis tent with our results, which show an apparent uncoupling of oxidative metabolism from myocardial perfusion in sympa thetically denervated myocardial territories. Figure 6 shows a 52% reduction in the slope of the relationship between the k2 for acetate and RPP in denervated relative to innervated myocardial regions. In contrast, the slope of the relationship between K) and RPP was reduced only by 5.6% in the denervated regions relative to innervated regions.

LE Control Studies

Group I

Group n

(N=10)

(N=5)

(N=5)

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