Relation of immediate and delayed thallium-201 distribution to localization of iodine-125 antimyosin antibody in acute experimental myocardial infarction

Relationof Immediateand Delayed Thallium-201 Distributionto Localizationof Iodine-l 25 Antimyosin Antibodyin Acute ExperimentalMyocardialInfarction BAN AN KHAW,

PhD, H. WILLIAM

STRAUSS,

GERALD M. POHOST, MD, JOHN T. FALLON, HUGO A. KATUS,

MD,

MD, PhD,

MD, and EDGAR HABER,

MD

Thallium-201 (Ti-201) distribution in acute experimental myocardiai infarction (MI) (n = 18) was compared with cardiac-specific antimyosin Fab (AM-Fab) uptake, a specific marker for myocardiai necrosis. When antimyosin was injected 4 hours after ligation with Ti-201 administered 23 hours 55 minutes later and measurement of myocardiai distribution determined 5 minutes after intravenous administration of Ti-201, (1) Ti-201 distribution closely correlated with microsphere regional blood flow, and (2) an inverse exponential relation to iodine-125 (i-125) AM-Fab uptake was apparent. in another group of 4 animals, Ti-201 and AM-Fab were administered intravenously 4 hours afler MI, and 38

hours later myocardiai distribution was measured. This delayed Ti-201 distribution had a close inverse linear correlation with i-125 AM-Fab uptake. This inverse linear relation also was apparent in 28hour-old MIS in dogs (n = 4) where collateral circulation had been established. Ti-201 was administered intravenously at 27 hours after MI, and Ti-201 distribution was determined 1 hour later. The present study demonstrated that whereas immediate Ti-201 distribution is Wow-limited, delayed Ti-201 distribution is a marker of ceil viability which, due to prolonged circulation time and redistribution, is not flow-limited.

Therapy to restore coronary blood flow in patients with acute myocardial infarction (MI) has been evaluated both by return of myocardial perfusion, demonstrated with thallium imaging, and the restoration of ventricular function. Since thallium-201 (Tl-201) uptake is believed to occur exclusively in nonnecrotic tissues1p2 and early thallium uptake is linearly related to blood flow,s5 thallium imaging may provide the most suitable marker for acute improvement. Yasuda et al6 demonstrated a lag in the return of function in several patients successfully treated with intracoronary streptokinase. The factors controlling the delayed distribution of thallium, however, are not as well defined. Studies by Pohost,4 Beller,7 and co-workers suggest that the con-

centration of Tl-201 in ischemic myocardium 2 to 3 hours after intravenous injection is not related to flow. It has been postulated that the late distribution of Tl-201 in myocardium is related to viable cell mass. Cardiac myosin-specific antibodies labeled with iodine-125 (I-125) localize in necrotic myocytes8 and thus are highly specific markers for MI.s-11 Studies were performed in experimental animals with acute coronary occlusion to compare the distribution of radiolabeled antimyosin antibody with immediate and delayed distribution of Tl-201.

From the Cardiac Unit, Massachusetts General Hospital, and the Department of Pathology, Harvard Medical School, Boston, Massachusetts. This study was supported in part by U.S. Public Health Service Grants HL-17665 and HL-21751 from the National Heart, Lung, and Blood Institute, the National Institutes of Health, Bethesda, Maryland. Manuscript received November 17, 1982; revised manuscript received January 26, 1983, accepted January 28, 1983. Address for reprints: Edgar Haber, MD, Cardiac Unit, Massachusetts General Hospital, Fruit Street, Boston, Massachusetts 02114.

method of Katz et all2 as described previously.g-ll Antisera were prepared in New Zealand White rabbits, and antibody specific for cardiac myosin was purified by affinity chromatography with canine cardiac myosin-Sepharose immunoadsorbent.s Anticardiac myosin Fab (AM-Fab) were prepared by papain digestion of purified whole antibody at 37°C for 1.5

Methods Preparation

of cardiac-specific

antimyosin

Fab

(AM-Fab): Purification of canine cardiac myosin was by the

hours at pH 7.0 by the method of Porter.13 Fab were separated from crystallizable fragment (Fc) and undigested antibody

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Volume51 May1, 1983 THEAMERICAN JOURNAL OFCARDIOLOGY

by chromatography on a protein-Sepharose immunoadsorbent.‘* (Protein-A binds Fc and undigested whole IgG but does not bind Fab.) AM-Fab thus obtained were concentrated by vacuum dialysis then dialyzed against 0.3 M phosphatebuffered saline solution, pH 7.0. Antibody activity of AM-Fab was tested by degree of binding to I-125 canine cardiac myosin. AM-Fab were iodinated with I-125 by the lactoperoxidase procedure of Marchalonis.15 I-125 (10 mCi) was used to iodinate 250 pg AM-Fab with a resultant 50 to 60% radioiodine incorporation at an enzyme to substrate ratio of 1:50. The radiolabeled AM-Fab were repurified utilizing a IO-ml myosin-Sepharose column. Active I-125-AM-Fab were desorbed from the immunoadsorbent with 5 M guanidine-hydrochloride and renatured by sequential dialysis with an excess of 3 M potassium chloride, then against phosphate-buffered saline solution. Approximately 1 to 1.5 mCi of repurified I-125-AM-Fab was obtained from each iodination for this study. Experimental MI: Eighteen mongrel dogs were anesthetized with 30 mg/kg of pentobarbitol, intubated, and placed on a Harvard pump respirator. A thoracotomy was performed on each dog at the fourth intercostal space and the heart suspended in a pericardial cradle.gJ6 The left anterior descending coronary artery (LAD) was dissected free at approximately two-thirds the distance from apex to base and occluded with a silk ligature. This ligation usually affected approximately 30% of the left ventricular myocardium as determined by the extent of cyanosis. The thoracotomy was closed, and the animals were allowed to recover. Experimental protocol: Three different experimental protocols (I to III) were employed to study the relation of Tl-201 distribution to viable myocardial cell mass. Each protocol was performed in 6 dogs; however, 2 dogs each from Groups I and III and 3 from Group II were lost due to ven-

DOG (Atns~12lID)

LAD 0CCL”SION CLOSE THOAACOTOMr

GAalP

I

I\

I

CmHJP

II

4 hr

I-125-AM-Fab i.". (200 pa)

23 hP

mouP

4 br I-12%AM-Fab

i.". (200 pcij

23 hr

III

4 tlr I-125-AM-Fab (20 ,,Cil + 200 pci n-201 i.".

35 hr

AND COUNT ENDO- .4ND EPICARDIAL SAMPLES IN SCINTILLATION COUNTER

FIGURE 1. Schematic diagram of the 3 experimental protocols employed to study TI-201 distribution with respect to viable myocardial mass as determined by cell death criteria of AM-Fab localization. i.v. = intravenously.

1429

tricular fibrillation after LAD occlusion. Figure 1 is a schematic diagram of the 3 protocols. The LAD was occluded in all dogs as described previously. At 4 hours after LAD occlusion, 200 &i of I-125-AM-Fab was administered intravenously. In Group I, each of the 4 living dogs received 100 &i Tl-201 by intravenous injection 23 hours after administration of the I-125-AM-Fab. One hour later, the animals were killed by pentobarbitol overdose. The hearts were excised immediately and cut into 1 cm-thick ventricular slices parallel to the atrioventricular groove. Each ventricular slice was cut into 8 to 10 equal pieces and then further subdivided into epicardial and endocardial samples. All tissue samples were counted in an automatic scintillation gamma counter (Packard) for relative distribution of Tl-201 and I-125 activities. Windows were set at 80 to 200 keV for Tl-201 counting and 15 to 50 keV for I-125 activity. Corrections were made for Tl-201 activity in the iodine window. Normal epicardial and endocardial samples (4 each) obtained from the posterior left ventricular myocardium were used for comparison with the rest of the myocardial samples for distribution of various radioisotopes. In Group II, the 3 living dogs were reanesthetized 23 hours after intravenous administration of the I-125-AM-Fab, and the thoracotomy was reopened. Forty-five minutes later, a bolus of 3 X 106 scandium-46 (SC-46) carbonized microspheres (8 to 10 pm, 3M, St. Paul, Minnesota) was injected into the left atrium for determination of relative regional blood flow. Ten minutes after microsphere injection, 100 &i of Tl-201 were administered by intravenous injection, and the animals were killed exactly 5 minutes later by pentobarbitol overdose. Relative distribution of I-125, Tl-201, and SC-46 activities in the various samples of the myocardium was determined as described for Group I with an additional window of 680 to 1,000 keV for SC-46 activity. The 4 living dogs in Group III were given 200 &i of I125-AM-Fab and 200 PCi of Tl-201 intravenously simultaneously at 4 hours of LAD occlusion. Thirty-six hours later, the animals were killed. Fifteen minutes before death, a bolus of 3 x lo6 SC-46 microspheres was injected into the left atrium as previously described. The hearts were excised, and distribution of I-125, Tl-201, and SC-46 activities in the myocardial samples was determined as described for Groups I and II. Comparison of Tl-201 with regional blood flow: Data obtained from Groups II and III were reanalyzed for comparison of Tl-201 distribution at 5 minutes and 36 hours after intravenous injection of Tl-201 to the corresponding regional myocardial blood flow. Computer curve fitting was performed as described later. Statistical analyses: At least 4 endocardial and 4 epicardial samples from the posterior left ventricular myocardium of each animal were employed as controls to determine mean uptake and distribution of I-125-AM-Fab and Tl-201. In studies involving comparison of relative antibody uptake to thallium distribution or relative regional blood flow, best-fit linear regression curves relating antibody uptake to Tl-201 or flow were determined by utilizing the formula y = a + bx, where y = log (percent blood flow) or log (percent Tl-201) and x = relative antibody uptake; or, in case of inverse relations, y = percent blood flow or percent Tl-201 and x = antibody uptake. Constants a and b were obtained by the method of Snedecor and Cochran.l7

Results The relation of I-125AM-Fab localization to 1 hour of Tl-201 distribution determined at 28 hours after LAD occlusion is demonstrated in Figure 2. There is an in-

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THALLIUM-201 TO ANTIMYOSIN UPTAKE IN MYOCARDIAL INFARCTION

/

Y=lli-12.9x R=-0935

140

r--

N=135 120

Y=107-12.2X R = - 0.828 N=86

.

.

8 Q s

60

Y 22.106-0.1242X R = 0.669 N=86

80-

x 60 -

t

20 c

Lil 2

.!

FIGURE 2. Relation between AM-Fab uptake and TI-201 distribution in 28-hour-old experimental MI: y = 111 - 12.9x, where y = percent flow and x = (AM-Fab)l/(AM-Fab)N.

verse relation between Tl-201 distribution and AM-Fab uptake. In the region of normal myocardium, determined by lack of an antibody uptake ratio above 2 (1.15 f 0.18, mean f standard deviation [SD]), Tl-201 distribution was 100 f 11.2% of average normal posterior left ventricular myocardial Tl-201 distribution. Only 1 sample point showed Tl-201 distribution at 67%. The correlation coefficient was highly significant at -0.935 (p 0.5) (Fig. 3). In regions of normal myocardium showing AM-Fab uptake of less than a ratio of 2 (1.22 f 0.3, mean f SD), mean regional blood

4

6

8

FIGURE 4. Simultaneous comparison of AM-Fab uptake and TI-201 distribution and regional myocardial blood flow. Left, relation between antibody uptake and TI-201 distribution 36 hours after antibody and TI-201 intravenous injection. Right, relation between antibody uptake and regional myocardial blood flow.

flow was 88.4 f 20.2% and mean Tl-201 distribution was 92 f 16.9% (p = not significant [NS]). When AM-Fab uptake at 36 hours of antibody circulation was compared with Tl-201 uptake and regional blood flow, Tl-201 distribution was inversely proportional to AM-Fab uptake (r = -0.828), whereas regional blood flow still showed an inverse exponential relation (r = -0.669,p
~ EARLY

T/-20f/5mml

i ATE n-20/

f36hr.l

140t Y:749+0936X

.

I

Y:245-0437X R=0757

Y=515-0534x R:-0854 N:1171

% FLOW

(AM-Fob4 /IAM-FobI,

FIGURE 3. Simultaneous comparison of AM-Fab uptake to TI-201 distribution and regional myocardial blood flow at 5 minutes of TI-201 distribution in 26hour-old experimental MI.

FIGURE 5. Comparison of immediate (5 minutes) TI-201 distribution to regional myocardial blood flow at (left) 28 hours of coronary occlusion: y = a + bx, where y = percent TI-201, x = percent flow, a = 7.49, b = 0.936. and r = 0.97 1, and (right) 36 hours of TI-20 1 distribution to regional myocardial blood flows in 40-hour-old experimental myocardial infarcts: y = a + bx, where y = In percent TI-201, x = percent In flow, a = 2.45, b = 0.437, and r = 0.757.

May I,1983

distribution to regional blood flow for 26 and 40hour-old infarcts with Tl-201 distribution determined at 5 minutes and 36 hours of circulation. As expected, 5-minute Tl-201 distribution is reflective of regional blood flow demonstrating a direct proportionality (r = +0.971). While 36-hour distribution is diphasic, with near normal Tl-201 uptake in tissue samples in which regional flow has been reduced to 40% of normal, a more drastic reduction of Tl-201 uptake is noted in regions of hypoperfused myocardium (r = 0.757).

Discussion We have demonstrated with primary neonatal murine myocytes in culture that antimyosin bound covalently to 1 pm polystyrene beads binds only to necrotic myocytes.18Jg This observation was confirmed by fluorescence-activated cell sorting as well as by scanning electron microscopy. We have also previously demonstrated that antimyosin is highly specific for myocardial necrosis by macro- and microautoradiography, and by histochemical and histologic criteria.a Based on these studies, where antimyosin localization occurs only in necrotic myocytes, we have now used antimyosin uptake as a standard for determination of cell necrosis for comparison with Tl-201 distribution. Tl-201 imaging, with injection either at rest or during exercise, has been used in clinical studies to identify ischemic or infarcted regions of myocardium.1-4,7 Although many studies support the concept that the initial distribution of thallium is a linear function of blood fl~w,~,~ at most clinically relevant blood flows, the factors controlling the redistribution of thallium have not been well defined. Recent studies4,20-22 suggest that redistribution is independent of blood flow. This study suggests that the distribution of Tl-201 several hours after administration in acute MI and labeled antimyosin localization are inversely proportional. These data support the contention that delayed thallium scans represent the distribution of viable myocardium (r = -0.935 and -0.828). There appears to be little difference between 1 hour and 36 hours of thallium distribution in this relation (slopes -12.35 and -12.27, p >0.5), suggesting that in an older MI where collateral circulation has already been established, serial distribution need not continue beyond 1 hour to allow evaluation of viability at least in experimental animals. However, in fresh MIS, 2 hours of Tl-201 redistribution may be required for evaluation of cell viability as observed by Pohost et a1.4.This difference in time may be due to the difference in the amount of collateral circulation. Immediate thallium distribution (at 5 minutes), on the other hand, results in a very different slope in relation to antibody uptake (-17.9, p
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1431

Thus, at 1 hour or 36 hours after thallium injection, the distribution of radionuclide is not limited by regional blood flow in older infarcts, but appears to reflect cell viability. We have previously demonstrated that antimyosin antibodies localize specifically in infarcted myocardium over and above the extent of nonspecific adsorption of control IgG.g-ll This specificity has also been demonstrated by microautoradiographys where only necrotic myocytes showed antimyosin localization, whereas normal myocardium did not show antimyosin adsorption. These studies indicated that antimyosin localizes only in necrotic cells. By this criterion of cell necrosis, mean thallium uptake in normal myocardium was 95.3 f 8.6%, whereas average flow in these same samples was 101.3 f 25% (Fig. 4). A possible explanation of the observed gradient of increasing antibody uptake and decreasing thallium uptake from normal tissue to the center of an infarcted region may well be related to a varying admixture of viable and necrotic cells. Near the periphery of the ischemic zone where the cells are largely viable, thallium is taken up, with some antibody uptake in a small number of necrotic cells. As one proceeds to the center of the infarct, there are more necrotic cells that take up antibody and fewer viable cells that take up thallium. If, however, ischemic cells take up thallium at a reduced concentration, then the cells in the periphery of the ischemic zone would show reduced thallium uptake, and since these are still viable cells, the antibody uptake would remain at about a ratio of 1. If such were the case, the relation between delayed thallium and antimyosin uptake would be similar to that of percent flow and antimyosin uptake. However, the data indicate an inverse linear relation between delayed thallium uptake and antimyosin localization, indicating that the region of MI is a mixture of viable and necrotic myocytes with maximum necrotic myocytes at the center of the MI. In immediate (5 minutes) thallium distribution, which appears to be flow-limited, there may be regions of viable cells that take up neither thallium nor antibody. Antibody is not taken up because it is excluded by intact cell membranes. Thallium is not taken up because there has been insufficient time for adequate concentration of this radionuclide to reach cells which have the potential of adequate uptake. The data presented in our report demonstrating delayed thallium distribution as an indicator of myocardial cell viability may be of importance in view of the recent studies utilizing intracoronary streptokinase, angioplasty, or both to recanalize occluded coronary vessels.24-2s These invasive interventions are aimed at salvaging jeopardized myocardium. Intravenous thallium distribution images obtained before recanalization provided images of the areas at risk, whereas thallium images obtained after recanalization should provide images including areas of viable myocardium in the jeopardized zones salvaged by the intervention. The differences between the pre- and postrecanalization thallium images should provide areas of salvaged myocardium. Return of cardiac function after recanalization has not been observed to be a sensitive parameter for determination of myocardial salvage.6 Whether Tl-201 images obtained immediately after

1432

THALLIUM-201 TO ANTIMYOSIN UPTAKE IN MYOCARDIAL INFARCTION

recanalization with a second dose of intravenous thallium would provide information regarding the salvaged myocardium is not clear; however, a delayed image after recanalization should provide areas of thallium uptake in viable myocardium. Comparison of pre- and postrecanalization thallium images should thus provide scintigraphic data demonstrating the outcome of the intervention. In conclusion, this study has demonstrated that (1) late thallium distribution reflects cell viability rather than regional blood flow; (2) immediate thallium distribution at 5 minutes of circulation is an indicator of relative blood flow, the analysis of which may significantly overestimate the area of irreversibly damaged myocardium; and (3) the comparison of immediate and delayed Tl-201 images could provide information concerning the zone of reversible ischemic compromise. Acknowledgment: We thank Rebecca Rubin for editorial assistance in the preparation of this manuscript.

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