JACC Vol. 9. No.6June 1987:1235-42
Relation Between Coronary Artery Stenosis and Myocardial PurineMetabolism, Histology and Regional Function in Humans
WILLEM FLAMENG, MD,* JOHAN VANHAECKE, MD,! HERMAN VAN BELLE,:j:MARCEL BORGERS, DRSC,:j: LUC DE BEER, DRSC,* JAAK MINTEN, DRSet
Leuven and Beerse, Belgium
1235
In 54 patients undergoing elective or emergency aortocoronary bypass grafting, angiographic and electrocardiographic changes were studied. Five patients with unstable angina and fivepatients with evolvingmyocardialinfarction were included. .High energy phosphate metabolism and the histologic appearance of the myocardium were analyzed in transmural biopsy specimens acquired at the time of surgery.
In patients without anterior infarction on the electrocardiogram, severe stenosis of the left anterior descending coronary artery resulted in a reduction of anterior wall motion that was associated with a partialdepletion of the adenylate pool. Mitochondrial function,however, remained intact: the adenosine diphosphate/adenosine triphosphate ratio, the energy chargeand the creatine phosphate/adenosine triphosphate ratiowere in the normal range . Histologic assessment demonstrated viable myocardium with a high incidence ofatrophic cells.
In evolving myocardial infarction, 170minutesof acute
The effect of acute coronary artery occlusion on high energyphosphate metabolism has been exten sively studied in experimental animals (1-5). Under these conditions a rapiddegradation of adenine nucleotides has been demonstratedand a critical level of adenosine triphosphate (ATP) wasproposed as the triggering mechan ism for irreversible myocardial cell damage (I). Acute coronary artery obstructionin humans, however , is usually preceded by a significantdegree of coronary artery stenosis and the influence of sucha chronic , progressive stenosis on purine metabolism, myo-
From the *Department of Cardiovascular Surgery and the t Department of Cardiology, University Clinic Gasthuisberg, Leuven and thet Department of Cell Biology, Janssen Pharinaceutica Research Laboratories, Beerse, Belgium. This study was supported by a grant from theNationaal Fonds voor Wetenschappelijk Onderzoek, Brussels. Belgium.
Manuscript received March 31, 1986; revised manuscript received November 19, 1986, accepted December 12, 1986.
Address for reprints: Willem Flameng , MD. Department of Cardiovascular Surgery. U.Z. Gasthuisberg . Herestraat 49, B-3000 Leuven, Belgium.
©1987 by the American College of Cardiology
coronary artery obstruction resulted in anterior wallakinesia associated with a decrease of the sum of theadenylates to 52% and of creatine phosphate to 16% oftheir normal value (p < 0.05). The nucleosides accumulated; their major fraction (91%) was inosine. Theadenosine diphosphate/adenosine triphosphate ratio increased from 0.14 ± 0.04 to 0.49 ± 0.20 (p < 0.01) andthe energy charge decreased from 0.924 ± 0.021 to 0.660± 0.169 (p < 0.01). Ultrastructure examination revealedirreversible cell damage in at least the subendocardiallayer.
These results suggest that the energetic base of reduced contractility due to severe coronary artery stenosisis different from that in acute coronary obstruction: thedecrease in contractile function cannot be explained bya reduced oxidative phosphorylation but may be relatedto an impaired energy utilizationat the myofibrillarlevel,in analogy with the postischemic reperfused stunnedmyocardium.
(J Am CoilCardiol1987;9:123S-42)
cardial function and cell viability has not been established.Recurrent episodes of regional ischemia associated with asevere degree of coronary stenosis may finally influencethese variables. Indeed. recent experimental evidence suggests that intermittent myocardial ischemia induces persistent contractile dysfunction (6) , changes in cell volume regulation (5) and depre ssion of the adenine nucleotide pool(4,5) despite the prevention of cell death by adequate reflow .
We studied high energy phosph ate metabolism and histology in the myocardium of patient s with coronary arterydisease at the time of surgery and related these findings tothe degree of coronary stenosis and to regional wall motionas measured on the preoperative angiogram. Because thestudy group included patients with stable angina pectoris,unstable angina pectori s and evolving or healed myocardialinfarction; a wide range of coronary stenosis progressing tocomplete occlusion was examined .
Of special interest are the observations on the rate ofadenine nucleotide degradation in patients with evolving
0735-1097/87 /$3 .50
1236 FLAMENG ET AL.MYOCARDIAL PURINE CATABOLISM IN CORONARY ARTERY DISEASE
JACC Vol. 9, No.6June 1987:1235-42
myocardial infarction because of possible discrepancies between experimental animals and human patients in terms ofischemia tolerance of the myocardium and time constraintsfor effective treatment after the onset of acute coronaryartery obstruction.
MethodsStudy patients. Fifty-four surgical patients with obstruc
tive coronary artery disease were included in the study. Thepatients (6 women and 48 men) were 47 to 68 years oldand all underwent aortocoronary bypass grafting. All patients had ventriculographic and coronary arteriographic examinations within 8 weeks before surgery. Five patients hadunstable angina pectoris. Another five had coronary bypasssurgery performed as an emergency procedure because ofevolving myocardial infarction; four of these patients developed their acute infarction during cardiac catheterizationor coronary angioplasty. The duration of occlusion of theleft anterior descending coronary artery before revascularization was 170 ± 25 minutes.
Catheterization and angiography. In each patient angiograms of the left ventricle were obtained in the 30° rightanterior oblique and the 45° left anterior oblique projection.Coronary angiograms were performed using the Sones technique. Ejection fraction was calculated and wall motion wasmeasured from the frontal end-diastolic and end-systolicoutlines of the left ventricle as described previously (7).The degree of stenosis in the left anterior descending coronary artery was determined by measuring the prestenotic,poststenotic and intrastenotic vessel diameter in both projections. The mean intraluminal pre- and poststenotic area(rnm') was calculated using the catheter diameter for calibration. The percent stenosis was calculated by expressingthe intraluminal area at the narrowest site of the stenosis asa percent of the mean pre- and poststenotic area.
Patient groups. The patients were classified into groupsaccording to the degree of stenosis of the left anterior descending coronary artery and electrocardiographic (ECG)signs of anterior infarction. Group I comprised 16 patientswith 70 to 89% stenosis. Group II comprised 27 patientswith 90 to 100% stenosis; of whom 5 had unstable angina.Only patients without electrocardiographic signs of previousanterior infarction were selected for groups I and II. GroupIII comprised five patients with evolving anterior infarction;acute occlusion of the left anterior descending coronaryartery was confirmed during surgery in all five. Group IVcomprised six patients with electrocardiographic signs ofprevious anterior infarction (healed infarction).
Myocardial biopsy. Two transmural needle biopsyspecimens measuring 1.5 mm in diameter (Tru-cut biopsyneedle, Travenol Laboratories) were obtained from the center of the area perfused by the left anterior descending coronary artery. These specimens were obtained when the patients were on cardiopulmonary bypass but before any of
the distal or proximal coronary anastomoses were performed. Each specimen used for microscopic studies wasdivided into a subepicardial and subendocardial sample.
Biochemicalanalysis. The transmural biopsy specimenswere immediately (within 3 seconds) cooled in liquid nitrogen and stored at - 80°C. Adenosine triphosphate andcreatine phosphate were determined by luminometry (LKB1250; ATP luminescence kit, CLS-Boehringer). Nucleotides, nucleosides and the purine bases were separated usinghigh pressure liquid chromatography in one single run (gradient on reversed phase Cl8 column) (8). Inorganic phosphate was measured using an automated malachite greenmethod. Tissue contents are calculated as micromoles pergram dry weight.
Myocardial histology. The biopsy specimens were immersed in fixative containing 3% glutaraldehyde and 90rnmol/liter oxalate adjusted to pH 7.4 with normal potassiumhydroxide. Further processing for light and electron microscopy was done as described previously (9).
In an attempt to quantify the degree of cell degenerationin the different layers of the myocardium, 100 myocardialcells that were sectioned through the nucleus were examined. This was done on semithick toluidine blue-stainedsections of the subepicardial and subendocardial parts of thebiopsy samples. The percent of normal cells was calculated(9).
Ultrastructural changes in the myocytes were analyzedin several electron micrographs that were taken at randomfor both areas (subepi- and subendocardial), No attempt wasmade to quantify the degree of subcellular myocardial damage.
Statistical methods. Biochemical, functional andhistologic data are expressed as mean values ± standarddeviation. For statistical analysis one way analysis of variance was performed between groups. Unpaired t test versusgroup I was performed when the F value was significant atthe 0.05 level.
ResultsCoronary artery stenosis and high energy phosphate
metabolism (Table 1). Data on myocardial creatine phosphate, inorganic phosphate, nucleotides, nucleosides andthe purine bases in the different groups are listed in Table1. As coronary artery stenosis progressed, a loss of highenergy phosphates was observed. With severe stenosis (groupII), the sum of adenylates decreased to 73% of the valuemeasured in patients with mild stenosis (group 1). Adenosinetriphosphate (ATP) content dropped to 74%. As long as thecoronary artery was not acutely obstructed, purine catabolism was not associated with increased tissue content ofnucleosides and purine bases (Fig. 1). However, when acuteocclusion of the coronary artery developed (group III), thesum of adenylates dropped to 54% of the value found withmild stenosis. Then adeny1ate catabolism was reflected by
JACC Vo!. 9, No.6June 1987:1235- 42
FLAMENG ET ALMYOCARDIAL PURINECATAROLISM IN CORONARY ARTERY DISEASE
1237
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30
Figure 2. Myocardial tissue content of adenine nucleotides, nuc1eosides and purine bases in group [ patients with 90 to 100%stenosis without infarction (open bars ) and in group III patientswith evolving infarction (hatched bars ). Values are means ±SEM. **p < 0.01 versus 90 to 100% stenosis; ***p < 0.001.A + I = adenosine + inosine; ADP = adenosine diphosphate;AMP = adenosine monophosphate; ATP = adenosine triphosphate; DW = dry weight; H + X = hypoxanthine + xanthine;NrNsB = sum of nucleotides, nucleosides and bases.
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1238 FLAMENG ET AL.MYOCARDIAL PURINE CATABOLISM IN CORONARY ARTERY DISEASE
JACe Vol. 9, No.6June 1987:1235-42
Figure 3. Relation between the degree of coronary stenosis andmyocardial mitochondrial function as assessed by the ADP/ATPratio (0) and theenergy charge (e). Values are means ± SEM.*** P < 0.001 versus group I. Abbreviations as in Figure 2.
only 0.1 % of the sum of nucleotides, nucleosides and basesin mild stenosis, increased during acute coronary arteryocclusion to 19%. The major fraction of the accumulatednucleosides was inosine: 2.65 ± 1.34 J.Lmollg or 91% ofthe nucleosides. The adenosine diphosphate (ADP)/ATP ratio, which increased slightly from 0.14 to 0.17 when thedegree of stenosisprogressedfrom mild to severe, increasedfurther to 0.49 in acute coronary occlusion (Fig. 3). Theenergy charge (ATP + 1/2 ADP)/(ATP + ADP + adenosine monophosphate [AMP]) (10) decreased slightly from0.921 to 0.904 whencoronarystenosisprogressed frommildto severe. After acute coronary occlusion the energy chargefell abruptly to 0.660 (Table 1 and Fig. 3).
Creatine phosphate decreased during acute occlusion tovery low levels: myocardial tissue contents decreased to17% of the value measured in mild stenosis (Fig. 1). Afterchronic infarction (group IV), purine content recovered tolevels comparable with those in severe stenosis without infarction as did creatine phosphate, ADP/ATP ratio and energy charge (Table 1).
Histologic findings related to coronary artery stenosis(Table 2). At the light microscopic level, myocardial cellchangeswere quantifiedin subepicardial and subendocardial
biopsy samples as described previously (9) and the percentof completely normal myocytes per sample was calculated.Myocardial cell abnormalities were characterizedmainly bya reduction of the volume fraction of the myofibrils, especially in the perinuclearzones, and by nuclear tortuosity.The frequency of altered cells was significantly increasedin patientsof group II (90 to 100%stenosiswithoutpreviousinfarction). However, interpatient variation is very high inthis group, whichcomprisespatientswithalmostcompletelynormal histologic findings as well as those with a highdegree of myocardial cell abnormalities. Tissue samplesfrom patients having ECG signs of previous anterior infarction (group IV) uniformly showed a low incidence ofnormal myocytes in the subepicardium and in the subendocardium: 32 and 17%, respectively.
Myocardial fibrosis wasobservedin only 11 % of samplesin group II. In group IV it was uniformly found in samplesfrom patients with ECG signs of previous infarction.
At the ultrastructural level, the myocardium in samplesfrom group I revealed a normal subcellular structure. Ingroup II, ultrastructural changes consisted of myofibrillaratrophy, presence of many small mitochondria, glycogenaccumulation and polymorphism of the nucleus (9, II) (Fig.4). In some cases ultrastructure was completely normal. Anormal subcellular structure was often associated with ahistory of unstable angina related to this area (Fig. 5).
Ultrastructural changes related to acute myocardial ischemia (1,12) were rare in all groups except group III(evolving myocardial infarction). In groups I, II and IV,only the lack of intramatricial granules in the mitochondriaand someswellingof the mitochondrial matrixwerenoticed.
In the group with evolving myocardial infarction (groupIll), ultrastructural degenerative changes were dramatic, inall subendocardial samplesirreversible myocardialcell damage was found: rupture of the sarcolemma, mitochondriacontaining flocculent densities, intracellularedema and nuclear chromatin clumping (Fig. 6). In contrast, subcellularinjury to the subepicardial cells was reversible: only mildto severe mitochondrial swelling without sarcolemmal andnuclear changes was observed (Fig. 6).
Regional left ventricular wall motion (Table 2). Whencoronarystenosisprogressedto 90%, segmentalwallmotionremained in the normal range. With stenosisof 90 to 100%,
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Table 2. Myocardial Regional Wall Motion and Histology in 49 Patients
%Normal Myocytes*
AWM* (%) Epi Endo Total Fibrosis
Group I (n = (6) 48.9 ± 8.9 83 ± 14 79 ± 18 82 ± 14 o of 16 (0%)Group II (n = 27) 34.9 ± 9.9* 59 ± 33:1: 51 ± 30:1: 55 ± 30:1: 3 of27 (11%)Group IV (n = 6) 24.8 ± 10.9* 32 ± 28* 17 ± 9* 24 ± 18* 6 of6 (100%)F value 5.5411 6.61 11 12.511 10.711
Statistics as in Table I. *Values are means ± SD. AWM = anterior wall motion; Epi = subepicardiallayer; Endo = subendocardial layer.
JACC Vol. 9, No.6June 1987: 1235-42
FLAMENG ET AI..MYOCARDIAL PURINE CATABOLISM IN CORONARY ARTERY DISEASE
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Figure 4. Electron micrograph from a subendocardial biopsy sample of a hypokinetic anterior wall in a patient from group II. Thissection shows a severely altered myocyte. There is considerablereduction of the myofibrillar volume fraction in the center of themyocardial cell. The sarcolemma appears intact whereas the nucleus (n) is tortuous; most mitochondria are small in size and largeareas of glycogen (gl) fill up the cytosol (original magnificationx 2,250, reduced by 25%).
Figure S. Electron micrograph from a subendocardial sample ofthe anterior wall in a patient from group II with unstable angina.In this sample, myocardial structure is normal except for someclearing of the mitochondrial matrix (arrows) (original magnification x 13,200, reduced by 25%).
however, wall motion declined to 72% of its initial value.With complete acute coronary artery occlusion, paradoxicalmovement of the segment occurred. This paradoxical movement was noted on the preoperative left ventriculogram inone case and was observed during surgery in the other four
Figure 6, a, Electron micrograph of a subendocardial sample ofthe anterior wall in a patient with an evolving infarction due toocclusion of the left anterior descending coronary artery. The myocardial cell is irreversibly damaged: the sarcolemma is disrupted(arrows) and the mitochondria show the typical flocculent densities(arrowheads) that are hallmarks of irreversibility. The nucleus(0) shows clumping and margination of chromatin. b, Electronmicrograph of a subepicardial sample of the anterior wall in thesame patient. The sample was taken 160 minutes after the onsetof coronary occlusion. Note that the ultrastructure of the myocyteis normal except for a very slight clearing of the mitochondrialmatrix. (Original magnification in a and b x 13,200, reduced by25%.)
cases. In these four cases a left ventriculogram was notperformed in the acute stage of evolving infarction. In groupIV (patients with ECG evidence of previous infarction),segmental wall shortening attained only 50% of normal values.
1240 FLAMENG ET AL.MYOCARDIAL PURINE CATABOLISM IN CORONARY ARTERY DISEASE
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DiscussionMyocardial purine metabolism in man. This study
deals with the relation between coronary artery stenosis andmyocardial purine metabolism , histology and regional function. In humans, this relation has not been previously established . Data on human myocardial tissue contents ofnucleotides are scarce, and usually restricted to the measurement of adenosine triphosphate (ATP) (13). In experimental animals, on the other hand, purine metabolism andits catabolism during ischemia are relatively well known(1-5). Our clinical results show that high energy phosphatemetabolism in humans is comparable with that found inexperimental animals. Under normoxic conditions, the composition of the adenine nucleotide pool is similar in thehuman and the canine heart. We found that the adenosinediphosphate (ADP) and monophosphate (AMP) content inthe human myocardium, expressed as a percent of the totaladenylate pool, is 12 and 2%, respectively, when coronaryartery stenosis does not exceed 90%. Comparable valuesare reported in experimental animals with normal coronaryvessels (3- 5, 14-17). The adenylate charge (10) in thehuman heart as reported in our study (0 .921 ± 0.028, mean± SD) is clearly in the same range as the values found inanimal hearts (0.919 to 0.928) (3,4,14-16). Also the ratiobetween creatine phosphate and ATP (1.50 in this report)is comparable with values reported in animal studies (1.29to 1.71) (3,4,14-16). Only values obtained with nuclearmagnetic resonance techniques seem to produce a slightlyhigher creatine phosphate/ATP ratio (18).
Relation between coronary stenosis, purine metabolism, histology and regional function. Our results indicatethat severe coronary stenosis is associated with a 20 to 30%reduction in myocardial nucleotide content. It may be assumed that in the presence of such severe stenosis, intermittent periods of myocardial ischemia lead to purine breakdown (15,17). Purine catabolism induces an accumulationof nucleosides and bases in the myocardium, but these substances are readily washed out as long as there is no completeocclusion of the vessel. Next , further catabolism to hypoxanthine represents an almost irreversible loss of the "backbones" ofthe adenylate pool. Such a washout of nucleosidesand purine bases was shown previously by de long andGoldstein (19) and Kugler (20) when they obtained highlevels of inosine and hypoxanthine in the coronary sinusblood during pacing and exercise-induced ischemic episodes . De novo synthesis of adenylates is an extremely slowprocess (4), and it is probably not optimal in the presenceof a severe coronary stenosis .
Severe left anterior descending artery stenosis was alsoassociated with a 30% reduction in regional wall motion .This confirms the results of a previous study (7). Our observation that the reduction in the adenylate pool parallelsthe reduction in local contractility does not necessarily imply
that the depression of local function is caused by this reduction in the adenylate pool. Indeed, the ADPIATP ratio,the energy charge and the creatine phosphate levels are inthe normal range and this is very suggestive for an intactmitochondrial function, that is, a normal energy production.We realize however that the ADP/ATP ratio, obtained byour methodology, is not an optimal estimate of mitochondrial function . The problem is that "cytosolic" ADP concentrations cannot be measured with any existing technology. Even with nuclear magnetic resonance techniques theADP concentration can only be estimated by subtracting theATP beta-phosphate peak from that of the gamma-phosphatepeak and the error associated with this measurement is relatively high (21). Nevertheless, our values for creatine phosphate and energy charge are accurate and they are acceptableindexes of mitochondrial function .
A near normal energy production may well be associatedwith postischemic myocardial dysfunction, reduced adenylate pools and normal or elevated creatine phosphate levels(21,22). Experimental evidence exists that this entity, calledmyocardial stunning, is due to a deficiency of the creatinephosphate shuttle , at the level of the myofibrillar isoenzymeof creatine kinase (23). The same phenomena, reduced wallmotion, reduced adenylate pool, normal mitochondrial function and normal creatine phosphate levels, were found inour group II patients; therefore , myocardial dysfunction mayrepresent a " stunned myocardium " induced by chronic intermittent ischemia (6). However , recurrent episodes ofischemia not only may induce stunning and purine loss butalso may activate proteolytic systems responsible for myofibrillar lysis. A certain degree of myofibrillar lysis wasfound in at least 50% of the myocytes in this subset ofpatients. We reported in previous studies (7,9,11) that thistype of cell degeneration correlates best with reduction inleft ventricular function. It was shown using immunohistochemical techniques (24) that fibers with myocytolysis areviable because they retain enzymes and other proteins (creatine kinase MB fraction, myoglobin, lactic dehydrogenase[LDH]-l , aspartate aminotransferase [AST]. Also using immunofluorescence staining Hayakawa et al. (25) demonstrated a similar labeling intensity for Ca2 + and Mg2 + adenosine triphosphatase and tropomyosin in myocytolytic cellsas in normal muscle cells. Thus, myocytolysis may be areversible form of myocardial alteration that , although itdoes not necessarily lead to cell death and eventual myocardial fibrosis (24), may well induce myocardial dysfunction.
Factors producing variations of findings in severe stenosis group. In the severe stenosis group there was a widescatter in the biochemical, histologic and functional data .Many factors may contribute to this variation : 1) A smallbiopsy specimen is assumed to be representative of a relatively large area. Hearse et al. (3) showed that there isalready a large variation in biochemical values when a large
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FLAMENG ET AL.MYOCARDIAL PURINE CATABOLISM IN CORONARY ARTERY DISEASE
1241
number of simultaneously obtained samples are removedfrom a homogeneously perfused subepicardial zone duringthe same cardiac cycle. 2) Analytic errors may be relatedto the small size of the biopsy specimens. 3) Assessmentof the degree of stenosis is not completely accurate becausethe length of the stenotic segment is not taken into account.4) The determination of regional wall motion as an indexof regional contraction may be inaccurate. Axis shorteningas such may not completely reflect segment movement, andprevious medication (beta-adrenergic blocking agents) mayinfluence contraction. There is also a time interval betweenangiography and surgery so that correct extrapolation of wallmotion data to the moment of tissue sampling remains questionable. 5) The absence of electrocardiographic evidenceof anterior infarction does not exclude the presence of anterior wall infarction, a factor that may account for someof the differences between the group with the highest gradestenosis and those with lesser degrees of stenosis. On theother hand, some of the variation may be real because coronary stenosis is a dynamic process: progression of the disease may be fast or slow, coronary spasm may influencethe degree of stenosis from one minute to another and theoxygen supply and demand balance of the heart is not stable.This group still comprises some patients with normal purinelevels, normal histologic features and normal wall motion.Therefore, actual biochemical and functional measurementsmay represent no more than a "snap shot" of an evolutionary process.
Myocardial purine catabolism during evolving infarction. Purine catabolism during evolving myocardial infarction in humans has not been described before. Our results indicate that, in patients with a history of coronaryartery disease, the rate of purine catabolism during morethan 2 hours of acute coronary occlusion is not the same asthat found experimentally in the dog heart. In patients, adenosine triphosphate decreased to 43% of its initial value (thatis, 95% stenosis). In contrast, in dogs, adenosine triphosphate dropped to 6% of its normal value after only 40 minutes of acute coronary artery occlusion (1). This differencein tolerance to ischemia is most probably due to the presenceof a better developed collateral circulation in patients withcoronary artery disease as compared with healthy experimental animals. However, the pattern of purine catabolismin patients with acute coronary obstruction is similar to thatin dogs with acute coronary occlusion. Adenosine monophosphate increases and the nucleosides and bases accumulate extensively (Fig. 2). The main component of thenucleosides during occlusion is inosine: this component represents 90% of the nucleoside pool, the remaining 10%being adenosine. The histologic findings in acute infarctionin humans are also comparable with those in dogs (1). Mainly,the subendocardial part of the myocardium is irreversiblydamaged. This implies that the reduction of ATP in theviable portion of the myocardium at risk will be less than
the evaluation of a transmural and partly necrotic samplemay suggest.
In the subclass of patients with healed anterior infarction, the same level of nucleotides was found as in patientswith severe stenosis. However, the incidence of cell degeneration was higher and myocardial fibrosis was uniformly observed. This fibrous replacement of myocytes mayexplain the extremely low values of regional contractilefunction in this subclass despite relatively high levels ofhigh energy phosphates.
Conclusions. Progressive coronary artery stenosis appears to result in a partial destruction of the adenylate pool,probably as a result of recurrent short periods of purinecatabolism. Mitochondrial function, as estimated by theADP/ATPratio, energy charge and creatine phosphate/ATPratio is not impaired so that the associated reduction inregional contractile function cannot be explained by a decreased production of high energy phosphates. The energeticbase of this type of hypokinesia is probably an impairedenergy utilization with reduced ATP splitting at the myofibrillar level as in the postischemic reperfused "stunned"myocardium (22,23). A reduced volume fraction of myofibrils, however, can be demonstrated histologically andmay be the basis for this partial loss of function. Suddencomplete obstruction of a coronary vessel, however, resultsin a massive degradation of high energy phosphates and anaccumulation of mainly inosine in the ischemic myocardium. Mitochondrial function is severely impaired, as reflected by a sudden complete loss of contractile function.The rate of adenine nucleotide degradation is much slowerthan would be expected from animal studies. After 170minutes of occlusion, ultrastructural evidence of cell necrosis occurs in at least the subendocardiai part of the myocardium at risk.
We express our gratitude to Agnes Goethuys for preparing the manuscript.
References1. Jennings RB, Hawkins HK, Lowe JE, Hill ML, Klotrnan S, Riemer
KA. Relation between high energy phosphate and lethal injury inmyocardial ischemia in the dog. Am J Pathol 1978;92:187-214.
2. Jennings RB, Reimer KA, Hill ML, Mayer SE. Total ischemia in doghearts, in vitro. I. Comparison of high energy phosphate production,utilization and depletion, and of adenine nucleotide catabolism in totalischemia in vitro vs severe ischemia in vivo. Circ Res 1981;49:892-900.
3. Hearse OJ, Yellon OM, Chappell OA, Wyse RKH, Ball GR. A highvelocity impact device for obtaining multiple, contiguous myocardialbiopsies. J Mol Cell Cardiol 1981;13:197-206.
4. Swain JL, Sabina RL, McHale PA, Greenfield JC Jr, Holmes EW.Prolonged myocardial nucleotide depletion after brief ischemia in theopen chest dog. Am J Physiol 1982;242:H818-26.
5. Jennings RB, Schaper J, Hill ML, Steenbergen C Jr., and ReimerKA. Effect of reperfusion late in the phase of reversible ischemicinjury. Changes in cell volume, electrolytes, metabolites and Ultrastructure. Circ Res 1985;56:262-78.
1242 FLAMENG ET AL.MYOCARDIAL PURINE CATABOLISM IN CORONARY ARTERY DISEASE
JACC Vol. 9, No.6June 1987: 1235-42
6. Heyndrickx GR, Millard RW, McRitchie RJ, Marolco PR, VatnerSF. Regional myocardial functional and electrophysiological alterations after brief coronary artery occlusion in conscious dogs. J ClinInvest 1975;56:978-85.
7. Schwarz F, F1ameng W, Thiedemann KU, Schaper W, Schlepper M.Effect of coronary stenosis on myocardial function, ultrastructure andaortocoronary bypass grafting hemodynamics. Am J Cardiol 1978;42:193-201.
8. Wynants J, Van Belle H. Single-run high-performance liquid chromatography of nucleotides, nucleosides and major purine bases andits application to different tissue extracts. An<\1 Biochem 1985;144:258-66.
9. Flarneng W, Wouters L, Sergeant P, Lewi P, Borgers M, Thone F,Suy R. Multivariate analysis of angiographic, histologic, and electrocardiographic data in patients with coronary heart disease. Circulation1984;70:7-17.
10. Atkinson DE. The energy charge of the adenyl ate pool as a regulatoryparameter: interaction with feedback modifiers . Biochemistry 1968;7:4030-4.
II. Flameng W, Suy R, Schwarz F, eta!' Ultrastructural correlates ofleft ventricular contraction abnormalities in patients with chronic ischemic heart disease: determinants of reversible segmental asynergypostrevascularization surgery. Am Heart J 1981;102:846-57.
12. F1ameng W, Borgers M, Daenen W, Stalpaert G.Ultrastructural andcytochemical correlates of myocardial protection by cardiac hypothermia in man. JThorac Cardiovasc Surg 1980;79:413-24.
13. Flameng W, Van der Vusse GJ, Borgers M. Methods for assessingpreservation techniques: invasive methods. In: Engelman R, LevitskyS, eds. A Textbook of Clinical Cardioplegia. New York: Futura, 1982:63-80.
14. Fellenius E, Samuelsson R. Effects of severe systemic hypoxia onmyocardial metabolism. Acta Physiol Scand 1973;88:256-66.
15. Isselhard W, Eitenrnuller J, Maurer W, eta!' Increase in myocardialadenine nucleotides induced by adenosine: dosage, mode of appli-
cation and duration, species differences. J Mol Cell Cardiol 1980;12:619-34.
16. Allison TB, Holsinger JW Jr. Myocardial metabolism and regionalmyocardial blood flow in the canine left ventricle following twentyminutes of circumflex artery occlusion and reperfusion. J Mol CellCardiol 1983;15:151-61.
17. Vial C, Font B, Goldschmidt D, Pearlman AS, Delaye J. Regionalmyocardial energetics during brief periods of coronary occlusion andreperfusion: comparison with S-T segment changes. Cardiovasc Res1978;12:470-6.
18. Rehr RB, Jackson JA, Peshock R, Nunnally RL, Willerson H. Phosphorous NMR evaluation of in vivo regional myocardial ischemia(abstr). Circulation 1983;68(suppl III):III-388.
19. de Jong JW, Goldstein S. Changes in coronary venous inosine concentration and myocardial wall thickening during regional ischemiain the pig. Circ Res 1974;35:111-6.
20. Kugler G. Myocardial release of lactate, inosine and hypoxanthineduring atrial pacing and exercise-induced angina. Circulation 1979;59:43-9.
21. Bailey lA, Seymour AL, Radda GK. A 31p_NMR study of the effectsof reflow on the ischaemic rat heart. BiochimBiophys Acta 1981;637:1-7.
22. Schaper W, Buchwald A, Hoffmeister HM, Ito BR. "Stunned" myocardium is a problem of energy utilization and not of energy supply(abstr). Circulation 1985;72(suppllII):III-1I9.
23. Greenfield RA, Swain JL. Dissociation of the creatine phosphate shuttle as a potential mechanism for post-ischemic dysfunction (abstr).Circulation 1984;70(suppl 11):11-82.
24. Edwalds GM, Said JW, Block MI, Herscher LL, Siegel RJ, FishbeinMC. Myocytolysis (vacuolar degeneration) of myocardium: immunohistochemical evidence of viability. Hum Pathol 1984;15:753-6.
25. Hayakawa BN, Jorgensen AD, Gotlieb AI, Zhao MS, Liew cc.Immunofluorescent microscopy for the identification of human necrotic myocardium. Arch Pathol Lab Med 1984;108:284-6.