American Journal ofPathology, Vol. 146, No. 6, June 1995Copyright X) American Society for Investigative Pathology

Short CommunicationDNA Fragmentation of Human InfarctedMyocardial Cells Demonstrated by the Nick EndLabeling Method and DNA AgaroseGel Electrophoresis

Gen Itoh,* Jun Tamura,* Minako Suzuki,*Yasuhiko Suzuki,* Hiroshi Ikeda,*Masahiko Koike,* Masanao Nomura,*Tong Jie,* and Katsuki ItotFrom the First Department ofPathology,* Aichi MedicalUniversity, Aichi-ken, and Second Department of Surgery,tNagoya University, School ofMedicine, 65, Nagoya, Japan

Myocardial tissue takenfrom 19 autopsy cases ofmyocardial infarction were examined botb by thenick end labeling method(NELM) andbyDNA aga-rose gel electrophoresis in order to demonstratethe localization ofceUls withfragmentedDNA andto confirm the internucleosomal cleavage ofDNAbiochemicaly. the nuclei corresponding to thosewith the histologicalfeatures ofacute myocardialinfarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positivewith the nick end labeling method. MyocardialceUls corresponding to those with nick end label-ing method-stained nuclei, on the other hand, hadmostly pyknotic and karyolytic nuclei and someunremarkable nuclei, even nuclear ghosts, andshowed degenerated cytoplasm, including con-traction band necrosis in H&E-stained prepara-tions. The agarose gel electrophoresis ofDNA ex-tractedfrom the corresponding areas mentionedabove showed the ladderpattern of internucleo-somal cleavage characteristic of apoptosis. Thepresent study revealed that infarcted myocardialceUs with nuclear outlines, even nuclear ghosts,showed a distinct DNA fragmentation with theladderpattern ofinternucleosomal cleavage. It isconcludedfrom this study that the damaged myo-cardial ceUls of acute myocardial infarction rep-

resent a coagulation necrosis having the bio-chemical nature of apoptosis. (Am J Pathol1995, 146:1325-1331)

It is widely acknowledged that there are two majordistinct modes of death in eukaryotic cells: necrosisand apoptosis. 1-5 These two processes differ in struc-ture, mechanism, and sequelae. Necrotic cells showdamaged organelles, ruptured plasma membranes,and dispersal of cytoplasmic elements into the ex-tracellular space.2 The various mechanisms neitherdepend upon continuing synthetic activity nor involveintracellular signaling pathways,4 and the process re-sults in an acute inflammatory reaction.4

Apoptosis, on the other hand, is the term used forprogrammed cell death and has characteristic struc-tural changes consisting of losing surface contactwith neighboring cells, cell shrinkage with remainingintact organelles, condensation of chromatin intocrescentic caps at the nuclear periphery, andeventual fragmentation of nucleus and cytoplasm intoapoptotic bodies.2-5 Apoptotic cells and bodies mayundergo extracellular degeneration or be phagocy-tosed by neighbors or macrophages and do not elicitan acute inflammation.4 The initiation of the apoptoticprocess, however, is associated almost exclusivelywith both internucleosomal chromatin cleavage andthe intracellular signaling mechanisms.2-5

Supported in part by the Murayama Foundation and by a grant-in-aid for a specific research project from Aichi Medical University.Accepted for publication February 21, 1995.

Address reprint requests to Dr. Gen Itoh, First Department of Pa-thology, Aichi Medical University, Nagakute-cho, Aichi-gun, Aichi-ken, 480-1 1, Japan.

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In the myocardium, ischemia causes myocardialinfarction, known as coagulation necrosis of myocar-dial cells.67 The sequence of the process of necrosishas been widely accepted at the light microscopiclevel from the beginning of coagulation necrosis (4 to12 hours) to the initiation of fibrovascular reaction inthe margins (10 days).69 In the acute stage, pyknosisand karyolysis of the nuclei, shrunken eosinophiliccytoplasm, and marginal contraction band necrosisare the characteristic microscopic changes of in-farcted myocardial cells.6,7 These changes are fol-lowed by loss of nuclei and cytoplasmic striations,heavy interstitial infiltration of neutrophils, and fibro-vascular reaction in margins.69

Recent accumulating evidence has shown that ap-optosis is caused by mild cellular injuries such as

those caused by hyperthermia,1012 hypoxia,1-3'13'14hepatic toxins,13-15 injurious agents including anti-cancer drugs,12'16 and ischemia.17 In liver cells invivo coagulation necrosis was induced by ischemiaand hepatotoxin of CC14,13 and DNA extracted fromtwo groups of hepatic cells (ischemia group andhepatotoxin group mentioned above) showing thetypical coagulation necrosis demonstrated the ladderpattern characteristic of apoptosis.13 No investigationto date, however, has ever demonstrated the local-ization of the apoptotic cells in tissue sections untilGavrieli et a1l8 developed a method, nick end labelingmethod (NELM) for in situ visualization of apoptoticcells at a single-cell level. Gold et a119 have recentlyreported that the NELM described by Gavrieli et al18can be preferentially used for the detection of apop-tosis in cultured cells of rat thymus and rat brain tis-sue.

In the present study, we examined myocardial cellspreserving cell outlines in the infarction foci of theheart, both by NELM18 and DNA agarose gel elec-trophoresis,20 to localize the myocardial cells withDNA fragmentation and to determine whether or notsuch cells with the histological appearance of acutemyocardial infarction show DNA fragmentation at a

single-cell level.

Materials and MethodsMyocardial Tissue SamplesA total of 19 autopsy cases of myocardial infarctionwere examined. There were 14 men and 5 women,

ranging in age from 58 to 93 years. The duration of theinfarcts ranged from 12 hours to several days, clini-cally and histologically. There were 3 cases with no

particular myocardial changes that were selectedand examined as controls.

All tissues for morphological examination were

fixed in 10% neutral buffered formalin, dehydrated ingraded alcohols, cleared in xylene, and embedded inparaffin. Sequential sections were cut 6 p thick forlight microscopic examination.

DNA Nick End Labeling MethodDNA nick end labeling was performed by the methodof Gavrieli et al.18 In brief, paraffin sections were af-fixed to slides. After being transferred three times toa xylene bath for 5 minutes and twice to an acetonebath for 5 minutes, the sections were washed bydouble-distilled water (DDW), incubated with 20pg/mI proteinase K for 15 minutes at room tempera-ture (RT), and then washed four times in DDW for 2minutes. After the endogenous peroxidase was inac-tivated by covering sections with 2% H202 for 5 min-utes at RT, the sections were rinsed with DDW, andimmersed in TDT buffer (30 mmol/L Tris-HCI buffer, pH7.2,140 mmol/L sodium cacodylate, 1 mmol/L cobaltchloride). Deoxynucleotidyl transferase (0.3 U/ml)and biotinylated dUTP in TDT buffer were then addedto cover the sections and incubated in a humid at-mosphere at 37 C for 60 minutes. The reaction was

terminated by transferring the slides to buffer con-

taining 300 mmol/L sodium chloride and 30 mmol/Lsodium citrate for 15 minutes at RT. The sections wererinsed with DDW, covered with 2% aqueous solutionof bovine serum albumin for 10 minutes at RT, rinsedin DDW, and immersed in phosphate-buffered salinefor 5 minutes. The sections were covered with strepta-vidin peroxidase, incubated for 10 minutes at 37 C,washed in DDW, immersed for 5 minutes inphosphate-buffered saline, and stained with 3,3'-diaminobenzidine tetrahydrochloride for approxi-mately 30 minutes at RT.

Detection of DNA Fragmentation ofCardiac Muscles from Autopsy Cases ofMyocardial InfarctionDNA extraction and electrophoresis were performedin principle by the method reported previously by

Figure 1. a: An 86-year-oldfemale patient with acute myocardial infarction. Infarcted myocardial cells preserve outlines with or without nuclei,and cytoplasmic striations become less evident or disappear. The myocardial cells around the small artery spare ischemic change, whereas those attwo thirds of upper portion show ischemic change, including contraction band necrosis. H&E stain; magnification, x85. b: Higher magnificationof the left upper portion of (a). Myocardial cells showing contraction band necrosis are found. H&E stain; x 170. c: The myocardial cells at thecorresponding area of(a). The nuclei ofdamaged cells are stained dark brown with NELM. NELM stain; X85. d: The myocardial cells at the cor-responding area of(b). The nuclei ofdamaged cells shouing contraction band necrosis are stained positive with NELM. NELM stain; X 170.

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Facchinetti et al.20 In brief, the specimen from theinfarcted portion of myocardium was homogenizedand suspended in 20 ml of buffer (10 mmol/L Tris-HCI,pH 7.5, 0.1 mol/L NaCI, 1 mmol/L EDTA), 1 ml of 10%sodium dodecyl sulfate was added, and they were

incubated with 200 p1 of 10 mg/ml proteinase K at 50C overnight. DNA was extracted twice with phenoland twice with phenol/chloroform (1/1); the upper

layer was collected, to which 5 mol/L NaCI was addedat a 1:50 (v/v) ratio and then precipitated overnight at-20 C in absolute ethanol. DNA was pelleted by cen-

trifugation at 3000 rpm at 4 C for 10 minutes, washedwith 70% ethanol, and dried. DNA thus obtained wasresuspended in TE (10 mmol/L Tris-HCI, 1 mM EDTA).Samples were prepared for a volume of 20 pi thatcontained 20 pg of DNA and subsequently incubatedwith 0.1 mg/ml DNAse-free RNAse21 at 37 C for 30minutes. A 4-p1 volume of 0.25% bromphenol blueand 0.25% xylene cyanol in 40% sucrose was addedto samples at 1:5 (v/v) ratio. Electrophoresis was per-

formed at 50 V in 1.6% agarose gels. DNA was vi-sualized with ethidium bromide.

Positive Control of Fragmented DNA

DNA extracted from thymus cells of 4-week-old mice,which were sacrificed 6 hours after sublethal irradia-tion with a 60Co source at 600 cGy, was used as posi-tive control.

Results

Light Microscopic Findings

The areas of acute myocardial infarction selected inhematoxylin and eosin (H&E) preparations were ex-

amined by light microscopy, in which the damagedmyocardial cells preserved cell outlines with or with-out nuclei and the cytoplasm appeared more eosi-nophilic, whereas cytoplasmic striations became lessevident or disappeared (Figure la). The contractionband necrosis was also present in margins of acuteinfarction foci (Figure 1 b). Myocardial cells with typi-cal coagulation necrosis examined were located incentral areas of infarction foci, had preserved cellularoutlines, and had more eosinophilic cytoplasm, but

their nuclei disappeared in H&E-stained sections(Figure 2a). Infiltration of neutrophils was observed inplaces in the margins of infarction foci (Figure 2a).The characteristic morphological features in apopto-sis, such as shrinkage of cells, condensation of chro-matin under the nuclear membrane, nuclear frag-ments, apoptotic body formation, and phagocytosisby neighbors, were never observed.The myocardial cells at the corresponding areas

above were examined in the sections stained withNELM by light microscopy, demonstrating that the nu-

clei of infarcted myocardial cells, including thoseshowing contraction band necrosis, stained darkbrown (Figure 1c, d). Even nuclear ghosts (unstainednuclei) of myocardial cells with typical coagulationnecrosis, which were not detected by H&E-stainedsections, but the location of which was estimated bylipofuscin granules in the myocardial cells, were

stained strongly positive (Figure 2b). Neutrophils in-filtrating the interstitial spaces of infarcted myocardialfoci were not stained with NELM (Figure 2b). Normalmyocardial cells adjacent to infarcted myocardialcells (Figure 2c) were never stained positive withNELM (Figure 2d).

Detection of DNA Fragmentation

DNA fragmentation of myocardial cells obtained froma case of acute myocardial infarction (72-year-old fe-male, with histological changes demonstrated in Fig-ure 2a, b) was detected by agarose gel electrophore-sis. DNA fragmentation appeared as multiples of 180-to 200-bp subunits in the acute myocardial infarctioncase. In contrast, no bands were detected in DNAsamples from control myocardial cells (Figure 3).

DiscussionIt is widely acknowledged that cell death may occur

by either necrosis or apoptosis and that these twoprocesses are distinct modes of cell death with fun-damentally different structures, mechanisms, andsignificance. -5 However, the accumulating evidencerevealed that the apoptosis was induced by agentsthat were also capable of producing necrosis, the

Figure 2. a: A 72-year-old female patient with acute myocardial infarction. Myocardial cells with typical coagulation necrosis preserve cellularoutlines and have more eosinophilic cytoplasm, but their nuclei show karyolytic appearance or disappear (nuclear ghosts). The location of nuclei,however, is estimated by the location of lipofuscin granules (arrowheads). Neutrophils infiltrate the interstitial space between necrotic myocardialcells. H&E stain; magnification, X330. b: The myocardial cells at the corresponding area of (a). Karyolytic cells and even nuclear ghosts (un-stained nuclei) are stained strongly positive with NELM. NELM stain; X330. C: A 72-year-old male patient with acute myocardial infarction. Nor-mal myocardial cells (left side) are adjacent to infarcted myocardial cells (right side). H&E stain; x330. d: The myocardial cells at the correspond-ing area (c). The nuclei of infarcted myocardial cells (right side) are stainedpositive with NELM, showing sharp contrast to the unstained nuclei ofnormal myocardial cells (left side). NELM stain; X330.

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1 2 P M bps

23130941665574361

23222027

13531078872

603

1: case with no myocardial changes2: case of myocardial infarctionP: positive controlM: marker DNAFigure 3. Agarose gel electrophoresis ofDNA extractedfrom an acutemyocardial infarction case (histological changes of this case are

demonstrated in Figure 2a, b) and a control case. The acute infarc-tion case shows the ladderpattern, whereas the control case does not.

type of cell death resulting from mild cellular injuries,such as hypoxia, 1-3,13,14 hypertherm ia,10,11 nutri-tional deficiency, 13 toxic agents, 1-3,12,16 and ische-mia.17

Fukuda et al13 reported that hepatoma cell linestreated with noxious agents demonstrated the ladderpattern of DNA fragmentation with the morphologicalchanges characteristic of apoptosis and that the ratliver tissue undergoing ischemic necrosis showed a

distinct DNA ladder pattern without the morphologytypifying apoptosis. In the rat brain, Tominaga et al17demonstrated that focal ischemic injury caused bothnonspecific and specific DNA cleavage at the inter-nucleosomal linker regions and that brain nuclearproteins have Ca2+-dependent endonuclease activ-ity. However, studies with either gel electrophoresis ofDNA and/or routine morphology, including electronmicroscopy, have failed to localize the apoptotic cellsin tissue sections.The NELM developed by Gavrieli et a118 enables in

situ visualization of the apoptotic cells (nuclei with in-ternucleosomal cleavage of double strands of DNA)at a single-cell level while preserving tissue architec-ture. According to them, conventional paraffin sec-

tions were nick end labeled with biotinylated polydUTP (deoxyuridine triphosphate) introduced by ter-minal deoxytransferase and then stained with avidin-conjugated peroxidase. Using this method, Gold etaI19 recently reported that cells undergoing apoptosiswere preferentially labeled in routine paraffin sectionsof rat brain tissue.

Lennon et al16 reported that a range of noxiousagents, including ultraviolet radiation, caused necro-sis at higher levels but that these induced apoptosisat lower levels. They suggested the possibility thatcells suffering mild injury have the capacity to activatean internally programmed suicide death mechanism,whereas cells receiving greater injuries apparently donot.

In our study, the infarcted myocardial cells thatwere located sporadically among normal myocardialcells or along boundaries between ischemic and non-ischemic areas were stained positive with NELM, sug-gesting that these myocardial cells might suffer notsevere but mild hypoxia22'23 and supposedly showthe specific DNA fragmentation with the ladder pat-tern on agarose gel electrophoresis.How then could the nuclei, even the ghosts of nu-

clei, of necrotic myocardial cells in the central portionof severe infarction foci be stained with NELM? It maywell be that myocardial cell death starts from its onsetas apoptosis, the infarction area expands with time,and the appearance of the nuclei changes from apyknotic to a karyolytic stage, even to that of nuclearghosts (unstained nuclei).The usual pathology textbooks state that the py-

knosis, karyolysis, karyorrhexis, shrunken eosino-philic cytoplasm, and marginal contraction band ne-crosis are typical features of cells at an acute stageof ischemic myocardial cell death69 and that py-knotic nuclei, followed by karyolysis or karyorrhexis,eventually disappear as a result of the hydrolytic ac-tion of DNAses of lysosomal origin.24

In this study, damaged myocardial cell nuclei fromthe stage of pyknosis to that of karyolysis, even to thatof nuclear loss (nuclear ghosts), stained positive withNELM. These findings suggest that the shape of nu-clei with DNA fragmentation remains unchanged fora sufficient time period in situ.

The evidence obtained from our study thus sug-gests that myocardial cell damage in the acute stageof myocardial infarction is due to a combination ofapoptosis and necrosis.

AcknowledgmentsWe thank Mr. M. Takeuchi, Mr. N. Igari, and Mrs. YMatsubara for their skillful technical assistance.

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