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Circulation. 1996;94:1665-1673

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(Circulation. 1996;94:1665-1673.)
© 1996 American Heart Association, Inc.

Articles

Apoptosis of Cardiac Myocytes During Cardiac Allograft Rejection

Relation to Induction of Nitric Oxide Synthase

Matthias Szabolcs, MD; Robert E. Michler, MD; Xiaochun Yang, MD; Walif Aji, MD; Dilip Roy, MD; Eleni Athan, PhD; Robert R. Sciacca, EngScD; Oktavjian P. Minanov, MD; Paul J. Cannon, MD

the Departments of Medicine, Division of Cardiology (X.Y., W.A., E.A., R.R.S., P.J.C.), Cardiothoracic Surgery (R.E.M., D.R., O.P.M.), and Pathology (M.S.), Columbia University College of Physicians and Surgeons, New York, NY.

Correspondence to Paul J. Cannon, MD, Department of Medicine, Division of Cardiology, Columbia University, 630 W 168th St, New York, NY 10032.


*    Abstract
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 *Abstract
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Background Apoptosis is a distinct form of programmed cell death characterized by activation of endonucleases that cleave nuclear DNA, condensation and fragmentation of nuclear chromatin, blebbing of intact membranes, and cell shrinkage and fragmentation. The mechanisms responsible are unclear, but nitric oxide (NO) generated by inducible NO synthase (iNOS) has been demonstrated to induce apoptosis in macrophages in vitro. This study investigated whether apoptosis occurs during cardiac allograft rejection and examined the relationship of apoptosis to iNOS expression.

Methods and Results Heterotopic abdominal transplantation from Lewis to Wistar-Furth rats was used as a model of cardiac allograft rejection; Lewis-to-Lewis grafts served as controls. Apoptosis was identified by DNA ladders after electrophoresis on agarose gels and by in situ labeling of DNA fragments; cell types were determined by immunohistochemistry. The number of apoptotic cardiac myocytes increased sharply from day 3 (0.31/mm2 ventricular tissue) to day 5 (1.27/mm2) after transplantation. At day 5, allografts showed a significant increase (P<.01) in apoptotic cardiac myocytes, macrophages, and endothelial cells compared with syngeneic grafts. The expression of iNOS mRNA, protein, and enzyme activity paralleled in time and extent the apoptosis of cardiac myocytes. iNOS immunostaining of infiltrating macrophages and cardiac muscle fibers increased significantly in the allografts at days 3 to 5 and was accompanied by immunostaining of both cell types for nitrotyrosine, which is indicative of peroxynitrite formation.

Conclusions Apoptosis of myocardial cells occurs during cardiac allograft rejection. Apoptosis during rejection parallels the expression of iNOS, which suggests that apoptosis may be triggered by NO and peroxynitrite.


Key Words: apoptosis • cells • transplantation • rejection


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
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There is a large body of data concerning the immunologic reactions that are involved in cardiac allograft rejection and the mechanisms of immune-mediated cardiac diseases.1 2 Nevertheless, the cellular and biochemical mechanisms responsible for contractile failure and for the death of cardiac myocytes during cardiac allograft rejection are incompletely understood.2 T lymphocyte–mediated cytotoxicity and delayed-type hypersensitivity are the major immune mechanisms of cellular allograft rejection.1 2 3 Cytotoxic CD8-positive T cells bind specifically to foreign class I major histocompatibility complex–bearing target cells and release agents, eg, granzyme B, which disrupt cell membranes and may also induce apoptosis as demonstrated in vitro.1 4 5 6 The effector cells of delayed-type hypersensitivity are macrophages activated by CD4-positive T lymphocytes.1 6 Although the mechanisms by which macrophages induce cell death and damage are largely unclear, recent work4 5 7 8 suggests that in certain circumstances, macrophages can produce both cell necrosis and apoptosis. Necrosis of cardiac myocytes occurs during rejection episodes and is characterized by cell swelling, disruption of internal and external cell membranes, and cell lysis, which invokes a marked inflammatory response.1 5 8 Apoptosis of cardiac myocytes during cardiac allograft rejection has not been reported heretofore.

Apoptosis is a morphologically and biochemically distinct form of programmed cell death.4 5 8 9 10 During apoptosis, there is condensation of the nuclear chromatin and cell shrinkage with preservation of organelles. In later stages of apoptosis, nuclear and cytoplasmic budding and fragmentation of the dying cell into membrane-bound "apoptotic bodies" that undergo phagocytosis without eliciting an inflammatory response are observed. In contrast to necrosis, apoptosis is an internally determined program of events leading to "suicide" of the cell that involves the activation of genes, proteases, and endonucleases that degrade chromosomal DNA into oligonucleosomal fragments that are multiples of 180 to 200 bp and that form so-called DNA ladders after electrophoresis on agarose gels.4 5 8 9 10 11

Apoptosis occurs during embryogenesis12 and normal tissue turnover,13 in the immune system during the deletion of autoreactive and nonfunctional lymphocytes,4 10 in response to hormones or their withdrawal in many tissues,14 15 and in response to various forms of cell injury that damage DNA.5 16 In the cardiovascular system, apoptosis has been observed during development of the conduction pathways, in the response of neonatal cardiac myocytes to hypoxia, in pressure overload, and in adult cardiac myocytes during reperfusion after ischemia.17 18 19 20 Apoptosis of leukocytes, macrophages, and smooth muscle cells has also been found in atherosclerotic lesions and atherectomy specimens.21 22

In a previous study that used a heterotopic cardiac transplantation model in rats, Yang et al23 found that cardiac allograft rejection is associated with a marked induction of the mRNA, protein, and enzyme activity of iNOS in myocardial homogenates and isolated purified cardiac myocytes from allografts. Immunohistochemical studies indicated that in the rejecting allografts, iNOS was expressed in endothelial cells, macrophages infiltrating the myocardium, and cardiac myocytes. Additional studies indicated that cytokine induction of iNOS in macrophages and cardiac myocytes was associated with NO-mediated killing of adult rat cardiac myocytes in vitro.24 However, the mechanisms responsible for cell death in those experiments were not explored. Albina et al25 and Cui et al26 recently reported that NO produced by iNOS in macrophages was capable of triggering apoptosis of the macrophages and was also capable of triggering apoptosis in some tumor cells in coculture experiments. Apoptosis of macrophages has also been induced by drugs that release NO.27 28

The objectives of the present study were to use the heterotopic rat cardiac transplantation model to investigate (1) whether apoptosis of cardiac myocytes and other cells occurs during cardiac allograft rejection and (2) whether the time course and intensity of apoptosis during cardiac allograft rejection parallels the induction of iNOS in the myocardium. Apoptosis was identified by DNA ladders and by in situ labeling of DNA fragments in the nuclei of apoptotic cells in histological sections from the syngeneic and allogeneic cardiac grafts.


*    Methods
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 *Methods
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Cardiac Transplantation
Male Lewis (TR-11) and Wistar-Furth (WF, RT-1u) rats weighing 180 to 200 g each were purchased from Harlan Sprague-Dawley Inc (Indianapolis, Ind). The animals were maintained on a 12-hour light/dark cycle and fed a commercially available rat chow diet and tap water ad libitum. Heterotopic abdominal heart transplantation was performed as previously described according to the method of Ono and Lindsay.29 Heart grafts in the abdomen were palpated daily; rejection was determined by detection of weakening of the heart beat and confirmed by inspection at laparotomy and by histological examination. Syngeneic Lewis-to-Lewis control abdominal heart transplants were performed by use of the same technique. Hearts were removed at days 1 to 5 for pathological or biochemical examination.

Determinations of NO Synthase Activity
The excised hearts were rinsed and perfused via the aorta with ice-cold saline to remove blood completely. Ventricular tissue was homogenized in ice-cold buffer containing 50 mmol/L Tris-HCl (pH 7.4); 0.05 mmol/L EDTA; 0.5 mmol/L DTT; 10 µg/mL antipain, leupeptin, pepstatin, and trypsin inhibitor; and 0.1 mg/mL phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 100 000g for 60 minutes at 4°C. Supernatants were adjusted to a protein content of 20 mg/mL with BCA protein assay (Pierce) and BSA used as standard and were used immediately for the enzyme activity assay. NO synthase activity was determined by incubation of the myocardial cytosolic enzyme preparation with 15 mmol/L HEPES (pH 7.4), 2 mmol/L L-arginine, 0.2 mmol/L NADPH, 2 mmol/L Mg(OAc)2, and 0.5 mmol/L DTT for 20 hours at 37°C; the production of NO2- was measured with the Greiss reagent.23

Western Blot Analysis
Aliquots of myocardial cytosolic enzyme preparation (45 µg per lane), as described in determination of NO synthase activity, were electrophoresed on 8% SDS-PAGE and transferred to nitrocellulose membranes.23 24 After overnight blocking in Tris-buffered saline with 3% nonfat dried milk and 2% BSA and subsequent washing, the blots were immunoblotted with the rabbit anti-mouse iNOS antiserum (1:2000), a generous gift from Dr Susan A. Gregory of G.D. Searle Company, St Louis. An anti-rabbit horseradish peroxidase (Amersham) was used as a secondary antibody. Blots were detected with the enhanced chemiluminescence method (Amersham).

Quantitative RT-PCR
To correct for vial-to-vial differences in the efficiency of RT and PCR amplification, rRNA was synthesized and used as the internal standard.30 Briefly, the primers for rRNA synthesis were constructed as the anti-sense primer containing a 71-bp deletion of the iNOS mRNA sequence but with a 5' end of 22-bp oligonucleotides that was identical to the anti-sense primer for sample target mRNA, thereby producing a 71-bp shorter PCR product. The sense primer was identical to the sense primer for sample target mRNA except for the addition of T7 RNA polymerase promoter sequences at its 5' end. We synthesized rRNA using the sample RNA as template for cDNA synthesis and subsequent PCR amplification, which eliminated any minor variations that might have existed in the target gene sequence. The T7 promoter appended to the primer enabled in vitro transcription of the PCR product into rRNA (T7-MEGA shortscript, Ambion). The rRNA was treated with RNase-free DNase I to remove the DNA template, purified with phenol/chloroform extraction (DNA contamination was tested by PCR), and measured by absorbance at 260 nm. rRNA was spiked into the sample RNA in a dilution series of known concentrations before co-RT and coamplification by RT-PCR and acted as a competitive template sharing the same primers. PCR products of rRNA were distinguished from the products of the sample target gene by their size difference.

Total RNA was isolated from transplanted rat hearts by use of the RNAzol B method (Tel-Test, Inc). For each sample, six to seven aliquots of total RNA (2.0 µg) were prepared, and a dilution series (in a 1:3 scheme) of the internal standard rRNA was spiked into these aliquots, except for one vial that contained sample RNA only.

Primers were designed from the Genbank mRNA sequence of mouse iNOS. The sequences were 5'-AAGCAGAATGTGACCATCATGC-3' for the sense primer and 5'-CAGTAGCAAAGAGGACTGAGGC-3' for the anti-sense primer. The predicted sizes of RT-PCR products from sample RNA and rRNA were 355 and 284 bp, respectively. They spanned nucleotide positions 1519 to 1874 of iNOS mRNA. The gene homology of this segment between mouse and rat is >98%. Single-strand cDNA was synthesized with superscript II RNase H- reverse transcriptase (BRL Life Technologies) and gene-specific anti-sense primer. Ten percent of the RT reaction mixture was used for subsequent PCR. PCR was performed for 25 cycles with Taq DNA polymerase at 95°C for 1 minute, 59°C for 1 minute, and 72°C for 1 minute with a 6-minute extension at the final cycle. Aliquots of the RT-PCR products were electrophoresed on 1.5% agarose gel and visualized by ethidium bromide staining. Both products of sample RNA and internal standard rRNA produced a single band at the appropriate molecular weights, even with high amplification (35 cycles). The identity of amplified cDNA was further verified with restriction enzyme analysis. To measure the amount of mRNA during the exponential phase of amplification, digital scans were performed with the use of photographs of the PCR products that were illuminated with UV light. After densitometric analysis, the amount of target mRNA was determined by measurement of the ratio of sample mRNA to internal standard rRNA and interpolation to the actual sample concentration with use of the two points with a ratio closest to one.

Histology
One coronal section from each of the transplanted and native hearts of each animal was fixed in 10% buffered formalin and embedded in paraffin, and 4-µm serial sections were cut and mounted on sialine-coated glass slides. One section of each specimen was stained with hematoxylin and eosin to determine the extent and severity of rejection according to the International Society of Heart and Lung Transplantation classification.31 The remaining sections were used for immunohistochemistry and in situ detection of apoptotic cells. For this purpose, sections were deparaffinized in xylene (2x15 minutes), rehydrated in a series of alcohols with decreasing concentration (100%, 100%, 95%, 70%, each for 5 minutes), and finally washed with TBS at pH 7.5. Proteinase K digestion (1 mg/mL in 0.05 mol/L Tris-HCl buffer with 2 mmol/L calcium acetate at pH 7.5 for 30 minutes at 37°C) was performed on sections that were then stained for apoptotic cells.

In Situ Labeling of Apoptotic Cells
Apoptotic cells were identified by either ISEL or ISNT of DNA fragments as previously described.32 33 For ISEL,34 we added biotinylated dUTP to the 3' end of DNA fragments by incubating sections in 0.05 mol/L Tris-HCl buffer (pH 7.6), 140 mmol/L potassium cacodylate, 1 mmol/L cobalt chloride with 125 U/mL terminal deoxynucleotide transferase (Tdt, Boehringer-Mannheim), and 1 mmol/L biotin-16-dUTP for 30 minutes at 37°C. The sections were rinsed in TBS (3x5 minutes). Endogenous peroxidase was blocked with 1.5% H2O2 in distilled H2O. Sections were rinsed with TBS (3x5 minutes) and covered with 2% blocking solution in 0.1 mol/L sodium maleate (Boehringer-Mannheim) to reduce background staining. The sections were then incubated with avidin-peroxidase complexes (Vector) in TBS (1:100) for 30 minutes and rinsed with TBS (3x5 minutes). Peroxidase activity was visualized with 3,3'-diaminobenzidine (Sigma Chemical Co) until the brown reaction product was clearly visible. The sections were then counterstained with hematoxylin.

For ISNT,32 33 we incorporated digoxigenin-11-dUTP into DNA fragments by incubating sections overnight at 4°C with the Klenow fragment of the DNA polymerase I (20 U/mL; Boehringer-Mannheim), which requires the complementary DNA strand as a template for adding nucleotides to the 3' end of the nicked DNA strand. The incubation buffer consisted of 0.05 mol/L Tris-HCl (pH 7.5) with 0.025% BSA, to which was added a nucleotide mix of 1 mmol/L each of dATP, dGTP, and dCTP; 0.66 mmol/L dTTP; and 0.33 mmol/L digoxigenin-11-dUTP. Afterward, the sections were washed in TBS, covered with 2% blocking solution (Boehringer-Mannheim) in 0.1 mol/L sodium maleate, and incubated with an alkaline phosphatase–conjugated anti-digoxigenin antibody (1:500, Boehringer-Mannheim) in 2% blocking solution. After the sections were rinsed twice with TBS for 5 minutes and 0.1 mol/L Tris-HCl (pH 9.5) for 2 minutes, alkaline phosphatase activity was visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Biogenex) until the blue reaction product was clearly visible in cell nuclei. Sections were then labeled immunohistochemically for either cardiac myocytes, endothelial cells, T lymphocytes, or macrophages by use of antibodies to muscle actin (HHF-35), CD34, CD3 (DAKO), or a glycoprotein (ED1) expressed on rat macrophages (Accurate), respectively.

Positive control sections for apoptosis consisted of slides from prostates taken from rats 3 days after castration, when the number of apoptotic cells is highest,15 normal rat heart for antibody to muscle actin (HHF35), and rat spleen for anti-CD34, anti-CD3, and ED1. Negative controls for ISEL and ISNT were obtained by omission of the enzyme. Omission of the respective antibody served as a negative control for immunolabeling.

Apoptosis by DNA Analysis
Genomic DNA was prepared from heart tissue specimens by a standard extraction procedure.35 For the detection of DNA laddering, we radiolabeled 0.5 to 1.0 µg of cellular DNA with 5 U of the Klenow fragment of DNA Pol I using only one radiolabeled nucleotide (0.5 µCi of 32P-dCTP; New England Nuclear), as previously described.36 The radiolabeled material was then electrophoresed on a 1.8% agarose gel for 2 hours at 100 V. After the gel was dried on 3-mm Whatman paper, the filter was exposed for autoradiography.37

Immunohistochemistry
Single and double labeling of sections previously stained for apoptotic nuclei by ISNT or ISEL were performed for each antibody (HHF35, anti-CD34, anti-CD3, and ED1) with the use of the Ventana ES automated immunostaining system (Ventana). The protocols are detailed in Table 1Down. Ventricular sections were also immunolabeled with a polyclonal antibody to iNOS (see above) and a monoclonal antibody to nitrotyrosine (Upstate Biotechnology) to detect nitration of amino acids by peroxynitrite derived from the interaction of NO and superoxide radicals.38 39 40 For antigen retrieval, sections stained for CD3 or nitrotyrosine were boiled for 20 minutes in 0.01 mol/L citrate buffer (pH 6.0) in a microwave oven before immunostaining. Single-stained sections were counterstained with hematoxylin, and double-stained sections were counterstained with ethyl green.


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  		Table 1. Parameters for Immunolabeling With an Automated Immunostaining System

Quantitative Evaluation
The numbers of apoptotic cardiac myocytes, endothelial cells, and mononuclear leukocytes (lymphocytes and macrophages) were determined from an entire coronal section of the heart of each specimen without observer knowledge of the type of graft (allograft versus syngeneic graft) or the time point after transplantation. Values are expressed as number of apoptotic cells per square millimeter.

Statistical Analysis
The time course of changes in iNOS mRNA and enzyme activity was analyzed by ANOVA. A log transform was used for the iNOS mRNA data because of the markedly nonnormal distribution of values. The degree of apoptosis at each of the time points was analyzed by use of the nonparametric Kruskal-Wallis procedure.


*    Results
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 *Results
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Histopathology
The pathological changes that occurred during Wistar-Furth:Lewis cardiac allograft rejection are detailed in Fig 1Down. (In this model, cessation of allograft function occurs at days 6 to 9.) At day 1, a few focal, predominantly perivascular, inflammatory infiltrates were present (rejection grade 1; Fig 1aDown). At day 3, the inflammatory infiltrate extended into the myocardial interstitium, where there was mild edema (rejection grade 2). In later stages of cardiac allograft rejection (days 4 and 5; Fig 1bDown), there was expansion of the inflammatory infiltrate with obvious myocyte damage and destruction of cardiac muscle fibers (rejection grades 3 to 5). Two of seven allografts at day 5 displayed areas of eosinophilic necrosis of cardiac myocytes associated with vasculitis and interstitial hemorrhage (rejection grade 6). There was no evidence of rejection in native hearts or in day 1 or day 5 syngeneic grafts. The inflammatory infiltrate of day 1 cardiac allografts was composed almost exclusively of CD3-positive T lymphocytes (Fig 2aDown). After day 3, there was a decline in lymphocytes and a notable influx of ED1-reactive macrophages that increased further at days 4 and 5, when they constituted >50% of all inflammatory cells (Fig 2bDown).




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  		Figure 1. Cardiac allografts 1 day (a) and 5 days (b) after transplantation, stained with hematoxylin and eosin. a, The day 1 allograft shows focal mild perivascular lymphocytic infiltrates with intact cardiac myocytes. b, At day 5, there is evidence of myocyte damage associated with a dense, macrophage-rich, inflammatory infiltrate and interstitial edema and hemorrhage (magnification x400).




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  		Figure 2. a, Immunolabeling of day 1 cardiac allograft for CD3 shows that the majority of inflammatory cells are T lymphocytes. b, At day 5, approximately half of the inflammatory cells are macrophages, as demonstrated by immunolabeling for ED1. The macrophage-rich inflammatory infiltrate contains numerous apoptotic bodies (arrow), some of which appear engulfed by macrophages (magnification x400).

Apoptosis of Cardiac Myocytes and Macrophages in Cardiac Allografts
Both ISEL and ISNT demonstrated apoptotic nuclei in the cardiac allografts. Both techniques labeled hyperchromatic and fragmented nuclei (morphological changes characteristic of apoptosis) as well as nuclei that appeared histologically intact, which most likely represent early apoptosis (Fig 3Down). Most of the apoptotic cells were found within or adjacent to macrophage-rich inflammatory infiltrates. Apoptosis was rarely present in association with the pure lymphocytic infiltrates of early allograft rejection (day 1).



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  		Figure 3. ISEL of apoptotic nuclei (brown) in day 5 cardiac allograft. Brown reaction product is present in apoptotic bodies within the inflammatory infiltrate (arrowheads, late apoptosis) as well as morphologically normal–looking nuclei in cardiac myocytes adjacent to the inflammatory infiltrate (arrow, early apoptosis) (magnification x400).

Labeling for apoptosis along with immunostaining with cell-type–specific antigens revealed the nature of the apoptotic cells. Fig 4Down shows apoptotic cardiac myocytes in a section from a day 5 allograft, in which apoptotic nuclei are identified by blue labeling and the presence of muscle actin (HHF-35) is demonstrated by brown cytoplasmic staining. Apoptotic cardiac myocytes were abundant in areas of the myocardium proximate to areas of macrophage-rich inflammatory infiltrate (Fig 4aDown) but also occurred in myocytes distant from the inflammatory infiltrates (Fig 4bDown). In these areas, the myocytes frequently appeared shrunken and fragmented. Mononuclear leukocytes, including macrophages, also contributed to apoptosis (Fig 5Down). In these sections, the nuclei with DNA fragmentation were identified (blue labeling) in cells in which the nuclei were surrounded by a rim of brown reaction product, representing immunoreactivity for ED1 (macrophages). Cell-specific immunohistochemistry failed to label cells with the extensive nuclear fragmentation characteristic of advanced apoptosis. Such apoptotic bodies engulfed by macrophages were frequently observed in days 4 and 5 of allograft rejection (Fig 5Down).




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  		Figure 4. Double labeling for apoptotic cardiac myocytes, depicted by blue nuclear labeling for apoptosis (ISNT) and brown cytoplasmic staining for muscle actin (HHF35). a, Apoptosis of cardiac myocytes was most abundant adjacent to macrophage-rich inflammatory infiltrates. b, Apoptosis of cardiac myocytes also occurred in the absence of an inflammatory infiltrate (magnification x400).



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  		Figure 5. Double labeling for apoptotic nuclei (blue, ISNT) and ED1 (brown) present on the cell membrane of macrophages in an allograft on day 5. Apoptotic bodies are found adjacent to or engulfed by macrophages (arrowheads). Occasional macrophages also undergo apoptosis (arrow). Magnification x400.

Determination of Apoptotic Cells in Cardiac Allograft Rejection
Apoptotic cardiac myocytes and macrophages were extremely rare in native hearts or in day 1 syngeneic grafts or day 1 allografts. During the course of cardiac allograft rejection, the number of apoptotic cardiac myocytes increased sharply from day 3 to day 5 in association with the increased infiltration of macrophages into the myocardial tissue (Fig 6Down) and coinciding with the increased severity of allograft rejection determined by histological grading (grades 3 through 6, Table 2Down). At day 1, the number of apoptotic cardiac myocytes/mm2 averaged 0.21 compared with 0.31 at day 3. This value rose sharply to 1.1 at day 4 and 1.27 at day 5. Similar increases in the number of apoptotic endothelial cells and mononuclear leukocytes were also observed at days 3 to 5 (Table 2Down).



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  		Figure 6. The time course of apoptosis of cardiac myocytes, endothelial cells, and mononuclear leukocytes during cardiac allograft rejection. Apoptosis increased significantly from day 3 to day 5.


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  		Table 2. Histological Features and Degree of Apoptosis in Allografts, Syngeneic Grafts, and Native Hearts

The number of apoptotic cardiac myocytes at day 5 was significantly higher in allografts than in syngeneic grafts or native hearts (Table 2Up). The day 5 allografts manifested an approximately ninefold increase of apoptotic cardiac myocytes, sixfold increase of apoptotic mononuclear leukocytes, and threefold increase of apoptotic endothelial cells over the day 5 syngeneic grafts or the values observed at day 1 (P<.01; Table 2Up). DNA from day 5 allografts, day 5 syngeneic grafts, and native hearts was extracted, radiolabeled with 32P-dCTP, and subjected to electrophoresis in 1.8% agarose gels. DNA ladders composed of DNA fragments that are multiples of 200 bp and characteristic of apoptosis were observed in the day 5 allografts but not in the syngeneic grafts or native hearts (Fig 7Down).



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  		Figure 7. DNA was extracted from myocardial tissue, labeled with 32P, and subjected to electrophoresis on 1.8% agarose gel. Laddering of DNA fragments that are multiples of 200 bp is apparent in the allograft but not the syngeneic graft or native heart.

iNOS Expression and Nitration of Tyrosine in the Cardiac Allografts
Figs 8Down and 9 show the time course and extent of the expression of iNOS in myocardial tissue from the rejecting cardiac allografts. The iNOS mRNA was induced with significant increases (P<.05) by day 3 compared with days 1 and 2 and continued to increase progressively (P<.05) on days 4 and 5 as determined by quantitative RT-PCR (Fig 8aDown). The iNOS enzyme protein, as assessed by Western blot (with an estimated molecular weight of 130 kD), was first detectable on day 3 and became more abundant by days 4 and 5 (Fig 9Down). The increase in iNOS enzyme activity from cytosolic myocardial preparations began on day 4 and became significant (P<.05) by day 5 compared with levels measured on days 1 through 3 (Fig 8bDown). The expression of iNOS mRNA, enzyme protein, and enzyme activity paralleled the time course of apoptosis in cardiac myocytes (see Fig 6Up).




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  		Figure 8. The expression of iNOS mRNA (in picograms per sample) and enzyme activity (in nanomoles per milligram of protein) during allograft rejection. a, Myocardial total RNA was isolated from allograft hearts (2 at each time point for a total of 10 hearts) and subjected to iNOS quantitative RT-PCR. iNOS mRNA was induced significantly on day 3 and thereafter. b, The increase in iNOS activity from myocardial cytosolic preparations began on day 4 and became significant (P<.05) by day 5 compared with the levels on days 1 through 3 (n=2 for days 2 and 5, n=4 for days 1, 3, and 4).



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  		Figure 9. The time course of induction of iNOS mRNA (top) and iNOS enzyme protein (bottom). Both iNOS mRNA and iNOS protein were first detected on day 3 and became abundant by days 4 and 5.

Cardiac myocytes and macrophages displayed weak immunostaining for iNOS at days 1 and 3 of allograft rejection (Fig 10aDown). The intensity of the iNOS immunostaining was increased in cardiac myocytes and macrophages at days 4 and 5 (Fig 10bDown). Immunoreactivity for iNOS was strongest in cardiac myocytes adjacent to macrophage-rich inflammatory infiltrates. The distribution of immunostaining for nitrotyrosine was similar to that of iNOS (Fig 11Down). No immunoreactivity for nitrotyrosine was discerned in day 1 allografts (Fig 11aDown), syngeneic grafts, or native hearts (data not shown). At day 3, myocytes and macrophages in allografts showed faint reactivity for nitrotyrosine in areas of inflammation (Fig 11bDown). Nitrotyrosine was most abundant in and near macrophages and damaged myocytes of day 5 allografts (Fig 11cDown).




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  		Figure 10. Immunolabeling of cardiac allografts for iNOS. a, At day 1, no immunoreactivity for iNOS was discerned in inflammatory cells or myocytes. b, At day 5, strong reactivity for iNOS was present in macrophages and adjacent cardiac myocytes (magnification x400).





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  		Figure 11. Immunolabeling of cardiac allografts for nitrotyrosine, representing the effects of peroxynitrite. a, At day 1, no reactivity for nitrotyrosine was discerned. b, At day 3, the presence of nitrotyrosine was confined to inflammatory infiltrates with weak nuclear and cytoplasmic reactivities in adjacent cardiac myocytes. c, There was strong cytoplasmic staining for nitrotyrosine in inflammatory cells and damaged cardiac myocytes. Intact cardiac myocytes showed heavy nitration of nuclear proteins (arrow). Apoptotic bodies were also present (arrow and arrowhead) (magnification x400).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The major finding of this study is that apoptosis of cardiac myocytes occurs during cardiac allograft rejection. Apoptosis was identified in sections from the rejecting allografts by in situ labeling of DNA fragments in cell nuclei by two different methods and also by the presence of DNA ladders on agarose gels labeled with 32P-dCTP. Many of the apoptotic cells were cardiac myocytes, distinguished by their characteristic striations and cytoplasmic immunoreactivity for muscle actin. The apoptotic cardiac myocytes frequently were shrunken and had intact membranes containing hyperchromatic nuclei, whereas necrotic cells were swollen with disruption of the sarcolemma and karyolysis.8 Apoptotic myocytes were localized adjacent to areas of inflammatory infiltration and also in areas devoid of a significant inflammatory infiltrate. The percentage of cells that were apoptotic was significantly higher in allografts than in native hearts or syngeneic grafts studied at the same time points. There was a small amount of apoptosis early after transplantation, when the cellular infiltrate was primarily composed of T lymphocytes, but there was more apoptosis and necrosis later (days 3 to 5), when the inflammatory infiltrate was largely monocyte/macrophages. At that time (days 3 to 5), both macrophages and cardiac myocytes showed strong immunoreactivity for iNOS, and there was positive immunostaining for nitrotyrosine in the tissue. The absolute numbers of apoptotic cardiac myocytes in the sections were small; nevertheless, the loss of cells is significant, because it is known that the time course for apoptotic cell death is only a few hours.41 In the rejecting allografts, there were also increased numbers of apoptotic leukocytes (macrophages and lymphocytes) and endothelial cells.

The occurrence of apoptosis in the cardiovascular system has not been widely documented. Tanaka and coworkers18 reported that hypoxia induces apoptosis of cultured neonatal rat cardiomyocytes along with expression of mRNA for the Fas antigen. Gottlieb and coworkers20 used ISEL, electron microscopy, and DNA ladders to demonstrate in rabbits that apoptosis of cardiac myocytes occurs in response to ischemia and reperfusion but not prolonged ischemia. They raised the possibility that the burst of oxidative free radicals on reperfusion may constitute a trigger for the apoptotic program of cell death in this situation. Recently, Kajstura and coworkers42 and Cheng and coworkers43 reported that apoptosis of cardiac myocytes occurs during postnatal maturation of the heart and also in papillary muscles in response to excessive stretch. In the latter situation, apoptosis appeared in association with increased cellular production of superoxide and an increase in the number of myocytes expressing the Fas protein. Both apoptosis and superoxide formation were inhibited by an NO donor that quenches superoxide.43 In blood vessels with atherosclerosis and after percutaneous transluminal coronary angioplasty or other injury, apoptosis of vascular smooth muscle cells and macrophages has also been reported.21 22 Data from several such studies have led to the suggestion that the balance between cell migration and proliferation and apoptotic cell death may be an important determinant of vascular cell number and the size of the intimal lesions in atherosclerosis and after balloon injury.

The genes and biochemical reactions involved in apoptosis are incompletely understood.5 8 9 The involvement of apoptosis in development and homeostasis and in many diseases in which the rate of apoptosis is increased (eg, AIDS and Alzheimer's disease) or decreased (eg, cancer, viral infections, and autoimmune diseases such as lupus erythematosus) has led to much investigation and to a search for therapeutic modalities that modulate the rate of cell death.4 5 9 10 Physiological stimuli that inhibit and stimuli that activate apoptosis have been identified.5 8 9 Inducers of apoptosis that have been identified include cytokines, the Fas ligand, tumor necrosis factor-{alpha}, viruses, bacterial toxins, oncogenes such as c-myc, tumor suppressor genes such as p53, oxygen radicals, and UV and gamma irradiation.5 8 9 44 45 Reactive oxygen species are thought to damage DNA, impede DNA repair, and activate endonucleases that degrade nuclear DNA, leading to the formation of DNA fragments identified by end labeling or the formation of 180- to 200-bp DNA ladders on agarose gels.9 11 In many cells, the p53 gene plays a pivotal role in initiating the program of apoptosis in response to DNA damage.45 Other genes, such as Bcl-2, prevent the induction of the apoptotic cell program.9 46 47

In our previous study,23 iNOS mRNA, protein, and enzyme activity were induced in the myocardium during allograft rejection in this model. The iNOS was induced both in macrophages infiltrating the myocardium and in cardiac myocytes, probably in response to cytokines released during the immune response to the grafted tissue. In the present study, the time course and intensity of iNOS induction (mRNA measured by RT-PCR, protein measured by Western blot and immunostaining, and enzyme activity measured by the production of NO2- from L-arginine) paralleled the time course and extent of apoptosis. This observation is compatible with but does not prove the hypothesis that NO produced by iNOS in this setting acts to trigger apoptosis.

Other data, however, suggest that NO is capable of triggering apoptosis in cardiac myocytes. NO is a reactive ionic species that can damage DNA.25 48 Pinsky et al24 induced iNOS in macrophages in vitro and studied them in coculture with isolated purified adult rat cardiac myocytes; they showed that NO released by macrophages could kill adjacent myocytes by an NO-dependent mechanism. In other experiments,24 the induction of iNOS by cytokines in isolated purified cardiac myocytes was autodestructive to the cardiac myocytes by an NO-dependent mechanism. Several groups demonstrated that when cells were exposed directly to NO gas, when NO was produced by iNOS in activated rat peritoneal macrophages, or when NO was released from a donor drug, NO was capable of triggering apoptosis of macrophages.25 26 27 28 Preliminary studies in our laboratory indicate that the drug S-nitroso-N-acetyl-penicillamine (SNAP), which releases NO, can trigger apoptosis of adult rat cardiac myocytes studied in vitro and that this effect can be inhibited by addition to the incubation media of reduced hemoglobin, which scavenges NO released by the drug.49

It is known that activated macrophages expressing iNOS synthesize large amounts of both NO and superoxide. NO and O2- form peroxynitrite, a powerful oxidant that not only degrades to toxic hydroxyl radicals capable of causing necrosis but also can produce nitration of proteins and can produce DNA damage and strand breaks.38 39 48 50 In the rejecting allografts, there were both necrosis and apoptosis of cardiac myocytes. Necrosis of the myocytes may have resulted from direct attack by immunocompetent cells, severe oxidant damage from high doses of peroxynitrite and hydroxyl radicals, ischemic damage, or other mechanisms.2 The parallelism of iNOS induction and apoptosis suggests that NO produced by iNOS in infiltrating macrophages and cardiac myocytes may be a trigger for the apoptotic response of cardiac myocytes and infiltrating macrophages. The observation that immunostaining for nitrotyrosine, a marker for peroxynitrite, also was markedly increased at days 3 to 5 suggests that peroxynitrite also may have contributed to induction of the apoptotic response. In addition, other mechanisms, such as interaction of cytotoxic T cells or cytokines (eg, tumor necrosis factor-{alpha}) with the Fas protein on macrophages and cardiac myocytes as well as activation of cysteine proteases by granzyme-ß released from CD8-positive T lymphocytes, may also have caused apoptosis.2 4 5 18 43

Although experiments with iNOS inhibitors will be required to test more fully the hypothesis that NO and peroxynitrite trigger apoptosis of cardiac myocytes during cardiac allograft rejection, the immunohistochemical evidence for iNOS and nitration of proteins of cardiac muscle cells in allografts 3 to 5 days after transplantation is compatible with this idea. Myocyte death by apoptosis, in addition to the known effects of NO in impairing cardiac muscle contraction,51 may contribute to the decline in ventricular function observed during rejection. One may speculate that during mild, low-level rejection, induction of iNOS and apoptosis of myocytes may be responsible, at least in part, for the slow decline of ejection fraction that is frequently observed. iNOS has been demonstrated in ventricular tissue from humans with idiopathic dilated cardiomyopathy.52 Conceivably, apoptosis of heart muscle cells triggered by iNOS may also occur in patients with dilated cardiomyopathy, myocardial infarction, and other inflammatory diseases of the heart. The present data, which indicate that apoptosis contributes to cardiac myocyte loss during heart transplantation rejection, raise the possibility that this component of cardiac myocyte death may be ameliorated by therapies that reduce NO formation or interfere with the cellular program leading to myocyte cell death.


*    Selected Abbreviations and Acronyms
 
bp = base pair
DTT = DL-dithiothreitol
iNOS = inducible nitric oxide synthase
ISEL = in situ end labeling
ISNT = in situ nick translation
NO = nitric oxide
PCR = polymerase chain reaction
rRNA = recombinant RNA
RT = reverse transcription
TBS = Tris-buffered saline


*    Acknowledgments
 
This work was supported in part by grants HL-21006 and HL-54764 from the NHLBI.

Received January 8, 1996; revision received April 5, 1996; accepted April 15, 1996.


*    References
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