Cardiovascular Research 1998 38(1):6-15; doi:10.1016/S0008-6363(98)00022-4
© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
The role of inducible nitric oxide synthase in cardiac allograft rejection
Paul J. Cannona,*,
Xiaochun Yanga,
Matthias J. Szabolcsc,
Stefano Ravallia,
Robert R. Sciaccaa and
Robert E. Michlerb
aDepartment of Medicine, Columbia University College of Physicians and Surgeons, New York, NY, USA
bDepartment of Surgery, Columbia University College of Physicians and Surgeons, New York, NY, USA
cDepartment of Pathology, Columbia University College of Physicians and Surgeons, New York, NY, USA
* Corresponding author. Department of Medicine, Division of Cardiology, Columbia University, 630 West 168th Street, New York, NY 10032, USA. Tel.: +1 (212) 305-9052; Fax: +1 (212) 305-4648.
Received 16 October 1997; accepted 30 December 1997
Cardiac transplantation is an effective therapy for end-stage heart failure with one and five year survival rates about 80% and 65% respectively [1]. Despite these good results, cardiac allograft rejection remains a problem which produces impaired ventricular performance and death of cardiac myocytes ultimately causing congestive heart failure, low cardiac output, hypotension and reduced pressor responses to catecholamines. Chronic allograft rejection also results in the development of transplant-associated coronary atherosclerosis, a vasculopathy that produces myocardial ischemia, infarction and sudden death and is the leading cause of death in cardiac transplant recipients after the first year [2]. Although there is a large body of evidence concerning the immunological interactions involved in cardiac allograft rejection, knowledge concerning the cellular and biochemical mechanisms responsible for contractile dysfunction and for death of heart muscle cells remains incomplete. Since an inflammatory reaction in the myocardium is an intrinsic component of the pathological changes observed during cardiac allograft rejection, our laboratory undertook an investigation of the participation of nitric oxide synthases, particularly the inducible nitric oxide synthase (iNOS), in the manifestations of cardiac allograft rejection.
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1 NO synthesis
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The five electron oxidation of
L-arginine to
L-citrulline and
biologically active nitric oxide (NO) is important to a large
variety of physiological and pathological processes
[3, 4].
NO synthesis is accomplished by the three isoforms of NOS, the
neuronal NOS (nNOS, NOS 1) originally identified in brain, inducible
NOS (iNOS, NOS 2) originally identified in macrophages and endothelial
NOS (eNOS, NOS 3) originally identified in endothelial cells.
Constitutive nitric oxide synthases (nNOS and eNOS) require
calcium and calmodulin as cofactors and generate low amounts
of NO. Small amounts of NO released by endothelial cells in
response to hormones or sheer stress interact with soluble guanylyl
cyclases to increase the formation of cyclic GMP in target cells
such as platelets, endothelial cells and vascular smooth muscle
cells promoting inhibition of platelet adhesion and aggregation,
inhibition of leukocyte adhesion and migration and vasodilation
respectively. The NO produced by nNOS acts as a neurotransmitter
in brain, in cells of the nonadrenergic, noncholinergic nervous
system and in skeletal muscle
[5].
The inducible NO synthases expressed in macrophages, endothelial cells, vascular smooth muscle cells and cardiac myocytes in response to cytokines (such as IL-1β, TNF-
, IFN-
, IL-6) or bacterial endotoxin do not require calcium and calmodulin as cofactors and generate substantially larger amounts of NO for long periods of time [3–6]. NO produced by iNOS in activated macrophages is cytotoxic and participates in their antimicrobial actions [4, 7]. NO produced by vascular smooth muscle cells and cardiac myocytes in response to endotoxin has been implicated in the pathogenesis of hypotension in association with bacterial infections [4]. The continuous large production of NO by iNOS in cardiac myocytes has been shown to influence myocardial contractile responses and to affect heart rate [6].
Other factors important in the synthesis of NO by iNOS include the availability of the substrate, L-arginine, and of the cofactors NADPH and tetrahydrobiopterin (THB4) [3–7]. When iNOS is induced by cytokines in macrophages, endothelial cells or cardiac myocytes, cationic amino acid transporter proteins, CAT 1, CAT 2B (both high affinity) and CAT 2A (low affinity) are coinduced which can increase the intracellular L-arginine concentration. GTP cyclohydrolase, the key enzyme in THB4 biosynthesis, is also coinduced with iNOS. Other experimental evidence obtained with nNOS and iNOS indicates that NO can exert a negative feedback on NO synthesis [6]. In activated macrophages NO can inhibit iNOS activity by reducing the availability of heme and its insertion into monomers, blocking formation of the dimers required for enzyme activity.
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2 iNOS induction in cardiac allograft rejection
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During cardiac allograft rejection there is significant release
of cytokines by activated T lymphocytes and macrophages which
participate in the immune response to foreign HLA and other
antigens present in endothelial and other cells of the transplanted
heart
[8]. Because cytokines, particularly those released by
the Th1 subset of lymphocytes, were reported to stimulate iNOS
expression in several cell systems, Yang et al. used a rat model
to investigate iNOS expression during cardiac allograft rejection
[9]. Heterotopic abdominal cardiac transplantation was performed
using Lewis strain donor hearts and Wistar–Furth strain
recipients. Lewis to Lewis transplants were performed as syngeneic
controls. Allograft rejection is complete at days 6–7
with this model. Cardiac allografts examined at day 5 manifested
reduced contractility and histological changes of severe rejection,
with an inflammatory myocardial infiltrate composed of lymphocytes
and macrophages, edema and damage and destruction of cardiac
muscle fibers. The mRNA for iNOS and iNOS protein were detected
in ventricular homogenates and also in isolated purified myocytes
from the rejecting cardiac allografts but not in ventricular
tissue or myocytes from the syngeneic control grafts. iNOS enzyme
activity and tissue cyclic GMP levels were also increased significantly
in the rejecting allografts. Immunostaining with a highly specific
antibody indicated that iNOS protein was present in macrophages
infiltrating the myocardium and also in cardiac myocytes of
the rejecting allografts. Some lymphocytes and microvascular
endothelial cells in the allografts also showed positive iNOS
immunostaining. The data indicated that myocardial iNOS mRNA,
protein and enzyme activity are induced in infiltrating macrophages
and cardiac myocytes of rejecting allografts.
Using a similar Lewis to ACI strain heterotopic rat cardiac transplant model Worrall and coworkers confirmed that iNOS mRNA and enzyme activity were increased in rejecting cardiac allografts [10]. In agreement with a previous report serum nitrite/nitrate levels were also significantly increased in the allograft recipients [10–12]. Immunostaining revealed iNOS protein in infiltrating macrophages but not in cardiac myocytes. Whether absent iNOS immunostaining in cardiac myocytes in the Worrall studies [10, 11]relates to rat strain differences, milder rejection or methodological problems is unclear. Treatment with aminoguanidine, a drug which inhibits iNOS activity (but which also has antioxidant properties, reduces advanced glycation end product formation and inhibits diamine oxidase and aldose reductase) improved allograft papillary muscle contractile performance, reduced the extent of pathological changes and prolonged allograft survival from 10 to 15 days [10, 11]. EPR signals in myocardial tissue from the rejecting grafts were indicative of the formation of nitrosylferromyoglobin and/or nitrosylferrohemoglobin and of nonheme iron–dinitrosyl complexes; these signals were also ameliorated by aminoguanidine [10]. Russell et al., using a Lewis-F344 rat cardiac transplantation model, also observed a prolonged increase in expression of iNOS mRNA and immunostaining of myocardial macrophages and of vascular smooth muscle cells in rejecting cardiac allografts [13]. In studies of serial endomyocardial biopsies of patients during the first year following heart transplantation, Lewis and coworkers found iNOS mRNA during the first 180 days [15]. iNOS protein was demonstrated by immunohistochemistry in cardiac myocytes. There was also increased myocardial cGMP. iNOS mRNA expression was not related to the ISHLT histological grade of rejection but was associated significantly with systolic and diastolic dysfunction of the left ventricle. Winlaw et al. in a study of Brown–Norway to Lewis allografts and Lewis–Lewis isografts found that urinary nitrite excretion in untreated allograft rejection was increased 8 fold above basal excretion of isografts and that peak nitrate excretion occurred early in rejection and was attenuated by immunosuppressive therapy [16].
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3 iNOS in transplant vasculopathy
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Allograft associated vasculopathy is considered to be a form
of chronic rejection
[2]. Transplant associated coronary artery
disease tends to be diffuse, to extend to small arteries and
arterioles and to involve a proliferative intimal response of
smooth muscle cells and to a lesser extent of macrophages. Russell
et al. and Akyurek et al. reported that eNOS was present in
endothelial cells and that iNOS expression was upregulated in
macrophages and smooth muscle cells in the intimal lesions found
in rat models
[14, 17]. Smooth muscle immunostaining was particularly
apparent at later time points in lesions from the rat. Ravalli
et al. used in-situ hybridization and immunostaining to examine
iNOS expression in human transplant coronary artery disease
[18]. Both iNOS mRNA and protein were detected in neointimal
macrophages, neointimal ‘foam’ cells and neointimal
smooth muscle cells. Immunostaining for nitrotyrosine was also
found in the same cell types. These findings are similar to
those found in human atherosclerotic lesions and in mouse models
of the disease
[19, 20]. Whether iNOS expression is pathogenetic
in promoting the vascular disease (e.g. by increasing oxidant
stress and the expression of oxidant sensitive genes) or is
protective against the development of vascular disease is unclear.
In the study of Aji et al. the administration of
L-arginine
supplements to LDL receptor knockout mice fed a high cholesterol
diet reduced xanthoma formation and the extent of atherosclerotic
aortic lesions by 40%
[20]. This beneficial effect was abrogated
when a NOS inhibitor was coadministered with the
L-arginine
supplement suggesting that NO produced by eNOS and iNOS was
responsible for lesion reduction. NO is known to inhibit platelet
and white cell adhesion and transmigration of white blood cells
across the endothelium and to block smooth muscle cell proliferation
[3, 4, 6]. Another possibility is that NO contributes to vascular
remodeling by its known effects to promote apoptosis of macrophages
and vascular smooth muscle cells
[21, 22]. A recent preliminary
report which indicated that transplant associated vasculopathy
was more extensive in allografts in iNOS deficient mice is also
consistent with a protective effect of iNOS in this disorder
[23].
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4 Relationships of NO, cytokines and cellular immune responses in cardiac allograft rejection
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Cellular immune responses constitute major components of acute
and chronic allograft rejection
[24]. Activated T lymphocytes,
both CD4+ and CD8+, along with macrophages and endothelial cells
expressing class II MHC antigens and multiple cytokines released
by macrophages and other cells (including IL-1, IL-2, IFN-
,
TNF-
, IL-6 and IL-10) are present in the myocardium and vessels
undergoing allograft rejection
[8, 25]. iNOS expression occurs
early in the rejection process as reflected by immunostaining
of myocardial tissues and also by urinary excretion of nitrite/nitrate
[9–11, 16]. The administration of immunosuppressive therapy
such as dexamethasone, cyclosporine A and FK506 alone or in
combination resulted in an amelioration of the pathological
changes in the myocardium and also a reduction of the levels
of iNOS mRNA, and (in another report) a delay in the onset and
peak rise in urinary excretion of nitrite/nitrate
[9–11, 16, 26].
As mentioned previously, the administration of aminoguanidine,
a somewhat selective iNOS inhibitor, also resulted in an improved
survival and a delay in the histological appearance of severe
rejection
[10, 11]. These data suggest that iNOS in macrophages
and cardiac myocytes plays a role in the pathogenesis of rejection
but this is not exclusive or necessarily the most important
role.
The specific signal which induces iNOS expression during allograft rejection has not been defined. Nevertheless, it is probable that cytokines produced by activated macrophages play an important role [8, 25]. Interferon-, TNF- and IL-1β have been reported to induce iNOS mRNA, protein and enzyme activity in macrophages, vascular smooth muscle cells and isolated rat cardiac myocytes in vitro [3, 4, 6]. It is also probable that interaction between the CD40 ligand expressed on CD4+ T-lymphocytes and its target molecule CD40 also plays a role. Interaction by cell contact between the CD40 ligand on T-cells and CD40 on macrophages and has been reported to induce iNOS in vitro [27]. In recent studies of human coronary arteries using immunohistochemistry, Szabolcs et al. found that in both transplant coronary artery disease and atherosclerosis CD40 ligand+ T-lymphocytes were present in the intimal lesions along with abundant positive immunostaining for CD40 on endothelial cells, macrophages, ‘foam’ cells and smooth muscle cells [28]. Larson et al. using a mouse model of heterotopic cardiac transplantation reported that treatment with an antibody to CD40 ligand (anti-gp39) at the time of transplantation markedly prolonged graft survival [29]. Allografts from treated recipients showed decreased expression of mRNA for iNOS but unaltered expression of transcripts for T-cell cytokines or the costimulatory molecules B7-1, B7-2 [29]. In a subsequent publication this group demonstrated that simultaneous therapy which blocked CD40 ligand-CD40 interactions with an antibody which inhibited CD28-B7 interactions aborted T-cell clonal expansion in vitro and in vivo and resulted in long term acceptance of cardiac and skin allografts and inhibition of the development of coronary vascular disease in the transplanted hearts [30].
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5 iNOS and ventricular contractile function
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Impaired ventricular contractile performance and cardiac myocyte
death are the hallmarks of cardiac allograft rejection. Much
evidence suggests that NO produced by iNOS contributes to allograft
contractile dysfunction. NO acts upon soluble guanylyl cyclase
to form cyclic GMP which is known to decrease cardiac myocyte
L-type calcium channel current and the contractile responses
of cardiac myofilaments to calcium
[6, 31]. Increased myocardial
cGMP has been found in guinea pigs treated with endotoxin and
in rat and human cardiac allografts
[9, 15, 32]. In studies
by Brady et al. the amplitude of contraction of isolated guinea
pig cardiac myocytes was reduced in cells from animals treated
with endotoxin to induce iNOS and in myocytes from normal guinea
pigs that were treated with media containing NO or the NO donor
drug sodium nitroprusside
[32, 33]. In studies of isolated rat
cardiac myocytes by Balligand, the induction of iNOS in the
myocytes by cytokines was not associated with a depression of
basal contractile function but with a reduction in the contractile
responses to β adrenergic agonists that could be reversed
by administration of NOS inhibitors
[34, 35]. Worrall et al.
reported that iNOS derived NO in early rejection of cardiac
allografts (prior to widespread cell death) was associated with
impaired contractility of isolated papillary muscles at baseline
and during β adrenergic, adenylate cyclase and calcium
stimulation
[10, 11, 36]. There were also associated membrane
dysfunctions
[36]. The impaired contractile responses were ameliorated
by aminoguanidine or therapy with corticosteroids which inhibited
iNOS expression. As mentioned previously, in studies of patients
following cardiac transplantation Lewis et al. found that iNOS
mRNA was inversely related to cardiac function, i.e. ejection
fraction
[15]. Paulus et al. found that in human allografts,
iNOS mRNA (determined by RT-PCR in endomyocardial biopsies)
was an independent variable relating a reduced peak contractile
response to dobutamine to simultaneous shortening of systole,
i.e. there was earlier onset of left ventricular relaxation
and reduction of left ventricular end-systolic pressure
[37].
The mechanisms by which NO produced by iNOS influences ventricular performance remain largely unexplored but are potentially multiple [6]. In addition to increasing cGMP, NO can produce auto-ADP ribosylation of glycolytic enzymes, inhibition of ribonucleotide reductase, and activation of poly ADP ribose synthetase which can lead to depletion of cellular energy stores. NO can also inhibit enzymes involved in the mitochondrial respiratory chain, reducing oxygen consumption and can inhibit the citric acid cycle enzyme aconitase. NO and its derivatives such as nitrososnium ion (NO+) can react with SH groups to form S nitrosoproteins which can in turn support additional transnitrosation reactions with other SH containing proteins influencing their activity. Peroxynitrite, the product of the interaction of NO and superoxide, is a strong oxidant which can cause autoperoxidation of lipids and proteins and decompose to form toxic hydroxyl radicals [38–40]. Peroxynitrite can also produce nitration of tyrosines in cell proteins. Nitration of contractile proteins such as actin may impede the contractile function of cardiac myofilaments [40]. As will be discussed subsequently NO in large doses may also contribute to death of cardiac myocytes.
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6 iNOS and cardiac myocyte death
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Death of cardiac myocytes is the hallmark of cardiac allograft
rejection. Because iNOS expression was apparent in macrophages
and cardiac myocytes in rejecting allografts and because the
generation of NO by iNOS in macrophages is important in the
immune defenses against invading viruses, bacteria and other
microorganisms, Pinsky et al. performed in vitro experiments
to investigate potential toxic effects of NO in the heart
[41].
In the first experiments, J774 macrophages were stimulated with
cytokines to induce iNOS. Stimulated and control macrophages
were cocultured with isolated rat cardiac myocytes using transwell
filter inserts to prevent physical contact between the two cell
types. There was significantly greater myocyte death (assessed
by creatine kinase release and by trypan blue dye exclusion)
in cocultures with stimulated macrophages expressing iNOS. The
death rate returned to control levels when cocultures with activated
macrophages were treated with
L-NMMA which inhibits NOS enzyme
activity. The data indicated that NO produced by iNOS in activated
macrophages can be lethal to adjacent cardiac myocytes. In additional
experiments they showed that rat cardiac myocytes treated with
TNF-
, IL-1β and IFN-
expressed iNOS mRNA, protein and enzyme
activity and also exhibited a higher death rate than control
myocytes
[41]. The increased death of cardiac myocytes was inhibited
by the administration of
L-NMMA to block NO formation and by
coincubation of the stimulated myocytes with TGF-β which
reduced iNOS expression. The data indicate that proinflammatory
cytokines can induce iNOS expression in adult cardiac myocytes
and that NO so produced can be autotoxic to the cardiac myocytes.
Taken together the experiments suggested that NO produced by
macrophages infiltrating the myocardium or by cardiac myocytes
in a cytokine rich milieu is potentially cytotoxic to heart
muscle cells. Such cardiotoxicity may be manifest not only in
allograft rejection but in other pathological states such as
myocardial infarction or cardiomyopathy in which macrophages
and cytokines are abundant and in which there is myocardial
expression of iNOS.
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7 Necrosis and apoptosis
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Although there is considerable information concerning immune
mechanisms of cardiac myocyte death, knowledge concerning mechanisms
of myocyte death during cardiac allograft rejection is incomplete
[24]. During advanced episodes of rejection there can be necrosis
of heart muscle cells. This is characterized by swelling of
the cells, disruption of internal and external cell membranes
and eventual lysis
[42]. Necrosis is often associated with a
marked inflammatory response. Recently, it has become increasingly
apparent that apoptosis of cardiac myocytes also occurs in a
variety of important cardiovascular diseases. Apoptosis is a
morphologically and biochemically distinct form of programmed
cell death
[42, 43]. During apoptosis there is condensation
of nuclear chromatin and cell shrinkage with preservation of
intracellular organelles. Subsequently there is blebbing of
nuclear and cytoplasmic membranes and fragmentation of the dying
cell into membrane bound ‘apoptotic’ bodies which
undergo phagocytosis. The process is relatively rapid lasting
2–12 hours. The sequence of events during apoptosis requires
energy and follows an internally determined program involving
the activation of endonucleases which degrade chromosomal DNA
into oligosomal fragments that are multiples of 180–200
base pairs in lengths (which form DNA ‘ladders’
after electrophoresis on agarose gels)
[42, 43]. The program
also involves the expression of multiple genes which promote
or hinder the death pathway (e.g. Bcl-2 or Bax) and the expression
of genes for proteases (e.g. CPP-32) involved in cell destruction
[44, 45].
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8 Apoptosis and iNOS expression in cardiac allograft rejection
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Albina and coworkers have reported that NO produced by iNOS
in cytokine-treated macrophages can trigger apoptosis of the
macrophages and also of certain tumor cells cocultured with
the macrophages
[22, 46]. Drugs that release NO have also promoted
macrophage apoptosis. Accordingly, Szabolcs et al. used the
Lewis to Wistar–Furth heterotopic heart transplantation
model to investigate whether apoptosis occurs during cardiac
allograft rejection and whether the time course of apoptosis
was related to the induction of iNOS
[47]. They found that significant
apoptosis of macrophages and of cardiac myocytes occurred during
cardiac allograft rejection. Apoptosis was identified by in
situ labeling of DNA fragments in cell nuclei by two methods
and also by the presence of DNA ladders on agarose gels. Apoptotic
cardiac myocytes were distinguished by their characteristic
striations, immunoreactivity for muscle actin and from the appearance
of intact shrunken myocytes with intact membranes and hyperchromatic
or fragmented nuclei (
Fig. 1a). In the rejecting allografts
the number of apoptotic nuclei increased sharply during days
3–5 after transplantation and at day 5 there was a significant
increase in apoptotic nuclei (macrophages, endothelial cells
and cardiac myocytes) in comparison to syngeneic grafts. The
expression of iNOS mRNA (RT-PCR), protein and enzyme activity
in the allografts paralleled in time and extent the apoptosis
of cardiac myocytes. iNOS immunostaining of macrophages and
cardiac myocytes also increased at days 3–5 and was accompanied
by positive immunostaining of cardiac muscle cells with an antibody
to nitrotyrosine which is indicative of the formation of peroxynitrite
(
Fig. 1b and c). The data are compatible with but do not prove
the hypothesis that NO (and/or peroxynitrite) can trigger apoptosis
of macrophages and cardiac myocytes in this setting. Additional
mechanisms such as interaction of cytotoxic T lymphocytes or
cytokines with the Fas protein or the activation of cysteine
proteases by granzyme-β released from CD8+ T-lymphocytes
may also contribute to apoptosis during rejection
[43, 45].
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Fig. 1 Rejection in rat heterotopic cardiac allograft labelled for apoptosis (blue nuclei) and cardiac myocytes (brown cytoplasm) (a), iNOS (brown) (b) and nitrotyrosine (brown) (c). a: Cardiac myocytes (brown) adjacent to macrophage-rich inflammatory infiltrates show the highest rate of apoptotic nuclei (blue) (arrow). Many apoptotic nuclei are already phagocytosed by macrophages, which prevents determination of their cellular origin. b: Numerous macrophages show strong reactivity for iNOS (brown). Adjacent cardiac myocytes also show weak iNOS labelling. c: Nitrotyrosine which is formed after exposure of cellular proteins to peroxynitrite is present in areas of myocyte damage and inflammation.
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Other more recent studies have indicated that the NO-donor drug
S-nitroso-N-acetyl-penicillamine (SNAP) but not NAP triggered
apoptosis of isolated rat cardiac myocytes in vitro
[48]. This
effect was confirmed by identification of oligonucleosomal DNA
fragments (‘ladders’) on agarose gel electrophoresis
and was inhibited by the addition to the incubation media of
reduced hemoglobin which scavenges NO released by the drug.
Treatment with SNAP (but not NAP or SNAP plus reduced hemoglobin)
was also associated with the expression of p53, the gene product
which can be activated by DNA strand breaks to initiate apoptosis.
NO has been shown to inhibit DNA synthesis and ribonucleotide
reductase and both NO and peroxynitrite have been observed to
cause DNA damage and strand breaks
[49, 50].
In order to investigate whether apoptosis and increased expression of iNOS occur in human cardiac allograft rejection Szabolcs et al. compared right ventricular endomyocardial biopsies from 30 patients with cardiac allograft rejection (ISHLT grade 3A/B) with 12 biopsies without rejection (ISHLT grade 0) using in situ end labeling of intranuclear DNA fragments and immunohistochemistry [51]. Apoptosis of both macrophages and of cardiac myocytes was observed in the rejecting allografts at a rate that was 30 fold higher than that observed in the non-rejecting controls (Fig. 2a). Apoptosis of myocytes was present in both areas with macrophage-rich inflammatory infiltrates and in areas with no inflammation. Positive immunostaining for iNOS was present in macrophages, mononuclear cells and cardiac myocytes in the rejecting allografts Fig. 2b). Immunostaining for nitrotyrosine, a marker of peroxynitrite, was found in the rejecting biopsies in regions where iNOS protein expression was also present (Fig. 2c). The data indicate that apoptosis is a major form of cardiac myocyte death during human cardiac allograft rejection and that it occurs in association with the expression of iNOS and nitration of proteins indicative of the presence of peroxynitrite.
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Fig. 2 Human cardiac allograft rejection labelled for apoptosis (blue nuclei) and cardiac myocytes (brown cytoplasm) (a), iNOS (brown) (b) and nitrotyrosine (brown) (c). a: Similar to the experimental results in rat apoptosis of cardiac myocytes (arrow) occurs predominantly adjacent to macrophage-rich inflammatory infiltrates in human allograft rejection. b: Macrophages (arrow) and adjacent cardiac myocytes show strong reactivity for iNOS (brown). c: Damaged cardiac myocytes (arrow) show immunoreactivity for nitrotyrosine (brown) representing exposure to peroxynitrate.
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9 iNOS expression in myocardial infarction and cardiomyopathy
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As discussed previously, Lewis et al. found that the expression
of iNOS mRNA in endomyocardial biopsies of human heart transplant
recipients was inversely related to left ventricular performance,
i.e. ejection fraction
[15]. Since apoptosis produces the gradual
loss of cardiac myocytes, it is possible that apoptotic myocyte
loss triggered by NO may contribute to the decline in ventricular
function which is observed in many cardiac allografts over time.
iNOS has also been demonstrated in ventricular tissues from
animals with reperfusion injury or myocardial infarction and
from patients with idiopathic dilated cardiomyopathy
[52–56].
In addition, recent studies have demonstrated that apoptosis
of cardiac myocytes occurred in iNOS-rich regions at the margins
of acute myocardial infarctions in rabbits
[52]. Other studies
have independently documented iNOS enzyme activity, iNOS mRNA
and protein in cardiac myocytes in myocardial tissues from patients
with idiopathic dilated cardiomyopathy many of whom have elevated
circulating levels of cytokines and a macrophage-rich myocardial
inflammatory infiltrate
[53, 54]. Additional recent reports
have documented that apoptosis of cardiac myocytes occurs in
ventricular tissue from patients with idiopathic dilated cardiomyopathy
who died of advanced congestive heart failure
[57, 58]. Conceivably
apoptosis of heart muscle cells triggered by iNOS may contribute
to the progression of ventricular failure in these and other
inflammatory diseases of the myocardium.
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10 Possible therapeutic implications in allograft rejection and other cardiac disorders
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Apoptosis of cardiac myocytes in association with iNOS expression
in allograft rejection may have several therapeutic implications.
First, it raises the possibility that drugs which selectively
inhibit iNOS enzyme activity or which interfere with NO production
by macrophages or cardiocytes might be beneficial in reducing
myocyte loss during allograft rejection or other diseases such
as dilated cardiomyopathy. In a rat model, aminoguanidine, a
semiselective inhibitor of NO production by iNOS but which also
has other effects, prolonged survival and reduced the pathological
changes in the grafts
[10, 11]. In preliminary studies of our
rat cardiac transplantation model, Yang et al. administered
to the allograft recipients CNI-1493, a tetravalent guanylhydrazone
compound which blocks
L-arginine transport into macrophages
inhibiting iNOS synthesis of NO and which also blocks the release
of TNF-
and other cytokines from macrophages
[59–61].
In comparison to vehicle treated control allografts, administration
of CNI-1493 was associated with prolongation of survival, significant
reduction of apoptosis, myocyte loss and macrophage infiltration
of the myocardium
[59]. Second, the finding of apoptosis of
cardiac myocytes in association with iNOS raises the possibility
that one could use molecular techniques such as transfection
of Bcl-2 to alter the apoptotic gene program favorably to preserve
myocyte viability
[62]. Finally, it suggests that one might
reduce cardiac myocyte death by developing inhibitors or inactivators
of the proteases (caspaces) such as CPP32 which are involved
in the final stages of apoptotic destruction of the cardiac
myocytes
[63, 64].
Time for primary review 29 days.
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Acknowledgements
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This work was supported in part by NIH grant HL 54764.
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1 NO synthesis
2 iNOS induction in...