Cellular and molecular basis of inflammatory myocardial disease

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Inflammatory disease of the myocardium can inducedysfunction of the vascular system and the cardiacmyocyte by both humoral and cellular mechanisms.1,2

Inflammatory processes are clinically encountered mostcommonly in patients with cardiac allograft transplantrejection and in viral myocarditis, but they are also pres-ent in other types of myocarditis including bacterial andautoimmune types of inflammation. This article reviewsthe inflammatory cells involved and the molecular mech-anisms that produce myocyte injury and dysfunction,focusing primarily on transplant rejection and viralmyocarditis.

CLINICAL DETECTION OF INFLAMMATORYMYOCARDIAL DISEASE

The gold standard for detection of myocardialinflammation remains the endocardial biopsy. In thistechnique a catheter bioptome is inserted into the rightventricle, usually via the right internal jugular vein, butoccasionally via the femoral vein, and small pieces ofmyocardium are obtained from the right ventricular sideof the interventricular septum. The specimens thusobtained are examined histologically for evidence ofinflammation, and there are specific histologic criteriafor the diagnosis of acute myocarditis,3 for transplantrejection,4 and for grading of the severity of theseprocesses.

Because of the invasive nature and associated risk anddiscomfort involved in endocardial biopsy, there has beenconsiderable effort devoted to developing noninvasivetechniques to monitor for rejection and/or myocarditis.5

The most promising of these are imaging with gallium 67cardiac imaging to detect inflammatory cell infiltration of

the heart6 and imaging with indium 111–labeled antimyosinantibodies to detect myocyte lysis.7 Unfortunately, at the pre-sent time, neither of these techniques nor other noninvasivemethods such as echocardiography and electrocardiogra-phy have proved to be sufficiently sensitive and specific toallow elimination of the need for endocardial biopsy.However, in the future, use of escort aptamers coupledwith radionuclides8 may substantially improve the accu-racy of noninvasive methods for detection of myocardialinflammation.

CELLULAR COMPONENTS OF MYOCARDIALINFLAMMATION

Examples of the types of inflammatory infiltratesthat occur in various animal models of transplant rejec-tion,9,10 viral myocarditis,11 and autoimmune myocardi-tis12 are shown in Table 1. The most numerous cellsinvolved are lymphocytes and macrophages, with poly-morphonuclear leukocytes generally constituting asmaller fraction of the inflammatory infiltrate. Althoughthe relative numbers of these inflammatory cells vary indifferent severities, stages, and types of immune reac-tions, and other cell types such as eosinophils and giantcells can also be found, these are the principal effectorcells. In the next sections, we discuss how these cellularcomponents of inflammatory response interact and themechanisms by which they produce myocyte injury, dys-function, and death.

LYMPHOCYTES

Lymphocytes consist of both so-called helper T lym-phocytes (HTLs, CD4+) and cytotoxic T lymphocytes(CTLs, CD8+). HTLs (CD4+), via their T-cell receptor-CD3 complex, recognize endocytosed peptides that arebound to major histocompatibility complex (MHC) classII molecules on antigen-presenting cells. CTLs (CD8+)recognize endogenously synthesized peptide antigensassociated with MHC class I molecules on the target cell.Natural killer (NK) lymphocytes recognize viral antigensand/or immunoglobulin G molecules on the surface oftarget cells. Binding of the HTL and CTL CD3 complex

TOPICS IN MOLECULAR BIOLOGY

Cellular and molecular basis of inflammatorymyocardial disease

William H. Barry, MD

From the Division of Cardiology, University of Utah Health SciencesCenter, Salt Lake City, Utah.

Reprint requests: William H. Barry, MD, Division of Cardiology,University of Utah Health Sciences Center, 50 N Medical Dr, SaltLake City, UT 84132; [email protected].

J Nucl Cardiol 2001;8:499-505.Copyright © 2001 by the American Society of Nuclear Cardiology.1071-3581/2001/$35.00 + 0 43/1/116166doi:10.1067/mnc.2001.116166

to MHC antigens is supported by accessory mole-cules.13,14 CD4+ HTLs are essential for cardiac rejec-tion15 in which the immune reaction is initiated whenHTLs recognize peptides expressed on MHC class IIreceptors primarily on endothelial cells in the donor vas-cular system.16 In the case of viral myocarditis, viral anti-gens expressed on the surface of the cell may be recog-nized. Recognition and binding to target cell surfaceantigens produce activation of the lymphocyte, a processthat involves an increase in phospholipase C activity anda rise in intracellular free Ca2+ ([Ca2+]i) due to both aninositol triphosphate (IP3)–induced Ca2+ release fromintracellular stores and an increase in Ca2+ influx via anintracellular Ca2+ store–dependent pathway.17 This risein Ca2+ activates a Ca2+-calmodulin–dependent phos-phatase, calcineurin, which dephosphorylates nuclearfactors of activated T cells (NFATs).18 These dephospho-rylated NFATs then enter the nucleus, where they activateimmune regulatory genes and initiate cytokine produc-tion. The commonly used immunosuppressant drugscyclosporin A and FK506 inhibit the effect of calcineurinon NFATs.19

There are 2 subgroups of HTL, which can be distin-guished by the cytokines they secrete. So-called TH1HTLs produce interleukin (IL) 2, interferon γ (INF-γ),and lymphotoxin, which promote CTL development anddelayed-type hypersensitivity responses. TH2 HTLs pro-duce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, whichcause humoral, allergic, and mucosal responses.20-22 IL-2 produced by the activated HTL cell in an autocrinefashion stimulates further production of IL-2 and also ofINF-γ. This cytokine stimulates expression of variousadhesion molecules on endothelial cells, which results inattachment of additional neutrophils, lymphocytes, andmonocytes, which then migrate through the vascularendothelial layer into the extravascular tissue space.Monocytes are stimulated by INF-γ to differentiate intomacrophages. CTLs, which are recruited to the tissue bythe above mechanisms, interact with the target cell MHCclass I surface antigen, the expression of which is upreg-ulated by INF-γ. The CTL and its cousin the NK cell,

which recognizes viral antigens expressed on the surfaceof the target cell, as well as macrophages and neutrophils,can cause injury and/or dysfunction of the myocyte by avariety of mechanisms.

MOLECULAR MECHANISMS OF MYOCYTEINJURY AND DYSFUNCTION

When CTL and NK cells are activated by binding toa specific antigen on the surface of the target cell, theycan produce injury of the cell by several different path-ways (Figure 1). After conjugation to the myocyte target,the CTL becomes activated by a rise in [Ca2+]i. Thiscauses cytotoxic granule exocytosis. Cytotoxic granulescontain perforin and serine esterase.23 Exocytosed per-forin monomers reassemble as a polymer in a calcium-dependent manner in the target cell membrane. Thispolymer forms an nonselective ion channel and alsoallows water to enter the cell and thus osmotically leadsto cell swelling and eventually cell lysis. Granzyme B, aserine protease, is exocytosed from CTL together withperforin and can cross the target cell membrane in a per-forin-independent, energy-dependent mechanism.24

Perforin is required for granzyme B to enter the nucleus,where it mediates apoptosis through an IL-1–convertingenzyme–dependent pathway. For discussion of the mech-anisms of apoptosis, see references 2 and 25.

Results from our laboratory are consistent with theimportance of CTL degranulation–dependent myocytetarget cell injury.26,27 Allosensitized CTLs were able todecrease the amplitude of myocyte motion, calcium tran-sients, and action potential amplitude in cultures ofdonor-type fetal ventricular myocytes, and the diastolicmembrane potential was decreased. These effects wereabolished by pretreating lymphocytes with 4,4´-diiso-thiocyano-2,2´ disulfonic acid stilbene (DIDS), aninhibitor of lymphocyte degranulation. This work is alsoconsistent with results from Binah et al,28 who demon-strated that granules from cytotoxic lymphocytes couldinduce similar electrophysiologic changes (shortening ofaction potential duration and reduction of resting poten-

500 Journal of Nuclear CardiologyJuly/August 2001

BarryCellular and molecular basis of inflammatory myocardial disease

Table 1. Inflammatory cells in myocarditis

Lymphocytes Macrophages NeutrophilsAuthors (%) (%) (%) Animal model

Tilney et al9 75 15-25 5 Rat heterotopic heart transplantationWagoner et al10 44 36 20 Mouse heterotopic heart transplantationHenke et al11 ~50* ~45 ~5 Coxsackievirus myocarditis in CD4 knockout

mouseSmith and Allen12 ~30 ~30 ~30 Murine myosin–induced myocarditis

*Of lymphocytes, 30% to 50% are NK cells.

tial and action potential amplitude followed by a pro-gressive contracture and elevation in cytosolic calciumassociated with cell lysis) in isolated adult ventricularmyocytes.

CTLs may also injure target cells via the Fas/Fas-ligand pathway. Fas ligand on the CTL can induce apop-tosis in Fas-bearing target cells. Evidence for additionalmechanisms of Fas-mediated myocyte damage comesfrom a recent study by Felzen et al.29 They showed the

presence of Fas on freshly isolated murine ventricularmyocytes. Alloreactive peritoneal exudate CTLs, derivedfrom perforin gene knockout mice and co-incubated withdonor-specific myocytes, caused decreased myocyte rest-ing membrane potential, decreased action potentialamplitude, prolonged action potential duration, diastolic[Ca2+]i increase, and delayed afterdepolarizations. All ofthese effects could be reproduced by an anti-Fas mono-clonal antibody, Jo2. Their data suggest that IP3 is a sec-

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501

Figure 1. Mechanisms of CTL-mediated myocyte injury. Binding of the CTL to the myocyte induces activation of theCTL associated with a rise in [Ca2+]i. This causes release of cytotoxic granules, containing perforin and serine esterase.Perforin induces depolarization, decreased contractility, and contracture, whereas serine esterase can induce myocyteapoptosis. The activated CTL also expresses Fas ligand, which interacts with the Fas receptor on the myocyte. This caninduce intracellular production of IP3, with a subsequent rise in myocyte [Ca2+]i and depolarization. See text for details.

Figure 2. Mechanisms of macrophage-mediated myocyte injury. Macrophages become activated largely by an effect ofINF-γ, produced by HTLs. Activated macrophages produce a variety of inflammatory cytokines. IL-1 (IL-1β) and IL-6cause production of cytokine inducible NO synthase (iNOS, or NOS2) within the cardiac myocyte. NOS produces NO,which activates guanylyl cyclase, producing cyclic guanosine monophosphate (cGMP). cGMP activates protein kinaseG, which phosphorylates components of the L-type Ca2+ channel and contractile elements, decreasing the Ca2+ current(ICa,L) and decreasing the sensitivity of contractile elements to [Ca2+]i, respectively. Macrophages also produce TNF-α. TNF-α can also induce NOS2 and also activates sphingomyelinase, which produces sphingosine. Sphingosine reducesCa2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). The [Ca2+]i transient is reduced by the decrease in theCa2+ current and the impaired SR Ca2+ release, and the reduced [Ca2+]i transient and the decreased sensitivity of the con-tractile elements to Ca2+ can decrease the force of contraction of the myocyte. Macrophages may also affect myocytesby production of free radicals and NO and may recruit neutrophil, which can also injure myocytes by free radical pro-duction. See text for details.

ond messenger in this Fas pathway: blocking IP3 withheparin prevented myocyte dysfunction induced via Fas,whereas applying IP3 by means of a patch pipette directlyinto the myocyte could reproduce Fas-induced changes.It has been shown that the injury produced by these inter-actions of CTLs with myocytes is initially reversible, butif allowed to proceed for a period of time, they cause irre-versible cell injury with cell lysis.26

Although it is well established that CTLs can producemyocyte injury in vitro,26,27 the significance of CTL-medi-ated injury in vivo is less certain. Although it has beendemonstrated in human cardiac transplant biopsy speci-mens that the expression of granzyme and perforin corre-lates with rejection,30 when heart infiltrating cells isolatedfrom cardiac allografts were used as effector cells, only asmall portion of myocyte cytotoxicity caused by heartinfiltrating cells could be prevented by CTL depletion.10

Also, Menon et al31 showed that although in vivo deple-tion of CTL improved function of the rejecting murineheart early (at 5 days after transplant) during the rejectionprocess, both Chan et al32 and Menon et al31 found that invivo depletion of CD8+ cells in the recipient animal,which virtually eliminated CTLs from the infiltrating cellpopulation, did not cause prolonged survival of the allo-graft. Taken together, these data clearly demonstrate thatCD8+ cells can cause myocyte injury but their contribu-tion to myocyte necrosis during advanced heart transplantrejection in vivo may be relatively minor; other injurymechanisms are certainly involved.

MACROPHAGE-DEPENDENT CELL INJURY

As shown in Table 1, macrophages are also presentin large numbers in myocardial inflammatory lesions,and considerable experimental work indicates that thesemay contribute to myocyte dysfunction and injury by avariety of mechanisms, as outlined in Figure 2.Christmas and MacPherson33 have demonstrated thatmacrophages infiltrating a rejecting transplanted ratheart inhibited spontaneous contractions of culturedneonatal rat ventricular myocytes. Balligand et al34 haveshown that impaired contractile responses of rat ventric-ular myocytes to catecholamine stimulation can beinduced by exposure to medium conditioned by acti-vated macrophages. This effect on catecholamineresponsiveness was blocked by the presence of the nitricoxide (NO) synthase inhibitor L-N-monomethyl-argi-nine. Balligand et al concluded that the cytokines pro-duced by activated macrophages induced NO synthesisin ventricular myocytes and that NO inhibited theinotropic response to catecholamines, possibly by acti-vation of soluble intracellular guanylate cyclase. Themechanisms whereby cytokines increase NO and by

which NO alters myocyte function have been well sum-marized by Kelly et al.35

Not all of the negative inotropic effects ofmacrophage-derived cytokines are due to enhanced pro-duction of NO. Yokoyama et al36 found a rapid directnegative inotropic effect caused by tumor necrosis factorα (TNF-α) in feline myocardium, in both intact ventri-cle and isolated cardiac myocytes. They reported that incat myocytes, this negative inotropic effect of TNF-αwas associated with a decrease in the calcium transientbut no change in the calcium current, and was not inhib-ited by NO synthase blockers. Oral et al37 were able toshow in isolated adult feline myocytes that TNF-α treat-ment led to a rapid increase of free sphingosine mediatedby a cytokine-induced activation of neutral sphin-gomyelinase. Sphingosine applied to isolated myocytesmimicked the immediate effect of TNF-α, and blockingsphingosine production with oleoylethanolamine com-pletely blocked the TNF-α effect. McDonough et al38

have demonstrated in adult and neonatal rat ventricularmyocytes by whole cell patch clamping that sphingosinedirectly inhibits L-type calcium current in a dose-depen-dent fashion. Furthermore, the threshold for ryanodinereceptor calcium-induced calcium release is increased,providing a mechanism for the decrease in the [Ca2+]itransient and the negative inotropic effect induced byTNF-α.

Macrophages can also induce lysis of cardiacmyocytes, an effect that may involve production of NO.Pinsky et al39 have shown that J774 macrophages acti-vated by exposure to INF-γ and lipopolysaccharidescause lysis of isolated adult rat cardiac myocytes.This was inhibited by addition of the competitive NOsynthesis inhibitor L-N-monomethyl-arginine to the cul-ture medium. These findings are consistent with thereport by Higuchi et al40 that TNF-α production and L-arginine–dependent (NO) mechanisms act synergisti-cally as a major cytolytic mechanism of isolatedmacrophages when assessed against L929 and P815target cells. Hibbs et al41 have shown that L-arginine–dependent NO production by activated macrophagescan induce inhibition of mitochondrial respiration insome target cells. Macrophages may also produce freeradicals, which can cause cell injury.42

It is not entirely clear which of these various effectsof macrophages is of the most importance in causing dys-function in intact myocardium. However, recent studiesfrom our laboratory have demonstrated that NOS inhibi-tion can almost completely inhibit the early decrease incardiac contractility that occurs during transplant rejec-tion,31 and myocytes isolated from acutely rejectingmurine heterotopic transplants, in which the contractilityis severely depressed, show an L-arginine (NO)–dependent



decrease in the sensitivity of the contractile elements toCa2+, with no decrease in the [Ca2+]i transient.43 Thesefindings suggest that, at least in this model, an NO-induced decrease in contractile element Ca2+ sensitivity(Figure 2) is a major factor in the depressed functionobserved in vivo during early rejection.

EFFECTS OF NEUTROPHILS ON MYOCYTES

As shown in Table 1, neutrophils also constitute aminor component of the inflammatory response in car-diac rejection and viral myocarditis. It is clear that neu-trophils can also cause direct injury of cardiac myocytes,and this effect may be modulated by cytokines. Exposureof adult canine cardiac myocytes to cytokines such as IL-1 and TNF-α produces surface expression of intercellularadhesion molecule 1 (ICAM-1) on the myocyte.44

Subsequent exposure to activated neutrophils allows aneutrophil-myocyte adhesion mediated by the CD18/ICAM-1 interaction. This induces myocyte contractureassociated with intracellular oxidation as monitored bythe fluorescent probe dichlorofluorescein.45 Fluorescenceand myocyte injury could be inhibited by the intracellu-lar hydroxyl radical scavenger dimethylthiourea. Thisexpression of the Mac-1 (CD11B/CD18) complex onneutrophil and ICAM-1 on the myocyte appears to resultin activation of neutrophil respiratory burst, which resultsin a compartmentalized iron-dependent myocyte oxida-tive injury. The combined effects of macrophages andneutrophils in inflamed tissue constitute a delayed-typehypersensitivity response.

RELATIVE IMPORTANCE OF DIFFERENT INJURYMECHANISMS IN REJECTION

It is not clearly apparent which of these various cel-lular injury mechanisms is most important in producingmyocyte necrosis during transplant rejection. As dis-cussed, it has been shown in transplant rejection thatdepletion in recipient animals of CD8+ lymphocytes pro-duces only a partial improvement in function early dur-ing the rejection process and does not prolong survival ofthe transplanted graft.31 In addition, it has been shownthat heart infiltrating cells isolated from rejecting heartsproduce a substantial lysis of cardiac myocytes in vitro,but only a small portion of this injury is attributable toCTLs present in the heart infiltrating cell population.10

These findings taken together suggest that although CTL-mediated myocyte injury occurs during rejection, it is notthe principal mechanism of cell injury and dysfunction.Although there is considerable evidence that NO is animportant mediator of early functional depression duringtransplant rejection,31,46 complete graft rejection can

occur later in the rejection process with complete loss offunction, despite inhibition of NOS.31,47

Studies in a variety of transgenic mouse models haveprovided information on the importance of different path-ways thought to be involved in the rejection process. Forexample, Schowengerdt et al48 showed that knockout ofICAM-1, a receptor involved in neutrophil-inducedmyocyte injury, did not affect cardiac allograft survival.Also, Djamali and Odorico49 showed that knockout ofFas did not prevent rejection of murine nonvascularizedcardiac allografts. Although Ullrich et al50 found thatknockout of cytokine inducible NOS (iNOS, or NOS2) inmice reduced (presumably cytokine-dependent) contrac-tile abnormalities induced by endotoxin, Laubach et al51

reported that mice lacking iNOS were not resistant tolipopolysaccharide-induced death. These studies arecomplicated by the possibility that genetic knockout ofone pathway may result in upregulation of an alternativepathway for producing injury during the immuneprocess, but these findings indicate that a multitude ofmechanisms, some still undefined, are involved inmyocyte injury and dysfunction during the compleximmune response associated with rejection.

RELATIVE IMPORTANCE OF DIFFERENT INJURYMECHANISMS IN VIRAL MYOCARDITIS

The above discussion has focused principally ontransplant rejection because a great deal of experimentaldata exist with respect to this model. However, it is clearthat other additional mechanisms can be involved inmyocardial functional abnormalities and injury thatoccur as a consequence of myocarditis. It has beenknown for some time that the virus infection itself canlyse cardiac myocytes52 and that myocytes can be injuredby CTLs that are sensitized to viral antigens.53 However,the relative importance of these 2 processes has been indoubt. Henke et al11 have studied the relative importanceof direct virus-induced myocyte injury and immune-mediated myocyte injury in coxsackievirus B3 infection.Immunocompetent C57 BL/6 mice were highly suscepti-ble to early lethal injury (within 1 week), whereas micerendered somewhat immune incompetent by knockout ofCD4 were less susceptible. In vivo depletion of CD8+

CTLs in CD4 knockout mice caused even less suscepti-bility to lethal injury, with a marked reduction inmyocarditis, but with an increase in myocardial viraltiters. These interesting findings suggest that CTLs (andNK cells) help eliminate virus from the infected heart,but in the process of doing so they induce a markedmyocarditis that is detrimental to cardiac function.

It has long been suspected that in a high percentageof cases, idiopathic dilated cardiomyopathy may be a



503

consequence of previous viral infection. Areas of cyto-toxic lymphocytes may be found in a high percentage ofpatients with idiopathic dilated cardiomyopathy,54 andviral genomic persistence may be documented by poly-merase chain reaction.55,56 Thus impaired function ofmyocardium in idiopathic dilated cardiomyopathy couldbe due in part to a persistent, low-level immune reactionmediated by CTLs. However, recent work by Knowltonand associates57 has suggested another possibility. Theseinvestigators showed that transgenic expression of repli-cation-restricted enteroviral genomes in heart muscleinduces a dilated cardiomyopathy in mice. Subsequentstudies have suggested that enteroviral protease2A–induced cleavage of the cytoskeletal protein dys-trophin may be the mechanism by which cardiomyopathyis produced.58 Interestingly, NO inhibits dystrophin pro-teolysis by protease 2A by an S-nitrosylation of theenzyme.59 Thus, although NO production during viral-induced myocardial inflammation may depress myocar-dial function by the mechanisms discussed earlier, it mayprotect from long-term deleterious effects of incorpora-tion of viral genomic material into the cardiac myocyte.

In summary, much progress has been made in under-standing the molecular mechanisms of myocyte injurythat occur during myocardial inflammation. Future use ofpowerful molecular biologic approaches to this problemshould further enhance the ability of clinicians to diag-nose, treat, and prevent cardiac dysfunction due tomyocardial inflammation associated with transplantrejection and myocarditis.

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