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J Mol Cell Cardiol 34, 11471161 (2002) doi:10.1006/jmcc.2002.2056, available online at http://www.idealibrary.com on 1 Review Article Cytotoxic Lymphocytes and Cardiac Electrophysiology Ofer Binah Rappaport Family Institute for Research in the Medical Sciences, Bruce Rappaport Faculty of Medicine, The Bernard Katz Minerva Center for Cell Biophysics, Technion-Israel Institute of Technology, Haifa 31096, Israel (Received 10 May 2002, accepted for publication 16 May 2002) O. BINAH. Cytotoxic Lymphocytes and Cardiac Electrophysiology. Journal of Molecular and Cellular Cardiology (2002) 34, 1147–1161. It is widely recognized that immune effector mechanisms contribute to cardiac dysfunction in major cardiac pathologies, such as myocarditis and the consequent dilated cardiomyopathy, Chagas’ disease and heart transplant rejection. Of the wealth of immune mechanisms known to affect cardiac function, this review will deal with the adverse effects caused by cytotoxic T lymphocytes (CTL, CD4 and CD8 T lymphocytes), which participate in a broad range of heart pathologies. The interaction between cytotoxic lymphocytes and their target cells can set off two different effector mechanisms: (1) The perforin/granzymes, and (2) The Fas/FasL. In this review, I will discuss these mechanisms, and present experimental evidence showing that both can adversely affect cardiac myocytes in vitro, in a way that can contribute to a decline in the overall cardiac function. # 2002 Elsevier Science Ltd. All rights reserved. Key Words: Cytotoxic lymphocytes; Ventricular myocytes; Perforin/granzymes; Fas receptor; 1,4,5-inositol triphosphate. Mechanisms of Lymphocytotoxicity Numerous studies have established the important role of immune cellular effector mechanisms in a variety of myocardial pathologies, such as Chagas’ disease, heart transplant rejection, myocarditis and the consequential dilated cardiomyopathy (DCM), in ischemic/reperfusion damage and in myocardial infarction. 1–8 Of the multiplicity of immune mechanisms known to affect cardiac function, this review will deal with the deleterious effects inflicted by cytotoxic T lymphocytes (CTL, CD4 and CD8 T lymphocytes), which contribute to a broad range of heart pathologies. The interaction between cyto- toxic lymphocytes and their target cells can set off two different effector mechanisms: 6,9–11 (1) The perforin/granzymes; (2) The Fas/FasL. In subse- quent sections I will discuss these mechanisms, and present experimental evidence showing that both can adversely affect cardiac myocytes in vitro, in a way that can contribute to a decline in the overall cardiac function. Which adverse effects of cytotoxic lymphocytes and ligands can affect cardiac electrophysiological properties? Alterations in myocardial electrophysiological properties (including excitability) and increased propensity to develop arrhythmias in a variety of disease state (e.g. ischemic myocardium), frequently result from changes in individual ion currents, which participate in action potential generation. For example, attenuation of potassium- repolarizing currents causes prolongation of action potential duration, which can lead to induction of early after depolarizations and triggered arrhyth- mias. Thus, as will be discussed shortly, activation of the Fas receptor in ventricular myocytes is compat- ible with this notion. However, while it is likely that Please address all correspondence to: Ofer Binah, PhD, Rappaport Institute, P.O.B. 9697, Haifa 31096, Israel. Tel: 972-4-8295262; Fax: 972-4-8513919; E-mail: [email protected] 0022–2828/02/091147 15 $35.00/0 # 2002 Elsevier Science Ltd. All rights reserved.

Cytotoxic Lymphocytes and Cardiac Electrophysiology

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Page 1: Cytotoxic Lymphocytes and Cardiac Electrophysiology

J Mol Cell Cardiol 34, 1147ÿ1161 (2002)

doi:10.1006/jmcc.2002.2056, available online at http://www.idealibrary.com on1

Review Article

Cytotoxic Lymphocytes and CardiacElectrophysiologyOfer Binah

Rappaport Family Institute for Research in the Medical Sciences, Bruce Rappaport Faculty ofMedicine, The Bernard Katz Minerva Center for Cell Biophysics, Technion-Israel Institute ofTechnology, Haifa 31096, Israel

(Received 10 May 2002, accepted for publication 16 May 2002)

O. BINAH. Cytotoxic Lymphocytes and Cardiac Electrophysiology. Journal of Molecular and Cellular Cardiology (2002)34, 1147±1161. It is widely recognized that immune effector mechanisms contribute to cardiac dysfunction inmajor cardiac pathologies, such as myocarditis and the consequent dilated cardiomyopathy, Chagas' disease andheart transplant rejection. Of the wealth of immune mechanisms known to affect cardiac function, this review willdeal with the adverse effects caused by cytotoxic T lymphocytes (CTL, CD4� and CD8� T lymphocytes), whichparticipate in a broad range of heart pathologies. The interaction between cytotoxic lymphocytes and their targetcells can set off two different effector mechanisms: (1) The perforin/granzymes, and (2) The Fas/FasL. In this review,I will discuss these mechanisms, and present experimental evidence showing that both can adversely affectcardiac myocytes in vitro, in a way that can contribute to a decline in the overall cardiac function.

# 2002 Elsevier Science Ltd. All rights reserved.

Key Words: Cytotoxic lymphocytes; Ventricular myocytes; Perforin/granzymes; Fas receptor; 1,4,5-inositoltriphosphate.

Mechanisms of Lymphocytotoxicity

Numerous studies have established the importantrole of immune cellular effector mechanisms in avariety of myocardial pathologies, such as Chagas'disease, heart transplant rejection, myocarditis andthe consequential dilated cardiomyopathy (DCM),in ischemic/reperfusion damage and in myocardialinfarction.1±8 Of the multiplicity of immunemechanisms known to affect cardiac function, thisreview will deal with the deleterious effects in¯ictedby cytotoxic T lymphocytes (CTL, CD4� and CD8� Tlymphocytes), which contribute to a broad rangeof heart pathologies. The interaction between cyto-toxic lymphocytes and their target cells can set offtwo different effector mechanisms:6,9±11 (1) Theperforin/granzymes; (2) The Fas/FasL. In subse-quent sections I will discuss these mechanisms,and present experimental evidence showing thatboth can adversely affect cardiac myocytes in vitro,

Please address all correspondence to: Ofer Binah, PhD, Rappaport InFax: 972-4-8513919; E-mail: [email protected]

0022±2828/02/091147�15 $35.00/0

in a way that can contribute to a decline in theoverall cardiac function.

Which adverse effects of cytotoxic lymphocytes andligands can affect cardiac electrophysiologicalproperties?

Alterations in myocardial electrophysiologicalproperties (including excitability) and increasedpropensity to develop arrhythmias in a varietyof disease state (e.g. ischemic myocardium),frequently result from changes in individual ioncurrents, which participate in action potentialgeneration. For example, attenuation of potassium-repolarizing currents causes prolongation of actionpotential duration, which can lead to induction ofearly after depolarizations and triggered arrhyth-mias. Thus, as will be discussed shortly, activation ofthe Fas receptor in ventricular myocytes is compat-ible with this notion. However, while it is likely that

stitute, P.O.B. 9697, Haifa 31096, Israel. Tel: 972-4-8295262;

# 2002 Elsevier Science Ltd. All rights reserved.

Page 2: Cytotoxic Lymphocytes and Cardiac Electrophysiology

1148 O. Binah

direct effects on ion channels can alter cardiacelectrophysiological properties, the question iswhether apoptosis, the `̀ traditional'' outcome ofcytotoxic lymphocyte assault, can also modify car-diac excitability. Whereas to the best of my know-ledge, this question has not been dealt withexperimentally, the following considerations sug-gest that apoptosis can also affect cardiac excitabil-ity: (1) Apoptosis is a slow process that may lastseveral hours,12 and therefore during its courseelectrophysiological properties (e.g. resting poten-tial) may change, thereby affecting tissue excitabil-ity. (2) Apoptosis is patchy in nature (few myocytesare randomly affected within the tissue13), andtherefore dead myocytes dispersed within a healthymatrix are likely to affect the electronic interactionsbetween adjacent myocytes, and thus the propertiesof the propagating action potential.14,15 Hence,these considerations suggest that apoptosis adverse-ly affects cardiac function not only by reducingthe number of mechanically-active myocytes, butalso by altering the electrophysiological propertiesof the myocardial tissue.

The perforin/granzymes mechanism of cytotoxicity

This cytotoxic mechanism, the intricacies ofwhich are still unfolding, involves co-secretion

Figure 1 Two major pathways of lymphocytotoxicity. [Righfrom cytotoxic lymphocytes of lytic granules containing lysthe pore-forming protein perforin, which can induce apoptotinon-secretory pathway: the Fas ligand (FasL) expressed on the(Fas/CD95/Apo-1) expressed by the target cell, an interaction wdamage. Fas activation can also be caused by soluble FasL (#

from lymphocytes of lytic granules containinglysosomal enzymes, serine esterases termed gran-zymes, and the pore-forming protein perforin (Fig. 1,right side). Following conjugate formation betweenlymphocytes and their target cells, and activation ofthe former, lytic granules migrate towards thelymphocyte plasma membrane, fuse with it andrelease their content into the lymphocyte±target cellintercellular cleft.16,17 A major active componentof lytic granules is perforin, a 70 kDa protein whichis structurally related to the C9 component of thecomplement system.18,19 Perforin is found in avariety of cell types, most commonly in naturalkiller (NK) cells, activated CTL (CD8�) and in themajority of cytotoxic CD4�.20±22 According to thetraditionally-held view [e.g. ref. 23], in the presenceof calcium ions, perforin polymerizes (5±20 mono-mers), into ring-like transmembrane channels(16±20 nm in diameter), through which granzymesand other lytic granule constituents can penetratethe affected cell. According to this paradigm,target cell demise is caused by: (1) a complement-like transmembrane damage causing target celldestruction; (2) granzymes which enter the targetcell through the perforin channel, activate theapoptotic machinery which causes unavoidablecell death.11 During the past several years, thismodel has been questioned, mainly based ontwo distinct observations: (1) the discovery that

t side] The secretory pathway which involves co-secretionosomal enzymes, serine esterases termed granzymes andc as well as non-apoptotic myocyte damage. [Left side] Thesurface of killer lymphocytes (#1) binds to the Fas receptorhich can result in apoptotic and in non-apoptotic myocyte

2), as well as by FasL expressed on the same cell (#3).

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1149Cytotoxic Lymphocytes and Cardiac Electrophysiology

perforin-induced membrane damage was notsuf®cient to cause apoptosis,24 which is charac-teristic of CTL assault; (2) the size of the perforinchannel is too small to allow passage of largemolecules, such as granzymes.25 Based on theseobservations as well as on other key studies, dif-ferent perforin-dependent and perforin-independentmodels for granzymes entry have been pro-posed.9,10,26±29 According to the perforin-dependentversion of this model,30 granzymes enter the cell viaa process termed ``reparative endocytosis''. As aresult of perforin incorporation into the membrane,and creation of a signal for the target cell to repairthe perforin-induced damage, the cell endocytosesthe perforin and the surrounding plasma mem-brane, including the granzymes present in proxi-mity to the perforin channel, which then trigger theapoptotic machinery. According to the perforin-independent model, granzymes can enter the targetcell by binding to their cation-independentmannose-6-P receptor (MPR), thereby activatingthe caspases cascade, consequently leading to apop-tosis.26 Although both models are supported byvalid experimental data, it should be stressed that:(1) the relative contribution of each of these modelsto actual granzymes entry into the target cell has notbeen de®ned; (2) since the studies supporting thesemodels were conducted in vitro, their physiologicalrelevance still needs to be established.

Are perforin/granzymes functional in vivo?

The important issues of whether the perforin/granzymes machinery is of physiological relevance,and the relative contribution of its cytotoxicity ina variety of conditions, have not been ®rmlyestablished. A detailed discussion of these topics isbeyond the scope of this article, and the interestedreaders are kindly referred to previous publica-tions.9,10,23,31 The main support for the conceptthat the perforin/granzymes mechanism partici-pates in cardiac pathologies, emanates from studiesdemonstrating that perforin and granzymes areexpressed by heart-in®ltrating lymphocytes, inexperimental disease models as well as in humanimmunopathologies.32±35 For example, in Coxsack-ievirus B3 (CVB3)-induced myocarditis, it wasshown that after viral invasion of the myocardium,the ®rst wave of in®ltrating cells are mainly NK cells,which express several cytokines as well as perforin.5

In®ltrating T lymphocytes are detected in the myo-cardium as the second wave of in®ltrating cells,which coincides with the most severe acute myocytedamage of the myocardium.36 Grif®ths and

co-workers37 have determined in graft-in®ltratinglymphocytes, granzyme A and perforin mRNA,which were utilized as markers for rejection aftercardiac transplantation. Alpert and co-workers38

analyzed biopsies obtained during the ®rst yearpost-transplantation, and found: (1) a correlationbetween the expression of granzyme A and perforin,and (2) that granzyme A expression was associatedwith the rejection score and with the decrease indiastolic function. Finally, a word of caution is inorder, although perforin-expressing lymphocyteshave been detected in vivo, and perforin pores havebeen claimed to be identi®ed in myocardial biopsiesfrom patients with post-viral myocarditis,39 a solidproof for the involvement of perforin-mediatedkilling in vivo is still lacking.

Effect of perforin/granzymes in ventricularmyocytes in vitro

Since perforin-containing lytic granules are poten-tial pathogenic effectors in cardiac pathologies, weexplored their effects on electrophysiological proper-ties of isolated guinea pig ventricular myocytes.Ventricular myocytes exposed to lytic granules orpuri®ed perforin undergo within minutes, typicalelectrophysiological changes, some of which areindicative of [Ca2�]i overload.40,41 These changesinclude decrease of the resting potential and actionpotential amplitude, shortening of action potentialduration, and generation of oscillatory poten-tials, reminiscent of cardiac glycosides- orcatecholamines-induced delayed afterdepolariza-tions (Fig. 2A). In this experimental setting, whereasgranzyme A alone was ineffective, it accelerated thedeleterious effects of perforin.41 These perforin-induced rapid electrophysiological changes inventricular myocytes, which occur in the absenceof granzymes, suggest that the proposed models forgranzymes/perforin action, may be applicable to theapoptotic effects of the lytic granules constituents,but not to the non-apoptotic, yet harmful effects ofperforin. Regardless the granzyme/perforin modelfavored, the elementary action of perforin is toassemble into large polyperforin transmembranechannels in the target cell membrane. Indeed,we found that in guinea pig ventricular myocytes,puri®ed perforin (as well as lytic granules) inducessingle channels with a duration of up to severalseconds, a mean conductance of 860 pS, anda reversal potential of ÿ8.2 mV (Fig. 2). Thesehighly non-selective channels, demonstrated inseveral CTL lines,42 enable massive ion ¯uxes(108±109 ions * secÿ1

* channel), which may result

Page 4: Cytotoxic Lymphocytes and Cardiac Electrophysiology

Figure 2 Electrophysiological effects of puri®ed perforinand perforin-containing lytic granules in guinea pigventricular myocyte. (A) Effect of lytic granules on thetransmembrane action potential. The action potential isshown in control (Tyrode's solution) and at variousintervals after application of lytic granules (5 ml ofgranules into a 0.5 ml recording bath). The bottomright panel is a recording at a slower sweep speed toillustrate oscillations in the membrane potential.Temperature�25�C, cycle length�5 sec. Similar effectswere induced by puri®ed perforin. (B) Single channelsinduced by puri®ed perforin in a guinea pig ventricularmyocyte. Note reversal of current polarity at positivemembrane potentials. Temperature � 25�C. (C) Current±voltage (I±V) relationships of channels induced by perforinin guinea pig ventricular myocyte. Each point representsthe mean (3±15 experiments) of the channel currentamplitudes measured at different membrane potentials.The conductance and reversal potential (Erev) calculatedby means of linear regression analysis are 860 pS andÿ8.2 mV, respectively (P , 0.05). Temperature�25�C.(Reprinted from Refs 40 and 41 with permission fromSpringer-Verlag and Elsevier Science, respectively.)

1150 O. Binah

in diminution of the electromechanical gradients,eventually leading to collapse of the affected cell.A support for this notion is derived by our study inwhich we demonstrated by means of fura 2 imaging,that in perforin-treated guinea pig ventricularmyocytes, [Ca2�]i was increased up to the micro-molar range,40 an effect highly toxic to cardiac

myocytes.43 Interestingly, similar channels areinduced by TNF-a,44 which has deleterious actionsin the myocardium.45 And although these ®ndingssuggest that perforin may cause myocyte damage,their interpretation is limited by the fact that it isunknown how much perforin is delivered to theintercellular space by the in®ltrating lymphocytes,in relation to the exogenous application on theisolated myocytes.

The Fas/FasL pathway of cytotoxicity

The Fas/FasL pathway is composed of two essentialelements: the Fas ligand (FasL), which is constitu-tively expressed by lymphocytes as well as by othercells including myocytes, and the target cell Fasreceptor (Fas/CD95/Apo-1).9,10,46 As a result of theimmune recognition between the effector and thetarget cell, FasL binds to Fas, an interaction whichcan lead to apoptotic as well as to non-apoptoticconsequences (Fig. 1, left side, #1). In brief, Fas is a48 kDa type I-membrane protein which belongs tothe tumor necrosis factor (TNF)/nerve growth factorreceptor superfamily. The Fas ligand (FasL) is amember of the TNF family, and has been identi®ed asa type ll membrane 36 kDa protein, or as a solublecytokine of 26 kDa. Importantly, recent reportssuggest that Fas-mediated effects in myocytes arenot restricted to myocardial diseases in¯icted directlyby cytotoxic lymphocytes, but may also be ofimportance in lymphocyte-independent diseases,such as ischemia/reperfusion injuries.47±49 In thisregard, it was recently proposed that soluble FasL(sFasL) cleaved from FasL by a metalloprotease, maycause apoptosis in susceptible cells (Fig. 1, leftside, #2). Therefore, blood sFasL which is elevatedin a variety of clinical and experimental patho-logies (for example, in advanced congestive heartfailure50) is a potential contributor to the ongoingdisease process. Finally, recent reports suggestthat Fas activation can be caused by FasL expressedon the same cell (Fig. 1, left side, #3).51

The Fas/FasL pathway in myocardial pathologies

Recent studies suggest that activation of theFas/FasL pathway is involved in important cardiacpathologies, such as heart transplant rejection,myocarditis and DCM. For example: Toyozaki andco-workers reported that serum levels of sFasL inhuman patients, increased with the severity of con-gestive heart failure.50 Ishiyama and co-workers52

showed that the Fas/FasL system is involved in the

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1151Cytotoxic Lymphocytes and Cardiac Electrophysiology

pathogenesis of myosin-induced autoimmune myo-carditis in rats. Nonetheless, it should be pointed outthat while there is strong evidence for the involve-ment of the Fas/FasL pathway in myocardialdiseases, the nature of the damage in¯icted by thispathway in vivo, has not been established.

Fas activation in ventricular myocytes in vitro

The prediction that activation of the Fas receptor inmyocytes will cause apoptosis, thereby affectingcardiac function including excitability, is based onnumerous reports showing that in a variety ofnormal and malignant cells, Fas activation causesapoptosis. We have recently tested this prediction(supported by ®ndings showing Fas expression inmyocytes,53,54) by exposing neonatal rat ventricu-lar myocytes (NRVM) to rFasL for 7 h, and measur-ing apoptosis immediately thereafter by the TUNELand DAPI assays. Thus, in contrast to the commonexperience, and in agreement with a recent reportby Wollert's group,53 Fas activation in normoxicmyocytes did not cause apoptosis.55

Figure 3 The adverse electrophysiological consequences(A) Interaction of perforin-de®cient CTL (Pÿ/ÿ CTL) with aaction potentials from a non-conjugated myocyte at t�0 and twith Pÿ/ÿ CTL (lower panel, top traces), immediately after conjtrace illustrates arrhythmogenic activity in a myocyte conjugadepicts a ventricular myocyte conjugated with several lymphoThe arrows indicate lymphocytes attached to a myocyte (1(B) Representative action potentials from control and Jo2-treatedin the absence (upper panel) or presence of 10 mg/ml Jo2 (lowmyocytes) are shown in control non-incubated myocytes (t�0)panel (180 min incubation with Jo2, two right-hand traces), theafter depolarization has developed. (Reprinted from Ref. 56 wit

As will be demonstrated shortly, in contrast to therefractoriness of ventricular myocytes to Fas-basedapoptosis, Fas activation in¯icts pronounced non-apoptotic effects. To study the functional conse-quences of Fas activation, we have developed anallogeneic model of murine ventricular myocytesinteracting with perforin-de®cient CTL (Pÿ/ÿ CTL),which thus affect their target cells via the Fas/FasLpathway.56 This unique experimental settingenabled us to record action potentials, membranecurrents, intracellular Ca2�([Ca2�]i) transients andmyocyte contraction, from a single ventricularmyocyte interacting with cytotoxic lymphocytes.Alternatively, the Fas receptor was stimulateddirectly by means of the activating Fas mAb Jo2.As seen by the representative experiments (Fig. 3),Fas activation markedly altered action potentialcharacteristics: resting potential and action poten-tial amplitude were reduced, action potential dur-ation was prolonged (mainly in Jo2-treated cells),and delayed or early afterdepolarizations developed.In a somewhat related study (although the immuneeffector mechanism was not determined), Ensleyand co-workers exposed spontaneously beating

of Fas activation in murine ventricular myocytes.ventricular myocyte. The ®gure depicts representative�60 min (upper panel), and from a myocyte conjugatedugate formation (t�0) and 60 min later. The lower rightted with Pÿ/ÿ CTL, 60 min after conjugation. The insetcytes, 30 min after lymphocytes were added to the bath.mM Epon section stained with alkaline toluidine blue).

myocytes. Myocytes were incubated for 180 min at 37�Cer panel). Action potentials (recorded from 5 different

and after 180 min incubation (upper panel). In the lowerlower trace depicts an action potential in which an early

h permission from Lippincot, Williams & Wilkins.)

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1152 O. Binah

myocytes from donor strain fetal mice to allosensi-tized CTL, and found a decrease in diastolic mem-brane potential and action potential amplitude, aswell as decreased amplitude of myocyte contractionand [Ca2�]i transients.57

The thought that the Fas/FasL pathway contri-butes to cardiac dysfunction in autoimmune myo-carditis, is supported by our study in which werecorded action potentials and membrane currentsfrom ventricular myocytes from mice with myosin-induced autoimmune myocarditis (Fig. 4).58 In thisexperimental model, the disease was characterizedby massive lymphocyte in®ltration and reduced tailartery blood ¯ow, which is indicative of cardiacfunction. Interestingly, lymphocytes were stronglyattached to isolated ventricular myocytes whichwere enzymatically dissociated from the diseasehearts (Fig. 4B), suggesting that they participate inthe on-going disease process. That the Fas/FasLpathway is involved in autoimmune myocarditis, issuggested by the marked resemblance between theelectrophysiological alterations in the diseased myo-cytes (Fig. 5), and those caused by Fas activation(Fig. 3): resting potential and action potentialamplitude were reduced, APD was increased, andearly afterdepolarizations, occasionally developinginto triggered arrhythmias were generated.

Figure 4 Assessment of the experimental model of myoslongitudinal ventricular sections from a control mouse ansectioned at 7 mm thickness, and stained with hematoxylin±myocyte dissociated from a mouse with myocarditis. The arsurface. (C) Tail artery blood ¯ow in control and diseased mice.laser±Doppler system. (Reprinted from Ref. 58 with permissio

Fas activation attenuates the transient outwardcurrent, Ito

To understand the ionic basis of Fas-mediated APDprolongation and the resulting arrhythmogenesis,we determined how Fas activation affects the tran-sient outward current (Ito) and the L type Ca2�

current (ICa,L), the major contributors to the repo-larization phase of murine ventricular actionpotential.59±62 As depicted in Figs 6A and 6B, Ito

was depressed by Jo2, an effect compatible withJo2-induced APD prolongation and generation ofearly afterdepolarizations. In further support ofthe involvement of the Fas/FasL pathway inmyocarditis, Ito was also attenuated in ventricularmyocytes from diseased mice (Figs 6C, D). Import-antly, these changes are consistent with previousreports on concomitant APD prolongation and Ito

attenuation in ventricular myocytes from cardio-myopathic Syrian hamsters,63 human patients withterminal heart failure,64 and dogs with pacing-induced heart failure,65 suggesting that the Fasactivation may be involved in these diseases aswell. Ito attenuation in Fas-affected myocytes wasfurther pursued by determining whether the effectsof Jo2 on Ito steady state activation and inactivationrelations can account for the observed changes in

in-induced autoimmune myocarditis. (A) Representatived from a mouse immunized with myosin.58 Hearts wereeosin. (B) A light microscopy photograph of a ventricularrows indicate mononuclear cells attached to the myocyteBlood ¯ow was measured in lightly anesthetized mice with an from Academic Press.)

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Figure 5 Electrophysiological alterations in ventricularmyocytes from mice with myosin-induced autoimmunemyocarditis. Representative action potentials are shownfrom control and diseased myocytes. Note that in diseasedmyocytes, action potential prolongation was associatedwith a large early afterdepolarization (middle panel) andtriggered arrhythmias (lower panel). The paced beats aremarked by black dots. (Reprinted from Ref. 58 withpermission from Academic Press.)

Figure 6 The effects of Fas activation with Fas mAb Jo2 and mventricular myocytes. In (A) and (B), recordings were obtainedwith Fas mAb Jo2. (A) Representative Ito traces from a control arelations in control and in Jo2-treated myocytes. (C) RepresenPeak Ito I±V relations in control and in diseased myocytes. Ito waofÿ80 mV, in response to test pulses ranging fromÿ90 mV to�seconds. Peak Ito was measured as the difference between peaktest pulse. (Reprinted from Futura Publishing Company and A

1153Cytotoxic Lymphocytes and Cardiac Electrophysiology

Ito I±V relations.54 As demonstrated in Figure 7A,Fas activation shifted the steady state activationrelations towards positive membrane potentials,and the inactivation relations towards negativemembrane potentials (for further details see the®gure legend and ref. 54). Collectively, these shiftsresult in decreased availability of Itochannels, whichin turn causes in Fas-activated myocytes, a smallerIto generation at depolarized membrane potentials.To determine whether these shifts can account forIto depression, we performed a numerical recon-struction of the fast inactivating component of Ito,which was based on applying the Hodgkin±Huxleytype of formalism to the macroscopic ionic cur-rent.54,59,66 As shown in Fig. 7B, the overall currentcon®guration of the calculated Ito, closely resemblesthe raw Ito traces. At VM��50 mV, Fas activationinduced a 57% reduction in the amplitude of thereconstituted Ito peak current, compared to a 74%reduction of the measured Ito amplitude. The smallerdecrease of peak Ito in the reconstructed vs the

yocarditis, on the transient outward current, Ito in murinefrom control myocytes or from myocytes incubated for 3 hrsnd Jo2-treated myocytes. (B) Peak Ito current±voltage (I±V)tative Ito traces from a control and a diseased myocyte. (D)s generated in myocytes held at a membrane potential (VM)40 mV or�50 mV, at 10 mV intervals, delivered every 10

and steady state outward currents at the end of the 500 mscademic Press, respectively.)

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Figure 7 (A) The effect of Fas activation with Fas mAb Jo2 on Ito steady state activation and inactivation relations.54 Ito

steady state activation relations were constructed by calculating the chord conductance (gk) from peak Ito amplitudescorrected for the driving force (VMÿEk) at each test pulse, and normalized to the maximal chord conductance (gK,max). Thechord conductance at each test pulse was obtained by dividing peak Ito by the driving force of the K� current (VMÿEk). Theexperimental data were ®tted using the Boltzmann equation: gk,max� gk/{1�exp[(V0.5ÿVM)/k]}, where V0.5 is the voltageat the half-maximally activated conductance, and k is the slope factor describing the steepness of the curve. The normalizedchord conductance was plotted against VM for both control and Jo2-treated cells. In control myocytes (n�10), V0.5 and kvalues were (mV):ÿ20.7�0.8 and 16.8�0.9, respectively. In Fas-treated myocytes (n�5), V0.5 and k values were (mV):ÿ6.0�0.6 and 11.8�0.5, respectively. Ito steady state inactivation relations were constructed as follows: currents wereelicited from VH� ÿ80 mV, by 400 ms test pulses ranging from ÿ100 to 0 mV (at 10 mV increments), followed by a400 ms test pulses of �100 mV (stimulation frequency of 0.1 Hz). The currents elicited by a test pulse (I) were normalizedto the maximal current (Imax), and ®tted to the Boltzmann equation, I/Imax�1/{1�exp[(VMÿV0.5)/k]}. In controlmyocytes (n�6), V0.5 and k values were (mV): ÿ51.0�0.7 and 7.4�0.7, respectively. In Fas-treated myocytes (n�5),steady state inactivation relations were shifted towards negative membrane potentials (P , 0.001), withV0.5�ÿ63.3�0.7 mV and k�7.3�0.6 mV. The relations were compared by means of 2-way ANOVA. (B) Numericalreconstruction of the fast inactivating component of Ito (see text for details). Calculated macroscopic Ito is a product of themaximal chord conductance, electromotive force, and activation and inactivation parameters. The numerical simulationwas performed for a holding potential of -80 mV, and for two representative command potentials, 0 and�50 mV. All ioniccurrents were computed for 1 pF of cell membrane capacitance. For further details please see ref. 54. (Reprinted fromRef. 54 with permission from Futura Publishing Company.)

1154 O. Binah

measured current can be partially explained byunderestimating the activation time constantsvalues used for calculating Ito.

Fas activation augments the L typeCa2� current, ICa,L

In contrast to the attenuating effect of Fas activationon Ito, ICa,L was markedly increased in Jo2-treatedmyocytes (Fig. 8); this effect contributes to APDprolongation, generation of afterdepolarizationsand to [Ca2�]i rise. Respecting ICa,L steady staterelations, whereas the activation relations wereunaffected by Jo2, the inactivation relations wereshifted towards positive membrane potentials, aneffect partially accounting for the increased ICa,L,especially at depolarized myocytes.

The 1,4,5-IP3 hypothesis: Involvement of 1,4,5-IP3 and[Ca2�]i rise in Fas-mediated myocyte dysfunction

An essential prerequisite for developing pharma-ceutical tools to control and modulate Fas-mediated

adverse actions, is understanding the signalingpathway(s) triggered by Fas activation. We soughtto understand these pathway(s), by testing theworking hypothesis that Fas activation causes gen-eration of 1,4,5-inositol trisphosphate (1,4,5-IP3),which by binding to 1,4,5-IP3-operated Ca2� -release channels in the sarcoplasmic reticulum(SR), elevates [Ca2�]i, which in turn contributes toFas-mediated adverse actions, including the electro-physiological perturbations (Fig. 9). The hypothesiswas tested by answering three key questions: (1) Is1,4,5-IP3 obligatory for Fas-mediated actions? (2) Is[Ca2�]i increased by Fas activation? (3) Is [Ca2�]i

rise necessary for Fas-mediated effects?

(1) Is 1,4,5-IP3 obligatory for Fas-mediated actions?

This question was addressed by testing whether Fas-mediated effects are (please refer to correspondingnumbers in Fig. 9): (1) duplicated by intracellularadministration of 1,4,5-IP3; (2) prevented by block-ing phospholipase C (PLC)Ðthe 1,4,5-IP3 producingenzyme; (3) prevented by blocking 1,4,5-IP3-operated SR Ca2�-release channel. (1) Intracellular

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Figure 8 The effect of Fas activation with Fas mAb Jo2 on the L type Ca2� current, ICa,L. (A) Representative ICa,L tracesfrom control and Jo2-treated myocytes were generated in myocytes maintained at VH�ÿ50 mV, in response to test pulsesranging fromÿ60 to�50 mV, at 10 mV interval, delivered every 15 sec. (B) ICa,L current±voltage (I±V) relations in controland Jo2-treated myocytes. (Reprinted from Ref. 54 with permission from Futura Publishing Company.)

Figure 9 A scheme summarizing the 1,4,5-IP3 hypothesis (please see text for details). Activation of the Fas receptor (Fas)with Fas ligand (FasL) leads to activation of phospholipase C (PLC) and to the consequent production of 1,4,5-IP3. Thehypothesis was supported by: (1) intracellular application of 1,4,5-IP3; (2) blocking PLC; (3) blocking IP3-operated SRCa2� release channels; (4) demonstrating [Ca2�]i rise; (5) modulating Ca2� release from intracellular stores.Abbreviations: Fas, Fas receptor; FasL, Fas ligand; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate;IP3R, inositol trisphosphate receptor; SERCA, sarcoplasmic-endoplasmic reticular Ca2�-ATPase; SR, sarcoplasmicreticulum; RyR, ryanodine receptor.

1155Cytotoxic Lymphocytes and Cardiac Electrophysiology

administration of 1,4,5-IP3. Intracellular administra-tion via the patch pipette of 1,4,5-IP3, but not of theinactive analogue 1,3,4-IP3, caused electrophysio-logical effects similar to those induced by Fasactivation: resting potential and action potentialamplitude were reduced, APD was prolonged, and

arrhythmias generated (Figs 10A, B).56 Additional-ly, intracellular 1,4,5-IP3 decreased Ito and slightlyincreased ICa,L (Figs 10C, D).54 (2) Blocking phospho-lipase C (PLC)Ðthe 1,4,5-IP3 producing enzyme.Further supporting the 1,4,5-IP3 hypothesis,the speci®c PLC blocker U73122 prevented all the

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Figure 10 Involvement of 1,4,5-IP3 in Fas-mediated electrophysiological changes in murine ventricular myocytes.(A) Representative traces of action potentials recorded at a slow sweep speed from a control ventricular myocyte and froma myocyte to which 2mM 1,4,5-IP3 was administered intracellularly. (B) Effect of intracellular administration of 1,4,5-IP3

on the action potential of a murine ventricular myocyte. Action potentials were recorded from a control myocyte and froma myocyte treated with 2 mmol/l 1,4,5-IP3. (C) and (D) The involvement of the IP3 cascade in the effect of Jo2 on Ito andICa,L. (C) The effect of pretreatment with U73122 (2mmol/l) or xestospongin C (10 mmol/l) on Jo2-induced decreaseof Ito. The ®gure also depicts the effect of intracellular administration of 1,4,5-IP3 (2mmol/l) on Ito. (D) The effect ofpretreatment with U73122 (2 mmol/l) on Jo2-induced increase of ICa,L. The ®gure also depicts the effect of intracellularadministration of 1,4,5-IP3 (2 mmol/l) on ICa,L. 1,4,5-IP3 was included in the patch pipette solution. (Reprinted fromRefs 54 and 56 with permission from Futura Publishing Company and Lippincot, Williams & Wilkins, respectively.)

1156 O. Binah

electrophysiological changes induced by Fas acti-vation, including diastolic [Ca2�]i rise and thearrhythmogenic activity.54,56 Speci®cally, asshown in Figs 10C and 10D, U73122 preventedJo2-induced depression of Ito and increased ICa, L. (3)Blocking the 1,4,5-IP3-operated SR Ca2�-release chan-nel. Heparin which blocks Ca2� release from intra-cellular stores, mainly the SR, and inhibits 1,4,5-IP3

binding to its SR receptors,67,68 attenuated Fas-mediated action potential changes, and the diastolic[Ca2�]i rise.54,56 Further, xestospongin C, a speci®cblocker of 1,4,5-IP3 channels,69 prevented Ito

decrease by Fas activation (Fig. 10C).54

(2) Is diastolic [Ca2�]i increased by Fas activation?

We addressed this question by measuring [Ca2�]i

transients (represented by the fura 2 ¯uorescenceratio, R� F340/F380) under four different experi-mental conditions in which the Fas receptor wasactivated in ventricular myocytes (#4 in Fig. 9).Diastolic [Ca2�]i was increased (by 125%) in murineventricular myocytes conjugated with Pÿ/ÿ CTLfor 60 min,56 in myocytes incubated for 3 h with FasmAb Jo2 (by 70%),56 or superfused with Fas mAbJo2 (by 47%, unpublished results) (Fig. 11A), and incultured neonatal rat ventricular myocytes incu-bated for 3 h with Jo2 (by �35%)55 (Fig. 11B).

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Figure 11 Fas activation causes diastolic [Ca2�]i rise in ventricular myocytes. (A) Acute effect of Fas mAb Jo2 on [Ca2�]i

transients (measured by fura 2 ¯uorescence ratio, R� F340/F380) in murine ventricular myocytes. Myocytes werecontinuously superfused with warmed (32�C) Tyrode's solution in the absence (Control) or presence of Fas mAb Jo2(2 mg/ml). Representative traces are shown at different times of superfusion: at the beginning (t�0 0), and at 15 and30 min. Myocytes were paced at 0.3 Hz. (B) Representative [Ca2�]i transients from a control culture of neonatal ratventricular myocytes, and from a culture treated for 3 hrs with rFas� enhancing Ab. Paced beats (at 1 Hz) are marked byasterisks. The diastolic [Ca2�]i level in the control culture is indicated by the dashed line.

1157Cytotoxic Lymphocytes and Cardiac Electrophysiology

(3) Is [Ca2�]i rise necessary for Fas-mediated effects?

By means of a variety of pharmacological tools, wedetermined that Ca2� release from intracellularstores is essential for Fas-mediated effects (#5 inFig. 9). Hence, we found that disrupting SR Ca2�

release with caffeine, ryanodine or thapsigargin,attenuated the electrophysiological perturbationsand diastolic [Ca2�]i elevation. Further, ryanodineand thapsigargin prevented Jo2-induced decrease ofIto and the increase in ICa,L (only ryanodinetested).54 In summary, the above ®ndings supportour hypothesis that engagement of the Fas receptoractivates the 1,4,5-IP3 cascade, resulting in Ca2�

release from intracellular stores, which contributesto the Fas-based electrophysiological perturbationsin ventricular myocytes.

In agreement with our work, previous relatedstudies suggested that Ca2� ions contribute tomyocyte dysfunction caused by cytotoxic lympho-cytes.70,71 In a study simulating CTL attack onvirus-infected myocytes, Hasin and co-workersdetermined the functional changes in mengovirus-infected neonatal rat ventricular myocytes.71 Basedon their observations that CTL decreased myocytecontraction, the oscillations in the action potentialplateau and the verapamil-sensitive rise in totalexchangeable calcium, the authors concluded thatthe cytotoxic effects resulted from altered calciumhandling causing increased [Ca2�]i. However, therelevance of their ®ndings to ours is not clear, sincethe immune pathway underlying CTL actionwas not established; perforin/granzymes, Fas/FasL,or both.

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1158 O. Binah

Does Fas-mediated apoptosis have a pathologic role inthe hypoxic/ischemic myocardium?

In previous sections I emphasized the intriguingfeature of Fas signaling in normoxic ventricularmyocytes; whereas Fas activation does not causeapoptosis, it alters myocytes electrophysiologicalproperties. Nonetheless, several considerations,among which are increased Fas expression inhypoxic cultured neonatal rat ventricular myocytes(NRVM),72 and augmented incidence of apoptosis inthe ischemic myocardium,73,74 suggested that inthe hypoxic/ischemic heart, Fas-mediated apoptosismay have a pathologic role. Therefore, we haverecently tested the hypothesis that hypoxia (22 h,1% O2) predisposes cultured NRVM to Fas-mediatedapoptosis by downregulating the expression ofantiapoptotic proteins and upregulating the expres-sion of proapoptotic proteins. In support of thishypothesis we found that: (1) hypoxia sensitizedhypoxic NRVM to Fas-mediated apoptosis; (2) hyp-oxia increased Fas expression by 200%, and theproapoptotic proteins ARTS and FADD by 323% and250%, respectively, and decreased the expression ofthe antiapoptotic proteins ARC and FLIP by 160%and 290%, respectively. Therefore, our main con-clusion from this study is that hypoxia predisposesventricular myocytes to Fas-mediated apoptosis,rendering this phenomenon an important patho-logical pathway in the ischemic heart.

Concluding Remarks

In this review I have focused on the two majoreffector mechanisms of cytotoxic lymphocytes: per-forin/granzymes and Fas/FasL, and presentedexperimental evidence showing that both adverselyaffect cardiac myocytes in vitro, in a way that canperturb cardiac function in situ. Respecting the Fas/FasL pathway which appears of major importancein cardiac pathologies, we propose the followingmodel for the dependency of Fas-mediated actionson the ambient oxygen level. In normoxic myocytes,whereas Fas activation does not cause apoptosis, itleads to caspase-independent, PLC activation, 1,4,5-IP3 generation and increased [Ca2�]i, which isresponsible for a variety of functional perturbations,including arrhythmias. On the other hand, underhypoxic conditions, hypoxia predisposes myocytesto the apoptotic effect of Fas activation, renderingthis phenomenon an important pathological path-way in the ischemic heart. The ®nding that in thenormoxic myocardium 1,4,5-IP3 is involved in Fas-induced myocyte dysfunction is of signi®cance to the

understanding of mechanisms of lymphocytotoxi-city as they relate to myocardial pathologies inwhich heart-in®ltrating lymphocytes play a keyrole, such as heart transplant rejection and DCM.Thus, an intracellular messenger (i.e. 1,4,5-IP3)mediating cytotoxic damage, may serve as a targetfor pharmaceuticals aimed at attenuating the injuryin¯icted to the affected heart by killer lymphocytes,thus restoring heart function.

Acknowledgments

Supported by grants from the U.S-Israel BinationalScience Foundation, The Minerva Foundation, andthe Rappaport Family Institute for Research in theMedical Sciences.

References

1. HERSHKOWITZ A, WOLFGRAM LJ, ROSE NR,BEISEL KW. Coxsackievirus B3 murine myocarditis.A pathologic spectrum of myocarditis in geneticallyde®ned inbred strains. J Am Coll Cardiol 1987; 9:1311±1319.

2. BARRY WH. Mechanisms of immune-mediatedmyocyte injury. Circulation 1994; 89: 2421±2432.

3. LESLIE K, BLAY R, HAISCH C, LODGE A, WELLER A, HUBER S.Clinical and experimental aspects of viral myocarditis.Clin Microbiol Rev 1989; 2: 191±203.

4. BINAH O. Immune effector mechanisms in hearttransplant rejection. Cardiovasc Res 1994; 28:1748±1757.

5. KAWAI C. From myocarditis to cardiomyopathy:mechanisms of in¯ammation and cell death.Circulation 1999; 99: 1091±1100.

6. BINAH O. Immune effector mechanisms in myocardialpathologies. Inter J Mol Med 2000; 6: 3±16.

7. SOARES MBP, DOS SANTOS RR. Immunopathology ofcardiomyopathy in the experimental Chagas disease.Mem Inst Oswaldo Cruz, Rio de Janeiro 1999; 94,Suppl 1: 257±262.

8. VARDA-BLOOM N, LEOR J, OHAD DG, HASIN Y, AMAR M,FIXLER R, BATTLER A, ELDAR M, HASIN D. CytotoxicT lymphocytes are activated following myocardialinfarction can recognize and kill healthy myocytesin vitro. J Mol Cell Cardiol 2000; 32: 2141±2149.

9. BERKE G. The Fas-based mechanism of lympho-cytotoxicity. Human Immunol 1997; 54: 1±7.

10. WALLACH D, VARFOLOMEEV EE, MALININ NL, GOLSTEV YV,KOVALENKO AV, BOLDIN MP. Tumor necrosis factor andFas signaling mechanisms. Ann Rev Immunol 1999;17: 331±367, 1999.

11. RUSSEL JH, LEY TJ. Lymphocyte-mediated cytotoxicity.Ann Rev Immunol 2002; 20: 323±370.

12. MARUYAMA R, TAKEMURA G, AOYAMA T, HAYAKAWA K,KODA M, KAWASE Y, QIU X, OHNO Y, MINATOGUCHI S,MIYATA K, FUJIWARA T, FUJIWARA H. Dynamicprocess of apoptosis in adult rat cardiomyocytesanalyzes using 48-hour videomicroscopy and

Page 13: Cytotoxic Lymphocytes and Cardiac Electrophysiology

1159Cytotoxic Lymphocytes and Cardiac Electrophysiology

electron microscopy. Am J Pathol 2001; 159:683±691.

13. CASSELLI D, JAKONIUK I, BARLUCCHI L, BELTRAMI P,HINTZE TH, NADAL-GINARD B, KAJSTURA J, LERI A,ANVERSA P. Oxidative stress-mediated cardiac celldeath is a major determinant of ventricular dys-function and failure in dog dilated cardiomyopathy.Circ Res 2001; 89: 279±286.

14. KLEÂBER AG, REIGGER CB, JANSE MJ. Electricaluncoupling and increase of extracellular resistanceafter induction of ischemia in isolated, arteriallyperfused rabbit papillary muscle. Circ Res 1978;61: 271±279.

15. WANG Y, RUDY Y. Action potential propagationin inhomogeneous cardiac tissue: safety factorconsiderations and ionic mechanisms. Am J PhysiolHeart Circ Physiol 2000; 278: H1019±H1029.

16. PODACK ER, HENGARTNER H, LICHTENHELD MG. Acentral role of perforin in cytolysis? Immunol Today1990; 11: 28±32.

17. TSCHOPP J, NABHOLZ M. Perforin-mediated target celllysis by cytolytic T lymphocytes. Ann Rev Immunol1990; 8: 279±302.

18. SCHMID DS, HORNUNG R, McGRATH KM, PAUL N,RUDDLE NH. Target cell DNA fragmentation ismediated by lymphotoxin and tumor necrosisfactor. Lymphokine Res 1987; 6: 195±202.

19. MASON D, TSCHOPP J. Isolation of a lytic, pore formingprotein (perforin) from cytolytic T-lymphocytes. J BiolChem 1985; 260: 9069±9072.

20. YOUNG JDE. Killing of target cells by lymphocytes.Physiol Rev 1988; 103: 37±51.

21. BERKE G. The functions and mechanisms of actionof cytolytic lymphocytes. In: Paul WE (ed.) Funda-mental Immunology. 1993: New York: Raven Press,965±1014.

22. SHINKAI Y, TAKIO K. Okumura K. Homology ofperforin to the ninth component of complement(C9). Nature 1988; 334: 525±527.

23. LIU CC, WALSH CM, YOUNG JDE. Perforin: structureand function. Immunol Today 1995; 16: 194±201.

24. KUANG AA, DIEHL GE, ZHANG J, WINOTO A. FADDis required for DR4- and D5-mediated apoptosis:lack of trail-induced apoptosis in FADD de®cientmouse embryonic ®broblasts. J Biol Chem 2000;275: 25065±25068.

25. BROWNE KA, BLINK E, SUTTON VR, FROELICH CJ,JANS DA, TRAPANI JA. Cytosolic delivery of granzymeB by bacterial toxins: evidence that endosomaldisruption, in addition to transmembrane poreformation, is an important function of perforin.Mol Cell Biol 1999; 19: 8604±8615.

26. MOTYKA B, KORBUTT G, PINKOSKI MJ, HEIBEIN JA,CAPUTO A, HOBMAN M, BARRY M, SHOSTAK I,SAWCHUK T, HOLMES CFB, GALDIE J, BLEACKLEY RC.Mannose 6-phosphate/insulin-like growth factor llreceptor is a death receptor for granzyme B duringcytotoxic T cell-induced apoptosis. Cell 2000; 103:491±500.

27. FROELICH CJ, ORTH K, TURBOV J, SETH P, GOTTLIEB R,BABIOR B, SHAH GM, BLEACKLEY RC, DIXIT VM, HANNA W.New paradigm for lymphocyte granule-mediatedcytotoxicity. Target cells bind and internalizegranzyme B, but an endosomolytic agent is necessaryfor cytolytic delivery and subsequent apoptosis.J Biol Chem 1996; 271: 29073±29079.

28. SHI L, MAI S, ISRAELS S, BROWNE K, TRAPANI JA,GREENBERG AH. Granzyme B (GraB) autonomouslycrosses the cell membrane and perforin initiatesapoptosis and GraB nuclear localization. J Exp Med1997; 185: 855±866

29. PINKOSKI MJ, HOBMAN M, HEBEIN JA, TOMASELLI K, LI F,SETH P, FROELICH CJ, BLEACKLEY RC. Entry and traf®ckingof granzyme B in target cells during granzymeB-perforin-mediated apoptosis. Blood 1998; 92:1044±1054.

30. PODACK ER. How to induce involuntary suicide: theneed for dipeptidyl peptidase l. Proc Natl Acad SciUSA 1999; 96: 8312±8314.

31. SMYTH MJ, KELLY JM, SUTTON VR, DAVIS JE, BROWNE KA,SAYERS TJ, TRAPANI JA. Unlocking the secrets ofcytotoxic granule proteins. J Leukoc Biol 2001;70: 18±29.

32. SEKO Y, SHINKAI Y, KAWASAKI A, YAGITA H, OKUMORA K,YAZAKI Y. Evidence of perforin-mediated cardiacmyocyte injury in acute murine myocarditis causedby Coxsackie virus B3. J Pathol 1993; 170: 53±58.

33. OKURA Y, YAMAMOTO T, GOTO S, INOMATA T, HIRONO S,HANAWA H, FENG L, WILSON CB, KIHARA I, IZUMI T,SHIBATA A, AIZAWA Y, SEKO S, ABO T. Characteriza-tion of cytokine and iNOS mRNA expressionin situ during the course of experimental autoimmunemyocarditis in rats. J Mol Cell Cardiol 1997; 29:491±502.

34. BADORFF C, NOUTSIAS M, KUHL U, SCHULTHEISS HP. Cell-mediated cytotoxicity in hearts with dilated cardio-myopathy: correlation with interstitial ®brosis andfoci of activated T lymphocytes. J Am Coll Cardiol1997; 29: 429±434.

35. SHULZHENKO N, MORGUN A, ZHENG XX, DINIZ RVZ,ALMEIDA DR, MA N, STORM TB, GERBASE-DELIMA M. Intragraft activation of genes encodingcytotoxic T lymphocytes effector molecules pre-cedes the histological evidence of rejection in humancardiac transplantation. Transplantation 2001; 72:1705±1708.

36. KISHIMOTO C, KURIBAYASHI K, MASUDA T, TOMIOKA N,KAWAI C. Immunologic behavior of lymphocytes inexperimental viral myocarditis: signi®cance ofT lymphocytes in the severity of myocarditis andsilent myocarditis in BALB/c-nu/nu mice.Circulation 1985; 71: 1247±1254.

37. GRIFFITHS GM, NAMIKAWA R, MUELLER C. Granzyme Aand perforin as markers for rejection in cardiactransplantation. Granzyme A and perforin as amarker for rejection in cardiac transplantation. Eur JImmunol 1991; 21: 687±693.

38. ALPERT S, LEWIS NP, ROSS H, FOWLER M, VALENTINE HA.The relationship of granzyme A and perforinexpression to cardiac allograft rejection and dys-function. Transplantation 1995; 60: 1478±1485.

39. YOUNG LHY, JOANG SV, ZHENG LM, LEE CP, LEE YS,YOUNG JDE. Perforin-mediated myocardial damage inacute myocarditis. Lancet 1990; 336: 1019±1021.

40. BINAH O, MAROM S, RUBINSTEIN I, ROBINSON RB,BERKE G, HOFFMAN BF. Immunological rejection ofheart transplant: how lytic granules from cytotoxicT lymphocytes damage guinea pig ventricularmyocytes. P¯uÈgers Arch 1992; 420: 172±179.

41. FELZEN B, BERKE G, ROSEN D, COLEMAN R, TSCHOPP J,YOUNG JDE, BINAH O. Effects of puri®ed perforin andgranzyme A from cytotoxic T lymphocytes on

Page 14: Cytotoxic Lymphocytes and Cardiac Electrophysiology

1160 O. Binah

guinea pip ventricular myocytes. Cardiovasc Res 28:643±649.

42. PERSECHINI PM, YOUNG JDE, ALMERS W. Membranechannel formation by the lymphocyte pore-formingprotein: comparison of susceptible and resistanttarget cells. J Cell Biol 1990; 110: 2109±2116.

43. KASS GEN, ORRENIUS S. Calcium signaling and toxicity.Environ Health Perspectives 1999; 107: 25±35.

44. KAGAN BL, BALWIN RL, MUNOZ D, WISNIESKI B. Form-ation of ion-permeable channels by tumor necrosisfactor-a. Science 1991; 255: 1427±1430.

45. FERRARI R. The role of TNF in cardiovascular disease.Pharmacol Res 1999; 40: 97±105.

46. SHARMA K, WANG KR, ZHANG LY, YIN DL, LUO XY,SOLOMON JC, JIANG RF, MARKOS K, DAVIDSON W,SCOTT DW, SHI YF. Death the Fas way: regulationand pathophysiology of CD95 and its ligand.Pharmacology and Therapeutics 2000; 88: 333±347.

47. FLISS H, GATTINGER D. Apoptosis in ischemic andreperfused rat myocardium. Circ Res 1996; 79:949±956.

48. YAOITA H, OGAWA K, MAEHARA K, MARUYAMA Y.Attenuation of ischemia/reperfusion injury in rats bya caspase inhibitor. Circulation 1998; 97: 276±281.

49. JEREMIAS I, KUPATT C, Martin-VILLALBA A, HABAZETTL H,SCHENKEL J, BOEKSTEGERS P, DEBATIN KM. Involvementof CD95/Apo1/Fas in cell death after myocardialischemia. Circulation 2000; 102: 915±920.

50. TOYOZAKI T, HIROE M, TANAKA M, NAGATA S, OHWADA H,MARUMO F. Level of soluble Fas ligand in myocarditis.Am J Cardiol 1998; 82: 246±248.

51. MOLLER P. Pathophysiological aspects of tumordevelopment. Stem Cells 1995; 1: 240±247.

52. ISHIYAMA S, HIROE M, NISHIKAWA T, SHIMOJO T, ABE S,FUJISAKI H, ITO H, YAMAKAWA K, KOBAYASHI N,KASAJIMA T, MARUMO F. The Fas/Fas ligand system isinvolved in the pathogenesis of autoimmune myo-carditis in rats. J Immunol 1998; 161: 4695±4701.

53. WOLLERT KC, HEINEKE J, WESTERMANNN J, LUÈ DDE M,FIEDLER B, ZIERHUT W, LAURENT D, BAUER MKA,SCHULZE-OSTHOFF K, DREXLER H. The cardiac Fas(APO-1/CD95) receptor/Fas ligand system. Relationto diastolic wall stress in volume-overload hyper-trophy in vivo and activation of the transcriptionfactor AP-1 in cardiac myocytes. Circulation 2000;101: 1172±1178.

54. SHILKRUT S, GEALIKMANN O, WOODCOCK E, BERKE G,BINAH O. Electrophysiological perturbations andarrhythmogenic activity caused by activation ofthe Fas receptor in murine ventricular myocytes: Therole of the inositol trisphosphate pathway. J CardiovascElectrophysiol 2001; 12: 185±195.

55. YANIV G, SHILKRUT M, LOTAN R, BERKE G, LARISCH S,BINAH O. Hypoxia predisposes neonatal rat ventricularmyocytes to apoptosis induced by activation of theFas (CD95/Apo-1) receptor: Fas activation andapoptosis in hypoxic myocytes. Cardiovasc Res 2002;54: 611±623.

56. FELZEN B, SHILKRUT M, LESS H, SARAPOV I, MAOR G,COLEMAN R, ROBINSON RB, BERKE G, BINAH O. Fas(CD95/Apo-1)-mediated damage to ventricular myo-cytes induced by cytotoxic T lymphocytes fromperforin-de®cient mice. Circ Res 1998; 82: 438±450.

57. ENSLEY DR, IVES M, ZHAO L, MCMILLEN M, SHELBY J,BARRY WH. Effects of alloimmune injury on contrac-tion and relaxation in cultured myocytes and

intact cardiac allografts. J Am Coll Cardiol 1994; 24:1769±1778.

58. LESS H, SHILKRUT M, RUBINSTEIN I, BERKE G, BINAH O.Cardiac dysfunction in murine autoimmune myo-carditis. J Autoimmun 1999; 12: 209±220.

59. CAMPBELL DL, RASMUSSON RL, QU Y, STRAUSS HC. Thecalcium-independent outward potassium currentin isolated ferret right ventricular myocytes. I.Basic characterization and kinetic analysis. J GenPhysiol 1993; 101: 571±601.

60. BENNDORF, K, MARKWARDT F, NILIUS B. Two types oftransient outward currents in cardiac ventricularcells of mice. P¯uÈgers Arch 1987; 409: 641±643.

61. WANG L, DUFF HJ. Developmental changes intransient outward current in mouse ventricle.Circ Res 1997; 88: 120±127.

62. ZHOU J, JERON A, LONDON B, HAN X, KOREN G. Charac-terization of a slowly inactivating outward currentin adult mouse ventricular myocytes. Circ Res1998; 83: 806±814.

63. THURINGER D, DEROUBAIX E, COULOMBE A, CORABOEUF E,MERCADIER JJ. Ionic basis of the action potentialprolongation in ventricular myocytes from Syrianhamster with dilated cardiomyopathy. CardiovascRes 1996; 31: 747±757.

64. BEUCKELMANN DJ, NAÈ BAUER M, ERDMANN E. Altera-tions of K� currents in isolated human ventricularmyocytes from patients with terminal heart failure.Circ Res 1993; 73: 379±385.

65. KAÈ AÈ B S, NUSS HB, CHIAMVIMONVAT N, O'ROURKE,PAK PH, KASS DA, MARBAN E, TOMASELLI GF. Ionicmechanism of action potential prolongation inventricular myocytes from dogs with pacing-inducedheart failure. Circ Res 1996; 78: 262±273.

66. LUO C, RUDY Y. A model of the ventricular cardiacaction potential: depolarization, repolarization, andtheir interaction. Circ Res 1991; 68: 1501±1526.

67. KOBAYASHI S, KITAZAWA T, SOMLYO AV, SOMLYO AP.Cytosolic heparin inhibits muscarinic and a-adrener-gic Ca2� release in smooth muscle. Physiologicalrole of 1,4,5-trisphosphate in pharmacologicalcoupling. J Biol Chem 1989; 264: 17997±18004.

68. RAO GH, FAREED J, WHITE JG. In¯uence of heparinon inositol 1,4,5-trisphosphate-induced calciummobilization in permeabilized human platelets.Biol Med Metabol Biol 1991; 5: 171±180.

69. GAFNI J, MUNSCH JA, LAM TH, CATLIN MC, COSTA LG,MOLINSKI TF, PESSAN IN. Xestospongins: potentmembrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 1997; 19: 723±733.

70. WOODLEY SL, MCMILLAN M, SHELBY J, LYNCH DH,ROBERTS LK, ENSLEY RD, BARRY WH. Myocyte injuryand contraction abnormalities produced by cytotoxicT lymphocytes. Circulation 1991; 83: 1410±1418.

71. HASSIN D, FIXLER R, SHIMONI Y, RUBINSTEIN E, RAZ S,GOTSMAN MS, HASIN Y. Physiological changesinduced in cardiac myocytes by cytotoxic T lympho-cytes. Am J Physiol 1987; 252: C10±C16.

72. TANAKA M, ITO H, ADACHI S, AKIMOTO H, NISHIKAWA T,KASAJIMA T, MARUMO F, HIROE M. Hypoxia inducesapoptosis with enhanced expression of Fas antigenmessenger RNA in cultured neonatal rat. Circ Res1994; 75: 426±433.

73. BIALIK S, GREENEN DL, SASSON IE, CHENG R, HORNER JW,EVANS SM, LORD EM, KOCH CJ, KITSIS RN. Myocytesapoptosis during acute myocardial infarction in

Page 15: Cytotoxic Lymphocytes and Cardiac Electrophysiology

1161Cytotoxic Lymphocytes and Cardiac Electrophysiology

the mouse localizes to hypoxic regions but occursindependently of p53. J Clin Invest 1997; 100:1363±1372.

74. TIAN YL, MA XL, WANG X, ROMANIC AM, LIU GL,LOUDEN C, GU J-L, KUMAR S, POSTE G, RUFFOLO JR RR,

FEUERSTEIN GZ. Possible involvement of stress-activatedprotein kinase signaling pathway and Fas receptorexpression in prevention of ischemia/reperfusion-induced cardiomyocyte apoptosis by carvedilol.Circ Res 1998; 82: 166±174.