Stretch-induced Programmed Myocyte Cell Death

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1 Stretchinduced Progrmmed Myocyte Cell Deth Wei Cheng,* Bosheng Li,* Jn Kjstur,* Peng Li,* Michel S. Wolin,* Edmund H. Sonnenblick,* Thoms H. Hintze,* Giorgio Olivetti,* nd Piero Anvers* Deprtments of *Medicine nd tphysiology, New York Medicl College, Vlhll, New York 1595; nd Deprtment of Medicine, Albert Einstein College of Medicine, New York 1461 Abstrct To determine the effects of loding on ctive nd pssive tensions, progrmmed cell deth, superoxide nion formtion, the expression of Fs on myocytes, nd sidetoside slippge of myocytes, ppillry muscles were exposed to 7 8 nd 5 mn/mm2 nd these prmeters were mesured over 3h period. Overstretching produced 21 nd 2.4fold increse in poptotic myocyte nd nonmyocyte cell deth, respectively. Concurrently, the genertion of rective oxygen species incresed 2.4fold nd the number of myocytes lbeled by Fs protein 21fold. Moreover, 15% decrese in the number of myocytes included in the thickness of the ppillry muscle ws found in combintion with 7% decrese in srcomere length nd the inbility of muscles to mintin stble levels of pssive nd ctive tensions. The ddition of the NOrelesing drug, C873754, prevented superoxide nion formtion, progrmmed cell deth, nd the ltertions in ctive nd pssive tensions with time of overloded ppillry muscles. In conclusion, overstretching ppers to be coupled with oxidnt stress, expression of Fs, progrmmed cell deth, rchitecturl rerrngement of myocytes, nd impirment in force development of the myocrdium. (J. Clin. Invest : ) Key words: biotinylted dutp lbeling * poptosis * overstretching Fs protein * morphometry * rective oxygen species Introduction Recently, the possibility hs been dvnced tht mechnicl forces produced by pthologic hemodynmic lods my led to myocyte cell deth in the myocrdium (1). This hypothesis hs been mde in n ttempt to understnd the chnges in crdic ntomy fter coronry rtery constriction nd globl myocrdil ischemi, nd coronry rtery occlusion nd myocrdil infrction (1, 2). However, no documenttion of this phenomenon hs been obtined. This is relevnt issue becuse cute reductions in wll thickness nd chmber diltion, s consequence of sudden increses in ventriculr enddistolic pressure, my involve murl trnsloction of cells, phenomenon commonly described s sidetoside slippge of myocytes (2). With this form of wll restructuring, the rings of cells tht move Address correspondence to Piero Anvers, Deprtment of Medicine, Vosburgh Pvilion, Room 32, New York Medicl College, Vlhll, NY Phone: ; FAX: Received for publiction 6 Mrch 1995 nd ccepted in revised form 17 July J. Clin. Invest. The Americn Society for Clinicl Investigtion, Inc /95/11/2247/13 $2. Volume 96, November 1995, rdilly towrds the epicrdil region must expnd to enclose lrger cvitry volume. Since lengthening of srcomeres nd elongtion of myocytes lone re indequte to explin the increse in dimension of the ventricle (24), single myocyte cell deth hs been postulted to occur to llow side by side trnsloction of cells within the wll. In the bsence of multiple cell deth, the sliding of myocyte bundles from the inner to the outer lyer of the wll would not tke plce, limiting murl thinning nd chmber diltion. Importntly, single myocyte cell deth hs been climed to be medited by mechnicl forces within the wll which my ctivte genes involved in poptotic cell deth (5). Therefore, experiments were conducted in n in vitro system in which bnorml resting tension levels were imposed on muscles obtined from norml dult rt herts. The possibility ws rised tht the physicl forces my induce poptotic cell deth in these muscles by enhncing the production of rective oxygen species (6). Overstretching my lso increse the expression of the cell surfce molecule, Fs (7, 8), which hs been found to be involved in poptotic myocyte cell deth (9). Since nitric oxide (NO) hs been shown to decrese progrmmed cell deth in other cell types (1) nd to rect with superoxide nion (11) nd perhps reduce oxidnt stress (12), the beneficil effects of n NOrelesing drug (13) were exmined in this setting. Methods Muscle mechnics. Mle SprgueDwley rts t 3 mo of ge were nesthetized with ether. Herts were rpidly excised nd plced in oxygented Thyrode solution contining potssium to induce distolic rrest. In ech experiment, two left posterior ppillry muscles were obtined from two nimls nd suspended sidebyside in muscle bth. The nontendinous end of ech ppillry muscle ws mounted to micrometer ssembly used to djust externl muscle length. The tendinous end of the ppillry muscle ws tied to steel wire with Ethicon 5 brided silk (Ethicon, Inc., Somerville, NJ). The wire ws ttched to 2 cm stinless lever connected to servocontrolled glvnometer for mesurement of isometric contrction. Muscles were perfused continuously with norml Thyrode solution mintined t 3C by using coiled glss het exchnger t the inflow line in conjunction with circulting wter bth. The solution ws gssed with 95% 2/5% CO2 (ph = 7.2). Preprtions were stimulted t.1 Hz by rectngulr depolrizing pulses 1 ms in durtion nd twice distolic threshold in intensity (14). After n equilibrtion period of 69 min, during which the muscles contrcted isometriclly t resting tension of 9.8 mn/mm2, the pssive nd ctive stressstrin reltions were determined. In one group of muscles (n = 12), resting muscle length ws incresed stepwise until mximl ctive tension ws developed (L.,.).' Resting length ws then chnged three times between L.,, nd 85% of L,,. to ssess reproductibility of the curve. In the other group of muscles (n = 14), similr procedure ws followed but initil muscle length ws tken beyond Lm,, until pssive nd ctive stressstrin reltions were estb 1. Abbrevition used in this pper: L,,., mximl ctive tension. Lod Dependentinduced Apoptosis 2247

2 lished between 16 nd 85% of Lmx. Step chnges in length were mde in reproducible sequence to minimize the effects of hysteresis. Prmeters dependent on muscle length were computed t intervls of 2% Lm, by liner interpoltion of the dt. At the completion of ech experiment, muscle length ws mesured with reticle in the eyepiece of dissecting microscope set t mgnifiction of 3. Muscle dimeter ws computed by verging five eqully spced microscopic mesurements throughout the length of the ppillry muscle. The crosssectionl re ws then clculted ssuming the geometry of circulr cylinder. This ltter prmeter ws lso confirmed by dividing muscle weight by its length ssuming tissue density of 1.65 grms/cm3 (14). Histologic detection of DNA strnd breks. For this portion of the study, nine left posterior ppillry muscles which hd not been stretched beyond Lm., were positioned in the muscle bth t fixed resting tension of nerly 78 mn/mm2. A second group of 1 left ppillry muscles, which hd been overstretched beyond Lm., were mintined t resting tension of 5 mn/mm2. These interventions were kept for 3h period during which muscles were stimulted s described bove. At the completion of these mechnicl impositions, ppillry muscles were fixed for 24 h in phosphte buffered 4% formlin. Tissue ws then embedded in prffin. Sections, 4 um in thickness, were obtined from ech ppillry muscle nd mounted on polyllysinecoted slides (Sigm Chemicl Co., St. Louis, MO). After deprffiniztion nd rehydrtion, tissue sections were incubted in PBS, contining.1% sponin nd 1 mm EGTA for 3 min (5). Subsequently, sections were covered with 5 /l of stining solution contining 5 U of terminl deoxynucleotidyl trnsferse, 2.5 mm CoCl2,.2 M potssium ccodylte, 25 mm TrisHCl,.25% bovine serum lbumin nd.5 nm 2' deoxyuridine5'triphosphte, coupled to biotin vi 16tom spcer rm (biotin 16dUTP). These regents were ll from Boehringer Mnnheim Biochemicls (Indinpolis, IN). Sections were incubted in this solution for 3 min t 37C in humidified chmber. After rinsing in PBS, sections were incubted for 3 min t room temperture in solution contining 4X concentrted SSC buffered nd 5% (wt/vol) nonft dry milk (Sigm Chemicl Co.). Finlly, the stining solution which contined 5 jg/ml of HITClbeled Extrvidin (Sigm Chemicl Co.), 4X concentrted SSC buffer,.1% Triton X1 nd 5% nonft dry milk ws pplied for 3 min. Positive nd negtive controls were included in the protocol to confirm the specificity of the ssy. As positive control, myocrdil sections were treted with DNse I to induce enzymticlly the formtion of DNA strnd breks. The tissue ws treted with this enzyme for 15 min t 37 C nd, fter wshing with PBS, the terminl deoxynucleotidyl trnsferse ssy ws performed. Negtive controls consisted of stining for DNA strnd breks sections similrly treted in which biotin16 dutp or terminl deoxynucleotidyl trnsferse ws not present. After terminl deoxynucleotidyl trnsferse rection, the sections were rinsed in PBS, nd stined for srcomeric ctin. The tissue ws incubted first t 37 C for 3 min with the primry ntibody (clone SC5; Sigm Chemicl Co.) diluted 1:2 in PBS, contining 1% got serum, nd subsequently with ntimouse IgG tetrmethylrhodmine B isothiocynte (TRITC) lbeled ntibody (Sigm Chemicl Co.), lso diluted 1:3 in PBS, contining 1% got serum. Sections were then stined with bisbenzimide, 5 ng/ml, for 15 min to visulize nuclei. After this procedure, sections were rinsed in PBS nd embedded in Vectshield (Vector Lbortories, Burlingme, CA) mounting medium. Quntittive nlysis of DNA strnd breks in myocytes nd nonmyocytes. The number of myocyte nuclei in the ppillry muscles lbeled by biotinylted dutp ws mesured by counting the number of stined nuclei per unit re of tissue sections in ech ppillry muscle. This nlysis ws performed with microscope working in epifluorescence mode, equipped with sets of excittion emission filters for FITC (excittion blue, emission green), which llowed the identifiction of incorported dutp in nuclei. The number of lbeled nuclei ws recorded nd the distinction between myocytes nd nonmyocytes ws obtined by detection of srcomeric ctin with the use of second set of filters specific for TRITC (excittion green, emission red). By this pproch, the number of myocytes nd nonmyocytes stined by dutp per mm2 of tissue ws determined in ech muscle. Sections were exmined t mgnifiction of 1,25 by viewing subsequent fields through the entire ppillry muscle. A squre tissue re equl to 6,84 nm2 ws delineted in the microscopic field by n oculr reticle (#15844; Wild Heerbrugg Instruments, Inc., Frmingdle, NY) nd n verge of 8 fields in ech muscle were exmined. Morphometric determintion of the number of dutplbeled myocytes nd nonmyocytes. The histologic preprtion described in the preceding section did not permit the determintion of the frction of myocyte nuclei lbeled by dutp. To obtin this prmeter the number of myocyte nuclei nd nonmyocyte nuclei per unit re of tissue ws determined by counting n verge of 3 fields, 6,84 Atm2 ech, t mgnifiction of 1,25 in ech ppillry muscle. This nlysis ws performed with microscope working in epifluorescence mode, equipped with sets of excittion emission filters for bisbenzimide H33258 (excittion UV, emission blue) nd TRITC (excittion green, emission red). In rndomly selected fields, nuclei were identified first (bisbenzimide stining), then excittion emission filters were chnged to visulize myocytes lbeled with nti srcomeric ctin. In this mnner, the numericl density of myocyte nd nonmyocyte nuclei in ech ppillry muscle ws obtined. By combining these dt with the preceding estimtions of dutplbeled myocyte nd nonmyocyte nuclei per unit re of myocrdium, the number of poptotic myocyte nd nonmyocyte nuclei per 16 nuclei ws determined. DNA gel electrophoresis. To confirm tht the histochemicl detection of DNA strnd breks corresponded to internucleosoml clevge, the presence of low moleculr weight DNA in ppillry muscles ws determined ( 15). Groups of three ppillry muscles ech were mechniclly homogenized on ice nd fixed for 24 h t 2'C in 7% ethnol. The tissue ws then centrifuged t 8 g for 5 min nd the ethnol ws thoroughly removed. The pellet ws resuspended in 4,ul of phosphtecitrte buffer, consisting of 192 prts of.2 M N2HPO4 nd 8 prts of.1 M citric cid (ph 7.8), t room temperture, for 1 h. After centrifugtion t 1, g for 5 min, the superntnt ws trnsferred to new tubes nd concentrted by vcuum in SpeedVc concentrtor (Svnt Instruments Inc., Frmingdle, NY) for 15 min. A 3Al liquot of.25% NP4 (Sigm Chemicl Co.) in distilled wter ws then dded, followed by 3 /l of solution of RNse, 1 mg/ml, lso in wter. After 3min incubtion t 37 C, 3 IL of solution of proteinse K, 1 mg/ ml (Boehringer Mnnheim Biochemicls), ws dded nd the extrct ws incubted for n dditionl 1 h t 37 C. After the incubtion, 12 Al of loding buffer,.25% bromophenol blue, 3% glycerol, ws dded nd smples were subjected to electrophoresis on 1% grose gel contining 5 Ag/ml ethidium bromide. The DNA in the gels ws visulized under UV light. For this portion of the study, six nonoverstretched muscles nd six overstretched muscles were utilized. Immunohistochemicl locliztion of Fs. Tissue sections mounted on polyllysinecoted glss slides were deprffinized by heting t 9 C followed by three wshes in xylene. After dehydrtion in bsolute ethnol for 15 s, slides were incubted in 2% (vol/vol) H22 in methnol for 345 s then wshed with 95% ethnol for 2 s, followed sequentilly by 7% ethnol for 2 s, distilled H2 for 1 min, nd PBS (12 mmol/liter NCl, 11.5 mmol/liter NH2PO4, 31.3 mmol/liter KH2PO4, ph ) for 5 min nd then heted by microwving for 15 min. After wshing twice in PBS for 5 min, tissue sections were preblocked for 45 min in TNK solution (1 mmol/liter Tris, ph , 55 mmol/liter NCl, 1 mmol/liter KCl) contining 2% (wt/vol) BSA (Sigm Chemicl Co.).1% Triton X1, nd 1% norml got serum. Antibodies were dded to the slide in the sme solution nd incubted overnight t room temperture. Antimouse Fs hmster monoclonl ntibody (clone Jo2; Phrmingen, Sn Diego, CA) ws used t 1:1 (vol/vol) dilution. In some cses, the primry ntibodies were omitted (negtive controls). Myelom IgG ntibody (Jckson ImmunoReserch Lbortories, Inc., West Grove, PA) ws lso used s second form of negtive control. After wshing with PBS, tissue sections were incubted for 1 h with 5 Ag/ml of biotinylted got ntirbbit ntibody (Vector Lbortories, Inc.) for the detection of Fs nd then wshed nd incubted for 345 min with n vidinbiotin complex regent contining horserdish peroxidse (Vector Lbortories, Inc.) in TNK. Color development ws chieved by incubtion for 1 minutes with 2248 Cheng et l.

3 solution contining 3,3'diminobenzidine (DAB) nd.1% (vol/vol) H22 in TNK buffer. The quntittive nlysis of the cell locliztion of Fs protein ws performed in 4 nd 5 ppillry muscles exposed to low nd high levels of tension, respectively. Five eqully spced res, long the longitudinl xis of ech muscle, were smples nd the number of lbeled nd unlbeled myocytes cross the dimeter of the muscle ws determined in ech region. These individul vlues were then verged to yield men vlue in ech muscle. Detection of superoxide nion. Ppillry muscle were exposed to levels of tension comprble to those described bove. Under these conditions, the formtion of superoxide nion ws exmined over period of 3 h during which loding ws kept constnt. At the end of this procedure, muscles were fixed in 4% phosphte buffered formlin nd embedded in prffin for the subsequent detection of progrmmed cell deth. Specificlly, chnges in force nd genertion were determined in single photon counting pprtus constructed in light tight box. In these experiments, stimulted muscles were incubted in Krebs' bicrbonte buffer contining.25 mm lucigenin in continuously gssed 1 cm2 spectrophotometer cuvette mounted in thermostted cell holder on the surfce of Lucite light guide (with shutter cover) directed into cooled Thorn EMI Photomultiplier Tube (model 9235B; Thorn EMI Electron Tubes, Ruslip, United Kingdom). A Thorn EMI mplifierdiscrimintor (model C64) nd photon counter (model C66) were used to quntitte chemiluminescence. The counts were integrted over 5s periods by the photon counter, nd n nlog signl of the integrted counts ws continuously recorded on (model 7 Polygrph; Grss Instrument Co., Quincy, MA) recorder together with chnges in force (16). This nlysis ws conducted in four nd five muscles t low nd high tension levels, respectively. The effects of superoxide dismutse on the genertion of superoxide nion in overstretched muscles were lso exmined (n = 4). Moreover, the bility of N2mercptopropionyl glycine to inhibit superoxide nion intrcellulry ws evluted in four overstretched ppillry muscles. For these two conditions, muscles were exposed to either superoxide dismutse, 3 jim, or N2mercptopropionyl glycine, 2 mm, for 15 min (17, 18) before the determintion of chemiluminescence levels (16). The dose of N2mercptopropionyl glycine used here ws estblished on the bsis of preliminry experiments showing tht the bility of this compound to ffect superoxide nion ws mximl t 2 mm without significnt increses up to 2 mm. Effects of CAS 936. The potentil beneficil influence of the NOrelesing drug C873754, metbolite of CAS 936 (13), on the genertion of superoxide nion nd the ctivtion of progrmmed cell deth ws exmined in ppillry muscles. This ws performed by superfusing muscles kept t low nd high tension levels (see bove), with solution contining 1 ktm C during 3h period. The formtion of rective oxygen species ws mesured throughout nd fter the histologic preprtion described bove, Fs nd dutp lbeling of the tissue ws determined. Five muscles of high tension levels were used for chemiluminescence mesurements, nd seven were used for evlution of DNA strnd breks. The effects of C on developed nd resting tensions with time were evluted in 1 ppillry muscles nd compred with n identicl number of muscles not exposed to the drug. Number of myocytes cross the muscle. Ppillry muscles exposed to low nd high levels of resting tension for 3h period were fixed in these conditions with solution contining 5% glutrldehyde nd 4% prformldehyde. Ppillry muscles were then removed nd sliced trnsversely into four pieces which were embedded in rldite. One frgment of tissue ws embedded longitudinlly for the mesurement of srcomere length. Semithin sections,.5 Hm in thickness, were cut from ech block perpendiculr to the long xis of the muscle nd showing its whole crosssectionl re. The trnsverse re ws determined by n intercting trcking device nd the men dimeter ws computed (Jndel Scientific, Corte Mder, CA). Subsequently, thin sections were obtined from these blocks nd low power electron microgrphs, four from ech of two blocks from ech ppillry muscle, were collected. These microgrphs were printed t finl mgnifiction of 4,5 nd the volume composition of the myocrdium nd the numericl density of myocyte profiles per unit re of tissue were mesured s previously described (2). The vilbility of ppillry muscle dimeter nd myocyte density llowed the clcultion of the number of cells cross the ppillry muscle (2). The men centertocenter distnce (d,c) between myocytes ws clculted from the number of profiles counted per unit re of tissue, (N(m)A) in trnsverse myocrdil sections by ssuming the tendency for these roughly cylindricl cells to pck in close hexgonl pttern (2): VN(m)A IN'.7A In hexgonl pttern, the spcing between plnes of djcent cells vries with the orienttion of the rry from mximum of d.c to minimum of dr, 6V/2 nd hs men vlue, d, representtive of rndom orienttion, given by 3dc 63 Ir/6 do dc =~~v~ 6d =.985 d ir O coso Thus, the number, N(m)t,,,, of myocytes cross the thickness of the ppillry muscle, W, cn be found from N(m)tin = W/d = W/ii(.)A This nlysis ws performed in six muscles t low nd six muscles t high tension levels. The block of tissue embedded prllel to the mjor xis of the ppillry muscle ws sectioned nd srcomere length in myofibrils ws determined in ech muscle from 1 mesurements mde t mgnifiction of 3,. These sections were longitudinlly oriented nd were cut perpendiculr to their length to void longitudinl compression. Dt collection nd nlysis. All tissue smples were coded, nd the code ws broken t the end of the experiment. Results re presented s mentsd computed from the verge mesurements obtined from ech muscle. Comprisons between two vlues were performed using twotiled Student's t test. Sttisticl significnce in multiple comprisons in which the nlysis of vrince nd the F test indicted the presence of significnt differences ws determined by the Bonferonni method. Vlues of P <.5 were considered significnt. Results Muscle mechnics. Fig. 1 illustrtes the pssive nd ctive length tension reltionships of ppillry muscles between 85 nd 1% of L,,,, nd between 85 nd 16% of L,,o. Muscles stretched up to 1% of L,,, showed tht the progressive increse in resting tension ws coupled with prllel increse in developed tension which peked t L., (Fig. 1 A). These muscles mintined their bility to generte expected levels of developed tension upwrd nd downwrd long the entire length tension curve. In contrst, ppillry muscles stretched beyond L,,, exhibited mrked increses in resting tension ssocited with decrese in isometric developed tension (Fig. 1 B). From 1 to 16% of L.,,, resting tension incresed from vlue of 19 mn/mm2 to vlue of 52 mn/mm2. In contrst, isometric developed tension decresed from vlue of 73 mn/mm2 to vlue of 61 mn/mm2. These differences were both sttisticlly significnt (P <.1). Moreover, overstretched ppillry muscles were unble to produce the sme developed tensions nd when moved downwrd long the length, tension reltionships produced less tensions throughout the rnge of physiologic lods exmined. The resting tension curve ws shifted to the right t ll lengths from 14 to 94% of Lml (P <.1), wheres the reductions in developed tension were sttisticlly significnt from 12 to 85% of L, (P <.1). Fig. 2 depicts the timedependent chnges in resting nd Lod Dependentinduced Apoptosis 2249

4 8 go 7 6 N., z 5 o 4 ml 3 E 2 Go A Muscle Length (% L..) B T/..I* I I I/ ,iNJ I" I/ I/,.I Muscle Length (% L.) Figure 1. Resting nd ctive length tension reltionships of left posterior ppillry muscles. Muscles extended beyond Lmx (n = 14) were not ble to mintin stble levels of resting nd developed tensions (B). This phenomenon ws not present in muscles extended up to Lmx (n = 12) (A). Results re presented s men±sd. (A) Corresponds to ctive tension development t incresing muscle lengths; (v) corresponds to ctive tension development t decresing muscle lengths; (A) corresponds to resting tension development t incresing muscle lengths; nd (v) corresponds to resting tension development t decresing muscle lengths. See text for sttistics nd more detils. developed tensions in ppillry muscles exposed to resting tension levels of 78 nd 5 mn/mm2, respectively. Modertely stretched ppillry muscles mintined comprble levels of pssive nd ctive tensions over 3h period. Conversely, overstretched muscles showed n initil increse in isometric developed tension followed by progressive decline with time. Resting tension in these muscles decresed continuously during the 3h intervl. In summry, overstretching ffected the bility of ppillry muscles to mintin stble levels of resting nd developed isometric tensions with time. Structurl chrcteristics. Semithin sections of ppillry muscles showed tht the norml structure of muscles ws preserved in ll cses. Thus, consistent with previous results ( 19), the size of the muscles used in this study did not limit oxygention of the core of the muscles producing cellulr dmge. This ws confirmed quntittively, since the volume frction of myocytes nd interstitil comprtment in muscles exposed to moderte levels of tension ws 86±1. nd 14 1., wheres the corresponding vlues in muscles kept t high tension were 87±2. nd 13+±2.. Mesurements of ppillry muscle crosssectionl re were obtined from these histologic preprtions for the subsequent evlution of ppillry muscle dimeter. High resting tension levels were ssocited with n 8% reduction in muscle dimeter which, however, ws not sttisticlly significnt (Fig. 3 A). The numericl density of myocyte profiles per unit re of ppillry muscle ws obtined by low power electron microscopy. Myocyte numericl density ws 5, in muscles mintined for 3 h t 78 mn/mm2 nd 4,857±444 in muscles kept for the sme time t 5 mn/mm2. These vlues, in combintion with the volume frction of myocytes in the tissue, were utilized to compute the verge crosssectionl re nd dimeter of myocytes. In comprison with muscles subjected to moderte lods, high lods were chrcterized by 13 nd 6% N z U,.P4 V Ed Tko mpvi 8o 7 F 6 _ 5 _ 4 _ 3 F 2 _ 1 AA, F B l 7 N z 6 5.P.4 4 o 1 2 Time (Hours) * * * 2 2 P Tir Figure 2. Effects of loding, 7 8 mn/mm2 (n = 9) nd 5 mn/ mm2 (n = 1) on resting nd developed tension with time. * Indi I I ctes vlue tht is sttisticlly significntly different from the ne (Hours) vlue obtined t 3 min, P < Cheng et l.

5 1.4 A S :t B 4 S 1. :S ob 4. 4 m S U P s 1.5 ID 1.6 w 1.6 i 1.4 di 1.2 c 78 MN/me' 6 mn/mm' 4.' U " z. D 75 mn/mm' 5 mn/mu Figure 3. Effects of loding, 78 mn/ mm2 (n 6) nd 5 mn/mm2 (n 6) = = for 3h period, on ppillry muscle dimeter (A), myocyte dimeter (B), srcomere length (C), nd number of myocyte profiles within the thickness of the ppillry muscle (D). Results re presented s men±sd. * Indictes vlue tht is sttisticlly significntly different, P <.5. greter myocyte trnsverse re nd dimeter, respectively. However, these chnges were not sttisticlly significnt (Fig. 3 B). On the other hnd, srcomere length mesured in longitudinlly oriented sections of myocytes ws 7% shorter in overloded muscle (Fig. 3 C) nd this difference ws sttisticlly significnt (P <.1). When the number of myocytes cross the dimeter of the ppillry muscle ws computed ccording to the eqution in the Methods section, muscles mintined t 5 mn/mm2 were found to possess 15% less myocyte profiles thn muscles kept t 78 mn/mm2 (Fig. 3 D). This difference ws sttisticlly significnt (P <.2). In summry, high tension levels reduced the number of myocyte profiles within the thickness of the ppillry muscle. Biotinylted dutp detection in the ppillry muscle. The specificity of the technique utilized to stin DNA strnd breks in different cell popultions ws documented by treting tissue sections with DNse I before the detection of DNA strnd breks by dutp lbeling (Fig. 4, A nd B). Conversely, DNA strnd breks were not detected when biotin16dutp (Fig. 4, C nd D) or terminl deoxynucleotidyl trnsferse ws not included in the enzymtic rection. An identicl effect ws lso obtined by stining sections which contined DNA strnd breks in the bsence of biotin16dutp or terminl deoxynucleotidyl trnsferse. Fig. 5 illustrtes the locliztion of DNA strnd breks in myocyte nuclei of left posterior ppillry muscles mintined for 3 h t 5 mn/mm2. Occsionl dutpstined myocyte nuclei were lso seen in muscles kept t 78 mn/mm2. However, the number of positively lbeled myocyte nuclei ws greter in muscles exposed to higher tension levels. Interstitil cells were lso found to be lbeled by dutp nd the number of poptotic nonmyocyte nuclei lso incresed in overstretched muscle preprtions. In contrst, fter tretment with C873754, dutp lbeling of myocytes nd nonmyocytes in muscles mintined t 5 mn/mm2 ws significntly reduced, reching vlues similr to those of muscles kept t 78 mn/mm2. To void potentil influences from the dmged ends of the muscle, this nlysis ws restricted to the midportion of ech ppillry muscle. Fig. 6 shows quntittively the effects of overstretching nd C on the number of myocyte nuclei nd nonmyocyte nuclei exhibiting DNA strnd breks in ppillry muscles. After 3h period of exposure of muscles to high tension levels, 6,4 myocyte nuclei per 16 nuclei exhibited DNA strnd breks. This vlue ws 21fold higher thn tht obtined in muscles kept t moderte tension, 3 myocyte nuclei per 16 nuclei. This difference ws sttisticlly significnt (P <.1). In ddition, overstretching ws ssocited with 2.4fold increse in the number of interstitil cells lbeled by dutp. This difference ws lso sttisticlly significnt (P <.2). Conversely, C ws cpble of mintining the extent of dutp lbeling of myocytes nd nonmyocytes within vlues comprble to those found t moderte tension, in spite of the overstretching. In summry, overstretching ws ssocited with 21fold nd 2.4fold increse in the mgnitude of dutp lbeling of myocytes nd nonmyocytes in ppillry muscles nd C prevented this phenomenon. Detection of DNA lddering in the myocrdium. The presence of DNA frgments of size equivlent to the mono or oligonucleosomes ws determined to confirm the detection of DNA strnd breks in myocyte nuclei. Becuse only reltive smll frction of myocytes ws undergoing poptosis in the overstretched ppillry muscles, procedure for extrction of low Lod Dependentinduced Apoptosis 2251

6 Figure 4. Detection of DNA strnd breks in nuclei fter tretment of sections of myocrdium with DNse I. (A) Illustrtes by bisbenzimide fluorescence, the nuclei included in the section, wheres (B) shows tht most nuclei were stined by biotinylted dutp fter DNse exposure. Omission of dutp from the rection results in negtive stining (compre C with D). The contrst in (D) ws enhnced to demonstrte more clerly the lck of dutp lbeling. (AD) x4. moleculr weight DNA from poptotic cells ws used (15). As illustrted in Fig. 7, extrcts obtined from the muscles exposed to 5 mnn/mm2 reveled the presence of DNA frgments consistent with poptosis. Stretches of DNA of size equivlent to 2 bp were the most bundnt. Moreover, 4, 6, nd 8bp frgments which correspond to two, three, nd four nucleosomes, respectively, were lso visible. In contrst, no degrdtion of DNA ws found in nonoverstretched ppillry muscles. In summry, DNA frgmenttion chrcteristic of poptotic cell deth ws observed in overstretched ppillry muscles. Chemiluminescent mesurement of superoxide nion genertion. To determine whether overstretching of the ppillry muscle ws ssocited with n enhnced formtion of rective oxygen species, lucigenindependent photoemission ws mesured in muscles kept for 3 h t 78 nd 5 mn/mm2. As illustrted in Fig. 8, ppillry muscles exposed to the lower level of tension exhibited no chnges in the formtion of superoxide nion during the 3h period of stretching. In contrst, overstretching resulted in mrked elevtion of superoxideelicited lucigenin chemiluminescence. When these two vlues were compred, 2.7fold higher ctivtion of superoxide formtion ws found in muscles t 5 mn/mm2 (P <.5) thn in muscles t 78 mn/mm2. To estblish whether NO ws ble to prevent stretchinduced superoxide genertion, seprte group of overstretched ppillry muscles ws exposed to 1,tM C As shown in Fig. 8, in the presence of the NOrelesing drug, 42% (P <.2) decrese in the formtion of superoxide nion ws noted. To confirm the intrcellulr origin of superoxide nion, the effects of superoxide dismutse nd N2mercptopropionyl glycine on overstretched muscles were mesured. Superoxide dismutse did not ffect the level of chemiluminescence generted by the overstretched muscles. This intervention decresed the formtion of superoxide nion by 2.65±4.51% (n = 4) which ws not sttisticlly significnt. In contrst, N2mercptopropionyl glycine reduced this prmeter by % (n = 4) nd this chnge ws sttisticlly significnt (P <.1). Fig. 9 illustrtes the vlues of ctive nd resting tensions of ppillry muscles exposed to lod of 5 mn/mm2, in the presence nd bsence of C This phenomenon ws exmined over 3h period. The NOrelesing drug ws ssocited with lower levels of developed tension thn those observed in the bsence of C873754, in spite of comprble mgnitude of prelod. Resting tension ws similr in both groups of muscles. Moreover, developed nd resting tension decresed with time in the bsence of the drug intervention, wheres no sttisti 2252 Cheng et l.

7 Figure 5. Detection of DNA strnd breks in myocyte (A) nucleus (rrowhed) by biotinylted dutp lbeling of n overstretched ppillry muscle. (B) Illustrtes the sme microscopic field by combintion of phse contrst nd bisbenzimide fluorescence. Myocytes were identified by lbeling with srcomeric ctin ntibody (C). (AC) x 1,2. Lod Dependentinduced Apoptosis 2253

8 to..p4 ) I z P ) ), p4 lo II VZZ1DvQQ I vies/~ Myocytes NonMyocytes Figure 6. Effects of loding, 78 mn/mm2 (n = 9) (open brs) nd 5 mn/mm2 (n = 1) (htched nd crosshtched brs) for 3hr period, in the presence (n = 7) (crosshtched brs) nd bsence (open nd htched brs) of CAS 936 on the number of dutplbeled nuclei per 16 cells in ppillry muscles. Results re presented s men±sd. * Indictes vlue tht is sttisticlly significntly different from the vlue in muscles exposed to nerly 78 mn/mm2, P <.5. * * Indictes vlue tht is sttisticlly significntly different from the vlue in muscles exposed to 5 mn/mm2, P <.5. clly significnt chnges in these prmeters occurred during the 3h intervl exmined with C In summry, overstretching ws chrcterized by enhnced formtion of superoxide nion intrcellulrly nd this effect ws ttenuted by n NO donor which lso mintined ctive nd pssive tension levels stble over time. Expression of Fs in ppillry muscles. Fig. 1, AC illustrte the locliztion of Fs protein in myocytes of muscles exposed to 78 nd 5 mn/mm2, respectively. Although immu _ l 1 7 8~~~ Figure 7. Electrophoretic pttern of DNA frgments extrcted from nonoverstretched (lnes 2 nd 3) nd overstretched (lnes 4 nd 5) ppillry muscles. Lnes I nd 6, moleculr weight mrkers. Ech lne corresponds to three ppillry muscles. Arrows correspond to 2, 4, 6, nd 8bp DNA frgments. P ) 3 m ) 4) o Figure 8. The reltive chnge in superoxide nionelicited lucigenin chemiluminescence fter 3h exposure of ppillry muscles to 78 mn/ mm' (n = 4) (open br), 5 mn/mm2 (n = 5) (htched br), nd 5 mn/mm2 in the presence of 1,uM CAS 936 (n = 7) (crosshtched br). Dt re presented s men±sd * Indictes vlue tht is significntly different from muscles exposed to 78 mn/mm2 (P <.5). * * Indictes vlue tht is significntly different from muscles exposed to 5 mn/mm2, P <.5. nostining for Fs ws only occsionlly observed in myocytes of muscles mintined t 78 mn/mm2 (Fig. 1 A), numerous myocytes were lbeled in muscles kept t 5 mn/mm2 (Fig. 1, B nd C). In contrst, Fs lbeling ws not detected in interstitil cells of the sme muscles. The specificity of immunostining for Fs ws confirmed by the lck of lbeling when sections were processed in the bsence of the primry ntibody (Fig. 1 D) or in the presence of myelom IgG ntibody (Fig. 1 E). It should be emphsized tht tissue sections could not be stined simultneously for DNA strnd breks nd Fs protein becuse dutp lbeling required.2 M ccodylte buffer which interferes with Fs immunolbeling. Conversely, retrievl of Fs ntigen requires microwving which induces DNA strnd breks (not illustrted). However, ttempts were mde to document Fs lbeling nd dutp stining in seril sections of the sme muscle. By this pproch, it ws possible to observe tht, t times, Fs overexpression nd DNA strnd breks involved the sme muscle cell profiles (Fig. 1, FI). Quntittively, the number of myocytes lbeled by Fs ws 67,±26, per 16 cells in overstretched muscles nd 3,2±2,4 per 16 cells in nonoverstretched muscles. This difference ws sttisticlly significnt (P <.3). Importntly, C tretment reduced the number of Fslbeled myocytes in overstretched muscle to 7,8±2,3 per 16 cells. The 88% decrese in Fs lbeling of myocytes ws sttisticlly significnt (P <.1). The ddition of C resulted in level of Fs lbeling in overstretched muscles similr to tht mesured in nonoverstretched muscles (P =.1). In summry, overstretching ws ssocited with n increse in the expression of Fs in myocytes which ws mrkedly ttenuted by n NO donor. Discussion The results of the current study indicte tht the force generting bility of ppillry muscles exposed to high levels of resting tension decresed long the entire length tension reltionship. Antomiclly, this phenomenon ws chrcterized by decrese * ** 2254 Cheng et l.

9 N 8 Z 6 A H FIT + [ N N1. 5 r 4 B I P4 IV F I I s 3 F Si 2 V E ba Figure 9. Effects of loding, 5 mn/mm2, in the bsence (n = 1) (open brs) nd presence (n = 1) (htched brs) of CAS 936 on ctive (A) nd pssive (B) tension with time. Results re presented s men+sd. * Indictes vlue tht is sttisticlly significntly different from the vlue obtined t 3 min, P <.5. in muscle dimeter, poptotic myocyte nd nonmyocyte cell deth, nd reduction in the number of muscle cells within the thickness of the ppillry muscle. Moreover, endogenous superoxide production ws incresed. At the moleculr level, n enhnced expression of Fs receptor protein ws documented in myocytes. Thus, the possibility my be rised tht pthologic ltertions of myocrdil lod my be coupled with the genertion of rective oxygen species nd the ctivtion of gene products implicted in progrmmed cell deth. In turn, this phenomenon my led to n rchitecturl rerrngement of the myocrdium involving sidetoside slippge of myocytes. Importntly, interventions resulting in the relese of NO prevented the formtion of superoxide nion, Fs protein, progrmmed cell deth nd the reltive reduction in muscle performnce ssocited with the imposition of bnorml mechnicl lods. Mechnicl performnce of the myocrdium. Along the scending limb of the crdic lengthtension curve, developed tension is length dependent nd this property is relted to the clcium ctivtion of myofilments medited by the troponintropomyosin protein regultory complex (2, 21). The overlpping of thin nd thick filments is unchnged in the positive portion of the FrnkStrling reltion (22), nd the chnges in resting length nd force development re relted to the extent of clcium myofilment interction (22, 23). However, less understood re the mechnisms implicted in the ltertions in resting nd ctive tensions observed in muscles exposed to levels of stretch beyond the pek of the lengthtension curve (22). Under this setting, excessive pssive forces re generted nd dmge hs been climed to occur to the ends of the muscle preprtions ffecting tension development (22). Mislignment of srcomeres nd/or slippge of myofibrils within the cells hve been considered to represent the in vivo counterprt of these in vitro conditions mimicking pthologic sttes of the hert (24). Observtions in the current study document tht high lods ltered the contrctile behvior of the myocrdium nd this phenomenon ws chrcterized by reduction in muscle dimeter. Importntly, srcomere length ws longer in muscles fixed t moderte thn high lods. Dmge to the myocyte nd nonmyocyte comprtments of the muscle ws not observed in either cse. In ddition, the lck of register between individul myofibrils in the cytoplsm ws consistent finding in both preprtions, rising questions on the role plyed by this structurl modifiction in the depression of force genertion in overstretched ppillry muscles. The decrese of srcomere length in muscles kept for 3h period t 5 mn/mm2 suggests tht the mechnicl imposition shifted the muscle downwrd on the length tension reltionship resulting in decrese in tension development. This notion is consistent with slight reduction in srcomere length t higher lods. Ventriculr diltion fter increses in filling pressure in the intct hert in vitro, cute nd chronic ventriculr dysfunction, cute nd chronic myocrdil infrction, sudden restrictions in coronry perfusion with globl myocrdil ischemi, nd during the ltertions in loding ssocited with the trnsition from the fetl to the dult circultory system (for review see reference 25) hs ll been considered to be chrcterized by structurl reorgniztion of the myocyte comprtment of the myocrdium, resulting in murl thinning nd expnsion in cvitry volume. This dpttion, consisting of sidetoside slippge of cells within the wll, ppers to be the principl determinnt of cute ventriculr diltion under these pthologic conditions of the hert. In few cses, decrese in the number of myocytes included in the thickness of the ventriculr wll hs been demonstrted quntittively (2, 26) in n ttempt to support the notion of murl trnsloction of cells. Findings in the current study demonstrte tht bnorml mechnicl forces resulted in reduction in the number of cells within the width of the ppillry muscle, providing the first documenttion tht lod nd myocyte slippge re cuslly relted. Progrmmed myocyte cell deth. Although very little is known concerning progrmmed cell deth in the myocrdium (5, 27), this phenomenon ffects vrious cell types nd is initited by vriety of environmentl stimuli (28). During this process, n importnt fctor is the ctivtion of n endogenous endonuclese which results in endonucleolysis (28). DNA degrdtion, triggered by this mechnism, is specific to the spcer regions, leving the DNA ssocited with the nucleosomes intct (29). The technique used here llowed n erly detection of DNA strnd breks in myocrdil cell nuclei by the terminl deoxynucleotydil trnsferse ssy (28). Moreover, the morphologic documenttion of poptotic cell deth ws confirmed by the demonstrtion of intrnucleosoml clevge by the DNA lddering ssy. Similr observtions hve been mde fter ischemic reperfusion injury of the rbbit hert in vivo (27), nd in neontl crdic myocytes in culture exposed to hypoxic condition (9). However, defects in oxygen diffusion to the ppillry muscle in our preprtion were unlikely, suggesting tht pro Lod Dependentinduced Apoptosis 2255

10 Figure 1. Photomicrogrphs illustrting Fs monoclonl ntibody lbeling of nonoverstretched (A) nd n overstretched (B nd C) ppillry muscle. Omission of the primry ntibody (D) or its substitution with myelom IgG ntibody (E) during the rection resulted in negtive stining of n overstretched muscle. Seril sections of n overstretched ppillry muscle showing dutp lbeling (F nd H) nd Fs lbeling (G nd I) of the sme cells (rrows). (A nd B) X4; (C) Xl,1; (DI) x6. grmmed cell deth occurred vi different mechnism. Ischemic myocyte cell deth is chrcterized by rupture of the srcolemml lining, contrction bnds in myofibrils, nd mitochondril swelling (283). Loss of membrne function (28) nd mitochondril swelling re distinct spects of necrotic cell deth in ll cell popultions (28, 3). In contrst, progrmmed cell deth typiclly occurs in the bsence of loss of plsm membrne integrity nd function (283). The morphology of mitochondri in poptotic cells is lso unchnged. Findings in this study, documenting the lck of ltertions t the level of the 2256 Cheng et l.

11 Figure 1 (Continued) plsm membrne, myofibrils nd mitochondri, indicte tht the protocols used did not produce necrotic cell deth in the ppillry muscles. In ddition, poptotic cell deth ppered to be independent from locl ischemi. DNA strnd breks in myocyte nd nonmyocyte nuclei were detected in the bsence of structurl bnormlities t the light nd electron microscopic levels, suggesting tht 3 h of mechnicl stretching produced lesions chrcteristic of the erly phses of poptotic cell deth. The observtion tht overstretching triggered progrmmed cell deth, involving nerly.5% of myocytes, rises the possibility tht loss of contrctile function in these poptotic cells my hve influenced the detrimentl impct of high loding sttes on isometric tension development of the myocrdium. Although the percentge of cells ffected by this phenomenon ws less thn the reduction in ctive tension, single cell deth my hve impinged upon the force generting bility of neighboring cells depressing more severely overll muscle performnce. DNA lddering clerly showed the presence of 2, 4, 6, nd 8bp frgments of DNA in the overstretched muscles, but the ctul mgnitude of progrmmed cell deth in Lod Dependentinduced Apoptosis 2257

12 the tissue cnnot be clculted from these observtions. Similrly, dutp lbeling of myocytes provides n estimtion of the erly events of poptosis only (28). Thus, the percentge of myocytes lbeled by dutp most likely represents n underestimtion of the rel extent of myocyte cell deth in the tissue (5). As indicted bove, myocyte slippge ws ccompnied by decrese in srcomere length, modifying the pssive nd ctive length tension reltionships. However, the reltive contribution of progrmmed myocyte cell deth nd myocyte slippge to the impirment in myocrdil performnce in overstretched ppillry muscles cnnot be determined t present. On the other hnd, these events chrcterize common pthophysiologic phenomenon in which bnorml lods trigger progrmmed myocyte cell deth which leds to sidetoside slippge of myocytes depressing muscle contrctile behvior. The mgnitude of poptotic myocyte deth in overstretched muscle ws lower thn the extent of myocytes expressing Fs protein on the surfce of the cell nd cytoplsm. Fs protein ws brely detectble in myocytes of modertely loded muscles, indicting tht its expression ws dependent upon the extent of lod imposed on the myocrdium. Although cuse nd effect reltionship between progrmmed myocyte cell deth nd the Fs molecule cnnot be climed, it is noteworthy tht Fs mrna increses in neontl crdic myocytes in culture undergoing progrmmed cell deth (9). The Fs gene belongs to the tumor necrosis fctor nd nerve growth fctor receptor fmily (79) nd poptosis cn be triggered by lignd ctivtion of the Fs receptor (8). Whether stressed myocytes cn generte the Fs lignd triggering their own suicide progrm is currently unknown. Although the lck of Fs lbeling in fibroblsts of overstretched muscles ws unexpected, this phenomenon ppers to be consistent with the moderte increse in poptotic cell deth of the interstitil cell popultion of the myocrdium under this setting. Rective oxygen species, NO, nd progrmmed cell deth. Impositions of high lods on the myocrdium re chrcterized by n incresed oxygen consumption nd this phenomenon my led to the genertion of superoxide nion which my ctivte the suicide progrm of myocytes nd interstitil cells (6). NO my counterct superoxide exerting protective influence ginst poptotic cell deth in vitro (1). The results of the current study re consistent with this contention, since the NOrelesing drug, C873754, ws cpble of preventing the formtion of DNA strnd breks in myocytes nd interstitil cells of ppillry muscles exposed to high levels of tension. In ddition, the preservtion of myocrdil structurl integrity ws ssocited with the mintennce of resting nd ctive tension vlues of overstretched muscles with time. Thus, NO slvged the myocrdium from the detrimentl impct of overstretching, functionlly nd morphologiclly. Although it is difficult to recognize, t present, the effector pthwy tht couples bnorml mechnicl lods, enhnced expression of Fs, progrmmed cell deth nd muscle restructuring, the observtions with NO relese point to the induction of superoxide s relevnt fctor in the initition of this cscde of events. Severl cellulr mechnisms cn form rective oxygen species, including the conversion of xnthine dehydrogense to xnthine oxidse in reperfusion injury (31), mixedfunction oxidse systems (32), nd electron trnsport in the respirtory chin (33). Whether one or multiple cellulr processes contributed to myocrdil injury in the overstretched ppillry muscle remins to be determined. NO my llevite oxidnt stress nd thereby protect from poptosis (34, 35). However, NO dministrtion mrkedly reduced the extent of tension development in the overloded ppillry muscle, nd this negtive inotropic effect my hve contributed to diminish the dmging impct of lod of 5 mn/mm2 on the structure nd function of myocytes. This reduction in muscle performnce is consistent with observtion in vivo (3638) nd in vitro (39, 4), indicting tht NO reduces oxygen consumption nd decreses myocrdil contrctility. In conclusion, bnorml levels of resting tension ppered to be ssocited with the ctivtion of the suicide progrm of myocytes nd reduction in muscle mechnicl performnce. In ddition, n rchitecturl rerrngement of the myocyte comprtment occurred, nd this phenomenon of restructuring, in combintion with poptotic cell deth, my hve contributed to depress the mechnicl behvior of the myocrdium. The intrcellulr formtion of superoxide nion nd the overexpression of Fs protein in myocytes my hve been the criticl fctors in the initition of the cscde of events leding to progrmmed cell deth nd ltertion in myocrdil contrctility. NO seemed to protect from these detrimentl effects of overstretching. Acknowledgments The expert technicl ssistnce of Mri Felicino nd the ssistnce of Dr. Pwel Kminski in dpting the superoxide quntittion methods re gretly pprecited. This work ws supported by grnts HL38132, HL3992, HL 4561, HL5142, HL5353, HL3169, nd PO1HL4323 from the Ntionl Hert, Lung nd Blood Institute. References 1. Cpsso, J. M., M. J. Jenty, T. Plckl, G. Olivetti, nd P. Anvers Ventriculr remodeling induced by cute nonocclusive constriction of coronry rtery in rts. Am. J. Physiol. 257:H1983H Olivetti, G., J. M. Cpsso, E. H. Sonnenblick, nd P. Anvers Sidetoside slippge of myocytes prticiptes in ventriculr wll remodeling cutely fter myocrdil infrction in rts. Circ. Res. 67: Ross, J., E. H. Sonnenblick, R. R. Tylor, H. M. Spotnitz, nd J. W. Covell Distolic geometry nd srcomere length in the chroniclly dilted cnine left ventricle. Circ. Res. 28: VitliMzz, L., P. Anvers, F. Tedeschi, R. Mstndre, V. Mvill, nd. Visioli Ultrstructurl bsis of cute left ventriculr filure from severe cute ortic stenosis in the rbbit. J. Mol. Cell. Crdiol. 4: Kjstur, J., M. Mnsukhni, W. Cheng, K. Reiss, S. Krjewski, J. C. Reed, F. Quini, E. H. Sonnenblick, nd P. Anvers Progrmmed cell deth nd the expression of the protooncogene bc12 in myocytes during postntl mturtion of the hert. Exp. Cell Res. 219: Hockenbery, D. M., Z. N. Olltvi, X. M. Yin, C. L. Millimn, nd S. J. Korsmeyer Bc12 functions in n ntioxidnt pthwy to prevent poptosis. Cell. 75: Itoh, N., S. Yonehr, A. Ishii, M. Yonehr, S. Mizushim, M. Smeshim, A. Hse, Y. Seto, nd S. Ngt The polypeptide encoded by the cdna for humn cell surfce ntigen Fs cn medite poptosis. Cell. 66: WtnbeFukung, R., C. I. Brnnn, N. J. Copelnd, N. A. Jenkins, nd S. Ngt Lymphoprolifertion disorder in mice explined by defects in Fs ntigen tht medites poptosis. Nture (Lond). 356: Tnk, M., H. Ito, S. Adchi, H. Akimoto, T. Nishikw, T. Ksjim, F. Mrumo, nd M. Hiroe Hypoxi induces poptosis with enhnced expression of Fs ntigen messenger RNA in cultured neontl rt crdiomyocytes. Circ. Res. 75: Mnnick, J. B., K. Asno, K. Izumi, E. Kieff, nd J. S. Stmler Nitric oxide produced by humn B lymphocytes inhibits poptosis nd Epstein Brr virus rectivtion. Cell. 79: Furchgott, R. F., M. T. Khn, nd K. D. Jothinndn Comprison of properties of nitric oxide nd endotheliumderived relxing fctor: some cutionry findings. In EndotheliumDerived Relxing Fctors, G. M. Rubnyi nd P. M. Vnhoutte, editors. Krger, Bsel Niu, X.F., C. W. Smith, nd P. Kubes Intrcellulr oxidtive stress induced by nitric oxide synthesis inhibition increses endothelil cell dhesion to neutrophils. Circ. Res. 74: Cheng et l.

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