Correlation between apoptosis and left ventricular remodeling in subacute phase of myocardial ischemia and reperfusion

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1 Wkyshi et l. EJNMMI Reserch (2015) 5:72 DOI /s ORIGINAL RESEARCH Open Access Correltion etween poptosis nd left ventriculr remodeling in sucute phse of myocrdil ischemi nd reperfusion Hiroshi Wkyshi 1*, Junichi Tki 1, Anri Inki 1, Kzuhiro Shi 2, Ichiro Mtsunri 3 nd Seigo Kinuy 1 Astrct Bckground: To investigte whether n poptotic process demonstrted y 99m Tc-nnexin-V ( 99m Tc-AV) uptke correltes with left ventriculr remodeling (LVR) fter myocrdil infrction, we ssessed 99m Tc-AV uptke in rt model of myocrdil ischemi nd reperfusion. Methods: The left coronry rtery (LCA) of 15 rts ws occluded for 20 to 30 min, followed y reperfusion. After 2 weeks, 99m Tc-AV ws injected, nd then 1 h lter, 201 Tl ws injected fter reocclusion of the LCA. Dul-trcer utordiogrphy ws performed to ssess 99m Tc-AV uptke nd the re t risk (AAR) y 201 Tl defect. 99m Tc-AV uptke rtio ws clculted y dividing the count density of the AAR y tht of the normlly perfused re. In short-xis LV slices, LV cvity diltion index (DI) ws clculted y dividing the re of LV cvity y tht of the whole LV re. LV wll-thinning rtio (WTR) ws clculted y dividing the LV wll thickness in the AAR y tht of the normlly perfused re. Results: Significnt 99m Tc-AV uptke in the AAR ws oserved in 10 rts. DI ws significntly higher in rts with positive 99m Tc-AV uptke thn in rts without uptke. WTR ws smller in rts with positive 99m Tc-AV uptke thn in rts without uptke. Conclusions: The dt suggest 99m Tc-AV uptke in injured myocrdium might correlte with LVR t 2 weeks fter myocrdil ischemi nd reperfusion. Keywords: 99m Tc-nnexin-V, Apoptosis, Myocrdil remodeling, Myocrdil ischemi Bckground Crdiomyocyte deth y poptosis during ischemireperfusion nd myocrdil infrction (MI) is ssocited with the progression of left ventriculr (LV) dysfunction nd remodeling [1 3]. The progressive loss of crdiomyocytes fter cute MI (AMI) nd ischemi-reperfusion my ply key role in the pthogenesis of hert filure. Apoptosis is triggered through two min pthwys: (1) the intrinsic pthwy involves mitochondri nd cytoplsmic reticulum, nd (2) the extrinsic pthwy utilizes cell surfce receptor. Cellulr stresses such s infrction nd ischemi-reperfusion induce poptosis through the intrinsic pthwy [4 7]. These stimuli led to * Correspondence: hiroshi @yhoo.co.jp 1 Deprtment of Nucler Medicine, Knzw University Hospitl, 13-1 Tkr-mchi, Knzw, Ishikw , Jpn Full list of uthor informtion is ville t the end of the rticle the relese of severl fctors into cytosol including cytochrome c, which ctivtes the inititor cspse-9 vi the poptosome followed y the ctivtion of effector cspses. The ctivtion of downstrem cspses leds to the clevge of numerous structurl nd regultory cellulr proteins, therey producing the poptotic phenotype chrcterized y cell shrinkge, chromtin condenstion, nucleus frgmenttion, nd externliztion of phosphtidylserine (PS) on the outside of the cell memrne tht serves s signl for phgocytes. Annexin-V, memer of phospholipid inding fmily of proteins, inds to PScontining sites on the cell surfce nd hs een suggested to e n erly mrker of poptosis. Then, rdioleled nnexin-v hs een used for the detection of poptosis s noninvsive imging tool [8]. The process of myocrdil tissue repir nd heling fter AMI is considered to consist of four phses sed 2015 Wkyshi et l. Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde.

2 Wkyshi et l. EJNMMI Reserch (2015) 5:72 Pge 2 of 6 on the pthologic findings: crdiomyocyte deth, cute inflmmtion, formtion of grnultion tissue, nd scr formtion [9]. After 2 weeks of MI, grnultion tissue formtion in the ischemic re is ongoing. The grnultion tissue is still rich in inflmmtory cells like lymphocytes nd mcrophges for helping to cler the cellulr deris. At this point, the prolonged 99m Tc-nnexin-V uptke y crdiomyocyte might correlte with vriety of tissue repir nd heling. Our previous studies showed tht 99m Tc-nnexin-V inding commenced t 0.5 h fter ischemi-reperfusion in the mid-myocrdium within the re t risk (AAR) nd expnded to the suendocrdil nd suepicrdil lyers t 6 h fter ischemi-reperfusion in rt models [10, 11]. The 99m Tc-nnexin-V inding diminished grdully over 3 dys nd continued till 2 weeks fter reperfusion. However, we could not ssess the reltion etween 99m Tc-nnexin-V uptke nd LV remodeling, ecuse we only hd investigted five rts with 20 min of ischemi t 2 weeks fter reperfusion. Therefore, we investigted whether poptosis or cell deth demonstrted y 99m Tc-nnexin-V uptke correlted with LV remodeling in rt models of myocrdil ischemi-reperfusion t 2 weeks fter reperfusion. Methods Animl model of cute ischemi nd reperfusion Mle Wistr rts 8 9 weeks old were nesthetized with intrperitonel dministrtion of pentoritl, 40 mg/kg, nd were ventilted mechniclly with room ir. After left thorcotomy nd exposure of the hert, 7-0 polypropylene suture on smll curved needle ws pssed through the myocrdium eneth the proximl portion of the left coronry rtery (LCA), nd oth ends of the suture were pssed through smll vinyl tue to mke snre. The suture mteril ws pulled tightly ginst the vinyl tue to occlude the LCA. The occlusion time ws 20 (n =9) or 30 min (n = 6) for mking differences in severity of ischemi [12, 13]. Myocrdil ischemi ws confirmed y ST-segment elevtion on electrocrdiogrphy nd y regionl cynosis of the myocrdil surfce. The snre ws left loose on the surfce of the hert for reocclusion of the LCA just efore scrificing the nimls to identify the AAR. At 2 weeks fter reperfusion, 99m Tc-nnexin-V ( MBq) ws injected vi til vein under nesthesi. After 1 h of the trcer injection, 0.74 MBq of 201 Tl ws injected just fter reocclusion of the proximl portion of the LCA for delinetion of the AAR. One minute lter, the rt ws euthnized y exsnguintion in ccordnce with institutionl guidelines nd the hert ws removed for nlysis. The hert ws rinsed in sline, emedded in methylcellulose, nd cooled in freezer. Seril short-xis hert sections of 20 μm thick were otined using cryostt to crete series of rings for utordiogrphy. Dul-trcer utordiogrphy Dul-trcer utordiogrphy of the left ventriculr shortxis slices ws performed to ssess 99m Tc-nnexin-V uptke nd AAR ( 201 Tl uptke). The first exposure on n imging plte (BAS-MS; Fuji Film) ws performed for min to visulize 99m Tc-nnexin-V distriution 1 2 h fter scrifice. Three dys lter (12 hlf-lives of 99m Tc), the second exposure ws mde for 24 h to imge the AAR expressed y 201 Tl distriution. All niml experimentl protocols were pproved y the Institute for Animl Studies of Knzw University. Rdioleling of nnexin-v Mutnt nnexin-v (nnexin V-117 mutnt, form of recominnt humn nnexin engineered to include inding site for technetium) ws prepred through expression in Escherichi coli. This mteril retins PSinding ctivity equivlent to tht of ntive nnexin-v. A specific ctivity of MBq ( μci)/μg of protein with rdiopurity of more thn 90 % ws chieved using rdioleling protocol [14]. Dt nlysis 99m Tc-nnexin-V ccumultion ws evluted in three myocrdil slices (20 μm thickness) t the midventriculr level spced 1 mm prt from one nother. Distriution of the trcers ws determined y nlysis of the digitized utordiogrphs. The photostimulted luminescence in ech pixel ( μm) ws determined using ioimging nlyzer (BAS-5000; Fuji Film). For quntittive nlysis, the uptke vlues for ech region of interest (ROI) were expressed s the ckground-corrected photostimulted luminescence per unit re (1 mm 2 ). A ckground ROI ws set djcent to the left ventricle. The AAR nd normlly perfused re were defined from the 201 Tl imge, nd these ROIs were pplied to the 99m Tc-nnexin-V imges to evlute uptke of 99m Tc-nnexin-V (Fig. 1). Regions of interest were drwn mnully over the re with esily identifile 99m Tc-nnexin-V uptke. The 99m Tcnnexin-V uptke rtio ws clculted y dividing the uptke vlue of the 99m Tc-nnexin-V uptke re y tht of the normlly perfused re. All prmeters otined from the three slices in ech rt were expressed s men vlue. Indexes of LV remodeling In ech three short-xis LV slices, LV cvity diltion index (DI) ws clculted y dividing the re of LV cvity y tht of the whole LV re. Then the verge of DIs from three slices ws considered s representtive vlue. LV wll-thinning rtio (WTR) ws clculted y dividing the LV wll thickness in the AAR y tht in the normlly perfused LV re. Ech re s wll thickness

Wkyshi et l. EJNMMI Reserch (2015) 5:72 Pge 3 of 6 201 Thllium 99m Tc-nnexin-V c d Fig. 1 Mesurement of prmeters.

3 Wkyshi et l. EJNMMI Reserch (2015) 5:72 Pge 3 of Thllium 99m Tc-nnexin-V c d Fig. 1 Mesurement of prmeters. The re t risk (AAR; curved yellow line) nd normlly perfused re (NA; lue pinting) were defined from the 201 Tl imge (, c), nd these ROIs were pplied to the 99m Tc-nnexin-V imges (, d). Regions of interest were drwn mnully over the re with esily identifile 99m Tc-nnexin-V uptke (d, red pinting). The 201 Tl imge demonstrtes the re t risk, while the 99m Tc-nnexin-V imge reflects the re nd intensity of poptosis. Ech re s wll thickness ws clculted s n verge of three rdil lines (c, ornge line) those eqully divided the re into four ws clculted s n verge of three rdil lines those eqully divided the re into four. Then the verge of WTRs from three slices ws clculted s representtive vlue (Fig. 1). Histopthologic exmintions with light microscopy Hemtoxylin- nd eosin-stined (HE) slices djcent to the slices used for utordiogrphy were exmined histopthologiclly y light microscopy ( 400). In situ detection of nucler DNA frgmenttion Rt hert tissues were exmined histopthologiclly for terminl deoxynucleotidyl trnsferse-medited dutp nick-end leling (TUNEL) immunorectivity to detect the presence of DNA frgmenttion. Sttisticl nlysis For ll sttisticl nlysis, we used sttisticl softwre pckge (JMP SAS Institute Inc., Cry, NC, USA). Contingency tle nlysis ws used for the reltions etween ischemic time nd 99m Tc-nnexin-V uptke. All results were expressed s men vlue ± stndrd devition (SD). Group comprisons were performed using nlysis of vrince. The Person product-moment correltion coefficient mesures the strength of the liner reltionship. A vlue of P < 0.05 ws considered sttisticlly significnt. Results The rtio of AAR to the whole LV re ws not significntly different etween 20 nd 30 min of ischemi (men vlue ± SD; 0.41 ± 0.05 vs ± 0.07, P = 0.2). Significnt 99m Tc-nnexin-V uptke ws (1) frequently documented in the AAR in 10 rts (66 % of ll rts), especilly in the cse of longer ischemi (100 % for 30 min of ischemi vs. 44 % for 20 min, P < 0.05) nd (2) correlted with LV DI (men vlue ± SD; 0.18 ± 0.06 vs ± 0.03, P = ) nd LV WTR (men vlue ± SD; 0.66 ± 0.10 vs ± 0.05, P < ), wheres it did not correlte with the rtio of AAR to the whole LV (men vlue ± SD; 0.51 ± 0.04 vs ± 0.06, P = 0.1). Only two rts with 30 min of ischemi hd 99m Tc-nnexin-V uptke inoththeaarndnormllyperfusedredjcent to the AAR. LV DI nd WTR were correlted with 99m Tc-nnexin-V uptke rtio [correlte coefficient = 0.70 (P = ) nd 0.81 (P = ), respectively] (Fig. 2). Representtive cses were shown in Fig. 3. In the rts with 20 nd 30 min of ischemi, light microscopic exmintion of the HE slices from frozen specimens showed tht necrotic myocrdium ws replced y grnultion tissues with inflmmtory cells. Scttered TUNEL-positive cells were detected in the AAR in rts with AV uptke, wheres TUNEL-positive cells were minimlly oserved in rts without 99m Tcnnexin-V uptke. Representtive HE nd TUNEL stining ws shown in Fig. 4.

4 Wkyshi et l. EJNMMI Reserch (2015) 5:72 Pge 4 of 6 Fig. 2 Comprison etween the 99m Tc-nnexin-V uptke nd indexes of LV remodeling. 99m Tc-nnexin-V uptke rtio correlted with LV DI [correlte coefficient = 0.70 (P = )] () nd WTR [correlte coefficient = 0.81 (P = )] () Discussion The present study confirmed tht 99m Tc-nnexin-V uptke ws oserved t 2 weeks fter LCA occlusion nd reperfusion in rts with dilted LV cvity nd thinned wll using utordiogrphy. Since 99m Tc-nnexin-V uptke might reflect ongoing cell deth of crdiomyocytes, the detection of poptosis cn e used s dignostic tool for predicting loss of crdiomyocytes in the vulnerle myocrdil res during the postinfrction recovery period. 99m Tc-nnexin-V uptke seems to depend on the severity of ischemi-reperfusion injury in the rt model. Previous investigtions demonstrted tht poptosis ws oserved t AAR fter short-time ischemi nd tht poptosis ws involved extensively in order zone fter long-time ischemi [15]. In this study, the size rtio of AAR to whole LV ws not different etween LCA occlusion time, ut 99m Tc-nnexin-V uptke in the AAR ws significntly more oserved in 30 min in the LCA occlusion model. Our results greed with the previous findings tht myocrdil poptosis ppered in ischemireperfusion re nd tht poptosis ws useful to evlute ischemi-reperfusion dmge. Loss of myocrdium cused y poptosis in the cute phse of MI contriuted to progressive myocrdil dysfunction in humn clinicl studies. Humn clinicl studies demonstrted tht incresed uptke of 99m Tcnnexin-V ws present in the infrct re in ptients with AMI fter percutneous coronry intervention 201 Thllium 99m Tc-nnexin-V c d Fig. 3 Autordiogrphy using of 201 Tl nd 99m Tc-nnexin-V. After 30 min (, ) nd 20 min (c, d) of ischemi, 99m Tc-nnexin-V ws injected t 2 weeks. Single mid-ventriculr slices re shown from representtive nimls. Significnt 99m Tc-nnexin-V uptke (lck rrow) nd morphologicl chnges re oserved in rt models with 30 min of ischemi

5 Wkyshi et l. EJNMMI Reserch (2015) 5:72 Pge 5 of 6 c d Fig. 4 HE nd TUNEL stining t 2 weeks fter LCA occlusion nd reperfusion. HE (, ) nd TUNEL (c, d) stining in the AAR in rts with (, c) nd without 99m Tc-nnexin-V uptke (, d) is presented. nd c s well s nd d re seril sections. Grnultion tissue with inflmmtory cells nd firosis re seen widespred (etween lck rrowheds) (, ). TUNEL-positive (lck rrow) cells re detected in crdiomyocytes in rts with positive 99m Tc-nnexin-V uptke (c). In rts with negtive 99m Tc-nnexin-V uptke, TUNEL-positive cells were rrely detected (d) [16, 17]. In these studies, timing of monitoring poptosis ws sed on the cute phse of MI, ecuse poptosis of myocrdium ws oserved most strongly in the cute phse fter ischemic dmge. Then, how long hs poptosis een oserved in rt model? Plojoki et l. [18] showed tht crdiomyocyte poptosis occurred continuously over n extended period of time in the vile order zones of infrct scrs in rt model of permnent ligtion. TUNEL-positive crdiomyocytes were numerous t 1 dy fter LCA ligtion in oth the vile order zones nd the centrl infrct res (verge vlue 4.39 nd 1.44 %). At lter time points, scttered TUNEL-positive crdiomyocytes were oserved in oth the order zones djcent to infrct scrs nd the remote myocrdium (verge vlue 0.34 nd 0.09 % t 4 weeks, 0.10 nd 0.04 % t 12 weeks, respectively). Crdiomyocyte poptosis occurs in dvnced hert filure fter the cute phse of MI. Olivetti et l. [19] demonstrted the existence of crdiomyocyte poptosis morphologiclly nd iochemiclly in ptients who underwent crdic trnsplnttion for intrctle congestive hert filure. Ate et l. [20] exmined n poptotic rte (AR), the rtio of the numer of crdiomyocytes co-expressing positive TUNEL nd cspse-3 on nucleted cells per field, in oth the infrct nd the remote re in ptients dying 10 dys fter AMI. The higher AR in the infrct re correlted with reltive LV cvity diltion strongly ut AR in the remote re did not. Apoptotic rte t the site of the infrction ws incresed nerly fourfold in ptients with symptomtic hert filure t the time of initil hospitliztion for AMI or susequently efore deth versus remining ptients (medin vlue 26.2 vs. 6.4 %). Our results consisted with their findings in tht the positive 99m Tc-AV uptke in the AAR correlted with LV cvity diltion nd thinned wll. Interestingly, the two rts demonstrted significnt uptke expnded over the order zone etween ischemic nd normlly perfused re fter ischemi nd reperfusion. Although poptotic crdiomyocytes in remote non-infrcted res contriute to the LV remodeling in rodent models of permnent ligtion [18, 21, 22], such n poptotic process over order zones in sucute phse hd not een reported. In this respect, further study would e wrrnted to determine the significnce of 99m Tc-AV uptke in the normlly perfused re. Limittions First, lthough 99m Tc-nnexin-V uptke should depend on the externlized PS on poptotic cells, the uptke lso might reflect necrotic cells. There might e little inding of nnexin-v to necrotic cells ecuse myocytes hve significnt intrcellulr content of (unleled) nnexin-v. In rt crdiomyocytes, nnexin-v is found predominntly on the srcolemm nd interclted disks

6 Wkyshi et l. EJNMMI Reserch (2015) 5:72 Pge 6 of 6 within the myocytes [23], nd its content is extremely high (round 130 μg/g of wet weight) [24] nd fr greter thn the concentrtion of the rdioleled nnexin-v, when 30 μg of leled nnexin-v re injected per rt (out 250 g of ody weight) in our experiment. Bsed on the reltive concentrtion grdient, exogenous rdioleled nnexin-v will not esily enter the intrcellulr environment nd ind to PS competitively. Our study lso demonstrted tht the distriution of nnexin-v ws consistent with the re of TUNEL stining. Therefore, we elieve tht 99m Tc-nnexin-V reflects poptosis considerly if not completely. Second, 99m Tc-nnexin-V uptke nd morphologicl chnges were evluted only y utordiogrphy, ecuse 99m Tc-nnexin-V inding t 2 weeks ws not expected to e high sed on previous studies [10]. Although the uptke vlue ws otined semiquntittively y utordiogrphy, other imging modlities including high-resolution niml SPECT, ultrsonogrphy, nd MRI my provide more ccurte ntomicl nd functionl informtion for monitoring seril chnge of 99m Tc-nnexin-V uptke simultneously. Conclusions 99m Tc-nnexin-V uptke in injured myocrdium correlted with LV remodeling t 2 weeks fter myocrdil ischemireperfusion. Ischemi-driven poptosis or cell deth demonstrted y 99m Tc-nnexin-V uptke in the sucute phse might e possile mrker of LV remodeling. Competing interests The uthors declre tht they hve no competing interests. Authors contriutions HW nd JT prticipted in the design of study, niml experiment, interprettion of the dt, nd drfting the mnuscript. AI prticipted in the study design, niml experiment, nd interprettion of the dt. IM nd KS were responsile for utordiogrphy, interprettion of the dt, nd sttisticl nlysis. SK prticipted in the design of study nd interprettion of the dt. All uthors red nd pproved the finl mnuscript. Acknowledgements The costs of puliction of this rticle were defryed in prt y the pyment of pge chrges. This work hs een supported y Grnts-in-Aid for scientific reserch (No nd ) from the Ministry of Eduction, Culture, Sports, Science, nd Technology, Jpn. Author detils 1 Deprtment of Nucler Medicine, Knzw University Hospitl, 13-1 Tkr-mchi, Knzw, Ishikw , Jpn. 2 Division of Trcer Kinetics, Advnced Science Reserch Centre, Knzw University, 13-1 Tkr-mchi, Knzw , Jpn. 3 The Medicl nd Phrmcologicl Reserch Centre Foundtion, Wo 32, Inoym, Hkui , Jpn. 3. Grg S, Nrul J, Chndrshekhr Y. Apoptosis nd hert filure: clinicl relevnce nd therpeutic trget. J Mol Cell Crdiol. 2005;38: Foo RS, Mni K, Kitsis RN. Deth egets filure in the hert. J Clin Invest. 2005;115: Hori M, Nishid K. Oxidtive stress nd left ventriculr remodeling fter myocrdil infrction. Crdiovsc Res. 2009;81: Tki J, Wkyshi H, Inki A, Mtsunri S, Kinuy S. Apoptosis imging in disesed myocrdium. In: Gholmreznezhd A, editor. The ook 12 Chpters on Nucler Medicine. Rijek: InTech; p Dorn 2nd GW. Apoptotic nd non-poptotic progrmmed crdiomyocyte deth in ventriculr remodeling. Crdiovsc Res. 2009;81: Korngold EC, Jffer FA, Weissleder R, Sosnovik DE. Noninvsive imging of poptosis in crdiovsculr disese. Hert Fil Rev. 2008;13: Blnkesteijn WM, Creemers E, Lutgens E, Cleutjens JP, Demen MJ, Smits JF. Dynmics of crdic wound heling following myocrdil infrction: oservtions in geneticlly ltered mice. Act Physiol Scnd. 2001;173: Tki J, Higuchi T, Kwshim A, Tit JF, Kinuy S, Murmori A, et l. Detection of Crdiomyocyte Deth in Rt Model of Ischemi nd Reperfusion Using 99mTc-Leled Annexin V. J Nucl Med. 2004;45: Tki J, Higuchi T, Kwshim A, Tit JF, Murmori A, Mtsunri I, et l. (99m)Tc-Annexin-V uptke in rt model of vrile ischemic severity nd reperfusion time. Circ J. 2007;71: Tki J, Inki A, Wkyshi H, Imnk-Yoshid K, Ogw K, Hiroe M, et l. Dynmic expression of tenscin-c fter myocrdil ischemi nd reperfusion: ssessment y 125I-nti-tenscin-C ntiody imging. J Nucl Med. 2010;51: Arheden H, Seed M, Higgins CB, Go DW, Ursell PC, Bremerich J, et l. Reperfused rt myocrdium sujected to vrious durtions of ischemi: estimtion of the distriution volume ofcontrst mteril with echo-plnr MR imging. Rdiology. 2000;215: Tit JF, Brown DS, Gison DF, Blnkenerg FG, Struss HW. Development nd chrcteriztion of nnexin V mutnts with endogenous cheltion sites for (99m)Tc. Bioconjug Chem. 2000;11: Krijnen PA, Nijmeijer R, Meijer CJ, Visser CA, Hck CE, Niessen HW. J Apoptosis in myocrdil ischemi nd infrction. Clin Pthol. 2002;55: Hofstr L, Liem IH, Dumont EA, Boersm HH, vn Heerde WL, Doevendns PA, et l. Visulistion of cell deth in vivo in ptients with cute myocrdil infrction. Lncet. 2000;356: Thimister PW, Hofstr L, Liem IH, Boersm HH, Kemerink G, Reutelingsperger CP, et l. In vivo detection of cell deth in the re t risk in cute myocrdil infrction. J Nucl Med. 2003;44: Plojoki E, Srste A, Eriksson A, Pulkki K, Klljoki M, Voipio-Pulkki LM, et l. Crdiomyocyte poptosis nd ventriculr remodeling fter myocrdil infrction in rts. Am J Physiol Hert Circ Physiol. 2001;280:H Olivetti G, Ai R, Quini F, Kjstur J, Cheng W, Nithr JA, et l. Apoptosis in the flling humn hert. N Engl J Med. 1997;336: Ate A, Biondi-Zocci GG, Bussni R, Dorin A, Cmilot D, Feroce F, et l. Incresed myocrdil poptosis in ptients with unfvorle left ventriculr remodeling nd erly symptomtic post-infrction hert filure. J Am Coll Crdiol. 2003;41: Sutton MG, Shrpe N. Left ventriculr remodeling fter myocrdil infrction: pthophysiology nd therpy. Circultion. 2000;101: Qin F, Ling MC, Ling CS. Progressive left ventriculr remodeling, myocyte poptosis, nd protein signling cscdes fter myocrdil infrction in rits. Biochim Biophys Act. 2005;1740: Luckcuck T, Trotter PJ, Wlker JH. Locliztion of nnexin V in the dult nd neontl hert. Biochem Biophys Res Commun. 1997;238: Mtsud R, Kneko N, Kikuchi M, Chiwki F, Tod M, Ieiri T, et l. Clinicl significnce of mesurement of plsm nnexin V concentrtion of ptients in the emergency room. Resuscittion. 2003;57: Received: 7 Septemer 2015 Accepted: 1 Decemer 2015 References 1. Gottlie RA, Burleson KO, Kloner RA, Bior BM, Engler RL. Reperfusion injury induces poptosis in rit crdiomyocytes. J Clin Invest. 1994;94: Kjstur J, Cheng W, Reiss K, Clrk WA, Sonnenlick EH, Krjewski S, et l. Apoptotic nd necrotic myocyte cell deths re independent contriuting vriles of infrct size in rts. L Invest. 1996;74:

Clinical Study Report Synopsis Drug Substance Naloxegol Study Code D3820C00018 Edition Number 1 Date 01 February 2013 EudraCT Number

Clinical Study Report Synopsis Drug Substance Naloxegol Study Code D3820C00018 Edition Number 1 Date 01 February 2013 EudraCT Number EudrCT Number 2012-001531-31 A Phse I, Rndomised, Open-lbel, 3-wy Cross-over Study in Helthy Volunteers to Demonstrte the Bioequivlence of the Nloxegol 25 mg Commercil nd Phse III Formultions nd to Assess