Activation of proteolysis by calpains and structural changes in human paroxysmal and persistent atrial fibrillation

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1 Crdiovsculr Reserch 54 (2002) locte/ crdiores Activtion of proteolysis by clpins nd structurl chnges in humn proxysml nd persistent tril fibrilltion * John J.L. Vn Der Wnt, Wiek H. vn Gilst, Hrry J.G.M. Crijns, Robert H. Henning,b, d b Binc J.J.M. Brundel, Jnnie Ausm, Isbelle C. vn Gelder, c e Deprtment of Clinicl Phrmcology, Groningen University Institute for Drug Explortion (GUIDE), Groningen, The Netherlnds b Deprtment of Crdiology, Thorxcenter University Hospitl, University of Groningen, Groningen, The Netherlnds c Deprtment of Cell Biology nd Electron Microscopy, University of Groningen, Groningen, The Netherlnds d Deprtment of Physiology, Crdiovsculr Reserch Institute Mstricht, University of Mstricht, Mstricht, The Netherlnds e Deprtment of Crdiology, Crdiovsculr Reserch Institute Mstricht, University of Mstricht, Mstricht, The Netherlnds Received 30 November 2001; ccepted 6 Februry 2002 Abstrct Objective: Atril fibrilltion (AF) is ccompnied by electricl, structurl nd ion-chnnel protein remodeling. We tested if proteolysis by clpin nd protesome is ctivted during AF, nd studied the reltion with the remodeling processes. Methods: Right tril ppendges were obtined from ptients with proxysml (n57) or persistent (n510) lone AF nd compred to controls (n510) in sinus rhythm undergoing coronry rtery bypss grfting (CABG). Proteolysis ws mesured using Suc-Leu-Leu-Vl-Tyr-7-mino-4-methylcoumrin. Protein expression of clpin I nd II ws ssessed by Western-blot nd clpin I locliztion by immunohistochemistry. Structurl chnges were quntified by counting tril myocytes with contrction bnds or hiberntion. Results: Clpin ctivity ws significntly incresed in proxysml AF (2-fold, P,0.001) nd persistent AF (3-fold, P,0.001), minly due to clpin I ctivtion. Incresed clpin I protein expression ws found in AF with Western blot nd immunohistochemistry. Myocytes from ll AF groups showed incresed contrction bnds, wheres hiberntion ws only found in persistent AF. Clpin ctivity correlted with L-type C chnnel nd Kv1.5 protein mounts (r520.80, P,0.001 nd r520.72, P,0.001, respectively), degree of structurl chnges (r50.90, P,0.001), shortening of tril effective refrctory period (AERP) (bsic cycle length 500 ms, r520.60, P,0.001) nd AERP rte dpttion (r520.80, P,0.001). Conclusions: Clpin ctivity is induced during AF nd correltes with prmeters of ion-chnnel protein, structurl nd electricl remodeling. The results suggest tht clpin ctivtion represents n importnt mechnism linking clcium overlod to cellulr dpttion mechnisms in humn AF Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Arrhythmi (mechnisms); Clcium (cellulr); Hiberntion; Remodeling; Suprventr. rrhythmi 1. Introduction [5 7]. To explin the electricl remodeling during AF, the expression level of ion-chnnels hve been investigted Atril fibrilltion (AF) is chrcterized by heterogeneity [8,9]. A reduction in protein levels of vriety of clcium in the electricl ctivtion pttern [1,2] nd loss of nd potssium ion-chnnels ws found [8], of which the contrctility [3,4]. Recent experimentl reserch hs dem- L-type C chnnel is believed to ply key role [6]. A onstrted tht AF induces electrophysiologicl chnges, reltionship between down-regultion of the L-type C influences protein expression nd leds to morphologicl chnnel nd chnges in AERP ws recently reported in ltertions which in turn increse the vulnerbility to AF humn AF [8]. The moleculr mechnism underlying chnges in expression of these proteins is poorly understood. An im- *Corresponding uthor. Deprtment of Rdition nd Stress Cell Biology, Deusingln 1, 9713 AV Groningen, The Netherlnds. Tel.: portnt clue my be obtined from the finding tht ; fx: E-mil ddress: b.j.j.m.brundel@med.rug.nl (B.J.J.M. Brundel). Time for primry review 17 dys / 02/ $ see front mtter 2002 Elsevier Science B.V. All rights reserved. PII: S (02)

2 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) reduction in protein expression occurs in bsence of rhythm undergoing coronry bypss grfting (CABG, n5 chnges in mrna in ptients with proxysml lone AF 10, Tble 1). The Institutionl Review Bord pproved the [8,9]. Such discrepncy ws lso observed by others study nd ptients gve written informed consent. Chnges [10,11] nd suggests ctivtion of protein degrdtion in electrophysiology were reported previously [8] by mechnism. An obvious cndidte is proteolysis invoked obtining intr-opertive AERP t five bsic cycle lengths by clcium dependent neutrl proteses, clpin I nd (BCL: ms). The djustment of AERP to BCL clpin II, s the cytosolic clcium increses during AF ws quntified by clculting the rte dpttion coefficient [12,13]. An incresed crdic clpin ctivity in crdic s the slope of the liner regression fter logrithmic myocytes hs been demonstrted following metbolic trnsformtion of BCL [8]. The mrna nd protein levels inhibition, crdic stunning nd clcium overlod [14,15]. of ion-chnnels in this popultion were lso reported Alterntively, ctivtion of the protesome my lso previously [8]. underlie incresed proteolysis in crdic cells, s shown for degrdtion of myosin hevy chin nd connexin43 in rt 2.2. Protein extrction [16,17]. To exmine whether proteolytic ctivtion plys role For nlysis of proteolysis, frozen RAAs were in humn AF, we determined the ctivity of clpins nd homogenized in buffer (100 mm Tris HCl, 145 mm the protesome in tril tissue of ptients with proxysml NCl, ph57.3) nd centrifuged t g (30 min, nd persistent lone AF nd of controls in sinus rhythm. An 4 8C). For Western-blot nlysis, prts of the sme RAAs incresed clpin-medited proteolysis ws found in AF. were homogenized in Rdio-Immuno-Precipittion-Assy Therefore, the protein levels of clpin I nd II were (RIPA) buffer [8]. Protein concentrtion ws determined determined nd locliztion of clpin I ws performed by using the DC ssy (Bio-Rd) with bovine lbumin immunohistochemistry. Since clpins degrde structurl stndrd. proteins [18], the structurl chnges of tril tissue were investigted by light nd electron microscopy. Finlly, we 2.3. Proteolytic ssy exmined the reltionship of clpin ctivtion with structurl chnges found in this study nd with chnges in the Suc-Leu-Leu-Vl-Tyr-7-mino-4-methyl-coumrin (AMexpression of vrious ion-chnnels nd electrophysiology C, Sigm) ws used s substrte. A 25 mg protein extrct s reported for the sme ptient popultion previously [8]. ws dded to 20 mm AMC in 300 ml Tris-buffered sline. AMC relese ws mesured by fluorometry (360-nm excittion; 430-nm emission, Spectrometer LS50B, Perkin 2. Mterils nd methods Elmer) fter incubtion for 30 min t 25 8C. Stndrd curves were generted using known concentrtions of Ptients mino-4 methyl-coumrin (Sigm) nd 25 mg het dentured protein. Mximl clpin ctivtion ws ssessed Right tril ppendices (RAAs) were obtined s de- fter reconstitution of clcium t 1 mm. E-64 (10 M, scribed before [8] from ptients with norml left ventricu- Roche), clpin I inhibitor (N-cetyl-Leu-Leu-norleucinl, lr function nd proxysml (n57) or persistent (n510) 10 M, Sigm) nd clpin II inhibitor (N-cetyl-Leu- lone AF undergoing MAZE surgery nd controls in sinus Leu-methioninl, 10 M, Sigm) were used to ssess Tble 1 Bseline chrcteristics of ptients with lone proxysml AF (PAF), lone persistent AF (CAF) nd control ptients in sinus rhythm (SR) Ptient chrcteristics SR PAF CAF N Age Previous durtion AF Medin, rnge (months) 12.5 (3 56) Durtion SR before surgery Medin, rnge (dys) 2 (0.5 12) Durtion lst proxysm Medin, rnge (h) 9 (1 ) Exercise tolernce NYHA Clss I NYHA Clss II Mediction Digitlis Clcium ntgonists Bet-blockers P,0.05 compred to SR.

3 382 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) clpin ctivtion. Lctcystin (10 M, Clbiochem) ws plemented with 1.5% K4Fe(CN) 6 in ccodylte buffer, ph used to investigte protesome ctivity. All inhibitors were 7.4) t 4 8C nd embedded in Epon. Light microscopy ws dded 30 min before mesuring AMC relese. The mount performed using semi-thin sections (1 mm) stined with of ntive protein chosen ws in the liner AMC relese 1% toluidine blue. Chnges were evluted in t lest 300 signl re. Protese inhibitors were used t concentrtions cells from six rndomly chosen regions with the nucleus in rendering mximl inhibition. Assys were performed in the plne of the section by n investigtor blinded for triplicte. Tissue clpin ctivity ws clculted s the ptient groups, who scored: (1) hibernting cells, showing mount of protese ctivity specificlly blocked by E-64. res of loss of srcomeres (.10% of the cell surfce) nd ple nuclei [7]; nd (2) cells displying intensely stined 2.4. Western blot contrction bnds (.10% of srcomere surfce) [19]. Ultrstructurl chnges were studied by electron micro- Protein levels of clpins were determined by Western scopy (Philips 201, 60 kv) on ultrthin sectons (60 nm) blot nd expressed s rtio to levels of GAPDH. Dentured stined with urnylcette nd led citrte. protein (10 mg) ws seprted by sodium dodecyl sulfte polycrylmide gel electrophoresis (SDS PAGE) nd 2.7. Sttisticl nlysis trnsferred to nitrocellulose membrnes (Strtgene) followed by stining with Ponceu S solution (Sigm). Results re expressed s men6s.d. One-wy ANOVA Membrnes were incubted with primry ntibody ginst ws used for multiple group comprisons. Correltion ws GAPDH (Affinity Regents, USA), lrge subunit of clpin determined with Spermn correltion test (SPSS 8.0). I or clpin II (both Reserch Dignostics, USA). Horse- P,0.05 ws considered sttisticlly significnt. rdish peroxidse-conjugted nti-mouse or nti-rbbit IgG (Snt-Cruz Biotechnology) ws used s secondry ntibody. Signls were detected by the ECL-detection method 3. Results (Amershm) nd quntified by densitometry. The mount of protein chosen ws in the liner immunorective signl 3.1. Proteolytic ctivity rnge. The specificity of the ntibodies ws checked by pre-incubtion with control peptide ntigen. Proteolytic ctivity ws significntly incresed in tissue from ptients with proxysml nd persistent AF compred 2.5. Immunohistochemistry to ptients in sinus rhythm (Fig. 1A, Tble 2). Proteolytic ctivity ws mesured in the presence of inhibitors to ssess the involvement of different pthwys Immunohistochemistry ws performed in six ptients with proxysml AF, eight with persistent AF nd eight (Tble 2). The non-selective clpin inhibitor E-64 (10 control ptients. Two groups of 5 mm thick frozen RAA M) nd clpin I inhibitor (10 M) significntly reduced sections were mde. Sections were ir dried before use or proteolytic ctivity to similr level in ll groups. Clpin immeditely fixed for 10 min in 4% prformldehyde in II inhibitor (10 M) only prtilly ttenuted the inphosphte buffered sline (PBS). After three wshes (PBS, cresed proteolytic ctivity observed in AF, while the 10 min) nd 30 min blocking with 1% BSA in PBS, protesome inhibitor lctcystin (10 M) did not reduce sections were incubted with nti-clpin I ntibodies proteolytic ctivity in ny group. To test whether incresed (1:100) (Reserch Dignostics, USA) overnight t room clpin ctivity ws due to clcium in the protein extrct, temperture. After three wshes with PBS (5 min), sections experiments were conducted in the presence of 10 mm were incubted with peroxidse conjugted rbbit-nti- EDTA. No differences were found in proteolysis in the mouse IgG (DAKO A/ S, Glostrup, Denmrk, 1 h). Three presence or bsence of EDTA (sinus rhythm: 96%68, wshes with PBS were followed by one with qu dest (5 proxysml AF: 93%67; persistent AF:101%610). min), peroxidse ctivity ws detected using buffer of 40 Mximl clpin ctivity ws determined by ddition of mg 3-mino-9-ethylcrbzole (Sigm), 10 ml N,N di- 1 mm clcium, resulting in n extr ctivtion of methylformmide (Merck), hydrogen peroxide 0.01% (v/ proteolysis cross ll ptient groups (Tble 2). The extr v) nd 190 ml 0.05 M sodium cette (ph 4.95). After ctivtion due to ddition of clcium ws bolished by stining for 10 min, sections were rinsed with wter, E-64, clpin I inhibitor nd clpin II inhibitor, but counterstined with hemtoxylin (Sigm) nd mounted unffected by lctcystin. Furthermore, tissue clpin with Kiser s glycerol geltin (Merck). ctivity cross ll groups correlted significntly with the mximl clpin ctivity (r50.89, P,0.001; Fig. 1B) Morphologicl evlution 3.2. Clpin protein levels Biopsies from RAAs were immeditely fixed for 2 h in 2% glutrldehyde (in 0.1 M ccodylte buffer, ph 7.4) To exmine the reltion between clpin ctivtion nd with post-fixtion for 2 h in 1% osmium tetroxide (sup- expression, proteins levels of clpins were determined by

4 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) Fig. 1. Significnt increse of tissue clpin ctivity (A) in RAAs of ptients with sinus rhythm (SR; s), proxysml AF (PAF; () nd persistent AF (CAF; d). (B) Significnt correltion for mximl nd tissue clpin ctivity. Clpin ctivity ws expressed s nm AMC/ mg protein/ 30 min. (C) Typicl Western-blot of clpin I expressed s rtio to GAPDH nd group protein rtios for clpin I. (D) Significnt correltion of clpin ctivity nd clpin I levels. * P,0.01 compred to SR. GAPDH levels did not differ between the groups (SR: , PAF: 10967, CAF: 98611, rbitrry units) Locliztion of clpin I Clpin I locliztion ws studied by immunohisto- chemistry to determine the cell type responsible for its increse in ctivity nd expression. Clpin I ws pre- dominntly loclized in the nucleus, interclted discs nd Western-blotting (Fig. 1C,D). Protein levels of clpin I were significntly incresed by 35% (P,0.01) in ptients with persistent AF compred to controls (Fig. 1C), wheres clpin II levels were similr in ll groups (sinus rhythm: ; proxysml AF: ; persistent AF: ). A positive correltion ws found between tissue clpin ctivity nd protein expression of clpin I (r50.61, P,0.001, Fig. 1D), wheres no correltion ws found with clpin II expression (r520.20, P50.33). Tble 2 Proteolytic ctivity (nm AMC/ mg protein/ 30 min) in tril tissue of ptients with lone proxysml AF (PAF), lone persistent AF (CAF) nd control ptients in sinus rhythm (SR) No clcium dded 11 mm clcium SR PAF CAF SR PAF CAF None E-64 (10 M) Clpin I inhibitor (10 M) Clpin II inhibitor (10 M) Lctcystin (10 M) P,0.05 compred to SR.

5 384 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) in the cytoplsm of tril myocytes (Fig. 2A,B). To obtin types of structurl chnges were found in the tril n indiction bout the mount of clpin I protein in the myocytes, i.e. contrction bnds (Fig. 3C) nd with myocytes, the stining intensity ws scored semi-quntit- hiberntion, chrcterized minly by reduced number of tively (Fig. 2C E). Stining intensity of the nucleus nd srcomeres (myolysis) (Fig. 3E). Atril myocrdium of the interclted discs incresed significntly from sinus sinus rhythm ptients showed normlly structured rhythm, vi proxysml AF to rech mximum in myocytes minly without myolysis (5%) nd contrction persistent AF (Fig. 2C,D). In contrst, stining intensity in bnds (12%) (Figs. 3A nd 4A). At the ultrstructurl the cytoplsm did not differ between the groups (Fig. 2E). level, highly orgnized srcomeric structure with mitochondri in between, nd typicl clustering of heteroch Structurl chnges romtin t the nucler membrne were found (Fig. 3B). In contrst, in ptients with proxysml AF, contrction Structurl nd ultr-structurl chnges in tril tissue bnds were bundntly present (Fig. 3C) nd these were exmined by light nd electron microscopy. Two myocytes showed pyknotic nuclei. Also secondry lyso- Fig. 2. (A) Immunohistochemicl locliztion of clpin I in myocytes in the nucleus (rrow) nd (B) t the interclted discs (rrow) (mgnifiction (A) 3400, (B) 3300). Intensity of the clpin I stining in ptients with PAF (n56; (), CAF (n58; d) nd SR (n58; s) in the nucleus (C), t the interclted discs (D) nd in the cytosol (E). 6 low stining intense stining; * P,0.05, ** P,0.01, compred to SR.

6 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) Fig. 3. Typicl exmples of structurl nd ultrstructurl chnges in tril myocytes. Control ptient exmined by light (A) nd electron microscopy (B), showing norml myocytes without myolysis, contrction bnds nd norml nucleus. Proxysml AF ptient show contrction bnds (C, rrow) nd degenertive fetures s clumping of nucler chromtin nd secondry lysosomelike bodies (D, rrow). Persistent AF ptient show extensive perinucler myolysis nd ple nuclei (E,F). Further, only rim of srcomeres is present t the border of the cell, glycogen (g) is visible nd the nucleus hs homogenously dispersed chromtin (F). (Mgnifiction A, C nd E 3250, B 34250, D nd F 33400).

7 386 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) ship ws observed (Fig. 4C). Atril myocytes of ptients with the shortest durtion of AF (,10 months) showed high mounts of contrction bnds nd low mounts of hibernting cells, wheres the opposite pttern ws found in ptients with the longest durtion of AF (.10 months) Reltion between clpin ctivtion, ion-chnnel expression nd AERP Previously we reported chnges in ion-chnnel mrna nd protein levels in tril tissue of this ptient popultion [8]. In brief, the reduction in protein expression of the L-type C chnnel, Kv1.5 nd HERG ws ccompnied with little or minor chnges in mrna levels. In contrst, reduction in both mrna nd protein levels, showing significnt correltion, ws observed for Kv4.3. To investigte potentil role of clpin ctivtion in reduction of these ion-chnnels, clpin ctivity ws correlted with ion-chnnel protein levels. A highly significnt correltion ws found between clpin ctivity nd reduction in L-type C -chnnel nd Kv1.5 protein, wheres correltion with HERG ws less prominent nd with Kv4.3 ws bsent (Fig. 5). Ptients with AF demonstrted significntly shorter AERPs nd poorer rte dpttion thn control ptients in sinus rhythm, s reported previously [8]. To investigte whether clpin ctivtion reltes to electricl remodeling, correltion between clpin ctivity, AERPs nd rte dpttion coefficient ws mde. Clpin ctivity showed significnt negtive correltion with AERP t different BCLs nd with the rte dpttion coefficient (Tble 3). 4. Discussion The min finding of this study is the ctivtion of clpin-medited proteolysis in tril tissue of ptients with proxysml nd persistent lone AF. Incresed clpin ctivity ws ccompnied by incresed expression of clpin I nd intensified stining of clpin I in tril myocytes. Furthermore, clpin ctivity correlted well with severl prmeters of electricl, structurl nd protein remodeling. Activtion of proteolysis by clpin explins the reduction of ion-chnnel proteins found in the presence of unchnged mrna levels in ptients with lone, prox- ysml AF [8], nd my represent one importnt moleculr event in vrious remodeling processes observed in AF Clpin ctivtion This study demonstrtes incresed proteolytic ctivity in tril myocytes of ptients with AF due to ctivtion of clpins. This is evidenced by the reduction of proteolysis to similr levels in ll groups by the non-selective clpin inhibitor E-64, even under high clcium conditions known to ctivte clpin II. In contrst, the selective inhibitor of Fig. 4. (A) Percentge structurl chnges of tril myocytes ffected by contction bnds or hiberntion in ptients with sinus rhythm (SR), proysml AF (PAF) nd chronic, persistent AF (CAF). (B) Significnt correltion between the totl mount of ffected cells nd clpin ctivity. (C) Significnt correltion between the type of structurl chnge nd the durtion of persistent AF. (d) contrction bnds nd (j) hiberntion. some like bodies were present t the ultrstructurl level (Fig. 3D). Ptients with persistent AF reveled both contrction bnds nd hiberntion (Fig. 3E,F). The number of myocytes with contrction bnds ws incresed 3-fold in both AF groups compred to controls (Fig. 4A), while the mount of hibernting cells ws only significntly incresed in persistent AF (Fig. 4A). Furthermore, correltion ws found between the clpin ctivity nd the totl number of ffected cells (either contrction bnds or hiberntion; r50.71, P,0.001; Fig. 4B). When the type of structurl chnge, i.e. contrction bnds or hiberntion, of ptients with persistent AF ws plotted ginst the durtion of AF n intriguing reltion-

8 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) Fig. 5. Correltion between tissue clpin ctivity nd expression of vrious plsm membrne ion-chnnels. (A) nti-l-type clcium chnnel, (B) nti-kv1.5, (C) nti-kv4.3 nd (D) HERG. (() indictes ptients with PAF, (d) CAF nd (s) SR. the protesome, lctcystin, did not influence proteolysis in However, clpin I inhibitor completely ttenuted the ny group. In ddition, Western-blot reveled incresed incresed proteolysis in AF both under bsl nd high protein levels of clpin I in persistent AF, but unchnged clcium conditions, wheres clpin II inhibitor did not. levels of clpin II. Finlly, intensity of clpin I stining Moreover, clpin I protein level ws incresed nd ws incresed in AF ptients. Tken together, these correlted with incresed clpin ctivity, in contrst to findings represent report of ctivtion of the clpin tht of clpin II. Therefore, our results suggest tht the pthwy in humn crdic disese. incresed clpin ctivity in AF is minly due to ctivtion The reltive contribution of clpin I nd II is difficult to nd up-regultion of clpin I, rther thn clpin II. estblish by using inhibitors, due to their prtil selectivity. The stining intensity of clpin I ws incresed t the interclted discs nd nucleus in ptients with AF, in ccord with the observed increse in ctivity nd protein Tble 3 expression. Incresed clpin I expression in AF seems Correltion of clpin ctivity with AERP nd rte dpttion coefficient confined to cellulr res tht re vitl for ction potentil BCL SR PAF CAF Correltion conduction nd structurl integrity of the tril myocytes. prmeters Under norml conditions, clpin is loclized diffusely in AERP (ms) r P the cytosol. After n increse in intrcellulr clcium, clpin rpidly trnsloctes to the inner surfce of the b b plsm membrne, ggregtes t the interclted discs, ,0.001 b which is followed by its ctivtion [20]. At the interclted discs, ctivted clpin I my degrde importnt ion NS chnnels, like the N -chnnel [] nd Kv1.5 [22], but Rte dpttion coefficient lso proteins involved in excittion-contrction coupling ,0.001 [23] nd conduction [10]. The incresed expression of NS, not significnt. clpin I t the nucleus might be suggestive for role in P,0.05. promoting cell deth [], indicting tht clpin I my b P,0.01. ply role in the degenertion observed in AF [25].

9 388 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) Structurl chnges in myocytes dditionl short-term dpttion mechnism through in- ctivtion of functionl C current [36]. This study in humn AF investigtes myocyte degener- Irrespective of whether clpin ctivtion is n inititor tion nd hiberntion in tril tissue. Contrction bnds or consequence of moleculr remodeling, its ctivtion is were incresed in equl mounts both in ptients with likely to contribute to further deteriortion of tril tissue. persistent nd proxysml AF. Contrction bnds were Therefore, interference with the clpin pthwy my previously observed in degenertive myocrdium [25,26]. represent n importnt tool to unrvel the sequence of In contrst, hibernting cells were only incresed in moleculr events in AF, nd perhps future strtegy for ptients with persistent AF. The ltter is in greement with phrmcologicl intervention. the experimentl got model for AF, in which hiberntion develops only t reltively lte stge of the rrhythmi 4.4. Possible clinicl relevnce [7]. Thus, these findings suggest tht contrction bnds represent n erly structurl bnormlity in humn lone The success rte of chemicl nd electricl crdioversion AF, wheres hiberntion develops only fter protrcted nd the time needed to recover tril contrctile function period of sustined AF. Similr ltertions in myocrdil depends on the AF durtion [4]. In ddition to electricl structure were described in ptients with tril rrhythmis remodeling, the structurl remodeling of tril myocytes of vrious etiology [27]. Moreover, in persistent AF, the my contribute to the intrctbility of long-term AF. After number of hibernting cells incresed with the durtion of restortion of norml sinus rhythm, it my tke the AF, while contrction bnds decresed. Hibernting crdiomyocyte certin period to normlize levels of myocrdium is suggested to be protected ginst degener- srcomeres, if possible t ll (J. Ausm et l. unpublished tion [28]. Tken together, the observed structurl chnges results). The erly structurl chnges observed in proxre indictive of substntil deteriortion of norml tissue ysml AF underscores the importnce to restore sinus rchitecture, likely to promote AF through heterogeneity of rhythm s soon s possible, thereby preventing the contril refrctoriness [1,2] nd slowed tril conduction tinution of the tril structurl, ion-chnnel protein nd [29 31]. electricl remodeling. Whether clpin inhibitors my serve to protect tri in this respect, remins to be investigted Clpin ctivtion nd remodeling 4.5. Limittions of the study Clpin ctivity correlted well with the observed structurl chnges nd previously mesured AERP djust- Idelly, the quntifiction of clpin I stining intensity ments. Furthermore, correltion with protein levels ws would need stining of reference protein to correct for present for three ion-chnnels, in which mrna levels did potentil differences during development. However, it is not ccount for reduction in chnnel protein [8]. The hrd to define suitble reference protein in AF, in view of significnt correltion with protein levels of L-type C the notble structurl chnges of myocytes. To void this chnnel nd Kv1.5 my be explined by the fct tht these experimentl bis, smples were cut, fixed, incubted nd undergo the most profound protein reduction nd tht both stined simultneously. The good greement between hve been described s trgets for clpin degrdtion upregultion of clpin I protein found in Western Blot nd [22,32]. in the stining, suggests but does not prove tht the As consequence, most prmeters show correltion method ws dequte. Therefore stining quntifiction with ech other. Therefore, it remins uncler whether dt should be interpreted with cre. clpin ctivtion cuses the moleculr chnges ssocited with remodeling, or merely reflects gross cellulr dmge. However, the up-regultion of clpin I protein seems References incomptible with generl cellulr dmge. Also on theoreticl grounds, clpin ctivtion represents likely [1] Rmnn H, Huer RNW, Wittkmpf FHM et l. Identifiction of cndidte to convey importnt cellulr chnges in AF. the substrte of tril vulnerbility in ptients with idiopthic tril Clpins hve been demonstrted to degrde cytoskeletl fibrilltion. Circultion 2000;101: [2] Freh S, Villemire C, Nttel S. Importnce of refrctoriness [18], contrctile [15] nd L-type C chnnel [32] heterogeneity in the enhnced vulnerbility to tril fibrilltion proteins tht re involved in tril remodeling in AF. The induction cused by tchycrdi-induced tril electricl remodeling. reduction in expression of L-type C chnnel is thought Circultion 1998;83: to ply mjor role in electricl remodeling in AF [6]. [3] Doud EG, Mrcovitz P, Knight B et l. Short-term effect of tril Clpins re redily ctivted by elevted cellulr clcium fibrilltion on tril contrctile function in humns. Circultion 1999;99: levels [33,34], while clcium overlod plys key role in [4] Mnning WJ, Silvermn DI, Ktz SE et l. Impired left tril the pthogenesis of AF [8,12,13,35]. Of note is tht mechnicl function fter crdioversion: reltion to the durtion of clcium overlod is lso responsible for the initition of tril fibrilltion. J Am Coll Crdiol 1994;23:

10 B.J.J.M. Brundel et l. / Crdiovsculr Reserch 54 (2002) [5] Wijffels MC, Kirchhof CJ, Dorlnd R, Allessie MA. Atril fibrill- Pontremoli S. Chnges in intrcellulr locliztion of clpsttin tion begets tril fibrilltion. A study in wke chroniclly in- during clpin ctivtion. Biochem J 1999;343: strumented gots. Circultion 1995;92: [] Cohen SA. Immunocytochemicl locliztion of rh1 sodium chn- [6] Yue L, Melnyk P, Gspo R, Wng Z, Nttel S. Moleculr mehnisms nel in dult rt hert tri nd ventricle. Presence in terminl underlying ionic remodeling in dog model of tril fibrilltion. interclted disks. Circultion 1996;94: Circ Res 1999;84: [22] Mys DJ, Foose JM, Philipson LH, Tmkun MM. Locliztion of [7] Ausm J, Wijffels M, Thone F et l. Structurl chnges of tril 1 the Kv1.5 K chnnel protein in explnted crdic tissue. J Clin myocrdium due to sustined tril fibrilltion in the got. Circul- Invest 1995;96: tion 1997;96: [23] Lflmme MA, Becker PL. G(s) nd denylylcyclse in trnsverse [8] Brundel BJJM, Vn Gelder IC, Henning RH et l. Ion chnnel tubules of hert: implictions for camp-dependent signling. Am J remodeling is relted to intr-opertive tril refrctory periods in Physiol 1999;277:H1841 H1848. ptients with proxysml nd persistent tril fibrilltion. Circultion [] Nkgw T, Yun J. Cross-tlk between two cysteine protese 2001;103: fmilies: ctivtion of cspse-12 by clpin in poptosis. J Cell [9] Brundel BJJM, Vn Gelder IC, Henning RH et l. Altertions in Biol 2000;150: potssium chnnel gene expression in tri of ptients with persis- [25] Aime-Sempe C, Folliguet T, Rucker-Mrtin C et l. Myocrdil cell tent nd proxysml tril fibrilltion. J Am Coll Crdiol deth in fibrillting nd dilted humn right tri. J Am Coll Crdiol 2001;37: ;34: [10] Vn der Velden HMW, Ausm J, Rook MB et l. Gp junctionl [26] Jennings RB, Gnote CE. Structurl chnges in myocrdium during remodeling in reltion to stbiliztion of tril fibrilltion in the cute ischemi. Circ Res 1974;34: got. Crdiovsc Res 2000;46: [27] Mry-Rbine L, Phm TD, Hordof A et l. The reltionship of [11] Goette A, Arndt M, Rocken C et l. Regultion of ngiotensin II humn tril cellulr electrophysiology to clinicl function nd receptor subtypes during tril fibrilltion in humns. Circultion ultrstructure. Circ Res 1983;52: ;101: [28] Ausm J, Thone F, Dispersyn GD et l. Dedifferentited cr- [12] Ausm J, Dispersyn GD, Duimel H et l. Chnges in ultrstructurl diomyocytes from chronic hibernting myocrdium re ischemiclcium distribution in got tri during tril fibrilltion. J Mol Cell tolernt. Mol Cell Biochem 1998;186: Crdiol 2000;32: [29] Morillo CA, Klein GJ, Jones D, Guirudom CM. Chronic rpid [13] Sun H, Chrtier D, Leblnc N, Nttel S. Intrcellulr clcium tril pcing. Structurl, functionl, nd electrophysiologicl chrcchnges nd tchycrdi-induced contrctile dysfunction in cnine teristics of new model of sustined tril fibrilltion. Circultion tril myocytes. Crdiovsc Res 2001;49: ;91: [14] Atsm DE, Bstinse EM, Jerzewski A, Vn Der Vlk LJ, Vn Der [30] Elvn A, Wylie K, Zipes DP. Pcing-induced chronic tril fibrill- Lrse A. Role of clcium-ctivted neutrl protese (clpin) in cell tion impires sinus node function in dogs: electrophysiologicl deth in cultured neontl rt crdiomyocytes during metbolic remodeling. Circultion 1996;94: inhibition. Circ Res 1995;76: [31] Gspo R, Bosch RF, Tljic M, Nttel S. Functionl mechnisms [15] Gorz L, Menbo R, Vitdello M, Bergmini CM, Di Lis F. underlying tchycrdi-induced sustined tril fibrilltion in Crdiomyocyte troponin T immunorectivity is modified by cross- chronic dog model. Circultion 1997;96: linking resulting from intrcellulr clcium overlod. Circultion [32] Belles B, Hescheler J, Trutwein W, Blomgren K, Krlsson JO. A 1996;93: possible physiologicl role of the C-dependent protese clpin nd [16] Eble DE, Sprgi ML, Ferguson A, Smrel AM. Srcomeric its inhibitor clpsttin on the C current in guine pig myocytes. myosin hevy chin is degrded by the protesome. Cell Tissue Res Pflugers Arch 1988;412: ;296: [33] Suzuki K, Imjoh S, Emori Y. Clcium-ctivted neutrl protese [17] Ling JG, Tdros PN, Sffitz J, Beyer EC. Proteolysis of nd its endogenous inhibitor. Activtion t the cell membrne nd connexin43-contining gp junctions in norml nd het-stressed biologicl function. FEBS Lett 1987;220: crdic myocytes. Crdiovsc Res 1998;38: [34] Mtsumur Y, Seki E, Otsu K et l. Intrcellulr clcium level [18] Ppp Z, Vn Der Velden J, Stienen G. Clpin-I induced ltertions required for clpin ctivtion in single myocrdil cell. J Mol in the cytoskeletl structure nd impired mechnicl properties of Cell Crdiol 2001;33: single myocytes of rt hert. Crdiovsc Res 2000;45: [35] Nttel S. New ides bout tril fibrilltion 50 yers on. Nture [19] Borgers M, Guo Shu L, Xhonneux R, Thone F, Vn Overloop P. 2002;415: Chnges in ultrstructure nd C distribution in the isolted [36] Rmirez RJ, Nttel S, Courtemnche M. Mthemticl nlysis of working rbbit hert fter ischemi. A time-relted study. Am J cnine tril ction potentils: rte, regionl fctors nd electricl Pthol 1987;126: remodeling. Am J Physiol Hert Circ Physiol 2000;279:H1767 [20] De Tullio R, Psslcqu M, Avern M, Slmino F, Melloni E, H1785.