Damage-induced arrhythmias: reversal of excitation contraction coupling

Transcription

1 Cardovascular Research 4 (1998) Revew Damage-nduced arrhythmas: reversal of exctaton contracton couplng * Henk E.D.J. ter Keurs, Yng Mng Zhang, Masahto Mura Departments of Medcne, Physology and Bophyscs, The Unversty of Calgary, Calgary, Alberta T2N 4N1, Canada Receved 1 July 1998; accepted 24 August Introducton The purpose of ths paper s to revew evdence for ths concept. In partcular, we wll consder the evdence n Sustaned or self-termnatng ventrcular arrhythmas support of a mechansm n whch both non-unform commonly arse as the oscllatory response of the mecha- contracton and ncreased Ca load of cells adjacent to nsms nvolved n re-entry to a premature beat n non- acutely damaged cells are essental n the spontaneous unform workng myocardum or conducton tssue. Non- generaton of Ca transents durng the relaxaton phase unformty of conducton n the human heart tself s often of the electrcally drven twtch. A novel aspect of ths a result of myocardal schema or nfarcton due to concept s that these Ca transents nduce propagatng coronary artery dsease. Ischemc non-unformty may Ca waves, whch travel nto the adjacent normal further be aggravated by cardac remodellng durng myocardum and cause after-depolarzatons, whch n turn development of congestve heart falure, thereby, provdng may cause premature beats. We assume that the mechansm a possble mechansm substrate for fatal arrhythmas whch of ntaton of the propagatng Ca waves nvolves often cause the demse of patents wth congestve heart feedback of rapd length or force changes to bndng of falure [1,2]. Most dscussons of these arrhythmas empha- Ca ons to the contractle flaments. Hence, consderasze the mportance of electrcal non-unformty. Neverthe- ton of these mechansms s of nterest n the context of less, t s evdent that myocardum that has been rendered ths ssue of Cardovascular Research. non-unform wll also exhbt varatons of mechancal stresses and strans from cell to cell as well as dfferences among cells n exctaton contracton couplng, n addton 2. After-contractons and after-depolarzatons and to electrcal non-unformty. Ca transents Too lttle s known about the role played by nonunform myocardal stress and stran dstrbutons and by It has been known for half a century [5,6], that two non-unform exctaton contracton couplng n mecha- types of oscllatons n membrane potental may follow an nsms underlyng premature beats that ntate an arrhyth- acton potental of cardac muscle: (1) early after-depolarma. Stll, ths knowledge s essental to the choce of zatons and (2) delayed after-depolarzatons (DAD). treatment amed at preventon of a premature beat versus Wdespread nterest n DAD resulted n the early 7 s from treatment amed at suppresson of re-entry. An especally the observaton that these after-depolarzatons could be clncally mportant scenaro n whch arrhythmas occur s caused by dgtals ntoxcaton [7 12]. Also, trggered that of schemc damage of myocardum. It s generally arrhythmas appeared to result from DAD emphaszng accepted that acute schemc damage of myocardum leads ther potental clncal mportance [13]. A clue to ther after 2 3 mn to Ca overload of the damaged cells mechansm was provded by the observaton that afterand ther neghbours [3]. The arrhythmas of schemc depolarzatons n Purknje fbers and the accompanyng myocardum that occur n ths phase may well be related to after-contractons occur when the muscle was loaded the Ca overload [4]. Hence, t s concevable that wth Ca. Both ntaton after the last stmulated twtch damage-nduced premature beats may ntate re-entry and the tme course of these transents accelerated f arrhythmas and that abnormal cellular Ca transport may [Ca ] or the stmulaton rate was ncreased. Dgtals play a crucal role n the ntaton of the premature beat. appeared to accelerate the after-depolarzatons even more than hgh [Ca ] and a hgh stmulus rate [14,15]. * Correspondng author. Tel.: ; fax: ; e-mal: Henk@cvrsun1.cvr.ucalgary.ca Tme for prmary revew 16 days / 98/ $ see front matter 1998 Elsever Scence B.V. All rghts reserved. PII: S8-6363(98)263-6 Downloaded from on January 218

2 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) Lke n any arrhythma, the acton potentals that trggered arrhythmas both result from the prevous mpulse and lead to subsequent mpulse generaton. It has been shown that the DADs are based on a spontaneous ncrease n ntracellular [Ca ] ([Ca ] ) leadng to a transent nward current on the one hand and to actvaton of the contractle flaments on the other hand [16,17]. Kass et al. have proposed that a [Ca ] transent assumed to be due to spontaneous Ca release from the sarcoplasmc retculum (SR) leads to a transent nward current [17]. Hence, a suffcently large Ca load of the SR would create an unstable state where the spontaneous Ca release may become so large that the resultng transent Fg. 1. Spatal changes n fluorescence sgnals durng a delayed after- nward current depolarzes the cells enough to trgger a depolarzaton (DAD), caused by a slowly propagatng Ca wave n a cardac myocyte. Top panel shows membrane potental, mddle shows new acton potental, whch perpetuates tself as a trggered spato-temporal changes n Fura 2 fluorescence sgnals, and bottom shows arrhythma [13]. cell length. Mddle: Focal fluorescence transents emerged spontaneously at the center of the myocyte and spread n both drectons after fluorescence transents were elcted by an acton potental. Propagatng patterns of fluorescence transents seemed to be waves. It should be noted that spontaneous fluorescence transents were occurrng concom- 3. Spontaneous Ca release from the SR and Ca tant wth a DAD n top (arrow) and a spontaneous contracton n bottom, waves n myocytes ST, electrcal stmulaton. Modfed from Ref. [3]. Spontaneous Ca release from the sarcoplasmc retculum has been well-documented n solated enzymatcal- Ca waves arse smultaneously from multple foc, the ly dspersed cardac cells usng confocal mcroscopy. resultant sarcolemmal depolarzaton s augmented to a These events range from Ca release from sngle Ca level that can nduce an acton potental [32]. These channels n the SR (Ryanodne receptors (RyR)) to Ca observatons are consstent wth the concept that durng a release by clusters of RyR n response to Ca enterng va DAD the ncrease n [Ca ] causes a transent nward 1 the L-type Ca channels called sparks [18]. Ca sparks current arsng from electrogenc Na / Ca exchange and may also trgger each other and cause Ca waves as s Ca -actvated non-selectve caton channels [17]. suggested by the observaton that Ca sparks lead the Ca waves usually start at one end of myocytes, where wave front of Ca waves [19]. The arrval of Ca - one mght envsage gap junctons, but sometmes multple senstve fluorescent dyes such as Fura 2, Indo 1, and Fluo and varable foc of Ca waves can been observed [29]. 3 has made t possble to record mages of the regonal When a Ca wave begns n a focus wthn a myocyte, t ncrease n [Ca ] that elct regonal contractle actvty. spreads at equal velocty n all drectons, so that the Clearly, solated cardac myocytes have proven to be an observer gets the mpresson of a wave spreadng n a pond mportant tool to study spontaneous Ca -release events [23]. Because such a wave wll reach the sdes of a and ther consequences for contractle actvty provdng cylndrcal cell frst, t spreads subsequently n both nsghts nto phenomena such as after-contractons, ar- drectons at equal speed whle the wave front becomes rhythmas, and depresson of systolc and dastolc functon flatter (Fg. 1). In addton to lnear propagaton, Ca n the state of [Ca ] overload [2]. Whle spontaneous waves may also propagate n sprals spnnng around actvty s most easly observed n rat myocytes, essentally ntracellular organelles [26]. Sometmes Ca waves the same s seen n myocytes from other mammals under emerge, upon ntaton at the end of a myocyte, as a condtons n whch [Ca ] s forced to an excess []. We dome-lke regon of spontaneously elevated [Ca ] (3 wll focus here on regonal Ca transents, whch have nm) approxmately 2 mm n dameter, and propagate as a been shown to appear as Ca waves that propagate localzed 1 mm wde band of elevated [Ca ] [23,28]. along a myocyte [ 29]. Fg. 1 shows a Ca wave after Ampltude and wdth of Ca waves are farly constant an acton potental nduced a synchronous Ca transent durng propagaton [23 25] and ther velocty of propagan a myocyte accompaned by an after-contracton and a ton s typcally about 1 mm/ s n unstmulated cells DAD. In ths case, the spontaneous Ca wave emerged at [ 26,28,33]. However, t has been observed that the the center of the myocyte and spread n both drectons propagaton velocty can be ncreased transently up to concomtant wth a DAD and a spontaneous contracton about 1 mm/ s after rapd electrcal stmulaton (Mura, [3]. Typcally, the nterval between the last stmulaton unpublshed observatons). and the onset of the frst Ca wave shortens and the Ca waves may start n multple stes n a myocyte as probablty of multple foc of Ca waves ncreases when s shown n Fg. 2. As a result they may appear to collde the stmulus frequency or [Ca ] s ncreased [31]. When whle travellng through the cell. Also, waves may be Downloaded from on January 218

3 446 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) Fg. 2. [Ca ] transents are followed by refractorness of Ca release by the SR. The fgure shows membrane potental (upper panel), spatal and temporal changes n fura-2 fluorescence ntenstes (mddle panel), and cell length (lower panel). When a [Ca ] wave (F(t)W ) precedes a Ca transent nduced by an acton potental (F(t)AP), the latter transent s smallest at the ste where the wave has just arrved. Conversely, the wave stops as a result of the occurrence of the acton potental. F(t)W, fluorescence transents due to calcum wave; F(t)AP, fluorescence transents nduced by an acton potental; Ex 38, exctaton wavelength at 38 nm; ST, electrcal stmulaton. Modfed from Ref. [34]. present n the cell at the moment that the cell s electrcally loadng [33] as does the number of foc of Ca waves excted. The behavour of the waves under these crcum- [31]. When two or more foc wth dfferent frequences stances s qute revealng about the process that generates perodcally generate Ca waves wthn a myocyte, a them. When Ca waves start from two or more foc Ca wave orgnatng from fastest focus can reset wave wthn a myocyte, the waves appear to collde wthout generaton at other foc and domnate actvaton of the augmentaton of the [Ca ]. After the collson, the whole myocyte [29]. [Ca ] declnes wthout evdence of further propagaton of the waves demonstratng refractorness of the propagaton mechansm (the left part of Fg. 2) [25 27]. Fg Trggered propagated contractons n cardac further llustrates that Ca waves are the consequence of muscle a process wth a refractory perod. The fgure shows that f the expermenter elcts an acton potental durng the Events related to spontaneous Ca release by the SR propagaton of a Ca wave, the ampltude of acton smlar to those n solated myocytes have been observed potental-nduced Ca transents and the accompanyng usng confocal mcroscopy under physologcal condtons twtch are reduced by the precedng Ca wave (the n ntact cardac muscle [37]. However, Ca waves occur mddle part of Fg. 2). The decrease of the Ca wave nfrequently and propagate at low veloctes ( 3 mm/s) nduced by the acton potental s more pronounced f the and over lmted dstances (,1 mm) n ntact cardac nterval wth the precedng Ca transent s shorter [34]. It muscle under physologcal condtons (378C and normal appears that the resultant twtch of the myocyte recovers free [Ca ] ). Specfc requrements, such as lowerng of wth a tme course smlar to that of the mechancal the temperature and/ or ncrease of [Ca ], have to be met resttuton curve,.e. full recovery as attaned at room n order to provoke sgnfcant spontaneous actvty n temperature after 12 2 ms [35]. Ths s suggestve, myocardum. Even then, spontaneous Ca release man- but ndrect, evdence that the spontaneous contracton and fests tself only as Ca waves that occur nsde cells and twtch generaton share the same mechansms nvolved n whch rarely (,2%) move from cell to cell. Only when ntracellular Ca cyclng [36]. cardac muscle s damaged locally, such as by mcroelec- Ca waves occur usually randomly wth a frequency trode mpalement or dssecton procedures, do we see n that may vary from less than.1 to 5 6 Hz, although and near the damaged regon the ntaton of Ca waves remarkably stable ntervals between spontaneous Ca waves can be observed [29]. In an ndvdual cell the frequency ncreases monotoncally wth ncreased SR Ca that propagate n a coordnated fashon nto adjacent tssue [38]. Several observatons suggest that after-contractons n multcellular preparatons occur as the combned result of Downloaded from on January 218

4 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) the mechancal effects and elevated cellular Ca levels the ntaton of premature beats and arrhythmas n the owng to regonal damage and may gve rse to premature adjacent myocardum. TPCs appeared to arse n the ends beats as well as trggered arrhythmas. Unque aspects of of trabeculae [32,39], whch are easly damaged by dssecthe after-contractons that arse n damaged regons of ton and mountng of the muscle. The damage by dsseccardac muscle are that they appear to be ntated by ton and mountng of the muscle s worrsome for studes stretch and release of the damaged regon durng the of muscle mechancs, but provdes a means to nduce and regular twtch and that they propagate nto neghbourng control the extent of the damage and to study the consemyocardum. Most of the observatons that wll be ds- quences of acute njury on myocardum. cussed here have been made on solated rat ventrcular and human atral trabeculae. These studes have suggested that the after-contractons may be caused by Ca release, frst 5. Trggered propagated contractons: damage and by the myoflaments and, then, by the SR n the cells near the ntatng event the damaged regon. Ths local release of Ca n the damaged regon causes a Ca transent that s conducted TPCs arse nvarably n damaged regons of cardac nto adjacent muscle by the combnaton of Ca dffuson muscle, as s shown n Fg. 3. Damage of a cardac cell and Ca -nduced Ca release. The propagatng [Ca ] causes loss of ntegrty of the cell membrane and allows a transent s accompaned by a local contracton, that we Ca entry nto damaged cells whch n ts turn wll nduce have denoted as trggered propagated contracton (TPC) Ca overload and asynchronous spontaneous actvty [32,39]. Fg. 3 shows a typcal example of a TPC. The [32]. A consequence of ths effect of damage s that Ca [Ca ] transent underlyng the TPC also causes a depo- wll dffuse va gap junctons nto adjacent cells n the larzaton of the cell membrane whch may reach threshold border zone where cells stll have an ntact sarcolemma. and become responsble for acton potental generaton Entry of Ca nto the border zone contnues, as long as [38]. Damage-nduced after-contractons may, therefore, the gap junctons reman open [4], whch depends on the serve as the mechansm that couples regonal damage wth cell s envronmental condtons ncludng temperature [41]. Fg. 3. Panel A: Sarcomere length (SL) recordngs at fve dfferent stes (each 3 mm apart) along a 2.94-mm long trabecula durng a TPC wth a propagaton velocty of 1.4 mm/ s. The nterval between peak sarcomere shortenng due to the TPC (vertcal dashed lnes) was constant from ste to ste, ndcatng that propagaton velocty remaned constant along the preparaton. F5force. [Ca ] 1. mm, temperature 8C. Intal sarcomere length vared less than.5 mm between the stes of measurement. Modfed from Ref. [41]. Panel B: Schematc drawng of a muscle n whch TPCs started from a centrally located damaged regon (marked by the astersk) near two cut sde branches. The bottom panel shows the sarcomere length (SL) recordngs at three stes of the preparaton, llustratng that the TPC propagated away from the damaged regon n both drectons. Modfed from Ref. [41]. Downloaded from on January 218

5 448 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) Spontaneous actvty n the damaged zone s random; fluenced by the manpulaton of the after-load. So, appar- hence, the accompanyng Ca transents are small and do ently the probablty of trggerng a TPC depends on the not propagate through the muscle, but can cause Ca degree of stretch of the damaged area durng the twtch or overload and spontaneous actvty n border zone. Ths on the shortenng durng the relaxaton phase of the twtch. process contnues untl the [Ca ] gradent between cells s Several prevous studes have suggested a smlar couplng too small or untl gap junctons close [32]. The exstence between the mechancal events durng the twtch and of spontaneous contractons ncreases restng tenson and membrane electrophysology. Frst, studes of both skeletal decreases twtch force [42,43]. Thus, the damaged cells and of cardac muscle have shown that a quck release of and the border zone may be weaker durng the twtch than the muscle durng contracton causes rapd release of Ca the central regon of trabeculae. So, durng twtch contrac- ons from contractle flaments [44]. Second, Kaufmann et ton the central regon of the trabeculae stretches the al. [45] have shown that a quck release of the muscle by damaged regon. Durng the rapd relaxaton phase of the the expermenter could nduce a propagated acton potwtch the stretched damaged regon shortens suddenly. tental and a contracton. M. Lab and collaborators have Stretch or quck release of damaged ends of trabeculae provded the lnk between these observatons by showng durng the electrcally drven twtch trgger TPCs [39] may that quck releases can nduce an ntracellular [Ca ] provde an explanaton for the trggerng mechansm. transent accompaned by an after depolarzaton [46]. Support for ths contenton comes from the observaton that elmnaton of the stretch of the damaged regon and border zone durng the twtch and hence elmnaton of the 6. Propagaton of TPCs quck release durng the relaxaton phase prevents the nducton of TPCs. Fg. 4 shows an example of such an Fg. 3 showed a characterstc TPC n a rat cardac experment wth servo control of the after-load on the trabecula. The dsplacement of the TPC occurs at a V prop muscle. When the after-load s reduced, stretch of the whch vares n these preparatons at room temperature damaged areas decreases but sarcomere shortenng n the from.1 to 15 mm/s [32] and correlated tghtly wth the center of the trabeculae ncreases, whle the TPC s ampltude of the twtch precedng the TPC suggestng that delayed. the Ca load of the SR dctates V prop [41]. In contrast, It appeared that reducton of the after-load below twenty sarcomere stretch, whch ncreases twtch force for any percent of the sometrc force elmnated the TPCs com- level of loadng of the SR, dd not ncrease V prop [39]. pletely; an effect that was mmedately reversed by forcng Studes of the effects of nterventons such as vared the muscle to contract agan aganst a hgh after-load. At [Ca ], Ca channel agonsts and antagonsts also the same tme, t was clear that as long as a TPC was support the dea that the Ca load of the SR s the man observed, the propagaton velocty (V prop) was not n- determnant of V prop [47]. On the other hand, nterventons, 2 1 whch cause a leak of Ca from the SR (caffene and ryanodne), ncrease V prop, suggestng that Vprop also de- pends on the dastolc cytosolc Ca level [47]. The rate of ntaton of TPCs s tghtly correlated wth Vprop when the Ca load of the SR s modulated suggestng that the trggerng process and the propagaton process share closely related mechansms. 7. Mechansms underlyng ntaton and propagaton of Ca waves and TPCs 7.1. Intaton of Ca waves n myocytes Fabato s work on the propertes of cardac SR has provded a potental explanaton for spontaneous Ca release n solated myocytes. He observed that n mechancally sknned cardac cells n whch the SR was Fg. 4. Sarcomere length (SL) and force (F ) recordngs of the last of the ntact, excessve Ca loadng of the SR caused spontaelectrcally stmulated twtches and a TPC at dfferent after-loads n one neous Ca release [48]. The mechansm for ncreased muscle. [Ca ] 2.5 mm. Intal sarcomere length 2.15 mm. All probablty of openng of the SR Ca channel when the sarcomere length recordngs were made at one ste of the preparaton. Both sarcomere length and force tracngs were artfcally shfted on the SR s heavly loaded wth Ca s stll uncertan, but vertcal axs. A decrease n after-load delayed the ntaton of the TPC suggests that the channel s drectly or ndrectly senstve sgnfcantly. Modfed from Ref. [39]. to the lumenal [Ca ] of the SR. Recently the probable ste Downloaded from on January 218

6 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) of the Ca sensor of the SR Ca channel has been suggests that these sparks may act as a trgger for Ca 2 dentfed [49]. The localzaton of the Ca sensor n waves. The transton from non-propagatng sparks to (glutamate 3885) the transmembrane doman of the chan- waves s possbly caused by an ncrease n senstvty of nel would make t sutable as a sensor of both lumenal and the SR Ca -release channel for actvaton by cytosolc of cytosolc [Ca ]. Intact cells wth a hgh SR Ca load Ca as a consequence of the greater amount of SR Ca show smlar phenomena [31,42]. Hence, the oscllatory loadng [58]. Although these observatons have provded a character of a trggered arrhythma n myocardum wth a reasonable framework for explanaton of propagated Ca hgh cellular Ca load may be due to further ncrease of waves, the model s stll only a workng model and many Ca entry nto the cells durng the acton potentals of the questons reman unanswered. For example t has been arrhythma causng even more Ca loadng of the SR. noted that n myocytes wthout Ca overload a local Consequently, as soon as the release process has recovered ncrease n [Ca ] usng caged Ca does not propagate after an electrcally nduced Ca release, the overloaded [59], and that a Ca wave nduced by local applcaton of SR agan releases a fracton of ts Ca. The requrement caffene decreases n both ampltude and velocty as t that the Ca release mechansm has to recover frst [5] propagates along the cell [6]. would explan the presence of a delay between aftercontractons and after-depolarzatons to the precedng twtch. A small degree of Ca depleton of the SR may 7.4. Propagaton of TPCs play an addtonal role n ths process [51]. TPCs propagate wth a constant velocty along a trabecula. Lke n solated myocytes, ths wave-lke charac Intaton of TPCs n multcellular preparatons ter s thought to be consstent wth a model of Ca - nduced Ca release from the SR medated by Ca The observaton that the TPCs always start shortly after dffuson along ts concentraton gradent to adjacent the rapd shortenng of the damaged areas suggests that t sarcomeres and adjacent cells [31,32,38,39,61 63]. Ths s actually the shortenng durng relaxaton that ntates a model s supported by work on saponn-sknned muscle TPC. Housman s [44] and others observaton [52 55] that fbers whch also exhbt propagatng local contractons rapd shortenng of a contractng muscle causes release of after rapd local exposure of the fber to a Ca -contanng Ca ons from the myoflaments provdes a canddate soluton suggestng that the SR, but not the cell membrane, mechansm for ntaton of TPCs. Ca ons that dssos essental for the phenomenon [38]. The observaton that cate from the contractle flaments due to the quck release nether ntaton of TPCs nor ther propagaton s affected of the damaged areas durng relaxaton could ntate a 31 by Gd ons suggests that stretch-actvated channels play TPC f Ca -nduced Ca release has recovered sufflttle or no role n the ntaton or propagaton of damage- cently [35] to allow amplfcaton of the ntal Ca nduced TPCs [64]. Ths s consstent wth the hypothess transent n the damaged regon and/or the border zone. that the TPCs depend on ntracellular organelles. As was shown by the lack of effects of vared after-load and vared 7.3. Propagaton of Ca waves n myocytes sarcomere length on V prop, t s unlkely that stretch of the myofbrls s essental to the propagaton process. The observaton that Ca waves travel at a constant The characterstcs of Ca waves are qute smlar to velocty and wth constant ampltude through solated those of TPCs n trabeculae. In addton, nether spontamyocytes and through multcellular preparatons s an neous actvty n sngle myocytes nor TPCs n trabeculae mportant clue about the mechansm of propagaton. requre an ntact sarcolemma and both are abolshed by Dffuson of Ca alone would clearly be too slow by at agents that nterfere wth SR Ca loadng or release [65]. least 2 3 orders of magntude and would be accompaned On the other hand, at frst glance, a strkng dfference by a declne of the observed wave ampltude. On the other between them s the propagaton velocty. The velocty of hand the propagaton of electrcal actvty s much faster, 1 Ca waves n unstmulated cells s about ten tmes lower m/ s for the acton potental n ventrcular myocardum. than V prop. However, t s mportant to realze that TPCs are Also, electrotonc conducton s too fast to be compatble generated n cardac muscle preparatons at a short nterval wth the observed values for V prop. The mechansm n- after the twtch so that ther propertes are affected by volved n the propagaton of Ca waves n cells has been resdual bndng of Ca to ntracellular lgands. Ths proposed whch conssts of dffuson of Ca due to the contrasts the stuaton n myocytes where the moment of local ncrease of [Ca ] and subsequent Ca -nduced appearance of Ca waves followng the twtch s both Ca release from adjacent SR [56,57]. Recently, the use random and usually later. Hence, elmnaton of Ca from of fluorescence magng and confocal mcroscopy has these lgands later durng dastole, when the Ca extru- revealed the presence of several spontaneous Ca sparks son processes have done ther work would cause slowng near the ste of ntaton of a Ca wave and a macro- of the propagaton velocty [32]. spark at the pont of ntaton [58]. Ths observaton We have nvestgated whch parameters of Ca dffu- Downloaded from on January 218

7 45 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) son and Ca -nduced Ca release are requred for the hgh propagaton velocty of TPCs n muscles by modelng the behavour of a myofbrl accompaned by ts SR durng a sudden focal Ca release [66] n a way represented n Fg. 5. The method of soluton nvolved wrtng the dffuson equaton as a dfference equaton n the spatal coordnates. The resultant coupled ordnary dfferental equatons n tme wth banded coeffcents were solved usng Gear s 6th order predctor corrector algorthm for stff equatons wth reflectve boundares. It appeared that Ca transents propagate through the cytosol (as s llustrated n Fg. 6) at a rate modfed by bndng to troponn and calmoduln and sequestraton by the SR, as well as by the rate of Ca release from adjacent release stes of the SR. Vprop ap- peared to ncrease n the model from.1 to 15 mm/ s Fg. 6. The Ca transents as a functon of poston along the preparaton at dfferent tmes. The propagatng nature of the Ca wave s evdent from the fgures. Openng of the Ca channel n the SR was assumed to follow an exponental tme course (tme constant 1/k rel); after 2/krel the channel was assumed to close wth the same tme constant, so that for krel5.1 ms the open tme of the channel would be.5 ms [Ca ] dast s the dastolc Ca concentraton. Vmax s the maxmal rate of Ca extruson ons from the cytosol. KD s the threshold for Ca release from the SR. Modfed from Ref. [66]. owng to the combned effects of a rse of: () the dastolc [Ca ] ; () the rate of Ca release and; () the amount Fg. 5. Dagram of the exctaton contracton couplng system n the of Ca released by the SR [66]. Ths combnaton of cardac cell, as well as ts role durng TPCs. The left panel shows the changes n Ca levels n the cytosol and n the SR would events durng the twtch. Durng the acton potental a large transent be expected to result from loadng of cardac cells wth Ca nflux enters the cells followed by a mantaned component of the slow nward current (dashed lne). Ca entry does not lead drectly to Ca durng repettve stmulaton as well as due to force development as the Ca that enters s rapdly bound to bndng exposure to hgh [Ca ] or Ca agonsts [32]. stes on the SR. The rapd nflux of Ca va the T-tubul s thought to Important to the vablty of the proposed model of nduce release of Ca from a release compartment n the SR, by TPCs was a hgh conductance of the Ca channel n the trggerng openng of Ca channels n the termnal csternae, thus SR. In addton, the expected open tme for the Ca actvatng the contractle flaments to contract. Rapd relaxaton follows because the cytosolc Ca s sequestered rapdly n an uptake compartn order to allow for the hghest V channel n the SR would have to be n the order of.5 ms, ment of the SR and partly extruded through the cell membrane by the prop. These propertes of 1 Na / Ca exchanger and by the low capacty hgh affnty Ca pump. the SR Ca channels seemed exceedngly rapd at the Ths process loads the SR. It s mportant to note that the process of tme of formulaton of the model. Nowadays, reports on 1 Na / Ca exchange s electrogenc so that Ca extruson through the the knetcs of RyR n blayers [49] and on extremely fast exchanger leads to a depolarzng current. The mddle panel shows the events near a damaged regon durng trggerng of the TPC. Rapd Ca transents such as sparks recorded usng confocal shortenng of ths regon occurs durng relaxaton of the twtch followng mcroscopy and fast and senstve fluorescent Ca dyes stretch by the normal, and therefore stronger myocardum, durng such as Fluo 3 [67], suggest that ths assumpton may not contracton. Ths rapd release of the sarcomeres leads to dssocaton of have been excessve. Nevertheless, t s clear that our Ca from the contractle flaments durng the relaxaton phase. The SR understandng of TPCs requres further study of Ca has recovered enough to respond to the ncrease n [Ca ] by Ca - nduced Ca release, leadng to an after-contracton. The resultant transents at a hgh spatal and temporal resoluton. elevaton of [Ca ] also causes dffuson of Ca to adjacent sarcomeres. The model of a myofbrl together wth SR over a length The rght panel shows that the arrval of dffusng Ca after release n of 5 sarcomeres predcted the propagaton veloctes the damaged regon leads to Ca -nduced Ca release by the SR n the observed n muscles accurately. Ths accuracy s somewhat adjacent sarcomeres. Ca dffuses agan nto the next sarcomere, whle surprsng because n the real muscle the Ca transent causng a local contracton as well as a delayed after- depolarzaton 1 (DAD) due to electrogenc Na / Ca exchange and actvaton of Ca - has to travel not only from sarcomere to sarcomere but senstve non-selectve channels n the sarcolemma. Dffuson of Ca also from cell to cell. The hgh propagaton velocty along ts gradent mantans the propagaton of the TPC. suggests that the barrer for Ca dffuson mposed by gap Downloaded from on January 218

8 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) junctons between cells s mnor compared to the other parameters n the model such as Ca bndng to lgands n the cell and Ca extruson and sequestraton processes. We tested the mportance of gap junctons to the propertes of the TPCs by exposng the trabeculae to the gap juncton blockers heptanol and octanol. Although these compounds lke many drugs have probably numerous sde effects, ther man effect s assumed to be a reducton of the open frequency of gap junctons. Exposure of the muscles to these alcohols showed a unque effect on TPCs that we have not encountered wth other drugs,.e.: the rate of ntaton and Vprop decreased dramatcally wth only a small decrease n twtch force [68]. Ths suggests that closure of gap junctons reduces the rate of ntaton and Vprop by reducng the effectve rate of dffuson from cell to cell. The model suggested that the rate of propagaton of TPCs should be reduced by actve extruson of Ca from the cytosol [66]. Therefore, we tested whether the rate of propagaton of contractons that can be nduced n sknned fbers would be equally fast as those n ntact trabeculae. Sknned trabeculae were exposed to a soluton, whch allowed loadng of the SR. Subsequently, Ca -nduced Ca release was nduced by usng a mcroelectrode n order to squrt a small amount of Ca -contanng soluton onto the fber. The rapd local exposure to hgh Ca led to a local contracton followed by a propagated contracton whch travelled at a velocty rangng from 5 to 3 mm/ s. We dd not succeed n creatng condtons under whch the contracton travelled faster than 3 mm/s, rrespectve of whether we enhanced or reduced the SR load wth Ca or whether we restrcted Ca dffuson to the bathng medum by placng the fbers n slcon ol. It s Fg. 7. Parallel changes n the characterstcs of TPC and DAD followng possble that a crucal condton for rapd propagaton s an two (mddle panel) and 15 (bottom panel) condtonng stmul. The top elevated [Ca ] concentraton around the myofbrls so that panel represents a trabecula wth ts ventrcular end postoned n a cradle more cytosolc Ca bndng stes are occuped; ths that was attached to a force transducer and the valvular sde attached to a possblty remans to be tested. hook. Sarcomere length and membrane potental were montored at two stes along the preparaton (X and Y). The records n the mddle and bottom panel show force (F ), sarcomere length (SL), and membrane 7.5. Propagated Ca release nduces premature beats potental (V ) of the last stmulated twtch and a subsequent TPC and DAD n a representatve muscle. The preparaton hyperpolarzed slghtly Whenever a TPC arses, t s accompaned by a depolar- durng stmulaton, accountng for the depolarzng drft upon whch zaton. These depolarzatons are remarkably smlar to DADs occurred. At the two measurng stes, membrane potental tracngs were vrtually dentcal. Wth an ncrease n the number of condtonng DADs, albet that ther duraton vared wdely. It appears stmul from two to 15, TPC propagaton velocty ncreased from 2.4 to that the duraton of the depolarzatons correlated exactly 9.5 mm/ s. Furthermore, TPC force and DAD ampltude ncreased, whle wth the tme durng whch the TPCs travel through the TPC latency, DAD latency, and duraton of both TPC force and DAD trabeculae. Ths observaton s consstent wth electrotonc decreased. Followng 15 stmul, a second TPC and DAD occurred. conducton along muscles that act lke a cable wth a [Ca ] 1. mm, temperature 2.88C, restng membrane potental 268 and 264 mv at stes X and Y respectvely. Muscle length 2.23 mm; ntal length constant of several mllmetres [38]. Also the sarcomere length 2.1 mm. From Ref. [38]. ampltude of the after-depolarzatons correlated exactly wth the ampltude of the TPCs [38]. Ths s shown n Fg. 7. trabeculae that we have used for these studes ths depolar- The tght correspondence between the tme course of zaton can be recorded over a dstance of a few ml- TPCs and those of the depolarzatons suggests that the lmetres wthout much decrement due to electrotonc depolarzaton s elcted by a Ca dependent current, conducton followng the cable propertes of these fbers whch exsts as long as the [Ca ] transent perssts, as [38]. Ths assumpton could be verfed expermentally by has been proposed by Kass and Tsen [17]. In the small nterruptng the propagaton process of the TPC by locally Downloaded from on January 218

9 452 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) heatng the muscle over a few hundred mcrometres length trotonc conducton of the DAD for whch the current by a few degrees as s shown n Fg. 8. generators are located n the regon wth elevated [Ca ]. Local heatng of the muscle caused the TPC to stop at Ths observaton clearly ndcates that the depolarzaton the ste of heatng. In contrast, the concomtant depolarza- cannot be the source of the TPC but must be nduced by ton could stll be measured at a dstance of about 1 mm the TPC. The effect of local heatng makes t also unlkely dstal of the heatng ste [38] agan as a result of elec- that TPCs are nduced as a result of a lnear gradent of Ca overload along the muscle from a maxmum n the damaged regon to a mnmum at the other end of the muscle. Such a gradent could potentally cause apparent propagaton of a contracton f Ca overload-nduced Ca release occurred along the muscle at a latency whch s small n the damaged regon and ncreased lnearly toward the other end of the muscle. 8. Premature beats and trggered arrhythmas resultng from TPCs TPCs always can be notced drectly followng damage to the muscle. Ths s usually evdent shortly after-mount- ng a muscle n an expermental setup. The acute Ca load to the damaged cells and ther neghbours s then apparently so large that vrtually every electrcally paced beat s followed by TPCs. In that case, we observed that the TPC was accompaned by a DAD, whch was suffcently large to elct an acton potental wth twtch as shown n Fg. 9A. As dscussed above, the acton potental trggered by the frst TPC may add so much Ca to the cell that a trggered arrhythma starts (Fg. 9B). Trggered arrhythmas ndeed occur n the damaged muscle when the Ca load of the SR s large. We have observed these trggered arrhythmas at room temperature durng the frst hour after damage to the muscle has occurred. In such a case, the full-blown arrhythma s usually preceded by the repeated occurrence of sngle premature beats. At 378C, the tme span over whch these damage-related events occur s much shorter and the TPCs, whch cause the premature beats, dsappear n 1 mn or less [69]. Under those condtons t s lkely that ther occurrence s lmted by rapd closure of gap junctons as a result of persstently Fg. 8. Effect of local heatng of the muscle on a TPC and DAD. The top elevated Ca levels n the damaged cells. In addton, the panel schematcally represents the muscle wth free rght ventrcular wall ph n these cells may be low due to the enormous attachment, postoned n the cradle of a force transducer (left), and metabolc load resultng from ntense on movement across valvular sde (rght). TPCs propagated n a drecton opposte to the superfusng flow. Measurements of sarcomere length were made both ther membranes or across membranes of adjacent cells. proxmal (X) and dstal (Y) to the hearng wre; membrane potental was The lowered ph may promote gap juncton closure only montored at Y. The mddle panel shows, durng the control phase, These observatons make t lkely that arrhythma ntatrecordngs of sarcomere length (SL) at both X and Y, force (F ), and ng premature beats n acutely damaged myocardum may membrane potental (V ) of the last stmulated twtch and a TPC and DAD, result from a trggered ntracellular Ca transent n the wth the local contracton n X precedng that n Y, ndcatve of the propagatng character of the TPC. Restng membrane potental, 264 mv. damaged regon. The resultng [Ca ] transent propagates [Ca ] 1.25 mm; temperature 19.88C. TPC propagaton velocty 3. nto adjacent myocardum and nduces DADs and TPCs. If mm/ s. Durng local heatng (bottom panel), a local contracton due to a the [Ca ] transent s large enough t wll lead to acton TPC stll occurred at ste X, but propagaton of the TPC was nterrupted potental formaton. Clearly, damage ntates a chan of at the heatng wre and no unstmulated contracton was vsble at Y, subcellular events leadng to oscllatons of [Ca ] that despte a local depolarzaton. Note the accelerated relaxaton of the last stmulated twtch and the decreased latency of both TPC and DAD. The may cause macroscopc arrhythmas n the damaged heart. decrease n dstance of TPC propagaton s responsble for the decrease n Few studes have addressed possble pharmacologcal TPC duraton and ampltude. From Ref. [38]. nterventons amed at ths source of premature beats. In Downloaded from on January 218

10 H.E.D.J. ter Keurs et al. / Cardovascular Research 4 (1998) evaluated. Fnally, the TPCs dscussed here have been found n stuatons where t was obvous that cardac muscle had been damaged mechancally. Whether njury results n smlar mechansms to those, whch result from schema or other causes of Ca overload, needs to be tested. Lastly, the authors hope that ths revew wll be a stmulus to others to study whether ths mechansm underlyng arrhythmas n vtro may play a role n the human heart. Acknowledgements Ths work was supported by grants from the Janssen Research Foundaton and the Alberta Heart and Stroke Foundaton. Dr. H.E.D.J. ter Keurs s a Medcal Scentst of the Alberta Hertage Foundaton for Medcal Research (AHFMR). References [1] Luu M, Stevenson WG, Stevenson LW, Baron K, Walden J. Dverse mechansms of unexpected cardac arrest n advanced heart falure. Crculaton 1998;8: [2] Stevenson WG, Stevenson LW, Mddlekauff HR, Saxon LA. Sudden Fg. 9. Spontaneous contractons cause development of twtches and death preventon n patents wth advanced ventrcular dysfuncton. arrhythmas. Panel A shows an example of the development of a Crculaton 1998;88: spontaneous twtch that s trggered by a propagatng after-contracton. [3] Esner DA, Nchols CG, O Nell SC, Smth GL, Valdeolmllos M. Note the acute ncrease of the rate of rse of force durng the development The effects of metabolc nhbton on ntracellular calcum and ph of the frst after-contracton (arrow), and also the smlarty of the tme n solated rat ventrcular cells. J Physol 1989;411: course of the subsequent twtch and that of the electrcally elcted twtch. [4] Dekker LRC, Folet JW, VanBavel E, et al. Intracellular Ca, Modfed from Ref. [32]. Panel B shows force (F ) and membrane ntercellular electrcal couplng, and mechancal actvty n schemc potental (V ) recordngs durng a tran of condtonng stmul (endng at rabbt papllary muscle. Crc Res 1998;79: the arrow) and a subsequent trggered arrhythma. Note the ntal slow [5] Bozler E. The ntaton of mpulses n cardac muscle. Am J Physol upstroke n both force and membrane potental of trggered twtches, 1943;138: suggestve of an underlyng TPC and DAD. The trggered arrhythma [6] Segers M. Le battement auto-entretenu du coeur. Arch Int Phar- termnated spontaneously wth an ncrease n the nterval between macodynam 1947;75: trggered beats, followed by a TPC and DAD. Temperature 2.48C; [7] Wttenberg SM. Acceleraton of ventrcular pacemakers by transent [Ca ] 2.25 mm. Restng membrane potental, 271 mv. Modfed from ncreases n heart rate n dogs durng ouaban admnstraton. Crc Ref. [41]. Res 197;26: [8] Morrs GL, Cheng HC, Colyer J, Wang JH. Phospholamban regula- ton of cardac sarcoplasmc retculum Ca Mg -ATPase. Mechaour hands, t was so far possble to reduce the chance of nsm of regulaton and ste of monoclonal antbody nteracton. J development of TPCs wthout smultaneously causng a Bol Chem 1991;266: negatve notropc effect usng R56865, a Na and Ca [9] Hogan PM, Wttenberg SM, Klocke FJ. Relatonshp of stmulaton frequency to automatcty n the canne Purknje fber durng overload nhbtor [7], or usng the gap juncton blockers ouaban admnstraton. Crc Res 1973;32: octanol and heptanol [68]. [1] Rosen MR, Gelband H, Hoffman BF. Mechansms of dgtals It s clear that many mechansms nvolved n the toxcty. Effects of ouaban on phase four of canne Purknje fber generaton of TPCs and subsequently arrhythmas requre transmembrane potentals. Crculaton 1973;47: further study. The mechansms nvolved n the speed at [11] Ferrer GR, Saunders JH, Mendez C. A cellular mechansm for the generaton of ventrcular arrhythmas by acetylstrophanthdn. Crc whch these contractons travel need further study n order Res 1973;32:6 69. to resolve how they can propagate at a speed, whch s [12] Hashmoto K, Moe GK. Transent depolarzatons nduced by uncommon for those who are used to observng propaga- acetylstrophanthdn n specalzed tssue of dog atrum and ventr- ton of Ca waves n sngle myocytes. The role of the cle. Crc Res 1973;32: sarcolemma deserves partcular attenton as the assumpton [13] Cranefeld PF. Acton potentals, afterpotentals, and arrhythmas. Crc Res 1977;41: that depolarzaton of the membrane does not play a role [14] Ferrer GR, Saunders JH, Mendez C. A cellular mechansm for the (see Fg. 5) needs to be tested. Furthermore, the nfluence generaton of ventrcular arrhythmas by acetylstrophanthdn. Crc of gap junctons on the propagaton process needs to be Res 1973;32:6 69. Downloaded from on January 218

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Bophys J tons between calcum waves and acton potental-nduced calcum 1984;45:94a. transents n gunea pg. Heart Vessels 1994;9: [57] Regrer SA, Tsaturyan AK, Chernaya GG. Mathematcal model of [35] Banjamal HS, Gao WD, MacIntosh BR, ter Keurs HEDJ. Force- propagaton of actvaton waves n an solated cardomyocyte. nterval relatons of twtches and cold contractures n rat cardac Bophyscs 1986;31: trabeculae. Influence of ryanodne. Crc Res 1991;69: [58] Cheng H, Lederer WJ, Cannell MB. Calcum sparks. Elementary [36] Capogross MC, Suarez-Isla BA, Lakatta EG. The nteracton of events underlyng exctaton contracton couplng n heart muscle. electrcally stmulated twtches and spontaneous contractle waves n Scence 1993;262: sngle cardac myocytes. J Gen Physol 1986;88: [59] O Nell SC, Mll JG, Esner DA. Local actvaton of contracton n [37] Wer G, ter Keurs HEDJ, Marban E, Gao WD, Balke CW. Ca solated rat ventrcular myocytes. Am J Physol 199;258:C1165 sparks and waves n ntact ventrcular muscle resolved by confocal C1168. magng. Crc Res 1997;81: [6] Trafford AW, Lpp P, O Nell SC, Nggl E, Esner DA. Propagatng Downloaded from on January 218