Real time, confocal imaging of Ca waves in arterially perfused rat hearts

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1 Cardovascular Research 53 (2001) locate/ cardores Real tme, confocal magng of Ca waves n arterally perfused rat hearts Andreas P. Baader, Lorenz Buchler, Llly Brcher-Lehmann, Andre G. Kleber * Unversty of Bern, Department of Physology, Buhlplatz 5, 3012 Bern, Swtzerland Receved 1 February 2001; accepted 30 July 2001 Abstract Objectve: The am of ths study was to characterze the spato-temporal dynamcs of slow Ca waves (SCW s) wth cellular resoluton n the arterally-perfused rat heart. Methods: Wster rat hearts were Langendorff-perfused wth Tyrode soluton contanng bovne-albumne and Dextran. The heart was loaded wth the Ca senstve dye Fluo-3 AM. Intracellular fluorescence changes reflectng changes n [Ca ] were recorded from subepcardal tssue layers usng a slt hole confocal mcroscope wth an mage ntensfed vdeo camera system at mage rates of up to 50/ s. Results: SCW s appeared spontaneously durng cardac rest or after trans of electrcal stmul. They were ntated preferentally n the center thrd of the cell and propagated to the cell borders, suggestng a relaton between the cell nucleus and wave ntaton. They were suppressed by Ca transents and ther probablty of occurrence ncreased wth the Ca restng level. Propagaton velocty wthn myocytes (40 to 180 mm/ s) decreased wth the restng Ca level. Intercellular propagaton was mostly confned to two or three cells and occurred b-drectonally. Intercellular undrectonal conducton block and facltaton of SCW s was occasonally observed. On average 10 to 20% of cells showed non-synchronzed smultaneous SCW s wthn a gven area n the myocardum. Conclusons: SCW s occurrng at ncreased levels of [Ca ] n normoxc or schemc condtons are mostly confned to two or three cells n the ventrcular myocardum. Spato-temporal summaton of changes n membrane potental caused by ndvdual SCW s may underle the generaton of trggered electrcal ectopc mpulses Publshed by Elsever Scence B.V. Keywords: Arrhythma (mechansms), Calcum (cellular), Conducton system, Sgnal transducton 1. Introducton reactvaton of L-type Ca currents durng a prolonged duraton of the acton potental plateau (early afterdepolar- Changes of cytoplasmc [Ca ] durng the cardac cycle zatons, EAD s) and as the result of electrogenc extruson are characterzed by a complex nterplay of Ca enterng of Ca va the Na/Ca exchanger after repolarzaton of from the extracellular space, Ca released from the the acton potental (delayed afterdepolarzatons, DAD s). ntracellular stores, Ca pumped nto ntracellular stores, Addtonally, Ca bound to Troponn C can nduce Ca and Ca transferred from the cytoplasm to the extracellu- transents n the case of rapd force changes [1]. Whle lar space. Snce some of these processes nduce changes n these events necesstate coordnated acton of many transmembrane voltage by affectng transmembrane onc myocytes to produce a source for electrcal exctaton, channels and transporters, fluctuatons n ntracellular Ca Ca changes n the cytoplasm have been reported to can contrbute to repettve depolarzatons, and conse- occur n dscrete enttes that appear to relate to the specal quently, to arrhythmas. Thus, oscllatons of transmem- compartment structure of the SR. The overall ncrease of brane potental have been reported as a consequence of cytoplasmc Ca durng a contracton cycle then results from the ndependent and dscrete summaton of local Ca releases (sparks). When reachng a crtcal cellular *Correspondng author. Tel.: ; fax: cytoplasmc [Ca ] of about 500 nm [2], local sparks can E-mal addresses: baader@pyl.unbe.ch (A.P. Baader), kleber@pyl.unbe.ch (A.G. Kleber). Tme for prmary revew 29 days / 01/ $ see front matter 2001 Publshed by Elsever Scence B.V. PII: S (01)

2 106 A.P. Baader et al. / Cardovascular Research 53 (2001) turn nto propagatng waves. Analyss of Ca waves, and myocytes of the whole heart over a relatvely large area of especally ther propagaton and co-ordnaton wthn and cardac tssue. among cells s mportant for our understandng of ntaton of arrhythmas. It was not untl recently that the frst studes descrbed Ca waves n multcellular cardac 2. Methods muscle preparatons [3 6]. In trabeculae muscles, propagatng waves were found to orgnate from mechancally The nvestgaton conforms wth the Gude for the Care damaged muscle fber regons [7 9]. These waves could and Use of Laboratory Anmals publshed by the US travel nto undamaged myocardum areas to cause trg- Natonal Insttutes of Health (NIH Publcaton no , gered propagated contractons (TCP s) of the sarcomeres. revsed 1996). But the probablty that ths ntercellular propagaton occurred was found to be rather low [10]. Spontaneous 2.1. Langendorff hearts and expermental setup Ca oscllatons were also reported n whole rat hearts. Mnamkawa et al. [5] descrbed multfocal waves, the After anesthesa 3 6-day-old Wster rats were decap- actvty of whch ncreased wth Ca overload and whch tated. The excsed hearts (weght: mg) were brought had propagaton veloctes of mm/ s. Hama et al. to a Langendorff-chamber. Tme from excson to cannula- [11] gave a qualtatve descrpton of wave propagaton n ton was less than 5 mn. In the perfuson chamber the ventrcular myocytes. Most recently, the nteractons be- heart was equlbrated wth perfuson soluton contanng tween Ca waves and transents and the ntra- and Tyrode soluton wth 1.8 mm Ca, bovne-albumne (2 ntercellular propagaton characterstcs of Ca waves g/ l) and Dextran (40 g/ l) [14]. The preparatons were were studed n perfused rat hearts (Kaneko et al. [12]) and stmulated at 2 Hz by means of two platnum wres (20 n Purknje cells [13]. mm dameter) nserted nto the ventrcular myocardum. In the present study, we nvestgated the occurrence of Temperature nsde the chamber was set to C. A slow Ca waves (SCW s) n arterally-perfused whole rat subsequent equlbraton perod of 30 mn allowed the hearts n dependence of dfferent cytosolc Ca. The am heart to recover and to adjust constant pressure and was to provde a detaled ntracellular characterzaton of temperature condtons n the chamber. the temporal and spatal dynamcs of SCW s. A slt hole The perfuson system has been descrbed prevously n confocal magng system was used for real tme acquston detal [15]. In bref, t conssted of a roller pump, a slcone of ntracellular Ca changes that had both a hgh sen- membrane gas exchanger and a Perspex recordng chamber stvty and a hgh speed of up to one frame every 20 ms. (Fg. 1). The roller pump generated a sustaned perfuson Ths allowed us to nvestgate Ca fluctuatons n pressure of 40 to 55 mm Hg. N -, CO - and O -levels n Fg. 1. Expermental setup for the recordng of myocardal Ca fluctuatons n rat hearts. The Langendorff-perfused heart s postoned left ventrcle upn a sealed recordng chamber under constant temperature and full H2O saturaton condtons. It s paced electrcally (El. Stm.) by means of platnum wres nserted nto the left ventrcle. The Ca senstve dye Fluo-3 AM s loaded nto the myocardum through the perfuson system. Fluorescence exctaton of Fluo-3 dye s provded by a 488-nm Argon laser and recorded through a slt hole confocal mcroscope by an mage-ntensfed vdeo camera system.

3 A.P. Baader et al. / Cardovascular Research 53 (2001) the soluton and n the surroundng H2O-saturated atmos- mage ntensfer was routnely adjusted at the begnnng of phere of the chamber were adjusted to mantan an oxygen each experment so that electrcally nduced Ca -transconcentraton of $99% and a ph of 7.4. In the perfuson ent-peaks produced non-saturated pxel values. Durng chamber, the heart was mounted on a wax platform wth mage acquston the laser beam contnuously llumnated the left ventrcle up. A 150 mm thck glass cover slp was recordng the preparaton. Interlaced vdeo mages were mounted n a 30 mm wde crcular openng n the ld, acqured n seres of up to 80 frames usng the vdeo drectly above the heart gently depressng the myocardal grabbng board and/ or were recorded contnuously on surface. It sealed the recordng chamber completely and vdeotape. Durng these (up to 3.2 s) recordng perods no prevented excessve movements. bleachng of sgnals were observed. Wth recordng tmes Neonatal rat hearts were chosen because loadng of the $5 s bleachng produced a gradual reducton n fluores- myocytes wth Ca -senstve dyes va arteral perfuson cence ntensty (see Fg. 2). Largest recorded regons of was faster and provded more homogenous stanng than nterest (ROI s) were mm wth the 403 adult rat hearts. Neonatal hearts dffer from adult myocar- objectve and mm wth the 633 objectve, dum by a somewhat dfferent cellular structure wth a and depended on the deflecton ampltude of the oscllatng smaller densty and a less regular organzaton of cells. mrror n the confocal mcroscope. From these mages, Although the dstrbuton of some membrane channels was sub-roi s were off-lne selected for analyss usng NIH also found to be dfferent [16,17], the major on channels Image software. Ca -nduced fluorescence changes were and gap junctons present n adult hearts are fully ex- calculated from mage seres. Background fluorescence pressed, and neonatal myocytes show a stable ntracellular (F back) was determned from a 144-pxel-area wthn the Ca concentraton between P1 (postnatal day 1) and P7 recordng regon where no myocyte was dscerned. Non- [17]. The hearts beat n normal snus rhythm and can be ratometrc estmaton of Ca changes was expressed overdrven by ventrcular stmulaton. ether as arbtrary unts (a.u.), derved drectly from the As another morphologcal characterstc neonatal rat 8-bt gray level contents of the mages after subtracton of myocytes are predomnantly mononuclec whle adult the background fluorescence, or as rato wth F/F 0 5 myocytes have two nucle per cell. We staned neonatal (F F )/(F 2 F ) or DF/F 5 (F F )/ max back base back max base ventrcles wth eosne and glutaraldehyde, and found (FbaseF back) [19]. Fmax s the peak fluorescence of the vrtually no bnucleate cells at P3 and a very low propor- Ca -medated sgnal and F represents the fluorescence ton of bnucleate myocytes n P6-ventrcles, the oldest level of the myocyte durng rest. For the calculaton of the postnatal state used n our experments. We also found that veloctes of ndvdual Ca waves a look-up table (LUT) all nucle were consstently located wthn the center thrd of 12 colours representng levels of F/F0 was appled. porton of the cells. These results are n agreement wth Conducton velocty was evaluated from the spatal proprevous morphometrc studes [18], whch report 2.94, gresson of the ntensty values n the steepest part of the and 50.6% bnucleate myocytes n left ventrcular wave front. Cell borders were determned from off-lne myocardum of P1, P5 and P11 postnatal rat hearts, averaged seres of mages. respectvely Confocal Ca magng 2.3. Expermental procedure base The whole chamber assembly was postoned on a Durng loadng of the myocardum wth Ca -senstve movable stage under an uprght slt detector confocal fluorescent dye (fluo 3-AM, Teflabs, 4 mmoles/ l n 20 ml mcroscope (Merdan Insght). Imagng was performed at perfusate), the temperature of the chamber and of the 403 (water mmerson, C-Apochromat, NA1.2, Zess) and perfuson soluton was lowered to 7 108C. Dependng on 633 (ol mmerson, Plan-Neofluar, NA1.25, Zess) magn- the flow rate cold loadng lasted for 30 to 40 mn. fcaton. Wth the 403 objectve confocal magng pro- Afterwards, the hearts were perfused wth dye-free soluvded a sharp mage n a depth of 61.5 mm around the ton at C. In most experments t was possble to focal plane. A CCD vdeo camera wth GEN II mage obtan mechancally stable recordngs. In 14 of the 29 ntensfer (MXRI, Admex Image Systems) recorded experments excessve contractons nterfered wth the nterlaced 8 bt mages at a frequency of 25 frames/ s and a optcal recordngs. In these cases 2,3-butanedone monoxsze of pxels. By de-nterlacng the vdeo me (BDM, 20 mm) was added to the soluton to mnmze sequences off-lne frame rates of 50 frames/ s could be contractons. acheved. Vdeo mages were acqured wth a Matrox SCW s appeared spontaneously n electrcally quescent Pulsar grabbng board on a Pentum II computer at a preparatons or they were nduced after bursts of rapd transfer rate of #60 MB/ s. Images could be acqured stmul. These trans modfed cytosolc free [Ca ]. A down to a depth of 150 mm from the surface of the gradual ncrease of cytosolc restng [Ca ] levels was also myocardum before blurrng occurred. A laser power of observed durng extended recordng perods of up to 1 h. 80% of the maxmum output (nomnally 500 mw) was Therefore, ths method was used to nvestgate the relatonused to produce fluorescence sgnals. The gan of the shp between SCW s and [Ca ]. In all measurements,

4 108 A.P. Baader et al. / Cardovascular Research 53 (2001)

5 A.P. Baader et al. / Cardovascular Research 53 (2001) when SCW s were measured rapd Ca transents reflect- ndcated by the whte crcle. Peak ntensty values of ng the cytosolc Ca ncrease durng normal electrcal F/F to 1.5 were reached after ms and exctaton were observed. Ths excluded electrcal conduc- decayed durng the subsequent 400 ms wth a half decay ton block due to Ca -nduced cell-to-cell uncouplng tme of 110 ms. These ntenstes were typcal for the durng the occurrence of SCW s. majorty of SCW s observed under normoxc condtons (see also Fg. 2). The mean propagaton velocty of ths wave was 130 mm/ s. A detaled spato-temporal ds- 3. Results trbuton of such a wave wthn a myocyte s shown n Fg. 3B. The frst phase started wth a crcumscrbed rse n 3.1. Calcum sgnals n ventrcular myocytes Ca near the center of the cell. Ths local spot ncreased n sze for about 40 ms untl t reached a certan con- Two dstnct types of Ca fluctuatons were observed: centraton level. In a second phase, two wave fronts (gray (1) Ca transents that appeared synchronzed across the arrow lnes n Fg. 3B) emerged from ths central ste to whole muscle tssue at the avalable tme resoluton and the perphery whle further ncreasng n ntensty. When that followed electrcal exctaton, and (2) local waves, the two centrfugal waves were at ther hghest ntensty, whch propagated wthn and/ or between myocytes (slow the Ca concentraton at the ste of orgn of the wave Ca waves, SCW s). In Fg. 2 fluorescence changes n a began to decrease. The thrd phase was characterzed by a small cardac area from a typcal experment are depcted. general Ca decrease throughout the whole cell. Although The pseudo color mages of the upper panel show three n ths example propagaton of Ca nto neghborng cells myocytes n the focal plane labeled as green to red areas at the same focal plane was not observed, t can not be and confned by dashed lnes. They are separated by cells ruled out that Ca spread occurred nto cell layers above not fully n the focal plane and by extracellular clefts (blue or below. A secondary small central Ca ncrease was areas). Mean ntensty changes (F/F 0) were recorded from vsble after some 200 ms at the ste of orgn of the wave a crcular 144-pxel-ROI (18 mm dameter, whte crcle n (black arrow), whch however dd not develop nto a the upper left mage). Wthn the frst 6 s of ths recordng, second wave. Whle n our experments most of the the heart was burst-paced at 2.3 Hz. Ca transents observed SCW s (79.1%, 43 observatons) orgnated n or appeared synchronzed over the entre muscle tssue. The near the center thrd of the cell, others could also start n recovery tme course (the tme at 50% maxmum am- the cell perphery (see also Ref. [10]). pltude) was 0685 ms (n595), and longer than the nter-stmulus nterval, and consequently, the Ca level 3.2. Dynamcs common to slow Ca waves remaned elevated throughout the stmulaton perod. After termnaton of the electrcal burst, restng Ca level As shown n Fg. 2, SCW s always appeared n perods decreased but remaned stll hgher than before burst between Ca transents, and preferentally, after multple stmulaton. Ths gave rase to a frst and second sponta- transents nduced by burst pacng. Ca transents sup- neous Ca transent after t59.3 and 11.2 s. At t513 s an pressed ongong slow wave actvty completely, no matter SCW developed, followed by addtonal SCW s and occa- whether they had been electrcally nduced or occurred sonal Ca transents. Durng ths long perod of laser spontaneously. As llustrated n Fg. 4A, the delay T W stmulaton (24 s) the average baselne fluorescence gradu- between a transent and the frst SCW n a gven myocyte ally decreased as a consequence of dye bleachng. depended on the ampltude of the precedng Ca tran- Spontaneous SCW s n the myocytes appeared as local sent. The hgher ts ampltude the longer was the tme spots before ther wave fronts propagated to the perphery nterval TW untl the frst slow wave was observed (Fg. (Fg. 3A). The values of the temporal dynamcs of the 4B). The regresson of measurements n 27 myocytes from wave n ths fgure were acqured from the round ROI 15 experments was R In general, restng Ca Fg. 2. Electrcally nduced and spontaneous Ca fluctuatons n myocytes of a perfused P6-rat-heart. Fluorescence changes (F/F 0) were calculated from vdeo mages taken every 40 ms durng 25 s. Selected pseudo color mages are shown at the tmes ndcated. Dashed lnes surround three myocytes n the focal plane. Measurements were taken from one cell at a 144-pxel-ROI (whte crcle n upper left mage). Durng the frst 6 s the preparaton was stmulated electrcally at 2 Hz (see stmulaton pattern n bottom trace), resultng n bref Ca transents. After termnaton of the electrcal stmulaton two spontaneous Ca transents (at t59.3 and 11.2 s) appeared, followed by a seres of lower ampltude Ca waves and another transent. The course of one such slow Ca wave s shown n detal n the lower contnuous mage sequence. No BDM was used n ths experment. Fg. 3. Intracellular propagaton characterstcs of sngle slow Ca waves. (A) Propagaton of a wave wthn a sngle ventrcular myocyte (outlned by the orange dotted lne). Pseudo color mages of 14 consecutve frames, every 40 ms. The local fluorescence changes n ths cell were measured at the pont ndcated by a whte crcle and ther average ntensty values durng sx waves are plotted n the graph below. (B) Typcal phases of wave propagaton wthn another myocyte (m, outlned by the orange dotted lne). Intensty changes were measured smultaneously every 20 ms at 15 non-overlappng 174-pxel-ROI s along the longtudnal mdlne of the cell. A central local Ca ncrease s followed by a Ca spread towards the perpheral membrane boundares, a central decrease and fnally a perpheral decrease of Ca. No BDM was used n ths recordng.

6 110 A.P. Baader et al. / Cardovascular Research 53 (2001) ncreased gradually after a perfuson perod of 30 mn. Durng ths gradual transton from normal to elevated restng Ca the dynamcs of SCW s and the transents followed the relatonshps shown n Fg. 4C n that the ampltude of Ca transents was nversely related to the restng Ca level (R50.55, 37 myocytes). Therefore, myocytes wth low restng Ca and consequently, hgh ampltude Ca transents were more effectve n suppres- sng SCW s than low ampltude Ca transents. Wth ncreasng restng Ca the slow waves became smaller n ampltude and eventually dsappeared. Analyss of the velocty of SCW s revealed an nverse relatonshp between restng Ca levels and propagaton speed (R5 0.72, N533, Fg. 5), a fndng that contradcts prevous reports (see Dscusson) Propagaton of Ca waves between myocytes In the majorty of the observatons, SCW s were confned to ndvdual myocytes. The waves often reached the cell borders at hgh ntensty values before they vanshed. Ths observaton suggested collson wth cell borders wthout propagaton nto neghborng cells. In many cases however, an SCW crossed the cell border and propagated nto a neghborng myocyte. Ths propagaton could assume varous characterstcs. In the example n Fg. 6 a seres of sequental mages depcts two myocytes (outlned by the dashed orange lnes). Durng the frst 120 ms, an SCW was generated n the left cell (frames [1 3) whle no vsble Ca changes were detected n the rght cell. In frame [4, the SCW crossed the cell border from left to rght, leadng to a SCW n the other myocyte (arrow n frame [4). Both waves then gradually vanshed (frames Fg. 4. The ampltude of Ca transents affects the onset of subsequent slow Ca waves and changes wth the cytosolc Ca load. (A) Example of a sngle Ca transent followed by slow local waves. Arrows ndcate tme ponts used to determne the delay TW between the transent and the frst subsequent appearance of a slow wave. (B) TW ncreases wth the ampltude (labeled Transent F/F ) of the prevous Ca 0 transent. Ths Ca transent ampltude n turn decreases wth an ncrease n the mean Fg. 5. Velocty of ntracellular slow Ca waves as a functon of cardac Ca load at rest (C). A 2nd order polynomal ft was used n B cytosolc Ca load. In 33 cells the wave speed tended to ncrease wth a and C. decrease n cellular Ca (R50.72, exponental ft).

7 A.P. Baader et al. / Cardovascular Research 53 (2001)

8 112 A.P. Baader et al. / Cardovascular Research 53 (2001) [7 12). Subsequently, a second smaller wave n the left s-perod. The black dots n Panel B denote the cells cell (frames [13 19) dd not reach the crossng pont and producng Ca waves at the gven tme nterval durng the hence dd not nduce Ca changes n the rght cell. perod. Panel C llustrates the results of four experments Interestngly, an SCW n the rght cell (frames [16 23) wth areas contanng at average 3166 myocytes. Between reached the contact pont wth hgh ntensty (see arrow n 10 and 20% of cells produced SCW s at each determned frame [18) but dd not propagate nto the left cell. Thus, nstant wthn the recorded area of mm. Ca spread from a frst nto a second cell was not always followed by a spread n the opposte drecton (undrectonal block). 4. Dscusson 3.4. Rhythmcty of slow Ca waves 4.1. Relatonshp between Ca transents and SCW s Slow Ca waves (SCW s) were consstently observed In most cases, SCW s were generated at random waveafter bursts of electrcal stmulaton. Durng normal stmuto-wave ntervals. However, dstnct waves were occasonlaton rates SCW s were only observed n schemc conally recorded wth a hgh degree of rhythmcty. Ths dtons (not shown). Ths ndcates that normal electrcal rhythmcty was always confned to one cell and apparently exctaton suppressed SCW s and that, n general, the dd not correspond to any actvty n other cells nor was appearance of SCW s was assocated wth an elevaton of there ever any propagaton nto neghborng cells observed. cytosolc Ca. Thus, our results are n accordance wth The cell shown n Fg. 7A and B produced regular Ca prevous studes descrbng the relaton between cytosolc waves every ms (mean6s.d.). Fg. 7C shows a Ca and propagatng cytosolc SCW s [5,12]. The tme plot of the frequency of such hghly rhythmc slow waves nterval between the last stmulated Ca transent and the n 12 dfferent preparatons versus the cytosolc dastolc frst appearance of a SCW was dependent on the restng Ca level of the respectve myocytes. Interestngly, there Ca level. A smlar result was descrbed n a recent study was no correlaton between frequency of ths wave type usng freshly solated gunea-pg myocytes [] that and the Ca loadng. showed that global Ca transents lead to cytosolc suppresson of local Ca events. It was found that Ca release from SR stores was requred to nduce ths suppres Spato-temporal occurrence of slow Ca waves n son. The duraton of the suppresson was reduced wth an the cellular network elevaton of [Ca ] and wth an ncrease n Ca SR contents (E. Nggl, personal communcaton). SCW s lead to changes n transmembrane potental, that are supposed to trgger new propagated acton potentals [20]. An arrhythmogenc effect of such local waves could 4.2. Intracellular ntaton and propagaton of SCW s be the consequence of both an ncreased local wave frequency and of the spatal frequency. The spato-tempo- In our experments, the majorty of Ca waves org- ral occurrence of slow Ca waves wthn an area of nated ntracellularly n an area correspondng to the center mm s shown n Fg. 8. Wthn such a small thrd of the cells and subsequently traveled to the perphery regon spatal electronc nteracton of the changes n (Fg. 3). It was proposed that the ntaton of SCW s s a transmembrane potental nduced by SCW s s lkely to consequence of a restrcted, localzed summaton of noccur (see Dscusson). Panel A shows the temporal dvdual Ca sparks that reach a crtcal concentraton occurrence of SCW s n one such lmted area contanng level [2,4,22] and nduce a propagated regeneratve Ca 39 myocytes durng an nterval of 4 s. The arrows ndcate response. In our experments, the locaton of the cell ntercellular propagaton of SCW s observed durng the nucleus could explan a central ntaton ste. An nter- whole nterval. In order to defne the potental effect of acton between cytosolc Ca and nuclear Ca has temporal summaton, the smultaneous occurrence of recently been reported n solated myocytes [23]. The Ca SCW s was assessed n 400 ms ntervals durng the 4 sparks per se, whch were occasonally observed at the Fg. 6. Intercellular un-drectonal propagaton of Ca waves. Frame-by-frame recordng (every 40 ms) of several myocytes (outlned by the black dotted lnes). The last frame shows the average ntensty dstrbuton durng ths sequence. The wave front n the left cell (a) reaches the cell border, leadng to a sudden Ca ncrease n the rght cell (b, arrow n frame [4). At the same contact pont, a subsequent wave n the rght cell does not spread nto the left cell (arrow n frame [18). C5capllary. Fg. 7. A. Ca nduced fluorescence changes recorded from the mddle cell at the ROI ndcated by a whte feld. B. Rhythmc pulsatory waves were observed as ther wave fronts passed through the ROI shown n A. The wave pulse frequences were n the range of Hz, and were found to be ndependent from the ntracellular Ca load (C).

9 A.P. Baader et al. / Cardovascular Research 53 (2001) Fg. 8. Spatal dstrbuton of Ca waves. A. Reconstructon of a smultaneous recordng of Ca waves from a mm ventrcular segment durng 4 s. Arrows ndcate ntercellular communcaton at any pont durng the 4 s-perod. B. Four 400 ms-frames recorded every 1.2 s n another preparaton. Black dots ndcate the number of waves observed durng the 400 ms-wndow n the respectve myocytes. C. Percentage of myocytes exhbtng smultaneous slow Ca waves durng subsequent 400 ms-frames n arbtrary mm square myocyte layers (four experments).

10 114 A.P. Baader et al. / Cardovascular Research 53 (2001) lmt of detecton wth our method (A. Baader, personal exhbtng SCW s and, probably DAD s, wll be eleccommuncaton), occurred anywhere n the cytosol. trotoncally nfluenced by neghborng cells. As n another The ntracellular propagaton velocty of SCW s n our ntaton of focal mechansms, a mnmal amount of tssue experments was n the range of 50 to 180 mm/ s. Ths s n needs to be actvated n a coordnated manner for an agreement wth prevous studes that reported values of exctatory effect [29]. As a major goal of ths study, we about 100 mm/ s [5,12,24]. Although n our experments therefore nvestgated the spato-temporal nteracton the probablty of SCW ntaton ncreased wth ncreasng among varous cells. Such nteracton could be caused by [Ca ], propagaton velocty of ndvdual waves de- (1) propagaton of SCW s across cell borders, and/ or (2) creased (Fg. 5). Ths contradcts prevous reports showng spato-temporal coexstence of SCW s n closely adjacent an ncrease n propagaton velocty wth ncrease n cells exertng a mutual electrotonc nfluence. cytosolc [Ca ] [5,12]. The reason for ths dscrepancy, Intercellular Ca waves were observed that showed a whch appeared to be sgnfcant (Fg. 5), remans to be clear spatal restrcton to two, or at most three cells. At elucdated. It may be speculated that Ca release from the ths very small scale, propagaton phenomena such as SR or from the nucleus would produce a hgher local Ca collson wth cell borders and undrectonal propagaton concentraton dfference at lower [Ca ] than at hgh occurred (Fg. 6). Also, the observed conflaton of SCW s cytosolc [Ca ] loads. Ths [Ca ] gradent may facltate n one cell by waves from neghborng cells (Fg. 8) could a hgher propagaton velocty of the nduced SCW. Besdes represent a mechansm of synchronzaton. The reason of beng a Ca ndcator, Fluo-3 has the sde effect to buffer the consstent restrcton of SCW s to only a few cells s cytosolc Ca to some degree. Ths mght have affected not fully evdent. Lamont et al. [10] nterpret t as a useful our results n Fgs. 4 and 5 wth respect to the propertes of mechansm to lmt arrhythmc actvty. It s known that both transents and SCW s. We have mnmzed ths Ca per se and n conjuncton wth cellular acdfcaton problem by keepng all loadng parameters absolutely decreases gap junctonal conductance [30,31]. Thus, Ca constant throughout our experments. ncrease mght be a self-lmtng factor for ntercellular In a recent study, slow Ca waves were defned and Ca dffuson. However, the SCW s occurred concomnamed on the bass of ther frequency [12]. Besdes SCW s tantly wth fast Ca transents and wth coordnated ( waves/mn Hz) whch were of the vsble mechancal actvty, ndcatng that the electrcal type also descrbed n Fgs. 2 and 3, these authors exctaton process was not markedly slowed n presence of descrbed a further type of Ca wave (so-called agonal SCW s. Ths argument speaks aganst a major degree of waves ), whch were characterzed by a hgh frequency cell-to-cell uncouplng durng the occurrence of SCW s at (133.1 waves/mn 2.2 Hz), a hgh degree of rhythmcty slghtly or moderately elevated cytosolc [Ca ]. Phenom- and an ntaton at hgh cytosolc [Ca ]. In our exper- ena such as collson and extncton of SCW s were ments relatvely frequent (1 5 Hz) and hghly regular nterpreted on the bases of 2-dmensonal measurements n waves were also observed, n coexstence wth the slower a gven focal plane. It s lkely that these phenomena were and relatvely rregular waves. However, the frequency of also affected by 3-dmensonal propagaton whch was not these waves showed no dependency on the cytosolc assessed n our experments. [Ca ]. A further type of regular Ca waves was Delayed afterdepolarzatons n heart occur mostly as descrbed n solated cardac myocytes as a consequence of relatvely fast and regular transent changes n membrane crcus movements wth re-entry around the cell nucleus potental. Spatal as well as temporal summaton (Fg. 8) servng as a fxed anatomcal obstacle [25]. In our experrather could explan that SCW s, whch as ndvdual waves were ments such an ntracellular crcus movement was not slow (2 5 Hz), mght lead to a relatvely rapd observed. electrcal exctaton. In the present experments, we select- ed an area ( mm) wth a dameter consderably smaller than the electrcal space constant, to nvestgate the 4.3. Spato-temporal occurrence of Ca waves and spato-temporal occurrence of SCW s. Wthn ths area potental relevance for arrhythmas 10 20% of the cells showed non-synchronzed SCW s. Both spatal summaton or nhbton mght occur n such a Delayed afterdepolarzatons (DAD s) are generally case, and summaton mght lead to a hgher overall thought to nvolve electrogenc Na/ Ca exchange [26,27] frequency. However, the rhythmcty of DAD s observed and, possbly, chlorde currents [28]. These mechansms n macroscopc tssue s not easly explaned by the result from the transducton of a chemcal Ca wave nto observed spato-temporal occurrence of SCW s, because a change n electrcal membrane potental. A propagated the slow Ca waves were not phase-locked. Ths suggests acton potental s generated, f the change n membrane that addtonal factors mght determne whether localzed 1 potental exceeds the threshold for Na nward current. SCW s lead to electrcal exctaton and arrhythmas. Whle ths mechansm s undsputed at the level of the In summary, our results show ntercellular dffuson and sngle cell, ts generaton n a network of coupled cells and a close spato-temporal nteracton of slow Ca waves. n tssue seems more complex. Ths s because sngle cells Summaton of such waves mght explan the producton

11 A.P. Baader et al. / Cardovascular Research 53 (2001) and the relatvely hgh overall frequency of trggered [14] Fleschhauer J, Lehmann L, Kleber AG. Electrcal resstances of acton potentals. However, the exact mechansm of ntersttal and mcrovascular space as determnants of the extracel- lular electrcal feld and velocty of propagaton n ventrcular synchronzaton remans to be determned. myocardum. Crculaton 1995;92: [15] Casco WE, X YG-J, Kleber AG. Passve electrcal propertes, mechancal actvty, and extracellular potassum n arterally perfused Acknowledgements and schemc rabbt ventrcular muscle. Crc Res 1990;66: [16] Cohen NM, Lederer WJ. Changes n the calcum current of rat heart We are very grateful to Peter Eggl MD, for hs advce ventrcular myocytes durng development. J Physol 1988;406:115 and help wth the hstologcal control experments Supported by the Swss Natonal Scence Foundaton [17] Gomez JP, Potreau D, Raymond G. Intracellular calcum transents and the Swss Heart Foundaton from newborn rat cardomyocytes n prmary culture. Cell Calcum 1994;15: [18] Anversa P, Olvett G, Loud AV. Morphometrc study of early References postnatal development n the left and rght ventrcular myocardum of the rat. I. Hypertrophy, hyperplasa, and bnucleaton of myocytes. Crc Res 1980;46: [1] Allen DG, Kurhara S. Calcum transents n mammalan ventrcular [19] Takahash A, Camacho P, Lechleter JD, Herman B. Measurement of muscle. Eur Heart J 1980:5 15. ntracellular calcum. Physol Rev 1999;79: [2] Cheng H, Lederer WJ, Cannell MB. Calcum sparks: elementary [20] Lakatta EG. Functonal mplcatons of spontaneous sarcoplasmatc events underlyng exctaton contracton couplng n heart muscle. retculum Ca release n the heart. Cardovasc Res 1992;26:193 Scence 1993;262: [3] Del Ndo PJ, Glynn P, Buenaventura P, Salama G, Koretsky AP. [] Del Prncpe F, Egger M, Nggl E. Calcum sgnallng n cardac Fluorescence measurement of calcum transents n perfused rabbt muscle: refractorness revealed by coherent actvaton. Nature Cell heart usng rhod 2. Am J Physol 1998;274: Bol 1999;1: [4] Wer WG, Ter Keurs HEDJ, Marban E, Gao WD, Balke CW. Ca [22] Cheng H, Lederer MR, Lederer WJ, Channell MB. Calcum sparks sparks and waves n ntact ventrcular muscle resolved by confocal and [Ca ] waves n cardac myocytes. Am J Physol magng. Crc Res 1997;81: ;270: [5] Mnamkawa T, Cody SH, Wllams DA. In stu vsualzaton of [23] Genka C, Ishda H, Ichmor K, Hrota Y, Tanaam T, Nakazawa H. spontaneous calcum waves wthn perfused whole rat heart by Vsualzaton of bphasc Ca dffuson from cytosol to nucleus n confocal magng. Am J Physol 1997;272: contractng adult rat cardac myocytes wth an ultra-fast confocal [6] Mura M, Boyden PA, Ter Keurs HEDJ. Ca waves durng magng system. Cell Calcum 1999;25: trggered propagated contractons n ntact trabeculae. Am J Physol [24] Takamatsu T, Wer WG. Calcum waves n mammalan heart: 1998;274: quantfcaton of orgn, magntude, waveform, and velocty. FASEB [7] Mura M, Boyden PA, Ter Keurs HE. Ca waves durng trggered J 1990;4: propagated contractons n ntact trabeculae. Determnants of the [25] Lpp P, Nggl E. Mcroscopc spral waves reveal postve feedback velocty of propagaton. Crc Res 1999;84: n subcellular calcum sgnallng. Bophys J 1993;65: [8] Zhang YM, Mura M, Ter Keurs HE. Trggered propagated contraccurrent [26] Kass RS, Tsen RW, Wengart R. Ionc bass of transent nward tons n rat cardac trabeculae. Inhbton by octanol and heptanol. nduced by strophantdn n cardac Purknje fbres. J Physol Crc Res 1996;79: ;281: [9] Ter Keurs HE, Zhang YM, Mura M. Damage-nduced arrhythmas: [27] Nggl E, Lederer WJ. Actvaton of Na Ca exchange current by reversal of exctaton contracton couplng. Cardovasc Res photolyss of caged calcum. Bophys J 1993;65: ;40: [28] Trafford AW, Daz ME, Esner DA. Ca-actvated chlorde current [10] Lamont C, Luther PW, Balke CW, Wer WG. Intercellular Ca and Na Ca exchange have dfferent tme courses durng sarcoplaswaves n rat heart muscle. J Physol 1998;512: mc retculum Ca release n ferret ventrcular myocytes. Pflugers [11] Hama T, Takahash A, Ichhara A, Takamatsu T. Real tme n stu Archv-Eur J Physol 1998;435: confocal magng of calcum wave n the perfused whole heart of the [29] Fozzard HA, Schoenberg M. Strength-duraton curves n cardac rat. Cell Sgnal 1998;10: Purknje fbres: effects of lmnal length and charge dstrbuton. J [12] Kaneko T, Tanaka H, Gyamada M, Kawata S, Takamatsu T. Three Physol (Lond) 1972;226: dstnct types of Ca waves n Langendorff-perfused rat heart [30] Noma A, Tsubo N. Dependence of junctonal conductance on revealed by real-tme confocal mcroscopy. Crc Res 2000;86:1093 proton, calcum and magnesum ons n cardac pared cells of gunea-pg. J Physol 1987;382: () () [13] Boyden P, Pu J, Pnto J, ter Keurs H. Ca transents and Ca [31] Frek L, Wengart R. Modfcaton of gap juncton conductance by waves n Purknje cells: role n acton potental ntaton. Crc Res dvalent catons and protons n neonatal rat heart cells. J Mol Cell 2000;86: Cardol 1995;27: