Heterogeneous changes in action potential and intracellular Ca in left ventricular myocyte sub-types from rabbits with heart failure

Transcription

1 Crdiovsculr Reserch 45 (2000) locte/ crdiores locte/ crdiores Heterogeneous chnges in ction potentil nd intrcellulr C in left ventriculr myocyte sub-types from rbbits with hert filure b, * M.A. McIntosh, S.M. Cobbe, G.L. Smith Institute of Biomedicl nd Life Sciences, West Medicl Building, University of Glsgow, Glsgow G12 8QQ, UK b Deprtment of Medicl Crdiology, Royl Infirmry, Glsgow University, Glsgow G31 2ER, UK Received 2 July 1999; ccepted 22 September 1999 Abstrct Objective: Myocrdil cellulr electrophysiology nd intrcellulr C regultion re ltered in hert filure. The extent of these chnges my vry within the lyers of the ventriculr wll. To exmine this, cell size, ction potentil nd intrcellulr C trnsient chrcteristics (Fur-2) were mesured in single crdic myocytes from sub-epicrdil, mid-myocrdil, nd sub-endocrdil regions of the left ventricle of rbbits with hert filure. Methods: Myocytes were isolted from nimls with hert filure induced by chronic coronry rtery ligtion nd from shm operted controls. Trns-membrne potentil ws mesured using high resistnce microelectrodes electrodes (30 MV; 2 M KCl). Fur-2 ws loded into cells by incubtion with the AM form. Subsequent fluorescence mesurements were used to mesure intrcellulr C concentrtion t rnge of stimulus frequencies. Results: Resting cell length ws significntly greter in the hert filure group; 115% of control vlues in sub-epicrdil nd mid-myocrdil cells, nd 108% in sub-endocrdil cells. Using criteri described by previous studies on other mmmlin herts, functionl M cells were identified by higher mximum rte of depolristion nd longer ction potentil durtion t 90% repolristion (APD 90) compred to the two other myocyte sub-types. In the hert filure group, APD90 nd C trnsient durtion (CD 50) were prolonged in sub-epicrdil nd M cells but shortened in sub-endocrdil myocytes. These chnges were significnt t lower stimulus frequencies, but the reltive effect diminished t higher frequencies (3 Hz). Pek systolic C ] ws reduced in sub-epicrdil nd M cells but incresed in sub-endocrdil cells in the hert filure group compred to controls. At higher stimulus frequencies, end distolic C levels were lower in sub-epicrdil cells but higher in sub-endocrdil myocytes of the hert filure group compred with controls. In generl, chnges were greter in hert filure nimls with more severe in vivo ventriculr dysfunction (ejection frction #44%). Conclusions: Hert filure ws ssocited with n incresed cell size throughout the left ventricle, but the form of the chnges in electrophysiology nd C trnsient were dependent on the myocyte sub-type. In prticulr sub-endocrdil cells displyed mrkedly different chnges compred to the other myocyte sub-types Elsevier Science B.V. All rights reserved. Keywords: Clcium (cellulr); Hert filure; Hypertrophy; Membrne potentil; Myocytes 1. Introduction Chnges in myocyte shpe depend on the nture of the mechnicl stress within the hert 4], nd there my lso In both humn hert filure nd niml models chronic be differentil chnges depending on the trnsmurl posimechnicl stress cuses enlrgement of myocytes (hy- tion of the myocyte 5]. Myocytes from the sub-endocrpertrophy) nd chnges in their electrophysiologicl nd dil nd sub-epicrdil regions of the mmmlin left mechnicl properties 1,2]. These chnges re thought to ventriculr wll differ in cell size, electricl nd mecontribute both to the mechnicl dysfunction nd the chnicl properties 6,7]. A third sub-type with distinct incresed risk of rrhythmis in filing myocrdium 3]. electrophysiologicl chrcteristics, the M cell 8], hs been found in the mid-myocrdil region of the ventricle in *Corresponding uthor. Tel.: ; fx: humn, dog, ct, rt nd guine-pig. No studies hve E-mil ddress: g.smith@bio.gl.c.uk (G.L. Smith) Time for primry review 27 dys / 00/ $ see front mtter 2000 Elsevier Science B.V. All rights reserved. PII: S (99) on June 2018

2 398 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) exmined whether the electrophysiologicl chnges during brnch of the left coronry rtery ws identified nd ligted hypertrophy re common to ll three ventriculr myocyte midwy between the left tril ppendge nd the crdic sub-types. Prolongtion of ction potentil durtion p- pex. This gives rise to lrge homogeneous infrct due to pers to be consistent feture of cells isolted from the the limited collterl circultion in the rbbit. Shm-opersub-epicrdil region of hypertrophied herts 9 12]. ted nimls underwent thorcotomy with the hert mn- However, conflicting results hve been reported for cells ipulted in similr fshion to the hert filure group but isolted from sub-endocrdil regions, rnging from the rtery ws not tied. prolongtion of ction potentil durtion 11,13], no Left ventriculr function ws ssessed by echocrdiogchnge 9] to mrked shortening 10]. rphy ] 22] 8 weeks fter surgery. The coronry ligted Abnormlities of intrcellulr C ] ccompny the nimls showed significnt hemodynmic dysfunction in electrophysiologicl chnges ssocited with myocyte hy- terms of incresed left ventriculr end-distolic dimension pertrophy nd my contribute to both the electricl nd (LVEDD) nd left tril dimension (LAD) nd decresed mechnicl dysfunction in hert filure. C trnsients of ejection frction (EF) (Tble 1). Evidence of congestion reduced mplitude nd prolonged time course re comweight ws mnifest in significnt increses in lung nd liver wet monly observed in humn hert filure 14,15] nd in present t post-mortem exmintion. Previous work niml models 16,17]. However, severl studies suggest hs shown tht this niml model shows significnt crdic either unchnged 18,19], or incresed intrcellulr C ] hypertrophy, evident s 20 30% increse in hert wet in filing myocrdium 16,20]. These inconsistencies my weight nd left ventriculr dry weight,22]. In vivo be ttributed to interspecies vrition or to different forms hemodynmic mesurements revel reduced crdic of response ccording to the underlying stimulus to output, rised end distolic pressure nd reduced response hypertrophy or filure. An lterntive hypothesis, tht there to n incresed pre-lod in this model ]. Incresed re trnsmurl differences in the hypertrophic response, is inducibility of rrhythmis nd lowered fibrilltion thresddressed in the present study. hold observed in vitro 25] suggest ccompnying elec- trophyiologicl dysfunction. In the present study, the hert filure group displyed rnge of hemodynmic dysfunction. EF rnged from 56 to 2. Methods 34% suggesting non-uniform infrct size t the end of the 8 weeks post-ligtion period. In seprte study, herts 2.1. Animl model from different cohort of nimls were sectioned nd the infrct perimeter studied. This did show lrge vrition, A well-chrcterised model of hert filure induced by which ws correlted with the severity of the in vivo LV chronic left ventriculr infrction in the rbbit ws used in dysfunction (Burton nd McPhden, unpublished observthis study 25]. Procedures were undertken in ccord- tion). Consequently, the hert filure group ws dichotomnce with the United Kingdom Animls (Scientific Pro- ised on the bsis of the medin vlue of EF for the whole cedures) Act 1986 nd conforms with the Guide for the group (44%). The sub-group with n EF#44% hd greter Cre nd Use of Lbortory Animls published by the US men LAD, LVEDD, liver nd lung weights thn the Ntionl Institutes of Helth (NIH Publiction No , sub-group with n EF.44%, lthough these differences revised 1996). New Zelnd White mle rbbits ged were not sttisticlly significnt. pproximtely 12 weeks nd weighing kg were nesthetised with fentnyl citrte (Hypnorm) nd min Cell isoltion tined with hlothne nd nitrous oxide/ oxygen. A left thorcotomy ws performed nd the lrge circumflex At 8 weeks post-opertion, the rbbits were given n Tble 1 Men echocrdiogrphic prmeters nd post-mortem orgn weights in shm nd hert filure groups Shm HF HF (EF.44%) HF (EF#44%) (n514) (n525) (n512) (n513) Body weight (kg) Ejection frction (%) * ** ** LAD (mm) ** ** ** LVEDD (mm) ** ** ** Liver wet weight (g) * * * Lung wet weight (g) * * * The hert filure (HF) group is subdivided on the bsis of ejection frction (EF) to those less thn or equl to 44% (EF#44%), nd those greter thn 44% (EF.44%). Left tril dimension (LAD) nd left ventriculr ends distolic dimension (LVEDD). Vlues re expressed s men6s.e.m. * Significnt difference between Shm nd HF group t the level of P,0.05. ** Significnt difference between Shm nd HF groups t the level of P, on June 2018

3 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) intrvenous injection of 500 U heprin together with n shpe were stble over the durtion of the mesurements overdose of sodium pentobrbitone(100 mg/ kg). Isolted suggesting tht the dilysis of the intrcellulr contents by herts were perfused retrogrdedly (25 ml/ min, 378C) with KCl from the recording microelectrode ws not significnt. nominlly C -free Krebs Henseleit solution for 10 In seprte group of experiments, simultneous ction min. This ws followed by perfusion with re-circulted potentil nd intrcellulr C ] trnsients were recorded Krebs Henseleit solution supplemented with 0.6 mg/ ml in myocytes from shm nd filing herts. Intrcellulr collgense (type 1, Worthington Chemicl Co.), 0.1 mg/ C ] ws mesured from Fur-2 fluorescence signls ml protese (type XIV, Sigm Chemicl Co) nd 80 mm using dul wvelength spectrophotometric method de- CCl2 for min. The left ventriculr free wll ws scribed previously 28]. Fur-2 ws incorported into the isolted, nd the infrct nd neighbouring myocrdium cells s the cetoxymethyl (AM) ester, by incubting them (2.5 3 mm border) ws crefully dissected wy. Bsed on with 3 mm Fur-2-AM t 378C for 20 min. Miniml Fur-2 mesurements of the size of the border zone from loding protocols were used nd the recorded C histologicl study 26], this procedure ensured tht the trnsients were verged to give sufficient signl-to-noise remining myocrdium did not contin myocytes from the rtio. This precution ws used to minimise the possibility peri-infrct zone 27]. The sub-endocrdil, mid-myocr- of intrcellulr C buffering by the indictor. The loded dil nd sub-epicrdil lyers of the free wll were cells were plced in the recording chmber nd superfused dissected from the remining tissue by dissecting with physiologicl sline. Fluorescence mesurements (t mm lyer from the epicrdil nd endocrdil surfces..500 nm) from sequentil illumintion with light t 340 These two lyers of tissue nd the remining intervening nd 380 nm t 60 Hz were mde using spinning wheel mid-myocrdil lyer were incubted seprtely for 5 min spectrophotometer (Cirn Reserch Ltd). The rtio mein enzyme solution contining 80 mm CCl2 nd 4% surement of fluorescence (340:380 nm) provides direct bovine serum lbumin (BSA, frction V, Sigm). The cell mesure of intrcellulr C ] 29]. The minimum nd suspensions obtined t the end of the incubtion period mximum fluorescence rtios (R min nd R mx) were de- were filtered into Krebs Henseleit solution contining 0.1 termined using previously published protocol 30]. The mm CCl nd 1.5% BSA, nd the C ] ws incresed vlues of R nd R were not significntly different in 2 min mx to 1.5 mm progressively over 30 min. The cells were cells from different regions or between shm nd hert trnsferred to Petri dishes contining Medium 199 (Gibco) filure groups. These vlues were R min(shm) ; plus supplements of turine (5 mm), cretine (5 mm) nd R min(hf) ; R mx(shm) ; R mx(hf)5 BSA (0.2%) nd kept t room temperture until use. No As with previous studies 17,31] the rnge of difference in the percentge yield of cells ws observed Rmin nd Rmx vlues represents much lower dynmic either between myocrdium from different regions or rnge thn tht mesured in vitro, possibly due to dditionbetween experimentl groups. l fluorescence components from non-cytosolic forms of the indictor. The intrcellulr C ] ws clculted 2.3. Experimentl procedures ssuming dissocition constnt of 200 nm 17,32] in ll cell types nd in both experimentl groups. The men pek Myocytes were superfused with the physiologicl slt systolic C nd minimum distolic C were mesured solution t C in chmber mounted on the stge of n inverted microscope. Trnsmembrne ction potentils were recorded using 2 M KCl filled glss microelectrodes mplitude (CD 50). with resistnces of MV. Micro-electrode with from ech cell nd expressed s men vlues. C trnsient durtion ws mesured t 50% of the trnsient Cells were stimulted by progressive step increses in similr chrcteristics hve been used by other groups to stimulus frequencies. Ech test frequency ws mintined study ction potentil chrcteristics of mmmlin crdic until ction potentil nd C trnsients reched stedy cells 9]; this configurtion minimises intrcellulr dilysis stte (1 5 mins), before incresing the test frequency. by the electrode solution. Action potentils were elicited in Only cells tht returned to stble bseline fter this bridge mode by injecting 2 5-ms threshold current pulses protocol were used. Mesurements were mde from ver- (Axoclmp 2A mplifier, Axon Instruments, Foster City, ges of 56 sequentil stedy stte records with 0.1, , CA, USA). Membrne potentil ws recorded to mgnetic 2.0 nd 3.0 Hz stimuli. As described in detil in the results, tpe (18.5 KHz bndwidth) for lter offline nlysis. cells isolted from the sub-endocrdil nd mid-myocrdil Action potentil durtion ws mesured from the rpid regions exhibited electrophysiologicl chrcteristics of upstroke of the AP following the stimulus to 90% (APD 90) either M cells or sub-endocrdil cells (see below). M cells repolristion. Resting membrne potentil, mximum were distinguished from other cell types by prolonged upstroke velocity (V mx) nd ction potentil mplitude ction potentil durtion t low stimulus rtes nd high were mesured from the digitised signl using Ntionl rtes of depolristion. Some cells isolted from the mid- Instruments A/ D bord (PC-Lb) controlled by CMAP softwre developed by Dr J. Dempster, (Strthclyde University). Resting membrne potentil nd ction potentil myocrdium showed non-m cell (possibly sub-endocrdil or sub-epicrdil) chrcteristics. These cells were not included in the electrophysiologicl nlysis. on June 2018

4 400 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) In prllel with electrophysiologicl studies, cell length were used to clculte the verge 6stndrd error of the nd width mesurements using n eye-piece grticule were men (S.E.M.) for ech experimentl group bsed on the mde on pproximtely 20 cells from the sub-endocrdil, number of nimls. This method ws used insted of mid-myocrdil nd sub-epicrdil regions in ech niml. pooling ll the mesurements together, s this ltter Cell dimension mesurements did not llow sub-endocr- technique would give unequl weight to the herts from dil cells to be distinguished from M cells (see bove). which the most mesurements were vilble 33]. Com- Therefore this group is termed mid-myocrdil myocytes prisons of vlues from different lyers nd from different to distinguish this group from cells which were identified experimentl groups were performed using one-wy s M-cells from their electrophysiologicl chrcteristics. ANOVA followed by Tukey Krmer multiple comprisons The dimensions of cells used for electrophysiologicl post test. Correltions were exmined by liner regression mesurements were recorded seprtely. nlysis. P,0.05 ws considered significnt Solutions 3. Results The Krebs Henseleit solution used in the cell isoltion contined, in mm: NCl, 130; KCl, 5.4; NH2PO 4, 0.4; 3.1. Cell size MgCl, 3.5; Hepes, 5; turine, 20; cretine, 10; nd glucose, 11.1 (ph 7.25, equilibrted with 100% O 2). The superfu- Cell length nd width were mesured in smples of cells ste solution used for the experiments contined, in mm: tken from herts from14 shm-operted nd 25 hert NCl, 144; KCl, 5.4; NH2PO4, 0.3; MgCl 2, 1; Hepes, 5; filure nimls. As described bove, the medin cell length Glucose, 11.1 nd CCl 2,1.8. All chemicls were obtined nd width for ech hert ws clculted nd men from Sigm with the exception of Fur-2 AM nd DMSO (6S.E.M.) of these vlues re expressed in Tble 2. The which were obtined from Moleculr Probes nd Fluk, cell dimensions for ech lyer were normlly distributed in respectively. the shm nd hert filure groups, indicting one underlying popultion of cell size from both groups. In shm 2.5. Sttisticl nlysis herts, sub-endocrdil cells were significntly longer thn mid-myocrdil nd sub-epicrdil cells. Cell width lso Mesurements were mde from cells from sub-endocr- tended to be greter but the differences were not signifidil, mid-myocrdil nd sub-epicrdil regions of ech cnt. In the hert filure group, verge cell lengths in the hert s described bove. The men vlues of the electro- sub-endocrdil, mid-myocrdil nd sub-epicrdil re- physiologicl nd C trnsient prmeters from two or gions were significntly greter thn in shms. The inmore cells from ech sub-group (typiclly three) were crese in cell length ws more pronounced in sub-epicrclculted for ech niml. These individul medin vlues dil nd mid-myocrdil cells ( 115%) thn in sub-endo- Tble 2 Averge left ventriculr cell dimensions from shm nd hert filure (HF) groups Cell dimensions (mm) Shm HF HF(EF.44%) HF(EF#44%) Sub-epicrdil cells n nc Length (mm) ** * ** Width (mm) * * * Mid-myocrdil cells n nc Length (mm) * * * Width (mm) Sub-endocrdil cells n nc Length (mm) * * * Width (mm) n is the number of nimls, nc is the totl number of cells in ech experimentl group. The men nd stndrd errors re clculted using niml number. The HF group is subdivided on the bsis of ejection frction (EF). Indictes significnt difference between myocyte sub-type nd sub-endocrdil cells P,0.05. * Significnt difference between Shm nd HF groups t the level of P,0.05. ** Significnt difference between Shm nd HF groups t the level of P, on June 2018

5 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) crdil myocytes ( 108%). The effect ws greter in cells 0.3 Hz (Tble 3). This pttern lso pplied when comprfrom nimls with severe left ventriculr dysfunction ing shm vlues with the hert filure sub-group with (EF#44%) for ll regions. A significnt increse in cell severe ventriculr dysfunction (EF#44%, results not width (to 110% of control) ws observed only in sub- shown). epicrdil cells. The dimensions of cells used for mesure- ments of membrne potentil nd intrcellulr C ] ( Upstroke velocity cells per group, results not shown) were not significntly As hs been reported in number of other mmmlin different to those mde on the lrger smples ( 350 cells species, the mximum rte of depolristion (V mx) ws per group) from ech of the three ventriculr regions. In significntly greter in M cells thn in the other two seprte study on the sme niml model (M. McIntosh, myocyte sub-types over the rnge 0.3 to 3 Hz. The vlues unpublished observtions), similr increses of cell length mesured t 0.3 Hz re shown in Tble 3. Similrly, V mx were ccompnied by significnt incresed cell electricl ws significntly greter in M cells thn the other two cell cpcitnce of both sub-endocrdil (shm: pf, sub-types in the hert filure group (Tble 3). There were n511; HF: pf, n55) nd sub-epicrdil cells no significnt differences in Vmx vlues between shm nd (shm: pf, n510; HF: pf, n55). This hert filure groups in corresponding ventriculr regions, supports the conclusion tht the cell shpe chnges re or in the sub-group with more severe ventriculr dysfuncresult of cellulr hypertrophy. tion (EF#44%, results not shown) Action potentil chrcteristics Action potentil durtion Fig. 1 shows trces of ction potentils nd C Electrophysiologicl mesurements were mde on 30 trnsients recorded t 0.3 nd 3 Hz from cells isolted from cells from ech region in the shm nd hert filure the three ventriculr lyers in the two experimentl groups. groups. About hlf of the cells in ech group were loded The records hve been normlised for mplitude to highwith Fur-2 AM for simultneous mesurement of intrcel- light the differences in ction potentil durtion (APD 90) lulr C ]. Comprison of ction potentil chrcteristics nd C trnsient durtion (CD 50). The men vlues re indicted no effects of loding cells with Fur-2. Resting shown in Fig. 2, with the results from the HF group membrne potentil ws not significntly different between dichotomised on the bsis of ejection frction. Compring the shm nd hert filure groups in ny myocyte sub-type APD90 vlues from different regions within the shm t ny frequency of stimultion, the results for 0.3 Hz group revels tht the APD90 in sub-endocrdil cells ws stimulus rte re shown in Tble 3. There were no longer thn in sub-epicrdil cells t Hz stimultion significnt differences in ction potentil mplitude (APA) frequency. The APD90 of M cells were longer thn those between the shm nd hert filure groups in ny region, of sub-epicrdil myocytes t stimultion frequencies 0.1 with the exception of the vlues in sub-epicrdil cells t 2 Hz nd longer thn those of sub-endocrdil myocytes t 0.1 nd 0.3 Hz. The prolonged APD90 t low frequencies (nd higher V mx) is chrcteristic of M cells in other Tble 3 mmmlin species. Averge single myocyte ction potentil chrcteristics There is n overll increse in APD90 in sub-epicrdil Shm HF cells nd decrese in sub-endocrdil cells in hert filure. Sub-epicrdil cells These chnges reduce the endo-epicrdil difference in n APD90 vlues. In the severe ventriculr dysfunction sub- E m (mv) group there ws no significnt difference in APD90 be- APA (mv) * V (mv/ s) tween cells from sub-epicrdil nd sub-endocrdil remx gions t ll stimultion frequencies prt from 0.1 Hz. At M cells n this frequency the norml pttern is reversed nd sub- E (mv) epicrdil APD90 is now significntly longer thn in subm APA (mv) endocrdil myocytes. M cell APD90 ws significntly V mx (mv/ s) longer thn sub-epicrdil nd sub-endocrdil cells t Sub-endocrdil cells stimulus frequencies #1 Hz. n Compring ech region, the men APD90 of sub-epicr- E m (mv) ] dil cells in the HF group (EF #44%) ws longer thn APA (mv) those from shm herts t ll stimultion frequencies prt V mx (mv/ s) from 3 Hz. A similr trend ws seen in the M cells from n is the number of nimls in ech experimentl group. The men nd the HF group (EF #44%) but the difference ws only stndrd errors re clculted using niml number. Indictes significnt difference between sub-group nd M cell group significnt t 1 nd 2 Hz. In contrst, the APD90 ws t the level of P,0.05. * Indictes significnt difference between Shm shorter in sub-endocrdil cells from the HF group com- nd HF groups t the level of P,0.05. pred with shms t 0.3 nd 0.1 Hz. on June 2018

6 402 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) Fig. 1. Averged nd normlised records of ction potentil nd ssocited C trnsients from single crdic myocytes isolted from the left ventricle of shm nd hert filure rbbit herts (d). In ll, 56 seprte records (1-s durtion) of simultneous recordings of membrne potentil nd Fur-2 fluorescence were verged from single cell during stimultion t: (i) 0.3 Hz, nd (ii) 3 Hz. Myocyte sub-types re shown bove the record: pnel A, sub-epicrdil; pnel B, M cell; nd pnel C, sub-endocrdil cell C trnsient durtion Pek systolic nd end distolic C ] Averged C trnsient records were normlised by Fig. 4 shows the overll men vlues (6S.E.M.) of pek mtching pek systolic nd end distolic fluorescence rtio systolic nd end distolic C ]. All cells from shm vlues. Superimposed records from sub-epicrdil, M nd herts showed n incresed pek systolic C ] nd end sub-endocrdil cells isolted from shm nd filing herts distolic C ] s stimultion frequency incresed. Comre illustrted in Fig. 1. Men durtions re shown in Fig. pring these prmeters from regions within the shm 3, with the results from the hert filure group dichotom- group reveled no significnt difference between the three ised on the bsis of ejection frction. Between different myocyte sub-types prt from significntly higher pek regions within the shm group, CD50 in sub-endocrdil systolic C ] t 0.1 nd 1 Hz in M cells. In the severe cells ws significntly longer thn in sub-epicrdil cells t ventriculr dysfunction sub-group the pek systolic C ] Hz stimultion rtes. The CD50 of M cells were ws significntly higher in sub-endocrdil cells thn sublonger thn sub-epicrdil myocytes nd sub-endocrdil epicrdil nd M cells. This shift in chrcteristics is due cells t frequencies #1 Hz. to the decresed pek systolic C ] in sub-epicrdil nd As with APD90 vlues, there is n overll increse in M cells in the severe hert filure group while pek CD in sub-epicrdil cells nd decrese in sub- systolic C ] is incresed in sub-endocrdil cells. In the 50 endocrdil cells in hert filure. These chnges reduce the sub-group of cells with low ejection frction, end distolic endo-epicrdil difference in CD50 vlues observed nor- C ] ws significntly reduced in sub-epicrdil cells mlly. In the sub-group with more severe ventriculr stimulted t 2 nd 3 Hz nd significntly higher in dysfunction there ws no significnt difference in the sub-endocrdil cells stimulted t 3 Hz. CD50 between cells from sub-epicrdil nd sub-endocrdil regions. However, CD50 of M cells were significntly 3.3. Correltion between electrophysiologicl prmeters longer thn tht of sub-epicrdil nd sub-endocrdil cells nd intrcellulr C ] t stimulus frequencies #1 Hz. Between corresponding regions, the men CD50 of As described bove, the APD90 nd CD50 show sub-epicrdil cells in the hert filure group (EF#44%) complex dependency on stimultion frequency in both ws longer thn those from shm herts only t 1 Hz. shm nd hert filure groups. In prticulr, non-uniform There ws no significnt difference in CD50 of M cells chnges in these vribles occur in sub-epicrdil nd from hert filure group. In contrst, the CD ws sub-endocrdil cells. At ny one stimultion frequency, 50 shorter in sub-endocrdil cells from the HF group com- the hert filure group hd n incresed APD nd CD pred with shms t 0.3 nd 0.1 Hz. in sub-epicrdil nd M cells nd decresed APD nd 90 on June 2018

7 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) Fig. 2. The reltionship between stimulus frequency nd ction potentil durtion t 90% repolristion (APD 90) in sub-epicrdil cells (pnel A), Fig. 3. The reltionship between stimulus frequency nd C trnsient M cells (pnel B) nd sub-endocrdil cells (pnel C). Dt re presented durtion t 50% mplitude (CD 50) in sub-epicrdil cells (pnel A), M s men (6S.E.M.) APD90 for shm group nd hert filure group cells (pnel B) nd sub-endocrdil cells (pnel C). Dt is presented s dichotomised on the bsis of ejection frction (EF). Indictes significnt men (6S.E.M.) for shm group nd hert filure group dichotomised on difference between myocyte sub-type nd sub-endocrdil cells. the bsis of ejection frction (EF). Indictes significnt difference Indictes significnt difference between sub-type nd M cells. between myocyte sub-type nd sub-endocrdil cells. Indictes signifi- * Indictes significnt difference between shm nd hert filure groups. cnt difference between sub-type nd M cells. * Indictes significnt Number nimls (n) re indicted in ech pnel. Note different scle of difference between shm nd hert filure groups. Number of nimls (n) y-xis in pnel B. re indicted in ech pnel. Note difference in scle of y xes in pnels A C. CD50 in sub-endocrdil cells compred to the shm group. This suggests linkge between APD90 nd CD50 hert filure group t one stimultion frequency (0.3 Hz) s in cells throughout the myocrdium, which ws confirmed shown in Fig. 5A. There is strong liner reltionship by plotting APD90 ginst CD50 for individul cells in the common to cells from ll three myocyte sub-types (r5 on June 2018

8 404 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) Fig. 5. Pnel A shows the reltionship between C trnsient durtion (CD 50) nd ction potentil durtion (APD 90) for myocytes isolted from the hert filure group. Mesurements were mde t stimulus rte of 0.3 Hz. Results from the three myocyte sub-types (sub-epicrdil, M nd endocrdil cells) re shown individully. The line through the vlues for ll three myocyte sub-groups represents the best fit to liner correltion (r50.93, P,0.001). Pnel B, the reltionship between pek systolic C ] nd C trnsient durtion (CD 50). Lines represent the best fit liner correltion through the vlues for ech myocyte sub-group: sub-epicrdil cells r520.83, P,0.001; M cells, r520.74, P,0.001; sub-endocrdil cells, r520.76, P, between the CD50 nd the mplitude of the C trn- Fig. 4. The reltionship between stimulus frequency nd pek systolic sient. As shown in Fig. 5B, higher pek systolic C ] nd end distolic C ] in sub-epicrdil cells (pnel A), M cells (pnel is correlted with shorter C durtion in cells from the B) nd sub-endocrdil cells (pnel C). Dt is presented s men three ventriculr regions. While sub-endocrdil cells nd (6S.E.M.) for shm group nd hert filure group dichotomised on the sub-epicrdil cells pper to hve similr reltionship, bsis of ejection frction (EF). Indictes significnt difference between myocyte sub-type nd sub-endocrdil cells. Indictes significnt differ- M cells hve significntly prolonged CD50 vlues for ence between sub-type nd M cells. * Indictes significnt difference comprble pek systolic C ]. between shm nd hert filure groups. Number of nimls (n) re indicted in ech pnel. 4. Discussion 0.93, P,0.001). A similr reltionship exists for cells from shm herts nd for both groups t other stimultion The im of this study ws to chrcterise nd interrelte frequencies, however the correltion ws weker t higher the effects of chronic myocrdil infrction on cell size, stimultion frequencies due to limited rnge of vlues. electrophysiology nd intrcellulr C ] in ventriculr Another ssocition to be mde is the reltionship myocyte sub-types. on June 2018

9 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) Cell size of APD 90, nd higher mximum rte of depolristion (V mx) thn in sub-epicrdil or sub-endocrdil cells. The epi-endocrdil cell length difference evident in These properties re chrcteristic of the M cell phenotype shm herts ws not present in the hert filure group observed in other species 5,8] but not previously seen in where cells from ll regions were mm long. So in isolted myocytes from the rbbit, lthough recordings this model, the hypertrophic stimulus to sub-epicrdil nd from M cells in the rbbit ventriculr slice preprtion mid-myocrdil cells results in greter increse in length hve been reported 44]. The finding of mixed popultions ( 115%) thn in sub-endocrdil cells ( 108%). The sme of M cells nd sub-endocrdil cells in the isoltes from bseline pttern nd extent of chnges in cell dimension the sub-endocrdil nd mid-myocrdil regions hs imhve been observed in the crdic hypertrophy model portnt implictions, since it cnnot be ssumed tht cells ssocited with hyperthyroid-induced volume overlod 5]. isolted from these regions form homogeneous popul- In contrst, pressure overlod hypertrophy in response to tion. Chrcteristion of ech cell by its ction potentil hypertension results in mrked increse in cell cross chrcteristics is necessry to void confusion. sectionl re but no increse in cell length 34 36], nd the increse in cross sectionl re ws gretest in the 4.3. Electrophysiology of hert filure myocytes. endocrdil region 35]. Therefore, the site nd form of cellulr hypertrophy ppers to be dependent upon the The most striking finding of the present study is the nture of the pthologicl stress. In humn hert filure, trnsmurl difference in the hypertrophic response to hert increses in both cell length nd width re observed 37] filure, resulting in opposite chnges in the chrcteristics with n pproximtely 60% lengthening of myocytes of sub-epicrdil nd M cells compred with sub-endocrobserved in dilted crdiomyopthy 38] greter degree dil cells. of hypertrophy thn tht observed in this study. As summrised in Tble 4, APD90 ws prolonged in sub-epicrdil nd M cells in hert filure. The effect ws 4.2. Electrophysiology of norml (shm) myocytes more pronounced in cells from herts with severe left ventriculr dysfunction (EF#44%). The results re con- The shorter APD90 in myocytes from the sub-epicr- sistent with epicrdil monophsic ction potentil medium compred to sub-endocrdil cells reported here surements mde in Lngendorff perfused preprtions of confirms erlier work on rbbit 39,40] nd other mm- this hert filure model 45]. The lrge vribility of M mlin species 8]. Work on lrger mmmls hs indicted cell APD90 reduces the bility to distinguish differences tht sub-epicrdil cells possess chrcteristic spike nd between cells from the shm nd hert filure groups. dome ction potentil morphology 5,41]. Although ction However, sttisticlly significnt prolongtion in APD 90 potentils with this morphology were recorded in rbbit ws observed t 1 nd 2 Hz in M cells belonging to the myocytes, they were not exclusive to the sub-epicrdil sub-group of rbbits with severe left ventriculr dysfuncregion. APD90 in sub-epicrdil cells remined signifi- tion. cntly shorter thn in sub-endocrdil cells over most of Prolongtion of APD is the most common observtion in the frequency rnge studied (0.1 2 Hz). Sub-epicrdil previous studies on myocytes isolted from niml models myocyte APD90 incresed with frequency of stimultion in nd from filing humn herts 2]. The origin of the this nd previous study in rbbit 42], in contrst to the myocytes is not specified in the mjority of these studies. flt rte dependence observed in sub-endocrdil cells However, the few niml studies using myocytes from 39,43]. specific ventriculr regions hve reported prolonged APDs A distinct myocyte type ws observed in smples tken in sub-epicrdil myocytes 9 12]. No previous study hs from the sub-endocrdil nd the mid-myocrdil regions. specificlly exmined M cells in hert filure. Cells from These cells were chrcterised by n extremely long the mid-myocrdium of mildly hypertrophied guine-pig APD90 t low stimulus frequencies, steep rte-dependence left ventricle exhibited prolonged APD, but s noted by Tble 4 Summry of chnges in ction potentil nd C trnsient chrcteristics in the rbbit model of hert filure Chnges observed in hert filure group Sub-epicrdil cells M-cells Sub-endocrdil cells Action potentil durtion * * * C trnsient durtion * * * Pek systolic C ] End distolic C ] Increses nd decreses in the prmeters re indicted by nd, respectively. indictes no significnt chnge. * Indictes tht significnt differences between shm nd hert filure group ws only evident t low stimulus frequencies (0.1 nd 0.3 Hz). Indictes tht significnt differences between shm nd hert filure groups were evident t high stimulus frequencies (2 3 Hz). on June 2018

10 406 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) the uthors 9], the ction potentil did not exhibit the stresses experienced in the sub-endocrdil nd sub-epicrincresed Vmx nd long APD chrcteristic of M cells. dil regions in situ compred to dissocited single cells. In contrst to the chnges in sub-epicrdil nd M cells, sub-endocrdil myocytes from the hert filure group in the present study showed consistently shorter APD vlues 4.5. Intrcellulr C trnsients in hert filure thn in the shm group. Sub-group nlysis indicted tht more severe filure ws ssocited with more profrom As summrised in Tble 4, sub-epicrdil nd M cells this rbbit model of hert filure pper to conform nounced shortening of APD 90 (sttisticlly significnt t 0.1 nd 0.3 Hz), while the APD90 vlues in mild filure to the generlly observed hypertrophic behviour of pro- were very similr to the shm vlues. Thus the shortening longed ction potentil nd C trnsient durtion, cou- of APD90 in sub-endocrdil cells is dependent on the pled with reduced pek systolic C ] nd poor severity of ventriculr dysfunction. A previous study hs inotropic response to incresed stimulus frequency 1]. reported shorter APD90 vlues ssocited with hypertrophy These results re in contrst to those from sub-endocrdil in sub-endocrdil cells of the rt 10]. cells in hert filure, which displyed shortened CD50 nd incresed pek systolic C ] in comprison to shm 4.4. Intrcellulr C trnsients in norml (shm) herts. Also, s indicted in Fig. 5B, the cells with myocytes shortened CD50 re ssocited with n incresed pek systolic C nd pper to belong to the sme reltion- The time-course of the C trnsients recorded in this ship observed in sub-epicrdil cells. This is thought to be study re similr to those observed in previous reports on due in prt to the reltionship between the rte of C isolted rbbit 46] nd guine-pig myocytes 17] using the uptke by the SR nd systolic C ] 30,50]. A combin- C indictor Fur-2. Aprt from n increse t 0.3 Hz, tion of decresed ction potentil durtion nd incresed CD50 vlues shortened s stimultion frequency ws C trnsient mplitude noted in sub-endocrdil cells incresed in ll cell types. This shortening of CD 50 from hert filure group is observed experimentlly in prlleled the APD90 shortening observed in M cells, but rised extrcellulr C 51]or isoprenline 52]. This ws in contrst to the lengthening of APD90 vlues in suggests tht n incresed C relese from the SR my sub-epicrdil cells nd the flt APD90-frequency response be the primry chnge tht lters the ction potentil in sub-endocrdil cells. The close correltion between durtion vi C ctivted currents. Further work is APD90 nd CD50 cross the frequency rnge for M cells required to determine the cellulr bsis of the ction is s expected from voltge clmp studies in isolted rt potentil nd C trnsient chnges observed in the submyocytes, which show tht shortening the clmp durtion endocrdil cells of this hert filure model. reduced the C trnsient durtion 47]. This effect is In summry, myocytes from the hert filure group 1 thought to be medited by the srcolemml N / C displyed mrked differences in APD90 nd CD50 when exchnger. However, other membrne currents pper to compred to the shm group (Tble 4). At higher stimulus be importnt in determining the reltionship between rtes (3 Hz), however, the differences were not significnt, APD90 nd CD50 t different stimulus frequencies in lthough smll differences my exist which cnnot be sub-epicrdil nd sub-endocrdil rbbit myocytes. detected becuse of the inherent vribility of single cell In ll myocyte sub-types, pek systolic C ] nd end mesurements. In support of this, epicrdil monophsic distolic C ] incresed s stimulus frequency incresed. ction potentil nd C trnsient durtion were pproxi- The vlues of end distolic C ] nd pek systolic mtely 20 ms longer in the hert filure group in Lngen- C ] (t 0.3 Hz stimultion) were similr to those dorff perfused herts from the sme rbbit model (3 Hz recorded t 0.5 Hz using Indo-1 in rbbit myocytes 48] stimultion rte) 22,45]. This is comprble to the differnd guine pig myocytes t (1 Hz stimultion) 17]. No ences in APD90 nd CD50 vlues observed in sub-epicrsignificnt differences in pek systolic nd end distolic dil cells in this study nd suggests tht smll APD nd C ] were obvious in ny of the shm myocyte sub- CD durtion differences persist in epicrdil cells t types, in greement with previous work 7]. However, physiologicl stimulus rtes. The present study indictes there ws tendency for lower systolic C ] in tht hert filure relted differences in APD persist in sub-endocrdil cells, which is consistent with lower sub-epicrdil cells isolted from the electrotonic interintrcellulr sodium concentrtion in sub-endocrdil cells ction of djcent res of myocrdium. Furthermore, the 40]. The results of the current study re in contrst to in improved opticl signls from single cell experiments situ intrcellulr C ] mesurements in rt hert 49] llowed direct quntifiction of distolic nd systolic indicting tht pek systolic C ] nd end distolic C ] indicting tht the APD differences were ccom- C ] ws higher in the sub-endocrdil region thn the pnied by chnges in distolic nd systolic intrcellulr sub-epicrdil region. This difference my hve bsis in C ] the mrked APD differences between rt nd rbbit Unlike APD nd CD mesurements, the differences myocytes, or lterntively, the differentil mechnicl in pek systolic C in the hert filure group were most on June 2018

11 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) prominent t the highest stimulus rtes. This study is the tht the C trnsient mplitude is incresed in sub- first to report contrsting chnges in pek systolic C in endocrdil cells in the hert filure group. However, C myocytes from different sites of filing myocrdium. Sub- trnsient mplitude ws reduced in sub-epicrdil nd M endocrdil cells from the hert filure group displyed n cells, so the overll chnge in the C trnsient within the incresed pek systolic C ] in contrst to the decresed vible myocrdium of the left ventricle will depend on the vlues observed in sub-epicrdil cells nd M cells. These proportion of these three myocyte types. Bsed on estiresults my explin the previous rnge of disprte results mtes from other species 55,56], M cells nd sub-epicrin the literture including both decresed 14,53,54] nd dil cells my constitute 60 75% of the left ventriculr enhnced 16,20] pek systolic C ]. Studies on free wll 8]. However, no estimtes exist for rbbit hert. trbecule or ppillry muscles would be using myocr- Also, the contrctility of the myocyte depends criticlly on dium tht is predomintely sub-endocrdil, while in the properties of the myofilments. However, studies on dissocited left ventricle, only pproximtely 15% of the the rbbit infrct model hve filed to revel ltered cells would be of sub-endocrdil origin 55,56]. myofilment C -sensitivity or force production 59]. In summry, the two novel findings of the present study 4.6. Electricl nd mechnicl consequences re: (1) In chronic myocrdil infrct model, sub-endocrdil cells of the left ventricle showed n incresed It is difficult to predict the net effect of differentil mplitude of the C trnsient nd shortened ction chnges in ventriculr ction potentils nd C trnsients potentil durtion. (2) M cells, identified by electrophysio- on the electricl stbility nd mechnicl function of the logicl criteri, showed decresed C trnsient mwhole hert. At physiologicl stimulus rtes ( 3 Hz), the plitude nd prolonged ction potentil durtion. Similr APD90 nd CD50 vlues were similr to norml. As chnges in C trnsient nd ction potentil chrcterisdiscussed bove, the inherent vribility of single cell tics were observed in sub-epicrdil cells. The extent of mesurements my prevent smll consistent chnges from these chnges ws relted to the severity of left ventriculr being observed. The results of this study indicte tht the dysfunction in vivo. Comprble informtion concerning norml endocrdil epicrdil differences in ADP90 would endocrdil-epicrdil differences in humn hert filure is be reduced in hert filure prticulrly t sub-physiologicl not vilble. The mrked regionl differences in rbbit hert rtes. This feture hs been observed in other hert myocrdium described in this study my represent filure models 9], the resulting ltered pttern of trns- distinct stge in hert filure reltively erly (8 weeks) murl repolristion my hve pro-rrhythmic conse- fter the formtion of n infrct nd in the bsence of quences. Among the postulted mechnisms for r- underlying hypertension. rhythmogenesis in hert filure re single cell rrhythmic The primry cuse of reduced intrcellulr C obmechnisms, prticulrly triggered ctivity due to erly or served in humn hert filure nd in most niml models is delyed fter depolristions 57]. These events my be thought to be depressed srcoplsmic reticulum (SR) more frequent in cells with incresed intrcellulr C ]. function 60], lthough vriety of C hndling proteins Bsed on the results presented in this study, the incresed re known to be ffected: in prticulr the N/ C intrcellulr C ] observed in sub-endocrdil cells in exchnger 61]. There is widespred interest in the SR s hert filure my predispose these cells to rrhythmic trget for therpeutic intervention in hert filure 62]. events. In support of this, Pogwizd 58] showed tht the The results of this study suggest tht up-regultion of sub-endocrdil region ws the site of premture ventricu- SERCA2 function my restore crdic contrctility by lr complexes in rbbit model of hert filure; seprtely incresing the mplitude of the C trnsient in M cells Vermeulen et l. 43] demonstrted delyed fter-depolri- nd sub-epicrdil cells. However, this might lso result in stions in surfce cells of ppillry muscles from filing SR C overlod in res (e.g. sub-endocrdium) where herts. The lrger thn norml C trnsients observed in function ws norml or even up-regulted, predisposing filing sub-endocrdil cells in the present study suggest these res to the development of fter-depolristions nd n incresed SR C content. This cn rise from ltered triggered rrhythmis. srcolemm C flux pthwys or n up-regultion of SR function. The significntly higher end distolic C ] observed t high (3 Hz) stimultion rtes in the hert Acknowledgements filure group suggests the former rther thn the ltter cuse. However, the inbility to distinguish significnt The uthors would like to thnk Dr Mrtin Hicks, nd differences in distolic C ] t lower stimulus rtes the technicl ssistnce of Dine Smillie, Greme Deuchr, between the two experimentl groups prevents more Anne Wrd nd Aileen Rnkin of the University of detiled nlysis. Glsgow for their ssistnce in the preprtion of the Previous reports in this model hve indicted tht left niml model. Dr Frncis Burton is thnked for his ventriculr contrctile function is reduced,22]. This comments. This work ws finncilly support by proinitilly ppers difficult to reconcile with the observtion grmme grnt funding from the Medicl Reserch Council. on June 2018

12 408 M.A. McIntosh et l. / Crdiovsculr Reserch 45 (2000) References ] Pye MP, Blck M, Cobbe SM. Comprison of in vivo nd in vitro hemodynmics function in experimentl hert filure use of echocrdiogrphy. Crdiovsc Res 1996;31: ] Dvies CH, Hrding SE, Poole-Wilson PA. Cellulr mechnisms of 22] Ng GA, Cobbe SM, Smith GL. Non-uniform prolongtion of contrctile dysfunction in humn hert filure. Eur Hert J intrcellulr C trnsients recorded from the epicrdil surfce of 1996;17: isolted herts from rbbits with hert filure. Crdiovsc Res 2] Hrt G. Cellulr electrophysiology in crdic hypertrophy nd 1998;37: filure. Crdiovsc Res 1994;28: ] Mhffey KW, Ry TE, Pennock GD, Morkin E, Goldmn S. Left 3] Pogwizd SM, Corr PB. Biochemicl nd electrophysiologicl lterventriculr remodeling in rbbits fter myocrdil infrction. Circutions underlying ventriculr rrhythmis in the filing hert. Eur ltion 1995;91: Hert J 1994;15: ] Litwin SE, Bridge JHB. Enhnced N -C exchnge in the 4] Anvers P, Olivetti G, Cpsso JM. Cellulr bsis of ventriculr infrcted hert. Circ Res 1997;81: remodeling fter myocrdil infrction. Am J Crdiol 1991;68:7D 25] Pye MP, Cobbe SM. Arrhythmogenesis in experimentl models of 16D. hert filure: the role of incresed lod. Crdiovsc Res 5] Cmpbell SE, Gerdes AM. Regionl chnges in myocyte size during 1996;32: the reversl of thyroid-induced crdic hypertrophy. J Mol Cell 26] Burton FL, Cobbe SM. Ventriculr fibrilltion threshold nd locl Crdiol 1988;20: dispersion of refrctoriness in isolted rbbit herts with left 6] Spotnitz HM, Sonnenblick EH, Spiro D. Reltion of ultrstucture to ventriculr dysfunction. J Crdiovsc Electrophysiol 1999 (subfunction in the intct hert: srcomere structure reltive to pressure mitted). volume curves of intct left ventricles of dog nd ct. Circ Res 27] Lict A, Aggrwl R, Robinson RB, Boyden P. Frequency depen- 1966;18: dent effects of Ci trnsients nd cell shortening in myocytes tht 7] Chmunorw JP, O Neill SC. Regionl differences in rest decy nd survive in the infrcted hert. Crdiovsc Res 1997;33: recoveries of contrction nd the clcium trnsient in rbbit ven- 28] Eisner DA, Nichols CG, ONeill SC, Smith GL, Vldeolmillos M. triculr muscle. Pflugers Arch 1995;430: The effects of metbolic inhibition on intrcellulr clcium nd ph 8] Antzelevitch C, Sicouri S, Litovsky SH, Luks A, Krishnn SC, in isolted rt ventriculr cells. J Physiol 1989;411: Diego JM, Ginnt GA, Liu D-W. Heterogeneity within the ventricu- 29] Grynkiewicz G, Poenie M, Tsien RY. A new genertion of C lr wll. Circ Res 1991;69: indictors with gretly improved fluorescence properties. J Biol 9] Brynt SM, Shipsey SJ, Hrt G. Regionl differences in electricl Chem 1985;260: nd mechnicl properties of myocytes from guine-pig herts with 30] Frmpton JE, Orchrd CH, Boyett MR. Distolic, systolic nd mild left ventriculr hypertrophy. Crdiovsc Res 1997;35: srcoplsmic reticulum (C ) during inotropic interventions in 10] Shipsey SJ, Brynt SM, Hrt G. Effects of hypertrophy on regionl isolted rt myocytes. J Physiol 1991;437: ction potentil chrcteristics in rt left ventricle. Circultion 31] Bssni JW, Bssni RA, Bers DM. Clibrtion of Indo-1 nd 1997;96: resting intrcellulr C] i in intct rbbit crdic myocytes. Biophys 11] McIntosh MA, Cobbe SM, Kne KA, Rnkin AC. Action potentil J 1995;68: prolongtion nd potssium currents in left ventriculr myocytes 32] Beuckelmnn DJ, Weir WG. Mechnism of relese of clcium from isolted from hypertrophied rbbit herts. J Mol Cell Crdiol srcoplsmic reticulum of guine-pig crdic cells. J Physiol 1998;30: ;405: ] Nbuer M, Beuckelmnn DJ, Uberfuhr P, Steinbeck G. Regionl 33] Mnor O, Krlin A. A comprtive study of four methods for differences in current density nd rte-dependent properties of the nlysing repeted mesures dt. Stt Med 1996;15: trnsient outwrd current in subepicrdil nd subendocrdil 34] Smith SH, McCslin M, Sreenn C, Bishop SP. Regionl myocyte myocytes of humn left ventricle. Circultion 1996;93: size in two-kidney, one clip renl hypertension. Mol Cell Crdiol 13] Billy P, Benith JP, Mouchoniere M, Vssort G, Lorente P. Regionl 1988;20: ltertion of the trnsient outwrd current in humn left ventriculr 35] Bishop SP, Opril S, Reynolds RH, Drummond JL. Regionl septum during compensted hypertrophy. Circultion myocyte size in normotensive nd spontneously hypertensive rts. 1997;96: Hypertension 1979;1: ] Gwthmey JK, Copels L, McKinnon R et l. Abnorml intrcellu- 36] Gerdes AM. Differences in regionl cpillry distribution nd lr clcium hndling in myocrdium from ptients with end-stge myocyte sizes in norml nd hypertrophic rt herts. Am J Ant hert filure. Circ Res 1987;61: ;156: ] Beuckelmnn DJ, Nbuer M, Erdmnn E. Intrcellulr clcium 37] Olivetti G, Melissri M, Blbi T et l. Myocyte cellulr hypertrophy hndling in isolted ventriculr myocytes from ptients with termi- is responsible for ventriculr remodelling in the hypertrophied hert nl hert filure. Circultion 1992;85: of middle ged individuls in the bsence of crdic filure. 16] Bing OHL, Brooks WW, Conrd CH, Sen S, Perreult CL, Morgn Crdiovsc Res 1994;28: JP. Intrcellulr clcium trnsients in myocrdium from spont- 38] Beltrmi CA, Finto N, Rocco M, Feruglio GA, Puricelli C. The neously hypertensive rts during the trnsition to hert filure. Circ cellulr bsis of dilted crdiomyopthy in humns. J Mol Cell Res 1991;68: Crdiol 1995;27: ] Siri FM, Krueger J, Nordin C, Ming Z, Aronson RS. Depressed 39] Fedid D, Giles WR. Regionl vritions in ction potentils nd intrcellulr clcium trnsients nd contrction in myocytes from trnsient outwrd current in myocytes isolted from rbbit left hypertrophied nd filing guine pig herts. Am J Physiol ventricle. J Physiol 1991;442: ;261:H514 H ] Cook SJ, Chmunorw JP, Lncster MK, O Neill SC. Regionl 18] Annd IS, Liu D, Chugh SS et l. Isolted myocyte contrctile differences in the regultion of intrcellulr sodium nd in ction function is norml in postinfrct remodeled rt hert with systolic potentil configurtion in rbbit left ventricle. Pflugers Arch Eur J dysfunction. Circultion 1997;96: Physiol 1997;433: ] Zugg CE, Wu ST, Lee RJ, Wikmn-Coffelt J, Prmley WW. 41] Litovsky SH, Antzelevitch C. Trnsient outwrd current prominent Intrcellulr C hndling nd vulnerbility to ventriculr fibrill- in cnine ventriculr epicrdium but not endocrdium. Circ Res tion in spontneously hypertensive rts. Hypertension 1997;30: ;62: ] Gibbs CL, Johnson EA. Effects of chnges in frequency of 20] Heller LJ. Augmented ftercontrctions in ppillry muscles from stimultion upon rbbit ventriculr ction potentil. Circ Res rts with crdic hypertrophy. Am J Physiol 1979;237:H649 H ;9: on June 2018