Cardivascular Research 58 (003) 89 98 www.elsevier.cm/ lcate/ cardires Calcium-activated transient utward chlride current and phase replarizatin f swine ventricular actin ptential a, a b a,b a a Gui-Rng Li *, Xin-Ling Du, Yaw L. Siw, Karmin O, Hung-Fat Tse, Chu-Pak Lau a Institute f Cardivascular Science and Medicine/Department f Medicine, The University f Hng Kng, Hng Kng SAR, China b Department f Pharmaclgy, Faculty f Medicine, The University f Hng Kng, Hng Kng SAR, China Received 7 July 00; accepted 7 December 00 Abstract Objective: It is unknwn whether 4-aminpyridine- (4-AP-) sensitive transient utward K current (I t) and/ r Ca -activated Ca.Cl transient utward Cl current (I r I ) cntribute(s) t phase replarizatin f pig ventricular actin ptential (AP). The purpse f the present study was t determine inic cntributin f the phase replarizatin f AP in pig ventricle. Methds: We used whle-cell patch techniques t recrd APs and membrane currents, and Western immunbltting analysis t detect expressin f It prtein (Kv4. r Kv4.3) in pig ventricular mycytes. Results: A transient utward current (I t) was activated upn deplarizatin vltage steps t between 0 and 60 mv frm 50 mv in pig ventricular cells, and the I was resistant t 4-AP applicatin, but sensitive t the t inhibitin by ryandine (0 mml/ l) and the Ca channel blckade, and the Cl channel blcker 4,49-diisthicyanstilben-,9disulfnic acid (DIDS, 50 mml/ l). The current was diminished by external Cl (Cl ) replacement and shwed a bell-shaped I V relatinship at rm temperature, typical f I. N difference in I was bserved in the reginal cells frm epicardium, midmycardium, and endcardium f left ventricle. APs shwed significant phase and spike and dme in pig ventricular mycytes. The phase and spike and dme f APs were nt affected by 4-AP (3 mml/l), but ablished by replacing Cl and by applicatin f 00 mml/ l DIDS, suggesting I cntributin. Western immunbltting analysis shwed n evidence fr the expressin f 4-AP- sensitive I channel prtein (Kv4. r Kv4.3) in pig ventricular cells. Cnclusin: The results indicate that 4-AP-sensitive I is nt t t expressed, and nly Ca -activated I is present in pig cardiac cells, which cntributes imprtantly t the phase replarizatin f ventricular APs in this species. 003 Eurpean Sciety f Cardilgy. Published by Elsevier Science B.V. All rights reserved. Keywrds: Cl-channel; In channels; K-channel. Intrductin sheep cardiac Purkinje fibers that was decreased by external Cl substitutin []. Subsequently, distinctive 4- Deplarizatin-activated transient utward current (I t) AP-sensitive cmpnent (transient utward K current) has been recgnized in cardiac Purkinje fibers since the and 4-AP-resistant cmpnent (Ca -activated transient 960s []. It was initially believed t be a Cl current, utward chlride current, I Cl.Ca) were described in sheep and subsequently demnstrated in sheep cardiac Purkinje Purkinje fibers [3], and in rabbit and dg cardiac mycytes fibers t be predminantly due t an increase in K [4 6]. The 4-AP-sensitive and 4-AP-resistant cmpnents cnductance. With 4-aminpyridine (4-AP), Kenyn and are ften termed It and I respectively, after Tseng Gibbns firstly identified a 4-AP-resistant cmpnent in and Hffman [7]. It and I play imprtant rles in the phase replarizatin f cardiac actin ptential (AP) [8 0]. *Crrespnding authr. Department f Physilgy L04-55, Labratry Blck Faculty f Medicine Building, The University f Hng Kng, Sassn Rad, Pkfulam, Hng Kng SAR, China. Tel.: 85-89- 830; fax: 85-855-9730. E-mail address: grli@hkucc.hku.hk (G.-R. Li). Experimental studies demnstrated that the distributin f It and I was species-dependent. Bth It and I Time fr primary review 3 days. 0008-6363/03/$ see frnt matter 003 Eurpean Sciety f Cardilgy. Published by Elsevier Science B.V. All rights reserved. di:0.06/ S0008-6363(0)00859-3 Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
90 G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 were fund in cardiac mycytes frm rabbit atrium [4,0],.. Slutins ventricle [6], and Purkinje fibers [], and canine cardiac cells [5,7,], and als in sheep cardiac Purkinje fibers The Tyrde slutin cntained (mml/ l): NaCl 36, [,3]. Only It was detected in mycardial cells in species KCl 5.4, MgCl.0, CaCl.8, NaHPO4 0.33, glucse 0 including rat [3], ferret [4], and humans [9]. Earlier and HEPES 5; ph adjusted t 7.4 with NaOH. The high- 4 studies demnstrated I in calf Purkinje fibers [5], and K strage medium cntained (mml/ l): KCl 0, KH PO elephant seal atrial fibers [6]. Hwever, neither It nr 0, glucse 0, K-glutamate 70, taurine 0, EGTA 0.5, I was present in guinea pig ventricular mycytes [7]. mannitl 0, albumin 0.%; ph adjusted t 7. with KOH. Pig is a species cmmnly used fr the study f The pipette slutin cntained (mml/l): KCl 0, K- cardivascular diseases [8]. Hwever, cellular elec- aspartate 0, MgCl.0, HEPES 0, EGTA 0.05, GTP trphysilgy f the pig heart has nt been well dcu- 0., Na -phsphcreatine 5.0, Mg -ATP 5.0; ph adjusted mented. Althugh a recent reprt described a significant t 7. with KOH. K in the pipette and bath slutin was phase replarizatin f ventricular APs in pig heart [9], replaced by equimlar Cs when K -free cnditins were inic mechanisms are unknwn. The present study was used (specified). Fr determinatin f I Cl.Ca (r I ), BaCl designed t study inic cntributin t the phase (0.5 mml/ l) was added r external K was mitted t replarizatin f ventricular APs in pig heart. The results inhibit inward rectifier K current (I K). The experiments demnstrated that I cntributed imprtantly t the phase were cnducted at rm temperature ( 8C fr I recrd and spike and dme f ventricular APs in pig heart, ing) r 36 8C (fr AP recrding, and Q determinatin f 0 different frm thse bserved in dg and humans [8,9,]. I )..3. Data acquisitin and analysis. Methds The whle cell patch-clamp technique was used... Cardiac cell preparatin Brsilicate glass electrdes (. mm O.D.) were pulled with a Brwn Flaming puller (mdel P-97), and had tip resistances f t 3 MV when filled with pipette slutin. Left ventricular tissues frm islated hearts were b- The tip ptentials were cmpensated befre the pipette tained via a left thractmy after pigs (45 80 kg, with the tuched the cell. A 3 M KCl agar bridge was used as Guide fr Care and Use f Labratry Animals f NIH reference electrde. After a giga-seal was btained, the cell publicatin n 85-3) were anesthetized with phentbarbimembrane was ruptured by gentle suctin t establish the tal (IV, 30 mg/ kg). The heart was initially placed in whle-cell cnfiguratin. Liquid junctin ptentials after xygenated Tyrde slutin, the left anterir descending membrane rupture between the external and pipette slucrnary artery was cannulated. Ventricular cells were tins (0.460.3 mv) were nt crrected except fr the enzymatically islated with a prcedure described previrecrding f AP. Data were acquired by the use f an usly [0]. Briefly, a free wall f the left anterir ventricle EPC-9 amplifier (Heka Elektrnik, Germany). Cmmand ( 30350 mm) was remved alng with the crnary artery pulses were generated by a digital-t-analg cnverter branch irrigating it. The free wall was perfused with cntrlled by Pulse sftware (Heka Elektrnik). Membrane xygenated, nminally Ca -free Tyrde slutin fr 0 capacity and series resistance was electrnically cmpen- 30 min, and the slutin was then changed t ne sated by the sftware. Recrdings were stred n the hard cntaining 00 300 U/ ml cllagenase (CLS II, Wrthdisk f an IBM cmpatible cmputer. ingtn Bichemical, Freehld, NJ) fr 90 50 min. Ventricular cells were separated frm the sftened tissue. The rat ventricular mycytes were prepared with a Langendrff.4. Western immunbltting analysis methd as previusly described []. All the cells were placed in a high-k strage slutin (see Slutins) and Membrane fractins were islated frm dissciated gently triturated with a Pasteur pipette. The islated cells single rat and pig ventricular mycytes using standard were kept in the medium at least h (at rm temperature) labratry prcedure. The Kv channel prtein was debefre use. termined by Western immunbltting analysis. Briefly, 40 A small aliqut f the slutin cntaining the islated mg f ttal membrane prteins frm the pig and rat cells was placed in an pen perfusin chamber ( ml) cardimycytes were separated by electrphresis n an munted n the stage f an inverted micrscpe. Cells 8% SDS plyacrylamide gel. Prteins in the gel were then were allwed t adhere t the bttm f the dish fr 5 t transferred t a nitrcellulse membrane, which was 0 min, and were then superfused at t 3 ml/ min with subsequently incubated with anti-kv4. r anti-kv4.3 Tyrde slutin. Only quiescent rd-shaped cells shwing antibdy (Almne Labs, Israel). Alkaline phsphataseclear crss-striatins were used. cnjugated secndary antibdy and the apprpriate sub- Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 9 strate were used t develp the membranes. Bands crre- ( 8C) in different pig ventricular cells with lw pipette spnding t Kv prteins were identified accrding t their EGTA (0.05 mml/ l) and nrmal bath Tyrde slutin relative mbility n the gel. The specific Kv prtein bands cntaining 0.5 mml/l Ba (t blck I K). An It was were analyzed with a gel dcumentatin system (Bi-Rad slwly activated after an initial inward ICa when vltage Gel Dc 000 and Multi-Analysis versin.). steps were psitive t 0 mv under cntrl cnditins Nnlinear curve-fitting prgrams (SigmaPlt, SPSS (Fig. A, D). The I was nt affected by the applicatin f t Science, Chicag, IL) and/ r Pulsefit sftware (Heka) 3 mml/ l 4-AP (Fig. B), and n 4-AP-sensitive I was t were used t perfrm curve-fitting prcedures. Paired and bserved (Fig. C). Hwever, the I was decreased by the t unpaired Student s t tests were used as apprpriate t applicatin f 0 mml/ l ryandine t inhibit Ca release evaluate the statistical significance f differences between frm sarcplasmic reticulum (SR) (Fig. E), and tw grup means, and analysis f variance (ANOVA) was ryandine-sensitive It was significant (Fig. F). Similar used fr multiple grups. Values f P,0.05 were cnsid- results were btained in a ttal f 5 cells fr each grup. ered t indicate statistical significance. Results are pre- These bservatins suggest that the It is nt 4-AP-sensi- sented as the mean6s.e. tive I, and may be Ca -activated I. 3.. Effect f blcking L-type Ca current n I t 3. Results If the It is Ca -activated I, it wuld be inhibited by 3.. It in pig ventricular cells the blckade f L-type Ca current (I Ca), and the current vltage (I V ) relatinship wuld be bell-shaped. T Fig. displays It tracings recrded at rm temperature study if the It wuld be decreased by blcking I Ca, Cd t Fig.. Transient utward current (I t) in pig ventricular mycytes. A. It was slwly activated after an inward ICa elicited by 00 ms vltage steps t between 40 and 60 mv frm 50 mv as shwn in the inset f E. B. Applicatin f 3 mml/l 4-AP did nt significantly affect the current. C. 4-AP-sensitive cmpnent btained by digital subtractin f currents befre and after applicatin 3 mml/ l 4-AP, and n 4-AP-sensitive It was bserved. D. It was recrded in anther cell with the same prtcl. E. The It was significantly inhibited by applicatin f 0 mml/l ryandine fr 0 min, indicating that the It is intracellular Ca -dependent. F. Ryandine-sensitive It was btained by digital subtractin f currents befre and after the additin f 0 mml/l ryandine. Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
9 G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 t was applied in the bath slutin. Fig. A illustrates I 3.3. Effect f lwering Cl r DIDS n I current elicited by the vltage prtcl shwn in the inset f Fig. B in cntrl. The It was fully inhibited by the Previus studies have demnstrated that I is charged applicatin f 00 mml/l Cd (Fig. B). Similar results by Cl in [4]. T further determine if Ca -activated I were btained in a ttal f 0 cells. Fig. C shws the I V was carried by Cl in pig ventricular cells, Cl was relatinship f mean values f time-dependent peak It reduced t frm 47 mml/ l by substituting bath NaCl under cntrl cnditins, and in the presence f Cd with equimlar Na-aspartate. Fig. 3 displays membrane (n5). The I V relatin curve shwed bell-shaped currents elicited by 00 ms vltage steps t 0, 0, and under cntrl cnditins, typical f I, indicating that 40 mv frm 50 mv befre and after Cl replacement. 4-AP-resistant, ryandine- and ICa-sensitive current is I was significant upn the deplarizatin ptentials Ca -activated I. The linear I V relatin curve in the under cntrl cnditins (Fig. 3A), and disappeared with presence f Cd indicated a nn-specific catin r Cl substitutin (Fig. 3B). Fig. 3C shws Cl -sensitive leakage current. I at crrespnding vltages btained by digital subtrac- tin f currents befre and after Cl replacement. A similar result was btained in a ttal f 9 cells. The change f liquid junctin ptential was very limited with the reference electrde f 3 M KCl agar bridge, and did nt significantly affect experimental data. The result indicates that the I is carried by Cl in pig ventricular cells. The density f Cl -sensitive I was.660.4,.560.6, and.960.7 pa/ pf at 0, 0, and 40 mv, respectively (P,0.0 amng ptentials). I is sensitive t the anin channel blcker DIDS []. We therefre bserved the effect f DIDS n I in pig ventricular cells. Fig. 3D illustrates I elicited by 00 ms vltage steps t 0, 30 and 50 mv frm 50 mv, and Fig. 3E shws the I inhibited by the additin f 50 mm DIDS in bath slutin. Fig. 3F displays DIDS-sensitive I current tracings btained at crrespnding vltages by digital subtractin f current befre and after DIDS applicatin. Similar results were btained in a ttal f 0 cells. DIDS-sensitive I was.60.5,.760.7, and.560.8 pa/pf at 0, 30, and 50 mv, respectively (P,0.0 amng ptentials). 3.4. Temperature-dependence f I It is well knwn that temperature may significantly affect the amplitude and kinetics f membrane currents [9,3]. Temperature-dependence f I was therefre determined under K -free cnditins at rm temperature ( 8C) and 36 8C with 00 ms steps t between 40 and 60 mv frm 50 mv. T directly analyze the temperature effect, we studied I at 8C and then repeated the measurements at 36 8C in the same cells t determine the temperature dependence f I amplitude in pig ventricular cells. The temperature cefficient (Q 0) [9,3] fr I density was calculated by using the equatin Q0 5 0(A A )/[A (T T )], where A and A are I Fig.. Inhibitin f It by blckade f Ca current. A. An initial inward density at different temperatures T and T. ICa was fllwed by the It recrded in a representative cell with the Fig. 4 displays the values f amplitude and density f prtcl shwn in the inset f B. B. Blckade f L-type Ca current I at 8C and 36 8C. Representative recrdings are with 00 mml/ l ablished the I t. C. I V relatinship f peak I t, shwing bell-shaped curve, typical f Ca -activated I (s), and linear shwn in Fig. 4A (at 8C) and Fig. 4B (at 36 8C) in the I V relatin in the presence f Cd measured frm the zer (arrw), same cell. I amplitude substantially increased at 36 8C. indicating the nnspecific catin r leakage current (d) (n5). I V relatinships f mean values f I density at and Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 93 Fig. 3. Effects f lwering Cl r applying DIDs n I.A.I was activated after inward ICa by 00 ms vltage steps t 0, 0 and 40 mv frm 50 mv in a representative cell. B. Substitutin f Cl with aspartate ablished I, nly inward ICa was seen. C. Cl -sensitive I btained by digital subtractin f currents befre and after Cl replacement. D. I was activated after inward ICa by 00 ms vltage steps t 0, 30, and 50 mv frm 50 mv in anther mycyte. E. I was significantly diminished by the applicatin f 50 mml/l DIDS in bath slutin fr 0 min, and nly ICa was bserved in the same mycyte. F. DIDS-sensitive I btained by digital subtractin f currents befre and after applicatin f DIDS at crrespnding vltages. 36 8C are shwn in Fig. 4C. Interestingly, the typical further suggests the lack f expressin f It in pig bell-shaped I V relatin f I at 8C became linear as ventricle. Hwever, the prminent phase and significant bath temperature was increased t 36 8C, and density f I spike-and-dme were diminished by lwering Cl (right significantly augmented at 36 8C. At 40 mv, I was panel f Fig. 5B), r by the applicatin f the Cl channel increased frm.60.3 t 5.360.9 pa/ pf (increased by blcker DIDS (00 mml/ l, right panel f Fig. 5C), 5%, n57, P,0.0). The calculated Q0 was.4560.05. indicating that the I cntributes t the phase replarizatin f ventricular APs. Similar results were btained in 3.5. Cntributin f I t phase replarizatin f 5 8 cells fr the different experimental grups. cardiac APs 3.6. I and phase f the AP in reginal mycytes The previus study frm Stamkvicva et al. [9] demnstrated that ventricular APs f pig hearts shwed a It and the phase f AP shw a significant regin- significant fast replarizatin phase (phase ). T study the dependent distributin in mycytes frm dg and human cntributin f I t the phase replarizatin f pig cardiac ventricles [8,9]. T study if I and the phase f ventricular APs, we recrded APs in current clamp mde. AP are regin-dependent in pig cardiac ventricle, epicar- Fig. 5 illustrates transmembrane APs recrded at Hz in dial, midmycardial, and endcardial mycytes were disscells frm pig left ventricular epicardium under cnditins ciated frm pig ventricular free wall with a prcedure as f nrmal Tyrde slutin at 36 8C, shwing a significant previusly described [9]. Vltage-dependent I was re- phase and spike and dme cnfiguratin in 3 different crded at 36 8C with the prtcl as in Fig. 4, while APs cells (left panels f Fig. 5A, 5B, and 5C). Fig. 5A displays were recrded with prtcl as in Fig. 5. I shwed linear that the phase and spike and dme were nt affected by I V relatinship, and n significant reginal difference was the applicatin f 3 mml/l 4-AP (right panel), which bserved in the current density (Fig. 6A). At 40 mv, I Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
94 G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 Fig. 5. Phase replarizatin f APs and I. A. APs recrded in a ventricular cell in current clamp mde under cntrl cnditins (left) and in the presence f 3 mml/l 4-AP (right). 4-AP did nt affect phase f the AP. B. APs recrded in a ventricular cell under cntrl cnditins Fig. 4. Temperature-dependent I. A. I recrded at rm temperature (left) and after replacing Cl (right). Phase f the AP was ablished by f 8C with the prtcl shwn in the inset. B.I was substantially Cl substitutin, indicating I cntributin. C. APs recrded in a augmented by increasing bath temperature t 36 8C in the same cell. C. ventricular cell under cntrl cnditins (left) and in the presence f 00 I V relatinships f I at 8C (s) and 36 8C (d). The bell-shaped mml/l DIDS. Phase and spike and dme f the AP were diminished I V curve f I became linear, and the current was significantly by DIDS. increased at 0 and 60 mv by the elevatin f bath temperature (n56, *P,0.05, **P,0.0 vs. 8C). dial cells. Representative recrdings are shwn in Fig. 6B. Similar results were btained in 6 t 8 cells in each grup. was 4.60.6, 3.960.8, and 3.460.6 pa/ pf in epicardial (n5) midmycardial (n50), and endcardial (n5) 3.7. Western immunbltting analysis f Kv4. and cells (P5NS). Kv4.3 The three reginal mycytes shwed similar AP mrphlgy with a prminent phase. Mst f the cells had It is believed that It is mlecularly ensemble t clned significant spike and dme. The phase and/ r spike Kv4. and Kv4.3 channels [4]. T further study whether and dme f APs were insensitive t 3 mml/ l 4-AP (data It is expressed in pig ventricular mycytes, we de- nt shwn), but diminished by the applicatin f 00 termined the presence f the channel prteins, i.e. Kv4. mml/ l DIDS in epicardial, midmycardial, and endcar- and Kv4.3 with Western immunbltting technique. We Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 95 Fig. 7. Lack f mlecular evidence fr It expressin in pig ventricular cells. A. N It was recrded under cnditins f blcking Ca current in pig ventricular cell. B. Significant It was recrded under identical Fig. 6. I and APs in reginal mycytes. A. I V relatin curves f cnditins in a rat ventricular cell. C. Immunbltting shwed that the I t channel prteins Kv4. and Kv4.3 were present in rat ventricular cells, but nt in pig ventricular cells. vltage-dependent I recrded at 36 8C with the prtcl as in Fig. 4 in mycytes islated frm epicardium (Epi, d, n5,), midmycardium (M, s, n50), and endcardium (End,., n5) f pig left ventricular wall. B. Actin ptentials recrded with the prtcl as in Fig. 5 in Epi, and 6 rat ventricular mycytes. Fig. 7C shws that the K M, and End mycytes shwed significant phase and spike and dme under cntrl cnditins (arrws, left panels), but the phase and spike channel prteins detected by specific antibdies. It was and dme f APs were diminished by the applicatin f 00 mml/l significant fr the K channel prteins specific against DIDS fr 8 min (arrws, right panels). Kv4. and Kv4.3 in the rat heart. In cntrast, these K channel prteins were either minimal r undetectable by the antibdy against Kv4. r Kv4.3 in the pig heart. The simultaneusly determined I, and Kv4. and Kv4.3 in rat result is cnsistent with electrphysilgical measurement, t ventricular mycytes as psitive cntrl under identical shwing that pig ventricle des nt express It channel. cnditins. N I was bserved in pig ventricular cells t under cnditins f 5 mml/l EGTA in pipette slutin (t buffer intracellular Ca ), and 00 mml/ l Cd (t blck 4. Discussin I Ca) and 0.5 mml/l Ba (t blck I K) in bath slutin (Fig. 7A). Hwever, significant 4-AP sensitive It was In the present study we have fund a significant It in bserved in rat ventricular cells under the identical cn- pig ventricular mycytes. This I is insensitive t the t ditins (Fig. 7B). Similar results were btained in pig applicatin f 4-AP, but sensitive t the blckade f I r Ca Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
96 G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 Ca release frm SR, inhibited by DIDS r Cl replace- temperature alteratin (Fig. 4). It is well knwn that I is ment, and shws a bell-shaped I V relatinship at rm ligand (i.e. intracellular Ca ) gated. The increase f I temperature, typical f Ca -activated I. The I plays reflects an elevatin f intracellular Ca cntent, while an imprtant rle in the phase replarizatin f pig the intracellular Ca is invlved in multiple mechanisms ventricular APs. including inward I Ca, reverse mde f Na Ca ex- changer, Ca -induced Ca release [,3], and a vlt- 4.. Cmparisn with previus reprts f pig cardiac age-sensitive Ca release mechanism [3]. The increased electrphysilgy I (by 5%) and the linear I V relatinship at 36 8C may be related t the enhanced activity f these mechanisms. Pig is a cmmnly used species in studies n cardiac diseases, e.g. ischemia, heart failure and/ r arrhythmia, 4.3. Distributin f cardiac It and I etc. [8]. In additin, the pig heart is being studied fr xentransplantatin [5]. Hwever, studies n cardiac It and I play imprtant rles in the phase replari- electrphysilgy were limited t AP recrding in islated zatin f the cardiac APs [8 0]. Hwever, the distribuhearts [6] r papillary muscles [7], and t study changes tin f It and I is species-dependent. The species with in APs and Ca current during transitin t heart failure expressin f bth It and I in their cardiac mycytes in islated cells [8]. Characterizatin f pig ventricular are rabbit [4,6], canine [5,7,], and sheep []. Only It is AP prperties was nly studied with transmural ventricular detected in mycardial cells in species including rat [3], slices [9] befre Stankvicva et al. [9]. Stankvicva ferret [4], and humans [9]. I is demnstrated in calf and clleagues [9] characterized in detail AP prperties in Purkinje fibers [5] and elephant seal atrial fibers [6]. The islated ventricular mycytes f pig heart, and demnpresent bservatin has demnstrated that It is lack f strated that M cells are present in pig ventricles. Als, the expressin, and nly I is present in pig ventricular data frm this grup revealed that significant phase mycytes. replarizatin f APs crrelated significantly with the J It plays an imprtant rle in phase replarizatin f wave f ECG, while the M cells with lngest AP duratin cardiac cells in species including humans [9]. Hwever, in are related t U wave frmatin f ECG in this species cardiac mycytes with bth It and I, the cntributin [9]. T date, there is limited infrmatin regarding inic f either It t the phase replarizatin f cardiac APs is current characterizatin and distributin f pig cardiac species-dependent. In dg, It cntributes mre t phase mycytes. A recent reprt described the rapid and slw and spike and dme f ventricular AP than I [], and cmpnents (IKr and I Ks) f delayed rectifier K current in is respnsible fr the frmatin f J wave f ECG [3]. pig sin-atrial cells [30]. In the present study, we have Blckade f It significantly diminished the phase and demnstrated fr the first time the nn-expressin f 4-AP- spike and dme, whereas inhibitin f I slightly sensitive I t, and the presence f Ca -activated I in pig affected the shape f the AP []. It is believed that I ventricular cells. The I cntributes imprtantly t the t shapes cardiac AP mrphlgy and duratin [0,]. In phase replarizatin f ventricular APs in this species. rabbit, hwever, I seems t cntribute mre t the replarizatin f cardiac AP due t the inactivatin f I 4.. Cmparisn with previus reprt f I in ther t at nrmal heart rates [33,34]. The present bservatin has species demnstrated that nly I is present, and is respnsible fr the phase and spike and dme f APs in pig 4-AP-resistant, Ca -activated I was initially assumed ventricular mycytes. N reginal difference in I was t be charged by K [3,7] and subsequently was demnbserved in cells frm specific regins: epicardium, strated t be carried by Cl in [4,]. The current shws midmycardium, epicardium f left ventricular wall. Blcthe characteristics with bell shaped I V relatinship at kade f I significantly diminished phase and spike rm temperature, sensitivity t the replacement f Cl, t and dme f cardiac APs. Therefre, I wuld cntribute the applicatin f anin channel blckers, and t the t the J waves bserved n the ECG in this species [9]. blckade f Ca release frm SR r ICa in rabbit and dg Neither It nr I is present in guinea pig cardiac cardiac cells [4,5]. The present bservatin has demnmycytes, and s n significant phase replarizatin f strated that pig ventricular I is insensitive t 4-AP (Fig. cardiac AP is bserved in this species [7]. ), inhibited by the applicatin f DIDS, ryandine r Cd, and ablished by replacing Cl, and exhibited the characteristic f bell-shaped I-V relatinship at 8C, 4.4. Ptential limitatins cnsistent with thse seen in ther species [4,5]. We als bserved that the bell-shaped I V relatin curve became The present bservatin fcused n determining I t linear at 36 8C, similar t the bservatin in rabbit ven- and/r I t study the inic mechanisms f the phase tricular cells [3]. The present study shwed that the Q replarizatin f pig ventricular APs. The transient ut- 0 t.ir f I was.45, indicating that I is very sensitive t the ward K current with inward rectificatin (I ) bserved Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 97 in dg and guinea pig cardiac cells [0,] was nt References determined. Althugh the cntributin f It.ir t early replarizatin f AP is limited in cardiac cells [0,], [] Dudel J, Peper K, Rudel R, Trautwein W. The dynamic chlride further experimental studies are required t clarify cellular cmpnent f membrane current in Purkinje fibers. Pflugers Arch electrphysilgy in this species. In additin, ther rep- Gesamte Physil Menschen Tiere 967;95:97. [] Kenyn JL, Gibbns WR. 4-Aminpyridine and the early utward larizatin currents, e.g. IKr and I Ks, shuld be characterized current f sheep cardiac Purkinje fibers. J Gen Physil 979;73:39 in the future in detail in the ventricle f this species. On 57. the ther hand, Ca transient was nt studied in the [3] Crabeuf E, Carmeliet E. Existence f tw transient utward present bservatin. Hwever, crrelatin f Ca tran- currents in sheep cardiac Purkinje fibers. Pflugers Arch sient t I was well studied in cardiac mycytes frm 98;39:35 359. ther species, e.g. rabbit, dg, and calf [,6,3]. [4] Zygmunt AC, Gibbns WR. Calcium-activated chlride current in rabbit ventricular mycytes. Circ Res 99;68:44 437. In view f undetectable It in pig ventricular mycytes, [5] Zygmunt AC. Intracellular calcium activates a chlride current in ne may reasnably questin whether it might be a result canine ventricular mycytes. Am J Physil 994;67:H984 f the experimental cnditins (e.g. cell islatin). T H995. exclude this pssibility we have determined Kv4. and [6] Hiraka M, Kawan S. Calcium-sensitive and insensitive transient Kv4.3 expressin in pig ventricular cells by Western utward current in rabbit ventricular mycytes. J Physil immunbltting analysis, in parallel with that in rat 989;40:87. [7] Tseng GN, Hffman BF. Tw cmpnents f transient utward ventricular cells under identical cnditins. Significant I t, current in canine ventricular mycytes. Circ Res 989;64:633 647. and Kv4. and Kv4.3 were bserved in rat ventricular [8] Liu DW, Gintant GA, Antzelevitch C. Inic bases fr electrcells, but n evidence fr Kv4. r Kv4.3 was bserved physilgical distinctins amng epicardial, midmycardial, and (Fig. 7), cnsistent with the electrphysilgical bserva- endcardial mycytes frm the free wall f the canine left ventricle. tin. Circ Res 993;7:67 687. [9] Li GR, Feng J, Yue L, Carrier M. Transmural hetergeneity f actin ptentials and It in mycytes islated frm the human right 4.5. Ptential significance ventricle. Am J Physil 998;75:H369 H377. [0] Hiraka M, Kawan S, Hiran Y, Furukawa T. Rle f cardiac chlride currents in changes in actin ptential characteristics and As described abve, pig is a cmmnly-used species in arrhythmias. Cardivasc Res 998;40:3 33. i [Ca ] i-dependent chlride current in single Purkinje cells frm anatmy f pig heart are clse t thse f human heart; rabbit heart. J Physil 993;468:64 667. ntwithstanding sme differences [35], it is being studied [] Zygmunt AC, Rbitelle DC, Eddlestne GT. It dictates behavir f fr xentransplantatin [5], Althugh the inic mecha- ICl(Ca) during early replarizatin f canine ventricle. Am J Physil studies n cardiac diseases [8]. The size and crnary [] Sipid KR, Callewaert G, Carmeliet E. [Ca ] transients and nisms fr replarizatin f ventricular APs f pig heart is 997;73:H096 H06. nt cmpletely clarified frm present study, ur results [3] Crabeuf E, Culmbe A, Derubaix E, Hatem S, Mercadier JJ. Transient utward ptassium current and replarizatin f cardiac demnstrate clearly the cntributin f I t phase cells. Bull Acad Natl Med 998;8:35 333. replarizatin f ventricular APs, and the nn-expressin [4] Campbell DL, Rasmussn RL, Qu Y, Strauss HC. The calciumf I t, which are different frm thse bserved in human independent transient utward ptassium current in islated ferret heart [9]. These imprtant differences shuld be taken int right ventricular mycytes. I. Basic characterizatin and kinetic cnsideratin when extraplating data frm pig studies n analysis. J Gen Physil 993;0:57 60. [5] Siegelbaum SA, Tsien RW. Calcium-activated transient utward electrical remdeling, assessing pharmaclgical efficacy current in calf cardiac Purkinje fibres. J Physil 980;99:485 506. fr treatment study, and/ r evaluating the pig heart fr [6] Maylie J, Mrad M. A transient utward current related t calcium xentransplantatin. release and develpment f tensin in elephant seal atrial fibres. J In summary, we have demnstrated fr the first time the Physil 984;357:67 9. lack f expressin f I, and nly I is present in pig [7] Sipid KR, Callewaert G, Prciatti F, Vereecke J, Carmeliet E. t [Ca ] i-dependent membrane currents in guinea-pig ventricular cells ventricular cells. I cntributes imprtantly t the phase in the absence f Na/ Ca exchange. Pflugers Arch 995;430:87 and spike and dme f APs in this species. 878. [8] Verduw PD, Wlffenbuttel BH. The effect f feldipine n ventricular fibrillatin after crnary artery ligatin in the anaesthetized pig. Br J Pharmacl 983;79:6 8. Acknwledgements [9] Stankvicva T, Szilard M, De S I, Sipid KR. M cells and transmural hetergeneity f actin ptential cnfiguratin in mycytes frm the left ventricular wall f the pig heart. Cardivasc The study was supprted in part by the grants frm Res 000;45:95 960. Research Grant Cuncil f Hng Kng (7338/0M), and [0] Li GR, Sun H, Nattel S. Characterizatin f a transient utward K frm Institute f Cardivascular Science and Medicine, current with inward rectificatin in canine ventricular mycytes. Am J Physil 998;74:C577 C585. HKU. The authrs thank Prfessr TM Wng fr his [] Li GR, Yang B, Sun H, Baumgarten CM. Existence f a transient supprt thrughut the study, and Ms Haiying Sun fr the utward K current in guinea pig cardiac mycytes. Am J Physil data analysis and excellent technical supprt. 000;79:H30 H38. Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07
98 G.-R. Li et al. / Cardivascular Research 58 (003) 89 98 [] Srta S. Insights int the structure, distributin and functin f the [9] Rdriguez-Sinvas A, Cinca J, Tapias A, Armadans L, Tresanchez cardiac chlride channels. Cardivasc Res 999;4:36 376. M, Sler-Sler J. Lack f evidence f M-cells in prcine left [3] Kiysue T, Arita M, Muramatsu H, Spindler AJ, Nble D. Inic ventricular mycardium. Cardivasc Res 997;33:307 33. mechanisms f actin ptential prlngatin at lw temperature in [30] On K, Shibata S, Iijima T. Prperties f the delayed rectifier guinea-pig ventricular mycytes. J Physil 993;468:85 06. ptassium current in prcine sin-atrial nde cells. J Physil [4] Tseng GN. Mlecular structure f cardiac It channels: Kv4., 000;54:5 6. Kv4.3, and ther pssibilities? Cardivasc Res 999;4:6 8. [3] Kawan S, Hirayama Y, Hiraka M. Activatin mechanism f [5] Lambrigts D, Sachs DH, Cper DK. Discrdant rgan xentrans- Ca -sensitive transient utward current in rabbit ventricular plantatin in primates: wrld experience and current status. Trans- mycytes. J Physil 995;486:593 604. plantatin 998;66:547 56. [3] Ferrier GR, Hwlett SE. Cardiac excitatin cntractin cupling: [6] Janse MJ. Electrphysilgical effects f mycardial ischaemia. rle f membrane ptential in regulatin f cntractin. Am J Relatinship with early ventricular arrhythmias. Eur Heart J 986;7 Physil 00;80:H98 H944. Suppl A:35 43. [33] Fermini B, Wang Z, Duan D, Nattel S. Differences in rate depen- [7] Berman ND, Wang LY, Ahmed A. The cellular electrpharmaclgy dence f transient utward current in rabbit and human atrium. Am J f mexiletine cmbined with stall in prcine papillary muscle and Physil 99;63:H747 H754. Purkinje fibre. I. Alteratin f mexiletine-induced Vmax depressin. [34] Wang Z, Fermini B, Feng J, Nattel S. Rle f chlride currents in Can J Cardil 990;6:46 4. replarizing rabbit atrial mycytes. Am J Physil 995;68:H99 [8] Mukherjee R, Hewett KW, Walker JD, Basler CG, Spinale FG. H00. Changes in L-type calcium channel abundance and functin during [35] Crick SJ, Sheppard MN, H SY, Gebstein L, Andersn RH. the transitin t pacing-induced cngestive heart failure. Cardivasc Anatmy f the pig heart: cmparisns with nrmal human cardiac Res 998;37:43 444. structure. J Anat 998;93:05 9. Dwnladed frm https://academic.up.cm/cardivascres/article-abstract/58//89/95738 n 0 December 07