Received 2000-04-16 Accepted 2000-06-25

This work was supported by the National Natural Science Foundation of China (No.39470269)

Corresponding author. Tel: 021-54231880. E-mail: Qixy@hotmail.com

Acta Physiologica Sinica

Oct. 2000, 52 (5), 360¡«364

A study on the electrophysiological heterogeneity of rabbit ventricular myocytesthe effect of ischemia on action potentials and potassium currents

QI Xiao-Yanª³ª³, SHI Wei-Bing1, WANG Hai-Hong1, ZHANG Zhi-Xiong, XU You-Qiu1

(Department of Physiology, Shanghai University of Traditional Chinese Medicine, Shanghai 200032;

1Department of Physiology, Shanghai Second Medical University, Shanghai 200025)

Abstract: With the whole-cell variant patch-clamp technique, action potentials (AP) and outward potassium currents of rabbit ventricular myocytes isolated from subendocardium and subepicardium were recorded and their changes were observed under normal and ischemia conditions. The results showed that (1) under normal condition, there were differences in the AP figures between ventricular subendocardial and subepicardial myocytes. Action potentials recorded from subepicardial myocytes had shorter action potential duration (APD) and a notch between phases 1 and 2, compared with those of subendocardial myocytes. The resting potential had no significant difference between these two populations of the action myocytes; (2) under ischemia condition, the notch of action potentials of subepicardial myocytes disappeared and the APD was shortened even more, compared with that of subendocardial myocytes; (3) under normal condition, the density of steady-state outward potassium currents of subepicardial myocytes was significantly greater than that of subendocardial myocytes; (4) under ischemia condition, the increase of steady-state outward potassium currents of subepicardial myocytes was greater than that of subendocardial myocytes. Glybenclamide could partly reverse the above changes. It is suggested that the increase of steady-state outward potassium currents during ischemia is mainly due to the opening of IK-ATP channels as a result of the deficiency of intracellular ATP caused by ischemia.

Key words: ventricular myocytes; electrophysiological heterogeneity; action potential; ischemia; outward potassium current

ÍÃÐÄÊÒ¼¡Ï¸°ûµçÉúÀíÒìÖÊÐÔÑо¿È±Ñª¶Ô¶¯×÷µçλºÍ¼ØÁ÷µÄÓ°Ïì*

ÆîСÑળª³, ʩμ±ò1, Íôº£ºê1, ÕÅÖ¾ÐÛ, ÐìÓÐÇï1

£¨ÉϺ£ÖÐÒ½Ò©´óѧÉúÀí½ÌÑÐÊÒ£¬ ÉϺ£ 200032£»

1ÉϺ£µÚ¶þÒ½¿Æ´óѧÉúÀí½ÌÑÐÊÒ£¬ ÉϺ£ 200025)

ÕªÒª: ʵÑéÓÃȫϸ°ûĤƬóé¼¼Êõ, ¹Û²ìÕý³£¼°È±ÑªÌõ¼þÏÂ, ÍÃÐÄÄÚĤÏÂÐÄÊÒ¼¡Ï¸°ûÓëÐÄÍâĤÏÂÐÄÊÒ¼¡Ï¸°ûµÄ¶¯×÷µçλºÍÎÈ̬ÍâÏò¼ØÁ÷¼°Æä±ä»¯¡£ ½á¹ûÏÔʾ: (1)Õý³£Ìõ¼þÏÂ, ÐÄÍâĤÏÂÐÄÊÒ¼¡Ï¸°ûÓëÐÄÄÚĤÏÂÐÄÊÒ¼¡Ï¸°û¶¯×÷µçλÐÎ̬ÓвîÒì, ÐÄÍâĤÏÂÐÄÊÒ¼¡Ï¸°û¶¯×÷µçλʱ³Ì(APD)½Ï¶Ì, ¸´¼«1ÆÚºóÓÐÃ÷ÏÔµÄÇм£, ¶¯×÷µçλÐÎ̬ÊÇ¡°·æºÍÔ²¶¥¡±, ¶øÐÄÄÚĤÏÂÐÄÊÒ¼¡Ï¸°û APD½Ï³¤, ²¢ÇÒûÓÐÉÏÊö¶¯×÷µçλÐÎ̬ÌØÕ÷¡£ÕâÁ½Ààϸ°û¾²Ï¢µçλÎÞ²îÒì¡£(2)ÔÚȱѪÌõ¼þÏÂ, ÐÄÍâĤϵÄÐÄÊÒ¼¡Ï¸°û¶¯×÷µçλ¸´¼«1ÆÚºóÇм£Ïûʧ, ÇÒAPDËõ¶Ì³Ì¶ÈÃ÷ÏÔ´óÓÚÐÄÄÚĤϵÄÐÄÊÒ¼¡Ï¸°û¡£(3)ÔÚÕý³£Ìõ¼þÏÂ, ÐÄÍâĤÏÂÐÄÊÒ¼¡Ï¸°ûÎÈ̬ÍâÏò¼ØÁ÷ÃܶÈÏÔÖø´óÓÚÐÄÄÚĤÏÂÐÄÊÒ¼¡Ï¸°û¡£(4)ÔÚȱѪÌõ¼þÏÂ, ÐÄÍâĤÏÂÐÄÊÒ¼¡Ï¸°ûµÄÎÈ̬ÍâÏò¼ØÁ÷µÄÔö¼Ó³¬¹ýÐÄÄÚĤϵÄÐÄÊÒ¼¡Ï¸°û, ÓÃÓŽµÌÇ¿ÉÒÔ²¿·ÖÄæתÉÏÊö±ä»¯¡£ÊµÑé½á¹ûÌáʾ: Ôö¼ÓµÄÎÈ̬ÍâÏò¼ØÁ÷´ó²¿·ÖÊÇÓÉÓÚȱѪÔì³Éϸ°ûÄÚATPȱ·¦, ÖÂʹ IK-ATP ͨµÀ¿ª·Å, ¼ØÍâÁ÷¡£

¹Ø¼ü´Ê: ÐÄÊÒ¼¡Ï¸°û; µçÉúÀíÒìÖÊÐÔ; ¶¯×÷µçλ; ȱѪ; ÍâÏò¼ØÁ÷

ѧ¿Æ·ÖÀàºÅ: Q463; Q424

Although the diversity of electrophysiological activities of mammalian ventricular myocytes has long been recognized, a systematic study of such diversification in different regions of the heart and their ionic basis is lacking. Recent studies have delineated several electrophysiological differences between subepicardial and subendocardial myocytes isolated from canine and feline heart[1¡«4]. By means of standard microelectrode and patch clamp techniques, it was demonstrated that subepicardium has shorter action potential duration and notch after phase 1 repolarization, showing the ¡°spike and dome¡±pattern[5]. It has been reported that the difference in action potential configuration and duration was due to the differences of variant ionic currents as transient outward potassium current, L-type calcium current, delayed rectifier potassium current, ATP-sensitive potassium current and ect. Previous experiments have also shown that the reaction of the two populations of myocytes to simulated ischemia was different[6,7]. The electrophysiological activity differences were most prominent in canine ventricular myocytes, and less in feline ventricular myocytes. However, the electrophysiological heterogeneity of rabbit ventricular myocytes needs to be further investigated. Whole-cell variant patch clamp techniques were used in the present investigation to compare the differences in action potential and repolarizing potassium current in subepicardial and subendocardial myocytes of rabbit ventricle under normal and simulated ischemic conditions.

1MATERIALS AND METHODS

1.1Solution and chemicals The following solutions were prepared: high potassium Tyrode solutions (in mmol/L): NaCl 137.0, KCl 27, CaCl2 1.8, MgCl2 1.0, NaHCO3 23.8, NaH2PO4 0.4, glucose 50, gassed with 95% O2 plus 5% CO2, pH 7.4. Normal Tyrode solution: NaCl 137.0, KCl 5.4, CaCl2 1.8, MgCl2 1.0, NaHCO3 23.8, NaH2PO4 0.4, glucose10, gassed with 95% O2 plus 5% CO2, pH 7.4. Simulated ischemia solution: NaCl 117.0, KCl 5.4, CaCl2 1.8, MgCl2 1.0, NaHCO3 3.8, NaH2PO4 0.9, sodium lactate 20, gassed with 95% N2 plus 5% CO2, pH 6.8. Action potential recording pipette solution: KCl 120, MgCl2 1.0, EGTA 10, HEPES 10, Na2ATP 10, pH 7.2 (KOH). Outward potassium current recording pipette solution: KCl 140, MgCl2 0.5, EGTA 10, HEPES 10, Na2ATP 5, pH 7.2 (KOH).

All the chemicals including collagenase IA, protease ¢ú¢ô, glybenclamide, EGTA, HEPES, Na2ATP, and verapamil were purchased from Sigma.

1.2Cell isolation Isolation of single subepicardial and subendocardial myocytes from the left ventricle of New Zealand rabbit was performed as perviously reported[7]. Rabbits of either sex (1.5¡«2.0 kg) were anesthetized with sodium pentobarbital (3 ml/kg, 1%) and anticoagulated with heparin (500 U/kg). The heart was excised quickly and placed in high potassium Tyrode solution at 4¡æ. The aorta was cannulated and the heart was retrogradely perfused on a Langendroff apparatus at 37¡æ. All of the perfusion solutions were equlibrated with 95% O2-5% CO2. To isolate single ventricular myocytes, the heart was purfused in turn with normal Tyrode for 10 min, with normal calcium-free Tyrode solution for 10 min, the same calcium-free Tyrode solution supplemented with 0.03% collagenase for 8¡«10 min, the same enzyme solution containing 0.008% protease ¢ú¢ô for 8¡«10 min and last with low calcium Tyrode solution (0.18 mmol/L CaCl2) for 10 min. Single myocytes were obtained from subepicardial and subendocardial myocardium after removing a thin layer muscular tissue. Firstly, the removed tissue was minced and gently agitated with low calcium Tyrode solution. The solution was filtered through a 200-¦Ìm nylon mesh, resuspended in the Tyrode¡äs solution in which the calcium concentration gradually increased to 1.8 mmol/L. The cells were stored in normal Tyrode solution at room temperature. Rod-shaped ventricular myocytes with clear striations were selected for experiments.

1.3Whole-cell patch clamp recording Myocytes were placed in a 1-ml chamber on the stage of an inverted microscope (Nikon). The chamber was continuously perfused with test solution at a speed of 2 ml/min at 37¡æ. Membrane current and action potential (AP) were recorded with whole-cell patch-clamp techniques (patch-clamp amplifier, Axopatch 1-C, Axon Instrument Co., USA). Micropipettes were pulled by a two-step vertical puller (PB-7, Narishige, Tokyo, Japan), which had a tip resistance of 2¡«4 M¦¸ when filled with pipette solution. After the whole-cell configuration was achieved, AP was recorded in current clamp mode and membrane current recorded in voltage clamp mode. The experimental protocol and data acquisition were performed with Pclamp 5.5 software (Clampex 5.5.0 and Clampfit £¶.0, Axon Instrument Co. USA) running on a personal computer.

1.4Statistics Statistical analysis was performed by Student¡äs t test for paired and group comparison. All results were expressed as mean¡ÀSE. P<0.05 was considered significant.

2RESULTS

2.1Characteristics of action potential

The action potential configuration was observed. At the same stimulation frequency (1 Hz), a notch of the subepicardial myocyte action potential appeared between phase 1 and phase 2, the configuration showed ¡°spike and dome¡±and a shorter action potential duration was compared with that of subendocardial myocytes (n=5, P<0.05) (Fig.1). After perfusing with simulated ischemia solution for 20 min, the notch which appeared after phase 1 repolarization of subepicardial myocytes disappeared, its action potential duration APD90 was shortened by 64.3¡À3.1% (n=5, P<0.005) and the APD90 of subendocardial myocytes shortened by 24.4¡À3.1%(n=5, P<0.01) (Fig.2). The resting membrane potential was not affected by perfusing with simulated ischemia solution (Table 1).

Fig.1.Representative recording of AP from subendocardial (A) and subepicardial (B) myocyte.

Fig.2. 2 is a representative record of AP from subendocardial (A) and subepicardial (B) myocytes after perfusion with simulated ischemia solution for 20 min, while 1 and 3 is a record of control and after washout for 30 min.

Table¡¡£±.Effect of ischemia on AP (n=5, mean¡ÀSE)

¡¼£Â£È£Ä£Æ£Ç4£¬£×£Ë7ZQ1£¬£×£Ë4,WK19£¬WK1,WK19W¡½[] Time

(min)[] Subendocardical myocytes (Endo)RP (mV) [] APD90 (ms)[][]Subepicardial myocytes¡¡(Epi)RP (mV) [] APD90 (ms) Control [] 20[] -81.3¡À1.5[]797.3¡À6.9 [][] -84.6¡À3.4[] 754.0¡À10.6*Ischemia []5 [] -81.3¡À1.3 [] 695.5¡À29.1* [][] -81.8¡À1.9 [] 604.0¡À31.9**+[] 10 [] -82.2¡À1.5 [] 667.1¡À26.8**[][] -79.8¡À3.5 [] 420.0¡À19.3**++ [] 15 [] -82.9¡À1.9 [] 641.2¡À22.9**[][] -81.7¡À4.6[] 333.0¡À10.6***++ [] 20 [] -82.6¡À1.6[] 602.7¡À20.8** [][] -82.2¡À5.2 [] 291.0¡À15.5***+++ Washout [] 30 [] -82.9¡À2.2 [] 776.0¡À11.0 [] [] -80.9¡À3.6 [] 454.0¡À24.9 *, paired comparison. P<0.05, *P<0.01, **P<0.005 vs control. +, group comparison. +P<0.05, ++P<0.01, +++P<0.005 vs subendocardial myocytes.

2.2Characteristics of outward repolarzing potassium current and steady-state current-voltage relation

The outward potassium currents were evoked by 400 ms step depolarization between -40 mV and +40 mV from a holding potential of -80 mV. A 100 ms prepulse to -40 mV was used to inactivate sodium channels. Verapamil (10-6 mol/L) was added to the perfusion solution to block L-type calcium channel. Under control condition outward repolarzing potassium currents of the subepicardial and subendocardial myocytes were observed and compared with each other. It was found that the steady-state outward repolarizing potassium current density of subepicardial myocytes was significantly greater than that of subendocardial myocytes (n=8, P<0.005) at +40 mV membrane potential. After perfusing with simulated ischemia solution for 20 min, the steady-state outward re-

Fig.3.Steady-state potassium outward current from subendocardial (A) and subepicardial (B) myocytes after perfusion with simulated ischemia solution for 30 min.

polarizing potassium current increased in both groups. In subendocardial myocytes, their density increased from control value 5.8¡À1.2 pA/pF to 7.7¡À1.4 pA/pF (n=8, P<0.01) and those of subepicardial myocytes increased from 7.3¡À0.9 pA/pF to 11.7¡À0.3 pA/pF (n=8, P<0.005). The increase of steady-state outward potassium current was much more prominent in subepicardial myocytes (n=7, P<0.005). The I-V curve position of subepicardial myocytes was higher than that of subendocardial myocytes. At different test potentials, the density of outward potassium current was significantly different in these two populations of ventricular myocytes. After perfusing with simulated ischemia solution for 20 min, the outward potassium current occurred from -30 mV to +40 mV in the two populations of ventricular myocytes, and the magnitude of outward potassium current was greater than that of control. The configuration of I-V curve was similar to that of the control (Figs. 3,4). But the change in the potassium current of subepicardial myocytes was more prominent than that of subendocardial myocytes (n=8, P<0.005).

Fig.4.Plot of the relationship between the mean steady-state potassium outward currents of the subendocardial (Endo) and subepicardial (Epi) myocytes after perfusion with simulated ischemia solution for 20 min. P<0.05, **P<0.005 vs control.

3DISCUSSION

In our experiments, it was observed that the configuration of the action potential was different in the two populations of myocytes. Compared with that of subendocardial myocytes, the action potential of subepicardial myocytes showed briefer APD and a notch between phase 1 and phase 2. It is well known that the length of APD is mainly dependent on L-type calcium current and delayed rectifier potassium current. The transient outward current also has great influence on APD. Action potential data suggest that the differences in the electrophysiological characteristics of subendocardial and subepicardial myocytes may be due to those three ionic currents. In the present study, the steady-state outward potassium current was observed. The density of the steady-state outward potassium current of subepicardial myocytes was significantly larger than that of subendocardial myocytes. Therefore, the explanation of the difference between the APD of the two populations of myocytes is that a larger repolarizing potassium current made the APD shorter in subepicardial myocytes than in subendocardial myocytes.

In simulated ischemia condition, the APD shortened in both myocytes, and the shortening was much prominent in subepicardial myocytes. The changes in steady-state outward potassium current were the sum of the changes of many potassium currents. One candidate is the ATP-dependent potassium current (IK-ATP). Under hypoxia and ischemia conditions, the IK-ATP channel will open due to the deficiency of intercellular ATP. But the activation of IK-ATP channel did not affect cardiac electrophysiology in normal condition. Using the specific IK-ATP channel blocker, it has been demonstrated that the increase of steady-state outward potassium current in our experiments is mainly due to the opening of IK-ATP channel. Glybenclamide (0.3 ¦Ìmol/L) could partly reverse the above changes. It is interesting that our preliminary observation showed the blocking effect of glybenclamide on the increasing outward potassium current was more prominent during ischemia in subepicardial ventricular myocytes compared with that in subendocardial ventricular myocytes. It suggests that the sensitivity of IK-ATP channel to ischemia in subepicardial myocytes was higher than that of subendocardium. The different sensitivity of the channel to [ATP]i contributes to the different degrees of APD shortening during ischemia.

During ischemia, there were some other factors affecting outward potassium current besides ATP sensitive potassium current. The inhibiting metabolism of cardiac myocytes causes high PCO2 and low pH that induces intercellular acidosis[8]. At the same time, potassium channel is activated by fatty acid and arachidonic acid in ventricular myocytes[9,10]. KNa channel can be activated by Na+-K+ pump suppression[11].

In summary, the outward potassium current of ventricular myocytes appeared to increase causing the shortening of APD. Our experiments demonstrate that subepicardial myocytes are more sensitive to ischemia than supendocardial myocytes. These differences suggest that ischemia induced electrophysiological inhomogeneities may facilitate reentrant arrthythmia. However, since only one kind of repolarizing outward potassium current has been studied in this paper, it should be noted that other ionic current may be involved in the difference in the sensitivity of subepicardial and sudendocardial myocytes to ischemia.

REFERENCES

[1]Litovsky SH, Antzelevitch C. Transient outward current prominent in canine ventricular epicardium but not endocardium. Circ Res, 1988, 62: 116¡«126.

[£²]Tseng GN, Hoffman BF. Two components of transient outward current in canine ventricular myocytes.¡¡Circ Res, 1989, 64: 633¡«647.

[£³]Fedida D, Giles WR. Regional variations in action potentials and transient outward current in myocytes isolated from rabbit left ventricular. J Physiol, 1991, 442: 191¡«209.

[£´]Antzelevitch C, Sicouri S, Litovsky SH et al. Heterogeneity within the ventricular wall-electrophysiology and pharmacology of epicardial, endocardial and M cell. Circ Res, 1991, 69: 1427¡«1449.

[£µ]Furukawa T, Myerburg RJ, Furukawa N et al. Differences in transient outward current of feline endocardial and epicardial myocytes. Circ Res, 1990, 67: 1287¡«1291.

[£¶]Liu DW, Gintant GA, Antzelevitch C. Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle. Circ Res, 1993, 72: 671¡«687.

[£·]Furukawa T, Kimura S, Furukawa N et al. Role of cardiac ATP-regulated potassium channels in different responses of endocardial and epicardial cells to ischemia. Circ Res, 1991, 68: 1693¡«1702.

[£¸]Cascio WE, Yan GX, Kleber AG. Early changes in extracellular potassium in ischemic rabbit myocardiumThe role of extracellular carbon dioxide accumulation and diffusion. Circ Res, 1992, 70: 409¡«422.

[£¹]Kim D, Clapham DE. Potassium channels in cardiac cells activated by arachidonic acid and phospholipids. Science, 1989, 224: 1174¡«1179.

[1£°]Carmeliet E. Cardiac ionic currents and acute ischemia: from channels to arrhythmia. Physiol Rev, 1999, 79: 917¡«1017.

[£±1]Kameyama M, Kakei M, Shibasaki T et al. Intracellular Na+ activates a K+ channel in mammalian cardiac cells. Nature, 1984, 309 (24): 354¡«356.