Skip Navigation

Cardiovascular Research 2004 62(1):96-104; doi:10.1016/j.cardiores.2004.01.007
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Chiang, C.-E.
Right arrow Articles by Wang, T.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chiang, C.-E.
Right arrow Articles by Wang, T.-M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Swelling-activated chloride current is activated in guinea pig cardiomyocytes from endotoxic shock

Chern-En Chiang*,a,b, Hsiang-Ning Lukc and Tsui-Min Wangb

aDivision of Cardiology, Taipei Veterans General Hospital and National Yang-Ming University, Taipei, Taiwan
bGraduate Institute of Medical Science, Taipei Medical University, Taipei, Taiwan
cDepartment of Anesthesiology, Chang-Gung Memorial Hospital-Kaohsiung, Taipei, Taiwan

* Corresponding author. Division of Cardiology, Taipei Veterans General Hospital 201, Sec 2, Shih-Pai Road, Taipei 112, Taiwan. Tel.: +886-2-2875-7602; fax: +886-2-2874-5422. Email address: cechiang{at}vghtpe.gov.tw

Received 10 July 2003; revised 26 December 2003; accepted 6 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 3.2. Effects of DIDS...
 4. Discussion
 References
 
Objective: Myocardial swelling occurs during endotoxic shock. The hypothesis that swelling-activated Cl– current (ICl,swell) activates during endotoxic shock was tested. Methods: Endotoxic shock was induced by intravenous lipopolysaccharides (10 mg/kg) in guinea pigs. The effects of ICl,swell blockers on the cardiac action potentials in papillary muscles and on the ICl,swell in single ventricular myocytes were tested. Results: Action potential duration (APD) at 90% of repolarization (APD90) was significantly shortened after 5-h endotoxic shock in guinea pig papillary muscles. ICl,swell blockers, 9-anthracene carboxylic acid (9-AC) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), dose-dependently prolonged the shortened APD90. Inducible nitric oxide synthase (iNOS) inhibitors, L-N6-(1-iminoethyl) lysine (L-NIL) and N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide (1400 W), also prolonged the APD90. Protein kinase C (PKC) activators, 4β-phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-didecanoate (PDD), also prolonged the APD. The addition of glibenclamide (an ATP-sensitive K+ channel blocker) on top of these ICl,swell blockers hastened the recovery of APD90 compared to the use of ICl,swell blockers alone. Whole-cell voltage-clamp study in single ventricular myocytes from endotoxic shock heart disclosed activation of a DIDS- and 9-AC-sensitive current. These currents displayed outward rectification with reversal potentials similar to the calculated Nernst potential for Cl–. The reversal potentials tracked the ECl closely when the Cl– gradient was changed, suggesting that Cl– was the major charged carrier. Conclusions: We have shown for the first time that ICl,swell activates in guinea pig heart in endotoxic shock. The change in this membrane current, together with the activation of ATP-sensitive K+ current, contributes to the electrophysiological derangement in endotoxic shock.

KEYWORDS Guinea pigs; Ion channels; Septic shock


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 3.2. Effects of DIDS...
 4. Discussion
 References
 
The hemodynamic changes in endotoxic shock were characterized by decreased vascular tone and depressed myocardial function [1]. Myocardial dysfunction was mainly caused by increased nitric oxide production and related to shortened action potential duration (APD) [2,3]. The ionic mechanisms responsible for the shortened APD were not fully understood. Decreased L-type Ca2+ channel currents was one of the proposed mechanisms [4]. We recently observed in guinea pig model that after 6-h endotoxic shock, the shortened APD recorded from papillary muscle could persist for more than 1 h in vitro, and glibenclamide could hasten the process of the recovery of APD to the control level, suggesting a role of ATP-sensitive K+ channel in the shortening of APD during endotoxic shock [3]. However, it took about 1 h to achieve the restoration of APD by glibenclamide. It is possible that other ionic mechanisms are involved.

Activation of swelling-activated Cl– current (ICl,swell) contributes to the shortening of APD during cell swelling in guinea pig ventricular myocytes [5], and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) partially antagonized the APD-shortening effect of cell swelling [5]. We hypothesized that ICl,swell activates during endotoxic shock and contributes to APD shortening.

In the present study, we used 9-anthracene carboxylic acid (9-AC) and DIDS to explore the role of ICl,swell in endotoxic shock. We found that during endotoxic shock, ICl,swell was activated in ventricular myocytes from guinea pig and might play a role in the APD-shortening and myocardial dysfunction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 3.2. Effects of DIDS...
 4. Discussion
 References
 
This study conforms to NIH Guidelines for the Care and Use of Laboratory Animals. Throughout the studies, all efforts were taken to minimize animal pain and suffering. Adult Hartley guinea pigs (300–450 g) of either sex were used in this study.

2.1. Lipopolysaccharide-induced endotoxic shock
We used the lipopolysaccharide (LPS)-induced endotoxic shock model in guinea pigs, which has been described previously [3]. In brief, guinea pigs were anesthetized with urethane (1.2 g/kg, intraperitoneal injection). Escherichia coli LPS (serotype 0127:B8) (10 mg/kg) was injected via right femoral vein [3,6]. Sham experiments were performed in the same way as the LPS-treated ones except that LPS was not given.

2.2. Recording of cardiac action potentials
Guinea pigs were sacrificed by cervical dislocation at the fifth hour after injection of LPS. A strand of free-running papillary muscle from left ventricle was dissected, mounted into a 2-ml perfusion chamber, and perfused with Tyrode solution oxygenated with a gas mixture of 3% CO2 and 97% O2 at 37 °C. The composition of normal Tyrode solution was (in mM) NaCl, 137, KCl, 4, CaCl2, 1.8, MgCl2, 0.5, NaH2PO4, 1, NaHCO3, 12, and glucose, 5, with pH titrated to 7.4. Conventional microelectrode technique applied to multicellular preparation from guinea pig papillary muscles was performed [3,7,8]. Borosilicate glass micropipettes were made by a pipette puller (David Kopf, Model 720, Tujunga, CA, USA). The tip resistance of pipette was around 20–40 M{Omega} when filled with 3 M KCl pipette solution. The tissue preparation is electrically driven using bipolar point stimulation. Square-wave electric stimulations with pulse duration of 2 ms and a frequency of 1 Hz were delivered from the stimulator (model S88, Grass, W. Warwick, USA). The resting membrane potential (RMP), Vmax of phase 0 upstroke of action potential (obtained by a differentiator), and action potential duration at 90% repolarization (APD90) were recorded. Data were also recorded by a digital data recorder (model VR-10B, Instrutech, Great Neck, NY, USA) for off-line analysis.

Action potentials were continuously monitored for 60 min without any pharmacological intervention (control) or under perfusion of different concentrations of ICl,swell blockers: 9-AC [9] and DIDS [10], to examine their effects on the action potentials. The role of inducible nitric oxide synthase (iNOS) in the shortening of the APD was also investigated by using specific iNOS inhibitors: L-N6-(1-iminoethyl) lysine (L-NIL) [11] and N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide (1400 W). We also tested the role played by protein kinase C (PKC) activation in the APD shortening in the endotoxic shock by using 4β-phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-didecanoate (PDD) [10,12].

2.3. Voltage-clamp experiments
Enzymatic digestion method was used to isolate single ventricular myocytes from the papillary muscles of guinea pig left ventricle after 5-h endotoxic shock [13,14]. Guinea pigs were killed by cervical dislocation. The heart was immediately excised. After the aorta was cannulated, the heart was mounted onto a Langendorff apparatus. All solutions used for the cell isolation procedure were oxygenated and maintained at 37 °C. The hearts were perfused retrogradely with normal Tyrode solution with the following composition (in mM): NaCl, 137, KCl, 4, CaCl2, 1.8, MgCl2, 0.5, HEPES, 10, and glucose, 5.5 (pH=7.4, titrated with NaOH). Immediately after the blood was washed out, the perfusate was switched to the nominally calcium-free Tyrode solution for 5 min. Subsequently, nominally calcium-free Tyrode solution containing 1 mg/ml collagenase (Class 2, Worthington, NJ, USA) and 0.04 mg/ml protease (type XIV, Sigma, St. Louis, MO, USA) were perfused for 2–10 min. The perfusion solution was then shifted to low-calcium (0.18 mM) enzyme-free Tyrode solution for 5 min. The papillary muscle was dissected into small pieces and gently agitated. The dispersed cells were stored in normal Tyrode solution, and only the quiescent ones with clear striation were used. We performed single-pipette, whole-cell, voltage-clamp technique with a patch clamp amplifier (Axopatch 1-D, Axon, Foster City, USA) to record membrane currents [15,16]. All experiments were performed at room temperature ({approx}22 °C) [9]. Cell capacitance was measured by integrating the capacitive transient evoked by applying a 5-mV hyperpolarizing step (from –40 to –45 mV) [12,16]. The cell capacitance and series resistance were electrically compensated by 60–90%.

The ICl,swell was recorded by stepped voltage-clamp pulses (200 ms) between 80 and –80 mV in 20-mV steps from a holding potential of –40 mV. The effects of different ICl,swell blockers were examined. The modified Tyrode solution for perfusion contains (in mM) NaCl, 130, CsCl, 5.4, MgCl2, 0.5, CdCl2, 1, HEPES, 10, glucose, 5, ouabain, 20 µM, and verapamil, 0.02, with a pH of 7.4 (titrated with CsOH). The pipette solution contains (in mM) CsCl, 110, TEA-Cl, 20, MgATP, 5, EGTA, 20, creatine, 5, and HEPES, 10, with a pH of 7.2 (titrated with CsOH). To test if Cl– is the charged carrier of the current, we replaced part of the CsCl in the pipette solution with Cs-aspartate to measure the reversal potential and to calculate the Nernst potential for Cl– (ECl=RT/Fln[Cl]i/[Cl]o) in some experiments [17]. The bath was ground via a 3 M KCl agar bridge. The final osmolarity of both solutions was between 292 and 296 mOsm/l.

2.4. Drugs and data analyses
E. coli LPS (serotype 0127:B8), 9-AC, DIDS, PMA, PDD, 4{alpha}-PDD, and glibenclamide were purchased from Sigma. L-NIL and 1400 W were purchased from Tocris (Northpoint, Avonmouth, UK). Lipopolysaccharide, DIDS, L-NIL, and 1400 W were dissolved in normal saline, while other agents were dissolved in DMSO. The final concentration of DMSO in the perfusate was <0.1%.

All values are expressed as mean±S.E.M. Two-way ANOVA was used to analyze the experimental data, with one way being between groups and the other way being within group. If significance was found, a post hoc analysis (Newman–Keul's) was used. The difference was considered statistically significant when P value was less than 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
3.2. Effects of DIDS...
 4. Discussion
 References
 
3.1. Effects of 9-AC on the action potentials in endotoxic shock
After 5-h endotoxic shock, the APD90 of LPS-treated guinea pigs was significantly shortened compared to those receiving sham experiments (146.2±2.3 vs. 195.1±1.9 ms, P<0.001). In the following 60-min perfusion of various drugs or normal Tyrode solution (defined as control), the APD90 was continuously monitored for a total of 60 min. Fig. 1A shows the original action potential tracings from guinea pig papillary muscles. The APD90 was progressively prolonged by 9-AC, and the effect was concentration dependent. The summary of the changes by 9-AC was shown in Fig. 1B. The APD90 also prolonged in the control group, but to a much lesser extent than that by 9-AC. The RMP did not changed significantly in the control group (0 min=–86.4±1.2 mV vs. 60 min=–85.7±1.5 mV; P>0.05), nor by 9-AC (for 300 µM, 0 min=–86.2±1.3 mV vs. 60 min=–86.5±1.4 mV; for 1 mM, 0 min=–86.1±1.1 mV vs. 60 min=–86.8±1.3 mV; for 3 mM, 0 min=–86.0±1.3 mV vs. 60 min=–87.2±1.5 mV; all P>0.05). The Vmax was not significantly changed in the control (0 min=208.1±6.8 V/s vs. 60 min=204.2±6.4 V/s; P>0.05), nor in those receiving 9-AC (for 300 µM, 0 min=204.5±5.2 V/s vs. 60 min=209.2±7.6 V/s; for 1 mM, 0 min=202.5±4.8 V/s vs. 60 min=213.2±8.9 V/s; for 3 mM, 0 min=208.2±7.2 V/s vs. 60 min=216.6±9.6 V/s; all P>0.05). There was a trend for the RMP to become more negative, and the Vmax became larger with higher doses of 9-AC.


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Effects of 9-AC on the APD90 of papillary muscles from guinea pigs with endotoxic shock. (A) Original action potential tracings. In control (a), the APD90 slightly prolonged. 9-AC dose-dependently and time-dependently prolonged the APD90 (b–d). There was slight hyperpolarization of RMP by 9-AC. (B) The time courses of the changes in APD90 by 9-AC. n=12, 10, 13, and 16 for control, 300 µM 9-AC, 1 mM 9-AC, and 3 mM 9-AC, respectively. *P<0.05 vs. control; +P<0.01 vs. control; #P<0.001 vs. control.

 

   3.2. Effects of DIDS on the action potentials in endotoxic shock
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 3.2. Effects of DIDS...
4. Discussion
 References
 
DIDS had similar effects as 9-AC. Fig. 2A shows the action potential tracings from guinea pig papillary muscle. DIDS dose-dependently prolonged APD90. Fig. 2B was the summary of the effects by DIDS. The APD90 also prolonged in the control group, but to a much lesser extent than that by DIDS. The RMP did not change significantly in the control group (0 min=–85.6±2.4 mV vs. 60 min=–86.1±2.1 mV; P>0.05), nor by DIDS (for 100 µM, 0 min=–85.8±1.6 mV vs. 60 min=–86.1±2.4 mV; for 300 µM, 0 min=–86.2±1.6 mV vs. 60 min=–86.8±1.5 mV; for 1 mM, 0 min=–86.1±1.7 mV vs. 60 min=–87.7±2.5 mV; all P>0.05). The Vmax was not significantly changed in the control (0 min=201.9±3.8 V/s vs. 60 min=203.1±4.9 V/s; P>0.05), nor in those receiving DIDS (for 100 µM, 0 min=205.8±5.2 V/s vs. 60 min=210.3±6.6 V/s; for 300 µM, 0 min=208.7±5.9 V/s vs. 60 min=217.8±9.4 V/s; for 1 mM, 0 min=207.9±7.3 V/s vs. 60 min=221.7±11.7 V/s; all P>0.05). Similarly, there was a trend for the RMP to become more negative, and the Vmax became larger with higher doses of DIDS.


Figure 2
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effects of DIDS on the APD90 of papillary muscles from guinea pigs with endotoxic shock. (A) Original action potential tracings. In control (a), the APD90 slightly prolonged. DIDS dose-dependently and time-dependently prolonged the APD90 (b–d). There was slight hyperpolarization of RMP by DIDS. (B) The time courses of the changes in APD90 by DIDS. n=12, 14, 13, and 15 for control, 100 µM DIDS, 300 µM DIDS, and 1 mM DIDS, respectively. *P<0.05 vs. control; +P<0.01 vs. control; #P<0.001 vs. control.

 
3.3 The effects of ICl,swell blockers on basal APD90
Fig. 3 shows the effects of 9-AC and DIDS on basal APD90. The baseline value (196.7±1.5 ms) is that before the sham operation. There was no significant change after the sham operation (197.7±2.1 ms). In the following 60-min perfusion with normal Tyrode solution containing ICl,swell blockers: 9-AC and DIDS, the APD90 prolonged to 223.3±4.6 ms (9-AC, 3 mM) and to 224.8±4.6 ms (DIDS, 1 mM), suggesting a basal activation of ICl,swell in guinea pig ventricular myocytes.


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 The effects of ICl,swell blockers on basal APD90. The number of experiment was shown in the parentheses. +P<0.01 vs. sham.

 
3.4. Effects of iNOS inhibitors on the action potentials in endotoxic shock
In order to define the involvement of iNOS in the action potential shortening in endotoxic shock, we used specific iNOS inhibitors, L-NIL and 1400 W, in this study. L-NIL (30 µM) and 1400 W (10 µM) had no effect on the APD90 in the sham group (Fig. 4), suggesting that there is no basal iNOS activity, or that basal iNOS activity did not play a role in the APD modulation. In the papillary muscle from guinea pig after 5-h endotoxic shock, 60-min perfusion of 30 µM L-NIL significantly prolonged the APD90 to a similar level as that in the sham group (Fig. 4). Lower concentration of L-NIL (3 µM) was less effective. Similarly, 1400 W, another specific iNOS inhibitor, concentration-dependently prolonged the APD90. These findings suggest that iNOS activation is involved in the APD shortening in endotoxic shock.


Figure 4
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effects of iNOS inhibitors on the APD90 in endotoxic shock. Specific iNOS inhibitors, L-NIL and 1400 W, had no effect on the APD90 in the sham group. In contrast, L-NIL and 1400 W dose-dependently prolonged APD90 in papillary muscles from endotoxic shock guinea pigs. The number of experiment was shown in the parentheses. *P<0.05 vs. LPS-treated group; +P<0.01 vs. LPS-treated group.

 
3.5. The role of PKC in the APD shortening in endotoxic shock
To assess the role of PKC activation in the APD shortening in endotoxic shock, phorbol esters, including PMA and PDD, were used in the present study. As shown in Fig. 5, 1 µM PMA significantly prolonged the APD90. PDD (1 µM) had similar effect. 4{alpha}-PDD, which has similar chemical structure as PDD but is ineffective in activating PKC, could not prolong APD90. These findings suggest that PKC activators are capable of prolonging APD, possibly through their inhibitory effect on ICl,swell.


Figure 5
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 The role of PKC in APD shortening in endotoxic shock. The number of experiment was shown in the parentheses. +P<0.01 vs. LPS-treated group.

 
3.6 Additive effects of ICl,swell blockers and glibenclamide on the action potentials in endotoxic shock
We previously reported that ATP-sensitive K+ channel current activated during endotoxic shock and contributed partly to the APD-shortening phenomenon [3]. We therefore tested if there is additive effect of blockers of ICl,swell and ATP-sensitive K+ current in reversing the shortening of APD. Fig. 6 shows that the time needed for the APD90 to recover to 190 ms was abbreviated further by the addition of glibenclamide to ICl,swell blockers, suggesting that both ICl,swell and ATP-sensitive K+ channel current contribute to the APD shortening in endotoxic shock.


Figure 6
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Additive effects of ICl,swell blockers and glibenclamide on the action potentials in endotoxic shock. Glibenclamide hastened the recovery of APD90, and the time needed for the APD90 to reach 190 ms is significantly shorter than ICl,swell blockers alone. n=16 and 10 for 3 mM 9-AC and 3 mM 9-AC+100 µM glibenclamide, respectively; n=15 and 11 for 1 mM DIDS and 1 mM DIDS+100 µM glibenclamide, respectively. *P<0.05.

 
3.7 ICl,swell activated in ventricular myocytes in endotoxic shock
The cell capacitance of the isolated ventricular myocytes from guinea pigs undergoing endotoxic shock was larger than those from the sham operation (268.3±10.3 vs. 224.1±8.1 pF, P<0.01; n=26 and 30, respectively). Fig. 7 shows that time-independent currents were recorded in myocytes from endotoxic shock (panel (a)). These currents were sensitive to 1 mM DIDS (panel (b)), and the DIDS-sensitive currents (panel (c)) showed outward rectification (panel (d)). DIDS was more effective in inhibiting the outward current than the inward current. The reversal potential of the DIDS-sensitive currents was –2.2 mV, very close to the calculated ECl (–1.4 mV). When we changed the chloride concentration of the pipette solution, the reversal potentials tracked the ECl, indicating Cl– is the major charged carrier (Fig. 8). Fig. 9 shows that these time-independent currents (panel (a)) induced in myocytes from endotoxic shock were also sensitive to 3 mM 9-AC (panel (b)). The 9-AC-sensitive currents (panel (c)) also demonstrated outward rectification characteristic (panel (d)).


Figure 7
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Original current tracings recorded in ventricular myocytes from endotoxic shock guinea pigs (n=26). (a) Currents were recorded by stepped voltage clamp pulses (200 ms) between 80 and –80 mV in 20-mV steps from a holding potential of –40 mV; (b) residual currents after 1 mM DIDS; (c) DIDS-sensitive currents (a–b) showing a time-independent characteristic; (d) Current–voltage (I–V) curve. The DIDS-sensitive current showed outward rectification. The reversal potential (–2.2 mV) was very close to the calculated ECl (–1.4 mV), suggesting that this DIDS-sensitive current, which was activated in endotoxic shock, was an ICl,swell. {blacksquare}: currents from panel (a); bullet: currents from panel (b); {circ}: currents from panel (c).

 

Figure 8
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 The correlation of the reversal potentials with the ECl. When the Cl– concentrations in the pipette solution were changed, the reversal potentials tracked the ECl closely. The number in the parentheses is the cell number.

 

Figure 9
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 9 Original current tracings recorded in ventricular myocytes from endotoxic shock guinea pigs (n=16). (a) Currents were recorded by stepped voltage clamp pulses (200 ms) between 80 and –80 mV in 20-mV steps from a holding potential of –40 mV; (b) residual currents after 3 mM 9-AC; (c) 9-AC-sensitive currents (a–b) showing a time-independent characteristic; (d) Current–voltage (I–V) curve. The 9-AC-sensitive current showed outward rectification. {blacksquare}: currents from panel (a); bullet: currents from panel (b); {circ}: currents from panel (c).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 3.2. Effects of DIDS...
 4. Discussion
References
 
We have shown for the first time that cardiac ICl,swell activates during endotoxic shock. The change in this membrane current may contribute to the electrophysiological and contractile derangement in endotoxic shock.

Histomorphometrical analysis in canine endotoxic shock revealed myocardial cell swelling and capillary compression [18]. In a porcine model of septic shock, created by intermitted application of E. coli LPS, the earliest lesions were ischemic alterations of the muscle fibers with myocardial swelling [19]. The breakdown of high-energy phosphates and macromolecules increases the osmolarity of cardiac tissue and partly explains the swelling of cardiac myocytes [20]. Similar findings occur in myocardial ischemia and reperfusion [21,22]. In the present study, the cell capacitance of myocytes from endotoxic shock was about 20% larger than that from sham operation, suggesting an increase in cell volume.

ICl,swell is stimulated by an increase in cell volume. Cardiac ICl,swell was initially identified by Tseng [23] and is expressed throughout the heart in many species. The current is time independent over the physiologic voltage range [12,24] and outwardly rectifying with either a physiologic [25] or a symmetric [17] Cl– gradient. ICl,swell reverses near ECl and tracks the Cl– gradient almost ideally. The current, which was activated by endotoxic shock in this study, has several characteristics. They were (1) dependent on Cl– gradient with the reversal potential near ECl, (2) exhibited outward rectification, and (3) were sensitive to ICl,swell blockers. These features match those of ICl,swell described by others [10,23].

At positive membrane potentials, ICl,swell is outwardly rectifying and contributes to enhancement of the plateau phase (phase 2) and the rapid repolarization phase (phase 3) of the action potential and will thus shorten the action potential [5,9]. The shortening of the action potential could be attenuated by ICl,swell blocker, such as DIDS [5]. In single cardiomyocytes isolated from the LPS-treated rabbits, decreased action potential duration was associated with impaired contractile function [26]. From the results in this study, it is possible that ICl,swell becomes a novel therapeutic target in the treatment of endotoxic shock.

At negative membrane potentials, ICl,swell is inwardly rectifying and causes membrane depolarization when activated [5]. In the present study and the previous one [3], we observed a slight but insignificant depolarization of RMP. In fact, in addition to ICl,swell, ATP-sensitive K+ current also activates in endotoxic shock [3]. The latter produces hyperpolarization of RMP, serving to limit the extent of depolarization of resting membrane potential. On the other hand, the co-activation of ATP-sensitive K+ current contributes further to the shortening of APD as shown in Fig. 6.

Activation of ICl,swell is a common response to cardiac stress [10]. ICl,swell is persistently activated in ventricular myocytes from a canine pacing-induced dilated cardiomyopathy model [9,27]. The current is also fully turned on in ventricular myocytes isolated from a rabbit aortic regurgitation model of dilated cardiomyopathy [10] or from a rat aortic constriction model of ventricular hypertrophy [28]. In LPS-treated rats, Cl– conductance increased in hepatocytes, and the voltage dependence and inhibitor specificity of this conductance were similar to that of a swelling-activated Cl– conductance [29]. Previously, we have shown that in guinea pigs undergoing endotoxic shock, the increase in plasma nitrate and tissue cGMP content correlated with the decrement of the APD in papillary muscles [3]. In the present study, we further demonstrated that specific iNOS inhibitors, L-NIL and 1400 W, prolonged the APD. Swelling-activated Cl– current can be modulated by NO and NO donors in rabbit portal vein myocytes through cGMP-dependent and cGMP-independent mechanisms [30]. The detail-controlling mechanisms in guinea pig ventricular myocytes remain to be determined.

It is proposed that ICl,swell is regulated by the balance between PKC-mediated phosphorylation of the channel and its dephosphorylation by serine/threonine protein phosphatases [31]. Inhibition of ICl,swell by PKC activation was observed in guinea pig atrial and ventricular myocytes [32]. In rabbit atrial myocytes, PDD, but not 4{alpha}-PDD, produced strong inhibition of ICl,swell [12]. Furthermore, we found that PKC activators, PMA and PDD, but not 4{alpha}-PDD, could reverse the APD shortening in endotoxic shock, in line with the hypothesis that PKC-mediated phosphorylation can inhibit ICl,swell.

In the present study, we measured the APD90 from papillary muscles by conventional microelectrode technique instead of from single ventricular myocytes by current clamp technique. This is based on the following reasons. First, for a given osmotic perturbation, isolated myocytes swell to a greater extent than do myocytes in intact tissue [20], i.e., 50–60% of predicted for isolated myocytes [33,34] compared to 25–30% for cells in situ [35]. This is because of the space constraints imposed by the relatively inelastic extracellular matrix [35,36] and pericardium. Since the extent of activation of ICl,swell correlates with the extent of cellular swelling [9,20], it is likely that the actual magnitude of ICl,swell activation in intact tissue is less than that from isolated cells. It seems appropriate to measure the APD90 from multicellular preparation such as papillary muscles. Second, the model-simulated action potential changes responding to stretch correlate better with action potential changes in guinea pig papillary muscle than with those from isolated myocytes [37]. For example, the resting APD90 for guinea pig ventricle calculated from modeling is 193 ms [37], very close to what we recorded from papillary muscle. On the other hand, the APD90 recorded by current clamp technique in isolated guinea pig ventricular myocytes ranged from 154 to 748 ms [5,38–40].

In the present study, we have demonstrated that ICl,swell blockers prolonged APD90 in sham group, suggesting a basal activation of ICl,swell. This might explain why the final APD90 after 60-min perfusion of ICl,swell blockers were longer than the averaged value from an intact guinea pig ventricle (about 190 ms when stimulated with 1 Hz) [16]. Indeed, basal activation of ICl,swell has been reported in both atrial and ventricular myocytes [12,23,25]. In addition, ICl,swell is the primary basal Cl– conductance [10].


   Acknowledgements
 
The study is supported, in part, by research grants from the National Science Council (NSC 91-2314-B-075-079) and Taipei Veterans General Hospital (VGH-92-C-242).


   Notes
 
Time for primary review 19 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 3.2. Effects of DIDS...
 4. Discussion
 References
 

  1. Natanson C., Fink M.P., Ballantyne H.K., et al. Gram-negative bacteremia produces both severe systolic and diastolic cardiac dysfunction in a canine model that simulates human septic shock. J. Clin. Invest. (1986) 78:259–270.[Web of Science][Medline]
  2. Balligand J.L., Cannon P.J. Nitric oxide synthases and cardiac muscle. Autocrine and paracrine influences. Arterioscler. Thromb. Vasc. Biol. (1997) 17:1846–1858.[Abstract/Free Full Text]
  3. Chen C.C., Lin Y.C., Chen S.A., et al. Shortening of cardiac action potentials in endotoxic shock in guinea pigs is caused by an increase in nitric oxide activity and activation of the adenosine triphosphate-sensitive potassium channel. Crit. Care Med. (2000) 28:1713–1720.[CrossRef][Web of Science][Medline]
  4. Zhong J., Hwang T.C., Adams H.R., et al. Reduced L-type calcium current in ventricular myocytes from endotoxemic guinea pigs. Am. J. Physiol. (1997) 273:H2312–H2324.[Web of Science][Medline]
  5. Vandenberg J.I., Bett G.C., Powell T. Contribution of a swelling-activated chloride current to changes in the cardiac action potential. Am. J. Physiol. (1997) 273:C541–C547.[Web of Science][Medline]
  6. Ruetten H., Thiemermann C. Effects of tyrphostins and genistein on the circulatory failure and organ dysfunction caused by endotoxin in the rat: a possible role for protein tyrosine kinase. Br. J. Pharmacol. (1997) 122:59–70.[CrossRef][Web of Science][Medline]
  7. Vanhoutte F., Vereecke J., Verbeke N., et al. Stereoselective effects of the enantiomers of bupivacaine on the electrophysiological properties of the guinea-pig papillary muscle. Br. J. Pharmacol. (1991) 103:1275–1281.[Web of Science][Medline]
  8. Groh W.J., Gibson K.J., McAnulty J.H., et al. β-Adrenergic blocking property of d,l-sotalol maintains class III efficacy in guinea pig ventricular muscle after isoproterenol. Circulation (1995) 91:262–264.[Abstract/Free Full Text]
  9. Clemo H.F., Stambler B.S., Baumgarten C.M. Swelling-activated chloride current is persistently activated in ventricular myocytes from dogs with tachycardia-induced congestive heart failure. Circ. Res. (1999) 84:157–165.[Abstract/Free Full Text]
  10. Baumgarten C.M., Clemo H.F. Swelling-activated chloride channels in cardiac physiology and pathophysiology. Prog. Biophys. Mol. Biol. (2003) 82:25–42.[CrossRef][Web of Science][Medline]
  11. Moore W.M., Webber R.K., Jerome G.M., et al. L-N6-(1-iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase. J. Med. Chem. (1994) 37:3886–3888.[CrossRef][Web of Science][Medline]
  12. Duan D., Fermini B., Nattel S. Alpha-adrenergic control of volume-regulated Cl– currents in rabbit atrial myocytes. Characterization of a novel ionic regulatory mechanism. Circ. Res. (1995) 77:379–393.[Abstract/Free Full Text]
  13. Mitra R., Morad M. A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates. Am. J. Physiol. (1985) 249:H1056–H1060.[Medline]
  14. Chiang C.E., Chen S.A., Chang M.S., et al. Genistein directly induces cardiac CFTR chloride current by a tyrosine kinase-independent and protein kinase A-independent pathway in guinea pig ventricular myocytes. Biochem. Biophys. Res. (1997) 235:74–78.[CrossRef]
  15. Hamill O.P., Marty A., Neher E., et al. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. (1981) 391:85–100.[CrossRef][Web of Science][Medline]
  16. Chiang C.E., Luk H.N., Wang T.M., et al. Effects of sildenafil on cardiac repolarization. Cardiovasc. Res. (2002) 55:290–299.[Abstract/Free Full Text]
  17. Vandenberg J.I., Yoshida A., Kirk K., et al. Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology. J. Gen. Physiol. (1994) 104:997–1017.[Abstract/Free Full Text]
  18. Bronsveld W., van Lambalgen A.A., van Velzen D., et al. Myocardial metabolic and morphometric changes during canine endotoxin shock before and after glucose–insulin–potassium. Cardiovasc. Res. (1985) 19:455–464.[Abstract/Free Full Text]
  19. Hauptmann S., Klosterhalfen B., Weis J., et al. Morphology of cardiac muscle in septic shock. Observations with a porcine septic shock model. Virchows Arch. (1995) 426:487–491.[Web of Science][Medline]
  20. Vandenberg J.I., Rees S.A., Wright A.R., et al. Cell swelling and ion transport pathways in cardiac myocytes. Cardiovasc. Res. (1996) 32:85–97.[CrossRef][Web of Science][Medline]
  21. Tranum-Jensen J., Janse M.J., Fiolet W.T., et al. Tissue osmolality, cell swelling, and reperfusion in acute regional myocardial ischemia in the isolated porcine heart. Circ. Res. (1981) 49:364–381.[Free Full Text]
  22. Steenbergen C., Hill M.L., Jennings R.B. Volume regulation and plasma membrane injury in aerobic, anaerobic, and ischemic myocardium in vitro. Effects of osmotic cell swelling on plasma membrane integrity. Circ. Res. (1985) 57:864–875.[Abstract/Free Full Text]
  23. Tseng G.N. Cell swelling increases membrane conductance of canine cardiac cells: evidence for a volume-sensitive Cl channel. Am. J. Physiol. (1992) 262:C1056–C1068.[Web of Science][Medline]
  24. Shuba L.M., Ogura T., McDonald T.F. Kinetic evidence distinguishing volume-sensitive chloride current from other types in guinea-pig ventricular myocytes. J. Physiol. (1996) 491(Pt. 1):69–80.[Abstract/Free Full Text]
  25. Sorota S. Swelling-induced chloride-sensitive current in canine atrial cells revealed by whole-cell patch-clamp method. Circ. Res. (1992) 70:679–687.[Abstract/Free Full Text]
  26. Hung J., Lew W.Y. Cellular mechanisms of endotoxin-induced myocardial depression in rabbits. Circ. Res. (1993) 73:125–134.[Abstract]
  27. Clemo H.F., Stambler B.S., Baumgarten C.M. Persistent activation of a swelling-activated cation current in ventricular myocytes from dogs with tachycardia-induced congestive heart failure. Circ. Res. (1998) 83:147–157.[Abstract/Free Full Text]
  28. Benitah J.P., Gomez A.M., Delgado C., et al. A chloride current component induced by hypertrophy in rat ventricular myocytes. Am. J. Physiol. (1997) 272:H2500–H2506.[Web of Science][Medline]
  29. Meng X.J., Carruth M.W., Weinman S.A. Leukotriene D4 activates a chloride conductance in hepatocytes from lipopolysaccharide-treated rats. J. Clin. Invest. (1997) 99:2915–2922.[Web of Science][Medline]
  30. Ellershaw D.C., Greenwood I.A., Large W.A. Dual modulation of swelling-activated chloride current by NO and NO donors in rabbit portal vein myocytes. J. Physiol. (2000) 528(Pt. 1):15–24.[Abstract/Free Full Text]
  31. Hume J.R., Duan D., Collier M.L., et al. Anion transport in heart. Physiol. Rev. (2000) 80:31–81.[Abstract/Free Full Text]
  32. Duan D., Cowley S., Horowitz B., et al. A serine residue in ClC-3 links phosphorylation–dephosphorylation to chloride channel regulation by cell volume. J. Gen. Physiol. (1999) 113:57–70.[Abstract/Free Full Text]
  33. Drewnowska K., Baumgarten C.M. Regulation of cellular volume in rabbit ventricular myocytes: bumetanide, chlorothiazide, and ouabain. Am. J. Physiol. (1991) 260:C122–C131.[Web of Science][Medline]
  34. Suleymanian M.A., Clemo H.F., Cohen N.M., et al. Stretch-activated channel blockers modulate cell volume in cardiac ventricular myocytes. J. Mol. Cell. Cardiol. (1995) 27:721–728.[Web of Science][Medline]
  35. Pine M.B., Brooks W.W., Nosta J.J., et al. Hydrostatic forces limit swelling of rat ventricular myocardium. Am. J. Physiol. (1981) 241:H740–H747.[Web of Science][Medline]
  36. Page E., Storm S.R. Cat heart muscle in vitro: IX. Cell ion and water contents in anisosmolal solutions. J. Gen. Physiol. (1966) 49:641–653.[Abstract/Free Full Text]
  37. Riemer T.L., Sobie E.A., Tung L. Stretch-induced changes in arrhythmogenesis and excitability in experimentally based heart cell models. Am. J. Physiol. Heart Circ. Physiol. (1998) 275:H431–H442.[Abstract/Free Full Text]
  38. Kocic I., Hirano Y., Hiraoka M. Ionic basis for membrane potential changes induced by hypoosmotic stress in guinea-pig ventricular myocytes. Cardiovasc. Res. (2001) 51:59–70.[Abstract/Free Full Text]
  39. Hume J.R., Uehara A. Ionic basis of the different action potential configurations of single guinea-pig atrial and ventricular myocytes. J. Physiol. (1985) 368:525–544.[Abstract/Free Full Text]
  40. Groh W.J., Gibson K.J., Maylie J.G. Hypotonic-induced stretch counteracts the efficacy of the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Cardiovasc. Res. (1996) 31:237–245.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
E. Grandi, M. Govoni, S. Furini, S. Severi, E. Giordano, A. Santoro, and S. Cavalcanti
Induction of NO synthase 2 in ventricular cardiomyocytes incubated with a conventional bicarbonate dialysis bath
Nephrol. Dial. Transplant., July 1, 2008; 23(7): 2192 - 2197.
[Abstract] [Full Text] [PDF]

Home page
 Eur J Heart FailHome page
A. d. de Tassigny, A. Berdeaux, R. Souktani, P. Henry, and B. Ghaleh
The volume-sensitive chloride channel inhibitors prevent both contractile dysfunction and apoptosis induced by doxorubicin through PI3kinase, Akt and Erk 1/2
Eur J Heart Fail, January 1, 2008; 10(1): 39 - 46.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Chiang, C.-E.
Right arrow Articles by Wang, T.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chiang, C.-E.
Right arrow Articles by Wang, T.-M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?