© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Reduced swelling-activated Cl– current densities in hypertrophied ventricular myocytes of rabbits with heart failure*
aDepartment of Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
bExperimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
* Corresponding author. Tel.: +31-20-566-4670; fax: +31-20-691-9319 m.m.vanborren{at}amc.uva.nl
Received 12 January 2001; accepted 9 October 2001
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Objective: Hypertrophied myocytes of failing hearts have prolonged action potential durations. It is unknown how the swelling-activated Cl– current (ICl,swell) affects the abnormal AP configuration. Methods: We studied ICl,swell in ventricular myocytes isolated from failing and age-matched normal rabbit hearts. We applied whole-cell patch-clamp methodology and activated ICl,swell by lowering tonicity of the superfusate. Results: Neither with ruptured-patch nor with amphotericin B perforated-patch, whole-cell clamp we found ICl,swell active under isotonic conditions in either the normal or the hypertrophied failing heart (HFH) myocytes. ICl,swell caused AP shortening and resting membrane potential (Vm) depolarization in an osmotic gradient-dependent fashion. However, in the HFH myocytes swelling-induced AP changes were significantly smaller, even though the cells underwent the same relative change in planar cell surface area. Voltage-clamp experiments revealed that in HFH myocytes ICl,swell current density was
50% reduced. Conclusion: Reduced ICl,swell densities in HFH myocytes cause limited AP shortening and Vm depolarization upon swelling of the cells.
KEYWORDS Cl-channel; Heart failure; Hypertrophy; Myocytes
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Heart cells subjected to excessive workloads undergo typical morphological and electrophysiological changes. Prolongation of the action potential (AP) is frequently observed in hypertrophied myocytes of failing hearts. As reviewed by Nabauer et al. [1], this is due to a reduction of a number of different K+ conductances. Because of this, AP configuration of hypertrophied cells may be expected to be more susceptible to membrane stretch-induced currents, among which is the swelling-activated Cl– current (ICl,swell). Moreover, because failing heart cells are chronically subjected to membrane stretch, density and properties of ICl,swell may be changed. As yet, two studies have addressed this issue. Clemo et al. [2] showed that in myocytes isolated from tachycardia-induced, failing dog hearts, ICl,swell was nearly maximally activated under isotonic conditions. Furthermore, they reported a 40% increase in ICl,swell current density. Bénitah et al. [3] showed the activity of time-independent Cl– current in rat ventricular myocytes isolated from pressure overload-induced, hypertrophied hearts, again under isotonic conditions. These observations lend credit to the hypothesis that a persistently activated ICl,swell of increased magnitude is present in hypertrophied ventricular myocytes of failing hearts. We aimed to test this hypothesis in our combined pressure and volume overload rabbit model of cardiac failure. Furthermore, the question as to how ICl,swell influences the abnormal AP configuration of hypertrophied cells of failing hearts remains to be answered. In view of the altered K+ current densities, effects of ICl,swell on the various AP parameters are difficult to predict. In this paper we also address this issue.
Here we report that ICl,swell caused AP shortening and Vm depolarization in an osmotic gradient-dependent fashion in both control myocytes and cells isolated from rabbits with heart failure. In the latter group, ICl,swell current density was significantly reduced and not active under isotonic conditions.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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2.1. Cell preparation
Cardiac failure was induced in eight New Zealand–White male rabbits by combined volume and pressure overload as described by Vermeulen et al. [4]. In short, in a first operation the aortic valve was mechanically damaged, whereas in a second operation 3 weeks thereafter, the abdominal aorta was partially ligated. Three or 9 months after this procedure the animals were sacrificed and the hearts excised. In the five rabbits that were sacrificed after 3 months, the abdominal aorta diameter was reduced to a greater degree and the aortic valve damaged more extensively, as compared to the three rabbits that stayed in the trial for 9 months. By the time the animals were sacrificed both groups had developed the same degree of heart failure (see below). Left ventricular myocytes were isolated by directly perfusing the coronary arteries with enzyme containing solutions [4]. Myocytes from normal hearts were enzymatically isolated by retrogradely perfusing the aorta, essentially as described by Tytgat [5]. Single cells were stored in Tyrode's solution at room temperature and were used within 8 h. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
2.2. Solutions
Compositions of the solutions used are listed in Table 1. To isolate ICl,swell in our voltage clamp experiments we replaced all cations from bath and pipette solutions by Mg2+ (solution 2) and NMDG+ (solution 4), respectively. By lowering the sucrose concentration of solution 2, its tonicity could be decreased while keeping the ionic strength similar to that of normal Tyrode's solution (solution 1). The solutions used to record action potentials were based on a normal Tyrode's solution in which the NaCl concentration was lowered to 55 mM and to which various amounts of sucrose were added to modify the tonicity (solution 3) [6]. The current-clamp experiments were conducted with a high K+, low Cl– pipette solution (solution 5). We verified osmolarity of all salines with a freezing point depression type osmometer (Knauer, Germany). Osmotic gradients (T) are expressed as the ratio of the osmolarity of the bathing solution relative to that of the pipette solution.
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2.3. Recording conditions, data acquisition and analysis
Rod-shaped myocytes having smooth surfaces with clear cross-striations were selected for electrophysiological measurements. Cells were superfused with pre-warmed solutions (35–37°C). Patch pipettes (2–5 M
) were pulled from borosilicate glass and heat polished. I–V relationships of the swelling-induced current density were obtained by subtracting the currents recorded under hypotonic conditions from the currents recorded under isotonic conditions. Experiments designed to investigate the presence of a DIDS-sensitive current under isotonic conditions were performed using both the perforated-patch and ruptured-patch whole-cell clamp configurations. The perforated-patch whole-cell clamp configuration was obtained by adding 52 µM amphotericin B to the pipette solution. A 1000x concentrated stock of amphotericin B dissolved in DMSO was prepared freshly at the day of the experiment and protected from bright light. Under ruptured-patch whole-cell clamp conditions, swelling might raise the hydrostatic pressure inside the myocyte that may provide a driving force for the movement of cell water into the patch pipette. To assess this possibility we swelled the cells while using patch pipettes filled with salines to which 0.5 mM of fluorescent probe carboxy-seminaphthorhodafluor-1 (SNARF, Molecular Probes, Oregon, USA) was added. During and after swelling, the fluorescent signals emanating from the sarcoplasm increased dramatically and remained high during the experiment (data not shown). We infer from this observation that the raise in hydrostatic pressure inside swollen cells, if any, is not enough to cause net flow of cell water into the pipette. We also conclude that during swelling part of the water that flows into the sarcoplasm comes from the pipette. Nevertheless, to limit water flow from the cell interior into the pipette we clamped the suction tube.
Voltage- and current clamp data were acquired and analyzed off-line with the software package pClamp6 (Axon Instruments, Foster City, CA). The signals were filtered online with a cut-off frequency of 2 kHz and digitized at 5 kHz. We monitored development of ICl,swell by repetitively stepping the pipette potential every 3 s from a holding potential of –80 to +40 mV for 50 ms. AP were elicited at a rate of 1 Hz by current pulses of 3 ms duration, that were applied via the patch pipette. Action potential duration (APD) was measured at 90% repolarization (APD90). Voltage-clamp data were corrected for both liquid-junction potential and voltage drop across the access resistance of the pipette to the whole-cell membrane. The data were normalized for cell size by dividing by the membrane capacitance, Cm.
2.4. Determination of planar cell surface area
To quantify changes in cell shape we recorded images of the myocytes with a CCD camera and stored them on videotape for off-line analysis. Increase in planar cell surface area was taken as a measure of cell swelling. To determine cell surface area we used the software package UTHSCSA Image Tool (San Antonio, TX, USA). The increase in planar cell surface area is expressed as percentage change relative to control.
2.5. Statistics
Results are expressed as mean±standard error of the mean (S.E.M.). Microsoft Excel® software was used to conduct statistical analyses. Action potential parameters were averaged from data obtained from ten consecutive action potentials. Two sets of data were considered significantly different if the P value of the (paired) Student's t-test or ANOVA was <0.05. The capital ‘N’ represents the number of hearts used whereas the lower case ‘n’ represents the number of cells measured.
2.6. Nine and 3 months combined pressure and volume overload causes heart failure
The rabbits whose hearts were subjected to 3 months ‘severe’ combined pressure and volume overload or for 9 months ‘moderate’ combined pressure and volume overload, were considered hemodynamically failing because in addition to gallop rhythm and ascites, firstly, their wet heart weight relative to body weight doubled, 4.9±0.6 (n=8) versus 2.4±0.3 g/kg (n=5). Secondly, their wet lung weight relative to body weight became 1.5 times higher, 3.9±0.7 versus 2.6±0.1 g/kg. Finally, their left ventricular end diastolic pressure was 6 times higher than in control animals, 19±2.7 versus 3.2±0.7 mmHg. Consistent with the gross anatomy, Cm of the hypertrophied failing heart (HFH) myocytes was more than doubled, 373±21 (n=44) versus 166±7 pF (n=73, P<10–4). Pooling of results limits the variability inherent to surgically-induced hypertrophic heart failure models. We justify pooling of results obtained with the ‘3 months HFH myocytes’ with the ‘9 months HFH myocytes’ because we observed no significant differences in the aforementioned indices of circulatory failure between the two groups of animals. Secondly, ANOVA analysis revealed no statistical significant differences between the ICl,swell–V relationships, obtained from ‘3 months HFH myocytes’ and ‘9 months HFH myocytes’.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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3.1. ICl,swell of rabbit ventricular myocytes
In a first series of ruptured-patch whole-cell voltage-clamp experiments we identified and characterized ICl,swell in our normal rabbit ventricular myocyte preparation. Exposure to an isotonic MgCl2 solution (solution 2) while dialyzing the sarcoplasm with solution 4, caused disappearance of all inward and outward currents. Subsequent application of hypo-osmotic conditions, elicited large, instantaneous, time-independent inward and outward currents (data not shown), the development of which showed a close relationship with cell volume as estimated by planar cell surface area (Fig. 1A). When the osmotic gradient was 0.48 T, we observed a delay of 104±13 s (n=4) between the abrupt
30% increase of planar cell surface area and activation of the membrane currents. Return to a 1.07-T osmotic gradient resulted in a brusque decrease of cell surface area and current amplitude back to control values (Fig. 1A). Before, during and after swelling, we applied a series of 20 voltage-clamp steps of 50 ms duration, from a holding potential of –80 mV first to –120 mV, and with 10-mV increments to +70 mV. The voltage-clamp steps were applied once every second. In the voltage range between –80 and +20 mV, the I–V relationships of the unswollen cells were essentially linear. Mean normalized conductance was 15±3 pS/pF (n=17) under 1.07 T isotonic conditions. It increased 20-fold to 338±74 pS/pF (n=21) when the osmotic gradient was lowered to 0.48 T. We also noticed that in the voltage range between +30 and +70 mV, slope of the I–V relationship was 90% greater, 644±137 pS/pF (n=21, P=0.001), than in the –80 and +20 mV interval. Furthermore, the swelling-induced currents reversed close to the expected equilibrium potential for Cl– ions. To ascertain the anion selectivity of the swelling induced currents we replaced all extracellular Cl– ions by SO42– ions. This caused the reversal potential to reversibly shift from –4.2±1.8 to 56±1.5 mV (n=3), confirming that under our experimental conditions mainly Cl– ions flow through the channels (Fig. 1B). Outwardly rectifying, swelling-activated Cl– currents are known for their sensitivity to inhibition by 4,4'-diisothiocyanostilbene-2,2'disulfonic acid (DIDS). In the presence of 0.5 mM DIDS (Sigma) inwardly and outwardly directed ICl,swell currents were inhibited by 40 and 70% (n=3, osmotic gradient of 0.57 T), respectively (Fig. 1C). The differential effect of the stilbene on inwardly and outwardly directed currents is another distinguishing propensity of swelling activated cardiac Cl– currents. Taken together, the delayed activation of the osmotic swelling induced, outwardly rectifying, time independent membrane currents, their Cl– selectivity and DIDS sensitivity, all are characteristic for ICl,swell currents. We thus conclude that our rabbit ventricular myocyte preparation shares this conductance with other cardiac cells.
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3.2. ICl,swell is not active under isotonic conditions in normal and HFH myocytes
In ventricular myocytes isolated from rapid pacing induced failing dog hearts, Clemo et al. [2] showed that ICl,swell was nearly maximally active under isotonic conditions. These authors applied perforated-patch whole-cell clamp methodology. We therefore asked the question whether in our HFH myocytes ICl,swell is active under isotonic conditions. To this end, myocytes obtained from five normal and four failing hearts were compared. The cells were exposed to the 1.0-T variant of solution 3, whereas the pipette was filed with solution 5. We employed both ruptured-patch whole-cell clamp and amphotericin B perforated-patch whole-cell clamp. Voltage clamp steps of 500 ms duration were applied. We used the average current measured at the end of the voltage step to construct the I–V relationships. As is shown in Fig. 2, irrespective of the method with which whole-cell recording conditions were created, the N-shaped I–V relationships typical of ventricular myocytes were obtained. At potentials more negative than
–30 mV inwardly rectifying K+ currents are active. At potentials positive to –30 mV sustained Ca2+ current and delayed rectifying K+ currents contribute to the I–V relationships. More importantly, 0.5 mM DIDS had no significant effect on the whole-cell membrane currents measured at the end of a 500-ms voltage step in both normal and HFH myocytes. Regardless of the method to obtain the whole-cell configuration, the drug did not reduce the voltage-dependent currents that are elicited under isosmotic condition. These observations suggest that ICl,swell is not active under isotonic conditions in both normal and HFH myocytes. Furthermore, these results show that patch rupture by itself does not cause activation of ICl,swell.
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3.3. HFH myocytes show relatively less APD90 shortening and Vm depolarization upon swelling
Next, we studied the effects of cell swelling on AP parameters. We continued with the ruptured-patch whole-cell clamp methodology. By dialyzing the cell interior with a pipette solution low in Cl– (solution 5), we prevented the shift in Cl– equilibrium potential towards less negative values, that otherwise would have occurred upon lowering the extracellular NaCl concentration. Indeed, preliminary experiments with amphotericin B perforated-patch whole-cell clamp revealed that swollen rabbit myocytes became inexcitable (data not shown). The standard solution changes were as follows. Firstly, we switched from normal Tyrode's solution (solution 1) to the isotonic variant of solution 3. Next, we lowered its sucrose concentration to achieve the appropriate extracellular tonicity. Finally, we added sucrose back to restore isotonicity. Fig. 3 illustrates the typical AP changes in two normal (A, C) and two HFH myocytes (E, G), when these cells were exposed to osmotic gradients of 0.77 T (A, E) and 0.50 T (C, G). The solution changes greatly affected AP duration and Vm. Also shown in Fig. 3 are the time courses of the changes in these parameters. The data of these and at least four other cells are summarized in Fig. 4.
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As is shown in Fig. 4A we observed the
60% action potential prolongation typical of failing heart cells. When bathed in normal Tyrode's solution, APD90 of control cells stimulated at 1 Hz was 230 ms, but 375 ms in the HFH myocytes. Lowering of the extracellular Na+ concentration and ionic strength by switching to the isotonic variant of solution 3, resulted in a significant shortening of the AP duration and hyperpolarization of Vm (Fig. 3B,D,F,H and Fig. 4A). Under these conditions APD90 and Vm of normal and HFH myocytes became almost indistinguishable (Fig. 4A). The underlying ionic mechanism was not investigated. Swelling of the cells further reduced ADP90 and led to an appreciable Vm depolarization. Both effects were partially reversible and their magnitudes were proportional to the osmotic gradient applied (Figs. 3B,D,F,H and Fig. 4A). ANOVA revealed that in all four solutions ADP90 of HFH myocytes was significantly higher than in control cells, on average 70% (Fig. 4A). Interestingly, the relative change in ADP90 upon swelling proved significantly smaller in the HFH myocytes (Fig. 4B). When these cells were exposed to the 0.77 and 0.50 T variant of solution 3, ADP90 fell by 14 and 22%, respectively. In contrast, in normal myocytes, ADP90 decreased by 26% and as much as 56% when exposed to the same osmotic gradients. Also the swelling-induced depolarization was less pronounced in the HFH myocytes. Differences in Vm did not reach statistical significance according to the ANOVA (Fig. 4A). But the relative change in Vm at 0.50 T was significantly smaller in the HFH myocytes (Fig. 4B). We next addressed the questions whether the limited effects of ICl,swell in the HFH myocytes could be explained by an altered ICl,swell density or changed resistance to cell swelling.
3.4. Swelling-induced membrane currents are reduced in HFH myocytes
To assess changes in membrane currents provoked by the 0.77 and 0.50 T osmotic gradients, we interrupted the current-clamp measurements and switched to a voltage-clamp protocol. Nineteen voltage-clamp steps of 500 ms duration were applied every 2 s from a holding potential of –60 mV, first to –140 mV and incremented with 10 mV. The quasi steady-state current recorded in the final 10 ms of a 500-ms voltage step was considered. By subtracting the appropriate families of currents, the I–V relationship of the swelling-activated current could be constructed. Fig. 5 summarizes these data. Typical of ICl,swell when measured under asymmetric Cl– gradient, all four I–V relationships showed an even more pronounced outward rectification, and reversed close to –50 mV. Magnitude of the currents was proportional to the osmotic gradient applied. ANOVA revealed that the density of the difference current at positive membrane potentials was significantly lower in the HFH myocytes. The reduction amounted to 40–50% at both osmotic gradients.
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In a subsequent series of ruptured-patch whole-cell voltage-clamp experiments, we optimized recording conditions to directly determine ICl,swell conductance. We filled pipettes with solution 4 and replaced normal Tyrode's solution by solution 2. Then, we lowered its sucrose content as to achieve osmotic gradients of 1.07, 0.88, 0.75, 0.57, and 0.48 T. When ICl,swell had reached plateau amplitudes (Fig. 1A), we applied the aforementioned voltage-clamp protocol. The data are summarized in Fig. 6A. Again we noticed a clear dependency of ICl,swell magnitude on the prevailing osmotic gradient. The higher the gradient, the higher the current density. Furthermore, ANOVA revealed that HFH myocytes possessed a significantly lower ICl,swell density. In all hypotonic solutions, normalized ICl,swell conductance proved on average 55% lower.
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3.5. Normal myocytes and HFH myocytes swell to the same extent
To investigate the hypothesis that the reduced ICl,swell conductance in HFH myocytes could be caused by a greater resistance to swelling, we estimated the percentage increase in planar cell surface area during exposure to hypotonic solutions. Normal cells showed a planar cell surface area increase of 37% per unit T change in the interval between 0.48 and 1.07 T, whereas HFH myocytes showed a planar cell surface area increase of 54% per unit T change in that same interval (Fig. 6B). This difference, however, was not statistically significant.
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4. Discussion |
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4.1. Overview
We studied ICl,swell in rabbit ventricular myocytes isolated from combined pressure and volume overloaded failing hearts and from age-matched control hearts. Goal of our experiments was to investigate ICl,swell current densities in our animal model of heart failure and to investigate effects of ICl,swell on the abnormal AP configuration. Our ruptured-patch, and perforated-patch whole-cell clamp experiments showed that ICl,swell was not active under isotonic conditions (Figs. 2 and 6A
). As expected, APD90 of HFH cells was prolonged by 60% under control conditions (Fig. 4A). ICl,swell caused AP shortening and Vm depolarization in an osmotic gradient dependent fashion (Figs. 3 and 4A). However, in HFH myocytes swelling-induced electrophysiological changes were smaller, even though the cells underwent the same relative change in planar cell surface area (Figs. 3, 4, and 6). Voltage-clamp experiments revealed that ICl,swell current density was a curvilinear function of the osmotic gradient in both groups (Figs. 5 and 6A). But in the HFH myocytes, ICl,swell density proved 50% reduced (Figs. 5 and 6A). This reduction offers a good explanation for the relatively limited swelling-induced electrophysiological changes in HFH ventricular myocytes.
4.2. ICl,swell of normal rabbit ventricular myocytes
Aside from ICl,swell a number of other prominent types of Cl– currents are present in heart cells. The distribution, characteristics and (patho)physiological role of cardiac chloride currents have been recently reviewed [7]. Purinergic receptor stimulation, and adrenoceptor stimulation activate the time-independent ICl,ATP, ICl,PKC and ICl,PKA currents, respectively. Because we omitted both ATP and catecholamines from all our bathing solutions, currents through these particular types of Cl– channels were assumed negligible. We have another argument in support of this assumption. The data summarized in Fig. 5 were obtained by subtracting two families of currents, one recorded before, and one recorded after swelling. The subtraction procedure eliminates interference of the time-independent Cl– currents with determination of ICl,swell current amplitude. Inasmuch as in our direct measurements of ICl,swell current density as summarized in Fig. 6 essentially the same results were obtained, we infer that also in these experiments interference of agonist-activated, time-independent Cl– currents was minimal. Both sarcoplasmic Ca2+ and voltage activate the time-dependent ICl,Ca2+. In rabbit ventricular myocytes ICl,Ca2+ is relatively small and active only in the first 30–50 ms after a depolarizing voltage clamp step positive to –40 mV [7]. Because the data summarized in Fig. 5 are derived from the quasi steady-state current at the end of a 500-ms voltage step, interference of ICl,Ca2+ with determination of ICl,swell current amplitude is unlikely. The voltage-clamp experiments summarized in Fig. 6 were conducted with pipette solutions to which EGTA was added. Furthermore, the bathing solution was free of Ca2+ thus preventing the Ca2+ induced Ca2+ release (CICR). The presence of intracellular EGTA but primarily blockage of CICR prevent activation of ICl,Ca2+. Indeed, we observed no time-dependent current transients which would have been indicative of an active ICl,Ca2+. Aside from ICl,swell one other swelling-induced current has been described in rabbit ventricular myocytes [8]. ICir,swell is a time-independent, inwardly rectifying cation current [9,10]. It activates at membrane potentials negative to –60 mV. Both Na+ and K+ permeate through ICir,swell channels but not NMDG+ or Mg2+ ions. In the experiments summarized in Fig. 5, the ionic conditions were such that ICir,swell currents potentially could have been detected. Indeed, at negative potentials there was a slight inward rectification, which could be ICir,swell. Inasmuch as in our direct measurements of ICl,swell current density (Fig. 6) all intracellular cations were replaced by NMDG+, whereas all extracellular cations were replaced by Mg2+, interference of ICir,swell with determination of ICl,swell current amplitude is not very probable. Taken together, the swelling-induced Cl– currents we investigated in this study were flowing predominantly through ICl,swell channels.
ICl,swell has been found in normal myocytes isolated from the heart chambers of many species. These include human atrium and ventricle [11], dog atrium [11–13] and ventricle [14], guinea pig atrium [15,16] and ventricle [6,15,17,18], chick cultured cells [19,20], rabbit atrium [21,22] and ventricle [8]. From our observations and those made by Clemo and Baumgarten [8], it can be inferred that ICl,swell of rabbit ventricular myocytes shares selectivity, time-independency, outward rectification, delayed activation, osmolarity-dependency and sensitivity to DIDS with the volume-activated anion currents found in other cardiac preparations.
In contrast to the well documented voltage-clamp properties of ICl,swell, as yet data on the effects of this current on normal and abnormal AP configuration are relatively scarce. We noticed that swelling caused a significant decrease in APD90. This observation is consistent with outwardly directed, repolarizing ICl,swell currents at membrane potentials positive to the Cl– equilibrium potential of –50 mV. Swelling also caused Vm to depolarize. This is consistent with inwardly directed ICl,swell currents at membrane potentials negative to the Cl– equilibrium potential. The swelling-induced AP changes agree to a great extent with observations made in guinea-pig ventricular myocytes [18].
4.3. ICl,swell in hypertrophied ventricular myocytes of failing hearts
In the HFH myocytes we observed the prolonged AP configuration characteristic of this condition. We have not rigorously looked at the ionic mechanisms in our model, but as reviewed by Nabauer et al. [1], AP prolongation associated with hypertrophy is often caused by a reduction in time-dependent K+ conductances. We now show that also ICl,swell current density may be reduced. Even though the ICl,swell-induced changes in the abnormal AP's are qualitatively analogous to those observed in normal cells, the relative shortening of AP duration is significantly less. This indicates that ICl,swell is reduced to a greater degree than the other conductances that are active during the plateau phase of the action potential.
To date, one study has investigated voltage-clamp properties of ICl,swell in an animal model of cardiac failure. Clemo et al. [2] used cells isolated from rapid pacing induced failing dog hearts. These authors showed that ICl,swell current density was increased by 40%. Furthermore, ICl,swell was nearly maximally active under isotonic conditions. They concluded that their hypertrophied cells behave like swollen cells, and they point to the possibility that ICl,swell channels may become a target for pharmacologic therapy. Bénitah et al. [3] found evidence for a 9-AC sensitive chloride current in hypertrophied rat ventricular myocytes isolated from pressure overloaded hearts. This chloride current was active under isotonic conditions and caused AP shortening but not Vm depolarization. Although the observations of Bénitah et al. [3], to a certain extent, agree with the presence of ICl,swell in hypertrophied rat cells, the exact nature of this current awaits further investigation.
Our results contrast with these two studies. There are a number of potential explanations for the discrepancies. Firstly, the species difference may account for the differences. Secondly, the methods used to induce heart failure may prove important. Thirdly, differences in the period of time the cells function in a damaged heart may contribute to the divergence. In our experimental protocols we used 3 and 9 months failing models, as opposed to the 1–2 months in the dog [2] and rat [3] model. For example, we can not exclude the possibility that in the early phases of the development of heart failure, in rabbits too ICl,swell density is increased and active under isotonic conditions. In any event, our study shows that in HFH myocytes ICl,swell density may not necessarily have to be increased at all time. Thus, the efficacy of pharmacologic interventions targeted at ICl,swell channels may depend on the pathophysiologic mechanisms that have led to cardiac failure, or on the timing of the intervention.
In conclusion, we have shown that influence of ICl,swell on normal rabbit cardiac AP and abnormal AP configuration in HFH myocytes, is scaled to the intensity of the osmotic stress. The smaller effects of ICl,swell on AP parameters in HFH myocytes are due to a reduction in ICl,swell density in the adapted cells.
Time for primary review 47 days.
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Acknowledgements |
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The authors thank Drs Antoni C.G. van Ginneken and E. Etienne Verheijck for critically reading the manuscript and fruitful discussions. Jan Zegers and Charly N.W. Belterman are gratefully acknowledged for their excellent technical assistance. Cees A. Schumacher, Berend de Jonge and Jan Bourier are acknowledged for their help with cell isolations. This work was supported by a grant from the Dutch Organization for Scientific Research (ALW 805-06-153) and the Dutch Heart Association (NHS 96.039).
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Notes |
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* For this manuscript Dr Steve Soroto acted as guest-editor.
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References |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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- Nabauer M., Kaab S. Potassium channel down-regulation in heart failure. Cardiovasc. Res. (1998) 37(2):324–334.
[Abstract/Free Full Text] - 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(2):157–165.
[Abstract/Free Full Text] - Benitah J.P., Gomez A.M., Delgado C., Lorente P., Lederer W.J. A chloride current component induced by hypertrophy in rat ventricular myocytes. Am. J. Physiol. (1997) 272(5):H2500–H2506.[Web of Science][Medline]
- Vermeulen J.T., McGuire M.A., Opthof T., et al. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc. Res. (1994) 28:1547–1554.
[Abstract/Free Full Text] - Tytgat J. How to isolate cardiac myocytes. Cardiovasc. Res. (1994) 28:280–283.
[Free Full Text] - 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]
- Hume J.R., Duan D., Collier M.L., Yamazaki J., Horowitz B. Anion transport in heart. Physiol. Rev. (2000) 80(1):31–81.
[Abstract/Free Full Text] - Clemo H.F., Baumgarten C.M. Swelling-activated Gd3+-sensitive cation current and cell volume regulation in rabbit ventricular myocytes. J. Gen. Physiol. (1997) 110:297–312.
[Abstract/Free Full Text] - Sorota S. Insights into the structure, distribution and function of the cardiac chloride channels. Cardiovasc. Res. (1999) 42(2):361–376.
[Abstract/Free Full Text] - Hiraoka M., Kawano S., Hirano Y., Furukawa T. Role of cardiac chloride currents in changes in action potential characteristics and arrhythmias. Cardiovasc. Res. (1998) 40(1):23–33.
[Abstract/Free Full Text] - Oz M.C., Sorota S. Forskolin stimulates swelling-induced chloride current, not cardiac cystic fibrosis transmembrane-conductance regulator current, in human cardiac myocytes. Circ. Res. (1995) 76:1063–1070.
[Abstract/Free Full Text] - 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] - Du X.Y., Sorota S. Cardiac swelling-induced chloride current depolarizes canine atrial myocytes. Am. J. Physiol. (1997) 272:H1904–H1916.[Web of Science][Medline]
- 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]
- Vandenberg J.I., Yoshida A., Kirk K., Powell T. 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] - Sakaguchi M., Matsuura H., Ehara T. Swelling-induced Cl– current in guinea-pig atrial myocytes: inhibition by glibenclamide. J. Physiol. (1997) 505:41–52.
[Abstract/Free Full Text] - Sasaki N., Mitsuiye T., Wang Z., Noma A. Increase of the delayed rectifier K+ and Na+–K+ pump currents by hypotonic solutions in guinea pig cardiac myocytes. Circ. Res. (1994) 75:887–895.
[Abstract/Free Full Text] - 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) 491:69–80.
[Abstract/Free Full Text] - Zhang J., Rasmusson R.L., Hall S.K., Lieberman M. A chloride current associated with swelling of cultured chick heart cells. J. Physiol. (1993) 472:801–820.
[Abstract/Free Full Text] - Hall S.K., Zhang J., Lieberman M. Cyclic AMP prevents activation of a swelling-induced chloride-sensitive conductance in chick heart cells. J. Physiol. (1995) 488:359–369.
[Abstract/Free Full Text] - Hagiwara N., Masuda H., Shoda M., Irisawa H. Stretch-activated anion currents of rabbit cardiac myocytes. J. Physiol. (1992) 456:285–302.
[Abstract/Free Full Text] - Duan D., Hume J.R., Nattel S. Evidence that outwardly rectifying Cl– channels underlie volume-regulated Cl– currents in heart. Circ. Res. (1997) 80:103–113.
[Abstract/Free Full Text]
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