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Cardiovascular Research 2002 55(3):660-671; doi:10.1016/S0008-6363(02)00454-6
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Pharmacological preconditioning in rabbit myocardium is blocked by chloride channel inhibition

Michelle Batthisha,d, Roberto J Diaza,b, He-Ping Zenga,b, Peter H Backxc,d and Gregory J Wilsona,b,d,*

aDivision of Cardiovascular Research, Research Institute, The Hospital for Sick Children; Department of Medicine, The Toronto Hospital, Toronto, Ontario, Canada
bDivision of Pathology, The Hospital for Sick Children, Department of Medicine, The Toronto Hospital, Toronto, Ontario, Canada
cDepartment of Medicine, The Toronto Hospital, Toronto, Ontario, Canada
dDepartment of Physiology, The University of Toronto, Toronto, Ontario, Canada

diazport{at}sickkids.on.ca

* Corresponding author. Division of Cardiovascular Research, McMaster Building, Room 7019C, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Tel.: +1-416-813-5965; fax: +1-416-813-7480

Received 7 November 2001; accepted 22 April 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: We have recently proposed that chloride (Cl–) channels contribute to ischemic preconditioning (IPC) in the myocardium. To further evaluate this hypothesis, we investigated the role of Cl– channels in pharmacological preconditioning. Methods: Isolated rabbit cardiomyocytes and isolated buffer-perfused rabbit hearts were initially preconditioned with a 10 min exposure to either an adenosine receptor agonist [2-chloro-N6-cyclopentyladenosine (CCPA, 200 nM) and/or N6-2-(4-aminophenyl)ethyladenosine (APNEA, 1 µM)] or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 µM) followed by a 10 or 20 min washout or not preconditioned (control). Cardiomyocytes or whole hearts were then subjected to prolonged ischemic period (45 min simulated ischemia or 40 min of regional myocardial ischemia, respectively) followed by 60 min reperfusion (resuspension in oxygenated medium or release of the transient coronary occlusion, respectively). Results: Indanyloxyacetic acid 94, a selective Cl– channel inhibitor that produced substantial inhibition of the regulatory volume decrease (RVD) when given at 10 µM concentration in cultured cardiomyocytes, was administered before ischemia to block RVD through Cl– channel inhibition. CCPA, APNEA and PMA significantly (P<0.01) reduced the % of dead cardiomyocytes (by trypan blue staining) after 45 min SI/60 min SR, as compared to controls, while IAA-94 abolished this protection but did not affect PKC{varepsilon} translocation by IPC. We confirmed that IAA-94 blocked IPC-, APNEA- and PMA-induced protection against infarction in the isolated heart model. Conclusions: These findings support our contention that Cl– channels are downstream effectors of IPC.

KEYWORDS Hypoxia/anoxia; Ischemia; Myocytes; Preconditioning


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Ischemic preconditioning (IPC) is the process by which one or more brief episodes of transient ischemia followed by a short period of reperfusion induces myocardial protection against subsequent prolonged ischemia and reperfusion injury [1]. We have shown in both freshly isolated cardiomyocytes and Langendorff perfused hearts that blockade of Cl– channels abolishes the protection of IPC [2]. Studies performed in cardiomyocytes by Vanden-Hoek et al. [3] also indicate that blockade of Cl– channels abolishes IPC protection. Heusch et al. [4] have been unable to reproduce these results. However, more recently, we have confirmed the role of Cl– channels in IPC [5].

The rationale for our examining Cl– channels in IPC [2,5] was that Cl– channels are thought to be involved in volume regulation in cardiomyocytes [6–8], and that cell swelling and the loss of cell volume regulation play important roles in ischaemic injury [9]. In fact, Steenbergen et al. [10] have shown that while myocyte swelling alone is incapable of rupturing plasma membranes under normoxic conditions, it causes disruption of the plasma membrane under anoxic conditions indistinguishable from that observed in cardiomyocytes lethally injured by ischemia. Therefore, we have hypothesized that IPC protects the myocardium by activation of Cl– channels which, in turn, enhance the ability of cardiomyocytes to regulate cell volume early during ischemia, thus reducing ischemic cell swelling and preventing cell death. The fact that IPC protection can be abolished both by bracketing the IPC or the long (index) ischemic period with Cl– channel inhibitors strongly support the notion of early activation by IPC of Cl– channels and their role in limiting ischemic injury [2].

If Cl– channels are indeed end-effectors of IPC, their blockade should also abolish the pharmacological preconditioning (PPC) protection induced by stimulation of known key receptors and mediators of IPC. Stimulation of adenosine receptors (A1 and A3), one of best characterized triggers in the protection of IPC [11], protects the myocardium against ischemia/reperfusion injury [12,13] and activates a Cl– channel conductance in guinea pig cardiomyocytes [14]. Furthermore, stimulation of PKC with phorbol esters mimics IPC protection [15] and activates a Cl– current in feline [16] and guinea pig cardiomyocytes [17]. Thus, in the present study, we sought to determine the role of Cl– channels in PPC induced through activation of adenosine A1/A3 receptors and direct stimulation of PKC against cell death caused by the index ischemia period in both freshly isolated rabbit cardiomyocytes and buffer-perfused rabbit hearts.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Animal protocols conformed to the Guide for the Care and Use of Laboratory Animals published by NIH (NIH publication No. 85-23, revised 1996).

2.1 Cell volume regulation studies
Since regulatory volume decrease (RVD), important in cell volume regulation, is mostly dependent on Cl– channels in rabbit cardiomyocytes [18], we used RVD as the end point to assess the inhibitory dose–response of Cl– channel inhibitors.

2.1.1 Primary culture of cardiomyocytes
Ventricular myocytes were isolated from New Zealand White rabbits (weight range, 3.0 to 3.5 kg) by enzymatic dissociation [2,19] and subsequently cultured [20]. Briefly, cardiomyocytes were placed in 35-mm laminin-coated petri dishes, with a density of 2.5x105 cells/dish, each containing culture medium 199 (Gibco, Burlington, Canada) with Earle's salts supplemented with 10% fetal bovine serum, 10 µM cytosine β-D-arabinofuranoside, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 0.08 mg/ml gentamicin. Cardiomyocytes were then incubated at 37 °C in a humidified 5% CO2–95% air mix for 24–48 h.

2.1.2 Experimental protocol for cultured cardiomyocytes
After 24 or 48 h in culture, each petri dish containing cardiomyocytes attached to laminin was placed on a heated (37 °C) stage of an inverted microscope (Axiovert 100, Zeiss). Only viable, rod-shaped cardiomyocytes with no blebs were used. Cardiomyocytes were initially exposed for 15 min to iso-osmotic conditions by replacing the culture medium 199 with an oxygenated (95% O2–5% CO2) Tyrode solution containing (mM): NaCl 68, MgSO4 0.53, KCl 4.7, NaHCO3 25, KH2PO4 1.2, CaCl2 1.2, Dextrose 10, pH 7.4. The osmolarity of the iso-osmotic solution was adjusted to 300 mOsM by adding mannitol. Subsequently, cardiomyocytes were exposed to either 30, 60, 90 or 120 min of hypo-osmotic stress (Tyrode buffer without mannitol, 200 mOsM) to induce cell swelling.

Cardiomyocytes were treated either with the known Cl– channel inhibitor indanyloxyacetic acid 94 (IAA-94, at 5, 10 or 50 µM) [21–23] or its vehicle (ethanol), following the same protocol as for control (untreated) cardiomyocytes, with the exception that either indanyloxyacetic acid 94 (IAA-94) or the vehicle was present 15 min prior and throughout the hypo-osmotic stress period. We administered 9-anthracenecarboxylic acid (9-AC, 500 µM), a known Cl– channel inhibitor [24], or its vehicle dimethyl sulfoxide (DMSO), during the hypo-osmotic period to create a positive control myocyte group to evaluate against the effect on cell volume regulation of IAA-94. We used each myocyte as its own control and reported the changes in cell volume as normalized percentage of cell swelling.

2.1.3 Cell volume measurements
A video-camera mounted on an inverted microscope (Axiovert 100, Zeiss, Germany) was used to acquire myocyte images (400x magnification) at short intervals (1 or 5 min) during the entire experimental protocol for subsequent analysis. Each image was then used to trace myocyte area, used as an index to measure changes in cell volume.

For validation, we measured changes in cell volume using confocal laser (one photon) scanning microscopy. Initially, 24–48 h cultured rabbit cardiomyocytes were simultaneously stained with calcein AM (2 µM) to stain the cytosol of intact cells and ethidium homodimer-1 (4 µM) to identify nuclei of cells with disrupted membranes. Each culture plate containing stained cardiomyocytes was placed on a heated (37 °C) stage of an inverted microscope (Axiovert 100, Zeiss). From each plate, a single rod-shaped viable myocyte was selected and then scanned using a multitrack beam path (argon 488 nm and HeNe1 543 nm), at baseline (iso-osmotic) conditions and after 5 or 10 min exposure to the same hypo-osmotic solution used in the RVD study. A three-dimensional image of each myocyte was obtained using optical cross-sections captured along the height of each myocyte. With these optical images, we were able to measure the length (y-axis), width (x-axis), and height (z-axis) of each cell to calculate volume (lengthxwidthxheight).

A regression analysis of the % cell swelling under hypo-osmotic conditions (200 mOsM) after 5 or 10 min determined by area vs. volume showed that the calculated % cell swelling as measured by area was closely related (r = 0.87) to the % cell swelling calculated based on laser scanning microscopy (LSM) measurements (volume) (Fig. 1), and that the slope of this relationship was 0.5. Thus, we were able to obtain a good estimate of the actual increase in the % cell swelling by volume (Fig. 1) by multiplying the % cell swelling (calculated from surface area measurements) by a factor of 2. From these % increases in cell volume estimates, the RVD for each myocyte was calculated as:


Figure 1
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  		Fig. 1 This graph shows a regression analysis of the % cell swelling under hypo-osmotic conditions (200 mOsM) after 5 or 10 min as determined by surface area vs. tri-dimensional (lengthxwidthxheight) calculation of cell volume by laser scanning microscopy (LSM). In this study, we found that the calculated % cell swelling as measured by area was closely related (r = 0.87) to the % cell swelling calculated based on LSM measurements of cell volume. The slope of this relationship was 0.5, which indicates that the increase in % cell swelling as measured by cell area represents about half of the total increase in % cell swelling as measured by cell volume.

 

Formula

where peak time refers to the maximum increase in cell volume occurring between 7 and 12 min of hypo-osmotic stress. RVD values are reported as decimal fraction; normalized increases in cell volume are presented as percentages.

2.2 Preconditioning studies in isolated cardiomyocytes
In these studies we assessed the role of Cl– channels as end-effectors of adenosine receptor (A1/A3) and PKC induced preconditioning as compared to IPC in freshly isolated rabbit cardiomyocytes.

2.2.1 Simulated ischemia (SI) and simulated reperfusion (SR)
Ischemia and reperfusion were simulated in cardiomyocytes as previously described [2,19]. Briefly, 1.5 ml of the cell suspension was placed in a 1.8-ml Eppendorff tube and centrifuged for 2 min at 45 g to form an 8- to 10-mm-thick cell pellet. The supernatant was discarded, except for a volume equivalent to about one third of the pellet thickness. The cell pellet and supernatant were covered by a 3- to 4-mm-thick mineral oil layer and incubated at 37 °C. To simulate reperfusion, the cell pellet was resuspended by removing the oil layer and remaining supernatant and adding 1.0 ml of oxygenated calcium-containing buffer supplemented with 0.1% bovine serum albumin (BSA) and incubated on a multidish cell culture plate at 37 °C with agitation in an O2 atmosphere.

2.2.2 Dose–response study
A dose–response for the Cl– channel inhibitor was performed to validate the concentration used in the PPC studies. The rationale was to determine a concentration of IAA-94 which was sufficient to block the protective effect of IPC in the model. Cardiomyocytes (n = 5 hearts) were allowed to stabilize in S-MEM buffer at 37 °C with agitation in an O2 atmosphere for 30 min. Control cardiomyocytes underwent an additional 30 min stabilization. IPC cardiomyocytes were subjected to 10-min SI followed by 20-min SR. All cardiomyocytes were then subjected to 45-min SI followed by 60-min SR. IAA-94 (2.5, 5 or 10 µM) or its vehicle (ethanol) were administered 10 min prior to the long SI (Fig. 2).


Figure 2
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  		Fig. 2 Experimental protocol used in freshly isolated rabbit cardiomyocytes and isolated buffer perfused hearts. Isolated cardiomyocytes were initially subjected to 30-min stabilization (cardiomyocytes suspended in oxygenated buffer at 37 °C) followed either by additional 30-min stabilization (control cardiomyocytes) or by pharmacological preconditioning (PPC) consisting of a 10-min exposure to either 2-chloro-N6-cyclopentyladenosine (CCPA, 200 nM), N6-2-(4-aminophenyl)ethyladenosine (APNEA, 1 µM) or phorbol 12-myristate 13-acetate (PMA, 1 µM) followed by a 20 min drug-free suspension. The adenosine antagonist, 8-sulfophenyl theophylline (8-SPT, 100 µM) and the PKC inhibitor, chelerythrine (CHE, 5 µM) were administered 10 min prior to PPC. Next, all cardiomyocytes were subjected to 45 min SI (pelleting under oil at 37 °C) followed by 60 min SR. Indanyloxyacetic acid 94 (IAA-94, 10 µM) was added 10 min before the long SI. Oxygenated baseline cardiomyocytes were incubated in an O2 atmosphere at 37 °C for 165 min. Myocyte viability was assessed at the following time points (*): end of stabilization, before the long SI and at the end of SR. Isolated buffer perfused hearts were initially subjected to 15 min of stabilization followed by a long period of regional myocardial ischemia (40 min) and reperfusion (60 min). Preconditioned hearts either received three cycles of 5 min/10 min of ischemia/reperfusion, pretreated for 5 min with APNEA (1 µM) followed by 10 min washout, or pretreated with PMA (a total dose of 2 nmol was given via a side port over 10 min) prior to the long ischemia/reperfusion episode. IAA-94 (10 µM) was given also through a side port starting 10 min prior to the long ischemia and ending at the end of the ischemic period.

 
2.2.3 Experimental protocol for isolated cardiomyocytes
Following an initial 30-min stabilization period, cardiomyocytes (n = 6 hearts in each study) were either pharmacologically preconditioned with a 10 min exposure to the adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 200 nM), the adenosine A1/A3 agonist N6-2-(4-aminophenyl)ethyladenosine (APNEA, 1 µM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 µM) followed by a 20-min washout. All three agonist (CCPA, APNEA and PMA), given at similar concentrations, have been shown to mimic IPC protection in cardiomyocytes [12,25,26]. Control cardiomyocytes (non-preconditioned) remained in the incubator for an additional 30 min. To verify that the protection of the agonist was specific to its expected pharmacological effect, cardiomyocytes were pretreated with either the non-specific adenosine receptor inhibitor 8-sulfophenyl theophylline (SPT, 100 µM) or the PKC antagonist chelerythrine (CHE, 5 µM) given 10 min prior to preconditioning. Next, both control and PPC cardiomyocytes were subjected to 45-min SI/60-min SR (Fig. 2).

Control (non-preconditioned), ischemically preconditioned (IPC), and oxygenated baseline (165 min incubation in an O2 atmosphere at 37 °C) cardiomyocytes were simultaneously treated with either the Cl– channel blocker indanyloxyacetic acid 94 (IAA-94, 10 µM) or its vehicle, and either CCPA, APNEA, PMA or each drug's vehicle given prior to the SI/SR protocol as shown in Fig. 2.

2.3 Preconditioning studies in isolated buffer-perfused hearts
2.3.1 Surgical preparation
Rabbits were prepared as previously reported [27]. Briefly, hearts were excised, mounted on a modified nonrecirculating Langendorff apparatus, and immediately perfused, at a constant pressure (75 mmHg) with Krebs–Henseleit buffer solution containing (in mM) NaCl 118.5, KCl 4.7, MgSO4 1.2, CaCl2 2.5, NaHCO3 24.8, KH2PO4 1.2 and glucose 10 (pH 7.4, oxygenated with 95% O2–5% CO2) at 37 °C. A branch of the left coronary artery was intermittently occluded to induce ischemia. An intraventricular latex balloon connected to a pressure transducer was placed into the left ventricle to assess left ventricular developed pressure (LVDP; systolic minus diastolic pressure) and heart rate. Coronary flow was also measured by collecting buffer dripping from each heart. Once instrumented, hearts were placed in a water-jacketed chamber and stabilized before each experiment began.

2.3.2 Experimental protocol for isolated hearts
All hearts were initially subjected to 15 min aerobic perfusion (stabilization period) followed by 40 min normothermic (37 °C) regional ischemia and 60 min reperfusion. Control hearts were also subjected to an additional 45 min aerobic perfusion before the long ischema while preconditioned hearts were subjected to either three cycles of 5-min regional ischemia followed by 10-mn reperfusion, a 5 min infusion of APNEA followed by a 10 min washout, or a 10 min infusion of PMA followed by a 20 min washout before the long ischemia (Fig. 2). The total amount of PMA admnistered (0.2 nmol in a volume of 2 ml of a Krebs buffer solution containing 0.1 nmol/ml of PMA) was administered over 10 min as previously reported [28]. To determine the maintenance phase of preconditioning (the index ischemia), hearts were subjected to the same control or preconditioning protocols with a 10 min infusion of 10 µM IAA-94 just before the long ischema.

2.3.3 Infarct size measurements
After each experiment, the coronary artery used for the intermittent ischemic episodes was reoccluded. Each heart was then perfused with 5 to 10 µm zinc-cadmium sulfide yellow fluorescent particles (Duke Scientific, Palo Alto, CA, USA) to identify the area at risk (without particles). Next, hearts were cross-sectioned into several slices and stained with tryphenyl tetrazolium chloride (TTC) as we have previously described [27]. TTC stains viable tissue brick-red while necrotic tissue looks white or tan. The areas of infarct, risk, and total areas from each heart slice were then traced and planimetered to calculate the % of infarct size.

2.3.4 Effect of IAA-94 on PKC{varepsilon} translocation signaling
Since Cl– channels may also be directly activated by stretch it is possible that opening of stretch-activated Cl– channels may trigger a downstream signaling via PKC. To rule out this possibility and to confirm that Cl– channel inhibition with IAA-94 does not prevent PKC signaling (translocation of PKC{varepsilon} from the cytosol to the cell membrane), we determined the effect of IAA-94 on IPC induced PKC{varepsilon} translocation in Langendorff perfused rabbit hearts using the same preconditioning protocols described above. After IAA-94 (10 µM) treatment, myocardial tissue samples from areas at risk, from both control and IPC hearts, were immediately frozen, then homogenized. Cytosolic and particulate fractions were obtained by differential centrifugation, protein concentrations measured and Western immunoblotting performed as described by Ping et al. [29].

2.4 Chemicals
All chemicals were obtained from Sigma–Aldrich Canada (Oakville, Canada) unless otherwise stated. For all experiments, APNEA, CCPA, 9-AC and PMA were dissolved in DMSO (final concentration was 0.33%, v/v, or less in isolated cardiomyocytes and less than 0.001%, v/v, for whole hearts), IAA-94 was dissolved in ethanol (final concentration was 0.03%, v/v, or less), and SPT was dissolved in water. All vehicle control experiments were performed using the same concentrations of vehicles included in each agonist or antagonist drug.

2.5 Statistical analysis
Data are expressed as mean±S.E.M. and were first tested for normality (Kolmogorov–Smirnoff test) and homogeneity of variance (Levene test). Since the criteria for parametric analysis were not met, we performed nonparametric analysis using the multigroup comparison Kruskal–Wallis test to assess for differences among the groups, followed by the Mann–Whitney test to determine whether a statistically significant difference (P<0.05) existed between two groups.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Cell volume regulation studies
The rate of RVD was significantly (P<0.0001) slower (0.76±0.05% RVD/min) in cardiomyocytes subjected to hypo-osmotic stress between 30 to 60 min of exposure as compared to the rate of volume regulation (2.95±0.07% RVD/min) between peak swelling and 30 min. This rate continued to decrease after 90 and 120 min of hypo-osmotic exposure (0.33±0.02 and 0.10±0.01% RVD/min, respectively). Since approximately 60% of the volume regulation occurred within the first 30 min, we selected 30 min as the maximum RVD time point for the inhibitory studies (Fig. 3A).


Figure 3
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  		Fig. 3 Effect of Cl– channel inhibition on the regulatory volume decrease (RVD) in 24–48 h cultured rabbit cardiomyocytes. After a 30 min exposure to the hypo-osmotic solution, control cardiomyocytes regulate volume by 0.58±0.02 (N = 6 hearts, n = 26 cells). RVD was not affected when 5 µM indanyloxyacetic acid 94 (IAA-94) was administered to inhibit Cl– channels (N = 5 hearts, n = 26 cells). However, 10 µM IAA-94 produced a significant (N = 5 hearts, n = 21 cells, *P<0.01) reduction in RVD to 0.35±0.06, representing a 40% blockade of the total RVD. Similarly, five times that concentration of IAA-94 (50 µM) also produced a significant (N = 4 hearts, n = 15 cells, **P<0.001) reduction in RVD to 0.26±0.01, a value that represents a RVD blockade of 54%. Inhibition of Cl– channels with 9-antracenecarboxylic acid (9-AC, 500 µM) produced a significant (N = 3 hearts, n = 15 cells, *P<0.001) reduction in RVD to 0.25±0.01% which represents a RVD blockade of 57%. Data are expressed as mean±S.E.M.

 
No difference in the response to hypo-osmotic stress was observed among untreated, DMSO-treated and ethanol-treated control groups. Therefore, we pooled the data from these control groups. Cell volume increased significantly (P<0.0001) in control cardiomyocytes (43.8±4.0%, N = 6 hearts, n = 26 cells) upon exposure to the 200 mOsM solution, reaching a peak value between 7 and 12 min. After 30 min in hypo-osmotic solution, control cardiomyocytes showed only a 20.2±1.6% increase in cell volume. This significant (P<0.001) reduction in cell swelling represents a RVD of 0.58±0.02 (Fig. 3). Using RVD as the end-point, we determined the concentration of IAA-94 that effectively inhibits Cl– channels. IAA-94, administering at 5 µM, had no effect on RVD relative to control (Fig. 3). Increasing doses of IAA-94 produced a dose-dependent block of RVD. At 10 µM or 50 µM, IAA-94 significantly (P<0.001) reduced RVD (0.35±0.06 and 0.26±0.01, respectively) as compared to vehicle-treated controls. To confirm that this effect of IAA-94 on RVD was indeed the result of Cl– channel inhibition, we also inhibited Cl– channels with 500 µM 9-AC. 9-AC produced a significant (P<0.001) reduction in RVD to 0.25±0.01, similar to IAA-94 (P = 0.23 vs. 10 µM IAA-94), when compared to untreated controls (Fig. 3). Both IAA-94 and 9-AC did not influence cardiomyocyte volume under iso-osmotic conditions.

3.2 Preconditioning studies in isolated cardiomyocytes
3.2.1 Effect of Cl– channel inhibition on pharmacological preconditioning in isolated cardiomyocytes
In isolated ventricular myocyte studies, myocyte viability was expressed as a percentage of dead cardiomyocytes for each group of cells during 45 min SI combined with 60 min SR (Fig. 5).There was no difference in the percentage of dead cardiomyocytes between untreated and treated (with each agonist or antagonist drug) oxygenated baseline cardiomyocytes, in each set of experiments (data not shown). Furthermore, no difference was observed in the percentage of dead cardiomyocytes among untreated control myocyte groups or among untreated preconditioned myocyte groups in all cardiomyocyte experiments at any time point. In each study, there was no difference in the percentage of dead cardiomyocytes before the index ischemia among all experimental groups.


Figure 5
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  		Fig. 5 Effect of chloride channel inhibition on the protection against cell death by adenosine A1 receptor stimulation, adenosine A1/A3 receptor stimulation or PKC stimulation. Treatment with 200 nM 2-chloro-N6-cyclopentyladenosine (CCPA, panel A), 1 µM N6-2-(4-aminophenyl)ethyladenosine (APNEA, panel B) or 1 µM phorbol 12-myristate 13-acetate (PMA, panel C) significantly (*P<0.01 vs. control, n = 6) reduced the percentage of dead cardiomyocytes after 45 min SI/60 min SR. This pharmacological preconditioning (PPC) protection by CCPA and APNEA was abolished by the non-selective adenosine receptor antagonist 8-sulfophenyl theophylline (SPT, 100 µM) while PPC induced by PMA was blocked with the PKC antagonist chelerythrine (CHE, 5 µM). Inhibition of Cl– channels with 10 µM indanyloxyacetic acid 94 (IAA-94) also blocked this pharmacological protection. SPT, CHE and IAA-94 had no effect on viability when given to control cardiomyocytes (C). Data are expressed as mean±S.E.M.

 
In the first set of experiments (Fig. 5, panel A), a 10 min exposure to 200 nM CCPA prior to the index ischemia (Fig. 2A) significantly (P<0.01) reduced the percentage of dead cardiomyocytes observed at the end of 60 mm SR, when compared to vehicle-treated (with 0.033%, v/v) controls. This protection was blocked by SPT (100 µM), the adenosine receptor antagonist, given 10 min prior to CCPA. IAA-94 (10 µM, a concentration selected based on our dose–response studies, Figs. 3 and 4Go), administered 10 min prior to the index ischemia, completely abolished the PPC protection by CCPA. Both SPT and IAA-94 had no effect on vehicle-treated controls. Similarly, 10 min exposure to 1 µM APNEA preconditioned cardiomyocytes to the same extent as CCPA, as compared to vehicle-treated controls. This PPC protection by APNEA was also blocked by SPT and IAA-94 (Fig. 5, panel B). Furthermore, transient (10 min) exposure to 1 µM PMA, followed by a 20 min washout, significantly (P<0.01) reduced the percentage of dead cardiomyocytes after 45 min SI/60 min SR, when compared to vehicle-treated controls (Fig. 5, panel C). This protection was blocked by chelerythrine (CHE, 5 µM), a specific PKC inhibitor, given 10 min prior to PMA. Cl– channel inhibition with 10 µM IAA-94 completely abolished the PPC protection by PMA against myocyte death. CHE and IAA-94 had no effect on controls.


Figure 4
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  		Fig. 4 Dose–response of Cl– channel inhibition by indanyloxyacetic acid 94 (IAA-94) on ischemic preconditioning protection against cell death caused by a period of 45 min SI/60 min SR in rabbit cardiomyocytes. There was no difference in the percentage of dead cardiomyocytes among all groups before the long ischemia. Preconditioning cardiomyocytes with one cycle of 10 min SI/10 min SR prior to the long SI/SR episode significantly (*P<0.001) reduced the % of dead cardiomyocytes when compared to non-preconditioned cardiomyocytes (control), IPC+vehicle 27.4±1.5% vs. control+vehicle 48.4±2.5%. In cardiomyocytes treated either with 2.5 or 5 µM AA-94 (present 10 min prior and during the long SI/SR), IPC significantly (*P<0.001 vs. control; P = 0.8 vs. IPC+vehicle) reduced the % of dead cardiomyocytes after the long SI/SR (IPC+IAA-94 28.2±3.0 and 33.0±2.6%, respectively). However, 10 µM IAA-94 completely abolished the IPC protection (IPC+IAA94 46.9±2.1% *P<0 001 vs. IPC+vehicle) while it had no effect on controls (C+IAA-94 45.5±1.1% vs. C+vehicle, P>0.32). Data are expressed as mean±S.E.M. These preliminary findings confirm that 40% inhibition of RVD with 10 µM IAA-94 was sufficient to abolish the protection of IPC against myocyte ischemic cell death. Since in the cardiomyocyte model Cl– channels may account for at least 58% of myocyte RVD, 40% RVD inhibition by 10 µM IAA-94 may actually represent a 70% Cl– channel blockade.

 
3.3 Effect of Cl– channel inhibition on pharmacological preconditioning in isolated buffer-perfused hearts
To confirm the results obtained in freshly isolated cardiomyocytes, we tested the effect of Cl– channel inhibition on PPC induced with APNEA and PMA in an isolated buffer-perfused heart model. Data for LVDP, heart rate and coronary flow are presented in Table 1. There was no significant difference among all groups, in terms of LVDP, heart rate, and coronary flow, after 15 min stabilization, before ischemia or after 60 min reperfusion.


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  		Table 1 Measurements of left-ventricular developed pressure (LVDP, mmHg) and heart rate (HR, beats/min) and coronary flow (CF, ml/min)

 
Although two vehicles (ethanol and DMSO) were used in these studies, they were not concomitantly administered at the same time in the same experiment. DMSO was only used during the PPC protocol and ethanol was used during the long ischemic episode (Fig. 2). There was no difference among control hearts treated with either vehicle. Therefore, the data were pooled and shown in Fig. 6. IPC significantly (P<0.01) reduced infarct size within the myocardium at risk. Cl– channel inhibition with 10 µM IAA-94 blocked the protection of IPC. IAA-94 neither altered infarct size in controls (non-preconditioned cardiomyocytes, Fig. 6) nor prevented translocation of PKC{varepsilon} from the cytosolic to the particulate fraction (as determined by Western immunoblotting), Fig. 7. Activation of adenosine A1/A3 receptors with APNEA (1 µM) or stimulation of PKC with a total dose of 2 nmol PMA (admnistered as previously used by other investigators [28] to precondition rabbit hearts, (Fig. 2B) mimicked the IPC protection while it had no effect on vehicle-treated controls. APNEA induced protection was partially blocked while PMA induced protection was abolished by 10 µM IAA-94 (Fig. 6). IAA-94 did not increase infarct size in vehicle-treated controls.


Figure 6
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  		Fig. 6 Infarct size measurements expressed as percentage of risk area for each group of hearts. IPC significantly (*P<0.01 vs. control) reduced infarct size after 40 min regional myocardial ischemia and 60 min reperfusion (IPC+vehicle 7.6±1.8%) as compared with controls (control+vehicle 27.0±3.8%). Pretreatment with N6-2-(4-aminophenyl)ethyladenosine (APNEA, administered for 5 min followed by 10 min washout) or with phorbol 12-myristate 13-acetate (PMA, administered for 10 min followed by 20 min washout) significantly (*P<0.01 vs. control+vehicle) limited infarct size (APNEA 4.4±0.9% and PMA 9.2±1.1%), as compared with controls (control+vehicle). Inhibition of Cl– channels with 10 µM IAA-94 completely abolished the protection of IPC and PMA against infarction (IPC+IAA-94 25.7±4.8% and PMA+IAA-94 20.2±3.9%) and partially blocked APNEA induced protection (APNEA+IAA-94 15.6±2.0%), whereas it did not have an effect in treated controls (control+IAA-94 23.1±2.1%). Vehicle=ethanol or DMSO. Data are expressed as mean±S.E.M.

 

Figure 7
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  		Fig. 7 (A) The upper figure shows a representative SDS–PAGE Western immunoblotting which determine the subcellular distribution of PKC{varepsilon} in both the cytosolic and the particulate fractions of hearts subjected either to 60 min of aerobic perfusion (non-preconditioned hearts, control) or three consecutive cycles of 5-min regional myocardial ischemia and 10 min reperfusion, either in the presence or absence of IAA-94 (10 µM) treatment in IPC hearts. In control hearts, most of the PKC{varepsilon} is expressed in the cytosolic fraction while in IPC hearts most of the PKC{varepsilon} is expressed in the particulate fraction. In this study, homogenization of heart samples was performed using a polytron (15 s). Cytosolic and particulate fractions were obtained by differential centrifugation. A 10-µg amount of protein derived from both cytosolic and the particulate fractions was electrophoresed on a 12% denaturing gel for 3–4 h at 30 mA per gel. Proteins were transferred onto polyvinylidene fluoride (PVDF) microporous membranes (Millipore, USA). Gel transfer efficiency was assessed by Ponceau-S staining of PVDF membranes and gel retention was determined by Coomassie blue staining of each gel. PKC isoform {varepsilon} antibodies were obtained from BD Transduction Laboratories, USA. Immunoblots were developed with the use of a chemiluminescent kit (ECL kit, Amersham). The cytosolic and particulate fractions of comparing groups were run in the same gel. In each group (n = 4 hearts/group), Western immunoblottings were performed in two separate samples from each heart and the assay repeated twice. (B) The densitometry data shown in the graph demonstrates that most of the PKC{varepsilon} expressed in the particulate fraction in IPC hearts was the result of a translocation from the cytosolic fraction. In untreated control hearts, the expression of PKC{varepsilon} in control hearts (n = 4), was significantly (*P<0.001) higher in the cytosolic fraction (1.00±0.1) compared to the particulate fraction (0.59±0.06 and 0.73±0.07, respectively), while in IPC hearts (n = 4) the PKC{varepsilon} was significantly ({dagger}P<0.03) higher in the particulate fraction (1.32±0.11) compared to the cytosolic fraction (0.47±0.06). In addition, the PKC{varepsilon} significantly (#P<0.0001) decrease in the cytosolic fraction of IPC hearts when compared to cytosolic fraction in non-preconditioned hearts. Similar results were obtained when hearts were treated with 10 µM IAA-94 in the same fashion as in the necrosis studies. The density ratios (particulate/cytosolic density) were obtained in each blot from densities measured in each column. Data are expressed as mean±S.E.M.

 
A regression analysis preformed on the infarct size data showed no relationship between risk area and infarct size for all control (r = 0. 162) and all preconditioned (r = 0.237) hearts.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The objective of this study was to assess the participation of Cl– channels, as volume regulatory proteins, in PPC protection against myocardial necrosis caused by a long (index) ischemia and reperfusion period. In freshly isolated rabbit cardiomyocytes, activation of adenosine A1 or A1/A3 receptors with CCPA or APNEA, respectively, protected cardiomyocytes to the same extent as IPC against cell death (Fig. 5, panels A and B). Similar protection was also observed in isolated cardiomyocytes by stimulating PKC with PMA before the index ischemia (Fig. 5, panel C). This PPC-induced protection produced by selectively targeting activation of these recognized IPC pathways was abolished by inhibiting Cl– channels with IAA-94 in isolated cardiomyocytes.

To demonstrate that the role of Cl– channels on IPC and PPC observed in freshly isolated cardiomyocytes is also present in whole hearts, similar experiments were performed in an isolated buffer-perfused rabbit heart model. IPC significantly (P<0.01) reduced infarct size compared to non-preconditioned hearts (Fig. 6), as we have previously reported [2,27]. An equivalent degree of protection was also observed in hearts pharmacologically preconditioned with APNEA or PMA. This PPC induced protection was significantly inhibited by IAA-94 (Fig. 6). Moreover, PKC{varepsilon} translocation from the cytosolic to the particulate fraction was unaffected by 10 µM IAA-94 (Fig. 7). These findings support the hypothesis that Cl– channels are downstream of PKC. Demonstration of a role for Cl– channels in IPC in an intact (in vivo) heart model is precluded, at the present time, by the lack of pharmacodynamic data for in vivo administration of specific Cl– channel inhibitors. Furthermore, no specific Cl– channel activators are available.

Our results confirm that Cl– channels play important roles in the protection produced by classical IPC as we previously proposed [2]. Whether Cl– channels have any role in the second window of protection by IPC is unknown. In this study, activation of the adenosine A1/A3 receptors and PKC has been shown to contribute to the protection produced by IPC and PPC, as previously demonstrated by other investigators in cardiomyocytes and whole hearts [12,15,30,31]. Some investigators have shown that sustained reperfusion (2 or more hours) may increase infarct size of the region at risk presumably because it allows borderline viable tissue to be defined as necrotic or viable on tetrazolium chloride staining. These observations raise the possibility that infarct size might be underestimated at shorter reperfusion periods. In this study, we limited the reperfusion period to 60 min. This was because in our model (Langendorff buffer perfused heart) each heart is retrograde perfused with oxygenated Krebs–Henseleit buffer without albumin or any other colloids. The absence of albumin or colloids renders hearts susceptible to developing interstitial edema if perfused for long periods. We have found prolonged perfusion using this type of buffer produces substantial reduction of coronary flow, LVDP and heart rate but only after 3.5–4 h. The total duration of our three-cycle regional myocardial ischemial reperfusion preconditioning protocol was 175 min (2 h and 55 min, Fig. 2). We point out that, in this study, there was no need to extend the duration of the reperfusion period beyond 60 min since IPC and PPC protection was already blocked in the IPC or PPC groups by administration of the antagonists used in the study and non-preconditioned (control) hearts received the same ischemia/reperfusion protocol as IPC hearts, making for a fair comparison.

A recent report by Heusch et al. [4] indicated that IAA-94, when dissolved in DMSO, did not abolish the protection of IPC in buffer-perfused rabbit hearts. This contrasts with our previous work [2] in which we reported that a concentration of 10 µM of IAA-94 abolishes the protection induced by IPC in both the isolated rabbit ventricular myocyte and buffer-perfused whole heart models. We have noted methodological weaknesses in these previous studies [2,4] but have recently confirmed that blockade of Cl– channels with IAA-94 (10 µM) does indeed abolish the protection of IPC [5]. In response, Heusch et al. [32] have acknowledged that improved cellular volume control related to swelling-induced chloride channel activation is an attractive effector mechanism of ischemic preconditioning.

It is known that the signaling pathway(s) associated with IPC also regulate(s) the activity of Cl– channels. Angiotensin II AT1 receptors [27] and adenosine A1/A3 receptors [31,33], two proposed triggers of IPC, activate a Cl– conductance in rabbit sinoatrial cells [34] and in guinea pig cardiomyocytes [14], respectively. Furthermore, activation of PKC with PMA, which mimics the protection of IPC (Fig. 5, panel C and Fig. 6), has been shown to activate a Cl– current in feline [16] and guinea pig cardiomyocytes [17]. In addition, there is evidence that a swell-activated Cl– current is triggered via protein tyrosine kinases [35], another postulated signaling mediator of IPC [36]. Although the precise identity of these Cl– channels (e.g., whether swell-, adenosine-. angiotensin II-, or PKC-activated channels) is still unknown, it is plausible that they could be involved in regulating cell volume early during the long ischemia and thus preventing cell death (IPC protection) by reducing ischemic cell swelling.

Indeed, in a recent study performed in collaboration with Drs. Ganote and Armstrong (Johnson City, TN, USA) (unpublished observations), we found that IPC and pharmacological preconditioning both enhance the ability of rabbit cardiomyocyte to regulate cell volume, resulting in marked reduction of hypo-osmotic induced cell swelling. We also found that cardiomyocyte RVD was dependent on both Cl– channel and PKC activation. This enhanced cell volume regulation observed under oxygenated conditions was preserved during simulated ischemia as ischemically preconditioning cardiomyocytes showed much less cell swelling during the index ischemia. Further support from this study for the importance of cell volume regulation in IPC was that the osmotic equivalent found for the IPC protection (50 to 60 mOsM), in cardiomyocytes, approximated the osmotic load during the index ischemia [37,38].

It is known that Cl– channels play important roles in volume regulation in cardiomyocytes [6,18,39]. Thus, we used the inhibitory effect of IAA-94 on the RVD in cardiomyocytes as an index of Cl– channel inhibition. In the present study, we found that when cardiomyocytes were exposed to a hypo-osmotic (200 mOsM) solution, cell volume increased to a maximum level within 7 and 12 min and then substantially decreased at 30 min. We have previously demonstrated that the same hypo-osmotic stress for 10 min prior to the index ischemia was sufficient to trigger cardioprotection of similar magnitude to IPC in cardiomyocytes [2]. These observations on cell volume are in agreement with a previous study performed on neonatal rat cardiomyocytes [40] and adult rat hearts [41]. Cl– channel inhibition by IAA-94 produced a 40% mean reduction of myocyte RVD (Fig. 3). Our results indicate that Cl– channel activation contributes significantly to rabbit ventricular myocyte RVD and that 40% inhibition of this RVD was sufficient to block IPC and PPC protection.

The mechanism by which RVD works is still not completely understood. However, volume regulatory proteins such as Na+/K+–2Cl– co-transporter and K+/Cl– co-transporter, in addition to K+ and Cl– channels, are thought to participate in cell volume regulation [8,39,42]. Thus, one would not expect that blockade of Cl– channels by IAA-94 would totally inhibit RVD since these other volume regulatory proteins are not expected to be affected by IAA-94. In fact, blockade of Cl– channels with a different inhibitor, 9-AC (500 µM), blocked RVD by 57% (Fig. 3), which appears to be the practical upper limit of the effect of Cl– channels on RVD. The relative role of the other volume regulatory proteins in IPC remains to be evaluated.

Since we have shown that IAA-94 is effective in substantially inhibiting RVD in cardiomyocytes and in blocking the protective effect of IPC and PPC, and since Cl– channel activation accounts for at least 57% of RVD in rabbit cardiomyocytes, one can infer that blockade of IPC and PPC is the result of effective Cl– channel inhibition. These findings are supported by our recent observations that IPC enhances cell volume regulation which substantially reduces subsequent myocyte ischemic swelling (unpublished data). The achievement of blockade of PPC with stimulation at both the cell membrane receptor level (adenosine) and intracellular mediator level (PKC) by IAA-94, with further demonstration that PKC{varepsilon} translocation by IPC is not affected by IAA-94, indicates a position for Cl– channel activity downstream of PKC, providing support for an end-effector role in IPC. The specific functional identity of Cl– channel(s) (swell-, adenosine-, angiotensin II-, or PKC-activated) involved in ischemic preconditioning, and their respective molecular identities, remain to be determined. Furthermore, as cardiomyocytes must maintain bulk electroneutrality across the plasma membrane to survive, an efflux of Cl– across the sarcolemma must be matched by an efflux of cations. Most likely, the key counter ion to chloride is potassium, the most abundant cation in the intracellular milieu.

Multiple end-effectors for IPC may well exist. The fact that diazoxide induced protection against infarction could not be abolished by IAA-94 in Langendorff perfused rabbit hearts [2] suggests an independent protective effect of mitochondrial KATP channels.

We conclude that Cl– channels play an important physiological role in the cardioprotection of IPC and PPC and are downstream of PKC, consistent with an end effector role. To be convincing, the claim for end effector status must be linked to an effect that is clearly cardioprotective. Our recent observations that IPC enhances cell volume regulation which substantially reduces cardiomyocyte swelling during subsequent index (long) ischemia period (unpublished data) suggest that the cardioprotective effect may be through improved cell volume regulation.

Time for primary review 29 days.


   Acknowledgements
 
We would like to thank Erin Williamson for her assistance on performing the digital video recordings and cell volume measurements. We also would like to thank Dr. Alina Hinek for her assistance in digesting and culturing cardiomyocytes. M.B. was supported by a Hospital for Sick Children Research Training Scholarship. This study was supported by a Ontario Heart and Stroke Foundation Grant (#T-4179).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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