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(Circulation Research. 2004;95:325.)
© 2004 American Heart Association, Inc.

Cellular Biology

Selective Inhibition of Inward Rectifier K⁺ Channels (Kir2.1 or Kir2.2) Abolishes Protection by Ischemic Preconditioning in Rabbit Ventricular Cardiomyocytes

Roberto J. Diaz^*, Carsten Zobel^*, Hee Cheol Cho, Michelle Batthish, Alina Hinek, Peter H. Backx, Gregory J. Wilson

From the Divisions of Cardiovascular Research (R.J.D., M.B., A.H., G.J.W.) and Pathology (G.J.W.), Research Institute, The Hospital for Sick Children; the Departments of Physiology (H.C.C., M.B., P.H.B., G.J.W.) and Medicine (P.H.B.), The University of Toronto; and the Division of Cardiology (C. Z., P.H.B.), University Health Network, Toronto, Canada. Present address of C.Z. is Laboratory of Muscle Research and Molecular Cardiology, Clinic III for Internal Medicine, University of Cologne, Cologne, Germany.

Correspondence to Dr Gregory J. Wilson, Division of Cardiovascular Research, McMaster Bldg, Rm 7019C, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario M5G 1X8, Canada. E-mail Diazport{at}sickkids.ca

	Abstract

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Volume regulatory Cl^– channels are key regulators of ischemic preconditioning (IPC). Because Cl^– efflux must be balanced by an efflux of cations to maintain cell membrane electroneutrality during volume regulation, we hypothesize that I_K1 channels may play a role in IPC. We subjected cultured cardiomyocytes to 60-minute simulated ischemia (SI) followed by 60-minute of simulated reperfusion (SR) and assessed percent cell death using trypan blue staining. Ischemic preconditioning (10-minute SI/10-minute SR) significantly (P<0.0001) reduced the percent cell death in nontransfected cardiomyocytes [IPC_CM 18.0±2.1% versus control (C_CM) 48.3±1.0%]. IPC protection was not altered by overexpression of the reporter gene (enhanced green fluorescent protein, EGFP). However, overexpression of dominant-negative Kir2.1 or Kir2.2 genes using adenoviruses (AdEGFPKir2.1DN or AdEGFPKir2.2DN) encoding the reporter gene EGFP prevented IPC protection [both IPC_CM+AdEGFPKir2.1DN 45.8±2.3% (mean±SEM) and IPC_CM+AdEGFPKir2.2DN 47.9±1.4% versus IPC_CM; P<0.0001] in cultured cardiomyocytes (n=8 hearts). Transfection of cardiomyocytes with AdEGFPKir2.1DN or AdEGFPKir2.2DN did not affect cell death in control (nonpreconditioned) cardiomyocytes (both C_CM+ AdEGFPKir2.1DN 45.8±0.7% and C_CM+AdEGFPKir2.2DN 46.2±1.3% versus C_CM; not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after I_K1 blockade with 20 µmol/L BaCl₂ plus 1 µmol/L nifedipine (to prevent Ba²⁺ uptake). Nifedipine alone neither protected against ischemic injury nor blocked IPC protection. Our findings establish that I_K1 channels play an important role in IPC protection.

Key Words: ischemic preconditioning • cardiomyocytes • ischemia • potassium channels • gene transfer

	Introduction

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Ischemic preconditioning (IPC) is a strong native protective mechanism that limits the amount of necrosis during a subsequent prolonged ischemia and reperfusion period.¹ Although much is known about the cell membrane receptors and intracellular pathways involved in IPC,^2–5 very little is known about the end effector(s) of IPC. Finding the end effector(s) of IPC is important to guide the development of new specific therapeutic approaches to protect the ischemic myocardium.

Our recent results in cardiomyocytes established that blockade of Cl^– channels inhibits cell volume regulation following hypoosmotic stress (regulatory volume decrease, RVD),^6,7 which results in loss of protection against necrosis induced by both ischemic⁸ and pharmacological preconditioning.⁶ Because accumulation of metabolic by-products is responsible for increasing the intracellular osmotic load in ischemia and thereby increasing water uptake in cardiomyocytes, and because IPC only produces a small reduction in the estimated osmotic load in ischemic cardiomyocytes,⁷ we hypothesized that enhanced cell volume regulation as a result of Cl^– channel activation plays a significant role in the protection induced by IPC. Consistent with this hypothesis, IPC substantially reduces hypoosmotic-induced and ischemia-induced cell swelling in cardiomyocytes.⁷ In order for Cl^– ion movement to effectively regulate cellular volume, electroneutrality dictates that positively charged ions must accompany chloride ions. Although any cation could act as a counter-ion for Cl^–, K⁺ is the most abundant intracellular cation. In addition, extracellular K⁺ increases by 2- to 3-fold during ischemia as a result of K⁺ efflux from cardiomyocytes.⁹ Passage of K⁺ out of the cardiomyocyte could occur through multiple K⁺ channels. Of these, voltage-gated channels such as for transient outward currents and delayed rectifier current are only open for relatively brief periods.¹⁰ Moreover, myocardium becomes electrically quiescent (ie, approaching resting membrane potential) during severe and persistent ischemia, thus minimizing the activation of voltage-gated K⁺ currents. On the other hand, inward rectifier potassium (IRK) currents remain open during quiescence that increases during ischemia as a result of elevated extracellular K⁺ levels. Several IRK currents exist in heart: strongly rectifying currents (I_K1), G protein-regulated currents (I_KAch), ATP-sensitive currents (I_KATP), and Na⁺-sensitive currents (I_KNa) K⁺ channels.^11,12 In the heart, I_KAch is primarily expressed in atrial and nodal cardiomyocytes,^10,11 whereas I_KNa channels only open when [Na⁺]_i reaches a very high level (30 mmol/L).^13,14 Thus, I_K1 and sarcolemmal I_KATP (sI_KATP) appear to be the most attractive candidates to allow K⁺ to move with Cl^– to regulate cardiomyocyte volume.

In the present study, we assessed whether I_K1 channels are involved in the protection induced by IPC against ischemia/reperfusion injury in fresh and cultured rabbit ventricular cardiomyocytes. We found that pharmacological inhibition of I_K1 abolished IPC cardioprotection and this finding does not appear to be related to nonspecific inhibition affecting sI_KATP. Moreover, knockdown of I_K1 channels by adenovirus transfection of either dominant-negative Kir2.1(C122S) gene (AdEGFPKir2.1DN) or the dominant-negative Kir2.2(C123S) gene (AdEGFPKir2.2DN) completely blocked the protection of IPC against cell death caused by the subsequent long period of simulated ischemia and reperfusion. To our knowledge, no previous published study has established a role for the I_K1 channel as a potential end effector in the cardioprotection of IPC.

	Materials and Methods

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Isolation of Ventricular Cardiomyocytes
Calcium tolerant-isolated cardiomyocytes used in these studies were obtained from New Zealand white rabbit (weight=3 to 3.5 kg) hearts by enzymatic dissociation using a method previously described^8,15 (for details of cardiomyocyte isolation, see the expanded Materials and Methods in the online data supplement available at http://circres.ahajournals.org). Animal protocols conformed to the Guide for the Care and Use of Laboratory Animals published by NIH (publication No. 85-23, 1996). All animals were obtained from Reimens Fur Ranches Ltd. (St. Agatha, Ontario, Canada).

Recombinant Adenoviruses
To define the participation of the I_K1 channels in the protection induced by IPC, we used three different type-5 recombinant adenoviruses, AdEGFP, AdEGFPKir2.1(C122S) (AdEGFPKir2.1DN), and AdEGFPKir2.2(C123S) (AdEGFPKir2.2DN), which have been extensively studied in our laboratory.^16,17 These recombinant adenoviruses encode a bidirectional construct, which includes a marker protein (enhanced green fluorescence protein, EGFP) used to confirm the transfer of each gene into cardiomyocytes. However, only the AdEGFPKir2.1DN and AdEGFPKir2.2DN viruses have a dominant-negative (DN) mutant, either the Kir2.1(C122S) or Kir2.2(C123S) gene, respectively, to knockdown the I_K1 channel (for details, see online data supplement).

Single Cell Electrophysiology Studies in Isolated and Cultured Cardiomyocytes
After ventricular cardiomyocytes were prepared as described, they were cultured on laminin-coated glass coverslips at a density of 4 to 5x10⁴ cells/35-mm dish in serum-free culture media 199 with Earle’s salts. After allowing 2 hours for cardiomyocyte attachment, the culture media was exchanged and the cells were either used directly for patch-clamp recordings (see later) or infected either with AdEGFP 25 TCID₅₀/cardiomyocyte, AdEGFPKir2.1DN 5 TCID₅₀/cardiomyocyte, or AdEGFPKir2.2DN 5 TCID₅₀/cardiomyocyte for patch-clamp recording after 48 hours in culture. Transfection efficiency using these viruses was 98% as assessed by EGFP phenotypic expression. After either 2 or 48 hours in culture, glass coverslips containing laminin-attached cardiomyocytes were transferred into a small recording chamber mounted on the stage of an inverted microscope (Olympus IX 70) and whole-cell patch-clamp recording of I_K1 current was performed as we have previously described¹⁶ (see online data supplement).

Ischemic Preconditioning Studies in Freshly Isolated Cardiomyocytes
After isolation (n=4 hearts), cardiomyocytes were divided in several groups and subjected to the following protocol (also see the online Figure in the online data supplement). First, all cardiomyocytes were transferred into separate wells in a multi-well dish and then incubated at 37°C in a 100% O₂ atmosphere for 30 minutes (stabilization period). Then, preconditioned cardiomyocytes were subjected to 10 minutes of simulated ischemia (SI, pelleting cells under an oil layer in a 1.5-mL Eppendorf tube and followed by incubation in a 37°C water bath) followed by 20 minutes of simulated reperfusion (SR, resuspension of cell pellet in oxygenated S-MEM medium). Control (nonpreconditioned) and baseline (incubation in S-MEM medium at 37°C without SI or SR) cardiomyocytes were incubated in S-MEM medium for another 30 minutes to match each group protocol in time. Next, each group of cardiomyocytes was subjected to 45-minute SI/60-minute SR. I_K1 channels were blocked with 20 µmol/L Ba²⁺ (used as BaCl₂), given during the 45-minute SI period. To avoid toxic effects and any other nonspecific effect derived from Ba²⁺ uptake into cardiomyocytes via L-type calcium channels, we concurrently administered the L-type calcium channel blocker nifedipine (1 µmol/L). The Ba²⁺ concentration used was based on the concentration-response electrophysiology study performed in freshly isolated rabbit ventricular cardiomyocytes (Figure 1) with the Ba²⁺ concentration estimated 20 µmol/L chosen to produce an estimated 80% to 90% inhibition of I_K1 under the conditions of simulated ischemia (see online data supplement for details). Cell viability (as measured by percentage of dead cardiomyocytes) was determined after 45-minute SI and 60-minute SR by trypan blue staining as previously described.⁸ We counted at least 300 cardiomyocytes (live and dead) at each time point in each group.

View larger version (28K): [in this window] [in a new window]   Figure 1. Left, Typical barium-subtracted IK1 currents (raw single traces) elicited by a voltage step to –110 mV for 500 or 2000 ms from a holding potential of –40 mV in freshly isolated cardiomyocytes are shown together with their respective averaged (mean±SEM) IK1 current densities (bar graph). BaCl2 (10 µmol/L) was added when indicated. *P<0.05 vs control. Right, Barium-subtracted IK1 currents (raw traces) elicited by a voltage step to –110 mV for 500 or 2000 ms from a holding potential of –40 mV in 48-hour cultured cardiomyocytes infected with either AdEGFP, AdEGFPKir2.1DN, or AdEGFPKir2.2DN are shown together with their respective averaged IK1 current densities (bar graph). *P<0.05 vs AdEGFP.

Primary Culture of Cardiomyocytes
Ventricular cardiomyocytes were cultured using a method we have previously described^6,7 (for details see online data supplement).

Ischemic Preconditioning Studies in Barium-Blocked I_K1 Channels in Cultured Cardiomyocytes
After 48 hours in culture, cardiomyocytes were subjected to the following experimental protocol (also see the online Figure in the online data supplement). After 30-minute stabilization in culture medium 199, cardiomyocytes were subjected to 60-minute simulated ischemia (SI) and 60-minute simulated reperfusion (SR). Prolonged ischemia was simulated by incubating cardiomyocytes in a severely hypoxic (8% O₂), HEPES-based solution (SI solution) containing (in mmol/L) 139 NaCl, 12.0 KCl, 0.5 MgCl₂, 0.9 CaCl₂, 5 HEPES, 10 2-deoxy-D-glucose, and 20 lactate, 0.1% BSA (pH 6.5, at 37°C), in a 100% N₂ atmosphere. Reperfusion was simulated by incubation of cardiomyocytes in oxygenated medium 199 (pH 7.4, at 37°C). KCl concentration was increased from physiological levels (4.7 mmol/L) to 12 mmol/L to simulate the high K⁺ concentration environment in myocardium during ischemia; lactate was added to the SI solution to simulate the build up of metabolic end products and low pH during ischemia. Cardiomyocytes were preconditioned by incubating them in an IPC solution (a solution similar to the SI solution with the exception that it has no lactate added, pH 7.4, and contained 4.7 mmol/L of KCl) for 10 minutes followed by a 10-minute SR before the 60-minute SI. Control cardiomyocytes were not preconditioned. I_K1 channels were blocked with Ba²⁺ (20 µmol/L BaCl₂) administered during the 60-minute SI and in the presence of 1 µmol/L nifedipine. All cardiomyocytes (control, preconditioned, and baseline) received the same treatments (BaCl₂+nifedipine, nifedipine alone, or no drug). Baseline cardiomyocytes were incubated in medium 199 for the duration of protocols to determine the effect of drugs on cell viability under oxygenated conditions. Cardiomyocyte viability was determined by trypan blue staining before ischemia and end of 60-minute SI/60-minute SR, as described in the online data supplement.

Ischemic Preconditioning Studies in I_K1 Channel Knockdown Cultured Cardiomyocytes
After 2 hours in culture, cardiomyocytes were infected with one of three recombinant adenoviruses: AdEGFP, AdEGFPKir2.1DN, or AdEGFPKir2.2DN. Culture dishes that did not meet the criteria for effective rate of cell transfection (98% of cardiomyocytes showing EGFP fluorescence) were not used for the study. Transfected cardiomyocytes were included in nine different groups: (1) EGFP-transfected control (C_CM+AdEGFP); (2) EGFP-transfected preconditioned (IPC_CM+AdEGFP); (3) EGFP-transfected baseline (Baseline_CM+AdEGFP); (4) AdEGFPKir2.1DN-transfected control (C_CM +AdEGFPKir2.1DN); (5) AdEGFPKir2.1DN-transfected preconditioned (IPC_CM+AdEGFPKir2.1DN); (6) AdEGFPKir2.1DN-transfected baseline (Baseline_CM+AdEGFPKir2.1DN); (7) AdEGFPKir2.2DN-transfected control (C_CM +AdEGFPKir2.2DN); (8) AdEGFPKir2.2DN-transfected preconditioned (IPC_CM+AdEGFPKir2.2DN); and (9) AdEGFPKir2.2DN-transfected baseline (Baseline_CM+AdEGFPKir2.2DN). Matched-control cardiomyocytes were used for nontransfected groups (preconditioned, control, and baseline). All cardiomyocytes were subjected to the same standard SI/SR and cell viability protocols described above for the barium studies in cultured cardiomyocytes. To establish an unbiased reproducibility of the results, we performed these studies in two stages: (1) nonblinded experiments to the observer (n=4 hearts) and (2) blinded experiments to the observer (n=4 hearts). Because the same outcome was observed in the two stages, we pooled these data.

Effect of Sarcolemmal I_KATP Channel Inhibition on Ischemic Preconditioning in Cardiomyocytes
To determine if sI_KATP channels may also have a role in IPC protection against ischemia/reperfusion injury in freshly isolated and 48-hour cultured cardiomyocytes, we inhibited sI_KATP channels with 30 µmol/L HMR1098 (a gift from Adventis Pharma Deutschland GmbH, Germany). All cardiomyocytes were subjected to the same control, preconditioning and baseline protocols described for the barium experiments. HMR1098 was added to the medium just before the 60-minute SI. Cardiomyocyte viability was determined as described in the barium studies.

Statistical Analysis
All data are summarized and expressed as mean±SEM. All cardiomyocyte data were first tested for normality and homogeneity of variance. ANOVA analysis followed by Scheffe post hoc test was performed to determine significant differences (P<0.05) among groups and between two groups, respectively. Where the criteria for parametric analysis were not met, we performed a nonparametric analysis (Kruskal-Wallis followed by Mann-Whitney U) to determine whether a significant difference (P<0.05) existed between two groups.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of I_K1 Conductance by Barium and Dominant-Negative Transfection With AdEGFPKir2.1DN
In these studies, inward rectifier K⁺ currents were inhibited in two ways. Inward rectifier K⁺ currents (I_K1) measured at –110 mV were inhibited by 90% at steady state with BaCl₂ (10 µmol/L) as shown in Figure 1 (see "Discussion" and online data supplement for an estimate of I_K1 inhibition of 86% to 87% under simulated ischemia at 20 µmol/L Ba²⁺ in the preconditioning protection studies). As expected, I_K1 current density decreased under culture conditions (Figure 1). Cardiomyocyte I_K1 conductance elicited at –110 mV was reduced by approximately 50% (control fresh cardiomyocytes –17.9±1.09 pA/pF versus control 48 hour-cultured cardiomyocytes –8.35±0.87 pA/pF; P<0.05) after 48 hours in culture. I_K1 currents were further reduced by transfection of cultured ventricular cardiomyocytes with recombinant adenovirus expressing Kir2.1DN or Kir2.2DN genes along with the EGFP. Adenovirus expressing EGFP alone (AdEGFP) were used as controls and AdEGFP had no effect on I_K1 current density after 48 hours in cultured cardiomyocytes compared with nontransfected control cardiomyocytes cultured for the same period of time (Figure 1). Transfection with either AdEGFPKir2.1DN or AdEGFPKir2.2DN resulted in a significant reduction (50%) of I_K1 current density (AdEGFPKir2.1DN –3.62±0.31 or AdEGFPKir2.2DN –4.49±0.21 versus AdEGFP –7.95±0.53; P<0.05) (Figure 1). BaCl₂, administered at a concentration of 10 µmol/L to AdEGFP transfected cardiomyocytes, blocked I_K1 current density to about the same extent as in freshly isolated cardiomyocytes (data not shown). Neither Ba²⁺ nor AdEGFPKir2.1DN/AdEGFPKir2.2DN transfection had any effect on average membrane potential (see online data supplement for details).

Blockade of Ischemic Preconditioning Protection by Pharmacological Inhibition of I_K1 Channels in Ventricular Cardiomyocytes
The effects of 20 µmol/L Ba²⁺, sufficient to inhibit I_K1 current by about 86% to 87% (see Discussion and online data supplement) on protection against cell death by IPC in freshly isolated cardiomyocytes is shown in Figure 2A. As expected, there was no difference in the percentage of dead cardiomyocytes either after the initial stabilization period or immediately before the long SI among all the groups. Similarly, no difference among all the baseline groups (with and without barium) was found with regard to the percentage of dead cardiomyocytes at the end of the experimental period. As we have previously shown,⁸ ischemic preconditioning (IPC) significantly (P<0.0001) reduced the percentage of dead cardiomyocytes after 45 minutes of SI and 60 minutes of SR, when compared with untreated control cardiomyocytes [IPC versus control (C)]. This amount of protection conferred by IPC in freshly isolated cardiomyocytes (46% reduction in mortality; Figure 2A) was similar to the amount of IPC protection observed in 48-hour cultured cardiomyocytes (49% reduction in mortality, Figure 2B) and with single-cycle IPC protection in vivo in rabbit hearts (42% reduction in infarct size)¹⁸ previously reported from our laboratory. Blockade of I_K1 channels with 20 µmol/L BaCl₂ (in the presence of 1 µmol/L nifedipine) significantly (P<0.0001) reduced the protection induced by IPC (IPC+BaCl₂), while having no effect on treated control cardiomyocytes (C+BaCl₂). Nifedipine alone did not protect against ischemia or block IPC-induced protection (C+NF and IPC+NF, respectively). None of the treatments used in these protocols affected cell mortality in time-matched nonischemic baseline cardiomyocytes (data not shown). Similar results were obtained in 48-hour cultured cardiomyocytes (Figure 2B).

View larger version (28K): [in this window] [in a new window]   Figure 2. These graphs show the effect of Ba2+-induced inhibition of IK1 channel activity on ischemic preconditioning (IPC) in freshly isolated (A) and 48-hour cultured cardiomyocytes (B). A, In freshly isolated cardiomyocytes, IPC significantly reduced the percent of dead cardiomyocytes after 45-minute SI/60-minute SR [IPC 26.3±1.1% vs control (nonpreconditioned) 47.8±1.1%]. BaCl2 (20 µmol/L), in the presence of 1 µmol/L nifedipine (NF), blocked IPC protection (IPC+BaCl2 46.7±1.3% vs IPC), although it did not affect controls (C+BaCl2 48.2±0.9% vs C, P=0.80). Nifedipine alone neither affected controls (C+NF 48.3±1.2% vs C, P=0.79) nor blocked IPC protection (IPC+NF 26.9±1.9% vs C+NF). *P<0.0001 vs C, C+NF; #P<0.0001 vs IPC. B, In 48-hour cultured cardiomyocytes, IPC significantly reduced the percent of dead cardiomyocytes after 60-minute SI/60-minute SR [IPCCM 22.2±1.8% vs control (CCM) 52.2±1.3%]. BaCl2 (20 µmol/L), in the presence of nifedipine (NF), abolished IPC protection (IPCCM +BaCl2 46.6±3.2% vs IPCCM), while it did not affect mortality in controls (CCM +BaCl2 50.9±1.4% vs CCM). Nifedipine alone neither affected controls (CCM+NF 50.8±1.2% vs CCM) nor blocked IPC protection (IPCCM+NF 21.5±2.8% vs CCM+NF). *P<0.0001 vs CCM, CCM+NF; #P<0.0001 vs IPC. All data presented in A and B were obtained from 4 to 5 cardiomyocyte isolations (n=4 to 5 hearts) in each cell model, respectively. Data are expressed as mean±SEM.

Blockade of Ischemic Preconditioning Protection by Targeted Knockdown of I_K1 Channel Subunits (Kir2.1 and Kir2.2) in Cultured Ventricular Cardiomyocytes
Because barium may not be completely specific for I_K1 channel blockade,¹⁹ we utilized a more definitive molecular approach to confirm the role of I_K1 channels in IPC. Cardiomyocytes were transfected with either a dominant-negative gene (AdEGFPKir2.1DN or AdEGFPKir2.2DN) to knockdown I_K1 channels or the AdEGFP gene as control. The percentage of dead cardiomyocytes did not differ among the different groups before the long SI/SR (Figure 3). In addition, the percentage of dead cardiomyocytes did not change over time (160 minutes) when cardiomyocytes were kept in oxygenated conditions at 37°C (data not shown). Knockdown of I_K1 channels by either AdEGFPKir2.1DN or AdEGFPKir2.2DN abolished IPC protection (Figure 3) when compared with cardiomyocytes transfected with the reporter gene (AdEGFP) alone or with nontransfected cardiomyocytes. Transfection of cardiomyocytes with AdEGFP neither changed cardiomyocyte mortality in controls (nonpreconditioned) nor affected IPC protection in cardiomyocytes.

View larger version (18K): [in this window] [in a new window]   Figure 3. This graph shows the effect of IK1 channels knockdown by adenovirus mediated transfection of mutants Kir2.1(C122S) gene (AdEGFPKir2.1DN) and Kir2.2(C123S) gene (AdEGFPKir2.2DN) on ischemic preconditioning (IPC) protection in 48-hour cultured cardiomyocytes. IPC significantly reduced cell mortality after 60-minute SI/60-minute SR in nontransfected cardiomyocytes [IPCCM 18.0±2.1% vs nontransfected controls (CCM) 48.3±1.0%]. IK1 channel knockdown with transfection of a dominant-negative gene, either AdEGFPKir2.1DN or AdEGFPKir2.2DN, abolished IPC protection (IPCCM+AdEGFPKir2.1DN 45.8±2.3% and IPCCM+ AdEGFPKir2.2DN 47.9±1.4% vs IPCCM, respectively), although it did not affect controls (CCM+AdEGFPKir2.1DN 45.8±0.7% and CCM+AdEGFPKir2.2DN 46.2±1.3% vs CCM, respectively). Overexpression of the reporter gene (AdEGFP) alone did not affect IPC protection or cell mortality in controls (IPCCM+AdEGFP 18.0±1.3% vs CCM+AdEGFP 48.9±0.9%). *P<0.0001 vs CCM, CCM+AdEGFP, IPCCM+AdEGFPKir2.1DN, IPCCM+ AdEGFPKir2.2DN. All pooled data were obtained from eight cardiomyocyte isolations (n=8 hearts). Data are expressed as mean±SEM.

Effect of Sarcolemmal I_KATP Channel Blockade on Ischemic Preconditioning Cardioprotection
To determine if in our cardiomyocyte culture model sI_KATP channels were important for IPC, we tested whether blockade of these channels eliminated IPC protection in both freshly isolated cardiomyocytes and 48-hour cultured cardiomyocytes. Selective blockade of sI_KATP channels with 30 µmol/L HMR1098 had no effect on the protection by IPC, as shown in Figure 4A and 4B. HMR1098 alone did not affect cell viability in baseline cardiomyocytes in either cell model (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 4. These graphs show the effect of sIKATP channel blockade by HMR1098 (HMR) on ischemic preconditioning (IPC) protection in freshly isolated (A) and 48-hour cultured cardiomyocytes (B). A, In freshly isolated cardiomyocytes, IPC significantly reduced the percent of dead cardiomyocytes after 45-minute SI/60-minute SR [IPC 25.8±2.1% vs control (nonpreconditioned, C) 49.1±0.9%]. HMR1098 (30 µmol/L) neither affected IPC protection (IPC+HMR 25.7±2.5% vs IPC) nor cell mortality in controls (C+HMR 48.9±2.6% vs C, P=0.94). *P<0.0001 vs IPC or IPC+HMR; #P=0.96 vs IPC. B, In 48-hour cultured cardiomyocytes, IPC significantly reduced the percent of dead cardiomyocytes after 60-minute SI/60-minute SR [IPCCM 17.6±1.6% vs control (CCM) 45.6±0.7%]. HMR1098 (30 µmol/L) neither affected IPC protection (IPCCM+HMR 21.4±4.4% vs IPCCM, P=0.74) nor cell mortality in controls (CCM+HMR 48.4±0.6% vs CCM, P=0.87). *P<0.0001 vs IPCCM or IPCCM+HMR; #P=0.97 vs IPCCM. All data presented in A and B were obtained from four cardiomyocyte isolations (n=4 hearts) in each cell model. Data are expressed as mean±SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study defines a role for I_K1 channels in the protection produced by ischemic preconditioning (IPC). We show that inhibition of I_K1 channels with Ba²⁺ or genetic knockdown of I_K1 channels using a dominant-negative approach abolishes IPC protection against ischemic cell death in cultured rabbit ventricular cardiomyocytes.

An initial concern in using Ba²⁺ to inhibit I_K1 currents was the possible toxic effects of this agent on cardiomyocytes. Indeed, in pilot studies, we observed barium-associated increase of necrosis in cardiomyocytes under baseline conditions. This necrosis above baseline levels was prevented by treatment with nifedipine to block L-type calcium channels. Importantly, nifedipine did not interfere with the protective response to preconditioning nor did it produce a protective effect under control conditions.

Although Ba²⁺ is a relatively nonspecific blocker of K⁺ currents, it is unlikely that the inhibition of IPC protection by Ba²⁺ application in our cardiomyocyte studies is a consequence of blockade of voltage-gated K⁺ channels because the cardiomyocytes were electrically quiescent. On the other hand, Ba²⁺ at levels used in our studies (20 µmol/L) can inhibit (40% to 50%) of I_KATP current at hyperpolarized membrane potentials (–100 mV) although there is minimal inhibition at membrane potentials close to the resting potential.¹⁹ Consistent with this conclusion, we found that inhibition of sI_KATP with HMR1098 did not abolish IPC protection under our experimental conditions, supporting that Ba²⁺-induced blockade of IPC was not the result of sI_KATP blockade by Ba²⁺. Because Ba²⁺ is known to be a potent inhibitor of cardiac I_K1 currents, our present cardiomyocyte studies suggest that I_K1 channels play a pivotal role in IPC.

Adenovirus overexpression of either Kir2.1DN or Kir2.2DN dominant-negative channels was equally effective at reducing I_K1 currents in cultured rabbit ventricular cardiomyocytes. The equivalent reduction of I_K1 currents observed with overexpression of these two dominant-negative genes are consistent with our previous study,¹⁶ suggesting that cardiac I_K1 channels appear to be predominantly composed of heterotetramers of Kir2.1 and Kir2.2 channels. Furthermore, because we previously showed that overexpression of these two dominant-negative genes did not affect voltage-gated K⁺ currents in cultured cardiomyocytes,¹⁶ it would appear that these dominant-negative genes caused specific reductions in I_K1 currents.

Because the amplitude of the I_K1 current is diminished in cardiomyocytes after 2 to 3 days in culture when compared with the I_K1 current in freshly isolated cardiomyocytes,^20–22 one may question whether 48-hour cultured cardiomyocytes are appropriate for studying the role of I_K1 channel in IPC. In fact, despite a significant reduction of I_K1 current density to about half that in control cardiomyocytes overexpressing the EGFP gene after 48 hours in culture (Figure 1), IPC protection against ischemic cell death was as potent as observed in freshly isolated cardiomyocytes (Figures 2A and 4A). These results suggest that a critical level of I_K1 current is necessary for IPC to occur and that only reductions below this critical "threshold" level will lead to inhibition of IPC. Consistent with this suggestion, the absolute level of I_K1 current remaining in freshly isolated cardiomyocytes after inhibition with Ba²⁺ during our IPC studies is estimated to be similar to that present in cultured cardiomyocytes after dominant-negative knockdown of I_K1. Specifically (also see online data supplement for further details), by accounting for the voltage-dependence of I_K1 block by Ba²⁺ as well as the changes in resting membrane potential of cardiomyocytes in our IPC studies (ie, –65 to –50 mV) due to K⁺ accumulation, I_K1 currents in our IPC studies with Ba²⁺ are estimated to be reduced by about 86% to 87% compared with a 75% to 80% reduction in I_K1 density recorded in cultured cardiomyocytes expressing dominant-negative Kir2.1/2 genes. It is possible that the extent of I_K1 inhibition necessary to block IPC protection may depend on the experimental conditions used (ie, fresh versus cultured cardiomyocytes), possibly as a result of a change in threshold of I_K1 current required to support IPC. Further studies will be necessary to fully unravel the dependence of IPC on the absolute level of I_K1 activity.

The results of the present study contrast with previous findings by Schultz et al²³ who showed that a bolus intravenous administration of terikalant, an I_K1 channel blocker, 15 minutes before the long ischemia in vivo did not block ischemic preconditioning in anesthetized rats. This discrepancy could be due either to species differences (rat versus rabbit) in the role of I_K1 channels or, more likely, to inadequate I_K1 blockade by terikalant in that study. Indeed, terikalant is a relatively weak blocker of inward rectifier K⁺ channels in cardiomyocytes²⁴ and Schultz et al²³ could only block 39% of I_K1 current in guinea-pig ventricular cardiomyocytes with 30 µmol/L terikalant, which was the concentration selected to avoid simultaneous blockade of I_K1 and sI_KATP channels. As described earlier, our data suggest that this level of inhibition is insufficient to block IPC protection. Furthermore, terikalant also blocks I_Kr at micromolar concentrations,²⁵ although this is unlikely to explain the inability of terikalant to block IPC-mediated cardioprotection.²³

The regulatory signaling underlying the protection produced by I_K1 currents against ischemic injury in our studies could involve several mechanisms. We have recently demonstrated that enhanced cell volume regulation is a key mechanism of IPC cardioprotection.⁷ Combining these results with our previous observations that Cl^– channel inhibition blocks the protection against myocardial necrosis of both ischemic⁸ and pharmacological⁶ preconditioning suggests that outward Cl^– movement is involved in protecting cardiomyocytes from ischemic injury. Because ionic balance (electroneutrality) is required for volume regulation, our results suggest that I_K1 currents are required for IPC as a consequence of contribution to volume regulation. The direct contribution of I_K1 channels to cell volume regulation in cardiomyocytes remains to be investigated and is beyond the scope of the present study. Recently, Irie et al²⁶ reported that preconditioning in single guinea pig cardiomyocytes produced a significant reduction in the average time for induction (ie, early activation) of outward K⁺ current in response to simulated ischemia. These results are consistent with I_K1 as a source for the outward K⁺ movement they have demonstrated.

To the extent to which enhanced cell volume regulation during ischemic stress is responsible for IPC protection against ischemia/reperfusion injury, transsarcolemmal K⁺ efflux in quiescent cultured cardiomyocytes could arise from background K⁺ currents other that I_K1, most notably sI_KATP. Addressing this issue, we found that sI_KATP blockade with HMR1098 did not inhibit IPC in freshly isolated or 48-hour cultured quiescent cardiomyocytes. Under in vivo conditions, the role of sI_KATP in IPC has been controversial. Consistent with the present study, blockade of sI_KATP with HMR1883, of which HMR 1098 is the sodium salt, did not abolish the protective effect of IPC in rabbit hearts.²⁷ Nevertheless, it has been shown that in Kir6.2 knockout mice lacking sI_KATP channels, IPC failed to protect from myocardial necrosis²⁸ and failed to improve postischemic functional performance.^29,30 A more intense and rapid contracture has been reported to occur during the index ischemia in the Kir6.2 knockouts than in control hearts with subsequent functional recovery much worse than control, in the absence of preconditioning.³¹ It is clear that knockout of sI_KATP channels results in a poor response of the myocardium to ischemic challenge under control conditions, likely shifting ischemic injury to higher levels. This makes it difficult to determine if sI_KATP is playing a specific role in IPC protection. It remains possible that both sI_KATP and I_K1, in combination, may be contributing to IPC protection in vivo through enhanced cardiomyocyte cell volume regulation. Because our experimental results are limited to fresh and cultured cardiomyocyte models, some caution is warranted in extrapolation to the whole heart. Other K⁺ channels and transporters might also contribute to K⁺ efflux under ischemic conditions. Also, the precise quantitative connection among K⁺ efflux, I_K1 density and preconditioning requires further investigation.

Other than sI_KATP, to date the putative ATP-sensitive mitochondrial K⁺ (mitoK_ATP) channel has been the primary focus of interest and controversy in regard to ion channels in IPC. Whatever the possible role of mitoK_ATP in IPC may be, it cannot, alone, account for the protection against necrosis afforded by IPC based on a recent review of the literature by Gross and Peart.³² Furthermore, there is no evidence that the I_K1 channel is expressed in mitochondria; therefore, involvement of mitochondria in the action of I_K1 in IPC, if any, would have to be indirect, conceivably through a signaling pathway terminating on the I_K1 channel.

In conclusion, our results indicate that I_K1 channels play an important role in the protection induced by ischemic preconditioning and support the I_K1 channel as a strong candidate for the role as one end effector in ischemic preconditioning of the myocardium.

	Acknowledgments

 
H.C. Cho was supported by the Canadian Institutes of Health Research. Dr C. Zobel was working in our laboratory on a fellowship from the Laboratory of Muscle Research and Molecular Cardiology, Clinic III for Internal Medicine, University of Cologne, Cologne (Germany), and was also supported by the VERUM Foundation for Behavior and Environment and the Deutsche Forschungsgemeinschaft (ZO 112/1–1). Dr P.H. Backx is a principal investigator at the Toronto Hospital, University Health Network and is a Career Investigator of the Heart and Stroke Foundation of Ontario. Dr Gregory J. Wilson is principal investigator at the Hospital for Sick Children. This study was also supported by Ontario Heart and Stroke Foundation Grants nos. T-4179 (G.J.W. and P.H.B.) and NA-5034 (P.H.B.).

	Footnotes

 
*Both authors contributed equally to this study.

Original received December 11, 2003; revision received June 16, 2004; accepted June 17, 2004.

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