We tested the hypothesis that the high-conductance calciumactivated potassium (KCa) channel is involved in the cardioprotection of preconditioning with ischemic insults. In the isolated perfused rat heart subjected to ischemia/reperfusion, effects of ischemic preconditioning (IPC) on infarct size and lactate dehydrogenase (LDH) release were abolished by 1 μM paxilline (Pax), an inhibitor of the KCa channel, administered 30 min before, but not during, ischemia. In isolated ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), preconditioning with MI/A increased their viability, and the effect was abolished by administering Pax before MI/A. Like IPC, 10 μM NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-trifluoromethyl-2Hbenzimidazol-2-one; NS), an opener of KCa channels, reduced infarct size and LDH release, effects attenuated by Pax. The harmful and protective effects of blockade and activation of the KCa channel were accompanied by impaired and improved left ventricular contractile functions, respectively. In addition, the effect of NS was not altered by 100 μM 5-hydroxydecanoate, an inhibitor of the KATP channel. Neither was the effect of 100 μM diazoxide, an activator of the KATP channel, altered by Pax. Furthermore, opening of the mitochondrial permeability transition pore (mPTP) with 20 μM atractyloside abolished the beneficial effects of IPC or NS in the isolated rat heart and myocyte. Inhibition of mPTP opening with 0.2 μM cyclosporin A decreased the infarct size and LDH release and improved the contractile function, effects not attenuated by Pax. In conclusion, the study provides evidence that the KCa channel triggers cardioprotection of IPC, which involves mPTP.
Ischemic preconditioning (IPC) confers cardioprotection (Murry et al., 1986). Although extensive studies have been performed, the mechanisms by which IPC protects the myocardium against ischemia/reperfusion-induced injury are still not fully understood. Recently, a high-conductance Ca2+-activated potassium (KCa) channel, located in the inner membrane of the mitochondrion, has been shown to mediate cardioprotection against ischemia/reperfusion (Xu et al., 2002), as does the mitochondrial ATP-sensitive potassium (mitoKATP) channel (Garlid et al., 1997). Opening of both channels increases mitochondrial uptake of potassium, which is required for optimal functioning of oxidative phosphorylation (Kowaltowski et al., 2001). Since activation of the mitoKATP channel participates in the cardioprotection of IPC, we hypothesized that activation of the KCa channel also participates in cardioprotection of IPC.
The mitochondrial permeability transition pore (mPTP) is a multiprotein complex formed at contact sites between the inner and outer mitochondrial membranes (Hunter and Haworth, 1979), the opening of which triggers apoptosis/necrosis (Kroemer et al., 1998; Crompton, 1999). The mPTP opens during the first few minutes of reperfusion (Griffiths and Halestrap, 1995), and this results in myocardial injury (Griffiths and Halestrap, 1995; Halestrap et al., 1998). It has been demonstrated that blockade of mPTP opening confers cardioprotection similar to that provided by IPC (Hausenloy et al., 2002; Javadov et al., 2003). Therefore, it is hypothesized that if IPC confers cardioprotection by activation of KCa, the mPTP may be involved.
To determine the role of the KCa channel in cardioprotection of IPC, we first determined the effects of preconditioning with ischemic insults on injury induced by ischemia/reperfusion upon blockade of the high-conductance KCa channel with a blocker of the channel, paxilline, and both the isolated perfused rat heart and isolated myocytes preparations. To delineate the role of the mPTP in the cardioprotection by activation of the KCa channel, we determined the effect of blockade of mPTP on cardioprotective effects of ischemic insults or activation of the KCa channel in the isolated perfused rat heart and isolated ventricular myocyte. The results showed that the KCa channel triggers the cardioprotection of IPC, and the mPTP is involved.
Materials and Methods
Isolated Perfused Heart Preparation. The protocols of this study were approved by the Committee on the Use of Experimental Animals for Teaching and Research, The University of Hong Kong.
Male Sprague-Dawley rats of 250 to 300 g body weight were anesthetized with 60 mg/kg sodium pentobarbitone i.p. and given 200 IU of heparin i.v. Hearts were excised rapidly and placed in ice-cold Krebs-Henseleit (K-H) perfusion buffer before being mounted on a Langendorff apparatus for perfusion at 37°C with K-H buffer at a constant pressure (100 cm of H2O) and equilibrated with 95% O2/5% CO2. The buffer contained: 118.0 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3, and 11.0 mM glucose. For hearts subjected to regional ischemia, a silk suture was placed around the left coronary artery to form a snare. The coronary artery was occluded by pulling the snare to produce ischemia. Reperfusion was achieved by releasing it. In the present study, the isolated heart was subjected to 30 min of ischemia followed by 120 min of reperfusion, which induced myocardial injury. IPC was achieved by two cycles of 5 min of ischemia followed by 5 min of reperfusion prior to the more sustained ischemia/reperfusion that caused myocardial infarction.
For the study of left ventricular contractile function, a balloon was inserted through the left atrium into the left ventricle, and the left ventricular end diastolic pressure (LVEDP) was adjusted between 4 and 8 mm Hg. Cardiac parameters, namely, heart rate, left ventricular developed pressure (LVDP; difference between left ventricular end systolic pressure and end diastolic pressure), and velocity of contraction and relaxation (±dp/dtmax) were monitored continuously. Coronary flow, expressed in milliliters per minute, was measured by timed collection of effluent at regular intervals using a calibrated tube.
Measurement of the Area of Risk. For determination of infarct size, the coronary artery was reoccluded at the end of reperfusion, and a solution with 2.5% Evans blue was perfused to delineate the area of risk. Hearts were then frozen and cut into slices, which were then incubated in a sodium phosphate buffer containing 1% (w/v) 2,3,5-triphenyl-tetrazolium chloride for 15 min to visualize the unstained infarcted region. Infarct and risk zone areas were determined by planimetry with the software Image/J from the National Institutes of Health. The infarct size was expressed as a percentage of the risk zone.
Determination of Myocardial Injury by Lactate Dehydrogenase (LDH) Efflux. The effluent from the isolated perfused heart was collected at 5 min of reperfusion, and LDH was spectrophotometrically assayed using a kit purchased from Sigma-Aldrich (St. Louis, MO). LDH activity was expressed as units per liter.
Preparation of Isolated Ventricular Myocytes. Single ventricular myocytes were prepared from the heart of male SpragueDawley rats by enzymatic dissociation. Immediately after decapitation, the heart was rapidly removed and rinsed in ice-cold Ca2+-free Tyrode's solution. The heart was perfused via a Langendorff apparatus with a 100% oxygenated, nonrecirculating Ca2+-free Tyrode's solution. Then, the perfusion solution was switched to a 100% oxygenated, recirculated, low-Ca2+ (50 μM) Tyrode's solution containing 0.03% collagenase and 1% bovine serum albumin for 10 min. The ventricles were cut, minced, and gently triturated with a pipette in the low-Ca2+ Tyrode's solution containing bovine serum albumin at 37°C for 10 min. The cells were filtered through 200-μm nylon mesh, resuspended in the Tyrode's solution, in which the Ca2+ concentration was gradually increased to 1.25 mM in 40 min. Only rod-shaped cells with clear cross-striations were used. For ischemic insults, myocytes were incubated for 5 min with a solution containing 10 mM 2-deoxy-d-glucose and 10 mM sodium dithionite, which induce metabolic inhibition and anoxia (MI/A) (Wu et al., 1999; Ho et al., 2002), two consequences of ischemia. This was followed by perfusion with normal K-H solution for 10-min reperfusion. For preconditioning with MI/A, cells were subjected to three cycles of 1-min superfusion with the MI/A solution, separated by 3 min of superfusion with normal Krebs' solution (Ho et al., 2002).
Cell Viability. Trypan blue exclusion was used as an index of the viability of the myocyte (Zhou et al., 1996; Hiebert and Ping, 1997). After cells were incubated with 0.4% trypan blue dye for 3 min, they were counted in a hemocytometer chamber under a light microscope. Dead cells are not able to exclude trypan blue and, thus, appear blue. The cell morphology was determined by microscopic examination (Zhou et al., 1996). Only rod-shaped (length/width ratio, >3:1) cells were used for data collection.
Experiment Protocol. Three series of experiments were performed in the isolated perfused heart. The first series was to determine whether blockade of the high-conductance KCa channel abolished cardioprotection of IPC. Paxilline (Pax; 1 μM) was administered either 30 min before ischemia or throughout the ischemia and 15 min into reperfusion (Fig. 1). In the second series of experiments, which determined whether opening of the high conductance KCa channel reduced injury induced by ischemia/reperfusion, 10 μM NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-trifluoromethyl-2Hbenzimidazol-2-one; NS) was perfused 15 min before ischemia for 10 min in the absence or presence of Pax, which was administered 30 min before ischemia (Fig. 2). The protocol for administration of NS was according to a previous study (Xu et al., 2002). To determine whether KCa channel was located up-stream of mPTP, the effect of NS was determined with atractyloside (Atr) administered from 25 min into ischemia to 15 min into reperfusion according to a previous study (Fig. 3) (Hausenloy et al., 2002). The effect of 0.2 μM cyclosporin A (CsA), an inhibitor of mPTP opening, administered from 25 min into ischemia to 15 min into reperfusion in the presence of Pax was also determined. The third series of experiments determined the relationship between the KCa and mitoKATP channels (Fig. 4). The effects of activation of one potassium channel were determined in the presence of the blocker of another channel. Diazoxide (Dz; 100 μM), activator of KATP channels or 10 μM NS, was administered 15 min before ischemia for 10 min (Fig. 4). 5-Hydroxydecanoate (5-HD; 100 μM), an inhibitor of KATP channels, or 1 μM Pax was administered 30 min before ischemia (Fig. 4).
Infarct size and LDH, determined at the end and 5 min into reperfusion, respectively, were used as parameters of injury. LDH was measured at 5 min into reperfusion when its release reached its peak (Cao et al., 2004).
In the isolated ventricular myocyte, two series of experiments were performed. The first was aimed at determining whether blockade of the KCa channel abolished cardioprotection of preconditioning with ischemic insults. Myocytes were incubated with Pax for 30 min before MI/A with and without preconditioning with MI/A or NS (Fig. 5). The second series of experiments addressed the relationship between the KCa channel and mPTP. The effects of activation of the KCa channel upon opening of mPTP and inhibition of mPTP upon blockade of the KCa channel were determined (Fig. 6). NS and CsA were added 15 min before MI/A for 10 min, whereas Pax and Atr were added 30 min before MI/A. Trypan blue exclusion was used as a parameter of cell viability.
Chemicals and Solutions. Collagenase (type I), bovine serum albumin, NS1619, Pax, Dz, 5-HD, Atr, CsA, 2-deoxy-d-glucose, sodium dithionite, fura-2/acetoxymethyl ester, trypan blue, and 2,3,5-triphenyl-tetrazolium chloride were purchased from Sigma-Aldrich. FK506 was purchased from Merck. The concentrations of Pax, NS (Xu et al., 2002), CsA, Atr (Hausenloy et al., 2002), Dz (Miura et al., 2000), and 5-HD (Auchampach et al., 1992) used in this study were based on previous studies.
Statistical Analysis. Values are presented as means ± S.E.M. Statistical comparisons were performed by one-way analysis of variance and the Newman-Keuls test. Differences of P < 0.05 were regarded as significant.
Studies in the Isolated Perfused Heart Subjected to Ischemia/Reperfusion
Effect of IPC on Injury upon Blockade of the KCa Channel by Paxilline. IPC significantly reduced the infarct size and LDH release in the isolated perfused rat heart subjected to 30-min ischemia and 120-min reperfusion compared with untreated control hearts (Fig. 1), in agreement with the well established cardioprotection by IPC.
When 1 μM Pax, an inhibitor of the KCa channel, was administered 30 min before ischemia, both the infarct size and LDH release after ischemia were the same as those in the control group, i.e., without IPC, indicating that cardioprotection of IPC had been abolished. On the other hand, when the inhibitor was administered only during ischemia and 15 min into reperfusion, both the infarct size and LDH release remained the same as those of the group subjected to IPC, indicating that cardioprotection of IPC was still present.
Effects of Activation of KCa Channel with NS on Injury. Pretreatment with 10 μM NS, known to activate KCa channel (Xu et al., 2002), significantly reduced the infarct size and LDH release in isolated perfused rat heart subjected to ischemia/reperfusion, an effect similar to that of IPC (Fig. 2). These results are in agreement with a recent study (Wang et al., 2004).
We next determined the relationship between the KCa channel and the mPTP, which is known to be involved in cardioprotection/injury (Hausenloy et al., 2002). Atr (20 μM), an opener of the mPTP, abolished the effects of IPC on myocardial infarct and LDH release (Fig. 3). Consistent with the role of the mPTP in ischemia/reperfusion injury, administration of 0.2 μM CsA, an inhibitor of the mPTP, during reperfusion significantly reduced the infarct size and LDH release in the isolated perfused rat heart subjected to ischemia/reperfusion (Fig. 3). FK506 (5 μM), which inhibits calcineurin, also an effect of CsA, but does not inhibit mPTP opening (Griffiths and Halestrap, 1993; Connern and Halestrap, 1994), did not affect the infarct size and LDH release when administered during reperfusion, indicating that the cardioprotective effect of CsA was due to its action on the mPTP.
To determine whether closure of the mPTP mediated cardioprotection upon activation of KCa channels by NS (10 μM), cardioprotection was assessed in the presence of Atr (20 μM). As shown in Fig. 2, the effects of NS on infarct size and LDH release were attenuated in the presence of Atr, indicating that activation of KCa channels confers cardioprotection via mPTP at least partly. On the other hand, the effect of inhibiting the mPTP opening with CsA was not altered by pretreatment with Pax, indicating that the mPTP is located downstream from the KCa channel.
Administration of Atr (20 μM) for 30 min did not alter the infarct size and LDH release (Fig. 2).
Effects of Dz on Injury. In agreement with the previous observation (Miura et al., 2000), pretreatment with 100 μM Dz, which opens the mitoKATP channel and confers cardioprotection, significantly reduced the infarct size and LDH release in the isolated perfused rat heart subjected to ischemia/reperfusion (Fig. 4). Inhibition of the mitoKATP channels with 5-HD (100 μM) abolished the protective effect both of Dz and IPC (Fig. 4). On the other hand, the protective effect of Dz was not altered by Pax, nor was the effect of NS affected by 5-HD (Fig. 4), indicating no interaction between the two potassium channels. 5-HD alone had no effect on infarct size or LDH release.
In agreement with the previous finding (Hausenloy et al., 2002), Atr (20 μM) abolished the protective effects of Dz. On the other hand, the protective effect of CsA (0.2 μM) was not altered by 5-HD (10 μM) (data not shown).
Effects of KCa Blockade on Ventricular Functions and Coronary Flow. In all groups, occlusion of the left anterior descending artery resulted in a marked decrease in LVDP, ±dP/dtmax, and coronary flow, and an elevation in LVEDP at the end of reperfusion (Fig. 5). The reductions in LVDP, ±dP/dtmax, and the elevation in LVEDP in the group treated with IPC were significantly attenuated, effects that were reduced by either 1 μM Pax or 20 μM Atr. On the other hand, in the group treated with 10 μM NS, reductions in LVDP and ±dP/dtmax during reperfusion were significantly attenuated, suggesting that NS improved the left ventricular contraction and relaxation following ischemic injury. The effects of NS were attenuated by Pax and Atr.
Inhibition of mPTP opening with CsA attenuated the reductions in LVDP and ±dP/dtmax and the elevation in LVEDP during reperfusion, which were not altered by Pax, indicating again that inhibition of the mPTP is downstream from the KCa channel.
Heart rate and coronary flow during ischemia and reperfusion were the same in these groups (Fig. 5).
Studies in Isolated Ventricular Myocytes Subjected to MI/A
Effect of Preconditioning with MI/A on Viability upon Blockade of KCa Channel with Pax. To confirm the observation from the isolated perfused heart preparation that blockade of KCa channel abolished cardioprotection of IPC by direct action on the myocardium, we determined the effects of preconditioning with ischemic insults, MI/A, on viability of the myocytes subjected to MI/A upon blockade of the channel. The percentage of rod-shaped unstained cells 10 min into reperfusion was significantly decreased in ventricular myocytes subjected to MI/A compared with that in control group (Fig. 6). In the cells subjected to MI/A, the percentage of rod-shaped unstained cells subjected to MI/A was significantly greater than that subjected to MI/AR only, in agreement with our previous finding (Liu et al., 2004). This increase was attenuated by pretreatment with Pax (1 μM) before MI/A.
NS (10 μM; Fig. 6) or 0.2 μM CsA (Fig. 7) restored the percentage of unstained cells of the MI/A cells to the level of the control group. Atr (20 μM) attenuated the effects of MI/A or NS (Fig. 7). Pax (1 μM) abolished the effect of NS, but not the effect of CsA (Fig. 7). Atr or Pax alone had no effect at all.
There are two important observations in the present study. First, cardioprotection conferred by IPC was abolished by blockade of the high-conductance KCa channel with Pax administered before ischemia, and the effect of Pax was not accompanied by alterations in coronary flow in the isolated perfused rat heart. Second, the beneficial effect of preconditioning with brief periods of metabolic inhibition and anoxia, two consequences of myocardial ischemia, on viability of isolated ventricular myocytes subsequently subjected to MI/A was also abolished by Pax administered before MI/A. Therefore, blockade of the KCa channel abolished cardioprotection of ischemic insult by direct action on myocardium. In agreement with a previous study (Wang et al., 2004), in the present study it was found that activation of the channel with its opener, NS, conferred cardioprotection, as did IPC or opening of the mitoKATP channel with diazoxide. These observations provide unequivocal evidence that the high-conductance KCa channel is involved in cardioprotection of preconditioning with ischemic insults. It is important to note that when the KCa channel was blocked with Pax 30 min before ischemia, the effect of IPC was abolished in the isolated perfused heart. On the other hand, when Pax was added during ischemia and reperfusion, the effect of IPC remained. This finding indicates that the KCa channel is a trigger, rather than a mediator, of cardioprotection of preconditioning.
Another important finding is the causal relationship between the high-conductance KCa channel and the mPTP, a crucial step in the cardioprotection of preconditioning (Hausenloy et al., 2002, 2004; Javadov et al., 2003). We discovered that opening the mPTP with Atr, which abolished the beneficial effect of IPC on the heart in terms of protection against injury and contractile function, also abolished the beneficial effects of activating the high-conductance KCa channel with NS. This is the first demonstration that mPTP plays a permissive role for cardioprotection due to activation of the KCa channel. The KCa channels involved are most likely those in the mitochondria because the channel has been shown to be mainly, if not exclusively, located on the membrane of this organelle (Xu et al., 2002).
We also found that the cardioprotection conferred by activation of mitoKATP channels with diazoxide was not altered by blockade of KCa channels nor vice versa, indicating that these channels act independently. It should be noted that activation of mitoKATP channels also confers cardioprotection by inhibiting mPTP opening (Hausenloy et al., 2002). Therefore, both potassium channels located in mitochondria confer cardioprotection via the same machinery, the mPTP.
One of the common features of these two channels is influx of K+ into mitochondria upon activation. That opening of these channels results in cardioprotection suggests that K+ influx may play a crucial role in cardioprotection. There is evidence that K+ influx into mitochondria may be important for oxidative phosphorylation, regulation of mitochondrial functions such as reactive oxygen species production, and mitochondrial volume regulation (Garlid, 2000; Garlid and Paucek, 2003). It is also interesting to note that the KCa channel acts as a trigger as shown in the present study, whereas mitochondrial KATP channel acts as a trigger as well as a mediator (Gross and Peart, 2003). Therefore, although both channels share a common feature, namely increased K+ influx into mitochondria upon activation, they may have unique properties other than influx of K+, which are responsible for different roles each of them plays. Further studies are warranted.
In the present study, we also observed that blockade of the KCa channel abolished the protective effects of IPC accompanied by impairment of contraction, whereas activation of the channel conferred cardioprotection accompanied by improved contractile functions. Further study is needed to determine whether improved/impaired contractile function is secondary to cardioprotection/injury upon activation/inactivation of the channel.
In conclusion, the study has provided the first evidence that activation of KCa channels triggers cardioprotection of preconditioning with ischemic insults. Cardioprotection of preconditioning is accompanied by improved left ventricular contractile function. The study has also provided the first evidence that mPTP plays a permissive role for cardioprotection due to activation of KCa channels.
We thank Iain C. Bruce for advice on the use of English, Gudrun H. Borchert for advice on viability study, and Chi Pei Mok for technical assistance.
- Received July 18, 2004.
- Accepted September 1, 2004.
This work was supported by a Cardiovascular Physiology Research Fund donated by L.C.S.T. (Holdings).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: IPC, ischemic preconditioning; KCa, Ca2+-activated potassium; mitoKATP, mitochondrial ATP-sensitive potassium; FK506, tacrolimus; mPTP, mitochondrial permeability transition pore; K-H, Krebs-Henseleit; LVEDP, left ventricular end diastolic pressure; LVDP, left ventricular developed pressure; LDH, lactate dehydrogenase; MI/A, metabolic inhibition and anoxia; MI/AR, metabolic inhibition and anoxia/reoxygenation; Pax, paxilline; NS, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-trifluoromethyl-2Hbenzimidazol-2-one (NS1619); Atr, atractyloside; CsA, cyclosporine A; Dz, diazoxide; 5-HD, 5-hydroxydecanoate.
- The American Society for Pharmacology and Experimental Therapeutics