Previous studies in our laboratory suggest that an acute inhibition of glycogen synthase kinase 3 (GSK3) by SB-216763 (SB21) is cardioprotective when administered just before reperfusion. However, it is unknown whether the GSK inhibitor SB21 administered 24 h before ischemia is cardioprotective and whether the mechanism involves ATP-sensitive potassium (KATP) channels and the mitochondrial permeability transition pore (MPTP). Male Sprague-Dawley rats were administered the GSK inhibitor SB21 (0.6 mg/kg) or vehicle 24 h before ischemia. Subsequently, the rats were acutely anesthetized with Inactin and underwent 30 min of ischemia and 2 h of reperfusion followed by infarct size determination. Subsets of rats received either the sarcolemmal KATP channel blocker HMR-1098 (6 mg/kg), the mitochondrial KATP channel blocker 5-hydroxydecanoic acid (5-HD; 10 mg/kg), or the MPTP opener atractyloside (5 mg/kg) either 5 min before SB21 administration or 5 min before reperfusion 24 h later. The infarct size was reduced in SB21 compared with vehicle (44 ± 2% vs. 61 ± 2%, respectively; P < 0.01). 5-HD administered either before SB21 treatment or 5 min before reperfusion the following day abrogated SB21-induced protection (54 ± 4% and 61 ± 2%, respectively). HMR-1098 did not affect the SB21-induced infarct size reduction when administered before the SB21 treatment (43 ± 1%); however, HMR-1098 partially abrogated the SB21-induced infarct size reduction when administered just before reperfusion 24 h later (52 ± 1%). The MPTP opening either before SB21 administration or 5 min before reperfusion abrogated the infarct size reduction produced by SB21 (61 ± 2% and 62 ± 2%, respectively). Hence, GSK inhibition reduces infarct size when given 24 h before the administration via the opening KATP channels and MPTP closure.
- ATP-sensitive potassium channel
- mitochondrial permeability transition pore
- infarct size
glycogen synthase kinase 3β (GSK3β) inhibition is an essential process necessary to elicit acute cardioprotection required for mechanical stimuli, such as ischemic preconditioning (IPC), and pharmacological stimuli, such as opioids (5, 16). Specific pharmacological inhibitors of GSK3, such as SB-216763 or SB-415286, reduce infarct size when administered at reperfusion, with GSK3β inhibition suggestive to be downstream of both phosphatidylinositol 3-kinase (PI3K) and target of rapamycin (TOR) (5). Both the activation of the ATP-sensitive potassium (KATP) channel and the inhibition of the mitochondrial permeability transition pore (MPTP) also contribute to the cardioprotective stimulus elicited by acute GSK3β inhibition (6, 10).
Delayed cardioprotection, also known as the second window of preconditioning, has tremendous therapeutic potential for patients undergoing nonemergency procedures such as percutaneous coronary intervention or coronary artery bypass grafting. A number of agents, including IPC, adenosine, bradykinin, opioids, volatile anesthetics, and KATP channel openers, are documented to reduce infarct size when administered 24 h before ischemia (1, 7). Hence, we investigated whether SB-216763, a direct pharmacological inhibitor of GSK3, could produce delayed preconditioning and reduce infarct size when administered 24 h before ischemia. Furthermore, whether GSK3-induced infarct size reduction is dependent on the KATP channel opening and MPTP closure was also determined.
The experimental procedures and protocols used in this study were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin and conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
The agents used for this study included the GSK3 inhibitor SB-216763 (Tocris), the selective sarcolemmal KATP channel inhibitor HMR-1098 (Aventis), the putative mitochondrial KATP channel inhibitor 5-hydroxydecanoic acid (5-HD; Sigma), and the MPTP channel opener atractyloside (BioMol). SB-216763 was dissolved in DMSO, whereas HMR-1098, 5-HD, and atractyloside were dissolved in water. To increase the solubility of atractyloside, the solution was also warmed in a water bath.
All agents given 24 h before ischemia were administered via tail-vein injection. Agents given before reperfusion were administered intravenously via the right jugular vein.
Male Sprague-Dawley rats (215–300 g) were obtained from Harlan (Indianapolis, IN) and used for an in vivo anesthetized intact rat model of ischemia and reperfusion. The general surgical protocol and the determination of infarct size were described previously (7). Rats underwent 30 min of ischemia and 2 h of reperfusion. The infarct size was expressed as a percentage of the area at risk as determined by tetrazolium staining.
The left common carotid artery was cannulated for blood pressure, heart rate, and blood gas measurements. Hemodynamics, including heart rate, mean arterial pressure, and rate pressure product, were quantified during baseline, 15 min into ischemia, and at 2 h of reperfusion for all experimental groups.
Infarct size studies.
Rats were randomly divided into groups (n = 6/group) receiving either SB-216763 (0.6 mg/kg) or vehicle 24 h before ischemia. Subsets of these groups received either HMR-1098 (6 mg/kg), 5-HD (10 mg/kg), or atractyloside (5 mg/kg) 5 min before SB-216763 or vehicle administration. An additional subset of groups pretreated with SB-216763 or vehicle 24 h earlier received the same doses of HMR-1098, 5-HD, or atractyloside administered 5 min before reperfusion 24 h later. The doses selected were based on those previously established for acute myocardial infarct size studies (5, 6, 13).
All values were denoted as means ± SE. Statistical significance was determined by performing a one-way ANOVA with Bonferroni's correction for multiplicity. Values significantly different from vehicle were indicated by P < 0.001.
Heart rate, mean arterial pressure, and rate pressure product for infarct size experiments are summarized in Table 1. No significant differences when compared with the vehicle group were found.
No differences between groups were seen for the ratio between the area at risk to left ventricle weight. Rats treated with SB-216763 produced a significant reduction in infarct size compared with those treated with vehicle (Fig. 1, A–C: 44 ± 2% vs. 61 ± 2%, respectively). HMR-1098 administration before SB-216763 did not affect infarct size, whereas HMR-1098 administered 5 min before reperfusion 24 h later abrogated the delayed cardioprotection afforded by SB-216763 (Fig. 1A, 43 ± 1% and 52 ± 1%, respectively). Additionally, 5-HD administration before SB-216763 or 5 min before reperfusion blocked the infarct size reduction (Fig. 1B, 54 ± 4% and 61 ± 2%, respectively). Atractyloside administered before SB-216763 or 5 min before reperfusion also abolished the SB-216763-induced infarct size reduction (Fig. 1C, 61 ± 2% and 62 ± 2%, respectively). No differences in infarct size were seen when HMR-1098, 5-HD, or atractyloside was administered 24 h before ischemia (Fig. 1, A–C: 58 ± 1%, 58 ± 2%, and 62 ± 2% vs. 61 ± 2%, respectively) or 5 min before reperfusion alone (Fig. 1, A–C: 64 ± 1%, 64 ± 2%, and 66 ± 3% vs. 61 ± 2%, respectively) compared with vehicle.
This is the first study to demonstrate that a pharmacologically induced delayed inhibition of GSK3 is cardioprotective, with the protection generated occurring 24 h after the administration during reperfusion. The mechanism of SB-216763-induced protection is mediated by MPTP inhibition and the KATP channel opening. Since GSK3β inhibition is a common mechanism involved in both pharmacological and IPC (5, 10, 16), perhaps these data indicate that the overall mechanism of either the pharmacological or ischemia-induced delayed cardioprotection involves the inhibition of a GSK-dependent signaling cascade at reperfusion.
These data are the first to indicate an essential role for the MPTP in mediating SB-216763-induced delayed cardioprotection, which is perhaps similar to a mechanism involved in GSK inhibition-induced acute cardioprotection (10). Interestingly, inhibition of the MPTP at reperfusion appears to be a common mechanism for acute cardioprotection either by mechanical or pharmacological stimuli (9). Our findings further suggest that MPTP inhibition is also a common mechanism involved as an end effector of pharmacological or mechanically induced delayed cardioprotection.
These data also indicate that an interaction occurs via the KATP channel and GSK3, since the protection afforded by the delayed GSK3 inhibition could be partially blocked by HMR-1098 or completely blocked by 5-HD at reperfusion. These data are consistent with results obtained from a previous study showing a blockade of acute SB-216763-induced infarct size reduction in the presence of KATP channel blockers administered just before reperfusion (6). However, our present findings suggest 5-HD, when given before reperfusion, abrogates to a greater extent a GSK-induced delayed infarct size reduction compared with the acute GSK-induced acute infarct size reduction observed in our previous study (6). The reason for the difference could be secondary to the different timing of SB-216763 administration (delayed vs. acute); however, more studies will be needed to address this possibility. These findings would also indicate that the pharmacological blockade of the mitochondrial KATP channel leads to an abrogation of additional events essential for the triggering of delayed cardioprotection via SB-216763, which will require further investigation to discern the downstream mechanism involved.
These findings also indicate that the sarcolemmal KATP channel is not involved as an initial trigger of delayed cardioprotection via pharmacological GSK inhibition since HMR-1098 administration before SB-216763 did not abrogate infarct size reduction. These findings differ from previous studies involving opioids (14) or delayed preconditioning (15), where the sarcolemmal KATP channel involvement is an integral component of the triggering mechanism. These data, in the context of prior studies, would suggest the sarcolemmal KATP channel opening occurs as an event upstream of GSK3 inhibition.
The mechanism of IPC-induced delayed cardioprotection is dependent on the stimulation of both PI3K and TOR (11). Both PI3K and TOR have also been implicated as being upstream of GSK3β and, when activated, result in GSK3β inhibition (5). Whether PI3K or TOR mediates protection via GSK3β inhibition is unknown; however, it is plausible within the context of prior publications (5, 11).
These present results must be interpreted within the realm of their potential limitations. The half-life of SB-216763 is unknown, and perhaps the delayed effect seen is secondary to a long half-life of the agent that is present even 24 h after the administration at reperfusion. However, two other selective GSK inhibitors, CHIR98023 and CHIR99021, have a reported half-life of 90 min, which may indicate that the plasma half-life of SB-216763 perhaps has a similar pharmacological profile (2). Furthermore, the half-life of 5-HD was reported to be about 7 min in dogs (12), and the half-life of HMR-1098 was reported to be between 60 and 90 min (H Goegelein; unpublished observation), suggestive that the acute inhibition of either the KATP channel at reperfusion or 5-HD before SB-216763 administration abrogates SB-216763-induced protection regardless of the half-life of SB-216763.
This study is also limited in that only one dose of SB-216763 was examined, which was selected based on an acute cardioprotective dose from a previous study (5). Future studies will be needed to discern whether an alternative dose can provide a more robust cardioprotective response. Moreover, SB-216763 has previously been reported in vitro to selectively inhibit GSK3 with little effect on the activity of phosphoinositide-dependent kinase, JNK, p70S6 kinase, or about 20 other kinases when examined in vitro (3). However, perhaps additional proteins could be targeted by SB-216763 that have yet to be determined.
In addition, the inhibition of GSK regulates a number of cellular processes, including transcription, metabolism, cell division, adhesion, and apoptosis (4). SB-216763 competitively binds to the ATP site of GSK3, which in turn inhibits GSK3 activity. Whether the delayed protective effect by SB-216763 is due to the inhibition of one or more of these cellular processes requires further study. Additional agents that inhibit GSK, such as FRATTIDE, which inhibits GSK by an alternative means as SB-216763, would aid in determining the specific components of the GSK protein responsible for cardioprotection. Moreover, further assessment of downstream components regulated by GSK inhibition would be necessary to further elucidate the mechanism involved in cardioprotection.
Recent evidence that 5-HD exerts a mitochondrial effect by acting as a possible bottleneck of β-oxidation via its conversion to l-3,d-5-dihydroxydecanoyl-CoA may question the validity of 5-HD as a mitochondrial KATP channel inhibitor (8). However, our findings still strongly indicate that a mitochondrial target of 5-HD is involved in GSK inhibition-induced delayed infarct size reduction.
In summary, these data suggest an essential role for both MPTP inhibition and the mitochondrial KATP channel opening both as a trigger and distal effector during reperfusion in mediating the cardioprotection afforded by delayed GSK3 inhibition. These data also suggest a role for the sarcolemmal KATP channel in mediating cardioprotection at reperfusion. These data extend the application of GSK inhibitors to the realm of delayed cardioprotection and generate an interesting area of research that requires further investigation.
This work was supported by NIH Grants H-08311 (to G. J. Gross) and H-074314 (to G. J. Gross).
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- Copyright © 2008 by the American Physiological Society