Published online before print May 19, 2008, doi: 10.1161/CIRCULATIONAHA.107.755066
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2008;117:2761-2768.)
© 2008 American Heart Association, Inc.
Molecular Cardiology |
Inhibition of GSK3β by Postconditioning Is Required to Prevent Opening of the Mitochondrial Permeability Transition Pore During Reperfusion
From INSERM U886, Université Claude Bernard Lyon (L.G., M.P., H.T., G.D., M.O.), and Hôpital Louis Pradel, Hospices Civils de Lyon (H.T., G.D., M.O.), Lyon, France.
Correspondence to Professor Michel Ovize, INSERM U886, "Cardioprotection," Laboratoire de Physiologie Lyon-Nord, 8, Avenue Rockefeller, 69373 Lyon, France. E-mail ovize{at}sante.univ-lyon1.fr
Received November 26, 2007; accepted March 20, 2008.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— Opening of the mitochondrial permeability transition pore (mPTP) is a crucial event in lethal reperfusion injury. Phosphorylation (inhibition) of glycogen synthase kinase-3β (GSK3β) has been involved in cardioprotection. We investigated whether phosphorylated GSK3β may protect the heart via the inhibition of mPTP opening during postconditioning.
Methods and Results— Wild-type and transgenic GSK3β-S9A mice (the cardiac GSK3β activity of which cannot be inactivated) underwent 60 minutes of ischemia and 24 hours of reperfusion. At reperfusion, wild-type and GSK3β-S9A mice received no intervention (control), postconditioning (3 cycles of 1 minute ischemia and 1 minute of reperfusion), the mPTP inhibitor cyclosporine A (CsA; 10 mg/kg IV), or the GSK3β inhibitor SB216763 (SB21; 70 µg/kg IV). Infarct size was assessed by triphenyltetrazolium chloride staining. The resistance of the mPTP to opening after Ca2+ loading was assessed by spectrofluorometry on mitochondria isolated from the area at risk. In wild-type mice, infarct size was significantly reduced by postconditioning, CsA, and SB21, averaging 39±2%, 35±5%, and 37±4%, respectively, versus 58±5% of the area at risk in control mice (P<0.05). In GSK3β-S9A mice, only CsA, but not postconditioning or SB21, reduced infarct size. Postconditioning, CsA, and SB21 all improved the resistance of the mPTP in wild-type mice, but only CsA did so in GSK3β-S9A mice.
Conclusion— These results suggest that S9-phosphorylation of GSK3β is required for postconditioning and likely acts by inhibiting the opening of the mitochondrial permeability transition pore.
Key Words: ischemia • myocardial infarction • reperfusion
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Ischemic postconditioning is a phenomenon in which brief episodes of ischemia-reperfusion performed immediately after reflow following a prolonged ischemic period attenuate infarct size1. This cardioprotective effect has now been reported in several animal preparations and in the human heart.1–5 Our group recently demonstrated that angioplasty postconditioning can reduce infarct size in patients with ongoing acute myocardial infarction.5 Unfortunately, angioplasty postconditioning cannot be applied to most patients with ongoing acute myocardial infarction, so we need a pharmacological postconditioning mimetic that would protect all acute myocardial infarction patients when administered at the onset of reperfusion.
Clinical Perspective p 2768
Recent reports suggest that opening of the mitochondrial permeability transition pore (mPTP), which plays a crucial role in lethal reperfusion injury, may be inhibited by ischemic postconditioning.2,6,7 Opening of the mPTP is favored by conditions set up by ischemia and reperfusion, including overproduction of reactive oxygen species, ATP depletion, and more specifically, accumulation of Ca2+ in the mitochondrial matrix. After this last phenomenon, Ca2+ stimulates the interaction of cyclophilin D (CypD) with a mPTP component, which triggers permeability transition.8–10 On the other hand, indirect evidence suggests that opening of the mPTP during reperfusion may be regulated by extramitochondrial activation/inhibition of several kinases, including the glycogen synthase kinase-3β (GSK3β).11–13
The constitutively active GSK3β is known as an important regulator of cellular function.14,15 Beside having a role in diabetes, inflammation, and cancer, GSK3β has been involved as a negative regulator of myocardial hypertrophy and as a key enzyme in the myocardial response to ischemia-reperfusion injury.15–17 Tong et al18 proposed that GSK3β was involved in preconditioning via its inactivation by phosphorylation at a specific serine residue (Ser9) located in its N-terminal domain. Gross et al19 reported that the preconditioning-like cardioprotective effect of morphine was mediated by GSK3β.
We hypothesized that GSK3β might be a key regulator of mPTP opening during postconditioning and might possibly act at the level of CypD. We therefore addressed whether GSK3β-S9A mice, the cardiac GSK3β activity of which cannot be inactivated, could undergo any infarct size reduction by postconditioning and whether GSK3β might regulate mPTP opening at the level of CypD.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Genotyping of GSK3β-S9A Mice
GSK3β-S9A mice were bred from pairs kindly provided by Antos et al,17 and wild-type (WT) mice C57BL/6J were obtained from Charles River Laboratories (L’Arbresle, France). Myosin heavy chain (MHC)–GSK3β transgene expression was assessed by polymerase chain reaction using genomic DNA isolated from transgenic and nontransgenic tail biopsies and primers: GSK, 5' TTG-GAC-TGT-GTA-GCC-GTC-TGC 3', and hGH, 5' CAC-TCC-AGC-TTG-GTT-CCC-CAA-TAG-ACC 3'. The GSK3β-S9A mutation was confirmed by the spot at 700 to 800 bp (according to the protocol for MHC-GSK3β genotyping by Antos et al17) (Figure 1).
|
In Vivo Model of Acute Myocardial Ischemia-Reperfusion Injury
WT and transgenic mice (male; 8 to 10 weeks old) were anesthetized by intraperitoneal injection of 0.3 mL/10 g body weight of a 1:1 mixture of fentanyl citrate (0.011 mg/mL) and midazolam (0.4 mg/mL) as previously described.20 The animals were orally intubated with a 22-gauge vinyl catheter and ventilated via a rodent ventilator (model 687, Harvard Apparatus, Holliston, Mass) with a tidal volume of 0.2 mL and a breath rate of 160 breaths per minute. Body temperature was maintained at 37°C. A left thoracotomy was performed in the fourth left intercostal space. The pericardium was opened, and the heart was exposed. An 8-0 polypropylene suture attached to a small curved needle was passed around the left anterior descending coronary artery. Successful left anterior descending coronary artery occlusion was confirmed by ST-segment shift on the ECG (78534C Monitor, Hewlett-Packard, Palo Alto, Calif) and the appearance of myocardial pallor. After surgery, the animals were allowed to recover from anesthesia, and the endotracheal tube was removed once spontaneous breathing resumed.
Pilot Study: Cardiac Phenotype of GSK3β-S9A Mice
Because GSK3β has been involved in the regulation of myocardial hypertrophy, which may alter the response to ischemia-reperfusion, we assessed myocardial wall thickness and left ventricular (LV) dimensions under baseline conditions in a subset of WT and transgenic mice (n=4 to 5 per group). Echocardiography was performed either with or without light anesthesia (ketamine 80 mg/kg IP). Images were acquired with a 13-MHz linear-array transducer with a digital ultrasound system (Vivid 7, GE Medical Systems, Waukesha, Wis). Conventional measurements were performed: LV end-diastolic diameter, LV end-systolic diameter, LV fractional shortening, interventricular thickness, and posterior wall thickness.
At baseline, WT and GSK3β-S9A mice had comparable heart rates (Table 1). LV end-diastolic diameter and wall thickness were similar in the 2 groups. LV end-systolic diameter, however, was significantly larger in the GSK3β-S9A than in the WT mice, with shortening fractions being significantly lower in GSK3β-S9A than in WT mice (Table 1).
|
In addition, 15 GSK3β-S9A and 15 WT mice were submitted to prolonged ischemia (60 minutes) and reperfusion (24 hours) to determine the cardiac phenotype under these stress conditions. During the ischemia-reperfusion protocol, the incidence of mortality averaged 60% in GSK3β-S9A mice and 30% in WT mice (P<0.05). Surviving GSK3β-S9A mice developed smaller infarcts than WT mice (P<0.05; Figure 2). We hypothesized that this infarct size limitation was related to the more pronounced heart rate reduction observed during ischemia because heart rate averaged 425±38 bpm in GSK3β-S9A mice and 557±24 bpm in WT mice (P<0.05; Table 1). This was confirmed when we paced both GSK3β-S9A and WT hearts at a frequency of 450 bpm with an electronic stimulator (HSE-D 7801-Hugstetten, Hugstetten, Germany) during the ischemic period. Pacing abolished the observed infarct size reduction in transgenic mice and did not affect infarct size in WT mice (Figure 2). Pacing also prevented the increase in the incidence of mortality in GSK3β-S9A mice, strongly suggesting that it was related to heart rate reduction–induced arrhythmias. We then decided to perform all further experiments by pacing the GSK3β-S9A and WT hearts at 450 bpm during the prolonged ischemia.
|
Experimental Design
All animals underwent 60 minutes of coronary artery occlusion (paced at 450 bpm) followed by 24 hours of reperfusion (Figure 3). Animals were randomly assigned to one of the following groups: control, postconditioning, cyclosporine A (CsA; a potent inhibitor of mPTP opening), and SB216763 (SB21; a GSK3β inhibitor). Control mice underwent no further intervention. Ischemic postconditioning consisted of 3 cycles of 1 minute of reperfusion and 1 minute of ischemia performed immediately after reflow. CsA, which has been demonstrated as being able to reduce infarct size when administered at the time of reflow, was used here to determine whether GSK3β-S9A mice retain a functional permeability transition pore and whether the mPTP opening is activated downstream of GSK3β during reperfusion. CsA was administered intravenously 5 minutes before reperfusion at a dose of 10 mg/kg. SB21 is a potent cell-permeant competitive inhibitor of the ATP binding site of GSK3β.21 It was expected to be able to reduce infarct size in WT but not GSK3β-S9A mice. SB21 was administered intravenously at a dose of 70 µg/kg 5 minutes before reperfusion. Sham animals received no intervention for the whole duration of the experimentation.
|
After this protocol, all mice had their chests closed and were returned to the animal facilities until the end of the reperfusion period. After 24 hours of reperfusion, mice were euthanized under deep anesthesia, and the hearts were excised for determination of infarct size (n=66) or for assessment of calcium-induced mitochondrial permeability transition (n=65).
Techniques
Area at Risk and Infarct Size Determination
At the end of the 24 hours of reperfusion, the coronary artery was briefly reoccluded, and 0.5 mg/kg Unisperse blue pigment (Ciba-Geigy, Hawthorne, NY) was injected intravenously to delineate the in vivo area at risk (AR) as previously described.3,20 The heart was excised and cut into five 1-mm-thick transverse slices parallel to the atrioventricular groove. Each slice was incubated for 15 minutes in a 1% solution of triphenyltetrazolium chloride at 34°C to differentiate infarcted (pale) from viable (brick red) myocardial area.22 Extent of the AR and area of necrosis (AN) was quantified by computerized planimetry and corrected for the weight of the tissue slices.
Preparation of Isolated Mitochondria
After 24 hours of reperfusion, hearts were excised while still beating and immediately placed in cold buffer, and AR myocardium was harvested for mitochondria isolation. Preparation of mitochondria was adapted from a previously described procedure.3 All operations were carried out in the cold at 4°C. Briefly, myocardial AR (25 to 35 mg) placed in isolation buffer A containing 70 mmol/L sucrose, 210 mmol/L mannitol, and 1 mmol/L EDTA in 50 mmol/L Tris/HCl, pH 7.4 (1 mL buffer/15 mg tissue), was finely minced with scissors and then homogenized with a Kontes tissue grinder (Fisher Scientific, Illkirch, France). The homogenate was centrifuged at 1300g for 3 minutes. The supernatant was centrifuged at 10 000g for 10 minutes. The mitochondrial pellet was suspended in isolation buffer B containing 70 mmol/L sucrose and 210 mmol/L mannitol in 50 mmol/L Tris/HCl, pH 7.4. Protein content was routinely assayed according to the Gornall et al23 procedure with BSA used as a standard.
Mitochondrial Oxidative Phosphorylation
Oxygen consumption in freshly isolated mitochondria was measured at 25°C with a Clark-type large-diameter Orbisphere oxygen electrode controlled chamber (Oroboros Oxygraph, Paar, Graz, Austria). Mitochondria (250 µg proteins) were incubated in buffer (pH 7.4) containing 80 mmol/L KCl, 50 mmol/L 4-morpholinepropanesulfonic acid, 1 mmol/L EGTA, 5 mmol/L KH2PO4, and 1 mg/mL defatted BSA. Pyruvate (3 mmol/L), malate (3 mmol/L), and glutamate (3 mmol/L) were used as substrates that donate electrons to complex I. State 3 (200 µmol/L ADP stimulated), state 4 (ADP limited), and respiratory control ratio were determined. Outer membrane intactness was calculated as the ratio of 8 µmol/L cytochrome c–stimulated respiration on maximally stimulated respiration by 2 mmol/L ADP.
Citrate Synthase Activity
Citrate synthase activity was quantified in cholate-solubilized frozen mitochondria by measuring the rate of 5,5'-dithiobis(nitrobenzoic acid)–reactive reduced coenzyme A (412 nm) at 37°C.
Electron Microscopy
Electron microscopy was performed at the end of the preincubation period (ie, before Ca2+ loading). Under these experimental conditions, samples of mitochondria were fixed for 2 hours in 2% glutaraldehyde and 100 mmol/L phosphate buffer, pH 7.4, and postfixed in 1% osmium tetroxide. Dehydration was performed in a series of ethanol and propylene oxide extractions before sample embedding in Epon. Ultrathin sections were examined with a Jeol 100 CX-II electron microscope at the Centre Commun d’Imagerie Laennec (Lyon, France).
Calcium Retention Capacity
Adapted from the description by Ichas et al,24 calcium retention capacity (CRC) was defined here as the amount of Ca2+ required to trigger a massive Ca2+ release by isolated cardiac mitochondria.25 It is used as an indicator of the resistance of the mPTP pore to opening after matrix Ca2+ accumulation and expressed as nmol CaCl2 per mg mitochondrial proteins. Extramitochondrial Ca2+ concentration was recorded with 0.5 µmol/L calcium green-5N, with excitation and emission wavelengths set at 500 and 530 nm, respectively. Isolated mitochondria (200 µg proteins) were suspended in 2 mL buffer (150 mmol/L sucrose, 50 mmol/L KCl, 2 mmol/L KH2PO4, and 5 mmol/L succinic acid in 20 mmol/L Tris/HCl, pH 7.4). At the end of the preincubation period (90 seconds), 5-nmol CaCl2 pulses were performed every 60 seconds. After sufficient calcium loading, extramitochondrial calcium concentration abruptly increased, indicating a massive release of calcium by mitochondria as a result of mPTP opening as previously described.3,26
Assessment of the mPTP resistance by CRC was performed in every experimental group (n=5 to 10 per group). Following the technique of Basso et al,27 we measured CRC in the absence [CRC(–CsA)] or after addition of 1 µmol/L of CsA in the cuvette [CRC(+CsA)]. These 2 measurements, which were both performed in each mitochondria sample, allowed us to address the following questions: Does the in vivo treatment (ie, postconditioning, CsA or SB21) increase the resistance of the mPTP? Any increase of CRC(-CsA) by a treatment administered in vivo indicates that this treatment has improved the mPTP resistance. Is the observed enhancement of mPTP resistance a result of an attenuation of the interaction of CypD with the mPTP? If CypD is involved, then the addition of CsA in vitro will mildly increase CRC (and to a lower extent than in sham hearts). On the other hand, if CypD is not involved, then the in vitro addition of CsA will move CRC(+CsA) to near sham values. Is GSK3β involved in any improvement in mPTP resistance? If so, whatever the treated group, this improvement will be present in WT but not in GSK3β-S9A mitochondria.
Pharmacological Agents
CsA was dissolved in a mixture of Cremophor and 94% ethanol at 50 mg/mL and resuspended in normal saline at 1 mg/mL for intravenous injection (10 mg/kg). After dilution in dimethyl sulfoxide, SB21 was dissolved in a mixture of Cremophor (BASF, Lyon, France) and 94% ethanol at 350 µg/mL and resuspended in normal saline at 7 µg/mL for intravenous injection (70 µg/kg).
Statistical Analysis
All values are expressed as mean±SEM. Comparisons among groups were performed with 1-way ANOVA. When a significant F value was obtained, means were compared by use of Tukey test. The difference in the relationship between infarct size and the AR was evaluated by ANCOVA, with the AN (expressed in mg) as the dependent variable and AR (expressed in mg) as the covariate. Statistical significance was defined as a value of P<0.05.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Mortality and Exclusions
One hundred thirty-one mice were included in the study: 66 in the infarct size protocol and 65 in the mitochondria protocol. The mortality rate in the infarct size protocol averaged 17% (7 of 41) in WT compared with 22% (9 of 41) in GSK3β-S9A mice. No difference among groups was found relative to the incidence of mortality.
Infarct Size in GSK3β-S9A
In WT mice, the AR was comparable among groups, ranging from 35±2% to 43±3% of the LV weight. As expected, postconditioned hearts developed significantly smaller infarcts, the size of which averaged 39±2% versus 58±5% in controls (P<0.05; Figure 4A). Both CsA and SB21 reduced infarct size to a comparable extent compared with postconditioning, averaging 35±5% and 37±4% of the AR weight, respectively (P<0.05 versus control; Figure 4A).
|
In GSK3β-S9A mice, AR was comparable among groups, ranging from 32±2% to 43±3% of the LV weight. In control GSK3β-S9A mice, infarct size averaged 66±7% of the AR and was comparable to that observed in WT control (P=NS; Figure 4B). CsA significantly reduced infarct size that averaged 36±7% of AR (P<0.05 versus control). In contrast, both postconditioning and SB21 failed to trigger protection in GSK3β-S9A mice, with infarct size averaging 51±5% and 61±7% of AR in postconditioning- and SB21-treated GSK3β-S9A mice, respectively (P=NS versus control; Figure 4B).
Study of Isolated Mitochondria
Balanced Isolation Procedure in Experimental Groups
Electron microscopy showed no morphological difference between cardiac mitochondria isolated from GSK3β-S9A and WT mice (Figure 5). The mitochondrial protein yield was comparable in WT and GSK3β-S9A hearts, averaging 19.7±2.3 and 18.2±1.3 µg protein per mg wet weight, respectively (P=NS). To address whether the recovery of mitochondria after the isolation procedure was similar in WT and GSK3β-S9A hearts, the enzyme cytology approach of Deduve28 was performed with the citrate synthase of the mitochondrial matrix used as a marker enzyme. The specific activity of citrate synthase was comparable between the 2 WT and GSK3β-S9A sham groups, averaging 2605±377 and 2189±130 mU/mg, respectively (P=NS). In addition, the outer membrane intactness, measured as the maximal rate of cytochrome oxidase activity, was comparable between WT and GSK3β-S9A mice (Table 2). Oxidative phosphorylation was similar in WT and GSK3β-S9A mice at baseline (Table 2). Taken together, these data indicate that WT and GSK3β-S9A mitochondria were comparable after the isolation procedure.
|
|
Calcium Retention Capacity
In WT animals, as expected, ischemia-reperfusion resulted in a significant reduction in CRC(–CsA) compared with sham (Figure 6). The postconditioning, CsA, and SB21 groups exhibited a significant increase in CRC(–CsA). In GSK3β-S9A mice, sham mitochondria displayed CRC(–CsA) comparable to that of WT mice. Ischemia-reperfusion resulted in a comparable decrease in CRC(–CsA) in GSK3β and WT mice. In contrast to WT mice, only CsA, but neither postconditioning nor SB21, improved CRC(–CsA).
|
P<0.05 vs CRC(+CsA) in the sham group (same strain). PostC indicates postconditioning; C, control.In WT mitochondria, CRC(+CsA) also was significantly decreased in control when compared with sham animals. In the 3 treated groups (postconditioning, CsA, SB21), CRC(+CsA) significantly increased to near sham values (P<0.05 versus control). In GSK3β-S9A sham mitochondria, CRC(+CsA) was very similar to that of WT. Unlike what was observed in WT, CRC(+CsA) improved to near sham values only in the CsA group, but not in the postconditioning or SB21 group (Figure 6). However, the difference between CRC(+CsA) and CRC(–CsA) was comparable among groups in GSK3β mice.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This study demonstrates that the inhibition of GSK3β by postconditioning is required for infarct size reduction and likely acts by preventing opening of the mPTP at reperfusion independently of CypD.
Inhibition of GSK3β Is Required for Infarct Size Reduction by Postconditioning
Studies have demonstrated that, after a prolonged ischemic insult, the phosphorylation of certain prosurvival kinases in the early minutes of reperfusion such as PI3K-Akt and the mitogen-activated protein kinase p42/p44 extracellular signal-regulated kinases 1 and 2 protects the heart against ischemia-reperfusion injury.13 Reports have proposed that GSK3β may be activated downstream of this signaling pathway and play a pivotal role in ischemic preconditioning. Tong et al18 demonstrated in the isolated rat heart model that ischemic preconditioning enhances the phosphorylation and decreases the enzymatic activity of GSK3β and that lithium and SB21, which inhibit GSK3β, can both mimic ischemic preconditioning. Nishihara et al29 showed that ischemic preconditioning increases phosphorylation of Akt and GSK3β at 5 minutes of reperfusion and subsequently reduces infarct size in the in vivo rat heart.
In the present study, results obtained in WT animals demonstrate that ischemic postconditioning can protect the mouse heart to an extent similar to that previously reported in other species, including humans.1,2,4 Pharmacological inhibition of GSK3β by SB21 was as efficient as ischemic postconditioning, in agreement with previous reports by Gross et al30 and Pagel et al31 in the rat and rabbit model, respectively. However, a direct implication of GSK3β in postconditioning had not been reported yet. We demonstrated here that GSK3β-S9A mice, which have a cardiac GSK3β activity that cannot be inhibited by phosphorylation at serine 9, cannot be protected by postconditioning. In addition, administration of SB21, which is a potent competitive inhibitor of the ATP binding site of GSK3β, at the time of reperfusion significantly limited infarct size in WT but not in GSK3β-S9A mice. This demonstrates that inhibition of GSK3β at the time of reflow is a key event in postconditioning.
We noticed that GSK3β-S9A mice exhibited a significant heart rate reduction, together with frequent supraventricular ectopic beats after anesthesia. They also displayed a slight increase in LV end-systolic diameter dimension and a mild alteration of contractile function, as already reported by Michael et al.32 These authors further showed that GSK3β-S9A mice cardiomyocytes displayed an increased cytosolic calcium concentration, together with a reduced speed of the decrease in Ca2+ concentration in diastole, likely related to an abnormal expression of SERCA2a.32 One may wonder whether this dysregulation of calcium handling may have played a role in the genesis of the rhythm disturbances that we observed in GSK3β-S9A mice. Although other differences might exist between GSK3β-S9A and WT mice that were not measured in the present study, cardiac pacing abolished both the baseline rhythm abnormalities and the bradycardia-induced anti-ischemic protection that we observed in our pilot experiments, indicating that this cardiac phenotype likely did not bias the results of our study.
Postconditioning Inhibits mPTP Opening Through Phosphorylation of GSK3β in a CypD-Independent Manner
mPTP opening is a crucial event in lethal reperfusion injury.33 We previously demonstrated that postconditioning inhibits opening of the mPTP, and inhibitors of mPTP opening (eg, CsA) can mimic postconditioning when administered at the time of reperfusion.2,3,6,11 Because GSK3β-S9A hearts could not be protected by postconditioning, we hypothesized that phosphorylation of GSK3β might reduce infarct size through the inhibition of mPTP opening. We analyzed the resistance of the mPTP to opening after Ca2+ overload in mitochondria isolated from the risk region in myocardium of mice from all experimental groups.
At baseline, mitochondria of GSK3β-S9A and WT mice displayed similar morphology on electron microscopy, together with comparable oxidative phosphorylation. WT and GSK3β-S9A sham mitochondria exhibited comparable resistance of the mPTP with or without the addition of CsA, indicating that the function of mitochondrial permeability transition was comparable in these 2 types of mice under normoxic conditions.
As previously demonstrated by Gomez et al,3 postconditioning significantly improved the resistance of the mPTP in WT mice. As expected, this protection was mimicked by the in vivo administration of the mPTP inhibitor CsA, which both reduced infarct size and enhanced mPTP resistance in isolated mitochondria. The GSK3β inhibitor SB21 ameliorated mPTP resistance in WT mitochondria to an extent similar to that achieved with postconditioning.
In WT mitochondria, because the addition of CsA in vitro further significantly improved the mPTP resistance in both postconditioning and SB21 groups, one can conclude that these 2 treatments were protective independently of any action on CypD. It is not surprising to see that the addition of CsA in vitro did not increase the mPTP resistance much in the CsA group because here CypD had been partially inhibited by the in vivo administration of this powerful inhibitor of mPTP opening.
In GSK3β-S9A mice, the addition of CsA in vitro improved the resistance of the mPTP to a comparable extent in control, postconditioning, and SB21 mice compared with the sham group, indicating the absence of action via CypD. This result also indicates that GSK3β-S9A mitochondria retain the ability of being protected against lethal reperfusion injury by mPTP inhibition and demonstrates that GSK3β is located upstream of the mPTP. Importantly, the resistance of the mPTP in the postconditioning and SB21 groups remained significantly less than that in corresponding WT mitochondria. This demonstrates that GSK3β regulates the mPTP opening independently of CypD. The fact that SB21 could not prevent mPTP opening in GSK3β-S9A mitochondria is in agreement with data from Juhaszova et al,12 who demonstrated that direct inhibitors of GSK3β, like lithium or SB21, were able to inhibit mPTP opening in WT but not in GSK3β-S9A cardiomyocytes.34
Our results demonstrate that phosphorylation of GSK3β is required to inhibit mPTP opening by postconditioning. It is worth noting that postconditioning and preconditioning share common protective mechanism in this regard. Juhaszova et al,12 who measured the time to mPTP opening after laser exposure in adult myocytes isolated from GSK3β-S9A mice hearts, reported that hypoxic preconditioning could not prevent the reduction of the time to mPTP opening in GSK3β-S9A mice as opposed to WT mice. Our results also are supported by a recent study by Park et al,35 who demonstrated, using both isolated rat hearts and isolated cardiomyocytes, that pharmacological postconditioning by bradykinin was associated with an increased phosphorylation of GSK3β and Akt and an inhibition of mPTP opening as assessed by the loss of the inner mitochondrial membrane potential after calcium loading.
How GSK3β may inhibit mPTP opening at the time of reperfusion remains unclear. Juhaszova et al12 proposed that GSK3β would be localized on mitochondria and possibly associated with the adenine nucleotide translocator and the voltage-dependent anion channel, which are supposed to be 2 major components of the permeability transition pore complex. Recently, Nishihara et al36 reported, using the isolated rat heart model, that ischemic preconditioning enhanced phosphorylation of GSK3β at 5 minutes after reperfusion and that the phosphorylated GSK3β cosegregates with adenine nucleotide translocator (but not voltage-dependent anion channel). Whether this also applies to postconditioning, how postconditioning may prevent interaction of these different components of the mPTP, and how this might prevent pore opening remain to be clarified.
Conclusions
Using a transgenic mouse model of reperfused myocardial infarction, we demonstrated that serine 9 phosphorylation of GSK3β is required for cardioprotection by postconditioning and likely acts by inhibiting opening of the mPTP at the time of reperfusion in a CypD-independent way. Because recent reports indicate that postconditioning can protect the human heart, the present data represent an encouraging background for the search for and future development of pharmacological agents that would attenuate lethal reperfusion injury in patients with ongoing AMI.
|
Acknowledgments |
|---|
We are very grateful to Eric Olson for the generous gift of the GSK3β-S9A transgenic mice. We thank Rhonda Bassel-Duby and Brigitte Chhin for technical help and advice in maintaining the GSK3β-S9A mice.
Source of Funding
This work was supported by a grant from the Fédération Française de Cardiologie and by a grant from the Fondation de France.
Disclosures
None.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Zhao Z-Q, Corvera JS, Halkos ME, Kerendi F, Wang N-P, Guyton RA, Vinten-Johansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol. 2003; 285: H579–H588.
2. Argaud L, Gateau-Roesch O, Raisky O, Loufouat J, Robert D, Ovize M. Postconditioning inhibits mitochondrial permeability transition. Circulation. 2005; 111: 194–197.
3. Gomez L, Thibault HB, Gharib A, Dumont JM, Vuagniaux G, Scalfaro P, Derumeaux G, Ovize M. Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice. Am J Physiol Heart Circ Physiol. 2007; 293: H1654–H1661.
4. Heusch G, Buchert A, Feldhaus S, Schulz R. No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol. 2006; 101: 354–356.[CrossRef][Medline] [Order article via Infotrieve]
5. Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L'Huillier I, Aupetit JF, Bonnefoy E, Finet G, Andre-Fouet X, Ovize M. Postconditioning the human heart. Circulation. 2005; 112: 2143–2148.
6. Hausenloy DJ, Duchen MR, Yellon DM. Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury. Cardiovasc Res. 2003; 60: 617–625.
7. Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion: a target for cardioprotection. Cardiovasc Res. 2004; 61: 372–385.
8. Halestrap AP, Kerr PM, Javadov S, Woodfield KY. Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart. Biochim Biophys Acta. 1998; 1366: 79–94.[Medline] [Order article via Infotrieve]
9. Crompton M, Virji S, Ward JM. Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore. Eur J Biochem. 1998; 258: 729–735.[Medline] [Order article via Infotrieve]
10. Duchen MR, McGuinness O, Brown LA, Crompton M. On the involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion injury. Cardiovasc Res. 1993; 27: 1790–1794.
11. Bopassa JC, Ferrera R, Gateau-Roesch O, Couture-Lepetit E, Ovize M. PI 3-kinase regulates the mitochondrial transition pore in controlled reperfusion and postconditioning. Cardiovasc Res. 2006; 69: 178–185.
12. Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, Olson EN, Sollott SJ. Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest. 2004; 113: 1535–1549.[CrossRef][Medline] [Order article via Infotrieve]
13. Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM. Ischemic preconditioning protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart Circ Physiol. 2005; 288: H971–H976.
14. Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. Biochem J. 2001; 359: 1–16.[CrossRef][Medline] [Order article via Infotrieve]
15. Harwood AJ. Regulation of GSK-3: a cellular multiprocessor. Cell. 2001; 105: 821–824.[CrossRef][Medline] [Order article via Infotrieve]
16. Hardt SE, Sadoshima J. Glycogen synthase kinase-3{beta}: a novel regulator of cardiac hypertrophy and development. Circ Res. 2002; 90: 1055–1063.
17. Antos CL, McKinsey TA, Frey N, Kutschke W, McAnally J, Shelton JM, Richardson JA, Hill JA, Olson EN. Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A. 2002; 99: 907–912.
18. Tong H, Imahashi K, Steenbergen C, Murphy E. Phosphorylation of glycogen synthase kinase-3beta during preconditioning through a phosphatidylinositol-3-kinase-dependent pathway is cardioprotective. Circ Res. 2002; 90: 377–379.
19. Gross ER, Hsu AK, Gross GJ. Opioid-induced cardioprotection occurs via glycogen synthase kinase beta inhibition during reperfusion in intact rat hearts. Circ Res. 2004; 94: 960–966.
20. Gomez L, Chavanis N, Argaud L, Chalabreysse L, Gateau-Roesch O, Ninet J, Ovize M. Fas-independent mitochondrial damage triggers cardiomyocyte death after ischemia-reperfusion. Am J Physiol Heart Circ Physiol. 2005; 289: H2153–H2158.
21. Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, Rausch OL, Murphy GJ, Carter PS, Roxbee Cox L, Mills D, Brown MJ, Haigh D, Ward RW, Smith DG, Murray KJ, Reith AD, Holder JC. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol. 2000; 7: 793–803.[CrossRef][Medline] [Order article via Infotrieve]
22. Vivaldi MT, Kloner RA, Schoen FJ. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am J Pathol. 1985; 121: 522–530.[Abstract]
23. Gornall AG, Bardawill CJ, David MM. Determination of serum proteins by means of the Biuret reaction. J Biol Chem. 1949; 177: 751–766.
24. Ichas F, Jouaville LS, Sidash SS, Mazat JP, Holmuhamedov EL. Mitochondrial calcium spiking: a transduction mechanism based on calcium-induced permeability transition involved in cell calcium signalling. FEBS Lett. 1994; 348: 211–215.[CrossRef][Medline] [Order article via Infotrieve]
25. Argaud L, Gateau-Roesch O, Chalabreysse L, Gomez L, Loufouat J, Thivolet-Bejui F, Robert D, Ovize M. Preconditioning delays Ca2+-induced mitochondrial permeability transition. Cardiovasc Res. 2004; 61: 115–122.
26. Gomez L, Raisky O, Chalabreysse L, Verschelde C, Bonnefoy-Berard N, Ovize M. Link between immune cell infiltration and mitochondria-induced cardiomyocyte death during acute cardiac graft rejection. Am J Transplant. 2006; 6: 487–495.[CrossRef][Medline] [Order article via Infotrieve]
27. Basso E, Fante L, Fowlkes J, Petronilli V, Forte MA, Bernardi P. Properties of the permeability transition pore in mitochondria devoid of cyclophilin D. J Biol Chem. 2005; 280: 18558–18561.
28. Deduve C. Enzyme Cytology. New York, NY: Academic Press; 1967; 1–26.
29. Nishihara M, Miura T, Miki T, Sakamoto J, Tanno M, Kobayashi H, Ikeda Y, Ohori K, Takahashi A, Shimamoto K. Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3beta. Am J Physiol Heart Circ Physiol. 2006; 291: H748–H755.
30. Gross ER, Hsu AK, Gross GJ. The JAK/STAT pathway is essential for opioid-induced cardioprotection: JAK2 as a mediator of STAT3, Akt, and GSK-3beta. Am J Physiol Heart Circ Physiol. 2006; 291: H827–H834.
31. Pagel PS, Krolikowski JG, Neff DA, Weihrauch D, Bienengraeber M, Kersten JR, Warltier DC. Inhibition of glycogen synthase kinase enhances isoflurane-induced protection against myocardial infarction during early reperfusion in vivo. Anesth Analg. 2006; 102: 1348–1354.
32. Michael A, Haq S, Chen X, Hsich E, Cui L, Walters B, Shao Z, Bhattacharya K, Kilter H, Huggins G, Andreucci M, Periasamy M, Solomon RN, Liao R, Patten R, Molkentin JD, Force T. Glycogen synthase kinase-3{beta} regulates growth, calcium homeostasis, and diastolic function in the heart. J Biol Chem. 2004; 279: 21383–21393.
33. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J. 1999; 341: 233–249.[CrossRef][Medline] [Order article via Infotrieve]
34. Murphy E. Inhibit GSK-3beta or there’s heartbreak dead ahead. J Clin Invest. 2004; 113: 1526–1528.[CrossRef][Medline] [Order article via Infotrieve]
35. Park S-S, Zhao H, Mueller RA, Xu Z. Bradykinin prevents reperfusion injury by targeting mitochondrial permeability transition pore through glycogen synthase kinase 3[beta]. J Mol Cell Cardiol. 2006; 40: 708–716.[CrossRef][Medline] [Order article via Infotrieve]
36. Nishihara M, Miura T, Miki T, Tanno M, Yano T, Naitoh K, Ohori K, Hotta H, Terashima Y, Shimamoto K. Modulation of the mitochondrial permeability transition pore complex in GSK-3[beta]-mediated myocardial protection. J Mol Cell Cardiol. 2007; 43: 564–570.[CrossRef][Medline] [Order article via Infotrieve]
 |
|
 |
|
  J. Xi, W. Tian, L. Zhang, Y. Jin, and Z. Xu Morphine prevents the mitochondrial permeability transition pore opening through NO/cGMP/PKG/Zn2+/GSK-3{beta} signal pathway in cardiomyocytes Am J Physiol Heart Circ Physiol, February 1, 2010; 298(2): H601 - H607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Omar, L. Wang, and A. S. Clanachan Cardioprotection by GSK-3 inhibition: role of enhanced glycogen synthesis and attenuation of calcium overload Cardiovasc Res, January 31, 2010; (2010): cvp421v2 - cvp421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Wong, A. M. Aponte, C. Steenbergen, and E. Murphy Cardioprotection leads to novel changes in the mitochondrial proteome Am J Physiol Heart Circ Physiol, January 1, 2010; 298(1): H75 - H91. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Bopassa, M. Eghbali, L. Toro, and E. Stefani A novel estrogen receptor GPER inhibits mitochondria permeability transition pore opening and protects the heart against ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, January 1, 2010; 298(1): H16 - H23. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Musiolik, P. van Caster, A. Skyschally, K. Boengler, P. Gres, R. Schulz, and G. Heusch Reduction of infarct size by gentle reperfusion without activation of reperfusion injury salvage kinases in pigs Cardiovasc Res, January 1, 2010; 85(1): 110 - 117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tamareille, N. Ghaboura, F. Treguer, D. Khachman, A. Croue, D. Henrion, A. Furber, and F. Prunier Myocardial reperfusion injury management: erythropoietin compared with postconditioning Am J Physiol Heart Circ Physiol, December 1, 2009; 297(6): H2035 - H2043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Heusch No RISK, no ... cardioprotection? A critical perspective Cardiovasc Res, November 1, 2009; 84(2): 173 - 175. [Full Text] [PDF]
|
|
|
|
|
|
P. Sicard, S. Jacquet, K. S. Kobayashi, R. A. Flavell, and M. S. Marber Pharmacological postconditioning effect of muramyl dipeptide is mediated through RIP2 and TAK1 Cardiovasc Res, July 15, 2009; 83(2): 277 - 284. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Gomez, B. Li, N. Mewton, I. Sanchez, C. Piot, M. Elbaz, and M. Ovize Inhibition of mitochondrial permeability transition pore opening: translation to patients Cardiovasc Res, July 15, 2009; 83(2): 226 - 233. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Juhaszova, D. B. Zorov, Y. Yaniv, H. B. Nuss, S. Wang, and S. J. Sollott Role of Glycogen Synthase Kinase-3{beta} in Cardioprotection Circ. Res., June 5, 2009; 104(11): 1240 - 1252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Miyamoto, M. Rubio, and M. A. Sussman Nuclear and mitochondrial signalling Akts in cardiomyocytes Cardiovasc Res, May 1, 2009; 82(2): 272 - 285. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Skyschally, P. van Caster, K. Boengler, P. Gres, J. Musiolik, D. Schilawa, R. Schulz, and G. Heusch Ischemic Postconditioning in Pigs: No Causal Role for RISK Activation Circ. Res., January 2, 2009; 104(1): 15 - 18. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Zhou, L. Zhang, J. Xi, W. Tian, and Z. Xu Ethanol Prevents Oxidant-Induced Mitochondrial Permeability Transition Pore Opening in Cardiac Cells Alcohol Alcohol., January 1, 2009; 44(1): 20 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Zhai and J. Sadoshima Overcoming an Energy Crisis?: An Adaptive Role of Glycogen Synthase Kinase-3 Inhibition in Ischemia/Reperfusion Circ. Res., October 24, 2008; 103(9): 910 - 913. [Full Text] [PDF]
|
|
|
|
|
|
G. Chanoit, S. Lee, J. Xi, M. Zhu, R. A. McIntosh, R. A. Mueller, E. A. Norfleet, and Z. Xu Exogenous zinc protects cardiac cells from reperfusion injury by targeting mitochondrial permeability transition pore through inactivation of glycogen synthase kinase-3{beta} Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1227 - H1233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Murphy and C. Steenbergen Does Inhibition of Glycogen Synthase Kinase Protect in Mice? Circ. Res., August 1, 2008; 103(3): 226 - 228. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||