Isoflurane and sevoflurane have been shown to elicit myocardial postconditioning, but the effect of desflurane remain unknown. The authors studied the mechanisms involved in desflurane-induced myocardial postconditioning.
Contracting isolated human right atrial trabeculae (34 degrees C, stimulation frequency 1 Hz) were exposed to 30-min hypoxia followed by 60-min reoxygenation. Desflurane at 3%, 6%, and 9% was administered during the first 5-min of reoxygenation. Postconditioning with 6% desflurane was studied in the presence of 1 microM calphostin C, a protein kinase C inhibitor; 800 mm 5-hydroxydecanoate, a mitochondrial adenosine triphosphate-sensitive potassium channels antagonist; 1 microM Akt inhibitor; 20 microM PD89058, an extracellular-regulated kinase 1/2 inhibitor; and 1 microM SB 202190, a p38 mitogen-activated protein kinase inhibitor. The force of contraction at the end of the 60-min reoxygenation period was compared (mean +/- SD). The p38 mitogen-activated protein kinase phosphorylation was studied using Western blotting.
Desflurane at 3% (77 +/- 10% of baseline), 6% (90 +/- 14% of baseline), and 9% (86 +/- 11% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (51 +/- 9% of baseline; P < 0.001). Calphostin C (55 +/- 3% of baseline), 5-hydroxydecanoate (53 +/- 3% of baseline), Akt inhibitor (57 +/- 8% of baseline), PD89058 (64 +/- 6% of baseline), and SB 202190 (61 +/- 3% of baseline) abolished desflurane-induced postconditioning. Western blot analysis showed that 6% desflurane increased p38 mitogen-activated protein kinase phosphorylation.
In vitro, desflurane postconditioned human atrial myocardium through protein kinase C activation, opening of mitochondrial adenosine triphosphate-sensitive potassium channels, Akt and extracellular-regulated kinase 1/2 activation, and p38 mitogen-activated protein kinase phosphorylation.
SIMILAR to the cardioprotection of volatile anesthetics given as preconditioning stimulus, these drugs can also protect the heart when given during reperfusion. This might result from anesthetic-induced postconditioning after the administration of the anesthetic at the very start of reperfusion. Therefore, isoflurane and sevoflurane administered during the first minute of reperfusion have been shown to reduce myocardial infarct size, in vivo , in rabbit and rats.1–4The protection afforded was similar to that observed after ischemic postconditioning.1Finally, a 15- to 30-min administration of desflurane at the time of reperfusion has been shown to protect myocardium in rabbit in vivo 5and in isolated rat heart.6
Mechanisms involved in volatile anesthetic–induced postconditioning remain incompletely studied. The activation of phosphatidylinositol-3-kinase and the opening of mitochondrial adenosine triphosphate–sensitive potassium (mitoKATP) channels have been identified as important mediators of sevoflurane- and isoflurane-induced postconditioning.1–3The phosphatidylinositol-3-kinase pathway results in activation of downstream targets such as protein kinase C (PKC), and survival protein kinases including Akt and extracellular-regulated kinase 1/2 (ERK1/2). PKC has been shown to play a key role in the signaling pathway of ischemic postconditioning, but its role in anesthetic postconditioning has not been studied.7,8On the other hand, isoflurane-induced postconditioning has been shown to be mediated, at least in part, by Akt and ERK1/2 activation.9,10Nevertheless, the role of Akt and ERK1/2 in desflurane-induced postconditioning remain unknown. p38 mitogen-activated protein kinase (MAPK) activation has been shown to be involved in ischemic postconditioning.11In contrast, it has been suggested that p38 MAPK inhibition was involved in attenuated apoptosis after hypoxic postconditioning.12The role of p38 MAPK in anesthetic-induced postconditioning remains unknown.
The current study examined the effect of desflurane-induced postconditioning in isolated human atrial myocardium, and studied the role of PKC, mitoKATPchannels, Akt, ERK1/2, and p38 MAPK in desflurane-induced postconditioning.
Materials and Methods
After the approval of local medical ethics committee (Comité de Protection des Personnes Nord Ouest III, Caen, France) and written informed consent, right atrial appendages were obtained during cannulation for cardiopulmonary bypass from patients scheduled for routine coronary artery bypass surgery and aortic valve replacement. All patients received total intravenous anesthesia with propofol, sufentanil, and pancuronium. Patients with chronic atrial arrhythmia and with diabetes mellitus treated with insulin or oral hypoglycemic agents were excluded from the study.
Right atrial trabeculae (one per appendage) were dissected and suspended vertically between an isometric force transducer (MLT0202; ADInstruments, Sydney, Australia) and a stationary stainless clip in a 200-ml jacketed reservoir filled with daily prepared Tyrode's modified solution containing 120 mm NaCl, 3.5 mm KCl, 1.1 mm MgCl2, 1.8 mm NaH2PO4, 25.7 mm NaHCO3, 2.0 mm CaCl2, and 5.5 mm glucose. The jacketed reservoir was maintained at 34°C by a thermostatic water circulator (Polystat micropros; Bioblock, Illkirch, France). The bathing solution was insufflated with carbogen (95% O2–5% CO2), resulting in a pH of 7.40 and a partial pressure of oxygen of 600 mmHg. Isolated muscles were field-stimulated at 1 Hz by two platinum electrodes with rectangular wave pulses of 5-ms duration 20% above threshold (CMS 95107; Bionic Instrument, Paris, France).
Trabeculae were equilibrated for 60–90 min to allow stabilization of their optimal mechanical performance at the apex of the length–active isometric tension curve (Lmax). The force developed was measured continuously, digitized at a sampling frequency of 400 Hz, and stored on a writable compact disc for analysis (MacLab; ADInstruments).
At the end of experiment, the muscle cross-sectional area was calculated from its weight and length assuming a cylindrical shape and a density of 1. To avoid core hypoxia, trabeculae included in the study should have a cross-sectional area less than 1.0 mm2, a force of contraction normalized per cross-sectional area (FoC) greater than 5.0 mN/mm2, and a ratio of resting force/total force less than 0.45.
At the end of the stabilization period, the trabeculae were randomly assigned (sealed envelopes) to one of the experimental groups. In all groups, hypoxia–reoxygenation was performed by replacing 95% O2–5% CO2with 95% N2–5% CO2in the buffer for 30 min, followed by a 60-min oxygenated recovery period.
In the control group (n = 10), trabeculae were exposed to the hypoxia reoxygenation protocol alone (fig. 1). In the desflurane treatment groups, desflurane was delivered to the organ bath by the gas flow passing through a specific calibrated vaporizer. Desflurane concentration in the carrier gas phase was measured with an infrared calibrated analyzer (Capnomac; Datex, Helsinki, Finland). Desflurane was administered at 3% (n = 6), 6% (n = 6), and 9% (n = 6) during the first 5 min of reoxygenation (fig. 1). These concentrations correspond to 0.5, 1.0, and 1.5 minimum alveolar concentration (MAC) desflurane in adult humans at 37°C, respectively.
Mechanisms involved in desflurane-induced postconditioning were studied in separate groups exposed to 6% desflurane in the presence of 1 μm calphostin C, a PKC inhibitor (n = 6); 800 μm 5-hydroxydecanoate (5-HD), a mitoKATPchannel antagonist (n = 6); 1 μm Akt inhibitor IV (n = 6); 20 μm PD98059, an ERK1/2 inhibitor (n = 6); 1 μm SB 202190, a p38 MAPK inhibitor (n = 6); and 0.1% dimethyl sulfoxide (DMSO; n = 6). Pharmacologic agents and DMSO were administered 5 min before, throughout, and 10 min after desflurane exposure.
In additional groups, muscles were exposed to 1 μm calphostin C (n = 6), 800 μm 5-HD (n = 6), 1 μm Akt inhibitor IV (n = 6), 20 μm PD98059 (n = 6), 1 μm SB 202190 (n = 6), and 0.1% DMSO (n = 6) 5 min before and in the first 15 min of reoxygenation (fig. 1).
Calphostin C, SB 202190, Akt inhibitor IV, and PD98059 dissolved in DMSO; the volume of DMSO never exceeded 0.1% of the total bath volume. The concentrations of inhibitors used have been validated in previous experimental studies in human myocardium.13,14
5-Hydroxydecanoate, calphostin C, and Akt inhibitor IV were purchased from Calbiochem (VWR International, Fontenay sous Bois, France); SB 202190, PD98059, and DMSO were purchased from Sigma Aldrich (Saint Quentin Fallavier, France). Desflurane was purchased from GlaxoWellcome (Marly-le-Roi, France).
Western Blot Analysis
The right atrial appendage was pinned in a chamber (5 ml) containing Tyrode's modified solution, oxygenated with 95% O2and 5% CO2, and maintained at 34°± 0.5°C (Polystat micropros; Bioblock). The preparation was stimulated at a frequency of 1 Hz.
In all groups, after a 90-min equilibration period, hypoxia was performed by replacing 95% O2–5% CO2with 95% N2–5% CO2in the buffer for 30 min, followed by a 5-min oxygenated recovery period (control; n = 5) or by 5-min exposure to 6% desflurane (desflurane; n = 5) (fig. 1). Then, atrial samples were frozen in liquid nitrogen and stored at −80°C before protein extraction and Western blot analysis. Frozen tissue sample were extracted into extraction buffer containing 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm ς-glycerophosphate, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin-pepstatin A-aprotinin and homogenized with a polytron. Homogenates were centrifuged at 10,000g for 30 min, the supernatant was decanted, and protein concentration was determined using the BCA protein assay (Bradford colorimetric method; Bio-Rad, Marnes-la-Coquette, France). Extracted protein samples were reduced with 100 mm DTT and denatured at 95°C for 5 min. Denatured proteins (30 μg/lane) from human atrial tissues were separated on 10% SDS-PAGE and transferred on nitrocellulose. Membranes were blocked for 1 h in TRISS-buffered saline Tween buffer (0.02 m Tris-HCl, pH 7.5, 0.15 m NaCl, and 0.05% Tween 20) containing 10% nonfat dry milk at room temperature.
The membranes were incubated with a rabbit monoclonal antibody recognizing p38 MAPK and phospho-p38 MAPK (Thr 180/Tyr 182, 1/1,000 dilution; Cell Signaling Technology, Ozyme, Saint Quentin Yvelines, France), one night at 4°C. After washing in TRISS-buffered saline Tween, the blots were incubated with a secondary antibody (goat anti-rabbit, 1/1,000 dilution) coupled to peroxidase (Santa Cruz Technology, Tebu-Bio, Le Perray en Yvelines, France) for 1 h at room temperature. The blots were washed again in TRISS-buffered saline Tween, and the bands were detected using chemiluminescence reagent (Pierce Perbio Science, Brebieres, France) before exposure to photography film. The Western blots of each group were stripped and probed again with an antibody against β-tubulin (Santa Cruz Technology) to ensure equivalent loading. The developed films were scanned, and the band densities were quantified using NIH Image J software (Research Service Branch, National Institutes of Mental Health, Bethesda, MD).
The endpoint of the study was the recovery of FoC at 60 min of reoxygenation (FoC60, expressed as percent of baseline). Power analysis calculated a group size of n = 4 to detect a difference of 40% (control and inhibitors group: FoC60= 50 ± 9% of baseline; and desflurane 6% group: FoC60= 90 ± 9% of baseline) with a power of 0.8 at an α level of 0.05. The number of experiments per group was calculated based on one-way analysis of variance with six control and inhibitor groups and one 6% desflurane group. Data are expressed as mean ± SD. Baseline values of main mechanical parameters, age, preoperative left ventricular ejection fraction, and FoC60were compared by univariate analysis of variance with group factor as the independent variable. If the P value was less than 0.05, a Bonferroni post hoc analysis was performed. Within-group data were analyzed over time using analysis of variance for repeated measures and Bonferroni post hoc analysis with group factor and time (baseline, hypoxia 5, 10, 20, 30 min, and reoxygenation 5, 10, 20, 30, 40, 50, and 60 min) as independent variables. The preoperative drug treatment repartition was analyzed using a chi-square test.
In Western blotting, band densities for protein of interest were then normalized to that of the band for β-tubulin in the same sample, and then normalized to the mean of control tissues, defined as 1 arbitrary unit ± SD. Statistical comparisons were made by use of analysis of variance for repeated measures and Bonferroni post hoc analysis. All P values were two-tailed, and a P value of less than 0.05 was required to reject the null hypothesis. Statistical analysis was performed using Statview 5 software (Deltasoft, Meylan, France) and PASS 2005 (NCSS Statistical and Power Analysis Software; Kaysville, UT).
There were no statistical differences between groups for patients' demographic data, preoperative treatments, and left ventricular ejection fraction (table 1). One hundred human right atrial trabeculae and 10 right atrial appendages were studied. There were no differences in baseline values for Lmax, cross-sectional area, ratio of resting force to total force, or FoC between groups (table 2).
Effects of Desflurane on Hypoxia Reoxygenation
In the control group, reoxygenation resulted in a partial recovery of FoC (FoC60: 51 ± 9% of baseline; fig. 2). As compared with the control group, 3% desflurane (77 ± 10% of baseline; P < 0.0001), 6% desflurane (90 ± 14% of baseline; P < 0.0001), and 9% desflurane (86 ± 11% of baseline; P < 0.0001) increased FoC60. There was no difference in FoC60measured in the 3%, 6%, and 9% desflurane groups (fig. 2).
Effects of 5-Hydroxydecanoate, Calphostin C, Akt Inhibitor, PD98059 and SB 202190
As shown in figure 3, the desflurane-induced enhanced recovery of FoC60(6% desflurane: 90 ± 14% of baseline) was abolished in the presence of calphostin C (Des + Cal: 55 ± 3%; P < 0.0001), 5-HD (Des + 5-HD: 53 ± 3% of baseline; P < 0.0001), Akt inhibitor IV (Des + AktInh: 57 ± 8% of baseline; P < 0.0001), PD98059 (Des + PD: 64 ± 6% of baseline; P < 0.0001), and SB 202190 (Des + SB: 61 ± 3% of baseline; P < 0.0001). DMSO (Des + DMSO: 84 ± 4% of baseline; P = 0.054) did not modify FoC as compared with the desflurane group. As compared with the control group (control: 51 ± 9% of baseline), calphostin C (Cal: 54 + 5% of baseline; P = 0.39), 5-HD (55 ± 3% of baseline; P = 0.29), Akt inhibitor IV (AktInh: 54 ± 5% of baseline; P = 0.50), PD98059 (PD: 55 ± 4% of baseline; P = 0.31), SB 202190 (SB: 56 ± 5% of baseline; P = 0.16), and DMSO (55 ± 7% of baseline; P = 0.27) did not significantly modify FoC60(fig. 3).
Phosphorylation of p38 Mitogen-activated Protein Kinase
Desflurane at 6% in the first 5 min of reperfusion significantly increased p38 MAPK phosphorylation (2.8 ± 0.5-fold increase in desflurane vs. control; P < 0.0001). As compared with the control group, desflurane did not modify protein expression of p38 MAPK (fig. 4).
The current study showed that desflurane postconditioned isolated human atrial myocardium exposed to hypoxia–reoxygenation. Furthermore, desflurane-induced postconditioning was dependent on Akt, ERK1/2, PKC, and p38 MAPK activation and opening of mitoKATPchannels. Finally, desflurane-induced postconditioning increased p38 MAPK phosphorylation.
Ischemic postconditioning (i.e. , brief cycles of alternating ischemia and reperfusion at the onset of reperfusion) was first established in 2003 by Zhao et al. 15It has been shown that ischemic postconditioning markedly decreased myocardial ischemia–reperfusion injury through recruiting prosurvival signaling pathways.16A decade ago, volatile anesthetics administered during the first 30 min of a 1-h reperfusion period were shown to decrease myocardial reperfusion injury.5,6We have previously shown that administration of desflurane during the first 15 min of a 3-h reperfusion reduced infarct size to the same extent that desflurane-induced preconditioning in rats in vivo .17However, this cannot be attributable strictly to pharmacologic postconditioning, which refers to brief interventions at the very start of the reperfusion. On the other hand, increasing evidence supports volatile anesthetic–induced postconditioning during myocardial ischemia–reperfusion injury. Therefore, a brief administration of isoflurane (1.0 MAC) at the beginning of reperfusion has been shown to reduce infarct size in rabbits.1,2Sevoflurane administered in early reperfusion was as effective as preconditioning in reducing infarct size in rat heart after ischemia–reperfusion.3,4The current study shows that 5-min administration of desflurane (3%, 6%, and 9%) at the onset of reoxygenation postconditioned human myocardium after 30-min hypoxia in vitro . The beneficial effect of desflurane-induced postconditioning was observed on the recovery of FoC during the reoxygenation period (fig. 2).
Although the recovery of FoC at the end of the reoxygenation measured in the presence of 3% desflurane was lower than that measured in the presence of 6% and 9% desflurane, the difference was not statistically significant. However, a statistical type II error cannot be ruled out. Previous studies have shown that 1.0 MAC isoflurane and sevoflurane was required to trigger myocardial postconditioning in vivo .1,2,18Furthermore, increasing concentrations of sevoflurane to 2.0 MAC did not further reduced infarct size.18As suggested for myocardial preconditioning, the threshold required to trigger the cardioprotective effect may vary among species, experimental models, and ischemia–reperfusion injury protocols. Further studies are required to determine whether there is a threshold and a concentration-dependent response in volatile anesthetic-induced postconditioning.
A growing body of evidence supports the concept that postconditioning triggers a cardioprotective cascade of molecular signaling events similar to that of ischemic preconditioning. Indeed, many of the triggers/mediators implicated in preconditioning seem to be involved in postconditioning.16Therefore, activation and translocation of PKC have been shown to be a key mediator of ischemic and pharmacologic preconditioning19and have been suggested to be also a mediator of ischemic postconditioning.7,8Zatta et al. 7showed that ischemic postconditioning increased PKC-ϵ expression and translocation but limited translocation of PKC-Δ to mitochondria in rat heart in vivo . In addition, activation of PKC just before reperfusion through phorbol 12-myristate 13-acetate decreased infarct size in rabbits in vivo .8The current investigation showed that desflurane-induced postconditioning was inhibited in the presence of calphostin C, suggesting that PKC was involved in desflurane-induced postconditioning in human myocardium. It has been shown that activation of PKC-ϵ led to phosphorylation and opening of mitoKATP20and formed a mitochondrial localized signaling complex with MAPKs.21Interestingly, the current study also showed that desflurane-induced postconditioning was inhibited by 5-HD and PD98059, suggesting that opening of mitoKATPchannels and ERK1/2 activation were important mediators of desflurane-induced postconditioning. Opening of the mitoKATPchannel has been shown to inhibit the opening of the mitochondrial permeability transition pore (mPTP), which is recognized as an important mediator of reperfusion injury.16The current results confirm and extend previous findings showing that mitoKATPchannel opening was involved in isoflurane- and sevoflurane-induced postconditioning in rabbit and rat myocardium.2,3The role of mPTP has also been suggested in isoflurane-induced postconditioning because isoflurane-induced postconditioning was inhibited by the mPTP opener atractyloside.2Furthermore, it has been shown that 5-HD inhibited the desflurane-induced resistance of the mPTP to calcium-induced opening, suggesting a close link between mitoKATPand the mPTP.22Finally, isoflurane prevented opening of the mPTP, at least in part, through inhibition of glycogen synthase kinase 3β after protein kinase B/Akt phosphorylation.23
In addition to PKC-ϵ, mitoKATPchannel openers have also been shown to activate ERK1/2.24In human myocardium, Sivaraman et al. 11showed that ischemic postconditioning enhanced the activity of ERK1/2 and Akt at the time of reperfusion. The current study strongly suggests that ERK1/2 and Akt activation are important mediators of volatile anesthetic–induced postconditioning in human myocardium. To our knowledge, only one study has reported that ERK1/2 activation mediated isoflurane-induced postconditioning in rabbits.9Similarly, few studies suggested the involvement of the phosphatidylinositol-3-kinase/Akt cascade in isoflurane-induced postconditioning.1,10
The role of p38 MAPK in ischemic and pharmacologic postconditioning remains poorly studied. Only one study has examined the role of p38 MAPK in ischemic postconditioning. Sun et al. 12showed that hypoxic postconditioning attenuated activation of p38 MAPK and JUN n-terminal kinase concomitantly with a reduction in apoptotic cardiomyocytes. The current study showed that the specific p38 MAPK inhibitor SB 202190 abolished the enhanced recovery of contractile force resulting from desflurane-induced postconditioning. Furthermore, we showed that phosphorylation of p38 MAPK was enhanced after desflurane administration at the onset of the reoxygenation. These results suggest that activation of p38 MAPK was involved in desflurane-induced postconditioning. However, the current study could not determine whether p38 MAPK activation was required during the triggering or mediating phase of desflurane-induced postconditioning. Furthermore, multiple isoforms of p38 MAPK exist. Of these, p38α and β are expressed in the myocardium and may have opposing functions.25Further studies are required to determine the precise role of p38 MAPK in desflurane-induced postconditioning both on myocardial infarct size and myocytes apoptosis.
The results of the current study must be interpreted with the knowledge of recent clinical studies suggesting the cardioprotective effects of ischemic postconditioning after coronary angioplasty for ongoing acute myocardial infarction26and adult cardiac valve surgery.27Importantly, ischemic postconditioning has recently been shown to provide short- and long-term myocardial beneficial effects in patients scheduled for primary angioplasty and stenting of acute myocardial infarction.28
Several limitations must be considered in the interpretation of the current results. First the effects of anesthetics drugs, diseases, or medical treatments received by the patients before obtaining the atrial appendages cannot be ruled out.29Furthermore, although age has been shown to attenuate volatile anesthetic-induced preconditioning,30there was no difference in patients' demographic data between groups. However, the patients included in this study are representative of those patients in whom desflurane may be used during anesthesia. Importantly, our investigation included a control group that would be equally affected by any of these potentially modifying factors. Second, our experiments were performed during moderate hypothermia (34°C) to ensure stability of trabeculae over time. Nevertheless, no data exist on the postconditioning effect hypothermia. On the other hand, hypothermia may have decreased mitoKATPchannel sensitivity.31However, during surgical procedures, moderate hypothermia may occur. Third, inhibition of PKC by calphostin C has no isoform specificity. Similarly, although SB 202190 and SB 203580 are widely used as p38 MAPK inhibitors, it has been suggested that they may also inhibit JUN n-terminal kinase at high concentration, whereas 1 μm was specific for the inhibition of p38 MAPK.32However, this occurred at a higher dose than that administered in the current study. Fourth, we studied isolated contracting human atrial trabeculae but not myocardial ventricular infarct size. Nevertheless, as described in preconditioning, the beneficial effects of postconditioning have also been described on reperfusion-induced arrhythmias33and myocardial conctractility.11
In conclusion, the current study showed that desflurane postconditioned isolated human myocardium as assessed by recovery of FoC. Furthermore, activation of PKC, Akt, ERK1/2, and p38 MAPK and opening of mitoKATPchannels were involved in desflurane-induced postconditioning.