Desflurane Preconditioning Induces Time-dependent Activation of Protein Kinase C Epsilon and Extracellular Signal-regulated Kinase 1 and 2 in the Rat Heart In Vivo 

The study was performed in accordance with the regulations of the German Animal Protection Law and was approved by the Bioethics Committee of the District of Düsseldorf, Nordrhein-Westfalen, Germany.

Desflurane was purchased from Baxter Deutschland GmbH (Unterschleissheim, Germany). Calphostin C and PD98059 were purchased from Sigma (Taufkirchen, Germany). The enhanced chemoluminescence protein detection kit was from Santa Cruz (Heidelberg, Germany). Phospho-PKC-ε and total PKC-ε polyclonal antibodies were from Upstate (Lake Placid, NY, USA). Phosho-ERK1/2 and total ERK1/2 polyclonal antibodies were from Cell Signaling (Frankfurt/M, Germany). Monoclonal anti-α-tubulin and antiactin antibodies were from Sigma (Taufkirchen, Germany). Peroxidase-conjugated donkey antirabbit and peroxidase-conjugated goat antimouse antibodies were from Jackson Immunolab (Dianova, Hamburg, Germany). All other materials were either purchased from Sigma or Merck-Eurolab (Munich, Germany).

Male Wistar rats (250–300 g) were anesthetized by intraperitoneal S(+)-ketamine injection (150 mg/kg). Further preparation and infarct size measurement by triphenyltertrazoliumchlorid staining were performed as described previously.14In summary, after tracheal intubation, the lungs were ventilated with oxygen-enriched air and a positive end-expiratory pressure of 2–3 cm H²O. Respiratory rate was adjusted to maintain partial pressure of carbon dioxide within physiologic limits. Body temperature was maintained at 38°C by the use of a heating pad. The right jugular vein was cannulated for saline and drug infusion, and the left carotid artery was cannulated for measurement of aortic pressure. Anesthesia was maintained by continuous α-chloralose infusion. A lateral left sided thoracotomy followed by pericardiotomy was performed and a ligature (5–0 prolene) was passed below the main branch of the left coronary artery. The ends of the suture were threaded through a propylene tube to form a snare, and the coronary artery was occluded by tightening the snare. Successful coronary artery occlusion was verified by epicardial cyanosis. The area of risk and the infarcted area were determined by planimetry using SigmaScan Pro 5®computer software (SPSS Science Software, Chicago, IL) and corrected for dry weight.

The study protocol is shown in figure 1. For infarct size measurement, 42 rats were randomly assigned to one of the six groups and all animals were subjected to 25 min of left coronary artery occlusion followed by 120 min of reperfusion. After surgical preparation, the rats were allowed to recover for 30 min and then received either vehicle (0.9% saline), the highly selective PKC inhibitor calphostin C (0.1 mg/kg)15in dimethyl sulfoxide 1% aqueous solution, or the ERK1/2 mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (1 mg/kg in dimethyl sulfoxide 1% aqueous solution)16,17in the presence or absence of 1 minimum alveolar concentration (MAC) desflurane (1 MAC equals 6.9 vol %).18 

In the preliminary phase of this study two groups (each n = 6) were subjected to the same ischemia/reperfusion protocol with or without 3 ml/kg dimethyl sulfoxide 1% infusion and the infarct size was not changed after dimethyl sulfoxide (control, 57.4 ± 5.2% of area at risk versus  dimethyl sulfoxide alone, 53.4 ± 21.9%). To investigate time-dependent activation of PKC-ε and ERK1/2, twelve additional groups received either vehicle, calphostin C or PD98059, and the hearts were excised at four different time points (fig. 1B): without further treatment (baseline), after the first period of 1 MAC desflurane (Des I), after the second period of 1 MAC desflurane (Des II), and after the last washout phase before infarct inducing ischemia (Wash II).

The aortic pressure signal was digitized using an analog-to-digital converter (PowerLab/8SP, ADInstruments Pty Ltd, Castle Hill, Australia) at a sampling rate of 500 Hz and continuously recorded on a personal computer using Chart for Windows v5.0(ADInstruments Pty Ltd).

Tissue specimens were prepared for protein analysis to investigate PKC-ε activation and distribution (membrane-, cytosolic-fraction) and ERK1/2 phosphorylation. The excised hearts were frozen in liquid nitrogen. The frozen tissue was pulverized and dissolved in lysis buffer containing Tris base, EGTA, NaF, and Na³VO⁴(as phosphatase inhibitors), a freshly added protease inhibitor mix (aprotinin, leupeptin and pepstatin), 100 μm/ml okadaic acid, and dithiothreitol. The solution was vigorously homogenized on ice (Homogenisator, IKA, Staufen, Germany) and then centrifuged at 1000g , 4°C, for 10 min to obtain the cytosolic fraction of the tissue. The supernatant, containing cytosolic fraction, was centrifuged again at 16,000g , 4°C, for 15 min to clean up the fraction. The remaining pellet was resuspended in lysis buffer containing 1% Triton × 100, incubated for 60 min on ice and vortexed. The solution was centrifuged at 16000g , 4°C, for 15 min and the supernatant was collected as membrane fraction.

After protein determination by the Lowry method19equal amounts of protein were mixed with loading buffer containing Tris-HCl, glycerol, and bromphenol blue. Samples were vortexed and boiled at 95°C before being subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were separated by electrophoresis and then transferred to a polyvinylidene difluoride membrane by tank blotting. Unspecific binding of the antibody was blocked by incubation with 5% fat dry milk powder solution in Tris buffered saline containing Tween for 2 h. Subsequently, the membrane was incubated overnight at 4°C with the respective first antibody (either phospho specific or total in the case of PKC-ε and ERK1/2, anti-α-tubulin, and antiactin as internal standard) at indicated concentrations. After washing in fresh, cold, Tween-containing Tris buffered saline, the blot was subjected to the appropriate horseradish peroxidase conjugated secondary antibody for 2 h at room temperature. Immunoreactive bands were visualized by chemoluminescence detected on autoradiography film (Hyperfilm ECL; Amersham, Freiburg, Germany) using the enhanced chemoluminescence system Santa Cruz. The blots were quantified by SigmaScan Pro 5® computer software (SPSS Science Software, Chicago, IL). The results were calculated as ratio of phosphorylated protein to total protein (to detect phosphorylation) or as ratio of total protein to α-tubulin (to detect translocation) and they were presented as absolute values of average light intensity.

Data are expressed as means ± SD. Statistical analysis of infarct size and PKC-ε or ERK1/2 measurements was performed by Student’s t  test with Bonferroni’s correction for multiple comparisons. Statistical analysis of the hemodynamic variables was performed by two-way repeated-measures analysis of variance for time and treatment effects. If an overall significance was found, comparisons between groups were done for each time point using one-way analysis of variance followed by Tukey post hoc  testing. Time effects within each group were analyzed by repeated-measures analysis of variance followed by Dunnett post hoc  test with the baseline value as the reference time point. P < 0.05 was considered statistically significant.

Table 1shows the time course of heart rate and mean aortic pressure during the experiments. No significant differences in heart rate and aortic pressure were observed between the experimental groups during baseline. Calphostin C or PD98059 administration did not influence heart rate or aortic pressure. Desflurane transiently reduced mean aortic pressure, and the values recovered to baseline during the washout periods. At the end of experiment, mean aortic pressure was significantly decreased compared with baseline in calphostin C treated groups.

Desflurane preconditioning reduced infarct size from 57.2 ± 4.7% in controls to 35.2 ± 16.7% (P < 0.05; fig. 2). Both calphostin C and PD98059 preinfusion abolished this cardioprotection (Calphostin + desflurane, 58.8 ± 13.2%; PD98059 + desflurane, 64.2 ± 15.4%; both P < 0.05 versus  desflurane-induced preconditioning). Calphostin C or PD98059 infused without desflurane-induced preconditioning had no effect on infarct size (58.8 ± 13.2% and 48.8 ± 11.6%, respectively; P = 0.27).

PKC-ε phosphorylation increased to the highest values after the second desflurane administration (1.54 ± 0.96 versus  1.10 ± 0.41; P < 0.05 versus  baseline; fig. 3, white columns) and returned to baseline after the last washout period (1.17 ± 0.82; P = 0.7 versus  baseline). The PKC blocker calphostin C abolished the increased phosphorylation at the respective time points. As evidenced by equal actin identifications, protein content in the different probes was similar, excluding the fact that the observed effect was attributable to different protein amounts.

PKC-ε in the membrane fraction increased during desflurane-induced preconditioning and a maximum increase was observed after the second washout period (0.79 ± 0.27 versus  0.63 ± 0.32; P = 0.1 versus  baseline; fig. 4, white hatched columns). Conversely, PKC-ε in the cytosolic fraction decreased (after the second washout period 0.50 ± 0.27 versus  0.62 ± 0.28 baseline; P = 0.2; fig. 4, white columns). The PKC blocker calphostin C abolished the respective changes in PKC content.

ERK1 phosphorylation (fig. 5, white columns) reached its maximum after the first desflurane-induced preconditioning (1.83 ± 1.20 versus  0.95 ± 0.53 in baseline; P < 0.01), slightly decreased after the second desflurane-induced preconditioning (1.56 ± 0.93; P < 0.01 versus  baseline), and further decreased toward baseline after the last washout period (1.23 ± 0.58, P < 0.05 versus  baseline).

As shown in figure 6, ERK2 phosphorylation (white columns) followed a similar time course pattern as ERK1 phosphorylation: 2.02 ± 1.22 after the first desflurane-induced preconditioning (P < 0.01 versus  baseline), 1.57 ± 1.06 after the second desflurane-induced preconditioning (P < 0.01 versus  baseline), and 0.77 ± 0.49 after the last washout (P < 0.05 versus  baseline) versus  0.50 ± 0.32 during baseline.

Phosphorylation of PKC-ε was blocked by PD98059 (fig. 3, black columns): 1.02 ± 0.75 after the first desflurane-induced preconditioning, 0.94 ± 0.58 after the second desflurane-induced preconditioning, and 0.80 ± 0.50 after the last washout versus  1.03 ± 0.59 during baseline. PD98059 also blocked the increase of PKC-ε in the membrane fraction (fig. 4, black hatched columns): 0.64 ± 0.33 after the first desflurane-induced preconditioning, 0.63 ± 0.26 after the second desflurane-induced preconditioning, and 0.68 ± 0.17 after the last washout versus  0.69 ± 0.28 during baseline. Similarly, PD98059 blocked the decrease of PKC-ε in the cytosolic fraction (fig. 4, black columns): 0.57 ± 0.48 after the first desflurane-induced preconditioning, 0.55 ± 0.31 after the second desflurane-induced preconditioning, and 0.54 ± 0.28 after the last washout versus  0.59 ± 0.16 during baseline.

In contrast, phosphorylation of ERK1 was not affected by the PKC blocker calphostin C (fig. 5, black columns): 1.91 ± 1.57 after the first desflurane-induced preconditioning (P < 0.05), 1.89 ± 1.38 after the second desflurane-induced preconditioning (P < 0.05), and 1.12 ± 0.72 after the last washout period versus  1.10 ± 0.90 during baseline. Phosphorylation of ERK2 (fig. 6, black columns) was also not blocked by calphostin C treatment: 1.10 ± 1.13 after the first desflurane-induced preconditioning (P < 0.05), 1.33 ± 1.50 after the second desflurane-induced preconditioning (P < 0.05), and 0.63 ± 0.52 after the last washout (P = 0.2) versus  0.40 ± 0.32 during baseline in calphostin C treated hearts. The failure of PKC blockade to attenuate ERK1/2 phosphorylation suggests that ERK1/2 phosphorylation during desflurane-induced preconditioning is not PKC dependent.

We investigated the role of PKC-ε and ERK1/2 mitogen activated protein kinase in desflurane-induced preconditioning in the rat heart in vivo . The new findings of the current study are, first, that both PKC and ERK1/2 are involved in desflurane-induced pharmacological preconditioning as proved by the loss of cardioprotection after infusion of specific PKC and MEK/ERK1/2 inhibitors calphostin C and PD98059, respectively. Second, during desflurane administration, cytosolic PKC-ε and ERK1/2 were phosphorylated in a time-dependent manner and returned toward the baseline level at the end of the preconditioning protocol when PKC-ε translocation from cytosol to membrane reached the highest level. Third, desflurane administration induced ERK1/2 phosphorylation even after PKC blockade, indicating that ERK1/2 activation during desflurane-induced preconditioning is not PKC-dependent. Taken together, we showed that both enzymes in active form are necessary for cardioprotection elicited by desflurane.

Ischemic and anesthetic-induced preconditioning share several steps in the signal transduction cascade. Besides opening of mitochondrial K^ATPchannels, activation of PKC plays a central role in anesthetic-induced preconditioning. Zaugg et al.  3showed that PKC represents an important signaling pathway upstream to the mitochondrial K^ATPchannels: volatile anesthetics seem to prime rather than directly increase the open state probability of mitochondrial K^ATPchannels, and this process is accomplished through multiple PKC-dependent pathways.3,5,20Using an in vivo  animal model, we previously demonstrated that PKC-ε phosphorylation was increased after three 5-min periods of isoflurane inhalation and that the PKC-ε inhibitor calphostin C abolished both the isoflurane-induced infarct size reduction and the increased phosphorylation of PKC-ε.21In the current study, we demonstrate that PKC-ε also plays an important role in desflurane-induced cardioprotection.

Interestingly, after the last washout period, we detected a translocation of PKC-ε to the membrane without an increase of cytosolic phosphorylation. This finding seemed to disagree with the existing data about structural and spatial regulation of PKC: to accomplish its biologic function, PKC must be processed by phosphorylation, have its pseudosubstrate exposed, and be localized at the correct intracellular location for signaling. On ligand binding at the membrane, phosphorylation at the activation loop triggers autophosphorylation of the two carboxyl-terminal sites and the fully phosphorylated form is then released into the cytosol where generation of diacylglycerol provides the allosteric switch of PKC.22,23 

Therefore, we investigated the time course of intracellular PKC-ε changes and found a strong time-dependent pattern of PKC-ε activation during desflurane-induced preconditioning, with a maximum increase of cytosolic phosphorylated PKC-ε after the second desflurane administration and a complete return to baseline before the infarct-inducing ischemia (fig. 3). During desflurane-induced preconditioning, PKC-ε progressively translocated from cytosol to the membrane fraction and we detected the maximum before the infarct-inducing ischemia (fig. 3). The specific PKC blocker calphostin C abolished the increased phosphorylation/translocation at the respective time points. These findings are in agreement with the mechanisms of PKC regulation mentioned above22,23and with the fact that prolonged PKC activation by phorbol ester results in complete dephosphorylation coupled to translocation to a detergent-insoluble cell fraction.22Anesthetic preconditioning was found to be dose-dependent,24,25and at least two cycles of short exposure to a volatile anesthetic followed by its washout enhance cardioprotection afforded by anesthetic preconditioning.26However, it is difficult to compare the strength of the stimulus we used (two 5-min periods of 1 MAC desflurane) to induce anesthetic preconditioning with the PKC activation by phorbol esters.

Activation of PKC affects mitogen activated protein kinases like the extracellular signal-regulated kinase (ERK1/2), an enzyme that has been described to be involved in preconditioning.9,10The existing data regarding the role of ERK1/2 in acute cardioprotection by ischemic preconditioning are controversial. Increased phosphorylation of mitochondrial ERK seems to mediate cardioprotection, in part by phosphorylation and inactivation of proapoptotic proteins.8,10MEK overexpression in isolated cardiomyocytes up-regulates ERK1/2 activation and induces cardiomyocyte hypertrophy (an equivalent for cardioprotection).27Isolated hearts from rats treated with the MEK/ERK1/2 inhibitor PD98059 failed to show complete functional recovery after ischemia/reperfusion.28Fryer et al.  29found that ERK1 and ERK2 are differentially regulated in the rat heart in vivo  and that cytosolic activation of ERK1 could be an important part in the signal transduction mediating acute cardioprotection by ischemic preconditioning or by opioid agonists.29Su et al.  30and Zhong et al.  31found that effects of isoflurane and halothane in vascular tissues in vitro  were reversed by inhibitors of PKC or ERK1/2. Using the specific MEK/ERK1/2 inhibitor PD98059, we clearly demonstrated that the cardioprotection after desflurane preconditioning was abolished (fig. 2), showing an important role of ERK1/2 in desflurane-induced preconditioning. In addition, we demonstrated an early increase of ERK1/2 phosphorylation after the first administration of the anesthetic that rapidly decreased after the second desflurane administration and after the second washout period (fig. 4). Taken together, our data clearly show an involvement of ERK1/2 in desflurane-induced preconditioning and a time-dependent activation of ERK1/2.

The activation of ERK1/2 is regulated by phosphorylation and this process was shown to be PKC-dependent11and it has been suggested that ERK1/2 is a downstream effector of PKC mediating effects of volatile anesthetics.30,31To investigate a possible cross-link between PKC and ERK1/2, phosphorylation of each enzyme was determined after blockade of the other enzyme. Our results show that desflurane increased ERK1/2 phosphorylation even after PKC blockade (figs. 5 and 6). In addition, phosphorylation of ERK1/2 occurred earlier (after the first desflurane period) compared with phosphorylation of PKC (after the second desflurane period). These data suggest that desflurane activates ERK1/2 independent of PKC and that in desflurane-induced preconditioning, ERK1/2 is not downstream of PKC. In contrast, blockade of ERK1/2 by PD98059 abolished PKC activation by desflurane (figs. 3 and 4), suggesting that ERK1/2 might be upstream of PKC. Alternative, PKC-independent activation pathways of ERK1/2 are already known from other settings such as α-adrenergic stimulation, when ERK1/2 activation occurs by a tyrosine kinase-dependent pathway.32In contrast to our results, ERK1/2 activation after isoflurane was found to be PKC-ε-dependent using either in vitro  vascular cell preparates30,31or an isolated rat model.12Better functional recovery after anesthetic preconditioning was not abolished by PD98059, but based on late activation during ischemia/reperfusion, ERK1/2 was supposed to play a role as a mediator, and not as a trigger, in anesthetic preconditioning.12We used an in vivo  model and the catecholamine release by desflurane could produce early activation of ERK1/2, providing evidence for ERK1/2 acting as a trigger in desflurane-induced preconditioning. Adrenergic stimulation triggers pharmacological preconditioning,33,34and the cardioprotective effects of desflurane in isolated human cardiac tissue involve α and β adrenoceptor stimulation.35In our setting, catecholamine release by desflurane might have resulted in early ERK1/2 activation via  adrenergic receptors and tyrosine kinases, and cardioprotection might have occurred after subsequent PKC-ε phosphorylation and translocation.

Aikawa et al.  36showed that the tyrosine kinase inhibitor genistein suppressed the ERK1/2 activation induced by hydroxyl radicals, whereas inhibitors of PKC did not influence the ERK1/2 activation. Treatment of cardiac myocytes either with a PKC activator or inhibitor did not affect H²O²-induced ERK activation, suggesting PKC-independent activation of ERK1/2.36Reactive oxygen species are also involved in anesthetic preconditioning,37,38and a direct generation of reactive oxygen species by volatile anesthetics could also explain a PKC-independent ERK1/2 activation.

For the induction of anesthesia, rats were anesthetized with S(+)-ketamine because it has been shown that S-enantiomer of ketamine does not interfere with the mechanisms of preconditioning.39Racemic ketamine and R(-)-ketamine,40but not S(+)-ketamine,39block the protective effects of preconditioning.

The absolute specificity and potential toxicity of PD98059 are difficult to evaluate. PD98059 prevents the activation and phosphorylation of MEK1 in vitro  and in vivo  16and does not interfere with the mechanisms of PKC activation.17PD98059 has a high level of specificity for MEK/ERK1/2 pathway and we can conclude that an effect of PD98059 on PKC is very unlikely. The pharmacokinetic profile of pharmacologic inhibitors affecting signaling pathways, such as PD98059, is normally not known and the in vivo  protocol has to be adapted with short periods between drug infusion and measurement points. Giving the dose we infused in the current study it is less probable that the concentrations of PD98059 before the last measurement point (40 min after infusion) are lower than the effective inhibitory concentration of the targeted kinase. The reason for choosing calphostin C instead of other widely used PKC inhibitors (such as chelerythrine) was the higher specificity in the used concentration. The 50% inhibitory concentration of chelerythrine chloride for PKC is approximately 150-fold and 260-fold less than for protein tyrosine kinase and protein kinase A, respectively,41whereas the 50% inhibitory concentration of calphostin C for PKC (0.05 μm) is more than 1000-fold less than for protein tyrosine kinase and protein kinase A.42Calphostin C acts on the regulatory domain of PKC, which is distinct from other protein kinases, and induces a more specific inhibition of PKC than other PKC inhibitors.42An inhibitor whose specificity for PKC is higher in the used concentration decreases the chance to inhibit other kinases probably also involved in preconditioning. There exist very few data about the pharmacokinetic features of calphostin C in the rat in vivo . In mice, calphostin C has a half-life time of 91.3 min after intraperitoneal injection.43In the current study, the last measurement point is 40 min after calphostin C infusion; therefore, a bolus injection should result in a sufficient concentration of the inhibitor during the preconditioning period to affect the targeted enzyme which is up-regulated in this period of time. This was confirmed by the western blot data showing a clear inhibition of the enzyme phosphorylation after inhibitor treatment.

The translocation of PKC-ε to the membrane was not statistically significant. This finding could be the result of an already existent membrane-bound fraction of PKC-ε; i.e. , the changes induced by desflurane are probably relative small when compared to the total membrane-bound PKC-ε, including constitutively translocated PKC-ε existing in the virgin myocardium. The first step in the physiologic posttranslational changes of PKC is represented by membrane binding in a conformation that favors the action of PKC kinase.22We did not present the absolute values of phosphorylated PKC-ε into the cytosolic fraction and we do not have a measure of this phenomenon in our settings. We detected an increase in the phosphorylated form of PKC-ε compared with the control conditions, and it might have been that this rather nonabrupt increase was also attributable to constitutively existent cytosolic phosphorylated PKC-ε.

In summary, the current study provides, for the first time evidence, that both PKC and ERK1/2 are involved in desflurane-induced pharmacological preconditioning and that both enzymes are necessary for cardioprotection. Phosphorylation of PKC-ε and ERK1/2 is time dependent and rapidly reversible, returning to baseline values before the infarct-inducing ischemia when PKC-ε translocation from cytosol to membrane reached the highest level. In contrast to our study hypothesis, desflurane induced ERK1/2 phosphorylation even after PKC blockade, indicating that ERK1/2 activation during desflurane-induced preconditioning occurs upstream of that of PKC.