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The Cardioprotective Effect of Sevoflurane Depends on Protein Kinase C Activation, Opening of Mitochondrial K+ATP Channels, and the Production of Reactive Oxygen Species

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The Cardioprotective Effect of Sevoflurane Depends on Protein Kinase C Activation, Opening of Mitochondrial K+ATP Channels, and the Production of Reactive Oxygen Species
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   The Cardioprotective Effect of Sevoflurane Depends onProtein Kinase C Activation, Opening of Mitochondrial K    ATP Channels, and the Production of Reactive Oxygen Species Wouter de Ruijter,  MD* , Rene´ J.P. Musters,  PhD† , Christa Boer,  PhD*† , Ger J. M. Stienen,  PhD† ,Warner S. Simonides,  PhD† , and Jaap J. de Lange,  MD ,  PhD**Department of Anesthesiology and †Laboratory for Physiology, Vrije Universiteit University Medical Center, Institute forCardiovascular Research Vrije Universiteit, Amsterdam, the NetherlandsSeveral studies suggest that the cardioprotective effectofsevofluranedependsonproteinkinaseC(PKC)acti-vation, mitochondrial K  ATP  channel (mitoK  ATP )opening,andreactiveoxygenspecies(ROS).However,evidence for their involvement was obtained in sepa-rateexperimentalmodels.Here,westudiedtherelativeroles of PKC, mitoK  ATP , and ROS in sevoflurane-induced cardioprotection in one model. Rat trabeculaeweresubjectedtosimulatedischemiabyapplyingmet-abolicinhibition(MI)throughbuffercontainingNaCN,followed by 60-min reperfusion. Recovery of activeforce(F a )wasassessedaspercentageofpre-MIforce.Intime controls, F a  amounted 60%  5% at the end of theexperiment.TherecoveryofF a afterMIwasreducedto28%    5% ( P    0.045 versus time control), whereassevoflurane reversed the detrimental effect of MI (F a recovery, 67%  8%;  P  0.01 versus MI). The PKC in-hibitor chelerythrine, the mitoK  ATP  inhibitor5-hydroxy decanoic, and the ROS scavenger N-(2-mercaptopropionyl)-glycine all completely abolishedtheprotectiveeffectofsevoflurane(recoveryofF a ,31%   8%, 33%    8%, and 24%    9% for chelerythrine,5-hydroxy decanoic, and N-(2-mercaptopropionyl)-glycine, respectively). In conclusion, PKC activation,mitoK  ATP  channel opening, and ROS production areall essential for sevoflurane-induced cardioprotection.These signaling events are arranged in series within acommonsignalingpathway,ratherthaninparallelcas-cades.Ourfindingsimplicatethattheperioperativeuseof sevoflurane preserves cardiac function by prevent-ingischemia-reperfusioninjury.(AnesthAnalg2003;97:1370–6)  S evoflurane (sevo) has cardioprotective proper-ties against perioperative ischemia and im-proved patient recovery after surgery (1–3). Thecardioprotective effects of sevo and other volatile an-esthetics depend on distinct downstream signalingand effector molecules, such as protein kinase C (PKC)and mitochondrial K ATP  channels (mitoK  ATP ) (2–6).The activation of PKC in the cardioprotective effect of sevo was demonstrated in an isolated guinea pig heartmodel, whereas K ATP  channel activation by sevo wasshown by means of the nonspecific K ATP  channel an-tagonist glyburide in pigs and dogs (2,4). Interest-ingly, more recent findings in rabbits also point toendogenous production of reactive oxygen species(ROS) in isoflurane-induced preconditioning (7).Until now, most signal transduction processes un-derlying sevo-induced cardioprotection have been in-vestigated in a variety of models, mostly focusing onsingular elements in the preconditioning pathway.However, the interplay between individual elementsin the protective downstream signaling cascade is asimportant as the individual roles of PKC, mitoK  ATP ,and ROS. Therefore, we determined the relative con-tribution of PKC activation, the opening of themitoK  ATP  channels, and the endogenous productionof ROS in sevo-induced preconditioning in one model,i.e., the isolated right ventricular rat trabecula. Thismodel, which is extensively used in our laboratory,has the advantage that it is not limited by oxygendiffusion problems and has been proven suitable tostudy intracellular signaling events in combinationwith force of contraction (8–10). Applying metabolicinhibition (MI) by using cyanide was chosen to simu-late the two main intracellular consequences of ische-mia: adenosine triphosphate (ATP) depletion and Supported, in part, by the Institute for Cardiovascular ResearchVrije Universiteit (ICaR-VU), Amsterdam, the Netherlands.Accepted for publication May 23, 2003.Address correspondence and reprint requests to Christa Boer,PhD, Department of Physiology, Vrije Universiteit University Med-ical Center, van der Boechorststraat 7, 1081 BT Amsterdam, theNetherlands. Address e-mail to boer@physiol.med.vu.nl.DOI: 10.1213/01.ANE.0000081786.74722.DA ©2003 by the International Anesthesia Research Society 1370  Anesth Analg 2003;97:1370–6 0003-2999/03  Ca 2  overloading. Investigating the three signalingevents, as mentioned above, within this well-definedfunctional, multicellular preparation provides the op-portunity to assess their relative contribution to thepreconditioning process. This may be the first step inunraveling the exact sequence of signaling moleculesin this sevo-induced signal transduction cascade un-derlying cardioprotection. Methods The use of animals in this study was approved by theInstitutional Animal Care and Use Committee. MaleWistar rats (275 – 420 g;  n    54) were anesthetized bypentobarbital sodium (60 mg/kg, intraperitoneally)and heparinized (1000 U, IV). Subsequently, the heartwas excised and retrogradely perfused at room tem-perature with Tyrode solution (95% O 2 / 5% CO 2 , witha pH value of 7.35). Standard solution contained (inmM): NaCl 120.0, KCl 5.0, CaCl 2  1.0, MgSO 4  1.22,NaH 2 PO 4  1.99, NaHCO 3  27.0, and glucose 10.1.Only during isolation of the trabecula was myocar-dial contraction prevented by 30 mM of 2,3- butanedione monoxime. A right ventricular trabec-ula, running from the atrioventricular ring to thefree wall, was dissected. Subsequently, the trabeculawas mounted between a force transducer (AE801,SensoNor, Norway), which was connected to a per-sonal computer, and a micromanipulator in aclosed, airtight muscle bath and superfused withstandard solution. The trabecula was paced using 2platinum electrodes (field stimulation, 5-ms dura-tion) (8 – 10).After mounting the trabeculae, the protocol startedwith a stabilization period of 45 min (27 ° C; 0.5 Hz).During the stabilization period, muscle diameter wasmeasured using a microscope (50  magnification) us-ing a calibrated reticule. The length of the trabeculaewas determined by a force-length relation and set atan equivalent of 85% of the optimum sarcomerelength, L0.85, as described by Schouten et al. (11). Inthe last phase of the stabilization period, the stimula-tion voltage was set at two times the stimulationthreshold. After 45 min of stabilization, the muscle bath temperature was decreased to 24 ° C, which wasmaintained throughout the experiment, and the pac-ing frequency to 0.2 Hz. After a subsequent 10-minstabilization period, initial active force of contraction(F a,start ) and maximal force (F max,start ) were deter-mined. F max,start  was determined (basal stimulationfrequency of 0.2 Hz) using a postextrasystolic poten-tiation (PESP) protocol, as described previously (12).Briefly, PESP is based on the addition of an increas-ing number of multiple, extra-systolic contractions,which result in a gradual and maximal filling of thesarcoplasmic reticulum (SR) with Ca 2  and thus inthe concurrent development of maximal contractileforce. Preparations were excluded when they didnot stabilize within the equilibration period, exhib-ited spontaneous contractions, or failed to showPESP.Figure 1 displays an overview of the experimentalprotocol for the different experimental groups. Ex-cept for time controls (Time), trabeculae were sub- ject to MI and 60 min of reperfusion. MI wasachieved by exposure to standard solution withoutglucose and containing 2 mM of NaCN and anincrease of the pacing frequency to 1 Hz. During MI,F a  decreased to zero, and a rigor contracture devel-oped (10). Here, we used a rigor period of 30 min,and at the end of the rigor period, the superfusionand pacing frequency were switched back to controlconditions. Subsequently, the trabeculae were su-perfused for 60 min, and the recovery of F a , F max ,and passive force were recorded. The recovery of force after reperfusion was expressed as the ratio of F a  and F max  after reperfusion to F a,start  and F max,start (initial force values before MI), respectively. Isomet-ric force was normalized to the cross-sectional areacalculated from muscle diameters in two perpendic-ular directions measured at the end of each experi-ment. Figure 2 shows two typical recordings froman MI and preconditioning experiment. In the timecontrol group, trabeculae were paced at 0.2 Hz andsuperfused with standard solution for an equal timeperiod as the MI and preconditioning group.During the preconditioning period, superfusionwas switched to standard solution (95% O 2 /5%CO 2 ). Sevo 3.8% was vaporized into the gas supply,and the buffer solution was equilibrated with thegas mixture (13). The concentration of the anestheticwas monitored using a calibrated anesthetic. Inthree distinct experimental groups, the PKCcatalytic-site inhibitor chelerythrine (chel) (2   M)(14), the highly selective mitoK  ATP  channel blocker5-hydroxy decanoic acid sodium (5-HD) (100   M),and the ROS scavenger N-(2-mercaptopropionyl)-glycine (MPG) (300   M) were applied. The inhibi-tors and scavenger were dissolved in water andremained present in the superfusion medium fromthe preincubation period until the onset of MI. Inthree additional control groups, the effects of chel,5-HD, and MPG on recovery after MI were investi-gated without pretreatment of sevo (blocker con-trols). All chemicals were obtained from Sigma-Aldrich (Zwijndrecht, Netherlands).Unless stated otherwise, the recovery of F a  is ex-pressed as percentage of the initial value F a,start  at thestart of the experiment. All data are expressed as mean   sem  of six trabeculae. Between-group comparisonswere performed using analysis of variance and eval-uated with Tukey  post hoc  tests. Differences were con-sidered to be significant at  P  0.05. ANESTH ANALG ANESTHETIC PHARMACOLOGY RUIJTER ET AL.  1371 2003;97:1370 – 6 MEDIATORS OF PRECONDITIONING BY SEVOFLURANE  Results Muscle dimensions (cross-sectional area) and F a,start were not different between groups (Table 1), althoughF a,start  varied between groups, being smallest in thesevo  MI  chel group (27.9  5.2 mN/mm 2 ) and larg-est in the MI  MPG group (48.8    11.3 mN/mm 2 ).Overall, passive force values at L0.85 before MI andafter reperfusion were not different between groups(mean amounts to 1.5    0.1 and 1.6    0.2 mN/mm 2 ,respectively; not significant). Furthermore, F max,start and the F a  at the end of the preincubation and wash-out phase were similar in all groups. Finally, in timecontrols the F max,start  decreased from 63.0    6.7 mN/mm 2 (initial value) to 46.3  7.9 mN/mm 2 at the endof the experiment. MI worsened this decrease (54.8  7.7 to 33.4  7.9 mN/mm 2 ), and preconditioning withsevo did not prevent this decrease in F max,start .At the onset of MI, the force typically decreased to alower level and decreased to zero, followed by a rapidincrease in passive force (rigor state; Fig. 2; time scaleof 155 min). Time to rigor (approximately 31 min) andmaximal rigor force (approximately 117% of F max,start )were comparable in all groups (Table 1). The volumepercentage of sevo in the vapor phase greater than theequilibrated solution was not different betweengroups.Sevo depressed the force by approximately 20%,which was completely reversed in the washout period(Fig. 3). In time controls, the force at the end of theexperiment amounted to 60%    5% of F a,start . MI re-duced the recovery of F a  to 28%    5%, which issignificantly less than in time controls ( P    0.045).Pretreatment with sevo preserved F a  after MI andreperfusion to 67%    8%, which was significantlyhigher compared with the MI group ( P    0.01) andsimilar to time controls. Thus, the depressant effect of MI was completely counteracted by pretreatment withsevo.Figure 4 shows the relative importance of PKC,mitoK  ATP  channels, and ROS in the protective action Figure 1.  Schematic overview of the experimental protocols. The protocol consisted of a 5-min preincubation period, a 15-min precondi-tioning period (preco), a 15-min washout period (wo), metabolic inhibition (MI), and a 60-min reperfusion period. Shaded areas indicatetreatments additional to time control in the respective experimental phase and group. Time  time control; sevo  3.8% sevoflurane; chel  chelerythrine; 5-HD  5-hydroxy decanoic acid sodium; MPG  N-(2-mercaptopropionyl)-glycine. 1372  ANESTHETIC PHARMACOLOGY RUIJTER ET AL. ANESTH ANALGMEDIATORS OF PRECONDITIONING BY SEVOFLURANE 2003;97:1370 – 6  of sevo (panel A). Recovery of F a  after preconditioningwith sevo in the presence of the PKC inhibitor chel(31%    8%) was not different from MI alone (28%   5%). Furthermore, inhibition of mitoK  ATP  channels by 5-HD (Sevo  MI  5-HD; F a  amounted 33%    8%)or ROS by MPG (Sevo  MI  MPG; F a  amounted 24%   9%) also abolished the sevo-induced cardioprotec-tive effect after MI. In three control groups withoutadministration of sevo, the effects of chel, 5-HD, andMPG alone on recovery of F a  after MI were assessed.After 60 min of reperfusion, F a  amounted to 32%   6%, 27%    7%, and 24%    6% for chel, 5-HD, andMPG, respectively. These values did not differ fromrecovery of F a  in the MI group. Discussion We demonstrated that sevo has potent cardioprotec-tive properties because it completely restored F a  totime-control values after MI and reperfusion in iso-lated rat trabeculae. Furthermore, it is shown that the Figure 2.  (A) Illustration of the stimulation protocol on a time scale of 155 min. Note the steady-state contractions during which initial activeforce (F a,start ) was obtained and postextrasystolic potentiation (PESP) to determine maximal force (F max,start ) during the control phase. (B andC) Typical examples of the development of active force (F a ; relative to F a,start ) during the course of an experiment from metabolic inhibition(MI) (panel B) and sevoflurane (sevo)  MI (panel C) groups (time scale 155 min). During MI, F a  decreases to zero, and subsequently, passiveforce increases. When passive force reaches 50% of F max,start  (dotted lines), the rigor period of 30 min begins (gray areas). Duringpreconditioning (panel C), sevo induces a small depression of force. Preconditioning (panel C) improves recovery of force because of MIcompared with MI alone (panel B). Table 1.  General Characteristics of the TrabeculaeCross-sectionalarea (mm 2 )F a,start (mN/mm 2 )F max,start (mN/mm 2 )F max,recovery (mN/mm 2 )Time torigor (min)Maximal rigorforce (%)volume%Time 0.026  0.01 41.6  6.0 63.0  6.7 46.3  7.9 - - -Metabolic inhibition (MI) 0.038  0.01 33.3  5.2 54.8  7.7 33.4  7.9 30  2 113  15 -MI  chel 0.022  0.01 45.6  5.1 56.1  8.4 32.2  6.8 28  4 133  17 -MI  5-HD 0.020  0.01 44.3  4.8 60.8  7.8 27.3  5.9 27  3 123  13 -MI  MPG 0.030  0.01 48.8  11.3 60.9  8.6 24.1  5.5 30  4 114  7 -Sevo  MI 0.014  0.00 34.8  8.1 48.5  6.8 34.8  6.1 34  4 100  7 3.7  0.06Sevo  MI  chel 0.026  0.01 27.9  5.2 42.9  7.6 23.6  5.0 34  3 131  11 3.8  0.03Sevo  MI  5-HD 0.032  0.02 33.9  6.1 57.5  12.2 36.6  9.6 33  3 134  41 3.7  0.09Sevo  MI  MPG 0.026  0.01 45.8  7.2 58.3  8.5 31.5  4.8 32  2 103  4 3.9  0.02 MI  metabolic inhibition; 5-HD  5-hydroxy decanoic acid sodium; MPG  N-(2-mercaptopropionyl)-glycine; chel  chelerytine.ANESTH ANALG ANESTHETIC PHARMACOLOGY RUIJTER ET AL.  1373 2003;97:1370 – 6 MEDIATORS OF PRECONDITIONING BY SEVOFLURANE  cardioprotective effect of sevo depends on PKC acti-vation, the opening of mitoK  ATP  channels, and theendogenous production of ROS. These results suggestthat all three of these signaling events are activewithin the common intracellular signaling cascade of sevo-induced cardioprotection.Pretreatment with sevo results in an improvementof postischemic contractile recovery in rat trabeculae,further strengthening clinical observations (1). Mostvolatile anesthetics render the heart resistant to thedepressant effects of ischemia/reperfusion injury in avariety of   in vivo  and  in vitro  experimental models,with different outcome variables (15). In an  in vivo  dogmodel, isoflurane and desflurane improved postisch-emic contractile performance (4,5), and sevo (3) reducedinfarct size. In contrast, Roscoe et al. (6) did not observean improved postischemic contractile recovery in hu-man trabeculae after preconditioning with halothane.The protective effects of sevo were also observed inperfused guinea pig hearts (2). Interestingly, the timepoint of administration determines the protection capac-ity against ischemia/reperfusion damage. Although ap-plication of sevo in the reperfusion period induces car-dioprotective effects as well, postischemic contractileand metabolic recovery was significantly better in heartsthat were preconditioned before ischemia (13).We used right ventricular rat trabeculae to study themechanisms underlying sevo-induced precondition-ing. This well-defined model has been extensivelyapplied in our laboratory, is not limited by oxygendiffusion, and can be used for intracellular signalingas well as for functional studies. Experiments wereperformed at 27 ° C to maintain a stable preparationover several hours and to prevent oxygen limitation(9 – 12). We used NaCN, a glucose-free buffer and anincreased stimulation frequency, to induce simulatedischemia. NaCN induces ATP depletion and Ca 2  overloading, which both characterize ischemia. Thismodel has been used in several studies performed byour group and provides similar results as comparedwith hypoxia-induced ischemia-reperfusion injury(9,10). We administered sevo as pretreatment beforethe simulated ischemia period, followed by a washoutperiod. Thus, the negative inotropic effect of sevo oncontractile force did not interfere with the MI period,and it can therefore be concluded that cardiodepres-sion during MI is not the cause of sevo-induced car-dioprotection. After 60 minutes of reperfusion, therecovery of force was sufficient to provide informationabout the ischemia-induced injury.There was no significant difference in F a  betweengroups at the end of the washout period, indicatingcomplete elimination of the anesthetic in the experi-mental setup before starting MI. In all groups, exceptfor time-controls, F max  was equally decreased, regard-less of the specific treatment of the preparation. Thus,preconditioning with sevo improves F a , but not F max ,after MI. This suggests that the number of functionalcontractile filaments may be reduced after MI (i.e.,necrosis). Furthermore, it implies that the saturation of force (F a /F max ) was less in all groups after MI, exceptfor time controls and the sevo  MI group.Chel, 5-HD, and MPG are common inhibitors forstudying PKC, mitoK  ATP , and ROS, respectively, andare frequently used in studies dealing with ischemicand pharmacological preconditioning. Therefore, weused concentrations of these inhibitors that are com-parable with concentrations used in other studies. Thepresent study shows a role for PKC activation in thepreconditioning process leading to sevo-inducedcardioprotection. Toller et al. (4) showed that isoform-unspecific PKC inhibition by a small dose of bisin-dolylmaleimide produces cardioprotection, andisoflurane further enhances this protection. However,a large dose of bisindolylmaleimide alone did notprevent the cardioprotective effect of isoflurane. Inaddition, Cope et al. (15) reported that the protectiveeffect of halothane in rabbits is abolished by chel.There are strong indications that PKC activationduring the preconditioning process opens mitoK  ATP channels through decreasing the ATP-binding affinityof these channels (16 – 18). Furthermore, the cardiopro-tective effects of sevo could be reversed by other non-selective inhibitors of the mitoK  ATP  channel thanused in this study (2,3,19). Direct evidence for sevo-induced opening of mitoK  ATP  channels was obtained by Kohro et al. (20) in guinea pig myocytes. Here,evidence was obtained supporting these data becausethe specific mitoK  ATP  channel inhibitor 5-HD abol-ished the protective effect of sevo entirely, whereaspretreatment with the inhibitor alone had no effect.The significance of mitoK  ATP  channel opening for Figure 3.  Recovery of active force (F a ) as percentage of F a,start  in thetime controls, metabolic inhibition (MI), and preconditioning(sevoflurane [sevo]) groups. MI decreased the recovery of F a  com-pared with time controls ( P    0.045), whereas sevo improved therecovery of F a  after MI ( P    0.01 sevo versus MI). All data areexpressed as mean  sem  of   n  6 per group. 1374  ANESTHETIC PHARMACOLOGY RUIJTER ET AL. ANESTH ANALGMEDIATORS OF PRECONDITIONING BY SEVOFLURANE 2003;97:1370 – 6
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