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(Circulation. 2006;114:I-226 – I-232.)
© 2006 American Heart Association, Inc.

Myocardial Protection and Vascular Biology

Cardioprotection Via Activation of Protein Kinase C- Depends on Modulation of the Reverse Mode of the Na⁺/Ca²⁺ Exchanger

R. Arthur Bouwman, MD; Kanita Salic, BSc; F. Gieneke Padding, BSc; Etto C. Eringa, PhD; Brechje J. van Beek-Harmsen, BSc; Toshio Matsuda, PhD; Akemichi Baba, PhD; René J.P. Musters, PhD; Walter J. Paulus, MD, PhD; Jaap J. de Lange, MD, PhD; Christa Boer, PhD

From Department of Anesthesiology (R.A.B., J.J.d.L., C.B.), Laboratory for Physiology (K.S., G.P., E.C.E., B.J.v.B.-H., R.J.P.M., W.J.P., C.B.), Institute for Cardiovascular Research Vrije Universiteit (ICaR-VU), VU University Medical Center (VUmc), Amsterdam, the Netherlands; Laboratory of Medicinal Pharmacology (T.M.), Laboratory of Molecular Neuropharmacology (A.B.), Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

Correspondence to R. Arthur Bouwman, VU University Medical Center, Department of Anesthesiology, de Boelelaan 1118, PO Box 7057, 1007 MB Amsterdam, the Netherlands. E-mail a.bouwman{at}vumc.nl

	Abstract

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Background— Pretreatment with the volatile anesthetic sevoflurane protects cardiomyocytes against subsequent ischemic episodes caused by a protein kinase C (PKC)- mediated preconditioning effect. Sevoflurane directly modulates cardiac Ca²⁺ handling, and because Ca²⁺ also serves as a mediator in other cardioprotective signaling pathways, possible involvement of the Na⁺/Ca²⁺ exchanger (NCX) in relation with PKC- in sevoflurane-induced cardioprotection was investigated.

Methods and Results— Isolated right ventricular rat trabeculae were subjected to simulated ischemia and reperfusion (SI/R), consisting of superfusion with hypoxic glucose-free buffer for 40 minutes after rigor development, followed by reperfusion with normoxic glucose containing buffer. Preconditioning with sevoflurane before SI/R improved isometric force development during contractile recovery at 60 minutes after the end of hypoxic superfusion (83±7% [sevo] versus 57±2% [SI/R];n=8; P<0.01). Inhibition of the reverse mode of the NCX by KB-R7943 (10 µmol/L) or SEA0400 (1 µmol/L) during preconditioning attenuated the protective effect of sevoflurane. KB-R7943 and SEA0400 did not have intrinsic effects on the contractile recovery. Furthermore, inhibition of the NCX in trabeculae exposed to sevoflurane reduced sevoflurane-induced PKC- translocation toward the sarcolemma, as demonstrated by digital imaging fluorescent microscopy. The degree of PKC- phosphorylation at serine⁶⁴³ as determined by western blot analysis was not affected by sevoflurane.

Conclusions— Sevoflurane-induced cardioprotection depends on the NCX preceding PKC- translocation presumably via increased NCX-mediated Ca²⁺ influx. This may suggest that increased myocardial Ca²⁺ load triggers the cardioprotective signaling cascade elicited by volatile anesthetic agents similar to other modes of preconditioning.

Key Words: anesthesia • calcium • ion channels • preconditioning • signal transduction

	Introduction

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The myocardium contains intrinsic protective mechanisms against ischemia/reperfusion (I/R) injury, which can be triggered by several stimuli, including volatile anesthetics like sevoflurane. The cardioprotective properties of sevoflurane depend on activating protein kinase C (PKC) and producing reactive oxygen species (ROS) similar to ischemic preconditioning and Ca²⁺ preconditioning. Cardioprotection induced by ischemic preconditioning and Ca²⁺ preconditioning is mediated via Ca²⁺ and reduction of cellular Ca²⁺ influx reduces PKC activation and subsequent protection against I/R-injury.¹

Sevoflurane reduces myocardial Ca²⁺ availability, but paradoxically increases sarcoplasmic reticulum (SR) Ca²⁺ content.² Changes in cellular SR Ca²⁺ load are associated with activation of survival and/or death signaling pathways,³ and therefore provide a potential mechanism for cardioprotective signaling. Sevoflurane reduces Ca²⁺ influx via the L-type Ca²⁺ channels,⁴ and therefore another Ca²⁺-loading mechanism may be involved. One of the key regulation proteins of myocardial Ca²⁺ loading is the Na⁺/Ca²⁺ exchanger (NCX) via NCX-dependent Ca²⁺ influx in exchange for Na⁺ (reverse mode of the NCX).⁵ Until now, it is unknown whether there is a particular contribution of the reverse mode of the NCX in sevoflurane-induced alterations in Ca²⁺ homeostasis. Furthermore, the possible role of the NCX in the activation of signaling proteins involved in sevoflurane preconditioning, like PKC, has not been addressed. In this study, we investigated the involvement of the reverse mode of the NCX in sevoflurane-induced cardioprotective signaling and specifically focused on the relation with the PKC- isoform, which previously was shown to be essentially involved in sevoflurane-induced preconditioning.⁶

	Materials and Methods

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The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.

Animals and Experimental Setup
This study was performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Vrije Universiteit University Medical Center. Our experimental setup has previously been described in detail.⁶ Briefly, male Wistar rats (250 to 400 grams; Harlan, Horst, The Netherlands) were anesthetized with sodium pentobarbital (80 mg/kg, intraperitoneally, Nembutal; Sanofi Sante BV) and the heart was subsequently removed. Trabeculae were isolated from the right ventricle (length 2 to 5 mm, diameter <0.2 mm) under contractile arrest conditions and mounted in an airtight organ bath developed for isometric force measurements. The muscle was superfused with oxygenated (95% O₂ and 5% CO₂) Tyrode buffer with the following composition: 120 mmol/L NaCl, 1.22 mmol/L MgSO₄·7H₂O, 1.99 mmol/L NaH₂PO₄, 27.0 mmol/L NaHCO₃, 5.0 mmol/L KCl, 1 mmol/L CaCl₂, and 10 mmol/L glucose. Subsequently, muscle length was set at 95% of the maximal length as determined by a force–length relation.

Experimental Protocol
After 60 minutes of equilibration (40 minutes: 27°C/0.5 Hz; and 20 minutes: 24°C/0.2 Hz), the initial developed force of contraction (F_dev) and the potentiated force (F_pot), as determined by a postextrasystolic potentiation protocol, were recorded. Postextrasystolic potentiation protocol determines the contractile reserve of trabeculae by maximally filling the SR with Ca²⁺. Trabeculae failing to stabilize, spontaneously contracting trabeculae, or failing to show postextrasystolic potentiation were excluded. Figure 1A depicts the design for all groups exposed to different the preconditioning protocols. Ischemia was simulated in trabeculae (except time controls) by superfusion with hypoxic Tyrode without glucose and increasing stimulation frequency to 1 Hz for 40 minutes after rigor development as described previously.⁶ Trabeculae were preconditioned for 15 minutes with normal Tyrode saturated with 3.8 vol% sevoflurane 30 minutes before simulated ischemia and reperfusion (SI/R). After washout of sevoflurane, trabeculae were superfused for 15 minutes with normal Tyrode until SI/R. In separate experimental groups, the preconditioning phase was preceded by addition of KB-R7943 (10 µmol/L, KBR; Tocris Bioscience) or SEA0400 (1 µmol/L, SEA; synthesized in Taisho Pharmaceutical, Saitama, Japan). After 60 minutes of reperfusion, the recovery of F_dev (F_dev,rec) was expressed as a percentage of F_dev before SI/R.

View larger version (18K): [in this window] [in a new window]   Figure 1. The role of the reverse mode of the NCX in sevoflurane-induced cardioprotection. A, Experimental design for trabeculae subjected to simulated ischemia and reperfusion (SI/R). B, The recovery of the developed force (Fdev,rec) after SI/R is expressed as percentage of the developed force just before SI/R. Pretreatment with sevoflurane improved the postischemic contractile force recovery, and this protective effect was attenuated when the NCX was inhibited during preconditioning with either KB-R7943 (KBR) (10 µmol/L) or SEA0400 (SEA) (1 µmol/L). KB-R7943 and SEA0400 did not affect the Fdev,rec after SI/R. C, Data are mean±SEM and represent 8 experiments, except for time control (n =6), SI/R+KBR (n =6), SI/R+SEVO + SEA (n=3), and SI/R+SEA (n=3). *P<0.05 vs time control. P<0.05 vs SI/R. P<0.05 vs SI/R+SEVO.

Effect of Sevoflurane on NCX-Mediated Ca²⁺ Loading
The contribution of Ca²⁺ influx via the reverse mode of the NCX during sevoflurane was evaluated by experimental design shown in Figure 2A. To study transsarcolemmal Ca²⁺ influx via the NCX, electrical stimulation was stopped and trabeculae were superfused with a low Na⁺ buffer consisting of normal Tyrode except the replacement of 120 mmol/L NaCl with 120 mmol/L tetramethylammonium chloride (TMA).⁷ After 60 seconds, superfusion was switched back to normal Tyrode and as superfusion with low Na⁺ buffer increases the electrochemical potential for Ca²⁺ uptake via the NCX, the developed force after resuming electrical stimulation was potentiated (F_TMA). F_TMA was normalized on the F_dev during basal stimulation conditions before the Ca²⁺ loading protocol and provides an estimate for the amount of Ca²⁺ loading. Trabeculae, exposed to 3.8vol% sevoflurane in the presence or absence of the NCX inhibitors KB-R7943 (10 µmol/L) or SEA0400 (1 µmol/L) were subjected to this Ca²⁺-loading protocol (Figure 2B).

View larger version (16K): [in this window] [in a new window]   Figure 2. Sevoflurane can increase Ca2+ influx via the reverse mode of the NCX. A, Overview of the experimental protocol studying the effects of sevoflurane on NCX-mediated Ca2+ loading. B, Developed force after low Na+ superfusion (FTMA) is normalized on the developed force during basal stimulation conditions (initial Fdev) before the Ca2+ loading protocol. During sevoflurane FTMA is increased compared with control trabeculae. This effect of sevoflurane was attenuated in trabeculae with inhibition of the NCX by KB-R7943 (10 µmol/L) as well as by SEA0400 (1 µmol/L). C, Effect of KB-R7943 (KBR) and SEA0400 (SEA) itself on low Na+-induced Ca2+-loading protocol (C). Data are expressed as mean±SEM (n=5, except SEVO + SEA and SEA, n=3). *P<0.05 vs control.

Immunofluorescent Microscopy of PKC- Distribution
Figure 3A demonstrates the experimental design for all groups exposed to analysis of PKC- localization. The sevoflurane-induced subcellular redistribution of PKC- before SI/R was studied by immunofluorescent staining and by digital imaging fluorescence microscopy, as described earlier.⁶ Briefly, trabeculae subjected to an experimental protocol were embedded in gelatin and cross-sections were subsequently fixed, stained for PKC- (Research & Diagnostic Antibodies), counterstained for the sarcolemma (10% v/v) wheat germ agglutinin (WGA; Molecular Probes, Invitrogen) and nuclei (DAPI)-containing mounting medium (Vector Laboratories). The sections were analyzed with a ZEISS Axiovert 200 Marianas inverted digital imaging microscope workstation using Slidebook software (Slidebook version 4.1; 3l Intelligent Imaging Inovations, Inc).

View larger version (36K): [in this window] [in a new window]   Figure 3. PKC- activation in response to sevoflurane. A, Schematic overview of the experimental protocol studying sevoflurane-induced PKC- activation. B, Digital imaging fluorescence microscopy analyses of PKC- translocation. In all panels, green represents specific PKC- staining and blue represents the nuclear DAPI staining. In panels a and d, red represents the wheat germ agglutinin staining of the sarcolemmal glycocalyx. In response to sevoflurane, PKC- translocated toward the sarcolemma (white arrows, panels a and b) as previously shown.6 Inhibition of the reverse mode of the NCX abolished this specific translocation of PKC- (panels c and d, white arrows indicate sarcolemma lacking PKC- staining). C, Sevoflurane did not increase PKC- phosphorylation on serine643. The ratio of serine643-phosphorylated PKC- and total PKC- is expressed as relative change compared with control in arbitrary units (AU). Data are mean±SEM, n=5. SEVO indicates sevoflurane; KBR, KB-R7943.

Western Blot Analyses for PKC- Phosphorylation
For PKC- phosphorylation analysis, trabeculae were subjected to different experimental protocols (Figure 3A). Proteins were separated by gel electrophoresis (10 µg per lane) blotted onto a nitrocellulose membrane and stained for phosphorylated PKC- at serine⁶⁴³ (Cell Signaling Technology) as well as for total PKC- (Research & Diagnostic Antibodies). The immunoreactive bands were visualized by chemiluminescence (Amersham, GE Health Care UK Ltd) and quantified using a charge-coupled device camera (Fuji Science Imaging Systems) in combination with AIDA Image Analyzer software (Isotopenmessgeräte; Raytest).

Statistical Analysis
Data were analyzed by analysis of variance (ANOVA), followed by either Tukey or Dunnett post-hoc test. For all analyses P<0.05 was considered to reflect a significant difference. All values are given as means±standard error of the mean (SEM).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of the Reverse Mode of the NCX Attenuates Sevoflurane-Induced Cardioprotection
The initial contractile parameters showed minor variation between the experimental groups (Table 1). The time to rigor development was nominally prolonged in the SI/R+SEA0400 group, but this did not affect contractile recovery in this experimental group. SI/R reduced the contractile recovery (F_dev,rec) to 57±2%, whether preconditioning with sevoflurane improved the myocardial force development to 83±7% (P<0.05 versus [SI/R]) (Figure 1B). Inhibition of the reverse mode of the NCX during preconditioning by either KB-R7943 or SEA0400 completely reversed the protective effect of sevoflurane on contractile recovery. Both KB-R7943 and SEA0400 did not show an intrinsic effect on the contractile recovery (Figure 1C). Although both inhibitors slightly induced positive inotropy, as was previously reported by others, this did not interfere with our study objectives.^8,9

View this table: [in this window] [in a new window]   General Characteristics of Trabeculae Subjected to Simulated Ischemia and Reperfusion

Sevoflurane Increases NCX-Mediated Ca²⁺ Loading
Low Na⁺-induced Ca²⁺ loading was used to study the effect of sevoflurane on Ca²⁺ influx via the reverse mode of the NCX (Figure 2). Sevoflurane increased force development (F_TMA) after 60 seconds of low Na⁺ superfusion, suggesting increased intracellular Ca²⁺ loading. This increased force development caused by sevoflurane was abolished in trabeculae treated with KB-R7943 or SEA0400.

Differential Involvement of the Reverse Mode of the NCX in Sevoflurane-Induced Translocation and Phosphorylation of PKC-
Figure 3B shows the translocation pattern of PKC- in response to sevoflurane and sevoflurane in combination with KB-R7943 in cross-sections of isolated trabeculae. In all panels, green represents specific PKC- staining, blue represents nuclear DAPI staining, and red shows sarcolemmal staining with wheat germ agglutinin. As previous reported, sevoflurane induced translocation of PKC- from the cytosolic compartment to the sarcolemma (Figure 3B, panels a and b).⁶ This sevoflurane-induced translocation of PKC- was effectively reduced in trabeculae treated with KB-R7943 (Figure 3B, panels c and d). This translocation most likely does not depend on phosphorylation of PKC- at serine⁶⁴³, because sevoflurane treatment did not affect the ratio of phosphorylated PKC- to total PKC-, as shown by Western blot analyses (Figure 3C).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sevoflurane-induced preconditioning is mediated via activation and translocation of PKC-, as was earlier shown in isolated rat trabeculae.⁶ The present study shows that sevoflurane-induced cardioprotection, and particularly the involvement of PKC- in this protective mechanism, depends on the activity of the reverse mode of the NCX. Several lines of evidence were provided for this role of the NCX in sevoflurane-induced protective signaling: (1) sevoflurane-induced cardioprotection was abolished by KB-R7943 and SEA0400 possibly via reduction of Ca²⁺ uptake via the NCX; (2) sevoflurane increased NCX-mediated Ca²⁺ loading, indicating that sevoflurane can facilitate Ca²⁺ influx via the NCX; and (3) pharmacological blockade of the reverse mode of the NCX attenuated sevoflurane-induced translocation of PKC-.

In various protective signaling cascades, an important role for Ca²⁺ has been demonstrated.¹ Cardioprotection induced by ischemic preconditioning and Ca²⁺ preconditioning can be abolished by Ca²⁺ influx inhibition, especially Ca²⁺ influx via the L-type Ca²⁺ channels or the NCX.^1,8 Interestingly, inhibition of the L-type channels as well as indirect inhibition of Na⁺/Ca²⁺ exchange coincided with a reduction of PKC translocation and PKC activity.¹⁰ Our present data show that inhibition of the reverse mode of the NCX during sevoflurane preconditioning attenuates cardioprotection and reduces PKC- translocation. This implies that increased NCX-mediated Ca²⁺ influx is upstream in sevoflurane-induced cardioprotective signaling, in parallel to other preconditioning stimuli.

Our data seem to be in contrast to previous literature, showing that volatile anesthetics reduce Ca²⁺ availability and inhibit the NCX.^11,12 However, sevoflurane can increase intracellular (SR) Ca²⁺ load,^2,13 and in this study we specifically demonstrate that sevoflurane augments force development after low Na⁺ superfusion, confirming that sevoflurane can facilitate NCX-mediated Ca²⁺ influx. Several explanations may account for these contradictory results. In the majority of studies, myocardial NCX function is evaluated in isolated cells and is not confirmed in functional experiments, as in our investigation. Furthermore, volatile anesthetic-induced alterations on myocardial Ca²⁺ handling and action potential depend on concentration and duration of exposure to the anesthetic.^14,15 The mechanism of sevoflurane-induced facilitation of reverse Na⁺/Ca²⁺ exchange may involve an increased Na⁺ load because of inhibition of the Na⁺/K⁺-ATPase and the unequal effect of sevoflurane on Na⁺ and Ca²⁺ entry during depolarization.^4,13,15 This changes the electrochemical driving force in favor of the Na⁺-dependent Ca²⁺ influx. In addition, Na⁺/Ca²⁺ exchange is under ionic control of Ca²⁺, Na⁺, and H⁺ via catalytic regulation of the intracellular loop of the NCX.⁵ In isolated mouse papillary muscles devoid of regulation by Ca²⁺ and Na⁺, the contribution of reverse Na⁺/Ca²⁺ exchange is increased, as shown by increased rest potentiation.¹⁶ H⁺ ions inhibit concentration-dependent ion transport via the NCX, but in cells devoid of ionic control by H⁺, inhibition of NCX ion transport is reduced even at physiological pH.⁵ Therefore, we speculate that sevoflurane may influence NCX activity by altering the ionic regulation of the NCX by Na⁺, Ca²⁺, and H⁺, thereby increasing NCX-induced myocardial Ca²⁺ loading. Another potential mechanism may involve a volatile anesthetic-induced increase in intracellular pH by direct stimulation of Na⁺/H⁺ exchange and thereby increase NCX ion transport.¹⁷ However, in our experimental model, alterations in hydrogen exchange may only minimally affect intracellular pH because of the buffer capacity of the bicarbonate-containing superfusion solution.¹⁸

The present data implicate that modulation of Ca²⁺-handling via the reverse mode of the NCX precede PKC- activation in the cardioprotective signal transduction pathway elicited by sevoflurane. However, it is unknown how PKC- is modulated by alterations in myocardial Ca²⁺ handling. Interestingly, PKC- is a Ca²⁺-independent PKC isoform, in contrast to the Ca²⁺-dependent isoforms PKC- and PKC-ß₁. However, it is well-described that several preconditioning stimuli that depend on Ca²⁺ induce translocation of both Ca²⁺-dependent and Ca²⁺-independent isoforms, suggesting isoform crosstalk.^1,10 The link between Ca²⁺ handling and PKC- modulation may involve ROS-dependent PKC- translocation as well as phosphorylation. We previously demonstrated in isolated rat trabeculae that sevoflurane-induced PKC- translocation depends on the production of ROS,⁶ which could be a result of altered Ca²⁺ handling in the myocardium. In particular, mitochondrial Ca²⁺ loading may induce ROS production. Increased Ca²⁺ influx via the reverse mode of the NCX may contribute to this mitochondrial ROS production, with PKC activation as result.

We showed that sevoflurane induces translocation, but not increased serine⁶⁴³ phosphorylation of PKC-. Interestingly, sevoflurane-induced translocation of PKC- was attenuated by NCX inhibition. The discrepancy between sevoflurane-induced PKC- translocation and phosphorylation may be caused by the presence of several PKC- (auto-)phosphorylation sites, and serine⁶⁴³ has been demonstrated to be an auto-phosphorylation site involved in the regulation of its enzymatic activity.¹⁹ Therefore, it may be that translocation of PKC- involves phosphorylation of a different site than serine⁶⁴³, which was not investigated in the present study.

Several limitations must be considered in the interpretation of the present results. Stable and reliable contractile performance was ensured by conducting the experiments at hypothermic conditions, which may affect ion exchange. However, we previously demonstrated in isolated trabeculae that results of similar experiments on NCX function were unaffected by temperature.⁷ Second, KB-R7943 was developed as a selective NCX inhibitor, and up to 10 µmol/L is reported to be specific for Ca²⁺ entry via NCX with little influence on Ca²⁺ efflux rate.²⁰ To exclude a specific effect of KB-R7943, experiments to inhibit the reverse mode of the NCX were repeated in the presence of SEA0400, a novel NCX inhibitor.²¹ In our trabeculae, consistent with previous publications, KB-R7943 as well as SEA0400 exerted a slight positive inotropic effect, suggesting some inhibitory effect on the forward mode of the NCX and were not easily washed out from the preparation.^8,22 However, the relative alteration in basal force development of trabeculae exposed to NCX inhibition did not induce cardioprotection and did not alter rigor development or final recovery characteristics. Finally, we have not directly shown activation of the NCX by sevoflurane by a direct measurement of intracellular Ca²⁺. However, our results do show that pharmacological inhibition of the reverse mode of the NCX attenuates sevoflurane-induced PKC- activation and subsequent cardioprotection.

In summary, we demonstrate that sevoflurane-induced cardioprotection is dependent on the reverse mode of the NCX preceding PKC- translocation, presumably because of facilitated Ca²⁺ uptake via the NCX. Our results imply that sevoflurane-induced facilitation of NCX-dependent Ca²⁺ influx serves as a trigger for myocardial protection against ischemic episodes similar to other modes of preconditioning. This essential, but novel, role of the NCX in cardioprotective signaling may provide possible clues for the clinical induction of cardioprotective strategies.

	Acknowledgments

 
We acknowledge the helpful comments of Ronald Vlasblom.

Sources of Funding

The Institute for Cardiovascular Research Vrije Universiteit (ICaR-VU, Amsterdam, the Netherlands) provided support for this study. R.A. Bouwman is a MD-clinical research trainee supported by ZonMw, the Netherlands Organization for Health Research and Development, the Hague, Netherlands.

Disclosures

None.

	Footnotes

 
Presented at the American Heart Association Scientific Sessions, Dallas, Tex, November 13–16, 2005.

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