Volatile anesthetics produce cardioprotective action by attenuating cellular Ca2+ overload. The Ca2+ paradox is an important model for studying the mechanisms associated with Ca2+ overload-mediated myocardial injury, and was recently found to be mediated by Ca2+ entry through the transient receptor potential canonical channels upon Ca2+ repletion. This study investigated the effect of sevoflurane on cellular mechanisms underlying the Ca2+ paradox.


The Ca2+ paradox was examined in fluo-3 or mag-fluo-4-loaded mouse ventricular myocytes using confocal laser scanning microscope, upon Ca2+ repletion after 15 min of Ca2+ depletion in the absence and presence of sevoflurane.


The Ca2+ paradox was evoked in approximately 65% of myocytes upon Ca2+ repletion, as determined by an abrupt elevation of cytosolic Ca2+ accompanied by hypercontracture. The Ca2+ paradox was significantly suppressed by sevoflurane administered for 3 min before and during Ca2+ repletion (Post) or during Ca2+ depletion and repletion (Postlong), and Postlong was more beneficial than Post application. The sarcoplasmic reticulum Ca2+ levels gradually decreased during Ca2+ depletion, and the Ca2+ paradox was readily evoked in myocytes with reduced sarcoplasmic reticulum Ca2+ levels. Postlong but not Post application of sevoflurane prevented decrease in sarcoplasmic reticulum Ca2+ levels by blocking Ca2+ leak through ryanodine receptors. Whole cell patch-clamp recordings revealed that sevoflurane rapidly blocked thapsigargin-induced transient receptor potential canonical currents.


Sevoflurane protects ventricular myocytes from Ca2+ paradox-mediated Ca2+ overload by blocking transient receptor potential canonical channels and by preventing the decrease in sarcoplasmic reticulum Ca2+ levels, which is associated with less activation of transient receptor potential canonical channels.

  • The Ca2+paradox is the rapid deterioration of the heart as a result of Ca2+overload on reperfusion after a period of Ca2+depletion, and is primarily mediated by Ca2+entry via  the transient receptor potential canonical channels

  • Sevoflurane blocks the transient receptor potential canonical channel in mouse ventricular myocytes at clinically relevant concentrations, thereby preventing the Ca2+paradox

  • Sevoflurane helps preserve sarcoplasmic reticulum Ca2+levels during Ca2+depletion by blocking its spontaneous release in mouse ventricular myocytes

A NUMBER of studies have demonstrated that volatile anesthetics such as sevoflurane and isoflurane protect the heart from ischemia/reperfusion injury when administered before ischemia (anesthetic preconditioning, APC)1,,4or even immediately upon reperfusion (anesthetic postconditioning, APoC).5,,7Several cellular mechanisms have been proposed to mediate the beneficial effects of APoC, such as activation of protein kinase C and phosphatidylinositol-3-kinase, or inhibition of glycogen synthase kinase-3β and mitochondrial permeability transition pore.6,8,,10Because myocardial reperfusion injury occurs within the first few minutes of reperfusion after ischemia,11it seems unlikely that the prosurvival signaling pathways implicated in APoC give rise to adequate protective effects on initial injury during reperfusion. On the other hand, an excessive accumulation of intracellular Ca2+occurs during the early reperfusion phase and leads to various cellular injuries associated with ischemia/reperfusion.12,13Experimental evidence has been presented to indicate that volatile anesthetics substantially attenuate Ca2+overload upon the onset of reperfusion and thereby produce an APoC action in the heart.5,7However, the mechanisms by which volatile anesthetics ameliorate cellular Ca2+overload have yet to be fully characterized.

The Ca2+paradox rapidly develops upon repletion of Ca2+to the heart after a brief period of Ca2+depletion.13,,15There are some similarities in the structural and functional alterations caused by the Ca2+paradox and those induced by reoxygenation after anoxia or ischemia, such as Ca2+overload-mediated hypercontracture, mitochondrial dysfunction, and electrical and contractile disorders. These findings suggest that the Ca2+paradox is an important experimental model for investigating the cellular mechanisms associated with Ca2+overload-mediated injury during reperfusion of ischemic myocardium.16 

The transient receptor potential canonical (TRPC) channels are Ca2+-permeable nonselective cation channels that can be activated by a variety of signals, including depletion of endoplasmic reticulum/sarcoplasmic reticulum (SR) Ca2+stores.17,,19TRPC channels have therefore been implicated in the store-operated Ca2+entry (SOCE) in various cell types. Recent studies suggest that stromal interacting molecule 1 and Orai1 interact with TRPC proteins to mediate SOCE in some cell types.20,21There is accumulating evidence that TRPC channels are involved in diverse physiologic and pathophysiologic processes in cardiac cells.22,23The human myocardium expresses multiple TRPC channel isoforms such as TRPC1, TRPC4, TRPC5, and TRPC6,24and TRPC-mediated Ca2+entry is implicated in the development of hypertrophic phenotype in humans24and experimental animal models.25,26Our recent study has demonstrated that in mouse ventricular myocytes, the Ca2+paradox is primarily mediated by Ca2+entry through TRPC channels (probably TRPC1) upon restoration of extracellular Ca2+.27Furthermore, recent evidence indicates that the SOCE-mediated Ca2+entry contributes to the occurrence of ischemia/reperfusion-induced Ca2+overload in the heart.28,29 

The current investigation examined the effect of sevoflurane on the cellular and electrophysiological mechanisms associated with Ca2+paradox-mediated Ca2+overload. Our results provide novel evidence that sevoflurane protects ventricular myocytes from the Ca2+paradox by two distinct but closely related mechanisms, namely by blocking the TRPC channel that enables Ca2+entry upon Ca2+repletion and by preventing the decrease in Ca2+levels in the SR during Ca2+depletion, which is associated with less TRPC activation.

Preparation of Mouse Ventricular Myocytes

All of the experimental protocols are in conformance with the Guide for the Care and Use of Laboratory Animals  published by the National Institutes of Health (National Institutes of Health Publication no. 85–23, revised 1996) and were approved by the institutional Animal Care and Use Committee of Shiga University of Medical Science (Otsu, Shiga, Japan; no. 2010–1–3). Ventricular myocytes were enzymatically dispersed from hearts of 7- to 10-week-old male C57BL/6J mice (body weight, 20–25 g), as described previously.27,30In brief, mice were killed by intraperitoneal injection of sodium pentobarbital overdose (300 mg/kg) with heparin (8,000 U/kg). The heart was excised and quickly cannulated via  the ascending aorta, and retrograde perfusion was performed in a Langendorff apparatus at 37°C initially with normal Tyrode solution for 3 min, then with cell isolation buffer supplemented with 0.4 mM EGTA, for 3 min, and finally with cell isolation buffer supplemented with 1 mg/ml collagenase, 0.06 mg/ml trypsin, 0.06 mg/ml protease, and 0.3 mM CaCl2for 6–10 min. The ventricles were cut, minced, and then gently agitated intermittently for 6–8 min in cell isolation buffer supplemented with 1 mg/ml collagenase, 0.06 mg/ml trypsin, 0.06 mg/ml protease, 0.7 mM CaCl2, and 2 mg/ml bovine serum albumin. The supernatant containing isolated myocytes was centrifuged (14 g  for 3 min) and the myocytes were suspended in cell isolation buffer supplemented with 1.2 mM CaCl2and 2 mg/ml bovine serum albumin. After 10 min of incubation, the cell suspension was again centrifuged and resuspended in normal Tyrode solution supplemented with 2 mg/ml bovine serum albumin and antibiotics (penicillin/streptomycin). This isolation procedure resulted in a high yield (70–80%) of rod-shaped quiescent ventricular myocytes in normal Tyrode solution. A previous study confirmed that ventricular myocytes isolated in this way have normal electrophysiologic and contractile properties.30 

Solutions and Chemicals

Normal Tyrode solution contained (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, and 5 HEPES (pH adjusted to 7.4 with NaOH). In some experiments, the concentration of Ca2+in Tyrode solution was reduced to 0.3, 0.7, or 1.2 mM. The nominally Ca2+-free Tyrode solution was prepared by simply omitting CaCl2(no added EGTA) from the normal Tyrode solution. The free Ca2+concentration in the nominally Ca2+-free Tyrode solution is estimated to be in the range of a few to 20 μM.31,32The extracellular solution used to measure whole cell TRPC currents was K+-free Tyrode solution supplemented with nisoldipine, which contained (in mM) 140 NaCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, 0.001 nisoldipine, and 5 HEPES (pH adjusted to 7.4 with NaOH). The pipette solution contained (in mM) 90 Cs-aspartate, 30 CsCl, 20 tetraethylammonium chloride, 2 MgCl2, 5 adenosine 5′-triphosphate (Tris salt), 0.1 guanosine 5′-triphosphate (dilithium salt), 5 EGTA, 2 CaCl2, and 5 HEPES (pH adjusted to 7.2 with CsOH). The concentration of free Ca2+in the pipette solution was calculated to be approximately 0.1 μM.33,34Reagents added to bathing solutions (normal Tyrode and/or nominally Ca2+-free Tyrode solution) included thapsigargin (Wako Pure Chemical Industries, Osaka, Japan), 2-aminoethoxydiphenyl borate (2-APB; Tocris Cookson Inc., Ellisville, MO), caffeine (Sigma Chemical Company, St Louis, MO), and tetracaine (Sigma Chemical Company). Concentrated stock solutions were made for thapsigargin (10 mM) and 2-APB (20 mM) in dimethyl sulfoxide and diluted in bathing solutions at a final concentration as indicated. Caffeine and tetracaine were directly added to bathing solutions. Fluo-3 acetoxymethyl ester (fluo-3 AM) was from Dojin Chemicals (Kumamoto, Japan), and mag-fluo-4 AM and pluronic F-127 were from Molecular Probes (Eugene, OR). The cell isolation buffer used for cell preparation contained (in mM) 130 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 22 glucose, 25 HEPES (pH adjusted to 7.4 with NaOH), and 50 U/ml bovine insulin. Collagenase (type 2) was obtained from Worthington Biochemical Corporation (Lakewood, NJ), and trypsin, protease, bovine serum albumin, and bovine insulin were from Sigma Chemical Company.

Fluo-3 and Mag-Fluo-4 Fluorescence Imaging with Laser Scanning Confocal Microscope

Ventricular myocytes were loaded at 37°C either with fluo-3 AM (5 μM) for 20 min or with mag-fluo-4 AM (5 μM) together with pluronic F-127 (0.1%) for 30 min, and were then washed to remove excess extracellular dye with normal Tyrode solution supplemented with 2 mg/ml bovine serum albumin. Fluo-3 or mag-fluo-4 loaded myocytes were then resuspended in normal Tyrode solution supplemented with bovine serum albumin for an additional 30 min to allow for intracellular hydrolysis of fluo-3 AM or mag-fluo-4 AM before experiments. An aliquot of fluo-3 or mag-fluo-4 loaded myocytes was allowed to settle onto the glass bottom of a recording chamber (0.5 ml in volume) mounted on the stage of an Eclipse TE2000-E inverted microscope (Nikon, Tokyo, Japan), equipped with a C1si spectral imaging confocal laser scanning system (Nikon). The chamber was continuously perfused with bathing solutions at a constant rate of 2–3 ml/min at room temperature (23–25°C). Fluo-3 or mag-fluo-4 loaded myocytes were excited with an argon laser beam (wavelength 488 nm) at 30-s or 10-s intervals, respectively, unless otherwise noted, and data were collected for emission intensity at wavelength of 515 nm. Fluorescence images were analyzed frame by frame using Nikon EZ-C1 software to calculate the average intensity in each myocyte, which was used as an estimate of cytosolic (fluo-3) or SR (mag-fluo-4) Ca2+levels. All intensity values were calculated by subtracting the background fluorescence. Changes in the cell morphology were also assessed by measuring a length/width ratio in each myocyte image, and the myocytes showing the length/width ratio of less than 2 were considered to be either hypercontracted or dead.35 

The periods of exposure to various reagents and changes in extracellular Ca2+concentrations are denoted by horizontal bars or boxes in the figures. The data (fluorescence intensity and length/width ratio) for initial superfusion with normal Tyrode solution was shown for the latter 2 min. Fluorescence images were obtained from quiescent (not paced) mouse ventricular myocytes because myocytes failed to respond to electrical stimulation when superfused with nominally Ca2+-free Tyrode solution. Ventricular myocytes that generated spontaneous Ca2+waves during initial superfusion with normal Tyrode and/or subsequent superfusion with nominally Ca2+-free Tyrode solution were excluded from the analysis (less than approximately 2% of the total viable myocytes).

Protocols for Evaluation of Ca2+Paradox in Fluo-3 Loaded Myocytes

To evoke the Ca2+paradox, ventricular myocytes loaded with fluo-3 were successively superfused, initially with normal Tyrode solution for 5 min, then with nominally Ca2+free-Tyrode solution for 15 min (Ca2+depletion), and again with normal Tyrode solution (Ca2+repletion).27Fluo-3 fluorescence images were obtained using a × 10 objective lens, and approximately 20 rod-shaped viable myocytes were observed within a single field of view in a typical experiment. The incidence of the Ca2+paradox was measured in each experiment as a percentage of hypercontracted myocytes due to increased cytosolic Ca2+levels (as assessed by fluo-3 fluorescence intensity) upon Ca2+repletion with reference to the total number of rod-shaped viable myocytes during initial superfusion with normal Tyrode solution. The effect of various interventions on the Ca2+paradox was assessed by the changes in the percentage incidence of the Ca2+paradox. Fluo-3 fluorescence intensity was expressed as arbitrary units, unless otherwise stated. It should be noted that the Ca2+paradox is consistently evoked upon Ca2+repletion in mouse ventricular myocytes after 10, 15, and 20 min of Ca2+depletion and that the percentage incidence of the Ca2+paradox is not significantly affected by these durations of Ca2+depletion.27 

Protocols for Assessment of SR Ca2+Levels during Ca2+Depletion in Mag-Fluo-4 Loaded Myocytes

Mag-fluo-4 fluorescence intensity was measured to evaluate the changes in SR Ca2+levels during 15 min of Ca2+depletion, using a superfusion protocol similar to that for fluo-3-loaded myocytes. Mag-fluo-4 fluorescence images were captured using a ×40 objective lens, and 1–3 of viable myocytes were usually observed within a single field of view. Mag-fluo-4 fluorescence intensity (F) was expressed as relative value (F/F0) compared with baseline value obtained just before Ca2+-free superfusion (F0), unless otherwise stated. The occurrence of Ca2+paradox was assessed in each myocyte by cellular hypercontracture upon Ca2+repletion. In experiments evaluating the changes in the mag-fluo-4 fluorescence during Ca2+-free superfusion with tetracaine, tetracaine was applied from the initial superfusion with normal Tyrode solution to avoid possible artifact arising from the fluorescence-quenching effect of tetracaine.36,37 

Protocols for Administration of Sevoflurane

Sevoflurane (Abbott Laboratories, North Chicago, IL) was applied for three different periods during the superfusion: for a period of initial superfusion with normal Tyrode (Pre), for a period of 3 min before and during Ca2+repletion (Post), and for a period of Ca2+depletion and repletion (Postlong). These three protocols were applied to examine the effect on the Ca2+paradox, whereas Post and Postlong protocols were used to assess the SR Ca2+levels.

Sevoflurane was equilibrated in normal Tyrode and/or nominally Ca2+-free Tyrode solutions, or nisoldipine-containing K+-free Tyrode solution in a reservoir by passing air (flow rate, 0.5 l/min) through a calibrated vaporizer for at least 15 min before superfusing ventricular myocytes. The concentration of sevoflurane in normal Tyrode solution at the recording chamber was measured by gas chromatography.

Whole Cell Patch-Clamp Recordings

Ventricular myocytes were voltage-clamped using the whole cell patch-clamp technique38with an EPC-8 patch-clamp amplifier (HEKA, Lambrecht, Germany). Fire-polished pipettes pulled from borosilicate glass capillaries (Narishige Scientific Instrument Laboratory, Tokyo, Japan) had a resistance of 2.0–3.5 MΩ when filled with the pipette solution. An aliquot of cell (ventricular myocyte) suspension was transferred to a recording chamber (0.5 ml in volume) mounted on the stage of a Nikon TMD-300 inverted microscope (Tokyo, Japan) and was allowed to adhere lightly to the glass bottom for at least 1–2 min. The chamber was continuously perfused at a constant rate of 2 ml/min with bathing solutions at 34–36°C. The voltage ramp protocol (dV /dt = ±0.25 V/s) was repeated every 8 s and consisted of three phases: an initial + 50 mV depolarizing phase from a holding potential of 0 mV, a second hyperpolarizing phase of −150 mV, and then a third phase returning to the holding potential. The current-voltage relationship was measured during the second hyperpolarizing phase. Voltage-clamp protocols and data acquisition were controlled with PATCHMASTER software (Version 1.03, HEKA), and current records were filtered at 1 kHz, digitized at 5 kHz through an LIH-1600 interface (HEKA), and stored on a Macintosh computer. Current amplitude was presented as current density (in pA/pF), obtained by normalizing with reference to cell membrane capacitance.

Statistical Analysis

The effects of experimental protocols and animals on the data were initially examined by the two-way layout ANOVA (GLM procedure; SAS 9.1.3, SAS Institute, Inc., Cary, NC). This statistical analysis reveals that the mean square for animals was much smaller than the mean square for experimental protocol, which indicates that the effect of animals on the data is negligibly small in the current experiments. Data are presented as means (95% CIs), with the number of animals (cell isolations) and experiments indicated by N  and n , respectively, whereas error bars in the figures represent 95% CIs with number of experiments (n ) given in parentheses. Statistical comparisons between two groups were evaluated by Mann–Whitney U  test and comparisons among multiple groups were performed by Kruskal-Wallis test followed by Mann–Whitney U  test with Bonferroni correction, using the GraphPad Prism 5 (La Jolla, CA). All statistical tests were two-tailed. A value of P < 0.05 was considered statistically significant.

Ca2+Paradox in Mouse Ventricular Myocytes and Its Suppression by Sevoflurane

Figure 1illustrates representative experiments showing the occurrence of the Ca2+paradox upon Ca2+repletion following 15 min of Ca2+depletion under control conditions (fig. 1A and C) and in the presence of 3% sevoflurane during Ca2+depletion and repletion (Postlong protocol; fig. 1B and D). In these experiments, time courses of changes in fluo-3 fluorescence intensity (panel a in fig. 1C and D) and length/width ratio (panel b) were measured for each of the fluo-3 fluorescence images of rod-shaped viable myocytes within the same field of view. Under control conditions, 12 out of the 21 myocytes (57.1%) rapidly underwent the Ca2+paradox upon reperfusion with normal Tyrode solution (fig. 1A), as determined by an abrupt elevation of fluo-3 fluorescence intensity (fig. 1C, panel a) accompanied by a marked decrease of length/width ratio to less than 2 (fig. 1C, b, hypercontracture). It should be noted that there was no recovery from hypercontracture during 10 min of Ca2+repletion (data not shown). In a total of 16 experiments, the incidence of the Ca2+paradox averaged 65.6% (56.5–74.6%; n = 16, N = 6) under control conditions.

The incidence of the Ca2+paradox was not affected by restoration of Ca2+at normal physiologic concentration of 1.2 mM, compared with that observed upon restoration of 1.8 mM Ca2+(see Supplemental Digital Content 1, fig. 1A and B, The Ca2+paradox was also examined during successive readmission of 0.3, 0.7, 1.2, and 1.8 mM Ca2+(see Supplemental Digital Content 1, fig. 1C, The incidence of Ca2+paradox was 67.1% (57.9–77.3%; n = 6, N = 3) upon restoration of 0.3 mM Ca2+after 15 min of Ca2+depletion and was little affected upon subsequent elevation of Ca2+to 0.7, 1.2, and 1.8 mM. Thus, it was found that restoration of 0.3 mM Ca2+similarly evokes the Ca2+paradox, compared with restoration of 1.8 mM Ca2+. The Ca2+paradox observed during restoration of successive Ca2+elevation was markedly suppressed by the TRPC channel blocker 2-APB (20 μM) applied for 3 min before and during Ca2+repletion (see Supplemental Digital Content 1, fig. 1C, These observations suggest that the Ca2+paradox observed during restoration of successive Ca2+elevation was evoked primarily by Ca2+entry through TRPC channels.

As demonstrated in fig. 1B and D, only 1 out of 16 myocytes (6.3%) underwent the Ca2+paradox upon Ca2+repletion after 15 min of Ca2+depletion during Postlong application of sevoflurane. The following experiments were therefore conducted to explore the cellular mechanisms underlying this protective effect of sevoflurane on the Ca2+paradox.

Figure 2A summarizes the results of the effect of three different protocols (Pre, Post, and Postlong) for the application of sevoflurane on the Ca2+paradox. The incidence of the Ca2+paradox was significantly reduced by sevoflurane applied in Post (36.2%, [26.6– 45.8%]; n = 16, N = 5) and Postlong (14.2%, [7.6–20.9%]; n = 11, N = 4) protocols but not in Pre protocol (62.3%, [51.4–73.1%]; n = 10, N = 4), when compared with control (65.6%, [56.5–74.6%]; n = 16, N = 6). As demonstrated in figure 2B, a linear correlation was observed between the volume percentage of sevoflurane delivered via  vaporizer and its millimolar concentrations in the superfusate (normal Tyrode solution), measured by gas chromatography. Figure 2C illustrates concentration-dependent effect of sevoflurane in Postlong application, which was the most protective protocol against the Ca2+paradox. The Ca2+paradox was suppressed by sevoflurane in Postlong protocol with an IC50of 0.14 mM (approximately1.0%, fig. 2B). Sevoflurane was thus found to effectively suppress the Ca2+paradox even at concentrations lower than 1 minimum alveolar concentration (2.3% in mouse).

Block of TRPC Channel by Sevoflurane

To elucidate the electrophysiologic and cellular mechanisms for the suppressive effect of sevoflurane on the Ca2+paradox, we examined the effect of sevoflurane on the TRPC channel current using whole cell patch-clamp method during superfusion with K+-free Tyrode solution containing 1 μM nisoldipine. It has been demonstrated in various cell types that thapsigargin activates the TRPC channel current by depleting SR Ca2+stores through the inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).39Bath application of thapsigargin (1 μM) gradually increased the membrane current during voltage ramps (fig. 3A and B), which exhibited a practically linear current-voltage relationship with a reversal potential of approximately 0 mV (fig. 3C, 2–1) and sensitivity to inhibition by 2-APB (20 μM, fig. 3C, 2–3). Similar properties have been reported for the TRPC1 current in submandibular gland acinar cells39and ventricular myocytes of mouse.27As demonstrated in figs. 3D-E, sevoflurane (3%) rapidly blocked the thapsigargin-induced TRPC current in a reversible manner. Similar results were obtained in four additional myocytes. These observations show that TRPC channels were blocked by sevoflurane and indicate that this blocking action could contribute to the suppressive effect of Post and Postlong applications of sevoflurane on the Ca2+paradox (fig. 1B and D and fig. 2A).

As illustrated in figure 4, the Ca2+paradox was markedly suppressed by 2-APB (20 μM) applied for a period of 3 min before and during Ca2+restoration, which again confirms the involvement of Ca2+entry via  TRPC channels in the Ca2+paradox.27Post application of sevoflurane together with 2-APB did not further improve the suppressive effect of 2-APB, which suggests that sevoflurane applied in Post protocol protects ventricular myocytes from the Ca2+paradox by the same mechanism as 2-APB, namely, by blocking the TRPC channels.

Preservation of SR Ca2+Content by Sevoflurane as Assessed by Mag-Fluo-4 Fluorescence

However, a significant difference was detected in the suppressive effect of sevoflurane on the Ca2+paradox between Post and Postlong application protocols (fig. 2A). The observation that Postlong application was more effective indicates that the presence of sevoflurane during Ca2+depletion period is important for producing a further protective effect against the Ca2+paradox. A number of studies have indicated that Ca2+decrease in the SR is one of the effective triggers for the activation of TRPC channels.17,,19In the next series of experiments, we assessed the hypothesis that sevoflurane applied during Ca2+-free superfusion prevents the decrease in SR Ca2+levels and thereby suppresses the subsequent activation of TRPC channels that enables rapid Ca2+entry leading to the Ca2+paradox upon Ca2+restoration.

In recent years, the low-affinity (K  Dof 22 μM) fluorescence Ca2+indicator mag-fluo-4 has proven useful in monitoring Ca2+levels in organelles, where free Ca2+levels are substantially high (submillimolar concentrations), such as SR and nuclear envelope.40To verify that mag-fluo-4 fluorescence largely reflects SR Ca2+levels in mouse ventricular myocytes, the effect of caffeine, a well-known ryanodine receptor agonist,41was examined (see Supplemental Digital Content 1, fig. 2, Addition of caffeine (10 mM) to nominally Ca2+-free Tyrode solution rapidly increased the fluo-3 fluorescence intensity (see Supplemental Digital Content 1, fig. 2A,, which indicates an abrupt elevation of the cytosolic Ca2+levels due to Ca2+release from the SR via  cardiac ryanodine receptor (RyR2). In contrast, mag-fluo-4 fluorescence was found to rapidly decrease to approximately 60% of the baseline levels by caffeine application (see Supplemental Digital Content 1, fig. 2B, A similar degree of decline in mag-fluo-4 fluorescence has been found during exposure to caffeine in primary cultured rat ventricular myocytes.40Therefore, the mag-fluo-4 fluorescence intensity can be regarded as primarily reflecting Ca2+levels in the SR in mouse ventricular myocytes.

In the experiment shown in figure 5, mag-fluo-4 fluorescence intensity was continuously measured to monitor SR Ca2+levels during 15 min of Ca2+-free superfusion. In control, mag-fluo-4 fluorescence was gradually decreased to approximately 80% of the initial levels measured just before Ca2+depletion (fig. 5A and B), indicating that there is a gradual decline of SR Ca2+levels during Ca2+depletion. This decline of mag-fluo-4 fluorescence was significantly reduced by the presence of tetracaine (1 mM), a drug that potently blocks the SR Ca2+leak via  RyR2,42suggesting that the gradual decline of SR Ca2+levels is ascribable to spontaneous Ca2+leak via  RyR2. On the other hand, mag-fluo-4 fluorescence was further decreased during Ca2+-free superfusion by the addition of the SERCA inhibitor thapsigargin (1 μM; fig. 5A and B), indicating that SR Ca2+depletion was facilitated by inhibiting Ca2+uptake into the SR. Thapsigargin was thus found to be effective at depleting SR Ca2+stores, which could be related to the activation of TRPC channels in the presence of this compound.39These results indicate that the SR Ca2+levels were decreased to some extent during Ca2+-free superfusion, due to the spontaneous Ca2+leak via  RyR2, and that this spontaneously released Ca2+was partly reuptaken via  SERCA.

Figure 6shows the effects of Post and Postlong applications of sevoflurane on the changes in SR Ca2+levels during Ca2+depletion. In Post application, the mag-fluo-4 fluorescence gradually declined during Ca2+depletion to an extent similar to control, whereas Postlong application significantly antagonized the decrease in mag-fluo-4 fluorescence (fig. 6A and B). These observations suggest that the SR Ca2+level was considerably preserved by the presence of sevoflurane during the Ca2+-free superfusion period. To elucidate the mechanism by which sevoflurane maintains the SR Ca2+levels during Ca2+depletion, changes in mag-fluo-4 fluorescence were examined under conditions where RyR2 was blocked by tetracaine (1 mM). As demonstrated in figures 6C and D, Postlong application of sevoflurane did not cause an additional increase in mag-fluo-4 fluorescence intensity during Ca2+depletion in the presence of tetracaine (fig. 6D), which suggests that sevoflurane maintains the SR Ca2+levels by blocking Ca2+leak via  RyR2. It was also found that mag-fluo-4 fluorescence intensity during the presence of tetracaine was not influenced by adding sevoflurane before tetracaine (fig. 6D). This observation further strengthens the view that sevoflurane shares the same mechanisms of action as tetracaine in maintaining the SR Ca2+levels, namely, by blocking RyR2.

Functional Role of SR Ca2+Levels in Suppression of Ca2+Paradox in Control, Post, and Postlong Applications of Sevoflurane

To clarify the dependence of Ca2+paradox on SR Ca2+levels, we analyzed the relationship between the mag-fluo-4 fluorescence value (F/F0) after 15 min of Ca2+-free superfusion and the occurrence of Ca2+paradox upon Ca2+restoration. Figure 7A illustrates the distribution of mag-fluo-4 F/F0for myocytes without (red squares) or with (blue squares) the development of Ca2+paradox, in control, Post, and Postlong applications of sevoflurane. In control applications, the fraction of myocytes that underwent the Ca2+paradox (15 myocytes) showed a significantly lower value for mag-fluo-4 F/F0, compared with that for the fraction of myocytes (6 myocytes) that did not undergo the Ca2+paradox (0.78, [0.74– 0.81]vs.  0.86 [0.79–0.92], P = 0.021; fig. 7A, a ), thus suggesting that the development of Ca2+paradox is significantly dependent on the SR Ca2+levels during Ca2+depletion. On the other hand, a significant difference could not be detected between myocytes without and with the development of the Ca2+paradox in Post application of sevoflurane (0.82, [0.77–0.86]vs.  0.76, [0.72–0.80], P = 0.20; fig. 7A, b).

The individual records for control and Post application of sevoflurane were arranged into three arbitrary groups according to the values for mag-fluo-4 F/F0, and the incidence of the Ca2+paradox was compared between data for control and Post application of sevoflurane (fig. 7B). Under control conditions, the incidence of Ca2+paradox was increased with a reduction of mag-fluo-4 F/F0, and reached 100% when mag-fluo-4 F/F0was decreased to less than 0.75. These results again suggest that the Ca2+paradox was more readily evoked in myocytes with lower Ca2+levels in the SR. Consistent with this notion, the incidence of the Ca2+paradox was markedly reduced by Postlong application of sevoflurane (1 out of 18 myocytes, 5.6%), where most of the myocytes showed a higher value of mag-fluo-4 F/F0(fig. 7Ac, see also fig. 6B). It is important to note that whereas there are no significant differences in the overall mean value (fig. 6B) and distribution of mag-fluo-4 F/F0(see Supplemental Digital Content 1, fig. 3, between control and Post application of sevoflurane, the incidence of the Ca2+paradox was reduced by Post application in all three arbitrary groups (fig. 7B). Thus, Post application of sevoflurane effectively protected ventricular myocytes from the Ca2+paradox (fig. 2A and fig. 7B) without appreciably affecting the SR Ca2+levels, as evidenced by the changes in mag-fluo-4 F/F0value (fig. 6A and B; see Supplemental Digital Content 1, fig. 3A, This beneficial effect of Post application of sevoflurane may be ascribable to its blocking action on TRPC channels (figs. 3and 4).

Our previous experiments demonstrated that tetracaine effectively prevents the occurrence of the Ca2+paradox with an IC50of 0.29 mM, a value close to that reported for the block of RyR2 by tetracaine (IC50of 0.26 mM),42and suggested that tetracaine prevents the Ca2+paradox primarily by preventing the decrease in SR Ca2+levels.27We examined the effect of Post application of sevoflurane (3%) on the Ca2+paradox in the presence of tetracaine at 0.3 mM, where the incidence Ca2+paradox was substantially reduced (see Supplemental Digital Content 1, fig. 4, Post application of sevoflurane was found to further suppress the Ca2+paradox. This observation again supports the view that Post application of sevoflurane prevented the Ca2+paradox by inhibiting the TRPC channels, which is a different mechanism of action of tetracaine (RyR2 block).

Recent studies have suggested that Ca2+influx associated with SOCE mediates the ischemia/reperfusion-induced Ca2+overload in the heart.28,29Because there is considerable evidence to indicate that TRPC channels contribute to SOCE in various cell types including cardiac myocytes,17,22,23these studies should suggest the important possibility that TRPC channel activation is actually involved in mediating the ischemia/reperfusion-induced Ca2+overload. It therefore appears worthwhile to examine the Ca2+paradox, which is primarily mediated via  Ca2+entry via  TRPC channels,27as an experimental model to assess the ischemia/reperfusion injury associated with the TRPC-mediated Ca2+overload. It should be noted that less Ca2+is stored in the SR during ischemia, probably due to the decreased rate of Ca2+uptake via  SERCA.43,44Furthermore, a recent study demonstrated that SR Ca2+level, as assessed by measuring the mag-fluo-4 fluorescence, is rapidly diminished at the onset of reperfusion in a Langendorff-perfused mouse heart model.45It may be reasonable to speculate that SR Ca2+depletion observed in ischemia and/or reperfusion is related to the activation of SOCE that may contribute to Ca2+overload after ischemia.28,29 

Block of TRPC Channels Contributes to the Preventive Action of Sevoflurane in Post and Postlong Applications against the Ca2+Paradox

The current experiments found that sevoflurane at clinically relevant concentrations blocks the TRPC channels (fig. 3D–F and fig. 4) and thereby prevents the Ca2+paradox in mouse ventricular myocytes (fig. 2A). Currently, it remains unclear whether the blocking action of sevoflurane on TRPC channels arises from direct binding to the channel proteins or is mediated indirectly by altering the behavior and dynamics of plasma membrane lipids. It is also probable that sevoflurane affects signal transduction processes associated with TRPC channel activation. It should, however, be noted that the blocking effect of sevoflurane is completely reversed within 1 min after washout (fig. 3D–F), which appears to be a favorable property for clinical applications. Future studies are required to elucidate the molecular and cellular signaling basis for the blocking action of sevoflurane on the TRPC channels.

Previous studies have demonstrated that sevoflurane blocks several types of ionic channels and transporters in cardiac ventricular myocytes, including the L-type Ca2+channel,46Na+/Ca2+exchanger,47and plasma membrane Ca2+pump,48while modestly affecting the inwardly rectifying K+channel.46The current data indicate that TRPC channels can be added to the list of cardiac ion channels modulated (inhibited) by sevoflurane.

Preservation of SR Ca2+by Postlong Application of Sevoflurane and Its Contribution to Better Protection against the Ca2+Paradox

The current experiments using a low-affinity Ca2+fluorescence indicator mag-fluo-4 found that SR Ca2+levels were significantly better preserved by the presence of sevoflurane during Ca2+depletion (Postlong), compared with conditions where sevoflurane was absent during Ca2+depletion (fig. 6A and B). This effect of sevoflurane appears to involve the block of spontaneous Ca2+leak via  RyR2, similar to the action of tetracaine (fig. 6C and D). Previous studies have also suggested that sevoflurane maintains SR Ca2+stores by blocking the spontaneous Ca2+release from the SR in rat ventricular myocytes.49It should be noted that sevoflurane exerts an inhibitory action on the plasma membrane Na+/Ca2+exchanger47and Ca2+pump,48which could also result in Ca2+loading into the SR through the inhibition of Ca2+extrusion from the myocytes,50so long as there is sufficient adenosine 5′-triphosphate for SERCA-mediated SR Ca2+uptake. Because Ca2+depletion in the SR may act as an effective trigger for the activation of TRPC channels,17,,19it is reasonable to propose that the better preservation of SR Ca2+levels by Postlong application of sevoflurane could result in less TRPC activation, and thereby contributes to a lower incidence of the Ca2+paradox, compared with Post application (fig. 2A and fig. 6A and B).

It was also found that the Ca2+paradox occurred in an all-or-none manner both in control conditions and in the presence of sevoflurane (fig. 1). The precise mechanism for this phenomenon is as yet undefined. As judged from the observation that TRPC channels are permeable to Ca2+and display time-independent kinetics,19,51it may be probable that once the channel opens due to SR Ca2+depletion, adequate Ca2+may enter the cell without delay to cause hypercontracture (Ca2+paradox). Even in the presence of sevoflurane applied in Postlong protocol, in myocytes where the SR Ca2+content was not maintained and/or TRPC channels were not blocked, TRPC channels may mediate sufficient Ca2+influx to cause “complete” Ca2+paradox. Additional studies are required to elucidate the ionic mechanisms associated with the occurrence of the Ca2+paradox in an all-or-none manner.

Protection of Ventricular Myocytes against Ca2+Paradox by Sevoflurane and Its Possible Clinical Implications

Although many cellular mechanisms have been put forward to explain the beneficial effects of APC and APoC, the attenuation of intracellular Ca2+overload appears to be one of the key end effects.2,5,7Because APC strategy is likely to be difficult to apply in clinical conditions where precise onset of ischemia is not well defined, APoC may be more preferable for those conditions. Assuming that the Ca2+paradox and/or TRPC activation are at least partly responsible for ischemia/reperfusion-induced Ca2+overload,28,29the anti-Ca2+paradox action of sevoflurane produced by Post and Postlong application (fig. 2A) can be included in its APoC action. Our experiments indicate the possible presence of novel targets for APoC action by sevoflurane, namely TRPC channels and RyR2, which are different from the previously known target proteins associated with prosurvival signaling pathways, such as protein kinase C, phosphatidylinositol-3-kinase, endothelial NO synthase, glycogen synthase kinase-3β, and mitochondrial permeability transition pore.6,8,,10 

The current experiments found that sevoflurane at clinically relevant concentrations is able to effectively prevent the Ca2+paradox that rapidly occurs after Ca2+restoration (figs. 1and 2). The difference in the protective effects of Post and Postlong applications of sevoflurane against the Ca2+paradox can be explained as follows: Post application of sevoflurane only inhibits the TRPC channels, whereas Postlong application in addition suppresses a factor for TRPC activation, namely SR Ca2+depletion. Administration of sevoflurane on clinical reperfusion, which corresponds to the current protocol of Post application, is expected to exert a protective action against Ca2+overload during reperfusion of ischemic myocardium, mediated at least partly through the SOCE mechanism.28,29Although it remains unknown as to whether and to what extent sevoflurane reaches the ischemic myocardium, continuous administration of sevoflurane even during the ischemic period (corresponding to Postlong application) is likely to produce additional beneficial effects against Ca2+overload-mediated reperfusion injury.

Limitations of the Study

Although the Ca2+overload-mediated ischemia/reperfusion injury may be at least partly reproduced by the Ca2+paradox phenomenon, some differences have also been shown to exist in the mechanisms involved in Ca2+paradox and ischemia/reperfusion injury.15For example, intracellular adenosine 5′-triphosphate is maintained during Ca2+depletion but is reduced during hypoxia/ischemia. In addition, intracellular Na+overload contributes substantially to ischemia/reperfusion injury but is not a prerequisite for the Ca2+paradox upon Ca2+repletion. Furthermore, the Ca2+paradox produces more severe damage to the heart than ischemia/reperfusion injury. It may also be noted that whereas APC effectively attenuates Ca2+overload during ischemia/reperfusion,2,5it remains to be elucidated whether APC protects the heart from the Ca2+paradox-mediated Ca2+overload. These two types of myocardial injury thus differ in their pathomechanisms,15whereas SOCE appears to be relevant to Ca2+overload in both injuries.27,,29Therefore, the current results obtained using the Ca2+paradox phenomenon cannot be directly extrapolated to myocardial Ca2+overload during ischemia/reperfusion. In addition, the current experiments conducted on isolated myocytes at room temperature (23–25°C) may not represent all of the features of the Ca2+paradox in the heart15where myocytes are sitting in the structured setting, connected by gap junctions (connexins) and surrounded by basal lamina. Because in clinical situations, the Ca2+paradox injury could occur during reperfusion after cardioplegic arrest or preservation with Ca2+-free cardioplegia,52,53it is important to examine whether TRPC activation has any relevance to possible cardioplegic ischemia/reperfusion injury, using a whole heart model.

In conclusion, sevoflurane markedly suppresses the Ca2+paradox-mediated Ca2+overload by two distinct but closely related cellular mechanisms, namely by blocking TRPC channels that mediates Ca2+entry upon Ca2+repletion associated with development of the Ca2+paradox and by preventing the SR Ca2+decrease during Ca2+depletion, which is associated with less TRPC activation. These mechanisms may be involved in the cardioprotective effect of APoC in the experimental setting and are likely to provide a promising and widely applicable strategy to protect the heart from Ca2+overload-mediated injury in clinical settings.

The authors thank Mr. Takefumi Yamamoto (Technical Instructor, Central Research Laboratory, Shiga University of Medical Science, Otsu, Shiga, Japan) and Mr. Yasuhiro Mori (Technical Instructor, Central Research Laboratory, Shiga University of Medical Science) for their assistance with use of the confocal laser scanning microscope, and Tadanori Sugimoto, Ph.D. (Dainippon Sumitomo Pharma Co., Ltd., Suita, Osaka, Japan), for his help with statistical analyses.

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