Protein Kinase C-ε Primes the Cardiac Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channel to Modulation by Isoflurane

All experiments were approved by the Animal Care and Use Committee of the Medical College of Wisconsin (Milwaukee, Wisconsin). Single cardiac ventricular myocytes were enzymatically isolated from adult guinea pigs (either sex) weighing 150–300 g as previously described,23using a modified isolation method of Mitra and Morad.24Briefly, the guinea pigs were intraperitoneally injected with heparin (1,000 U/ml) and anesthetized with pentobarbital sodium (250 mg/kg). After thoracotomy, the hearts were quickly removed and mounted on a Langendorff apparatus. They were perfused retrogradely through the aorta with warm (37°C) oxygenated Joklik medium (Sigma-Aldrich, St. Louis, MO) containing 2.5 U/ml heparin. After blood had been washed out, the hearts were perfused for 8–12 min by an enzyme solution containing Joklik medium, 0.3 mg/ml collagenase type II (Gibco BRL; Invitrogen, Grand Island, NY), 0.1 mg/ml protease type XIV (Sigma-Aldrich), and 0.1 mg/ml bovine serum albumin (Serelogicals Corporation, Kankakee, IL), at pH 7.23. The ventricles were then minced and incubated for an additional 3–10 min in the enzyme solution. Isolated myocytes were washed and stored in a Tyrode solution at room temperature (22°C) and used for experiments within 12 h of the isolation procedure.

The modified Tyrode solution contained 132 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl², 1 mm CaCl², 10 mm HEPES, and 5 mm glucose, at pH 7.3 adjusted with NaOH. The intracellular pipette solution contained 60 mm l-glutamic acid, 50 mm KCl, 10 mm HEPES, 1 mm MgCl², 11 mm EGTA, 1 mm CaCl², and 0.5 mm K²ATP, at pH 7.4 adjusted with KOH. The external bath solution contained 132 mm N -methyl-d-glucamine, 2 mm MgCl², 5 mm KCl, 1 mm CaCl², and 10 mm HEPES, at pH 7.4 adjusted with HCl. Nisoldipine, a gift from Miles-Pentex (West Haven, CT), was added to the external solution at a concentration of 200 nm to block the L-type calcium channels. The 1-mm stock solution of nisoldipine was made in polyethylene glycol. The 10-mm stock solution of pinacidil (Lilly Laboratories, Indianapolis, IN), a K^ATPchannel opener, was prepared in dimethyl sulfoxide. The 1-mm stock solution of glibenclamide (Sigma-Aldrich), a K^ATPchannel blocker, was also prepared in dimethyl sulfoxide. 5-Hydroxydecanoate (5-HD; Sigma-Aldrich), a specific blocker of the mitoK^ATPchannels, was prepared as a 10-mm stock solution in water.

To investigate the effects of PKC-ε and PKC-δ on the sarcK^ATPchannel, membrane permeable peptides (conjugated to the antennapedia carrier) of the PKC-ε and PKC-δ translocation activators and inhibitors, including a scrambled PKC-ε peptide, were used. These peptides were obtained from Daria Mochly-Rosen, Ph.D. (Professor and Chair, Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California).25The 1-μm stock solutions of these peptides were prepared in the external bath solution, stored at −20°C, and thawed before use. The specificity of each PKC peptide is high. The peptide activator mimics the isozyme-specific receptors for activated C-kinase (RACK) binding site and causes the conformational change of a specific PKC isoform, allowing it to translocate to its corresponding RACK. The peptide inhibitor inhibits the translocation of an activated PKC isozyme by blocking the binding site of its corresponding RACK.

Isoflurane (Abbott Laboratories, North Chicago, IL) was dispersed in the external bath solution, kept in glass-syringe reservoirs to ensure a constant concentration, and delivered to the recording chamber via  syringe pumps. At the end of each experiment, solution samples were collected, and the concentrations of isoflurane in the recording chamber were measured using gas chromatography (Shimadzu, Kyoto, Japan), as previously reported.23The concentration of isoflurane used in this study was 0.88 ± 0.05 mm, which is equivalent to 1.86 vol% (1.55 minimum alveolar concentration [MAC]) at 22°C.

Sarcolemmal K^ATPchannel current (I^KATP) was measured in the whole cell configuration of the patch clamp technique, using an EPC-7 patch clamp amplifier (List, Darmstadt-Eberstadt, Germany) and Digidata 1322A interface (Axon Instruments, Foster City, CA). pClamp8 software (Axon Instruments) was used for data acquisition and analysis. Patch pipettes with resistances ranging from 2 to 3 MΩ were pulled from borosilicate glass (Garner Glass, Claremont, CA) using a programmable micropipette puller (Sachs-Flaminh PC-84; Sutter Instruments, Novato, CA). Pipette tips were heat polished using a microforge MF-83 (Narishige, Tokyo, Japan). The cells suspended in Tyrode solution were placed in the recording chamber on the stage of an inverted microscope (Diaphot 300; Nikon, Tokyo, Japan). Only quiescent, rod-shaped cells with distinct striations were selected for experiments.

After a gigaohm seal was formed and the whole cell configuration was established by membrane rupture, the series resistance was adjusted to give the fastest possible capacitance transient without causing ringing. Whole cell I^KATPwas monitored during a 100-ms depolarizing voltage step to 0 mV applied every 15 s from a holding potential of −40 mV. Current signal was sampled at 10 kHz and low-pass filtered at a cutoff frequency of 3 kHz. Current amplitude was measured at the end of the voltage step. To allow for comparisons among cells, currents were normalized to cell capacitance and reported as current density (pA/pF).

Left ventricular tissue samples of guinea pigs hearts obtained from the control and the anesthetic preconditioned groups were snap-frozen in liquid nitrogen and stored at −70°C. Cryosections (10 μm) of the tissue were mounted on positively charged microscope slides. Sections were fixed for 10 min in 100% acetone at −20°C and rinsed with phosphate-buffered saline (PBS). Subsequently, the sections were incubated with 1:50 dilutions of rabbit polyclonal primary antibodies in PBS against PKC-ε and PKC-δ (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 37°C. The slides were then washed three times for 5 min in PBS, and the sections were incubated with a 1:1,000 dilution of anti-rabbit biotin conjugated antibody (Santa Cruz Biotechnology) in PBS for 30 min at 37°C. The sections were then washed again for three times for 5 min in PBS at room temperature. To visualize the protein expression of PKC-ε and PKC-δ, each section was incubated in 10 μg/ml labeled fluorescein isothiocyanate (Pierce, Rockford, IL) at 37°C for 15 min. Nuclear staining was achieved with 1 mm To-Pro-3 (Molecular Probes, Eugene, OR) at 37°C for 3 min. The protein expression of each section was incubated in 10 μg/ml labeled fluorescein isothiocyanate at 37°C for 15 min. Images were obtained at a magnification of 400× using a laser fluorescence imaging system and a confocal microscope (Nikon). A krypton–argon laser was used for excitation at wavelengths of 488 and 633 nm. Emitted fluorescence was determined after long-pass filtering at corresponding wavelengths of 520 and 661 nm for fluorescein isothiocyanate and To-Pro 3, respectively.

Data were analyzed using the following software: pClamp8 (Axon Instruments), Origin 6 (Originlab, Northampton, MA), and StatView version 5.0 (SAS Institute, Cary, NC). Results are reported as mean ± SD. Statistical analysis was performed using the Kruskal-Wallis test with Scheffé methods. Differences were considered significant at P < 0.05.

As previously reported, under whole cell conditions where the intracellular ATP concentration was set nominally at 0.5 mm ATP, isoflurane alone was unable to elicit sarcK^ATPchannel activity.26This is demonstrated in figure 1A. Approximately 25 min of control recordings were obtained to allow for the diffusional exchange of the pipette solution with the intracellular milieu. The subsequent exposure to isoflurane did not result in the activation of an outward current. Pinacidil (20 μm) was added to the bath solution at the end of the recording period to confirm the availability of functional sarcK^ATPchannels.

In contrast, pretreatment with a specific PKC-ε agonist peptide PP106 resulted in the opening of the sarcK^ATPchannel by isoflurane. A representative time-course experiment is demonstrated in figure 1B. PP106 (200 nm) was applied in the bath solution for 10 min before exposing the ventricular myocyte to isoflurane. In the presence of isoflurane (0.88 mm), a time-independent outward current was then elicited. The effect of isoflurane was sustained despite washout of the volatile anesthetic. The inhibition of the current by glibenclamide (0.5 μm) confirmed it as I^KATP. The mean I^KATPdensity in the presence of isoflurane after pretreatment with PP106 was 40.4 ± 18.2 pA/pF (n = 7).

Because the PKC-ε agonist peptide seemed to prime the opening of the sarcK^ATPchannel by isoflurane, whether the isoflurane-activated I^KATPamplitude was dependent on the concentration of the peptide was tested. Using the experimental protocol described above, 100 nm PP106 resulted in a markedly smaller I^KATPin the presence of isoflurane. The mean isoflurane-activated K^ATPcurrent density was 6.5 ± 6.0 pA/pF (n = 7), which was significantly smaller than the one obtained from the pretreatment with the higher concentration of the PKC-ε agonist peptide. Therefore, these results showed a concentration-dependent effect of PKC-ε translocation on the modulation of the sarcK^ATPchannel by isoflurane.

To confirm that the observed effects of PP106 were due to the translocation of PKC-ε, a specific PKC-ε translocation inhibitor peptide, PP93 (200 nm), was applied before exposing the myocyte to PP106. The result is depicted in figure 2A. The time-course experiment demonstrates that the inhibitor peptide abolished the effects of the agonist peptide, whereby isoflurane was unable to elicit I^KATP. Subsequent activation of I^KATPby pinacidil confirmed the functional availability of the sarcK^ATPchannels. Similar results were obtained in n = 7 cells. In addition, a scrambled peptide of the PKC-ε agonist, PP105, was used to further confirm the observed effects of PKC-ε. As shown in figure 2B, pretreatment with PP105 did not elicit I^KATPin the presence of isoflurane. Similar observations were made in n = 7 cells.

In addition to the PKC-ε isozyme, PKC-δ has also been implicated in the mechanism underlying preconditioning of the myocardium. Therefore, the effect of PKC-δ on the modulation of the sarcK^ATPchannel by isoflurane was also investigated. Similar to the experiments described above, a specific translocation activator peptide of PKC-δ, PP114, was used. As depicted in figure 3A, PP114 (200 nm) was applied extracellularly for approximately 10 min before exposure to 0.88 mm isoflurane. As shown in the figure, pretreatment with the PKC-δ activator did not elicit I^KATPin the presence of isoflurane. This was in contrast to the activation of I^KATPobserved in figure 1Bafter pretreatment with the PKC-ε activator. In n = 8 cells tested, 6 did not result in the activation of I^KATP. However, in the remaining two cells, I^KATPactivation was observed (data not shown). Due to the activation of I^KATPin two of the eight cells, the mean I^KATPdensity was 12.5 ± 22.1 pA/pF, which was significantly different from zero current (fig. 3B).

A higher concentration of PP114 was also tested. When cells were pretreated with 400 nm PP114, I^KATPwas elicited by isoflurane in four out of seven cells. Despite the observation that the higher concentration of PP114 resulted in a relatively more consistent activation of I^KATPin the presence of isoflurane, the mean I^KATPdensities after pretreatment with 200 or 400 nm PP114 were not significantly different (fig. 3B). With 400 nm PP114, I^KATPdensity was 5.7 ± 4.1 pA/pF. However, I^KATPdensities after the pretreatment with either 200 or 400 nm PP114 were significantly less than those recorded after the pretreatment with 200 nm PP106, the PKC-ε activator peptide. Furthermore, to confirm that the observed effects were due to the translocation of the PKC-δ isoform, the effects of a specific PKC-δ inhibitor peptide, PP101, were determined. Pretreatment with PP101 (200 nm) before the application of the PP114 activator (400 nm) did not elicit I^KATPactivation by isoflurane in n = 7 cells (data not shown).

Recent reports provide strong evidence that the mitoK^ATPchannel plays a pivotal role during the initial phases of cardioprotection by IPC and APC. The mitoK^ATPchannel is also modulated by PKC.27Consequently, the potential role of the mitoK^ATPchannel in the priming effect of PKC-ε on the sarcK^ATPchannel was investigated. 5-HD (200 μm), the putative blocker of the mitoK^ATPchannel, was added before the pretreatment of the cells with the PKC-ε activator PP106 (200 nm) and was subsequently present throughout the course of the experiment. As demonstrated in figure 4A, in the presence of 5-HD, the isoflurane-activated I^KATPdensity of 4.8 ± 2.9 pA/pF (n = 7) after pretreatment with PP106 was markedly smaller than that observed in the absence of 5-HD. Figure 4Bsummarizes the effects of the PKC-ε activator on the isoflurane-activated I^KATPin the absence and presence of 5-HD.

In the patch clamp experiments described above, specific PKC-ε and PKC-δ activators were used to induce the translocation of the isozymes to their respective anchoring proteins localized in the membrane. To assess the endogenous expressions of PKC-ε and PKC-δ, immunohistochemical analysis was performed. In these experiments, hearts were obtained from control guinea pigs and those that were previously exposed to 1 MAC isoflurane for 30 min to simulate an anesthetic-preconditioning period. Thoracotomy was performed to extract the heart tissues. For the guinea pigs that underwent APC, thoracotomy was performed after a 30-min recovery period after isoflurane exposure. The results are summarized in figure 5. After the APC protocol, the translocation of PKC-ε was localized to both the mitochondrial and sarcolemmal membranes, although translocation to the nuclei was also likely. In contrast, the translocation of PKC-δ was localized to the mitochondria but not the sarcolemma after APC. Both isozymes of PKC were detected in the cytosol in the control group.

A novel finding from this study is that a specific isozyme, PKC-ε, primed the sarcK^ATPchannel to open by isoflurane. The effect of PKC-ε on the sarcK^ATPchannel was concentration dependent. PKC-δ was significantly less effective in priming the sarcK^ATPchannel. Although studies have demonstrated that PKC activation by phorbol esters can increase channel activity28or prime the channel for activation,22isozyme-specific effects on the sarcK^ATPchannel have not been previously demonstrated. The results from this study demonstrate that the translocation of PKC-ε to the sarcolemma would likely result in the anchoring of the isozyme near its substrate and facilitate the modulation of its target protein, e.g. , the sarcK^ATPchannel. The difference in the effectiveness of PKC-ε and PKC-δ to prime the sarcK^ATPchannel was likely not due to differential expressions of the endogenous isozymes, because both were detected in the cytosol in the control group, as assayed by immunohistochemistry. In addition, the immunohistochemical analysis showed that PKC-ε but not PKC-δ translocated to the sarcolemma in the APC group, which supports the findings from the patch clamp experiments that PKC-ε, more so than PKC-δ, plays a greater role in the priming of the sarcK^ATPchannel to isoflurane.

A study by Uecker et al.  20showed that in rat hearts, PKC-ε translocated to the nuclei, sarcolemma, and intercalated disks, whereas PKC-δ translocated to the nuclei and mitochondria after preconditioning with isoflurane. However, a recent study by Ludwig et al.  29reported that in rat hearts, isoflurane induced translocation of PKC-ε and PKC-δ to the mitochondrial and sarcolemmal membranes, respectively. Furthermore, in human atrial tissue, preconditioning with sevoflurane resulted in the translocation of PKC-ε to the mitochondria, nuclei, and intercalated disks and PKC-δ to the sarcolemma.30Our results from the guinea pig hearts demonstrated that isoflurane translocated PKC-ε to the sarcolemma and mitochondria and PKC-δ was translocated to the mitochondria.

Currently, it is evident that translocations of PKC are pivotal steps involved in cardioprotection by preconditioning.31The PKC family contains 12 distinct isozymes, which are classified into three subfamilies: conventional (α, β¹, β², γ), novel (δ, ε, η, θ), and atypical (λ, ι, ζ, μ). Among these, PKC-δ and PKC-ε seem to play critical roles in the signaling cascade underlying preconditioning.19,20,32Because the types and levels of PKC isozyme expression vary among species,31reports on translocated PKC isozymes in preconditioning have been inconsistent.32–35Furthermore, the different types of preconditioning, pharmacologic35,36and ischemic,33,35,36may contribute to differential roles for the various PKC isozymes. For example, studies on simulated ischemia in isolated myocytes confirm a pivotal role for PKC-ε in ischemic preconditioning whereby a selective peptide inhibitor of the PKC-ε isozyme, εV1-7, abolished preconditioning.37Activation of PKC-ε that leads to cardioprotection has also been shown to couple to recruitment of PKC-ε–associated proteins.17,38In isolated perfused rat hearts, inhibition of PKC-δ prevented reperfusion injury and activation of PKC-ε mimicked ischemic preconditioning.18In opioid-induced preconditioning, activation of PKC-δ was shown to be essential.39In contrast, PKC-δ was found to be detrimental, whereas PKC-ε activation was required for ethanol-induced cardioprotection.36 

For volatile anesthetic–induced preconditioning, the translocation of specific PKC isozymes may be dependent on the animal model and the choice of anesthetic agent. Inhibition of PKC-δ by rottlerin, a specific blocker, abolished APC in an isolated rat heart model of isoflurane-induced preconditioning.20In this model, IPC was also inhibited by rottlerin. However, in isolated guinea pig hearts, a PKC-ε inhibitor peptide abolished sevoflurane-induced preconditioning. In contrast, a PKC-δ inhibitor peptide had no effect.19 

The importance of the mitoK^ATPchannel in APC and IPC has been demonstrated in numerous studies, but the role of the sarcK^ATPchannel has not been clearly defined. A recent study showed that the efficacy of IPC was significantly attenuated in knockout mice deficient in the gene encoding the pore-forming region of the sarcK^ATPchannel, Kir6.2.14Functional mitoK^ATPchannel activity was assessed by monitoring flavoprotein fluorescence in the knockout mice. Therefore, despite functional mitoK^ATPchannels, the absence of the sarcK^ATPchannel diminished the cardioprotective effects of IPC. These results were also supported by a study using transgenic mice expressing a mutant Kir6.2 with diminished ATP sensitivity.40Hearts from the transgenics did not exhibit postischemic recovery after IPC. A more recent study using Kir6.2-knockout mice demonstrated that diazoxide did not improve functional recovery in these mice.41Moreover, the cardioprotective effects of diazoxide on the wild-type mice were abolished by HMR 1098, suggesting that the sarcK^ATPchannels were involved.41Other studies have demonstrated that the opening of the mitoK^ATPmore so than the sarcK^ATPchannel plays a prominent role during preconditioning of isolated human right atria by sevoflurane.42However, in that study, block of the sarcK^ATPchannel by a putative blocker, HMR1098, significantly attenuated the recovery force at the end of a 60-min reoxygenation period.42 

In some cases, preconditioning may require both mitoK^ATPand sarcK^ATPchannels. The protective effects of desflurane were found to involve both the mitoK^ATPand sarcK^ATPchannels because either 5-HD or HMR1098 abolished the preconditioning effects of the anesthetic in anesthetized dogs.13These channels may also have separate, distinct roles in preconditioning. During adenosine-enhanced ischemic preconditioning, the mitoK^ATPchannel reduced infarct size during ischemia, whereas the sarcK^ATPchannel modulated functional recovery during ischemia and reperfusion.43Similar roles for the K^ATPchannels were reported in an hypoxia/reoxygenation model using isolated myocytes.21In a recent study on the delayed-protection by isoflurane, 5-HD and HMR 1098 partially attenuated the anesthetic-induced cardioprotective effects.44However, a combination of both 5-HD and HMR 1098 abolished the isoflurane-induced cardioprotection, suggesting that both mitoK^ATPand sarcK^ATPchannels are involved in the delayed protection by isoflurane.44 

The results obtained in our study support the hypothesis that both the mitoK^ATPand sarcK^ATPchannels are involved in APC in the guinea pig model. The modulation of the sarcK^ATPchannel may occur subsequent to changes in the signaling cascade triggered by mitoK^ATPchannel opening. In isoflurane-induced preconditioning, the opening of the mitoK^ATPchannel was found to trigger the cardioprotective signaling cascade by generating reactive oxygen species.45Downstream of the generated reactive oxygen species, PKC activation, in particular PKC-ε, has been shown to occur in sevoflurane-induced preconditioning.19Based on our results, the priming of the sarcK^ATPchannel by PKC-ε, following the opening of the mitoK^ATPchannel, would sensitize this channel to isoflurane. Other intracellular pathways linked to cardioprotection have also been shown to modulate the sarcK^ATPchannel. For example, the protein tyrosine kinase and phosphatase signaling pathway can modulate the sensitivity of the sarcK^ATPchannel to isoflurane.46Therefore, activation of the mitoK^ATPchannel, particularly during the initial phase of preconditioning, seems to be a critical step in triggering the signaling cascade that leads to cardioprotection. Although the role of the sarcK^ATPchannel has not been defined, it may contribute as an effector, whereby the channel is modulated by the signaling components activated by the opening of the mitoK^ATPchannel. In particular, as discussed above, the opening of the sarcKATP channel during reperfusion after a prolonged ischemic period may facilitate functional recovery of the myocardium. One recent study demonstrated that opening of the sarcK^ATPchannel can modulate the Na–Ca exchanger, resulting in a decreased influx of Ca²⁺ions and attenuation of Ca²⁺overload.47 

An intriguing result from our study was the attenuation of the priming effects of PKC-ε on the sarcK^ATPchannel by 5-HD. This implied that the mitoK^ATPchannels might mediate the priming of the sarcK^ATPchannel. However, the contribution of the mitoK^ATPchannel is unclear. Under the whole cell recording conditions, the intracellular ATP concentration is set by the solution in the recording pipette, which in this case was nominally 0.5 mm ATP. Consequently, cross-talk between the mitochondria and sarcK^ATPchannel, whereby ATP consumption by uncoupled mitochondria leads to opening of the sarcK^ATPchannel, is unlikely to have occurred. One possibility, though, is that PKC-ε may lead to mitoK^ATPchannel activation,27resulting in the generation of reactive oxygen species that was sufficient enough to prime the sarcK^ATPchannel downstream. Another possibility is the inhibition of the sarcK^ATPchannel by 5-HD.48However, in control experiments, 5-HD did not affect I^KATPelicited by pinacidil in our studies (data not shown). Yet another possibility is that the effects of 5-HD may be due to its mitoK^ATPchannel-independent targets. 5-HD has recently been demonstrated to serve as a substrate for acyl-CoA (coenzyme A) synthetase, resulting in 5-hydroxydecanoyl–CoA, which then can act on other intracellular targets.49Long-chain acyl-CoA esters have been shown to increase sarcK^ATPchannel activity by reducing the sensitivity of the channel to ATP.49This scenario is unlikely based on our observations because in this case, 5-HD would have increased channel activity. Nevertheless, the observed effect of 5-HD suggests a possible coupling between the mitochondria and the sarcK^ATPchannel. Further experiments would be needed to elucidate the mechanism underlying the 5-HD effect on the priming of the sarcK^ATPchannel by PKC-ε.

We cannot exclude the possibility that a brief hypoxic condition induced during thoracotomy before extraction of cardiac tissues resulted in the translocation of specific PKC isozymes. In addition, the cell isolation procedure using collagenase may also activate PKC isozymes. However, despite these possibilities, the immunohistochemical data suggest that “additional” translocation of the PKC-ε isozyme to the mitochondria and sarcolemma occurred after the APC protocol. Furthermore, translocation of PKC-δ to the mitochondria was also detected after APC.

In summary, the results from this study show a priming effect of PKC-ε on the sarcK^ATPchannel. After pretreatment with a specific PKC-ε translocation activator, isoflurane elicited the opening of the sarcK^ATPchannel. The ability of PKC-δ to prime the sarcK^ATPchannel was significantly less than that of PKC-ε. Our results suggest that the modulation of the sarcK^ATPchannel by PKC-ε may occur downstream of mitoK^ATPchannel activation.