Translocation of Protein Kinase C Isoforms to Subcellular Targets in Ischemic and Anesthetic Preconditioning (original) (raw)

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Pain Medicine| July 2003

Marcus C. Schaub, M.D., Ph.D.;

Marcus C. Schaub, M.D., Ph.D.

§Professor of Pharmacology, Institute of Pharmacology and Toxicology, University of Zurich.

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Michael Zaugg, M.D., D.E.A.A.

‖Head of Cardiovascular Anesthesia Laboratory, Institute of Anesthesiology, University Hospital Zurich.

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Received from the Institute of Anesthesiology, University Hospital Zurich, Zurich, Switzerland.

Anesthesiology July 2003, Vol. 99, 138–147.

Background

Translocation of protein kinase C (PKC) to subcellular targets is a pivotal signaling step in ischemic preconditioning (IPC). However, to date, it is unknown whether PKC isoforms translocate in anesthetic preconditioning (APC).

Methods

The PKC blockers chelerythrine and rottlerin and the adenosine triphosphate-dependent potassium (K(ATP)) channel blockers HMR-1098 and 5-hydroxydecanoate were used to assess the role of PKC and K(ATP) channels in isolated perfused rat hearts subjected to IPC or APC (1.5 minimum alveolar concentration isoflurane) followed by 40 min of ischemia and 30 min of reperfusion. Immunohistochemical techniques were used to visualize PKC translocation after preconditioning. In addition, the phosphorylation status of PKC isoforms was assessed.

Results

Chelerythrine, rottlerin, and 5-hydroxydecanoate blocked IPC and APC with respect to functional recovery, albeit IPC at higher concentrations. HMR-1098 did not affect IPC or APC. PKCdelta and PKCepsilon translocated to nuclei in both IPC and APC, which was inhibited by chelerythrine and rottlerin. PKCdelta translocated to mitochondria but not to the sarcolemma, and PKCepsilon translocated to the sarcolemma and intercalated disks but not to mitochondria. Interestingly, PKCepsilon was accumulated at the intercalated disks in control and preconditioned hearts. Phosphorylation of PKCdelta on serine643 was increased in IPC and APC and blocked by chelerythrine and rottlerin, whereas phosphorylation of PKCdelta on threonine505 was increased only in IPC and not blocked by chelerythrine or rottlerin. PKCepsilon on serine729 did not change its phosphorylation status.

Conclusions

This study indicates that translocation of PKCdelta plays a pivotal role in IPC and APC and suggests that phosphorylation of PKCdelta on serine643 may be of particular relevance in transferring the APC stimulus to mitochondrial K(ATP) channels.

KEY signaling pathways in preconditioning elicited by volatile anesthetics were unraveled recently by means of specific blockers for individual signaling components. From these experimental studies, it became clear that anesthetic preconditioning (APC) and ischemic preconditioning (IPC) share many fundamental steps including activation of G protein–coupled receptors, 1,2multiple kinases, 3and adenosine triphosphate–dependent potassium channels (KATPchannels). 4One of the most prominent kinases associated with the preconditioned state is protein kinase C (PKC) with at least 11 isoforms. The class of novel PKCs including δ and ε isoforms appears to be of particular relevance in transferring the preconditioning stimulus to the end effectors. PKC acts as a signal amplifier and is translocated to subcellular targets in an isoform-specific and cytoskeleton-mediated manner, ultimately leading to phosphorylation and activation of the sarcolemmal KATP(sarcKATP) and the mitochondrial KATP(mitoKATP) channels. 5Consistent with a possible role of PKC translocation in APC is the notion that colchicine, which disrupts microtubules, prevents infarct size reduction in APC. 6

We recently demonstrated that in adult rat ventricular myocytes volatile anesthetics enhance and accelerate activation of mitoKATPchannels through multiple PKC-coupled signaling pathways. 7Using nonbeating cardiomyocytes in a cellular model for simulated ischemia, we further showed that cardioprotection by isoflurane and sevoflurane is dependent on PKC and mitoKATPbut not sarcKATPchannels. 7However, so far, it is not known whether volatile anesthetics modify the phosphorylation status of PKC or promote translocation of PKC isoforms to subcellular targets including the mitochondria, the sarcolemma, the intercalated disks, and the nuclei. The data collected in this investigation now provide evidence that isoform-specific PKC translocation is an integral component of the mitoKATPchannel–mediated protection against ischemia in APC.

Materials and Methods

This study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Zurich.

Isolated Perfused Rat Hearts

Hearts from decapitated and heparinized (500 U intraperitoneally) male Wistar rats (250 g) were quickly removed and perfused in a noncirculating Langendorff apparatus with Krebs–Henseleit buffer containing the following ingredients: 155 mm Na+, 5.6 mm K+, 138 mm Cl−, 2.1 mm Ca2+, 1.2 mm PO43−, 25 mm HCO3−, 0.56 mm Mg2+, 11 mm glucose, and 13 mm sucrose. The buffer was saturated with 95% O2/5% CO2(pH 7.4, 37°C). Hearts were perfused at a constant pressure of 80 mmHg. A water-filled balloon-tipped catheter was inserted into the left ventricle through the left atrium. Left ventricular end-diastolic pressure was adjusted to 3–7 mmHg during the initial equilibration, and the volume of the balloon was not changed thereafter. The distal end of the catheter was connected to a performance analyzer (Plugsys Modular System; Hugo Sachs, March-Hugstetten, Germany) by way of a pressure transducer. Perfusion pressure, epicardial electrocardiogram, and coronary flow (Transit Time Flowmeter type 700; Hugo Sachs) were simultaneously recorded on the same performance analyzer. All recorded data were digitized and processed on a personal computer using IsoHeart software (Hugo-Sachs). Hearts were allowed to beat spontaneously in all experiments.

APC was induced by administration of 15 min of isoflurane at 2.1% (vol/vol) (corresponding to 1.5 minimum alveolar concentration [MAC] in rats at 37°C) until 10 min before prolonged test ischemia. Buffer solution was equilibrated with isoflurane using an Isotec 3 vaporizer (Datex-Ohmeda, Tewksbury, MA) with an air bubbler. The delivered vapor concentration of isoflurane was continuously controlled by the infrared gas analyzer Capnomac Ultima (Datex-Ohmeda). The applied concentration of isoflurane was also measured in the buffer solution using a gas chromatograph (Perkin-Elmer, Norwalk, CT): isoflurane at 2.1% (vol/vol), 0.53 ± 0.03 mm. IPC was induced by three cycles of 5-min ischemic episodes interspersed by 5 min of reperfusion. In IPC and APC experiments with specific blockers, the blockers were administered from 3 min before until 3 min after the preconditioning stimulus (fig. 1). The concentrations of the blockers used in the final experiments were determined in separate experiments. The following blockers were used at the indicated concentrations: chelerythrine, 5 and 10 μm; rottlerin, 0.1 and 0.2 μm; HMR-1098, 30 and 60 μm; and 5-hydroxydecanoate, 200 and 500 μm. Hearts were subjected to 40 min of test ischemia and 30 min of reperfusion according to the protocols depicted in figure 1. For each experimental group, seven hearts were prepared, and functional parameters were recorded.

Fig. 1. Schematic diagram of the preconditioning protocols. Ischemic preconditioning (IPC ) was induced by three times 5 min of ischemia interspersed by 5 min of reperfusion applied 10 min before test ischemia. Isoflurane was administered (for 15 min) 10 min before test ischemia. IPC and anesthetic preconditioning (APC ) were bracketed with chelerythrine, rottlerin, HMR-1098, and 5-hydroxydecanoate. Short-term protocols were used to assess translocation of protein kinase C and the phosphorylation status after the administration of the preconditioning stimulus. Hatched boxes = ischemia, open boxes = 1.5 minimum alveolar concentration (MAC ) isoflurane, black boxes = blocker.

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Analyses of Myocardial Tissue Samples

Separate experiments were performed to assess the effects of the preconditioning stimulus on translocation of PKC isoforms to subcellular targets and on the phosphorylation status of PKCδ on serine643 and threonine505 and of PKCε on serine729. For this purpose, tissue was collected after the administration of the preconditioning stimulus (short protocols without test ischemia and reperfusion). Six hearts in each experimental group were prepared for immunohistochemical analyses, and five hearts in each experimental group were prepared for Western blot analyses. To evaluate the impact of the isolation procedure itself on the phosphorylation status of PKC and on the translocation of PKC isoforms, additional hearts (n = 6) were directly taken from the animals without prior perfusion on the Langendorff apparatus (CTLLD= control hearts with time-matched perfusion on the Langendorff apparatus; CTLAN= control hearts directly taken from the animals without prior perfusion).

Immunohistochemical Analysis

Translocation of PKC isoforms in response to preconditioning was assessed by immunofluorescence staining. 8Briefly, left ventricular tissue samples were placed in optimal cutting temperature embedding medium (Tissue-Tek; Sakura Finetek Inc., Torrance, CA), frozen in liquid nitrogen, and stored at −70°C. Cryosections (5 μm) were prepared with a cryostat (Cryo-star HM 560 M; Microtom, Kalamazoo, MI) and collected on slides precoated with gelatin. All sections were fixed for 10 min in acetone, 100%, at −20°C, rinsed with phosphate-buffered saline, and incubated in normal goat serum, 10%, for 30 min to block nonspecific binding. Sections were incubated for 1 h at room temperature with primary antibodies. Rabbit polyclonal antibodies to PKCδ and PKCε (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in phosphate-buffered saline (1:500) containing normal goat serum, 2%, were added. Antibodies to PKC were combined with mouse monoclonal α-prohibitin-1 antibody (Research Diagnostics, Flanders, NJ) (1:25) as a mitochondrial marker, guinea pig polyclonal m-dystrophin (DYS12) antibodies to the N-terminal portion of the molecule 9(1:1,000) as a sarcolemmal marker, and mouse monoclonal α-myomesin antibodies as a marker for cardiomyocytes (gift from Hans M. Eppenberger, Ph.D., Professor of Cell Biology, Department of Cell Biology, Swiss Institute of Technology Zurich, Zurich, Switzerland) (1:5). All sections were then washed with PBS two times for 5 min each and incubated for 1 h with a mixture of secondary antibodies conjugated to Alexa Fluor 555 goat α-rabbit, Alexa Fluor 488 goat α-mouse, or Alexa Fluor 488 goat α-guinea pig (Molecular Probes, Eugene, OR) (1:500) and, in some experiments, with 4′,6-diamino-2-phenylindole (10 ng/ml) (Sigma, St. Louis, MO) in phosphate-buffered saline at room temperature. After washing with phosphate-buffered saline, sections were protected with coverslips using DAKO mounting medium (DAKO Corporation, Carpinteria, CA).

Sections were analyzed by epifluorescence microscopy using an upright microscope (Axioplan 2; Zeiss, Jena, Germany) with appropriate filter blocks for the detection of fluorescein isothiocyanate, tetramethyl rodamine isothiocyanate, and ultraviolet fluorescence. In addition, confocal images were obtained with an LSM Pascal confocal microscope (Zeiss) using the appropriate laser lines and filter blocks (fluorescein isothiocyanate: excitation, 488 nm; emission, 525 nm; tetramethyl rodamine isothiocyanate: excitation, 540 nm; emission, 570 nm; ultraviolet: excitation, 360 nm; emission, 461 nm). Five randomly chosen fields (at ×400 magnification) of sections from samples from each group (including control hearts directly taken from animals [CTLAN] and untreated hearts taken after time-matched perfusion on the Langendorff apparatus [CTLLD]) were examined for translocation of PKCδ and PKCε to sarcolemma (colocalization with dystrophin) or to mitochondria (colocalization with prohibitin-1). Specific translocation was measured and quantified using Imaris/Colocalization Version 3.1.2 software (Bitplane Inc., Zurich, Switzerland). This software generates a map of locations at which multichannel images have a common event as defined by a signal of a given intensity found in two or more channels at the same spatial location. The number of pixels of these events was calculated and served to express the degree of colocalization as x-fold increase in the various groups as compared with the control groups. Moreover, confocal imaging was used to quantify PKCδ and PKCε translocation to nuclei of cardiomyocytes (colocalization with 4′,6-diamino-2-phenylindole). Five randomly chosen fields at ×400 magnification from sections of samples from the various groups that were collected immediately before test ischemia were analyzed for colocalization of PKCδ and PKCε with nuclei (4′,6-diamino-2-phenylindole staining). Double staining with myomesin was used to assure that only PKCδ and PKCε translocation to cardiomyocytes was counted. The number of PKCδ- and PKCε-positive nuclei was expressed as percentage of total nuclei of myocytes per field. Colocalization analyses of the samples were performed without prior knowledge of the treatment.

Western Blot Analysis

The tissue was crushed in liquid nitrogen, and lysis buffer, 1 m dithiothreitol solution, and protease inhibitor cocktail (Complete Mini protease inhibitor cocktail; Roche, Basle, Switzerland) were added. After homogenization and heating for 7 min at 100°C in a water bath, the samples were centrifuged for 5 min at 20,000 g , and the supernatants were used for Western blotting. Gel electrophoresis was performed on a 10% sodium dodecyl sulfate–polyacrylamide gel. The samples were electroblotted overnight at 4°C onto a nitrocellulose membrane (Osmotics Inc., Westborough, MA). Primary antibodies were diluted in nonfat dry milk, 5%, in Tris buffered saline and incubated for 2.5 h. The following antibodies were used: monoclonal mouse antibody to total actin (Chemicon, Temecula, CA); phospho-PKCε (serine729), polyclonal rabbit antibody (Upstate Biotechnology, Lake Placid, NJ); phospho-PKCδ (serine643), polyclonal rabbit antibody (Cell Signaling Technology, Beverly, MA); and phospho-PKCδ (threonine505), polyclonal rabbit antibody (Cell Signaling Technology). The membrane was washed in Tris buffered saline–Tween 20 and incubated in horseradish peroxidase–labeled secondary antibody (goat antibody to rabbit and goat antibody to mouse IgG-HRP; Perbio Science, Bonn, Germany) for 1 h. The membrane was then incubated with chemiluminescence substrate (Super Signal; Perbio Science) and exposed to an x-ray film (New RX; Fuji, Tokyo, Japan). Quantitative analysis of the band density was performed using simultaneously blotted actin density to correct for protein content (MCID; Imaging Inc., Fonthill, Ontario, Canada).

Statistical Analysis

Data are expressed as mean ± SD (or SEM for hemodynamic parameters). Functional parameters at identical time points were compared for the groups by unpaired t tests. Repeated-measures ANOVA was used to evaluate differences over time between groups for hemodynamic parameters. P values were multiplied by the number of comparisons that were made (Bonferroni correction), and corrected P < 0.05 was considered statistically significant. StatView Version 4.5 (Abacus Concepts, Berkeley, CA) was used for the statistical analysis.

Results

Chelerythrine and the PKCδ-specific Blocker Rottlerin Inhibit IPC and APC in Isolated Perfused Rat Hearts

Administration of isoflurane at 1.5 MAC over 15 min before prolonged test ischemia significantly improved postischemic recovery when compared with untreated hearts (fig. 2, A ; n = 7 for each group). Postischemic left ventricular developed pressure and coronary flow were increased, and left ventricular end-diastolic pressure was decreased in APC. Concomitant treatment with chelerythrine at 5 μm or rottlerin at 0.1 μm, bracketing isoflurane administration, abolished isoflurane-induced protection. To allow direct comparison with classic preconditioning elicited by ischemia (IPC), all experiments were also performed with ischemia (three times for 5 min) as the preconditioning stimulus. Similar to APC, IPC was inhibited by chelerythrine and rottlerin, albeit at higher concentrations (chelerythrine, 10 μm; rottlerin, 0.2 μm) (fig. 2, B ). Chelerythrine and rottlerin alone did not affect postischemic functional recovery (table 1). The results of these experiments indicate that activation of PKC and, in particular, of PKCδ plays a pivotal role in IPC and APC.

Fig. 2. Hemodynamic changes in hearts subjected to ischemia or reperfusion. The functional improvement by anesthetic preconditioning (APC ) and ischemic preconditioning (IPC ) was completely abolished by the protein kinase C (PKC ) inhibitors chelerythrine (CHE ) and rottlerin (ROT ) as well as by the mitochondrial KATP(mitoKATP) channel blocker 5-hydroxydecanoate (5HD), albeit at different concentrations. IPC and APC were not blocked by HMR-1098. Effects of chelerythrine and rottlerin in APC (A ) and IPC (B ). Effects of 5HD and HMR-1098 in APC (C ) and IPC (D ). Numbers behind individual blockers indicate concentrations in μm. Values are mean ± SEM (n = 7 for each group). For comparisons between groups during the treatment period and the effects of the blockers alone on postischemic functional improvement, see table 1. CTL = control, EDP = end-diastolic pressure, sarcKATP= sarcolemmal KATP.

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Table 1. Summary of Preischemic and Postischemic Hemodynamic Variables in the Various Treatment Groups

Data are mean ± SEM (n = 7 for each group); isoflurane concentration = 1.5 minimum alveolar concentration. Baseline was recorded at 10 min. Treatment was recorded after APC and IPC. Reperfusion was recorded after 30 min of reperfusion.

*P < 0.05 vs. CTL.

†P < 0.05 vs. ischemia.

‡P < 0.05 vs. APO.

§P < 0.05 vs. IPC.

APC = anesthetic preconditioning; CF = coronary flow; CHE = chelerythrine; CTL = control; DP = left ventricular developed pressure; EDP = left ventricular end-diastolic pressure; 5HD = 5-hydroxydecanoate; HMR = HMR-1098; HR = heart rate; IPC = ischemic preconditioning; ROT = rottlerin.

Table 1. Summary of Preischemic and Postischemic Hemodynamic Variables in the Various Treatment Groups

MitoKATPbut Not SarcKATPChannels Mediate IPC and APC in Isolated Perfused Rat Hearts

To evaluate whether isoflurane-induced preconditioning is mediated by mitoKATPor sarcKATPchannels, separate experiments were performed with 5-hydroxydecanoate, a mitoKATPchannel–specific blocker, and HMR-1098, a sarcKATPchannel–specific blocker. In both types of preconditioning, 5-hydroxydecanoate abolished isoflurane-induced postischemic functional improvements, whereas HMR-1098 did not affect IPC and APC at even high concentrations (60 μm) (fig. 2, C and D ; n = 7) (table 1). Interestingly, APC was inhibited at a lower concentration of 5-hydroxydecanoate (200 μm) than was IPC (500 μm), indicating a less strong preconditioning stimulation by APC than by IPC at the used concentration of isoflurane. 5-Hydroxydecanoate and HMR-1098 alone did not affect postischemic functional recovery (table 1).

PKCδ Is Translocated to Mitochondria but Not to the Sarcolemma in IPC and APC

To evaluate whether PKCδ is translocated to subcellular targets in response to IPC and APC, colocalization of PKCδ with prohibitin, a highly specific marker of mitochondria, and dystrophin, a marker of the sarcolemma, was determined in control hearts and preconditioned hearts. For these experiments, separate hearts were prepared (n = 6 for each group). No colocalization of PKCδ with prohibitin or dystrophin was observed in control hearts with time-matched perfusion on the Langendorff apparatus (CTLLD). The immunohistochemical analyses further revealed clear translocation of PKCδ to mitochondria (fig. 3, A and B ) but not to the sarcolemma (fig. 3, C ) in IPC and APC. Chelerythrine and rottlerin abolished this translocation. The results of these experiments visualize for the first time PKCδ translocation to mitochondria, the predominant site of mitoKATPchannel–induced protection against ischemia, in response to APC.

Fig. 3. Protein kinase C δ (PKCδ ) colocalization with subcellular targets in response to ischemic preconditioning (IPC ) and anesthetic preconditioning (APC ). (A and B ) There was clear translocation of PKCδ (red ) to mitochondria (green ) in IPC and APC (colocalization indicated in yellow ), which was inhibited by chelerythrine (CHE ) (IPC, 10 μm; APC, 5 μm) and rottlerin (ROT) (IPC, 0.2 μm; APC, 0.1 μm). (C ) No translocation of PKCδ (red ) to the sarcolemma (green ) was observed in both types of preconditioning. Note that there was no translocation of PKCδ to mitochondria and sarcolemma in control hearts (CTLLD= hearts with time-matched perfusion on the Langendorff apparatus). Pictures represent epifluorescence micrographs with ×400 magnification. *Significantly increased compared with CTLLD, P < 0.05. †Not significantly different from CTLLD.

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PKCε Is Translocated to the Sarcolemma but Not to Mitochondria in IPC and APC

PKCε markedly colocalized with dystrophin in IPC and APC, indicating translocation to the cell membrane (fig. 4, A and B ). Chelerythrine abolished this translocation in both IPC and APC. Moreover, the staining experiments also revealed constitutively translocated PKCε to the intercalated disks in control (including CTLLDand CTLAN) and preconditioned hearts (fig. 4). This clearly shows that PKCε translocation was present in hearts directly taken from the animals. In contrast to PKCδ, PKCε did not colocalize with prohibitin, indicating no translocation to mitochondria (fig. 4, C ).

Fig. 4. Protein kinase C ε (PKC ε) colocalization with subcellular targets in response to ischemic preconditioning (IPC ) and anesthetic preconditioning (APC ). (A and B ) There was clear translocation of PKCε (red ) to sarcolemma (green ) in IPC and APC (colocalization indicated in yellow ), which was inhibited by chelerythrine (CHE ) (IPC, 10 μm; APC, 5 μm). (C ) No translocation of PKCε (red ) to the mitochondria (green ) was observed in both types of preconditioning. There was constitutively translocated PKCε to the intercalated disks in control hearts (CTLLD= hearts with time-matched perfusion on the Langendorff apparatus and hearts directly taken from the animals without prior perfusion [CTLAN]). Pictures represent epifluorescence micrographs with ×400 magnification. *Significantly increased compared with CTLLDor CTLAN, P < 0.05. †Not significantly different from CTLLDor CTLAN.

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PKCδ and PKCε Are Translocated to the Nuclei in IPC and APC

To further evaluate whether IPC and APC induce translocation of PKC to nuclei of cardiomyocytes, additional staining experiments were performed with the DNA-specific 4′,6-diamino-2-phenylindole (n = 6 for each group). Concomitant staining with myomesin was used to assure that only translocation of PKC isoforms to nuclei of cardiomyocytes was counted. These experiments allowed quantification of PKC translocation by counting the percentage of PKC-positive nuclei in the various experimental groups. IPC and APC translocated PKCδ and PKCε to nuclei, albeit IPC exhibited more pronounced translocation than APC. The observed translocations were abolished in the presence of chelerythrine (fig. 5).

Fig. 5. Translocation of protein kinase C δ (PKCδ ) and protein kinase C ε (PKC ε) to nuclei of cardiomyocytes in response to ischemic preconditioning (IPC ) and anesthetic preconditioning (APC ) in the presence or absence of chelerythrine (CHE ) (IPC, 10 μm; APC, 5 μm). Double staining with myomesin (green ) was used to ensure that only PKCδ or PKCε (red ) translocation to cardiomyocytes was counted. The number of positive nuclei was expressed as the percentage of total nuclei of myocytes per field. (A,B ) Percentages of positive nuclei in the various groups. (C ) Representative PKCε-positive nuclei after APC. (D ) Same image with concomitant myomesin staining (green ). Data are mean ± SD. *Significantly increased compared with control (CTLLD= hearts with time-matched perfusion), P < 0.05. †Not significantly different from CTLLD.

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IPC and APC Elicit a Distinctive Phosphorylation Status in PKC Isoforms

To explore the phosphorylation status of PKC isoforms in IPC and APC, key phosphorylation sites of PKCδ on serine643 and threonine505 and of PKCε on serine729 were indexed on the basis of Western blot analyses in the presence and absence of chelerythrine and rottlerin (n = 5 for each group). IPC and APC increased phosphorylation of PKCδ but not phosphorylation of PKCε (fig. 6). Although IPC increased phosphorylation on serine643 and threonine505, APC increased phosphorylation exclusively on serine643. Chelerythrine or rottlerin did not block IPC-induced phosphorylation on threonine505. To further test whether the isolation procedure and subsequent perfusion on the Langendorff apparatus affected the phosphorylation status of PKC isoforms, separate experiments were performed with hearts directly taken from the animals (n = 5). PKCδ on threonine505 and PKCε on serine729 exhibited the same degree of phosphorylation in these hearts, indicating a negligible impact of the isolation procedure. In addition, although phosphorylation of PKCδ on serine643 was increased by instrumentation, IPC and APC further markedly enhanced this phosphorylation, which was inhibited by chelerythrine and rottlerin.

Fig. 6. Phosphorylation status of protein kinase C (PKC ) isoforms was determined after ischemic preconditioning (IPC ) and anesthetic preconditioning (APC ) in the presence or absence of chelerythrine (CHE ) (IPC, 10 μm; APC, 5 μm) or rottlerin (ROT ) (IPC, 0.2 μm; APC, 0.1 μm) using Western blot analysis. (A ) Phosphorylation of PKCδ on threonine505. (B ) Phosphorylation of PKCδ on serine643. (C ) Phosphorylation of PKCε on serine729. Data are mean ± SD. *Significantly increased or decreased compared with control (CTLLD= hearts with time-matched perfusion), P < 0.05. Scale on ordinate denotes relative change compared with CTLLD. CTLAN= hearts directly taken from the animals.

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These data suggest that phosphorylation of PKCδ on serine643 may be important in transferring the preconditioning stimulus to the end effectors. In addition, these results provide the first evidence of distinctive differences in the phosphorylation status of PKCδ between IPC and APC.

Discussion

The principal new findings of this study are as follows. Isoflurane-induced preconditioning, similar to IPC, is dependent on PKC activity and is predominantly mediated by mitoKATPchannels in isolated perfused rat hearts. Blockade of isoflurane-induced postischemic functional improvement by rottlerin, a PKC inhibitor with a 30-fold higher specificity for PKCδ, supports the pivotal role of this isoform in APC in the rat heart model. This study further visualized for the first time PKC isoform–specific translocation to subcellular targets, i.e. , mitochondria, the sarcolemma, intercalated disks, and the nuclei, in response to isoflurane administration. Translocation of PKC isoforms was previously reported for ischemic 10and pharmacologic 11preconditioning. However, to date, no study has evaluated the effect of volatile anesthetics on this key signaling step in preconditioning. Importantly, although PKCδ and PKCε translocated to nuclei and PKCε also translocated to the sarcolemma, there was specific translocation of PKCδ to mitochondria, the predominant site of protection against ischemia. 5,12Finally, distinctive differences in the phosphorylation status of PKCδ were observed between IPC and APC, suggesting that phosphorylation of PKCδ at serine643 may be of particular relevance in transferring the APC stimulus to the end effectors. Collectively, the results of these experiments confirm and extend our previous findings in isolated adult rat ventricular myocytes.

Previous studies have shown that PKC is an important signal amplifier of G protein–linked receptors in preconditioning and that intact PKC activity is a prerequisite in many animal models for the protection afforded by preconditioning. Accordingly, blockade or down-regulation of PKC activity annihilates diazoxide- and ischemia-induced preconditioning. 11,13,14Using a Langendorff rabbit model, Cope et al. 15showed that chelerythrine inhibited the preconditioning effect by halothane. Similarly, high doses of bisindolylmaleimide, a less specific PKC inhibitor, blocked isoflurane-enhanced recovery of canine stunned myocardium. 3Isoflurane and halothane are known to affect PKC activity. PKC-induced coronary vasoconstriction is inhibited by halothane but enhanced by isoflurane. 16Another study indicated that neither isoflurane nor halothane inhibited PKC-induced alterations in coronary vascular tone. 17Conversely, inhibitory effects of both isoflurane and halothane were reported in PKC-induced hepatic vascular tone. 18These controversial results need further investigation. The precise mechanisms by which volatile anesthetics activate KATPchannels, the putative end effectors of ischemic and pharmacologic preconditioning, 5are unknown. However, volatile anesthetics, similar to phorbol esters, 19selectively activate PKC and subsequently prime sarcKATP20as well as mitoKATP7channels in cardiomyocytes, probably by increasing their phosphorylation status. Primed, i.e. , phosphorylated, KATPchannels would then allow earlier and more intense opening at the initiation of ischemia. 21The results of our experiments clearly confirm the pivotal role of PKC in APC at the whole organ level.

Translocation of PKC isoforms has been implicated in mechanisms involved in heart failure, 22myocardial hypertrophy, 23and preconditioning. PKC isoforms are activated by phosphorylating enzymes such as G proteins and are modified in enzyme activity by phospholipids, diacylglycerol, increased Ca2+, nitric oxide, and superoxide anions. This is followed by translocation in an isoform-specific and cytoskeleton-mediated manner to subcellular targets, which can be directly visualized by immunohistochemical methods. Only 10 min of ischemia 24or brief administrations of pharmacologic agents 11,25may elicit significant PKC translocation. Recent evidence indicates that translocation is dependent on PKC binding to a family of proteins called receptors of activated C kinase. 26These anchoring proteins are highly specific, and each PKC isoenzyme can bind to only one receptor of activated C kinase. Thus, different PKC isoforms may be linked to distinctive aspects of myocardial function, and this functional segregation may be mediated by the localization of the isoform-specific receptors of activated C kinase on defined subcellular structures. Notably, translocation of PKC isoforms is highly species-dependent and also determined by the type of preconditioning, as evidenced by a characteristic translocation of PKC isoforms in Ca2+, 8pharmacologic, 11or the various protocols of ischemic preconditioning. 14,27,28The results of our experiments confirm for the first time that PKC translocation, indeed, occurs in APC and, in particular, that translocation of the δ isoform is important in IPC and APC in the isolated perfused rat heart model. The latter observation is supported by the fact that the δ isoform exclusively translocated to the mitochondria, the main site of protection, while PKCε did not. Although mitoKATPchannel activity was not directly measured, the results of this study suggest that PKC translocation is essential for anesthetic-induced activation of mitoKATPchannels, which was previously reported for IPC. 5Since PKCε translocation to the sarcolemma was observed in APC and IPC, some role of PKCε and its putative target, the sarcKATPchannel, cannot be totally dismissed in APC and IPC. However, isoflurane effects were completely blocked in our experiments by 5-hydroxydecanoate, a specific blocker for the mitoKATPchannel. In accordance, previous studies demonstrated that shortening of the action potential by opening of sarcKATPchannels was not responsible for the protection by IPC 29and that APC exerts protection in nonbeating cardiomyocytes 7and under cardioplegic arrest. 30

Although we observed constitutively active translocated PKCε to the intercalated disks in control hearts, PKC activity in intercalated disks appeared to be further enhanced by IPC and APC. 31The translocation of PKCε to intercalated disks and gap junctions (mainly connexin 43) in myocytes may modulate intercellular communication and be involved in the regulation of ischemia-induced necrotic and apoptotic cell death. 32It is suggested that in preconditioned hearts PKC and other kinases phosphorylate connexin 43, leading to a decreased intercellular communication. Conversely, in nonpreconditioned hearts, increased Na+flux through gap junctions, specifically at the initiation of reperfusion, leads to propagation of hypercontracture to adjacent myocytes and cell death. Taken together, modified chemical communication through gap junctions by IPC and APC could significantly decrease myocardial injury. Moreover, isoflurane-induced PKCδ and PKCε translocation to nuclei points to a possible role of altered gene expression by APC similar to IPC and suggests that these isoforms may initiate transcriptional changes, as previously described in late preconditioning. 33However, preliminary data in a dog model reject the existence of a late phase or “second window” of protection in APC. 34

PKC isoforms are synthesized as inactive precursors and require stepwise phosphorylation for activation either by upstream kinases or by autophosphorylation. 35At least three phosphorylation sites and multiple intramolecular phosphorylations of neighboring amino acids are involved in the complex mechanisms of isoenzyme-selective and stimulation-specific regulation of PKCs. On the basis of site-directed mutagenesis experiments, key residues of catalytic and regulatory domains that define PKC activity were previously identified. 36,37Accordingly, PKCα requires phosphorylation on threonine497 and PKCβII requires phosphorylation on threonine500 to become functional enzymes. 35Conversely, PKCδ on threonine505, the corresponding site in the δ isoform, does not appear to be essential for a catalytically competent PKC conformation. 36This is in accordance with our observation that phosphorylation of threonine505 in PKCδ was not a prerequisite for effective preconditioning by isoflurane. Consistent with previous reports, we detected the threonine505-phosphorylated PKCδ at a molecular weight of 60 kDa instead of 78 kDa. Proteolytic degradation following activation is a common feature of many PKC isoforms. 35,38In contrast to phosphorylation on threonine505, phosphorylation on serine643 was obligatory for effective IPC and APC and may therefore be essential to establish the preconditioned state. 37No alteration in the phosphorylation state by IPC or APC was observed in PKCε on serine729. However, this does not exclude changes in the phosphorylation state on other sites in PKCε but is consistent with a high degree of phosphorylation in constitutively active translocated PKCε, as observed in control hearts. 31

Altered PKC activity may be in part responsible for the loss of protection afforded by preconditioning in aged and diseased myocardium. 27In the aged heart, PKC isoforms are insufficiently translocated in response to IPC. Whether this applies also to APC is not clear yet. However, a recent clinical study of patients undergoing percutaneous transluminal angioplasty that compared IPC in younger and elderly patients suggested that IPC is attenuated in the aged myocardium most probably due to inhibitory effects upstream of the mitoKATPchannel. 39Similar mechanisms may be operative in diabetic and remodeled myocardium. Results for muscle slices of human right atrial appendages from patients with left ventricular ejection fractions of less than 30% or diabetes indicated that failing and diabetic human myocardium is much less amenable to IPC and that the mechanism for this phenomenon may lay in elements of the signal transduction pathways. 40In the current study, a standard Krebs–Henseleit solution containing 11 mm glucose and 13 mm sucrose was used. Recently, high glucose concentrations (30 mm) were shown to inhibit the protective effects of diazoxide-induced preconditioning in an in vivo dog model of regional ischemia. 41However, these inhibitory effects could be offset by isoflurane concentrations of greater than 1 MAC. 42

The following specific comments should be added. First, we used immunohistochemical methods to evaluate translocation of PKC isoforms. Immunohistochemical analysis allows the determination of colocalization of PKC with subcellular targets selectively within cardiomyocytes. Conversely, PKC levels in subcellular fractionations from whole tissue samples do not differentiate between individual cell types, i.e. , fibroblasts, endothelial cells, or myocytes. Considering that approximately 70% of all cardiac cells are not cardiomyocytes, the chosen experimental approach bears an important methodologic advantage. Second, no infarct size was determined in the various blocker experiments. However, unlike in other species, functional recovery and cell damage are closely correlated in preconditioning experiments with the isolated perfused rat heart model. 43Third, 5-hydroxydecanoate was recently reported to have other intracellular targets than mitoKATPchannels. 44However, the significance of these findings with respect to cardioprotection needs further investigation. Fourth, although we have unraveled PKCδ and PKCε activation as an integral component in IPC and APC, we cannot exclude the involvement of additional PKC isoforms or other kinases in APC. It may well be that the complex signaling cascade of APC involves more than one single kinase. Some studies propose activation of extracellular signal-regulated kinase, c-Jun-N-terminal kinase, p38-mitogen–activated protein kinase, or tyrosine kinases as additional candidate members of key protein kinases in preconditioning. 45The concept that alternate and additional signaling pathways may be recruited depending on the preconditioning stimulus studied is supported by our observation that IPC and APC were abolished at different concentrations of the specific blockers, suggesting that the two types of preconditioning may not lead to strictly comparable activation of the various signaling pathways.

In summary, this is the first study to demonstrate a PKC isoform–specific translocation in APC. PKCδ was translocated to mitochondria in response to isoflurane treatment, suggesting the importance of this isoform in mitoKATPchannel–mediated cardiac protection by isoflurane. Our data further provide the first evidence of distinctive differences in the phosphorylation status of PKCδ between IPC and APC.

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Translocation of Protein Kinase C Isoforms to Subcellular Targets in Ischemic and Anesthetic Preconditioning (original) (raw)