Propofol-induced Activation of Protein Kinase C Isoforms in Adult Rat Ventricular Myocytes

All experimental procedures and protocols were approved by The Cleveland Clinic Foundation Institutional Animal Care and Use Committee (Cleveland, Ohio).

Freshly isolated adult ventricular myocytes from rat hearts were obtained as previously described.16,17Immediately after the rats were killed, the hearts were rapidly removed and perfused in a retrograde manner at a constant flow rate (8 ml/min) with oxygenated (95% oxygen–5% carbon dioxide) Krebs-Henseleit buffer (37°C) containing the following: 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl², 1.2 mm KH²PO⁴, 1.2 mm CaCl², 37.5 mm NaHCO³, and 16.5 mm dextrose, pH 7.35. After a 5-min equilibration period, the perfusion buffer was changed to a Ca²⁺-free Krebs-Henseleit buffer containing collagenase type II (309 U/ml). After digestion with collagenase (20 min), the ventricles were minced and shaken in Krebs-Henseleit buffer, and the resulting cellular digest was washed, filtered, and resuspended in phosphate-free HEPES-buffered saline (23°C) containing the following: 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl², 1.25 mm CaCl², 11.0 mm dextrose, 25.0 mm HEPES, and 5.0 mm pyruvate, pH 7.35.

Fractionation of cardiac myocytes was performed as previously reported with slight modifications.16After treatment with the interventions, cardiomyocytes were quickly pelleted by centrifugation (45 s at 800g ) and washed with ice-cold HEPES-buffered saline. Myocytes were resuspended in relaxing solution (4 mm MgATP, 100 mm KCl, 10 mm imidazole, 2 mm EGTA, 1 mm MgCl², 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 10 mm benzamidine, and 0.01 mm leupeptin) containing 20% glycerol and 0.05% Triton X-100. Cell permeabilization and membrane solubilization were facilitated by sonication for 5 min on ice. After centrifugation at 800g  for 2 min, a nuclear, mitochondrial, and undigested cellular pellet was isolated. The supernatant was collected and labeled as SUP 1 (supernatant). The pellet was resuspended in the same relaxing solution containing 20% glycerol and 0.05% Triton X-100 and sonicated for 5 min on ice. The supernatant was collected and added to SUP1. SUP1 was centrifuged at 100,000g  for 30 min at 4°C, producing a pellet (designated as the total cellular membrane fraction) and a supernatant (designated as the cytosolic fraction).

After treatment with propofol, cardiomyocytes ere centrifuged at 1,000g  for 1 min, and the pellet was resuspended in 0.5–1 ml (depending on pellet size) of mammalian protein extraction reagent with added protease inhibitor cocktail that includes 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, bestatin, leupeptin, and aprotinin (Sigma Chemical Co., St. Louis, MO). The pellet was homogenized (5°C, 20 min) and then centrifuged at 1,000g . The supernatant (whole cell lysate) was removed and assayed for protein content according to the methods of Bradford.21PKC was immunopurified from control and propofol-treated ventricular whole cell lysates using the AminoLink Plus immobilization kit (Pierce, Rockford, IL), per the manufacturer’s instructions. Briefly, the column was equilibrated with 5 ml phosphate-buffered saline (pH = 7.2) and coupled with a polyclonal anti-PKC antibody (H-300; Santa Cruz Biotechnology, Santa Cruz, CA) corresponding to a segment of the C4 conserved region which recognizes all PKC isoforms. The column was then equilibrated at room temperature for 60 min. Next, 1.5 ml cell lysate (diluted 1:1 in sample buffer containing 0.1 m phosphate, 0.15 NaCl, pH 7.2) was applied to the column and allowed to equilibrate for 30 min. The immunopurified PKC was eluted with 8–10 ml of immunopure immunoglobulin G elution buffer (0.1 m glycine HCl). Elution fractions (0.5 ml) were collected and monitored for protein content (absorbance at 280 nm). The fractions containing protein were pooled and used for assessing total (membrane and cytosolic) PKC activity.

Immunoblot analysis was performed on the cytosolic and membrane fractions as previously described.16Protein concentration was assessed using the Bradford method.21All samples were adjusted to a protein concentration of 1–2 mg/ml in sample buffer, boiled for 5 min, and then kept at −20°C until use. Equal amounts of protein (50 μg) from each fraction were electrophoresed on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific binding was blocked with Tris-buffered saline solution (0.1%; vol/vol) Tween-20 in 20 mm Tris base, 137 mm NaCl adjusted to pH 7.6 with HCl, containing 3% (wt/vol) bovine serum albumin for 1 h at room temperature. Monoclonal and polyclonal antibodies against PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ were diluted 1:1,000 in Tris-buffered saline solution containing 1% bovine serum albumin for immunoblotting (2 h). After washing in Tris-buffered saline solution three times (10 min each), filters were incubated for 1 h at room temperature with horseradish peroxidase–linked secondary antibody (ovine anti-mouse; 1:5,000 dilution in Tris-buffered saline solution containing 1% bovine serum albumin). Filters were again washed and bound antibody detected by the enhanced chemiluminescence method. Protein content was analyzed via  densitometry using NIH Image (National Institutes of Health, Bethesda, MD).

The activity of purified PKC from whole cell lysates obtained from control and propofol-treated cardiomyocytes was measured using a colorimetric PKC activity assay kit (Stressgen Bioreagents, Victoria, British Columbia, Canada), per manufacturer’s instructions. Briefly, a readily PKC phosphorylated substrate (cyclic adenosine monophosphate response element binding protein) is precoated on the wells of a PKC substrate microtiter plate provided in the kit. The purified PKC samples are added to the wells, and a PKC phosphorylation reaction is initiated with addition of adenosine triphosphate. After a 90-min incubation at 30°C, the reaction is terminated by emptying the contents of each well. A phosphospecific substrate antibody was added to each well followed by a peroxidase conjugated anti-rabbit immunoglobulin G secondary antibody. After incubation (30 min, 23°C) and four washes, tetramethylbenzidine substrate was added to develop the reaction. After incubation at room temperature for 45 min, the developing reaction was terminated with acid stop solution (2N HCl). The intensity of the color was measured on a microplate reader at 450 nm, and the relative kinase activity (compared with untreated, baseline controls) of the samples was calculated from the absorbance measurements.

Freshly isolated cardiomyocytes were placed on laminin-coated coverslips and allowed to adhere to the glass undisturbed for 1 h. After treatment with propofol, cardiomyocytes were fixed (acetone:methanol, 1:1; −80°C) for 20 min. Nonspecific binding was blocked using 5% milk in a phosphate buffered saline solution for 1 h at room temperature. After a single wash with phosphate buffered saline, cardiomyocytes were incubated with primary antibody in phosphate buffered saline–0.3% Triton solution. The following dilutions were used for the antibodies: PKC-α, 1:500; PKC-δ, 1:500; PKC-ϵ, 1:500; PKC-ζ, 1:250; and desmin, 1:200. Primary antibodies were incubated on the slides for 1 h at room temperature, or overnight at 4°C. Coverslips were washed four times (5, 10, 10, and 10 min, respectively) with phosphate-buffered saline before being placed into a light-insulated container. Fluorescein-5-isothiocyanate (1:200) or tetramethylrhodamine isothiocyanate (1:200) secondary antibodies were incubated for 1 h at room temperature. After washing as described above, the coverslips were placed on slides with a 20% 4,6-diamidino-2-phenylindole and 80% Vectashield solution (Vector Laboratories, Burlingame, CA). Images were acquired using a Leica TCS AOBS SP2 laser scanning spectral confocal microscope (Heidelberg, Germany) with a 63× oil immersion objective (numerical aperture = 1.4) at zoom 2. Three hundred fifty-six–nanometer (ultraviolet), 488-nm (argon), and 568-nm (krypton) lasers were used to excite 4,6-diamidino-2-phenylindole, fluorescein isothiocyanate–labeled PKC isoforms, and tetramethylrhodamine isothiocyanate–labeled desmin, respectively. For background removal, a photomultiplier tube offset value was chosen and kept constant eliminating any issues of image variability. Emission was collected between 400 and 500 nm for 4,6-diamidino-2-phenylindole, 500 and 550 nm for fluorescein-5-isothiocyanate, and 580 and 750 nm for tetramethylrhodamine isothiocyanate. For single focal plane acquisitions, a 1,024 × 1,024 (120 × 120-μm field size at zoom 2) pixel central slice (z-axis) was chosen for each myocyte. For z-axis positioning, a focal plane was selected in which the nuclei were most clearly visible. For maximum intensity projections, 1,024 × 1,024 pixel slices were taken through each myocyte at regular 1-μm intervals.

Immunoblot analysis was performed on subcellular fractions of cardiomyocytes treated with propofol alone (10 μm), phorbol myristate acetate (PMA, 1 μm), or bisindolylmaleimide I (1 μm; 10 min pretreatment) plus propofol. Cytosolic and membrane fractions were prepared and proteins were separated and transferred to nitrocellulose for immunoblotting. Rat brain lysate was used as a positive control for the PKC isoforms.

Suspensions of ventricular myocytes were exposed to propofol (1, 10, and 30 μm) for 10 min at 37°C in the presence or absence of a pretreatment with the PKC inhibitor bisindolylmaleimide I (1 μm; 10 min). Cardiomyocytes were lysed and PKC was immunopurifed by affinity chromatography. Activity of the immunopurified PKC was assessed by colorimetric determination of cyclic adenosine monophosphate response element binding protein phosphorylation. Parallel studies were performed using the Ca²⁺-dependent and novel PKC isoform activator PMA (1 μm). Results are expressed as percent change in relative kinase activity from baseline.

Immunocytochemical analysis was performed on propofol-treated (10 μm, 10 min) cardiomyocytes. Cardiomyocytes were incubated with monoclonal antibodies against PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ with and without z-line counterstains. Intracellular localization was assessed using confocal microscopy.

For assessment of PKC activity, each experimental protocol was performed in triplicate with myocytes obtained from the same heart and averaged to obtain a single value. All experimental protocols were repeated in myocytes obtained from at least five different hearts. Results obtained from each heart were averaged so that all hearts were weighted equally. Changes in PKC activity or translocation from cytosolic to membrane fraction, compared with baseline values in untreated control cardiomyocytes, were assessed using one-way analysis of variance with repeated measures and the Bonferroni post hoc  test. Comparisons between groups were made using two-way analysis of variance. Differences were considered statistically significant at P < 0.05. All results are expressed as mean ± SD.

Collagenase was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Monoclonal antibodies for PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ, as well as secondary antibodies, were purchased from Upstate Biotechnology (Charlottesville, VA). Propofol, bisindolylmaleimide I, PMA, antidesmin, and anti-sarcomeric actin were purchased from Sigma Chemical Co. The mammalian protein extraction reagent was purchased from Pierce, and the pan–anti-PKC (H-300) was obtained from Santa Cruz Biotechnology. Fluorescein-5-isothiocyanate and tetramethylrhodamine isothiocyanate were obtained from Jackson Laboratories (West Grove, PA). Vectashield and 4′,6-diamidino-2-phenylindole, dihydrochloride was obtained from Vector Laboratories.

Immunoblot analyses of PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ in cardiomyocyte cytosolic and membrane fractions are shown in figures 1and "2A. PKC-α, PKC-δ, and PKC-ζ were primarily associated with the cytosolic fraction but had some association with the membrane fraction in untreated cardiomyocytes. PKC-ϵ was exclusively associated with the cytosolic fraction. After treatment with propofol (10 μm), all four isoforms translocated from the cytosolic to membrane fraction. Pretreatment with bisindolylmaleimide I (1 μm) attenuated the propofol-induced translocation of all four isoforms. PMA (1 μm) stimulated translocation of all four isoforms from cytosolic to membrane fraction. Summarized data depicting the relative association of PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ with the cytosolic and membrane fractions, before and after treatment with propofol alone, PMA alone, or bisindolylmaleimide I plus propofol, are also depicted in figures 1, 2B, and 2C.

Summarized data representing the effects of propofol and PMA in the presence or absence of pretreatment with bisindolylmaleimide I (1 μm) on PKC activity are shown in figure 3. Propofol (1, 10 and 30 μm) increased PKC activity in a dose-dependent manner. PKC inhibition with bisindolylmaleimide I reduced the propofol-induced (30 μm) and PMA-induced (1 μm) increase in PKC activity by 93 ± 12% (P = 0.003) and 96 ± 9% (P = 0.002), respectively.

Immunocytochemical analysis of PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ was performed on untreated and propofol-treated (10 μm) cardiomyocytes. The nuclei were stained with 4′,6-diamidino-2-phenylindole, and antidesmin was used to localize the z-line. PKC-α primarily localized at the intercalated discs with diffuse deposition in the cytoplasm of untreated myocytes (fig. 4A, left). Propofol stimulated a greater association of PKC-α with the intercalated discs and also with structures associated with the z-line near the t-tubule (fig. 4A, right). PKC-δ localized primarily in cone regions near the nucleus of untreated myocytes (fig. 4B, left). Propofol stimulated a redistribution of PKC-δ to the perinuclear region of the cell (fig. 4B, right). PKC-ϵ had a diffuse localization throughout the cytosol with some association near the intercalated discs, similar to PKC-α (fig. 5A, left). Propofol stimulated translocation of PKC-ϵ to the sarcolemma and structures associated with the z-line near the t-tubule (fig. 5A, right). PKC-ζ displayed diffuse deposition in the cytoplasm of untreated myocytes (fig. 5B, left). Propofol stimulated the appearance of PKC-ζ in the nucleus (fig. 5B, right).

This is the first study to directly assess the effects of propofol on PKC activation in isolated cardiac muscle cells and to identify the intracellular localization of individual PKC isoforms before and after exposure to propofol. Previous studies, using pharmacologic inhibitors of PKC in the setting of myocardial ischemia and reperfusion injury, have demonstrated an important role for PKC activation in the cardioprotective effects of general anesthetics.22–24Previous studies from our laboratory using pharmacologic inhibitors of PKC have demonstrated a propofol-induced, PKC-dependent decrease in cardiomyocyte intracellular free Ca²⁺concentration ([Ca²⁺]ⁱ) and shortening.20We also demonstrated that propofol stimulates PKC-dependent phosphorylation of contractile proteins16and an Na⁺-H⁺–dependent increase in pHⁱ¹⁷causing an increase in the sensitivity of the myofilaments to [Ca²⁺]ⁱ. Propofol also attenuates the β-adrenoreceptor–mediated increase in cardiomyocyte function via  a PKC-dependent pathway.18The key finding of the current study is that propofol activates PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ, resulting in their translocation to distinct intracellular sites in cardiomyocytes. A schematic diagram illustrating the relevant cellular pathways and potential cellular targets of propofol in cardiomyocytes is depicted in figure 6.

A subcellular translocation of PKC isoforms from the cytosolic to membrane fraction is associated with activation of PKC isoforms, and translocation is facilitated by receptors for activated C kinases.25, 26The binding of the PKC isoform to its specific receptor for activated C kinase is also critical to the phosphorylation of substrate proteins that are specific for that PKC isoform. Most previous studies have confirmed that the major PKC isoforms expressed in the adult rat heart are PKC-α PKC-δ, PKC-ϵ, and PKC-ζ, with some controversy over the existence of PKC-βII.2–4In this study, we identified the presence of PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ. PKC-ϵ was entirely associated with the cytosolic fraction in untreated myocytes, whereas PKC-α, PKC-δ, and PKC-ζ had some association with the membrane fraction. Propofol caused translocation of all four PKC isoforms from cytosolic to membrane fraction, providing indirect support for their activation. These data also indicate that propofol activates multiple PKC-dependent pathways simultaneously, and likely explain our previous findings demonstrating propofol-induced, PKC-dependent alterations in [Ca²⁺]ⁱ, Na⁺–H⁺exchange, cyclic adenosine monophosphate production, phosphorylation of myofibrillar proteins and myofibrillar actomyosin adenosine triphosphatase.16–18,20A functional role for PKC-α, PKC-δ, PKC-ϵ, and PKC-ζ in mediating propofol-induced increases in cardiomyocyte contractility during α^1a-adrenoreceptor activation has recently been demonstrated by our laboratory.19Similar signaling pathways and cellular mechanisms of regulation are likely involved in propofol-induced myocardial protection.

Although translocation of PKC isoforms from cytosolic to membrane fraction has been used as the classic example of PKC activation, translocation can occur without activation, and activation can occur without translocation.27,28In the current study, we demonstrated that propofol caused a dose-dependent increase in PKC activity, as assessed by cyclic adenosine monophosphate response element binding protein phosphorylation, that is abolished by the PKC inhibitor bisindolylmaleimide I. These data provide the first direct evidence that propofol activates PKC in cardiac muscle cells and is consistent with our previous findings demonstrating that pharmacologic inhibition of PKC reduces propofol-induced modifications of cellular mechanisms regulating [Ca²⁺]ⁱand myofilament Ca²⁺sensitivity.16–18,20Propofol has been shown to activate purified brain PKC in reconstituted systems.29,30However, the extent to which propofol activates PKC in intact cardiac muscle cells causing substrate phosphorylation has not been examined.

Translocation of PKC isoforms from cytosolic to membrane fraction indicates movement of the protein from one intracellular domain to another. Previous studies examining the intracellular localization of individual PKC isoforms in cardiomyocytes, before and after an intervention, have been useful for identifying the potential cellular targets of each isoform.27,28,31Identifying the cellular target of the isoforms provides important information for predicting changes in cellular regulation and function that may likely occur. This study is the first to demonstrate the intracellular redistribution of PKC isoforms after exposure to propofol. Propofol caused translocation of PKC-α and PKC-ϵ to structures at or near the z-line. In the heart, t-tubular membranes penetrate the cardiomyocytes near the z-line. L-type Ca²⁺channels, nitric oxide synthase, receptors for activated C kinases, and A-kinase anchoring proteins have been found to be localized in the t-tubules.32Such a position adjacent to sarcoplasmic reticulum ryanodine receptors and myofibrils creates the potential for an intricate modulation of excitation–contraction coupling via  the regulation of [Ca²⁺]ⁱand myofilament Ca²⁺sensitivity.33,34Moreover, PKC-α and PKC-ϵ localization at the intercalated discs is consistent with the finding that PKC-α and PKC-ϵ mediate gap junction conductance in cardiomyocytes through interactions with connexin 43.35 

Protein kinase C δ has previously been shown to translocate to the perinuclear region under various stimulation conditions.31,36Our study also demonstrates a propofol-induced translocation of PKC-δ to the perinuclear region. Lamin B, a protein found in the inner nuclear membrane, has been shown to be involved in the regulation of cell apoptosis37and is a substrate for PKC-δ. The translocation of PKC-δ to the perinuclear region may be indicative of a role for phosphorylation of lamin B as a protective mechanism against cellular apoptosis. In contrast to PKC-δ, propofol stimulated translocation of PKC-ζ into the nucleus. The nuclear transcription factor nuclear factor κB is a substrate for PKC-ζ that is involved in gene regulation, suggesting that propofol could modulate gene expression in ventricular myocytes. Furthermore, PKC-ζ has been shown to translocate into the nucleus during ischemia, possibly regulating apoptosis in the ischemic heart.38Our finding that phorbol myristic acid stimulated translocation of PKC-ζ from the cytosolic to membrane fraction was intriguing, considering that PKC-ζ is a member of the atypical PKC isoforms that are not activated by phorbol esters. This effect could be explained by a PKC-dependent activation of phospholipase A², which liberates arachidonic acid, a known activator of the atypical PKC isoforms.39Taken together, our data indicate that propofol activates multiple PKC isoforms that likely have distinct roles in regulating cardiomyocyte function, adding to the complexity in understanding the roles of the individual isoforms in mediating anesthetic-induced cardiac depression and myocardial protection.

Activation of PKC in cardiomyocytes is important for protection from myocardial injury, providing an important and beneficial role for anesthetics in myocardial preservation and cell survival, in addition to anesthesia. It is believed that when myocardial metabolism is depressed, adenosine triphosphate production is limiting and cellular Ca²⁺homeostasis becomes impaired, causing abnormalities in cardiac muscle function and increasing the risk for cellular injury and muscle failure. Previous studies from our laboratory have demonstrated a propofol-induced, PKC-dependent decrease in sarcolemmal Ca²⁺influx with a concomitant PKC-dependent increase in myofilament Ca²⁺sensitivity.16–18,20Together, these changes could provide a mechanism to reduce the potential for intracellular Ca²⁺overload that could lead to injury and failure while preserving and maintaining adequate cardiac contractility. This may be a fundamentally important cellular mechanism of anesthesia-induced myocardial protection in the setting of ischemia–reperfusion injury. Moreover, the activation of PKC by anesthetics may serve as an important molecular mechanism of general anesthesia. Increasing our knowledge of these mechanisms may be used to improve the efficacy of anesthetic agents in patients with limited or compromised cardiovascular reserve, ischemic heart disease, or other cardiomyopathies.

As always, extrapolation of data obtained from laboratory-based studies to the clinical situation is difficult. The use of rat cardiomyocytes as a model for human cardiomyocytes is a limitation, because species differences in PKC isoform expression patterns and levels likely exist. However, the propofol-induced activation of PKC in this study may actually underestimate the effect of propofol in the clinical setting. This study used pure propofol solubilized in dimethyl sulfoxide instead of the commercially available 10% Intralipid™ emulsion (KabiVitrum, Alameda, CA) that contains fatty acids combined with glycerol and lecithin to create micelles for transporting propofol. Although this allowed us to directly assess the effects of propofol, independent of the vehicle, on PKC activity, fatty acids have been shown to activate PKC directly and act synergistically in combination with diacylglycerol.40,41Therefore, our results may underestimate the effect that propofol has on increasing the activity of myocardial PKC isoforms in the clinical setting.

Propofol stimulates activation of several PKC isoforms in cardiomyocytes. Translocation of multiple PKC isoforms to distinct intracellular sites likely provides a mechanism to coordinate and fine-tune specific cellular and/or molecular events. Moreover, activation of unique subsets of PKC isoforms by different anesthetic agents may be important in defining anesthetic specificity in protecting against distinct mechanisms of myocardial injury. Activation of these pathways may be important in mediating anesthesia-induced myocardial protection as well as the molecular mechanisms of anesthesia.