Ann Thorac Surg 2006;82:664-671
© 2006 The Society of Thoracic Surgeons
Original article: Cardiovascular
Differential Effects of Protein Kinase C Isoform Activation in Endothelin-Mediated Myocyte Contractile Dysfunction With Cardioplegic Arrest and Reperfusion
Kimberly A. Apple, MD,
Julie E. McLean, BS,
Christina E. Squires, BS,
Brooke Schaeffer, BS,
Jeffrey A. Sample, BS,
Rebecca L. Murphy,
Anne M. Deschamps, BS,
Amy H. Leonardi, BS,
Claire M. Allen, BS,
Jennifer W. Hendrick, BS,
Robert E. Stroud, MS,
Rupak Mukherjee, PhD,
Francis G. Spinale, MD, PhD*
Division of Cardiothoracic Surgery, Medical University of South Carolina, and The Ralph H. Johnson Veteran's Affairs Medical Center, Charleston, South Carolina
Accepted for publication March 7, 2006.
* Address correspondence to Dr Spinale, Cardiothoracic Surgery, Room 625, Strom Thurmond Research Building, 770 MUSC Complex, Medical University of South Carolina, 114 Doughty Street, Charleston, South Carolina 29425. (Email: wilburnm{at}musc.edu).
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Abstract
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BACKGROUND: Increased myocardial interstitial levels of endothelin (ET) occur during cardioplegic arrest (CA) and may contribute to contractile dysfunction. Endothelin receptor transduction involves the protein kinase-C (PKC) family comprised of multiple isoforms with diverse functions. Which PKC isoforms may be involved in ET-induced contractile dysfunction after CA remains unknown.
METHODS: Shortening velocity was measured in isolated left ventricular porcine myocytes and randomized (minimum of 30 per group): normothermia (cell culture media for 2 hours at 37°C); CA (2 hours in CA solution [4°C, 24 mEq K+] followed by reperfusion in cell media); ET/CA (100 pM ET incubated during CA and reperfusion). These studies were carried out in the presence and absence of PKC inhibitors (500 nM) and directed against members of the classical PKC subfamily (beta I, beta II, gamma) and the novel subfamily (epsilon, eta).
RESULTS: Cardiac arrest reduced shortening velocity by approximately 50%, which was further reduced in the presence of ET. Inhibition of either the beta II or gamma PKC isoform significantly increased shortening velocity from ET/CA as well as CA only values. In separate studies (n = 3), total beta II and phosphorylated beta II increased by over 150% with ET/CA (p < 0.05). Taken together, these results suggest that a predominant intracellular effector for the negative contractile effects mediated by ET in the context of CA is the PKC isoform beta II.
CONCLUSIONS: Targeted inhibition of specific PKC isoforms relieves the negative inotropic effects of ET after simulated CA. These findings provide important mechanistic support for the development of targeted inhibitory strategies with respect to ET signaling and myocyte contractile dysfunction in the context of CA and reperfusion.
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Introduction
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Transient left ventricular (LV) dysfunction can occur in the context of cardiac surgery and has been attributed to the brief periods of ischemia and reperfusion requisite for the conduct of the procedure. Most notably, global hypothermic cardioplegic arrest and cardiopulmonary bypass support, which is still utilized for a number of cardiac surgical procedures, is associated with a transient period of LV myocardial dysfunction after reperfusion and separation from cardiopulmonary bypass [1–3]. During this perioperative period, significant neurohormonal activation occurs and has been implicated to contribute to, and (or) exacerbate, the degree of postoperative LV dysfunction [3–5]. The signaling molecules, which are evoked during this process, include catecholamines, cytokines, and bioactive peptides such as endothelin (ET) [4, 5]. Experimental and clinical studies have shown a cause-effect relationship between ET release and the degree of ischemia-reperfusion injury [5–8]. Moreover, increased ET release within the myocardial interstitium has been demonstrated to occur in patients both during and after cardiac surgery [9]. Finally, increased ET levels have been associated with adverse postoperative outcomes in cardiac surgery patients [10]. Identifying the downstream signaling pathways, which contribute to the effects of ET on LV contractility, after cardioplegic arrest, holds clinical relevance. The overall goal of this study was to identify the initial intracellular signaling pathway contributing to contractile dysfunction after ET exposure in an isolated myocyte system of cardioplegic arrest (CA).
The most abundant ET receptor on the cardiac myocyte is the ET-A receptor [11]. After ligand binding, a cascade of intracellular events occurs which includes activation of phospholipase C, which in turn forms inositol triphosphate and diacylglycerol, and ultimately activation and mobilization of protein kinase-C (PKC) [12, 13]. The PKC family consists of 12 closely related serine-threonine kinases, which have been classified into three broad subfamilies: classical, novel, and atypical [13–17]. The classical PKC isoforms are activated through the G-protein coupled pathway and require calcium (Ca+2) for full activation. The novel PKC isoforms are Ca+2 independent. The activational cascade and downstream targets for the novel PKC subfamily remains unclear. In animal and human myocardium, isoforms of the classical and novel PKC subfamilies have been identified [17, 18]. The biological significance of these different isoforms has only recently gained appreciation with respect to myocyte contractile function. Past studies have demonstrated that ET receptor occupancy, and activation of PKC, causes diverse effects on contractile function [19]. Therefore, it is likely that exposure of myocytes to ET causes activation of a number of PKC isoforms, which in turn would cause diverse effects on contractility.
It is now known that PKC activation and translocation to an effector target site are dependent upon specific protein sequences that are highly selective for PKC isoforms [20–22]. These intracellular receptors for activated PKC, receptors for activated C-kinase (RACK) proteins, have been sequenced and cloned [20, 21]. The discovery of these PKC anchoring proteins have provided an important step in the elucidation of PKC-mediated signaling and the effects of specific PKC isoforms on myocyte contractile processes [18, 20, 21]. Studies have demonstrated that small peptide fragments, corresponding to the RACK binding site of each PKC isoform, can result in selective loss of function of the targeted isoform [20–22]. Therefore, these RACK binding proteins provide the opportunity for identifying the potential role of PKC isoforms with respect to modulating myocyte contractile function after ET exposure in the context of CA. This study utilized an isolated myocyte model of CA and ET exposure and RACK inhibitory proteins targeted against specific PKC isoforms, in order to identify which PKC isoforms potentially contribute to the contractile dysfunction operative in this model.
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Material and Methods
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Experimental Overview and Rationale
The present study utilized an isolated porcine myocyte model of CA that has been well-characterized previously [6, 23]. In the present study, isolated myocyte function was measured under normothermic steady state conditions or after simulated CA in the presence and absence of ET. The concentration of ET utilized in the present study (100 pM) was based upon previous concentration-response studies by this laboratory demonstrating that this concentration of ET yielded a consistent negative effect on contractile function after simulated CA [6]. Moreover, past studies have demonstrated that this effect of ET was primarily due to occupancy of the ET-A receptor [11]. In the present study, measurements were performed after incubation with inhibitors of a specific PKC isoform. Past studies have identified potential roles for both the classical and atypical PKC isoforms within the myocardium [13–19]. Accordingly, this study focused upon dissecting out the role of specific PKC isoforms from these subfamilies, which may be operative with respect to ET signaling after CA. The PKC isoforms from the classical subfamily (beta I, beta II, gamma), in addition to the novel subfamily (eta and epsilon), were inhibited by incubation with corresponding selective RACK inhibitory proteins [21, 22]. A consensus sequence corresponding to the binding domain for the classical PKC isoforms have been identified and a corresponding RACK inhibitory peptide developed [21, 22, 24]. A consensus sequence for the transactivation domain for the classical PKC isoforms has also been identified [21, 22]. Accordingly, additional studies were performed with inhibition of the entire classical PKC subfamily.
Myocyte Isolation and Contractile Function Measurements
Myocytes were isolated from the left ventricular free wall of pigs (Yorkshire, n = 22, 25 to 30 Kg) using a collagenase digestion method previously described in detail [6, 11, 23]. All animals were treated and cared for in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (National Research Council, Washington, 1996). The liberated myocytes were plated onto coverslips, which were previously coated with a basement membrane substrate (Matrigel, Collaborative Research Inc, Bedford, MA). These were stabilized at 37°C in oxygenated media for 60 minutes and then randomly assigned to the experimental protocols described in the subsequent paragraph. The yield of viable myocytes from all isolations was greater than 60%. Isolated myocyte contractile measurements were performed using computer-assisted videomicroscopy as described previously [6, 11, 23]. Myocytes were thermostatically maintained at 37°C and electrically field-stimulated at a frequency of 1 Hz. Myocyte shortening velocity (µm/s) was determined from the digitized contraction profiles as the average value obtained from 20 consecutive contractions.
PKC Isoform Inhibition and PKC Activation
In these studies, specific RACK proteins that corresponded to the specific binding domains for the activated PKC isoform were used. Binding of specific activated PKC isoforms to these RACK proteins inhibits translocation of the activated PKC isoform and thereby prevents phosphorylation of downstream targets [21, 22, 24, 25]. For these studies, the RACK inhibitory peptides (provided by KAI Pharmaceuticals, San Francisco, CA) corresponding to the classical PKC isoforms beta I (KIBI31-1), beta II (KIBII31-1), gamma (KIG31-1), and to the novel PKC isoforms eta (KIET1-1) and epsilon (KAE1-1), were utilized. In addition, a RACK inhibitory peptide for the classical PKC subfamily (KIC1-1) and an activation peptide for this subfamily (KAC1-1) were used. Based upon past reports utilizing these inhibitory peptides [25], and initial titration studies, it was determined that incubation of the myocyte systems, with a final concentration of 500 nM, provided inhibition of the PKC isoform without a change in myocyte viability. Viable myocytes included those that retained a rod shape, excluded trypan blue, and remained quiescent in culture.
Experimental Design
Myocytes were first randomly assigned to be maintained under normothermic control conditions (incubation in a physiologic solution [Medium 199, Gibco; Life Technologies, Inc, Rockville, MD] at 37°C and in a 95% oxygen environment for 2 hours) or subjected to simulated CA (incubation in Ringer's solution at 4°C containing 24 mEq/L K+ and 30 mEq/L HCO3
–, and stored at 4°C for 2 hours [O2 saturation: 120 to 150 Torr]). After completion of the respective incubation periods, myocytes were resuspended with normothermic cell culture media and contractile studies performed. Subsets of myocytes were then randomized to be exposed to ET (100 pM) throughout the incubation and resuspension period, as well as incubated with the respective PKC isoform inhibitor-activator described in the previous paragraph.
PKC Isoform Expression
In a separate set of studies (n = 3), myocyte suspensions (105 cells/mL) were exposed to the conditions described in the preceding paragraph and then immediately flash frozen and subjected to immunoblotting for specific PKC isoforms. The cell pellets were homogenized on ice and equivalent amounts of protein (10 µg) were separated electrophoretically on a 4% to 12% bis-tris gel (Invitrogen, Carlsbad, CA), and transferred to a nitrocellulose membrane (TransBlot; BioRad, Hercules, CA). After vigorous washing, the membrane was incubated (25°C for 60 minutes) with polyclonal antisera directed against the PKC isoforms beta I, beta II, gamma, eta, and epsilon (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA). In addition, membranes were incubated with antisera corresponding to the phosphorylated form of PKC beta II (1:1,000; Abcam Inc, Cambridge, MA). The membranes were then washed and incubated with an anti-rabbit horseradish conjugate (1:5,000, Vector Laboratories, Inc, Burlingame, CA) and the immunoreactive signal detected by chemiluminescence (Western Lightning, PerkinElmer Inc, Boston, MA). The signals were digitized and analyzed (Gel Pro Analyzer, Media Cybernetics, Silver Spring, MD) in which the integrated optical density (IOD) values were obtained. Positive controls were included in every procedure and negative controls also were included, which entailed substitution of the primary antisera with nonimmune sera.
Sample Size and Data Analysis
The dependent variable in this study was the index of myocyte contractility, which is the velocity of shortening. The LV myocytes from each isolation were randomized to the different treatment groups utilizing a split-plot analysis of variance (ANOVA) design. The first level was normothermia or CA. The second level was the presence or absence of ET. Finally, the third level was the presence or absence of a PKC inhibitor-activator. In this study, 3,523 individual myocytes were randomized and subjected to contractile function analysis. A minimum of 30 myocytes were included in each treatment subplot. The parameters of contractile function conformed to a Gaussian distribution. Therefore, parametric statistics were utilized in which a multiway ANOVA was first performed, and mean separation utilized Bonferroni-corrected pairwise t tests. For the immunoblotting results, IOD values were analyzed as a percent of normothermic control values. The percent change from normothermic values were subjected to a one sided t test, where normothermic values were set to 100%. For pairwise comparisons, a multiway ANOVA with Tukey post-hoc comparisons were employed. A p value of less than 0.05 was considered statistically significant. All statistical procedures were performed using STATA statistical software (STATA Intercooled V 8.0; College Station, TX). Results are presented as mean ± standard error of the mean.
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Results
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Inhibition of the Classical PKC Subfamily: Effects of Endothelin and Cardioplegic Arrest
Consistent with past reports, myocyte contractile function was reduced by approximately 50% after CA (Fig 1) [6, 23]. Incubation with ET caused a reduction in contractile function under normothermic conditions and a further reduction after CA. Exposure of normothermic control myocytes to the inhibitor for the classical PKC subfamily had no effect on myocyte contractility. In contrast, incubation of myocytes with the classical PKC inhibitor, during and after CA, increased myocyte contractility when compared with CA only values. Coincubation with both ET and the classical PKC inhibitor increased myocyte function from ET only respective values under both normothermic conditions and after CA. These experiments demonstrated that ET exacerbates myocyte contractility after CA. In addition, inhibition of the classical PKC subfamily attenuated the negative contractile effects of ET.
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Fig 1. Myocyte contractility was measured under normothermic conditions and after simulated cardioplegic arrest (CA), as well as in the presence and absence of endothelin (ET). Additional experiments were performed using a specific peptide that caused inhibition of the classical subfamily of protein kinase-C (PKC) isoforms. Myocyte shortening velocity was reduced after CA and reperfusion and was further reduced in the presence of ET. Incubation with an inhibitor of this entire PKC subfamily increased contractility after CA. Inhibition of the classical PKC subfamily in the presence of ET normalized myocyte contractility under normothermic conditions and was increased significantly after CA. (*p < 0.05 versus normothermic baseline, + vs ET only values, and & versus CA baseline; black bars = normothermic; striated bars = CA.)
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Inhibition of Specific Isoforms of the Classical PKC Subfamily; Effects of Endothelin and Cardioplegic Arrest
In the presence of the beta I inhibitory peptide, the effects of ET under normothermic conditions appeared similar to ET only values (Fig 2A). However, after CA, inhibition of the beta I isoform of PKC increased myocyte contractility from ET only values. Coincubation with ET and the beta II PKC inhibitor normalized contractile function under normothermic conditions and increased myocyte contractile function from CA only values by almost twofold. Under normothermic conditions, coincubation with ET and the gamma PKC inhibitor yielded similar results to that obtained with ET alone. However, incubation with ET and the gamma PKC inhibitor after CA markedly improved contractile function when compared with CA only values. Thus, inhibition with the selective classical PKC isoforms resulted in differential effects on ET-mediated contractile dysfunction under normothermic conditions and after CA. Moreover, in the context of CA, inhibition of either the beta II or the gamma PKC isoform reversed the effects of ET from one of a negative inotropic effect to a positive inotropic effect.
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Fig 2. (A) Myocyte contractility was measured under normothermic conditions and after simulated cardioplegic arrest (CA) in which myocytes were exposed to endothelin (ET) and an inhibitory peptide specific for the isoforms of the classical protein kinase-C (PKC) subfamily: beta I, beta II, and gamma. The baseline and ET only histograms have been transposed from Figure 1 for reference purposes. Myocyte contractility in the presence of ET and the beta I isoform yielded a variable response, but contractility appeared higher under normothermic and CA conditions when compared with ET only values. Inhibition of the beta II isoform significantly increased myocyte contractility after CA. Inhibition of the gamma isoform yielded similar results to ET only under normothermic conditions, but significantly higher values after CA. (B) Coincubation with either of the inhibitors for the novel PKC isoforms and ET yielded results similar to that obtained with ET only. However, there was a high degree of variation in the presence of epsilon isoform inhibition under normothermic conditions. Coincubation with ET and the eta inhibitory peptide during and after CA increased contractility from ET and CA-only values. (*p < 0.05 versus normothermic baseline, +versus ET only values, and & versus CA baseline; black bars = normothermic; striated bars = CA).
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Inhibition of Specific Isoforms of the Novel PKC Subfamily: Effects of Endothelin and Cardioplegic Arrest
Coincubation studies were performed with ET and inhibitory peptides specific for the novel PKC isoforms, eta and epsilon (Fig 2B). Under normothermic conditions, inhibition of either isoform did not affect myocyte contractility when compared with ET only values. However, the results for the epsilon inhibitory peptide were associated with a greater degree of variation. After CA, coincubation with ET and these inhibitory peptides yielded shortening velocity values similar to CA only values. However, coincubation with the eta inhibitory peptide yielded results higher than ET and CA only values.
Specific PKC Isoforms in Cardiac Myocytes; Effects of Endothelin and Cardioplegic Arrest
In a second series of studies, myocytes were treated under the identical conditions described above, and the relative levels of specific PKC isoforms determined. A strong immunoreactive signal for the PKC isoforms beta I and beta II were observed in the cardiac myocyte preparations and increased significantly with CA and coexposure to ET (Figs 3. Moreover, the phosphorylated form of beta II robustly increased with CA and ET. A specific immunoreactive signal was detected in cardiac myocyte preparations for the PKC isoforms gamma, eta, and epsilon, but relative levels remained unchanged from normothermic values with CA or ET (Fig 4).
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Fig 3. Relative levels of the protein kinase-C (PKC) isoforms beta I, beta II, and the phosphorylated form of beta II were examined in isolated myocytes under normothermic (N) conditions and after simulated cardioplegic arrest (CA), as well as in the presence and absence of endothelin (ET). The results are summarized for three independent sets of experiments and normothermic values used for relative comparisons. Robust immunoreactive signals for these PKC isoforms were detected in the myocyte preparations. After CA and ET exposure, total levels of beta I and II were increased from normothermic values. A significant increase in the phosphorylated form of beta II was identified after CA and ET exposure. (*p < 0.05; black bars = normothermic; grey bars = CA.)
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Fig 4. Relative levels of the protein kinase-C (PKC) isoforms gamma, eta, and epsilon were examined in isolated myocytes under normothermic (N) conditions and after simulated cardioplegic arrest (CA), as well as in the presence and absence of endothelin (ET). A clear and specific immunoreactive signal was detected for these PKC isoforms in the cardiac myocyte preparations. Total abundance for these PKC isoforms appeared unchanged with CA or ET when compared with normothermic values.
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Comment
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Transient myocardial dysfunction can occur in the early period after cardioplegic arrest (CA). Contributory factors for this perioperative contractile deficit likely include the local release of bioactive peptides that in turn change critical molecular pathways within the myocyte. Endothelin has been shown to be released within the myocardial interstitium during and after CA, cause receptor mediated changes in myocyte contractility, and is associated with postoperative outcomes [5–10]. A fundamental intracellular signaling pathway for ET is the activation and mobilization of the large family of kinases, the PKCs [12–14]. However, the relative contribution of specific PKC isoforms, which may be evoked with ET-mediated contractile dysfunction in the context of CA, has not been examined. The present study utilized an isolated ventricular myocyte system, simulated CA and rewarming, specific PKC inhibitory peptides, and immunoblotting procedures in order to begin to address this complex issue. The unique and important findings from the present study were threefold. First, the effects of ET and activation of specific PKC isoforms appear to be condition specific. Notably, ET receptor activation and the recruitment of specific PKC isoforms appear to be different under normothermic, steady-state conditions to that after CA and reperfusion. Second, PKC isoforms of the classical subfamily (beta I, beta II, and gamma) and novel subfamily (eta and epsilon) are robustly expressed in mammalian cardiac myocytes and are selectively up-regulated in the presence of ET and CA. Third, the negative contractile effects of ET after CA appear to be predominantly due to activation of specific PKC isoforms belonging to the classical PKC subfamily, particularly that of beta II. These results demonstrated that targeted inhibition of these PKC isoforms relieved the negative inotropic effects of ET and improved myocyte contractile performance after CA. These findings provide important mechanistic support for the development of targeted inhibitory strategies with respect to ET signaling and myocyte contractile dysfunction in the setting of CA and reperfusion.
PKC Activation and RACKs
The activation of PKCs is a multistep process and it is this activational cascade that was utilized to develop the initial classification scheme of the classical, atypical, and novel subfamilies. These subfamilies, under which a number of specific isotypes-isoforms exist, have specific subcellular distribution patterns and downstream substrates [15–18, 25]. The specificity of the PKC isoform for targeted downstream substrates was fully appreciated when the RACKs were sequenced and cloned [20, 21, 24]. The PKC isoform specificity appears to be due to the fact that the activated isoform docks to a specific RACK sequence on intracellular structures [20, 21, 24]. This led to the development of specific RACK binding peptides, which would specifically inhibit the interaction of an activated PKC isoform to dock to putative substrates [21, 22, 25]. These RACK antagonists have been utilized in isolated myocyte systems previously in order to demonstrate that specific PKC isoforms are operative in ischemia-reperfusion injury and preconditioning [14, 22, 25]. For example, past in vitro and in vivo studies have demonstrated that the activation of the novel PKC isoform, epsilon, is an important intracellular event in the type of preconditioning which involves short periods of ischemia-reperfusion prior to a prolonged period of ischemia [22, 25]. These past findings emphasize the multiple and divergent biological roles of PKC isoforms in the context of myocardial protection. The present study examines the putative role of different PKC isoforms in ET-mediated contractile dysfunction that occurs in the context of CA and reperfusion. The findings from this study provide mechanistic evidence that the ET-mediated exacerbation of myocyte contractile dysfunction, which occurs after simulated CA and reperfusion, is due at least in large part to activation of members of the classical PKC subfamily.
Activation of Classical PKC Isoforms and Myocyte Dysfunction
The present study examined the contributory role of the classical PKC subfamily with respect to ET-mediated contractile dysfunction in several ways. First, an inhibitory RACK peptide, which inhibited all activated classical PKC isoforms, was utilized and demonstrated a significant attenuation in the myocyte contractile dysfunction mediated by ET and CA. Second, selective RACK inhibitory peptides for the classical PKC isoforms beta I, beta II, and gamma were also examined in the context of ET stimulation and CA. While inhibition of the beta I PKC isoform improved myocyte function from ET only values, inhibition of either the beta II or gamma PKC isoform significantly relieved the negative contractile effects of ET in the context of CA and reperfusion. Third, the present study demonstrated a robust increase in the relative abundance of the beta I and beta II isoform with ET and CA, and this was accompanied by a significant increase in the phosphorylated form of beta II- indicative of heightened activation. It has been demonstrated previously that myocyte intracellular Ca+2 levels increase significantly in this model of simulated CA and reperfusion [23]. The classical PKC isoforms require changes in intracellular Ca+2 for full activation and translocation to target substrates [13, 14]. The results from the present study demonstrated that ET exposure during CA and reperfusion cause heightened activation of the classical PKC isoform beta II. The downstream substrates for the classical PKC isoforms include the troponin complex, the L-type calcium channel, potassium channels, and the sodium-hydrogen exchanger [15–18, 26]. While it was beyond the focus of the present study to identify which of these entities are phosphorylated by specific PKC isoforms induced by ET and CA, the net effect would be to reduce the myocyte inotropic state by modifying components of the excitation-contraction coupling process.
Novel PKC Isoforms and Myocyte Contractility
In marked contrast to the potential negative inotropic effects that activation of classical PKC isoforms may impart, evidence indicates that activation of novel PKC isoforms may enhance myocyte inotropic response. Specifically, activation of the novel PKC isoform epsilon phosphorylates residues on the troponin I complex, which improves Ca+2 sensitivity of the actin-myosin complex [18, 21, 26]. In the present study, inhibition of the classical PKC pathway in the presence of ET, particularly during CA and reperfusion, caused a substantial increase in myocyte contractile performance. This may have been due in part to the fact that ET activates both the classical and novel PKC subfamilies. Therefore, inhibiting the classical PKC pathway unmasked the positive inotropic effects of the novel PKC pathway. In the present study, inhibition of the novel PKC isoforms had modest effects on myocyte contractile function in the presence of ET with CA and reperfusion. The PKC epsilon has been shown to impart protective effects in ischemia-reperfusion and is likely involved in the preconditioning response [21, 22, 25]. The present study demonstrated that with exposure to ET, inhibition of the classical PKC pathway improved myocyte contractility with CA and reperfusion, while inhibition of the novel PKC isoform epsilon yielded minimal effects upon contractility. The present study also demonstrated that there was no appreciable change in the relative abundance of the novel PKC isoforms epsilon or eta with either ET or CA. Taken together, the results from past studies and the present report suggest that the negative effects of ET stimulation during CA and reperfusion with respect to myocyte contractility are mediated through the classical PKC isoforms such as beta I and beta II. Whether, and to what degree, ET may increase the activational state of novel PKC isoforms and in turn influence other cellular processes independent of the acute effects on myocyte contractility remain to be established.
Summary and Conclusion
The present study focused upon representative PKC isoforms from the classical and novel subfamilies, which were predicated upon past studies as well as practical considerations. First, the classical PKC subfamily has been shown to be activated after ET receptor stimulation and is Ca+2 dependent [12, 19]. Therefore, studies of this particular transduction pathway utilizing the myocyte system of CA and reperfusion were appropriate. Second, an important role of particular novel PKC isoforms such as epsilon have been demonstrated in past myocardial protection studies [22, 25]. Third, the activator-inhibitor RACK peptides employed in the present study have been utilized in past in vitro studies and, therefore, the specificity and concentration profiles have been established previously [21, 25, 27]. However, it must be recognized that the present study was not comprehensive in that certain PKC isoforms of the classical subfamily (alpha) and the novel subfamily (delta, theta) were not examined. This was due to the fact that inhibitory peptides for these PKC isoforms are either not available or the specificity has not been fully characterized. Moreover, the present study did not examine the potential role of the atypical PKC subfamily with respect to ET-mediated contractile dysfunction with CA and reperfusion. The expression and role of the atypical PKC isoforms within the myocardium remain poorly understood, and whether and to what degree ET causes activation of this PKC subfamily remains to be defined. While continued research is necessary to define further the function of these PKC isoforms under more relevant in vivo conditions, the present study provides evidence that the ET-mediated contractile dysfunction that occurs in the context of CA and reperfusion is through specific PKC isoforms. More specifically, it is likely that the effects of ET receptor stimulation in the context of CA and reperfusion causes the induction and activation of classical PKC isoforms such as beta II, which in turn alters fundamental components of the contraction process (Fig 5). Thus, a more selective strategy for interrupting the negative effects of ET in the context of CA and reperfusion would be to target this specific postreceptor transduction pathway.
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Fig 5. A schematic of the endothelin (ET) receptor transduction pathway and subsequent activation of the classical protein kinase-C (PKC) isoforms in a cardiac myocyte. After binding of ET to the ET receptor (primarily the ET-A subtype), which is a G-protein coupled receptor, a cascade of intracellular events occurs which includes activation of phospholipase C (PLC) which in turn forms inositol triphosphate (IP3) and diacylglycerol (DAG), and ultimately activation and mobilization of PKC. The PKC family consists of three broad subfamilies: classical, novel, and atypical. The classical PKC isoforms are activated through the G-protein coupled pathway and require calcium (Ca+2) for full activation and, thus, increased intracellular Ca+2 that occurs with CA and ET exposure would likely cause induction-activation of the classical PKC isoforms. The results from the present study confirm this and identify that the specific classical PKC isoform, beta II, is induced and activated with CA and ET exposure. Beta II PKC activation can, in turn, modify a number of intracellular events critical to myocyte contractile behavior which include changes in myofilament sensitivity to Ca+2, changes in Ca+2 reuptake by the sarcoplasmic reticulum (SR), and modifying the function of the voltage sensitive L-type Ca+2 channel. The present study demonstrated that using a selective inhibitory peptide for the beta II isoform, the negative contractile effects associated with CA and ET exposure were attenuated. (PLB = phospholamban; SERCA = sarcoplasmic reticulum calcium-adenosine triphosphatase, both components of the SR.)
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Acknowledgments
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This study was supported by NIH grants HL56603, HL57952, and a Merit Review Award from the Veterans' Affairs Health Administration.
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Abstract
Introduction
