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CARDIOVASCULAR
II Peptide Inhibitor Exerts Cardioprotective Effects in Rat Cardiac Ischemia/Reperfusion Injury
Department of Pathology, Microbiology, and Immunology, Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania
Received December 10, 2004; accepted April 4, 2005.
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Abstract |
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II peptide inhibitor was used to test the hypothesis that PKC II inhibition could attenuate PMN-induced cardiac dysfunction by suppression of superoxide production from PMNs and increase NO release from vascular endothelium. The effects of the PKC II peptide inhibitor were examined in isolated ischemic (20 min) and reperfused (45 min) rat hearts with PMNs. The PKC II inhibitor (10 µM; n = 7) significantly attenuated PMN-induced cardiac dysfunction compared with I/R hearts (n = 9) receiving PMNs alone in left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dtmax) cardiac function indices (p < 0.01). The PKC II inhibitor at 10 µM significantly increased endothelial NO release from a basal value of 1.85 ± 0.18 pmol NO/mg tissue to 3.49 ± 0.62 pmol NO/mg tissue from rat aorta. It also significantly inhibited superoxide release (i.e., absorbance) from N-formyl-L-methionyl-L-leucyl-L-phenylalanine-stimulated rat PMNs from 0.13 ± 0.01 to 0.02 ± 0.004 (p < 0.01) at 10 µM. Histological analysis of the left ventricle of representative rat hearts from each group showed that the PKC II peptide inhibitor-treated hearts experienced a marked reduction in PMN vascular adherence and infiltration into the postreperfused cardiac tissue compared with I/R + PMN hearts (p < 0.01). These results suggest that the PKC II peptide inhibitor attenuates PMN-induced post-I/R cardiac contractile dysfunction by increasing endothelial NO release and by inhibiting superoxide release from PMNs.
). However, reperfusion results in a marked degree of cardiac contractile dysfunction and myocardial cell injury. A harmful cascade of events accelerates structural and functional changes in endothelial cells, resulting in a progressive decrease in micro-circulatory flow (Forman et al., 1989; Tsao et al., 1990; Lefer and Lefer, 1996). These events occur sequentially and include a decreased endothelial release of NO; up-regulation of adhesion molecules on the endothelial surface, leading to enhanced leukocyte-endothelium interaction; infiltration of polymorphonuclear leukocytes (PMNs) into the myocardium; and subsequent release of superoxide radicals, which are largely responsible for producing cardiac dysfunction and enhanced necrosis (Tsao and Lefer, 1990; Entman et al., 1992; Lefer and Lefer, 1996).
The time course of events is similar in the ex vivo and in vivo myocardial I/R models within the first 30 min of reperfusion with respect to PMN/endothelial interaction. However, the in vivo model requires a longer reperfusion period (i.e., 270 min) to accumulate PMNs (Tsao and Lefer, 1990; Tsao et al., 1990; Ma et al., 1993; Young et al., 2001).
PMN activation by chemotactic substances such as interleukin-8 and complement fragment C5a results in the release of cytotoxic substances (i.e., oxygen-derived free radials such as superoxide) from the PMNs (Tsao and Lefer, 1990; Ma et al., 1991; Tsao et al., 1992). Damage to the endothelium by these free radicals is what contributes to the decrease in the endothelium-derived relaxing factor, NO (Tsao et al., 1990; Lefer and Lefer, 1996). As well as decreasing the availability of NO, they contribute to reperfusion injury by initiating lipid peroxidation through H2O2 formation, and they alter membrane permeability to ions such as Ca2+ (Lucchesi and Mullane, 1986; Rubanyi and Vanhoutte, 1986). Attenuation of the harmful effects of superoxide results in improved cardiac contractile function on reperfusion of ischemic tissue. Superoxide reduces the bioavailability of NO by combining with it to produce the peroxynitrite anion, thereby inhibiting the vasodilating effects of NO and resulting in endothelial dysfunction (Rubanyi et al., 1989; Tsao et al., 1992).
Protein kinase C (PKC) plays an important role in neutrophil activation. PKC activation results in an increase of superoxide release because it phosphorylates the cytosolic factor p47phox that is required for NADPH oxidase activation to produce superoxide from PMNs (Xiao et al., 1998; Babior, 1999; Li et al., 2000). It also down-regulates the activity of endothelial nitric-oxide synthase, leading to a reduction of endothelium-derived NO and augments superoxide release from endothelial cells (Rubanyi et al., 1989; Hirata et al., 1995; Meyer et al., 1999).
PKC activity and expression are increased during ischemia and reperfusion in acute myocardial I/R models (Strasser et al., 1992). Therefore, PKC inhibition at the postischemic coronary endothelium will lead to the attenuation of superoxide production as a result of postischemia reperfusion injury and the preservation of NO bioavailability (Young et al., 2001; Peterman et al., 2004; Phillipson et al., 2005).
Six isoforms of PKC have been identified in rat neonatal cardiomyocytes (Dang et al., 1995). These isoforms include PKC 
, PKC I, PKC II, PKC 
, PKC 
, and PKC 
. Rat PMNs possess all of these isoforms except PKC (Korchak and Kilpatrick, 2001). PKC II is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion and is, in part, responsible for superoxide release in activated PMNs (Dekker et al., 2000; Korchak and Kilpatrick, 2001). However, the role of PKC II inhibition has not been previously characterized in myocardial ischemia/reperfusion (I/R).
In this study, the hypothesis being tested is that PKC II inhibition will attenuate PMN-induced cardiac contractile dysfunction after I/R. In addition, the hypothesis would predict that PKC II inhibition would attenuate PMN superoxide release, PMN adhesion, and transmigration into the postischemic myocardium and augment endothelial NO release. The selective PKC II peptide inhibitor (mol. wt. = 1300) used in these assays is myristolated (fatty acid moiety) to allow for rapid cell permeability (within 10 s). Mechanistically, the selective PKC II peptide inhibitor may improve cardiac function by disrupting the binding of PKC II to the receptor for activated C kinase (RACK-1) region (Korchak and Kilpatrick, 2001).
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Materials and Methods |
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II peptide inhibitor (control hearts), or plasma containing different concentrations of PKC II peptide inhibitor (1–10 µM) to be directly infused into the coronary inflow line. Coronary flow was monitored by a flowmeter (T106; Transonic Systems Inc., Ithaca, NY). Left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dtmax) were monitored using a pressure transducer (SPR-524; Millar Instruments Inc., Houston, TX), which was positioned in the left ventricular cavity. Coronary flow, LVDP, and +dP/dtmax were recorded using a Powerlab Station acquisition system (ADInstruments, Grand Junction, CO) in conjunction with a computer. Figure 1 illustrates a schematic diagram of the protocol for ischemia/reperfusion in the isolated perfused rat heart. LVDP, +dP/dtmax, and coronary flow were measured every 5 min for 15 min to equilibrate the hearts and obtain a baseline measurement. LVDP was defined as left ventricular end-systolic pressure minus left ventricular end-diastolic pressure.
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II peptide inhibitor N-Myr-SLNPEWNET (mol. wt. = 1300) (Genemed Synthesis, Inc., San Francisco, CA) was added to plasma at a final concentration of 1, 5, or 10 µM. Data were recorded every 5 min for 45 min postreperfusion. After each experiment, the left ventricle was isolated, fixed in 4% paraformaldehyde, and stored at 4°C for later histological analysis.
Groups of Isolated Perfused Hearts. Table 1 indicates the nine groups (control and treatment conditions) of isolated perfused rat hearts used in the study. Sham I/R hearts were not subjected to ischemia and were not perfused with PMNs. Previous studies showed that sham I/R hearts given PMNs exhibited no changes from initial control values (Peterman et al., 2004). In some sham I/R hearts, PKC II peptide inhibitor (10 µM) was dissolved in plasma and infused at a rate of 1 ml/min for 5 min after 35 min of perfusion.
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Isolation of Plasma. To more closely simulate the conditions in vivo, the plasma used for infusion with the PMNs was isolated from the same rat from which the heart was isolated in each cardiac perfusion experiment. Blood was collected from the aorta in citrate phosphate buffer (Sigma Chemical Co., St. Louis, MO) over a period of 1 min just before isolation of the rat heart. The blood was centrifuged at 10,000g for 10 min at 4°C. Then, the plasma was decanted and used for infusion in the I/R hearts. Five milliliters of plasma collected from a single rat was used for each perfused heart.
Isolation of PMNs. Male Sprague-Dawley rats (350–400 g; Ace Animals), used as PMN donors, were anesthetized with ethyl ether and were given a 16-ml i.p. injection of 0.5% glycogen (Sigma Chemical Co.) dissolved in phosphate-buffered saline (PBS). The rats were reanesthetized with ethyl ether 16 to 18 h later, and the PMNs were harvested by peritoneal lavage in 30 ml of 0.9% NaCl, as previously described (Young et al., 2001). The peritoneal lavage fluid was centrifuged at 250g for 20 min at 4°C. The PMNs were then washed in 20 ml of PBS and centrifuged at 250g for 10 min at 4°C. Thereafter, the PMNs were resuspended in 2.5 ml of PBS and a total of 10 to 12 samples were pooled before use in cardiac perfusion experiments. The PMN preparations were >90% pure and >95% viable, according to microscopic analysis and exclusion of 0.3% trypan blue, respectively.
Determination of PMN Vascular Adherence and Infiltration into the Cardiac Tissue. In this I/R model, cardiac injury results from PMNs infiltrating the myocardium within the 45-min reperfusion period. Another component of the cardioprotective effects offered by the PKC II peptide inhibitor may be associated with an inhibition of PMN infiltration and adherence to vascular endothelium. Three representative rat hearts from each of the nine experimental groups were used for histological analysis. These hearts were representative of each group because their individual cardiac function data (LVDP and +dP/dtmax) were closest to the mean values of the entire group. The hearts were dehydrated in graded ice-cold acetone washes (50–100%). The heart tissue was then embedded in plastic and sectioned into 2.5-µm serial sections and placed onto glass slides. Sections were then stained with hematoxylin and eosin as described previously (Young et al., 2001). The number of PMNs was counted by light microscopy in 10 areas of the left ventricle. To determine the effect of PKC II peptide inhibitor on PMN adherence, the intravascular PMNs that adhered to the coronary vascular endothelium were counted and expressed as adhered PMNs per square millimeter. The effect of PKC II peptide inhibitor on PMN transmigration was determined by counting the total intravascular and infiltrated PMNs, and it was expressed as total intravascular and infiltrated PMNs per square millimeter area of cardiac tissue. Slides of I/R + PMN control and PKC II peptide inhibitor-treated hearts were viewed on a Nikon E800 epifluorescent microscope, and the images were captured with the Spot RT camera and analyzed with the Phase 3 Image Pro plus 4.5 imaging software.
Measurement of NO Release from Rat Aortic Segments. NO release from rat aortic endothelium was measured to determine whether PKC II peptide inhibition provides cardioprotection by a mechanism involving increased endothelial NO release. Rat aortas were isolated after anesthetizing the rats with 60 mg/kg pentobarbital sodium. The aortic tissue came from the same rats that were used for hearts in the cardiac perfusion experiment. The excised aortas were immersed in warm oxygenated (95% O2, 5% CO2) Krebs-Henselit (K-H) buffer solution. The K-H buffer had the following composition: 10 mM dextrose, 119 mM NaCl, 12.5 mM NaHCO3, 2.5 mM CaCl2, 4.8 mM KCl, 1.2 mM KH2PO4, and 1.2 mM MgSO4. The aortas were cleaned of adherent fat and connective tissue, and rings 6 to 7 mm in length (i.e., 10-mg wet weight) were prepared. The aortic rings were cut, spread opened, and fixed by pins with the endothelial surface facing up in 24-well culture dishes containing 1 ml of K-H solution. After equilibration at 37°C, NO released into the buffer solution was measured after administration of PKC II peptide inhibitor (1–10 µM) to the aortic segments. NO release from rat aortic endothelium was compared with that from samples containing no drug. Acetylcholine (500 nM) was used as a positive control to assess the viability of the endothelium for NO production/release (Tsao et al., 1992). Basal rat aortic endothelial NO release is determined by placing the NO electrode in a well containing only K-H buffer and then placing the NO electrode in a well containing aortic tissue. The difference between the two readings determines the basal NO release for that aortic endothelial segment. After basal NO release is determined, the effect of 500 nM acetylcholine and PKC II peptide inhibitor are then determined. Thereafter, 400 µM L-NAME is added to the K-H buffer solution, and NO release is reassessed 30 min later in the presence of 500 nM acetylcholine or 10 µM PKC II peptide inhibitor. The NO release was measured using a calibrated NO meter (Iso-NO; WPI, Sarasota, FL) connected to a polygraph internally shielded NO electrode (Guo et al., 1996). NO released into the medium was reported in picomoles per milligram of aortic tissue. Between 5 and 17 trials were performed for each group.
Measurement of Superoxide Radical Release from Rat PMNs. Another mechanism that may contribute to the cardioprotective effects (i.e., LVDP) of the PKC II peptide inhibitor may be inhibition of PMN superoxide release. The superoxide anion release from PMNs was measured spectrophotometrically (model 260 Gilford; Nova Biotech, El Cajon, CA) by the reduction of ferricytochrome c (Young et al., 2000). The PMNs (5 x 106) were resuspended in 450 µl of PBS and incubated with 100 µM ferricytochrome c (Sigma Chemical Co.) in a total volume of 900 µl of PBS for 15 min at 37°C in spectrophotometric cells. PKC II peptide inhibitor was added to the 900-µl PMN/ferricytochrome c suspension and mildly vortexed to yield a final concentration of 1, 5, 10, or 20 µM and incubated at 37°C for 15 min in spectrophotometric cells. Control samples did not contain PKC II peptide inhibitor. The PMNs were stimulated with 200 nM fMLP (Sigma Chemical Co.) in a final reaction volume of 1.0 ml. Positive control samples were given 10 µg/ml superoxide dismutase (SOD) just before addition of fMLP. Absorbance at 550 nm was measured every 30 s up to 90 s (peak response) (Young et al., 2000), and the change (
) in superoxide anion release from PMNs was determined from time 0.
Statistical Analysis. All data in the text and figures are presented as means ± S.E.M. The data were analyzed by analysis of variance using post hoc analysis with the Bonferroni/Dunn test. Probability values of 
0.05 are considered to be statistically significant.
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II peptide inhibitor (10 µM), and I/R + PMN groups. It illustrates the changes in LVDP during the 80-min perfusion period.
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However, the hearts in the I/R + PMN group exhibited severe cardiac contractile dysfunction, only recovering to 43 ± 5% of initial baseline values by the end of reperfusion. By contrast the hearts in the I/R + PMN + PKC II peptide inhibitor (10 µM), although initially showing a depression in LVDP of 61 ± 10% of initial baseline values at 15 min into reperfusion, recovered to 82 ± 9% of baseline.
To establish whether the PKC II peptide inhibitor produced any direct inotropic effects on cardiac contractile function, sham I/R hearts were perfused with 10 µM PKC II peptide inhibitor, which was the highest dose administered in this study. This group served as one of the controls for the study. These hearts did not show any significant change in LVDP (Fig. 3) or +dP/dtmax (Fig. 4) at the end of the 80-min reperfusion period, hence indicating that at this dose the PKC II peptide inhibitor has no direct effect on cardiac contractile function.
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II peptide inhibitor (10 µM), I/R, and I/R + PKC II peptide inhibitor (10 µM) groups. However, there was a significant difference between the initial and final values of LVDP and +dP/dtmax for the I/R + PMN group (p < 0.01). This group only recovered to 43 ± 5% in LVDP and 42 ± 6% in +dP/dtmax from initial baseline at 45 min postreperfusion.
The presence of the PKC II peptide inhibitor at a 5 and 10 µM dose attenuated the decrease in LVDP and +dP/dtmax associated with the postischemic reperfusion with PMNs. The 10 µM dose was the most cardioprotective because the hearts in the I/R + PMN + PKC II peptide inhibitor (10 µM) recovered to 82 ± 9 and 79 ± 10% of initial baseline at 45 min postreperfusion for LVDP and +dP/dtmax, respectively. These values were significantly different from I/R + PMN at 45 min postreperfusion (p < 0.01). The 5 µM dose was also cardioprotective, although not to the same extent as the 10 µM dose, because the hearts in the I/R + PMN + PKC II peptide inhibitor (5 µM) recovered to 69 ± 7 and 63 ± 7% for LVDP and +dP/dtmax of initial baseline at 45 min postreperfusion, respectively. The LVDP values for the 5 µM dose were significantly different from I/R + PMN at 45 min postreperfusion (p < 0.05). Although not shown in the results, hearts treated with 2.5 µM(n = 2) showed similar recovery with the hearts in the 5 µM group. The 1 µM dose of PKC II peptide inhibitor was not cardioprotective because the hearts in the I/R + PMN + PKC II peptide inhibitor (1 µM) group only recovered to 57 ± 4 and 53 ± 6% for LVDP and +dP/dtmax, respectively. The final values of LVDP and +dP/dtmax at the 1 µM dose group were not significantly different from the final values of the I/R + PMN group. The cardioprotective effects of the PKC II peptide inhibitor (10 µM) were blocked by the presence of L-NAME (50 µM) in the IR + PMN + PKC II peptide inhibitor (10 µM) + L-NAME (50 µM) group, because the LVDP and +dP/dtmax values at the end of the 45-min reperfusion period were only 56 ± 2 and 53 ± 5% of the initial baseline values, respectively, and were not significantly different from the final values of the IR + PMN group (Figs. 3 and 4).
Figure 5 shows that segments of the endothelium treated with PKC II peptide inhibitor generated significantly more NO compared with the basal NO release for those segments [5 µM (p < 0.05) and 10 µM (p < 0.01)]. The basal value of NO release was measured at 1.85 ± 0.18 pmol NO/mg tissue. There was a definite dose-response effect of stimulating the endothelium with PKC II peptide inhibitor as the 1, 2.5, 5, and 10 µM produced an increase in NO release above basal of 0.75 ± 0.19, 1.91 ± 0.44, 2.54 ± 0.29, and 3.49 ± 0.62 pmol NO/mg tissue, respectively. Acetylcholine (500 nM) was used as a positive control in this assay and stimulated the endothelium, causing an increase of 3.75 ± 0.58 pmol NO/mg tissue above the baseline basal value. The nitric-oxide synthase inhibitor L-NAME was used as another control to decrease basal release of NO to zero. Both the acetylcholine and PKC II peptide inhibitor-stimulated release of NO were completely inhibited by treating the endothelium with 400 µM L-NAME.
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II peptide inhibitor significantly inhibited superoxide release (i.e., absorbance) from suspensions of fMLP-stimulated rat PMNs from 0.13 ± 0.01 to 0.05 ± 0.009 (p < 0.01), 0.02 ± 0.004 (p < 0.01), and 0.02 ± 0.007 (p < 0.01) for 5, 10, and 20 µM, respectively (Fig. 6). There was no significant inhibition of superoxide at the 1 µM dose. SOD (10 µg/ml) was used as a positive control, and it scavenged the superoxide released by the fMLP-stimulated rat PMNs, reducing the response to 0.0016 ± 0.0006.
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During reperfusion, a significant number of PMNs transmigrated into the myocardium, increasing from less than 20 PMN/mm2 to more than 150 PMN/mm2 at the end of the reperfusion period in I/R + PMN hearts (Fig. 7). In the control groups sham, sham + PKC II peptide inhibitor, IR, and IR + PKC II peptide inhibitor, the number of PMNs per square millimeter determined through histological analysis was 18.2 ± 3.1, 19.6 ± 3.5, 27.3 ± 1.2, and 25.9 ± 4.3, respectively. In the IR + PMN group, this number increased to 155.4 ± 5.6 PMNs/mm2. However, in the treated groups of I/R + PMNs + PKC II peptide inhibitor at 1, 5, and 10 µM, the number of PMNs per square millimeter decreased to 88.9 ± 17.7, 80.5 ± 14.1, and 76.3 ± 14.2, respectively. Compared with I/R + PMN hearts, PKC II peptide inhibitor-treated hearts experienced a 43 ± 11, 48 ± 9, and 51 ± 9% reduction in PMN infiltration into the postreperfused cardiac tissue at 1, 5, and 10 µM doses, respectively (Fig. 7). In the I/R + PMN + PKC II peptide inhibitor 10 µM + L-NAME group, where NO was inhibited, the number of PMNs per square millimeter was 158.9 ± 20.5, which is almost identical to the number of PMNs per square millimeter in the I/R + PMN group.
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In the control groups, sham, sham + PKC II peptide inhibitor, IR, and IR + PKC II peptide inhibitor, the number of adhered PMNs per square millimeter determined through histological analysis was 11.2 ± 1.9, 10.5 ± 1.2, 11.2 ± 1.9, and 16.1 ± 3.9, respectively. In the IR + PMN group, this number increased to 53.9 ± 9.4 PMNs/mm2. But in the treated groups of I/R + PMNs + PKC II peptide inhibitor at 1, 5, and 10 µM, the number of PMNs/mm2 decreased to 21 ± 3.6, 23.8 ± 3.1, and 20.3 ± 2.8, respectively. Again compared with I/R + PMN hearts, PKC II peptide inhibitor-treated hearts experienced a 61 ± 7, 56 ± 6, and 62 ± 5% reduction in the number of adherent PMNs at 1, 5, and 10 µM doses, respectively (Fig. 8). In the presence of L-NAME, the number of PMNs adhered was 67.2 ± 6.3 PMNs/mm2. Figure 9 is a photograph of a representative untreated and PKC II-treated I/R + PMN heart that shows the relative differences in total intravascular and infiltrated PMNs in Fig. 9, A and C. PKC II treatment attenuated PMN adherence and infiltration indicated in Fig. 9, B and D.
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Discussion |
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II peptide inhibitor-treated rat hearts (10 µM) exerted maximal restoration of postreperfusion cardiac function (i.e., LVDP and +dP/dtmax) and were associated with 2) maximal attenuation of intravascular PMN adherence/infiltration in postreperfused myocardium. 3) Both of these effects were blocked in the presence of L-NAME, which was similar to I/R + PMN control hearts. 4) NO release in rat aortic segments was significantly increased dose dependently in the presence of PKC II inhibitor (1–10 µM), and this effect was blocked by L-NAME. 5) fMLP-stimulated PMN superoxide release was significantly inhibited by 85% in the presence of PKC II inhibitor (10 µM).
PKC II Peptide Inhibitor Effects on Cardiac Function. The most effective dose of the PKC II peptide inhibitor for attenuating the effects of PMN-induced cardiac contractile dysfunction was the 10 µM dose. The dose regiment used in this study was determined from the results of previous studies that found this range to be most effective in inhibiting the PKC II isoform in cardiac myocytes (Ron et al., 1995). The I/R + PMN hearts + PKC II peptide inhibitor clearly show a dose-response effect in improving cardiac function (i.e., LVDP and +dP/dtmax) with the concentrations used in this study (1–10 µM; Figs. 3 and 4).
I/R hearts that were perfused with PMNs and treated with 10 µM PKC II peptide inhibitor exhibited maximal restoration of postreperfusion LVDP and +dP/dtmax, and there was no significant difference between initial and final (45-min postreperfusion) LVDP and +dP/dtmax values for hearts in this group (Figs. 3 and 4).
Hearts treated with the 5 µM dose also exhibited cardioprotection but to a lesser extent (i.e., 69 ± 7% for LVDP), compared with the 10 µM dose. However, the effects of a 1 µM dose showed minimal postreperfusion recovery (i.e., 57% for LVDP) and were not significantly different from the hearts in the I/R + PMN control group (Figs. 3 and 4).
The hearts in the I/R + PKC II peptide inhibitor (10 µM) and the sham I/R + PKC II peptide inhibitor (10 µM) showed no significant difference between initial baseline and final LVDP and +dP/dtmax values (45-min postreperfusion), establishing that there were no cardiodepressant or cardiotonic effects of the PKC II peptide inhibitor on cardiac contractile function. However, another study showed the PKC II peptide inhibitor reversed PKC-activated human cardiac Na+ channel current depression in Xenopus oocytes (Shin and Murray, 2001), suggesting that PKC II may be involved in electrophysiological regulation of cardiac function. Although differences in the regulation of cardiac Na+ channels between the whole organ preparation (i.e., isolated perfused heart) and cardiac Na+ channel expression in Xenopus oocytes may account for the apparent discrepancy between the two studies.
Mechanism of Action Related to Cardioprotection. Endothelial-derived NO release. This study established that PKC II peptide inhibition results in cardioprotection of the isolated perfused rat heart from PMN-induced I/R injury. The significant restoration of postreperfusion LVDP and +dP/dtmax is a direct indicator of these cardioprotective effects, which can be attributed to enhanced release of endothelium derived NO, which quenches superoxide, improves vasodilation, and inhibits PMN aggregation (Lefer and Lefer, 1996; Pabla et al., 1996). This is further demonstrated by the absence of these cardioprotective effects in the I/R + PMN + PKC II peptide inhibitor (10 µM) + L-NAME group of hearts because the NO release was blocked by the presence of L-NAME (Phillipson et al., 2005). The significant reduction in PMN adherence to the vascular endothelium and infiltration into the postreperfused myocardial tissue also demonstrates these cardioprotective effects (Scalia et al., 1996; Xiao et al., 1998) and suggests that the cardioprotection may be mediated, in part, by a NO mechanism, since L-NAME treatment in heart and aortic tissue resulted in significant increases in postreperfusion PMN vascular adherence and infiltration and decreases in PKC II peptide inhibitor stimulated NO release, respectively.
Preserving endothelial NO release attenuates endothelial dysfunction and inhibits adherence and infiltration of PMNs into the coronary vasculature and surrounding tissue, thus attenuating cardiac contractile dysfunction (Ma et al., 1993; Pabla et al., 1996). The aortic NO release data (Fig. 5) show a direct relationship between the dose of PKC II peptide inhibitor used to significantly augment NO release and cardioprotection of the hearts (Figs. 3 and 4) and establishes a correlation between the two assays.
PMN superoxide release. The inhibition of PKC II peptide inhibits the activation of PMNs, thereby inhibiting the release of superoxide and attenuating the cardiac contractile dysfunction associated with its release (Young et al., 2001). Similar to the NO release data, the doses of the PKC II peptide inhibitor that significantly attenuated fMLP-stimulated PMN superoxide release (Fig. 6) show a direct relationship to the cardioprotective doses (Figs. 3 and 4) and suggest that the mechanism of cardioprotection may be due either to enhanced NO release (Fig. 5) or attenuated superoxide release (Fig. 6), or to a combination of both (Lefer and Lefer, 1996; Yan and Novak, 1999; Phillipson et al., 2005). Oxygen-derived free radicals such as superoxide up-regulate endothelial cell adhesion molecules and quench endogenous NO (Patel et al., 1991; Davenpeck et al., 1994). NO inhibits the leukocyte-endothelial cell interaction by suppressing up-regulation of endothelial cell adhesion molecules (Davenpeck et al., 1994; Lefer and Lefer, 1996). Therefore, the PKC II peptide inhibitor that attenuates superoxide production from PMNs and increases the bioavailability of NO would attenuate the expression of endothelial cell adhesion molecules, and this would effectively diminish the transmigration of PMNs into cardiac tissue and the subsequent release of superoxide radicals from transmigrated PMNs at or near cardiomyocytes (Young et al., 2000).
In this study, it is evident that the number of PMNs present in the postischemic reperfused myocardial tissue (Figs. 7, 8, 9) is associated with cardiac contractile dysfunction in untreated I/R + PMN hearts, and this effect is attenuated by the PKC II peptide inhibitor treatment (Fig. 9).
Role of PKC Isozymes in Cardiac Ischemia/Reperfusion. During the early reperfusion period (5 min) of acute myocardial ischemia, PKC activation and cell membrane expression are enhanced (Strasser et al., 1992). PKC isozymes translocate on activation from one cellular compartment to another. RACKs are the receptor proteins responsible for translocation and subsequent function of a PKC enzyme (Ron and Mochly-Rosen, 1995). Activated PKC II binds to the RACK-1 binding site, which increases the PKC II isoform phosphorylation of substrates (e.g., NADPH oxidase and endothelial nitric-oxide synthase), by stabilizing the active form of PKC II and translocating it to the cell membrane (Ron et al., 1995). NADPH oxidase activation in both PMNs and endothelial cells generates reactive oxygen species (e.g., superoxide), which cause oxidative damage and perpetuates cardiac contractile dysfunction (Ma et al., 1991; Hansen, 1995). The PKC II peptide inhibitor occupies the RACK-1 binding site, preventing the binding of PKC II and thereby inhibiting the translocation of the activated PKC II isoform and its function of activating NADPH oxidase (Korchak and Kilpatrick, 2001).
Other PKC isoforms have been shown to be either beneficial (e.g., PKC ) or injurious (e.g., PKC ) when activated in cardiac and endothelial function (Hu et al., 2000; Das, 2003; Phillipson et al., 2005). PKC activation has been associated with recovery of postreperfusion LVDP (Inagaki et al., 2003), but it requires pretreatment of PKC activator before ischemia to elicit cardioprotection in contrast to the PKC II inhibitor given at the beginning of reperfusion. PKC has been implicated in superoxide release in PMNs and endothelial cells (Dang et al., 2001; Frey et al., 2002), adhesion molecule up-regulation (Rahman et al., 2000), and restoration of cardiac function in I/R (Peterman et al., 2004; Phillipson et al., 2005). However, the PKC II inhibitor restores early postreperfusion LVDP (i.e., 10 min) sooner and does not exhibit cardiodepressant effects at higher doses compared with compounds that inhibit the PKC isoform (Peterman et al., 2004; Phillipson et al., 2005). An interesting prospective study would be to characterize the potential cardioprotective effects of a combination of PKC and PKC II peptide inhibitors on cardiac and endothelial function.
In summary, these results show a cardioprotective effect of a selective PKC II peptide inhibitor on LVDP and +dP/dtmax on PMN-induced myocardial ischemia/reperfusion injury in the isolated perfused rat heart. The cardioprotection seems to be related to an increase in endothelial-derived NO along with inhibition of PMN-generated superoxide release and PMN adherence to the vascular endothelium, resulting in fewer PMNs infiltrating into the cardiac tissue. The resulting decrease in superoxide radical release at the level of cardiomyocytes leads to an attenuation of cardiac contractile dysfunction in PMN-induced I/R injury.
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Footnotes |
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ABBREVIATIONS: PMN, polymorphonuclear leukocyte; PKC, protein kinase C; I/R, ischemia/reperfusion; RACK-1, receptor for activated C kinase-1; LVDP, left ventricular developed pressure; +dP/dtmax, maximal rate of development of pressure; PBS, phosphate-buffered saline; K-H, Krebs-Henselit; L-NAME, N
-nitro-L-arginine methyl ester; fMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, SOD, superoxide dismutase.
Address correspondence to: Dr. Lindon H. Young, Department of Pathology, Microbiology, and Immunology, Philadelphia College of Osteopathic Medicine, 4170 City Ave., Philadelphia, PA 19131. E-mail: lindonyo{at}pcom.edu
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