In human patients and animal models, the most effective means of limiting myocardial damage and restoring ventricular function is through early reperfusion. 1 However, myocardial ischemia followed by reperfusion initiates deleterious biochemical and structural changes that result in cardiac arrhythmias, prolonged left ventricular dysfunction, and myocardial cell injury. 2–4 Reperfusion injury is characterized by a decrease in endothelial release of nitric oxide, up-regulation of endothelial surface adhesion molecules, increased leukocyte-endothelium interaction, and transmigration of polymorpho-nuclear leukocytes (PMNs) into the myocardium producing cardiac dysfunction. 4–6 A rapid decrease in endothelium-derived nitric oxide (NO) begins 2.5 to 5 minutes postreperfusion and PMN infiltration of the myocardium is observed 30 minutes postreperfusion. 5,7
PMNs activated by plasma factors trigger endothelial and myocardial damage by releasing oxygen-derived free radicals, inflammatory cytokines, and proteolytic enzymes. 8–10 The reactive oxygen species released by PMNs represent the primary cause of myocardial necrosis and contractile dysfunction following postischemic reperfusion. 3,8 These free radicals not only inactivate NO, but also initiate lipid peroxidation and alter membrane permeability to ions. 11 Inactivation of NO is significant because a decrease in basal release of NO promotes PMN adherence to the coronary endothelium and subsequent transmigration into inflamed (ie, ischemic/reperfused) tissues. 12,13 In contrast, inhibition of superoxide release from PMNs is associated with decreased adherence to the endothelium, as well as diminished transmigration into postischemic tissue. 8,14,15
Protein kinase C (PKC) is an important mediator of PMN activation (eg, superoxide release and chemotaxis) and endothelial adhesion molecule up-regulation and superoxide release. 16–19 PKC inhibition of the postischemic coronary endothelium preserves basal endothelial NO release and inhibits the subsequent adherence and transmigration of PMNs into the postischemic cardiac tissue. 11,20
PMNs possess 5 isoforms of PKC, including α-PKC, βI-PKC, βII-PKC, δ-PKC, and ζ-PKC. 21 The PKC family of isozymes plays an important role in the regulation of NADPH oxidase activity via phosphorylation of phox 47, a component of the NAPDH oxidase enzyme. 18 Previous studies have shown that the βII-PKC isoform is responsible for superoxide release from activated PMNs. 22,23 However, the βII-PKC superoxide release only accounts for about 50% of the pharbol-12-myristate-13-acetate (PMA)-stimulated PMN-generated superoxide production. 23 The other 50% of superoxide release may be generated by the Ca2+ and diacylglycerol (DAG) insensitive ζ-PKC isoform. 18 ζ-PKC, which is abundantly expressed in human and rat PMNs, has been implicated in the regulation of PMN superoxide release and chemotaxis. 16,18 It has also been demonstrated that ζ-PKC regulates tumor necrosis factor (TNF)-α–induced activation of NADPH oxidase in endothelial cells, leading to superoxide production. 24 In addition, ζ-PKC can induce intercellular adhesion molecule 1 (ICAM-1) expression on vascular endothelium. 17 ICAM-1 expression is necessary for PMN adhesion to vascular endothelium prior to PMN transmigration. 17 Therefore, blocking ζ-PKC in addition to βII-PKC could prove to be cardioprotective by inhibiting the generation of reactive oxygen species by activated PMNs.
Gö 6983 (M.W. = 442.5) is a bisindolylmaleimide compound, which passes into cells by simple diffusion. Unlike other broad-spectrum PKC inhibitors previously studied in myocardial ischemia/reperfusion (ie, staurosporine), Gö 6983 also inhibits ζ-PKC (IC50 = 60 nM) in addition to the other 4 PKC isoforms in PMNs. 25,26 Gö 6983 has not previously been evaluated in myocardial I/R injury, and may offer greater cardioprotection than other broad-spectrum PKC inhibitors in postischemic reperfusion injury due to the additional inhibition of ζ-PKC. Staurosporine and other staurosporine analog compounds (ie, bisindolylmalemidies) previously tested in MI/R 20,27 do not effectively inhibit the ζ-PKC isoform within the same concentration range as inhibition of the other PKC isoforms. 28,29 Therefore, ζ-PKC inhibition may further attenuate PMN superoxide release and adherence to post reperfused vascular endothelium. Other non-staurosporine analog PKC inhibitor compounds such as chelerythrine and calphostin C have shown to be cardioprotective in MI/R 20,27; however, these compounds inhibit other enzymes in addition to PKC such as alanine aminotransferase and phospholipase D, respectively. 30 Therefore, the cardioprotective effects of chelerythrine and calphostin C in MI/R may be independent of PKC inhibition. It could be hypothesized that, by inhibiting ζ-PKC, Gö 6983 may prevent further PMN adhesion, superoxide release, and transmigration into the postischemic myocardium and the subsequent release of oxygen-derived free radicals.
The purposes of the present study were to (1) determine the extent of cardioprotection by Gö 6983 on cardiac contractile function in the isolated perfused rat heart after PMN-induced I/R injury, (2) establish the concentration-response relationship of Gö 6983 in this model, and (3) investigate the mechanism of any observed cardioprotective effect of Gö 6983 relevant to PMN superoxide radical release.
METHODS
The Institutional Animal Care and Use Committee (IACUC) of Philadelphia College of Osteopathic Medicine approved all animal protocols performed in this study.
Isolation of Polymorphonuclear Leukocytes and Plasma
Sprague Dawley rats (350–400 g), used as PMN donors, were anesthetized with ethyl ether and were given a 16 mL i.p. injection of 0.5% glycogen (Sigma) dissolved in phosphate buffered saline (PBS). The rats were re-anesthetized with ethyl ether 16 to 18 hours later, and the PMNs were harvested by peritoneal lavage in 30 mL of 0.9% NaCl, as previously described. 31 The peritoneal lavage fluid was centrifuged at 250 × g for 20 minutes at 4°C. The PMNs were then washed in 15 mL of PBS and centrifuged at 250 × g for 10 minutes at 4°C. Thereafter, the PMNs were resuspended in 2.5 mL of PBS and a total of 10 to 12 samples were pooled prior to 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.
The plasma used for infusion with the PMNs was isolated from the same rat from which the heart was isolated from for each cardiac perfusion experiment to more closely simulate the conditions in vivo. Blood was collected from the aorta in citrate phosphate buffer (Sigma Chemical Co., St. Louis, MO) over a period of 1 minute just before isolation of the rat heart. The blood was centrifuged at 10,000 × g for 10 minutes. 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.
Isolated Rat Heart Preparation
Male Sprague Dawley rats (275–325 g) were anesthetized with 60 mg/kg pentobarbital sodium intraperitoneally (i.p.). Sodium heparin (1000 U) was also administered i.p. The hearts were rapidly excised, the ascending aortas were cannulated, and retrograde perfusion of the heart was initiated with a modified Krebs buffer maintained at 37°C at a constant pressure of 80 mm Hg. The Krebs buffer had the following composition (in mmol/l): 17 dextrose, 120 NaCl, 25 NaHCO3, 2.5 CaCl2, 0.5 EDTA, 5.9 KCl, and 1.2 MgCl2. The perfusate was aerated with 95% O2-5% CO2 and equilibrated at a pH of 7.3–7.4. The 2 side arms in the perfusion line proximal to the heart inflow cannula allowed PMNs, plasma, or plasma containing various concentrations of Gö 6983 to be directly infused into the coronary inflow line. Coronary flow was monitored by a flowmeter (T106, Transonic System, Inc., Ithaca, NY). LVDP and +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 (Gateway).
Left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dtmax), +dP/dtmax, and coronary flow were measured every 5 minutes for 15 minutes to equilibrate the hearts and obtain a baseline measurement. LVDP was defined as left ventricular end-systolic pressure minus left ventricular end-diastolic pressure.
After 15 minutes, the flow of the Krebs buffer was reduced to zero for 20 minutes to induce global ischemia. Throughout the entire ischemic period the heart remained immersed in a water-jacketed reservoir containing 37°C Krebs buffer, and the temperature was maintained by a circulating water bath (ThermoHaake P5, Fisher Scientific, Pittsburgh, PA). At reperfusion, hearts were infused for 5 minutes with 200 × 106 PMNs resuspended in 5 mL of Krebs buffer plus 5 mL of plasma at a rate of 1 mL/min. The control I/R and sham hearts were also reperfused with plasma at a rate of 1 mL/min for the first 5 minutes of reperfusion or after 35 minutes perfusion in sham I/R hearts. In some experiments, Gö 6983 (Calbiochem, Inc., La Jolla, CA) was added to plasma at a final concentration of 25, 50, 100, or 200 nM. One group of sham I/R hearts were not subjected to ischemia and were not perfused with PMNs, and another group of sham I/R hearts were not subjected to ischemia but were perfused with PMNs for 5 minutes after 35 minutes of perfusion. Previous studies showed that sham I/R hearts given PMNs exhibited no changes from initial control values. 31 In some sham I/R hearts, Gö 6983 (100 nM) was infused at a rate of 1 ml/min for 5 minutes after 35 minutes of perfusion. Data were recorded every 5 minutes for 45 minutes of postreperfusion. After each experiment, the left ventricle was isolated, fixed in 4% paraformaldehyde, and stored at 4°C for subsequent histologic analysis.
Groups of Isolated Perfused Hearts
The following groups of isolated perfused rat hearts were used:
Group 1: sham I/R. Hearts not subjected to ischemia and not perfused with PMNs (n = 7).
Another control group of sham I/R hearts was not subjected to ischemia but was perfused with PMNs (n = 6).
Group 2: sham I/R+Gö 6983. Hearts not subjected to ischemia and not perfused with PMNs but infused with Gö 6983 (100 nM) (n = 7).
Group 3: I/R. Hearts subjected to ischemia but reperfused without PMNs (n = 9).
Group 4: I/R+Gö 6983. Hearts subjected to ischemia but reperfused with Gö 6983 (100 nM) without PMNs (n = 9).
Group 5: I/R+PMNs. Hearts subjected to ischemia and reperfused with PMNs (n = 9).
Group 6: I/R+PMNs+Gö 6983 (25 nM). Hearts subjected to ischemia and reperfused with PMNs and Gö 6983 (25 nM) (n = 7).
Group 7: I/R+PMNs+Gö 6983 (50 nM). Hearts subjected to ischemia and reperfused with PMNs and Gö 6983 (50 nM) (n = 8).
Group 8: I/R+PMNs+Gö 6983 (100 nM). Hearts subjected to ischemia and reperfused with PMNs and Gö 6983 (100 nM) (n = 7).
Group 9: I/R+PMNs+Gö 6983 (200 nM). Hearts subjected to ischemia and reperfused with PMNs and Gö 6983 (200 nM) (n = 8).
Determination of Polymorphonuclear Leukocytes Vascular Adherence and Infiltration into the Cardiac Tissue
Three rat hearts from each of the 9 experimental groups were used for histologic analysis. Ten similar areas of each heart section, ranging from the endocardium throughout the myocardium to the epicardium of the left ventricle, were counted for PMN vascular adherence and infiltration. 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 placed in 100% ethanol for 5 minutes to remove the plastic and rehydrated in tap water for 1 minute. Subsequently, hematoxylin was applied to the sections for 7 minutes and the sections were rinsed under running tap water for 30 seconds. Eosin stain was then applied for 2 minutes, followed by a second rinse under running tap water for 30 seconds. The number of PMNs was counted by light microscopy. To determine the effect of Gö 6983 on PMN adherence, the intravascular PMNs that adhered to the coronary vascular endothelium were counted. These results are expressed as intravascular and infiltrated PMNs/mm2 area of cardiac tissue.
Measurement of Superoxide Radical Release from Rat Polymorphonuclear Leukocytes
The superoxide anion release from PMNs was measured spectrophotometrically (model 260 Gilford, Nova Biotech, El Cajon, CA) by the reduction of ferricytochrome C. 32 The PMNs (5 × 106) were resuspended in 450 μL PBS and incubated with ferricytochrome C (100 μM, Sigma) in a total volume of 900 μL of PBS for 15 minutes at 37°C in spectrophotometric cells. Gö 6983 was added to the 900 μL PMN/ferricytochrome C suspension and mildly vortexed to yield a final concentration of 10, 25, 50, 100, or 200 nM and incubated at 37°C for 15 minutes in spectrophotometric cells. Control samples did not contain Gö 6983. The PMNs were stimulated with 15 nM PMA (Sigma) in a final reaction volume of 1.0 mL. Positive control samples were given superoxide dismutase (SOD) (10 μg/ml) just prior to addition of PMA. Absorbance at 550 nm was measured every 30 seconds up to 360 seconds (peak response) and the change (Δ) in superoxide anion release from PMNs was determined from time zero.
Statistical Analysis
All data in the text and figures are presented as means ± SEM. The data were analyzed by ANOVA using post hoc analysis with Bonferroni/Dunn test. Probability values of <0.05 were considered to be statistically significant.
RESULTS
Coronary flow (ml/min) data for the 9 groups is presented in Figure 1. The initial coronary flow values for all study groups were not significantly different from each other. Sham I/R hearts that were not subjected to ischemia and not perfused with PMNs did not show any significant differences between initial and final coronary flow values. However, the remaining 7 groups of hearts subjected to 20 minutes of ischemia followed by 45 minutes of reperfusion in the presence or absence of PMNs experienced a significant reduction in final compared with initial coronary flow values (P < 0.01). Furthermore, the final coronary flow values from the 7 groups of hearts subjected to 20 minutes of ischemia were not significantly different from each other. Sham I/R hearts that were not subjected to ischemia but perfused with PMNs also experienced a significant reduction (P < 0.05) between initial (20.3 ± 1.4 mL/min) and final (12.7 ± 0.7 mL/min) coronary flow values (data not shown in graph); however these hearts did not experience a significant reduction in LVDP or +dP/dtmax be tween initial (90.8 ± 2.5 mm Hg, 2366.4 ± 61.4 mm Hg/s) and final (83.2 ± 3.5 mm Hg, 2201.8 ± 78.9 mm Hg/s) values, respectively.
;)
FIGURE 1.:
Initial and final coronary flow expressed in ml/min in isolated perfused rat hearts before ischemia (I) and after reperfusion (R). Ischemic hearts were perfused in the presence or absence of PMNs. All values are expressed as mean ± SEM. Numbers of hearts are at the bottom of the bars. NS, not significant.
The time course of cardiac contractile function (ie, LVDP) is shown in Figure 2. The data from the sham I/R, I/R, I/R+PMN+Gö 6983 (100 nM), and I/R+PMN groups illustrate the relative changes in LVDP during the 80-minute perfusion period. As shown, the sham I/R remained at or near 100% of initial baseline values of LVDP for the entire perfusion period. The I/R hearts experienced a depression in LVDP at the beginning of reperfusion, but recovered to 97 ± 1% of initial baseline values by the end of reperfusion. However, the I/R+PMN hearts suffered severe cardiac contractile dysfunction, recovering to only 55 ± 3% of initial baseline values by 45 minutes postreperfusion. In contrast, the I/R+PMN+Gö 6983 (100 nM) hearts recovered to 67 ± 6% of initial baseline values by 15 minutes postreperfusion and continued to significantly improve throughout the reperfusion period to 89 ± 4% at 45 minutes postreperfusion.
FIGURE 2.:
Time course of LVDP in sham, I/R, I/R+PMNs, and I/R+PMN+Gö 6983 (100 nM) perfused rat hearts. LVDP data at initial (baseline) and reperfusion from 0 to 45 minutes after 20 minutes of ischemia. The I/R+PMN group (n = 9) exhibited a significant and sustained reduction in LVDP compared with I/R (n = 9) and I/R+PMN+Gö 6983 (100 nM) (n = 7) groups. All values are expressed as mean ± SEM. *P < 0.05 and **P < 0.01 from I/R+PMNs.
To determine whether Gö 6983 produced direct inotropic effects on cardiac contractile function, nonischemic sham I/R hearts were perfused with Gö 6983 (100 nM). Treatment of sham I/R hearts with Gö 6983 did not result in any significant change in LVDP (Fig. 2) or +dP/dtmax (Fig. 3) during the 80-minute perfusion period, demonstrating that Gö 6983 exerts no direct effect on cardiac contractile function. Furthermore, perfusion of untreated I/R hearts without PMNs did not result in prolonged cardiac contractile dysfunction, indicating that global ischemia was not the cause of sustained contractile dysfunction in this model of I/R. In addition, I/R hearts treated with Gö 6983 (100 nM) recovered similarly to untreated I/R hearts, furthering the claim that Gö 6983 exerts no direct effect on cardiac contractile function.
FIGURE 3.:
Initial and final LVDP expressed in mm Hg from isolated perfused rat hearts before ischemia (I) (initial) and after 45 minutes of reperfusion (R) (final). Hearts were perfused in the presence or absence of PMNs. PMNs induced a significant contractile dysfunction, which was attenuated by Gö 6983. All values are expressed as mean ± SEM. Numbers of hearts are at the bottom of the bars. *P < 0.05 and **P < 0.01 from I/R+PMNs. NS, not significant.
Figures 3 and 4 show the initial and final values for LVDP and +dP/dtmax from isolated perfused rat hearts. The initial baselines are similar for all groups. However, the final LVDP and +dP/dtmax (45 minutes postreperfusion) is significantly decreased (P < 0.01) for the I/R hearts perfused with PMNs compared with its initial baseline. The Gö 6983 middle concentrations (50 and 100 nM) significantly attenuated the decrease in LVDP and +dP/dtmax associated with postischemic perfusion with PMNs. Although the 100 nM concentration showed the greatest improvement in LVDP and +dP/dtmax, the 50 nM concentration of Gö 6983 also was significantly different from final I/R+PMN values at 45 minutes postreperfusion. These hearts recovered to 78 ± 1% for LVDP and 70 ± 1% for +dP/dtmax as compared with initial baseline values. The 25 nM Gö 6983-treated hearts were not significantly different from I/R+PMN control hearts, recovering to only 62 ± 3% and 54 ± 4% for LVDP and +dP/dtmax, respectively. Similarly, the 200 nM Gö 6983-treated hearts showed only a modest 45 ± 5% and 39 ± 1% improvement for LVDP and +dP/dtmax, respectively, when compared with the recovery of the I/R+PMN control hearts.
FIGURE 4.:
Initial and final +dP/dtmax expressed in mm Hg/s in isolated perfused rat hearts before ischemia (I) (initial) and after 45 minutes of reperfusion (R) (final). Hearts were perfused in the presence or absence of PMNs. PMNs induced a significant contractile dysfunction, which was attenuated by Gö 6983. All values are expressed as mean ± SEM. Numbers of hearts are at the bottom of the bars. *P < 0.05 and **P < 0.01 from I/R+PMNs. NS, not significant.
In this I/R model, the cardiac injury associated with I/R results from the substantial number of PMNs infiltrating the myocardium within the 45-minute reperfusion period. During reperfusion, a significant number of PMNs transmigrated into the myocardium, increasing from less than 20 PMN/mm2 initially to more than 170 PMN/mm2 at the end of the reperfusion period. As opposed to I/R+PMN hearts, Gö 6983-treated hearts experience a 51 ± 2% and 55 ± 2% reduction in PMN infiltration into the postreperfused cardiac tissue in the 50 and 100 nM range, respectively (Fig. 5A). The attenuation of neutrophils infiltrating the myocardium is an important factor in preserving cardiac contractile function during I/R injury.
FIGURE 5.:
A, Histologic assessment of total intravascular and infiltrated PMNs in isolated perfused rat heart samples taken from 3 rats per group and 10 areas per heart. **P < 0.01.
Another component of the cardioprotective effects offered by Gö 6983 may be associated with an inhibition of PMN adherence to vascular endothelium. As seen in Figure 5B, the number of adherent PMNs is substantially reduced in Gö 6983-treated hearts in comparison with untreated I/R+PMN hearts. Both the 50 and 100 nM concentrations of Gö 6983 reduce the adherence of PMNs to the vascular endothelium by 45 ± 4% and 61 ± 1%, respectively (Fig. 5B).
FIGURE 5.:
B, Histologic assessment of intravascular PMNs that adhered to the coronary vasculature in isolated perfused rat heart samples taken from 3 rats per group and 10 areas per heart. All values are mean numbers of PMNs/mm2 of heart area ± SEM. The numbers of PMNs infiltrated into postreperfusion cardiac tissue and adhering to coronary vasculature was significantly attenuated by Gö 6983. **P < 0.01.
One of the possible mechanisms of the cardioprotective effects of Gö 6983 may be due to inhibition of superoxide release from infiltrated PMNs. Supporting this concept, Gö 6983 significantly inhibited superoxide release from suspensions of PMA-stimulated rat PMNs by 29 to 90% in the 25 to 100 nM range (Fig. 6). The superoxide scavenger, SOD (10 μg/ml), was used as a positive control in the superoxide assays. SOD scavenged the superoxide release produced by the PMA-stimulated rat PMNs by 99% (P < 0.01;Fig. 6). Gö 6983 (100 nM) attenuated superoxide release by 90%, whereas SOD scavenged practically all of the superoxide radicals generated by activated rat PMNs.
FIGURE 6.:
Superoxide release from rat PMNs. Superoxide release was measured from 5 × 106 PMNs after PMA (15 nM) stimulation. SOD (10 μg/ml) was employed as a positive control. The change in absorbance (Δ) was measured 360 seconds after PMA addition (peak response). Superoxide release was significantly inhibited by Gö 6983. *P < 0.05, **P < 0.01. All values are means ± SEM; numbers at bottom of bars are numbers of separate experiments.
DISCUSSION
In this study, we demonstrated the cardioprotective effects exerted by the compound Gö 6983 against PMN-induced I/R injury in the isolated perfused rat heart. The cardioprotective effects of Gö 6983 were characterized by a significant restoration of postreperfusion LVDP and +dP/dtmax in isolated PMN perfused rat hearts. These effects of the PKC inhibitor Gö 6983 are most likely due to a significant reduction in PMN adherence to the vascular endothelium, thereby leading to a substantial reduction of infiltrating PMNs into postreperfused cardiac tissue. 33 In addition, the significant reduction in cardiac function associated with the I/R + PMN group (only recovered to 55% of initial baseline for LVDP) is most likely not attributed to a decrease in postreperfused coronary flow (50% of initial baseline) since the I/R + PMN + Gö 6983 treated group (100 nM) recovered to 89% of initial baseline value for LVDP despite having a decrease in postreperfused coronary flow (46% of initial baseline). Furthermore, Sham I/R hearts perfused with PMNs recovered to 93% of initial baseline for LVDP despite a decrease in postreperfused coronary flow (63% of initial baseline). Previous studies have also shown that Sham I/R hearts perfused with PMNs do not result in an increase in post-I/R cardiac infiltration. 31 These results suggest that the combination of I/R reperfused with PMNs are required for the cardiac dysfunction in this MI/R model since significant reductions in coronary flow or PMN infusion in the absence of I/R do not result in cardiac contractile dysfunction separately.
The cardioprotective effects of Gö 6983 are associated with a decrease in PMN adherence to the vascular endothelium and PMN infiltration into postreperfused cardiac tissue, which may be mediated in part by suppressing superoxide release from PMNs. 8,34,35
In this regard, previous studies have shown that oxygen-derived free radicals up-regulate endothelial cell adhesion molecules (eg, P-selectin) and quench endogenous NO. 36,37 NO acts as a physiologic inhibitor of leukocyte-endothelial cell interaction by suppressing up-regulation of endothelial cell adhesion molecules. 6,37,38 In addition, Gö 6983 may also attenuate superoxide radical release from PMNs that have already migrated into the myocardium, and thus diminish the cardiotoxic effect of oxygen-derived free radicals on cardiomyocytes. 10 Therefore, antioxidative substances like Gö 6983 that reduce superoxide production from PMNs would tend to attenuate expression of endothelial cell adhesion molecules, effectively diminishing transmigration of PMNs into cardiac tissue and subsequent release of superoxide radicals from transmigrated PMNs at or near cardiomyocytes. 15
Broad-spectrum inhibitors of PKC (ie, staurosporine) also inhibit superoxide release from PMNs. 34–36 PKC activation is enhanced during acute myocardial ischemia particularly during the early reperfusion period, 39 and is associated with decreased NO release from the coronary endothelium, 40 as well as an increase in superoxide and hydrogen peroxide release from endothelial cells, 41 and up-regulation of the endothelial cell adhesion molecules. 33
Findings in this study demonstrate that Gö 6983 inhibits the superoxide release from PMNs in vitro and suggest a possible mechanism for the cardioprotective effects of Gö 6983 in this study. Gö 6983 is a bisindolymaleimide that is structurally similar to the potent PKC inhibitor staurosporine. 26 Inhibition of PMN superoxide release can significantly retard the cardiodepressant effects of PMN-derived superoxide radicals directly on cardiac myocytes. 20,42 Similar to staurosporine, Gö 6983 enters the cell via simple diffusion and inhibits PKC by binding to its ATP binding site. 20,43 However, Gö 6983 is reported to be a more selective inhibitor of the ζ-PKC isoform than staurosporine 26,28 and may provide better cardioprotection. 19 The cardioprotective effect of staurosporine in previous myocardial I/R studies is most likely attributed to inhibiting βII-PKC. 19,20,23 However, inhibiting βII-PKC only accounts for approximately 50% inhibition of PMN superoxide release. 19,23
The present results show that Gö 6983 (25–100 nM) concentration-dependently inhibited PMA-stimulated PMN superoxide release from 29 to 90%. Gö 6983 (100 nM) effectively inhibits both βII-PKC and ζ-PKC isoforms and most likely accounts for the 90% inhibition of PMN superoxide release in the current study. The effective concentration for inhibiting the ζ-PKC isoform (IC50 = 60 nM) 26 corresponds with significant recovery of postischemic LVDP and +dP/dtmax cardiac function and 60 to 90% PMN superoxide release inhibition in the 50 and 100 nM Gö 6983 concentrations. At 25 nM, Gö 6983 only modestly inhibited PMN superoxide release by 29% and was not effective in attenuating PMN-induced cardiac contractile dysfunction. However, Gö 6983 (200 nM) had a reduced cardioprotective effect compared with the cardioprotective Gö 6983 concentrations (50 and 100 nM) despite inhibiting PMN superoxide release by 99%. At the high concentration of 200 nM Gö 6983, selectivity for PKC may be lost. This could lead to an increase in cell death, considering only 90% PMNs were viable as determined by 0.3% trypan blue exclusion when exposed to 200 nM Gö 6983 compared with 95% PMN viability at all other concentrations (25–100 nM). Therefore, it is plausible that there may be an increase in coronary endothelial cell and cardiac myocyte cell death at 200 nM Gö 6983 as well. These effects would also lead to an increase in cytokine release from these cell types causing more PMN-induced cardiac contractile dysfunction when exposed to 200 nM Gö 6983.
Recent studies have shown that ζ-PKC plays a role in superoxide release, adhesion molecule expression, and chemotaxis. 16–18 ζ-PKC is expressed abundantly in PMNs and upon activation is translocated from the cytosol to the PMN plasma membrane where it participates in the regulation of NADPH oxidase. 16,18 An obligatory step in superoxide generation by NADPH oxidase is the phosphorylation of p47phox on serines 303/304 and 315 by ζ-PKC. 18 Therefore, by inhibiting ζ-PKC, the cardiodepressant effects resulting from activated PMNs may be significantly attenuated.
Involvement of ζ-PKC in the signaling pathway leading to neutrophil adhesion and chemotaxis has also been previously demonstrated. 16 ICAM-1 is expressed basally in endothelial cells, but during reperfusion injury, expression of ICAM-1 greatly increases in response to inflammatory cytokines. 17 ζ-PKC stimulates ICAM-1 expression thereby allowing for firm adhesion of PMNs to vascular endothelium. 16,17 Therefore, inhibiting ζ-PKC activation would prevent the pro-inflammatory effects leading to PMN adherence and transmigration into the cardiac tissue. This would significantly limit the cytotoxic effects induced by PMN extravasation during the postreperfusion inflammatory response.
The novelty in this study exists in the use of the bisindolylmaleimide compound Gö 6983 in myocardial I/R. Gö 6983 has been reported to differ from other broad spectrum staurosporine-like compounds (bisindolylmaleimides) in that it inhibits the ζ-PKC isoform in addition to the classic (α-PKC, βI-PKC, βII-PKC) and novel (δ-PKC) isozymes of PKC present in PMNs. 26,28 This is the first study in which a broad-spectrum PKC inhibitor also effecting ζ-PKC was used to attenuate PMN-induced I/R injury in the isolated perfused rat heart model. The findings suggest that specifically targeting ζ-PKC in addition to the other isoforms of PKC in PMNs (ie, βII-PKC) results in significant attenuation of cardiac contractile dysfunction following PMN-induced I/R injury. Due to the marked cardioprotective effects associated with Gö 6983, an interesting prospective for research might focus on specifically inhibiting the ζ-PKC isozyme to prevent injury induced by PMN activation.
CONCLUSION
In summary, these results are the first to show a cardioprotective effect of Gö 6983 on PMN-induced myocardial ischemia-reperfusion injury in the isolated perfused rat heart. The cardioprotective effects of Gö 6983 were associated with a reduction in PMN adherence to the vascular endothelium and PMN infiltration into postreperfused cardiac tissue, which may be related in part to decreased superoxide production. The net result would be that Gö 6983 attenuates cardiac contractile dysfunction in PMN-induced I/R injury.
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