Protein Kinase C βII Peptide Inhibitor Exerts Cardioprotective Effects in Rat Cardiac Ischemia/Reperfusion Injury

Abstract

Ischemia followed by reperfusion (I/R) in the presence of polymorphonuclear leukocytes (PMNs) results in a marked cardiac contractile dysfunction. A cell-permeable protein kinase C (PKC) ␤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 M; n ϭ 7) significantly attenuated PMN-induced cardiac dysfunction compared with I/R hearts (n ϭ 9) receiving PMNs alone in left ventricular developed pressure (LVDP) and the maximal rate of LVDP (ϩdP/ dt max ) cardiac function indices (p Ͻ 0.01). The PKC ␤II inhibitor at 10 M significantly increased endothelial NO release from a basal value of 1.85 Ϯ 0.18 pmol NO/mg tissue to 3.49 Ϯ 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 Ϯ 0.01 to 0.02 Ϯ 0.004 (p Ͻ 0.01) at 10 M. 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 ϩ PMN hearts (p Ͻ 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.In the setting of myocardial ischemia, it is imperative that blood flow be restored as soon as possible. Early reperfusion remains the most effective way of limiting myocardial necrosis and improving ventricular function in experimental models and human patients (Forman et al., 1989). 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 microcirculatory flow (Forman et al., 1989;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 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 Ma et al., 1993;Young et al....

Apocynin Exerts Dose-Dependent Cardioprotective Effects by Attenuating Reactive Oxygen Species in Ischemia/Reperfusion

“…The SO release from Poly Morphonuclear Leukocytes (PMNs) was measured spectrophotometrically (model 260 Gilford, Nova Biotech; El Cajon, CA) by the reduction of ferricytochrome c as previously described [23]. The PMNs (5 × 10 6 ) were resuspended in 450 μl PBS and incubated with ferricytochrome c (100 µM, Sigma Chemical) in a total volume of 900 μl PBS in the presence or absence of varying concentrations of apocynin (MW = 166 g/mol; Sigma …”

“…Regarding NADPH oxidase and eNOS uncoupling, activation of Protein kinase C beta II isoform is mostly responsible for NADPH oxidase activation and inhibition of eNOS activity [23,33]. Our previous studies found that a selective PKC beta II peptide inhibitor significantly attenuated oxidative stress in hind limb I/R and restored post-reperfused cardiac function in isolated I/R hearts [23,33,34].…”

Direct Measurement of Hydrogen Peroxide (H2O2) or Nitric Oxide (NO) Release: A Powerful Tool to Assess Real-time Free Radical Production in Biological Models

“…Doing so facilitates study of the process in the intact animal. Several publications have shown that the isolated rat aortic segment model is a prognostic indicator of whole organ function (15,(24)(25)(26)(27). Overall, the experimental setup is cost effective in that small amount of reagents, tissues, or cells are needed to generate reliable data.…”

“…At the conclusion of the experiment, the aortic segments are blotted and weighed, and results are usually reported as moles of NO released per gram of tissue (15,(23)(24)(25)(26)(27). Figure 3A is an illustration depicting the experimental setup for rat aortic segment tissue.…”

“…Real-time measurement of H 2 O 2 and NO can also be performed on isolated tissue bathed in physiologic buffers or on cultured cells in tissue culture medium (15,(22)(23)(24)(25)(26)(27). Previous studies have even utilized electrodes in organ chambers to record NO concentration (28).…”