Harsh Patel

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

Ischemia/reperfusion results in cardiac contractile dysfunction and cell death partly due to increased reactive oxygen species and decreased endothelial-derived nitric oxide bioavailability. NADPH oxidase normally produces reactive oxygen species to facilitate cell signalling and differentiation; however, excessive release of such species following ischemia exacerbates cell death. Thus, administration of an NADPH oxidase inhibitor, apocynin, may preserve cardiac function and reduce infarct size following ischemia. Apocynin dose-dependently (40 μM, 400 μM and 1 mM) attenuated leukocyte superoxide release by 87 ± 7%. Apocynin was also given to isolated perfused hearts after ischemia, with infarct size decreasing to 39 ± 7% (40 μM), 28 ± 4% (400 μM; p < 0.01) and 29 ± 6% (1 mM; p < 0.01), versus the control’s 46 ± 2%. This decrease correlated with improved final post-reperfusion left ventricular end-diastolic pressure, which decreased from 60 ± 5% in control hearts to 56 ± 5% (40 μM), 43 ± 4% (400 μM; p < 0.01) and 48 ± 5% (1 mM; p < 0.05), compared to baseline. Functionally, apocynin (13.7 mg/kg, I.V.) significantly reduced H2O2 by nearly four-fold and increased endothelial-derived nitric oxide bioavailability by nearly four-fold during reperfusion compared to controls (p < 0.01), which was confirmed in in vivo rat hind limb ischemia/reperfusion models. These results suggest that apocynin attenuates ischemia/reperfusion-induced cardiac contractile dysfunction and infarct size by inhibiting reactive oxygen species release from NADPH oxidase.

Myristoylated protein kinase C beta II peptide inhibitor exerts dose-dependent inhibition of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced leukocyte superoxide release

Phosphorylation of polymorphonuclear leukocyte (PMN) NADPH oxidase by protein kinase C (PKC) is essential to generate superoxide (SO) release. Inhibition of PMN SO release attenuates inflammation mediated vascular tissue injury during myocardial ischemia/ reperfusion (MI/R) injury. PMNs express five isoforms of PKC (alpha (α), beta I (βI), beta II (βII), delta (δ) and zeta (ζ)) and their role regulating this response have not been fully elucidated. PKC α, βII and ζ are thought to positively regulate PMN SO release, whereas PKC δ negatively regulates PMN SO release. [1,2] PKC βI, in contrast to the other four isoforms, translocates to the nucleus after second messenger stimulation [1]. PKC βII, a classical isoform, is activated by calcium and diacylglycerol (DAG) following PMN chemotactic receptor stimulation with fMLP peptide (Figure 1) [1]. Activated PKC βII will phosphorylate PMN NADPH oxidase to produce SO. Selective PKC βII peptide inhibitor has been developed based on its binding sites to receptor for activated C kinase (RACK) domain (Figure 2) [3]. RACK shuttles cytosolic PKC βII to interact with cell membrane substrates (e.g., NADPH oxidase). Myristoylation of peptides is known to be an effective strategy to enable simple diffusion through cell membranes to affect PKC function [4,5].The cell permeable myristoylated (myr) PKC βII peptide inhibitor is known to inhibit PMN SO release at doses that correlated with restoration of postreperfused cardiac function following global MI(20 min)/R(45 min) in leukocyte mediated cardiac MI/R dysfunction and more recently in prolonged MI(30 min)/R(90 min) in isolated rat hearts [1,6,7]. However, a full dose-response curve with Myr-PKC βII peptide inhibitor has not been indicated previously. Characterizing the full dose-response effects is essential in identifying putative mechanisms responsible for attenuating vascular and tissue injury following I/R.

Comparison of the Effects of Myristoylated and Transactivating Peptide (TAT) Conjugated Mitochondrial Fission Peptide Inhibitor (P110) in Myocardial Ischemia/Reperfusion (I/R) Injury

Mitochondrial dynamics, mitochondrial fusion and fission may be involved in myocardial ischemia/reperfusion (MI/R) injury. In particular, mitochondrial fission is associated with mitochondrial fragmentation and decreased ATP production leading to cardiac contractile dysfunction and increased infarct size in MI/R [1-3]. During ischemic events, coronary blood flow is restricted causing cardiomyocytes to enter a hypoxic state. This change in cellular respiration causes a buildup of lactic acid and a decrease in pH. The acidic conditions developed during ischemia prevent the opening of the mitochondrial permeability transition pore (MPTP) and cause cardiomyocyte hypercontracture. When blood flow and oxygen delivery are restored during reperfusion, reactive oxygen species (ROS) are generated which leads to the loss of mitochondrial membrane potential and opening of the MPTP, which potentiates mitochondrial fission in MI/R (Figure 1). Therefore inhibiting mitochondrial fission, which results from the vital act of reperfusion, may be a strategy to salvage damaged cardiomyocytes and protect them from MI/R injury. P110 (DLLPRGT) is a mitochondrial fission peptide inhibitor that acts by selectively inhibiting the interaction between human fission protein (Fis1), which is located on the outer mitochondrial membrane and dynamin related protein 1 (Drp1), a GTPase (Figure 2).

Comparing the effectiveness of TAT and Myristoylation of gp91ds on Leukocyte Superoxide (SO) Release

SO release from leukocytes via NADPH oxidase activation contributes to oxidative stress under various diseases, such as ischemia/reperfusion (I/R) injury and vascular complications in diabetes. NADPH oxidase has seven isoforms with NOX2 being the predominant isoform of NADPH oxidase in polymorphonuclear leukocytes (PMNs). Activation of NOX2 requires the assembly of cytosolic subunits (p47, p40, p67, Rac) to plasma membrane subunits (gp91 and p22) [1]. NADPH oxidase is activated during I/R injury via cytokine receptor stimulation or chemotactic factor (N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, MW= 438 g/mol) and utilizes molecular oxygen to produce SO [2] (Figure 1).

Protein Kinase C Beta II (PKC ßII) Peptide Inhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

Coronary heart disease is the leading cause of death worldwide, and is primarily attributable to the detrimental effects of tissue infarct after an ischemic insult. The most effective therapeutic intervention for reducing infarct size associated with myocardial ischemia injury is timely and effective reperfusion of blood flow back to the ischemic heart tissue. However, the reperfusion of blood itself can induce additional cardiomyocyte death that can account for up to 50% of the final infarction size. Currently, there are no effective clinical pharmacologic treatments to limit myocardial ischemia/reperfusion (MI/R) injury in heart attack patients [1]. Reperfusion injury is initiated by decreased endothelialderived nitric oxide (NO) which occurs within 5 min of reperfusion [2], and may in part be explained by PKC II mediated activation of NADPH oxidase, which occurs upon cytokine release during MI/R [3]. PKC II activity is increased in animal models of MI/R and known to exacerbate tissue injury [4,5]. PKC II is known to increase NADPH oxidase activity in leukocytes, endothelial cells and cardiac myocytes via phox47 phosphorylation, and decrease endothelial NO synthase (eNOS) activity via phosphorylation of Thr 495 [6-8]. NADPH oxidase produces superoxide (SO) and quenches endothelial derived NO in cardiac endothelial cells. Moreover, PKC II phosphorylation of p66Shc at Ser 36 leads to increased mitochondrial reactive active oxygen species (ROS) production, opening of the mitochondrial permeability transition pore (MPTP), and pro-apoptotic factors leading to cell death and increased infarct size [9] (Figure 1 left). Therefore, using a pharmacologic agent that inhibits the rapid release of PKC II mediated ROS, would attenuate endothelial dysfunction and downstream pro-