Robert Barsotti

Effects of Mitochondria-Targeted Antioxidants on Real-time Blood Nitric Oxide and Hydrogen Peroxide Release in Hind Limb Ischemia and Reperfusion

We hypothesized that the femoral I/R vein will exhibit increased levels of H2O2 in the blood when compared to the sham vein in the same anesthetized rat. Moreover, we predict that there will be a concurrent decrease in levels of NO released in the femoral I/R vein compared to the sham vein. When mitoQ or SS-31 is given at reperfusion, we expect that the I/R limbs will show decreased H2O2 blood levels and increased NO blood levels compared to the non-drug treated saline controls. As a result, there will be a decrease in ROS and I/R injury. When mitoQ or SS-31 is given at the beginning of reperfusion, there is a significant reduction of blood H2O2 and a significant increase in endothelial-derived NO bioavailablity compared to saline controls. The results of this study support our hypothesis that mitochondria-targeted antioxidant agents can significantly attenuate reperfusion induced ROS release and lead to an increase in NO bioavailability. Collectively, the data suggests that mitoQ or SS-31 can be effective tools in the clinical setting for attenuating post-ischemic insult and endothelial dysfunction. The results also suggest that mitochondrial derived ROS significantly contributes to increased blood H2O2 levels and decreased NO blood levels during reperfusion. Moreover, the mitochondria-targeted antioxidant agents mitoQ or SS-31 were able to attenuate the changes in blood H2O2 and NO levels suggesting that mitochondrial derived ROS are major contributors to oxidative stress in I/R injury. Male Sprague-Dawley (SD) rats (275-325 grams, Charles River, Springfield, MA) were anesthetized with an induction dose of 60mg/kg and maintenance dose 30mg/kg of sodium pentobarbital intraperitoneally (i.p.). The rats also received sodium heparin (1000 USP units/mL) i.p. to act as an anticoagulant. We measured blood H2O2 or NO release from femoral veins in real-time: one vein was subjected to I/R while the other was used as a non-ischemic sham control. The H2O2 or NO microsensors (100 μm, WPI Inc., Sarasota, FL) were connected to a free radical analyzer (Apollo 4000, WPI Inc.) and were inserted into a catheter placed in each femoral vein. Ischemia was induced by clamping the femoral artery/vein of one limb for 30 min followed by 45 min of reperfusion. MitoQ (2 mg/kg), SS-31 (2.5 mg/kg), or saline (for non-drug control group) was administered as a bolus injection via the jugular vein at the beginning of reperfusion. We continuously recorded the H2O2 or NO release and collected the data at 5 min intervals during a 15 min baseline period, 30 min ischemia and 45 min reperfusion. The changes in H2O2 or NO release during reperfusion (in picoamps) are expressed as relative change to baseline after correction to the calibration curve of H2O2 (μM) or NO (nM) microsensors. Experimental groups were compared with Student‘s t-test or ANOVA using post hoc analysis with the Student-Newman-Keuls test.

Effects of Selective NADPH Oxidase Inhibitors on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemia

We hypothesized that acute hyperglycemia (200 mg/dL) would increase H2O2 and decrease NO release in blood relative to saline control. By contrast, gp91ds-tat (RKKRRQRRR-CSTRIRRQL-Amide, MW=2452 g/mol, 1.2 mg/kg, Genemed Synthesis Inc., San Antonio, Tx), a cell-permeable peptide that selectively inhibits NADPH oxidase assembly/activation, would attenuate acute hyperglycemia-induced vascular dysfunction. Furthermore apocynin (MW=166 g/mol, Sigma Chemicals), another type of NADPH oxidase inhibitor, should have similar effects on H2O2 and NO blood levels as gp91ds-tat compared to hyperglycemia control, attenuating acute hyperglycemia-induced vascular dysfunction. We found that acute hyperglycemia significantly reduced blood NO compared to saline control. The addition of gp91ds-tat or apocynin with hyperglycemia significantly improved blood NO levels, similar to saline control. Meanwhile we found acute hyperglycemia maintained a higher level of H2O2 in blood compared to saline control. By contrast, gp91ds-tat or apocynin with hyperglycemia reduced blood H2O2 levels significantly compared to hyperglycemia. These results suggest that NADPH oxidase is a significant source of ROS overproduction and vascular endothelial dysfunction under acute hyperglycemic conditions, and that supplementation with gp91ds-tat or apocynin may be beneficial to attenuate hyperglycemia induced vascular endothelial dysfunction. Moreover, blood H2O2 levels in gp91ds-tat or apocynin treated groups were still significantly higher compared to that in saline groups. This may indicate that other sources of ROS exist such as in mitochondria, which will be further investigated. Results Male Sprague-Dawley rats (275 to 325g, Charles River, Springfield, MA) were anesthetized with 60 mg/kg of pentobarbital sodium with 1000 unit heparin via intraperitoneal (i.p.) injections. The jugular vein is catheterized in order to infuse intravenously with saline, 20% D-glucose, 20% D-glucose with 1.2 mg/kg gp91ds-tat, or 20% D-glucose with 14 mg/kg apocynin (see figure 3). The continuous infusion of 20% D-glucose solution is to maintain hyperglycemia at 200 mg/dL for about 180 min. gp91ds-tat and apocynin will be added to 20% glucose to reach approximately 20 μM and 1 mM in blood, respectively. Both femoral veins will be exposed and catheterized in order to place the calibrated NO and H2O2 microsensors (100μm, WPI Inc., Sarasota, FL) at random into each femoral vein (see figure 4). These microsensors will then be connected to the Apollo 4000 free radical analyzer (WPI Inc., Sarasota, FL) to measure for blood NO and H2O2 levels in real-time. NO, H2O2, and glucose levels will then be recorded at baseline and every 20 minutes intervals throughout 180 minutes infusion period. The changes of blood NO and H2O2 levels will be expressed as the relative change to the baseline. Blood NO and H2O2 recording in picoAmps pA will be converted to the concentration (nM for NO and μM for H2O2) according to the corresponding calibration curve. All the data are represented as a mean ± SEM. The data was then analyzed by ANOVA using post hoc analysis with the Student Newman Keuls. P<0.05 was considered as significant.

Cardioprotective Effects of Selective Mitochondrial-Targeted Antioxidants in Myocardial Ischemia/Reperfusion (I/R) Injury

Cardioprotective Effects of Mitochondrial-Targeted Antioxidants in Myocardial Ischemia/Reperfusion (I/R) Injury Reactive oxygen species (ROS) generated during myocardial I/R contribute to post-reperfused cardiac contractile dysfunction. Damaged cardiomyocyte mitochondria are major sites of excess ROS generation during reperfusion. We hypothesized that reducing mitochondrial ROS formation should attenuate myocardial I/R injury and thereby improve function of isolated perfused rat hearts subjected to I(30min)/R(45min) compared to untreated I/R hearts. Mitoquinone (MitoQ, MW=579g/mol; complexed with cyclodextrin (MW=1135g/mol) to improve water solubility, total MW=1714g/mol), a coenzyme Q derivative, and SS-31 (Szeto-Schiller) peptide ((D-Arg)-Dmt-LysPhe-Amide, MW=639g/mol, Genemed Synthesis, Inc., San Antonio, TX), an alternating cationic-aromatic peptide, are selective mitochondrial ROS inhibitors which significantly improved post-reperfused cardiac function compared to untreated I/R controls in this study (p<0.05). MitoQ elicits antioxidant effects through the recycling of ubiquinone to ubiquinol, whereas SS-31 utilizes an antioxidant dimethyltyrosine residue. Improvement in postreperfused cardiac function by MitoQ or SS-31 was associated with a significant decrease in myocardial tissue infarct size compared to untreated I/R hearts (p<0.01). These results suggest mitochondrial-derived ROS are important contributors to I/R injury, and MitoQ or SS-31 when administered at reperfusion may potentially augment the benefits of angioplasty or

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.

Effects of Mitochondrial-Targeted Antioxidants on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemia Rats

We hypothesized that acute hyperglycemia (200 mg/dL) would increase H2O2 and decrease NO levels in blood relative to saline group. By contrast, MitoQ and the SS-31 peptide would attenuate acute hyperglycemiainduced oxidative stress (e.g., H2O2) and improve vascular function (e.g., increase NO production) under acute hyperglycemia. We found that acute hyperglycemia significantly reduced blood NO levels compared to saline group. The administration of MitoQ or SS-31 during hyperglycemia significantly improved blood NO levels, similar to saline control. Meanwhile we found acute hyperglycemia maintained a higher level of H2O2 in blood compared to saline group. By contrast, MitoQ or SS-31 during hyperglycemia significantly reduced blood H2O2 levels compared to those under hyperglycemia. Moreover, SS-31 treatment showed a trend to reduce blood H2O2 levels more than those in MitoQ treatment, but was not significant. These results suggest that mitochondrial derived SO is a significant source of oxidative stress and vascular endothelial dysfunction under acute hyperglycemic conditions. Moreover, treatment with mitochondrial-targeted antioxidants, MitoQ or SS-31, may be beneficial to attenuate hyperglycemia induced oxidative stress and vascular endothelial dysfunction. Male Sprague-Dawley rats (275 to 325g, Charles River, Springfield, MA) were anesthetized with 60 mg/kg of pentobarbital sodium with 1000 unit heparin via intraperitoneal (i.p.) injections. The jugular vein was catheterized to allow for the infusion of saline, 20% D-glucose, or 20% D-glucose with 1.86 mg/kg MitoQ (MW=600 g/mol; complexed with cyclodextrin to improve water solubility, total MW=1714 g/mol) or with 2.7 mg/kg SS-31 (MW=640 g/mol, Genemed Synthesis, Inc., San Antonio, TX). The continuous infusion of 20% D-glucose solution was to maintain hyperglycemia around 200 mg/dL for about 180 min. MitoQ or SS-31 was added to 20% glucose to reach approximately 13 μM and 50 μM in blood, respectively. Both femoral veins will be exposed and catheterized in order to place the calibrated NO and H2O2 microsensors (100 μm, WPI Inc., Sarasota, FL) at random into each femoral vein. These microsensors were then connected to the Apollo 4000 free radical analyzer (WPI Inc., Sarasota, FL) to measure for blood NO and H2O2 levels in real-time. NO, H2O2, and glucose levels will then be recorded at baseline and at 20 minute intervals throughout the 180 minute infusion period [2]. The changes of blood NO (nM) and H2O2 (M) levels were expressed as the relative change to the baseline or to saline group, respectively. All the data was represented as a mean ± SEM. The data were then analyzed by ANOVA using post hoc analysis with the Student Newman Keuls. p<0.05 was considered as significant.

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-

Cardioprotective Effects of Cell Permeable NADPH oxidase inhibitors in Myocardial Ischemia/Reperfusion Injury

Cardioprotective Effects of Cell Permeable NADPH oxidase inhibitors in Myocardial Ischemia/Reperfusion (I/R) Injury Issachar Devine, Qian Chen, Regina Ondrasik, William Chau, Katelyn Navitsky, On Say Lau, Christopher W. Parker, Kyle D. Bartol, Brendan Casey, Robert Barsotti, Lindon H. Young Department of Pathology, Microbiology, Immunology & Forensic Medicine, Philadelphia College of Osteopathic Medicine