Regina Ondrasik

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.

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.

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