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 faci

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

During myocardial ischemia, coronary blood flow interruption deprives cardiomyocytes of oxygen, glucose and fatty acids. Ischemic damage is exacerbated by a burst of reactive oxygen species (ROS) generated at reperfusion when oxygen interacts with damaged mitochondrial electron transport chains (ETC), especially uncoupled complexes I and III (Fig. 1,2). Nicotinamide adenine dinucleotide phosphate oxidase (Nox) activity can also release ROS, inducing additional tissue/organ damage. Surgical intervention or thrombolytic treatments can restore coronary blood flow. However, as blood flow reestablishes, oxidative stress leads to I/R injury. Clinical treatment remains a challenge as no pharmaceutical agents effectively limit I/R-induced damage. Mitochondria are implicated in I/R as a major source of ROS3,4,5. Excess ROS leads to mitochondrial and cardiac contractile dysfunction6. Conventional antioxidants have limited efficacy in myocardial I/R because they are not targeted selectively to where most I/R damage occurs, in mitochondria (Fig. 3)3,4,5. Mitoquinone (mitoQ, MW=600 g/mol), a coenzyme Q analog, easily crosses phospholipid bilayers and is driven by the large electrochemical membrane potential to concentrate mitoQ several hundred-fold within mitochondria. The respiratory chain reduces mitoQ to its active ubiquinol antioxidant form to limit myocardial I/R injury5. The SS-31 (Szeto-Schiller) peptide ((D-Arg)-Dmt-Lys-Phe-Amide, MW=640 g/mol, Genemed Synthesis, Inc., San Antonio, TX) is also of interest since it is cellpermeable, specifically targeted to inner mitochondrial membranes based on its alternating cationic aromatic residue sequence, with an antioxidant dimethyltyrosine moeity. SS peptides scavenge ROS in I/R models. Although mitochondrial-targeted antioxidant pretreatment can effectively limit I/R injury, pretreatment is not always possible in cases of myocardial infarction. Therefore, evaluating cardioprotective efficacy of mitochondrialtargeted antioxidants when given at reperfusion is of high significanc

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

During myocardial ischemia/reperfusion (I/R), the generation of reactive oxygen species (ROS) contributes to post-reperfused cardiac injury and contractile dysfunction. Activation of NADPH oxidase (NOX) during reperfusion generates ROS, and exacerbates I/R injury. We hypothesize that reducing ROS formation through inhibition of NOX will attenuate myocardial I/R injury in isolated perfused rat hearts subjected to I(30min)/R(45min) compared to untreated I/R hearts. The cell-permeable NOX inhibiting peptide, gp91 ds/tat (RKKRRQRRR-CSTRIRRQL-Amide, MW=2452 g/mol, 20μM, n=5), significantly improved post-reperfused cardiac function compared to controls (n=15,

Combinational Effects of Apocynin and Mitoquinone in Reducing Myocardial Ischemia/reperfusion (MI/R) Injury

Reactive oxygen species (ROS) is generated due to the influx of oxygen during MI/R and contributes to post-reperfused cardiac contractile dysfunction and increased infarct size. Damaged mitochondria and NADPH oxidase activation are major sites of ROS in MI/R. In prior studies, apo, a NADPH oxidase inhibitor, and mitoQ, a mitochondrial-targeted antioxidant, dose-dependently improved post-reperfused left ventricular developed pressure (LVDP) and reduced infarct size in rat hearts subjected to I(30min)/R(45min). This led us to question whether low doses of apo and mitoQ given together can act synergistically to improve post-reperfused LVDP and reduce infarct size compared to either drug alone or control? Early data shows that the combination of apo (40μM) + mitoQ (1 μM ) (n=7) reduced infarct size to 27 ± 11% compared to mitoQ (1 μM) (n= 6) 54 ± 6% (

Combinational Effects of gp91 ds-tat and SS-31 in Reducing Myocardial/ischemia Reperfusion (MI/R) Injury

MI/R injury results in cardiac contractile dysfunction, and increased cell death principally due to the reperfusion of blood following ischemia. This injury is initiated in part by a decrease in endothelial derived nitric oxide bioavailability and an increase in reactive oxygen species (ROS). Two key sources of ROS are NADPH oxidase and damaged mitochondria. We’ve shown that gp91 ds-tat, a NADPH oxidase assembly inhibitor peptide and SS-31, a mitochondrial targeted antioxidant, dose dependently improved post-reperfused left ventricular developed pressure (LVDP) and reduced infarct size in rat hearts subjected to I (30min)/R (45min). This led us to the question, whether the combinational effects of low dose gp91 ds-tat and SS-31 would act synergistically to improve LVDP and reduce infarct size compared to the independent effects of each peptide and untreated control? Early data show that the combination of gp91 ds-tat (5μM) + SS31 (10μM) (n=3) improved post-reperfused LVDP by 63 ± 11% of baseline and reduced infarct size (IS) to 32 ± 9% compared to control (44 ± 10% of baseline, n=7; 42 ± 5% IS) and to SS-31 10μM (25 ± 1% of baseline, n=3; 41 ± 10% IS); gp91 5μM (56 ± 9% of baseline, n=6; 28 ± 5% IS). Although this combination improved post-reperfused LVDP more so than either drug alone or control, at these concentrations the combinations didn’t act synergistically. Other combinational doses will be tested in the future

The cardioprotective effects of a NOX1 inhibitor, ML171, on myocardial ischemia/reperfusion (I/R) injury

Oxidative stress is a major cause of I/R injury. NADPH oxidase is an important source of oxidative stress during I/R, and it consists of NOX1-5 and DOUX1-2 isoforms. The role of NOX1 in myocardial I/R injury is unclear. In this study, a specific NOX1 inhibitor, ML171 (MW=241.31 g/mol, IC50 for NOX1=0.25 μM), was tested in isolated perfused rat hearts following I (30 min)/R (45 min). We found that left ventricular developed pressure (LVDP) and the peak of the first derivative of left ventricular pressure (dP/dtmax) only recovered to 42 ± 4% and 30 ± 3% of baseline values, respectively, at 45 min post-reperfusion in control I/R hearts (n=8). By contrast, ML171 (1 μM, n=5) given at reperfusion for 5 min significantly restored LVDP and dP/dtmax to 90 ± 6% and 67 ± 7% of baseline values, respectively (both p\u3c0.01). ML171 also significantly reduced infarct size to 18 ± 3% compared to 41 ± 2% in control I/R hearts (p\u3c0.01). Furthermore, a selective mitochondrial ATP-dependent K+ channel (mitoKatp) inhibitor, 5- hydroxydecanoate (100 μM, n=4); and a heme oxygenase-1 inhibitor, SnPP (20 μM, n=3), significantly abolished the cardioprotective effects of ML171 (all p\u3c0.05). We also confirmed that ML171 (0.1-1 μM, n=4) did not inhibit phorbol 12-myristate 13-acetate (30 nM) induced superoxide release from isolated rat neutrophils. These results suggest that NOX1 inhibition improves post-reperfused contractile function and reduces infarct size, possibly by opening mitoKatp channels and activating heme oxygenase-1