Myocardial infarction is a form of ischemia/reperfusion (I/R) injury that causes cardiac contractile dysfunction and cell death. I/R injury is mediated, in part, by decreased endothelial-derived nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS) resulting in cell death. Cytokines released from I/R tissue activate G-protein coupled receptors that in turn stimulate NADPH oxidase to produce ROS. Thus, administration of a NADPH oxidase peptide inhibitor, Nox2ds-tat (formerly known as gp91ds-tat), may be a rational approach to attenuate I/R injury. Nox2ds-tat dose-dependently inhibited (10 μM -80 μM; n=5) phorbol 12-myristate13-acetate (n=21) induced polymorphonuclear leukocyte superoxide production up to 37 ± 7% (p<0.05; Fig. 3). Similarly, Nox2ds-tat dose-dependently attenuated I/R induced cardiac contractile dysfunction as evidenced by improved post-reperfused left ventricular developed pressure (LVDP) which recovered up to 77 ± 7% (5 μM-80 μM; p<0.05; n=6-7) of initial values (pre-ischemic values) at 45 min post-reperfusion when compared to control I/R hearts (n=14) that only recovered to 46 ± 6% from initial values for LVDP in isolated perfused rat hearts subjected to global I(30 min)/R(45 min) (Table 1). I/R control hearts exhibited an infarct size of 46 ± 2.1%, whereas I/R + Nox2ds-tat hearts exhibited infarct sizes of 30 ± 4% (5 μM), 15 ± 1.4% (10 μM), 23 ± 2.0% (40 µM), and 19 ± 1.6% ( 80µM) (p<0.01 vs. control I/R hearts; Figure 4, Panel A-B). Regarding in vivo assessments, Nox2ds-tat (4.1 mg/kg, IV) significantly reduced blood H 2 O 2 (1.4 µM) and increased endothelial-derived blood NO (127 nM) at 45 min reperfusion

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I/R injury induces cell death and organ dysfunction in part due to a burst of reactive oxygen species that occurs upon the reintroduction of oxygen into the ischemic area, leading to endothelial dysfunction: decreased blood NO and increased hydrogen peroxide (H2O2 ) levels. We’ve previously shown in isolated rat hearts subjected to I/R injury, gp91ds‐tat attenuated cardiac contractile dysfunction and reduced infarct size compared to controls presumably by the inhibition of NADPH oxidase induced superoxide release. Superoxide can quench NO via the formation of peroxynitrite and also be converted to H2O2 in blood. We attempted to confirm this hypothesis using a rat hind limb I/R model that permitted real time measurements of changes in blood NO and H2O2. NO or H2O2 microsensors were inserted into both femoral veins in anesthetized male rats. One limb’s femoral artery/vein is subjected to I(30min)/R(45min) while the other served as a non‐ischemic sham. Preliminary results show blood NO release significantly increased by the end of reperfusion in gp91ds‐tat treated rats (1.2 mg/kg, MW 2452g/mol, n=5) compared to saline treated rats (n=3;p<0.05). The preliminary data suggest that gp91ds‐tat attenuates I/R induced endothelial dysfunction. The results of gp91ds‐tat on blood H2O2 levels are forthcoming.

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.

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