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

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 H 2 O 2 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.

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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% (p<0.05); apo (40 μM) (n=6) 40 ± 8% and control I/R hearts (n=7) 42 ± 5%. Reduced infarct size of the apo + mitoQ combination correlated with an improvement in LVDP (61 ± 4% of baseline) compared to control hearts (44 ± 10% of baseline) and apo 40 μM and mitoQ 1 μM (58 ± 9% and 44 ± 12% of baseline), respectively. Although the combination reduced infarct size and improved post‐reperfused LVDP more so then either drug alone or control, at these concentrations the combinations did not act synergistically. Other combinations will be tested to further establish the dose‐dependent effects.