HNO Protects the Myocardium against Reperfusion Injury, Inhibiting the mPTP Opening via PKCε Activation.

Donors of nitroxyl (HNO), the one electron-reduction product of nitric oxide (NO), positively modulate cardiac contractility/relaxation while limiting ischemia-reperfusion (I/R) injury. The mechanisms underpinning HNO anti-ischemic effects remain poorly understood. Using isolated perfused rat hearts subjected to 30 min global ischemia/1 or 2 h reperfusion, here we tested whether, in analogy to NO, HNO protection requires PKCε translocation to mitochondria and K channels activation. To this end, we compared the benefits afforded by ischemic preconditioning (IPC; 3 cycles of I/R) with those eventually granted by the NO donor, diethylamine/NO, DEA/NO, and two chemically unrelated HNO donors: Angeli's salt (AS, a prototypic donor) and isopropylamine/NO (IPA/NO, a new HNO releaser). All donors were given for 19 min before I/R injury. In control I/R hearts (1 h reperfusion), infarct size (IS) measured via tetrazolium salt staining was 66 ± 5.5% of the area at risk. Both AS and IPA/NO were as effective as IPC in reducing IS [30.7 ± 2.2 (AS), 31 ± 2.9 (IPA/NO), and 31 ± 0.8 (IPC), respectively)], whereas DEA/NO was significantly less so (36.2 ± 2.6%, < 0.001 vs. AS, IPA/NO, or IPC). IPA/NO protection was still present after 120 min of reperfusion, and the co-infusion with the PKCε inhibitor (PKCV1-2500 nM) prevented it (IS = 30 ± 0.5 vs. 61 ± 1.8% with IPA/NO alone, < 0.01). Irrespective of the donor, HNO anti-ischemic effects were insensitive to the K channel inhibitor, 5-OH decanoate (5HD, 100 μM), that, in contrast, abrogated DEA/NO protection. Finally, both HNO donors markedly enhanced the mitochondrial permeability transition pore (mPTP) ROS threshold over control levels (≅35-40%), an action again insensitive to 5HD. Our study shows that HNO donors inhibit mPTP opening, thus limiting myocyte loss at reperfusion, a beneficial effect that requires PKCε translocation to the mitochondria but not mitochondrial K channels activation.