A new low molecular weight, MnII-containing scavenger of superoxide anion protects cardiac muscle cells from hypoxia/reoxygenation injury

Reperfusion injury after oxygen starvation is a key pathogenic step in ischemic diseases. It mainly consists in oxidative stress, related to mitochondrial derangement and enhanced generation of reactive oxygen species (ROS), mainly superoxide anion (O-2(center dot 2)), and peroxynitrite by cells exposed to hypoxia. This in vitro study evaluates whether Mn-II (4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diacetate).2H(2)O, or Mn-II(Me(2)DO2A), a new low molecular weight, Mn-II-containing O-2(center dot) scavenger, has a direct protective action on H9c2 rat cardiac muscle cells subjected to hypoxia and reoxygenation. Mn-II(Me(2)DO2A) (1 and 10 mu mol/l) was added to the culture medium at reoxygenation and maintained for 2 h. In parallel experiments, the inactive congener Zn-II(Me(2)DO2A), in which Zn-II replaced the functional Mn-II center in the same organic scaff old, was used as negative control. Mn-II(Me(2)DO2A) (10 mu mol/l) signifi cantly increased cardiac muscle cell viability (trypan blue assay), improved mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test, membrane potential Delta psi), reduced apoptosis (mitochondrial permeability transition pore opening, caspase-3, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay), decreased intracellular ROS levels (2',7'-dichlorodihydrofl uorescein diacetate and MitoSOX assays), and decreased protein nitroxidation (nitrotyrosine [ NT] expression) and DNA oxidation (8-hydroxy-deoxyguanosine levels). Of note, Zn-II(Me(2)DO2A) had no protective eff ect. The mechanism of Mn-II(Me(2)DO2A) relies on concentration-dependent removal of harmful O-2(center dot) generated at reoxygenation from dysfunctional mitochondria in hypoxia-induced cells, as indicated by the MitoSOX assay. This study suggests that Mn-II(Me(2)DO2A) is a promising antioxidant drug capable of reducing O-2(center dot)-mediated cell oxidative stress which occurs at reoxygenation after hypoxia. In perspective, Mn-II(Me(2)DO2A) might be used to reduce ischemia-reperfusion organ damage in acute vascular diseases, as well as to extend the viability of explanted organs before transplantation.