Nitric oxide (NO-) donors can mimic ischaemic preconditioning (IP) through the formation of reactive nitrogen species (RNOS). The involvement of nitroxyl (HNO/NO–), the one-electron reduction product of NO-, in IP has been previously suggested (Pagliaro et al. 2001). Nitroxyl can be produced by L-arginine oxidation by NO synthase under certain conditions (Schmidt et al. 1996). At physiological pH, HNO/NO– may be generated by Angeli’s salt (AS, Na2N2O3). We tested the hypothesis that AS provides IP-like effects that are significantly more potent than those observed with an NO- donor. Finally, we wanted to determine whether HNO/NO–-mediated myocardial protection might be attributed to a ‘pro-oxidant’ mechanism, inhibitable by switching myocardial redox conditions.
Wistar rats were anaesthetised with 1 g kg-1 urethane I.P. 10 min after heparin (2500 U, I.M.). Hearts were excised and retrogradely perfused with oxygenated Krebs-Henseleit solution at 37 °C. After 20 min stabilisation they were assigned to one of the six following treatment groups (n = 12 in each group): (1) hearts treated with intracoronary 1 mM AS; (2) with 0.5 mM diethylamine/NO (DEA/NO), an NO- donor; (3) with vehicle (100 nM NaOH), or (4) with buffer for 19 min prior to ischaemia/reperfusion (I/R) injury (30 min global ischaemia, 30 min reperfusion). (5) To test the influence of the redox state, AS was co-infused with N-acetyl-L-cysteine (NAC, 4 mM). In hearts of group (6), IP was induced by three cycles of 3 min of ischaemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury was assessed by changes in left ventricular pressure (LVP), lactate dehydrogenase (LDH) release and quantification of infarct size. Mean ± S.E.M. data were analysed by Student’s unpaired t test. P < 0.05 was considered significant.
In control hearts LDH release was 686 ± 14 U g -1 and infarct size was 39 ± 2 % of the left ventricle mass. AS and IP were more effective than DEA/NO in limiting cardiac injuries. In fact, in AS and IP groups LDH release and infarct size were reduced by 50Ð55 and 60Ð65 %, respectively. In the DEA/NO group LDH and infarct size were reduced by only 40 and 30 %, respectively. Post-ischaemic LVP recovery, which was 36 ± 6 % of pre-ischaemic levels in control hearts, was 55 ± 3, 53 ± 5 and 43 ± 4 % in AS, IP and DEA/NO groups, respectively. AS-induced protection was redox sensitive, as it was abolished by NAC co-infusion.
Data show that HNO/NO–, generated by AS, affords myocardial protection akin to IP and greater than NO-, suggesting that RNOS like HNO/NO– are not only necessary but also sufficient to trigger myocardial protection against I/R through a pro- ‘oxidative’ and/or ‘nitrosative’ stress mechanism
We thank ‘Compagnia di San Paolo’ and the Italian Ministry of University.
All procedures accord with current national legislation.