Exogenous hydrogen sulfide induces pharmacological postconditioning in anesthetized rabbits through AKT/PKG/PLB activation, independently of mitoKATP channels opening and JAK/STAT pathway

Abstract

Background: The signal transduction pathways which are recruited during early reperfusion (rep), include the NO/cGMP/PKG/KATP pathway, the rep injury salvage kinase pathway (PI3K/Akt, ERK½, GSK3β), and the JAK/STAT pathway, all targeting the inhibition of mPTP opening. The production of H2S plays a role in myocardial pre-and post-conditioning and several in vitro studies support that exogenous H2S emerges cardioprotection via KATP channels. However, the underlying molecular mechanism of cardioprotection against ischemia (isc)/rep injury in vivo is entirely obscure. Preliminary findings of our group indicate that therapeutic administration of the H2S donor sodium hydrosulphide NaHS reduces the infarct size in anesthetized rabbits. Aim: To elucidate the molecular mechanism underlying the induced cardioprotection from exogenous H2S administration in vivo. Methods: Anesthetized rabbits were divided into 6 groups and were subjected to 30 min regional isc of the heart and 3 hours rep with additional interventions: 1) Control group, no further intervention, 2) NaHS group was treated with NaHS at a dose of 100 μg kg-1 bolus on the 20th min of isc followed by infusion of 1mg kg-1 h-1 for the next 120 min, 3) NaHS +DT2 group, treated with NaHS and the PKG inhibitor DT2 at a dose of 0.25 mg kg-1 10 min before isc 4) NaHS+5-Hydroxydecanoic acid (5-HD) treated with NaHS and the mitoKATP channels inhibitor 5-HD at a dose of 5 mg.kg-1 40 min before isc, 5) NaHS+ L-NAME treated with NaHS and the synthase of NO inhibitor L-NAME bolus at a dose of 10 mg.kg-1 on the 19th min of isc and 6) NaHS+ Wortmannin treated with NaHS and the PI3/AKT inhibitor wortmannin at a dose of 60μg kg-1 on the 19th min of isc. The infarct size and the area at risk were estimated (%I/R). In a second series of experiments, we determined the activation of tissue AKT, ERK½, eNOS, GSK3β, STAT3 and phopsholamban (PLB) from Control, NaHS and NaHS+DT-2 groups. Results: NaHS reduced %I/R compared to Control (12.3±3.3% vs 46.4±1.8%, p<0.05), the addition of the DT2 abrogated this benefit (39.8±3.4%), 5-HD and L-NAME did not alter the infarct size reduction (14.1±2.0% and 14.7±2.2% respectively, p=NS vs NaHS) whilst wortmannin reversed it (41.8±1.4%, p=NS vs Control). Phosphorylation of AKT, ERK½, and PLB was higher in NaHS vs Control and NaHS+DT-2 groups with no eNOS and GSK3β activation. STAT-3 activation was higher in NaHS and NaHS+DT-2 vs Control. Conclusion: Exogenous administration of H2S induces pharmacological postconditioning via AKT/PKG/PLB and PKG/ ERK½ activation independently of eNOS, GSK3β, JAK/STAT and mitoKATP activation.