Abstract
Cardiac tissue undergoes changes during ischemia-reperfusion (I-R) that compromise its normal function. Cell death is one of the consequences of such damage, as well as diminution in nitric oxide (NO) content. This signaling molecule regulates the function of the cardiovascular system through dependent and independent effects of cyclic guanosine monophosphate (cGMP). The independent cGMP pathway involves post-translational modification of proteins by S-nitrosylation. Studies in vitro have shown that NO inhibits the activity of caspases and calpains through S-nitrosylation of a cysteine located in their catalytic site, so we propose to elucidate if the regulatory mechanisms of NO are related with changes in S-nitrosylation of cell death proteins in the ischemic-reperfused myocardium. We used two compounds that increase the levels of NO by different mechanisms: Prolame, an amino-estrogenic compound with antiplatelet and anticoagulant effects that induces the increase of NO levels in vivo by activating the endothelial nitric oxide synthase (eNOS) and that has not been tested as a potential inhibitor of apoptosis. On the other hand, S-Nitroso-N-acetylpenicillamine (SNAP), a synthetic NO donor that has been shown to decrease cell death after inducing hypoxia-reoxygenation in cell cultures. Main experimental groups were Control, I-R, I-R+Prolame and I-R+SNAP. Additional groups were used to evaluate the NO action pathways. Contractile function represented as heart rate and ventricular pressure was evaluated in a Langendorff system. Infarct size was measured with 2,3,5-triphenyltetrazolium chloride stain. NO content was determined indirectly by measuring nitrite levels with the Griess reaction and cGMP content was measured by Enzyme-Linked ImmunoSorbent Assay. DNA integrity was evaluated by DNA laddering visualized on an agarose gel and by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling assay. Activities of caspase-3, caspase-8, caspase-9 and calpain-1 were evaluated spectrophotometrically and the content of caspase-3 and calpain-1 by western blot. S-nitrosylation of caspase-3 and calpain-1 was evaluated by labeling S-nitrosylated cysteines. Our results show that both Prolame and SNAP increased NO content and improved functional recovery in post-ischemic hearts. cGMP-dependent and S-nitrosylation pathways were activated in both groups, but the cGMP-independent pathway was preferentially activated by SNAP, which induced higher levels of NO than Prolame. Although SNAP effectively diminished the activity of all the proteases, a correlative link between the activity of these proteases and S-nitrosylation was not fully established.
Highlights
Nitric oxide (NO) is a short-life molecule produced after the oxidation of the guanidine group of L-arginine by NO synthases (NOS) (Moncada, Palmer & Higgs, 1991; Loscalzo & Welch, 1995)
The enhanced chemiluminescence detection system was from Millipore Corporation (Bedford, MA, USA) and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); In situ Cell Death Detection Kit, Fluorescein (11-684-795-910) was from Roche; Cyclic GMP Enzyme-linked Immunosorbent Assay Kit was purchased from Cayman Chemical (Ann Arbor, MI, USA); whereas Pierce S-Nitrosylation Western Blot Kit was from Thermo Scientific. 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) was from Calbiochem (Darmstadt, Germany) and 17β-(3-hydroxy-1-propylamino)1,3,5(10)-estratrien-3-ol, Prolame, was synthesized and chemical purity established as previously reported (Fernández-G et al, 1985)
Prolame and SNAP maintain cardiac function and reduce infarct size in reperfused hearts Cardiac function expressed as the double product (DP) was maintained in the Control group during 110 min of constant reperfusion
Summary
Nitric oxide (NO) is a short-life molecule produced after the oxidation of the guanidine group of L-arginine by NO synthases (NOS) (Moncada, Palmer & Higgs, 1991; Loscalzo & Welch, 1995). NO modulates cardiac function by regulating vascular tone, excitation-contraction coupling (Hammond & Balligand, 2012), platelet aggregability (Ikeda et al, 2000) and mitochondrial function (Zhao et al, 2005) Such effects are mainly associated either with the activation of soluble guanylate cyclase (sGC) that produces cyclic guanosine monophosphate (cGMP) and stimulates protein kinase G (PKG) or, with redox reversible modification of cysteine residues that results in the formation of S-nitrosothiols (SNO) in a process named S-nitrosylation (Stamler, Lamas & Fang, 2001). We and others have reported that NO-mediated cardioprotection might be regulated by S-nitrosylation signaling. Sun et al (2013) demonstrated that blockage of the sGC/cGMP/PKG signaling pathway did not affect ischemic postconditioning-mediated cardioprotection and this finding was correlated with increased SNO levels; whereas our group described the partial recovery of heart function when the NO donor: (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate (DETA-NO) was administrated to postconditioned hearts in which sGC was inhibited (Correa et al, 2015)
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