Nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease 2A through S-nitrosylation: A protective mechanism against enteroviral cardiomyopathy.

BACKGROUND: Infection with enteroviruses like coxsackievirus B3 (CVB3) as well as genetic dystrophin deficiency can cause dilated cardiomyopathy. We recently identified cleavage and functional impairment of dystrophin by the viral protease 2A during CVB3-infection as a molecular mechanism that may contribute to the pathogenesis of enterovirus-induced cardiomyopathy. Nitric oxide (NO) is elevated in human dilated cardiomyopathy, but the relevance of this finding is unknown. In mice, NO inhibits CVB3 myocarditis. Therefore, we investigated the effects of NO on the coxsackieviral protease 2A. METHODS AND RESULTS: In vitro, NO donors like PAPA-NONOate inhibited the cleavage of human and mouse dystrophin by recombinant coxsackievirus B protease 2A in a dose-dependent manner (IC(50), 51 micromol/L). In CVB3-infected HeLa cells, addition of the NO donor SNAP inhibited protease 2A catalytic activity on dystrophin. Because this inhibitory effect was reversed by the thiol-protecting agent DTT, we investigated whether NO S:-nitrosylates the protease 2A. In vitro, NO nitrosylated the active-site cysteine (C110) of the coxsackieviral protease 2A, as demonstrated by site-directed mutagenesis. Within living COS-7 cells, SNAP-induced S:-nitrosylation of this site was confirmed with electron spin resonance spectroscopy. CONCLUSIONS: These data demonstrate inactivation of a coxsackieviral protease 2A by NO through active-cysteine S:-nitrosylation in vitro and intracellularly. Given that the enteroviral protease 2A cleaves mouse and human dystrophin, NO may be protective in human heart failure with an underlying enteroviral pathogenesis through inhibition of dystrophin proteolysis.

Detection of nitrosyl-iron complexes by proton-electron-double-resonance imaging.

The nitrogen monoxide radical (NO*) forms paramagnetic mono- and dinitrosyl-iron complexes in biologic tissues. To establish a noninvasive technique for in vivo NO* imaging, we evaluated the suitability of these complexes as magnetic resonance (MR) contrast agents, making use of the ability of the unpaired electrons of the complexes to enter into dynamic nuclear polarization with water protons and hence produce enhancement on images generated by the technique of proton-electron-double-resonance imaging (PEDRI). Phantom solutions of synthetic nitrosyl-iron complexes (NICs) altered the signal intensity of PEDRI images. The dinitrosyl-iron complex (DNIC) with serum albumin induced a significantly larger signal alteration than the mononitrosyl-iron complex (MNIC) with dithiocarbamate. Exposure of rat liver to sodium nitroprusside (SNP) by ex vivo and in situ perfusion induced a composite X-band electron spin resonance (ESR) spectrum of the isolated liver characteristic of a MNIC and DNIC. On storage of the tissue, the MNIC signal disappeared and the DNIC signal intensity increased. Correspondingly, in cross-sectional PEDRI images taken at room temperature, the SNP-exposed livers initially exhibited a weak signal that strongly increased with time. In conclusion, NICs can be detected using PEDRI and could be exploited for in vivo NO* imaging.

MR imaging of nitrosyl-iron complexes: experimental study in rats.

PURPOSE: To assess the influence of several nitrosyl-iron complexes on proton nuclear spin relaxation rates to establish a magnetic resonance (MR) imaging technique for nitric oxide. MATERIALS AND METHODS: The influence of aqueous phantom solutions of nitrosyl-iron complexes on proton relaxation rates was analyzed for signal enhancement at conventional 1.5-T MR imaging. To induce formation of nitrosyl-iron complexes in a biologic tissue, isolated rat liver was perfused with a saline solution of the NO donor sodium nitroprusside (SNP), and the MR signal intensity was examined thereafter. RESULTS: All investigated nitrosyl-iron complexes shortened the longitudinal, or T1, and transverse, or T2, relaxation times in a concentration-dependent fashion. Relaxivities were highest with a dinitrosyl-iron complex bound to albumin and with a water-soluble mononitrosyl-iron dithiocarbamate complex. The contrast properties of 240 micromol/L of a paramagnetic nitrosyl-iron complex were sufficient to substantially enhance the signal intensity of SNP-perfused rat livers at hydrogen 1 MR imaging. CONCLUSION: Nitrosyl-iron complexes exhibit a contrast effect at MR imaging that can be exploited for NO imaging in living animals and patients with conventional (1)H MR imaging techniques.

Nitric oxide inhibits caspase-3 by S-nitrosation in vivo.

In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 by S-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody. S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO by N-methyl-D-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely prevented S-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 microM) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with beta-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-L-cysteine (50 microM). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity via S-nitrosation of the Cys-163 residue of caspase-3.