Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

Abstract

Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg 2+ -ATPase activity, reaching 50% inhibition with 46.7 ( 8.3 µM SIN-1 for 8.7 µM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg 2+ -ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca 2+ -dependent and K + /EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg 2+ -ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys 707 and Cys 697 ), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5′-dithiobis(2-nitrobenzoic acid) and by the parallel decrease of Cys 707 labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg 2+ -ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADP‚V 1 to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.

Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation

“…These results pointed out that F-actin is between 10-and 20-fold more sensitive to peroxynitrite than other proteins of relevance to muscle bioenergetics reported so far, like creatine kinase [27], myosin [24], and sarcoplasmic reticulum Ca 2+ -ATPase [21], and point out that actin filaments can be seen as a primary target in the impairment of muscle contraction by oxidative insults promoting a rise of peroxynitrite. …”

“…1C), and (iii) hydrogen peroxide produced during SIN-1 decomposition in the buffer used in these experiments was lower than 0.5 lM and catalase did not prevent against the depolymerization of F-actin by exposure to SIN-1 (results not shown). From the kinetics of peroxynitrite production during SIN-1 decomposition under these experimental conditions [15,24] it follows that: (1) exposure of F-actin to peroxynitrite fluxes as low as 50-100 nM during 1-2 h produced nearly 50% F-actin depolymerization, and (2) the IC 50 for F-actin depolymerization takes place at a molar ratio of peroxynitrite/actin monomer close to 2.…”

Alterations to myofibrillar protein function in nonischemic regions of the heart early after myocardial infarction

“…Both are located in a critical ␣-helix in myosin (34) where covalent modifications of either cysteine typically lead to complete inhibition of myosin (24,35). Tiago et al (39) reported that myosin may be inactivated by ONOO Ϫ through oxidative modification of one of the reactive cysteines, perhaps to sulfenic acid (1), leading to partial unfolding of the protein. Consistent with this, our data show a decrease in reactive cysteines in myosin heavy chain purified from remote-zone samples.…”

A change of heart: oxidative stress in governing muscle function?

“…Free radicalinduced oxidative modifications of proteins can alter their functional or structural properties (Bolli and Marbán 1999;Kloner and Jennings 2001a, b;Tiago et al 2006;Solaro 2007;Murphy et al 2008;Hertelendi et al 2008;Ding et al 2011;Gao et al 2012;Alegre-Cebollada et al 2014). These modifications modulate muscle force production and regulate passive stiffness in vitro (Bolli and Marbán 1999;Kloner and Jennings 2001a, b;Tiago et al 2006;Solaro 2007;Murphy et al 2008;Hertelendi et al 2008;Ding et al 2011;Gao et al 2012;Alegre-Cebollada et al 2014). Protein oxidation generally occurs at the reactive thio-moieties of cysteine residues (or to a lesser extent methionine), which can be modified through either reversible or irreversible oxidative states (Fig.…”

“…The myosin ATPase activity is the driving enzyme of the cross-cycle and was shown to be modified upon S-glutathionylation at different MHC sites (Passarelli et al 2008). Indeed, oxidation of two cysteines near the catalytic centre of the subfragment 1 (S1)-domain in MHC (Cys 697 and Cys 707 ) results in a strong inhibition of S1-ATPase activity and reduces maximal force (Tiago et al 2006). Elevated levels of glucose adducts (glycosylation) have been found on the MHC in post-mortem hearts of diabetic patients, and carbonylation of MHC as a result of reactive carbonyl species (RCS) has been consistently found in rat hearts during diabetes (Shao et al 2010).…”