Copyright © 2005, European Society of Cardiology
Peroxynitrite like Pan?
Division of Cardiology, Johns Hopkins Hospital, Baltimore, MD, USA
* 835 Ross Bldg, 720 Rutland Avenue, Baltimore, MD 21205, USA. Tel.: +1 1 410 955 4813; fax: +1 1 410 502 2558. Email address: npaoloc1{at}jhmi.edu
Received 24 May 2005; accepted 31 May 2005
See also article by Borbély et al. [9] (pages 225–233) in this issue.
Peroxynitrite is a reactive nitrogen species (RNS) generated by the rapid, diffusionally controlled reaction of nitric oxide (NO) and superoxide [1]. Its formation has been implicated in many pathophysiological conditions such as ischemia–reperfusion, sepsis, and congestive heart failure (CHF). Nitrotyrosine formation has been claimed as a "footprint" for peroxynitrite presence and induced damage. This alteration is due to covalent protein modification derived from the addition of a nitro (–NO2) group onto one of the equivalents on the carbons of the aromatic rings of tyrosine residues (NO2Tyr) [2]. Nitrotyrosine production is not only indicative of oxidative/nitrosative stress, but is also a process whereby many proteins might be post-translationally modified. Protein function, catalytic activity, and protein–protein interactions can be altered by nitration [3], particularly those proteins rich in tyrosine residues.
In vitro studies have provided the majority of data supporting tyrosine nitration in the heart, whereas in vivo or ex vivo evidence is less clear. In isolated hearts, the amount of nitrated proteins correlates with the reduction in cardiac pump function [4]. Moreover, in the whole heart, exogenous peroxynitrite appears to reduce myofibrillar in vitro Ca2+ responsiveness in a cGMP-protein kinase-dependent manner [5] and affects the ability of the heart to utilize ATP [6]. Accordingly, the in vitro nitration of the 40-kDa myofibrillar isoform of creatine kinase is likely one major contributor to the altered ATP-to-mechanical work conversion, at least in some CHF models [7]. In vivo, nitrotyrosine production can be significantly increased in the myocardium of interleukin-1-β-treated dogs. In this model, a linear relationship between myocardial superoxide production and nitrotyrosine concentrations was found [8]. When iNOS activity was blocked by aminoguanidine, this resulted in a marked improvement in left ventricular ejection fraction, alluding to endogenous peroxynitrite production. However, this study does not provide any hint about the "topography" of in vivo peroxynitrite-induced damage in that overall nitrotyrosine myocardial content (as per HPLC analysis) was the only index used to evaluate cardiac nitration. So, while some in vivo evidence and in vitro studies (obtained using pharmacological boluses of peroxynitrite) may convey the idea of a rather "broad", almost ubiquitous, nitrotyrosine production within the myocardium under oxidative/nitrosative stress conditions, this pattern may not be the sole or even the most important for nitration-based changes in the heart.
In the current issue of the Journal, Borbély and colleagues [9] report the effects of exogenous peroxynitrite on permeabilized human ventricular myocytes. They measured isometric force generation and used immunoblot analysis to reveal the localization of the ultrastructural alterations, namely protein nitration, following the exposure to synthetic boluses of peroxynitrite. These authors found that after peroxynitrite, the maximal Ca2+-activated isometric force decreased in a dose-dependent fashion, whereas no differences before and after peroxynitrite were noted in terms of Ca2+ sensitivity of force production, in contrast to a previous report [5]. Likewise, no appreciable changes in the steepness of the Ca2+–force relationship as well as in the actin–myosin turnover kinetics were observed. Along with this, the authors observed that peroxynitrite had a major, deteriorating impact on muscular cross-striation pattern with a modest but significant increase in the passive force component. Peroxynitrite appeared to selectively nitrate a protein with an apparent molecular weight of about 100 kDa that, with subsequent immunoprecipitation, was identified as 
-actinin. This cytoskeletal, tyrosine-rich protein provides a scaffold for sarcomeric proteins and is crucial for the maintenance of the Z line and for the integrity of the sarcomeres. Moreover, its appearance precedes the recruitment of both -actin and myosin during the formation of the nascent myofibrils [10], thereby having potential implications for heart development as well. Yet, peroxynitrite-triggered ("experimental") or CHF-induced ("naturally occurring") nitrotyrosine formation is unlikely confined to a single myofibrillar protein and/or enzymatic activity within the contractile machinery.
The present concept of a more "focused" target for protein nitration is in agreement with the recent study of Lokuta and colleagues [11], performed in explanted human hearts. These authors reported nitrotyrosine levels to be almost doubled in idiopathic dilated cardiomyopathy (DCM) as compared to age-matched controls groups. Remarkably, however, nitrotyrosine staining was present in a single protein with the molecular weight of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a isoform) [11].
By pin-pointing to the fact that -actinin and SERCA2a are potential, "selective" targets for nitration, these studies provide initial explanations for depressed cardiac mechanical function in hearts exposed to oxidative/nitrosative stress, by affecting Ca2+-dependent myofibrillar force and inactivating SERCA2a, respectively. Concomitantly, these studies bring to light additional intriguing questions and yet unresolved issues.
First, the specificity of "targeted nitration" could be a function of the amount of exogenously introduced or endogenously produced nitrating agents. In the present study, relatively low (50 µM) doses of peroxynitrite preferentially targeted -actinin, displaying a positive correlation between the extent of the actual structural change and the resulting decay in isometric force. However, with higher peroxynitrite concentrations more proteins with molecular weights above and below that of -actinin (100 kDa) were recruited for nitration. On the other hand, -actinin nitration was not detected with anti-nitrotyrosine antibody in DCM human hearts [11]. On one hand, this evidence highlights one potential limitation of any study employing exogenous peroxynitrite, i.e. the possibility that "actual" concentrations of endogenously formed peroxynitrite may not be high enough to reproduce, entirely or in part, the alterations procured by "nominal" concentrations of the exogenous one, including its "nitrating potential" [12]. On the other, very high concentrations of synthetic RNS may mask other major modulatory and/or signaling features of peroxynitrite/RNS that may work by different biochemical mechanisms. For instance, very recently it has been shown that peroxynitrite (10–50 µM) can also activate SERCA2a and increase sarcoplasmic reticulum Ca2+-uptake activity in vascular smooth muscle via reversible S-glutathiolation of cysteine residues [13].
It is plausible that in vivo protein nitration is a "dynamic" process, not always culminating in irreversible alterations. In this sense, the fate of in vivo nitrated proteins, their metabolism, and the reversibility of either free or protein-associated tyrosine residues (i.e. proteolytic degradation and/or denitration [3]) constitute another possible challenge for a more complete understanding of the role of in vivo nitration in cardiac diseases. Accordingly, the availability of proteomics-based strategies, by revealing the extent and exact location of nitration of specific amino acids, should help in monitoring this process that is meanwhile unfolding in chronic cardiac diseases such as CHF.
Lastly, the formation of NO2 (and consequent functional alterations) is increasingly viewed as being a contribution of several pathways [3,14], whereby triggers other than peroxynitrite may prominently be at play. In the present study, a major role for hydrogen peroxide-dependent conversion of nitrite to NO2 has been convincingly ruled out, as well as the possible participation of nitrite/nitrate as the natural decomposition products of peroxynitrite. Still, whether other potential nitrating agents are involved in vivo remains to be established. Myeloperoxidase and other peroxidases are other potential major sources of nitration potential, and their activity is abundantly present in myocardial areas subjected to ischemia and inflammation [15].
With a mythological take, it could be tempting to compare "peroxynitrite, the ugly" with Pan, the horrible-looking Greek god of forests, famous for his quick, horrifying, and ubiquitous appearances in the woods and countryside. However, it is now increasingly evident that peroxynitrite action might not be so ubiquitous and, perhaps, not always ugly. Moreover, since "pan" in Greek also means "all", we can now postulate that in vivo nitration and its functional consequences are unlikely "pan-peroxynitrite"-induced effects. Instead, it is more conceivable that nitration-induced structural and functional changes are due to the "concert" of more than one endogenously dancing "proteiform satyr".
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- Reiter C.D., Teng R.J., Beckman J.S. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem (2000) 275:32460–32466.
[Abstract/Free Full Text] - Gow A.J., Farkouh C.R., Munson D.A., Posencheg M.A., Ischiropoulos H. Biological significance of nitric oxide-mediated protein modifications. Am J Physiol Lung Cell Mol Physiol (2004) 287:L262–L268.
[Abstract/Free Full Text] - Schopfer F.J., Baker P.R., Freeman B.A. NO-dependent protein nitration: a cell signaling event or an oxidative inflammatory response? Trends Biochem Sci (2003) 28:646–654.[CrossRef][ISI][Medline]
- Ferdinandy P., Danial H., Ambrus I., Rothery R.A., Schulz R. Peroxynitrite is a major contributor to cytokine-induced myocardial contractile failure. Circ Res (2000) 87:241–247.
[Abstract/Free Full Text] - Brunner F., Wolkart G. Peroxynitrite-induced cardiac depression: role of myofilament desensitization and cGMP pathway. Cardiovasc Res (2003) 60:355–364.
[Abstract/Free Full Text] - Ferdinandy P., Panas D., Schulz R. Peroxynitrite contributes to spontaneous loss of cardiac efficiency in isolated working rat hearts. Am J Physiol (1999) 276:H1861–H1867.[ISI][Medline]
- Mihm M.J., Coyle C.M., Schanbacher B.L., Weinstein D.M., Bauer J.A. Peroxynitrite induced nitration and inactivation of myofibrillar creatine kinase in experimental heart failure. Cardiovasc Res (2001) 49:798–807.
[Abstract/Free Full Text] - Cheng X.S., Shimokawa H., Momii H., Oyama J., Fukuyama N., Egashira K., et al. Role of superoxide anion in the pathogenesis of cytokine-induced myocardial dysfunction in dogs in vivo. Cardiovasc Res (1999) 42:651–659.
[Abstract/Free Full Text] - Borbély A., Tóth A., Édes I., Virág L., Papp J.G., Varró A., et al. Peroxynitrite-induced -actinin nitration and contractile alterations in isolated human myocardial cells. Cardiovasc Res (2005) 67:225–233.
[Abstract/Free Full Text] - Bird S.D., Doevendans P.A., van Rooijen M.A., Brutel D.L.R., Hassink R.J., Passier R., et al. The human adult cardiomyocyte phenotype. Cardiovasc Res (2003) 58:423–434.
[Abstract/Free Full Text] - Lokuta A.J., Maertz N.A., Meethal S.V., Potter K.T., Kamp T.J., Valdivia H.H., et al. Increased nitration of sarcoplasmic reticulum Ca2+-ATPase in human heart failure. Circulation (2005) 111:988–995.
[Abstract/Free Full Text] - Pfeiffer S., Mayer B. Lack of tyrosine nitration by peroxynitrite generated at physiological pH. J Biol Chem (1998) 273:27280–27285.
[Abstract/Free Full Text] - Adachi T., Weisbrod R.M., Pimentel D.R., Ying J., Sharov V.S., Schoneich C., et al. S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide. Nat Med (2004) 10:1200–1207.[CrossRef][ISI][Medline]
- Tarpey M.M., Wink D.A., Grisham M.B. Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am J Physiol Regul Integr Comp Physiol (2004) 286:R431–R444.
[Abstract/Free Full Text] - Kin H., Zhao Z.Q., Sun H.Y., Wang N.P., Corvera J.S., Halkos M.E., et al. Postconditioning attenuates myocardial ischemia–reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res (2004) 62:74–85.
[Abstract/Free Full Text]
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