Peroxynitrite-induced inhibition and nitration of cardiac myofibrillar creatine kinase

Abstract

Although cardiac peroxynitrite formation and attendant protein nitration is an established event in both acute and chronic settings of cardiac failure, the putative intracellular targets involved remain incompletely defined. We have recently shown that the myofibrillar isoform of creatine kinase (a critical energetic controller of cardiomyocyte contractility) may be a particularly sensitive target of peroxynitrite-induced nitration and inactivation in vivo. However, the kinetic and mechanistic aspects of this interaction remain undefined. Here we tested the hypothesis that myofibrillar creatine kinase is sensitive to inhibition by peroxynitrite, and investigated the mechanistic role for tyrosine nitration in this process. Peroxynitrite potently and irreversibly inhibited myofibrillar creatine kinase capacity (Vmax), at concentrations as low as 100 nM, while substrate affinity (Km) was unaffected. Concentration-dependent nitration of myofibrillar creatine kinase was observed. The extent of nitration was linearly related to peroxynitrite concentration and highly correlated to the extent of myofibrillar creatine kinase inhibition. This inhibition was not reversible by treatment with free cysteine (250 μM), but pre-incubation with substrate (phosphocreatine and/or ATP) provided significant protection of MM-CK from both nitration and inhibition. These results suggest that myofibrillar creatine kinase is a highly sensitive target of peroxynitrite-mediated inhibition, and that nitration may mediate this inhibition.

Introduction

Peroxynitrite (ONOO^–) is a biologically relevant, highly cytotoxic reactive nitrogen species (RNS) formed in the diffusion-rate limited, bimolecular reaction of nitric oxide and superoxide anion [1]. ONOO^– exerts many of its cytotoxic effects through its avid capacity to alter protein structure and function via sulfhydral oxidation and/or nitration of tyrosine residues, which results in the formation of 3-nitro-l-tyrosine (3NT) [2]. ONOO^– has been demonstrated to inhibit enzymatic processes in vitro; both nitration and oxidative chemistries have been implicated as mechanisms of this enzyme inhibition [3], [4], [5], [6].

A wealth of recent data has demonstrated that cardiac ONOO^– formation may be a universal phenomenon in both acute and chronic settings of cardiac failure [7], [8], [9], [10], [11]. However, its mechanistic significance in the initiation and/or progression of cardiac failure, as well as the putative cellular targets involved, remain incompletely defined. We have recently demonstrated that the myofibrillar compartment (the protein structure that mediates the mechanical work of cardiomyocyte contraction) is a predominant site of ONOO^–-mediated protein nitration in multiple settings of cardiac failure, and that the myofibrillar isoform of creatine kinase (MM-CK) demonstrates high levels of protein-3NT formation relative to other myofibrillar protein constituents both in vitroand in vivo—MM-CK may represent a particularly relevant target of ONOO^–-induced protein oxidative events during cardiac failure [8], [9], [10].

The creatine kinases (CK) are critical energetic controllers that regulate cardiac myocyte contractility in both health and disease [12], [13]. Two isoforms of creatine kinase, octomeric mitochondrial creatine kinase and homodimeric myofibrillar creatine kinase (MM-CK), form an energy shuttle system that vectors mitochondrial ATP production to myofibrillar utilization sites using creatine as a high energy phosphate carrier—MM-CK is directly responsible for supplying high energy phosphate to the active site of myosin ATPase to energize cross-bridge cycling and myocyte contraction [14], [15]. Impairment of the CK shuttle system has been demonstrated in multiple settings of cardiac failure, including humans, with significant reductions in MM-CK activity, content and gene expression observed [16], [17], [18], [19], [20]. Although impairment of the CK system during cardiac failure has been recognized for over 15 years, the mechanism(s) by which these events develop remains poorly understood.

We have observed nitration and inactivation of MM-CK as an early event in multiple acute and chronic settings of cardiac myocyte dysfunction and failure, in both in vivo and in vitro settings [8], [9], [10]. Despite recent suggestion of CK isoform sensitivity to cellular redox state, the kinetic and mechanistic effects of ONOO^– on the myofibrillar isoform of creatine kinase have not been evaluated [21], [22]. Here we tested the hypothesis that ONOO^– has potent direct inhibitory effects on MM-CK function and investigated kinetic and mechanistic aspects of this inhibition.