Peroxynitrite-induced inhibition and nitration of cardiac myofibrillar creatine kinase
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.
Section snippets
Creatine kinase activity
All chemicals used were purchased from Sigma Chemical (St. Louis, MO), except where noted. Pure MM-CK (Type III, bovine heart CK) was suspended in 50 mM imidazole buffer, pH 6.8 (local pH for myofibrillar microenvironment). CK activity was determined spectrophotometrically, as previously described [9]. CK velocities at various phosphocreatine (PCr, 0.1–45 μM) concentrations were fit to the Michaelis–Menton equation, and MM-CK Vmax and Km were determined for all the treatment groups. Each
MM-CK activity after ONOO– administration
Bolus administration of ONOO– to MM-CK (250 or 5 μg/ml) resulted in significant inhibition of enzyme capacity (Vmax) at concentrations as low as 100 nM ONOO– (Fig. 1). Enzyme affinity (Km) was not affected (Fig. 1). The three control groups employed in these studies (No addition, pH control, degraded ONOO–) were not statistically different at each enzyme concentration studied, and were therefore pooled for statistical comparisons (121 ± 2.5, 112 ± 2.1, 109 ± 2.5 μmol/mg MM-CK/min, respectively,
Discussion
RNS formation in the heart may have important pathophysiological consequences, mediated by the distinct reactivities of RNS relative to more traditionally recognized reactive oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical, etc.) [1], [2], [26]. Tyrosine nitration may be the predominant reactivity of RNS in vivo, and protein-3NT formation has been demonstrated to be a potent structural and functional protein modification [27]. We have recently shown that the myofibrillar
Conclusion
These data suggest that MM-CK may be a particularly sensitive protein target for ONOO–-mediated inhibition, and support a role for tyrosine nitration in this process. When combined with the considerable evidence that MM-CK is nitrated during cardiac failure, these studies strongly implicate protein nitration as a central mechanism of impairment in the CK shuttle in vivo. Moreover, these findings support the emerging hypothesis that cardiac ONOO– formation is a meaningful participant in the
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (HL59791, DK55053, HL63067) and an award from the American Heart Association, Ohio Valley Affiliate.
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