© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Peroxynitrite induced nitration and inactivation of myofibrillar creatine kinase in experimental heart failure
Division of Pharmacology/College of Pharmacy and OSU Heart and Lung Research Institute The Ohio State University, 412 Riffe Building, 500 West Twelfth Street, Columbus, OH 43210, USA
* Corresponding author. Tel.: +1-614-292-1614; fax: +1-614-292-9083 bauer.140{at}osu.edu
Received 6 September 2000; accepted 23 November 2000
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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Objective: Oxidative stress is implicated in the initiation and progression of congestive heart failure, but the putative reactive species and cellular targets involved remain undefined. We have previously shown that peroxynitrite (ONOO–, an aggressive biological oxidant and nitrating agent) potently inhibits myofibrillar creatine kinase (MM-CK), a critical controller of contractility known to be impaired during heart failure. Here we hypothesized that nitration and inhibition of MM-CK participate in cardiac failure in vivo. Methods: Heart failure was induced in rats by myocardial infarction (left coronary artery ligation) and confirmed by histological analysis at 8 weeks postinfarct (1.3±1.4 vs. 37.7±3.2% left ventricular circumference; sham control vs. CHF, n = 10 each). Results: Immunohistochemistry demonstrated significantly increased protein nitration in failing myocardium compared to control (optical density: 0.58±0.06 vs. 0.93±0.09, sham vs. CHF, P<0.05). Significant decreases in MM-CK activity and content were observed in failing hearts (MM-CK kcat: 6.0±0.4 vs. 3.0±0.3 µmol/nM M-CK/min, P<0.05; 6.8±1.3 vs. 4.7±1.2% myofibrillar protein, P<0.05), with no change in myosin ATPase activity. In separate experiments, isolated rat cardiac myofibrils were exposed to ONOO– (2–250 µM) and enzyme studies were conducted. Identical to in vivo studies, selective reductions in MM-CK were observed at ONOO– concentrations as low as 2 µM (IC50=92.5±6.0 µM); myosin ATPase was unaffected with ONOO– concentrations as high as 250 µM. Concentration dependent nitration of MM-CK occurred and extent of nitration was statistically correlated to extent of CK inhibition (P<0.001). Immunoprecipitation of MM-CK from failing left ventricle yielded significant evidence of tyrosine nitration. Conclusion: These data demonstrate that cardiac ONOO– formation and perturbation of myofibrillar energetic controllers occur during experimental heart failure; MM-CK may be a critical cellular target in this setting.
KEYWORDS Contractile function; Free radicals; Heart failure; Infarction; Nitric oxide
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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Congestive heart failure (CHF) is a progressive syndrome deriving from multiple etiologies, and is defined by impaired cardiac contractility, multiorgan failure and death [1,2]. The incidence of this condition continues to steadily increase despite consistent reductions in the incidence of other cardiovascular disease states in the last 20 years (e.g. hypertension, coronary artery disease, etc.) [3]. The molecular mechanisms by which CHF develops are multifactorial and incompletely understood, and currently available therapies have only modest impact on patient survival (
50% of CHF patients die within 5 years of diagnosis) [4]. Increased oxidative stress has been implicated in the etiology and pathology of multiple cardiovascular disease states, including CHF, with a variety of reactive oxidative species implicated [5]. One of the most toxic is peroxynitrite (ONOO–), formed in the nearly-instantaneous reaction of nitric oxide with superoxide anion [6]. ONOO– can participate in multiple oxidative chemistries, and has a high affinity to nitrate tyrosine residues relative to other biological oxidants, resulting in posttranslational modification of protein-bound tyrosine to 3-nitrotyrosine (3NT) [7]. We and others have demonstrated that protein nitration is a relevant phenomenon in cardiovascular disease, and have found that the extent of protein nitration is statistically correlated to extent of cardiac dysfunction in vivo [8,9]. Although protein nitration is known to mediate some toxic effects of ONOO– in vitro, including enzyme inhibition, the putative cellular targets of ONOO– in vivo are just now beginning to be investigated [10].
Recent evidence suggests that impaired cardiac energy production and transduction may also participate in the pathogenesis of CHF [11]. Significant impairments in the creatine kinase energy shuttle have been demonstrated in multiple experimental settings of heart failure [12,13]. Human studies confirm these results, as creatine depletion and decreased creatine kinase activity are associated with CHF from multiple etiologies [14]. Despite the recognition of severe impairment in the creatine kinase system during CHF over two decades ago, the mechanisms by which this impairment occurs remain poorly understood and therapeutic strategies for intervention are undefined. While multiple in vitro studies suggest that reactive oxygen species may mediate this energetic dysfunction, a causal link has not been firmly established [15,16].
The primary myofibrillar energetic controllers are the myofibrillar isoform of creatine kinase (MM-CK) and myosin ATPase. Both have been shown in vitro to be susceptible to a variety of oxidative insults in the absence of mitochondrial dysfunction [17]. Recent studies have demonstrated that the myofibrillar isoform of creatine kinase is highly sensitive to inactivation by in vitro exposures to ONOO– at physiologically relevant concentrations, and that the apparent mechanism is via nitration of critical tyrosine residues at the active site [18]. Here we used a relevant animal model of CHF to test the hypothesis that ONOO– is formed during CHF and results in cardiac protein nitration. We then tested the hypothesis that cardiac myofibrillar energetic controllers, namely myofibrillar CK and myosin ATPase, were impaired during this model of CHF, and investigated a mechanistic role for ONOO– and attendant protein nitration in this process.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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2.1 Rat model of CHF
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 82-23, revised 1996). Congestive heart failure was produced in healthy male Sprague–Dawley rats (300–325 g) secondary to ligation of the left coronary artery, as previously described [19]. Animals were allowed to recover from surgery for at least 6 weeks, producing a fully healed myocardial infarct. Complete occlusion of the left coronary artery in surviving rats produced transmural infarct at the apex and anterior free wall of the left ventricle. Overall mortality of this procedure was approximately 40% during the 6–8 week recovery.
2.2 General histology and immunohistochemistry
Animals were sacrificed with 100 mg/kg i.p. pentobarbital sodium (Abbott Laboratories, Chicago, IL, USA). The apical portion of the heart was bisected distal to the mitral valve and immersed in 10% formalin. Tissues were evaluated for general histology (Masson's trichrome) and immunostained for 3-nitrotyrosine (anti-3-NT, Upstate Biotechnology, Lake Placid, NY, USA; 1:400) as previously described [8].
2.3 Image capture and digital image analysis
Images were captured using a Polaroid digital camera (Polaroid, Cambridge, MA, USA) and transferred into research-based digital image analysis software (Image Pro Plus, Media Cybernetics, Silver Spring, MD, USA). Extent of immunoreactivity in the LV was determined by measuring the optical density of diaminobenzidine signal in each tissue. Multiple images were randomly captured from non-infarcted left ventricular tissue (5–6 per cross-section) under identical lighting and optical settings as to encompass <40% of total non-infarcted left ventricular area. Images were then segmented to eliminate background and nuclear counterstain from analysis. Integrated optical densities were determined for each image as a measure of staining intensity. A total of 132 images were captured and analyzed by this procedure. Intra- and inter-observer variability were each less than 2%.
Trichrome staining of cardiac slices was assessed to estimate left ventricular infarct size according to Rubin et al. [20]. Ratios of scar length to total left ventricular circumference were determined for each section, using both epicardial and endocardial measures. Area measurements were not used because these may underestimate infarct size due to tissue resorption and wall thinning [21].
2.4 Myofibrillar isolations
Tissue was isolated from infarcted hearts distal from the scar regions (uninvolved anterior and/or posterior LV regions collected for homogenates; locations matched those taken from sham controls). LV myofibrillar fractions were prepared as previously described [8]. Light microscopy was used to observe isolated myofibrils in solution. Additionally, CK activity was measured from the supernatant of the final isolation wash. This analysis yielded activity measures less than 3% of the enriched myofibrillar fraction. Myofibrillar protein content was determined by the Bradford assay at 25°C. Protein content was normalized for each sample before treatment.
2.5 MM-creatine kinase activity
MM-CK activity from isolated fibrillar fractions was determined spectrophotometrically by the method of Oliver [22]. This enzyme, in its native form, is a functional homo-dimer of two M-CK subunits. Briefly, a three enzyme system was used to measure ATP formation indirectly by NADPH formation. The formation of NADPH was monitored spectrophotometrically at 340 nm, and the rate of change of absorbance was directly proportional to CK activity. Myofibrillar fractions were added to the reaction mixture at 25°C, then various concentrations of PCr (0.1–35 mM) were added to start the reaction. Change in absorbance was monitored for 6 min and reflects CK velocity. Each treatment group was investigated at each concentration in triplicate. In preliminary experiments, ATP formation was demonstrated to be rate-limiting at all CK concentrations analyzed by varying the CK concentration from 2.5 mg/ml to 5 g/ml.
2.6 Myosin ATPase activity
Calcium dependent myosin ATPase activity was assayed from cardiac myofibrillar fractions according to Malhotra et al. [23]. Reaction mixtures consisted of 0.3 M KCl, 0.01 M CaCl2 in 50 mM Tris–maleate buffer, pH 7.6 at 25°C. Reactions were initiated by the addition of Na2ATP (5 mM, confirmed to produce maximal ATPase velocity) and terminated 70 min later by addition of 30% trichloroacetic acid. Inorganic phosphate was then determined by the methods of Fiske and SubbaRow as a measure of ATPase activity [23]. Each treatment group was investigated in triplicate. ATPase activity was calibrated to an inorganic phosphate standard curve and expressed as µmol Pi formed/mg myofibrillar protein/min.
2.7 Immunoprecipitation and Western blot analyses for M-CK
Immunoprecipitation of M-CK (reduced monomer of homodimeric MM-CK) was performed according to Hemmer et al. [24]. Briefly, 200 µg of myofibrillar isolate was incubated with 2 µg of polyclonal anti-M-CK for 1 h at 4°C in RIPA buffer (100 mM Tris, 300 mM NaCl, 10 mM EDTA, 2% Triton X-100, pH 7.5). Isolates were then treated with Protein G Sepharose overnight, then spun at 200 g. Pellet was washed three times in ice-cold RIPA buffer, then suspended in Western blotting sample buffer, and loaded for SDS–PAGE analysis.
Acrylamide gel electrophoresis and western blotting methods were used to isolate MM-CK and quantify 3NT immunoprevalence after ONOO– treatment, as previously described [8]. Blots were scanned using a Hewlett-Packard Scanjet 6200c capable of 1290x960 resolution (Palo Alto, CA, USA) and transferred into research-based digital image analysis software (Image Pro Plus) for analysis. Images were background corrected then set to grayscale, and band areas and average pixel intensity were determined. The product of these two parameters gave a measure of 3NT or M-CK immunoprevalence for each band. In nitration studies, this product was corrected for blot-to-blot variability by dividing by 3NT signal for the nitrated BSA internal standard of the respective blot. Untreated and tetranitromethane-treated M-CK were used to develop standard curves of DAB signal vs. micrograms M-CK or moles 3NT (determined by assuming full nitration of 10 available tyrosine residues per M-CK, respectively). Comparison of triplicate standard curves developed with tetranitromethane-treated MM-CK and reduced by tetranitromethane-treated internal standard yielded a detection limit of approximately 10 pmol 3NT. Detection limit for M-CK standard curve was approximately 100 ng. Coefficients of variation for this method were <5% for intra-blot variability, and equal to 16% for inter-blot variability.
2.8 Treatment of isolated myofibrils with ONOO–
Myofibrillar fractions were isolated from normal rat hearts as described above and treated in vitro with ONOO–. ONOO– (Upstate Biotechnology, 5 mM) was diluted in 0.3 M NaOH in ultrapure water. Immediately before addition, ONOO– concentrations were confirmed by spectroscopy (
302=1.62 mM–1cm–1). The half-life of ONOO– in 0.3 M NaOH was experimentally determined to be 3.3 h. Various concentrations (2–250 µM final concentration) of ONOO– were achieved. ONOO– solutions were rapidly stirred into myofibrillar fraction solutions (1 mg/ml total protein) as bolus additions (5% of total volume). After addition, the solution was stirred for an additional 10 min to allow for complete reaction (t1/2 1 s for ONOO at pH 6.8) [7], and assayed for CK or myosin ATPase activity as described above. Three controls were used for ONOO– treatment: no addition control, ONOO– background electrolyte control (0.3 M NaOH) and degraded ONOO– control (250 µM ONOO– titrated to pH 7 and degraded for 30 min). In all experiments, all three controls were not statistically different; treatments were pooled as one control for further statistical comparisons.
2.9 Data handling and statistics
MM-CK and myosin ATPase velocity data were fit by Michaelis–Menton kinetics, yielding Vmax and Km parameters, using GRAPHPAD PRIZM software. Significant differences were determined using two-tailed Student's t-tests or one-way analyses of variance on ranks for non-parametic comparisons (relative 3NT immunoprevalence), with posthoc Newman–Keuls tests to evaluate significant comparisons. Correlation analysis performed using Spearman's non-parametric correlation. P<0.05 described statistical significance.
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3 Results |
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3.1 Rat model of heart failure
CHF rats demonstrated left ventricular dilation and significant scarring in the infarcted region (Fig. 1). Left ventricular infarct size was significantly increased compared to sham operated control (37.7±3.2% vs. 1.3±1.4%; CHF vs. sham, P<0.05), indicative of moderate heart failure [25].
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, P<0.05 compared to preadsorbed control.3.2 Cardiac immunohistochemistry
Shown in Fig. 1 are representative photomicrographs of left ventricular immunohistochemical staining for 3NT. Immunohistochemical staining demonstrated significant increases in cardiac immunoprevalence for 3NT in failing hearts compared to sham controls (Fig. 1, upper panel). The pattern of immunoreactivity observed was diffuse throughout the left ventricle and was apparently not resultant from focal immune cell infiltration nor regionally confined to the infarction scar.
Also shown in Fig. 1 is the integrated optical density analysis of 3NT immunoprevalence in CHF versus sham treated left ventricles. Failing cardiac tissue demonstrated a statistically significant increase in 3NT immunoprevalence compared to sham control (P<0.05, n = 8 hearts for each group). Further examination of myocardial 3NT staining (1000x magnification by oil immersion light microscopy) suggested that immunoprevalence may parallel the myofibrillar architecture.
3.3 MM-CK and myosin ATPase activity in CHF
We observed significantly decreased MM-CK activity in failing hearts compared to sham controls. Left ventricular MM-CK activity was decreased by 58% during CHF compared to sham control (Fig. 2, left panel). In contrast, myosin ATPase activity was not impaired during CHF. Myosin ATPase activity in failing left ventricle was not statistically different compared to sham control, and actually demonstrated a trend toward increased activity (Fig. 2, middle panel). The kinetic ratio of MM-CK velocity to ATPase velocity was also significantly reduced by 67% in failing hearts compared to sham controls (Fig. 2, right panel, P<0.05).
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3.4 MM-CK content in cardiac myofibrils during experimental heart failure
Standard curves for M-CK content were constructed using pure M-CK (0.3–5 µg) probed for M-CK immunoprevalence (Fig. 3A). Immunoreactive response was linear over the protein loads studied (Fig. 3B). Myofibrillar samples were analyzed using identical methods and applied to the M-CK standard curve (Fig. 3C). Induction of heart failure resulted in loss of M-CK content (reduced 29%) in CHF rats compared to sham control (Fig. 3D, left panel, P<0.05). However, this result incompletely explained the observed impairment in MM-CK activity, as kcat was also significantly reduced (Fig. 3D, right panel, 6.0±0.4 vs. 3.0±0.3 µmol/nM M-CK/min, sham vs. CHF, P<0.05).
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3.5 Myofibrillar MM-CK activity after ONOO– administration
Administration of ONOO– to left ventricular myofibrils resulted in statistically significant inhibition of MM-CK activity at concentrations as low as 2 µM, with a fitted IC50=92.5±6.0 µM (Fig. 4, upper panel, statistics performed on raw data). The four control groups employed (No Addition, pH Control, 50 µM and 250 µM degraded ONOO–, 19.7±1.52, 18.5±0.62, 16.8±0.66, 19.6±0.56 µmol/mg myofibrillar protein/min) were not statistically different, and were pooled.
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Myosin ATPase activity was also assayed following ONOO– exposure. Again, no statistically significant differences were seen between any of the controls employed, and these values were pooled (2.1±0.15, 1.8±0.34, 2.0±0.31 µmol/mg myofibrillar protein/min, No Addition, pH Control, and 250 µM degraded ONOO–, respectively, P = NS). Similar to the in vivo results, ONOO– had no significant inhibitory effect on ATPase Vmax at concentrations as high as 250 µM (Fig. 4, middle panel).
The ratio of MM-CK to ATPase velocity was assessed at each ONOO– concentration studied (Fig. 4, lower panel). Significant decreases in this ratio occurred at ONOO– concentrations of 25 µM and above, and was reduced 250% at 250 µM ONOO.
3.6 Western blotting analysis of cardiac myofibrils following ONOO– exposure
Myofibrils exposed to ONOO– demonstrated extensive nitration of M-CK. Equal protein load was verified in each lane by FastBlot protein stain (data not shown). A representative blot is shown in Fig. 5, where 3NT immunoprevalence can be seen at all ONOO– concentrations studied. At low concentrations, M-CK was the only nitrated protein band detectable. 3NT immunoprevalence was quantified using digital image analysis, then converted to moles 3NT using a tetranitromethane-treated-M-CK calibration curve. Moles 3NT formed in myofibrillar M-CK was related to ONOO– in a concentration-dependent manner (Fig. 4, middle panel). While this concentration dependency approached linearity, nitration efficiency (moles 3NT formed/moles ONOO– administered, i.e. slope of the 3NT-ONOO– curve) was higher at lower ONOO– concentrations, suggesting that nitration chemistries may be favored in MM-CK at lower ONOO– concentrations.
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Correlation analysis was applied to investigate the dependency of MM-CK inhibition on nitration. Extent of protein nitration of M-CK was highly correlated to extent of inhibition of MM-CK activity as pmol 3NT formed was negatively correlated to MM-CK Vmax by Spearman's non-parametric correlation (Fig. 5, lower panel, P<0.001).
3.7 Nitration of myofibrillar creatine kinase following experimental heart failure
MM-CK enzyme content immunoprecipitated from myofibrillar isolates was not significantly different between sham and failing left ventricles. We observed a significant elevation in the extent of tyrosine nitration in myofibrillar creatine kinase from failing versus sham left ventricle (Fig. 6). Basal tyrosine nitration was detectable in M-CK from sham controls, consistent with immunohistochemistry. However, the extent of tyrosine nitration was elevated 50% in failing left ventricles compared to control (Fig. 6, lower panel). In addition, a statistically significant relationship between the extent of nitration and inactivation of MM-CK was detected, as nitration intensity of M-CK was negatively correlated to MM-CK kcat (Spearman's correlation: r = 0.80, P<0.01).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionAcknowledgmentsReferences |
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Here we employed a relevant animal model of CHF to test the hypothesis that cardiac protein nitration, suggestive of ONOO– formation, occurs during cardiac failure. We then demonstrated that CHF was associated with impaired myofibrillar energetic control and investigated the role of protein nitration in this process, testing the hypothesis that ONOO– contributes to energetic impairments during CHF.
The rat model of CHF produced by myocardial infarction is well established and has been studied by many investigators. This model mimics most of the biochemical and functional changes seen in human heart failure. For example, reductions in both diastolic and systolic performance are observed in this experimental model [25]. Neurohumoral alterations are also remarkably similar [26]. This rat model develops drug-related complications identical to humans and is highly predictive of clinical outcomes as well [19]. This model therefore provides an excellent paradigm for the mechanistic study of oxidant and myocyte energetic interactions during CHF.
Using immunohistochemical analysis, we observed striking evidence of cardiac 3NT formation in failing ventricles. Staining was widespread throughout the left ventricle and not isolated to focal immune cell infiltrates or the infarction scar. Quantitative analysis of 3NT immunoprevalence demonstrated a statistically significant increase in cardiac 3NT formation in failing hearts, although sham controls did demonstrate some evidence of protein nitration. These results are consistent with the emerging view that tyrosine nitration may be a dynamic posttranslational signaling event that, under certain conditions, may be a regulated and/or controlled modulator of protein structure and enzymatic function [27]. Further study into the controllers of protein nitration and the thresholds at which basal protein nitration escalates into a pathogenic phenomenon are warranted, and are currently ongoing in our laboratory.
High magnification analysis of 3NT immunostaining in the failing left ventricle suggested that 3NT immunoprevalence paralleled the striated architecture of the myofibrillar structure. We therefore investigated the state of the primary energetic controllers bound to the myofibril, namely myosin ATPase and MM-CK. We found that failing hearts exhibited a 58% decrease in maximal velocity of myofibrillar ATP formation (MM-CK activity) compared to sham controls. Interestingly, we observed no decrease in calcium-activated myosin ATPase activity in failing hearts compared to sham controls. This differential inhibition suggests that MM-CK is preferentially sensitive to the energetic disruption that occurred during cardiac failure. The ratio of MM-CK activity to ATPase activity was drastically altered in failing left ventricle, reduced by 67% compared to sham controls. The functional consequences of a diminished ratio include increased myocardial stiffening, diastolic dysfunction and increased myocardial workloads, all early indicators and symptoms of CHF. While significant decreases in myofibrillar M-CK content were observed in failing left ventricles compared to sham control, the magnitude of these changes do not completely explain the observed impairment in MM-CK activity since kcat was more drastically reduced.
We followed these biochemical measures with mechanistic studies to determine the potential contribution of ONOO– to the observed alterations in energetic control. Isolated myofibrillar studies assessed the relative consequences of ONOO– exposure to the intact structural environment of MM-CK and myosin ATPase. While regional myocardial differences at the infarct borders may exist, we tried to limit this complication by avoiding these tissue regions. Thus our findings probably reflect global alterations in myocyte performance rather than local changes at infarct edges. The pattern of ONOO–-induced inhibition observed was identical to that observed during heart failure — selective diminution of MM-CK with no alteration of ATPase activity. We observed potent inhibition of MM-CK at concentrations as low as 2 µM, with no decrease in ATPase Vmax at any of the ONOO– concentrations studied. Similarly, the MM-CK/ATPase ratio was significantly decreased with increasing ONOO– concentration, falling from approximately 10 to 2.5 at 250 µM ONOO–. Importantly, this organelle demonstrates extremely high sensitivity to ONOO– compared to other subcellular structures previously reported, most notably mitochondria and mitochondrial CK. Studies on isolated mitochondria required exposure to ONOO– concentrations <200 µM before significant inhibition of Mi-CK was observed, and ONOO– concentrations <500 µM before alterations in oxidative respiration were detected [28]. Here, using an analogous method of administration (bolus administration to intact organelles), we observed significant inhibition of MM-CK at ONOO– concentrations 100-fold lower than employed in such studies, within expected pathologically relevant ONOO– concentrations. These findings are consistent with previous studies suggesting that the myofibrillar structure may be a highly sensitive target of reactive oxygen species during cardiac failure [29]. In achieving significant inhibition at ONOO– concentrations as low as 2 µM, MM-CK is 1000 and 100-fold more sensitive to ONOO– than to H2O2 and superoxide anion, respectively, two other oxidants implicated in the pathology of CHF which have been evaluated as toxicants to myofibrillar bound CK activity [29]. These data illustrate that the myofibrillar isoform of CK may be a uniquely sensitive target of the myofibrillar structure. Other investigators have demonstrated that compensatory upregulation of alternative CK isoforms (particularly cytosolic BB-CK isoform, usually silent in cardiac myocytes) occur in this model [30]. However, high energy phosphate flux (by in vivo 31P-NMR) remains severely disrupted, likely due to the importance of the intracellular location of MM-CK, covalently bound to the M-line of the myofibril and functionally coupled to the myosin ATPase (MB- and BB-isoforms do not possess this binding capacity) [31]. Our findings are consistent with these investigations and support the importance of the MM-CK isoform in this setting.
In vitro myofibrillar exposures caused ONOO– concentration-dependent nitration of MM-CK. Moreover, at low concentrations of ONOO– (2 µM), MM-CK was the only fibrillar protein demonstrating detectable 3NT immunoprevalence (limit of detection=10 pmol 3NT). While the relationship between ONOO– and MM-CK 3NT formation approached linearity, nitration efficiency appeared to be higher at low ONOO– concentrations. Correlation analysis demonstrated that extent of nitration of MM-CK was statistically correlated to extent of MM-CK inhibition. 3NT immunoprevalence was negatively correlated to MM-CK Vmax. An essential TYR residue is believed to be critical for the orientation of ADP during catalysis, and may be an important site of nitration in this enzyme [24]. These data, in combination with our previous isolated MM-CK studies, suggest that the potent inhibition of MM-CK observed both as pure enzyme and bound to its functional environment may be mediated by tyrosine nitration. Other investigators have employed in vitro methodologies to evaluate ONOO– interactions with other CK isoforms, concluding that nitration occurs only at high ONOO– concentrations (100s of micromolar) and/or nitration does not likely contribute mechanistically to inhibition [16,28]. These authors suggested that oxidation of critical sulfhydrals are mechanistically involved. However, those studies employed purified enzyme, fully dissociated from its native environment in its monomer form. While we cannot exclude the contributions of sulfhydral oxidation, we observed a striking linear relationship between extent of inhibition and nitration (consistent with one biochemical mechanism) throughout the wide range of enzyme activities studied. Therefore, the experiments described herein may more appropriately reflect biologically relevant conditions; nitration of this isoform may play a primary mechanistic role.
Finally, myofibrillar isolates from LV of heart failure rats demonstrated significant increases in tyrosine nitration. These results are consistent with our in vitro studies implicating ONOO–-mediated tyrosine nitration as a potent event in the impairment of myofibrillar creatine kinase, and suggest that nitration of MM-CK may contribute to the observed inhibition during decompensated cardiac failure. While our data strongly suggest that MM-CK is an important and vulnerable target of ONOO–, they do not preclude the importance of other biological oxidants in the contractile and energetic impairments that occur during heart failure.
In summary, using a relevant model of CHF, we observed significant evidence of increased ONOO– formation during cardiac failure. This nitration appeared to predominate in the myofibrillar structure, and failing hearts demonstrated specific and severe alteration in myofibrillar CK activity, with no change in myosin ATPase activity. Direct administration of ONOO– to isolated myofibrils paralleled this failing energetic profile, and nitration of MM-CK was correlated to extent of MM-CK dysfunction. Finally, MM-CK from failing left ventricle demonstrated significant tyrosine nitration. These data suggest that cardiac ONOO– formation has deleterious effects on important myofibrillar energetic controllers, specifically potent inhibition of MM-CK, during congestive heart failure, and that tyrosine nitration may be an important event in this setting. Further study of the implications of these interactions in the pathogenesis of heart failure appears warranted.
Time for primary review 23 days.
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Acknowledgments |
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This work was supported in part by the American Heart Association (Ohio-West Virginia Affiliates) and the National Institutes of Health (HL59791, HL63067).
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