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CARDIOVASCULAR
Division of Cardiology, Department of Medicine (N.P., C.G.T., D.A.K.) and Department of Pathology (M.H., P.M.C.), Johns Hopkins Medical Institutions, Baltimore, Maryland; Department of Clinical Medicine, Section of General Pathology, University of Perugia, Perugia, Italy (N.P., R.B.); Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Rome, Italy (B.T.); Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute of Aging, National Institutes of Health, Baltimore, Maryland (Y.A.G.); Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Catania, Italy (A.M.A., G.L.); and Department of Medicine, New York Medical College, Valhalla, New York (J.K.)
Received November 28, 2005; accepted January 23, 2006.
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GATP), and phosphocreatine while increasing free [ADP], AMP catabolites (nucleoside-total purines), and lactate. PD166793 reversed most of these changes, in part due to its inhibition of AMP deaminase. MMP activation may influence cardiac diastolic function by mechanisms beyond modulation of extracellular matrix. Interaction between MMP activation and HEP metabolism may play an important role in mediating diastolic dysfunction. Furthermore, these data highlight a potential major role for increased AMP deaminase activity in diastolic dysfunction.
). Such stiffening can result from alterations in the extracellular matrix and matrix turnover (Norton et al., 1997), the latter controlled by metalloproteinases (MMP) (Thomas et al., 1998). MMP activation is often more prominent in late stage CHF and accompanied by oxidative stress and activation of the renin-angiotensin (AII) simulation (McElmurray et al., 1999). We have reported that AII infusion combined with short-term rapid ventricular pacing in the dog (AII+P) induces systolic dysfunction with exacerbated diastolic stiffening and marked activation of cardiac MMPs (Senzaki et al., 1998, 2000). The latter two appeared coupled, but not via changes in cardiac interstitial fibrosis (Senzaki et al., 2000). This is consistent with other studies where MMP inhibition improved compliance without lowering collagen content (Brower and Janicki, 2001) or in which increased myocardial stiffness was not associated with changes in collagen volume fraction (Norton et al., 1996). These results suggest that other mechanisms may link MMP activation and diastolic compliance.
A potential alternative mechanism is that MMP activation influences HEP metabolism (Iwaki-Egawa et al., 2001), increasing free cytosolic ADP that could exacerbate diastolic stiffness (Tian et al., 1997a,b). HEP stores decline with CHF (Ingwall and Weiss, 2004) often coupled to oxidative stress from neurohumoral stimulation. Increased Mg2+-ADP competes with ATP-myosin binding to favor strongly bound cross-bridges that increase stiffness and slow motor velocity. Cross-activity of MMP9 on AMP catabolism (Iwaki-Egawa et al., 2001) could contribute to the formation of oxypurines, such as uric acid, which itself is correlated with diastolic dysfunction in heart-failure patients (Cicoira et al., 2002). Accordingly, the present study examined the effect of a broad MMP inhibitor on cardiac function, the extracellular matrix, and HEP stores and enzymatic activities involved in AMP catabolism in a canine model of subacute heart failure (AII+P) exhibiting prominent diastolic dysfunction.
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Materials and Methods |
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Animal Preparation. Studies employed the AII+P model as described previously (Senzaki et al., 1998, 2000). In brief, adult mongrel dogs received angiotensin II (AII, 15 ± 4 ng · kg–1 · min–1 i.v. x 7 days) superimposed with right ventricular tachypacing (240 base beats/min) during the final 48 h. In group 1 (n = 8), no further interventions were made, whereas group 2 animals (n = 9) received the broad-spectrum MMP inhibitor PD166793 [(S)-2-(4-bromo-biphenyl-4-sulfonylamino-3-methyl butyric acid; 5 mg/kg/day p.o.] (Spinale et al., 1999; Peterson et al., 2001) for 2 weeks, starting 1 week before and continuing during the AII+P protocol. This PD166793 dose yields plasma levels well below the IC50 for neutral endopeptidase, angiotensin-converting enzyme, or other protease inhibition (Ye et al., 1994). Animals were instrumented with indwelling high-fidelity left ventricular (LV) pressure transducers, sonomicrometers for LV cross-sectional area, inferior vena caval cuff occluders, right atrial and aortic indwelling catheters, and pacing leads as described previously (Senzaki et al., 1998, 2000). At terminal study, all animals were euthanized, hearts were excised, and tissue was stored in liquid N2, optimal cutting temperature compound, or formalin for analysis.
In Vitro and in Situ Zymography. MMP abundance was assessed by in vitro gelatin zymography, with 10 to 40 µg of protein/lane loaded onto 10% polyacrylamide gels containing 0.1% gelatin (Novex, San Diego, CA), and gels were stained with 0.5% Coomassie Blue. In situ MMP activation was assessed in freshly frozen 5-µm myocardial slices incubated with 0.1 mg/ml gelatin-Oregon green (Invitrogen, Carlsbad, CA). Gelatin lysis was visualized by emitted fluorescence, and coincubation with 50 mmol/EDTA or anti-MMP9 antibody (Calbiochem, San Diego, CA) was used to confirm general and specific activity.
Collagen Evaluation. Collagen subtypes and content were assessed by quantitative immunohistochemistry on left ventricle sections (4 µm each), fixed in 10% phosphate-buffered formalin, and paraffin-embedded (Cesselli et al., 2001). Collagen cross-linking was determined by cyanogen bromide (CNBr) digestion (Vasan et al., 1996), with final aliquots of CNBr digests equivalent to 4 µg of hydroxyproline analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions using 7.5% separating gel/5% stacking gel and visualized by silver stain.
Assessment of Redox and Energy Metabolism. At the time of sacrifice, animals underwent thorachotomy and hearts from both study groups and a separate set of normal controls (n = 7) were removed under ice-cold cardioplegia (100 mEq K+; Plegisol; Abbott Laboratories, Abbott Park, IL). Transmural samples from the LV (100–200 mg each) were harvested from the left ventricle far from the K+-injected region, immediately frozen in liquid nitrogen, and stored at –80°C until use. At the time of redox and energy status assessment, each heart specimen was processed according to a recent organic deproteinization procedure suitable for the determination of water-soluble low-molecular weight compounds and representative of both tissue oxidoreductive and energy status (Lazzarino et al., 2003). High-energy phosphates, oxypurines (hypoxanthine, xanthine, and uric acid), nucleosides (inosine and adenosine), ascorbic acid, malondialdehyde, and reduced and oxidized glutathione were measured by ion-pairing HPLC (Lazzarino et al., 2003) using a Kromasil C-18, 250 x 4.6-mm, 5-µm particle size column, provided with its own guard column (Eka Chemicals AB, Bohus, Sweden). The HPLC apparatus consisted of a SpectraSystem P2000 pump system and highly sensitive UV6000LP diode array detector (both instruments from ThermoFinnigan Italia, Rodano, Milan, Italy) equipped with a 5-cm light path flow cell and set up between 200 and 300 nm wavelength. Data acquisition and analysis were performed using the ChromQuest software package (Thermo Electron Corporation, Waltham, MA) (Lazzarino et al., 2003). Pi was determined colorimetrically from the same extracts, and phosphocreatine (PCr) and creatine were measured enzymatically using a Jasco-650 UV-visible double-beam spectrophotometer (Jasco, Tokyo, Japan). PCr can rapidly degrade during the biopsy/freezing process, sometimes rendering lower polymerase chain reaction/ATP values than those derived from noninvasive 31P magnetic resonance measures. The biopsy approach was nonetheless used here because the biopsies provide samples to assess both energetics and oxidative stress (by HPLC) and, more importantly, because NMR could not have easily been performed in these animals with chronically implanted electronic pacemakers. Moreover, such a systematic effect, if present, would similarly affect biopsies from control and PD16673 hearts. However, the polymerase chain reaction/ATP ratio we found (
1) was not that dissimilar from that reported in some NMR studies (1.3) (Horn et al., 1998).
Free cytosolic [ADP] was calculated from the creatine kinase equilibrium reaction: free [ADP] = [ATP] [free creatine]/[PCr] [H+] 1.66 x 109 (Lawson and Veech, 1979). The free energy available (G
ATP) from the hydrolysis of ATP was calculated as follows: GATP = G0 + RT log[ADP] [Pi]/[ATP], where GATP is free energy of ATP, G0 is standard free energy change, R is the universal gas constant, and T is absolute temperature (Gibbs, 1985).
Assessment of PD166793 Effects on AMP Deaminase Activity. To determine whether PD166793 had direct effects on enzymes involved with AMP and purine catabolism, 20% tissue homogenates from control canine myocardium (n = 3) were prepared in 150 mM KCl and centrifuged at 20,600g for 15 min at 4°C, and aliquots (20 µl) were incubated for 45 min in 100 mM KCl, 20 mM NH +4COOH, and 2 mM AMP (250 µl of final volume), with PD166793 added at 0 to 10 µM concentrations. Incubation was stopped by the addition of 750 µl of HPLC-grade acetonitrile and deproteinized, and IMP was assessed by HPLC to index AMP deaminase activity, as described previously (Tavazzi et al., 2000).
Statistical Analysis. Data are presented as mean ± S.E.M. unless otherwise specified. Between- and within-group analysis was performed by repeated-measures analysis of variance, with a Tukey test for multiple comparisons. In addition, specific within-group-paired data were analyzed by nonpaired Student's t test for between-group treatment effect determination.
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, 2000). In group 2 animals, diastolic stiffening was largely prevented (see Fig. 1, B and D), whereas systolic function and active relaxation were depressed similar to group 1 dogs (Table 1).
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PD166793 Reduces Cardiac MMP Abundance and Activation. In situ myocardial tissue analysis of MMP activity revealed minimal basal activation but marked stimulation by AII+P (Fig. 2, A and B). MMP activation was substantially inhibited by coincubation with an MMP9-blocking antibody (Fig. 2C), consistent with prior reports (Senzaki et al., 1998, 2000). Similar results were obtained using an MMP2-blocking antibody (data not shown), supporting the involvement of this species as well. MMP activation was also absent in chronic PD166793-treated animals (Fig. 2D). In vitro zymography (Fig. 2E) yielded marked gelatin-lysis at 92 and 72 kDa (MMP9 and MMP2, respectively) induced by AII+P, with double-banding patterns consistent with activation. This was also prevented by PD166793 treatment, indicating reduced MMP abundance and activity.
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MMP Inhibition by PD166793 Does Not Reduce Collagen or Collagen Cross-linking. Both type I and III collagen increased with AII+P, with collagen I/III ratio declining (all p < 0.001; Fig. 2, F–G; Table 2). Collagen cross-linking also increased as revealed by reduced soluble collagen by CNBr assay. As shown in Fig. 2J, the percentage of collagen soluble after CNBr digestion decreased from 15.8 ± 1.65 in controls to 10.16 ± 1.3 in AII+P hearts (p = 0.01, n = 5 for each group), indicating increased cross-linked collagen. However, this was not further altered by PD166793 treatment (10.68 ± 1.19, n = 5, p = not significant versus AII+P). Although these changes would be consistent with diastolic stiffening, they were not reversed by PD166793 treatment (Fig. 2, H–I; Table 2) despite reduced chamber stiffness. An alternative cause of chamber stiffness is tissue edema, and it was possible that MMP inhibition reduced myocardial water content. This was assessed by wet-dry weight ratio, but these ratios were nearly identical between group 1 (3.57 ± 0.15) and group 2 (3.55 ± 0.12) animals (n = 4–5).
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PD166793 Restores PCr and GATP while Reducing Elevated Free ADP from AII+P. AII+P induced marked abnormalities in HEP metabolism, including a 50% decline in myocardial ATP, and a 4-fold increase in AMP: 22.5 ± 2.3 in controls (n = 5) to 97 ± 16 nmol/g wet weight in AII+P (n = 8, p < 0.001; Fig. 3). Total adenine nucleotides declined because of increased AMP catabolism as reflected by increased oxypurines (hypoxanthine, xanthine, and uric acid) and nucleosides (inosine and adenosine). IMP nearly doubled: from 32.02 ± 2.3 in controls to 57 ± 5 nmol/g wet weight in AII+P hearts (p < 0.005). The sum of oxypurines/nucleosides and adenine nucleotides (ATP + ADP + AMP) remained unchanged. Thus, HEP depletion was paralleled by catabolite accumulation, indicating an altered phosphorylation-dephosphorylation balance.
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In group 2 PD166793-treated dogs (n = 9), total adenine nucleotide levels remained near control levels. Whereas ATP levels remained reduced (Fig. 3), total ADP and AMP increased (p < 0.001 versus group 1) and AMP catabolites and IMP declined (p < 0.001 versus group 1). Thus, one particular target of PD166793 effects was AMP catabolism. The sum of adenine nucleotides, oxypurines, and nucleosides remained unaltered.
It is noteworthy that, from the perspective of diastolic stiffening, group 2 animals had reduced estimated levels of free ADP. The majority of ADP is protein-bound in myocytes, with the biologically relevant form in the free cytosolic fraction. Calculated free ADP increased nearly 80% (0.34 ± 0.01 versus 0.54 ± 0.14 µM, p < 0.01), with AII+P paralleled by a 150% rise in Pi. PCr and GATP declined while lactate and lactate/pyruvate ratio increased (Fig. 4), the latter supporting reduced oxidative glycolysis. These changes were absent in MMP inhibitor-treated hearts.
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Dose-Dependent Inhibition of AMP Deaminase by PD166793. Given the inhibition of myocardial AMP catabolism in group 2 dogs, we tested whether PD166793 had any direct, previously unknown action on purine catabolism. AMP deaminase activity was 0.139 ± 0.028 IU/g wet weight in control canine myocardium (n = 3). PD166793 modestly and dose-dependently inhibited this activity, with 20% inhibition at 0.1 µM. This was specific, because PD166793 had no effect on adenosine-deaminase, 5'-nucleotidase, purine-nucleoside-phosphorylase, or xanthine oxidoreductase activities (data not shown).
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GATP, and AMP catabolism to in vivo diastolic stiffening. Lastly, our study suggests that targeting AMP deaminase activity to reduce adenine nucleotide catabolism, responsible for the increase in nucleosides/oxypurines and decrease in myocardial energy state, is a potential new strategy to counter heart-failure features, particularly diastolic stiffening.
Collagen, MMPs, and Diastolic Dysfunction. Prior studies have tended to focus on changes in matrix proteins and tertiary structure related to MMP activation in explaining alterations in myocardial geometry and function. MMPs degrade matrix, although their activation also reflects remodeling and matrix turnover, and net increases in matrix have been observed. Likewise, variability in matrix change with MMP inhibition has been reported. In some models, increased collagen content from MMP inhibition worsened diastolic stiffening (Spinale et al., 1999), whereas transgenic models targeting specific MMPs (e.g., MMP9) reported reduced collagen accumulation (Ducharme et al., 2000), although compensatory changes in other MMPs (e.g., MMP13) may explain this observation. It is noteworthy that PD166793 has itself been shown previously to reduce LV dilation in aged spontaneously hypertensive heart failure (SHHF) rats while improving diastolic compliance (Peterson et al., 2001). In the AII+P canine model, expression and activity of myocardial MMPs (notably MMP9, MMP2, and interstitial collagenase MMP1) increase due to sympatho-stimulation, because they are prevented by 
-blockade (Senzaki et al., 2000). However, suppression of MMP activity by -blockade did not lower collagen content, although it prevented diastolic stiffening. This finding hinted that MMP activation may be central to the behavior but work by an alternative mechanism.
High-Energy Phosphate and AMP Deaminase Contributions to Diastolic Dysfunction. Abnormal HEP metabolism is thought to contribute to myocardial dysfunction in CHF (Ingwall and Weiss, 2004). This is characterized by reduced total ATP and free energy of ATP hydrolysis (Ingwall and Weiss, 2004), a fall in PCr or energy storage (Shen et al., 1999), and shift from oxidative glycolysis. ATP-dependent processes may influence systolic function and active diastolic relaxation, and it is intriguing in this regard that these functional abnormalities were similar in group 1 and group 2 hearts, along with ATP depletion levels. However, other abnormalities, such as free ADP levels, increased AMP catabolism and increased lactate, and lactate/pyruvate were nearly fully prevented by MMP inhibition; these latter abnormalities may play a more important role in diastolic stiffening.
Most ADP is bound in myocytes, but the smaller pool of free ADP (3–4% total ADP) is the one that is metabolically active and coupled to diastolic function (Tian et al., 1997a,b). ADP release is a rate-limiting step in the actomyosin ATPase reaction. Increases in free [ADP] compete for ATP binding to myosin, reducing cross-bridge cycling rate, enhancing the formation of strongly bound cross-bridges, and lowering the free energy due to ATP hydrolysis (GATP) (Senzaki et al., 1998). Our data support a role for free ADP on diastolic stiffening and, importantly, provide the first demonstration of this relationship in vivo, with increases in cytosolic free [ADP] in the current model indeed comparable with those observed previously in vitro (Tian et al., 1997a,b).
Elevated free [ADP] reduced GATP, which also can contribute to diastolic dysfunction (Katz, 1998). The decline in GATP observed with AII+P (58 to 53 kJ/mol) is probably significant, because it is in the range required by ATPases, such as sarco(endo)plasmic reticulum Ca2+ ATPase 2a and maximal contractile function in isolated hearts (Tian and Ingwall, 1996), although its restoration appeared only to improve diastolic stiffening in the present model. Because adaptive responses moderate energetically inefficient changes in cytosolic free [ADP] and GATP over time, the subacuity of the present model probably explains, at least partially, the magnitude of the bioenergetic abnormalities. With more chronic tachypacing, a gradual loss of ATP/total purines occurred over 7 to 9 weeks with an early loss of creatine (Shen et al., 1999). The latter is likely a compensatory mechanism to minimize the increase in free [ADP] and reduced GATP with sustained ATP loss. The current acute model allowed for less compensation, enabling more direct evaluation of the mechanical consequences of energetic abnormalities before adaptive processes intervened.
AMP deaminase (AMPD) is a ubiquitous AMP-catabolizing enzyme constitutively active in cardiac muscle (Barsacchi et al., 1979). Cardiac AMPD may help regulate adenine nucleotide catabolism during myocardial ischemia (Thakkar et al., 1994) under oxidative stress conditions (Tavazzi et al., 2001), increased ADP (Chung and Bridger, 1976), and sustained -stimulation (Hohl, 1999). In skeletal muscle in vivo, AMPD activity increase during muscular work is mediated by ADP, AMP, and pH (Wheeler and Lowenstein, 1979). Neurohumoral/mechanical stimulation with AII+P probably activated AMPD as reflected by increased adenine nucleotide catabolism and loss in phosphorylation potential. Intriguingly, AMP catabolites, particularly uric acid, are associated with worsened CHF prognosis and independent predictors of elevated diastolic pressure (Cicoira et al., 2002). Among the proposed mechanisms for this are increases in xanthine oxidase expression and activity that enhance purine catabolism and urate synthesis and serve as a source for oxidative stress (Doehner et al., 2002). Although oxidative stress was clearly induced by AII+P, it was not prevented by MMP inhibition, whereas AMP catabolism largely was. This suggested a more direct effect of the MMP inhibitor on AMPD, and modest direct effects were demonstrated.
PD166793 partially inhibited AMPD directly and, although the exact magnitude of inhibition in vivo remains unclear, even partial decreases could have played an important role in AMP reaccumulation. The latter combined with higher total ADP could lead to an improved phosphorylation potential and GATP. Furthermore, although not directly tested in our study, higher concentrations of AMP might also increase AMP kinase activity, an enzyme up-regulated in several conditions with potential benefits in the energy-stressed heart (Young et al., 2005).
Still, it seems unlikely that this mechanism fully explains all of the observed changes, for example, the restoration of normal lactate and lactate/pyruvate ratios and recovery of PCr. Cross-reactivity of a MMP inhibitor on AMPD is consistent with the latter being a zinc metalloenzyme (Ranieri-Raggi et al., 2003), and PD166793 like other MMP inhibitors act as zinc chelators in the catalytic site. Further studies are needed to establish whether this is a feature common to all MMP inhibitors or extends to other zinc metalloenzymes. Likewise, it remains to be determined whether PD166793 displays a dual dose-dependent action, whereby at low doses, it may chiefly target AMPD activity, although at higher doses, it may interfere with other enzymatic activities along the same AMP-catabolic pathway.
Experimental Limitations and Future Directions. We used HPLC rather than NMR approaches to assess HEP metabolism. This was due in part given the complexity of the chronic instrumentation that precluded NMR studies and our desire to assess both redox conditions and HEP in the same tissue. PCr/ATP ratios by HPLC were somewhat lower than those reported by NMR (Kantor et al., 1986), perhaps reflecting PCr decline during tissue procurement (flash-freezing not employed). However, values were consistent, and all of the tissues were processed identically, reducing bias.
The present study does not establish a causal link between the energetic changes and diastolic stiffening with AII+P, nor does it prove that this was due to MMP inhibition or effects from an MMP inhibitor. Even accepting the latter, the results are pharmacologically important because much of our understanding of MMPs and their inhibition was derived from data employing this agent. The results certainly highlight another pathway that may be profoundly influenced by MMP inhibition and play an important role in regulating cardiac function. Ongoing efforts are testing whether more selective MMP inhibitors (Peterson, 2004) are endowed with the same or superior AMPD inhibitory activity. This is pharmacologically and functionally relevant in the light of current lack of "selective" (Kasibhatla et al., 2001) and cell-permeable AMPD inhibitors and AMPD-proposed role in CHF unfolding (Kalsi et al., 2003).
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Acknowledgements |
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This work was presented as an abstract in Paolocci N, Tavazzi B, Biondi R, Gluzband YA, Amorini AM, Tocchetti C.G., Donzelli S, Crow MT, Lazzarino G, and Kass DA (2004) Metalloproteinase inhibition prevents diastolic stiffening, AMP-breakdown, and xanthine/uric acid accumulation in accelerated heart failure. J Am Coll Cardiol 43 (Suppl 5):236A; 2004 March 7–10, American College of Cardiology 53rd Annual Scientific Session, New Orleans, LA.
N.P. and B.T. contributed equally to this work.
ABBREVIATIONS: CHF, congestive heart failure; AII, angiotensin II; AMPD, AMP deaminase; HEP, high-energy phosphates; MMP, metalloproteinase; P, tachypacing; LV, left ventricular; CNBr, cyanogen bromide; PD166793, (S)-2-(4-bromo-biphenyl-4-sulfonylamino-3-methyl butyric acid; HPLC, high-performance liquid chromatography.
Address correspondence to: Dr. Nazareno Paolocci, Ross 835, Division of Cardiology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205. E-mail: npaoloc1{at}jhmi.edu
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
Barsacchi R, Ranieri-Raggi M, Bergamini C, and Raggi A (1979) Adenylate metabolism in the heart. Regulatory properties of rabbit cardiac adenylate deaminase. Biochem J 182: 361–366.[Medline]
Brower GL and Janicki JS (2001) Contribution of ventricular remodeling to pathogenesis of heart failure in rats. Am J Physiol 280: H674–H683.
Cesselli D, Jakoniuk I, Barlucchi L, Beltrami AP, Hintze TH, Nadal-Ginard B, Kajstura J, Leri A, and Anversa P (2001) Oxidative stress-mediated cardiac cell death is a major determinant of ventricular dysfunction and failure in dog dilated cardiomyopathy. Circ Res 89: 279–286.
Chung L and Bridger WA (1976) Activation of rabbit cardiac AMP aminohydrolase by ADP: a component of a mechanism guarding against ATP depletion. FEBS Lett 64: 338–340.[CrossRef][Medline]
Cicoira M, Zanolla L, Rossi A, Golia G, Franceschini L, Brighetti G, Zeni P, and Zardini P (2002) Elevated serum uric acid levels are associated with diastolic dysfunction in patients with dilated cardiomyopathy. Am Heart J 143: 1107–1111.[CrossRef][Medline]
Doehner W, Schoene N, Rauchhaus M, Leyva-Leon F, Pavitt DV, Reaveley DA, Schuler G, Coats AJ, Anker SD, and Hambrecht R (2002) Effects of xanthine oxidase inhibition with allopurinol on endothelial function and peripheral blood flow in hyperuricemic patients with chronic heart failure: results from 2 placebo-controlled studies. Circulation 105: 2619–2624.
Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, et al. (2000) Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Investig 106: 55–62.[Medline]
Gibbs C (1985) The cytoplasmic phosphorylation potential. Its possible role in the control of myocardial respiration and cardiac contractility. J Mol Cell Cardiol 17: 727–731.[Medline]
Hohl CM (1999) AMP deaminase in piglet cardiac myocytes: effect on nucleotide metabolism during ischemia. Am J Physiol 276: H1502–H1510.
Horn M, Frantz S, Remkes H, Laser A, Urban B, Mettenleiter A, Schnackerz K, and Neubauer S (1998) Effects of chronic dietary creatine feeding on cardiac energy metabolism and on creatine content in heart, skeletal muscle, brain, liver and kidney. J Mol Cell Cardiol 30: 277–284.[CrossRef][Medline]
Ingwall JS and Weiss RG (2004) Is the failing heart energy starved? On using chemical energy to support cardiac function. Circ Res 95: 135–145.
Iwaki-Egawa S, Watanabe Y, and Matsuno H (2001) Correlations between matrix metalloproteinase-9 and adenosine deaminase isozymes in synovial fluid from patients with rheumatoid arthritis. J Rheumatol 28: 485–489.[Medline]
Kalsi KK, Yuen AH, Rybakowska IM, Johnson PH, Slominska E, Birks EJ, Kaletha K, Yacoub MH, and Smolenski RT (2003) Decreased cardiac activity of AMP deaminase in subjects with the AMPD1 mutation–a potential mechanism of protection in heart failure. Cardiovasc Res 59: 678–684.
Kantor HL, Briggs RW, Metz KR, and Balaban RS (1986) Gated in vivo examination of cardiac metabolites with 31P nuclear magnetic resonance. Am J Physiol 251: H171–H175.
Kasibhatla SR, Bookser BC, Xiao W, and Erion MD (2001) AMP deaminase inhibitors. 5. Design, synthesis and SAR of a highly potent inhibitor series. J Med Chem 44: 613–618.[CrossRef][Medline]
Kass DA, Bronzwaer JG, and Paulus WJ (2004) What mechanisms underlie diastolic dysfunction in heart failure? Circ Res 94: 1533–1542.
Katz AM (1998) Is the failing heart energy depleted? Cardiol Clin 16: 633–644.[CrossRef][Medline]
Lawson JWR and Veech RL (1979) Effect of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions. J Biol Chem 254: 6528–6537.
Lazzarino G, Amorini AM, Fazzina G, Vagnozzi R, Signoretti S, Donzelli S, Di Stasio E, Giardina B, and Tavazzi B (2003) Single-sample preparation for simultaneous cellular redox and energy state determination. Anal Biochem 322: 51–59.[CrossRef][Medline]
McElmurray JH 3rd, Mukherjee R, New RB, Sampson AC, King MK, Hendrick JW, Goldberg A, Peterson TJ, Hallak H, Zile MR, et al. (1999) Angiotensin-converting enzyme and matrix metalloproteinase inhibition with developing heart failure: comparative effects on left ventricular function and geometry. J Pharmacol Exp Ther 291: 799–811.
Norton GR, Candy G, and Woodiwiss AJ (1996) Aminoguanidine prevents the decreased myocardial compliance produced by streptozotocin-induced diabetes mellitus in rats. Circulation 93: 1905–1912.
Norton GR, Tsotetsi J, Trifunovic B, Hartford C, Candy GP, and Woodiwiss AJ (1997) Myocardial stiffness is attributed to alterations in cross-linked collagen rather than total collagen or phenotypes in spontaneously hypertensive rats. Circulation 96: 1991–1998.
Peterson JT (2004) Matrix metalloproteinase inhibitor development and the remodeling of drug discovery. Heart Fail Rev 9: 63–79.[CrossRef][Medline]
Peterson JT, Hallak H, Johnson L, Li H, O'Brien PM, Sliskovic DR, Bocan TM, Coker ML, Etoh T, and Spinale FG (2001) Matrix metalloproteinase inhibition attenuates left ventricular remodeling and dysfunction in a rat model of progressive heart failure. Circulation 103: 2303–2309.
Ranieri-Raggi M, Martini D, Sabbatini AR, Moir AJ, and Raggi A (2003) Isolation by zinc-affinity chromatography of the histidine-proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase. Evidence that the formation of a protein-protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme. Biochim Biophys Acta 1645: 81–88.[Medline]
Senzaki H, Gluzband YA, Pak PH, Crow MT, Janicki JS, and Kass DA (1998) Synergistic exacerbation of diastolic stiffness from short-term tachycardia-induced cardiodepression and angiotensin II. Circ Res 82: 503–512.
Senzaki H, Paolocci N, Gluzband YA, Lindsey ML, Janicki JS, Crow MT, and Kass DA (2000) -Blockade prevents sustained metalloproteinase activation and diastolic stiffening induced by angiotensin II combined with evolving cardiac dysfunction. Circ Res 86: 807–815.
Shen W, Asai K, Uechi M, Mathier MA, Shannon RP, Vatner SF, and Ingwall JS (1999) Progressive loss of myocardial ATP due to a loss of total purines during the development of heart failure in dogs: a compensatory role for the parallel loss of creatine. Circulation 100: 2113–2118.
Spinale FG, Coker ML, Krombach SR, Mukherjee R, Hallak H, Houck WV, Clair MJ, Kribbs SB, Johnson LL, Peterson JT, et al. (1999) Matrix metalloproteinase inhibition during the development of congestive heart failure: effects on left ventricular dimensions and function. Circ Res 85: 364–376.
Tavazzi B, Amorini AM, Fazzina G, Di Pierro D, Tuttobene M, Giardina B, and Lazzarino G (2001) Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP deaminase. J Biol Chem 276: 48083–48092.
Tavazzi B, Di Pierro D, Amorini AM, Fazzina G, Tuttobene M, Giardina B, and Lazzarino G (2000) Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress. Eur J Biochem 267: 684–689.[Medline]
Thakkar JK, Janero DR, Sharif HM, Hreniuk D, and Yarwood C (1994) Cardiac adenylate deaminase: molecular, kinetic and regulatory properties under phosphate-free conditions. Biochem J 300: 359–363.
Thomas CV, Coker ML, Zellner JL, Handy JR, Crumbley AJ 3rd, and Spinale FG (1998) Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation 97: 1708–1715.
Tian R, Christe ME, Spindler M, Hopkins JC, Halow JM, Camacho SA, and Ingwall JS (1997a) Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart. J Clin Investig 99: 745–751.[Medline]
Tian R and Ingwall JS (1996) Energetic basis for reduced contractile reserve in isolated rat hearts. Am J Physiol 270: H1207–H1216.
Tian R, Nascimben L, Ingwall JS, and Lorell BH (1997b) Failure to maintain a low ADP concentration impairs diastolic function in hypertrophied rat hearts. Circulation 96: 1313–1319.
Vasan S, Zhang X, Zhang X, Kapurniotu A, Bernhagen J, Teichberg S, Basgen J, Wagle D, Shih D, Terlecky I, et al. (1996) An agent cleaving glucose-derived protein cross-links in vitro and in vivo. Nature (Lond) 382: 275–278.[CrossRef][Medline]
Wheeler TJ and Lowenstein JM (1979) Adenylate deaminase from rat muscle. Regulation by purine nucleotides and orthophosphate in the presence of 150 mM KCl. J Biol Chem 254: 8994–8999.
Ye QZ, Johnson LL, Nordan I, Hupe D, and Hupe L (1994) A recombinant human stromelysin catalytic domain identifying tryptophan derivatives as human stromelysin inhibitors. J Med Chem 37: 206–209.[CrossRef][Medline]
Young LH, Li J, Baron SJ, and Russell RR (2005) AMP-activated protein kinase: a key stress signaling pathway in the heart. Trends Cardiovasc Med 15: 110–118.[CrossRef][Medline]
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) as well as systolic function was similarly reduced.
, p < 0.05 versus Con; 
, p < 0.03 versus MMPi; #, p < 0.01 versus both.