| | Selective activation of N-Acyl-D-glucosamine 2-epimerase expression in failing human heart ventricular myocytes☆☆☆Received 30 May 2002; received in revised form 28 October 2002 and 31 October 2002 Abstract Background: O-linked N-acyl-glycosylation may regulate protein function by competing with phosphorylation of serine residues. Availability of substrate for this process is regulated, in part, by N-Acyl-D-glucosamine 2-epimerase (NAGE), which interconverts N-acetyl-glucosamine (GlcNAc) and N-acetylmannosamine (ManNAc). NAGE is also a putative renin-binding protein. This study tested the hypothesis that NAGE is present in the human heart and that NAGE expression is increased in the failing human heart. Methods and Results: Ribonuclease protection assays (RPAs) demonstrated increased NAGE gene expression in failing hearts from subjects with idiopathic dilated and ischemic cardiomyopathies compared with nonfailing hearts. In situ reverse transcriptase–polymerase chain reaction, using primers designed to localize NAGE mRNA, demonstrated that, in nonfailing hearts, NAGE gene expression was restricted to endothelial cells and not detectable in cardiac myocytes. However, in failing human hearts NAGE gene expression was selectively activated in cardiac myocytes, but not endothelial cells. Immunohistochemistry confirmed that the pattern of NAGE protein expression corresponded to the pattern of gene expression. Conclusions: NAGE gene and protein expression were selectively activated in left ventricular myocytes from end-stage failing human hearts.
Posttranslational modification by O-linked N-Acyl-glycosylation has been shown to occur on nuclear, cytoplasmic, and cytoskeletal proteins and to compete with serine phosphorylation sites.1 A growing body of evidence suggests that this process regulates protein function and alters protein turnover.2 Although phosphorylation events mediated by several classes of kinases, including PKA, PKC, MAPK, and JAK/STAT kinases, play a critical role in the pathophysiology of heart failure, virtually nothing is known about O-linked N-acetylglucosamine (O-GlcNAc) posttranslational protein modification as a potential counterregulatory mechanism in this disease.
Availability of substrate for O-linked N-acyl-glycosylation is regulated, in part, by N-Acyl-D-glucosamine epimerase (NAGE), which interconverts N-acetylglucosamine (GlcNAc) and N-acetylmannosa-mine (ManNAc). Historically, NAGE was first identified as a renin binding protein (RnBP) by virtue of its ability to form a heterodimer with renin. In vitro, RnBP was shown to decrease renin activity, but it did not appear to do so in vivo, raising questions as to its importance in the regulation of the renin-angiotensin system.3
Subsequently, RnBP was shown to be an NAGE.4, 5, 6 By sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of human recombinant NAGE/RnBP was estimated to be 45 kDa; however, the theoretic molecular weight for human NAGE/RnBP is 47 kDa.7 The structure of this protein is of note for a leucine zipper motif, which is necessary for the RnBP/Renin interaction.8, 9 Rat NAGE/RnBP gene expression has been demonstrated predominantly in kidney, lung, adrenal gland, brain, spleen, ovary, testis, and heart.10 Porcine NAGE/RnBP mRNA abundance was also found to be greatest in the kidney.8 The human NAGE gene has been mapped to the distal Xq28 chromosomal region and spans 10kB with 11 exons.11, 12, 13
In this study, we show, for the first time, an increase in NAGE gene and protein expression in failing compared to nonfailing (NF) human heart left ventricles. In NF human hearts, NAGE gene expression was restricted to endothelial cells, but in failing human hearts NAGE gene and protein expression were selectively activated in ventricular myocytes. Furthermore, in failing human heart, NAGE was redistributed from a cytosolic to a sarcolemmal/sarcomeric fraction, suggesting a potential role of this enzyme in modification of cardiac cytoskeletal proteins.
Materials and methods  Patient characteristics Hearts obtained from 8 patients with end-stage idiopathic dilated cardiomyopathy (IDC), 6 patients with end-stage ischemic cardiomyopathy (ISC), and 6 organ donors were used for total RNA extraction and NAGE ribonuclease protection assays. The IDC and ISC hearts were obtained at the time of cardiac transplantation. The cardiac function of 1 organ donor was significantly decreased and was not used for cardiac transplantation for this reason. This organ donor was classified as having donor heart dysfunction (DHD), which is a form of acute heart failure that occurs in approximately 10% to 20% of subjects with severe brain injury.14 The hearts from the other 5 donors demonstrated normal left ventricular systolic function by echocardiography and met criteria for use as NF controls, as previously described.15 The reasons for not using the NF hearts for cardiac transplantation were as follows: nonobstructive coronary artery disease (a 20% to 30% left anterior descending artery stenosis) was found in 2 donors who underwent coronary angiography as part of the cardiac donor evaluation; 1 donor had a history of Wegener's granulomatosis and it was not known if there was any cardiac involvement; there was an ABO blood type or donor/recipient size mismatch in 2 cases. A description of patient characteristics and cardiac hemodynamics is shown in Table 1.
| | |  | Sample | Type | Age | Gender | EF | PW | CI | RA | PA | Medications | |
|---|
| 1 | IDC | 24 | M | 20 | 22 | NA | NA | 30 | ACE-I, Lasix | |
| 2 | IDC | 53 | M | 10 | 29 | 1.12 | 15 | 45 | Dobt, Dig, Lasix, Coumadin | |
| 3 | IDC | 18 | F | 10 | 35 | 1.4 | 19 | 46 | Dobt, Dig, Coumadin | |
| 4 | IDC | 61 | M | 5 | 24 | co 3.4 | 16 | 32 | ACE-I, Dig, Lasix | |
| 5 | IDC | 60 | M | 15 | 30 | co 2.4 | 17 | 47 | Dig, Lasix | |
| 6 | IDC | 64 | F | 8 | 16 | 1.45 | 2 | 23 | ACE-I, Dobt, Dig, Lasix | |
| 7 | IDC | 40 | F | 12 | 22 | 1.69 | 15 | 40 | ACE-I, Dig, Lasix | |
| 8 | IDC | 46 | M | 13 | 24 | 1.17 | 8 | 24 | ACE-I, Dopa, Dig, Lasix | |
| 9 | NF | 49 | F | 65 | NA | NA | 8 | NA | Dobt | |
| 10 | DHD | 31 | F | 25 | NA | NA | 8 | NA | Dopa | |
| 11 | NF | 57 | M | nl | NA | NA | 7 | NA | Dopa | |
| 12 | NF | 55 | F | 65 | NA | NA | 5 | NA | Dopa | |
| 13 | NF | 22 | M | nl | NA | NA | 15 | NA | ACE-I | |
| 14 | NF | 54 | F | 73 | 13 | NA | NA | 30/16 | Dopa | |
| 15 | ISC | 53 | F | 10 | 20 | 1.96 | 6 | 39 | Dig, Lasix | |
| 16 | ISC | 51 | M | 10 | 28 | 1.7 | 15 | 49 | ACE-I, Coumadin | |
| 17 | ISC | 60 | M | 20 | 10 | 2.4 | 2 | 17 | ACE-I, Lasix, Coumadin | |
| 18 | ISC | 57 | M | 15 | 4 | co 2.3 | 2 | 14 | ACE-I, Dig, Lasix, Coumadin | |
| 19 | ISC | 65 | M | 15 | 22 | 1.76 | 14 | 8.5 | Dig, Lasix, Coumadin | |
| 20 | ISC | 59 | M | 30 | 11 | NA | 4 | 20 | ACE-I, Dig, Lasix, Coumadin | |
| | | | | |
In one NF heart (subject 14), significant left ventricular hypertrophy was noted. Three patients with IDC and 1 donor were receiving dobutamine at the time of organ acquisition. One patient with IDC and 4 donors were receiving low-dose dopamine. Twelve patients with heart failure were receiving Lasix, 7 were receiving coumadin, and 11 were taking digoxin. Five patients with IDC, 4 with ISC, and 1 donor had been receiving an angiotensin-converting enzyme inhibitor. All cardiac tissue samples for mRNA analyses were obtained within 5 minutes of organ explantation and placed immediately in liquid nitrogen in the operating room. Samples for Western blotting and immunohistochemistry were prepared within 20 minutes of explantation. There were no differences in handling of NF and failing hearts. This study was performed under the auspices of an Institutional Review Board approved human heart tissue repository. Preparation of total RNA Cardiac tissue was obtained from the left ventricular free wall within 5 minutes of explantation and frozen in liquid nitrogen. Tissue was stored at −80° C until used. Total RNA was extracted using the acid guanidinium thiocyanate method.16 The frozen tissue was disrupted with a polytron homogenizer directly in RNA-Stat 60 (Tel-test, Inc., Friendswood, TX). Polymerase chain reaction amplification of riboprobe A 177-bp cDNA fragment of human NAGE/RnBP (bp 704-880; GenBank accession #D10232) was amplified by reverse transcriptase–polymerase chain reaction (RT-PCR) from NF human heart total RNA and subcloned for use as a riboprobe in pGEM-T (Promega Corp., Madison, WI). PCR amplification was performed using the forward primer 5'-TGCTGGAGAATGTGTCAGAGGG-3' and the reverse primer 5'-CGGAGTGGAAGGGCAACAATAG-3'. The PCR method was 94° C for 1 minute, 69° C for 1 minute, and 72° C for 2 minutes for 35 cycles. The subcloned fragment was verified as human NAGE/RnBP by automated DNA sequencing. Ribonuclease protection assays Vector containing the partial human NAGE/RnBP cDNA was restriction digested with Sal 1. Agarose gel electrophoresis was performed followed by extraction of the digested plasmid with Qiaex II (Qiagen, Inc., Valencia, CA). Antisense riboprobe was generated using the Maxiscript in vitro transcription kit from Ambion, Inc. (Austin, TX). Transcription was performed using the T7 promoter in the presence of α-32P-UTP (specific activity 800 Ci/mmol, NEN Research, Inc., MA). Ribonuclease protection assays were performed by incubating 20 μg total RNA per specimen with the labeled NAGE/RnBP riboprobe (1.4 × 106 cpm). An 18S RNA riboprobe (1.2 μg) labeled to a low specific activity was added to the initial hybridization to allow normalization to 18S RNA (Ambion, Inc., Austin, TX) according to the method of Pende et al.17 After digestion with RNAse, the protected fragments were separated on an 8% acrylamide 8 mol/L urea gel using a Sequi-Gen Sequencing Cell (Bio-Rad, Hercules, CA) at 55 W for 4 hours. The gel was mounted on Whatman paper, covered with plastic wrap, and placed in an autoradiography cassette with intensifying screens and Kodak XAR-5 imaging film. The film was exposed for 2 days at −80° C. Densitometry to quantify signal intensity was performed using an IS-1000 Digital Imaging System (Alpha Innotech, Corp., San Leandro, CA). In situ RT-PCR To examine the localization of NAGE mRNA expression in human left ventricular myocardium, we performed in situ RT-PCR as previously described with the GeneAmp In Situ PCR System 1000 (Perkin Elmer, Norwalk, CT).15 The NAGE forward primer was 5'-TGCTGGAGAATGTGTCAGAGG G-3' and the NAGE reverse primer was 5'-CGGAGTGGAAGGGCA ACAATAG-3' Detection of von Willebrand factor (Factor VIII) mRNA was performed in a similar fashion, using FVIII forward primer 5'-AAATGCTCCCCCAGGAAGTG-3' and FVIII reverse primer 5'-AGCAGAACA-TGCAGAGGACTGG-3'. Western blot analysis To verify specificity of the anti-human NAGE/RnBP antibody,10 and to examine the intracellular distribution of NAGE/RnBP, Western blot analyses were performed on cytosolic and crude sarcolemmal/sarcomeric fractions of failing and non-failing human heart preparations. Recombinant human NAGE/RnBP10 was used to demonstrate a linear relationship between the autoradiograph signal intensity and NAGE/RnBP concentration. For analysis of human heart tissue, one-half gram of left ventricular myocardium was disrupted under liquid nitrogen and homogenized at 4° C in 5 mL buffer (5 mmol/L Tris pH7.5, 7.5 mmol/L NaCl, 0.01 mmol/L Pefabloc, 0.1 μg/mL Aprotinin, 1 μmol/L Leupeptin, and 0.04 mmol/L EDTA). The homogenate was centrifuged at 10,000× g for 30 minutes. The pellet was resuspended in 5% SDS. Protein concentration for both the pellet and supernatant was determined on trichloroacetic acid (TCA)–precipitated aliquots according to the method of Lowry.18 This allowed equal protein loading for SDS-PAGE, which was performed using a 10% acrylamide gel and a 3% stacking gel and that was verified by Coomassie staining. Transfer to a nitrocellulose membrane was performed overnight at 4° C in transfer buffer (0.025 mol/L Tris base, 0.2 mol/L glycine, 20% methanol). After blocking with phosphate-buffered saline (PBS) 5% milk, and rinsing with 0.1% Tween-20 in PBS, incubation with rabbit anti-human NAGE (renin-binding protein) was performed.12 The membranes were rinsed with 0.1% Tween-20 in PBS and then incubated with 125I labeled protein A, as previously described.9 Imaging film (Kodak X-Omat Blue XB-1) was exposed to the membranes for 72 hours at minus 80° C. Densitometry was performed to quantify signal intensity. Immunohistochemistry To examine the localization of NAGE protein expression in human left ventricular myocardium, immunohistochemistry was performed. Tissue preparation Sections were fixed, processed, and embedded as described previously.15 Four-micron–thick sections were mounted on silane-coated slides, deparaffinized in xylene, rehydrated through graded reagent alcohol, and washed in Tris-buffered saline. Sections underwent antigen retrieval by microwave pretreatment in 0.1 M citrate (pH 6) for 18 minutes. Antibody detection Nonspecific binding was blocked by incubating the sections for 20 minutes in 10% bovine serum albumin (Sigma, St. Louis, MO). Primary antibody to NAGE/ RnBP (rabbit anti-human), nonimmunized swine-blocking serum (dilution 1/10), multilink swine anti-goat, mouse rabbit biotinylated secondary antibody (dilution 1/50), streptavidin AP (dilution 1/100), and biotin blocking system (DAKO A/S, Glostrup, Denmark) were applied following manufacturers' instructions. New fuchsin was used as substrate. Sections were counterstained with dilute Gill's hematoxylin. Statistical analysis Analysis of variance (ANOVA) was used to compare the relative mRNA abundance of NAGE in left ventricular cardiac tissue from the NF, IDC, and ISC groups. To control for nonspecific variation of target signal, the NAGE signal was normalized to the 18S RNA signal. To ensure that the results were not dependent on the method of normalization, statistical analysis was also performed on the natural logarithm transformed NAGE/18S RNA ratios. In addition, linear regression of the NAGE signal versus the 18S RNA signal was performed to determine the variation of NAGE gene expression “explained by” nonspecific variation. It has been demonstrated that the higher the correlation between the normalization signal of the control gene and the signal of the experimental gene, the more powerful the control gene is in detecting nonspecific variation of the experimental gene.19 The Student-Newman-Keuls test for multiple comparisons was used to detect differences between groups. Two-way ANOVA for interaction was used to examine the distribution of NAGE in failing and NF hearts between supernatant (containing cytosolic elements) and pellet fractions (containing sarcolemmal and sarcomeric components). Correlation analysis was performed to determine the relationship between NAGE gene expression and hemodynamic parameters. Statistical significance was set at P ≤ .05. Results Densitometric signal intensity corresponding to the NAGE mRNA protected fragment normalized to 18S RNA was significantly increased in the failing compared to NF human heart left ventricles. The ribonuclease protection assay for human NAGE mRNA is shown in Fig. 1A.
The NAGE mRNA/18s RNA signal ratio in NF, IDC, and ISC left ventricles was 1.65 ± 0.40 (NF), 3.31 ± 0.44 (IDC), 4.41 ± 0.72 (ISC), respectively ( P < .05, IDC and ISC versus NF) ( Fig. 1B). The NAGE mRNA/18S RNA ratio in the DHD LV was 1.24. Adding this value to the NF group did not alter the finding of a significant difference between the groups. Interestingly, the highest NAGE mRNA/18S RNA signal ratio in the NF group was found in subject 14, who had significant left ventrical hypertrophy (LVH). Inclusion or exclusion of this heart from the analysis did not alter the findings. When the data from the DHD and LVH hearts were both excluded, the NAGE mRNA/18S RNA signal ratio in the NF group was 1.47 ± 0.33. No significant difference between the IDC and ISC groups with regard to the NAGE mRNA/18s RNA ratio was found. The natural logarithms of the NAGE mRNA/18S RNA signals were also significantly different: 0.35 ± 0.30 (NF), 1.15 ± 0.11 (IDC), and 1.42 ± 0.17 (ISC) (mean ± standard error of the mean, P < .01). This analysis demonstrated that the increased NAGE gene expression was not dependent on the method of normalization. Linear regression of the NAGE signal vs. the 18S signal demonstrated a significant correlation between the two signals (r2 = .65, P < .0001). This analysis indicated that the variation in 18S signal “explained” 65% of the variation of the NAGE signal. Accordingly, 35% of NAGE signal variation was explained by differences in NAGE gene expression between the failing hearts and the NF hearts. In situ RT-PCR demonstrated localization of gene expression to microvascular cells in NF hearts; this pattern was similar to that of vWF gene expression that was used as a marker for endothelial cells in this assay (Fig. 2A, B).
In contrast, NAGE gene expression was activated in ventricular myocytes from the failing human hearts examined. The signal for NAGE mRNA notably outlined sarcomeres of left ventricular myocytes sectioned longitudinally in the failing hearts ( Fig. 2C). Immunohistochemistry confirmed that the pattern of protein expression between the failing and NF hearts reflected the observed differences in gene expression (Fig. 3).
These data suggest that selective activation of NAGE gene expression occurs in ventricular myocytes from failing human hearts and that NAGE protein localization is determined, at least in part, by the pattern of NAGE gene expression. Western blot analysis of increasing concentrations of recombinant human NAGE is shown in Fig. 4A (60, 180, 300, 420 pg loaded in lanes 1–4; 560, 680, 780, and 900 pg lanes 5–8, respectively).
These data were used to demonstrate a linear relationship between NAGE protein concentration and signal intensity of the autoradiogram ( r2 = .94; P < .0001, Figure 4B). Western blot analysis of NAGE in failing and NF human hearts demonstrated a redistribution of NAGE from a cytosolic fraction in NF human heart left ventricles to a fraction containing elements of the sarcolemma and sarcomere in failing LVs from IDC hearts (n = 4) ( Fig. 4C and D). The values in picograms for each group were as follows: IDC supernatant, 163.3 ± 6.7; IDC pellet, 287.7 ± 28.64; IDC total, 450.9 ± 21.9; NF supernatant, 166.2 ± 131.6; NF pellet, 49.9 ± 5.8; NF total, 216.1 ± 137 (mean ± SD, P < .05, IDC pellet versus IDC supernatant; P < .01 IDC pellet versus NF pellet). A trend toward increased total IDC NAGE protein was observed but did not reach statistical significance ( P = .07). NAGE protein in picograms correlated with NAGE gene expression (NAGE mRNA/18S; r = .95, P = 0.04). NAGE gene expression was found to correlate with PCWP in the IDC group (Pearson R = .9, r2 = .81, P = .002, Fig. 5).
A similar correlation was not found in the ISC group, which may have been related to the smaller n, and higher standard deviation in this group. There was no significant correlation between NAGE gene expression and left ventricular ejection fraction, pulmonary artery (PA) pressure, right atrial (RA) pressure, or cardiac index. Discussion In this study, NAGE gene expression was increased in failing IDC and ISC hearts compared with NF human hearts. Linear regression of the RPA used to quantify relative differences in NAGE gene expression between these groups demonstrated a significant correlation between the NAGE signal and the control gene (18S RNA) signal. Therefore, there is a high level of confidence that the increase in NAGE signal normalized to the 18S signal reflects a true difference in gene expression between the IDC and ISC groups as compared with the NF groups.19 This increase may be explained by selective activation of NAGE gene expression in left ventricle myocytes of failing human hearts as demonstrated by in situ PCR. Furthermore, the selective and specific increase in NAGE gene expression in ventricular cardiac myocytes translated to a similar pattern of protein expression. In NF ventricular myocardium, NAGE gene expression was restricted to the microvascular endothelium. Historically, NAGE was first described as a renin-binding protein.20, 21 It was found to form a heterodimer with renin and to inhibit renin activity in vitro.7 A large body of evidence supports the role of a local cardiac renin-angiotensin system in the pathophysiology of heart failure. It has been reported that cardiac myocytes can synthesize angiotensin II internally and that angiotensin II is stored in granules before secretion from the cell.22 It has also been demonstrated that renin gene expression is activated in cardiac myocytes subjected to stress, and this in vitro finding has been confirmed in both animal models and in pathologic human heart specimens.23, 24, 25 Thus the selective activation of NAGE in ventricular myocytes from end-stage failing hearts represents a potential mechanism for localizing renin to these cells, where its expression is also stimulated by stress. However, the recently described NAGE/RnBP knockout mouse did not show increased circulating or tissue renin activity.3 The absence of a physiologically apparent interaction between NAGE/RnBP and renin suggests that these 2 proteins are segregated in different subcellular compartments. Therefore, it is likely that the more relevant action of NAGE/RnBP in vivo is to modulate N-Acylglycosylation via the interconversion of N-ace-tylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc).4, 5 O-linked N-Acylglycosylation has recently emerged as a dynamic process analogous to and competitive with O-phosphorylation of serine and threonine residues.2 The enzyme responsible for this modification is O-GlcNAc transferase, which is highly expressed in skeletal muscle and heart.26, 27 O-GlcNAc transferase uses UDP-GlcNAc, the end-product of hexosamine biosynthesis, to catalyze this modification. By converting N-acetyl mannosamine to N-Acetyl-glucosamine, NAGE provides substrate for GlcNAC-kinase to form N-Acetylglucosamine 6-phosphate, an intermediary in the formation of UDP-GlcNAc.28 O-linked N-Acylgly-cosylation has been shown to play a dynamic role in the regulation of transcription, translation, and protein degradation.29, 30 In addition, GlcNAc bearing molecules in the extracellular matrix may regulate neuronal architecture in the myocardium and play a role in the posttranslational modification of heat shock proteins.31, 32, 33 The heat shock protein alpha B-crystallin has recently been shown to play an important role in cardioprotection.34 In addition, a dominant negative form of alpha B-crystallin caused a desmin-associated cardiomyopathy in mice.35 Both dynamic O-linked N-Acylglycosylation and phosphorylation of alpha B-crystallin have been shown to occur.32, 36 Therefore, a study of the functional effects of O-linked N-acylglycosylation on alpha B-crystallin, and the potential regulatory effects of increased NAGE expression on alpha B-crystallin, is planned. Western blot analysis provided evidence of a relative redistribution of NAGE from the cytosol in NF left ventricles to a fraction containing elements of the sarcolemma and the sarcomere in failing left ventricles. As demonstrated by in situ PCR and immunohistochemistry, this redistribution occurs in the context of selective activation of NAGE gene and protein expression in ventricular myocytes from failing human heart. It may be that, normally, NAGE in the heart is primarily expressed in endothelial cells as a cytosolic protein or a protein secreted into the circulation, but in failing human heart its expression is activated in ventricular myocytes, where it is associated with components of the sarcolemma or sarcomere. The significant correlation between NAGE gene expression and PCWP in the IDC group suggests a relationship between NAGE expression and the degree of heart failure. These data and the finding that NAGE gene expression was relatively high in the heart with LVH suggest that NAGE expression may be activated by increased wall stress. However, whether NAGE itself contributes to the pathogenesis of heart failure or is activated as a counterregulatory cardioprotective response to heart failure is not known. Limitations This study did not determine the precise NAGE-protein interactions in ventricular myocytes. However, a shift in NAGE distribution in failing compared with NF hearts from endothelial cells to myocytes and from a cytosolic to a sarcomeric/sarcolemmal fraction was observed. Whether it was appropriate to include the DHD heart in the NF control group is uncertain. G-protein uncoupling has been found in some DHD hearts; such altered signal transduction could change gene expression even over the short time course in which DHD occurs. Nevertheless, inclusion or exclusion of the DHD heart did not alter the findings of this study. Conclusions To our knowledge, this is the first demonstration that NAGE mRNA is produced in the human heart, and that NAGE gene and protein expression are selectively activated in ventricular myocytes in failing human heart. Further study will be required to determine in more detail the cellular components with which NAGE associates, and the functional role of NAGE in cardiac myocytes. References 1.
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Albany, New York Denver, Colorado Akita, Japan From the ‡The Heart Institute, Albany Medical College, Albany, New York; *University of Colorado Health Sciences Center, Denver, Colorado; and †Akita Research Institute of Food and Brewing, Akita, Japan ☆ Reprint requests: Lawrence S. Zisman, MD, The Heart Institute, MC55, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. ☆☆ This study was supported by NIH grant HL03404 awarded to Dr. Zisman. PII: S1071-9164(02)25406-8 doi:10.1054/jcaf.2003.6 © 2003 Published by Elsevier Inc. | |
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