circulation

ISSN: 1524-4539 Copyright © 2008 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online 72514 Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX DOI: 10.1161/CIRCULATIONAHA.107.737031 2008;117;2626-2636; originally published online May 12, 2008; Circulation Wolin, Nicholas J. Alp, Nazareno Paolocci, Hunter C. Champion and David A. Kass Marc K. Halushka, James B. Mitchell, Shyam Biswal, Keith M. Channon, Michael S. Gianfranco Cormaci, Elizabeth A. Ketner, Maulik Majmudar, Kathleen Gabrielson, An L. Moens, Eiki Takimoto, Carlo G. Tocchetti, Khalid Chakir, Djahida Bedja, Therapeutic Strategy Tetrahydrobiopterin: Efficacy of Recoupling Nitric Oxide Synthase as a Reversal of Cardiac Hypertrophy and Fibrosis From Pressure Overload by http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.737031/DC1 Data Supplement (unedited) at: http://circ.ahajournals.org/cgi/content/full/117/20/2626 located on the World Wide Web at: The online version of this article, along with updated information and services, is http://www.lww.com/reprints Reprints: Information about reprints can be found online at journalpermissions@lww.com 410-528-8550. E-mail: Fax:Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050. Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters http://circ.ahajournals.org/subscriptions/ Subscriptions: Information about subscribing to Circulation is online at by on April 28, 2011 circ.ahajournals.orgDownloaded from Reversal of Cardiac Hypertrophy and Fibrosis From Pressure Overload by Tetrahydrobiopterin Efficacy of Recoupling Nitric Oxide Synthase as a Therapeutic Strategy An L. Moens, MD; Eiki Takimoto, MD, PhD; Carlo G. Tocchetti, MD, PhD; Khalid Chakir, PhD; Djahida Bedja, MS; Gianfranco Cormaci, MD; Elizabeth A. Ketner, MS; Maulik Majmudar, MD; Kathleen Gabrielson, DVM, PhD; Marc K. Halushka, MD; James B. Mitchell, PhD; Shyam Biswal, PhD; Keith M. Channon, MD, PhD; Michael S. Wolin, PhD; Nicholas J. Alp, MD, PhD; Nazareno Paolocci, MD, PhD; Hunter C. Champion, MD, PhD; David A. Kass, MD Background—Sustained pressure overload induces pathological cardiac hypertrophy and dysfunction. Oxidative stress linked to nitric oxide synthase (NOS) uncoupling may play an important role. We tested whether tetrahydrobiopterin (BH4) can recouple NOS and reverse preestablished advanced hypertrophy, fibrosis, and dysfunction. Methods and Results—C57/Bl6 mice underwent transverse aortic constriction for 4 weeks, increasing cardiac mass (190%) and diastolic dimension (144%), lowering ejection fraction (�46%), and triggering NOS uncoupling and oxidative stress. Oral BH4 was then administered for 5 more weeks of pressure overload. Without reducing loading, BH4 reversed hypertrophy and fibrosis, recoupled endothelial NOS, lowered oxidant stress, and improved chamber and myocyte function, whereas untreated hearts worsened. If BH4 was started at the onset of pressure overload, it did not suppress hypertrophy over the first week when NOS activity remained preserved even in untreated transverse aortic constriction hearts. However, BH4 stopped subsequent remodeling when NOS activity was otherwise declining. A broad antioxidant, Tempol, also reduced oxidant stress yet did not recouple NOS or reverse worsened hypertrophy/fibrosis from sustained transverse aortic constriction. Microarray analysis revealed very different gene expression profiles for both treatments. BH4 did not enhance net protein kinase G activity. Finally, transgenic mice with enhanced BH4 synthesis confined to endothelial cells were unprotected against pressure overload, indicating that exogenous BH4 targeted myocytes and fibroblasts. Conclusions—NOS recoupling by exogenous BH4 ameliorates preexisting advanced cardiac hypertrophy/fibrosis and is more effective than a less targeted antioxidant approach (Tempol). These data highlight the importance of myocyte NOS uncoupling in hypertrophic heart disease and support BH4 as a potential new approach to treat this disorder. (Circulation. 2008;117:2626-2636.) Key Words: antioxidants � heart failure � hypertrophy � nitric oxide synthase � reactive oxygen species � remodeling � therapeutics Sustained pressure overload stimulates pathological car-diac hypertrophy and dysfunction,1 and reversing such maladaptations has emerged as an important therapeutic goal.2 A prominent pathway is activation of reactive oxygen species (ROS), which contributes to chamber remodeling and contractile failure.3 Although treatment with ROS scavengers has not been very effective to date,4,5 suppression of key ROS generators in the myocardium may prove more so. Myocar- dial ROS sources include xanthine and NADPH oxidases, mitochondrial electron transport, and nitric oxide synthase (NOS), and among these, some recent evidence suggests that NOS may be particularly important to more advanced dilative disease. NOS behaves somewhat like Jekyll and Hyde, generating NO to provide antioxidant and antihypertrophic effects yet contributing to cardiovascular pathobiology if it becomes functionally uncoupled.6 This occurs if the normal flow of electrons from NADPH in the reductase domain to heme in the amino-terminus oxidase domain is disturbed, Received September 4, 2007; accepted March 7, 2008. From the Division of Cardiology, Department of Medicine (A.L.M., E.T., C.G.T., K.C., D.B., G.C., E.A.K., M.M., K.G., N.P., H.C.C., D.A.K.) and Department of Pathology (M.K.H.), Johns Hopkins Medical Institutions, Baltimore, Md; Department of Environmental Health Sciences, Bloomberg School of Public Health (S.B.), John Hopkins University, Baltimore, Md: Department of Physiology, New York Medical College, Valhalla (M.S.W.); Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Md (J.B.M.); and Department of Cardiovascular Medicine, University of Oxford, Oxford, UK (K.M.C., N.J.A.). The online Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.737031/DC1. Correspondence to David A. Kass, MD, Johns Hopkins Medical Institutions, Division of Cardiology, Ross Research Bldg, Room 858, 720 Rutland Ave, Baltimore, MD 21205. E-mail dkass@jhmi.edu © 2008 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.107.737031 2626 by on April 28, 2011 circ.ahajournals.orgDownloaded from liuhuibin Underline liuhuibin Underline limiting NO synthesis and favoring superoxide generation by dissociation of the ferrous-dioxygen complex.6,7 Endothelial NOS (eNOS) uncoupling has been documented in hyperten- sion,8 diabetes,9 and atherosclerosis10 and may have a prom- inent role in cardiac hypertrophic remodeling.11 Clinical Perspective p 2636 A major cause of eNOS uncoupling is depletion and/or oxidation of tetrahydrobiopterin (BH4).12,13 BH4 is an obli- gate cofactor for the 3 aromatic amino acid hydroxylases,14 and insufficiency of phenylalanine hydroxylase causes phe- nylketonuria, a genetic disorder characterized by progressive mental retardation. Affected individuals must follow a phenylalanine-restricted diet, and BH4 replacement therapy can reduce phenylalanine levels and appears to be useful for treating a substantial number of these patients.15 BH4 also is required for normal NOS function (reviewed elsewhere16). It is synthesized de novo from guanosine triphosphate (GTP),17 with the rate-limiting enzyme being GTP cyclohydrolase-1 (GCH). Models in which GCH is genetically enhanced in endothelial cells show suppressed diabetic and atherosclerotic vasculopathy.9,10 Effective BH4 levels also depend on redox state because the oxidized form of BH4 (BH2) does not serve as an NOS cofactor. BH4 levels decline in pressure-overload hypertrophy in conjunction with NOS uncoupling,11 but whether BH4 supplementation can treat already established advanced disease and whether this involves targeted eNOS recoupling are unknown. Here, we demonstrate that exoge- nous BH4 can indeed recouple NOS and reverse advanced hypertrophy/dilation more effectively than a less specific antioxidant strategy. Methods General Experimental Model Seventy-six male mice (C57BL/6; age, 8 to 9 weeks; weight, 22 to 24 g) underwent transverse aortic constriction (TAC) as previously described.11,18 Animals were screened by echocardiography at 4 weeks for hypertrophy and an ejection fraction (EF)�70% (chamber dilation). Of these animals, 10 were killed for tissue analysis, and the remaining were randomized to receive BH4, Tempol, or vehicle treatment during 5 more weeks of TAC. At 9 weeks, subsets of these animals were randomly selected and used for molecular, cellular, and enzymatic assays; histopathology; or in vivo function analysis. BH4 (200 mg · kg�1 · d�1; Schircks Laboratories, Jona, Switzerland) or vehicle was mixed in soft diet, and Tempol (10 mg/g food; 1.67 g · kg�1 · d�1)19 or vehicle was premixed in solid food logs (Bio- Serv, Frenchtown, NJ). All animal protocols were approved by the Animal Care and Use Committee of Johns Hopkins University. Endothelial GTP Cyclohydrolase Transgene Overexpression GTP cyclohydrolase transgenic (GCH-Tg) mice (n�24) and non- transgenic control littermates (n�16)9 were subjected to 12 weeks of TAC. Age-matched sham controls also were generated. Serial echocardiography and final sacrifice tissue analysis were performed. Cardiac Function and Geometry In vivo cardiac geometry and function was serially assessed by transthoracic echocardiography (Acuson Sequoia C256, 13-MHz transducer, Siemens Medical Systems, Malvern, Pa) in conscious mice. M-mode left ventricular (LV) end-systolic and end-diastolic dimensions were averaged from 3 to 5 beats, and data were analyzed by investigators blinded to heart condition as described.11 In a subset of mice, LV function was assessed by pressure-volume relations (SPR 839, Millar Instruments Inc, Houston, Tex) in anesthetized animals as described.11 Histology Myocardium was fixed in 10% formalin and stained with hematox- ylin and eosin, periodic acid–Schiff methenimine silver, or Masson’s trichrome to determine myocyte cross-sectional diameter (mean, 40 cells from 3 slices in 4 to 5 different hearts) and interstitial fibrosis. Fibrosis was scored 0 to 3 by a pathologist blinded to heart condition. Whole-Cell Myocyte Shortening and Calcium Transients Adult myocytes were isolated from left ventricles, and cell shorten- ing and calcium transient changes (Indo-1-AM) were determined by fluorescence microscopy (Diaphot 200, Nikon, Inc, Melville, NY) equipped with image/analysis (IonOptix, MyoCam, Milton, Mass) as described.20 Data were assessed in control and 9-week TAC hearts with or without BH4 treatment. eNOS Monomer-to-Dimer Ratio and Activity Cold SDS-PAGE Western blot analysis was performed in self-made 7% to 4% SDS-Tris gels run overnight on ice and then transferred for 3 hours to nitrocellulose membranes. Primary eNOS antibody (1:350, Santa Cruz Technology, Inc, Santa Cruz, Calif) was detected by enhanced chemiluminescence (Pierce, Rockford, Ill). NOS activ- ity was measured from myocardial homogenates (80 �g protein) by C14 arginine to citrulline conversion (Stratagene, La Jolla, Calif).11 cGMP-Dependent Protein Kinase Activity cGMP-dependent protein kinase (PKG)-1 activity was assayed from whole-heart protein lysates by ELISA (CycLex-PKG assay kit, MBL, Woburn, Mass) and immunoblot for PKG-phosphorylated vasodilator-stimulated protein with a monoclonal antibody to pS239 vasodilator-stimulated protein (Alexis, Lausen, Switzerland) at 1:1000 dilution.20 Superoxide Determination Myocardial superoxide was measured by dihydroethidine fluorescent microtopography and lucigenin-enhanced chemiluminescence. Fresh-frozen 8-�m LV slices were incubated for 1 hour at 37°C with dihydroethidine (2 �mol/L, Invitrogen, Carlsbad, Calif) and fluores- cence imaged as described.11 For lucigenin analysis, fresh-frozen myocardium was homogenized and centrifuged at 4000 RPM for 30 seconds; lucigenin (5 �mol/L) and NADPH (100 �mol/L) were added to the supernatant; and chemiluminescence was measured by scintillation counter (LS6000IC, Beckman Instruments, Fullerton, Calif) at 37°C. Data are reported as counts per minute per 1 mg protein after background subtraction. Microarray Analysis Microarrays for 9 weeks of TAC with and without delayed BH4 and Tempol treatment were performed with the Mouse Genome 430 2.0 array chip (Affymetrix, Santa Clara, Calif). Details are provided in Methods section of the online Data Supplement. Polymerase Chain Reaction Analysis Quantitative polymerase chain reaction was performed with an Applied Biosystems Prism 7900HT Sequence Detection System with the TaqMan universal polymerase chain reaction master mix accord- ing to the manufacturer’s specifications (Applied Biosystems Inc, Foster City, Calif). The Mann-Whitney U test was used to compare the different groups (SigmaStat, Systat Software, Inc, San Jose, Calif). Details are provided in the supplemental Methods. Myocardial BH4/BH2 Analysis Myocardial BH4 and BH2 levels were determined by direct high- performance liquid chromatography analysis of frozen tissue homog- enates. Details are provided in the supplemental Methods. Moens et al Recoupling eNOS and Cardiac Remodeling 2627 by on April 28, 2011 circ.ahajournals.orgDownloaded from Statistical Analysis All data are expressed as mean�SEM. Group data were compared by use of 1- and 2-way ANOVA. Nonparametric data were analyzed with the Kruskall-Wallis test and the Mann-Whitney U test. Re- ported probability values were Bonferroni or Tukey test adjusted for multiple comparisons (3 to 5 comparisons, depending on the data analyzed). The minimum sample size was 4 for any group; other specific details are provided in the text. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written. Results BH4 Reverses Chronic Hypertrophic Remodeling and Fibrosis Four weeks of TAC induced substantial left ventricular remodeling, increasing cardiac mass by 190% and chamber end-diastolic dimension by 140% and lowering fractional shortening by 44% (Figure 1A and 1B). EF declined from 87.4�0.5% to 45.7�1.6% (P�0.001). Hypertrophy reversed and heart function improved in mice that subsequently received BH4 for 5 weeks of continued TAC (Figure 1A and 1B). Chamber dilation was arrested at levels present at the onset of treatment. In contrast, all these features worsened in vehicle-treated mice. Myocyte enlargement and interstitial fibrosis were present at 4 weeks of TAC and were reversed by BH4 treatment (Figure 1C), whereas both remained elevated or worsened in untreated 9-week-TAC mice. BH4 Prevents Progressive Deterioration of Myocardial Function Pressure-volume relations were obtained to better assess LV function (Figure 2A and the Table). Rest conditions are reflected by the most rightward pressure-volume loop of each set. At 4 weeks of TAC, hearts were dilated and had increased end-systolic elastance (arrow) typical of hypertrophy. After 9 weeks of TAC, they became markedly dilated and had depressed function (reduced slope [Ees] and right shift of end-systolic pressure-volume relation; the Table). These changes were prevented by BH4, with end-systolic pressure- volume relations maintaining their position at 4 weeks of TAC (ie, onset of treatment; summary data on the right and the Table). Importantly, BH4 did not alter ventricular after- load assessed by peak systolic pressure (Figure 2A, top left) or total resistive load (P�0.3; data not shown). To further assess the effect of BH4 on myocardial function, myocytes were isolated from treated and untreated 9-week- TAC hearts (Figure 2B). The rate of sarcomere shortening and relengthening improved with BH4 treatment and was associated with higher peak calcium transients and a faster transient decay, consistent with improved calcium cycling. BH4 Recouples eNOS As previously reported, 3 to 4 weeks of TAC results in NOS uncoupling indexed by eNOS homodimer instability, reduced Ca2�-dependent NOS activity, and increased NOS-derived ROS.11 Here, we show data for homodimer instability (higher ratio of monomers to dimers in cold SDS nonreducing gels; Figure 3A). Untreated 9-week-TAC mice had persistent instability, with a marked decline in NOS activity and increased NOS-dependent ROS generation (Figure 3B). These behaviors were restored to normal with BH4 treatment. Total eNOS (monomer plus dimer) was unchanged. In 6 additional animals, BH4 treatment was initiated at the onset of TAC and continued for 9 weeks. After 1 week of TAC, hearts developed nondilated hypertrophy, which was not suppressed by BH4; however, the progressive rise in LV mass and chamber dilation and the decline in EF observed thereafter in controls were prevented by BH4 treatment (Figure 3C; P�0.001 for treatment, time, and treatment-by- time interaction for each parameter based on 2 way- ANOVA). This result was consistent with the time course of reduced NOS activity. After 1 week of TAC, in vitro NOS activity remained at control levels, whereas it declined by �50% after 3 weeks (Figure 3D), consistent with our earlier report,11 and even more by 9 weeks (Figure 3B). Thus, BH4 became effective once NOS activity otherwise started to decline. Effect of BH4 on PKG Activity Improved eNOS activity could potentially suppress hypertro- phy by stimulating downstream PKG.18,21 PKG activity rose after 9 weeks of TAC as previously reported with 3 weeks of TAC18 but was not further enhanced by BH4 treatment (Figure 3E). This was demonstrated by both in vitro activity and phosphorylated vasodilator–stimulated protein immuno- blot (Figure 3E, top and bottom, respectively). Antioxidant Effect of BH4 and Comparison With Tempol Another potential mechanism of BH4 efficacy is its targeting upstream signaling from NO, the NO-ROS interaction, or ROS itself. Dihydroethidium fluorescent microtopography (Figure 4A) revealed marked ROS generation at 4 and 9 weeks of TAC that fell to nearly control levels with delayed BH4 treatment. This result was confirmed by lucigenin chemiluminescence (Figure 4B). Given this potent antioxidant effect, we tested whether BH4 therapeutic benefits could be duplicated with a broad antioxidant. With the same delayed-treatment TAC protocol, mice received control diet or food premixed with the nitrox- ide Tempol (30 to 50 mg/d), a superoxide dismutase mimetic that also suppresses hydroxyl, hydrogen peroxide, and other radicals.22 Both Tempol and BH4 were equally effective in scavenging superoxide in vitro (Figure 4C, left), and Tempol reduced myocardial superoxide potently and similarly to BH4 in TAC hearts (Figure 4C, right; see also Figure 4B). Yet, Tempol did not reverse or prevent progressive hypertrophy (Figure 4D) or affect fibrosis from sustained TAC, and myocyte size declined less than with BH4 (Figure 4E). Tempol increased EF (Figure 4D) by reducing end-systolic dimensions (3.8�0.4 versus 2.8�0.4 mm; P�0.05), so some systolic improvement resulted, although it did not restore eNOS coupling (Figure 4F). To further probe differences between these therapies, gene-expression microarrays were performed (Table I of the online Data Supplement). Quantitative reverse-transcription polymerase chain reaction was performed on a subset of genes to confirm array results. The 9-week TAC principally 2628 Circulation May 20, 2008 by on April 28, 2011 circ.ahajournals.orgDownloaded from liuhuibin Highlight liuhuibin Highlight liuhuibin Underline liuhuibin Highlight liuhuibin Underline liuhuibin Underline liuhuibin Note BH4未通过PKG改善NOS的活性