Reversal of Cardiac Hypertrophy and Fibrosis From Pressure Overload by Tetrahydrobiopterin: Efficacy...

Reversal of Cardiac Hypertrophy and Fibrosis FromPressure 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 stresslinked 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 oxidativestress. Oral BH4 was then administered for 5 more weeks of pressure overload. Without reducing loading, BH4 reversedhypertrophy and fibrosis, recoupled endothelial NOS, lowered oxidant stress, and improved chamber and myocytefunction, whereas untreated hearts worsened. If BH4 was started at the onset of pressure overload, it did not suppresshypertrophy over the first week when NOS activity remained preserved even in untreated transverse aortic constrictionhearts. 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 sustainedtransverse 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 toendothelial cells were unprotected against pressure overload, indicating that exogenous BH4 targeted myocytes andfibroblasts.

Conclusions—NOS recoupling by exogenous BH4 ameliorates preexisting advanced cardiac hypertrophy/fibrosis and ismore effective than a less targeted antioxidant approach (Tempol). These data highlight the importance of myocyte NOSuncoupling 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 therapeuticgoal.2 A prominent pathway is activation of reactive oxygenspecies (ROS), which contributes to chamber remodeling andcontractile failure.3 Although treatment with ROS scavengershas not been very effective to date,4,5 suppression of key ROSgenerators 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 thatNOS may be particularly important to more advanced dilativedisease. NOS behaves somewhat like Jekyll and Hyde,generating NO to provide antioxidant and antihypertrophiceffects yet contributing to cardiovascular pathobiology if itbecomes functionally uncoupled.6 This occurs if the normalflow of electrons from NADPH in the reductase domain toheme 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, BloombergSchool 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 Departmentof 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 [email protected]© 2008 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.107.737031

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limiting NO synthesis and favoring superoxide generation bydissociation of the ferrous-dioxygen complex.6,7 EndothelialNOS (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 2636A 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 progressivemental retardation. Affected individuals must follow aphenylalanine-restricted diet, and BH4 replacement therapycan reduce phenylalanine levels and appears to be useful fortreating a substantial number of these patients.15 BH4 also isrequired for normal NOS function (reviewed elsewhere16). Itis 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 inendothelial cells show suppressed diabetic and atheroscleroticvasculopathy.9,10 Effective BH4 levels also depend on redoxstate because the oxidized form of BH4 (BH2) does not serveas an NOS cofactor. BH4 levels decline in pressure-overloadhypertrophy in conjunction with NOS uncoupling,11 butwhether BH4 supplementation can treat already establishedadvanced disease and whether this involves targeted eNOSrecoupling are unknown. Here, we demonstrate that exoge-nous BH4 can indeed recouple NOS and reverse advancedhypertrophy/dilation more effectively than a less specificantioxidant strategy.

MethodsGeneral Experimental ModelSeventy-six male mice (C57BL/6; age, 8 to 9 weeks; weight, 22 to24 g) underwent transverse aortic constriction (TAC) as previouslydescribed.11,18 Animals were screened by echocardiography at 4weeks for hypertrophy and an ejection fraction (EF) �70% (chamberdilation). Of these animals, 10 were killed for tissue analysis, and theremaining were randomized to receive BH4, Tempol, or vehicletreatment during 5 more weeks of TAC. At 9 weeks, subsets of theseanimals were randomly selected and used for molecular, cellular, andenzymatic assays; histopathology; or in vivo function analysis. BH4(200 mg · kg�1 · d�1; Schircks Laboratories, Jona, Switzerland) orvehicle was mixed in soft diet, and Tempol (10 mg/g food; 1.67g · kg�1 · d�1)19 or vehicle was premixed in solid food logs (Bio-Serv, Frenchtown, NJ). All animal protocols were approved by theAnimal Care and Use Committee of Johns Hopkins University.

Endothelial GTP CyclohydrolaseTransgene OverexpressionGTP cyclohydrolase transgenic (GCH-Tg) mice (n�24) and non-transgenic control littermates (n�16)9 were subjected to 12 weeks ofTAC. Age-matched sham controls also were generated. Serialechocardiography and final sacrifice tissue analysis were performed.

Cardiac Function and GeometryIn vivo cardiac geometry and function was serially assessed bytransthoracic echocardiography (Acuson Sequoia C256, 13-MHztransducer, Siemens Medical Systems, Malvern, Pa) in consciousmice. M-mode left ventricular (LV) end-systolic and end-diastolicdimensions were averaged from 3 to 5 beats, and data were analyzedby 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 anesthetizedanimals as described.11

HistologyMyocardium was fixed in 10% formalin and stained with hematox-ylin and eosin, periodic acid–Schiff methenimine silver, or Masson’strichrome to determine myocyte cross-sectional diameter (mean, 40cells 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 andCalcium TransientsAdult myocytes were isolated from left ventricles, and cell shorten-ing and calcium transient changes (Indo-1-AM) were determined byfluorescence microscopy (Diaphot 200, Nikon, Inc, Melville, NY)equipped with image/analysis (IonOptix, MyoCam, Milton, Mass) asdescribed.20 Data were assessed in control and 9-week TAC heartswith or without BH4 treatment.

eNOS Monomer-to-Dimer Ratio and ActivityCold SDS-PAGE Western blot analysis was performed in self-made7% to 4% SDS-Tris gels run overnight on ice and then transferred for3 hours to nitrocellulose membranes. Primary eNOS antibody(1:350, Santa Cruz Technology, Inc, Santa Cruz, Calif) was detectedby enhanced chemiluminescence (Pierce, Rockford, Ill). NOS activ-ity was measured from myocardial homogenates (80 �g protein) byC14 arginine to citrulline conversion (Stratagene, La Jolla, Calif).11

cGMP-Dependent Protein Kinase ActivitycGMP-dependent protein kinase (PKG)-1 activity was assayed fromwhole-heart protein lysates by ELISA (CycLex-PKG assay kit,MBL, Woburn, Mass) and immunoblot for PKG-phosphorylatedvasodilator-stimulated protein with a monoclonal antibody to pS239vasodilator-stimulated protein (Alexis, Lausen, Switzerland) at1:1000 dilution.20

Superoxide DeterminationMyocardial superoxide was measured by dihydroethidine fluorescentmicrotopography and lucigenin-enhanced chemiluminescence.Fresh-frozen 8-�m LV slices were incubated for 1 hour at 37°C withdihydroethidine (2 �mol/L, Invitrogen, Carlsbad, Calif) and fluores-cence imaged as described.11 For lucigenin analysis, fresh-frozenmyocardium was homogenized and centrifuged at 4000 RPM for 30seconds; lucigenin (5 �mol/L) and NADPH (100 �mol/L) wereadded to the supernatant; and chemiluminescence was measured byscintillation counter (LS6000IC, Beckman Instruments, Fullerton,Calif) at 37°C. Data are reported as counts per minute per 1 mgprotein after background subtraction.

Microarray AnalysisMicroarrays for 9 weeks of TAC with and without delayed BH4 andTempol treatment were performed with the Mouse Genome 430 2.0array chip (Affymetrix, Santa Clara, Calif). Details are provided inMethods section of the online Data Supplement.

Polymerase Chain Reaction AnalysisQuantitative polymerase chain reaction was performed with anApplied Biosystems Prism 7900HT Sequence Detection System withthe 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 comparethe different groups (SigmaStat, Systat Software, Inc, San Jose,Calif). Details are provided in the supplemental Methods.

Myocardial BH4/BH2 AnalysisMyocardial 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.

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Statistical AnalysisAll data are expressed as mean�SEM. Group data were comparedby use of 1- and 2-way ANOVA. Nonparametric data were analyzedwith the Kruskall-Wallis test and the Mann-Whitney U test. Re-ported probability values were Bonferroni or Tukey test adjusted formultiple comparisons (3 to 5 comparisons, depending on the dataanalyzed). The minimum sample size was 4 for any group; otherspecific details are provided in the text.

The authors had full access to and take full responsibility for theintegrity of the data. All authors have read and agree to themanuscript as written.

ResultsBH4 Reverses Chronic Hypertrophic Remodelingand FibrosisFour weeks of TAC induced substantial left ventricularremodeling, increasing cardiac mass by 190% and chamberend-diastolic dimension by 140% and lowering fractionalshortening by 44% (Figure 1A and 1B). EF declined from87.4�0.5% to 45.7�1.6% (P�0.001). Hypertrophy reversedand heart function improved in mice that subsequentlyreceived BH4 for 5 weeks of continued TAC (Figure 1A and1B). Chamber dilation was arrested at levels present at theonset of treatment. In contrast, all these features worsened invehicle-treated mice. Myocyte enlargement and interstitialfibrosis were present at 4 weeks of TAC and were reversed byBH4 treatment (Figure 1C), whereas both remained elevatedor worsened in untreated 9-week-TAC mice.

BH4 Prevents Progressive Deterioration ofMyocardial FunctionPressure-volume relations were obtained to better assess LVfunction (Figure 2A and the Table). Rest conditions arereflected by the most rightward pressure-volume loop of eachset. At 4 weeks of TAC, hearts were dilated and had increasedend-systolic elastance (arrow) typical of hypertrophy. After 9weeks of TAC, they became markedly dilated and haddepressed function (reduced slope [Ees] and right shift ofend-systolic pressure-volume relation; the Table). Thesechanges were prevented by BH4, with end-systolic pressure-volume relations maintaining their position at 4 weeks ofTAC (ie, onset of treatment; summary data on the right andthe 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 shorteningand relengthening improved with BH4 treatment and wasassociated with higher peak calcium transients and a fastertransient decay, consistent with improved calcium cycling.

BH4 Recouples eNOSAs previously reported, 3 to 4 weeks of TAC results in NOSuncoupling indexed by eNOS homodimer instability, reducedCa2�-dependent NOS activity, and increased NOS-derivedROS.11 Here, we show data for homodimer instability (higherratio of monomers to dimers in cold SDS nonreducing gels;Figure 3A). Untreated 9-week-TAC mice had persistentinstability, 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 theonset of TAC and continued for 9 weeks. After 1 week ofTAC, hearts developed nondilated hypertrophy, which wasnot suppressed by BH4; however, the progressive rise in LVmass and chamber dilation and the decline in EF observedthereafter 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 ofreduced NOS activity. After 1 week of TAC, in vitro NOSactivity remained at control levels, whereas it declined by�50% after 3 weeks (Figure 3D), consistent with our earlierreport,11 and even more by 9 weeks (Figure 3B). Thus, BH4became effective once NOS activity otherwise started todecline.

Effect of BH4 on PKG ActivityImproved eNOS activity could potentially suppress hypertro-phy by stimulating downstream PKG.18,21 PKG activity roseafter 9 weeks of TAC as previously reported with 3 weeks ofTAC18 but was not further enhanced by BH4 treatment(Figure 3E). This was demonstrated by both in vitro activityand phosphorylated vasodilator–stimulated protein immuno-blot (Figure 3E, top and bottom, respectively).

Antioxidant Effect of BH4 and ComparisonWith TempolAnother potential mechanism of BH4 efficacy is its targetingupstream signaling from NO, the NO-ROS interaction, orROS itself. Dihydroethidium fluorescent microtopography(Figure 4A) revealed marked ROS generation at 4 and 9weeks of TAC that fell to nearly control levels with delayedBH4 treatment. This result was confirmed by lucigeninchemiluminescence (Figure 4B).

Given this potent antioxidant effect, we tested whetherBH4 therapeutic benefits could be duplicated with a broadantioxidant. 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 mimeticthat also suppresses hydroxyl, hydrogen peroxide, and otherradicals.22 Both Tempol and BH4 were equally effective inscavenging superoxide in vitro (Figure 4C, left), and Tempolreduced myocardial superoxide potently and similarly to BH4in 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, andmyocyte size declined less than with BH4 (Figure 4E).Tempol increased EF (Figure 4D) by reducing end-systolicdimensions (3.8�0.4 versus 2.8�0.4 mm; P�0.05), so somesystolic improvement resulted, although it did not restoreeNOS coupling (Figure 4F).

To further probe differences between these therapies,gene-expression microarrays were performed (Table I of theonline Data Supplement). Quantitative reverse-transcriptionpolymerase chain reaction was performed on a subset ofgenes to confirm array results. The 9-week TAC principally

2628 Circulation May 20, 2008



stimulated genes controlling collagen synthesis/degradationand tissue growth factor-� signaling and reduced the expres-sion of genes controlling metabolism. Intriguingly, none ofthese were significantly offset by BH4 or Tempol. Instead,

BH4 increased the expression of genes regulating lipidmetabolism (eg, fatty-acid-binding protein 1, apolipoproteinA-1, major urinary protein 1,2) and kallikrein signaling (eg,plasminogen, fibrinogen). Only 8% of these genes were

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Figure 1. A, BH4 treatment reverses advanced hypertrophy caused by sustained pressure overload. TAC-stimulated increases in heartweight at 4 weeks were reversed by the subsequent addition of oral BH4, whereas untreated hearts continued to enlarge. Right, Pairedchanges in LV mass between weeks 4 and 9 (treatment period) between untreated and BH4-treated hearts. These were significantlydifferent (P�0.01). B, Example M-mode echocardiograms showing increased dilation, wall thickening, and reduced fractional shorteningafter 4 weeks of TAC. They improved in mice treated with BH4. *P�0.001 vs control; †P�0.001, ‡P�0.05 vs 9 weeks of TAC;#P�0.001 vs 4 weeks of TAC. C, BH4 treatment reverses myocyte enlargement and fibrosis after 4 weeks of TAC. Panels show hema-toxylin and eosin staining (top) and periodic acid–Schiff/methenimine silver staining (bottom). Summary data are provided on the right.Color coding follows the legend at the top. *P�0.001 vs control; †P�0.001 vs 9 weeks of TAC; #P�0.01 vs 4 weeks of TAC. ES indi-cates end systole; ED, end diastole.




similarly affected by BH4 alone (without TAC), and none ofthese related to lipid metabolism. A more complete list ofBH4 modified genes is provided in supplemental Table II.Tempol altered virtually none of the same genes as BH4 butmodestly lowered expression of a different set, eg, phospho-lipase C �4, �1, AMP-kinase �2, GSK3�, mitogen-activatedprotein-4k3, PKG-1, and flavin-containing monooxygenase 2(the only gene with similar changes from BH4). Thus,suppressing TAC-induced ROS, more broadly or by a NOS-targeted strategy, resulted in very different gene profiling andphenotype.

Nonendothelial BH4 Is Central for ItsAntihypertrophic EffectsExogenous BH4 can diffuse into myocytes, fibroblasts, andthe vascular endothelium, and because endothelial cellscontain 80% of eNOS in the myocardium, this might be thepresumed primary target. To test this, we studied mice thatoverexpressed GCH only in endothelial cells by use of aTei-2 promoter.9 GCH is the rate-limiting enzyme involved inde novo BH4 synthesis, and in this model, isolated myocyteBH4 levels are unaltered,9 whereas total myocardial levelsrise �4 fold (5.2�3.5 to 19.3�4.9 pmol/mg protein), similarto the rise achieved by exogenous BH4 (40.5�19.1 pmol/mgprotein). Intriguingly, chamber hypertrophy, fibrosis, myo-

cyte enlargement, heart function, and dilation changed iden-tically during 12 weeks of TAC in the hearts of GCH-Tg andlittermate controls (Figure 5). However, superoxide declinedin GCH-Tg myocardium (P�0.05), suggesting a role ofendothelial NOS uncoupling to myocardial ROS that is lessassociated with cardiac hypertrophic remodeling. These dataindicate that the effectiveness of BH4 to ameliorate pressure-overload cardiac dysfunction and remodeling lies in itstargeting of NOS uncoupling in myocytes (and perhapsfibroblasts) rather than in the endothelium.

DiscussionThe ability of exogenous BH4 to reverse advanced hypertro-phic remodeling and to ameliorate heart and myocyte func-tion despite ongoing pressure overload is unusual amongexisting therapies and suggests that targeting uncoupledeNOS may be a potent and useful strategy for treatinghypertrophic heart disease. Few experimental studies involv-ing established advanced disease models have shown thecapacity of an intervention to reverse the process. Much ofthe recent work has relied on genetically engineered modelsin which the manipulation is generated at or before birth andinterventions are initiated at or shortly after the induction ofmyocardial stress. In clinical trials, however, advanced dis-ease often is required, making the present results notable from

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Figure 2. A, BH4 treatment improves intact heart function. Pressure-volume loops are measured before and during transient inferiorvena cava occlusion, with the most rightward loop reflecting rest conditions. TAC for 4 weeks induced moderate dilation, reduced EF,and increased chamber end-systolic elastance (slope of the end-systolic pressure-volume relation [Ees]; arrow) consistent with hyper-trophy but contractile compensation. At 9 weeks of TAC, the loops shifted rightward (V100�end-systolic volume at an end-systolic pres-sure�100 mm Hg indexes this shift) and Ees declined. BH4-treated hearts had far less dilation (V100 remained small) and improved EFand relaxation time constant (�). Peak systolic pressure (Psys) was similar among groups. *P�0.05 vs 9 weeks of vehicle. B, BH4improves sarcomere shortening kinetics and calcium transients. Left, Example tracings; right, summary data. #P�0.05, control vs TACat 9 weeks; *P�0.05, TAC at 9 weeks vs TAC at 9 weeks plus BH4.




a translational perspective. We specifically targeted patholog-ical hypertrophy coupled to cardiac decompensation, a periodwhen ROS generation may be particularly important. Drugssuch as angiotensin-converting enzyme and receptor blockersblunt disease progression,23 but the capacity of BH4 toreverse this pathobiology is striking and supports a detrimen-tal role of NOS uncoupling and the nitroso-redox imbalance24

that ensues.NOS uncoupling impairs NO synthesis and its downstream

effecter signaling (ie, cGMP and PKG) while concomitantlyincreasing ROS generation. Both aspects can trigger myocar-dial hypertrophy and remodeling.3 Myocardial PKG signalingcoupled to natriuretic peptides25,26 or modulation of cGMPcatabolism18 blunts cardiac hypertrophy and can improveheart function. This is thought to be due in part to suppressionof calcineurin and nuclear factor of activated T-cell activa-tion21; other pathways are likely important also.18,27 BecauseNO stimulates soluble guanylate cyclase to generate cGMPand thus activate PKG, improved NOS function by BH4could potentially involve this antihypertrophic mechanism.Yet, enhanced PKG activity was not observed, and there areseveral potential reasons for this. BH4 did not hyperstimulateNOS but returned its activity to normal control levels forwhich corresponding PKG activation is typically low. PKGactivity could have already been maximal, although this isunlikely because enhancing cGMP via PDE5a inhibition (eg,sildenafil) during TAC can potently activate PKG further.18

PKG also was activated more with 9 weeks of TAC aloneover control (similar to results after 3 weeks of TAC18)despite reduced NOS activity, indicating that alternativemechanisms such as cGMP generated by natriuretic peptides(both ANP and BNP expression rose with TAC; see supple-mental Table I) or oxidant stress28 could have played a role.Although BH4 therapy might lower one source such asoxidant stress, it could raise another (ie, eNOS), leaving netPKG activation unchanged.

An alternative to cGMP/PKG mechanisms is the modula-tion of NO and NO-ROS interactions by BH4. This wouldinclude S-nitrosylation, which can alter cardiac contractileregulation,29 or local interaction of NOS-derived ROS withNO (forming peroxynitrite)30 perhaps in a particularly vul-nerable subcellular compartment. Recoupling NOS wouldsteer superoxide-derived ROS formation away from peroxide,which may be important. Restored NOS activity would notitself be expected to stimulate peroxynitrite in the absence ofoxidant stress because this is not observed in normal hearts,and if anything, BH4 appears to lower peroxynitrite inoxidant stress disorders.31

The present data support a growing notion that ROSsignaling is compartmentalized32 and that targeting specificoxidant generators may be more efficacious than broaderantioxidant scavengers. Although both Tempol and BH4provided similar in vitro and tissue (lucigenin) antioxidanteffects, their effects on hypertrophy, fibrosis, and NOS

Table. Invasive Hemodynamic Analysis of TAC and TAC Plus Delayed BH4 Treatment Based on Pressure-Volume Analysis

Baseline(n�5)

4 Weeks of TAC(n�6)

9 Weeks ofTAC�Vehicle

(n�6)TAC 9 Weeks�BH4

(n�5)P, 4

Groups

P, 9 Weeks ofTAC-P vs 9Weeks ofTAC�BH4

P, 4 Weeks ofTAC-P vs 9Weeks ofTAC�BH4

Hemodynamics

Heart rate, bpm 523�14 520�13 540�15 490�20 0.17 � � � � � �

Heart mass, mg 117.6�3.7 232.8�6.8 303.7�32.8 204.6�14.2 �0.001 0.01 0.20

LV peak pressure, mm Hg 107�2.2 179.9�3.2 168.5�3.4 157�6.1 �0.001 0.14 0.018

LV end-diastolic pressure, mm Hg 5.4�0.6 7.1�1.4 6.1�0.8 8.0�0.4 0.1 � � � � � �

LV end-systolic volume, �L 10.2�1.0 23.3�3.3 55.9�7.5 16.9�4.5 0.001 0.006 0.36

LV end-diastolic volume, �L 29.0�2.0 38.8�3.4 68.4�7.3 33.5�6.0 0.006 0.01 0.59

Stroke volume, �L 18.9�1.4 15.5�0.6 12.5�0.7 16.6�2.6 0.056 0.20 0.71

Cardiac output, mL/min 9.9�0.7 8.0�0.3 6.7�0.3 8.2�1.5 0.047 0.36 0.86

EF, % 65.1�2.1 41.3�3.6 18.7�2.2 52.0�5.3 �0.001 0.006 0.10

Systolic function

dP/dtmax, mm Hg/s 13 368�370 12 602�620 9836�421 10 210�618 0.001 0.36 0.006

Ees, mm Hg/�L 4.5�0.72 16.1�2.9 7.8�1.4 10.3�1.8 0.008 0.36 0.27

Ves-100, �L 9.9�1.1 18.8�2.6 46.4�5.5 11.5�4.8 0.001 0.006 0.004

MSW, mm Hg 79.5�4.1 120.75�12.6 79.2�8.4 113.9�13.7 0.01 0.028 1

Diastolic function

dP/dtmin, mm Hg/s �10 728�236 �10 508�500 �8550�189 �9908�813 0.008 0.10 0.46

�(norm), ms 3.6�0.1 4.4�0.2 5.6�0.1 4.1�0.2 �0.001 0.006 0.27

PFR/EDV, s�1 37.1�5.6 24.4�1.4 14.5�2.2 31.4�4.0 0.003 0.018 0.10

dP/dtmax indicates peak rate of pressure rise; Ees, LV end-systolic elastance (stiffness); V100, end-systolic volume at end-systolic pressure of 100 mm Hg; MSW,

slope of stroke work–end-diastolic volume relationship; dP/dtmin, peak rate of pressure decline; �(norm), time constant of relaxation normalized to heart rate; andPFR/EDV, peak filling rate normalized to end-diastolic volume. Four-way group analysis was performed by the Kruskal-Wallis test; post hoc comparisons between 2groups were done by the Mann–Whitney U test.




4wk 9wk 9wk BH4

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Figure 3. A, Left, eNOS dimer/monomer gel electrophoresis. Control (Con) tissue has principally dimers in the gel, whereas monomersare more apparent at 4 and 9 weeks of TAC. BH4 restored the control appearance. Relative dimer/monomer density normalized to con-trol is shown in the summary. B, Left, NOS Ca2�-dependent arginine-citrulline conversion (NOS activity) is reduced after 9 weeks ofTAC and restored to normal by BH4. Right, NOS-dependent superoxide determined as the relative lucigenin chemiluminescent signalreduced after blocking NOS (NG-nitro-L-arginine methyl ester 100 �mol/L). *P�0.01 vs control and 9 weeks treated. C, Effect of BH4treatment from the onset of TAC over a 9-week period. Hypertrophy generated after 1 week is unaltered, but thereafter, chamber dila-tion and hypertrophy progression are blocked by BH4 treatment. Statistics from Tukey test based on 2-way ANOVA: *P�0.001 vs con-trol, 1 week, and 9 weeks of TAC, P�0.021 vs untreated and 3 weeks of TAC; **P�0.001 vs other groups and 9 weeks untreated;†P�0.05 vs control, P�0.005 vs 3 and 9 weeks of TAC; ‡P�0.005 vs all other groups, P�0.001 vs untreated; #P�0.02 vs control;§P�0.001 vs control and 9 weeks of TAC; ¶P�0.001 vs untreated. D, NOS activity measured in untreated TAC hearts at 1 and 3weeks; data shown are normalized to normal control. E, PKG activity increases with 9 weeks of TAC similarly with or without BH4treatment. Results for in vitro assay (top) and phosphorylated vasodilator-stimulated protein (P-VASP) immunoblot (bottom) are shown.




recoupling were quite different. This is not likely due to aninsufficient Tempol dose because the dose used was fairlyhigh (equivalent to 58 mmol/L).19 It also is the highest dosethat mice will tolerate in their food because of the taste.Although clinical trials testing broad antioxidant strategieshave been fairly unimpressive to date,4,5 this may be analo-gous to continuously applying sponges to blot up water froman open faucet versus turning the faucet off. The latter, ie,suppressing a strategic ROS source, might well provide moreeffective results.

There are other oxidant sources in the heart besidesuncoupled NOS, although the impact of their inhibition onhypertrophic remodeling remains unclear. Xanthine oxidase–derived free radicals have been found to play a role in dilatedcardiac failure, and allopurinol and its active metaboliteoxypurinol, which block xanthine oxidase, also improvemyocardial efficiency, NO-ROS balance, and myofilamentcalcium sensitization.33 However, in clinical trials, thesedrugs did not improve symptoms or exercise capacity.34

Furthermore, their role in hypertrophic disease has not beenestablished. NADPH oxidases also have been widely stud-ied.35 Genetic studies in mice lacking NOX2 (gp91phox)

found hypertrophic responses resulting from aortic bandingsimilar to those in controls36,37 but somewhat less fibrosis.Other NOX oxidases such as NOX4 may be important,although this remains to be confirmed. Importantly, small-molecule inhibitors remain scant, and none are clinicallyviable or sufficiently selective at present. Finally, ROSleakage linked to mitochondrial electron transport may alsocontribute to cardiac failure,38 although the involvement withpressure-overload hypertrophy remains to be established.

This study has several limitations. Although our datademonstrate that BH4 restores NOS coupling even in ad-vanced hypertrophic heart disease, it does not prove that thisis the sole or necessarily primary mechanism underlying thedecline in ROS stimulation or amelioration of hypertrophicremodeling and cardiac function. However, the finding thatBH4 administered from the onset of TAC did not suppresshypertrophy during the first week (when control heart NOSactivity was still preserved) yet prevented progression afterthat (when NOS uncoupling and reduced activity otherwiseoccurred) further supports such a link. A second limitationregards the comparison between BH4 and Tempol. Becausewe did not use a full dose-ranging study, the possibility

C

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Figure 4. A, BH4 reversed myocardial oxidant stress. Example of dihydroethidine (DHE) fluorescence images show increased ROSat 4 and 9 weeks of TAC that is reversed with BH4 therapy. Right, Summary data. B, Lucigenin chemiluminescence (CL) assayconfirms a marked rise in O2

� at 9 weeks of TAC that is reversed to control levels by BH4 treatment. C, Left, BH4 and Tempolhave similar dose-dependent antioxidant effects in vitro. Assay used xanthine/xanthine oxidase (X/XO) O2

� generator system.Right, O2

� generation in myocardium at 9 weeks of TAC is reduced by delayed Tempol treatment, similar to that with BH4. D,Tempol does not reduce LV mass, but EF shows a borderline increase (P�0.08). *P�0.001 vs 9 weeks of TAC; †P�0.08 vs TACuntreated. E, Tempol does not reduce TAC-stimulated fibrosis and more modestly reduces myocyte size compared with BH4(P�0.0001). Top, Masson’s trichrome stain; bottom, hematoxylin and eosin. F, Tempol does not reverse eNOS uncouplingreflected in dimer/monomer ratios on immunoblot.




remains that different pharmacology for the 2 compoundsand/or alternative signaling not revealed by the data obtainedcould explain some of the disparate effects.

The present findings suggest that NOS-derived ROS playsa particularly important role in decompensated hypertrophicheart disease. The existing clinical viability of BH4 as a drug,albeit for a different disorder at present,15 should helpfacilitate clinical translation and testing of the present find-

ings to human heart disease. An intriguing potential patientpopulation is individuals with heart failure and a preservedEF, often called diastolic heart failure. This disorder affectsnearly half of all patients with heart failure worldwide, mostoften elderly women with hypertension and ventricular hy-pertrophy,39 and its prevalence is rising.40 If the presentfindings can be translated to humans, BH4 might provide anovel and potent therapy to treat this common heart disease.

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Figure 5. A, Mice overexpressing GCH in endothelial cells develop progressive hypertrophy with TAC similar to littermate (non-transgenic [NTG]) controls. Right, Summary data for ratio of heart weight to body weight. B, Transgenic animals develop intersti-tial fibrosis (*P�0.05 vs no TAC) and myocyte enlargement (*P�0.001 vs no TAC) similar to controls. C, TAC-induced decline inEF and increases in wall thickness (WT) and left ventricular end-diastolic and end-systolic dimensions (LVEDD, LVESD) were virtu-ally identical in GCH-Tg and littermate controls. D, Superoxide increases significantly more in NTG than in GCH-Tg mice sub-jected to 12 weeks of TAC.




AcknowledgmentsWe thank Leslie-Anne Boxill, Azeb Haile, Norman Barker, andPawel Kaminski for their technical assistance in performing thestudy.

Sources of FundingThe work was supported by the Belgian-American Education Foun-dation (Collen), the PhD program at the University of Antwerp, andan American Heart Association Fellowship Grant (Dr Moens); AHAScientist Development Grants (Drs Champion and Takimoto);HL081205 (Dr Biswal); NIEHS center grant P30 ES 03819,HL31069, HL43023, and HL66331 (Dr Wolin); and AG-18324,HL-47511, and HL-59408, The Peter Belfer Laboratory Fund, and anAbraham and Virginia Weiss Professorship (Dr Kass).

DisclosuresNone.

References1. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intra-

cellular signalling pathways. Nat Rev Mol Cell Biol. 2006;7:589–600.2. McKinsey TA, Kass DA. Small-molecule therapies for cardiac hypertro-

phy: moving beneath the cell surface. Nat Rev Drug Discov. 2007;6:617–635.

3. Takimoto E, Kass DA. Role of oxidative stress in cardiac hypertrophyand remodeling. Hypertension. 2007;49:241–248.

4. Vivekananthan DP, Penn MS, Sapp SK, Hsu A, Topol EJ. Use ofantioxidant vitamins for the prevention of cardiovascular disease: meta-analysis of randomised trials. Lancet. 2003;361:2017–2023.

5. Yusuf S, Dagenais G, Pogue J, Bosch J, Sleight P. Vitamin E supple-mentation and cardiovascular events in high-risk patients: the HeartOutcomes Prevention Evaluation Study Investigators. N Engl J Med.2000;342:154–160.

6. Forstermann U, Munzel T. Endothelial nitric oxide synthase in vasculardisease: from marvel to menace. Circulation. 2006;113:1708–1714.

7. Vasquez-Vivar J, Kalyanaraman B, Martasek P. The role of tetrahydro-biopterin in superoxide generation from eNOS: enzymology and physi-ological implications. Free Radic Res. 2003;37:121–127.

8. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM,Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads touncoupling of endothelial cell nitric oxide synthase in hypertension.J Clin Invest. 2003;111:1201–1209.

9. Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, RockettKA, Channon KM. Tetrahydrobiopterin-dependent preservation ofnitric oxide-mediated endothelial function in diabetes by targetedtransgenic GTP-cyclohydrolase I overexpression. J Clin Invest. 2003;112:725–735.

10. Alp NJ, McAteer MA, Khoo J, Choudhury RP, Channon KM. Increasedendothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and ath-erosclerosis in ApoE-knockout mice. Arterioscler Thromb Vasc Biol.2004;24:445–450.

11. Takimoto E, Champion HC, Li M, Ren S, Rodriguez ER, Tavazzi B,Lazzarino G, Paolocci N, Gabrielson KL, Wang Y, Kass DA. Oxidantstress from nitric oxide synthase-3 uncoupling stimulates cardiacpathologic remodeling from chronic pressure load. J Clin Invest. 2005;115:1221–1231.

12. Gorren AC, Mayer B. Tetrahydrobiopterin in nitric oxide synthesis: anovel biological role for pteridines. Curr Drug Metab. 2002;3:133–157.

13. Moens AL, Kass DA. Tetrahydrobiopterin and cardiovascular disease.Arterioscler Thromb Vasc Biol. 2006;26:2439–2444.

14. Fitzpatrick PF. Tetrahydropterin-dependent amino acid hydroxylases.Annu Rev Biochem. 1999;68:355–381.

15. Levy HL, Milanowski A, Chakrapani A, Cleary M, Lee P, Trefz FK,Whitley CB, Feillet F, Feigenbaum AS, Bebchuk JD, Christ-Schmidt H,Dorenbaum A. Efficacy of sapropterin dihydrochloride (tetrahydro-

biopterin, 6R-BH4) for reduction of phenylalanine concentration inpatients with phenylketonuria: a phase III randomised placebo-controlledstudy. Lancet. 2007;370:504–510.

16. Stuehr DJ. Structure-function aspects in the nitric oxide synthases. AnnuRev Pharmacol Toxicol. 1997;37:339–359.

17. Thony B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regen-eration and functions. Biochem J. 2000;347(pt 1):1–16.

18. Takimoto E, Champion HC, Li M, Belardi D, Ren S, Rodriguez ER,Bedja D, Gabrielson KL, Wang Y, Kass DA. Chronic inhibition of cyclicGMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy.Nat Med. 2005;11:214–222.

19. Mitchell JB, Xavier S, DeLuca AM, Sowers AL, Cook JA, Krishna MC,Hahn SM, Russo A. A low molecular weight antioxidant decreases weightand lowers tumor incidence. Free Radic Biol Med. 2003;34:93–102.

20. Takimoto E, Belardi D, Tocchetti CG, Vahebi S, Cormaci G, KetnerEA, Moens AL, Champion HC, Kass DA. Compartmentalization ofcardiac beta-adrenergic inotropy modulation by phosphodiesterasetype 5. Circulation. 2007;115:2159 –2167.

21. Fiedler B, Lohmann SM, Smolenski A, Linnemuller S, Pieske B, SchroderF, Molkentin JD, Drexler H, Wollert KC. Inhibition of calcineurin-NFAThypertrophy signaling by cGMP-dependent protein kinase type I in cardiacmyocytes. Proc Natl Acad Sci U S A. 2002;99:11363–11368.

22. Soule BP, Hyodo F, Matsumoto K, Simone NL, Cook JA, Krishna MC,Mitchell JB. The chemistry and biology of nitroxide compounds. FreeRadic Biol Med. 2007;42:1632–1650.

23. Opie LH, Commerford PJ, Gersh BJ, Pfeffer MA. Controversies inventricular remodelling. Lancet. 2006;367:356–367.

24. Zimmet JM, Hare JM. Nitroso-redox interactions in the cardiovascularsystem. Circulation. 2006;114:1531–1544.

25. Knowles JW, Esposito G, Mao L, Hagaman JR, Fox JE, Smithies O,Rockman HA, Maeda N. Pressure-independent enhancement of cardiachypertrophy in natriuretic peptide receptor A-deficient mice. J ClinInvest. 2001;107:975–984.

26. Holtwick R, van Eickels M, Skryabin BV, Baba HA, Bubikat A, BegrowF, Schneider MD, Garbers DL, Kuhn M. Pressure-independent cardiachypertrophy in mice with cardiomyocyte-restricted inactivation of theatrial natriuretic peptide receptor guanylyl cyclase-A. J Clin Invest. 2003;111:1399–1407.

27. Fiedler B, Feil R, Hofmann F, Willenbockel C, Drexler H, Smolenski A,Lohmann SM, Wollert KC. cGMP-dependent protein kinase type Iinhibits TAB1-p38 mitogen-activated protein kinase apoptosis signalingin cardiac myocytes. J Biol Chem. 2006;281:32831–32840.

28. Burgoyne JR, Madhani M, Cuello F, Charles RL, Brennan JP, SchroderE, Browning DD, Eaton P. Cysteine redox sensor in PKGIa enablesoxidant-induced activation. Science. 2007;317:1393–1397.

29. Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS. ProteinS-nitrosylation: purview and parameters. Nat Rev Mol Cell Biol. 2005;6:150–166.

30. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in healthand disease. Physiol Rev. 2007;87:315–424.

31. Kase H, Hashikabe Y, Uchida K, Nakanishi N, Hattori Y. Supplemen-tation with tetrahydrobiopterin prevents the cardiovascular effects ofangiotensin II-induced oxidative and nitrosative stress. J Hypertens.2005;23:1375–1382.

32. Madamanchi NR, Moon SK, Hakim ZS, Clark S, Mehrizi A, Patterson C,Runge MS. Differential activation of mitogenic signaling pathways inaortic smooth muscle cells deficient in superoxide dismutase isoforms.Arterioscler Thromb Vasc Biol. 2005;25:950–956.

33. Berry CE, Hare JM. Xanthine oxidoreductase and cardiovascular disease:molecular mechanisms and pathophysiological implications. J Physiol.2004;555:589–606.

34. Cleland JG, Coletta AP, Clark AL. Clinical trials update from the HeartFailure Society of America Meeting: FIX-CHF-4, selective cardiacmyosin activator and OPT-CHF. Eur J Heart Fail. 2006;8:764–766.

35. Cave AC, Brewer AC, Narayanapanicker A, Ray R, Grieve DJ, Walker S,Shah AM. NADPH oxidases in cardiovascular health and disease.Antioxid Redox Signal. 2006;8:691–728.

36. Byrne JA, Grieve DJ, Bendall JK, Li JM, Gove C, Lambeth JD, Cave AC,Shah AM. Contrasting roles of NADPH oxidase isoforms in pressure-overload versus angiotensin II-induced cardiac hypertrophy. Circ Res.2003;93:802–805.

37. Maytin M, Siwik DA, Ito M, Xiao L, Sawyer DB, Liao R, Colucci WS.Pressure overload-induced myocardial hypertrophy in mice does notrequire gp91phox. Circulation. 2004;109:1168–1171.




38. Tsutsui H, Ide T, Kinugawa S. Mitochondrial oxidative stress, DNAdamage, and heart failure. Antioxid Redox Signal. 2006;8:1737–1744.

39. Melenovsky V, Borlaug BA, Rosen B, Hay I, Ferruci L, Morell CH,Lakatta LE, Najjar SS, Kasuya H. Cardiovascular features of heart failurewith preserved ejection fraction versus nonfailing hypertensive left ven-

tricular hypertrophy in the urban Baltimore community. J Am CollCardiol. 2007;49:198–207.

40. Owan TE, Hodge DO, Herges RM, Jacobsen SJ, Roger VL, RedfieldMM. Trends in prevalence and outcome of heart failure with preservedejection fraction. N Engl J Med. 2006;355:251–259.

CLINICAL PERSPECTIVESustained pressure overload induces profound ventricular remodeling and is a leading cause of cardiac failure. Animportant mechanism for this maladaptive response is stimulation of reactive oxygen species, and recent studies indicatethat functional uncoupling of nitric oxide synthase (NOS) plays an important role in this pathophysiology. A keydeterminant of NOS coupling, and thus its generation of NO versus O2

�, is the level and redox state of its cofactortetrahydrobiopterin (BH4), which becomes compromised from pressure-overload stress. Here, we tested whetherexogenous administration of BH4 could restore NOS coupling and function, reduce oxidant stress, and block or reversemaladaptive remodeling in hearts with already advanced disease induced by pressure overload. In mice subjected toproximal aortic constriction, BH4 stopped progressive chamber dilation and dysfunction, reversed fibrosis and hypertro-phy, and improved myocyte function and calcium handling. NOS became recoupled, and oxidant stress potently declined.The effects of BH4 were linked to NO–reactive oxygen species interactions and became manifest when NOS activitystarted to decline after induction of pressure overload. Parallel studies performed with a broad antioxidant (Tempol) didnot replicate BH4 effects on reverse remodeling even though oxidant stress was reduced. Furthermore, selectiveenhancement of BH4 in endothelial cells did not mimic the response to exogenous BH4, highlighting the importance ofmyocyte NOS uncoupling. These findings support the potential therapeutic utility of BH4 as a treatment for advancedhypertrophic heart disease. They also highlight the notion that not all antioxidant strategies are equivalent and that targetingNOS uncoupling could prove to be a particularly potent approach.




Nazareno Paolocci, Hunter C. Champion and David A. KassJames B. Mitchell, Shyam Biswal, Keith M. Channon, Michael S. Wolin, Nicholas J. Alp,

Cormaci, Elizabeth A. Ketner, Maulik Majmudar, Kathleen Gabrielson, Marc K. Halushka, An L. Moens, Eiki Takimoto, Carlo G. Tocchetti, Khalid Chakir, Djahida Bedja, Gianfranco

StrategyTetrahydrobiopterin: Efficacy of Recoupling Nitric Oxide Synthase as a Therapeutic

Reversal of Cardiac Hypertrophy and Fibrosis From Pressure Overload by

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2008 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/CIRCULATIONAHA.107.737031

2008;117:2626-2636; originally published online May 12, 2008;Circulation.

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Reversal of Cardiac Hypertrophy and Fibrosis From Pressure Overload by Tetrahydrobiopterin: Efficacy...

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