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Am J Physiol Heart Circ Physiol 293: H620-H627, 2007. First published April 6, 2007; doi:10.1152/ajpheart.01236.2006
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Cardiomyocyte-restricted restoration of nitric oxide synthase 3 attenuates left ventricular remodeling after chronic pressure overload

Emmanuel S. Buys,1,* Michael J. Raher,1,* Sarah L. Blake,1,3 Tomas G. Neilan,1,2 Amanda R. Graveline,1 Jonathan J. Passeri,2 Miguel Llano,2 Teresa M. Perez-Sanz,2 Fumito Ichinose,1,3 Stefan Janssens,4 Warren M. Zapol,3 Michael H. Picard,2 Kenneth D. Bloch,1,3,* and Marielle Scherrer-Crosbie1,2,*

1Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown and 2Cardiac Ultrasound Laboratory, Cardiology Division, Department of Medicine and 3Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; and 4Cardiology Division and Center for Transgene Technology and Gene Therapy, University of Leuven, Campus Gasthuisberg, Leuven, Belgium

Submitted 10 November 2006 ; accepted in final form 30 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although nitric oxide synthase (NOS)3 is implicated as an important modulator of left ventricular (LV) remodeling, its role in the cardiac response to chronic pressure overload is controversial. We examined whether selective restoration of NOS3 to the hearts of NOS3-deficient mice would modulate the LV remodeling response to transverse aortic constriction (TAC). LV structure and function were compared at baseline and after TAC in NOS3-deficient (NOS3–/–) mice and NOS3–/– mice carrying a transgene directing NOS3 expression specifically in cardiomyocytes (NOS3–/–TG mice). At baseline, echocardiographic assessment of LV dimensions and function, invasive hemodynamic measurements, LV mass, and myocyte width did not differ between the two genotypes. Four weeks after TAC, echocardiographic and hemodynamic indexes of LV systolic function indicated that contractile performance was better preserved in NOS3–/–TG mice than in NOS3–/– mice. Echocardiographic LV wall thickness and cardiomyocyte width were greater in NOS3–/– mice than in NOS3–/–TG mice. TAC-induced cardiac fibrosis did not differ between these genotypes. TAC increased cardiac superoxide generation in NOS3–/–TG but not NOS3–/– mice. The ratio of NOS3 dimers to monomers did not differ before and after TAC in NOS3–/–TG mice. Restoration of NOS3 to the heart of NOS3-deficient mice attenuates LV hypertrophy and dysfunction after TAC, suggesting that NOS3 protects against the adverse LV remodeling induced by prolonged pressure overload.

echocardiography; heart failure; hypertrophy; mice

CHRONIC LEFT VENTRICULAR (LV) pressure overload, such as that due to systemic hypertension, is a major contributor to congestive heart failure (16). The ventricular response to chronic pressure overload involves concentric LV hypertrophy (5), which may progress to LV dilation and subsequent heart failure (21). Changes in LV mass, size, and geometry, which define LV remodeling, are major prognostic factors in patients with essential hypertension (15, 27).

Nitric oxide (NO) influences many of the processes involved in the pathophysiology of LV remodeling. In addition to acting as a potent vasodilator that can reduce cardiac load, NO stimulates angiogenesis, reduces cardiomyocyte hypertrophy, and limits production of extracellular matrix proteins by cardiac fibroblasts (14, 17, 22). NO is produced from the conversion of L-arginine to L-citrulline by three nitric oxide synthase (NOS) isoforms, NOS1 (neuronal NOS or nNOS), NOS2 (inducible NOS or iNOS), and NOS3 (endothelial NOS or eNOS) (20). NOS3 is constitutively expressed both in the vascular endothelium and in cardiomyocytes (2). NOS3 is synthesized as monomers and dimerizes in the presence of heme (8). Dimerization is necessary for the production of NO by NOS3. In the setting of increased hemodynamic or metabolic stress, NOS3 may generate superoxide rather than NO, a situation known as uncoupling (for a review, see Ref. 8). Failure of NOS3 to form dimers or monomerization of existing dimers has been implicated in the production of reactive oxidant species (ROS) in the heart (26).

Several studies suggest that NOS3-derived NO limits the development and progression of LV remodeling and failure after myocardial infarction (MI) (12, 13, 24). Scherrer-Crosbie et al. (24) reported that LV remodeling after occlusion of the left anterior descending artery was exacerbated in NOS3-deficient (NOS3–/–) mice compared with wild-type (WT) mice. Jones and colleagues (13) demonstrated that overexpression of NOS3 within the systemic and pulmonary vascular endothelium of mice prevented cardiac and pulmonary dysfunction in a murine model of heart failure due to MI. Janssens and colleagues (12) further demonstrated that cardiomyocyte-restricted overexpression of NOS3 (in NOS3TG mice) limited the degree of LV dysfunction and ventricular remodeling after MI.

Whether NOS3 has a beneficial role in pressure overload-induced ventricular remodeling is controversial. Ichinose et al. (10) and Ruetten et al. (23) observed that constriction of the thoracic and abdominal aorta, respectively, caused greater LV dysfunction in NOS3–/– mice than in WT mice. In contrast, Takimoto and colleagues (26) reported that NOS3–/– mice have preserved LV morphology and function after transverse aortic constriction (TAC) compared with WT mice and that the deleterious effect of NOS3 was due to its uncoupling after TAC, as reflected in part by an increase in cardiac superoxide production and a decrease in the ratio of NOS3 dimers to monomers.

The aim of the present study was to evaluate the effect of restoration of cardiac NOS3 on the LV remodeling response to TAC in NOS3–/– mice. We compared LV structure and function before and after TAC in NOS3–/– mice and NOS3–/– mice carrying a transgene that specifies cardiomyocyte-restricted expression of NOS3 (NOS3–/–TG mice). We report that restoration of NOS3 in the cardiomyocytes of NOS3–/– mice attenuated LV hypertrophy and dysfunction induced by TAC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. All animal procedures were conducted in accordance with guidelines published in the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington, DC, 1996) and were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Transgenic mice with cardiomyocyte-restricted overexpression of human NOS3 (NOS3TG mice) were generated as described previously (12). Cardiac NOS enzyme activity is 30-fold greater in NOS3TG mice than in WT mice (12). NOS3–/– and WT mice were obtained from Jackson Laboratory (Bar Harbor, ME). Both NOS3–/– and NOS3TG mice were backcrossed 10 generations onto a C57BL/6 background. NOS3–/– and NOS3TG mice were mated, and resulting NOS3+/–, NOS3TG mice were crossed with NOS3–/– mice to obtain NOS3–/– mice with cardiomyocyte-restricted NOS3 overexpression (NOS3–/–TG). Eight- to ten-week-old male C57BL/6 (WT), NOS3–/–, and NOS3–/–TG mice were studied.

TAC. TAC was performed as previously described (10). Briefly, mice were anesthetized with intraperitoneal administration of 100 mg/kg ketamine and 5 mg/kg xylazine and subsequently ventilated. TAC was performed by ligation (7-0 prolene) of the aorta between the innominate and left common carotid arteries with an overlying 27-gauge needle, followed by removal of the needle.

Echocardiographic analysis. Murine transthoracic echocardiography was performed with a 13-MHz probe (Vivid 7, GE Medical Systems, Milwaukee, WI) in lightly sedated mice (50 mg/kg ketamine ip) at baseline (control) and 14 and 28 days after surgery, as previously described (10, 24). Measurements were made by an observer who was blinded to the experimental group. Heart rate (HR), LV end-diastolic internal diameter (LVIDED), LV end-systolic internal diameter (LVIDES), posterior end-diastolic wall thickness (PWT), and fractional shortening of the LV (FS) were measured.

In vivo hemodynamics. NOS3–/– and NOS3–/–TG unoperated (control) mice (3 mo of age) and mice 28 days after TAC were anesthetized (250 µg/kg fentanyl and 50 mg/kg ketamine ip) and mechanically ventilated. HR, LV end-systolic pressure (LVESP), LV end-diastolic pressure (LVEDP), maximum first derivative of the developed LV pressure (dP/dtmax), and minimum first derivative of the developed LV pressure (dP/dtmin) were obtained with a 1.4-Fr high-fidelity SPR-671 Millar pressure catheter (Millar Instruments, Houston, TX) advanced into the LV, as described previously (10, 24). The time constant of isovolumic relaxation ({tau}) was calculated according to the method of Weiss. A left carotid arterial catheter was placed to measure systolic arterial pressure (SAPL) and to calculate the transstenotic pressure gradient (TSPG).

Tissue analysis. Mice were euthanized after invasive hemodynamic measurements were obtained, and the LV was blotted, weighed, and then fixed and embedded in paraffin. Sections were stained with reticulin to determine myocyte width. Twenty measurements were obtained at the level of the nucleus in longitudinally sectioned myocytes in each section (viewed at a magnification of x200). To assess the degree of ventricular fibrosis, sections were stained with Sirius red. The ratio of collagen deposition (indicated by red staining) to total myocardial area was outlined and quantified by an automated analysis program (IP Lab Spectrum; Signal Analytics, Vienna, VA). To visualize capillaries, LV sections were incubated with biotinylated Griffonia simplicifolia lectin I and stained with the Vectastain ABC immunoperoxidase system (Vector Laboratories, Burlingame, CA). The number of capillaries per square millimeter was counted. Eight 300 µm x 200 µm fields were analyzed for each mouse at a magnification of x250.

Determination of NOS3 dimer-to-monomer ratio. NOS3 dimerization was measured by low-temperature PAGE as previously described (7). Briefly, the supernatants of LV homogenates [extracted in 150 mM NaCl, 20 mM Tris pH 7.6, 1 mM CaCl2, 1 mM MgCl2, 1% NP-40, 10% glycerol, and 1% protease inhibitor cocktail (Sigma)] were cleared with protein G-agarose beads (Roche). The supernatants were mixed with a 4x sample buffer (0.25 M Tris pH 7.5, 8% SDS, 4% beta-mercaptoethanol, 40% glycerol, 0.2% bromophenol blue) and loaded, with or without boiling, onto 6% Tris-glycine gels (Invitrogen, Carlsbad, CA) for SDS-PAGE. In the boiled supernatants, all NOS3 is monomerized. Proteins were electroblotted onto Hybond-P membrane (Amersham UK) by semidry transfer. Membranes were blocked in 5% nonfat milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST milk) and incubated with an anti-NOS3 antibody (BDTransduction, Bedford, MA; 1:2,500) in TBST milk. Bound antibodies were detected with horseradish peroxidase (HRP)-labeled anti-mouse antibody (Cell Signaling, Charlottesville, VA; 1:1,000) in TBST milk and visualized with chemiluminescence with ECL Plus (Amersham Biosciences, Piscataway, NJ). To confirm that equal amounts of protein were loaded onto gels, membranes were subsequently incubated with anti-tubulin antibody (BDTransduction; 1:1,000) in TBST milk, and bound antibody was detected as above. Densitometric analysis was performed with the ImageJ software package [National Institutes of Health (NIH)].

Measurement of gene expression. Total RNA was extracted from the LV with TRIzol reagent (Invitrogen), and cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (Invitrogen, La Jolla, CA). NOS1, NOS2, and 18S ribosomal RNA (rRNA) transcript levels were measured by real-time PCR with an ABI Prism 7000 (Applied Biosystems, Foster City, CA) and primers for NOS1 (5'-CAAAGACCAGCCATTAGCAGT-3', 5'-CCACACCATTAGCCTGGGA-3'), NOS2 (TaqMan, Applied Biosystems), and 18s rRNA (5'-CGGCTACCACATCCAAGGAA-3', 5'-GCTGGAATTACCGCGGCT-3'). Changes in gene expression normalized to levels of 18S rRNA were determined with the relative threshold cycle method (Applied Biosystems).

Detection of reactive superoxide. Superoxide production was measured by lucigenin-enhanced chemiluminescence in freshly harvested cardiac tissue from NOS3–/– and NOS3–/–TG mice before and 7 and 28 days after TAC. Cardiac tissue (2 x 2-mm tissue blocks of the LV) was preincubated in Krebs-HEPES buffer containing 10 µM NADPH for 45 min. Tissues were then transferred into wells of a 96-well plate containing 300 µl of Krebs-HEPES buffer supplemented with 10 µM NADPH and 10 µM lucigenin [9,9'-bis-(N-methylacridinium nitrate)] (18). Chemiluminescence was recorded by a multilabel counter (Victor3, Perkin Elmer) reporting relative light units (RLU) emitted over 60 s. Addition of superoxide dismutase (200 U/ml) to the reaction buffer abolished the chemiluminescence signal, confirming the specificity of the assay (data not shown). Background chemiluminescence (RLU count of lucigenin-containing buffer without tissue) was subtracted from the RLU count with tissue. Superoxide production is expressed as counts per minute per milligram of dry tissue.

Measurement of phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPase protein levels and phospholamban phosphorylation. Protein extracts from LV tissue homogenates were microcentrifuged for 20 min at 20,000 g. Supernatant proteins (15 µg) were fractionated on 4–20% gradient gels and transferred to nitrocellulose membranes. Membranes were blocked for 1 h in TBST milk and incubated overnight with primary antibodies against sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) (diluted 1:500 in TBST; Affinity Bioreagents), total phospholamban (PLB) (1 µg/ml; Affinity Bioreagents), PLB phosphorylated at Ser16 (diluted 1:500 in TBST; Upstate Group), or PLB phosphorylated at Thr17 (diluted 1:5,000 in TBST; Badrilla). Bound antibody was detected with a HRP-linked antibody directed against rabbit IgG (diluted 1:2,500; Cell Signaling Technology) or mouse IgG (diluted 1:2,500; Promega) and was visualized with chemiluminescence with ECL Plus (Amersham Biosciences). Densitometric analysis was performed with the ImageJ software package (NIH).

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was done with the JMP statistical package (SAS Institute, Cary, NC). Analysis of variance (ANOVA) was used to compare hemodynamic, pathological, and molecular parameters between genotypes in mice studied before and after TAC. If the ANOVA was significant, unpaired Student's t-tests were used. For comparison of echocardiographic parameters over time, results were analyzed with an ANOVA for repeated measurements. If the interaction of time and genotype was significant, post hoc comparisons were performed with Dunnett's test. Unpaired Student's t-tests were used to compare echocardiographic parameters between genotypes at the same time point. A probability value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Echocardiographic measurements at baseline and after TAC. WT (n = 10), NOS3–/– (n = 11), and NOS3–/–TG (n = 7) mice were studied before and after TAC with serial echocardiography. Mice were matched for age and weight. Comparison of the LV remodeling response to TAC in WT and NOS3–/– mice (Table 1) confirmed our previous results that NOS3 deficiency exacerbates TAC-induced LV remodeling, dysfunction, and hypertrophy (10). LV systolic function, dimensions, and PWT were similar in NOS3–/– and NOS3–/–TG mice before TAC (Table 1). In NOS3–/– mice, TAC induced progressive LV hypertrophy, a trend toward LV dilation, and a decrease in FS (Table 1). In NOS3–/–TG mice, TAC induced concentric hypertrophy without LV dilation or a decline in FS. Twenty-eight days after TAC, both LV hypertrophy and the reduction of FS were less marked in NOS3–/–TG than NOS3–/– mice (Table 1). TAC-induced changes in LV systolic function and dimensions were similar in WT and NOS3–/–TG mice (Table 1). Representative M-mode echocardiograms of NOS3–/– and NOS3–/–TG mice obtained before and 28 days after TAC are shown in Fig. 1.


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  		Table 1. Echocardiographic analysis

 

Figure 1
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  		Fig. 1. Representative M-mode echocardiograms of the parasternal short-axis view at the level of the midpapillary muscle obtained from a nitric oxide synthase (NOS)3-deficient (NOS3–/–; left) mouse and a NOS3–/– mouse with transgene-directed cardiomyocyte-restricted overexpression of human NOS3 (NOS3–/–TG; right) before (top) and 28 days after (bottom) transverse aortic constriction (TAC). Twenty-eight days after TAC, NOS3–/– mice have increased posterior end-diastolic wall thickness (PWT) and reduced fractional shortening compared with NOS3–/–TG mice. IVS, interventricular septum; LVIDED, left ventricular (LV) end-diastolic internal diameter; LVIDES, LV end-systolic internal diameter.

 
Invasive hemodynamic measurements in control mice and mice after TAC. Hemodynamic parameters did not differ between control NOS3–/– and NOS3–/–TG mice (Table 2). In particular, SAPL was similar between genotypes (Table 2). Twenty-eight days after TAC, systolic function, as reflected by dP/dtmax, and diastolic function, as assessed by dP/dtmin, were decreased in both genotypes. In NOS3–/– mice, impaired diastolic function was accompanied by an increase in LVEDP and a prolongation of {tau}, the time constant of isovolumic relaxation. LVESP did not differ in unoperated control NOS3–/– mice and NOS3–/– mice 28 days after TAC, but SAPL was much less in the latter. In contrast, LVESP was greater in NOS3–/–TG mice 28 days after TAC than in unoperated control NOS3–/–TG mice, whereas LVEDP was unchanged. LVESP, SAPL, dP/dtmax, and dP/dtmin were greater in NOS3–/–TG mice than in NOS3–/– mice 28 days after TAC. Lower TSPGs were noted in NOS3–/– mice than in NOS3–/–TG mice at 28 days after TAC.


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  		Table 2. Hemodynamic parameters

 
LV weight, myocyte size, myocardial fibrosis, and capillary density. In control mice, LV weight-to-body weight ratio (LV/BW) and cardiomyocyte width did not differ between the two genotypes (Table 3). Twenty-eight days after TAC, both parameters increased in NOS3–/– and NOS3–/–TG mice. However, LV/BW and cardiomyocyte width were less in NOS3–/–TG mice than in NOS3–/– mice (Table 3). At baseline, only very low levels of fibrosis were observed in the myocardium (<0.5% of total myocardial area). At 28 days after TAC, abundant fibrosis was detected in the hearts of both NOS3–/– and NOS3–/–TG mice (Fig. 2, top). However, no difference in the degree of interstitial fibrosis was noted between NOS3–/– and NOS3–/–TG mice (3.6 ± 0.5% and 3.7 ± 0.4% of total myocardial area, respectively). Twenty-eight days after TAC, the density of capillaries in the myocardium was less in NOS3–/– than in NOS3–/–TG mice (1.5 ± 0.2 vs. 1.8 ± 0.1 x 103 capillaries/mm2; P < 0.05; Fig. 2, bottom).


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  		Table 3. Anatomic findings

 

Figure 2
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  		Fig. 2. Representative sections showing fibrosis (Sirius red staining, top) or capillary density (Griffonia simplicifolia lectin I staining, bottom) in the ventricles of NOS3–/– (left) and NOS3–/–TG (right) mice 28 days after TAC. Twenty-eight days after TAC, myocardial collagen deposition was similar in both NOS3–/– and NOS3–/–TG mice (viewed at a magnification of x100). The capillary density in the myocardium was less in NOS3–/– than in NOS3–/–TG mice 28 days after TAC (viewed at a magnification of x250).

 
NOS3 dimerization. TAC did not decrease the ratio of NOS3 dimers to monomers in NOS3–/–TG mice (Fig. 3). The NOS3 dimer-to-monomer ratio before and 7 days after TAC was 142 ± 10% vs. 163 ± 10%, respectively (n = 3 in each group). We also failed to observe a change in the NOS3 dimer-to-monomer ratio in WT mice subjected to TAC (data not shown).


Figure 3
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  		Fig. 3. Immunoblot analysis of NOS3 in boiled (lane 3) and unboiled LV extracts from NOS3–/–TG mice before TAC (lane 1) and 7 days after TAC (lane 2). To confirm equal loading of protein, expression of tubulin was measured (data not shown). Top band represents NOS3 dimers; bottom band represents NOS3 monomers. The ratio of NOS3 dimers to monomers did not decrease 7 days after TAC.

 
Cardiac NOS1 and NOS2 expression. Ventricular levels of mRNAs encoding NOS1 (Fig. 4, top) or NOS2 (Fig. 4, bottom) were not different between NOS3–/– and NOS3–/–TG mice before or 7 or 28 days after TAC.


Figure 4
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  		Fig. 4. Cardiac NOS1 (top) and NOS2 (bottom) gene expression in NOS3–/– and NOS3–/–TG mice before and 7 and 28 days after TAC. NOS1 and NOS2 mRNA levels were normalized to 18S rRNA levels and are expressed as fold change vs. expression levels in NOS3–/– before TAC. Numbers of animals in experimental groups are indicated above respective bars.

 
Oxidative stress in NOS3–/– and NOS3–/–TG mice. No difference in cardiac superoxide production was detected between genotypes at baseline. Seven days after TAC, a threefold increase in lucigenin-enhanced chemiluminescence in the LV of NOS3–/–TG mice was detected. Similarly, 28 days after TAC, superoxide production tended to be greater in the LV of NOS3–/–TG mice than in the LV of NOS3–/– mice. In contrast, there was no increase in chemiluminescence in NOS3–/– mice at either 7 or 28 days after TAC (Fig. 5).


Figure 5
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  		Fig. 5. Cardiac superoxide production before and 7 and 28 days after TAC in NOS3–/–and NOS3–/–TG mice. Cardiac tissue was incubated in the presence of lucigenin, and superoxide-induced chemiluminescence was measured. Superoxide production is expressed as relative light units (RLU) per milligram of cardiac tissue (dry weight) per minute. *P < 0.01 vs. baseline NOS3–/–TG; #P < 0.01 vs. NOS3–/– 7 days after TAC; {dagger}P = 0.15 vs. NOS3–/– 28 days after TAC. Numbers of animals in groups are indicated above respective bars.

 
Expression and phosphorylation of SERCA and PLB. Expression levels of both SERCA and PLB in cardiac tissue extracts did not differ between genotypes at baseline or after TAC (Fig. 6, A and B, respectively). Twenty-eight days after TAC, LV SERCA protein levels were increased similarly in both NOS3–/– and NOS3–/–TG mice, but PLB levels did not change. Phosphorylation of PLB was less in hearts of NOS3–/–TG mice than in those of NOS3–/– mice, both at Ser16 (before and 28 days after TAC) and Thr17 (before and 7 and 28 days after TAC) (Fig. 6, C and D, respectively). Seven days after TAC, phosphorylation of PLB at Ser16 tended to increase in NOS3–/–TG mice and levels of Ser16 phosphorylation did not differ in NOS3–/–TG and NOS3–/– mice.


Figure 6
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  		Fig. 6. Immunoblot analysis of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA; A), phospholamban (PLB; B), PLB phosphorylated at Ser16 (PLBSer16; C) or PLB phosphorylated at Thr17 (PLBThr17; D) in LV protein extracts from NOS3–/– and NOS3–/–TG mice before and 7 and 28 days after TAC. Results are normalized to NOS3–/– mice before TAC. Numbers of animals in groups are indicated above respective bars. Representative blots from 2 mice in each group are shown. *P < 0.01 vs. baseline same genotype; {dagger}P < 0.01 vs. NOS3–/– before TAC; #P < 0.01 vs. NOS3–/– at same time point; AP = 0.11 vs. NOS3–/–TG before TAC.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that selective restoration of NOS3 in the cardiomyocytes of congenitally NOS3-deficient mice attenuates the LV hypertrophy and dysfunction induced by TAC.

There was no echocardiographic or hemodynamic difference in LV size and function between NOS3–/– and NOS3–/–TG mice before TAC. These findings are consistent with those of Janssens et al. (12), who reported that cardiac-specific overexpression of NOS3 had no effect on either echocardiographic or hemodynamic parameters at baseline. Serial echocardiography revealed that TAC induced marked LV hypertrophy and dysfunction in NOS3–/– mice, as we reported previously (10). Presence of the NOS3 transgene was associated with reduced LV hypertrophy and improved LV systolic function in NOS3–/– mice. Invasive hemodynamic measurements revealed that TAC-impaired systolic function in NOS3–/– mice associated with a marked reduction in dP/dtmax and SAPL, as well as failure to maintain an elevated LVESP in the face of aortic constriction. Although TAC also impaired LV systolic function in NOS3–/–TG mice, dP/dtmax, LVESP, SAPL, and TSPG were all greater in NOS3–/–TG mice than in NOS3–/– mice. Together, these echocardiographic and hemodynamic findings indicate that restoration of cardiac NOS3 expression partially preserved contractile performance in NOS3–/– mice after TAC.

We considered the possibility that NOS3–/–TG mice developed less LV remodeling than NOS3–/– mice because of a reduced afterload in NOS3–/–TG mice. In support of this hypothesis, a decreased afterload was suggested by Jones and colleagues (13) to explain the improved cardiac function and survival after MI in mice with systemic NOS3 overexpression compared with WT mice. In the present study, however, no difference in aortic blood pressure was found between the two genotypes at baseline. This finding suggests that cardiomyocyte-restricted overexpression of NOS3 does not have a significant effect on peripheral vascular tone and suggests that local NO synthesis by cardiomyocyte NOS3 was responsible for attenuating LV remodeling. However, since the transgene we used specifies cardiac NOS3 levels that are much higher than those found in WT mice (12), a pharmacological effect of NO on LV remodeling cannot be excluded.

Although previous in vivo studies suggest that the absence of NOS3 leads to deleterious LV remodeling (10, 24), other investigators have reported differing results. Takimoto and colleagues (26) recently reported that LV hypertrophy and dysfunction induced by TAC were less marked in NOS3–/– mice than in WT mice. These investigators provided evidence that when WT mice were subjected to TAC cardiac NOS3 was fully monomerized, leading to an increase in cardiac ROS production (26). In our study, TAC increased cardiac superoxide generation in NOS3–/–TG mice without inducing monomerization of NOS3. These findings suggest that the presence of the NOS3 transgene attenuated TAC-induced LV hypertrophy and dysfunction in NOS3-deficient mice despite causing an increase in cardiac superoxide production.

The mechanisms by which restoration of NOS3 to the heart inhibits LV remodeling remain unknown. The preservation of LV function and partial attenuation of LV remodeling observed in NOS3–/–TG mice after TAC may be attributable to the chronic inhibition of beta-adrenergic stimulation, similar to that observed in WT mice carrying the NOS3 transgene (12). Inhibition of beta-adrenergic stimulation, in turn, could decrease the progression of LV hypertrophy (1, 25). Although "compensatory" hypertrophy in response to increased afterload initially facilitates LV systolic performance by normalizing wall stress, this stress-response pathway may become maladaptive, leading to ventricular dilation and systolic dysfunction (3, 9). Our finding that NOS3–/–TG mice had attenuated LV hypertrophy after TAC, as reflected by reduced LV mass and myocyte width, raises the possibility that NOS3 may directly modulate detrimental hypertrophic growth of the myocardium, for example, by modulating inflammatory cytokines or matrix metalloproteinases (14, 19). Interestingly, whereas systemic NOS3 deficiency was accompanied by an increased degree of cardiac fibrosis after TAC (10, 23), overexpression of NOS3 activity in cardiomyocytes did not reduce the degree of LV fibrosis in our study. The dissociation between the degree of fibrosis and LV dysfunction suggests that fibrosis does not play a prominent role in the cardiac dysfunction caused by TAC in NOS3–/– mice. Moreover, the presence of fibrosis in NOS3–/–TG mice suggests that NOS3 in cardiomyocytes did not reduce cardiac fibrosis and that the absence of NOS3 in nonmyocyte cells may be responsible for fibrosis after TAC in both NOS3–/– and NOS3–/–TG mice. The finding that capillary density was modestly greater in NOS3–/–TG mice than in NOS3–/– mice 28 days after TAC likely reflects the smaller cardiomyocyte size in the former. It is conceivable that relative myocardial ischemia due to decreased capillary density may contribute to the greater impairment of LV function in NOS3–/– mice than in NOS3–/–TG mice after TAC.

Functional alterations in Ca2+ regulatory proteins present in the sarcoplasmic reticulum (SR) have been implicated in the pathogenesis of heart failure (4). Phosphorylation of one of these proteins, PLB, decreases its ability to inhibit SERCA activity and therefore modulates SR Ca2+ handling parameters. Our observation that phosphorylation of PLB, both at Ser16 (before and 28 days after TAC) and at Thr17 (before and 7 and 28 days after TAC), was less in LV extracts of NOS3–/–TG mice than in those of NOS3–/– mice suggests that altered Ca2+ handling may be involved in attenuating LV dysfunction in NOS3–/–TG mice. In a recent study, we observed (11) that overexpression of NOS3 was associated with decreased phosphorylation of PLB at Ser16 and Thr17 in cardiomyocytes, as well as reduced SR Ca2+ load, SR Ca2+ leak, and Na+/Ca2+ exchanger activity. The tendency to augment PLB phosphorylation at Ser16 in NOS3–/–TG mice (7 days after TAC), but not in NOS3–/– mice, may increase SERCA activity, potentially attenuating the LV remodeling response to TAC (6). However, further investigations are required to ascertain whether or not differences in cardiomyocyte Ca2+ handling can account for the ability of the NOS3 transgene to attenuate LV remodeling in response to TAC.

In summary, restoration of cardiac NOS3 expression in NOS3–/– mice limits LV dysfunction and remodeling after TAC. These data provide evidence for a beneficial role of enhanced NOS3 activity in cardiomyocytes in preventing cardiac dysfunction associated with sustained pressure overload. Clinical strategies designed to augment myocardial NOS3 activity may provide a promising treatment to prevent deleterious LV remodeling.


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 MATERIALS AND METHODS
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 DISCUSSION
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This study was supported by postdoctoral fellowship awards of the American Heart Association Northeast Affiliate (E. S. Buys and T. G. Neilan) and a Scientist Development Grant (M. Scherrer-Crosbie) from the American Heart Association, as well as National Heart, Lung, and Blood Institute Grants HL-42397, HL-70896, and HL-71987. S. Janssens is a basic clinical investigator of the Fund for Scientific Research, Flanders, Belgium.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Scherrer-Crosbie, Cardiac Ultrasound Laboratory, 55 Fruit St., Blake 254, Boston, MA 02115-2696 (e-mail: marielle{at}crosbie.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* E. Buys and M. J. Raher contributed equally to this manuscript; K. D. Bloch and M. Scherrer-Crosbie contributed equally to this manuscript as senior authors. Back


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