| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Anaesthesia and Critical Care, the 2 Cardiac Ultrasound Laboratory in the Cardiology Division and the 3 Cardiovascular Research Center of the Department of Medicine, Massachusetts General Hospital, Boston, MA, USA
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 13 December 2005;
accepted after revision 6 March 2006; first published online 9 March 2006)
Corresponding author M. Scherrer-Crosbie: Cardiac Ultrasound Laboratory, Department of Medicine, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA. Email: marielle{at}crosbie.com
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In vitro, nitric oxide (NO) attenuates cardiomyocyte hypertrophy induced by angiotensin II, adrenergic stimulation and endothelin (Calderone et al. 1998; Ritchie et al. 1998; Heineke et al. 2003). Nitric oxide is synthesized from L-arginine by NO synthases (NOSs) 1, 2 and 3, which may play differing roles in cardiac hypertrophy. Nitric oxide synthase 1 is constitutively expressed in neuronal cells and has recently been identified in cardiomyocytes (Xu et al. 1999; Barouch et al. 2002). Nitric oxide synthase 2, or inducible NOS, was first identified in macrophages but has since been detected in a wide variety of cells (including cardiac myocytes), typically after exposure to endotoxin and/or cytokines. Nitric oxide synthase 2 activity is predominantly regulated at the levels of transcription and protein stability. Nitric oxide synthase 3 is constitutively expressed in the endothelial cells, endocardial cells and cardiac myocytes. The enzyme is stimulated by increased intracellular calcium concentrations via activation and binding of calmodulin and by post-translational modifications which control its activity and intracellular localization (Moncada & Higgs, 1993; Dimmeler et al. 1999).
Nitric oxide synthase 3 limits LV hypertrophy and dysfunction occurring after myocardial infarction (Scherrer-Crosbie et al. 2001). Its role after chronic pressure overload is controversial (Ichinose et al. 2004; Ruetten et al. 2005; Takimoto et al. 2005). The importance of NOS2 in the regulation of LV hypertrophy remains uncertain. Increased NOS2 levels have been demonstrated in hypertrophied cardiomyocytes after aortic banding (Brookes et al. 2001; Dai et al. 2001). This increase was accompanied with a bioenergetic defect of the hypertrophied heart which was reversed by NOS2 inhibition, suggesting that NOS2 plays a role in cardiac abnormalities associated with hypertrophy (Dai et al. 2001). Chronic treatment with a selective NOS2 inhibitor decreases cellular hypertrophy in rats after myocardial infarction (Saito et al. 2002). However, NOS2-deficient (NOS2–/–) mice and wild-type (WT) mice have similar LV mass 1 and 6 months after myocardial infarction (Feng et al. 2001; Sam et al. 2001; Jones et al. 2005). The contribution of NOS2 to myocardial contractile response after haemodynamic stress has also been investigated. Nitric oxide synthase 2-deficient mice have better cardiac function than WT mice both early and late after myocardial infarction (Feng et al. 2001; Sam et al. 2001). However, Jones et al. (2005) recently reported that NOS2 deficiency did not protect mice from severe heart failure after myocardial infarction. Inducible overexpression of cardiac NOS2 is associated with heart failure (Mungrue et al. 2002) whereas constitutive overexpression of NOS2 is not (Heger et al. 2002). Nitric oxide synthase 2-deficient mice are partly protected from endotoxin-induced LV dysfunction (Ullrich et al. 2000). Thus, the role of NOS2 in modulating cardiac systolic function remains controversial.
The purpose of the present study was to evaluate the contribution of NOS2 to changes in LV structure and function induced by prolonged pressure overload. We compared the LV remodelling response to transverse aortic constriction (TAC) in WT and NOS2–/– mice. Although NOS2 was detected after aortic banding, there were no differences in LV volume, wall thickness, load-dependent and -independent indices of contractility, cardiomyocyte hypertrophy and cardiac fibrosis between WT and NOS2–/– mice.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
A total of 20 male C57BL/6 WT and 21 male NOS2–/– mice (3 months old, mean body weights of 27 ± 1 and 29 ± 1 g, respectively) were used for the study. Serial echocardiograms were performed; 18 WT and 20 NOS2–/– mice survived 28 days after banding and completed the study. A subgroup of eight WT and eight NOS2–/– mice were followed for 42 days after banding and underwent an echocardiogram at that time point. The NOS2–/– mice were backcrossed 10 generations onto a C57BL/6 background (Jackson Laboratories, Bar Harbour, ME, USA). The protocol received institutional approval by the Massachusetts General Hospital Subcommittee on Research Animal Care and was performed 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).
Surgical procedure
Transverse aortic constriction was performed as previously described (Rockman et al. 1994). Briefly, mice were anaesthetized using xylazine (30 mg kg–1 administered intraperitoneally) and ketamine (100 mg kg–1 administered intraperitoneally), orally intubated and mechanically ventilated (Scherrer-Crosbie et al. 1999). The chest was opened, and TAC was performed between the left common carotid artery and the brachiocephalic trunk by tying a 7–0 (0.05 mm) silk suture against a 27 gauge needle. The needle was removed after banding the aorta, the chest was closed, and the mouse was allowed to recover from anaesthesia.
Echocardiography
After light sedation (ketamine, 80 mg kg–1 intraperitoneally), transthoracic echocardiography was performed using a linear array 15 MHz transducer with a frame rate of 166 MHz and a depth of 1 cm (Sequoia, Acuson, Siemens, Mountain View, CA, USA). Two-dimensionally guided M-mode images were obtained at the level of the papillary muscles. Echocardiographic measurements of end-systolic and end-diastolic LV internal dimensions (LVIS and LVID) and anterior wall and posterior wall thickness (AWT and PWT) were obtained at baseline and 7, 14 and 28 days after the surgical procedure. In eight WT and eight NOS2–/– mice, echocardiography was obtained 42 days after banding. Fractional shortening (FS) and LV mass were calculated as previously described (Collins et al. 2001).
Haemodynamic measurements
Haemodynamic measurements were obtained in 11 WT and nine NOS2–/– mice 28 days after TAC. Mice were anaesthetized (ketamine, 50 mg kg–1 and fentanyl, 250 µg kg–1 intraperitoneally), and a fluid-filled catheter was introduced into the left carotid artery (distal to the constriction). A 1.4 (0.42 mm) French Millar pressure catheter (SPF671, Millar Instruments, Houston, TX, USA) was advanced through the right carotid artery. After the pressure gradient between both arteries was measured, the Millar catheter was advanced into the LV. Left ventricular end-systolic and end-diastolic pressures, maximum rate of developed LV pressure (dP/dtmax) and maximum rate of LV pressure decay (dP/dtmin), as well as time constant of isovolumic relaxation (
), were obtained.
In seven WT and six NOS2–/– mice studied 42 days after TAC, a 1.4 (0.42 mm) French conductance catheter (SPR 839, Millar Instruments) was used for determination of a load-independent index of contractility. Reductions in end-systolic internal diameter and systolic pressure were produced by transient mechanical occlusion of the inferior vena cava through a small laparotomy, and pressure–volume loops were obtained. The maximal slope of the pressure–volume relationship (Emax) was calculated (PVAN software, Millar Instruments).
Pathology
After the haemodynamic measurements were obtained, the animals were killed by injection of pentobarbital (100 mg kg–1 intravenously). The LV was weighed, and the basal half and apical half separated. The basal half of the LV was embedded in gel (Cryomatrix, Thermoshandon, Pittsburgh, PA, USA) and frozen. Five-micrometer-thick sections were obtained at midventricular level. Myocyte width was measured 28 days after TAC in sections treated with Gomorri reticulin stain and Haematoxylin. Twenty measurements were obtained in each animal at the level of the nucleus in longitudinally sectioned myocytes, at a magnification of x200. Myocardial interstitial fibrosis was visually assessed using Sirius Red staining.
Ventricular NOS2 mRNA
RNA was isolated from ventricular tissues using the Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and cDNA was generated with MMLV reverse transcriptase (Promega, Madison, WI, USA) and random primers (Promega). Quantitative PCR was performed with the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using primers for murine NOS2 ( 5'-TCTTTGACGCTCGGAACTGTAG-3', 5'-TGATGGCCGACCTGATGTT-3') and SYBR Green PCR Master Mix (Applied Biosystems). Ribosomal RNA 18S was detected with 18S VIC MGB primers (Applied Biosystems) and Taqman Universal PCR Master Mix (Applied Biosystems). Changes in NOS2 gene expression normalized to 18S ribosomal RNA levels were determined using the relative quantification method.
Statistical analysis
All data are expressed as means ±S.E.M. Data were analysed using ANOVA for repeated measures with mixed effects models with the JMP statistical software package (Cary, NC, USA). Contrast analysis was used when the ANOVA was significant. P < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Nitric oxide synthase 2 mRNA was detected using quantitative RT-PCR in the LV of WT mice before and 7 and 28 days after banding. However, pressure-overload did not alter ventricular NOS2 gene expression.
Effect of TAC on LV structure and function
At baseline, there was no difference between WT and NOS2–/– mice in heart rate or in the echocardiographic parameters of LV size and systolic function (Table 1). In both genotypes, LV internal diameter was unchanged 7 and 14 days after TAC, but LV dilatation was noted 28 days after banding. After TAC, LV wall thickness and overall mass increased to a similar degree in NOS2–/– and WT mice. Fractional shortening was similar for both genotypes before aortic constriction, and it decreased in both genotypes 7 days after the procedure. Fractional shortening returned to normal values in WT mice at 14 and 28 days after banding but remained decreased in NOS2–/– mice. The overall changes of fractional shortening over time by ANOVA were not statistically different between the two genotypes. In a subgroup of mice studied until 42 days after TAC, there was no difference in echocardiographic parameters (Fig. 1).
|
|
At 28 and 42 days post-TAC, the gradients across the aortic constriction were similar in both genotypes (Table 2). At these time points, heart rate, mean aortic pressure, and end-systolic and end-diastolic LV pressures were similar for both genotypes. There were no differences between WT and NOS2–/– mice in dP/dtmax 28 and 42 days after TAC or in maximal elastance measured 42 days after TAC (119 ± 21 mmHg a.u.–1 in WT versus 106 ± 21 mmHg a.u.–1 in NOS2–/– mice). Diastolic function, as evaluated by dP/dtmin and at both 28 and 42 days after TAC did not differ between genotypes.
|
There was no difference in the ratio of LV weight to body weight of WT and NOS2–/– mice at 28 days after TAC (4.2 ± 0.5 mg g–1 in WT versus 4.2 ± 0.5 mg g–1 in NOS2–/– mice) or at 42 days after TAC (4.5 ± 0.3 mg g–1 in WT versus 4.7 ± 0.3 mg g–1 in NOS2–/– mice). Twenty-eight days after TAC, there was no difference in the cardiomyocyte width between strains (20 ± 1 µm in both WT and NOS2–/– mice).
Left ventricular fibrosis
There was no visual difference in the amount of perivascular and interstitial fibrosis between WT and NOS2–/– mice 28 days after TAC (data not shown).
Survival
A total of four mice (2 WT and 2 NOS2–/–) died after TAC. Three mice died during the first week but after 24 h of TAC. One NOS2–/– mouse died 28 days after TAC, before the haemodynamic measurements.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
A role of NOS2 in the development of cardiomyocyte hypertrophy was suggested by the increase in protein expression and activity after TAC found in rat cardiomyocytes (Dai et al. 2001). In the present study, NOS2 gene expression was detected in ventricular tissue after TAC. No difference was found in ventricular NOS2 mRNA levels in WT mice before and after TAC; however, this finding does not preclude the possibility that TAC alters cardiac NOS2 activity via a change in protein stability and/or post-translational modification or that the expression of NOS2 changes at later time points after TAC.
No effect of NOS2 was found on the degree of ventricular hypertrophy developed in mice in response to pressure overload. To our knowledge, no other study assessing the effect of NOS2 on LV hypertrophy has been performed using a pressure-overload model in vivo. Several studies, however, have examined the effect of NOS2 on remodelling and hypertrophy after myocardial infarction. No difference in LV hypertrophy was found between NOS2–/– and WT mice within weeks (Feng et al. 2001; Sam et al. 2001; Jones et al. 2005) or 6 months (Sam et al. 2001) after a myocardial infarction. Interestingly, Saito et al. (2002) reported that a selective NOS2 inhibitor decreased cardiac hypertrophy 2 months after rats were subjected to large myocardial infarctions accompanied by overt heart failure. It is possible that the beneficial effect of NOS2 inhibition on LV hypertrophy may be attributable to the ability of the inhibitor to improve cardiac function in failing hearts.
In the present study, there was no significant LV dysfunction in WT animals after TAC, which precluded the detection of a beneficial effect of NOS2 on LV function. Studying isolated, perfused hearts, Dai et al. (2001) reported that the LV pressure developed in response to acute pacing was decreased in hypertrophied rat hearts compared to hearts without hypertrophy, and that this decrease was reversed by specific NOS2 inhibitors. It is possible that a similar effect, with protection of the LV function in the NOS2–/– mice, would have been detected if the hearts had been stressed by pacing or exercise after TAC.
A limitation of the present study is that no sham group was used. However, the cardiac phenotype of the NOS2–/– mice after a sham operation (both LV function and morphometric measurements) has been previously studied and was similar to that of the WT up to 4 months after the sham operation (Sam et al. 2001).
We did not study whether NOS1 or NOS3 are changed in NOS2–/– mice after TAC and compensate for a beneficial or deleterious effect of NOS2. Nitric oxide synthase 1 has been recently reported to limit ventricular remodelling after myocardial infarction (Dawson et al. 2005; Saraiva et al. 2005) but its role has not been studied in ventricular function post-TAC. Similarly, NOS3 has been shown to have a role in ventricular function and remodelling post-TAC, although its effects are controversial and may depend on the degree and technique of TAC (Ichinose et al. 2004; Ruetten et al. 2005; Takimoto et al. 2005). A recent paper (Sun et al. 2005) reported that NOS3 expression tends to increase to a greater extent in the heart of NOS2–/– mice subjected to uninephrectomy and salt-induced hypertension than in similarly treated WT mice.
In conclusion, NOS2 does not appear to have an important role in modulating the development of LV hypertrophy after chronic pressure overload. Studies assessing the impact of additional stresses on the function of hypertrophied or failing hearts may be required to identify a role for NOS2 in LV remodelling after aortic constriction.
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Brookes PS, Zhang J, Dai L, Zhou F, Parks DA et al. (2001). Increased sensitivity of mitochondrial respiration to inhibition by nitric oxide in cardiac hypertrophy. J Mol Cell Cardiol 33, 69–82.[CrossRef][Medline]
Calderone A, Thaik CM, Takahashi N, Chang DL & Colucci WS (1998). Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts. J Clin Invest 101, 812–818.[Medline]
Collins KA, Korcarz CE, Shroff SG, Bednarz JE, Fentzke RC, Lin H et al. (2001). Accuracy of echocardiographic estimates of left ventricular mass in mice. Am J Physiol 280, H1954–H1962.
Dai L, Brookes PS, Darley-Usmar VM & Anderson PG (2001). Bioenergetics in cardiac hypertrophy: mitochondrial respiration as a pathological target of NO*. Am J Physiol 281, H2261–H2269.
Dawson
D, Lygate
CA, Zhang
MH, Hulbert
K, Neubauer
S
&
Casadei
B (2005). nNOS gene deletion exacerbates pathological left ventricular remodeling and functional deterioration after myocardial infarction. Circulation
112, 3729–3737.
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R & Zeiher AM (1999). Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601–605.[CrossRef][Medline]
Feng
Q, Lu
X, Jones
DL, Shen
J
&
Arnold
JM (2001). Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice. Circulation
104, 700–704.
Heger
J, Godecke
A, Flogel
U, Merx
MW, Molojavyi
A
et al. (2002). Cardiac-specific overexpression of inducible nitric oxide synthase does not result in severe cardiac dysfunction. Circ Res
90, 93–99.
Heineke
J, Kempf
T, Kraft
T, Hilfiker
A, Morawietz
H, Scheubel
RJ
et al. (2003). Downregulation of cytoskeletal muscle LIM protein by nitric oxide: impact on cardiac myocyte hypertrophy. Circulation
107, 1424–1432.
Ichinose F, Bloch KD, Wu JC, Hataishi R, Aretz HT et al. (2004). Pressure overload-induced LV hypertrophy and dysfunction in mice are exacerbated by congenital NOS3 deficiency. Am J Physiol 286, H1070–H1075.
Jones SP, Greer JJ, Ware PD, Yang J, Walsh K & Lefer DJ (2005). Deficiency of iNOS does not attenuate severe congestive heart failure in mice. Am J Physiol 288, H365–H370.
Moncada
S
&
Higgs
A (1993). The L-arginine-nitric oxide pathway. New Engl J Med
329, 2002–2012.
Mungrue IN, Gros R, You X, Pirani A, Azad A, Csont T et al. (2002). Cardiomyocyte overexpression of iNOS in mice results in peroxynitrite generation, heart block, and sudden death. J Clin Invest 109, 735–743.[CrossRef][Medline]
Ritchie RH, Schiebinger RJ, LaPointe MC & Marsh JD (1998). Angiotensin II-induced hypertrophy of adult rat cardiomyocytes is blocked by nitric oxide. Am J Physiol 275, H1370–H1374.
Rockman
HA, Ono
S, Ross
RS, Jones
LR, Karimi
M, Bhargava
V
et al. (1994). Molecular and physiological alterations in murine ventricular dysfunction. Proc Natl Acad Sci U S A
91, 2694–2698.
Ruetten
H, Dimmeler
S, Gehring
D, Ihling
C
&
Zeiher
AM (2005). Concentric left ventricular remodeling in endothelial nitric oxide synthase knockout mice by chronic pressure overload. Cardiovasc Res
66, 444–453.
Saito T, Hu F, Tayara L, Fahas L, Shennib H & Giaid A (2002). Inhibition of NOS II prevents cardiac dysfunction in myocardial infarction and congestive heart failure. Am J Physiol 283, H339–H345.
Sam
F, Sawyer
DB, Xie
Z, Chang
DLF, Ngoy
S, Brenner
DA
et al. (2001). Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction. Circ Res
89, 351–356.
Saraiva
RM, Minhas
KM, Raju
SV, Barouch
LA, Pitz
E, Schuleri
KH
et al. (2005). Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium. Circulation
112, 3415–3422.
Scherrer-Crosbie M, Steudel W, Ullrich R, Hunziker PR, Liel-Cohen N, Newell J et al. (1999). Echocardiographic determination of risk area size in a murine model of myocardial ischemia. Am J Physiol 277, H986–H992.
Scherrer-Crosbie
M, Ullrich
R, Bloch
KD, Nakajima
H, Aretz
HT, Lindsey
ML
et al. (2001). Nitric oxide synthase 3 limits left ventricular remodeling after myocardial infarction in mice. Circulation
104, 1286–1291.
Sun
Y, Carretero
OA, Xu
J, Rhaleb
NE, Wang
F, Lin
C
et al. (2005). Lack of inducible NO synthase reduces oxidative stress and enhances cardiac response to isoproterenol in mice with deoxycorticosterone acetate-salt hypertension. Hypertension
46, 1355–1361.
Takimoto E, Champion HC, Li M, Ren S, Rodriguez ER, Tavazzi B et al. (2005). Oxidant stress from nitric oxide synthase-3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load. J Clin Invest 115, 1221–1231.[CrossRef][Medline]
Ullrich
R, Scherrer-Crosbie
M, Bloch
KD, Ichinose
F, Nakajima
H, Picard
MH
et al. (2000). Congenital deficiency of nitric oxide synthase 2 protects against endotoxin-induced myocardial dysfunction in mice. Circulation
102, 1440–1446.
Xu
KY, Huso
DL, Dawson
TM, Bredt
DS
&
Becker
LC (1999). Nitric oxide synthase in cardiac sarcoplasmic reticulum. Proc Natl Acad Sci U S A
96, 657–662.
|
Acknowledgements |
|---|
This article has been cited by other articles:
|
|
 |
|
  X. Loyer, T. Damy, Z. Chvojkova, E. Robidel, F. Marotte, P. Oliviero, C. Heymes, and J.-L. Samuel 17{beta}-Estradiol Regulates Constitutive Nitric Oxide Synthase Expression Differentially in the Myocardium in Response to Pressure Overload Endocrinology, October 1, 2007; 148(10): 4579 - 4584. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Thibault, L. Gomez, E. Donal, G. Pontier, M. Scherrer-Crosbie, M. Ovize, and G. Derumeaux Acute myocardial infarction in mice: assessment of transmurality by strain rate imaging Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H496 - H502. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
