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Cardiovascular-Renal Mechanisms in Health and Disease
1Institut National de la Santé et de la Recherche Médicale U689, Centre de Recherche Cardiovasculaire Inserm Lariboisière, Paris, France; 2Université Denis Diderot, Paris; and 3Hôpital Lariboisière, Assistance Publique-Hôpital de Paris, Paris, France
Submitted 27 July 2007 ; accepted in final form 18 September 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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caveolae; hypertrophy; heart failure
NO is produced by NO synthases (NOS); the three isoforms are described as follows: two calcium dependent and constitutively expressed [endothelial NOS (or NOS3) and neuronal NOS (or NOS1)] and one calcium independent and inducible (NOS2). All three NOS isoforms may be expressed within the myocardium according to the pathophysiological status (22). In the failing heart, in both humans and rats, myocardial NOS activity is maintained through a distinct regulation of constitutive NOS that includes an increase in NOS1 expression and activity together with a decrease in NOS3 expression and activity (3, 10). One of the major mechanisms regulating the activity of calcium-dependant NOS is through interactions with their allosteric regulators caveolins, the structural proteins of caveolae. Caveolin-1 interacts with NOS3 in endothelium, whereas caveolin-3 interacts with NOS1 in failing cardiomyocytes (10, 22). In isolated and cultured arteries, NOS1 is rapidly induced by increases in intraluminal pressure (13). To get some insight into the mechanisms involved in the cardiac NO production, we investigated the constitutive NOS activity and the regulatory mechanism according to sex differences and pressure overload-induced cardiac hypertrophy. The present study showed that the respective expression of constitutive NOS and caveolins is differentially regulated according to sex in a rat model of pressure overload. The results not only highlight the sex difference of cardiac adaptation to pressure overload, they emphasize the unique mechanisms of NOS activity regulation, that of NOS3 being dependent on caveolin-1 level and NOS1 activity being related to the level of enzyme expression.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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After anesthesia with intraperitoneal xylazine (50 mg/kg) and ketamine (100 mg/kg), thoracic aortic constriction (TAC) was performed in male (n = 8–10) and female (n = 8–10) 25-day-old Wistar rats (Iffa Credo) by placement of a clip (Atrauclip, Pliling) on the ascending aorta as previously described (1). Additional male (n = 8–10) and female (n = 8–10) age-matched animals underwent sham operation to serve as controls (sham group). Animals were studied by 5 days or 2, 14, or 20 wk after operation. At 2 and 20 wk, echocardiography was performed as described (3).
Echocardiographic, hemodynamic, and anatomic parameters. Transthoracic echocardiography was performed on anesthetized rats with an echocardiograph (Vivid 7, General Electric, Fairfield, CT) equipped with a 10- to 14-MHz linear transducer. Data were transferred online to a computer for blind analysis. The left ventricle (LV) was measured in the parasternal long-axis view in M-mode. LV dimensions were measured during either systole (LVDs) or diastole (LVDd) and used to calculate myocardial performance (% of fractional shortening), calculated as [(LVDd – LVDs)/LVDd] x 100. Each group included 8–10 animals.
Under anesthesia, LV hemodynamic parameters [LV systolic pressure and LV end-diastolic pressure (LVEDP)] were measured before death. Briefly, LV pressure was measured by transparietal route using a needle (n = 8–10 animals per group) and a catheter connected to a pressure transducer. The heart was then removed, dissected, and weighed. Left and right ventricles were separated and weighed, along with the lungs, to determine signs of cardiac hypertrophy and failure. To determine LVH in the animal cohort, we chose to express LVH as the percent increase over LV weight of the respective sham groups (1). Briefly, for each TAC animal, the LVH was calculated according to the formula %LVH = [(TAC LV weight) – (mean LV weight of sham age-matched animals)]/(mean LV weight of sham age-matched animals) x 100, at each time point of the study. The classical heart weight-to-body weight ratio was not considered because of the large variations in body weight over the study in all groups (Table 1), as pointed out by Douglas et al. (12). For the same reason, heart weight normalization to tibia length cannot be used in such long-term studies.
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Protein extractions.
LV fragments (20–30 mg) were lysed with an Ultraturax (Polytron) in Triton X-100 buffer for NOS expression [1% Triton X-100, 50 mM Tris·HCl, pH 7.4, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM 
-glycerophosphate, 0.2 mM sodium orthovanadate, and Complete protease inhibitor cocktail (Roche)] and stored on ice for 1 h. After centrifugation at 12,000 rpm for 10 min at 4°C, supernatants, corresponding to the Triton X-100 soluble fraction, were stored at –20°C. For caveolin analysis (caveolin-1 and caveolin-3), proteins were solubilized in SDS buffer [1% SDS, 10 mM Tris·HCl, pH 7.4, 1 mM orthovanadate-sodium, and Complete protease inhibitor cocktail (Roche)]. Lysates were warmed for 15 s in a microwave (900 W) and centrifuged at 12,000 rpm at 15°C for 5 min. Supernatants obtained were stored at –20°C.
To analyze protein-protein interactions, an N-octylglucoside (OG; Roche) lysis buffer was employed. Briefly, LV fragments were lysed with an Ultraturax (Polytron) in OG buffer [600 mM OG, 10 mM Tris·HCl, pH 8, 150 mM NaCl, 10 mM NaF, 1 mM orthovanadate, Complete protease inhibitor cocktail (Roche)]. After centrifugation (12,000 rpm, 5 min, 15°C), supernatants were collected. Protein concentrations were measured by bicinchoninic acid protein assay (Pierce) by comparison with a BSA standard curve.
Western blotting. Proteins samples (n = 8–10 per group) were separated by electrophoresis on 12% and 7.5% SDS-PAGE gels (for caveolin-1 and -3 and for NOS3 and NOS1, respectively) and transferred to nitrocellulose (Schleicher & Schuell) membranes in 25 mM Tris, 192 mM glycine, 0.01% SDS, and 15% ethanol. The equal loading was verified by Ponceau S red (Sigma) staining (data not shown) and by probing the immunoblot samples with MAb against GAPDH (1:5,000; Chemicon). The membranes were blocked with a Tris-buffered saline (pH 7.4) with 0.1% Tween 20 (Sigma) and 5% nonfat dry milk for 1 h at room temperature before incubation with each antibody: anti-caveolin MAb (1:8,000; Transduction Lab), anti-caveolin-3 MAb (1:2,000; Transduction Lab), anti-NOS3 polyclonal Ab (1:1,000; Santa Cruz), and anti-NOS1 polyclonal Ab (1:1,000; Affinity Bioreagent). After they were washed, membranes were incubated for 1 h at room temperature with an anti-mouse IgG or an anti-rabbit IgG conjugated to horseradish peroxidase (1:5,000; Amersham). After samples were washed, bands were revealed by chemiluminescence using ECL+ (Amersham) and quantified by using a computer system (Image Gauge) and normalized to actin (Ponceau S red) and GAPDH.
Coimmunoprecipitation assays. For coimmunoprecipitation, OG lysate proteins (300 µg) were incubated overnight at 4°C with different antibodies according to the protein complex studied. To investigate NOS3-caveolin-1 interactions, OG lysates were incubated overnight at 4°C with caveolin-1 MAb at a final concentration of 5 µg/ml in 400 µl of immunoprecipitation buffer (1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris·HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, and protease inhibitor cocktail). Anti-mouse IgG-conjugated agarose (Sigma) was added at 4°C for 3 h. After centrifugation, the supernatant was stored, and aliquots (10 µl) were used for Western blotting. The pellets were washed in immunoprecipitation buffer, and the immunoprecipitates were eluted in Laemmli buffer and separated in two parts for Western blot analysis. Coimmunoprecipitation and supernatant samples were analyzed by electrophoresis and immunoblotted with NOS3 polyclonal Ab (1:1,000). For coimmunoprecipitation between NOS1 and caveolin-3, OG lysates were incubated overnight with anti-NOS1 polyclonal Ab (5 µg/ml in 400 µl of immunoprecipitation buffer). Protein A sepharose (Amersham) was added at 4°C for 3 h. After centrifugation, the protocol was similar to that described above, and membranes were incubated with caveolin-3 MAb (1:1,000). To investigate NOS3-caveolin-3 interactions, OG lysates were incubated overnight with caveolin-3 MAb (5 µg/ml in 400 µl of immunoprecipitation buffer). Anti-mouse IgG-conjugated agarose (Sigma) was added at 4°C for 3 h. After centrifugation, the fractions were processed as described above. The membranes were immunoblotted with NOS3 polyclonal Ab (1:1,000). Of note, to verify that the amount of supernatant protein loaded did not vary, the membranes were probed with GAPDH (1:5,000; Chemicon).
Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed with Statview. ANOVA followed by a Bonferroni-Dunn correction was performed. P < 0.05 after Bonferroni correction is considered significant. For comparison between two groups, Student's t-test was used.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Echocardiographic analysis, performed at 2 and 20 wk, also indicated that males exhibited earlier signs of cardiac dysfunction in response to TAC than females (Fig. 1). By 2 wk after TAC, percent fractional shortening was decreased in males (–50%; P < 0.05 vs. male sham group) and did not vary afterward. In contrast, females exhibited moderate signs of cardiac dysfunction only by 20 wk after TAC (Fig. 1A; %fractional shortening: –30%; P < 0.05 vs. results for female TAC). Further echocardiography parameter analysis revealed that more pronounced percent fractional shortening alterations in males were mainly due to the increase of LV cavity size during systole without any changes in LV cavity size during diastole (Table 1).
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Cardiac NOS activities according to sex difference and LVH status. We choose 2 and 20 wk as early and late time points, respectively, to study total and specific NOS activities in the LV extracts (Fig. 2).
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As shown in the Fig. 2B, the cardiac-specific NOS1 activity of sham animals was relatively low and stable for both male and female rats. By 2 and 20 wk after TAC, the cardiac NOS1 activity markedly increased in males (+122%; P < 0.05 vs. male sham group). In contrast, in females, the cardiac NOS1 activity increased only at the late point (+313%; P < 0.05 vs. female sham group), being at that time significantly higher than in males with TAC.
As shown in Fig. 2C, the specific NOS3 activity was similar in the sham groups. By 2 wk after TAC in males, NOS3 activity dramatically decreased (–54%; P < 0.05 vs. male sham group) and remained stable afterward. It is worth noting that, in female rats, NOS3 activity declined only by 20 wk after TAC (–74%; P < 0.05 vs. respective sham).
These results indicate that 1) total cardiac NOS activity is stable whatever the cardiac status and sex and 2) after TAC, specific changes in NOS3 and NOS1 activities occurred earlier in males than in females.
NOS3 and NOS1 expression in the heart according to sex and hypertrophic status. Figure 3, A and C, shows that in all sham animals NOS1 expression was stable, as previously reported (14). After TAC was performed, cardiac NOS1 expression increased as early as after 5 days and reached a plateau in male rats (+65%; P < 0.05 vs. male sham animals). In contrast, in females with TAC, NOS1 level was moderately enhanced by 2 wk (+15%) and further increased at the late time points (+70%; P < 0.05 vs. female sham group).
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Cardiac expression of caveolins-1 and -3 according to sex and hypertrophic status. As shown in Fig. 4, A and C, TAC induced a decrease in caveolin-1 expression in male hearts by day 5 (–35%; P < 0.05 vs. respective shams), whereas in females caveolin-1 level fell only at 20 wk (–65%; P < 0.05). In all sham animals, caveolin-1 expression was stable.
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Interactions between caveolin and NOS isoforms. The relative levels of interactions between NOS3 and caveolin-1, on one hand, and between NOS1 and caveolin-3, on the other hand, were investigated by coimmunoprecipitation by 2 and 20 wk (Fig. 5, A and B). The relative amount of NOS3 bound to caveolin-1 complexes remained stable whatever the experimental conditions (Fig. 5A). In contrast, the relative amount of NOS1-caveolin-3 complexes was two- to fourfold higher in females than in males (Fig. 5B; P < 0.05 vs. male rats). In response to TAC, NOS1-caveolin-3 complexes significantly increased by wk 2 in male groups only (P < 0.05 vs. male sham group). This difference did not exist by 20 wk.
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Regulation of specific NOS activities. Correlation analysis allowed us to exclude any relationship between the specific NOS activities and their respective levels of interactions with caveolins (NOS3-caveolin-1 or NOS1-caveolin-3; data not shown). In contrast, we found that NOS3 activity was related to caveolin-1 level (R2 = 0.81) (Fig. 6A), whereas NOS1 activity was correlated to the NOS1 level (R2 = 0.82) (Fig. 6B). These correlations emphasized the distinct mechanisms involved in the control of NOS activity in the heart.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The present data confirm that females, in response to pressure overload, have a greater hypertrophic reserve than males (12, 29). The delay in the occurrence of maladaptive hypertrophy, observed herein with female rats, could be due to estrogen effects as previously proposed in both rats and humans (24). To further explore the sex differences in the susceptibility to develop HF, we analyzed the NOS activities and the expression of the two constitutive NOS isoforms together with their major allosteric regulators.
An increase in NOS1 activity and expression has been demonstrated in experimental and human HF (3, 10), in aged hypertensive rats (23), and soon after ANG II infusion (28). Herein, we provide the first evidence that the upregulation of NOS1 in the heart is a primary response to pressure overload. Indeed in male rats, NOS1 expression is a very early (5 days) and sustained response to TAC. In female rats, the onset of NOS1 induction is delayed (
2 wk), but NOS1 expression rises with time. The TAC-induced increase in wall stress occurs earlier in males than in females (12). Therefore, we might hypothesize that increase in wall stress is a major trigger of NOS1 expression. Furthermore, in females, the further development of cardiac hypertrophy tends to decrease the wall stress, and when the limits of adaptive hypertrophy are reached, NOS1 is induced. Further investigations are needed to verify this hypothesis. It is worthy to note that NOS1 activity is tightly related to the enzyme level of expression (Fig. 5B). In line with this concept, NOS1 expression and activity have been shown to increase in response to mechanical loading in skeletal muscle (30, 32). As a result, in both cardiac and skeletal muscles, the mechanosensitive pathway triggers NOS1 expression and activity.
Conversely to ischemia-reperfusion or HF situations during which NOS1 is translocated to caveolae through interactions with caveolin-3 (3, 10, 27), pressure overload did not alter significantly the subcellular distribution of NOS1 in the rat heart (Fig. 5). These data raise the question of the role of NOS1 in the heart following TAC. It has been proposed that NOS1 could be beneficial for cardiac function in HF secondary to myocardial infarction (3, 11, 25). Here, we demonstrated that the NOS1 induction preceded but did not prevent the occurrence of cardiac dysfunction. Therefore, it is tempting to speculate that the stretch-induced NOS1 contributes to the ultimate phase of myocyte hypertrophy (5, 20) when the process became maladaptive (15).
Regarding NOS3, with the exception of age-dependent changes in females, the protein expression was remarkably stable whatever the cardiac status, as observed in males (21). The regulation of caveolin-1, the major partner of NOS3, differed. Indeed, the caveolin-1 levels were downregulated in response to TAC, the temporal pattern of expression being sex specific, that is, early and sustained in males and delayed in females. The fact that NOS3 activity was closely related to caveolin-1 expression (Fig. 6A) emphasizes not only the enzyme location at the level of endothelial caveolae but the importance of caveolin-1 in the NOS3-derived NO production in heart. It is worthy to note that NOS3-caveolin-3 interactions are minute (i.e., <5% of NOS3) and unmodified by age, sex, or hypertrophy status. This result points out the modest NOS3 distribution in muscle cells.
It therefore appears that the occurrence of endothelial dysfunction associated with the decreased NOS3 activity is sex difference dependent, highlighting the estrogen protection of the endothelium through the control of caveolin expression (16, 24) in the heart in response to a pathological situation.
Limitations of the study. It should be stressed that study of animals of different ages and body size changes is complex. Accordingly, we chose to normalize data to same-sex control values (LVH: the ratio between TAC LV weight to sham LV weight mean), as proposed by Douglas et al. (12). Indeed, as outlined by these authors, in long-term study, neither LV weight-to-body weight ratio nor LV weight-to-tibia length ratio can be used to normalize hypertrophy process because of the limitations of growth, age, and sex.
Another change related to development observed in the study was the differences in NOS activity with time in sham female animals compared with sham males. The data relative to age-related variations of NOS activity according or not to sex differences are rather few. There are reports on aging and on neonatal development (4), which have indicated a decrease in NOS3 activity from birth to adult and or senescent stages. In addition, Esberg et al. (14) observed that young female rats have higher NOS activities than males. Furthermore, we have demonstrated that estrogen stimulates NOS3 activity (19). Therefore, considering that, at 2 wk after surgery, rats were 40 days old, an age close to sexual maturity in females, we might propose that estradiols play a role in the higher NOS3 expression and activity observed in juvenile females vs. that shown in with age-matched male or older female animals. Such age-related variations in NOS3 activity in female animals must be considered in future studies.
The respective functions of NOS1 and NOS3 and of NOS1/3-derived NO were not addressed in the present study. It has been suggested that NOS3-derived NO, or exogenous NO, may be involved in preventing the development of cardiac hypertrophy (6). The present data indicate that the early decrease in NOS3-derived NO in male rats was not sufficient to allow the development of cardiac hypertrophy with time comparatively to female rats. Rather, such a drop in NOS3 activity underlined the endothelial dysfunction in male, which in turn participates in the development of HF (26). In females, through the beneficial effect of estrogens (24), the long lasting maintenance of endothelial function likely prevents the occurrence of HF. With regard to NOS1, it has been proposed that the enzyme modulates calcium homeostasis and -adrenergic response (7). Our results demonstrating active NOS1 in all hypertrophic myocardium are in line with a protrophic role of NOS1, as recently proposed (5, 20). Furthermore, NOS1-caveolin-3 complexes in females could protect cardiomyocytes, as proposed by Sun et al. (27).
To conclude, it emerged that, although total NOS activity in the heart remained unchanged against severe pressure overload, specific changes in NO compartmentation due to NOS1 or NOS3, respectively, might be involved in the development of cardiac hypertrophy and failure. Finally, the study emphasized the specific regulation of constitutive NOS isoform activities according to sex and the pathophysiological situation in the heart by two distinct pathways, the activity of NOS1 depending on transcriptional mechanism and that of NOS3 controlled through allosteric regulators.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
1 This paper was presented at the 9th Cardiovascular-Kidney Interactions in Health and Disease Meeting at Amelia Island Plantation, Florida, on May 26–29, 2006. 
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2-adrenergic receptor exhibit differences in ischemia/reperfusion injury: role of nitric oxide. Cardiovasc Res 53: 662–671, 2002.-estradiol on caveolin-1 mRNA and protein in bovine aortic endothelial cells. Am J Physiol Heart Circ Physiol 281: H1327–H1333, 2001. Estradiol regulates constitutive nitric oxide synthase expression differentially in the myocardium in response to pressure overload. Endocrinology 148: 4579–4584, 2007.
1 subunit and reduced ischemia/reperfusion injury. Circ Res 98: 403–411, 2006.This article has been cited by other articles:
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  X. Loyer, A. M. Gomez, P. Milliez, M. Fernandez-Velasco, P. Vangheluwe, L. Vinet, D. Charue, E. Vaudin, W. Zhang, Y. Sainte-Marie, et al. Cardiomyocyte Overexpression of Neuronal Nitric Oxide Synthase Delays Transition Toward Heart Failure in Response to Pressure Overload by Preserving Calcium Cycling Circulation, June 24, 2008; 117(25): 3187 - 3198. [Abstract] [Full Text] [PDF]
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 T. C. Gillebert and N. R. Van de Veire About left ventricular torsion, sex differences, shear strain, and diastolic heart failure Eur. Heart J., May 2, 2008; 29(10): 1215 - 1217. [Full Text] [PDF]
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P < 0.05 vs. male TAC (M-TAC) group. B: LVH is expressed as percentage of respective sham animals at each time point of the study. 
, female TAC (F-TAC) group; 
, male (M) TAC group. In males, n = 8 at 5 days and n = 9 at 2, 14, and 20 wk. In females, n = 10 at 5 days and n = 8 at 2 wk, 10 by 14 wk, and 9 by 20 wk. *P < 0.05 vs. F-TAC at the same time point.
P < 0.05 vs. M-TAC. C: specific NOS3 activity. *P < 0.05 vs. respective sham groups. cpm, Counts per minute.
, M-sham; 
, F-sham; 
