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Cardiovascular Research 2003 57(1):158-167; doi:10.1016/S0008-6363(02)00654-5
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Copyright © 2003, European Society of Cardiology

Discoordinate re-expression of cardiac fetal genes in N{omega}-nitro-L-arginine methyl ester (L-NAME) hypertension

Ying Zhang, Daniel Carreras and Adolfo J. de Bold*

Cardiovascular Endocrinology Laboratory, University of Ottawa Heart Institute, 40 Ruskin St., H-247 Ottawa, Ontario K1Y 4W7, Canada

* Corresponding author. Tel.: +1-613-761-4265; fax: +1-613-761-1597. adebold{at}ottawaheart.ca

Received 12 June 2002; accepted 29 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Hypertension produced by chronic inhibition of nitric oxide (NO) synthase by N{omega}-nitro-L-arginine methyl ester (L-NAME) was used to determine the effect of severe pressure overload with or without left ventricular (LV) hypertrophy on the transcriptional activation of the cardiac fetal genes encoding for the natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), and for β-myosin heavy chain (MHC) in both atrial and ventricular muscle. A previously reported association of LV hypertrophy with the activation of cardiac renin and angiotensin-converting enzyme (ACE) in this hypertension model was also investigated. Methods: Male Sprague–Dawley rats received L-NAME (75 mg/kg/day) or were left untreated for 4 (n=12) or 8 (n=12) weeks. Results: L-NAME-treated rats became severely hypertensive in both treatment groups but only five out of 12 8-week treatment animals showed a significantly increased LV weight to body weight (BW) ratio (LVW/BW). LV ANF mRNA, but not LV BNP mRNA, correlated significantly with LVW/BW only in animals showing LV hypertrophy. No changes were observed in atrial gene expression or plasma concentration of ANF or BNP. A significant correlation was found between LVW/BW and LV renin mRNA and LV ACE activity in rats with LV hypertrophy. LV β-MHC mRNA levels were significantly increased in the LV of rats with or without LV hypertrophy at both 4 and 8 weeks of treatment. Conclusions: It is concluded that pressure overload per se does not promote NP or cardiac renin–angiotensin system gene expression while increased β-MHC expression is a marker of LV pressure overload even in the absence of LV hypertrophy. It is apparent that L-NAME causes a disruption in the coordinated transcriptional activation of cardiac fetal genes expected of hypertrophic stimuli acting on the LV.

KEYWORDS Gene expression; Hypertension; Hypertrophy; Natriuretic peptide; Nitric oxide


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac hypertrophy resulting from chronic hemodynamic overload may be viewed as a basic adaptive response anatomically evidenced by an increase in the relative weight of a cardiac chamber. At the molecular level, ventricular hypertrophy is characterized by the re-expression of genes that are expressed at relatively high levels during fetal life such as the natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) as well as β-myosin heavy chain (β-MHC). The detailed mechanism underlying the transduction of chronic mechanical overload into the hypertrophic phenotype is the subject of much research given the importance of the development of left ventricular (LV) hypertrophy for subsequent cardiovascular morbidity [1].

At the present time it is not clear what is the relative contribution of overload per se to the transcriptional activation of ventricular genes leading to hypertrophy in vivo. Of interest to the study of the relationship between hypertrophy and the expression of fetal genes in vivo is the fact that administration of nitric oxide synthase inhibitors such as N{omega}-nitro-L-arginine methyl ester (L-NAME) and other L-arginine analogues can cause severe chronic hypertension with little or no anatomical cardiac hypertrophy [2–9] but it is not known whether this hemodynamic overload induces the re-expression of the cardiac fetal gene program.

In the present work, we determined the effect of chronic pressure overload on the cardiac expression of ANF, BNP, {alpha}- and β-MHC as well as components of the circulating and cardiac renin–angiotensin system. The latter has been previously associated with the development of hypertrophy when it occurs in L-NAME-treated animals [8].

We report that fetal gene expression is not upregulated in hypertensive L-NAME-treated rats in which anatomical hypertrophy did not develop with the exception of β-MHC, which thus appears as an indicator of pressure overload in the absence of anatomical hypertrophy. L-NAME-treated rats that did develop LV hypertrophy showed LV upregulation of ANF but not BNP. This upregulation occurred together with increased expression of the cardiac renin–angiotensin system. These results suggest that L-NAME causes a disruption in the coordinated transcriptional activation of the ventricular fetal genes studied.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
All procedures utilizing animals were carried out in accordance with the guidelines of the Canadian Council for Animal Care. Forty-eight male Sprague–Dawley rats weighting 180–200 g were divided into four groups as follows. (1) Four-week L-NAME group (n=12). These animals received L-NAME (Sigma) in tap water for 4 weeks at a concentration adjusted according to the body weight so that they received a dose of 75 mg/kg/day. (2) Four-week control group (n=12). These animals were given tap water. (3) Eight-week L-NAME group (n=12). The animals received L-NAME at the same dose as above in tap water for 8 weeks. (4) Eight-week control group (n=12). Animals were given tap water. No significant differences in the amount of fluid intake were noted.

Systolic blood pressure (SBP) was measured every 2 weeks by tail plethysmography. At the end of the treatment period, the rats were killed by decapitation. Trunk blood was collected in ice-chilled tubes containing EDTA and aprotinin for the measurement of plasma concentration of ANF, BNP, Ang I and Ang II. No aprotinin was added to blood samples destined to the measurement of plasma renin and ACE activities. Blood samples were centrifuged at 3000xg for 15 min at 4 °C. After centrifugation, the plasma samples were stored at –80 °C until used in the different assays. After blood collection, the heart was excised, rapidly weighed and dissected in cold saline into right and left atrium and right and left ventricles with their respective septa as part of the left chambers. After weighing, the tissue was quickly wrapped in aluminum foil and snap-frozen in liquid nitrogen.

Blood pressure, blood analytes and body weights were determined in all animals (n=12, each group at 4 and 8 weeks). Due to the nature of the procedures carried out in cardiac tissue, half of the hearts from each group (n=6 at 4 weeks and n=6 at 8 weeks) were processed for mRNA extraction while the other half were processed for the remainder of measurements.

2.2 Extraction of plasma and tissue samples
Plasma samples were acidified by adding 100 µl/ml of 1.0 M HCl and passed through Sep-Pak C18 cartridges (Millipore). The cartridges were prewetted with 5 ml of 80% acetonitrile in 0.1% trifluoroacetic acid (TFA) and 10 ml of 0.1% TFA. The cartridges with the absorbed peptides were washed with 20 ml of 0.1% TFA and eluted with 3 ml of 80% acetonitrile in 0.1% TFA. Tissue samples were homogenized in 10 volumes of an extracting mixture consisting of 0.1 N HCl, 1.0 M acetic acid, and 1% NaCl and centrifuged at 10,000xg for 30 min at 4 °C. The supernatants were then processed through Sep-Pak C18 cartridges as described above for plasma. The eluates from tissue or plasma were freeze-dried and processed for RIA to measure ANF, BNP, Ang I and Ang II.

2.3 Radioimmunoassays
Plasma and cardiac tissue concentrations of ANF and BNP were measured by radioimmunoassay, as previously described [10]. Antisera raised against rat ANF99–126 and rat BNP64–95 were obtained from Peninsula Laboratories. Cross-reactivity of ANF and BNP antisera with BNP and ANF peptides was less than 0.01%. Plasma renin activity (PRA) was measured by determining the level of Ang I generated during 1 h of incubation at 37 °C in the presence of dimercaprol and 8-hydroxyquinoline (NEN Life Science Products). Standards and antisera for Ang I and Ang II measurements were purchased from Advanced ChemTech. Both [125I]Ang I and [125I]Ang II were from Peninsula Laboratories. The Ang I antisera showed <0.01% cross-reactivity with Ang II peptide, and the Ang II antisera showed 1.7% cross-reactivity with Ang I peptide.

2.4 Measurement of plasma and cardiac tissue ACE activity
Plasma and left ventricular ACE activity were measured by the rate of generation of His–Leu from Hip–His–Leu substrate using the fluorometric assay described by Cushman and Cheung [11] and Reneland and Lithell [12]. The relative fluorescence intensity of His–Leu was measured using a spectrofluorometer with an excitation wavelength of 365 nm and an emission wavelength of 485 nm. Plasma ACE activity was expressed as nmol/ml/min and tissue ACE activity was expressed as nmol/g/min using a standard curve constructed from His–Leu (Sigma).

2.5 Reverse transcription-polymerase chain reaction (RT-PCR)
RNA samples (2 µg of total RNA) were reverse transcribed with Super Script II RNase H– Reverse Transcriptase and oligo (dT)12–18 primers with the use of a reverse transcription kit (GIBCO BRL). After RT, an aliquot of the cDNA product was used for PCR amplification with renin and β-actin primers. The latter amplification product was used as housekeeping transcript to express relative abundance of the renin transcripts. The sequence of renin and β-actin primers is shown in Table 1. PCRs were carried out in a final volume of 50 µl containing PCR buffer (10 mmol/l Tris–HCl and 50 mmol/l KCl, pH 8.3), 2 mmol/l MgCl2, 1.25 units of Ampli Taq DNA Polymerase (Perkin-Elmer), and 0.15 µmol/l of the respective primers. The reaction was heated at 95 °C for 10 min and cycled 35 times through a 60-s denaturing step at 95 °C, a 30-s annealing step at 62 °C and a 60-s extension step at 72 °C. After the final cycle, a 7-min extension step at 72 °C was included. Aliquots of the PCR product were electrophoresed on a 2% agarose gel and were visualized by ethidium bromide staining. To confirm the identity of the PCR products, restriction analyses were carried out using MobI, KpnI and MspI (GIBCO BRL). The renin PCR product sequence was confirmed by direct sequencing in a Big Dye Terminator Cyclosequencing (Applied Biosystems). A standard curve made with dilutions of total RNA was used to ascertain that the comparisons to the housekeeping gene were made in the linear amplification range. The quantification of renin and β-actin mRNA band intensities was done using a phosphoimager and the Quantity One software package (BIO RAD).


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  		Table 1 Primer sequences used in PCR

 
2.6 RNA extraction and Northern blot analysis
Total RNA extraction and Northern blot analysis were performed as previously described [13] using 32P-labeled probes. The cDNA probes used were (1) a 900-bp EcoRI/HindIII fragment containing the full-length rat ANF cDNA, (2) a 595-bp SalI fragment containing full-length rat BNP cDNA, (3) a 5-kb EcoRI/SalI fragment of the mouse 28S rRNA cDNA probe, and (4) a 2-kb BamHI/BglII fragment of the mouse phosphoglycerate kinase (PGK) gene cDNA. The two oligonucleotide probes were 39- and 24-base fragments specific for unique regions in the 3' untranslated regions of the rat {alpha}- and β-MHC genes. Autoradiographs were scanned with an Ultrascan XL laser densitometer (LKB Produckter) and LKB 2400 Gelscan XL software package. The scanning values of ANF, BNP and {alpha}-MHC and β-MHC mRNA were normalized to 28S rRNA or PGK mRNA as internal controls to correct for differences in the amount of RNA applied and transfer efficiency.

2.7 Statistical analysis
Results were expressed as mean±S.E.M. Statistical comparisons between groups were performed by unpaired Student's t-test. Correlations between two variables were determined by linear regression analysis using Systat®. Statistical significance was accepted at a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The anatomic and biochemical data described below are summarized in Table 2.


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  		Table 2 Summary of anatomic and biochemical data for L-NAME hypertensive rats and their respective controls

 
3.1 Systolic blood pressure and heart weights
Administration of L-NAME increased SBP in a time-dependent manner. SBP was 140±3 mmHg at the end of 4 weeks, and 173±6 mmHg at the end of 8 weeks. The values were significantly higher (P<0.001) than the SBP in control rats (105±1 mmHg and 106±2 mmHg), respectively.

Despite the increase in SBP, the average LV weight to body weight (BW) ratio (LVW/BW) of rats given L-NAME was not statistically different from those of controls in both the 4- and the 8-week groups, though LVW/BW was higher (2.22±0.08 mg/g) in 8-week L-NAME treatment group than that in control (2.03±0.04 mg/g). Right ventricular (RV) weight to body weight ratio (RVW/BW) was significantly decreased in both the 4-week and 8-week treatment groups.

After 8 weeks of L-NAME treatment, LVW/BW correlated with SBP (n=12, r=0.75, P<0.05, Fig. 1). Inspection of the raw data revealed that a subgroup of five of the 12 rats had LV hypertrophy as measured by LVW/BW, which averaged 2.50±0.10 compared to 2.03±0.04 in the control group (P<0.01). SBP was considerably higher (P<0.01) in the subgroup with LV hypertrophy (192±5 mmHg, n=5) than in those without (160±6 mmHg, n=7).


Figure 1
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  		Fig. 1 Correlation between LVW/BW and SBP for control animals (bullet) and after 8 weeks of L-NAME treatment ({blacktriangleup}) (n=12 each group).

 
3.2 Renin–angiotensin system
Plasma renin activity (PRA) was decreased in the 4-week L-NAME-treated group as compared to controls for the same time period (2.95±0.54 vs. 7.59±0.74, P<0.001) and had a tendency to be increased in 8-week L-NAME but the difference did not reach statistical significance. LVW/BW correlated with PRA in the group of rats treated for 8 weeks with L-NAME (P<0.05, Fig. 2). Plasma and LV Ang II were not significantly different in the 4- or 8-week L-NAME treatment groups from their respective control groups although LV Ang II showed a tendency to decrease at 4 weeks (80.1±18.9 vs. 42.7±2.6). This decrease was not statistically significant given the high variance of the control group values.


Figure 2
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  		Fig. 2 Correlation between LVW/BW and PRA for control animals (bullet) and after 8 weeks of L-NAME treatment ({blacktriangleup}) (n=12 each group).

 
3.3 LV renin gene expression
Relative LV renin mRNA concentration was not significantly different in the 4- or 8-week treatment groups compared to controls (0.29±0.02 vs. 0.34±0.01 and 0.49±0.08 vs. 0.93±0.31, respectively). However, LV renin mRNA concentration correlated with the LVW/BW (P<0.05, Fig. 3) after 8 weeks of L-NAME treatment. In addition, animals in the 8-week group that showed LV hypertrophy had a 3.6-fold increase in average of the ratio of LV renin mRNA/β actin (0.5±0.06) as compared to controls (1.8±0.4).


Figure 3
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  		Fig. 3 Correlation between LVW/BW and LV renin mRNA for control animals (bullet) and after 8 weeks of L-NAME treatment ({blacktriangleup}) (n=6 each group).

 
3.4 Plasma and cardiac tissue ACE activities
No changes were observed in plasma or LV ACE activity in the 4-week treatment group when compared to its control group. After 8 weeks of treatment a significant increase in plasma ACE activity (8.33±0.08 vs. 7.43±0.08, P<0.001) and LV ACE activity (13.48±1.09 vs. 8.32±0.36, P<0.01) was observed. LV ACE activity but not plasma ACE activity, correlated with the LVW/BW in the 8-week L-NAME treatment group (P<0.05, Fig. 4).


Figure 4
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  		Fig. 4 Correlation between LVW/BW and LV ACE activity for control animals (bullet) and after 8 weeks of L-NAME treatment ({blacktriangleup}) (n=6 each group).

 
3.5 Plasma and cardiac tissue concentration of ANF and BNP
In the 4-week L-NAME treatment group, left atrium (LA) ANF was decreased (115.0±11.9 vs. 182.5±18.9, P<0.05). LV BNP was increased (20.5±1.7 vs. 10.0±0.6, P<0.01). LA BNP and LV ANF were comparable to controls. No significant changes in ANF and BNP were observed in plasma or in the right atrium (RA) or RV. In the 8-week L-NAME treatment group, levels of immunoreactive ANF (irANF) and immunoreactive BNP (irBNP) were similar to those of controls in plasma, atria and ventricles.

3.6 ANF and BNP cardiac mRNA levels
Relative ANF mRNA levels were significantly decreased both in RA (0.61±0.08 vs. 1.30±0.25, P<0.05) and LA (0.71±0.11 vs. 1.43±0.20, P<0.01) in the 4-week L-NAME treatment group but no significant changes were found after 8 weeks of treatment. BNP mRNA levels were comparable to control levels in both the 4- and 8-week L-NAME treatment groups. RV ANF and BNP mRNA were not significantly different in the 4- or 8-week L-NAME groups compared to control groups. Fig. 5 shows the Northern blot results for LV ANF and BNP mRNA. In the 8-week treatment group, ANF mRNA level correlated with LVW/BW (P<0.05, Fig. 6). No such correlation was found for BNP mRNA.


Figure 5
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  		Fig. 5 Individual Northern blots and group scanning data for LV ANF and BNP mRNA.

 

Figure 6
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  		Fig. 6 Correlation between LVW/BW and LV ANF mRNA for control animals (bullet) and after 8 weeks of L-NAME treatment ({blacktriangleup}) (n=6 each group).

 
3.7 {alpha}-MHC and β-MHC cardiac gene expression
LV {alpha}-MHC mRNA was significantly elevated (3.89±0.24 vs. 2.75±0.19, P<0.05) in the 4-week L-NAME group but no change was detected in the 8-week L-NAME group. RV {alpha}- and β-MHC mRNA levels were comparable to controls both in the 4- and in the 8-week L-NAME treatment groups. LV β-MHC mRNA was significantly increased at 4 weeks (0.66±0.10 vs. 0.18±0.03, P<0.01) and at 8 weeks of treatment (0.91±0.07 vs. 0.25±0.05, P<0.001) and the ratio of LV β-MHC to {alpha}-MHC (β-MHC/{alpha}-MHC) were significantly increased both after 4 (0.17±0.02 vs. 0.07±0.01, P<0.01) and 8 weeks (0.38±0.05 vs. 0.12±0.03, P<0.01) of L-NAME treatment (Fig. 7). No β-MHC mRNA was detected in atrial muscle.


Figure 7
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  		Fig. 7 Individual Northern blots and group scanning data for LV {alpha}- and β-MHC mRNA.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Inhibition of NO synthase by L-NAME induces a severe type of hypertension that is unique in that the hemodynamic burden is associated with lack of or relatively diminished LV hypertrophic response despite sustained high blood pressure levels. The determinant of whether or not some degree of LV hypertrophy develops in L-NAME hypertension is unknown but this heterogeneity of response may be based on the genetic make up of individual animals in an outbred strain. Nevertheless, L-NAME hypertension is a useful model to identify genes whose expression is modified by pressure overload in the absence of detectable anatomical hypertrophy. In general, our findings agree with previous studies that have demonstrated that changes in LV mass in L-NAME hypertension are absent or modest, ranging from 9% to 30% [8,14–17]. In the present study, LV hypertrophy as measured by the group average LVW/BW ratio, did not appear to develop after either 4 or 8 weeks of L-NAME treatment. However, a subgroup (42%) of rats treated for 8 weeks developed LV hypertrophy (LVW/BW>control mean +2 S.D.) [8] when compared to controls. In rats with LV hypertrophy, SBP values were significantly higher than that of their group mates showing that in L-NAME hypertension there is a shift of the hypertrophy–SBP response curve rather than an inhibition of growth altogether.

Re-expression of ventricular ANF, BNP and genes that are expressed predominantly in the fetal ventricles is regarded as unfailing evidence of the molecular adaptation to chronic hemodynamic overload. In the present work, we observed that 4-week L-NAME treatment (hypertension without hypertrophy) had no effect on ANF or BNP gene expression. In the 8-week treatment group however, LV ANF mRNA levels correlated significantly with the LVW/BW. These findings demonstrate that LV ANF gene expression is related to cardiac hypertrophy and not necessarily related to pressure overload and are consistent with our previous findings in the hypertrophic LV of hypertensive, aortic-banded rats that suggested that the expression of ventricular ANF is at least partly independent of pressure load [13].

In contrast to the results obtained for LV ANF mRNA, LV BNP mRNA levels did not correlate with relative LV weight. This is unexpected because BNP gene expression in hypertrophic ventricles increases at least as much as, and often significantly more, than ANF gene expression in most other hypertension models that result in LV hypertrophy [18,19] suggesting that L-NAME may have a particular inhibitory effect on ventricular BNP gene expression.

The amount of ANF and BNP and their messages present in the atria of L-NAME-treated animals did not vary appreciably from controls at the end of 8 weeks of treatment. ANF mRNA levels, but not BNP mRNA levels, were significantly decreased in the RA and LA in the 4-week group while the peptide stores remain unchanged. We have previously observed that a depletion of NP occurs after sub-acute as well as chronic stimulation of the atrial ANF and BNP stores but this is accompanied by significant increases in peptide message [18,20]. The decrease in atrial ANF production at 4 weeks of L-NAME administration is thus clearly different from the pattern observed following stimulation by other means but its basis is not apparent from this work. The fact that the levels of NP and their messages appear recovered at 8 weeks suggests that an alternative signaling pathway comes into play sometime after 4 weeks of NO production inhibition. Nevertheless, neither the changes in atrial peptide storage seen at 4 weeks of L-NAME treatment nor the changes in ventricular natriuretic gene expression seen at 8 weeks appeared to be of enough magnitude to significantly modify NP plasma levels, which were not significantly different from controls at both time points. Unchanging or increased ANF plasma levels in L-NAME hypertension have been previously reported [8,21].

Coincidental with the decrease in atrial ANF gene expression at 4 weeks of L-NAME treatment, we observed that PRA was significantly decreased (Table 2). This decrease could be attributed either to an increase in renal perfusion pressure or to the suppression of the potential direct stimulation of renin release by NO [22,23]. By 8 weeks, rats without LV hypertrophy had PRA values comparable to controls but the rats with LV hypertrophy had increased PRA, this latter finding being reflected in a significant statistical correlation. We further found that LV renin gene expression in rats treated for 8 weeks with L-NAME correlated with relative left ventricular weight (Fig. 3) and increased 3.6-fold in rats with hypertrophy but did not change in rats without hypertrophy. These findings agree with those of previous studies demonstrating that RAS is actively modulated in L-NAME hypertension [6,15,16,24–27] and are consistent with the view [8,28] that RAS plays a significant role in the development of hypertrophy. The rise in PRA in rats treated for 8 weeks with L-NAME may be the results of kidney disease [28,29] but renal pathology is not likely to explain the increased LV renin gene expression found only in hypertrophic ventricles. In addition, 8 weeks of L-NAME treatment caused a significant increase in plasma and LV ACE activity in rats with hypertrophy. These findings agree with previous reports [7,8] showing that a subset of rats made hypertensive with L-NAME developed cardiac hypertrophy with increased cardiac ACE activity. While these results strongly suggest that the activation of plasma and cardiac ACE is involved in the development of cardiac hypertrophy in the L-NAME hypertension model, we found that plasma and LV Ang II were not significantly different in the 4- or 8-week L-NAME treatment groups and no significant correlation was found between plasma or LV Ang II and LV hypertrophy. However, numerous studies have demonstrated that hypertension and cardiac hypertrophy caused by long-term inhibition of NO synthesis can be prevented or reversed by treatments with angiotensin converting enzyme (ACE) inhibitors [15,16,24–27] and at least partially reverted with the Ang II antagonist losartan [8,21] although it has been suggested that the mechanism by which ACE inhibitors exert beneficial effects on cardiovascular remodeling in this model may be related to an inhibition of hydrolysis of kinin [30].

Increased expression of β-MHC, a rodent fetal isoform of MHC, is another marker of hypertrophy at the molecular level. Changes in the relative amount of {alpha}- and β-MHC are believed to be responsible in part for altered cardiac performance during hemodynamic overload and cardiac hypertrophy. It has been proposed that a high proportion of {alpha}-MHC is associated with a faster, but less economical force development, whereas, a high proportion of β-MHC is associated with a relatively slow, economical tension development during isometric contraction [31,32]. In the present work, we found that LV β-MHC mRNA and the LV β-MHC mRNA/{alpha}-MHC mRNA ratio were significantly increased both in 4-week L-NAME rats (hypertension without hypertrophy) and in 8-week L-NAME rats (hypertension with or without hypertrophy), suggesting that pressure overload alone is a stimulator of increased β-MHC gene expression (Fig. 7). This result is consistent with our previous finding [13] that in the suprarenal aortic banding (pressure overload) rats, the increased ventricular β-MHC gene expression did not respond to low-dose ramipril, a treatment that reverses or prevents LV hypertrophy but not blood pressure. Hence, these findings suggest that pressure overload can induce β-MHC gene expression before any evidence of cardiac hypertrophy is evident. This view is in line with other work [33] demonstrating that following pressure overload produced by aortic coarctation, the first adaptation response of the heart is MHC isoform transition; cardiac hypertrophy being a later response to hemodynamic overload. Our data shows that enhanced β-MHC mRNA response to increased hemodynamic overload was not associated with any change of {alpha}-MHC gene expression after 8 weeks of L-NAME treatment. This finding is consistent with the observation [34] that changes in isogene predominance could be accounted for a preferential increase in β-MHC isogene expression, with no change in {alpha}-MHC.

We did not detect MHC isoform change in the atria of L-NAME-treated animals. This is consistent with our previous observations [18] in DOCA-salt hypertension, which was found associated with isoform switch in the ventricles and upregulation of atrial NP stores and mRNA in the absence of changes in atrial β-MHC mRNA levels.

β-MHC mRNA did not change in the RV of L-NAME-treated rats, suggesting that the change in β-MHC gene expression is limited to the involved ventricle in terms of pressure overload. This finding is supported by the observation that in aortic coarctated- and pulmonary artery-banded rats, significant induction of the β-MHC mRNA and corresponding protein are limited to the overloaded ventricles only [35]. From the observations presented here, increased β-MHC gene expression appears as a marker of pressure overload in the absence of cardiac hypertrophy. These results are in contrast to those reported in another study [36] in which no increase in mRNA levels for LV β-MHC was found after 6 weeks of L-NAME treatment although a strong induction of LV ANF was found even though none of the animals developed anatomical hypertrophy of the LV. As stated earlier, we found increased LV ANF gene expression only in those animals that exhibited LV hypertrophy. There are differences in animal strain and L-NAME dosage schedule between this and the present study as well as in the method of transcript measurement. Nevertheless, the reasons for these differences are not apparent from this study.

Taken together, the findings presented above show that in L-NAME hypertension, the subset of experimental animals that develop anatomical LV hypertrophy and induction of cardiac RAS, show a partial re-induction of some of the fetal genes such as ANF in the ventricle. However, pressure overload per se does not produce this re-induction except in the case of β-MHC, which thus appears as a marker of pressure overload even in the absence of ventricular hypertrophy.

Time for primary review 22 days.


   Acknowledgements
 
The authors wish to thank Amalia Ponce and Carole Frost for their technical support. This work was supported by grants from the Canadian Institutes for Health Research and the Heart and Stroke Foundation of Ontario.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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