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(Circulation. 2000;101:423.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Chronic N^G-Nitro-L-Arginine Methyl Ester–Induced Hypertension

Novel Molecular Adaptation to Systolic Load in Absence of Hypertrophy

Jozef Bartunek, MD, PhD; Ellen O. Weinberg, PhD; Minori Tajima, MD, PhD; Susanne Rohrbach, BA; Sarah E. Katz, BA; Pamela S. Douglas, MD; Beverly H. Lorell, MD

From the Charles A. Dana Research Institute and the Harvard-Thorndike Laboratory, Beth Israel Deaconess Medical Center, and Department of Medicine, Cardiovascular Division, Harvard Medical School, Boston, Mass.

Correspondence to Beverly H. Lorell, MD, Cardiovascular Division, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215. E-mail blorell{at}caregroup.harvard.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Chronic N^G-nitro-L-arginine methyl ester (L-NAME), which inhibits nitric oxide synthesis, causes hypertension and would therefore be expected to induce robust cardiac hypertrophy. However, L-NAME has negative metabolic effects on protein synthesis that suppress the increase in left ventricular (LV) mass in response to sustained pressure overload. In the present study, we used L-NAME–induced hypertension to test the hypothesis that adaptation to pressure overload occurs even when hypertrophy is suppressed.

Methods and Results—Male rats received L-NAME (50 mg · kg^-1 · d^-1) or no drug for 6 weeks. Rats with L-NAME–induced hypertension had levels of systolic wall stress similar to those of rats with aortic stenosis (85±19 versus 92±16 kdyne/cm). Rats with aortic stenosis developed a nearly 2-fold increase in LV mass compared with controls. In contrast, in the L-NAME rats, no increase in LV mass (1.00±0.03 versus 1.04±0.04 g) or hypertrophy of isolated myocytes occurred (3586±129 versus 3756±135 µm²) compared with controls. Nevertheless, chronic pressure overload was not accompanied by the development of heart failure. LV systolic performance was maintained by mechanisms of concentric remodeling (decrease of in vivo LV chamber dimension relative to wall thickness) and augmented myocardial calcium–dependent contractile reserve associated with preserved expression of - and ß-myosin heavy chain isoforms and sarcoplasmic reticulum Ca²⁺ ATPase (SERCA-2).

Conclusions—When the expected compensatory hypertrophic response is suppressed during L-NAME–induced hypertension, severe chronic pressure overload is associated with a successful adaptation to maintain systolic performance; this adaptation depends on both LV remodeling and enhanced contractility in response to calcium.

Key Words: nitric oxide • calcium • NG-nitroarginine methyl ester • hypertrophy • remodeling

	Introduction

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Nitric oxide (NO) and its donors increase cyclic GMP and cause vasorelaxation, whereas a withdrawal of constitutive NO induces vasoconstriction and causes severe hypertension.¹ This would be expected to induce cardiac hypertrophy as the fundamental compensatory response that maintains left ventricular (LV) systolic performance in the presence of chronic systolic pressure overload and prevents development of heart failure.² In addition, the NO-cyclic GMP pathway inhibits cell growth in in vitro systems.^{3 4} Thus, it would be expected that chronic systemic NO inhibition will induce exuberant cardiac hypertrophy; however, L-N-nitro-L-arginine methyl ester (L-NAME) treatment appears to suppress the expected increase in LV mass.^{5 6 7 8 9 10 11 12} This recognized inhibitory effect on growth is independent of effects on tissue NO synthesis^{13 14 15 16} and mediated by effects on amino acid delivery and utilization by competing with amino acid transporters and by altering ornithine metabolism.^{13 14 15 16 17 18} Taken together, this suggests that chronic L-NAME treatment provides a powerful tool to experimentally increase systolic load and to simultaneously suppress compensatory hypertrophy.

It is questionable whether the suppression of LV hypertrophy is beneficial or deleterious in pathologic pressure overload because suppression of hypertrophy might be expected to cause heart failure. Paradoxically, none of the previous studies which used L-NAME to cause hypertension reported the development of heart failure. Therefore, in the present study we used L-NAME–induced hypertension to test the hypothesis that successful molecular adaptation to chronic severe pressure overload occurs even when hypertrophy is suppressed.

	Methods

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Preparation of Animals
Male Wistar rats (300 g, Charles River Breeding Laboratories, Wilmington, Mass) received no drug (controls, n=22) or L-NAME (Sigma Chemicals, St. Louis, Mo) at a dose of 50 mg · kg^-1 · d^-1 (n=26) in drinking water for 6 weeks.^{6 7 8 9} An additional group of rats with 6 weeks ascending aortic stenosis was created^{19 20 21 22 23} to compare the levels of systolic wall stress and extent of LV remodeling for the same duration of pressure overload.

In Vivo Measurements
In vivo tail-cuff systemic blood pressure was measured weekly by a single animal handler.^{19 20} At the end of the treatment period, rats from each group were randomly selected for echocardiographic measurements of LV dimensions, LV posterior wall thickness, and relative wall thickness (ratio of 2xposterior wall thickness/LV diastolic diameter).^{19 20} In vivo LV pressure measurements were performed before euthanasia, and LV meridional systolic wall stress (kdyn/cm²) was estimated.^{19 21}

Calcium-Dependent Contractile Reserve
The contractile reserve in isolated hearts from control (n=7) and L-NAME–treated rats (n=7) was evaluated using the isovolumic buffer-perfused rat heart preparation with constant coronary flow.^{19 21} To assess calcium-dependent contractile reserve, LV systolic pressure development was studied at 3 different perfusate calcium concentrations (0.6, 1.2, and 3.0 mmol/L) as previously described.^{20 21}

To investigate the contractile reserve in isolated myocytes, LV myocytes were prepared from additional control and L-NAME–treated rats as previously described (n=7 to 8 per group).^{22 23} In isolated myocytes, [Ca²⁺]_i was measured with the Ca²⁺-sensitive fluorescence indicator Fluo-3 as previously described.^{24 25 26} Myocytes were studied at 37°C and paced at 0.5 Hz. Simultaneous measurements of cell shortening and [Ca²⁺]_i were measured after 5 minutes of perfusion with 1.2 and 3.5x10 mmol/L Ca²⁺. In addition, the long axis myocyte area was quantified in quiescent isolated myocytes from control and L-NAME–treated rats using the NIH Image software.

Measurements of Tissue Cyclic GMP
The LV and aortic cyclic GMP were determined by enzymatic assay as previously described.^{25 27}

Left Ventricular RNA Measurements
Northern blot analyses were performed using 20 µg total RNA as previously described.^{19 21} Probes used were the cDNA fragment encoding the SR Ca2⁺ ATP-ase (SERCA-2, provided by D.H. MacLennan, University of Toronto), the cDNA fragment encoding the rat GAPDH, an 84-bp synthetic oligonucleotide complementary to the coding region of rat ANF, a 20-bp synthetic oligonucleotide complementary to the rat ß-myosin heavy chain (MHC) gene, and a 24-bp oligonucleotide fragment encoding the rat skeletal -actin.

Ribonuclease Protection Assay of angiotensin-converting enzyme (ACE) mRNA
LV ACE mRNA levels were quantified as previously described.²¹ The rat ACE probe was derived from clone pRace622 (provided by Dr M.A. Lee, Harvard Medical School, Boston, MA) which after linearization with Ava II yielded a 250-bp fragment. The rat ß-actin probe was derived from clone pSKrBac and yielded a 150-bp fragment after linearization with XhoI.

MHC iso-mRNA Analysis by Nuclease S1 Protection Assay
S1 nuclease protection assay of the myosin heavy chain (MHC-iso) mRNA was performed as described by Waspe et al.²⁸ The probe was a 61-base synthetic oligonucleotide that was designed to be complementary to a 41-nucleotide common coding sequence at the carboxyl end of both - and ß-MHC iso-mRNAs²⁸ and complementary to the final 15 nucleotides of ß-MHC iso-mRNA that significantly differs from those of -MHC iso-mRNA.²⁸ The probe was labeled with [-³²P dideoxyl] ATP (Amersham Corp, Arlington Heights, Ill) at 3' end and hybridized with 20 µg of total RNA in molar excess. S1 digestion was performed using Multi-NPA kit (Ambion, Austin, Tex) and was followed by separation of the protected fragments on a 5% polyacrylamide gel.

Western Analysis of SERCA-2 Protein Level
SERCA-2 protein levels were analyzed by Western analysis as previously described.²⁶

Statistical Analysis
All data are expressed as mean±SEM. Student’s unpaired t test was used where appropriate. Comparison between groups was performed by ANOVA comparison or ANOVA for repeated measures, where appropriate, followed by Fisher’s protected least significance test for post hoc analyses. P<0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of L-NAME on Blood Pressure and LV Mass
Chronic L-NAME treatment caused persistent severe hypertension (Figure 1). L-NAME treatment was associated with a lower body weight and no effect on tibial length, an index of body growth independent of the body mass (Table 1). LV weight and LV/body weight ratio were similar between L-NAME rats and controls. In addition, the long-axis myocyte area (>50 myocytes per rat from 3 to 4 rats per group) was similar between L-NAME rats and controls.

View larger version (12K): [in this window] [in a new window]   Figure 1. Tail cuff systolic arterial blood pressure in control and L-NAME–treated rats. L-NAME administration induced sustained severe systolic pressure overload over the entire treatment period.

View this table: [in this window] [in a new window]   Table 1. LV Hypertrophy and Blood Pressure

In Vivo Measurements
No L-NAME rat showed clinical signs of failure (tachypnea or edema). To investigate in vivo LV systolic function, we performed echocardiographic measurements in control and L-NAME rats before sacrifice (Table 2). In addition, we compared echocardiographic and hemodynamic indices of the L-NAME rats with the cohort of 6 weeks ascending aortic stenosis rats (Figure 2).^{19 20 21 22 23}

View this table: [in this window] [in a new window]   Table 2. In Vivo Hemodynamic and Echocardiographic Measurements

View larger version (42K): [in this window] [in a new window]   Figure 2. Representative examples of in vivo M-mode (top) and LV pressure (bottom) measurements in control (left), L-NAME–treated (center) and aortic stenosis (right) rats. Both L-NAME rats and aortic stenosis rat show similar increase in LV systolic pressure and preserved LV fractional shortening as compared with control rat. At this stage of compensatory LV hypertrophy in the aortic stenosis rat, LV diastolic pressure is already elevated. Remarkably, severe hypertrophy is present in the aortic stenosis rat, whereas no increase in the wall thickness or LV mass is observed in the L-NAME rat.

In L-NAME rats, both LV systolic and developed pressure per gram were significantly higher as compared with controls. Midwall fractional shortening, which is relatively independent of loading conditions,^{29 30} was also preserved as compared with controls (0.25±0.01% versus 0.23±0.01%, P=NS). In addition, the LV end-diastolic pressure (LVEDP) was not elevated as compared with controls. Thus, in spite of the absence of an increase in LV mass, L-NAME–induced hypertension was not associated with the depression of LV systolic pressure or elevation of LVEDP. Second, the level of LV systolic wall stress was elevated and similar in L-NAME and aortic stenosis rats; however, there was a marked difference in LV mass (Table 2). The relative wall thickness^{2 29 30} was similar between aortic stenosis rats and L-NAME rats. However, the increase in relative wall thickness in aortic stenosis rats reflects a marked increase in the wall thickness associated with an increase in LV mass; in contrast, in L-NAME rats, it reflects chiefly a decrease in internal LV dimension.

Isolated Heart and Myocytes Studies: Contractile Reserve
To study whether an increase in contractility also contributes to preserved in vivo LV systolic function in the L-NAME rats, we performed in vitro studies of the LV pressure-calcium relationship^{19 21} in isolated hearts (n=7 per group) and the shortening calcium relationship in myocytes^{22 26} (n=7 to 8 per group). At the identical LV balloon volume, comparable levels of LVEDP (10 mm Hg), heart rate and coronary flow per gram (data not shown), LV systolic pressure was similar at the low baseline calcium concentration of 0.6 mmol/L (Figure 3). However, at higher calcium concentrations of 1.2 and 3.0 mmol/L, the relationship between LV systolic pressure and calcium was shifted upward in L-NAME–treated rats compared with controls.

View larger version (11K): [in this window] [in a new window]   Figure 3. The LV pressure-calcium relationship in isolated buffer-perfused hearts from control and L-NAME–treated rats. At baseline calcium level (0.6 mmol/L), LV systolic pressure (LVSP) was similar between hearts from control and L-NAME–treated rats. In response to high calcium concentrations, L-NAME rats showed an upward shift in pressure-calcium relation as compared with controls.

To further examine calcium-dependent contractile function, LV-isolated myocytes from control and L-NAME–treated rats were paced at 0.5 Hz, and myocyte fractional shortening and [Ca²⁺]_i was measured in response to 1.2 and 3.5 mmol/L CaCl₂ at 37°C. Figure 4 shows the relationship between fractional myocyte shortening and peak systolic [Ca²⁺]_i in response to elevated perfusate calcium. There was no difference in baseline peak systolic [Ca²⁺]_i or fractional shortening between myocytes from L-NAME–treated rats and controls. Similar to the response of the isolated hearts, there was an upward shift in the relationship between myocyte shortening and peak systolic [Ca²⁺]_i at high perfusate calcium in myocytes from L-NAME–treated rats compared with control myocytes (P=0.09).

View larger version (19K): [in this window] [in a new window]   Figure 4. Relationship between myocyte shortening and peak systolic [Ca2+]i at baseline (1.2 mmol/L) and high (3.5 mmol/L) perfusate calcium concentrations in myocytes from control and L-NAME–treated rats. Peak systolic [Ca2+]i was similar between both groups at baseline and high perfusate calcium. There was a trend for enhanced myocyte shortening in response to calcium related to an upward shift in the cell-shortening peak systolic [Ca2+]i relationship consistent with an enhanced myofilament responsiveness.

Tissue Cyclic GMP Levels
Tissue cyclic GMP content was determined in aortic and LV tissues. As expected, L-NAME treatment caused a decrease in aortic cyclic GMP content (253±83 versus 746±103 fmol/mg, P<0.01). In contrast, LV cyclic GMP was unchanged in L-NAME rats compared with controls (87±10 versus 73±9 fmol/mg, P=NS).

Effects of L-NAME on LV Gene Expression
We then examined whether L-NAME–induced hypertension is associated with the load-induced changes in LV gene expression despite the absence of LV hypertrophy (Table 3). L-NAME treatment was associated with a 4-fold increase of LV ANF mRNA levels and a 5.5-fold increase in -skeletal actin LV mRNA levels. Unexpectedly, LV mRNA levels of ß-MHC and LV ACE mRNA were unchanged in L-NAME–treated rats. This suggests a dissociation between gene induction associated with pressure overload and hypertrophic growth per se. In addition, LV mRNA levels of SERCA-2 were increased; however, this did not translate into an increase in LV SERCA-2 proteins relative to controls (127±14% versus 99.9±6%, P=NS).

View this table: [in this window] [in a new window]   Table 3. LV Gene Expression

To investigate whether the preserved LV function in vivo in the presence of L-NAME–induced pressure overload is related to relative changes of steady state - and ß-MHC iso-mRNA levels, we performed quantitative S1 endonuclease assay in LV tissue of control, L-NAME rats, and aortic stenosis rats (Figure 5). Consistent with previous studies,^{27 28} control hearts expressed predominantly -MHC iso-mRNA (69±5% of total MHC). In LV tissue from aortic stenosis rats, there was a relative reduction in -MHC iso-mRNA and an increase in ß-MHC iso-mRNA (relative amount of -MHC iso-mRNA 24±9% of total MHC, P<0.05 versus controls). In contrast, in LV tissue from L-NAME rats, there was no significant change in the relative - and ß-MHC expression (relative amount of -MHC 60±4% of total MHC, P=NS versus controls). Thus, L-NAME–induced pressure overload is not associated with a switch in MHC isoform expression.

View larger version (28K): [in this window] [in a new window]   Figure 5. Representative blots of S1 endonuclease assay of MHC iso-mRNA expression. -MHC iso-mRNA is predominantly expressed in adult control LV, whereas in the AS rat, there is an upregulation of ß-isoform and decrease of -isoform. In L-NAME rats, there was a slight increase in the expression of ß-MHC iso-mRNA and no change in -MHC-iso-mRNA. AS indicates aortic stenosis.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study used chronic treatment with L-NAME as a potent tool to simultaneously induce hypertension and suppress adaptive LV hypertrophic growth to test the hypothesis that adaptation to pressure overload occurs even when hypertrophy is absent. L-NAME–induced pressure overload is associated with a distinct pattern of LV remodeling characterized by a decrease in LV chamber size relative to wall thickness in the absence of an increase in LV mass. Despite the lack of compensatory hypertrophy, L-NAME hypertension for 6 weeks is associated with the absence of heart failure and preserved in vivo LV function related to an enhanced pressure development and cardiomyocyte shortening in response to calcium.

Dissociation Between LV Hypertrophy and Induction of Fetal Genes
A novel feature of the present study was the comparison of hypertrophic growth in response to pressure overload in L-NAME and aortic stenosis animals. It is striking that hypertrophy did not develop in 6-week L-NAME–treated animals despite an elevated and similar LV systolic wall stress sufficient to cause an 2-fold increase in LV mass in 6-week aortic stenosis animals. Our comparison of the change in LV mass for a similar increase in LV pressure overload in 6-week L-NAME versus aortic stenosis rats supports the interpretation of earlier studies that the LV growth response is inappropriately low in L-NAME rats.^{5 6 7 8 11 12} The present study does not exclude the possibility of changes in LV mass in response to more prolonged L-NAME–induced hypertension. Some previous studies with higher doses or more prolonged treatment have observed a relative increase in LV mass ranging from 9% to 30%.^{9 10 31 32} Taken together, our study and these previous studies show that changes in LV mass are absent or modest in the face of severe sustained hypertension.

Several mechanisms are likely to account for blunted hypertrophic response. First, in addition to NO inhibition, L-NAME modulates amino acid delivery and polyamino acid synthesis.^{13 14 15 16 17 18} Second, in contrast to expected inhibitory effects of L-NAME on tissue cyclic GMP content,^{1 6 7 33 34} LV cyclic GMP content in response to chronic L-NAME treatment remained unchanged.⁶ Third, we did not observe changes in LV ACE mRNA expression that could result in increased angiotensin II production and modulation of the hypertrophic growth response.^{7 10 19 21} This contrasts with the observation of Takemoto et al,¹⁰ who (using higher doses of L-NAME) observed an upregulated systemic and local angiotensin system and a relative increase in LV mass. Fourth, the absence of hypertrophic growth may be related to L-NAME–induced vasoconstriction with myocardial ischemia;^{9 35} however, the absence of depressed LV systolic function in vivo or in the isolated heart preparation strongly argues against chronic ischemia.

Adaptation of the Adult Heart to Pressure Overload in Absence of Hypertrophy
The absence of compensatory hypertrophy in the presence of severe pressure overload would be expected to promote early cardiac dilatation and heart failure.² Our study indicates several mechanisms by which the heart can adapt to high systolic load without a pathologic increase in LV mass. The first mechanism is concentric geometric remodeling with a reduction of the LV chamber size relative to wall thickness that increases relative wall thickness (the ratio of posterior wall to the LV diastolic diameter), an adaptation which preserves LV pump function.^{29 30 36} Our observations are consistent with a recent study of Matsubara et al,¹² who also observed a decrease in LV volume, absence of LV hypertrophy, and preserved LV function in response to L-NAME–induced hypertension. The mechanisms which underlie this geometric remodeling are unclear because the decrease in LV dimension did not appear to be related to a change in myocyte size. It is a possibility that the decrease in LV dimension is related in part to the slight reduction in body mass or change in venodilation in L-NAME rats. We also did not address the possibility of differences in myocyte size in subendocardium versus subepicardium.

A second compensatory mechanism is an enhanced LV contractile reserve in response to calcium, which we observed in isolated hearts. Although we did not examine histological changes in matrix composition, other studies have demonstrated an increase in collagen in this model.^{9 10} Because changes in collagen deposition and matrix can alter the contractile response in the intact heart in vivo or in isolated hearts, we also examined the contractile response at the level of isolated myocytes. The enhanced pressure development in isolated hearts and enhanced myocyte shortening in response to calcium implicate an increased myocardial responsiveness to calcium. We²³ and others^{37 38} have shown that the acute depression of contractility by NO in rat myocytes is predominantly related to the depression of myofilament calcium responsiveness. Thus, we postulate that the augmentation of contractility in L-NAME–treated rats is related in part to the withdrawal of these mild depressant effects of constitutive NO on contractility and myofilament calcium sensitivity. In addition, in the present study, severe hypertension was not associated with an isoform switch of - and ß-MHC iso-mRNAs, typical for sustained mechanical pressure overload.^{39 40} The absence of such isoform switch could also contribute to preserved contractile function in vivo in the presence of pressure overload.⁴¹ Of interest, studies in transgenic animals with increased SERCA-2 expression have reported enhanced calcium transients and myocardial contractility.⁴² However, alterations in SERCA-2 expression do not appear to play an adaptive role in the present study because protein levels of SERCA-2 and calcium transients were unchanged by L-NAME treatment.

These observations in L-NAME hypertensive rats contrast with the molecular adaptation of aortic stenosis animals to pressure overload, which is characterized by a relative increase in ß-MHC expression and reduction in -MHC expression, as well as reduction in SERCA-2 expression. In contrast to L-NAME hypertensive rats, we²² and others⁴³ have previously shown that aortic banded rats at the stage of early concentric hypertrophy do not exhibit an enhanced myocyte contractile response to calcium, whereas this relationship is depressed during progression to failure.

Limitations and Conclusions
This study does not determine whether this early adaptation after 6 weeks of L-NAME–induced pressure overload will be successful in preventing the progression to heart failure during a longer period of L-NAME–induced hypertension. Second, determination of beneficial or adverse effects of chronic in vivo NO inhibition with L-NAME on contractile performance and hypertrophic growth in the conditions associated with excessive NO production, such as advanced heart failure, will require further investigation. Third, the potential use of L-NAME to suppress pathologic hypertrophy is limited by confounding vasoconstriction. Nonetheless, the present study supports the possibility that novel pharmacologic measures that suppress hypertrophic growth may be associated with beneficial geometric and molecular adaptations in pathologic pressure overload which suppress the progression to heart failure.

	Acknowledgments

 
This study was supported by a grant from National Heart, Lung, and Blood Institute Grant HL-38189 (to B.H.L. and E.O.W.), an award from NASA (to B.H.L.), and by the US Fogarty International Fellowship Award (NIH, F05 TW05261-01 [to J.B.]). We thank Lois Wiltberger for assistance in preparing this manuscript.

	Footnotes

 
Current affiliation of J.B. is Cardiologisch Centrum, Aalst, Belgium; current affiliation of M.T. is Tohoku University, Sendai, Japan.

Received March 5, 1999; revision received July 28, 1999; accepted August 11, 1999.

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