Chronic l-Arginine Administration Attenuates Cardiac Hypertrophy in Spontaneously Hypertensive Rats
Abstract Nitric oxide inhibits proliferation and migration of vascular smooth muscle cells and contractility of cardiomyocytes in vitro. In spontaneously hypertensive rats (SHR), evidence suggests intrinsic abnormalities of the l-arginine–nitric oxide axis, such as low cGMP-dependent protein kinase in the heart and abnormal l-arginine metabolism. To investigate the in vivo effect of l-arginine on cardiac hypertrophy, 30 SHR and 30 Wistar-Kyoto rats (WKY) were randomly grouped to receive l-arginine (7.5 g/L in drinking water) or vehicle for 12 weeks. l-Arginine treatment did not affect body weight or arterial pressure in either strain. In vehicle-treated animals, the heart/body weight ratio was significantly higher in SHR than in WKY (P<.01). l-Arginine treatment decreased the heart/body weight ratio in SHR (P<.05) but did not affect it in WKY. Expression of skeletal α-actin mRNA, known to be expressed in the hypertrophied myocardium, was attenuated in l-arginine–treated SHR compared with vehicle-treated SHR. Cardiac cGMP content and nitrate/nitrite content were less in SHR than WKY. l-Arginine treatment increased these levels only in SHR, suggesting enhanced nitric oxide production. Thus, chronic l-arginine administration attenuated cardiac hypertrophy independently of blood pressure and increased myocardial content of cGMP and nitrate/nitrite. Our results suggest that abnormality of the cardiac l-arginine–nitric oxide axis may play an important role in the pathogenesis of cardiac hypertrophy in SHR.
The lack of a close and parallel relation between blood pressure level and degree of cardiac hypertrophy has been demonstrated in both clinical studies and experimental animal models.1 2 3 In SHR, cardiac hypertrophy occurs before blood pressure elevation.2 3 Mechanisms of pressure-independent cardiac hypertrophy remain to be elucidated.
NO stimulates soluble guanylate cyclase to increase intracellular levels of cGMP and causes a reduction in intracellular calcium.4 5 6 NO inhibits mitogenesis and proliferation of smooth muscle cell7 8 and contraction of cardiac myocytes mediated through cGMP.9 10 11 12 13 In the heart, two distinct isoforms (inducible and constitutive) of NOS have been identified in cardiac myocytes.11
Abnormalities of the l-arginine–NO axis have been suggested in SHR. Plasma l-arginine concentration decreased significantly after stress stimuli in adult SHR with no changes in normotensive WKY, although basal concentrations of l-arginine were similar.14 Kuo et al15 found lower cardiac levels of cGMP and cGMP protein kinase in SHR than in normotensive rats. Furthermore, it is well known that endothelium-dependent vasodilation is impaired in SHR.16 Thus, it is possible that cardiac hypertrophy of SHR may be caused by an abnormal NO generating system.
In this study, we investigated whether the abnormal l-arginine–NO axis may play a role in cardiac hypertrophy by examining the effects of chronic l-arginine administration on blood pressure, heart weight, myocardial expression of the fetal isoform of α-actin mRNA, and myocardial contents of cGMP and NOx.
Thirty male SHR and 30 male WKY (7 weeks old) (Charles River Japan, Kanagawa) were randomly grouped to receive either l-arginine (Sigma Chemical Co) in drinking water (43 mmol/L, resulting in a daily intake of ≈0.63 mmol/kg) or distilled water as vehicle. Each of the four groups consisted of 15 weight- and age-matched rats. Throughout the study period, animals were housed in a room in which constant temperature (25±1°C) and humidity (60±5%) were maintained. The room was lighted automatically from 7 am to 7 pm. Rats were provided free access to water and rat chow (CE-2, Clea Japan) that consisted of 110 mmol of sodium, 280 mmol of potassium, and 89 mmol of arginine per kilogram. Animal care and treatment were conducted in conformity with institutional guidelines that are in compliance with international laws and policies (EEC Council Directive 86/609, OJL 358, December 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985).
After 12 weeks of oral administration of l-arginine or vehicle, rats were weighed and anesthetized with pentobarbital (50 mg/kg), and a catheter (Intramedic PE10 connected to PE50) was passed into the lower abdominal aorta via the right femoral artery as previously described.17 After rats recovered from anesthesia, MAP and HR were recorded (model TP-101T, Nihon Koden) for 1 hour; the reported pressures and HR represent the average during the last 15 minutes of the recording period. After direct measurement of MAP and HR, rats were killed by pentobarbital overdose, and the hearts were immediately removed and weighed. The heart was immediately frozen by liquid nitrogen and was saved for primer extension analysis of the expression of cardiac and skeletal α-actin mRNAs, cardiac cGMP and NOx content, and cardiac norepinephrine measurement.
Primer Extension Analyses
Total RNA was extracted from the myocardium by use of a previously described method.18 Expressions of cardiac and skeletal α-actin mRNA in cardiac myocytes were measured simultaneously in the same RNA sample by the primer extension method.18 The oligonucleotide used for the reaction was the 18-mer primer (5′ CGACCCACGATGGATGGG 3′) complementary to codons 31 through 37 in exon 2, which is identical in the two forms of sarcomeric actin mRNAs, synthesized at Takara Industry. The oligonucleotide was end-labeled with [γ-32P]ATP by use of T4 polynucleotide kinase and then extended with reverse transcriptase. The extension product was loaded onto denatured 6% acrylamide/urea gels and fractionated by electrophoresis. Gels corresponding to autoradiographic bands were excised, and their 32P content was measured by scintillation counting.
Measurements of Cardiac cGMP, NOx, and Norepinephrine
The frozen heart was minced rapidly and placed in ice-cold 0.3N perchloric acid (total volume, 12 mL). Immediately after the heart was homogenized, the homogenate was centrifuged at 20 000g for 20 minutes, and the supernatant was stored at −85°C until analyzed. Cardiac cGMP level was determined in duplicate by use of a radioimmunoassay kit (Yamasa). Cardiac NOx content was measured by a colorimetric assay based on Griess reaction.19 Cardiac norepinephrine content was determined by high-performance liquid chromatography with electrochemical detection (HLC-725CA, Tohso).
All data are expressed as mean±SEM unless otherwise indicated. Experimental groups were compared by ANOVA and, when appropriate, with Scheffé’s test for multiple comparisons.
Hemodynamics, Heart Weight
Fig 1A⇓ shows MAP after 12 week-administration of l-arginine. MAP was significantly higher in SHR than in WKY, whereas MAP was identical between vehicle-treated and l-arginine–treated groups, indicating chronic l-arginine treatment had no depressor effect in WKY and SHR. HR was also higher in SHR than in WKY, although it was similar between vehicle-treated and l-arginine–treated groups (vehicle-treated WKY, 270±8; l-arginine–treated WKY, 271±9; vehicle-treated SHR, 313±12; and l-arginine–treated SHR, 311±12 beats per minute; P<.05 between strains).
Although body weight was lower in SHR than in WKY at the same age, l-arginine treatment for 12 weeks did not affect body weight in either strain (vehicle-treated WKY, 389±10; l-arginine–treated WKY, 383±8; vehicle-treated SHR, 357±10; and l-arginine–treated SHR, 354±8 g; P<.05 between strains).
Cardiac weight (1.09±.03 versus 1.34±.03 g, vehicle-treated WKY versus vehicle-treated SHR, P<.01) and heart/body weight ratio (P<.01; Fig 1⇑) were significantly higher in vehicle-treated SHR than in vehicle-treated WKY, indicating the presence of cardiac hypertrophy in SHR. Although l-arginine treatment did not affect cardiac weight (1.09±.03 versus 1.08±.02 g, vehicle-treated WKY versus l-arginine–treated WKY, P=NS) and heart/body weight ratio (P=NS; Fig 1⇑) in WKY, l-arginine treatment significantly decreased cardiac weight (1.34±.03 versus 1.16±.02 g, vehicle-treated SHR versus l-arginine–treated SHR, P<.01) and heart/body weight ratio (P<.05; Fig 1⇑) in SHR.
Expression of Skeletal and Cardiac α-Actin mRNA
Expression of skeletal α-actin mRNA of myocytes from the heart was observed in vehicle-treated SHR (Fig 2A⇓). Chronic administration of l-arginine markedly attenuated expression of skeletal α-actin mRNA (Fig 2A⇓) and significantly decreased the expression ratio of skeletal/cardiac α-actin mRNA (P<.05, n=7; Fig 2B⇓) in SHR.
Cardiac cGMP, NOx, and Norepinephrine Content
In vehicle-treated animals, cardiac cGMP content tended to be less in SHR than in WKY, although this difference was not statistically significant (P=NS, n=9; Fig 3⇓). Chronic l-arginine administration significantly increased cardiac content of cGMP in SHR (P<.05, n=9; Fig 3⇓), whereas cardiac cGMP content in l-arginine–treated WKY was equivalent to that of vehicle-treated WKY (n=9; Fig 3⇓). Cardiac NOx content was significantly less in vehicle-treated SHR than in vehicle-treated WKY (P<.05, n=9; Fig 3⇓). Chronic l-arginine administration significantly increased cardiac content of NOx in SHR (P<.05, n=9; Fig 3⇓), whereas the difference in cardiac NOx content between l-arginine–treated and vehicle-treated WKY was not statistically significant (P=NS, n=9; Fig 3⇓). Cardiac norepinephrine content was similar between the two groups of SHR (4.3±0.4 versus 5.1±0.3 nmol/mg, vehicle-treated SHR versus l-arginine–treated SHR, n=7, P=NS).
In the present study, chronic l-arginine treatment attenuated cardiac hypertrophy in SHR without changes in blood pressure, whereas l-arginine treatment did not affect heart weight or blood pressure in WKY. Our results indicate that l-arginine administration attenuated cardiac hypertrophy, specifically in SHR, independently of blood pressure. Cardiac hypertrophy is characterized not only by an increase in heart weight but also by reexpression of fetal isoform of contractile proteins such as skeletal α-actin.18 20 The significant decrease in skeletal α-actin mRNA in l-arginine–treated SHR may indicate that l-arginine administration attenuated hypertrophy of the myocyte. The amount of myocardial cGMP and NOx was greater in l-arginine–treated SHR than in vehicle-treated SHR, and myocardial norepinephrine content was similar between two groups of SHR. Our results may suggest that the attenuation of cardiac hypertrophy by l-arginine was mediated by the l-arginine–NO pathway of the myocyte irrespective of hemodynamic and neural effects.
In this experiment, l-arginine did not affect blood pressure in SHR. Although acute administration of l-arginine reduces blood pressure in hypertensive humans21 and animals,22 chronic administration of l-arginine did not affect the development of hypertension in SHR22 or stroke-prone SHR.23 It is not known why chronic administration of l-arginine does not reduce blood pressure in SHR. It is possible that compensatory mechanisms may take over during the chronic phase of l-arginine administration.
l-Arginine attenuated cardiac hypertrophy only in SHR but did not affect cardiac weight in WKY. Thus, the effect of l-arginine was specific for SHR. Stier et al23 reported that l-arginine administration did not attenuate hypertrophy in stroke-prone SHR. The reasons for the different effects of l-arginine on cardiac hypertrophy between SHR in our study and stroke-prone SHR in the study of Stier et al are not clear. We administered l-arginine for 3 months, whereas they did so for only 1 month. Therefore, the different results from the previous study23 may have been due to the period of l-arginine administration. The other possibility may have been the different vehicles used to dissolve l-arginine; we dissolved l-arginine in distilled water, whereas Stier et al used 1% NaCl, which may exacerbate hypertensive organ damage.24 It is also possible that higher blood pressure in stroke-prone SHR may have masked the protective effects of l-arginine on cardiac hypertrophy.
In several experimental models, the development of cardiac hypertrophy induced mechanically20 and humorally18 is accompanied by the reexpression of fetal isoforms of contractile protein genes. In this experiment, we investigated the expression of skeletal α-actin mRNA of the myocardium, which is known as a fetal form of sarcomeric actin mRNA and which is reexpressed in the hypertrophied myocardium.18 Chronic administration of l-arginine attenuated the expression of skeletal α-actin mRNA and significantly decreased the expression ratio of skeletal/cardiac α-actin mRNA. Thus, our results may indicate that administration of l-arginine attenuated hypertrophy of the cardiac myocytes of SHR.
In cardiac myocytes, both constitutive and inducible forms of NOS have been shown to be expressed in mammalian hearts.11 To examine mechanisms by which chronic l-arginine administration attenuated hypertrophy of the myocytes of SHR, we measured myocardial content of cGMP, which mediates the intracellular signal transduction in response to NO by reducing cytosolic free Ca2+.4 5 6 Since cGMP is a second messenger of other substances, such as atrial natriuretic peptide,6 increased cGMP content does not necessarily indicate increased production of NO. We therefore measured myocardial content of NOx as an index of NO production. In vehicle-treated animals, myocardial content of cGMP tended to be less, and cardiac NOx contents were significantly less in SHR than in WKY, suggesting impaired myocardial NO production in SHR. Chronic l-arginine treatment significantly increased cardiac cGMP and NOx content in SHR but not in WKY. Thus, our results suggest that the attenuation of cardiac hypertrophy by l-arginine may be mediated by increased myocardial production of NO.
Several lines of evidence suggest that SHR have intrinsic abnormalities of the l-arginine–NO axis. Endothelium-dependent vasodilatory responses to various stimuli are impaired.16 An impairment of l-arginine metabolism after stress stimuli has been suggested in SHR.14 Furthermore, it has been shown that SHR have lower levels of cardiac cGMP and cGMP-dependent protein kinase than do normotensive rats.15 Possible mechanisms of dysfunction of the l-arginine–NO axis have been proposed to occur at several levels16 and may involve (1) altered expression of receptors, (2) impaired signal transduction mechanism, (3) decreased activity of NOS, (4) reduced intracellular availability of l-arginine, (5) increased breakdown of NO formed from l-arginine, (6) reduced responsiveness of target cell to endogenous NO, and (7) increased formation of an inhibitor of the l-arginine–NO pathway, such as asymmetrical dimethyl arginine. The decreased basal cardiac content of NOx and cGMP in SHR compared with WKY may suggest decreased activity of NOS and/or reduced intracellular availability of l-arginine in the heart. It is conceivable that chronic l-arginine administration enhanced production of NO in SHR and hence attenuated cardiomyocyte hypertrophy.
Recently, several laboratories reported the effects of NO on cardiac hypertrophy in vitro25 and in vivo.26 27 28 Harding et al25 demonstrated that neither interleukin-1β–induced endogenous NO synthesis nor exogenous nitroglycerin inhibited protein synthesis in cultured cardiac myocytes from normotensive rats under basal or phenylephrine-stimulated conditions. Chronic inhibition of NO synthesis in normotensive animals only partially induced cardiac hypertrophy despite significant blood pressure elevation.26 27 In the present study, l-arginine, a substrate of NO, did not affect either cardiac weight or cardiac NO production as assessed by cardiac cGMP and NOx content in WKY. Therefore, NO may not be involved in the hypertrophic process in the normal heart. On the other hand, Arnal et al28 demonstrated that chronic NO synthesis inhibition increased left ventricular weight significantly in SHR but not in WKY. In the present study, chronic l-arginine treatment attenuated cardiac hypertrophy with concomitant increases in cardiac content of cGMP and NOx in SHR. Thus, an abnormality of the l-arginine–NO pathway may be involved in cardiac hypertrophy in SHR.
Since acute inhibition of NO synthesis is known to activate the sympathetic nervous system,29 30 it is possible that chronic administration of l-arginine may have attenuated cardiac hypertrophy via sympathetic inhibition. However, this possibility is unlikely, because myocardial norepinephrine content was similar between the two groups of SHR.
It has been demonstrated that NO modulates the release and effect of several vasoactive substances that possess trophic effects, such as renin.31 l-Arginine may have putative effects on the cardiac renin-angiotensin system or other autocrine-paracrine systems and may inhibit cardiac hypertrophy. These possibilities were not examined in the present study.
Although we think that l-arginine treatment attenuated the development of hypertrophy, it is possible that it produced regression of existing hypertrophy, since it is well known that cardiac hypertrophy is already present in SHR at 7 weeks of age.2
As ventricular hypertrophy progresses, it is accompanied by cardiac fibrosis.1 Although we did not examine the amount of fibrosis in the present study, it is of interest whether chronic l-arginine treatment reduced cardiac fibrosis, since nitric oxide inhibits collagen synthesis in vitro.8 This possibility remains to be elucidated.
In conclusion, we demonstrated that basal cardiac cGMP and NOx content was less in SHR than in WKY and that chronic l-arginine administration attenuated cardiac hypertrophy independently of blood pressure and increased myocardial content of cGMP and NOx in SHR. Our results suggest that abnormality of the cardiac l-arginine–NO axis may play an important role in the pathogenesis of cardiac hypertrophy in SHR.
Selected Abbreviations and Acronyms
|MAP||=||mean arterial pressure|
|SHR||=||spontaneously hypertensive rats|
This study was supported in part by a grant from Kimura Memorial Heart Foundation, Kurume, and a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture.
Reprint requests to Hidehiro Matsuoka, MD, Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830, Japan.
- Received September 5, 1995.
- Revision received September 22, 1995.
- Accepted September 22, 1995.
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