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(Hypertension. 1996;28:627-634.)
© 1996 American Heart Association, Inc.

Articles

Role of Angiotensin-Converting Enzyme, Adrenergic Receptors, and Blood Pressure in Cardiac Gene Expression of Spontaneously Hypertensive Rats During Development

Kensuke Ohta; Shokei Kim; Hiroshi Iwao

the Department of Pharmacology, Osaka (Japan) City University Medical School.

Correspondence to Shokei Kim, MD, Department of Pharmacology, Osaka City University Medical School, 1-4-54 Asahimachi, Abenoku, Osaka 545, Japan.

	Abstract

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We undertook this study to investigate the regulatory mechanism of cardiac gene expression in spontaneously hypertensive rats (SHR) during development. We measured cardiac mRNAs by Northern blot analysis. In 9-week-old SHR at the very early stage of cardiac hypertrophy, the expression of various cardiac genes related to the regulation of cardiac contraction and relaxation was already significantly changed compared with control Wistar-Kyoto rats, indicating that cardiac molecular changes are responsible for cardiac remodeling or the modulation of cardiac performance in SHR. We gave various types of antihypertensive drugs, at oral doses causing a mild and comparable hypotensive effect, to 27-week-old SHR to examine the effects on the altered cardiac gene expression. Imidapril, an angiotensin-converting enzyme inhibitor, normalized the increased gene expression of atrial natriuretic polypeptide and collagen types I and III and the decreased expression of -myosin heavy chain in SHR heart. Atenolol (a ß₁-blocker) combined with doxazosin did not affect cardiac ANP and -myosin heavy chain expression of SHR but normalized the increased collagen expression. In contrast, despite a hypotensive effect comparable to these two drug treatments, doxazosin (an ₁-blocker) alone or manidipine (a calcium antagonist) did not normalize these altered gene expressions of SHR. These results show that the cardiac renin-angiotensin system is involved in the altered cardiac gene expression in SHR. The ß₁- but not ₁-adrenergic receptor is also responsible for the increased cardiac collagen expression in SHR.

Key Words: hypertrophy • angiotensin-converting enzyme • receptors, adrenergic • aging • hypertension, genetic

	Introduction

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Accumulating evidence indicates that LVH is characterized not only by an increase in myocyte size but also by qualitative alterations in myocytes and interstitium.^{1 2} Qualitative changes in cardiac myocytes include increased expression of "fetal" genes, such as ß-MHC, skeletal -actin, and ANP.^{1 2} Furthermore, the gene expression of SR Ca²⁺-ATPase, a potent regulator of intracellular calcium handling, is reported to be decreased in progressed LVH and failing hearts.^{3 4} On the other hand, changes in the interstitium in LVH include structural remodeling (fibrosis), which is at least partly due to the altered expression of extracellular matrix components.^{5 6 7} The proteins encoded by fetal contractile protein genes (ß-MHC and skeletal -actin) are known to be different in contractile property from the corresponding adult isoforms (-MHC and cardiac -actin, respectively).^{1 8} Furthermore, the increase in interstitial collagen volume is responsible for the abnormal myocardial stiffness and therefore the diastolic dysfunction that often accompanies progressed LVH.^{5 6} Thus, qualitative changes in LVH, including altered cardiac gene expression, are involved in the modulation of cardiac performance or onset of cardiac failure.

In humans, hypertension is one of the most important diseases causing LVH.⁹ However, the molecular mechanism of the modulation of cardiac performance in hypertension is poorly understood. In SHR, the most popular model of human essential hypertension, some of the above-mentioned qualitative changes in the LV, eg, enhanced expression of ANP,^{10 11} skeletal -actin,¹¹ and extracellular matrix components,¹² occur at the early stage of development. Moreover, recent works on SHR or stroke-prone SHR by us^{11 12 13} and other researchers¹⁴ suggest that the altered cardiac gene expression in LVH may be not only due to hypertension but also to the renin-angiotensin system. However, no comprehensive study concerning cardiac gene expression of SHR during development has been reported. Furthermore, little information is available for direct comparison of ACE inhibitors with other types of antihypertensive drugs with respect to their effects on cardiac gene expression.

In the present study, we first compared cardiac gene expression in SHR and WKY during development to examine the effects of age and hypertension on cardiac gene expression. Second, we evaluated the effects of various types of antihypertensive drugs on cardiac gene expression in SHR to elucidate the mechanism of the altered gene expression in hypertensive LVH.

	Methods

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Drugs and Rats
Imidapril, an ACE inhibitor,¹⁵ was donated by Tanabe Seiyaku Co, Ltd. Doxazosin, an ₁-adrenoreceptor blocker,¹⁶ was donated from Pfizer Pharmaceutical Inc. Atenolol, a ß₁-adrenoreceptor blocker,¹⁷ was a gift from ZENECA Yakuhin KK. Manidipine, a calcium antagonist,¹⁸ was donated by Takeda Chemical Industries, Ltd.

Male WKY and SHR were purchased from Japan SLC (Shizuoka, Japan). They were fed standard laboratory chow (CE-2, Clea Japan) and given tap water ad libitum.

Experimental Procedures
All procedures were in accordance with institutional guidelines for animal research. In the first experiments, we used SHR and WKY at the ages of 5, 9, 17, and 27 weeks (each group, n=5) to examine the effects of age and hypertension on LV gene expression. Blood pressure and heart rate of rats were measured by the tail-cuff method (TK-370A, Neuroscience Inc). For extraction of cardiac RNA, rats were killed by decapitation; the whole heart was rapidly excised and rinsed with cold saline; and the LV was separated from the RV and atria. After being weighed, the LV tissues were immediately frozen in liquid nitrogen and stored at -80°C until extraction of total RNA.

In the second experiments, to evaluate the effects of antihypertensive drugs on cardiac gene expression of SHR, we randomly separated 27-week-old SHR with established hypertension into five groups as follows: (1) treatment with vehicle (5% gum arabic solution) (n=6), (2) treatment with imidapril (5 mg/kg per day) (n=5), (3) treatment with doxazosin (5 mg/kg per day) (n=5), (4) treatment with a combination of atenolol and doxazosin (200 and 5 mg/kg per day, respectively) (n=6), and (5) treatment with manidipine (3 mg/kg per day) (n=7). Additionally, 27-week-old WKY were treated with vehicle (n=7). All the drugs were suspended in 5% gum arabic solution and were given orally by gastric gavage every morning for 7 days (from the age of 27 to 28 weeks). Preliminary experiments showed that the above-mentioned dose of each drug causes comparable and mild hypotensive effects in SHR. Systolic pressure and heart rate were measured before and after drug administration. Blood pressure was measured at the time when each drug showed the maximal hypotensive effect on SHR by a single administration (ie, 1 to 3 hours after dosing of doxazosin¹⁶ and manidipine¹⁸ and 3 to 6 hours after dosing of imidapril¹⁵ ). After 7 days of drug treatment, rats were decapitated, and the LV and RV were immediately excised, frozen in liquid nitrogen, and stored at -80°C, as described above.

Oligonucleotide and cDNA Probes
For measurement of mRNA levels for - and ß-MHCs¹⁹ and skeletal and cardiac -actins,²⁰ oligonucleotides complementary to their unique 3' flanking sequences were synthesized and used as probes as previously described.²¹ The oligonucleotide probes were 5' end-labeled with [³²P]ATP (specific activity, 6000 Ci/[mmol/L]). cDNA probes used were as follows: Rat ₁(I) collagen cDNA, a 1.3-kb Pst I–BamHI fragment²² ; mouse ₁(III) collagen, a 1.8-kb EcoRI–EcoRI fragment²³ ; ₁(IV) collagen, a 0.83-kb Ava I–Pst I fragment²⁴ ; rat TGF-ß1, a 1.2-kb HindIII–Xba I fragment²⁵ ; rabbit SR Ca²⁺-ATPase, a 2.0-kb BamHI fragment²⁶ ; and rat GAPDH, a 1.3-kb Pst I–Pst I fragment.²⁷ An 825-bp rat ANP cDNA probe was synthesized in our laboratory as previously described.¹¹ Each cDNA probe was labeled with [³²P]dCTP (specific activity, 3000 Ci/[mmol/L], EI du Pont de Nemours & Co, Inc) by a random primer extension method.

Northern Blot Hybridization
RNA extraction and Northern blot hybridization were performed as previously described in detail.²⁸ In brief, total RNA was extracted from the individual LV by the acid guanidinium thiocyanate/phenol/chloroform method,²⁹ dissolved in 0.1% diethyl pyrocarbonate–treated water, and stored at -80°C until use. The RNA concentration was spectrophotometrically determined at 260 nm.

Total RNA (20 µg) was denatured by incubation with 1 mol/L deionized glyoxal/50% dimethyl sulfoxide at 50°C for 1 hour and electrophoresed on a 1% agarose gel at 50 V. The 28S and 18S rRNAs in gels were stained with ethidium bromide for confirmation of the integrity of applied RNA and verification that the same amounts of RNA were applied to each lane. RNAs in the gel were then transferred to a nylon membrane (GeneScreen Plus, EI du Pont de Nemours & Co, NEN Products), followed by hybridization with each specific oligonucleotide or cDNA probe and autoradiography. The density of each mRNA band, obtained from autoradiography, was digitized by an optical scanner (Epson GT-8000, Seiko) and measured by use of the public domain Image program (National Institutes of Health). For all RNA samples, the density of an individual mRNA band was divided by that of GAPDH mRNA, a housekeeping gene, to correct for the difference in RNA loading and transfer to a nylon membrane as well as to verify the specificity of the change in mRNA levels. The same membrane was rehybridized with another oligonucleotide or cDNA probe after the previous probe was stripped off by boiling for 20 minutes in 0.1x SSC solution containing 1% sodium dodecyl sulfate.

Statistical Analysis
All data are expressed as mean±SE. Age-related change in body weight, blood pressure, heart rate, LV weight, and cardiac mRNA levels in SHR and WKY were evaluated by two-way ANOVA, followed by the least-squares means test. The effects of antihypertensive drugs on the above-mentioned parameters of SHR were evaluated by use of one-way ANOVA followed by Duncan's multiple range test. The differences were considered statistically significant at a value of P<.05.

	Results

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Age-Related Changes in Blood Pressure, Heart Rate, and LV Weight
As shown in Fig 1, blood pressure of SHR was increased with age and reached 224±4 mm Hg at 27 weeks of age. Heart rate of 5-week-old SHR was not significantly different from that in WKY of the same age. Heart rate of WKY decreased with aging, whereas that of SHR did not. Consequently, heart rate of SHR at 17 and 27 weeks was higher than that of age-matched WKY. LV weight, corrected for body weight, of SHR did not increase at 9 and 17 weeks of age, whereas that of WKY decreased with age, in good agreement with a previous report.¹⁷ Consequently, LV weight–body weight ratio of SHR was greater than in age-matched WKY at 9, 17, and 27 weeks.

View larger version (26K): [in this window] [in a new window]   Figure 1. Age-related changes in blood pressure (A), heart rate (B), body weight (C), and ratio of LV weight to body weight (D) of WKY () and SHR (). Each plot (n=5) represents mean±SE. *P<.05, P<.01 vs age-matched WKY.

Age-Related Changes in Cardiac Gene Expression
Marker Genes for Myocyte Phenotype
As shown in Figs 2 and 3, in the LV of WKY, aging decreased -MHC mRNA levels (47% decrease at 27 compared with 5 weeks of age, P<.001), whereas aging increased ß-MHC mRNA levels (3.8-fold increase at 27 compared with 5 weeks of age, P<.001) (Fig 3A and 3B). -MHC expression in SHR was higher (P<.01) at 9 weeks and lower (P<.01) at 27 weeks compared with that in age-matched WKY (Fig 3A). On the other hand, no significant difference in ß-MHC expression was observed between WKY and SHR at all ages examined (Fig 3B). The ratio of ß- to -MHC mRNAs did not differ between WKY and SHR at 5, 9, and 17 weeks of age but was 2.1-fold higher (P<.01) in 27-week-old SHR than in WKY because of the significant decrease in -MHC mRNA (Fig 3C).

View larger version (52K): [in this window] [in a new window]   Figure 2. Typical autoradiograms of LV mRNAs for -MHC (A), ß-MHC (B), ANP (C), skeletal -actin (Sk-actin) (D), cardiac -actin (Ca-actin) (E), SR Ca2+-ATPase (F), and GAPDH (G) from 5-, 9-, 17-, and 27-week-old WKY and SHR. Sizes of mRNA bands were 7.1 kb for -MHC, 7.1 kb for ß-MHC, 0.9 kb for ANP, 1.7 kb for Sk-actin, 1.7 kb for Ca-actin, 4.5 kb for SR Ca2+-ATPase, and 1.4 kb for GAPDH.

View larger version (32K): [in this window] [in a new window]   Figure 3. Age-related changes in LV mRNA levels for sarcomeric contractile proteins, ANP, and SR Ca2+-ATPase in WKY (open bars) and SHR (shaded bars). Abbreviations are as in Fig 2 legend. In individual RNA samples, the density of each autoradiographic band was corrected for that of the GAPDH band. Each bar (n=5) represents mean±SE. Mean value in 5-week-old WKY was represented as 1. P<.01.

In the LV of WKY, ANP gene expression increased with age (3.3-fold increase at 27 compared with 5 weeks of age, P<.001) (Fig 3D), whereas that of skeletal -actin was unchanged with age, except for a 51% decrease at 9 weeks (P<.05 compared with 5 weeks) (Fig 3E). In the LV of SHR, not only ANP but also skeletal -actin markedly increased with age, and ANP and skeletal -actin mRNA levels were higher in SHR at 9, 17, and 27 weeks compared with age-matched WKY (Fig 3D and 3E). On the other hand, cardiac -actin gene expression was almost unaffected by age in the LV of both WKY and SHR (Fig 3F).

The expression of SR Ca²⁺-ATPase in the LV of WKY increased 1.3-fold at 17 weeks of age compared with 9-week-old WKY (P<.001), whereas that in the LV of SHR increased at an earlier age (9 weeks) (P<.001) (Fig 3G). Consequently, SR Ca²⁺-ATPase expression in SHR was 1.3-fold higher than in WKY at 9 weeks, but there was no difference between the strains at 5, 17, and 27 weeks.

Extracellular Matrix Component and TGF-ß1 Gene Expressions
As shown in Figs 4 and 5, collagen types I, III, and IV and TGF-ß1 mRNA levels in the LV of WKY significantly decreased with age (by 71%, 82%, 66%, and 40%, respectively, at 27 compared with 5 weeks of age, P<.001). In the LV of SHR, all these collagen mRNA levels were lower than those in WKY at 5 weeks, higher than those in WKY at 9 weeks, and were not significantly different from those in WKY at 17 weeks (Fig 5A through 5C). At 27 weeks, collagen types I and III mRNA levels were 1.8- and 2.2-fold higher, respectively, in SHR than WKY, but no significant difference was found in collagen type IV between SHR and WKY (Fig 5A through 5C). TGF-ß1 mRNA levels did not differ between SHR and WKY at all ages examined (Fig 5D).

View larger version (67K): [in this window] [in a new window]   Figure 4. Typical autoradiograms of LV mRNA for collagen (Co) types I, III, and IV; TGF-ß1; and GAPDH from 5-, 9-, 17-, and 27-week-old WKY and SHR. Sizes of mRNA bands were 4.7 and 5.7 kb for Co I, 5.9 kb for Co III, 6.8 kb for Co IV, 2.5 kb for TGF-ß1, and 1.4 kb for GAPDH.

View larger version (43K): [in this window] [in a new window]   Figure 5. Age-related changes in LV mRNA levels for collagen (Co) types I, III, and IV and TGF-ß1 in WKY (open bars) and SHR (shaded bars). In individual RNA samples, the density of each autoradiographic band was corrected for that of the GAPDH band. Each bar (n=5) represents mean±SE. Mean value in 5-week-old WKY was represented as 1. *P<.05, P<.01.

Short-term Effects of Antihypertensive Treatment on Blood Pressure, Heart Rate, LV and RV Weights, and Cardiac Gene Expression in SHR
In the present study, we gave various types of antihypertensive drugs to SHR for 7 days (from 27 to 28 weeks) to evaluate the effects on cardiac gene expression. As shown in the Table, treatment of SHR with imidapril, doxazosin, atenolol combined with doxazosin, and manidipine lowered blood pressure only to a mild extent. Blood pressure did not differ significantly among the four SHR groups treated with antihypertensive drugs. Heart rate was not affected by imidapril or doxazosin but slightly increased by manidipine. The combination of atenolol and doxazosin significantly decreased heart rate of SHR to levels similar to those of WKY. These drugs did not significantly decrease the LV weight of SHR, although imidapril alone or atenolol combined with doxazosin tended to decrease LV weight.

View this table: [in this window] [in a new window]   Table 1. Blood Pressure, Heart Rate, Body Weight, and Ratios of Left and Right Ventricular Weights to Body Weight in Study Rats Treated With Vehicle or Antihypertensive Drugs for 7 Days

As shown in Figs 6 and 7, cardiac -MHC mRNA level in the LV of vehicle-treated SHR was lower by 30% than in WKY, whereas skeletal -actin, ANP, and collagen types I and III mRNAs in vehicle-treated SHR were 2.1-, 2.0-, 1.6-, and 1.8-fold, respectively, higher than in WKY. These results in vehicle-treated 28-week-old SHR were similar to those in 27-week-old SHR used to evaluate the age-related change (Fig 3). In addition, cardiac ß-MHC, SR Ca²⁺-ATPase, collagen type IV, and TGF-ß1 mRNA levels did not differ significantly between vehicle-treated SHR and WKY (data not shown), consistent with the results obtained for 27-week-old rats (Fig 3).

View larger version (75K): [in this window] [in a new window]   Figure 6. Typical autoradiograms of LV mRNAs from SHR treated with vehicle (V), imidapril (I), doxazosin (D), atenolol combined with doxazosin (D/A), or manidipine (M) for 7 days from 27 to 28 weeks of age. WKY were treated with vehicle for 7 days from 27 to 28 weeks of age. SK-actin indicates skeletal -actin; Co I, Co III, collagen types I and III.

View larger version (52K): [in this window] [in a new window]   Figure 7. Effects of 7 days of antihypertensive treatment on LV mRNA levels of SHR. In individual RNA samples, the density of each autoradiographic band obtained from Northern blot analysis was corrected for that of the GAPDH band. Each bar (n=5-7) represents mean±SE. Mean value in WKY was represented as 1. Abbreviations as in Fig 6 legend. *P<.05, P<.01 vs vehicle.

Treatment of SHR with imidapril normalized the reduced expression of -MHC and enhanced expression of ANP and collagen types I and III (Fig 7). In contrast, doxazosin or manidipine, despite hypotensive effects comparable to those with imidapril, did not normalize these altered gene expressions in the LV of SHR (Fig 7). Atenolol in combination with doxazosin significantly suppressed the enhanced cardiac expression of collagen types I and III in SHR but failed to normalize the changes in -MHC and ANP expression (Fig 7). The increased skeletal -actin in SHR could not be significantly suppressed by any drugs, although imidapril tended to suppress it. In addition, none of the drug treatments used in the present study affected the cardiac expression of ß-MHC, cardiac -actin, SR Ca²⁺-ATPase, collagen type IV, or TGF-ß1 in SHR (data not shown).

As shown in Fig 8, collagen type I mRNA level was 1.5-fold higher in the RV of vehicle-treated SHR compared with WKY. However, mRNA levels for -MHC, skeletal -actin, ANP, and collagen type III did not significantly differ between the RV of vehicle-treated SHR and WKY. As in the case of the LV, -MHC expression in the RV of SHR was significantly increased by treatment of SHR with imidapril, and collagen types I and III expression was decreased by both imidapril and the combination of doxazosin and atenolol. However, in contrast to the case in the LV, imidapril did not significantly affect ANP expression in the RV of SHR. On the other hand, treatment with doxazosin alone or manidipine did not significantly affect expression of these genes in the RV of SHR, similar to their effects in the LV tissue (Fig 8).

View larger version (45K): [in this window] [in a new window]   Figure 8. Effects of 7 days of antihypertensive treatment on RV mRNA levels of SHR. In individual RNA samples, the density of each autoradiographic band obtained from Northern blot analysis was corrected for that of the GAPDH band. Each bar (n=5-7) represents mean±SE. Mean value in WKY was represented as 1. Abbreviations as in Fig 6 legend. *P<.05, P<.01 vs vehicle.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
ß-MHC, ANP, and skeletal -actin are well-known markers of fetal phenotype in the rat heart.^{1 2} In the LV of WKY, we found a decrease in -MHC (adult phenotype) mRNA and an opposite increase in ß-MHC mRNA with aging, thereby showing that cardiac development in WKY is associated with the shift of MHC to a fetal phenotype at the molecular level. Interestingly, in contrast to the age-dependent increase in ANP and skeletal -actin mRNAs in the LV of SHR compared with WKY, ß-MHC mRNA levels in SHR did not differ from those in WKY throughout development. On the other hand, previous work showed that ß-MHC mRNA is dramatically increased in the LV of acute pressure-overloaded rat induced by aortic banding,³⁰ thereby suggesting that the responses of cardiac ß-MHC may differ between chronic and acute hemodynamic overload. In the present study, the expression of collagen types I and III in the LV of SHR was already increased at 9 weeks of age. Previous work shows that the increase in interstitial collagen volume fraction and increased myocardial diastolic stiffness are already present in SHR at 14 weeks of age.⁶ Therefore, the increased collagen gene expression in SHR at the early stage of hypertension may be responsible for the abnormal myocardial stiffness in SHR. Furthermore, SR Ca²⁺-ATPase expression in SHR was also significantly increased at 9 weeks of age. This early increase in SR Ca²⁺-ATPase expression in SHR may indicate a molecular adaptation to the increased hemodynamic overload, because SR Ca²⁺-ATPase plays a major role in the regulation of cardiac relaxation via intracellular Ca²⁺ uptake into SR.^{3 4} Thus, cardiac molecular changes, which already occurred in young SHR, may play an important role in the modulation of cardiac performance in SHR. However, further study is needed to elucidate the significance of the age-related changes in cardiac gene expression in WKY and SHR.

Previous in vitro studies show that angiotensin II and - or ß-adrenergic receptor contribute to cardiac hypertrophy and gene expression.^{31 32} However, the regulatory mechanism of cardiac gene expression in SHR remains to be elucidated. In the present study, to determine the mechanism responsible for the increased skeletal -actin, ANP, and collagen types I and III expression and for the decreased -MHC expression in the LV of 27-week-old SHR with established hypertension, we compared the effects of various types of antihypertensive drugs, at doses causing mild hypotension, on cardiac gene expression in this age of SHR. To determine whether the effects of each drug on cardiac gene expression are due to a hypotensive action of the drug, we examined mRNA levels not only in the LV but also in the RV because the RV is not significantly affected by arterial hypertension, as shown by no difference in RV weight between SHR and WKY.

Interestingly, in both the LV and RV of SHR, imidapril (an ACE inhibitor), at a dose that decreased blood pressure in SHR only by about 30 mm Hg, significantly increased -MHC expression and decreased collagen types I and III expression. On the other hand, doxazosin (an ₁-adrenergic blocker) or manidipine (a calcium antagonist) did not affect these mRNA expressions in the LV or RV of SHR despite similar blood pressure–lowering effects in response to imidapril. These observations show that the effects of imidapril on -MHC and collagen types I and III in the LV of SHR can be attributed to its direct rather than its hypotensive action.

Recently, we have examined in detail the cardiac gene expression of transgenic TGR(mRen-2)27 rats, a new chronic hypertension model that carries the mouse Ren-2 gene and expresses the renin transgene in the heart.^{33 34 35} We have found that the pattern of altered cardiac gene expression in TGR(mRen-2)27 rats is similar to that in SHR examined in the present study and that the local renin-angiotensin system in the heart is responsible for the altered cardiac gene expression of this transgenic rat, thereby providing evidence for a critical role of the local renin-angiotensin system in cardiac gene expression in vivo.³⁵ Furthermore, our previous work indicates that in vivo infusion of angiotensin II to rats stimulates various fetal and collagen gene expressions independent of blood pressure.²¹ Therefore, the above-mentioned effects of imidapril on cardiac -MHC and collagen types I and III of SHR seem to be mainly due to inhibition of the local renin-angiotensin system.

Imidapril also decreased ANP mRNA in the LV of SHR, in contrast to no inhibition by other antihypertensive drugs. This observation, taken together with our previous data that even blood pressure reduction in SHR to about 150 mm Hg by hydralazine did not lead to ANP suppression in the LV,¹¹ suggests the possible involvement of ACE in the increased ANP in the LV of SHR. However, ANP mRNA in the RV of SHR was not affected by imidapril, suggesting a possible difference in the regulation of ANP expression between the LV and RV. Further study is needed to elucidate the regulatory mechanism of ANP expression in the LV and RV of SHR.

In this study, to examine the role of ß₁-adrenergic receptors in cardiac gene expression, we gave atenolol to SHR in combination with doxazosin because atenolol alone cannot lower blood pressure in SHR.³⁶ Treatment of SHR with atenolol (a ß₁-adrenergic blocker) combined with doxazosin failed to affect -MHC and ANP expression but decreased collagen types I and III expression in both the LV and RV of SHR. We have previously demonstrated that in vivo infusion of isoproterenol to rats induces enhanced expression of collagen types I and III without elevating blood pressure.³⁷ This enhanced collagen gene expression could not be suppressed by an ACE inhibitor or angiotensin type 1 receptor antagonist.³⁷ Furthermore, Bhambi and Egbahli³⁸ reported that norepinephrine can directly act on cardiac fibroblasts, causing cell proliferation. These observations suggest that the suppressive effects of atenolol on cardiac collagen I and III expression in SHR in the present study might be due to the direct inhibition of cardiac ß-adrenergic receptor, independent of either blood pressure or the renin-angiotensin system. Alternatively, it is also possible that the response of collagen gene expression to atenolol might be secondary to the prevention of myocardial necrosis.³⁹ The previous reports on rats with myocardial infarction⁴⁰ and acute banding of the aorta^{41 42} have shown that myocardial necrosis is accompanied by increased TGF-ß1 expression. Indeed, TGF-ß1 expression is significantly increased in the LV of SHR that are older than 18 months, with marked cardiac necrosis.³⁹ However, in the 5- to 27-week-old SHR examined in the present study, cardiac TGF-ß1 mRNA was not increased compared with that in WKY, despite higher collagen mRNA levels in SHR. Furthermore, the inhibition of collagen expression by atenolol was not associated with the inhibition of TGF-ß1 expression. Thus, it is probably unlikely that the inhibitory effects of atenolol on cardiac collagen expression in SHR were mediated by the inhibition of cardiac necrosis.

On the other hand, only the increased cardiac skeletal -actin mRNA levels in SHR were not significantly suppressed by the above-mentioned antihypertensive drugs. However, in our previous study, when blood pressure of SHR was reduced to about 150 mm Hg by imidapril or hydralazine (a vasodilator), skeletal -actin mRNA levels in the LV of SHR were significantly suppressed.¹¹ Thus, the failure to suppress skeletal -actin expression in the present study seems to be due to mild antihypertensive effects, and the enhanced expression of skeletal -actin in the LV of SHR seems to be mainly due to hypertension.

In conclusion, the pattern of cardiac gene reprogramming in SHR significantly differed from that in the model of pressure overload induced by aortic constriction. Not only hypertension but also cardiac ACE seems to play a key role in both the modulation of cardiac performance and cardiac remodeling in SHR via the regulation of cardiac gene expression. Furthermore, ß₁- but not ₁-adrenergic receptor may also be responsible for cardiac remodeling in SHR.

	Selected Abbreviations and Acronyms

 

-MHC, ß-MHC = - and ß-myosin heavy chain ACE = angiotensin-converting enzyme ANP = atrial natriuretic peptide LV = left ventricle, left ventricular LVH = left ventricular hypertrophy RV = right ventricle, right ventricular SHR = spontaneously hypertensive rat(s) SR = sarcoplasmic reticulum TGF-ß1 = transforming growth factor-ß1 WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid for Scientific Research (07672471 and 05670100) from the Ministry of Education, Science, and Culture and by the Osaka City University Medical Research Foundation Fund for Medical Research. The authors are grateful to Dr Kazuo Takaori for critical evaluation of the manuscript. We also thank Eriko Gomi, Akiko Motoi, and Yuko Era for their excellent technical assistance.

Received May 17, 1996; first decision June 5, 1996; accepted June 5, 1996.

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