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(Circulation. 1996;94:785-791.)
© 1996 American Heart Association, Inc.

Articles

Contribution of Local Renin-Angiotensin System to Cardiac Hypertrophy, Phenotypic Modulation, and Remodeling in TGR(mRen2)27 Transgenic Rats

Kensuke Ohta, MD; Shokei Kim, MD; Hideki Wanibuchi, MD; Detlev Ganten, MD; Hiroshi Iwao, MD

the Department of Pharmacology (K.O., S.K., H.I.) and the First Department of Pathology (H.W.), Osaka (Japan) City University Medical School, and the Max Delbruck Center for Molecular Medicine (D.G.), Berlin-Buch, Germany.

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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Background The transgenic rat TGR(mRen2)27, carrying the mouse Ren-2 gene, is a new model to elucidate the role of the local renin-angiotensin system in vivo. However, the role of the local renin-angiotensin system in the heart remains to be determined in TGR(mRen2)27.

Methods and Results TGR(mRen2)27 were treated with various antihypertensive drugs for 6 weeks to examine the effects on cardiac hypertrophy and gene expression. Cardiac mRNAs were examined by Northern blot analysis. In TGR(mRen2)27, left ventricular hypertrophy was associated with a decrease in -myosin heavy chain expression of 31% and an increase in skeletal -actin and atrial natriuretic polypeptide expression by 2.6- and 21-fold, respectively (P<.05), thereby showing the shift of myocardium to a fetal phenotype. Furthermore, cardiac collagen and laminin expressions were increased in TGR(mRen2)27 (P<.05), suggesting the occurrence of cardiac remodeling. Although treatment of TGR(mRen2)27 with a high dose of TCV-116 (angiotensin AT₁ receptor antagonist) or manidipine (calcium antagonist) combined with atenolol (ß₁-adrenergic receptor blocker) completely normalized blood pressure, TCV-116 regressed cardiac hypertrophy and suppressed the changes in cardiac mRNA levels of TGR(mRen2)27 much more potently than manidipine with atenolol. Furthermore, the inhibitory effects of a low dose of TCV-116 on cardiac hypertrophy and altered gene expressions of TGR(mRen2)27 were greater than those of doxazosin (₁-adrenergic receptor blocker) combined with atenolol, despite their similar hypotensive effects.

Conclusions Our present observations provide evidence that the cardiac renin-angiotensin system in TGR(mRen2)27 is responsible for cardiac hypertrophy, phenotypic modulation, and remodeling.

Key Words: genes • renin • angiotensin • hypertrophy • remodeling

	Introduction

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The critical role of the RAS in the pathogenesis of LV hypertrophy in human essential hypertension has been supported by the beneficial effects of angiotensin-converting enzyme inhibitors on LV hypertrophy.¹ In the SHR, which is an excellent genetic model of human essential hypertension, the RAS may contribute to the development of LV hypertrophy not only indirectly by elevating blood pressure or activating other mechanisms, including the sympathetic nervous system, but also by the direct action of Ang II on the heart.^{2 3} However, the complex genetic background of the SHR has made it difficult to elucidate the role of the RAS in cardiac hypertrophy in detail.

TGR(mRen2)27, carrying the murine Ren-2 gene,⁴ is a transgenic hypertensive rat whose hypertension can be normalized by the AT₁ receptor antagonist.⁵ TGR(mRen2)27 is characterized by no increase in plasma and renal renin concentrations and by high adrenal renin concentration.⁴ Therefore, as in the case of SHRs, the local RAS is considered to play an important role in hypertension of TGR(mRen2)27.^{5 6} Furthermore, very recently, Ren-2 transgene has been demonstrated to be expressed in the cardiac tissue of TGR(mRen2)27.⁷ However, it is unknown whether cardiac hypertrophy of TGR(mRen2)27 is indeed mediated by the local RAS.

Accumulating evidence has demonstrated that cardiac hypertrophy is accompanied not only by the growth of myocytes but also by the qualitative alterations of cardiac myocytes and interstitium. The qualitative alterations of myocytes include the altered expression of MHC and -actin isogenes and of ANP gene,⁸ whereas those of the cardiac interstitium include the altered expression of extracellular matrix components.⁹ Indeed, we previously found that many of the above-mentioned genes are significantly altered in the LV of SHRs^{10 11} and SHRSPs.¹² These findings, taken together with the important role of these genes in cardiac performance,^{9 13} suggest that the qualitative changes of myocytes and interstitium may be responsible for the development of cardiac dysfunction.

In the present study, to characterize cardiac hypertrophy of TGR(mRen2)27 in detail, we examined not only cardiac weight but also the cardiac gene expression of this transgenic rat. Furthermore, to elucidate the mechanism of cardiac hypertrophy, we examined the effects of different types of antihypertensive drugs on cardiac weight and gene expression of TGR(mRen2)27.

	Methods

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Drugs
TCV-116, a nonpeptide selective AT₁ receptor antagonist,¹⁴ and manidipine, a long-acting calcium antagonist,¹⁵ were donated by Takeda Chemical Industries, Ltd. Doxazosin, an ₁-adrenergic receptor blocker,¹⁶ was a gift from Pfizer Pharmaceutical Inc. Atenolol, a cardioselective ß₁-adrenergic receptor blocker,¹⁷ was donated by ZENECA Yakuhin K.K. All these drugs were suspended in 5% gum arabic solution for oral administration.

Animals
Male TGR(mRen2)27 and male SD Hannover rats were used in the present study as normotensive genetic controls. The animals were fed standard laboratory chow (CE-2, Clea Japan) and given tap water ad libitum throughout the experiments.

Experimental Procedure
All procedures were in accordance with the institutional guidelines for animal research. TGR(mRen2)27 at the age of 11 weeks were randomly separated into seven groups and treated with (1) 5% gum arabic solution (vehicle) (n=28), (2) doxazosin alone (5 mg·kg^-1·d^-1) (n=10), (3) atenolol alone (200 mg·kg^-1·d^-1) (n=11), (4) a low dose of TCV-116 (0.1 mg·kg^-1·d^-1) (n=10), (5) a combination of doxazosin and atenolol (15 and 20 mg·kg^-1·d^-1, respectively) (n=9), (6) a high dose of TCV-116 (1.5 mg·kg^-1·d^-1) (n=10), or (7) a combination of manidipine and atenolol (10 and 20 mg·kg^-1·d^-1, respectively) (n=9). Additionally, the age-matched male SD rats were treated with vehicle (n=20). All the drugs were given orally by gastric gavage every morning for 6 weeks.

The previous works on the hypotensive effects of doxazosin^{18 19} and atenolol¹⁷ in SHRs show that 5 mg·kg^-1·d^-1 doxazosin and 200 mg·kg^-1·d^-1 atenolol are sufficient doses to obtain their maximal hypotensive effects. Therefore, these doses of doxazosin and atenolol were used in this study. To inhibit AT₁ receptor in vivo, we used TCV-116, since this compound is an extremely potent and selective AT₁ receptor antagonist, as shown by previous reports that the inhibitory effects of TCV-116 on Ang II–induced pressor response is 50-fold greater than those of losartan¹⁴ and that even a dose of 0.1 mg·kg^-1·d^-1 of TCV-116 can significantly lower blood pressure of SHRs.²⁰ In our preliminary experiments, we found that the hypotensive effects of doxazosin on TGR(mRen2)27 are moderate and comparable to those of 0.1 mg·kg^-1·d^-1 TCV-116. Therefore, in the present study, 0.1 mg·kg^-1·d^-1 of TCV-116 was used to compare with doxazosin with respect to the effects on cardiac hypertrophy and gene expression. In addition to the group treated with doxazosin alone, another group was treated with doxazosin in combination with atenolol to prevent the increase in heart rate caused by doxazosin. Furthermore, TGR(mRen2)27 were treated with 1.5 mg·kg^-1·d^-1 of TCV-116 or 10 mg·kg^-1·d^-1 of manidipine, because our preliminary studies indicated that these doses of drugs completely normalized blood pressure of TGR(mRen2)27. Manidipine was combined with atenolol to block the increase in heart rate.

Systolic blood pressure and heart rate were measured under light ether anesthesia by the tail-cuff method (PS-8000, Riken Development Co, Ltd). Blood pressure was measured at the time period when the drugs showed the maximum hypotensive effects on hypertensive rats by a single administration, ie, 3 to 5 hours after the administration of TCV-116²⁰ and 1 to 3 hours for doxazosin,¹⁸ atenolol,²¹ and manidipine.¹⁵ After 6 weeks of drug treatments, animals were killed by decapitation, and the trunk blood was collected in polyethylene tubes with 1 mg/mL EDTA. Then the plasma was separated and stored at -20°C to determine the plasma renin concentration. The whole heart was rapidly excised and rinsed with cold saline, and the free wall of the LV was separated from the whole heart and weighed. The tissues were immediately frozen in liquid nitrogen and stored at -80°C until the extraction of total RNA. Adrenal glands were removed, rinsed with saline, and stored at -80°C for the measurement of adrenal renin concentration.

Oligonucleotide and cDNA Probes
For the measurement of mRNA levels for - and ß-MHC²² and for skeletal and cardiac -actin,²³ oligonucleotides complementary to their unique 3' flanking sequences were synthesized as the probe, as previously described.^{11 24} The sequence of synthesized probe for rat vascular -SMA was 5'-CACAAAACATTCACAGTTGTGT-3'.²⁵ The probes were 5' end-labeled with [-³²P]ATP (specific activity, 6000 Ci/mmol) by use of a commercially available kit (Takara), followed by purification with a Bio-Spin 6 chromatography column (Bio-Rad).

The cDNA probes for extracellular matrix components and TGF-ß₁ were as follows: rat ₁-(I) collagen, a 1.3-kb Pst I/BamHI fragment²⁶ ; mouse ₁-(III) collagen, a 1.8-kb EcoRI/EcoRI fragment²⁷ ; mouse ₁-(IV) collagen, a 0.83-kb Ava I/Pst I fragment²⁸ ; mouse laminin B₁ chain, a 0.65-kb BamHI/EcoRI fragment²⁹ ; mouse laminin B₂ chain, a 1.7-kb EcoRI/Xba I fragment³⁰ ; rat GAPDH, a 1.3-kb Pst I/Pst I fragment³¹ ; and rat TGF-ß₁, a 1.2-kb HindIII/Xba I fragment.³² An 825-bp specific cDNA probe for rat ANP³³ was synthesized in our laboratory as previously described.¹¹ Each cDNA probe was labeled with [³²P]dCTP (specific activity, 3000 Ci/mmol, E.I. du Pont de Nemours & Co, Inc) by the random primer extension method with a commercially available kit (Takara), followed by purification with a Bio-Spin 30 chromatography column (Bio-Rad).

Extraction of Total RNA
Total RNA from the individual LV was extracted by the acid guanidinium thiocyanate–phenol-chloroform method,³⁴ as previously described.³⁵ The RNA pellet was finally dissolved in 0.1% diethyl pyrocarbonate–treated water and stored at -80°C until use. The RNA concentration was spectrophotometrically determined at 260 nm.

Northern Blot Hybridization
Twenty micrograms of total RNA from the LV 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, as described.³⁵ The 28S and 18S ribosomal RNAs in gels were stained with ethidium bromide to confirm the integrity of applied RNA and to verify that the same amounts of RNA were applied to each lane. RNAs in the gel were then transferred to a nylon membrane (Gene Screen Plus, E.I. du Pont de Nemours & Co, NEN Products).

Prehybridization and hybridization with oligonucleotide probes were performed as previously described.^{11 24} After the hybridization, the membrane was washed in 2xSSC for 10 minutes at room temperature, followed by washing twice in 2xSSC containing 1% SDS for 20 minutes at the indicated temperatures as follows: 55°C for -MHC, 53°C for ß-MHC, 57°C for skeletal -actin, 51°C for cardiac -actin, and 55°C for -SMA. Finally, the membrane was washed in 0.1xSSC at room temperature for 5 minutes, followed by autoradiography.^{11 24} For hybridization with cDNA probes, the membrane was prehybridized, hybridized, and washed, followed by autoradiography, as described.³⁵

For measurement of tissue mRNA levels, an optical scanner (Epson GT-8000, Seiko) was used to digitize the autoradiographic bands. The digitized images of autoradiographic bands were measured for their densities with the public domain NIH Image program.³⁵ 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 total RNA loading and transfer to a nylon membrane and to verify the specificity of the change in mRNA levels. To determine each mRNA value correctly, for all autoradiographs, the exposure time of x-ray film was carefully adjusted for mRNA autoradiograms so as not to be oversaturated. The membrane was rehybridized with another oligonucleotide or cDNA probe after the previous probe was stripped off by boiling in 0.1xSSC solution containing 1% SDS for 20 minutes.

Determination of Plasma and Adrenal Renin Concentrations
Plasma renin concentrations were measured as the rate of Ang I generation from rat plasma angiotensinogen, as previously described.³⁶ In brief, plasma samples were incubated with 48-hour nephrectomized rat plasma containing an excess of renin substrate (2 µmol/L angiotensinogen) at 37°C in 0.1 mol/L sodium phosphate buffer (pH 7.0) containing protease inhibitors, and the generated Ang I was measured by radioimmunoassay. For the measurement of adrenal renin concentration, adrenal tissue was homogenized in 1 mL of 75 mmol/L phosphate buffer (pH 7.0) containing protease inhibitors. The homogenates were centrifuged, and the supernatants were measured for renin concentration as described.³⁶

Statistical Analysis
All values obtained were expressed as mean±SEM. Statistical significance was determined by ANOVA and Bonferroni's multiple-range test. Differences were considered statistically significant at a value of P<.05.

	Results

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Blood Pressure and Heart Rate of TGR(mRen2)27
As shown in Fig 1A, the blood pressure of vehicle-treated TGR(mRen2)27 was significantly higher than that of SD rats (the genetic control) during the experiments. Conversely, as shown in Fig 1B, heart rate did not differ between TGR(mRen2)27 and SD rats. Treatment with atenolol alone did not significantly affect blood pressure and heart rate of TGR(mRen2)27, although it tended to decrease them. Doxazosin alone lowered blood pressure of TGR(mRen2)27 to a similar level in response to a low dose (0.1 mg·kg^-1·d^-1) of TCV-116 but, unlike TCV-116, significantly increased heart rate. However, doxazosin combined with atenolol, whose hypotensive effects were comparable to those of doxazosin alone or a low dose of TCV-116, did not increase the heart rate of TGR(mRen2)27 at all, as in the case of TCV-116. High doses (1.5 mg·kg^-1·d^-1) of TCV-116 and the combination of manidipine and atenolol completely normalized the blood pressure of TGR(mRen2)27 without affecting heart rate, and there was no significant difference in blood pressure and heart rate between these two treatments.

View larger version (22K): [in this window] [in a new window]   Figure 1. Blood pressure (A) and heart rate (B) of TGR(mRen2)27. Each value represents mean±SEM of SD rats (, n=20) and of TGR(mRen2)27 treated with vehicle (, n=25), 0.1 mg·kg-1·d-1 TCV-116 (, n=10), 1.5 mg·kg-1·d-1 TCV-116 (, n=10), a combination of doxazosin and atenolol (15 and 20 mg·kg-1·d-1, respectively) (, n=9), a combination of manidipine and atenolol (10 and 20 mg·kg-1·d-1, respectively) (, n=9), 5 mg·kg-1·d-1 doxazosin alone (, n=10), or 200 mg·kg-1·d-1 atenolol alone (, n=11). *P<.05 vs vehicle-treated TGR(mRen2)27, P<.05 vs SD rats.

LV Weight Corrected for Body Weight of TGR(mRen2)27
As shown in Fig 2, the LV weight corrected for body weight of vehicle-treated TGR(mRen2)27 was greater than that of SD rats (2.87±0.06 versus 2.16±0.04 mg/g, P<.05). Both low and high doses of TCV-116 significantly regressed LV hypertrophy in a dose-dependent manner. In contrast, although doxazosin combined with atenolol and manidipine combined with atenolol had comparable effects on blood pressure and heart rate responses of TGR(mRen2)27 to low and high doses of TCV-116, respectively, these treatments did not significantly regress LV hypertrophy. In addition, the treatment with doxazosin or atenolol alone also did not regress cardiac hypertrophy of TGR(mRen2)27 at all.

View larger version (87K): [in this window] [in a new window]   Figure 2. LV weight, corrected for body weight, of TGR(mRen2)27. Bar graph represents mean±SEM. SD indicates SD rats treated with vehicle (n=16). TGR(mRen2)27 were treated with vehicle (V, n=18), 0.1 mg·kg-1·d-1 TCV-116 [T(L), n=10], a combination of doxazosin and atenolol (15 and 20 mg·kg-1·d-1, respectively) (D/A, n=9), 1.5 mg·kg-1·d-1 TCV-116 [T(H), n=8], a combination of manidipine and atenolol (10 and 20 mg·kg-1·d-1, respectively) (M/A, n=9), 5 mg·kg-1·d-1 doxazosin alone (D, n=6), or 200 mg·kg-1·d-1 atenolol alone (A, n=7). *P<.05 vs vehicle-treated TGR(mRen2)27 (V), P<.05 between T(H) and M/A.

Plasma and Adrenal Renin Concentrations
As shown in Table 1, plasma renin concentrations of TGR(mRen2)27 showed no significant difference from those of SD rats. Treatment with high doses of TCV-116 elevated plasma renin concentrations of TGR(mRen2)27 by about ninefold, but any other treatment did not change them significantly. Adrenal renin concentrations in TGR(mRen2)27 were about 17-fold higher than those in SD rats, consistent with a previous report,⁴ and were not significantly affected by any drug treatment.

View this table: [in this window] [in a new window]   Table 1. Plasma and Adrenal Renin Concentrations

Expression of Marker Genes for Cardiac Myocyte Phenotype
As shown in Figs 3 and 4, the expression of -MHC was decreased by 31% in the LV of TGR(mRen2)27 compared with SD rats, whereas the expression of ß-MHC did not show a significant difference between the two groups of rats. Skeletal and cardiac -actin mRNA levels in the LV of TGR(mRen2)27 were 2.6- and 1.2-fold, respectively, higher than in SD rats. As indicated by Table 2, these changes in MHC and -actin expression led to the increase in the ratios (arbitrary ratios, not the true molecular ratios) of ß/-MHC and skeletal/cardiac -actin in TGR(mRen2)27 by 1.7- and 2.2-fold, respectively, compared with SD rats. The expression of ANP and -SMA was increased by 21- and 1.2-fold, respectively, in TGR(mRen2)27 compared with SD rats. As shown in Fig 3, treatment with doxazosin or atenolol alone did not affect the altered cardiac gene expression of TGR(mRen2)27 at all. As shown in Figs 3 and 4, all the changes in cardiac gene expression of TGR(mRen2)27 were almost normalized by the treatment with high doses (1.5 mg·kg^-1·d^-1) of TCV-116. Conversely, treatment with both manidipine and atenolol, the effects of which on blood pressure and heart rate were comparable to those of high doses of TCV-116, inhibited only the enhanced expression of ANP and cardiac -actin and did not significantly affect expression of other genes. Moreover, the inhibitory effect of manidipine with atenolol on ANP expression was significantly less potent than high doses of TCV-116. Furthermore, even low doses (0.1 mg·kg^-1·d^-1) of TCV-116 significantly prevented the changes in skeletal -actin, cardiac -actin, and ANP. In contrast, treatment with the combination of doxazosin and atenolol, whose effects on blood pressure and heart rate were comparable to those of low doses of TCV-116, did not affect expression of these genes at all. In addition, the increased arbitrary ratios of ß/-MHC and skeletal/cardiac -actin mRNA levels were suppressed only by TCV-116 (Table 2).

View larger version (73K): [in this window] [in a new window]   Figure 3. Typical autoradiograms of Northern blot analysis of LV mRNAs. Sk-actin indicates skeletal -actin; Ca-actin, cardiac -actin; and SD, SD rats treated with vehicle. TGR(mRen2)27 were treated with vehicle (V), 1.5 mg·kg-1·d-1 TCV-116 [T(H)], 5 mg·kg-1·d-1 doxazosin (D), 200 mg·kg-1·d-1 atenolol (A), 0.1 mg·kg-1·d-1 TCV-116 [T(L)], a combination of doxazosin and atenolol (15 and 20 mg·kg-1·d-1, respectively) (D/A), or a combination of manidipine and atenolol (10 and 20 mg·kg-1·d-1, respectively) (M/A). The size of the mRNA band was 7.1 kb for -MHC, 7.1 kb for ß-MHC, 1.7 kb for Sk-actin, 1.7 kb for Ca-actin, 0.9 kb for ANP, 1.6 kb for -SMA, and 1.4 kb for GAPDH.

View this table: [in this window] [in a new window]   Table 2. Ratios of ß-MHC to -MHC and Skeletal to Cardiac -Actin mRNA Levels

View larger version (61K): [in this window] [in a new window]   Figure 4. LV mRNA levels for -MHC, ß-MHC, Sk-actin, Ca-actin, ANP, and -SMA. Bar graph represents mean±SEM. The density of each autoradiogram was measured densitometrically and corrected for GAPDH mRNA value. The mean value in SD rats was represented as 1. SD indicates SD rats treated with vehicle (n=16). TGR(mRen2)27 were treated with vehicle (n=18), 0.1 mg·kg-1·d-1 TCV-116 (n=10), a combination of doxazosin and atenolol (15 and 20 mg·kg-1·d-1, respectively) (n=9), 1.5 mg·kg-1·d-1 TCV-116 (n=8), or a combination of manidipine and atenolol (10 and 20 mg·kg-1·d-1, respectively) (n=9). *P<.05 vs vehicle-treated TGR (V), P<.05 between indicated groups. Abbreviations as in Fig 3.

Expression of Marker Genes for Cardiac Remodeling
Figs 5 and 6 show the Northern blot analysis of extracellular matrix components and TGF-ß₁. In the LV of TGR(mRen2)27, the expression of collagen types I and III was increased significantly, by 1.4- and 1.4-fold, respectively, compared with SD rats. Collagen type IV and laminin B₂ chain mRNAs, the main basement membrane components, were also increased, by 1.3- and 1.4-fold, respectively. Moreover, a 1.2-fold increase in expression of TGF-ß1 was found in TGR(mRen2)27. As shown in Fig 5, these enhanced expressions of extracellular matrix components and TGF-ß₁ in the LV of TGR(mRen2)27 were not at all suppressed by treatment with doxazosin or atenolol alone. In contrast, as shown in Figs 5 and 6, high doses of TCV-116 completely inhibited all these enhanced gene expressions of TGR(mRen2)27. In contrast, despite similar hypotensive effects of high doses of TCV-116, the combination of manidipine and atenolol suppressed only the expression of collagen III and laminin B₂. Even low doses of TCV-116 significantly suppressed collagen types III and IV. Despite hypotensive effects similar to those of low doses of TCV-116, doxazosin combined with atenolol suppressed collagen III but not collagen IV.

View larger version (83K): [in this window] [in a new window]   Figure 5. Typical autoradiograms of Northern blot analysis of LV mRNAs. Co I represents collagen I; Co III, collagen III; Co IV, collagen IV; LB1, laminin B1 chain; and LB2, laminin B2 chain. Rat groups and abbreviations as in Fig 3. The size of mRNA was 4.7 and 5.7 kb for Co I, 5.9 kb for Co III, 6.8 kb for Co IV, 6.0 kb for LB1, 8.0 kb for LB2, 2.5 kb for TGF-ß1, and 1.4 kb for GAPDH.

View larger version (60K): [in this window] [in a new window]   Figure 6. LV mRNA levels for collagen I, collagen III, collagen IV, laminin B1, laminin B2, and TGF-ß1. Bar graph represents mean±SEM. The density of each autoradiogram was measured densitometrically and corrected for GAPDH mRNA value. The mean value in SD rats was represented as 1. Rat groups and abbreviations as in Figs 3 and 5. *P<.05 vs vehicle-treated TGR (V), P<.05 between indicated groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Despite the absence of elevation of plasma active renin,⁴ hypertension of TGR(mRen2)27 can be normalized by AT₁ receptor antagonists,⁵ as in the case of SHRs²⁰ or SHRSPs.¹² Moreover, TGR(mRen2)27 have high adrenal renin activity, which is also the case in SHRs³⁷ and SHRSPs.³⁶ These similarities between TGR(mRen2)27 and SHR/SHRSPs led us to examine the cardiac gene expression of TGR(mRen2)27 in detail. Among the cardiac genes examined in the present study, the change in the ratio of ß- to -MHC^{13 38} and/or of skeletal to cardiac -actin³⁹ in the heart is known to significantly affect cardiac contractility. Furthermore, the increased deposition of extracellular matrix components in the cardiac interstitium not only contributes to the diastolic dysfunction of the heart⁹ but also may affect differentiation and growth of myocytes.⁴⁰ In addition, TGF-ß₁ is known to provoke the induction of fetal genes in cultured myocytes^{8 41} and to potently stimulate extracellular matrix production by fibroblasts.⁴² Thus, the changes in these cardiac gene expressions significantly affect both systolic and diastolic functions of the heart.

Cardiac hypertrophy was found in TGR(mRen2)27, which is in accordance with the previous report.⁴³ Furthermore, we found that the change in cardiac gene expression of TGR(mRen2)27 is characterized by the increase in the ratio of ß- to -MHC due to the decrease in -MHC gene expression. In addition, the ratio of skeletal to cardiac -actin, as well as ANP and -SMA expression, was increased in the LV of TGR(mRen2)27, thereby showing the shift of myocytes to the fetal phenotype.^{8 44} Interestingly, the lack of increase in ß-MHC expression in the LV of TGR(mRen2)27 was in contrast to the significant increase in ß-MHC expression in previous in vitro models of myocyte hypertrophy provoked by mechanical stretch⁴⁵ and various humoral or growth factors, including ₁-agonist,^{46 47 48} TGF-ß₁,^{8 41} and basic fibroblast growth factor.^{8 41} As in the case of these in vitro models, the load-induced rat cardiac hypertrophy in vivo is accompanied by the upregulation of ß-MHC as well as ANP and skeletal -actin.^{38 49 50} Furthermore, our recent data show that the marked induction of ß-MHC expression is also found in the acute rat cardiac hypertrophy induced by Ang II infusion.²⁴ Conversely, no increase in ß-MHC expression is seen in the hypertrophied heart of 11-week-old SHRs.¹¹ Furthermore, we recently found that the more progressed cardiac hypertrophy in 27-week-old SHRs is indeed characterized by the decrease in -MHC expression, no alteration of ß-MHC, and an increase in skeletal -actin and ANP, consistent with our present observations on TGR(mRen2)27 (unpublished data). Therefore, ß-MHC expression may be preferentially induced in acute models of cardiac hypertrophy (whether load- or agonist-induced) by comparison with more chronic models such as SHRs and TGR(mRen2)27. Moreover, our previous data show that collagen types I, III, and IV and laminin mRNA levels are significantly elevated in the LV of SHRs¹⁰ and SHRSPs,¹² which is also consistent with the case of TGR(mRen2)27. These findings suggest that the pattern of cardiac gene expression in TGR(mRen2)27 is similar to that of SHRs or SHRSPs.

To elucidate the mechanism responsible for LV hypertrophy and the altered cardiac gene expression of TGR(mRen2)27, we examined the effects of various antihypertensive drugs on these parameters. Of note are the observations that, despite the comparable effects on blood pressure and heart rate, LV hypertrophy of TGR(mRen2)27 was regressed by AT₁ receptor antagonist (TCV-116) but not by the combination of ß₁-adrenergic receptor blocker (atenolol) with either ₁-adrenergic receptor blocker (doxazosin) or calcium antagonist (manidipine). Furthermore, the altered expressions of -MHC, skeletal -actin, -SMA, collagen types I and IV, and TGF-ß₁ in TGR(mRen2)27 were suppressed by TCV-116 but not by normalization of blood pressure with the combination of manidipine and atenolol. In addition, the enhanced expression of ANP was suppressed by manidipine combined with atenolol less potently than by high doses of TCV-116. Thus, taken together with the previous findings that the circulating RAS is not activated in TGR(mRen2)27⁶ and that the Ren-2 transgene is indeed expressed in cardiac tissue of TGR(mRen2)27,⁷ cardiac hypertrophy and the above-mentioned hypertrophy-related gene expression in TGR(mRen2)27 are mediated mainly by cardiac (local) RAS rather than by hemodynamic factors.

In contrast, the enhanced expressions of cardiac -actin, collagen III, and laminin B₂ chain were suppressed by the combination of manidipine and atenolol to a similar extent in response to high doses of TCV-116. In addition, cardiac collagen III expression was also normalized by the combination of doxazosin and atenolol. Thus, the increased expression of cardiac -actin, laminin B₂ chain, and collagen III in TGR(mRen2)27 seems to be mediated primarily by hemodynamic stress. Conversely, it has been demonstrated that Ang II stimulates sympathetic nervous activity, and adrenergic receptors play a critical role in myocyte hypertrophy or gene expression.^{3 46 47 48} Therefore, it cannot be excluded that the suppressive effects of the combination of doxazosin and atenolol or of manidipine and atenolol on these gene expressions (particularly on collagen III expression) might be due in part to the blockade of (cardiac) adrenergic receptors, although our present results on the minor effects of doxazosin or atenolol alone indicate that the sympathetic nervous system is not the major factor in cardiac gene expression of TGR(mRen2)27. However, further studies are required to elucidate the detailed mechanism.

Treatment of TGR(mRen2)27 with high doses of TCV-116 markedly elevated plasma renin concentration, as did another AT₁ receptor antagonist, losartan.⁵ Therefore, negative feedback regulation of renin release via AT₁ receptor exists in TGR(mRen2)27 as well as in normal rats. Furthermore, in the present study, sympatholytic drugs or a calcium antagonist did not significantly affect plasma renin concentration, thereby indicating that renin release in TGR(mRen2)27 is specifically regulated by the AT₁ receptor.

In conclusion, we have demonstrated that cardiac hypertrophy of TGR(mRen2)27 is associated with significant changes in various cardiac mRNA levels, whose pattern is similar to that in SHRs. Furthermore, we obtained evidence that cardiac (local) RAS plays an important role in these changes in TGR(mRen2)27. Thus, TGR(mRen2)27 is an excellent new model to elucidate the role of local RAS in cardiac pathophysiology in vivo.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II ANP = atrial natriuretic polypeptide AT1 = angiotensin II type 1 receptor LV = left ventricular MHC = myosin heavy chain RAS = renin-angiotensin system SD = Sprague-Dawley SHR = spontaneously hypertensive rat SHRSP = stroke-prone SHR -SMA = -smooth muscle actin TGF-ß1 = transforming growth factor-ß1

	Acknowledgments

 
This work was supported in part by grants-in-aid for scientific research (07672471 and 05670100) from the Ministry of Education, Science, and Culture and by Osaka City University Medical Research Foundation Fund for Medical Research. The authors are grateful to Dr Kazuo Takaori for critical evaluation of the manuscript, and they also thank Rumiko Fujioka, Miki Kasamatsu, Terumi Numasawa, and Masatoshi Nakata for their excellent technical assistance.

Received November 9, 1995; revision received February 20, 1996; accepted February 21, 1996.

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