TABLE I

Primer sequences for mRNA analyses Sequences are listed 5' to 3'.

Quantitative Real-time PCR by SYBR Green Detection Method-The mRNA levels of angiotensin II type la receptor (AT1aR), angiotensin II type 2 receptor (AT2R), and transforming growth factor-1 (TGF-1) were evaluated by quantitative real-time PCR. In brief, total RNA isolated from heart tissue was used for cDNA synthesis. A cDNA was prepared from 1 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen), used for random hexamers, dNTPs, and first-strand cDNA synthesis, according to the manufacturer's instructions. The amplification reaction was performed in a final volume of 20 µl in 96-well optical reaction plates (Applied Biosystems, Foster City, CA). The PCR mixture contained 50 ng of cDNA, 1 µl of 0.1 nM forward and reverse primer mix, and 13 µl of SYBR Green PCR Master Mix (Applied Biosystems), which contains the fluorescent dye SYBR Green. Assays were performed with an ABI Prism 7500 Sequence Detector (Applied Biosystems). The dye exhibits fluorescence enhancement upon binding to double-stranded DNA, and the enhancement of fluorescence is proportional to the initial concentration of the cDNA. Amplification included one stage of 2 min at 50 °C and one stage of 10 min at 95 °C followed by 40 cycles of a three-step loop: 30 s at 95 °C, 30 s at 54 °C, and 30 s at 72 °C. A melting curve was used to identify a temperature where only the amplicon, and not primer dimers, accounted for the SYBR green-bound fluorescence. Results were analyzed with the SDS7500 software, and all values were normalized to levels of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Oligonucleotide primers used in this study are shown in Table I.

Statistical Analysis-Values for each parameter within a group were expressed as a mean ± S.E. For comparisons among groups, statistical significance was assessed using a one-way analysis of variance, and the significance of each difference was determined by post hoc testing using Tukey-Kramer's method. These analyses were performed on an Apple Macintosh computer with the use of Excel (Microsoft X) and Stat View statistical package (Stat View 5.0, SAS Institute Inc.) (17). Statistical significance was considered at p

RESULTS

Blood Pressure and Heart Rate-Basal levels of systolic blood pressure in WT mice were similar to those of ARKO mice. Systolic blood pressure levels in both WT and ARKO mice were significantly increased by Ang II stimulation, but there was no statistical difference between the two groups (Table II). There was no significant difference in heart rate between WT and ARKO mice, and Ang II stimulation did not affect heart rate in either group (Table II).

TABLE II

Systolic blood pressure, heart rate, and heart-to-body weight ratio Values are means ± S.E. Each group, n = 40.

Reduced Cardiac Mass in ARKO Male Mice with or without Ang II Stimulation-Gross appearance of the isolated heart was smaller in ARKO mice than in WT mice (Fig. 1A), and the HW/BW was significantly lower in ARKO mice than in WT mice (Table II), whereas there was no obvious difference in cardiac size or HW/BW between these mice until the age of 6 weeks (data not shown). Although Ang II stimulation caused an increase in both cardiac size (Fig. 1, A and B) and the HW/BW ratio (Table II) in WT and ARKO mice, these values were still lower in ARKO mice than in WT mice. Cross-sections of the heart demonstrated that LV volume and wall thickness in ARKO mice were reduced compared with those in WT mice (Fig. 1B). In addition, Ang II stimulation could elicit a prominent cardiac hypertrophy in WT mice, whereas the cardiac LV volume and wall thickening after Ang II stimulation was still lower in ARKO mice compared with those in WT mice (Fig. 1B).

FIG. 1.

Macro- and microscopic analyses in WT and ARKO male mice with or without Ang II stimulation. A, appearance of the isolated hearts; B, Masson-Trichrome-stained LV cross-sections on the papillary muscle level; C, histological sections of Masson-Trichrome-stained cardiac tissue for the determination of the cross-sectional area and interstitial fibrosis; D, myofibrillar cross-sectional areas; E, interstitial collagen fractions in WT (white bars) and ARKO (black bars) male mice. Values are expressed as mean ± S.E. *, p n = 12.

Histomorphometric analyses of the LV tissues showed that the cross-sectional area of cardiomyocytes without Ang II stimulation was significantly smaller in ARKO mice than in WT mice (Fig. 1D). Although Ang II stimulation caused an increase in the cross-sectional area of the cardiomyocytes in both WT and ARKO mice, the cross-sectional area after Ang II stimulation was again smaller in ARKO mice than in WT mice (Fig. 1D).

Exacerbated Cardiac Fibrosis by Ang II Stimulation in ARKO Male Mice-The collagen fraction volume ratio calculated from histomorphometric analyses of Masson-Trichrome-stained specimens revealed that cardiac fibrosis was faintly present in LV tissues of WT and ARKO mice without Ang II stimulation. Ang II stimulation elicited a markedly enhanced fibrotic change in LV tissues of ARKO mice compared with those of WT mice (Fig. 1, B, C, and E). However, Ang II administration did not cause an obvious progression of cardiac fibrosis in both WT and ARKO mice when the experiments were conducted at 8 weeks instead of 25 weeks of age (data not shown).

Analyses of Cardiac Structure-Echocardiographic studies revealed that Ang II stimulation caused a significant increase in the values of AW and PW in both WT and ARKO mice (mean ± S.E. mm; AW: 0.86 ± 0.03 to 1.15 ± 0.01 in WT and 0.60 ± 0.02 to 0.70 ± 0.02 in ARKO mice; PW: 0.88 ± 0.03 to 1.13 ± 0.03 in WT and 0.68 ± 0.02 to 0.72 ± 0.02 in ARKO mice), whereas there was no statistically significant change in LVM/BW and RWT by Ang II stimulation in ARKO mice. Furthermore, although the values of LVDd and LVDs were decreased in Ang II-stimulated WT mice due to their concentric hypertrophy, these values did not change or even slightly increase in ARKO mice by Ang II stimulation (Fig. 2).