Moderate cardiac-selective overexpression of angiotensin II type 2 receptor protects cardiac functions from ischaemic injury

Abstract

We hypothesized that moderate cardiac-selective overexpression of the angiotensin II type 2 receptor (AT2R) would protect the myocardium from ischaemic injury after a myocardial infarction (MI) induced by coronary artery ligation. For in vitro studies, adenoviral vector expressing genomic DNA of AT2R and enhanced green fluorescence protein (EGFP) was used to overexpress AT2R in rat neonatal cardiac myocytes. Expression of AT2R, measured by real-time PCR and immunostaining, demonstrated efficient transduction of AT2R in a dose-dependent pattern. The AT2R constitutively induced apoptosis in rat neonatal cardiac myocytes in dose-dependent patterns. For in vivo studies, 4 × 10(10) vector genomes (vg) of recombinant adeno-associated virus serotype 9 (rAAV9)-chicken β actin promoter-AT2R was injected into the left ventricle of 5-day-old Sprague-Dawley rats. At 6 weeks of age, hearts were harvested and expression of AT2R determined by real-time PCR and Western blotting. Expression was increased onefold over control hearts, and no apoptosis was detected. Two subsequent in vivo studies were performed. In a prevention study, 4 × 10(10) vg of rAAV9-CBA-AT2R was injected into the left ventricle of 5-day-old Sprague-Dawley rats and MI was induced at 6 weeks of age. For a post-treatment study, 4 × 10(10) vg of rAAV9-CBA-AT2R was administrated to the peri-infarcted myocardium area immediately after MI in 6-week-old animals. For both in vivo studies, cardiac functions were assessed using echocardiography and haemodynamic measurements 4 weeks after coronary artery ligation. In the in vivo studies, the rats subjected to MI showed significant decreases in fractional shortening and rate of change of left ventricular pressure, with increased left ventricular end-diastolic pressure and ventricular hypertrophy. For the prevention study, the moderate cardiac-selective overexpression of AT2R attenuated these MI-induced impairments and also caused a decrease in ventricular wall thinning. In the post-treatment study, the overexpression of AT2R partly reversed the MI-induced cardiac dysfunction. Myocardial infarction also induced the upregulation of angiotensin II type 1 receptor, angiotensin-converting enzyme and collagen I mRNA expression, all of which were attenuated by the overexpression of AT2R. It is concluded that moderate cardiac-selective overexpression of AT2R protects heart function from ischaemic injury, which may be mediated, at least in part, through modulation of components of the cardiac renin-angiotensin system and collagen levels in the myocardium.

Figure 1

Basal and adeno viral vector mediated expression of AT2R in RNCM. PBS (left) was used as negative control. RNCM were transduced with either 5 ifu/cell (middle) or 50 ifu/cell (right) of Ad-G-AT2R-EGFP for 48 h. Viral transduction was confirmed by detection of EGFP fluorescence and AT2R immunoreactivity using an anti-AT2R antibody. Representative fluorescence micrographs from Ad-G-AT2R-EGFP transduced cells showing GFP (green fluorescence), AT2R immunostaining (red fluorescence), and merge (GFP+AT2R). Scale bars, 20 µm.

Figure 2

(a), RNCM were transduced with four different dose of Ad-CMV-EGFP or Ad-G-AT2R-EGFP (0.5 ifu/cell, 5 ifu/cell, 50 ifu/cell, and 100 ifu/cell) for 48 h. PBS was used as negative control and Ad-CMV-EGFP was used as viral control. (b), Apoptosis was detected using the DeadEnd Colorimetric TUNEL System kit. The brown-colored nuclei as indicated by arrow head represent TUNEL-positive (apoptotic) cells. (c), Representative fluorescence micrographs from RNCM transduced with 50 ifu/cell of Ad-CMV-EGFP or Ad-G-AT2R-EGFP. (d), AT2R mRNA level was quantified by realtime RT-PCR. (P<0.05, * 24h vs 0h and 6h; # 36h vs 0h, 6h, 12h, and 72h; $ 48h vs 0h,6h,12h,24h,60h, and 72h;** 60h vs72h.). Scale bars, 50 µm.

Figure 3

(a) Semi-quantitative Real-Time RT-PCR detection of AT2R mRNA level presented in heart tissues. (b) AT2R in the rat ventricles was detected by western blotting. (c) The relative density of AT2R was determined following densitometric measurements of the protein bands and normalization against the GAPDH signals. (d) Apoptosis was determined in the rAAV9-AT2R transduced rats by DeadEnd Colorimetric TUNEL System kit at 6 weeks after vial administration. Scale bars, 20 µm. (** p<0.05, rAAV9-AT2R, MI+AT2R(PT), and MI+AT2R(P), vs control, MI and rAAV9-GFP)

Figure 4

Effects of cardiac-selective AT2R overexpression on cardiac functions. (a), Fractional shortening is analyzed using echocardiography. (P<0.05, * : MI vs control, rAAV9-AT2R,rAAV9-GFP, and MI+AT2R(P); #: MI+AT2R(PT) vs control, rAAV9-AT2R, and rAAV9-GFP;**: MI+AT2R(P) vs control, rAAV9-AT2R, and MI). (b), Hemodynamic analysis: % of left ventricle end-diastolic pressure (LVEDP) changes.(P<0.05, &: MI vs control, rAAV9-AT2R, rAAV9-GFP, and MI+AT2R(P). Control LVEDP was 4.0 ± 1.5 mmHg. (c) and (d), % of +dP/dt and –dP/dt (maximal and minimal peak rate of left ventricular pressure). (P<0.05, +: MI vs all other groups). Control value for dP/dt_max and dP/dt_min were in the 8000 and 5000mmHg ranges, respectively.

Figure 5

Effects of moderate cardiac-selective overexpression of AT2R on ventricular remodeling. (a), Cardiac hypertrophy was evaluated by the ratio of ventricular weight (g) to tibia length (cm) (P<0.05, &: MI vs all other groups). (b), Left ventricular wall thickness was quantified by the percentage of control. (P<0.05, * MI, MI+AT2R(PT), and MI+ AT2R(P) vs control, rAAV9-GFP, and rAAV9-AT2; # MI+rAAV9-AT2R(PT) and MI+rAAV9-AT2R(P) vs MI).

Figure 6

Effects of cardiac-selective overexpression of AT2R on mRNA levels of the RAS, collagen I, and collagen III. Realtime RT- PCR was used to measure (a) AT1 receptor, (b) Mas receptor, (c) ACE, (d) ACE2, (e) Collagen I, and (f) Collagen III levels in the left ventricles of hearts. AT2R increased the ratio of heart (g) Mas/AT1R and (h) ACE2/ACE mRNA levels. Data are expressed as mean ± SD. (P<0.05, * MI versus all other groups, # MI vs control, rAAV9-GFP, and MI+AT2R(P); ** MI+AT2R(P) vs all other groups.)