Effect of renin inhibition and AT1R blockade on myocardial remodeling in the transgenic Ren2 rat

Abstract

Angiotensin II (Ang II) stimulation of the Ang type 1 receptor (AT(1)R) facilitates myocardial remodeling through NADPH oxidase-mediated generation of oxidative stress. Components of the renin-angiotensin system constitute an autocrine/paracrine unit in the myocardium, including renin, which is the rate-limiting step in the generation of Ang II. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo renin inhibition and/or AT(1)R blockade in a rodent model of chronically elevated tissue Ang II levels, the transgenic (mRen2)27 rat (Ren2). The Ren2 overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, and cardiovascular damage. Young (6- to 7-wk-old) heterozygous (+/-) male Ren2 and age-matched Sprague-Dawley rats were treated with the renin inhibitor aliskiren, which has high preferential affinity for human and mouse renin, an AT(1)R blocker, irbesartan, or placebo for 3 wk. Myocardial NADPH oxidase activity and immunostaining for NADPH oxidase subunits and 3-nitrotyrosine were evaluated and remodeling changes assessed by light and transmission electron microscopy. Blood pressure, myocardial NADPH oxidase activity and subunit immunostaining, 3-nitrotyrosine, perivascular fibrosis, mitochondrial content, and markers of activity were significantly increased in Ren2 compared with SD littermates. Both renin inhibition and blockade of the AT(1)R significantly attenuated cardiac functional and structural alterations, although irbesartan treatment resulted in greater reductions of both blood pressure and markers of oxidative stress. Collectively, these data suggest that both reduce changes driven, in part, by Ang II-mediated increases in NADPH oxidase and, in part, increases in blood pressure.

Fig. 1.

Systolic Blood Pressure (SBP) in the transgenic Ren2 rat. SBP was measured prior to the experimental protocol being started and on days 19 and 20 prior to the rats being killed (day 21). Values are presented as means ± SE. *P < 0.05 compared with Sprague-Dawley control (SD-C); **P < 0.05 compared with Ren2 control (Ren2-C); #P < 0.05 compared with aliskiren-treated Ren2 (Ren2-A). SD-A, aliskiren-treated Sprague-Dawley; Ren2-I, Ren2 rats treated with irbesartan.

Fig. 2.

Myocardial NADPH oxidase in the transgenic Ren2 rat NADPH oxidase activity (A) and immunostaining of subunits p47^phox and Rac1 and quantification of converted signal intensities in average grayscale intensities to the bottom (B and C). *P < 0.05 compared with SD-C; **P < 0.05 compared with Ren2-C; #P < 0.05 compared with aliskiren-treated Ren2 (Ren2-A). Scale bar, 50 um.

Fig. 3.

Myocardial oxidative stress. A: 3-nitrotyrosine staining representative of oxidative and nitroso stress in left ventricle sections and quantification of converted signal intensities in average grayscale intensities to the right. *P < 0.05 compared with SD-C; **P < 0.05 compared with Ren2-C; #P < 0.05 compared with Ren2-A. Scale bar, 100 um.

Fig. 4.

Myocardial remodeling in the transgenic Ren2 rat. Perivascular fibrosis visible by VVG staining (A) and quantification by %area to the right (B). *P < 0.05 compared with SD-C. **P < 0.05 compared with Ren2-C; scale bar, 100 um. C: remodeling of the intercalated discs (ID) in left ventricle tissue. 10K images demonstrate increased convolutions and duplication of the ID in Ren2-C that are not present in SD-C or treated animals (Ren2-A or SD-A). Scale bar, 500 nm.

Fig. 5.

Myocardial mitochondria in the transgenic Ren2 rat. A: left ventricular sections immunostained for mitochondrial complex 4 subunit 1. B: quantification of converted signal intensities in average grayscale intensities to the right. C: mitochondrial biogenesis within the left ventricle of the Ren2 on transmission electron microscopy. Ren2-C ventricle possesses increased numbers of mitochondria compared with SD-C or treated animals (Ren2-A and SD-A). Scale bar, 500 nm.