Exercise training attenuates age-dependent elevation of angiotensin II type 1 receptor and Nox2 signaling in the rat heart

Highlights

• Angiotensin II type 1 receptor (AT1R), a Nox2 mediator, is elevated in aged heart.

• Exercise training reduces AT1R, Nox2, and superoxide release in the old heart.

• Exercise mitigates elevation of TGF-ß, Smad2/3, myofibroblasts, and collagen I.

Abstract

Fibrosis of the aging heart impedes cardiac function and increases the risk of arrhythmias and heart disease. Previously, we demonstrated that exercise-induced reduction of collagen I in the aging heart was linked to a suppression of oxidative stress and transforming growth factor-beta (TGF-ß). The renin–angiotensin II system (RAS) increases oxidative stress via NADPH oxidase-2 (Nox2) and thus elevates TGF-ß and collagen accumulation. Therefore, we tested the hypothesis that exercise training would alleviate age-related upregulation of the angiotensin II receptor I (AT1R) and NADPH oxidase-2 (Nox2), concomitant with suppression of TGF-β and fibrosis. Young (3 months, n = 20) and old (31 months, n = 20) Fischer 344 × Brown Norway F1 (FBNF1) hybrid rats were assigned into sedentary and exercise groups, with exercise training rats training on a treadmill 45 min/day, 5 days/week for the next 12 weeks. Exercise training mitigated age-related upregulation of AT1R, Nox2 activity, and Nox2 subunits gp91phox and p47phox. Exercise training also attenuated TGF-ß positive staining and downstream effectors of fibrosis in the aging heart: connective tissue growth factor, phosphorylation of Smad2 at Ser423, myofibroblast proliferation, and collagen I-positive staining. Our results are consistent with the hypothesis that exercise training protects against age-dependent cardiac fibrosis by suppressing AT1R and Nox2 as part of a RAS–Nox2–TGF-β pathway.

Introduction

Reduction in heart function during the aging process is largely mediated by structural remodeling. Characteristics of age-related remodeling include cardiomyocyte hypertrophy and accumulation of extracellular matrix (ECM) proteins (Centurione et al., 2003). Increased ECM mass during the aging process is manifested by an accretion of fibrotic collagen fibers or “fibrosis” (Centurione et al., 2003). Fibrosis alters the cardiac mechanical environment by increasing wall stress, elevating stiffness, and decreasing elasticity. Thus fibrosis increases internal work of the heart and impedes filling.

Transforming growth factor-beta1 (TGF-ß1) is a proliferative autocrine cytokine and an important regulator of cardiac fibrosis (Biernacka and Frangogiannis, 2011, Li et al., 2011). Downstream effectors of TGF-ß that trigger proliferation of fibroblasts and drive fibrosis include Smad proteins and connective tissue growth factor (CTGF) (Biernacka and Frangogiannis, 2011). Indeed, suppression of TGF-ß levels reduced Smad proteins and CTGF in the heart, thus ameliorating fibrosis (Bujak and Frangogiannis, 2007, Rosenkranz, 2004, Schultz et al., 2002).

Upstream signaling candidates in the aging heart that trigger upregulation of TGF-ß and thus collagen accumulation include oxidative stress and renin–angiotensin II-system (RAS) (Biernacka and Frangogiannis, 2011). Furthermore, elevation of RAS and TGF-β1 has been proposed as an important stimulus in eliciting cardiac remodeling that occurs with aging (Biernacka and Frangogiannis, 2011, Campbell and Katwa, 1997, Wang et al., 2010).

Elevation of reactive oxygen species (ROS) and thus oxidative stress may also trigger fibrosis and remodeling in the heart (Arnold et al., 2001, Irani et al., 1997). NADPH oxidase is a family of membrane-bound oxidoreductase complex and has been proposed as a source of oxidative stress in the heart (Heymes et al., 2003, Ushio-Fukai et al., 1996). The Nox2 isoform of NADPH oxidase is increased during heart failure and associated with cardiac remodeling and fibrosis (Heymes et al., 2003, Murdoch et al., 2006). Moreover, inhibition of Nox2 improved cardiac function in a rabbit model of heart failure, directly linked to diminution of cardiac fibrotic remodeling (Liu et al., 2010). However, the sources of redox signaling that contribute to cardiac fibrosis in the aging heart have not been elucidated.

Upregulation of the angiotensin II type 1 receptor (AT1R) is central to increased RAS, and has been previously linked to acceleration of collagen formation and cardiomyocyte hypertrophy (Campbell and Katwa, 1997, Cave et al., 2006). Increased expression of AT1R can be upstream of Nox2 activation (Bendall et al., 2002, Byrne et al., 2003, Cave et al., 2006). Indeed, Nox2 activity was shown to be increased when angiotensin II signaling is elevated (Heymes et al., 2003, Ushio-Fukai et al., 1996). Furthermore, Wang et al. (2010) showed that angiotensin II and AT1R induce fibrosis and cardiomyocyte hypertrophy via upregulation of NADPH oxidase (Wang et al., 2010). Importantly, inhibition of Nox2 via genetic ablation of gp91phox reduced AT1R-dependent cardiac hypertrophy, thus establishing a causal relationship between AT1R and Nox2 (Bendall et al., 2002).

Regular exercise training improves heart function in elderly populations by increasing stroke volume, ejection fraction, cardiac output, and cardiac index (Schulman et al., 1996, Stratton et al., 1994). Previously, our laboratory (Kwak et al., 2006, Kwak et al., 2010) and other investigators (Thomas et al., 2000, Thomas et al., 2001) demonstrated that exercise training attenuates age-related collagen content and collagen cross-linking in rats. Recently, we found that exercise training also reduces TGF-ß1 in the aging heart (Kwak et al., 2010). However, the mechanisms underlying exercise training-induced protection of age-related fibrosis remains poorly understood. Furthermore, the role of habitual exercise in targeting a RAS–Nox2 pathway in the aging heart is unknown.

Therefore, the purpose of the current study is to test the hypothesis that twelve weeks of exercise training in old FBNF1 rats would ameliorate a RAS–Nox2 pathway, linked to suppression of TGF-β1 and its downstream effectors of cardiac fibrosis (Smad2/3 phosphorylation, CTGF, myofibroblasts).