Temporal changes in cardiac matrix metalloproteinase activity, oxidative stress, and TGF-β in renovascular hypertension-induced cardiac hypertrophy

Abstract

Cardiovascular remodeling found in later phases of two-kidney, one-clip (2K1C) hypertension may involve key mechanisms particularly including MMP-2, oxidative stress, transforming growth factor-β (TGF-β), and inactivation of the endogenous MMP inhibitor, the tissue inhibitor of MMP (TIMP)-4. We examined whether temporal cardiac remodeling resulting from 2K1C hypertension occurs concomitantly with alterations in cardiac collagen, MMP activity, MMP-2, TIMP-4, TGF-β, and reactive oxygen species (ROS) levels during the development of 2K1C hypertension. Sham-operated and 2K1C hypertensive rats were studied after 15, 30, and 75 days of hypertension. Systolic blood pressure was monitored weekly. Left ventricle (LV) morphometry and fibrosis were evaluated in hematoxylin/eosin and picrosirius red-stained sections, respectively. Cardiac MMP-2 levels/activity was determined by gelatin zymography, immunofluorescence, and in situ zymography. TIMP-4 levels were determined by western blotting. Cardiac TGF-β levels were evaluated by immunofluorescence and ROS levels were evaluated with a dihydroethidium probe. 2K1C hypertension induced LV hypertrophy associated with augmented gelatinolytic activity at an early phase of hypertension and further increased after 75 days of hypertension. These alterations were associated with increased cardiac MMP-2, TGF-β, and ROS in hypertensive rats. Higher TIMP-4 levels were found in hypertensive rats only after 75 days after surgery. Our findings show that increased MMP-2 activity is associated with concomitant development of LV hypertrophy and increased TGF-β and ROS levels.

Introduction

Hypertension is widely known to induce cardiomyocyte hypertrophy and cardiac remodeling (Berk et al., 2007, Opie et al., 2006) leading to abnormal myocardial stiffness and impaired left ventricular (LV) function (Lopez et al., 2006, Rossi, 1998, Rossi and Peres, 1992). In this respect, increased matrix metalloproteinase (MMP) activity has been implicated in the degradation and reorganization of cardiac extracellular matrix (ECM) that usually results in abnormal collagen deposition or fibrosis (Gajarsa and Kloner, 2011, Matsusaka et al., 2006). Importantly, MMP-2 is particularly relevant in pressure overload-induced cardiac remodeling (Iwanaga et al., 2002, Matsusaka et al., 2006, Rizzi et al., 2010, Spinale et al., 2000, Tozzi et al., 2007), and increased MMP-2 expression was sufficient to induce severe ventricular remodeling and systolic dysfunction in absence of any superimposed injury (Bergman et al., 2007), possibly explained by intracellular targets being degraded by MMP-2 (Schulz, 2007). These findings have supported the suggestion that MMP inhibition is an important pharmacological target in LV hypertrophy (Rizzi et al., 2010, Spinale, 2002, Spinale, 2007, Spinale et al., 2000).

MMP activity is inhibited by endogenous mediators, the tissue inhibitors of MMPs (TIMPs) (Clark et al., 2008, Fanjul-Fernandez et al., 2010, Schulz, 2007) and impaired TIMP function promotes cardiac dysfunction and remodeling during pressure overload (Kandalam et al., 2011). Although four TIMPs (TIMP-1 to -4) have been identified, TIMP-4 is abundantly found in myocardial tissue (Schulz, 2007) and this inhibitor has exerts potent effects on MMP-2 activity (Bigg et al., 1997). Importantly, increased levels of reactive oxygen species (ROS), which are implicated in cardiac remodeling (Rizzi et al., 2011, Tsutsui et al., 2011, Zhao et al., 2008), may result in impaired TIMP-4 activity and MMP-2 activation (Donnini et al., 2008, Kandasamy et al., 2009, Luchtefeld et al., 2005), thus promoting enhanced net MMP activity and modify ECM.

Collagen is a major component of myocardial ECM and its turnover is primarily regulated by fibroblast (Berk et al., 2007). In hypertension, these cells proliferate in response to increased levels of key mediators including angiotensin II and transforming grown factor-β (TGF-β), and therefore fibroblasts become myofibroblasts resulting in increased collagen deposition (Berk et al., 2007, Rosenkranz, 2004). This is of particular importance since angiotensin II exerts hypertrophic effects via increased ROS and TGF-β levels (Rizzi et al., 2011, Rosenkranz, 2004, Sadoshima and Izumo, 1993, Zhao et al., 2008), and both factors promote MMP activation.

Experimental two kidney, one clip (2K1C) hypertension is clearly dependent on the activation of the renin–angiotensin–aldosterone system (Doggrell and Brown, 1998). While increased MMP activity and fibrosis has been shown in later phases of cardiac hypertrophy in 2K1C hypertension (Rizzi et al., 2010, Rizzi et al., 2011), it not known whether imbalanced MMPs activity is present in early phases of 2K1C hypertension-induced cardiac remodeling. In addition, the relation between MMP activity (especially MMP-2) and the levels of the key players discussed above (TIMP-4, TGF-β, ROS levels) are not known. Our general hypothesis was that these alterations would occur concomitantly and at early phases of 2K1C hypertension. In the present study, we examined the cardiac remodeling and the cardiac changes in collagen, MMP-2, TIMP-4, TGF-β, and ROS levels during the development of 2K1C hypertension.