Aldosterone increases plasminogen activator inhibitor-1 synthesis in rat cardiomyocytes

Abstract

Plasminogen activator inhibitor-1 (PAI-1) is an anti-thrombolytic factor that also promotes tissue fibrosis. Under certain conditions, exposure to aldosterone can result in cardiac fibrosis by an unknown mechanism. In the current study, we tested the hypothesis that PAI-1 is a mediator of aldosterone's fibrotic effects. Aldosterone increased levels of PAI-1 mRNA and protein in the H9c2 rat cardiac cell line, responses that could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone. Confocal microscopy confirmed an effect of aldosterone to increase PAI-1 expression with reversal by spironolactone. Aldosterone also increased PAI-1 expression in neonatal rat cardiomyocytes, which was again blocked by spironolactone. In the neonatal cardiomyocytes (but not the H9c2 cells), anti-transforming growth factor-β1 antibody inhibited the PAI-1 response to aldosterone. In summary, aldosterone directly increased PAI-1 expression in two different cardiac muscle cell types, an effect that was dependent on MR. In the neonatal cells, there appeared to be a requirement for transforming growth factor-β1.

Introduction

In the heart, under conditions of high salt intake, aldosterone produced fibrosis and ventricular hypertrophy in several different rat models (Brilla et al., 1990, Rocha et al., 1998, Rocha et al., 2000). In studies of transgenic mice overexpressing 11 β-hydroxysteroid dehydrogenase type 2 in cardiomyocytes, thereby protecting mineralocorticoid receptors (MR) in heart from occupancy by glucocorticoids in favor of aldosterone, fibrosis and hypertrophy developed and eventually heart failure resulting in premature death (Qin et al., 2003). A relationship between aldosterone and cardiac function was evident in two clinical trials of heart failure where treatment with MR antagonists reduced cardiovascular morbidity and mortality (Pitt et al., 1999, Pitt et al., 2003). The mechanism for how aldosterone has these effects on the heart is unknown.

Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of thrombolysis (Saksela and Rifkin, 1988), is known to promote fibrosis (Eddy, 2002, Kaikita et al., 2001, Loskutoff and Quigley, 2000), an effect which could contribute to its known relationship to cardiovascular disease risk (Hamsten et al., 1985, Hamsten et al., 1987, Schneiderman et al., 1992). Angiotensin II (Ang II) stimulates PAI-1 synthesis (Brown et al., 1998, Pahor et al., 2002, Ridker et al., 1993, Vaughan et al., 1995) and there is emerging evidence that aldosterone, whose secretion is stimulated by Ang II, may mediate some of Ang II's effects. Aldosterone and Ang II acted synergistically to increase PAI-1 expression in studies using rat aortic smooth muscle and human umbilical vein endothelium (Brown et al., 2000a). Aldosterone has also been shown to increase PAI-1 synthesis in isolated human neutrophils (Calo et al., 2004). Treatment with spironolactone, an MR antagonist, lowered blood levels of PAI-1 in both animal (Brown et al., 2000b) and human studies (Sawathiparnich et al., 2002). On the other hand, PAI-1 deficient mice [PAI-1 (−/−)] infused with Ang II and in addition treated with the NO synthase inhibitor N^g-nitro-l-arginine methyl ester (l-NAME) were unprotected from myocardial injury whereas spironolactone was protective (Oestreicher et al., 2003). Thus, the role of PAI-1 as a mediator of aldosterone's deleterious effects on the heart remains controversial. Because of the potential for PAI-1 to promote fibrosis, we looked further at its role as a mediator of aldosterone's actions in the heart by exploring for direct influences of aldosterone on PAI-1 expression in myocardial cells.