Captopril prevents myosin light chain phosphatase isoform switching to preserve normal cGMP-mediated vasodilatation

Abstract

Congestive heart failure (CHF) is characterized by abnormal vasoconstriction and an impairment in nitric oxide (NO)-mediated vasodilatation. We have previously demonstrated that the decrease in sensitivity to NO lies at least partially at the level of the smooth muscle and is due to a reduction in the relative expression of the leucine zipper positive (LZ⁺) isoform of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase. We hypothesized that since the attenuated vasodilatory response to NO in CHF has been shown to be secondary to an increased activity of the renin–angiotensin system, angiotensin converting enzyme (ACE) inhibition could affect MYPT1 isoform expression. To test this hypothesis, a rat myocardial infarction (MI) model of CHF was used; following left coronary artery ligation, rats were divided into control and captopril-treated groups. A third group of rats was given prazosin for 4 weeks. In the untreated control group, left ventricular function (LVF) was reduced at 2 weeks post-MI and remained at this level. Captopril treatment attenuated the fall in LVF. In the control aorta and iliac artery, the expression of the LZ⁺ MYPT1 isoform fell 44–52% between 2 and 4 weeks post-MI, whereas in animals treated with captopril, MYPT1 isoform expression did not change. A decrease in the sensitivity to cGMP-mediated smooth muscle relaxation occurred coincident with the decrease in LZ⁺ MYPT1 expression. The change in LZ⁺ MYPT1 expression was not due to the decrease in afterload, as prazosin therapy produced an improvement in LVF but did not increase the relative expression of LZ⁺ MYPT1 isoform. These data suggest that ACE inhibition, unique from pure afterload reduction, prevents MYPT1 isoform switching, which would preserve normal flow, or NO-mediated vasodilatation.

Introduction

Congestive heart failure (CHF) is a clinical syndrome, which is associated with a vascular abnormality, characterized by an elevated baseline vascular tone and impaired response to NO-mediated vasodilatation [1]. While neuroendocrine activation of the renin–angiotensin system has been implicated in contributing to the abnormal vasoconstriction in CHF [2], [3], recent studies have shed more light on angiotensin II (Ang II) as a pleiotropic mediator in modulating the endothelium and regulating vascular remodeling [4]. In vitro studies on cultured vascular smooth muscle cells (VSMCs) have demonstrated that Ang II increases superoxide production by activation of NADH/NADPH oxidases, which leads to enhanced NO catabolism and decreased availability [5]. Additionally, the reactive oxygen species, superoxide anions, and hydrogen peroxide, may also stimulate hypertrophy and hyperplasia of vascular smooth muscle cells [6], [7], [8]. Although decreased NO bioavailability would seem to be a likely explanation for enhanced vascular tone, other animal models and human studies have shown that endothelium dependent as well as independent vasorelaxation are impaired in CHF [1], [9], [10].

We have previously demonstrated that cGMP-mediated smooth muscle relaxation is attenuated in CHF and this abnormality lies at least partially at the level of the smooth muscle [11]. Similarly, others [12] have shown in a rat model of heart failure that the impairment in NO-mediated vasodilatation is present despite intact baseline NO production. Further, this group [12] demonstrated that Ang II receptor blockade restored the renal vasodilatory response to acetylcholine (ACh). These studies are consistent with the inherent flaw in abnormal vascular reactivity in CHF lying not only at the level of the endothelium where NO is synthesized, but also deeper at the level of the vascular smooth muscle where a remodeling phenomena is taking place possibly secondary to the influence of the renin–angiotensin cascade.

At the molecular level, vascular tone is determined by the level of smooth muscle myosin regulatory light chain (MLC₂₀) phosphorylation, and thus tone can be modulated by either an increase or decrease in MLC₂₀ phosphorylation [13]. Nitric oxide acts via the cGMP-dependent protein kinase Iα (PKGIα) pathway to not only decrease intracellular Ca²⁺ but also to activate myosin light chain phosphatase (MLCP), which dephosphorylates MLC₂₀, resulting in smooth muscle relaxation [14], [15], [16], [17]. MLCP is a heterotrimeric protein consisting of a 110–130 kDa myosin targeting subunit (MYPT1), a catalytic subunit, and a 20 kDa subunit of unknown function [13], [15]. MYPT1 has four isoforms that are generated via alternative splicing of a central and a 3′ exon [15]. Interestingly, the exclusion of the 31 bp 3′ exon results in a C-terminal leucine zipper (LZ) motif while inclusion of the exon encodes a MYPT1 lacking the LZ [18]. PKGIα has been demonstrated to bind to MYPT1 [14]. The C-terminal LZ motif has been demonstrated to be required for PKGIα-mediated activation of MLCP activity and smooth muscle relaxation [19], and the sensitivity to cGMP-mediated smooth muscle relaxation has been demonstrated to be proportional to LZ⁺/LZ⁻ MYPT1 expression [11], [18], [19].

We have previously demonstrated in the rat infarct model that at 8 weeks post-infarction there is a significant decrease in the expression of the leucine zipper positive (LZ⁺) MYPT1 isoform, which results in a decrease in sensitivity to cGMP-mediated smooth muscle relaxation [11]. This suggests that the impairment in NO-mediated vasodilatation characterizing CHF lies in part at the level of the smooth muscle and is due to the decrease in LZ⁺ MYPT1 isoform expression. In the present study, we tested the hypothesis that the fall in LZ⁺ MYPT1 occurs as soon as left ventricular function begins to deteriorate. In addition, since others have demonstrated that Ang II blockade restores ACh-mediated vasodilatation in an animal model of CHF [12], we tested whether pharmacologic intervention with an ACE inhibitor (captopril) to block the activation of the renin–angiotensin cascade can modulate vascular smooth muscle remodeling by altering MYPT1 isoform expression.