Role of reactive oxygen species in the regulation of cardiac contractility

Abstract

Increased production of reactive oxygen species (ROS) has been linked to the pathogenesis of contractile dysfunction in heart failure. However, it is unclear whether ROS can regulate physiological cellular processes in the myocardium. Here, we characterized the role of endogenous ROS production in the acute regulation of cardiac contractility in the intact rat heart. In isolated perfused rat hearts, endothelin-1 (ET-1, 1 nmol/L) stimulated ROS formation in the left ventricle, which was prevented by the antioxidant N-acetylcysteine and the NAD(P)H oxidase inhibitor apocynin. N-acetylcysteine, the superoxide dismutase mimetic MnTMPyP, and apocynin significantly attenuated ET-1-mediated inotropic effect, which was accompanied by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Moreover, the mitochondrial K_ATP channel blocker 5-HD, and the mitochondrial large conductance calcium activated potassium channel blocker paxilline, but not the sarcolemmal K_ATP channel blocker HMR 1098 attenuated the inotropic response to ET-1. However, ET-1-induced ROS generation was not abolished by inhibiting mitochondrial K_ATP channel opening. In contrast to ET-1 stimulation, the positive inotropic effect of β₁-adrenergic receptor agonist dobutamine (250 nmol/L) was significantly augmented by N-acetylcysteine and apocynin. Moreover, dobutamine-induced phospholamban phosphorylation was markedly enhanced by apocynin. In conclusion, NAD(P)H oxidase-derived ROS play a physiological role in the acute regulation of cardiac contractility in the intact rat heart. Our results reveal that ET-1-induced increase in cardiac contractility is partially dependent on enhanced ROS generation, which in turn, activates the ERK1/2 pathway. On the other hand, β-adrenergic receptor-induced positive inotropic effect and phospholamban phosphorylation is enhanced by NAD(P)H oxidase inhibition.

Introduction

Oxygen by-products, such as superoxide anion (O₂·⁻) and hydrogen peroxide (H₂O₂), are produced as a consequence of normal aerobic metabolism. These molecules are referred to as reactive oxygen species (ROS) and they are highly reactive with other biological molecules. Under normal physiological conditions, ROS production is balanced by an efficient system of antioxidants, molecules that are capable of scavenging ROS [1]. However, ROS are present in relative excess in various pathological states, including congestive heart failure (CHF). It is well established that oxidative stress in CHF triggers a variety of changes such as cardiac hypertrophy, loss of functional myocytes due to enhanced apoptosis and necrosis, and interstitial fibrosis ultimately leading to pump dysfunction [1], [2], [3], [4]. Enhanced ROS production may also directly contribute to contractile dysfunction in this setting. Excessive levels of ROS can alter the activity of different proteins involved in excitation–contraction coupling, including the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the SR Ca²⁺ ATPase, and the L-type Ca²⁺ channel, by modifying sulfhydryl groups of cysteine residues [5].

In contrast to pathological conditions, less is known about the role of ROS in the regulation of normal cardiac function. Recently a relationship has been suggested between contractile activity, oxidative metabolism and ROS production. Increases in contraction frequency are accompanied by enhanced oxygen consumption and ROS formation in isolated cardiomyocytes [6], [7]. However, the effect of ROS on contractile function is controversial. Some studies suggest that ROS may have no effect [8] or exert a negative inotropic effect [9], [10], whereas others propose that ROS may indeed augment contractile force [11], [12], [13].

To resolve the existing discrepancy, the objective of the present study was to characterize the role of endogenous ROS production in the acute regulation of cardiac contractility by G protein-coupled receptor (GPCR) agonists under physiological conditions in the intact rat heart. Moreover, we studied the potential cellular sources of ROS and the molecular mechanisms by which ROS affect contractility. To accomplish these goals, we used two characteristically distinct inotropic stimuli, endothelin-1 (ET-1) [14], [15], acting mainly via ET_A receptors [16], [17] and the β₁-adrenergic receptor agonist dobutamine [15]. Stimulation of cardiac β₁-adrenergic receptors activates the G_s protein–adenylyl cyclase–cAMP–protein kinase A (PKA) pathway that mediates the evolutionarily conserved “fight-or-flight” response to stress [18]. In contrast, ET-1 increases contractile force via extracellular signal regulated kinase 1/2 (ERK1/2)−p90 ribosomal S6 kinase−Na⁺-H⁺ exchanger-1 pathway [19] and may contribute to the Gregg effect [17] and the Frank–Starling response [20].