5-Methoxytryptophan attenuates postinfarct cardiac injury by controlling oxidative stress and immune activation

Highlights

• Cardiac 5-MTP biosynthesis is impaired following myocardial infarction (MI).

• 5-MTP treatment within 24 h of MI improves left ventricular systolic function.

• 5-MTP administered within 24 h of MI effectively attenuated cardiac inflammation.

• 5-MTP promotes mitochondrial stabilization and controls redox imbalance.

• 5-MTP protected against H₂O₂-induced cardiomyocyte and endothelial cell death.

Abstract

Aims

Myocardial infarction (MI) remains a major cause of heart failure. 5-Methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of L-tryptophan, exerts anti-inflammatory and antifibrotic effects, but MI impairs the biosynthesis of cardiac 5-MTP. Therefore, we evaluated the effect of exogenous 5-MTP administration on rescuing post-MI cardiac injury.

Methods and results

After a detailed pharmacokinetic analysis of 5-MTP, Sprague Dawley rats that had undergone left anterior descending coronary artery ligation received intraperitoneal administration of either 17 mg/kg 5-MTP or saline at 0.5 and 24 h after MI. Cardiac systolic function, infarction size, and fibrosis were evaluated using echocardiography, triphenyltetrazolium chloride staining, and Masson trichrome staining, respectively. Myocardial apoptosis was analyzed by staining for caspase-3 and cardiac troponin I. 5-MTP treatment decreased the infarct area and myocardial apoptosis; attenuated systolic dysfunction and left ventricular dilatation; and reduced cardiomyocyte hypertrophy, myocardial fibrosis, and infarct expansion. Crucially, 5-MTP alleviated oxidative stress by preserving mitochondrial antioxidant enzymes and downregulating reactive oxygen species–generating NADPH oxidase isoforms and endothelin-1. Consequently, 5-MTP-treated MI rat hearts exhibited lower levels of chemokines and cytokines, namely interleukin (IL)-1β, IL-18, IL-6, C-C motif chemokine ligand (CCL)-2, and CCL5, accompanied by reduced infiltration of CD11b⁺ cells and CD4⁺ T cells. Notably, 5-MTP protected against H₂O₂-induced damage in HL-1 cardiomyocytes and human umbilical vein endothelial cells in vitro.

Conclusion

5-MTP prevented post-MI cardiac injury by promoting mitochondrial stabilization and controlling redox imbalance. This cytoprotective effect ameliorated macrophage and T-cell infiltration, thus reducing the infarct size, attenuating fibrosis, and restoring myocardial function.

Introduction

Cardiac remodeling and subsequent heart failure (HF) after myocardial infarction (MI) cause long-term morbidity and mortality [1]. Timely revascularization can reduce myocardial injury and progressive ventricular remodeling [2,3], but this standard therapeutic modality is not available in all medical centers. Moreover, despite epicardial recanalization, myocardial hypoperfusion related to microvascular injury continues to be a challenge [4]. Revascularization may also induce reperfusion injury, which limits the benefit of preserving ventricular function [5]. In the present study, we identified a novel therapeutic strategy to alleviate cardiac injury after nonreperfused MI.

Apoptosis and inflammation following acute MI are critical pathophysiological processes that lead to cardiac remodeling and dysfunction [6]. Apoptosis of cardiomyocytes and nonmyocytes occurs in infarct and peri-infarct zones immediately after MI [7,8]. Excessive apoptosis can lead to progressive myocardial loss, fibrosis, and functional deterioration [9]. Inflammation is essential in the repair process after an acute cardiac insult [10,11], but exaggerated inflammatory responses can cause tissue damage, cardiac remodeling, and fibrosis [10]. At the site of ischemic injury, CXC motif chemokine ligand 2 (CXCL2) and CC motif chemokine ligand 2 (CCL2) play crucial roles in regulating the migration and infiltration of neutrophils and monocytes/macrophages [12,13]. Besides, several proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, are also expressed to various degrees [14].

The elevated production of reactive oxygen species (ROS) causes oxidative stress. In the infarcted myocardium, a major source of ROS is the infiltrating neutrophils and monocytes involved in the coordinated activation of NADPH oxidase (NOX) and the neutrophil-derived heme enzyme myeloperoxidase (MPO) [15]. Following MI, NOX activation and mitochondrial dysfunction trigger a ROS burst [16]. Three NOX isoforms (NOX1, NOX2, and NOX4) are expressed in heart tissues and contribute to ROS genesis [17]. Moreover, activation of NOX can stimulate endothelin-1 (ET-1) release [18], and inhibition of ET-1 in rats has been reported to confer cardioprotection [19]. Overall, this redox dysregulation is critical in the pathogenesis of HF after MI [20]. Therefore, the development of innovative therapeutic approaches to control inflammation and oxidative stress remains an urgent need.

5-Methoxytrptophan (5-MTP) was first identified as a tryptophan metabolite synthesized by and released from human fibroblasts [21]. Enzymatic catalysis of 5-MTP in human cells involves two enzymes: tryptophan hydroxylase (TPH), which catalyzes the conversion of L-tryptophan to 5-hydroxytryptophan (5-HTP), and hydroxyindole O-methyltransferase, which converts 5-HTP to 5-MTP [22]. Of the two TPH isoforms, only TPH-1, which mediates 5-MTP synthesis, is expressed in human cells [22]. Furthermore, a defect in the TPH-1 gene was demonstrated to cause cardiac dysfunction [23].

The anti-inflammatory properties of 5-MTP have been reported both in vitro and in vivo [24]. Vascular endothelial cells produce and secrete 5-MTP, which acts in a paracrine and autocrine manner to protect endothelial barrier function, control the expression of adhesion molecules, and inhibit leukocyte transmigration [25,26]. Furthermore, 5-MTP inhibits the expression of lipopolysaccharide (LPS)-induced cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) and the release of proinflammatory cytokines and chemokines in macrophages [27]. Mechanistically, 5-MTP exerts anti-inflammatory effects on endothelial cells, macrophages, and vascular smooth muscle cells by blocking p38 and nuclear factor (NF)-κB activation [27,28]. In a carotid artery ligation model, 5-MTP was observed to prevent arterial injury–induced intimal hyperplasia [27]. 5-MTP also ameliorates renal ischemia/reperfusion injury and suppresses bleomycin-induced pulmonary fibrosis through an antifibrotic effect [29,30]. Regarding cardioprotective effects, 5-MTP was found to rescue rat cardiomyocytes from H₂O₂-induced oxidative damage [31]. Moreover, clinically, 5-MTP appears to be a useful early marker of HF after MI [32]. Therefore, it is reasonable to hypothesize that 5-MTP protects against post-MI injury and dysfunction by controlling inflammation and fibrosis.

In this study, we investigated this hypothesis, and our findings provide evidence in support of it. 5-MTP administration to a rat coronary artery ligation model reduced infarct size, attenuated myocardial inflammation, alleviated cardiac fibrosis, and preserved cardiac function.