Original article
Cytokine expression profiling of the myocardium reveals a role for CX3CL1 (fractalkine) in heart failure

https://doi.org/10.1016/j.yjmcc.2008.05.009Get rights and content

Abstract

Several lines of evidence suggest that inflammatory processes mediated by cytokines are involved in the pathogenesis of heart failure (HF). However, the regulation of cytokine expression and the role of cytokines during HF development are not well understood. To address this issue, we have examined alterations in gene expression during HF progression by microarray technology in non-infarcted left ventricular (LV) murine tissue at various time points after myocardial infarction (MI). The highest number of regulated genes was found five days after MI. In total, we identified 14 regulated genes encoding cytokines with no previous association to HF. The strongest up-regulation was found for the chemokine fractalkine (CX3CL1). In human failing hearts we detected a 3-fold increase in CX3CL1 protein production, and both cardiomyocytes and fibrous tissue revealed immunoreactivity for CX3CL1 and its specific receptor CX3CR1. We also found that the circulating level of CX3CL1 was increased in patients with chronic HF in accordance with disease severity (1.6-fold in NYHA II, 2.2-fold in NYHA III and 2.9-fold in NYHA IV). In vitro experiments demonstrated that CX3CL1 production could be induced by inflammatory cytokines known to be highly expressed in HF. CX3CL1 itself induced the expression of markers of cardiac hypertrophy and protein phosphatases in neonatal cardiomyocytes. Given the increased CX3CL1 production in both an experimental HF model and in patients with chronic HF as well as its direct effects on cardiomyocytes, we suggest a role for CX3CL1 and its receptor CX3CR1 in the pathogenesis of HF.

Introduction

Although treatment strategies for heart failure (HF) have improved over the last decades, this clinical syndrome is still a major cause of death. This indicates that important pathogenic mechanisms are not modified by the present treatment modalities [1]. Several lines of evidence suggest that persistent inflammation and immune activation could represent such mechanisms affecting development of HF [2], [3]. Moreover, the complex regulation of inflammatory mediators and their differential expression in myocardial tissue during HF development are far from completely understood. To address these issues, we have in this study performed microarray analysis of non-infarcted left ventricular (LV) tissue from mice subjected to left coronary artery ligation. In total, we discovered 14 cytokines which had not previously been assigned a role in HF progression.

CX3CL1 is a unique member of the CX3C chemokine subfamily and differs from most other cytokines in that it can function both as a circulating cytokine (soluble form) and in a membrane-bound form [4]. The full-length, membrane-bound form is thought to act mainly as an adhesion molecule promoting retention of leukocytes, while the soluble form resembles a conventional chemokine exhibiting chemotactic activity on monocytes, natural killer cells and T cells [4], [5]. In any form, the activity of CX3CL1 depends upon binding to the receptor CX3CR1, which originally was shown to be coupled to and act through pertussis toxin-sensitive Gαi protein [6]. Later, CX3CR1 has also been shown to signal through other G-proteins such as Gβγ[7], [8].

Previously, CX3CL1 has been suggested to play a role in atherogenesis [9] and two specific mutations in the CX3CR1 gene have been shown to be associated with reduced risk for future coronary events [10]. Furthermore, accumulating evidence indicates that CX3CL1/CX3CR1 are involved in the pathogenesis of other inflammatory disorders such as glomerulonephritis [11], rheumatoid arthritis [12], cardiac allograft rejection [13] and HIV infection [14]. To our knowledge, the potential role of CX3CL1 in the pathogenesis of myocardial hypertrophy or HF has not previously been studied.

Here we report that CX3CL1 synthesis is increased in cardiac tissue, not only in experimental HF, but also in the myocardium of HF patients. The increased myocardial production is accompanied by enhanced serum levels of CX3CL1, in accordance with disease severity. We also show that CX3CR1 is abundantly expressed in cardiomyocytes, and that CX3CL1 can act on cardiomyocytes to stimulate expression of genes involved in development of myocardial hypertrophy and HF.

Section snippets

Animal model of experimental HF

Myocardial infarction (MI) was induced in C57BL/6 mice (Taconic, Skensved, Denmark) [15], and the mice were sacrificed after 3, 5, 7 or 14 days. MI-mice included in the study had an infarcted area > 30% of total endocardial area in addition to significantly increased lung weight and left atrial diameter, indicating HF (see supplementary material) [15]. The animal experiments and housing were in accordance with institutional guidelines and national legislation. The study was approved by the local

Microarray screening for detection of regulated cytokines after MI

Alterations in gene expression in non-infarcted LV tissue following MI were analyzed at 3, 5, 7 and 14 days after ligation of the left coronary artery. Compared to the sham-operated animals, the mice with MI had a significant increase in lung weight, being highest at 14 days (see supplementary material). Similarly, LV fractional shortening was reduced and left atrial diameter was increased at all time points compared to the sham-operated animals, indicating cardiac failure in MI-mice. The

Discussion

A few other studies [26], [27] have examined gene expression in the non-infarcted myocardium with array technology at several time points after MI. While Stanton et al. [26] reported alterations in genes encoding cell signaling molecules and transcription factors, Sehl et al. [27] showed altered expression of genes involved in the wound healing process. Our study, focusing on inflammatory and anti-inflammatory mediators, explores alterations in expression of a larger number of genes in the

Acknowledgments

We thank Bjørg Austbø, Hilde Dishington, Geir Florholmen and Annlaug Ødegaard for excellent technical assistance.

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    S.N. and A.V.F contributed equally to this work.

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