Inhibition of microRNA-23b prevents polymicrobial sepsis-induced cardiac dysfunction by modulating TGIF1 and PTEN

Abstract

Cardiovascular dysfunction is a major complication associated with sepsis induced mortality. Cardiac fibrosis plays a critical role in sepsis induced cardiac dysfunction. The mechanisms of the activation of cardiac fibrosis is unclarified. In this study, we found that microRNA-23b (miR-23b) was up-regulated in heart tissue during cecal ligation and puncture (CLP)-induced sepsis and transfection of miR-23b inhibitor improved survival in late sepsis. Inhibition of miR-23b in the myocardium protected against cardiac output and enhanced left ventricular systolic function. miR-23b inhibitor also alleviated cardiac fibrosis in late sepsis. MiR-23b mediates the activation of TGF-β1/Smad2/3 signaling to promote the differentiation of cardiac fibroblasts through suppression of 5′TG3′-interacting factor 1 (TGIF1). MiR-23b also induces AKT/N-Cadherin signaling to contribute to the deposition of extracellular matrix by inhibiting phosphatase and tensin homologue (PTEN). TGIF1 and PTEN were confirmed as the targets of miR-23b in vitro by Dual-Glo Luciferase assay. miR-23b inhibitor blocked the activation of adhesive molecules and restored the imbalance of pro-fibrotic and anti-fibrotic factors. These data provide direct evidence that miR-23b is a critical contributor to the activation of cardiac fibrosis to mediate the development of myocardial dysfunction in late sepsis. Blockade of miR-23b expression may be an effective approach for prevention sepsis-induced cardiac dysfunction.

Introduction

Sepsis is identified as a systemic deleterious inflammatory response to infection or injury [1]. The severity of sepsis and septic shock is associated with high mortality rate, which mainly results from dysfunction and failure of vital organs [1,2]. Cardiovascular dysfunction is a major complication associated with sepsis induced mortality [3]. Survivors of severe sepsis also have high risk of cardiovascular events [4]. Cardiac fibrosis, an important hallmark of maladaptive hypertrophy, plays a critical role in sepsis induced cardiac dysfunction [5,6], which is characterized by the adverse accumulation of collagens and other extracellular matrix (ECM), resulting in myocardial stiffness, cardiac remodeling, and eventual heart failure [7]. However, the mechanisms inducing the activation of cardiac fibrosis remain unclarified in severe sepsis.

The activation of transforming growth factor-β1 (TGF-β1) signaling mediates cardiac fibrosis by accumulation of fibroblasts and fibroblast-to-myofibroblast transition (FMT) [8]. TGF-β1 promotes profibrotic signaling by activating Smad2/3 canonical pathway [9]. In addition, TGF-β1 signal can also orchestrate through non-canonical pathways including PI3K/AKT, ERK1/2, and p38 MAPK, which can coordinate with the Smad-dependent canonical pathway to induce fibrosis [9,10]. Therefore, the negative regulators of the TGF-β1/Smad2/3 pathway are well-defined to protect against fibrosis [11]. 5′TG3′-interacting factor 1 (TGIF1) is the transcriptional repressor of TGF-β1 signaling via the Smad-dependent pathway [12]. Phosphatase and tensin homologue (PTEN) is a dual protein which dephosphorylates focal adhesion kinase (FAK) and suppresses the activation of PI3K/AKT signaling [13].

Sepsis is initiated by a hyperinflammatory reaction and shifts within a few days to a protracted state of anti-inflammation and immunosuppression, which is associated with increased production of immunosuppressive cytokines, including TGF-β1 [14]. Inhibition of TGF-β1/Smad2/3 signal may improve the host immunosuppression following sepsis [15], and deletion of Smad2/3 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling [16]. The complex pathogenesis of sepsis-induced cardiomyopathy involves a combination of dysfunction of cardiomyocytes, cardiac fibroblasts and/or endothelial cells [17]. Cardiac fibroblasts are important pathogenesis in inflammation and fibrosis in the heart during sepsis and lead to cardiac dysfunction that would affect the outcome of sepsis by elevating adhesion to ECM and adhesive signaling [18].

microRNAs (miRNAs) are dominant players in different aspects of cardiac remodeling, including fibrosis [19,20]. microRNA-23b (miR-23b) is emphasized recently as a multiple functional miRNA because of the prominent effects on immunology and inflammatory signal in sepsis and autoimmune diseases [[21], [22], [23]]. Iaconetti et al reported that miR-23b as a regulator of vascular smooth muscle cells (VSMC) phenotypic switch and revealed that miR-23b suppressed urokinase-type plasminogen activator, SMAD family member 3, and transcription factor forkhead box O4 (FoxO4) expression in phenotypically modulated VSMCs [25]. However, the role of miR-23b in sepsis-induced cardiac dysfunction is not known yet.

Knockdown of miR-23b cluster miRNAs in fetal and newborn liver can block or revert TGF-β1-induced liver fibrosis [26]. miR-23b-3p was significantly up-regulated in keloid fibroblasts (KFs) contributing to the etiology of keloids by affecting several pro-fibrotic signaling pathways [27]. These studies suggest that the upregulation of miR-23b is associated the activation of pro-fibrotic signal. Therefore, we hypothesized that the induction of miR-23b in polymicrobial sepsis might emerge as a modifier to regulate fibrotic remodeling in the heart.