Original article
Death receptor 5 contributes to cardiomyocyte hypertrophy through epidermal growth factor receptor transactivation

https://doi.org/10.1016/j.yjmcc.2019.08.011Get rights and content

Highlights

  • Death Receptor (DR) 5 is expressed in cardiomyocytes and activation does not result in classical death receptor signaling

  • DR5 activation in isolated cardiomyocytes and heart leads to ERK1/2 activation and results in hypertrophy

  • ERK1/2 activation by DR5 occurs through EGFR-transactivation and GATA4 leading to pro-hypertrophic gene transcription.

Abstract

Cardiomyocyte survival and death contributes to many cardiac diseases. A common mechanism of cardiomyocyte death is through apoptosis however, numerous death receptors (DR) have been virtually unstudied in the context of cardiovascular disease. Previous studies have identified TNF-related apoptosis inducing ligand (TRAIL) and its receptor, DR5, as being altered in a chronic catecholamine administration model of heart failure, and suggest a role of non-canonical signaling in cardiomyocytes. Furthermore, multiple clinical studies have identified TRAIL or DR5 as biomarkers in the prediction of severity and mortality following myocardial infarction and in heart failure development risk suggesting a role of DR5 signaling in the heart. While TRAIL/DR5 have been extensively studied as a potential cancer therapeutic due to their ability to selectively activate apoptosis in cancer cells, TRAIL and DR5 are highly expressed in the heart where their function is uncharacterized. However, many non-transformed cell types are resistant to TRAIL-induced apoptosis suggesting non-canonical functions in non-cancerous cell types. Our goal was to determine the role of DR5 in the heart with the hypothesis that DR5 does not induce cardiomyocyte apoptosis but initiates non-canonical signaling to promote cardiomyocyte growth and survival. Histological analysis of hearts from mice treated with a DR5 agonists showed increased hypertrophy with no differences in cardiomyocyte death, fibrosis or function. Mechanistic studies in the heart and isolated cardiomyocytes identified ERK1/2 activation with DR5 agonist treatment which contributed to hypertrophy. Furthermore, epidermal growth factor receptor (EGFR) was activated following DR5 agonist treatment through activation of MMP and HB-EGFR cleavage and specific inhibitors of MMP and EGFR prevented DR5-mediated ERK1/2 signaling and hypertrophy. Taken together, these studies identify a previously unidentified role for DR5 in the heart, which does not promote apoptosis but acts through non-canonical MMP-EGFR-ERK1/2 signaling mechanisms to contribute to cardiomyocyte hypertrophy.

Introduction

Cardiovascular disease is a leading cause of morbidity and mortality worldwide [1]. A common characteristic and main contributor to most cardiac diseases is cell death, which occurs through two main mechanisms, necrosis and apoptosis [2]. There are two main pathways through which apoptosis occurs, the intrinsic pathway that uses mitochondrial pathways and has been extensively studied in the heart, and extrinsic pathway that occurs through cell surface death receptors [3]. Some extrinsic pathways, such as tumor necrosis factor (TNF)-α, are also well characterized in the heart however, there are numerous other death receptors that have been virtually unstudied in the context of cardiovascular disease and heart failure [4].

Whole transcriptome analysis in a chronic catecholamine administration model of heart failure identified TNF-related apoptosis inducing ligand (TRAIL; also known as Tnfsf10 and CD253) and its receptor DR5 (TRAIL-R2) as being altered with cardioprotective β-adrenergic receptor signaling in mouse hearts [5]. There have been virtually no studies investigating the role of TRAIL/DR5 in the heart. However, TRAIL and its three receptors, DR4, DR5 and DcR1, are moderately to highly expressed at the transcript and protein level in the human and chimpanzee heart [6,7]. Rodents express a single death domain containing receptor for TRAIL, DR5, which is homologous to human DR4 and DR5 and also have high DR5 expression at the mRNA level in the heart [8]. Furthermore, in the last several years, multiple clinical studies have identified TRAIL or DR5 as biomarkers in the prediction of severity and mortality following myocardial infarction and in heart failure development risk [[9], [10], [11]]. In a study employing proteomics on serum of patients following acute myocardial infarction, DR5 levels were found to be one of the most powerful biomarkers in predicting long-term mortality with low levels of circulating DR5 being associated with worsened outcome [9]. Additionally, in a longitudinal study spanning over a decade, DR5 was identified as a biomarker for predicting the development of heart failure in elderly patients and associated with worsened left ventricular function with high levels of circulating DR5 being associated with worsened systolic function [10].

The majority of TRAIL research has focused on therapeutically targeting the TRAIL system for the treatment of cancer due to its ability to selectively induce apoptosis in cancer cells [12]. Many non-transformed cell types are resistant to TRAIL-induced apoptosis, suggesting non-canonical TRAIL/DR5 functions in non-cancerous cell types [13]. Pre-clinical and phase I clinical trials assessing the safety of TRAIL administration have demonstrated no toxicity however, the cardiac effects were not carefully examined and treatment durations were short [12,[14], [15], [16]]. While the literature is sparse, there is evidence to suggest that TRAIL signaling in relevant cell types to the heart do not lead to apoptosis. Dermal fibroblasts and umbilical artery smooth muscle cells are resistant to TRAIL induced apoptosis in spite of expressing both DR4 and DR5 [17]. In skeletal myoblasts, TRAIL has a non-canonical function of promoting differentiation [18]. In a separate study, a number of human fibroblast cell lines were found express DR4 and DR5 but to be resistant to TRAIL-induced apoptosis due to differences in the ability to activate caspase 8 compared with cancer cell lines [19]. It is not clear what causes resistance to TRAIL in non-transformed cell types, it is likely that alterations in the apoptotic machinery attribute to these differences [17]. These findings demonstrate the necessity of understanding the function of TRAIL outside of tumor cells since TRAIL is secreted by most cells types [20], including cardiomyocytes [5], and it is becoming increasingly apparent that the role of TRAIL differs in non-transformed cell types. Furthermore, while minimal research has been performed in cell types closely relevant to the heart, there have been no investigation into the function of the TRAIL/DR5 system in the heart, in spite of its high expression and strong correlation with cardiovascular disease in humans [[9], [10], [11]]. The purpose of this study was to investigate the role of DR5 activation in the heart with the hypothesis that DR5 does not induce cardiomyocyte apoptosis but initiates non-canonical signaling to promote cardiomyocyte growth and survival. Using DR5 agonist administration in vitro and in vivo, cardiomyocyte death was not observed, but increased hypertrophy occurred through the activation of ERK1/2. EGFR transactivation occurred with DR5 activation and specific inhibitors of matrix metalloprotease (MMP) and EGFR prevented ERK1/2 activation and hypertrophy. Hypertrophy occurred through the ERK1/2-mediated activation of the transcription factor GATA4 to promote pro-hypertrophic gene transcription. Taken together, these findings identify novel role for DR5 in cardiomyocytes where activation does not promote cell death, but results in hypertrophy through ERK1/2-EGFR-GATA4 dependent mechanisms.

Section snippets

Animals

C57BL/6 mice (male; 8–12 weeks old) were randomly assigned to treatment group and administered MD5–1 antibody (BioXCell, catalog # BE0161; 2 mg/kg) or IgG control (BioXCell, catalog # BE0091; 2 mg/kg) via intraperitoneal injection every 3 days. Another group was administered vehicle (10% DMSO in sterile saline) or bioymifi (5 mg/kg/d) via osmotic minipump (Alzet). Mice were euthanized after 2 weeks administration, hearts were excised and flash frozen in liquid nitrogen for use in biochemical

DR5 activation does not induce cardiomyocyte death

Few studies have examined DR5 expression in the heart however, it is thought to be moderately to highly expressed at the transcript and protein level in the human and chimpanzee heart [6,7] and highly expressed at the mRNA level in the murine heart [8]. To confirm DR5 protein expression in mouse and rat, hearts from C57BL6 mice were subjected to enzymatic digestion to isolate cardiomyocyte and non-cardiomyocyte cells of the heart and DR5 expression was examined by immunoblot. DR5 expression was

Discussion

Cardiomyocyte growth, survival and death are critical contributors to the outcome of heart failure. The TRAIL/DR5 system has been implicated in multiple types of heart failure [[9], [10], [11]], but their role in the heart has never been established. Initial characterization of DR5 expression in mice identified high expression in heart, lung and kidney and moderate to high expression in the human and chimpanzee heart [6,7], however, very little is known about the role of TRAIL and DR5 in the

Funding

This work was supported by American Heart Association Scientific Development Grant 17SDG33400114 (L.A.G.).

Declaration of Competing Interest

The authors have declared that no conflict of interest exists.

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