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

Journal Pre-proof Death receptor 5 contributes to cardiomyocyte hypertrophy through epidermal growth factor receptor transactivation

Miles A. Tanner, Toby P. Thomas, Laurel A. Grisanti PII:

S0022-2828(19)30172-5

DOI:

https://doi.org/10.1016/j.yjmcc.2019.08.011

Reference:

YJMCC 9053

To appear in:

Journal of Molecular and Cellular Cardiology

Received date:

19 April 2019

Revised date:

26 August 2019

Accepted date:

27 August 2019

Please cite this article as: M.A. Tanner, T.P. Thomas and L.A. Grisanti, Death receptor 5 contributes to cardiomyocyte hypertrophy through epidermal growth factor receptor transactivation, Journal of Molecular and Cellular Cardiology(2018), https://doi.org/ 10.1016/j.yjmcc.2019.08.011

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

Journal Pre-proof Death Receptor 5 Contributes to Cardiomyocyte Hypertrophy Through Epidermal Growth Factor Receptor Transactivation Miles A. Tanner1, Toby P. Thomas 1 and Laurel A. Grisanti, PhD 1,* [email protected] 1

Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri,

Columbia, MO, USA *

Corresponding author at: Department of Biomedical Sciences, University of Missouri-Columbia

of

1600 E. Rollins Street, Columbia, MO 65211, Room W158

ro

Abstract:

-p

Cardiomyocyte survival and death contributes to many cardiac diseases. A common

re

mechanism of cardiomyocyte death is through apoptosis however, numerous death receptors (DR) have been virtually unstudied in the context of cardiovascular disease. Previous studies

lP

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

na

non-canonical signaling in cardiomyocytes. Furthermore, multiple clinical studies have identified

ur

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.

Jo

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

Journal Pre-proof 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 DR5mediated 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 noncanonical MMP-EGFR-ERK1/2 signaling mechanisms to contribute to cardiomyocyte

of

hypertrophy.

-p

ro

Keywords: Death Receptor 5, hypertrophy, ERK1/2, EGFR transactivation

Introduction:

re

Cardiovascular disease is a leading cause of morbidity and mortality worldwide [1]. A

lP

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

na

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

ur

through cell surface death receptors [3]. Some extrinsic pathways, such as tumor necrosis

Jo

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

Journal Pre-proof 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 [9-11]. In a study employing proteomics on serum of patients following acute myocardial infarction, DR5 levels were found to

of

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

ro

study spanning over a decade, DR5 was identified as a biomarker for predicting the

-p

development of heart failure in elderly patients and associated with worsened left ventricular

re

function with high levels of circulating DR5 being associated with worsened systolic function [10].

lP

The majority of TRAIL research has focused on therapeutically targeting the TRAIL

na

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,

ur

suggesting non-canonical TRAIL/DR5 functions in non-cancerous cell types [13]. Pre-clinical

Jo

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, 14-16]. 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

Journal Pre-proof 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

of

correlation with cardiovascular disease in humans [9-11]. The purpose of this study was to investigate the role of DR5 activation in the heart with the hypothesis that DR5 does not induce

ro

cardiomyocyte apoptosis but initiates non-canonical signaling to promote cardiomyocyte growth

-p

and survival. Using DR5 agonist administration in vitro and in vivo, cardiomyocyte death was not

re

observed, but increased hypertrophy occurred through the activation of ERK1/2. EGFR transactivation occurred with DR5 activation and specific inhibitors of matrix metalloprotease

lP

(MMP) and EGFR prevented ERK1/2 activation and hypertrophy. Hypertrophy occurred through

na

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

ur

where activation does not promote cell death, but results in hypertrophy through ERK1/2-EGFR-

Methods:

Jo

GATA4 dependent mechanisms.

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

Journal Pre-proof frozen in liquid nitrogen for use in biochemical assays. For MD5-1 treatment followed by removal studies, mice were administered IgG or MD5-1 for 2 weeks as outlined above then 2 weeks of no treatment. All animal procedures and experiments were carried out according to the National Institutes of Health Guidelines on the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee.

of

Neonatal Rat Ventricular Myocyte Isolations:

ro

Primary neonatal cell cultures were prepared from 1 to 2 days old Sprague Dawley rat

-p

pups (Charles River) by enzymatic digestion as previous described [21]. In brief, hearts were excised, cleaned and ventricles digested using collagenase II (Worthington) and pancreatin.

re

Neonatal rat ventricular myocytes (NRVMs) were separated via pre-plating for 2h. Following

lP

isolation, NRVMs were cultured overnight in F-10 media containing 10% horse serum, 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5%

na

CO2. The following day, media were replaced with F-10 media containing 5% FBS and 1% PSF.

ur

NRVMs were pretreated with antagonist/inhibitor (1 µM AG1478, 10 µM GM6001, 10 µM PD98059 for 10 min or 5 µg/mL actinomycin D for 1h). Pretreatment antagonists/inhibitors

Jo

continued for the duration of TRAIL (100 ng/mL; R&D Systems) or bioymifi (10 µM; Caymen Chemicals) treatment, which were 24h in duration for hypertrophy and gene expression studies or 30 min for signal transduction experiments. Doses were chosen based on previous studies [22] and dose response curves (Supplemental Figure 1A-D). Small interference RNAs (siRNAs) for rat DR5 (Dharmacon catalog # CTM-489781), GATA4 (Dharmacon catalog # J-090725-09-0002) or control non-targeting (Dharmacon catalog # D-001810-03) siRNA were transfected as previously described [23]. In brief, siRNA was purchased from Dharmacon and NRVMs were transfected 24h post-seeding with Dharmafect 1

Journal Pre-proof (2.5 µL/mL media; Dharmacon) and the appropriate siRNA (25 nM) according to the manufacturer’s protocol.

Echocardiography: Cardiac function was assessed via transthoracic 2D echocardiography performed at

of

baseline and at weekly intervals following treatment using a 12-mHz probe on mice anesthetized with isoflurane (1.5%) as previously described [21]. M-mode echocardiography

ro

was performed in the parasternal short-axis view to assess several cardiac parameters

-p

including left ventricular (LV) end-diastolic dimension, wall thickness, and LV fractional

lP

re

shortening.

Immunoblotting:

na

NRVM or LV samples were homogenized and resolved for immunoblot using standard

ur

protocols as previously described [5]. Membrane preparations were performed as previously described [5]. In short, NRVMs or tissue were homogenized in lysis buffer (25 mM Tris pH 7.4, 5

Jo

mM EDTA, HALT protease inhibitor cocktail and phosphatase inhibitor cocktail) and incubated on ice 15 min. Lysate was centrifuged at 3000xg for 5 min and the supernatant was spun at 20,000xg for 25 min. Supernatant was saved as the cytosolic fraction while the pellet was resuspended in RIPA and saved as the membrane fraction. Immunoblotting was performed with diluted antibodies against DR5 (1:2000; R&D Systems, catalog # AF721), GATA4 (1:1000; eBioscience, catalog # 50-139-53), GAPDH (1:1000; Cell Signaling, catalog #2118), Na+/K+ATPase (1:1000; Novus Biologicals, catalog # NB300147), phospho-EGFR (1:1000; Cell Signaling, catalog # 2231), total-EGFR (1:3000; Novus, catalog # NB12010414, phosphoERK/12 (1:1,000; Cell Signaling, catalog # 9101), total-ERK1/2 (1:1,000; Cell Signaling, catalog

Journal Pre-proof # 4696), phospho-Akt (1:1,000; Cell Signaling, catalog # 4060), total-Akt (1:1,000; Cell Signaling, catalog # 2920), phospho-p38 (1:1,000; Cell Signaling, catalog # 9216), total-p38 (1:1,000; Cell Signaling, catalog # 8690), phospho-p65 (1:1,000; Cell Signaling, catalog # 3036) or total-p65 (1:1,000; Cell Signaling, catalog #8242). After washing, membranes were incubated with the appropriate diluted secondary antibody and bound antibody was detected using the Azure Imaging System. Phosphorylated protein intensities were normalized to corresponding

ro

of

total protein intensities while other protein intensities were normalized to GAPDH.

-p

Reverse Transcription-Quantitative PCR:

re

cDNA was synthesized from the total RNA of NRVM or LV samples using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-qPCR was performed with

lP

PowerUP SYBR Master Mix (Applied Biosystems) in triplicate for each sample using primers listed in Supplemental Table 1 at an annealing temperature of 60.1 °C. All RT-qPCR data were

na

analyzed using the Applied Biosystems Comparative CT Method (ΔΔCT). Gene expression

ur

analysis was normalized to translationally controlled tumor protein 1 (TPT1) and expressed as

Jo

2-CT with min/max indicated for range.

Enzyme-Linked Immunosorbent Assay: Levels of HB-EGF were detected using an HB-EGF DuoSet® ELISA kit (R&D Systems, catalog # DY8239-05) according to manufacturer’s instructions. Media was concentrated using Amicon Ultra-15 centrifugal filters (3,000 NMWL, Millipore). 96 well plates were coated overnight with Capture Antibody then blocked with Reagent Diluent. Plates were incubated with concentrated media followed by incubations with Detection Antibody, Streptavidin-HRP and substrate solution. Absorbance was measured at 450 nm with a 570 nm wavelength correction.

Journal Pre-proof

Immunohistochemistry: Excised hearts were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5-μm thickness. To quantify fibrosis, hearts were stained for Masson’s trichrome (SigmaAldrich). Cardiomyocyte area was measured using Fluorescein conjugated wheat germ agglutinin (WGA; 20 µg/mL; Sigma-Aldrich) staining. Sections were visualized at 20X

of

magnification using a Nikon Eclipse microscope and staining was quantified in ImageJ from 10

ro

fields/heart.

-p

An In Situ Cell Death Detection Kit, TMR Red (Roche Diagnostics) was used to measure apoptosis via terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL).

re

DNA strand breaks were labeled according to the manufacturer's instructions using tetra-methyl-

lP

rhodamine-dUTP. Hearts were counterstained with troponin I (TNNI, 1:100; Cell Signaling, catalog #4002) to identify cardiomyocytes. Coverslips were mounted on glass slides using

na

Prolong® Gold Antifade Reagent (Invitrogen). Sections were visualized at 20X magnification

ur

using a Nikon Eclipse microscope and the percentage of TUNEL-positive nuclei for 10

Jo

fields/heart were calculated in relation to the number of DAPI-stained nuclei.

Cell Viability Assay:

NRVM viability was assessed using a CellTiter 96® Non-Radioactive Cell Proliferation (MTT) Assay (Promega). NRVMs were plated 20,000/well in a 96-well plate. Following treatment, Dye Solution was added to the plate followed by the Solubilization/Stop Solution after a 1h incubation at 37°C. Absorbance was measure at 570 nm with a 750 nm reference wavelength.

Journal Pre-proof Caspase 3/7 Activity Assay: Caspase 3/7 activity was measured using a Caspase-Glo® 3/7 Assay according to the manufacturer's instructions (Promega; Madison, WI). In brief, NRVMs were plated 20,000/well of a white walled 96-well plate. After treatment, 100 μL of Caspase-Glo® 3/7 Reagent was added to each well. Plates were incubated for 1h prior to reading.

of

Luciferase Assay:

ro

NRVMs were co-transfected with a plasmid encoding the Gal4 DNA binding domain fused with the wild-type (WT) GATA4 (Gal4-GATA4) or GATA4 S105A, an ERK1/2 activation

-p

site deficient mutant [24], and the Gal4-luciferase reporter, pGL2-GAL4-UAS-Luc [25] as

re

previously described [24]. In brief, 24h after plating, NRVMs were transfected using 1 µg

lP

plasmid/1x106 cells with a 3:1 Transfection Reagent to DNA ratio according to the manufacturer’s protocol. Gal4-GATA4 or Gal4-GATA4 S105A were co-transfected into NRVMs

na

with pGL2-GAL4-UAS-Luc using X-tremeGENE 9 DNA Transfection Reagent (Roche). Gal4GATA4 and Gal4-GATA4 S105A were kindly provided by Jeffery Molkentin [24]. pGL2-GAL4-

ur

UAS-Luc was a gift from Martin Walsh (Addgene plasmid # 33020) [25]. Cells were treated for

Jo

24h and luciferase activity was measured using the Bright-Glo™ Luciferase Assay System (Promega) according to the manufacturer’s protocol.

Lactate Dehydrogenase Assay: NRVM cell death was measured using a Lactate Dehydrogenase (LDH) Colorimetric Assay Kit according to manufacturer's instructions (Pierce; Rockford, IL). Release of LDH was determined in media from NRVMs treated with TRAIL or bioymifi. Absorbance was measured by a spectrophotometer at 490 and 680 nm. LDH activity was expressed as fold of vehicle.

Journal Pre-proof Electrophoretic Gel Mobility Shift Assays: IRDye 700 end labeled oligonucleotides were incubated with 2.5 µg NRVM nuclear extracts. For antibody-mediated supershift assay, 1 µg antibody was incubated in reaction mixture. Protein-DNA complexes were separated on a 4% non-denaturing polyacrylamide gel and visualized using an Azure imaging system. Electrophoretic gel mobility shift assays (EMSAs) were quantified by measuring the intensity of the shifted band normalized to the total

ro

of

lane intensity.

-p

Statistics:

re

Data presented are expressed as mean ± SEM. Statistical analysis was performed using

lP

unpaired Student t tests, one-way ANOVA with a Tukey’s multiple comparison test, or two-way repeated-measures ANOVA where appropriate using Prism 5.0 software (GraphPad Software),

Jo

Results:

ur

na

with p values indicated in the figure legends.

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 readily detected in adult mouse cardiomyocytes with lower expression in non-cardiomyocyte cells of the heart (Figure 1A).

Journal Pre-proof Furthermore, DR5 expression was examined by immunoblot in NRVMs and neonatal rat ventricular fibroblasts (NRVF) and similarly showed higher expression in cardiomyocytes compared with cardiac fibroblasts (Figure 1B). Isolation of membranes from adult mouse heart demonstrated enrichment of DR5 levels in the plasma membrane fraction compared to whole heart lysate (Figure 1C). Fractionation of NRVMs into plasma membrane and cytosolic compartment also demonstrated expression of DR5 specifically on the plasma membrane with

of

no expression apparent in the cytosolic fraction (Figure 1D).

ro

Classical DR5 signaling induces apoptosis through the extrinsic pathway [26]. Noncancer cells tend to be resistant to DR5 activated cell death however, their function in non-

-p

transformed cell types, including cardiomyocytes, is not well established. To determine the role

re

of DR5 in initiating apoptosis in cardiomyocytes, NRVMs were treated with the endogenous

lP

DR5 ligand, TRAIL, or a small molecular DR5 agonist, bioymifi, over time. A caspase 3/7 activity assay was used to measure activation of apoptosis and demonstrated that caspase 3/7

na

activation did not occur following treatment with TRAIL or bioymifi (Figure 1E). TRAIL treated thymocytes, which are known to undergo apoptosis following DR5 activation [27] is shown as a

ur

positive control. Furthermore, LDH release (Figure 1F) and propidium iodide staining

Jo

(Supplemental Figure 1E), measures of membrane integrity damage, established that cell death mechanisms were not activated with DR5 agonist treatment in NRVMs. Treatment of NRVMs with H2O2 is shown as a positive control. Additionally, assessment of cell survival confirmed no alterations in NRVM viability occurred with TRAIL or bioymifi treatment (Supplemental Figure 1F). Taken together, these findings establish that DR5 activation in NRVMs does result in the canonical function of DR5, induction of apoptosis, but may serve a different role.

DR5 agonists activate ERK1/2 in cardiomyocytes

Journal Pre-proof To determine the signaling pathways activated by DR5 agonists in cardiomyocytes, a number of alternative signal transduction pathways that are potentially activated by TRAIL were investigated including various MAPK pathways, NF-κB and Akt over time [26]. Of the pathways investigated, only ERK1/2 phosphorylation was increased with TRAIL (Figure 2A) or bioymifi (Figure 2B) treatment whereas other MAPK pathways, Akt and NF-κB were unaltered with TRAIL or bioymifi treatment (Supplemental Figure 2A-F). Activation of ERK1/2 by TRAIL (Figure

of

2C) and bioymifi (Figure 2D) was inhibited by the ERK1/2 inhibitor PD98059, confirming the specificity of this response. NRVMs were transfected with control or DR5 siRNA to knockdown

ro

DR5 expression, which was confirmed by immunoblot (Supplemental Figure 3A). DR5

-p

knockdown prevented ERK1/2 activation in response to TRAIL (Figure 2E) or bioymifi (Figure

re

2F) treatment, demonstrating the importance of DR5 in the ERK1/2 response to TRAIL and

lP

bioymifi.

na

DR5 activation causes hypertrophy in NRVMs

ERK1/2 plays many roles in the heart including activation hypertrophic signaling [28]. To

ur

determine the impact of DR5 activation on hypertrophy, NRVMs were treated with TRAIL or

Jo

bioymifi, stained with troponin I to identify cardiomyocytes and cell area was quantified. Cardiomyocytes treated with TRAIL or bioymifi treatment were significantly larger than control NRVMs (Figure 3A and B). To determine the involvement of ERK1/2 activation in DR5-mediated cardiomyocyte hypertrophy, cells were pretreated with an ERK1/2 inhibitor prior to TRAIL or bioymifi treatment. Pre-incubation with PD98059 prevented TRAIL and bioymifi induced cardiomyocyte hypertrophy, while having no effect alone (Figure 3A and B). These findings identify that DR5 activation in cardiomyocytes promotes hypertrophy through ERK1/2. To confirm the involvement of DR5 in the hypertrophic responses observed with TRAIL and bioymifi treatment, NRVMs transfected with control or DR5 siRNA were treated with TRAIL and bioymifi

Journal Pre-proof and cell area was quantified (Figure 3C and D). Knockdown of DR5 prevented the increase in NRVM size with TRAIL or bioymifi incubation.

DR5 activation initiates cardiac hypertrophy in vivo To confirm that DR5 activation produces hypertrophy in vivo, C57BL/6 mice were

of

administered the DR5 agonist antibody, MD5-1, or IgG control for two weeks according to the treatment regime outlined in the methods. Decreased thymus weight (Supplemental Figure 4A)

ro

and increased thymocyte apoptosis (Supplemental Figure 4B) was assessed to confirm MD5-1

-p

administration activated DR5 in vivo since DR5 agonist administration is known to cause

re

thymocyte apoptosis [27]. Immunoblot analysis of ERK1/2 activation in hearts from mice injected with MD5-1 or IgG control showed an increase in phospho-ERK1/2 levels with MD5-1

lP

treatment (Figure 4A). Mice administered MD5-1 had increased hypertrophy as indicated by the heart weight to tibia length (HW/TL) ratio (Figure 4B). This was confirmed at the cellular level by

na

WGA staining and cell area analysis (Figure 4C and D). Echocardiography confirmed increases

ur

in left-ventricular mass and thickening of the left-ventricular walls with no changes in internal

Jo

dimeters (Supplemental Table 2). Changes in hypertrophy were not associated with alterations in stroke volume or cardiac output, but there were increases in the left-ventricular ejection fraction (Supplemental Table 2) and fractional shortening (Figure 4E) indicating that hypertrophy was not producing maladaptive effects on contractility. TUNEL staining was used to identify apoptotic cells and Masson’s trichrome staining was used to assess cardiac fibrosis (Supplemental Figure 5A-C). Importantly, no changes in cardiomyocyte apoptosis (Supplemental Figure 5B) or cardiac fibrosis (Supplemental Figure 5C) were observed confirming that DR5 is not inducing apoptosis in vivo nor is promoting fibrosis. While bioymifi has been predicted to have good bioavailability, this has never been demonstrated in vivo. Increasing doses of bioymifi decreased thymic weight and increase thymocyte apoptosis

Journal Pre-proof indicating that bioymifi was distributed and bioavailable (Supplemental Figure 6A and B). In accordance with activation of DR5 with MD5-1, bioymifi administration increased cardiac hypertrophy at the organ (Supplemental Figure 6C) and cellular level (Supplemental Figure 6D and E) without affecting cardiomyocyte death (Supplemental Figure 6D and F) or cardiac fibrosis (Supplemental Figure 6D and G). These findings are in accordance with NRVM results and establish that DR5 activation promotes cardiomyocyte hypertrophy in vivo.

of

There are two types of cardiomyocyte hypertrophy, concentric (pathological), that is

ro

detrimental to cardiac function and eccentric (physiological), which is adaptive and produces no or beneficial effects on cardiac function [28]. Pathological hypertrophy is often associated with

-p

increases in cardiomyocyte death, cardiac fibrosis and induction of the fetal gene program

re

whereas physiological hypertrophy occurs through unique pathways and is reversible. MD5-1

lP

administration produced minor to no alterations in fetal gene expression in TRAIL or bioymifi treated NRVMs (Supplemental Figure 5D) or hearts from MD5-1 treated mice (Supplemental

na

Figure 5E). These findings, along with the increases in contractility and lack of cardiomyocyte death and fibrosis, would suggest DR5 activation promotes physiological hypertrophy. To

ur

determine if DR5-mediated hypertrophy is reversible, mice were treated for 2 weeks with IgG or

Jo

MD5-1 followed by 2 weeks of treatment removal. After MD5-1 treatment and removal, there were no alterations in the HW/TL ratio (Figure 5F) or cell size (Figure 5G and H). Similar to what was observed following 2 weeks MD5-1 treatment, no alterations were observed in fibrosis (Supplemental Figure 5F-G), but the fractional shortening was elevated following 2 weeks MD51 treatment and remained elevated even after treatment cessation (Figure 5I) whereas the LV mass, as determined by echocardiography, was significantly elevated 2 weeks following MD5-1 treatment, but returned to control levels upon termination of treatment (Figure 5J). Taken together, these findings suggest that DR5 activation results in physiological hypertrophy.

Journal Pre-proof DR5 promotes ERK1/2 activation through the transactivation of EGFR DR5 and EGFR are both known to contribute to the pathogenesis of some cancers and interaction between the signaling of both receptors has been identified [30-34]. In the heart, transactivation of EGFR by numerous receptors, cumulates in ERK1/2 signaling, which can impact cardiomyocyte growth [35] and studies in cardiomyocytes have suggested a role for EGFR in TRAIL signaling [5]. To determine the involvement of EGFR-transactivation in DR5mediated activation of ERK1/2, NRVMs were pretreated with the EGFR inhibitor, AG1478, prior

of

to TRAIL or bioymifi treatment. Inhibition of EGFR prevented DR5-mediated ERK1/2 activation

ro

in response to TRAIL (Figure 5A) or bioymifi (Figure 5B) treatment.

-p

EGFR transactivation is known to occur through different mechanisms including

re

activation of MMP- or a disintegrin and metalloprotease (ADAM)-mediated cleavage of

lP

extracellular EGFR ligands including HB-EGF [35]. DR5 has been shown in cancer cells to activate MMPs to promote metastasis [36]. To identify the mechanism of EGFR-transactivation

na

with TRAIL or bioymifi treatment, NRVMs were incubated with the MMP inhibitor, GM6001, prior to DR5 agonist treatment. GM6001 prevented phosphorylation of ERK1/2 in response to TRAIL

ur

(Figure 5C) and bioymifi (Figure 5D) treatment. Furthermore, HB-EGF was measured in the

Jo

media from bioymifi treated NRVMs and increased following bioymifi treatment (Figure 5E) as well as phosphorylation of membrane localized EGFR at Tyr845 was increased (Figure 5F). Additionally, EGFR phosphorylation is elevated in membranes of hearts from mice injected with MD5-1 (Figure 5G) verifying that DR5 activation results in EGFR transactivation in the heart in vivo. Inhibition of MMPs by GM6001 pretreatment in NRVMs prevented bioymifi-induced HBEGF release (Figure 5H) and EGFR phosphorylation (Figure 5I). These findings confirm the involvement of DR5-mediated EGFR-transactivation in activating ERK1/2. To confirm the involvement of EGFR-transactivation in DR5-mediated hypertrophy, NRVMs were pretreated with GM6001 or AG1478 prior to TRAIL or bioymifi treatment. NRVMs

Journal Pre-proof were stained with troponin I (Figure 6A) and cardiomyocyte area was quantified (Figure 6B and 6C). As demonstrated, TRAIL and bioymifi treatment increased cardiomyocyte area, which was prevented with EGFR (Figure 6B) or MMP inhibition (Figure 6C), confirming the involvement of EGFR-dependent signaling in DR5-mediated hypertrophy.

DR5-mediated EGFR-transactivation promotes hypertrophic gene transcription

of

ERK1/2 is known to activate transcription factors to influence cardiomyocyte hypertrophy. To confirm the importance of transcription in hypertrophic responses to DR5

ro

agonist treatment, NRVMs were pre-incubated with the transcriptional inhibitor actinomycin D

-p

prior to TRAIL or bioymifi treatment. Cardiomyocytes were stained with troponin I (Figure 7A)

re

and cell area was quantified (Figure 7B). Actinomycin D pretreatment prevented TRAIL and bioymifi-mediated increases in cell size (Figure 7A and B), confirming the necessity of gene

lP

transcription in cardiomyocyte response to DR5 activation.

na

Transcription is initiated by transcription factor binding to DNA. Hypertrophic signaling by ERK1/2 can occur through a number of transcription factors. To identify transcription factor

ur

activation by DR5 agonist treatment, EMSAs were performed for pro-hypertrophic transcription

Jo

factors known to potentially be activated by ERK1/2 including GATA4 [29]. In NRVMs treated with bioymifi, GATA4 transcription factor binding was increased with GATA4 activation peaking at 60 min (Figure 7C). GATA4 binding was supershifted using a GATA4 antibody and competitively inhibited using unlabeled GATA4 probe demonstrating the specificity of GATA4 binding with TRAIL or bioymifi treatment (Supplemental Figure 7A; Uncropped EMSAs are shown in Supplemental Figures 7B-E). To investigate if GATA4 binding influences transcriptional activation, constructs containing the WT GATA4 activation domain or a mutant GATA4 with the ERK1/2 activation site altered to prevent ERK1/2-mediated GATA4 activation (S105A) fused to the GAL4 DNA binding domain [24] were co-transfected into NRVMs with the

Journal Pre-proof GAL4-luciferase reporter, pGL2-GAL4-UAS-Luc [25]. Treatment with TRAIL or bioymifi induced GATA4 transcriptional activation in WT GATA4, which did not occur in NRVMs expression GATA4 S105A (Figure 7D). Additionally, prevention of ERK1/2 signaling and inhibition of EGFR or MMPs abrogated GATA4 transcription factor binding (Figures 7E, G, I) and transcriptional activity (Figures 7F, H, J) with bioymifi treatment. Similar to bioymifi treatment, TRAIL increased transcriptional activation of GATA4 through MMP/EGFR/ERK1/2-dependent mechanisms

of

(Supplemental Figure 8A-C). To confirm the importance of GATA4 activation in DR5-mediated cardiomyocyte hypertrophy, NRVMs were transfected with control or GATA4 siRNA

ro

(Supplemental Figure 3B) followed by treatment with TRAIL or bioymifi. Cell area was quantified

-p

and demonstrated that GATA4 knockdown prevented TRAIL and bioymifi-induced hypertrophy

re

(Figure 8A and B). These results demonstrate the importance of pro-hypertrophic gene

lP

transcription in DR5-mediated cardiomyocyte growth through the activation of GATA4.

na

Discussion:

Cardiomyocyte growth, survival and death are critical contributors to the outcome of

ur

heart failure. The TRAIL/DR5 system has been implicated in multiple types of heart failure [9-

Jo

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 heart [37]. TRAIL is secreted by most cell types [20], including cardiomyocytes [5], making it important to understand the role of TRAIL/DR5 in the heart. Circulating mononuclear cells have increased TRAIL expression following myocardial infarction and could also be an important source of TRAIL for activating DR5 in cells of the heart [38]. Our findings demonstrate that DR5 plays a non-canonical role in cardiomyocytes by not initiating apoptosis or cell death, but promoting cardiomyocyte hypertrophy through a novel mechanism of EGFR-transactivation, ERK1/2 activation and GATA4 transcription. In particularly, DR5

Journal Pre-proof promotes physiological hypertrophy since our findings demonstrate many of the hallmarks of physiological hypertrophy including minimal changes in fetal gene expression, no negative effects on cardiomyocyte survival, cardiac fibrosis or cardiac function and contrarily, moderate improvements in contractility were observed in with MD5-1 treatment. Furthermore, changes in hypertrophy were reversible upon discontinuation of MD5-1 treatment. Surprisingly, the hypercontractile state initiated by MD5-1 treatment was maintained following MD5-1 removal.

of

While the mechanism of this change is not known, synthesis of contractile proteins occurs, sometimes through GATA4 activation, during physiological hypertrophy, which might be slower

ro

to reverse [29]. If this hypercontractility is maintained at later time points, if these changes occur

re

effects on contractility remains to be studied.

-p

through EGFR-transactivation and GATA4 or other indirect mechanisms and if DR5 has direct

lP

Initial studies identified TRAIL as selectively inducing apoptosis in cancerous cells while have little or no effect in non-transformed cell types and making it an attractive therapeutic

na

target for cancer [12]. Indeed, clinical trials using DR5 activating antibodies have proven to have no overt negative effects [39]. Thus, it is not unexpected for DR5 activation to not result in

ur

apoptosis in cardiomyocytes, as demonstrated in our study. In other muscle cell types including

Jo

smooth muscle cells and skeletal myoblasts, TRAIL also serves a non-canonical role of promoting proliferation or differentiation [17, 18]. While the role of TRAIL in the heart has not been studied, TRAIL and DR5 expression are known to be altered with cardioprotective βadrenergic receptor signaling in mouse hearts [5]. These alterations in TRAIL and DR5 occurred specifically in cardiomyocytes, with no change in cardiac fibroblasts, suggesting a differential regulation between cell types, which is in line with our in vivo findings of DR5 activation promoting cardiomyocyte hypertrophy without affecting cardiac fibrosis. TRAIL and DR5 have not been directly linked with hypertrophy however, in a model of pulmonary hypertension, TRAIL levels were associated with right ventricular hypertrophy, which

Journal Pre-proof was attributed to effects on systolic pressure and the direct effects of TRAIL on the heart were not examined [40]. Furthermore, mice lacking TRAIL have decreased smooth muscle cell hypertrophy in response to ovalbumin-induced chronic allergic airway disease however, this was attributed to the effects of TRAIL on inflammation rather than smooth muscle cells [41]. However, the role of ERK1/2 in the heart has been extensively studied where is has been characterized to influence hypertrophy [29].

of

While the ability of TRAIL to promote ERK1/2 phosphorylation has not previously been

ro

demonstrated in the heart, TRAIL is known to activate ERK1/2 in certain cell types through caspase-independent mechanisms to promote protective effects [42], which has been shown to

-p

occur through an unidentified tyrosine kinase [43]. The ability of ERK1/2 activation to promote

re

both pathological and physiological hypertrophy is well established [45]. Studies in transgenic

lP

mice demonstrate a role for ERK1/2 activation in concentric cardiac hypertrophy and protection from hypertrophic stimuli [29]. However, differences in the response to ERK1/2 activation can

na

occur depending on the external stimuli and may depend on level, frequency or duration of ERK1/2 activation or subcellular localization [44]. These differences may also account for

ur

downstream ERK1/2 target specificity since ERK1/2 is reported to phosphorylate a number of

Jo

transcriptional effectors including Elk-1, Ets1, Sap1a and c-Myc [45]. ERK1/2 is known to activate GATA4 [24], a well-established regulator of cardiac hypertrophy that is also characterized to contribute to both physiological and pathological hypertrophy [46-48]. Transgenic mice expressing an activated MEK1 fail to develop hypertrophy when crossed with GATA4 null mice, demonstrating the importance of GATA4 in the hypertrophic effects of ERK1/2 [49]. ERK1/2 activation is known to occur in the heart via EGFR transactivation in response to a number of receptors including the angiotensin II type 1A receptor [50, 51], urotensin II receptor [52] and β-adrenergic receptor [53, 54]. While many of the studies examining cardiac

Journal Pre-proof EGFR transactivation have focused primarily on G protein-coupled receptors [35], in noncardiac cell types, numerous receptor families are capable of inducing EGFR transactivation including TNF receptors [55-58], which are highly homologous to DR5. While this is the first study demonstrating the ability of DR5 to transactivate EGFR, in some cancer cells, there is crosstalk between TRAIL and EGFR signaling [32-34] and DR5 is known to activate MMPs [36], which are involved in HB-EGF cleavage and EGFR transactivation [35]. While EGFR

of

transactivation has been extensively characterized as a regulator of pathological hypertrophy, the role of EGFR in physiological hypertrophy has not been defined. However, EGFR is

ro

structurally and functionally similar to the insulin-like growth factor-1 (IGF1) receptor, which has

-p

been extensively defined in physiological hypertrophy [59]. Furthermore, crosstalk and

re

interactions between EGFR and IGF1R have been shown to occur to regulate signaling and outcomes in cancer cells [60]. However, if this occurs in cardiac cell types has not been

lP

investigated.

na

At present, it is unclear of the role of DR5 changes between the healthy and diseased heart. Changes in cardiomyocyte structure that occur during heart failure may alter DR5

ur

signaling, thus changing its function. Cardiomyocyte injury or stress may alter TRAIL receptor

Jo

expression. Furthermore, alterations in membrane localization of receptors occurs in heart failure. Some TRAIL receptors are confined to lipid rafts, which directs their signaling. Disruption of plasma membrane domains, such as what occurs during heart failure, may allow for TRAIL receptors to associate with alternative signal transduction mechanisms or change accessibility to decoy receptors. Additionally, there are known changes in expression of the TRAIL decoy receptor OPG. A major regulatory mechanism of TRAIL signaling occurs through altered expression of decoy receptors [8], which we anticipate might be responsible for the differences in the magnitude of ERK1/2 activation between TRAIL and bioymifi since TRAIL will bind to all TRAIL receptors whereas bioymifi is selective for DR5 [22]. While DcR1 and DcR2 have not

Journal Pre-proof been examined in the context of the heart, OPG can also act as a TRAIL decoy receptor and its role in the heart has been examined [61]. Alterations in OPG are associated with multiple forms of cardiovascular disease [62-66]. OPG knockout mice have exacerbated cardiac hypertrophy with aging [67]. These mice have elevations in TRAIL and DR5 expression and ERK1/2 activation suggesting that lack of the decoy receptor function of OPG for TRAIL may contribute to its role in the heart. However, unlike our current findings, OPG knockout mice displayed

of

increased cardiomyocyte death, fibrosis and contractile dysfunction with aging, indicating further mechanisms are occurring with OPG knockout. However, decoy receptor expression levels are

ro

not the sole regulatory mechanism for apoptotic TRAIL signaling and in smooth muscle cells,

-p

resistance to TRAIL-induced apoptosis occurs through other mechanisms [17].

re

In summary, we have identified a novel mechanism of DR5 signaling in cardiomyocytes

lP

that does not result in cell death, but activation of ERK1/2-mediated signaling through the transactivation of EGFR, resulting in GATA4 activation and pro-hypertrophic gene transcription

na

(Figure 8C).

ur

Funding: This work was supported by American Heart Association Scientific Development

Jo

Grant 17SDG33400114 (L.A.G.).

Conflict of Interest: The authors have declared that no conflict of interest exists.

Literature Cited: [1] Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R et al. Heart Disease and Stroke Statistics-2017 Update: A Report from the American Heart Association. Circulation 2017; 135: e146-e603.

Journal Pre-proof [2] Konstantinidis K, Whelan RS, Kitsis, RN. Mechanisms of cell death in heart disease. Arterioscler Thromb Vasc Biol 2012; 32: 1552-152. [3] Crow MT, Mani K, Nam YJ, Kitsis RN. The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res 2004; 95: 957-970. [4] Mann DL. Recent insights into the role of tumor necrosis factor in the failing heart. Heart Fail Rev 2001; 6: 71-80.

of

[5] Grisanti LA, Talarico JA, Carter RL, Yu JE, Repas AA, Radcliffe SW et al. Beta-Adrenergic receptor-mediated transactivation of epidermal growth factor receptor decreases

ro

cardiomyocyte apopotsis through differential subcellular activation of ERK1/2 and Akt. J

-p

Mol Cell Cardiol 2014; 72: 39-51.

re

[6] Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997; 277: 815-818.

lP

[7] Spierings DC, de Vries EG, Vellenga E, van den Heuvel FA, Koornstra JJ, Wesseling J et

na

al. Tissue distribution of the death ligand TRAIL and its receptors. J Histochem Cytochem 2004; 52: 821-831.

ur

[8] Ozoren N, El-Deiry WS. Cell surface Death Receptor signaling in normal and cancer cells.

Jo

Semin Cancer Biol 2003; 13: 135-147. [9] Skau E, Henriksen E, Wagner P, Hedberg P, Siegbahn A, Leppert J. GDF-15 and TRAIL-R2 are powerful predictors of long-term mortality in patients with acute myocardial infarction. Eur J Prev Cardiol 2017; 24: 1576-1583. [10] Stenemo M, Nowak C, Byberg L, Sundström J, Giedraitis V, Lind L et al. Circulating proteins as predictors of incident of heart failure in the elderly. Eur J Heart Fail 2017; 20: 55-62. [11] Bernardi S, Bossi F, Toffoli B, Fabris B. Roles and Clinical Applications of OPG and TRAIL as Biomarkers in Cardiovascular Disease. Biomed Res Int 2016; 2016: 1752854. [12] Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 1999; 104: 155-162.

Journal Pre-proof [13] MacFarlane M. TRAIL-induced signalling and apoptosis. Toxicol Lett 2003; 139: 89-97. [14] Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999; 5: 157163. [15] Harbst RS, Eckhardt SG, Kurzrock R, Ebbinghaus S, O’Dwyer PJ, Gordon MS et al. Phase I dose-escalation study of recombinant human Apo2L/TRAIL, a dual proapoptotic

of

receptor agonist, in patients with advanced cancer. J Clin Oncol 2010; 28: 2839-2846. [16] Soria JC, Smit E, Khayat D, Besse B, Yang X, Hsu CP et al. Phase 1b study of dulanermin

ro

(recombinant human Apo2L/TRAIL) in combination with paclitaxel, carboplatin, and

-p

bevacizumad in patients with advanced non-squamous non-small-cell lung cancer. J Clin

re

Oncol 2010; 28: 1527-1533.

[17] van Dijk M, Halpin-McCormick A, Sessler T, Samali A, Szegezdi E. Resistance to TRAIL in

lP

non-transformed cells is sdue to multiple redundant pathways. Cell Death Dis 2013; 4:

na

e702.

[18] Freer-Prokop M, O’Flaherty J, Ross JA, Weyman CM. Non-canonical role for the TRAIL

ur

receptor DR5/FADD/caspase pathway in the regulation of MyoD expression and skeletal

Jo

myoblast differentiation. Differentiation 2009; 78: 205-212. [19] Crowder RN, Dicker DT, El-Deiry WS. The Deubiquitinase Inhibitor PR-619 Sensitizes Normal Human Fibroblasts to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Cell Death. J Biol Chem 2016; 291: 5960-5970. [20] Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995; 3: 673-682.

Journal Pre-proof [21] Grisanti LA, Thomas TP, Carter RL, de Lucia C, Gao E, Koch WJ et al. Pepducin-Mediated

Cardioprotection via β-Arrestin-Biased β2-Adrenergic Receptor-Specific Signaling. Theranostics 2018; 8: 4664-4678. [22] Wang G, Wang X, Yu H, Wei S, Williams N, Holmes DL et al. Small-molecule activation of the TRAIL receptor DR5 in human cancer cells. Nat Chem Biol 2013; 9: 84-89. [23] Sato PY, Musa H, Coombs W, Guerrero-Serna G, Patiño GA, Taffet SM et al. Loss of

of

plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes. Circ Res 2009; 105: 523-526.

ro

[24] Liang Q, Wiese RJ, Bueno OF, Dai YS, Markham BE, Molkentin JD. The transcription factor

-p

GATA4 is activated by extracellular signal-regulated kinase 1- and 2-mediated

re

phosphorylation of serine 105 in cardiomyocytes. Mol Cell Biol 2001; 21: 7460-7469. [25] Nishio H, Walsh MJ. CCAAT displacement protein/cut homolog recruits G9a histone lysine

lP

methyltransferase to repress transcription. Proc Natl Acad Sci USA 2004; 101: 11257-

na

11262.

[26] Shirley S, Morizot A, Micheau O. Regulating TRAIL receptor-induced cell death at the

ur

membrane: a deadly discussion. Recent Pat Anticancer Drug Discov 2011; 6: 311-323.

Jo

[27] Hernandez JB, Newton RH, Walsh CM. Life and death in the thymus--cell death signaling during T cell development. Curr Opin Cell Biol 2010; 22: 865-871. [28] McMullen JR, Jennings GL. Differences between pathological and physiological cardiac hypertrophy: novel therapeutic strategies to treat heart failure. Clin Exp Pharmacol Physiol 2007; 34: 255-262. [29] Mutlak M, Kehat I. Extracellular signal-regulated kinases 1/2 as regulators of cardiac hypertrophy. Front Pharmaol 2015; 6: 149. [30] Yan D, Ge Y, Deng H, Chem W, An G. Gefitinib upregulates death receptor 5 expression to mediate rmhTRAIL-induced apoptosis in Gefitinib-sensitive NSCLC cell line. Onco Targets Ther 2015; 8: 1603-1610.

Journal Pre-proof [31] Xu L, Zhang Y, Liu J, Qu J, Hu X, Zhang F et al. TRAIL-activated EGFR by Cbl-b-regulated EGFR redistribution in lipid rafts atagonises TRAIL-induced apoptosis in gastric cancer cells. Eur J Cancer 2012; 48: 3288-3299. [32] Shrader M, Pino MS, Lashinger L, Bar-Eli M, Adam L, Dinney CP et al. Gefitinib reverses TRAIL resistance in human bladder cancer cell lines via inhibition of AKT-mediated Xlinked inhibitor of apoptosis protein expression. Cancer Res 2007; 67: 1430-1435.

of

[33] Bremer E, Samplonius DF, van Genne L, Dijkstra MH, Kroesen BK, de Leij LF et al. Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and

ro

enhanced activation of tumor necrosis factor-related apoptosis-inducin ligand (TRAIL)

-p

receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity

re

for human EGFR. J Biol Chem 2005; 280: 10025-10033. [34] Dolloff NG, Mayes PA, Har LS, Dicker DT, Humphreys R, El-Deiry WS. Off-target lapatinib

na

Transl Med 2011; 3: 86ra50.

lP

activity sensitizes colon cancer cells through TRAIL death receptor up-regulation. Sci

[35] Grisanti LA, Guo S, Tilley DG. Cardiac GPCR-Mediated EGFR Transactivation: Impact and

ur

Therapeutic Implications. J Cardiovasc Pharmacol 2017; 70: 3-9.

Jo

[36] Takahashi K, Takeda K, Saiki I, Irimura T, Hayakawa Y. Functional roles of tumor necrosis factor-related apoptosis-inducig ligand-DR5 interaction in B16F10 cells by activating the nuclear factor-kappaB pathway to induce metastatic potential. Cancer Sci 2013; 104: 558-562. [37] Wu GS, Burns TF, Zhan Y, Alnemri ES, El-Deiry WS. Molecular cloning and functional analysis of the mouse homologue of the KILLER/DR5 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor. Cancer Res 1999; 59: 2770-2775. [38] Nakajima H, Yanase N, Oshima K, Sasame A, Hara T, Fukazawa S et al. Enhanced expression of the apoptosis inducing ligand TRAIL in mononuclear cells after myocardial infarction. Jpn Heart J 2003; 44: 833-844.

Journal Pre-proof [39] Bellail AC, Qi L, Mulligan P, Chhabra V, Hao C. TRAIL agonists on clinical trails for cancer therapy: the promises and the challenges. Rev Recent Clin Trials 2009; 4: 34-41. [40] Liu H, Yang E, Lu X, Zuo C, He Y, Jia D et al. Serum levels of tumor necrosis factor-related apoptosis-inducing ligand correlate with the severity of pulmonary hypertension. Pulm Pharmacol Ther 2015; 33: 39-46. [41] Collison A, Li J, Pereira de Siqueira A, Zhang J, Toop HD, Morris JC et al. Tumor necrosis

of

factor-related apoptosis-inducing ligand regulates hallmark features of airways remodeling in allergic airways disease. Am J Respir Cell Mol Biol 2014; 51: 86-93.

ro

[42] Tran SE, Holmstrom TH, Ahonen M, Kahari VM, Eriksson JE. MAPK/ERK overrides the

-p

apoptotic signaling from Fas, TNF, and TRAIL receptors. J Biol Chem 2001; 276: 16484-

re

16490.

[43] Lee MW, Bach JH, Lee HJ, Lee DY, Joo WS, Kim YS et al. The activation of ERK1/2 via a

lP

tyrosine kinase pathway attenuates trail-induced apoptosis in HeLa cells. Cancer Invest

na

2005; 23: 586-592.

[44] Mebratu Y, Tesfaigzi Y. How ERK1/2 activation controls cell proliferation and cell death: Is

ur

subcellular localization the answer? Cell Cycle 2009; 8: 1168-1175.

Jo

[45] Bueno OF, Molkentin JD. Involvement of extracellular signal-regulated kinases ½ in cardiac hypertrophy and cell death. Circ Res 2002; 91: 776-781. [46] Liang Q, De Windt LJ, Witt SA, Kimball TR, Markham BE, Molkentin JD. The transcription factors GATA4 and GATA6 regulate cardiomyocyte hypertrophy in vitro and in vivo. J Biol Chem 2001; 276: 30245-30253. [47] Bisping E, Ikeda S, Kong SW, Tarnavski O, Bodyak N, McMullen JR et al. Gata4 is required for maintenance of postnatal cardiac function and the protection from pressure overloadinduced heart failure. Proc Nat Acad Sci USA 2006; 103: 14471-14476.

Journal Pre-proof 48. Oka T, Maillet M, Watt AJ, Schwartz RJ, Aronow BJ et al. Cardiac-specific deletion of Gata4 reveals its requirement for hypertrophy, compensation, and myocyte viability. Circ Res 2006; 98: 837-845. [49] van Berlo JH, Elrod JW, Aronow BJ, Pu WT, Molkentin JD. Serine 105 phosphorylation of transcription factor GATA4 is necessary for stress-induced cardiac hypertrophy in vivo. Proc Natl Acad Sci USA 2011; 108: 12331-12336.

of

[50] Thomas WG, Brandenburger Y, Autelitano DJ, Pham T, Qian H, Hannan RD. Adenoviraldirected expression of the type 1A angiotensin receptor promotes cardiomyocyte

ro

hypertrophy via transactivation of the epidermal growth factor receptor. Circ Res 2002;

-p

90: 135-142.

re

[51] Murasawa S, Mori Y, Nozawa Y, Gotoh N, Shibuya M, Masaki H et al. Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kiase activation is mediated by

na

Res 1998; 82: 1338-1348.

lP

Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor. Circ

[52] Onan D, Pipolo L, Yang E, Hannan RD, Thomas WG. Urotensin II promotes hypertrophy of

Jo

2354.

ur

cardiac myocytes via mitogen-activated protein kinases. Mol Endocrinol 2004; 18: 2344-

[53] Grisanti LA, Repas AA, Talarico JA, Gold JI, Carter RL, Koch WJ et al. Temporal and gefitinib-sensitive regulation of cardiac cytokine expression via chronic beta-adrenergic receptor stimulation. Am J Physiol Heart Circ Physiol 2015; 308: H316-330. [54] Noma T, Lemaire A, Naga Prasad SV, Barki-Harrington L, Tilley DG, Chen J et al. Betaarrestin-mediated beta1-adrenergic receptor transactivation of the EGFR confirs cardioprotection. J Clin Invest 2007; 117: 2445-2458.

Journal Pre-proof [55] Argast GM, Campbell JS, Brooling JT, Fausto N. Epidermal growth factor receptor transactivation mediates tumor necrosis factor-induced hepatocyte replication. J Biol Chem 2004; 279: 34530-34536. [56] Hobbs SS, Goettel JA, Liang D, Yan F, Edelblum KL, Frey MR et al. TNF transactivation of EGFR stimulates cytoprotective COX-2 expression in gastrointestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 2011; 301, G220-229.

of

[57] Kakiashvili E, Dan Q, Vandermeer M, Zhang Y, Waheed F, Pham M et al. The epidermal growth factor receptor mediates tumor necrosis facto-alpha-induced activation of the

ro

ERK/GEF-H1/RhoA pathway in tubular epitelium. J Biol Chem 2011; 286, 9268-9279.

-p

[58] Yamaoka T, Yan F, Cao H, Hobbs SS, Dise RS, Tong W et al. Transactivation of EGF

re

receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis. Proc Natl Acad Sci USA 2008; 105: 11772-11777.

lP

[59] Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2010; 141:

na

1117-1134.

[60] van der Veeken J, Oliveira S, Schiffelers RM, Storm G, van Bergen En Henegouwen PM,

ur

Roovers RC. Crosstalk between epidermal growth factor receptor- and insulin-like growth

Jo

factor-1 receptor signaling: implications for cancer therapy. Curr Cancer Drug Targets 2009; 9: 748-60.

[61] Montagnana M, Lippi G, Danese E, Guidi GC. The role of osteoprotegrin in cardiovascular disease. Ann Med 2013; 45: 254-264. [62] Tschiderer L, Klingenschmid G, Nagrani R, Willeit J, Laukkanen JA, Schett G et al. Osteoprotegerin and Cardiovascular Events in High-Risk Populations: Meta-Analysis of 19 Prospective Studies Involving 27 450 Participants. J Am Heart Assoc 2018; 7: e009012.

Journal Pre-proof [63] Tschiderer L, Willeit J, Schett G, Kiechl S, Willeit P. Osteoprotegerin concentration and risk of cardiovascular outcomes in nine general population studies: Literature-based medaanalysis involving 26,442 participants. PLoS One 2017; 12, e0183810. [64] Roysland R, Masson S, Omland T, Milani V, Bjerre M, Flyvbjerg A et al. Prognostic value of osteoprotegerin in chronic heart failure: The GISSI-HF trial. Am Heart J 2010; 160: 286293.

of

[65] di Giuseppe R, Biemann R, Wirth J, Menzel J, Isermann B, Stangl GI et al. Plasma osteoprotegerin, its correlates, and risk of heart failure: a prospective cohort study. Eur J

ro

Epidemiol 2017; 32: 113-123.

-p

[66] Ueland T, Jemtland R, Godanf K, Kjekshus J, Hognestad A, Omland T et al. Prognostic

Cardiol 2004; 44: 1970-1976.

re

value of osteoprotegerin in heart failure after acute myocardial infarction. J Am Coll

lP

[67] Hao Y, Tsuruda T, Sekita-Hatakeyama Y, Kurogi S, Kubo K, Sakamoto S et al. Cardiac

na

hypertrophy is exacerbated in aged mice lacking the osteoprotegerin gene. Cardiovasc

Jo

Figure Legends:

ur

Res 2016; 110: 62-73.

Figure 1: DR5 is expressed in cardiomyocytes and does not activate pro-death signaling. A. Representative immunoblot and quantification of DR5 expression in cardiomyocytes (CM) and non-myocytes (non-CM) isolated from adult mouse heart. GAPDH is shown as a loading control. n=4; t test, * p < 0.05 versus CM. B. Representative and quantified immunoblot of DR5 expression in NRVMs and neonatal rat cardiac fibroblasts (NRVFs). GAPDH is shown as a loading control. n=4; t test, * p < 0.05 versus NRVMs. C. Immunoblot of whole heart lysate (heart) and membrane fractions of adult mouse hearts blotted for DR5. Na+/K+ ATPase β1 is shown as a membrane marker and GAPDH is shown as a cytosolic marker. D. NRVMs were

Journal Pre-proof fractionation into membrane (mem) and cytosolic (cyt) fractions and blotted for DR5. Na+/K+ ATPase β1 is shown as a membrane marker and GAPDH is shown as a cytosolic marker. E. A caspase 3/7 activity assay was used to measure induction of apoptosis in NRVMs treated temporally with TRAIL or bioymifi. Bioymifi treated thymocytes (Thy) are shown as a positive control. Data is expressed as fold over vehicle (Veh) treated cells. n=5 independent experiments; one-way ANOVA, * p<0.05 versus Veh. F. An LDH assay was used to measure

of

overall NRVM death after TRAIL or bioymifi treatment over time. Treatment of NRVMs with H2O2 is shown as a positive control. Data is expressed as fold over Veh treated cells. n=4

-p

ro

independent experiments; one-way ANOVA, * p<0.05 versus Veh.

re

Figure 2: DR5 agonist administration in NRVMs activates ERK1/2. Lysates from NRVMs

lP

treated temporally with TRAIL (A) or bioymifi (B) were immunoblotted for phospho (P)-ERK1/2 and total (T)-ERK1/2. P-ERK1/2 intensity was normalized to T-ERK1/2 intensity and expressed

na

as fold over Veh. n=10; one-way ANOVA, * p < 0.05 versus Veh. ERK1/2 phosphorylation was examined by immunoblot in NRVM lysates pretreated with PD98059 followed by TRAIL (C) or

ur

bioymifi (D) treatment. P-ERK1/2 levels were normalized to T-ERK1/2 and expressed as fold

Jo

over Veh. n=10; one-way ANOVA, * p < 0.05. E. Representative P-ERK1/2 immunoblot and quantification of NRVM lysates transfected with scrambled (Scr) or DR5 siRNA and treated with TRAIL. P-ERK1/2 levels were normalized to T-ERK1/2 and expressed as fold over Scr Veh. n=5; one-way ANOVA, * p < 0.05. F. Representative immunoblot and quantification of Scr or DR5 siRNA transfected NRVMs treated with bioymifi and blotted for P- and T-ERK1/2. PERK1/2 intensities were normalized to T-ERK1/2 levels and expressed as fold over Scr Veh. n=5; one-way ANOVA, * p < 0.05.

Journal Pre-proof Figure 3: DR5-mediated ERK1/2 activation promotes cardiomyocyte hypertrophy. A. Representative troponin I (green) staining of NRVMs pretreated with PD98059 followed by TRAIL or bioymifi. DAPI (blue) was used to identify the nuclei of cells. B. Quantification of cardiomyocyte area of NRVMs treated with vehicle, TRAIL or bioymifi in the presence or absence of PD98059. Values are expressed as fold over vehicle. n=5 independent experiments; one-way ANOVA, * p < 0.05. C. Representative troponin I (green) staining of NRVMs

of

transfected with scrambled (Scr) or DR5 siRNA followed by treatment with TRAIL or bioymifi. Dapi (blue) was used to identify the nuclei of cells. D. Quantification of cardiomyocyte area of

ro

control (Scr siRNA) or with DR5 knockdown (DR5 siRNA) treated with Veh, TRAIL or Bioy.

-p

Values are expressed as fold over Scr siRNA Veh. n=5 independent experiments; one-way

lP

re

ANOVA, * p < 0.05.

Figure 4: DR5 activation promotes cardiac hypertrophy in vivo. A. Immunoblot analysis for P-

na

ERK1/2 and T-ERK1/2 of hearts from MD5-1 or IgG injected control mice. P-ERK1/2 levels were normalized to T-ERK1/2 expression and expressed as fold over IgG. n=6; t test, p < 0.05 versus

ur

IgG. B. Gravimetric analysis of HW normalized to TL for mice treated for 2 weeks with IgG or

Jo

MD5-1. n=6; t test, p < 0.05 versus IgG. Representative WGA staining (C) and quantification of cardiomyocyte area (D) in stained heart sections from IgG and MD5-1 treated animal hearts. n=6; t test, * p < 0.05. E. Echocardiography was used to measure left ventricular fractional shortening (FS) over time in mice treated with IgG or MD5-1. n=6; two-way ANOVA, * p < 0.05 versus IgG. F. Gravimetric analysis of HW normalized to TL for mice treated for 2 weeks with IgG or MD5-1 followed by 2 weeks of treatment cessation. n=6. Representative WGA staining (G) and quantification of cardiomyocyte area (H) in stained heart sections from IgG and MD5-1 treated animal hearts. n=6. Echocardiography was used to measure fractional shortening (I) and

Journal Pre-proof left ventricular mass (J) in mice receiving 2 weeks IgG or MD5-1 followed by 2 weeks treatment removal. n=6; two-way ANOVA, * p < 0.05 versus IgG. Figure 5: DR5 activates ERK1/2 through EGFR-transactivation. Immunoblot analysis for PERK1/2 and T-ERK1/2 in NRVMs pretreated with AG1478 followed by TRAIL (A) or bioymifi (B). P-ERK1/2 expression was normalized to T-ERK1/2 levels and expressed as fold over vehicle. n=10; one-way ANOVA, * p < 0.05. ERK1/2 phosphorylation was assessed in TRAIL (C) or

of

bioymifi (D) treated NRVMs with or without GM6001. P-ERK1/2 levels are normalized to T-

ro

ERK1/2 and expressed as fold over vehicle. n=6; one-way ANOVA, p < 0.05. E. HB-EGF expression was measured in the media of NRVMs treated with bioymifi over time using an

-p

ELISA. n=6; one-way ANOVA, * p < 0.05 versus vehicle. F. Immunoblot analysis of membrane

re

localized EGFR phosphorylation in NRVMs treated with bioymifi over time. P-EGFR levels were

lP

normalized to T-EGFR and expressed as fold over vehicle. n=6; one-way ANOVA, * p < 0.05. G. Immunoblot of P-EGFR levels in heart membranes from mice injected with IgG or MD5-1. P-

na

EGFR intensity was normalized to T-EGFR expression and expressed as fold over IgG. n=7, t test, * p < 0.05 versus IgG. H. HB-EGF release was assessed in the media of NRVMs treated

ur

with bioymifi in the presence or absence of GM6001 via ELISA. n=6; one-way ANOVA, * p <

Jo

0.05. I. Representative and quantified immunoblot of P-EGFR from membranes of NRVMs pretreated with GM6001 followed by bioymifi. P-EGFR levels were normalized to T-EGFR and expressed as fold over vehicle. n=5; one-way ANOVA, * p < 0.05. Figure 6: DR5-mediated EGFR-transactivation stimulates hypertrophy. A. Representative troponin I (green) staining of NRVMs pretreated by AG1478 or GM6001 followed by TRAIL or bioymifi. DAPI (blue) was used to label the nuclei. B. Quantification of cell area for NRVMs treated with TRAIL or bioymifi in the presence or absence of AG1478. Values are expressed as fold over vehicle treated NRVMs. n=5; one-way ANOVA, * p < 0.05. C. Cardiomyocyte area

Journal Pre-proof measurements for NRVMs pretreated with GM6001 followed by TRAIL or bioymifi. n=6, oneway ANOVA, * p < 0.05.

Figure 7: DR5 activation promotes hypertrophy through GATA4 transcription factor activation. A. Representative troponin I (green) staining of NRVMs pretreated with actinomycin D followed by 24h TRAIL or bioymifi treatment. Cells were counterstained with DAPI (blue) to identify

of

nuclei. B. Quantification of cell area for TRAIL or bioymifi treated NRVMs with or without preincubation with actinomycin D. Values are represented as fold over vehicle treated cell area.

ro

n=6, one-way ANOVA, * p < 0.05. C. Representative EMSA and quantification of nuclear lysates

-p

of NRVMs treated temporally with bioymifi and incubated with a GATA4-specific probe. Intensity

re

of GATA4 transcription factor binding to GATA4-specific IR680 labelled DNA sequences were normalized to total lane intensity and expressed as fold over vehicle. n=7; one-way ANOVA, * p

lP

< 0.05 versus vehicle. D. NRVMs were co-transfected with a plasmid encoding for the WT or

na

S105A mutant GATA4 transcriptional activation domain fused to a GAL4 DNA binding domain and the GAL4-luciferase reporter, pGL2-GAL4-UAS-Luc. NRVMs were stimulated with vehicle,

ur

TRAIL or bioymifi for 24h and luminescence was measured. n=6; one-way ANOVA, * p < 0.05.

Jo

E. Representative GATA4 EMSA and quantification from nuclear lysates of NRVMs preincubated with PD98059 then bioymifi. n=5; one-way ANOVA, * p < 0.05. F. Luciferase assay of GATA4 transcriptional activation following bioymifi treatment in the presence or absence of PD98059. n=5; one-way ANOVA, * p < 0.05. G. Representative and quantified EMSA from NRVM nuclear lysates from cells treated with bioymifi in the presence or absence of AG1478. n=5; one-way ANOVA, * p < 0.05. H. Luciferase assay of GATA4 transcriptional activation following bioymifi treatment in the presence or absence of AG1478. n=5; one-way ANOVA, * p < 0.05. I. Representative and quantified GATA4 EMSA from nuclear lysates of NRVMs preincubated with GM6001 then bioymifi. n=5; one-way ANOVA, * p < 0.05. J. Luciferase assay of

Journal Pre-proof GATA4 transcriptional activation following bioymifi treatment in the presence or absence of GM6001. n=5; one-way ANOVA, * p < 0.05.

Figure 8: A. Representative troponin I (green) staining of NRVMs transfected with scrambled (Scr) or GATA4 siRNA and treated with TRAIL or bioymifi. Cells were counterstained with DAPI (blue) to identify nuclei. B. Quantification of cardiomyocyte size for TRAIL or bioymifi treated

of

NRVMs with or without GATA4 knockdown. n=6, one-way ANOVA, * p < 0.05. C. Summary of the mechanisms involved in DR5-mediated hypertrophy in cardiomyocytes. Activation of DR5

ro

transactivates EGFR through activation of MMPs and cleavage of HB-EGF. EGFR

-p

transactivation leads to ERK1/2 activation and increased pro-hypertrophic gene transcription

Jo

ur

na

lP

re

through activation of the transcription factor GATA4.

Journal Pre-proof Highlights:

of ro -p re lP na



ur



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 activates GATA4 leading to pro-hypertrophic gene transcription

Jo

