Cell culture

The cardiac-derived myoblast cell line, H9c2 cells (Sigma-Aldrich, #88092904-1VL, St. Louis, MO, USA) and HEK293T cells (ATCC, CRL-3216TM, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, #12-604F, Basel, Switzerland) supplemented with 10% FBS (v/v) (Gibco, #1099-141, Massachusetts, USA) and 1% L-glutamine (v/v) (Sigma-Aldrich, #G7513-100ML, St. Louis, MO, USA) at 37 oC in a humidified atmosphere containing 5% (v/v) CO2 cells.

Neonatal rat ventricular myocytes

All animal procedures had ethical approval (Western Sydney Local Health District animal ethics protocol 4332) and were performed in accordance with the National Health and Medical Research Council (NHMRC) Code for the care and use of animals for scientific purposes. NRVMs were isolated from day 3 (D3) neonatal Wistar rats via enzymatic dissociation of ventricles using trypsin and collagenase as previously described [21]. Isolated NRVMs were seeded at 2–4 × 105 cells/well in 24 well plates. Twenty-four hours later, cells were washed with DPBS (Lonza, #12001-664, Basel, Switzerland). On D2 after cell plating, FBS was reduced to 2% and media was changed on cultures every second day.

Human induced pluripotent stem cells

The hiPSC line SCVI 8 used in this study was obtained from Professor Joseph Wu (Stanford Cardiovascular Institute, USA). Stem cells were maintained on Matrigel (Corning, #354277, New York, USA) coated 60 mm cell culture dishes using the mTeSRTM Plus kit (STEMCELL Technologies, #05825, Vancouver, Canada). Upon confluence, cells were passaged as colonies using gentle cell dissociation reagent (STEMCELL Technologies, #07174, Vancouver, Canada) every 6-7 days.

For cardiomyocyte differentiation, cells were dissociated from confluent dishes on D-2 using TrypLETM Express Enzyme (ThermoFisher Scientific, 12604-021, Massachusetts, USA), then seeded into Matrigel coated 12 well plates at 7 × 105 cells/well using mTeSRTM Plus supplemented with Y-27632 (STEMCELL Technologies, #72304, Vancouver, Canada). On D-1, a media change was performed to remove the Y-27632. On D0, the differentiation was commenced for both the STEMdiff (SD) kit method and the Small Molecule (SM) method. For the SD method, differentiation was commenced using the STEMdiff Cardiomyocyte Differentiation Kit (STEMCELL Technologies, #05010, Vancouver, Canada) according to manufacturer’s instructions until the point of maintenance in STEMdiff Cardiomyocyte Maintenance Medium (CMM). Cells were maintained on CMM until differentiation completion on D15. For the SM method, differentiation was commenced by addition of 10 μM CHIR-99021 (Tocris Bioscience, #4423, Bristol, United Kingdom), 1x B27-Insulin supplement (Life Technologies, #A1895601, California, USA) and 1x penicillin-streptomycin solution (Life Technologies, #15140122, California, USA) in RPMI 1640 (Life Technologies, #21870092, California, USA). The next day, the media was replaced with RPMI containing 1x B27-Insulin and 1x penicillin-streptomycin solution. On D3, the media was exchanged for RPMI containing 1x B27-Insulin, 1x penicillin-streptomycin and 5 μM IWP-2 (Tocris Bioscience, #3533, Bristol, United Kingdom). On D5, the media was exchanged for RPMI containing 1 x B27-Insulin and 1x penicillin–streptomycin without supplemental cytokines. From D7 onwards, the cultures were fed every 2 days with RPMI plus 1x B27 supplement (Life Technologies, #17504001, CA, USA) and 1x penicillin–streptomycin until differentiation completion on D15.

Once differentiation was finished, cells were replated for further experiments. After 1 h of incubation with 10 µM Y-27632, differentiated cardiomyocytes were dissociated using TrypLETM Express supplemented with 2 µg/mL DNase I (STEMCELL Technologies, #07900, Vancouver, Canada). Dissociated cells were then replated into GeltrexTM (Gibco, #A14133-02, Massachusetts, USA) coated 24 well plates at 5 × 10 5 cells/well using RPMI 1640 media and B27 supplement accompanied with Y-27632.

Molecular cloning

A gene block was synthesised (Integrated DNA Technologies Pte. Ltd, Singapore, Republic of Singapore) to contain the human ghrelin sequence (Genbank: BC025791.1). The ghrelin cDNA (full length preproghrelin) was cloned into the lentiviral vector plasmid pRRLsin18.cPPT.CMV.GFP.Wpre (ppt.CMV.GFP, Inder Verma, The Salk Institute for Biological Studies, California, USA) after removal of GFP. The new vector was named ppt.hGhre. A control vector was also generated, by including LacZ coding sequence in place of the ghrelin transgene (ppt.LacZ).

A truncated “minighrelin” vector (ppt.mini.Ghre) was produced in an attempt to circumvent the requirement for PC1/3 mediated cleavage of preproghrelin into the mature ghrelin peptide. The proximal 150 bp of the ghrelin coding sequence was cloned with an in-frame stop codon into the lentiviral vector plasmid, as described for the ppt.hGhre vector (see also Supplementary Fig. 3).

Lentiviral vector production

Lentiviral vectors were produced by calcium phosphate transfection into HEK293T cells. Vector containing supernatant was collected at 48 and 72 h after transfection, then filtered and concentrated by ultrafiltration (100,000 MWCO PES, sartorius, #VS2042, Gottingen, Germany). Concentrated virus (LV.LacZ, LV.miniGhre and LV.hGhre) was aliquoted and stored at −80 °C. Transduction titre was assigned on concentrated supernatant by assessing transgene expression in HEK293T cells using a limiting dilution assay in the presence of polybrene 8 μg/mL (Sigma-Aldrich, #H9268-10G, St. Louis, MO, USA) four days after transduction. For transduction experiments, concentrated vector stock was used at the indicated multiplicity of infection (MOI) in the presence of polybrene 8 μg/mL. Vector was applied for 18 h, and media changed to 2% FBS in DMEM the following morning.

Western blots

For analysis of intracellular ghrelin, protein was extracted from cell pellets using RIPA buffer and 25x protease inhibitor (Sigma-Aldrich, #P8340-1ML, St. Louis, MO, USA), then quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, #23227, Massachusetts, USA). Protein samples were diluted in 4x laemmli sample buffer, denatured at 95 °C for 5 min, then loaded onto a Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad, #4561094, CA, USA), along with a 10–250 kDa protein ladder, with SDS-PAGE at 100 V for 1 h in 1× running buffer. Proteins were transferred onto a nitrocellulose membrane in 1X transfer buffer, using a Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad, #1703930, CA, USA) at 70 V for 1.5 h.

Afterwards, the membrane was temporarily stained with Ponceau S to observe the total protein transferred to the membrane. The membrane was then blocked for 1 h in 5% skim milk, followed by overnight incubation with the primary mouse anti-ghrelin (MERCK Millipore, MAB10404, New Jersey, USA) antibody. The membrane was subsequently washed 3 × 10 min with PBST, followed by incubation with the rabbit anti-mouse (Sigma-Aldrich, #A9044-2ML, St. Louis, MO, USA) secondary antibody for 1 h. The membrane was then washed 3 × 10 min with PBST then imaged via chemiluminescence using the SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, #34095, Massachusetts, USA). The membrane was stripped with 0.5 M NaOH, then incubated with a rabbit anti-β-actin antibody (abcam, #ab8227, Massachusetts, USA), goat anti-rabbit secondary antibody and imaged as previously described. Immunoblots were quantified using ImageJ.

To determine whether biologically active mature ghrelin was being secreted into the supernatant of H9c2 cells transduced with LV.hGhre or LV.miniGhre, 12 mL of supernatant was collected from transduced cells and concentrated using a Vivaspin 20 (3000 MWCO PES, sartorius, #VS2091, Gottingen, Germany) at 3500 × g and 4 °C for 15 min (or until approximately 200 µL of supernatant remained). A Western blot was performed on this concentrated supernatant as previously described.

Amplification of ghrelin, PSCK1 and GAPDH transcript by RT-PCR

RNA was extracted from H9c2 cells, NRVMs and U87-MG cells using the ISOLATE II RNA Mini Kit (Bioline, #BIO-52072, Tennessee, USA) then quantified using a NanoDrop™ 2000 Spectrophotometer (ThermoFisher Scientific, #ND2000, MA, USA). For cDNA synthesis, Random Primers (500 ng/μL) were added to the extracted RNA (1 μg), followed by incubation at 70 °C for 5 min. M-MLV Reverse Transcriptase Buffer (5×), M-MLV Reverse Transcriptase (Promega, #M1701, Wisconsin, USA), 10 mM dNTPs (New England Biolabs, #N0447S, Massachusetts, USA) and RNasin® Ribonuclease Inhibitor (Promega, #N2111, Wisconsin, USA) were then added to the reaction mixture, followed by incubation under the following conditions: 25 °C for 10 min, 37 °C for 1 h then 70 °C for 15 min.

Primers were designed for both human and rat PCSK1, and GAPDH as a loading control, as described in Table 1. Each PCR product, along with HyperLadder™ IV (Bioline, #BIO33030, TN, USA) as a molecular weight marker, was loaded onto a 2% agarose gel in 1× TAE and run at 100 V for 1 h, then imaged using a Gel Doc EZ Imager (Bio-Rad, #1708270, CA, USA).

Table 1 Primer sequences for amplification of PCSK1 and GAPDH.

A relative qPCR was performed on the synthesised cDNA using the SensiFAST™ SYBR® No-ROX Kit (Bioline, #BIO-98020) on the Rotor-GeneTM 3000 (Corbett Research), with results normalised to GAPDH as a housekeeping gene.

Induction of oxidative stress in vitro

To investigate the mechanisms of ghrelin protection, LV.LacZ or LV.hGhre was used to transduce H9c2s (MOI 100) or NRVMs (MOI 20). Four days post transduction, cells were injured with a dose titration of H2O2, whilst some cells were uninjured (0 µM H2O2) as a control. After 24 h, cell viability was assessed using the CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega, #G4000, WI, USA), hereafter abbreviated to “MTT assay”, according to manufacturer’s instructions.

TMRE assay to assess mitochondrial function

Transduced H9c2 cells which had undergone injury with H2O2 were assessed together with uninjured control cells using the TMRE-Mitochondrial Membrane Potential Assay kit (abcam, #ab113852, Massachusetts, USA). Briefly, TMRE dye was added to each well at a final concentration of 200 nM, followed by incubation at 37 oC for 30 min. Subsequently, cells were dissociated using trypsin, then washed in 2% FBS in PBS. Following staining with DAPI (0.1 mg/μL), cells were analysed for TMRE fluorescence using a Zeiss Axiovert 200 M live-cell imaging microscope (Zeiss, Oberkochen, Germany) and BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA).

Flow cytometry

Cells were dissociated using TrypLETM Express, then washed with Dulbecco’s Phosphate Buffered Saline without calcium and magnesium. Cells were then stained using the Zombie NIRTM Fixable Viability Kit (Biolegend, #423105, San Diego, USA). After washing, cells were stained with BV650 conjugated rat anti-CD90 antibody (Biolegend, #202533, San Diego, USA), then fixed in 4% PFA for 30 min, then washed and further stained with BV421 conjugated mouse anti-cTnT antibody (BD Biosciences, #565618, San Diego, USA) for 2 h. Upon further washing, cells were analysed on the FACSCantoTM II Cell Analyzer or LSRFortessaTM and data recorded using FACSDivaTM Software (BD Biosciences, Franklin Lakes, NJ, USA). Analysis was subsequently performed using FlowJo (FlowJo LLC, Oregon, USA) version 10.

Statistical analysis and software

Depending on the data, different statistical analyses detailed in the figure legends were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). All data are mean ± SEM. For all used tests, significance was represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SnapGene software (www.snapgene.com) version 5.1.3 was used to design cloning strategies to demonstrate the rationale behind the two versions of the ghrelin vectors. Western blots and agarose gels were visualised using Image Lab software (Bio-rad Laboratories, Inc), version 6.1.0.