Switching of hypertrophic signalling towards enhanced cardiomyocyte identity and maturity by a GATA4-targeted compound

Background The prevalence of heart failure is constantly increasing, and the prognosis of patients remains poor. New treatment strategies to preserve cardiac function and limit cardiac hypertrophy are therefore urgently needed. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as an experimental platform for cardiac in vitro studies. However, in contrast to adult cardiomyocytes, hiPSC-CMs display immature morphology, contractility, gene expression and metabolism and hence express a naive phenotype that resembles more of a foetal cardiomyocyte. Methods A library of 14 novel compounds was synthesized in-house and screened for GATA4-NKX2-5 reporter activity and cellular toxicity. The most potent compound, 3i-1262, along with previously reported GATA4-acting compounds, were selected to investigate their effects on hypertrophy induced by endothelin-1 or mechanical stretch. Morphological changes and protein expression were characterized using immunofluorescence staining and high-content analysis. Changes in gene expression were studied using qPCR and RNA sequencing. Results The prototype compound 3i-1262 inhibited GATA4-NKX2-5 synergy in a luciferase reporter assay. Additionally, the isoxazole compound 3i-1262 inhibited the hypertrophy biomarker B-type natriuretic peptide (BNP) by reducing BNP promoter activity and proBNP expression in neonatal rat ventricular myocytes and hiPSC-CMs, respectively. Treatment with 3i-1262 increased metabolic activity and cardiac troponin T expression in hiPSC-CMs without affecting GATA4 protein levels. RNA sequencing analysis revealed that 3i-1262 induces gene expression related to metabolic activity and cell cycle exit, indicating a change in the identity and maturity status of hiPSC-CMs. The biological processes that were enriched in upregulated genes in response to 3i-1262 were downregulated in response to mechanical stretch, and conversely, the downregulated processes in response to 3i-1262 were upregulated in response to mechanical stretch. Conclusions There is currently a lack of systematic understanding of the molecular modulation and control of hiPSC-CM maturation. In this study, we demonstrated that the GATA4-interfering compound 3i-1262 reorganizes the cardiac transcription factor network and converts hypertrophic signalling towards enhanced cardiomyocyte identity and maturity. This conceptually unique approach provides a novel structural scaffold for further development as a modality to promote cardiomyocyte specification and maturity. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-023-03623-x.


General procedure I: Synthesis of aldoximes 2a-g
Aldehyde 1a-g, hydroxylammonium chloride (1.1 equiv) and pyridine (1.1 equiv) were dissolved in anhydrous ethanol and stirred at room temperature for 1-5 h.The reaction was quenched with a saturated solution of NH4Cl in water and the resulting mixture was extracted with dichloromethane (DCM) or ethyl acetate (EtOAc).Combined organic layers were dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated under reduced pressure.The crude product was purified by flash chromatography on silica gel cartridges using increasing gradient of EtOAc in n-heptane as indicated.
General procedure II: Synthesis of isoxazoles 3a-g via 1,3-dipolar cycloaddition Aldoxime 2a-g and ethyl 2-butynoate (1.5 equiv) were dissolved in a 2:1 mixture of acetonitrile (MeCN) and water at 0 °C.(Diacetoxyiodo)benzene (DIB, 1.2 equiv) was dissolved into equal volume of a 2:1 mixture of MeCN and water, and added dropwise to the reaction mixture during 5 min.The reaction mixture was stirred for 40-120 min, after which time the organic solvent was evaporated under reduced pressure.The aqueous phase was washed with DCM and the resulting organic phase was concentrated under reduced pressure.The crude oil was purified with flash chromatography on silica gel cartridges using increasing gradient of EtOAc in n-heptane as indicated.

General procedure III: Synthesis of carboxylic acids 4a-g via ester hydrolysis
Carboxylic acid ester 3a-g was dissolved in equal volumes of THF, MeOH and water at room temperature.Lithium hydroxide (LiOH, 1.5-2.0equiv) was added and the mixture was stirred at room temperature for 1-3 days.The mixture was diluted with water and a 1 M solution of NaOH in water, and the aqueous phase was washed with EtOAc or DCM, followed by the addition of EtOAc.The aqueous phase was acidified with a 1 M solution of HCl in water, and the organic phase was washed with water and concentrated under reduced pressure.Resulting carboxylic acids 4a-g were used in the next step without further purification.

AlphaScreen
The AlphaScreen (Amplified Luminescent Proximity Homogenous Assay) method was pre-validated by optimising the following parameters: amount of plasmids in transfections, the lysis buffers, expression levels of tagged proteins and their cross-titrations for optimal signal intensity for GATA4-NKX2-5 interaction.Shortly, COS-1 cells were seeded onto 6-well plates at 300,000 cells per well and transfected next day with 3 µg of pDEST40-GATA4-C-V5 or 2.4 µg of pcDNA™5/FRT/TO-NKX2-5-N-SH using FuGene6 (Promega) in 3:1 ratio to DNA.After 24 h the cells were detached by trypsin and counted with hemocytometer.Medium was removed by centrifugation 200 g at 4 °C for 4 min and the cells were washed once with phosphate-buffered saline (PBS).Finally, the cells were suspended into non-denaturing lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100, 2.5 mM sodium pyrophosphate, pH 7.5) with phosphatase inhibitors (1 mM βglycerophosphate, 1 mM Na3VO4, 50 mM NaF) and protease inhibitors (Protease Inhibitor Mini Tablets, #88666, Pierce) to contain 4,000 cells per µL.The cells were disrupted by vortexing vigorously for 20 s followed by centrifugation at 15 000 g for 20 min at 4 °C.The supernatant containing the total proteins was transferred into clean sample tube.GATA4 and NKX2-5 protein lysates were diluted into AlphaScreen sample buffer (50 mM Tris-HCl pH7.4,150 nM NaCl, 0.1% BSA) added to ½ AreaPlate-96 (#6002290, Perkin Elmer) in addition to dilution series of compound or DMSO into AlphaScreen sample buffer and incubated at 4 °C for 60 min.Each sample well contained proteins from 1,000 cells with GATA4 overexpression and 2,500 cells with NKX2-5 overexpression.V5-acceptor beads (AL129, Perkin Elmer) and Strep-Tactin donor beads (AS106, Perkin Elmer) were added 20 µg/mL in final concentration and incubated at room temperature covered from light for 60 min.The plate was analysed using Enspire Alpha plate reader (Perkin Elmer).

Immunofluorescence staining
The cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min.Non-specific binding sites were blocked with 4% FBS in PBS for 45 min followed by the addition of primary antibodies diluted in 4% FBS in PBS: cardiac troponin T (cTnT) antibody (Abcam Cat# ab45932) at 1:800, proBNP antibody (Abcam Cat# ab13115) at 1:200, BrdU antibody (Abcam Cat# ab6326) at 1:250, and GATA4 antibody (Cell Signaling Technology Cat# 36966) at 1:400.After a 60-min incubation at room temperature, the cells were washed 3 × 5 min with PBS and incubated with Alexa Fluor®conjugated secondary antibodies (Life Technologies, Eugene, Oregon) at 1:200, and 4ʹ,6-diamidino-2phenylindole (DAPI; Sigma-Aldrich) at 1 µg/mL at room temperature for 45 min.The cells were then washed 3 × 5 min with PBS and stored at 4 °C in PBS until imaged.

RNA sequencing
RNA sequencing (RNAseq) was performed as single-end sequencing for a read length of 75 bp with an Illumina NextSeq 500 sequencer (Illumina, San Diego, CA, USA) in high output runs using a NEBNext Ultra Directional RNA Library Prep kit (New England Biolabs, Ipswich, MA, USA) including rRNA depletion.Data quality was analysed by FastQC, and quality trimming was applied to the data with Trimmomatic software. 1The sample reads were aligned against the Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.p13,GCA_000001405.28) reference with Spliced Transcripts Alignment to a Reference (STAR). 2 Mapping quality was assessed with Qualimap. 3Read quantification was created with featureCounts 4 , and differential expression with quality assessment was performed with DESeq2. 5

Western blotting
The effects of endothelin-1 (ET-1; 100 nM) and compound 3i-1262 (30 µM) on α-actinin, cardiac troponin T, and pro-B-type natriuretic peptide (proBNP) protein expression in hiPSC-CMs was investigated with western blotting.hiPSC-CMs were plated on 6-well plates at a density of 2 × 10 6 cells / well and maintained in RB+ until day 30 of differentiation.The cells were treated with 3i-1262 or vehicle 1 h prior to addition of ET-1.Brefeldin A was added to the cells three hours before cell lysis.After the 24 h exposure, cells were lysed with 1% sodium dodecyl sulphate in 50 mM Tris-HCl (pH 7.5).
To shear the genomic DNA, a 25G needle was used.Protein concentrations of the samples were determined with the Pierce TM BCA Protein Assay -kit (Thermo Scientific).Fifteen micrograms of total protein were loaded on 10% Mini-PROTEAN® TGX Stain-Free™ Protein gels (Bio-Rad) and proteins were separated by electrophoresis.To activate the reaction between trihalo compounds and proteins, which enables the visualisation of total proteins, the gels were exposed to UV-light for 5 min.Next, proteins were transferred to a nitrocellulose membrane by Trans-Blot® Turbo™ transfer system (Bio-Rad).Nonspecific background was blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1 % Tween 20 (TTBS) for 1 h at RT.The membranes were incubated with primary antibody 1:1000 solution (anti α-actinin (Abcam, ab7811), anti-cTnT (Abcam, ab45932) and anti-proBNP (Abcam, ab13115)) in 5% milk-TTBS overnight at 4°C.The membranes were washed with TTBS, followed by a 1-hour incubation in the 1:2000 secondary antibody solutions (anti-mouse (Cell Signaling Technology, 7076); anti-rabbit (Cell Signaling Technology, 7074S)) conjugated with Precision Protein StrepTactin-HRP Conjugate (Bio-Rad).α-actinin and cTnT bands were detected with an enhanced chemiluminescent substrate SuperSignal TM West Pico PLUS (Thermo Scientific) whereas proBNP bands were detected with SuperSignal TM West Femto Maximum Sensitivity Substrate (Thermo Scientific) using a ChemiDoc TM MP Imaging System (Bio-Rad).Optical densities of the bands were quantified using the ImageLab 6.0.1.software (Bio-Rad).The densities of the protein bands were normalised to the total protein of the sample.S2.Summary chart of study compounds measured for GATA4-NKX2-5 synergy in luciferase reporter assay and viability in human induced pluripotent stem cells (hiPSC).S3.KEGG pathways enriched in upregulated genes in response to 3i-1262 in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).