GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line

Abstract

Background Pharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration. Methods Transcription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression. Results GATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression. Conclusions Collectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.

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

“…Our previous GATA4-NKX2-5 screening campaign produced the first generation of GATA4-acting compounds with cellular activity in a low micromolar range [ 26 , 27 ]. Our previous results show that modulation of the TF machinery can reduce the hypertrophic growth of cardiac myocytes [ 26 ], enhance myocardial repair after myocardial infarction and other cardiac injuries in rodent models [ 29 , 30 ], and promote atrial and ventricular cell fate [ 28 ]. However, to enhance the efficacy and to reduce cell toxicity encountered with our previous lead compound 3i-1000 [ 30 ], we synthesised and tested a novel set of structurally optimised compounds.…”

“…We have previously identified GATA4-acting compounds that inhibit the GATA4-NKX2-5 interaction and cause downstream effects on cardiac gene expression, such as BNP and myosin light chain 2 [ 26 – 28 ]. We have further reported that in rodent models by modulating GATA4-NKX2-5 synergy, we were able to reduce the hypertrophic growth of cardiac myocytes [ 26 ] and enhance myocardial repair after myocardial infarction and other cardiac injuries [ 29 , 30 ].…”

Nkx2.5: a crucial regulator of cardiac development, regeneration and diseases

“…Numerous previous studies have demonstrated that Nkx2.5 is regulated at the transcriptional level by many transcription factors and cis-regulatory elements upstream of the Nkx2.5. These transcriptions can regulate the expression of Nkx2.5 at different times and in different tissues ( Table 1 ) ( 41 ).…”

Cardiac stem cells: Current knowledge and future prospects

“…This SCA-1 + population is different from hematopoietic SCs as they lack CD45, CD34, c-KIT, LIM domain only 2, GATA2, VEGF receptor 1, and T-cell acute lymphoblastic leukemia 1/SC leukemia proteins. SCA-1 + cells are also distinct from endothelial progenitor cells and express cardiac lineage transcriptional factors such as GATA4, MEF2C, and translation elongation factor 1 yet lack transcripts for cardiomyocytic structural genes such as BMP1r1 and α-, β-MHC [ 47 , 48 ]. Although this population exhibits the endothelial marker CD31, it is suggested to be due to the contaminating endothelial CD31 + /SCA-1 + cells.…”