During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1 1 precursors. Embryos deficient for Nkx2-5 in the Isl1 1 lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.

Two types of distinct cardiac progenitor cell populations can be identified during early heart development: the first heart field (FHF) and second heart field (SHF) lineage that later form the mature heart. They can be characterized by differential expression of transcription and signaling factors. These regulatory factors influence each other forming a gene regulatory network. Here, we present a core gene regulatory network for early cardiac development based on published temporal and spatial expression data of genes and their interactions. This gene regulatory network was implemented in a Boolean computational model. Simulations reveal stable states within the network model, which correspond to the regulatory states of the FHF and the SHF lineages. Furthermore, we are able to reproduce the expected temporal expression patterns of early cardiac factors mimicking developmental progression. Additionally, simulations of knock-down experiments within our model resemble published phenotypes of mutant mice. Consequently, this gene regulatory network retraces the early steps and requirements of cardiogenic mesoderm determination in a way appropriate to enhance the understanding of heart development.

The anti-inflammatory and immunomodulatory abilities of oral selective phosphodiesterase 4 (PDE4) inhibitors enabled the approval of roflumilast and apremilast for use in chronic obstructive pulmonary disease and psoriasis/psoriatic arthritis, respectively. However, the antifibrotic potential of PDE4 inhibitors has not yet been explored clinically. BI 1015550 is a novel PDE4 inhibitor showing a preferential enzymatic inhibition of PDE4B. In vitro, BI 1015550 inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and phytohemagglutinin-induced interleukin-2 synthesis in human peripheral blood mononuclear cells, as well as LPS-induced TNF-α synthesis in human and rat whole blood. In vivo, oral BI 1015550 shows potent anti-inflammatory activity in mice by inhibiting LPS-induced TNF-α synthesis ex vivo and in Suncus murinus by inhibiting neutrophil influx into bronchoalveolar lavage fluid stimulated by nebulized LPS. In Suncus murinus, PDE4 inhibitors induce emesis, a well-known gastrointestinal side effect limiting the use of PDE4 inhibitors in humans, and the therapeutic ratio of BI 1015550 appeared to be substantially improved compared with roflumilast. Oral BI 1015550 was also tested in two well-known mouse models of lung fibrosis (induced by either bleomycin or silica) under therapeutic conditions, and appeared to be effective by modulating various model-specific parameters. To better understand the antifibrotic potential of BI 1015550 in vivo, its direct effect on human fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) was investigated in vitro. BI 1015550 inhibited transforming growth factor-β-stimulated myofibroblast transformation and the mRNA expression of various extracellular matrix proteins, as well as basic fibroblast growth factor plus interleukin-1β-induced cell proliferation. Nintedanib overall was unremarkable in these assays, but interestingly, the inhibition of proliferation was synergistic when it was combined with BI 1015550, leading to a roughly 10-fold shift of the concentration–response curve to the left. In summary, the unique preferential inhibition of PDE4B by BI 1015550 and its anticipated improved tolerability in humans, plus its anti-inflammatory and antifibrotic potential, suggest BI 1015550 to be a promising oral clinical candidate for the treatment of IPF and other fibro-proliferative diseases.

Nintedanib inhibits proliferation of pulmonary MVECs from controls, but not from PAH patients. While in rats with experimental PH nintedanib has no effects on the pulmonary vascular pathology, it has favorable effects on right ventricular remodeling.

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