Cardiac fibrosis is a pathological feature in >90% of heart failure patients, and thus a desirable but so far elusive target for therapeutic intervention. Building on existing evidence, our data implicates endothelin signalling in cardiac fibrosis leading to contractile dysfunction that ultimately lead to heart failure [1]. Although centrally implicated in cardiac pathologies, clinical trials targeting endothelin receptors were unsuccessful, likely due to the pleiotropic functions of endothelin across multiple organ systems. We propose targeting the downstream mediators of endothelin signalling which may provide avenues for discerning cardiac-specific therapeutics for heart failure. Here, we utilise our transformative stem-cell approaches with phosphoproteomics technologies [2] to define the pathological fibrosis signalling network.
Pluripotent stem cell-derived human cardiac organoids (hCO) represent a controlled functional model that is ideal for investigating intra- and inter-cellular signalling mechanisms in contractile heart tissue. hCOs are stimulated with a panel of pro-fibrotic mediators, such as endothelin-1, in addition to other contractile mediators, such as histamine, to pinpoint converging and diverging pathways that underpin fibrosis-induced cardiac dysfunction. The direct effects of contractile and pro-fibrotic stimuli on contractile force, rate and contraction kinetics were analysed from 10 second videos using custom Matlab scripts. Endothelin and histamine induced potent increases in force and rate, but only endothelin displayed prolonged relaxation time, indicative of diastolic dysfunction. EasyPhos-enabled phosphoproteomics identified 6,239 phosphopeptides across 1,806 proteins in pooled organoid samples, where stimulation by endothelin and histamine revealed differential regulation of sarcomere and contractility-related proteins that could account for different functional responses. Incorporation of motif-based kinase library analysis predicted the responsible kinases based on surrounding phosphosite sequence propensities, revealing discrepancies in pathway activation between the two stimuli.
Our functional and initial phosphoproteomics data suggest divergent signalling responses for endothelin-1 and other cardiac ligands. We are now pursuing the mechanisms that underpin these differences using systems biology approaches including time-resolved global phosphoproteomics and network modelling. These studies will uncover downstream endothelin signalling nodes that could represent new targets for cardiac dysfunction in heart failure.