Molecular and metabolomic characterization of hiPSC-derived cardiac fibroblasts transitioning to myofibroblasts

Abstract

1.AbstractMechanical stress and pathological signaling trigger the activation of fibroblasts to myofibroblasts, which accelerates extracellular matrix turnover, disrupts normal wound healing, and can generate deleterious fibrosis. Myocardial fibrosis independently promotes cardiac arrhythmias, sudden cardiac arrest, and contributes to the severity of heart failure. Fibrosis can also alter cell-to-cell communication and increase myocardial stiffness. Human induced pluripotent stem cell derived cardiac fibroblasts (hiPSC-CFbs) have the potential to enhance clinical relevance in precision disease modeling by facilitating the study of patient-specific phenotypes. However, it is unclear whether hiPSC-CFbs can be activated to become myofibroblasts akin to primary cells, and the key signaling mechanisms in this process remain unidentified. We hypothesize that the passaging of hiPSC-CFbs, similar to primary cardiac fibroblasts, induces specific genes required for myofibroblast activation and increased mitochondrial metabolism. Passaging of hiPSC-CFbs from passage 0 to 3 (P0 to P3) and treatment of P0 with TGFβ1 was associated with a gradual recruitment of genes to initiate the activation of these cells to myofibroblasts, including collagen, periostin, fibronectin, and collagen fiber processing enzymes with concomitant downregulation of cellular proliferation markers. Most importantly, canonical TGFβ1 and Hippo signaling component genes including TAZ were influenced by passaging hiPSC-CFbs. Seahorse assay revealed that passaging and TGFβ1 treatment increased mitochondrial respiration, consistent with fibroblast activation requiring increased energy production, whereas treatment with the glutaminolysis inhibitor BPTES completely attenuated this process. Based on these data, we suggest that hiPSC-CFb passaging enhanced fibroblast activation, influenced fibrotic signaling pathways, and enhanced mitochondrial metabolism approximating what has been reported in primary cardiac fibroblasts. Thus, hiPSC-CFbs may provide an accurate in vitro preclinical model for the cardiac fibrotic condition, which may facilitate the identification of putative anti-fibrotic therapies, including patient-specific approaches.HighlightsPassaging promotes the activation of fibroblast and transition towards myofibroblasts.TGFβ1 treatment activates the fibroblasts, but their expression profile was uniquely different from myofibroblasts.High energy requiring fibroblast activation is depended on glutaminase based mitochondrial metabolism.Passaging influences TGFβ1 and Hippo signaling pathways in activated fibroblasts and myofibroblasts.Graphical Abstract CaptionProbing the activation of fibroblasts to myofibroblast is key in ECM remodeling process to avoid fibrosis related adverse complications, and to better understand the disease pathology. Here we report that passaging of hiPSC-derived cardiac fibroblasts promotes fibroblast activation along with gradual shift in gene expression and metabolic changes towards myofibroblasts. TGFβ1 treatment activates the non-passaged fibroblasts, but they are not completely similar to myofibroblasts. Energy demanding fibroblast activation to myofibroblast conversion process is dependent on glutaminase mediated mitochondrial metabolism, such process is prevented by treatment with GLS-1 inhibitor BPTES. Our work demonstrates that hiPSC-CFs can offer a better preclinical model to offer patient specific novel anti-fibrosis drug screening and disease management.