Glutamine uptake and catabolism is required for myofibroblast formation and persistence

• Published in: Journal of molecular and cellular cardiology 2022-11, Vol.172, p.78-89
• Main Authors: Gibb, Andrew A., Huynh, Anh T., Gaspar, Ryan B., Ploesch, Tori L., Lombardi, Alyssa A., Lorkiewicz, Pawel K., Lazaropoulos, Michael P., Bedi, Ken, Arany, Zolt, Margulies, Kenneth B., Hill, Bradford G., Elrod, John W.
• Format: Article
• Language: English
• Subjects: Animals; Cells, Cultured; Collagen - metabolism; Epigenetics; Fibroblasts - metabolism; Fibrosis; Glutamine; Glutamine - metabolism; Heart failure; Humans; Metabolism; Mice; Myofibroblast; Myofibroblasts - metabolism
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2022.08.002

Fibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. Stable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1−/−) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. Carbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFβ-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. These results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis. [Display omitted] •Glutamine is the primary fuel/carbon source contributing to αKG bioavailability.•αKG is necessary for histone demethylation mediating myofibroblast differentiation and maintenance.•Pharmacological/genetic inhibition of GLS1 prevents myofibroblast formation.•Inhibition of GLS1 in fibroblasts isolated from HF patients impedes myofibroblast differentiation and ECM expression.