Abstract
Background: Although metabolic changes occur during activation of cardiac fibroblasts, it remains unclear how different metabolic pathways influence fibroblast phenotype and function. Substrates used for biosynthetic pathways could be particularly important to the fibrotic response because activated fibroblasts require higher anabolic output to provide biomolecular building blocks for cell division and ECM secretion. Hence, elucidating how metabolism influences fibroblast differentiation and activation could provide valuable information for understanding mechanisms of fibrosis. Hypothesis: We hypothesized that the availability of specific metabolic substrates regulate myofibroblast differentiation. Methods and Results: Naïve cardiac fibroblasts were isolated from wildtype, male C57BL/6J mice and treated with TGFβ or bFGF for 48 h. To examine metabolic genes that respond to profibrotic stimuli, we utilized RNA sequencing, which indicated that TGFβ significantly increases transcription of numerous glycolytic and glutamine metabolism genes as well as transporters for branched-chain amino acids (BCAAs), glutamine, and lactate. Guided by these results, we used an αSMA-reporter assay to examine how lactate, glutamine and BCAA availability influence TGFβ-induced myofibroblast differentiation. Fibroblasts were treated with deficient media supplemented with increasing concentrations of substrate and challenged with TGFβ for 48 hours, followed by assessment of αSMA positivity. In a dose-dependent manner, fibroblasts treated with lactate exhibited decreased αSMA reporter activity (EC50=6.17mM). Furthermore, extracellular flux analysis revealed that the presence of lactate (1mM–10mM) significantly reduced basal oxygen consumption rate and extracellular acidification rate in TGFβ-treated cells (n=2, p<0.05). In contrast, αSMA expression was blunted with diminishing concentrations of glutamine (n=3, p<0.05) and BCAAs, indicating suffcient supply of these substrates is essential for fibroblast differentiation. In addition, immunoblotting data of TGFβ-treated fibroblasts revealed that low extracellular BCAA levels (0 or 100μM) decreased abundance of col1a1 and periostin compared with treatment with BCAAs within the physiological range (400μM; n=4, p<0.05). Conclusion: These preliminary findings provide insight into how specific alterations of glycolysis, glutaminolysis, and BCAA metabolism modulate fibroblast activation in vitro. Additional studies that include alterations to other critical substrates (glucose, fatty acids, ketone bodies, etc.) and enzymes of ancillary biosynthetic pathways of glucose metabolism could provide additional insights into how metabolism regulates fibroblast function. Ultimately, the goal of these studies is to discover potential genetic and pharmacological interventions that positively influence wound healing in the infarcted heart and limit the burden of cardiac fibrosis. NIH and AHA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Published Version
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