Redox Biol. 2026 Oct 16. pii: S2213-2317(26)00440-4. [Epub ahead of print]98
104441
Fibroblast differentiation into matrix-producing, contractile myofibroblasts is accompanied by metabolic reprogramming, but whether these changes precede differentiation or arise from the activated phenotype remains unclear. We integrated time-resolved extracellular flux analysis, transcriptomics, stable isotope-resolved metabolomics, and metabolic interventions to define the relationship between metabolism and myofibroblast differentiation in adult mouse cardiac fibroblasts stimulated with transforming growth factor-β (TGFβ). TGFβ increased glycolysis within 1.5 h, before broad transcriptional remodeling and induction of periostin and α-smooth muscle actin. Mitochondrial respiration initially declined but increased at 48 h as the phenotype emerged, accompanied by smaller, less elongated mitochondria and greater respiratory capacity. [U-13C6]-glucose tracing revealed progressive incorporation of glucose-derived carbon into glycolytic intermediates, nucleotide sugars, and glycerol 3-phosphate, followed later by increased labeling of tricarboxylic acid cycle intermediates. TGFβ-treated fibroblasts also demonstrated enhanced insulin-stimulated glycolysis. Replacing glucose with galactose attenuated collagen and periostin induction, whereas glyceraldehyde 3-phosphate dehydrogenase inhibition prevented induction of collagen, periostin, and α-smooth muscle actin. Conversely, glucose transporter 1 or mutant PFK2 (GlycoHi) overexpression increased glycolysis without inducing myofibroblast markers. Together, these loss- and gain-of-function studies indicate that glycolytic activity is required for complete differentiation but is not sufficient to initiate the phenotype. Inhibition of mitochondrial Complex I or ATP synthase preferentially reduced collagen abundance, while glycolytic and Complex I inhibition diminished TGFβ-induced collagen gel contraction. These findings define a phasic metabolic program in which early glycolytic activation precedes transcriptional and phenotypic remodeling, whereas mitochondrial respiratory capacity increases later and distinct metabolic nodes selectively regulate collagen abundance and contractile behavior.
Keywords: Biosynthesis; Fibroblast; Fibrosis; Glycolysis; Mitochondria; Stable isotope