Phasic metabolic remodeling coordinates cardiac myofibroblast differentiation and function

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 (Glyco^Hi) 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.