Nitric Oxide Attenuates TGF-β-Induced Myofibroblast Differentiation Through Reversible Mitochondrial Bioenergetic Reprogramming Despite Compensatory Glycolytic Activation

Fibroproliferative disorders such as hypertrophic scars and sclerotic skin diseases are characterized by excessive fibroblast activation and differentiation into contractile myofibroblasts. Transforming growth factor-β (TGF-β) is a central driver of this process, promoting α-smooth muscle actin (α-SMA) expression, extracellular matrix production, and increased cellular energy demand. Nitric oxide (NO) is a key regulator of mitochondrial function and cellular bioenergetics and has been reported to exert both pro- and anti-fibrotic effects depending on the cellular context. However, the mechanisms linking NO-dependent metabolic regulation to fibroblast differentiation remain incompletely understood. Here, we demonstrate that exogenous NO, delivered by the long-acting donor DETA/NO, potently suppresses TGF-β-induced myofibroblast differentiation in primary human dermal fibroblasts. DETA/NO markedly reduced α-SMA, EDA-fibronectin, and collagen I expression while simultaneously inhibiting mitochondrial respiration, ATP-linked oxidative phosphorylation, and maximal respiratory capacity. Although NO elicited a pronounced compensatory increase in glycolytic ATP production, this response failed to preserve total cellular ATP availability, resulting in a substantial reduction in overall ATP production. Importantly, these metabolic alterations were fully reversible following removal of the NO donor, indicating transient metabolic regulation rather than irreversible cellular injury. Notably, enhanced glycolytic ATP production occurred despite profound inhibition of myofibroblast differentiation, demonstrating that increased glycolytic ATP production alone is insufficient to sustain the fibrogenic phenotype when cellular ATP availability is compromised. Inhibition of soluble guanylate cyclase by ODQ failed to reverse the anti-differentiative effects of NO, indicating that canonical NO–cGMP signaling is not required for this response. Conversely, inhibition of mitochondrial ATP synthesis by oligomycin phenocopied both the metabolic and anti-differentiative effects of NO. Collectively, our findings identify NO as a reversible regulator of mitochondrial bioenergetics and fibroblast activation and demonstrate that maintenance of cellular ATP homeostasis, rather than enhanced glycolytic ATP production per se, is a critical metabolic requirement for efficient myofibroblast differentiation. Targeting cellular bioenergetics may therefore represent a promising strategy for preventing pathological fibrosis and hypertrophic scar formation.