Single-nucleus RNA sequencing combined with fluorescence in situ hybridization reveals Stat3 as a therapeutic target for ventilator-induced diaphragm dysfunction

snRNA-seq revealed that MV induced significant changes in the cellular composition and transcriptional profiles of diaphragm tissue. Specifically, fibroblasts and type IIb myofibers showed increased proportions, while type IIx myofibers and other cell types, such as fibro/adipogenic progenitors (FAPs), showed a decrease in their representation. Transcriptionally, MV led to the upregulation of key muscle atrophy-related genes such as Atrogin-1 and Murf-1, as well as fibrosis-related genes including collagen and fibronectin. Stat3 emerged as a central player, with its expression significantly elevated in both myofibers and fibroblasts. Further analysis of gene regulatory networks demonstrated that Stat3 activation was associated with disrupted muscle metabolism, increased fibroblast activity, and enhanced fibrotic signaling pathways. FISH confirmed the spatial localization of these transcriptional changes, corroborating the snRNA-seq findings. The increased activity of Stat3, particularly in promoting fibrosis and muscle degradation, suggests that it is a key regulator in the pathogenesis of VIDD. Overall design: To induce anesthesia in mice, a 1% solution of pentobarbital (Sigma-Aldrich, St. Louis, MO) was administered via intraperitoneal injection. After shaving and disinfecting the neck area, a tracheostomy was performed, and the mouse's trachea was connected to a small animal ventilator (VentElite, Harvard Apparatus, USA) for controlled mechanical ventilation. The ventilator settings during MV were as follows: a tidal volume of 10 mL/kg, a respiratory rate (RR) of 150 breaths per minute, and a positive end-expiratory pressure (PEEP) of 2 cm H2O1. Throughout the entire process of MV, mouse body temperature was maintained at approximately 37°C using a mouse-specific temperature maintenance device. Continuous anesthesia was provided to the mice. The control group of mice did not undergo MV.