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Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation.

Lei Li1, Feng Jiang1, Wenyan Hao2

  • 1Department of Critical Care Medicine, Heping Hospital Affiliated to Changzhi Medical College, 110 South Yan'an Road, Luzhou District, Changzhi City, 046012, China.

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Mechanical ventilation causes diaphragm dysfunction by increasing fibroblast proliferation and immune cell infiltration, leading to fibrosis. Key genes like Pdgfd and Cxcr2 are implicated in this early-stage damage.

Keywords:
Diaphragm fibrosisEndothelial-mesenchymal transitionFibro-adipogenic progenitorHigh dimensional weighted gene coexpression network analysisSingle-nucleus RNA sequencing

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Area of Science:

  • Biomedical research
  • Cellular and molecular biology
  • Respiratory medicine

Background:

  • Mechanical ventilation can lead to ventilator-induced diaphragmatic dysfunction (VIDD), characterized by diaphragm atrophy and weakness.
  • The precise mechanisms driving VIDD, particularly diaphragm fibrosis, remain incompletely understood.

Purpose of the Study:

  • To investigate the impact of 24-hour mechanical ventilation on diaphragm cellular changes, including fibro-adipogenic progenitor (FAP) proliferation, endothelial-mesenchymal transition (EndMT), and immune cell infiltration.
  • To identify key genes and pathways involved in the early stages of diaphragm fibrosis induced by mechanical ventilation using single-nucleus RNA sequencing.

Main Methods:

  • A rabbit model was used, with diaphragm tissue analyzed via 10X Genomics single-nucleus RNA sequencing (snRNA-seq).
  • Bioinformatic analyses included differential gene expression (DEGs), Kyoto Encyclopedia of Genes and Genomes (KEGG), pseudotime, and high-dimensional weighted gene coexpression network analysis (hdWGCNA).
  • Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to validate snRNA-seq findings.

Main Results:

  • Mechanical ventilation increased the proportion of fibroblasts and decreased myofibers in the diaphragm.
  • Key fibrotic processes identified were FAP proliferation, EndMT, and immune cell infiltration, regulated by genes such as Pdgfd, Sema3a, and Cxcr2.
  • Glycolysis (Pfkfb3) and genes like Negr1 and Mef2c were also identified as important factors in fibrosis, nerve ending loss, and atrophy.

Conclusions:

  • Acute mechanical ventilation promotes cellular changes in the diaphragm that contribute to fibrosis.
  • FAP proliferation and immune cell infiltration are suggested as crucial early mechanisms in ventilator-induced diaphragm fibrosis.
  • Further research is necessary to confirm these findings and elucidate the underlying mechanisms of VIDD.