Mechanical ventilation causes diaphragm dysfunction in newborn lambs

Abstract

Background: Diaphragm weakness occurs rapidly in adult animals treated with mechanical ventilation (MV), but the effects of MV on the neonatal diaphragm have not been determined. Furthermore, it is unknown whether co-existent lung disease exacerbates ventilator-induced diaphragmatic dysfunction (VIDD). We investigated the impact of MV (mean duration = 7.65 h), either with or without co-existent respiratory failure caused by surfactant deficiency, on the development of VIDD in newborn lambs.

Methods: Newborn lambs (1-4 days) were assigned to control (CTL, non-ventilated), mechanically ventilated (MV), and MV + experimentally induced surfactant deficiency (MV+SD) groups. Immunoblotting and quantitative PCR assessed inflammatory signaling, the ubiquitin-proteasome system, autophagy, and oxidative stress. Immunostaining for myosin heavy chain (MyHC) isoforms and quantitative morphometry evaluated diaphragm atrophy. Contractile function of the diaphragm was determined in isolated myofibrils ex vivo.

Results: Equal decreases (25-30%) in myofibrillar force generation were found in MV and MV+SD diaphragms compared to CTL. In comparison to CTL, both MV and MV+SD diaphragms also demonstrated increased STAT3 transcription factor phosphorylation. Ubiquitin-proteasome system (Atrogin1 and MuRF1) transcripts and autophagy indices (Gabarapl1 transcripts and the ratio of LC3B-II/LC3B-I protein) were greater in MV+SD relative to MV alone, but fiber type atrophy was not observed in any group. Protein carbonylation and 4-hydroxynonenal levels (indices of oxidative stress) also did not differ among groups.

Conclusions: In newborn lambs undergoing controlled MV, there is a rapid onset of diaphragm dysfunction consistent with VIDD. Superimposed lung injury caused by surfactant deficiency did not influence the severity of early diaphragm weakness.

Keywords: Lung injury; Mechanical ventilation; Neonatal; Surfactant deficiency; Ventilator-induced diaphragmatic dysfunction (VIDD).

Fig. 1

Inflammatory signaling in mechanically ventilated newborn diaphragms. a Representative immunoblot images of phosphorylated and total STAT3 protein in the diaphragm. b Group mean ratio of phosphorylated to total STAT3 protein in the diaphragm, expressed as fold change relative to CTL. c Group mean transcript levels of IL-6, depicted as fold change relative to CTL group values. *p < 0.05 for CTL (n = 5) versus MV (n = 6) or MV+SD (n = 6)

Fig. 2

Proteolysis pathways in mechanically ventilated newborn diaphragms. a Transcript levels of muscle-specific E3 ubiquitin ligases (Atrogin1, MuRF1) and SIRT1 in the diaphragm. b Transcript levels of autophagy genes LC3B and Gabarapl1, expressed as fold change relative to CTL values. c Representative immunoblot and group mean values for LC3B-I and LC3B-II protein in the diaphragm. *p < 0.05 versus CTL; ^#p < 0.05 for MV versus MV+SD

Fig. 3

Indices of oxidative stress in mechanically ventilated newborn diaphragms. a Representative immunoblot images of carbonyl groups and 4-hydroxynonenal (4-HNE) in diaphragm proteins extracted from CTL, MV, and MV+SD groups. Group mean quantification of b carbonyls and c 4-HNE in the diaphragm under the above experimental conditions

Fig. 4

Diaphragm muscle fiber phenotype in mechanically ventilated newborn lambs. a Transcript levels of MyHC isoforms in the diaphragm. b Representative immunohistochemical staining for slow type 1 (blue) and fast type 2a (green) MyHC isoforms. c Group mean diaphragm muscle fiber size (Feret’s minimal diameter) for type 1 and type 2a fibers in the three experimental groups. *p < 0.05 versus CTL; ^#p < 0.05 for MV versus MV+SD

Fig. 5

Diaphragm muscle fiber function in mechanically ventilated newborn lambs. a Representative examples of plots of absolute isometric force generation after exposure to calcium by diaphragm myofibrils isolated from CTL, MV, and MV+SD lambs. b Group mean maximal isometric specific force (normalized to cross-sectional area). c Group mean rate of force development (Kact). d Group mean rate of force redevelopment after acute shortening (Ktr). e Group mean rate of relaxation (Krel). *p < 0.05 for CTL (n = 5) versus MV (n = 4) or MV+SD (n = 6)