Diaphragm muscle function in a mouse model of early-onset spasticity

Abstract

Spasticity is a common symptom in many developmental motor disorders, including spastic cerebral palsy (sCP). In sCP, respiratory dysfunction is a major contributor to morbidity and mortality, yet it is unknown how spasticity influences respiratory physiology or diaphragm muscle (DIAm) function. To investigate the influence of spasticity on DIAm function, we assessed in vivo transdiaphragmatic pressure (Pdi - measured using intraesophageal and intragastric pressure catheters under conditions of eupnea, hypoxia/hypercapnia and occlusion) including maximum Pdi (Pdi_max via bilateral phrenic nerve stimulation), ex vivo DIAm-specific force and fatigue (using muscle strips stimulated with platinum plate electrodes), and type-specific characteristics of DIAm fiber cross sections (using immunoreactivity against myosin heavy chain slow and 2A) in spa and wildtype mice. Spa mice show reduced Pdi_max, reduced DIAm specific force, and altered fatigability and atrophy of type IIx/IIb fibers. These findings suggest marked DIAm dysfunction may underlie the respiratory phenotype of sCP.NEW & NOTEWORTHY Developmental motor control dysfunctions, including spastic cerebral palsy (sCP) often have respiratory components. Spa mutant mice exhibit a spastic phenotype closely resembling sCP symptoms. Using the spa mouse model of spastic cerebral palsy (sCP), we quantified transdiaphragmatic pressure deficits, diaphragm muscle weakness, and fiber type-specific atrophy, improving our understanding of respiratory dysfunctions in sCP.

Keywords: fatigue; muscle fiber type; muscle-specific force; spasticity; transdiaphragmatic pressure.

Graphical abstract

Figure 1.

A: representative Pdi measures (cmH₂O) of inspiratory-related (eupnea, hypoxia/hypercapnia, and occlusion) and maximal (Pdi_max) behaviors in WT (top row) and spa (bottom row) mice. B: bar plots with individual data points for spa and WT mice (means ± 95% CI) of Pdi across behavior. In spa mice compared with WT, inspiratory-related activities were unchanged. However, a marked reduction in Pdi_max is observed in spa compared with WT mice (two-way ANOVA with Bonferroni post test, P < 0.05). In all experiments, n = 6 for WT (3 females and 3 males) and n = 4 for spa (2 females and 2 males), with females indicated by white filled symbols. Pdi, transdiaphragmatic pressure; Pdi_max, maximum transdiaphragmatic pressure; WT, wildtype. *P < 0.05.

Figure 2.

A: representative DIAm maximum specific force (Po) from WT and spa mice. B: bar plots with individual data points for spa and WT mice (means ± 95% CI) show reduced DIAm Po (N/cm²) in spa compared with WT mice (Mann–Whitney U test, P < 0.05). In all experiments, n = 11 for WT (5 females and 6 males) and n = 5 for spa (2 females and 3 males), with females indicated by white filled symbols. DIAm, diaphragm muscle; WT, wildtype. *P < 0.05.

Figure 3.

Relationship between DIAm force and stimulation frequency. Both plots show increasing DIAm specific force with increasing stimulation frequency in WT and spa mice. A: reduced DIAm specific force in spa mice compared with WT from 75 to 125 Hz (two-way ANOVA with Bonferroni post tests, *P < 0.05). B: no difference in DIAm relative force between spa and WT mice. In all experiments, n = 11 for WT (5 females and 6 males) and n = 5 for spa (2 females and 3 males). DIAm, diaphragm muscle; P_o, specific force; WT, wildtype.

Figure 4.

A: representative fatiguing DIAm contractions in WT (top) and spa (bottom) mice. B: progression of fatigue index with time in WT (circles) and spa (squares) shows greater fatigue index in spa mice from 60 s (two-way ANOVA with Bonferroni post tests, *P < 0.05). C: bar plots with individual data points for spa and WT mice (means ± 95% CI) show reduced fatigue index in spa compared with WT mice (Student’s unpaired t test, *P < 0.05). D: progression of DIAm-specific force with time in WT (light gray circles) and spa (dark gray squares) shows initial differences at time 0 s in WT compared with spa mice that disappear with time (two-way ANOVA with Bonferroni post tests, *P < 0.05). E: bar plots with individual data points for spa and WT mice (means ± 95% CI) show unchanged residual DIAm Po (N/cm²) following 120 s of fatiguing contraction in WT and spa mice (Student’s unpaired t test, P = 0.37). In all experiments, n = 11 for WT (5 females and 6 males) and n = 5 for spa (2 females and 3 males), with females indicated by white filled symbols. DIAm, diaphragm muscle; Po, DIAm-specific force; WT, wildtype.

Figure 5.

A: pictomicrographs showing DIAm fiber immunoreactivity to MyHC_slow (green) and MyHC_2A (red) in WT and spa mice. Fiber borders are delineated by immunoreactivity with laminin (blue). B: bar plots with individual data points for WT and spa mice (means ± 95% CI) show unchanged fiber type % in spa compared with WT mice (two-way ANOVA with Bonferroni post test). C: bar plots with individual data points for WT and spa mice (means ± 95% CI) show reduced DIAm fiber cross-sectional area (µm²) of IIx/IIb fibers in spa compared with WT mice (two-way ANOVA with Bonferroni post test, *P < 0.05). D: bar plots with individual data points for WT and spa mice (means ± 95% CI) show increased contributions to total DIAm mass of type IIa fibers and reduced contributions to total DIAm mass of type IIx/IIb fibers in spa compared with WT mice (two-way ANOVA with Bonferroni post test, *P < 0.05). In all experiments, n = 6 for WT (3 females and 3 males) and n = 5 for spa (2 females and 3 males), with females indicated by white filled symbols. Scale bar = 30 µm. DIAm, diaphragm muscle; WT, wildtype.