Respiratory defects in the Crtap KO mouse model of osteogenesis imperfecta

Abstract

Respiratory disease is a leading cause of mortality in patients with osteogenesis imperfecta (OI), a connective tissue disease that causes severely reduced bone mass and is most commonly caused by dominant mutations in type I collagen genes. Previous studies proposed that impaired respiratory function in OI patients was secondary to skeletal deformities; however, recent evidence suggests the existence of a primary lung defect. Here, we analyzed the lung phenotype of Crtap knockout (KO) mice, a mouse model of recessive OI. While we confirm changes in the lung parenchyma that are reminiscent of emphysema, we show that CrtapKO lung fibroblasts synthesize type I collagen with altered posttranslation modifications consistent with those observed in bone and skin. Unrestrained whole body plethysmography showed a significant decrease in expiratory time, resulting in an increased ratio of inspiratory time over expiratory time and a concomitant increase of the inspiratory duty cycle in CrtapKO compared with WT mice. Closed-chest measurements using the forced oscillation technique showed increased respiratory system elastance, decreased respiratory system compliance, and increased tissue damping and elasticity in CrtapKO mice compared with WT. Pressure-volume curves showed significant differences in lung volumes and in the shape of the curves between CrtapKO mice and WT mice, with and without adjustment for body weight. This is the first evidence that collagen defects in OI cause primary changes in lung parenchyma and several respiratory parameters and thus negatively impact lung function.

Keywords: Crtap; collagen; lung; osteogenesis imperfecta; respiratory function.

Fig. 1.

Crtap expression and postnatal lung histology. A: Western blot analysis of wild-type (WT) whole lung lysates at different ages probed with a rabbit polyclonal antibody against CRTAP. A lysate of a 1-mo-old CrtapKO [knockout (KO)] lung was also included as the negative control. B: densitometry of the band products shown in A expressed as a ratio between Crtap and β-actin expression levels. NB, newborn; P5, 5 days old; P10, 10 days old; m, age in months. Results are representative from n = 1 sample/age and three technical replicates. C: Western blot showing CRTAP expression in adult human lung tissue. D, E, and G: histological sections of WT and CrtapKO lungs collected at P5, 3 mo, and 8–9 mo of age stained with hematoxylin and eosin (H&E) or Masson’s. F: quantification of the lung parenchyma defect at 3 mo and at 7–11 mo of age (n = 5 or 6 and n = 4 or 5 mice/genotype, respectively; 10 fields analyzed/mouse at ×20 magnification; Student’s t test) using the mean linear intercept (L_m) method.

Fig. 2.

Histochemical staining of elastin and collagen fibers in wild-type (WT) and Crtap knockout (KO) lung sections. Representative images of WT (top) and CrtapKO (bottom) lung sections at 3 mo of age, stained with the Verhoeff-Van Gieson protocol. Elastic fibers are stained blue-black (black arrows) while collagen fibers are stained in red (light blue arrows). Images at ×40 are magnifications corresponding to the red boxed areas.

Fig. 3.

Collagen mass spectrometry from wild-type (WT) and Crtap knockout (KO) lung fibroblasts. A: Western blot of primary lung fibroblast lysates from CrtapKO and WT mice and probed with antibody against Crtap. B: electrophoretic migration of collagen extracted from media of WT and CrtapKO lung primary fibroblasts (n = 3). The gel was stained with Coomassie blue and shows electrophoretic migration delay of α1(I) and α2(I) chains in CrtapKO samples, characteristic of collagen overmodification. +C, positive control, is collagen extracted from rat tail (1 µg/µL). C–E: mass-spectrometry results showing loss of type I collagen 3Hyp at Pro¹¹⁵³ (previously referred to as Pro⁹⁸⁶ for processed Col1a1) in CrtapKO versus WT fibroblasts (C). D: a volcano plot showing the Linear Models for Microarray Data –log10 FDR adjusted P values and log2-fold changes for all peptides with a 3Hyp modification on prolines that were identified by mass spectrometry. E: a hierarchical cluster heat map displaying the scaled normalized peptide intensity values for each sample and significantly modified peptide. Peptides were considered significant with a fold change >2 and an FDR adjusted P value <0.05. These peptides are also visualized on the volcano plot. The sequence contains the position of the modified amino acid within the protein ± 7 amino acids. The proline in red and purple indicates the modified proline that is either over- or undermodified in WT versus CrtapKO. The numbering of amino acids starts from the first amino acid in the protein sequence.

Fig. 4.

Results derived from the Snapshot 150 and the Quick prime-3 maneuvers using the forced oscillation technique (FOT). A: respiratory system elastance (E_rs) and compliance (C_rs) were significantly different (increased and decreased, respectively), while overall resistance (R_rs) of the respiratory system (i.e., including lungs, airways, and chest wall) did not reach statistical significance. B: tissue damping (G) and tissue elasticity (H) were both significantly increased; however, airways resistance (i) and tissue hysteresivity (G/H) were not changed in Crtap knockout (KO) compared with wild-type (WT)/Het mice. N = 8–10 each genotype. Student’s t test.

Fig. 5.

Results derived from deep inflation and pressure-volume (P-V) maneuvers. A: partial P-V loops and the related parameters that were derived from these curves (C). C_st, static compliance; A, an estimate of inspiratory capacity; k, shape constant that describes the curvature of the deflation arm; area, area between the inflation and deflation curves. B: inspiratory capacity as measured with the deep inflation maneuvers. D: full-range P-V loops and the derived parameters, including total lung capacity (TLC), vital capacity (VC), residual volume (RV), compliance (C), volume at 10 cmH₂O as percentage of TLC (V10_TLC), and an estimate of functional residual capacity (FRC). All parameters were significantly decreased in Crtap knockout (KO) compared with wild-type (WT)/Het mice. N = 8–10 each genotype. Student’s t test.