Effects of elastase-induced emphysema on muscle and bone in mice

Abstract

Chronic obstructive pulmonary disease (COPD) causes sarcopenia and osteoporosis. However, the mechanisms underlying muscle and bone loss as well as the interactions between muscle and bone in the COPD state remain unclear. Therefore, we herein investigated the effects of the COPD state on muscle and bone in mice intratracheally administered porcine pancreatic elastase (PPE). The intratracheal administration of PPE to mice significantly reduced trabecular bone mineral density (BMD), trabecular bone volume, trabecular number, cortical BMD and cortical area. It also significantly decreased grip strength, but did not affect muscle mass or the expression of myogenic differentiation-, protein degradation- or autophagy-related genes in the soleus and gastrocnemius muscles. Among the myokines examined, myostatin mRNA levels in the soleus muscles were significantly elevated in mice treated with PPE, and negatively related to grip strength, but not bone parameters, in mice treated with or without 2 U PPE in simple regression analyses. Grip strength positively related to bone parameters in mice treated with or without PPE. In conclusion, we showed that a PPE model of COPD in mice exerts dominant effects on bone rather than skeletal muscles. Increased myostatin expression in the soleus muscles of mice in the COPD state may negatively relate to a reduction in grip strength, but not bone loss.

Fig 1. Effects of the intratracheal administration of PPE in mice.

(A) Body weight of and food intake by control mice and mice treated with PPE. Body weight was measured 8 weeks after the intratracheal administration of saline or PPE (0.1 to 2 U). Food intake was measured for 3 days on days 54 to 56 after the intratracheal administration of saline or PPE and shown as a representative of average daily food intake. Fat mass in the whole body was assessed by QCT 8 weeks after the intratracheal administration of saline or PPE. The tissue weight of epididymal WAT was measured 8 weeks after the intratracheal administration of saline or PPE. (B, D) Representative images of lung sections stained with hematoxylin/eosin 8 weeks after the intratracheal administration of saline or PPE. Scale bars indicate 100 μm. (C, E) Mean linear intercepts of hematoxylin/eosin-stained lung sections were measured 8 weeks after the intratracheal administration of saline or PPE. Data represent the mean ± SEM. n = 8 (Control), 11 (0.1 and 0.5 U) and 7 (2 U) mice (A, C, E). **p <0.01 and *p <0.05. Cont; Control.

Fig 2. Effects of the intratracheal administration of PPE on bone microstructural parameters measured with μCT.

(A) BV/TV, trabecular BMD (TbBMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and connectivity density (Conn.D) at the distal femurs of mice were assessed by μCT 8 weeks after the intratracheal administration of saline or PPE (0.1 to 2 U). (B) Cortical BMD (CtBMD), cortical thickness (Ct.Th) and cortical area (Ct.Ar) at the femurs of mice were assessed by μCT 8 weeks after the intratracheal administration of saline or PPE. n = 8 (Control), 11 (0.1 and 0.5 U) and 7 (2 U) mice. Data represent the mean ± SEM. **p <0.01 and *p <0.05.

Fig 3. Effects of the intratracheal administration of PPE on muscle strength and muscle mass.

(A) The grip strength of the four limbs in mice was measured by a grip strength meter 8 weeks after the intratracheal administration of saline or PPE (0.1 to 2 U). (B) Muscle mass in the whole body and in the lower limbs was assessed by QCT 8 weeks after the intratracheal administration of saline or PPE. (C) The tissue weights of the soleus or gastrocnemius muscles were measured in mice 8 weeks after the intratracheal administration of saline or PPE (2 U). Data represent the mean ± SEM. n = 8 (Control), 11 (0.1 and 0.5 U) and 7 (2 U) mice. **p <0.01 and *p <0.05.

Fig 4. Effects of the intratracheal administration of PPE on muscle-related parameters in mice.

Total RNA was extracted from the soleus (A) and gastrocnemius (B) muscles of mice 8 weeks after the intratracheal administration of saline or PPE (2U). A real-time PCR analysis of MyoD, myogenin, MHC-I, MHC-IIb, atrogin-1, MuRF1, Beclin-1, LC3B, Gabarapl and 18S rRNA was performed. n = 8 (Control), 11 (0.1 and 0.5 U) and 7 (2 U) mice. Data are expressed as relative values to 18S rRNA levels and represent the mean ± SEM. *p < 0.05.

Fig 5. Effects of the intratracheal administration of PPE on myokines in muscles.

Total RNA was extracted from the soleus (A) and gastrocnemius (B) muscles of mice 8 weeks after the intratracheal administration of saline or PPE (2 U). A real-time PCR analysis of myostatin, TGF-β, follistatin, Fndc5, IGF-1, FGF2, IL-6, osteoglycin, OLFM1 and 18S rRNA was performed. Data are expressed as relative values to 18S rRNA levels and represent the mean ± SEM. n = 8 (Control), 11 (0.1 and 0.5 U) and 7 (2 U) mice. *p <0.05.

Fig 6. Effects of the intratracheal administration of PPE on TNF-α.

(A) Total RNA was extracted from the gastrocnemius and soleus muscles of mice 8 weeks after the intratracheal administration of saline or PPE (0.1 to 2 U). A real-time PCR analysis of TNF-α and 18S rRNA was performed. Data are expressed as relative values to 18S rRNA levels. (B) Serum samples were collected from mice 8 weeks after the intratracheal administration of saline or PPE (0.1 to 2 U). Serum TNF-α levels were analyzed. Data represent the mean ± SEM. n = 8 (Control), 11 (0.1 and 0.5 U) and 7 (2 U) mice. *p <0.05.