Phenotypic and metabolic features of mouse diaphragm and gastrocnemius muscles in chronic lung carcinogenesis: influence of underlying emphysema

Abstract

Background: Muscle wasting negatively impacts the progress of chronic diseases such as lung cancer (LC) and emphysema, which are in turn interrelated.

Objectives: We hypothesized that muscle atrophy and body weight loss may develop in an experimental mouse model of lung carcinogenesis, that the profile of alterations in muscle fiber phenotype (fiber type composition and morphometry, muscle structural alterations, and nuclear apoptosis), and in muscle metabolism are similar in both respiratory and limb muscles of the tumor-bearing mice, and that the presence of underlying emphysema may influence those events.

Methods: Diaphragm and gastrocnemius muscles of mice with urethane-induced lung cancer (LC-U) with and without elastase-induced emphysema (E-U) and non-exposed controls (N = 8/group) were studied: fiber type composition, morphometry, muscle abnormalities, apoptotic nuclei (immunohistochemistry), and proteolytic and autophagy markers (immunoblotting) at 20- and 35-week exposure times. In the latter cohort, structural contractile proteins, creatine kinase (CK), peroxisome proliferator-activated receptor (PPAR) expression, oxidative stress, and inflammation were also measured. Body and muscle weights were quantified (baseline, during follow-up, and sacrifice).

Results: Compared to controls, in U and E-U mice, whole body, diaphragm and gastrocnemius weights were reduced. Additionally, both in diaphragm and gastrocnemius, muscle fiber cross-sectional areas were smaller, structural abnormalities, autophagy and apoptotic nuclei were increased, while levels of actin, myosin, CK, PPARs, and antioxidants were decreased, and muscle proteolytic markers did not vary among groups.

Conclusions: In this model of lung carcinogenesis with and without emphysema, reduced body weight gain and muscle atrophy were observed in respiratory and limb muscles of mice after 20- and 35-week exposure times most likely through increased nuclear apoptosis and autophagy. Underlying emphysema induced a larger reduction in the size of slow- and fast-twitch fibers in the diaphragm of U and E-U mice probably as a result of the greater inspiratory burden imposed onto this muscle.

Keywords: Lung carcinogenesis-induced muscle mass loss; Muscle apoptotic nuclei; Muscle-specific proteins; PPARs proteolytic and autophagy markers; Respiratory and limb muscle atrophy and structural abnormalities; Underlying emphysema.

Fig. 1

a Graphical time-line representation of the elastase–urethane group of mice. b Graphical time-line representation of the urethane group of animals. c Graphical time-line representation of the control group of rodents

Fig. 2

a Graphical representation of the progression of body weight in mice from all experimental groups over the study period in the 20-week time-cohort. The following signs have been used in each group: control (diamonds), U group (squares), and E–U group (triangles). b Graphical representation of the progression of body weight in mice from all experimental groups over the study period in the 35-week time-cohort. The following signs have been used in each group: control (diamonds), U group (squares), and E–U group (triangles). E–U elastase–urethane, U urethane

Fig. 3

Representative images of micro-CT scan analyses in all study groups in the 20- (left panel) and 35-week (right panel) cohorts of mice. Lung nodules (orange triangles) are shown in micro-CT slices of lung parenchyma for U (middle panel) and E–U (bottom panel) groups of mice. E–U elastase–urethane, U urethane

Fig. 4

Representative examples of histological sections stained with hematoxylin and eosin (×5 and ×20) of whole individual tumors in U (left panels) and E–U (right panels) groups of mice. E–U elastase–urethane, U urethane

Fig. 5

a Representative examples of stained muscle fibers in diaphragm and gastrocnemius muscles of control (top panel), U group (middle panel) and E–U group (bottom panel) in the 20-week cohort of animals. Myofibers positively stained with the anti-MyHC type II antibody appear in brown color (calibration bar 200 μm). Type I fibers appear in white color (not stained). b Representative examples of stained muscle fibers in diaphragm and gastrocnemius muscle of control (top panel) and U group (middle panel) and E–U group (bottom panel) in the 35-week cohort of animals. Myofibers positively stained with the anti-MyHC type II antibody appear in brown color (calibration bar 200 μm). Type I fibers appear in white color (not stained). anti-MyHC anti-myosin heavy chain, E–U elastase–urethane, U urethane

Fig. 6

Schematic representation of the distribution of the cross-sectional areas of the muscle fibers in the diaphragm and gastrocnemius muscles in both 20- and 35-week time cohorts of animals (a–d, respectively). Values of cross-sectional areas in the control mice are represented in light grey bars, while those detected in U and E–U animals are represented in dark grey and black respectively in all the histograms (a–d)

Fig. 7

a Mean values and standard deviation of the percentage of abnormal fraction (white bars), cellular inflammation (grey bars) and internal nuclei (black bars) in the diaphragm muscle of the 20-week cohort animals. Statistical significance is represented as follows: *p ≤ 0.05 and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. b Mean values and standard deviation of the percentage of abnormal fraction (white bars), cellular inflammation (grey bars) and internal nuclei (black bars) in the gastrocnemius muscle of 20-week cohort animals. Statistical significance is represented as follows: n.s.: non-significant and *p ≤ 0.05 between any of the intervention groups (U and E–U) and control mice. c Representative examples of muscle structural abnormalities in diaphragm of 20-week cohort animals (calibration bar 100 μm). A representative image of muscles in U group (right top image), in which black arrows point towards inflammatory cells, and a representative image of muscles in E–U group (right bottom image), in which black arrows point towards inflammatory cells and internal nuclei. d Representative examples of muscle structural abnormalities in gastrocnemius of 20-week cohort animals (calibration bar 100 μm). A representative image of muscles in U group (right top image), in which black arrows point towards inflammatory cells, and a representative image of E–U group (right bottom image), in which black arrows point towards internal nuclei. E–U elastase–urethane, U urethane

Fig. 8

a Mean values and standard deviation of the percentage of abnormal fraction (white bars), cellular inflammation (grey bars) and internal nuclei (black bars) in the diaphragm muscle of 35-week cohort animals. Statistical significance is represented as follows: **p ≤ 0.01 and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. b Mean values and standard deviation of the percentage of abnormal fraction (white bars), cellular inflammation (grey bars) and internal nuclei (black bars) in the gastrocnemius muscle of 35-week cohort. Statistical significance is represented as follows: *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. c Representative examples of muscle structural abnormalities in diaphragm of 35-week cohort animals (calibration bar 100 μm). A representative image of muscles in U group (right top image), in which black arrows point towards internal nuclei, and a representative image of E–U group (right bottom image), in which black arrows point towards inflammatory cells and internal nuclei. d Representative examples of muscle structural abnormalities in gastrocnemius of 35-week cohort animals (calibration bar 100 μm). A representative image of muscles in U group (right top image), in which black arrows point towards internal nuclei and a representative image of E–U group (right bottom image), in which black arrows point towards inflammatory cells and internal nuclei. E–U elastase–urethane, U urethane

Fig. 9

a Mean values and standard deviation of the percentage of positively stained nuclei for the TUNEL assay in diaphragm (white bars) and gastrocnemius (black bars) muscles of the 20-week cohort of animals. Statistical significance is represented as follows: **p ≤ 0.01 and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. b Representative examples of positively nuclei (red arrows) stained for the TUNEL assay in the diaphragm (top panel) and gastrocnemius (bottom panel) muscles of the 20-week cohort of the different study groups of mice (calibration bar 100 μm)

Fig. 10

a Mean values and standard deviation of the percentage of positively stained nuclei for the TUNEL assay in diaphragm (white bars) and gastrocnemius (black bars) muscles of the 35-week cohort of animals. Statistical significance is represented as follows: ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. b Representative examples of positively nuclei (red arrows) stained for the TUNEL assay in the diaphragm (top panel) and gastrocnemius (bottom panel) muscles of the 35-week cohort of the different study groups of mice (calibration bar 100 μm). E–U elastase–urethane, U urethane

Fig. 11

a Mean values and standard deviation of skeletal muscle actin, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. b Mean values and standard deviation of MyHC, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant, **p ≤ 0.01, and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. c Mean values and standard deviation of creatine kinase (CK), in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: *p ≤ 0.05 and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. d Mean values and standard deviation of carbonic anhydrase-3, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. a.u. arbitrary units, E–U elastase–urethane, MyHC myosin heavy chain, OD optical densities, U urethane

Fig. 12

a Mean values and standard deviation of myogenin, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. b Mean values and standard deviation of PPAR-α, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant, *p ≤ 0.05 and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. c Mean values and standard deviation of PPAR-gamma, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: *p ≤ 0.05 and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. d Mean values and standard deviation of PGC-1α, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant between any of the intervention groups (U and E–U) and control mice. a.u. arbitrary units, E–U elastase–urethane, OD optical densities, PPAR- peroxisome proliferator-activated receptor alpha; PPAR- peroxisome proliferator-activated receptor gamma, PGC-1 peroxisome proliferator-activated receptor gamma coactivator-1α, U urethane

Fig. 13

a Mean values and standard deviation of MDA, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: (i) n.s.: non-significant and **p ≤ 0.01 between any of the intervention groups (U and E–U) and control mice. b Mean values and standard deviation of SOD1, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. c Mean values and standard deviation of SOD2, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant, **p ≤ 0.01 and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. d Mean values and standard deviation of catalase, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. a.u. arbitrary units, E–U elastase–urethane, MDA malondialdehyde, OD optical densities, SOD1 superoxide dismutase isoform 1, SOD2 superoxide dismutase isoform 2, U urethane

Fig. 14

a Mean values and standard deviation of TNF-α in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by picogram per milliliter (pg/mL). Statistical significance is represented as follows: n.s.: non-significant and *p ≤ 0.05 between any of the intervention groups (U and E–U) and control mice. b Mean values and standard deviation of IL-6 in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by picogram per milliliter (pg/mL). Statistical significance is represented as follows: n.s.: non-significant and ***p ≤ 0.001 between any of the intervention groups (U and E–U) and control mice. E–U elastase–urethane, IL-6 interleukin-6, TNF- tumor necrosis factor-alpha, U urethane

Fig. 15

a Mean values and standard deviation of total protein ubiquitination in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: (i) n.s.: non-significant between any of the intervention groups (U and E–U) and control mice. b Mean values and standard deviation of E2_14K, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant any of the intervention groups (U and E–U) and control mice. c Mean values and standard deviation of MURF-1, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant between any of the intervention groups (U and E–U) and control mice. d Mean values and standard deviation of calpain-1, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant between any of the intervention groups (U and E–U) and control mice. a.u. arbitrary units, E–U elastase–urethane, E2 _14k ubiquitin-conjugating enzyme E2 (14k), MURF-1 muscle ring finger protein 1, OD optical densities, U urethane

Fig. 16

a Mean values and standard deviation of p62, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: (i) n.s.: non-significant and *p ≤ 0.05 between any of the intervention groups (U and E–U) and control mice. b Mean values and standard deviation of Beclin-1, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: n.s.: non-significant and **p ≤ 0.01 between any of the intervention groups (U and E–U) and control mice. c Mean values and standard deviation of LC3 II/LC3 I levels, in diaphragm (white bars) and gastrocnemius (black bars) muscles as measured by optical densities. Statistical significance is represented as follows: *p ≤ 0.05 between any of the intervention groups (U and E–U) and control mice. a.u. arbitrary units, E–U elastase–urethane, LC3 light chain 3, OD optical densities, p62 nucleoporin p62, U urethane