Diaphragm atrophy and contractile dysfunction in a murine model of pulmonary hypertension

Abstract

Pulmonary hypertension (PH) causes loss of body weight and inspiratory (diaphragm) muscle dysfunction. A model of PH induced by drug (monocrotaline, MCT) has been extensively used in mice to examine the etiology of PH. However, it is unclear if PH induced by MCT in mice reproduces the loss of body weight and diaphragm muscle dysfunction seen in patients. This is a pre-requisite for widespread use of mice to examine mechanisms of cachexia and diaphragm abnormalities in PH. Thus, we measured body and soleus muscle weight, food intake, and diaphragm contractile properties in mice after 6-8 weeks of saline (control) or MCT (600 mg/kg) injections. Body weight progressively decreased in PH mice, while food intake was similar in both groups. PH decreased (P<0.05) diaphragm maximal isometric specific force, maximal shortening velocity, and peak power. Protein carbonyls in whole-diaphragm lysates and the abundance of select myofibrillar proteins were unchanged by PH. Our findings show diaphragm isometric and isotonic contractile abnormalities in a murine model of PH induced by MCT. Overall, the murine model of PH elicited by MCT mimics loss of body weight and diaphragm muscle weakness reported in PH patients.

Figure 1. Progressive decrease in body weight and unchanged food intake in mice treated with monocrotaline to induce pulmonary hypertension (PH).

A) Images of sample mice in each group at the end of study illustrating that PH mice are smaller than controls. B) Body weight expressed as percentage of value at the onset of study. C) Food intake expressed as mg per g body weight per day in each week of the study period. Data are from mice receiving control and monocrotaline injections for 8 weeks (N = 4 for Control; N = 5 for PH). * P<0.05 for control vs. PH.

Figure 2. Diaphragm fiber cross sectional area and isometric force are decreased in mice with pulmonary hypertension (PH).

A) Cross sections taken from diaphragm muscle fiber bundles of sample control and PH mice. B) Diaphragm fiber cross sectional area of 200–300 fibers per muscle, from 3–4 mice per group. C) Specific Force, force normalized to bundle cross-sectional area. Data are from diaphragm bundles of controls and mice receiving monocrotaline for 6–8 weeks to induce PH (N = 10/group). * P<0.05 for PH vs. control.

Figure 3. Pulmonary hypertension impairs isotonic contractile properties of isolated diaphragm bundles.

A) Force-velocity relationship from sample control (open circles) and PH mice (closed circles). Force is relative to maximum isometric tetanic force (F _o). Shortening velocity is normalized to optimal bundle length for twitch force (L₀). Solid and dashed lines are best fit from Hill equation. B) Maximal shortening velocity (V_max) determined from extrapolation of shortening velocity to zero force using Hill equation (see Panel A for example). C) Force-power relationship of sample control and PH mice. Power is calculated as specific force (kN/m²) multiplied to shortening velocity (L₀/s) shown in panel A. D) Peak Power from all mice in each group. Data are mean ± SE. Data in panels C and D are from n = 6/group. * P<0.05 vs. control.

Figure 4. Protein carbonyls are unchanged in diaphragm from PH mice.

These findings suggest that PH does not increase protein oxidation in mouse diaphragm. Total protein carbonyls are normalized to corresponding α-tubulin and expressed relative to the mean for the control group (n = 4/group). Image shows membranes probed for protein carbonyls and α-tubulin. Lanes are examples of control and PH diaphragm.

Figure 5. Myofibrillar protein abundance is similar in control and PH mice.

A) Images show protein bands corresponding to myosin heavy chain (∼220 kDa) in examples of control and PH mice. MHC data were normalized for total protein measured as optical density of entire lane. Molecular weights were determined from protein standards (not shown). B and C) Images are examples of lanes from membranes probed for sarcomeric actin (B) and troponin T (C) in whole-muscle homogenates. Optical density of actin and troponin T were normalized to α-tubulin in the respective membranes. All bar graphs show mean ± SE of results expressed relative to mean of control (N = 4/group).