Diaphragm and ventilatory dysfunction during cancer cachexia

Abstract

Cancer cachexia is characterized by a continuous loss of locomotor skeletal muscle mass, which causes profound muscle weakness. If this atrophy and weakness also occurs in diaphragm muscle, it could lead to respiratory failure, which is a major cause of death in patients with cancer. Thus, the purpose of the current study was to determine whether colon-26 (C-26) cancer cachexia causes diaphragm muscle fiber atrophy and weakness and compromises ventilation. All diaphragm muscle fiber types were significantly atrophied in C-26 mice compared to controls, and the atrophy-related genes, atrogin-1 and MuRF1, significantly increased. Maximum isometric specific force of diaphragm strips, absolute maximal calcium activated force, and maximal specific calcium-activated force of permeabilized diaphragm fibers were all significantly decreased in C-26 mice compared to controls. Further, isotonic contractile properties of the diaphragm were affected to an even greater extent than isometric function. Ventilation measurements demonstrated that C-26 mice have a significantly lower tidal volume compared to controls under basal conditions and, unlike control mice, an inability to increase breathing frequency, tidal volume, and, thus, minute ventilation in response to a respiratory challenge. These data demonstrate that C-26 cancer cachexia causes profound respiratory muscle atrophy and weakness and ventilatory dysfunction.

Keywords: C-26; limb muscle; muscle function; respiratory muscles; single fiber.

Figure 1.

Diaphragm fiber size, fiber type distribution, and atrophy-related gene expression in control and C-26 tumor-bearing mice. A) Body weight of control mice and tumor-free body weight of C-26 mice at study endpoint (when largest tumor reached 1.5 cm in diameter). B, C) Representative cross sections taken from the diaphragm of control and C-26 mice. Sections were H&E stained (B) or incubated with an anti-laminin antibody to allow for visualization of muscle fibers (red) and anti-MyHC type I (blue) and anti-MyHC type IIa (green) antibodies; black fibers represent type IIb/x fibers (C). D) Mean CSA of all fibers and of each muscle fiber type. E) Percentage of each diaphragm muscle fiber type in control and C-26 mice. F) Relative mRNA levels of atrogin-1, MuRF1, Bnip3, and cathepsin L (CthL) from the diaphragm of control and C-26 mice. Bars represent means ± se for 6 muscles/group. *P < 0.05 vs. control.

Figure 2.

Diaphragm isometric contractile properties in control and C-26 tumor-bearing mice. A, B) Specific force-frequency relationship (A) and maximum specific force (B) in diaphragm strips of control and C-26 mice. C–H) In permeabilized fibers from control and C-26 mice, we measured absolute maximum calcium activated force (C), single fiber CSA (D), maximal specific calcium-activated force (sF_o max; panel E), pCa that elicited 50% of maximal calcium-activated force (pCa₅₀; panel F), the slope of the force-pCa relationship (n_H; panel G) and, the rate of tension redevelopment (k_tr; panel H). Bars represent means ± se for 6 muscles/group. *P < 0.05 vs. control.

Figure 3.

Diaphragm MyHC and actin protein expression in control and C-26 tumor-bearing mice. A) Representative gel identifying MyHC and Western blot for actin in the diaphragm of control and C-26 mice. B) Densitometric quantification of MyHC expression, normalized to total protein within the solubilized fraction. Bars represent means ± se for 6 muscles/group. *P < 0.05 vs. control.

Figure 4.

Diaphragm isotonic contractile properties in control and C-26 tumor-bearing mice. A) Relationship between specific force and shortening velocity from a representative control and C-26 mouse. B) Power-velocity relationship calculated from data shown in A. C) Maximal shortening velocity determined from each individual force-velocity relationships. D) Peak power determined from each individual power-velocity relationship. Bars represent means ± se for 5–6 mice/group. *P < 0.05 vs. control.

Figure 5.

Breathing pattern in control and C-26 tumor-bearing mice. All data were collected during baseline conditions (21% O₂) and challenge (hypoxia, 10% inspired O₂) in unanesthetized mice at study endpoint. A) Representative airflow traces to demonstrate the pattern of breathing. B) Breathing frequency. C) Tidal volume. D) Minute ventilation in control and C-26 mice. Bars represent means ± se for 8 mice/group. *P < 0.05 vs. control baseline; ^†P < 0.05 vs. control challenge.