Mitochondrial degeneration precedes the development of muscle atrophy in progression of cancer cachexia in tumour-bearing mice

Abstract

Background: Cancer cachexia is largely irreversible, at least via nutritional means, and responsible for 20-40% of cancer-related deaths. Therefore, preventive measures are of primary importance; however, little is known about muscle perturbations prior to onset of cachexia. Cancer cachexia is associated with mitochondrial degeneration; yet, it remains to be determined if mitochondrial degeneration precedes muscle wasting in cancer cachexia. Therefore, our purpose was to determine if mitochondrial degeneration precedes cancer-induced muscle wasting in tumour-bearing mice.

Methods: First, weight-stable (MinStable) and cachectic (MinCC) Apc^Min/+ mice were compared with C57Bl6/J controls for mRNA contents of mitochondrial quality regulators in quadriceps muscle. Next, Lewis lung carcinoma (LLC) cells or PBS (control) were injected into the hind flank of C57Bl6/J mice at 8 week age, and tumour allowed to develop for 1, 2, 3, or 4 weeks to examine time course of cachectic development. Succinate dehydrogenase stain was used to measure oxidative phenotype in tibialis anterior muscle. Mitochondrial quality and function were assessed using the reporter MitoTimer by transfection to flexor digitorum brevis and mitochondrial function/ROS emission in permeabilized adult myofibres from plantaris. RT-qPCR and immunoblot measured the expression of mitochondrial quality control and antioxidant proteins. Data were analysed by one-way ANOVA with Student-Newman-Kuels post hoc test.

Results: MinStable mice displayed ~50% lower Pgc-1α, Pparα, and Mfn2 compared with C57Bl6/J controls, whereas MinCC exhibited 10-fold greater Bnip3 content compared with C57Bl6/J controls. In LLC, cachectic muscle loss was evident only at 4 weeks post-tumour implantation. Oxidative capacity and mitochondrial content decreased by ~40% 4 weeks post-tumour implantation. Mitochondrial function decreased by ~25% by 3 weeks after tumour implantation. Mitochondrial degeneration was evident by 2 week LLC compared with PBS control, indicated by MitoTimer red/green ratio and number of pure red puncta. Mitochondrial ROS production was elevated by ~50 to ~100% when compared with PBS at 1-3 weeks post-tumour implantation. Mitochondrial quality control was dysregulated throughout the progression of cancer cachexia in tumour-bearing mice. In contrast, antioxidant proteins were not altered in cachectic muscle wasting.

Conclusions: Functional mitochondrial degeneration is evident in LLC tumour-bearing mice prior to muscle atrophy. Contents of mitochondrial quality regulators across Apc^Min/+ and LLC mice suggest impaired mitochondrial quality control as a commonality among pre-clinical models of cancer cachexia. Our data provide novel evidence for impaired mitochondrial health prior to cachectic muscle loss and provide a potential therapeutic target to prevent cancer cachexia.

Keywords: Cachexia; Cancer; MitoTimer; Mitochondrial quality; Muscle wasting; ROS.

Figure 1

mRNA content of mitochondrial quality controllers in quadriceps muscle of B6 and APC/Min⁺ mice. (A) mRNA content of mitochondrial biogenesis controllers. (B) mRNA content of mitochondrial dynamic controllers. (C) mRNA content of mitophagy regulators. N of 5–6 was utilized for each group. Lettering denotes statistical significance at an alpha set at P < 0.05.

Figure 2

Oxidative muscle fibres and mitochondrial content across a time course progression of cancer cachexia in Lewis lung carcinoma tumour‐bearing mice. (A–D) SDH staining performed in TA muscle. (A) Representative images for SDH stain across the different experimental groups. Scale bar is 50 μM long. (B) % SDH+ fibres throughout the progression of cancer cachexia. (C) Cross‐sectional area distribution of SDH+ fibres. (D) Cross‐sectional area distribution of SDH− fibres. (E–G) Immunoblotting in gastrocnemius muscle. (E) and (F) Immunoblot quantification of mitochondrial content markers COX‐IV (E) and VDAC (G). (G) Representative immunoblot images. N of 10 for each group was used for SDH analysis, and N of 8 for each group was utilized for immunoblot analysis. Lettering denotes statistical significance at an alpha set at P < 0.05.

Figure 3

Mitochondrial degeneration precedes muscle wasting in cancer cachexia in Lewis lung carcinoma tumour‐bearing mice. MitoTimer is a mitochondrially targeted variant of DsRed validated by Laker et al. 15 to emit green fluorescence when mitochondria are healthy and shift to red when mitochondria are damaged. (A–C) MitoTimer in FDB muscle. (A) Representative MitoTimer images taken at ×100 magnification. Scale bar is 20 μM in length. (B) Quantification of MitoTimer red:green ratio. (C) Quantification of pure red puncta in MitoTimer. Locations of pure red puncta co‐localize with LC3 and appear to represent completely degenerated mitochondria targeted for autophagy.15 (D–E) Mitochondrial function and ROS emission in plantaris muscle. (D) Respiratory control ratio (ratio of state 3:state 4 respiration) of permeabilized plantaris muscle. (E) Mitochondrial H₂O₂ production in permeabilized plantaris muscle. N of 12–24 per group was utilized for MitoTimer, while N of 12/group was utilized for respiration and ROS production analysis. Lettering denotes statistical significance at an alpha set at P < 0.05.

Figure 4

Immunoblot analysis of mitochondrial quality control regulators during progression of cancer cachexia in gastrocnemius muscle of Lewis lung carcinoma tumour‐bearing mice. (A, B) Immunoblot quantification (A) and representative immunoblots (B) of mitochondrial biogenesis regulators PGC‐1α, PPARα, PPARδ, and TFAM. (C, D) Immunoblot quantification (C) and representative immunoblots (D) of mitochondrial dynamic regulators MFN1, MFN2, OPA1, DRP1, and Fis1. (E, F) Immunoblot quantification (E) and representative immunoblots (F) of mitophagy regulators BNIP3, PINK1, p‐PARKIN, and PARKIN. N of 7–8 per group was utilized for immunoblot analysis. Lettering denotes statistical significance at an alpha set at P < 0.05.

Figure 5

Content of antioxidant enzymes during progression of cancer cachexia in gastrocnemius muscle of Lewis lung carcinoma tumour‐bearing mice. (A) Representative images of antioxidant enzymes. No statistically significant findings were found. N of 7–8 per group was utilized for immunoblot analysis.

Figure 6

Summary of functional mitochondrial derangements preceding cachectic muscle wasting in tumour‐bearing mice.