PDK4 drives metabolic alterations and muscle atrophy in cancer cachexia

Abstract

Cachexia is frequently accompanied by severe metabolic derangements, although the mechanisms responsible for this debilitating condition remain unclear. Pyruvate dehydrogenase kinase (PDK)4, a critical regulator of cellular energetic metabolism, was found elevated in experimental models of cancer, starvation, diabetes, and sepsis. Here we aimed to investigate the link between PDK4 and the changes in muscle size in cancer cachexia. High PDK4 and abnormal energetic metabolism were found in the skeletal muscle of colon-26 tumor hosts, as well as in mice fed a diet enriched in Pirinixic acid, previously shown to increase PDK4 levels. Viral-mediated PDK4 overexpression in myotube cultures was sufficient to promote myofiber shrinkage, consistent with enhanced protein catabolism and mitochondrial abnormalities. On the contrary, blockade of PDK4 was sufficient to restore myotube size in C2C12 cultures exposed to tumor media. Our data support, for the first time, a direct role for PDK4 in promoting cancer-associated muscle metabolic alterations and skeletal muscle atrophy.-Pin, F., Novinger, L. J., Huot, J. R., Harris, R. A., Couch, M. E., O'Connell, T. M., Bonetto, A. PDK4 drives metabolic alterations and muscle atrophy in cancer cachexia.

Keywords: C2C12 myotubes; chemotherapy; energy metabolism; mitochondria; skeletal muscle atrophy.

Figure 1

PDK4 is elevated in an in vitro model of cancer-induced atrophy. A) Representative images of C2C12 myotubes exposed to control medium (25% UCM) or 25% C26 CM for up to 48 h. The cell layers were stained for MitoTracker Red CMXRos, and the quantification of red fluorescence intensity (polarized mitochondria), normalized vs. DAPI (nuclei) was expressed as arbitrary units (a.u.). Myotube size was determined by measuring the minimum diameter of 250–350 myotubes per experimental condition (n = 3). Scale bar, 100 μm. B) Representative Western blotting for PDK4 in whole-protein extracts from C2C12 myotubes exposed to C26 CM (n = 3). Tubulin was used as loading control. Data (means ± sd) are expressed as fold change vs. UCM. *P < 0.05, ***P < 0.001.

Figure 2

The C26 tumors cause significant metabolic derangements. A) Assessment of PDH and SDH enzymatic activities in gastrocnemius muscle of control and C26 tumor–bearing mice (C26). Data are expressed as mU/ml for PDH or mU/μl for SDH. B) Representative Western blotting for PDK4 in whole-muscle protein extracts from mice bearing the C26 tumor (n = 8). Tubulin was used as loading control. Data (means ± sd) are expressed as fold change vs. control. *P < 0.05, **P < 0.01.

Figure 3

The PDK4 activator WY-14643 determines myotube atrophy. A) Assessment of myotube size in C2C12 cultures treated with WY-14643 for up to 48 h. On average, 250–350 myotubes were measured per experimental condition in triplicate. Green staining: myosin heavy chain. Scale bar, 100 μm. B) Representative Western blotting for PDK4 in whole protein extracts from C2C12 treated with WY-14643 (n = 3). Tubulin was used as loading control. Data (means ± sd) are expressed as fold change vs. control. *P < 0.05, ***P < 0.001.

Figure 4

In vivo WY-14643 administration induces a cachectic phenotype. A–C) Cumulative food intake (A), body weight curves (B) and body weight change (i.e., body weight at time of euthanization vs. initial body weight) (C) in mice fed a diet enriched with 0.1% WY-14643. D) Body composition assessment in mice fed WY-14643 was performed by means of EchoMRI. Data are expressed in grams of fat or lean tissue at time of euthanization. E) Representative image of hindlimb musculature in mice fed a control or WY-14643–enriched diet. F) Skeletal muscle weights (tibialis anterior, gastrocnemius, and quadriceps) in mice fed control or WY-14643 diets. Weights were normalized to the initial body weight (IBW) and expressed as weight/100 mg IBW; n = 8. Data are means ± sd. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Figure 5

WY-14643 administration causes muscle atrophy and mitochondrial alterations. A) Representative images of hematoxylin and eosin (H&E)– and SDH-stained 10 μm-thick sections of tibialis anterior muscle excised from control and WY-14643–treated mice. Scale bar, 100 μm. B) CSA in tibialis anterior muscle sections from mice fed a diet enriched with WY-14643. C) Quantification of SDH integrated density in control and WY-14643–treated mice. Data are expressed as arbitrary units (a.u.). D) Number of oxidative (dark blue) and glycolytic (light blue) fibers (expressed as percentage of control). E) SDH and PDH enzymatic activities in the gastrocnemius muscle of control and WY-14643–treated animals. Data are expressed as mU/μl for SDH or mU/ml for PDH. F) Representative Western blotting for PDK4, PGC1α, OPA1, Mitofusin 2, and cytochrome c in whole-muscle protein extracts from mice fed control or WY-14643 diets. Tubulin was used as loading control. G) Gene expression levels for atrogin-1 and MuRF1 ubiquitin ligases performed by real-time quantitative PCR. Gene expression was normalized to TATA-binding protein levels. Data (means ± sd) are expressed as fold change vs. control; n = 8. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

PDK4 overexpression affects C2C12 myotube size and metabolism. A) Representative Western blotting and quantification for PDK4 (shown with both low and high exposure times), PGC1α, OPA1, Mitofusin 2, cytochrome c, and ubiquitin-labeled proteins in C2C12 myotubes infected with Ad-GFP or Ad-GFP-mPDK4 (n = 3). Tubulin was used as loading control. B) Myosin heavy chain (MyHC) immunofluorescent staining in C2C12 myotubes infected with Ad-GFP or Ad-GFP-mPDK4 (n = 3) and myotube size quantification. On average, 250–350 myotubes were measured per experimental condition, in triplicate. Scale bar, 100 μm. C) Pyruvate dehydrogenase (PDH) and succinate dehydrogenase (SDH) enzymatic activities in C2C12 myotubes infected with Ad-GFP and Ad-GFP-mPDK4. Data are expressed as milliunits per millilter for PDH, or milliunits per microliter for SDH. D) Mitochondrial respiration and oxygen consumption rate (OCR) in C2C12 myotubes infected with Ad-GFP or Ad-mPDK4 (n = 3). ATP, adenosine triphosphate; BR, basal respiration; MR, maximal respiration; PL, proton leak; SRC, spare respiratory capacity. Data (means ± sd) were expressed in picomoles per minute. Data (means ± sd) are reported as fold change vs. Ad-GFP. *P < 0.05, **P <0.01, ***P < 0.001 vs. Ad-GFP (significance of the differences).

Figure 7

PDK4 deletion protects C2C12 myotubes from tumor-induced atrophy. A) Representative images of myosin heavy chain–stained C2C12 myotubes infected with either Ad-shScr or Ad-shPDK4 alone or in combination with either 25% control medium (25% UCM) or 25% C26 CM for up to 48 h. Data (means ± sd) are expressed as micrometers. On average, 250–350 myotubes were measured per experimental condition in triplicate. Scale bar, 100 μm. B) Representative Western blotting for PDK4 in whole protein extracts from C2C12 myotubes infected with Ad-shScr or Ad-shPDK4 and exposed to C26 CM for up to 48 h. Tubulin was used as loading control. C) PDH enzymatic activity in C2C12 myotubes infected with Ad-shScr or Ad-shPDK4 and exposed to C26 CM for up to 48 h. Data (means ± sd) are expressed as mU/ml. **P < 0.01, ***P < 0.001 vs. UCM, ^$P < 0.05, ^$$$P < 0.001 vs. Ad-shScr + UCM, ^#P < 0.05, ^###P < 0.001 vs. Ad-shScr + C26 CM.