Repeated eccentric contractions positively regulate muscle oxidative metabolism and protein synthesis during cancer cachexia in mice

Abstract

Cancer-induced wasting is accompanied by disruptions to muscle oxidative metabolism and protein turnover that have been associated with systemic inflammation, whereas exercise and stimulated muscle contractions can positively regulate muscle protein synthesis and mitochondrial homeostasis. In preclinical cancer cachexia models, a single bout of eccentric contractions (ECCs) can induce protein synthesis and repeated ECC bouts prevent myofiber atrophy. The cellular mechanisms providing this protection from atrophy have not been resolved. Therefore, the purpose of this study was to determine whether repeated stimulated ECC bouts affect basal muscle oxidative metabolism and protein synthesis during cancer cachexia, and if these changes were associated with plasma IL-6 levels. Male Apc^Min/+ (MIN; n = 10) mice initiating cachexia and healthy C57BL/6 (B6; n = 11) control mice performed repeated ECC bouts over 2 wk. MIN mice exhibited body weight loss and elevated plasma IL-6 before and during repeated ECC bouts. Control MIN muscle demonstrated disrupted signaling related to inflammation, oxidative capacity, and protein synthesis regulation, which were all improved by repeated ECC bouts. With cachexia, plasma IL-6 levels were negatively correlated with myofiber cross-sectional area, oxidative capacity, and protein synthesis. Interestingly, ECC improvements in these outcomes were positively correlated with plasma IL-6 levels in MIN mice. There was also a positive relationship between muscle oxidative capacity and protein synthesis after repeated ECC bouts in MIN mice. Collectively, repeated ECC bouts altered the cachectic muscle phenotype independent of systemic wasting, and there was a strong association between muscle oxidative capacity and protein synthesis in this adaptive response.

NEW & NOTEWORTHY Cancer-induced muscle wasting is accompanied by disruptions to muscle oxidative metabolism and protein turnover regulation, whereas exercise is a potent stimulator of muscle protein synthesis and mitochondrial homeostasis. In a preclinical model of cancer cachexia, we report that cachectic muscle retains anabolic and metabolic plasticity to repeated eccentric contraction bouts despite an overall systemic wasting environment. The attenuation of muscle atrophy is linked to improved oxidative capacity and protein synthesis during cancer cachexia progression.

INTRODUCTION

Cancer cachexia is a complex metabolic wasting syndrome characterized by skeletal muscle loss and accounts for 40% of all cancer-related deaths (10, 47). Cancer-induced muscle wasting promotes patient intolerance to cancer therapy, increases treatment toxicity, and accelerates morbidity and mortality (5, 16, 31). Hallmarks of cancer cachexia include systemic inflammation, whole body and organ-specific metabolic disruptions, and muscle proteostatic imbalance (47), all of which have the ability to influence skeletal muscle wasting and dysfunction. Although pharmaceutical agents targeting these mechanisms have been identified and investigated in clinical trials, many have failed because of either the singular specificity or adverse side effects (12). Thus, a better understanding of the mechanistic underpinnings of cancer-induced muscle wasting is required to develop treatment strategies that can enhance cancer patient outcomes and quality of life.

Preclinical cancer cachexia models have had utility in dissecting the cellular processes regulating the initiation and progression of cachexia. Disruptions to protein synthesis and oxidative metabolism have established roles in the etiology of cancer-induced muscle wasting (24). Basal protein synthesis rates are reduced during the initial stages of body weight loss (<5%) and further suppressed throughout cachexia progression (52). Perturbations in protein synthetic capacity are accompanied by the dysregulation of mechanistic target of rapamycin complex-1 (mTORC1) signaling (37, 52). Chronically elevated plasma interleukin-6 (IL-6) has been implicated in the suppression of muscle protein synthesis and mTORC1 signaling through the activation of signal transducer and activator of transcription 3 (STAT3) and 5′ AMP-activated protein kinase (AMPK) during cancer cachexia progression (52, 53). Systemic overexpression of IL-6 is sufficient to suppress muscle protein synthesis and mTORC1 signaling in healthy and tumor-bearing mice (22, 53), whereas inhibition of muscle IL-6/gp130 receptor and downstream signaling can attenuate muscle wasting in tumor-bearing animals (6, 37, 52, 53). Thus, strategies that modify the muscle’s sensitivity to systemic IL-6 and/or disrupt intracellular STAT3 signaling downstream of the IL-6R/gp130 receptor complex may serve as therapeutic targets to preserve muscle mass during cachexia progression.

Disrupted muscle oxidative metabolism also coincides with suppressed protein synthesis and mTORC1 signaling during cancer cachexia (51, 52, 54). Cancer-induced muscle wasting is accompanied by dysregulation of mitochondrial content, function, fission, fusion, and autophagy (11). Decreased mitochondrial content and function is associated with reduced transcription factor and coactivator expression that regulates mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in cachectic muscle (13, 44, 48, 51). Cachexia disrupts mitochondrial quality control by increasing fission (e.g., mitochondrial fission protein 1, Fis1) and decreasing fusion (e.g., mitofusin-1, Mfn1) protein expression (51, 54). Cachexia-induced disruptions to mitophagy are associated with the accumulation of damaged or dysfunctional mitochondria, and dysregulated expression of critical autophagy-regulating proteins (e.g., Atg, LC3B, p62) has been reported in cachectic skeletal muscle (2, 40, 41, 52). Evidence also suggests that mitochondrial dysfunction may precede cachexia development in some preclinical cancer models (9), which supports a potential role for oxidative metabolism as an early driver of muscle wasting (11). Mitochondrial function, autophagy/mitophagy, and protein synthesis regulation in skeletal muscle are interconnected and have the ability to alter muscle mass and metabolism (24). Although cancer can disrupt all of these critical processes, we have a limited understanding of how cancer-induced disruptions to this critical regulatory network are integrated to drive muscle wasting. Importantly, exercise training in tumor-bearing mice can improve the regulation of these pathways (e.g., mitochondrial biogenesis, mitophagy) and overall oxidative metabolism (45, 54). Similarly, promoting a slow muscle phenotype or targeting oxidative metabolism can attenuate wasting processes during cancer cachexia (20, 36). We have reported that repeated eccentric contraction (ECC) bouts can improve myofiber oxidative capacity and attenuate myofiber atrophy (23), but the molecular mechanisms underlying these adaptations have not been established. Ultimately, therapeutic strategies that simultaneously improve oxidative metabolism and protein synthesis have the potential to slow the progression of muscle wasting and thus enhance cancer patient quality of life and survival.

Exercise and neuromuscular electrical stimulation are nonpharmacological treatment approaches that have shown promise for improving health outcomes across many disease populations, including cancer (8, 25, 29, 30, 38). However, further investigation is needed to determine whether these beneficial adaptations are achievable in the cachectic cancer patient (18, 19). Understanding the effects of muscle contraction independent of whole body exercise after cachexia initiation has value, since cachectic mice have a limited capacity for performing vigorous, voluntary exercise (4). To this end, cachectic muscle’s anabolic and metabolic response to contraction has been examined with established models of low- and high-frequency electrical stimulation (21, 44). We have previously shown that cachexia can disrupt the signaling and transcriptional response to a single bout of concentric muscle contractions elicited by low-frequency electrical stimulation (44). In contrast, a single ECC bout elicited by high-frequency electrical stimulation induced protein synthesis and mTORC1 signaling (21), whereas repeated ECC bouts prevented muscle and myofiber atrophy in tumor-bearing mice (1, 23, 46). A remaining question is whether the ECC training response in cachectic muscle is accompanied by alterations to muscle signaling involving inflammation, oxidative metabolism, and protein synthesis, which have established roles in cancer-induced wasting. Therefore, the purpose of this study was to determine whether repeated stimulated ECC bouts affect basal muscle oxidative metabolism and protein synthesis during cancer cachexia and whether these changes are associated with plasma IL-6 levels. Overall, our results demonstrate that cachectic muscle retains plasticity to repeated ECC bouts despite the cancer systemic environment and elevated plasma IL-6. In addition, our results support a link between improved oxidative capacity and enhanced protein synthesis regulation during the attenuation of myofiber atrophy.

MATERIALS AND METHODS

Animals.

Male Apc^Min/+ (MIN) mice on a C57BL/6 (B6) background were originally purchased from Jackson Laboratories (Bar Harbor, ME) and bred at the University of South Carolina’s Animal Resource Facility. All mice used in the present study were obtained from the investigator’s breeding colony within the Center for Colon Cancer Research Mouse Core. Mice were individually housed and kept on a 12:12-h light-dark cycle and had access to standard rodent chow (no. 8604 Rodent Diet; Harlan Teklad, Madison, WI) and water ad libitum. Body weight measurements were taken weekly, and the percentage of body weight loss from peak body weight (~10–14 wk of age) was calculated. Daily food consumption was monitored during the treatment period, and mice were fasted for 5 h before tissue harvest. Mice lacking the Apc allele mutation (B6 mice) served as controls for all experiments. The University of South Carolina’s Institutional Animal Care and Use Committee approved all animal experimentation in this study.

Experimental design.

Male MIN (n = 10) mice were subjected to repeated ECC bouts (8 total sessions) after the initiation of cachexia (Fig. 1A). ECC bouts were separated by at least 48 h (e.g., Monday, Wednesday, Friday), and mice were euthanized 48 h after the last ECC bout, as previously described (23). We have found that basal protein synthesis is not affected by a single ECC bout at this time point (data not shown), and thus any alterations in ECC muscles would represent an adaptation to training. Mice were fasted for 5 h before tissue harvest and given an intraperitoneal injection of puromycin (0.040 µmol/g BW dissolved in 100 µL of PBS) 30 min before tissue harvest (17a). Age-matched B6 mice (n = 11) served as healthy controls undergoing the same experimental protocol.

Fig. 1.

Cachexia progression during repeated eccentric contraction (ECC) bouts. A: experimental design. At 16 wk of age male C57BL/6 (B6) and Apc^Min/+ (MIN) mice performed repeated ECC bouts. Each ECC bout consisted of 60 ECCs (100 Hz, 10 contractions, 6 sets). Repeated ECC bouts were separated by 48 h, and mice were killed 48 h after the last ECC bout. Mice were fasted 5 h before tissue harvest. B: body weight change pre- and post-ECC. C: plasma interleukin-6 (IL-6) levels pre- and post-ECC. D: tissue mass of MIN mice expressed as % of B6 control (CON). E: relationship between body weight (BW) and tissue mass (expressed as % of B6 CON) to plasma IL-6 levels in MIN mice. Data are means ± SE. A 2-way ANOVA was performed to determine differences between treatment groups when appropriate. Post hoc analyses were performed with Student–Newman–Keuls methods when appropriate. Student’s t test was performed to determine differences between 2 groups. Pearson correlation coefficients (r) were computed to determine the relationship between 2 variables when appropriate. Statistical significance was set at P < 0.05. BF, brown fat; EPI, epididymal fat; LABC, levator ani-bulbocavernosus muscle; ND, not detected; SV, seminal vesicles; TA, tibialis anterior. *Different from B6. †Different from MIN pre-ECC.

Eccentric contractions.

Eccentric contractions (ECCs) of the tibialis anterior (TA) muscle were induced by high-frequency electrical stimulation of the sciatic nerve as previously described, with slight modifications (3, 23). Mice were anesthetized via isoflurane (2% in O₂ with 1.5% maintenance), the stimulated leg was shaved at the hip region, and two needle electrodes were placed subcutaneously to stimulate the sciatic nerve. Tetanic muscle contractions of the hindlimb were generated with a Grass Stimulator (Grass Instruments) for 10 sets of 6 contractions (100 Hz, 6–12 V, 1-ms duration). Ten seconds of rest was given between contractions, and 50 s of rest was given between set of contractions. Mice were given an intraperitoneal injection of warm saline after each stimulation procedure and returned to cages upon complete recovery. The stimulation protocol recruits all motor units and results in net plantar flexion of the ankle (3, 55–57). Since the dorsiflexors [TA and extensor digitorum longus (EDL)] produced approximately one-third of the force of the plantar flexors (gastrocnemius, soleus, and plantaris) (55–57), the TA muscle performs ECCs against the plantar flexors undergoing concentric muscle contractions. This experimental approach results in muscle enlargement with training (3, 57) and increased protein synthesis and mTORC1 signaling following a single bout in rodent skeletal muscle (3, 56). The nonstimulated TA muscle served as an intra-animal control (CON) in all experiments. Our laboratory and others have demonstrated that repeated ECC bouts, but not concentric contractions, can induce muscle and myofiber growth in rodents (1, 3, 23, 57). Therefore, the TA was examined in all experiments.

Tissue collection.

Mice were anesthetized by a subcutaneous injection of ketamine-xylazine-acepromazine cocktail (1.4 mL/kg body wt) at the time of tissue harvest. The TA muscles were rapidly excised, cleared of excess connective tissue, rinsed in PBS, weighed, and snap frozen in liquid nitrogen. Blood was collected before muscle collection via retroorbital eye bleed with heparinized capillary tubes, placed on ice, and centrifuged (10,000 g for 10 min at 4°C). The supernatant was removed and stored for plasma IL-6 analysis. Plasma and tissue samples were stored at −80°C until further analysis.

Western blotting.

Western blot analysis was performed as previously described (21). Frozen TA muscle was homogenized in ice-cold Mueller buffer, and protein concentration was determined by the Bradford method. Crude muscle homogenates were fractionated on 6–5% SDS-polyacrylamide gels and transferred to PVDF membranes, and then membranes were stained with Ponceau red to verify equal loading and transfer. Membranes were blocked at room temperature (RT) for 1–2 h in 5% bovine serum albumin (BSA)-Tris-buffered saline with 0.1% Tween 20 (TBST). Primary antibodies for puromycin (Millipore catalog no. MABE343, 1:2,000), phospho-P70S6K (T389) (catalog no. 9205, 1:1,000), total P70S6K (catalog no. 2708, 1:1,000), phospho-RPS6 (S240/244) (catalog no. 2215, 1:500), total RPS6 (catalog no. 2708, 1:1,000), phospho-Akt (S473) (catalog no. 4060, 1:1,000), total Akt (catalog no. 9272, 1:2,000), phospho-STAT3 (Y750) (catalog no. 9145, 1:1,000), total STAT3 (catalog no. 4904, 1:2,000), phospho-AMPK (T172) (catalog no. 2535, 1:2,000), total AMPK (catalog no. 2603, 1:1,000), phospho-acetyl CoA carboxylase (ACC) (S59) (catalog no. 3661, 1:1,000), total ACC (catalog no. 3662, 1:1000), PGC-1α (Abcam catalog no. ab54481, 1:1,000), cytochrome-c oxidase (COX)IV (catalog no. 4844, 1:1,000), p62 (catalog no. 5114, 1:1,000), LC3B (catalog no. 2775, 1:1,000), and GAPDH (catalog no. 2118, 1:10,000) were incubated overnight in 5% BSA-TBST. Membranes were then incubated in 5% BSA-TBST containing anti-rabbit (catalog no. 7074, 1:5,000) IgG horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Exceptions to the aforementioned procedures were that 1% BSA-TBST was used for puromycin primary antibody incubation and a horseradish peroxidase-conjugated rabbit anti-mouse IgG2a antibody (Life Technologies catalog no. 610220, 1:5,000) in 5% BSA-TBST was used for secondary antibody incubation. All antibodies were from Cell Signaling Technology unless otherwise stated. TA protein extracts from a mouse that did not receive puromycin at death was included on all puromycin gels as a negative control. Enhanced chemiluminescence (ECL) (GE Healthcare Life Sciences) was used to visualize the antibody-antigen interactions. Immunoblot images were collected with an imager (SynGene GBox) and quantified by densitometry with imaging software (ImageJ; NIH). The activation of signaling molecules was determined by the ratio of phosphorylated and total protein expression when appropriate. For total protein expression, values were corrected to GAPDH. Each gel contained samples from all groups (e.g., B6/MIN, CON/ECC) and data were normalized to the B6 CON group when appropriate.

Cytochrome-c oxidase activity.

Cytochrome-c oxidase (COX) activity was assessed in TA muscles as previously described (17). Muscles were homogenized in extraction buffer (0.1 M KH₂PO₄/Na₂HPO₄ and 2 mM EDTA, pH 7.2), and COX enzyme activity was determined by measuring the maximal rate of oxidation of fully reduced cytochrome c and the change in absorbance at 550 nm.

Succinate dehydrogenase activity.

Succinate dehydrogenase (SDH) enzyme activity was performed as previously described (23). Briefly, frozen cross sections were air-dried for 10 min, followed by incubation in a solution containing 0.2 M phosphate buffer (pH 7.4), 0.1 M MgCl₂, 2.4 mM nitroblue tetrazolium (NBT), and 0.2 M succinic acid for 45 min at 37°C. Sections were then washed in dH₂O for 3 min, dehydrated in 50% ethanol for 2 min, and mounted for viewing with mounting medium. Images of each section at ×25 magnification and fibers were manually traced with imaging software (ImageJ; NIH). Whole TA muscle cross sections were examined since fibers with high oxidative capacity are more abundant in the deep region of the muscle compared with the superficial region. The images were converted to 8-bit grayscale (range of gray levels 0–255) images, and an integrated optical density was created by subtracting the background intensity from each myofiber. Thresholds corresponding to SDH enzyme activity were set manually and uniformly across all images, and myofibers were classified as having high or low SDH enzyme activities. The cross-sectional areas of high- and low-SDH enzyme activity myofibers were quantified. The analyses were performed by an investigator blinded to the treatment groups.

Plasma interleukin-6 concentration.

Plasma IL-6 concentrations were determined as previously described (28). A commercially available IL-6 enzyme-linked immunosorbent assay kit was obtained from BD Biosciences, and the manufacturer’s protocol was followed. Briefly, clear 96-well plates were coated and incubated overnight with an IL-6 capture antibody. The next morning the plate was blocked with assay diluent buffer and washed, and equal volumes of standards and plasma samples were added in duplicate. After a 2-h incubation the plate was washed and sAV-HRP reagent was added to each well. After several washes, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added and the reaction was developed for 20 min. The reaction was stopped with sulfuric acid, and absorbance was read at 450 nm with an iMark microplate absorbance reader (Bio-Rad Laboratories).

Statistical analysis.

Results are reported as means ± standard error. A two-way repeated-measures ANOVA (e.g., genotype × treatment) with Student–Newman–Keuls post hoc methods was performed when appropriate. Student’s t tests were performed to determine differences between two groups when appropriate. Pearson correlation coefficients (r) were computed to determine the relationship between two variables. The accepted level of significance was set at P < 0.05 for all analyses. Statistical analysis and figure generation were performed with Prism 5 for Mac OS X (GraphPad Software Inc).

RESULTS

Repeated ECC bouts attenuate muscle wasting despite the presence of a systemic cachectic environment.

Male B6 and MIN mice performed repeated ECC bouts and were euthanized 48 h after the last bout (Fig. 1A). MIN mice had initiated cachexia before the first ECC bout and continued to lose body weight during the training period (Fig. 1B, Table 1). Similarly, plasma IL-6 levels were elevated before the first ECC bout and increased during the training period in MIN mice (Fig. 1C). At the end of the study MIN mice displayed several key features of cachexia, which included body weight loss, adipose tissue depletion, muscle atrophy, elevated plasma IL-6 levels, and hypogonadal features [levator ani-bulbocavernosus (LABC) and seminal vesicle atrophy] (Fig. 1D, Table 1). Plasma IL-6 levels were highly correlated with body weight loss and tissue wasting in MIN mice (Fig. 1E). Although cachexia decreased the mass of the nonstimulated TA muscle, repeated bouts of ECC increased TA muscle irrespective of cancer (Table 1). There were no differences in daily food intake during the treatment period or tibia length, a measure of body size, at the end of the study between B6 and MIN mice (Table 1). These findings support our previous observations that repeated ECC bouts can attenuate muscle wasting despite the presence of a systemic cachectic environment (23, 46).

Table 1.

Animal characteristics

	B6	MIN
No. of mice	11	10
BW, g
Peak	27.5 ± 0.3	25.5 ± 0.4
Pre	27.6 ± 0.3	23.5 ± 0.5*†
Post	27.8 ± 0.3	22.6 ± 0.5*†‡
% Change, peak to postpeak	1.1 ± 0.5	−11.4 ± 1.5*
Tibialis anterior, mg
Control	47.7 ± 1.0	32.6 ± 1.6#
ECC	49.8 ± 1.0§	36.0 ± 1.5#§
Epididymal fat, mg	309 ± 27	69 ± 21*
Spleen, mg	72 ± 2	593 ± 28*
Testes, mg	198 ± 3	136 ± 12*
LABC, mg	89 ± 2	46 ± 5*
Seminal vesicle, mg	234 ± 18	53 ± 7*
Tumor number	0 ± 0	68 ± 9*
Food intake, g food/g BW	0.11 ± 0.003	0.12 ± 0.003
Tibia length, mm	16.9 ± 0.1	16.8 ± 0.1

Data are means ± SE. BW, body weight; B6, C57BL/6 mice; ECC, eccentric contraction; LABC, levator ani-bulbocavernosus muscle; MIN, Apc^Min/+ mice. A 2-way repeated-measures ANOVA with Student–Newman–Keuls post hoc methods was performed when appropriate. Student’s t test was performed when appropriate. Statistical significance was set at P < 0.05.

^*Significantly different from B6.

^†Significantly different from peak BW.

^‡Significantly different from pre BW.

^#Main effect of MIN.

^§Main effect of ECC.

Repeated ECC bouts improve oxidative metabolism regulation in cachectic MIN mice.

Chronically elevated IL-6 and the activation of muscle gp130 and downstream signaling have been implicated in disrupted oxidative metabolism and protein synthesis regulation during cancer cachexia progression. Although muscle STAT3 activation (phospho-to-total ratio) was increased in MIN mice, repeated ECC bouts reduced STAT3 activation irrespective of cancer (Fig. 2A). Similarly, AMPK and its downstream target ACC were elevated in MIN mice, which was reduced by repeated ECC bouts irrespective of cancer (Fig. 2A). It has been suggested that the activation of AMPK during late-stage cachexia may be related to energy stress/deficit caused by decreased mitochondrial content and function (24). Therefore, we examined whether repeated ECC bouts preserved mitochondrial content and improved mechanisms related to mitochondrial quality control. The reduction in PGC-1α protein expression was not improved by ECC in MIN mice (Fig. 2B). In contrast, repeated ECC bouts prevented the suppression of COXIV protein expression in MIN mice (Fig. 2B). Cachectic MIN muscle demonstrated enhanced expression of proteins related to autophagy regulation (LC3B-II, p62), which was reduced by repeated ECC bouts irrespective of cancer (Fig. 2C). Although muscle cytochrome-c oxidase (COX) enzyme activity, an established index of mitochondrial content, was reduced in MIN mice, repeated ECC bouts increased COX activity irrespective of cancer (Fig. 2D). Plasma IL-6 levels were negatively correlated with muscle COX activity; however, this relationship was abrogated by repeated ECC bouts (Fig. 2E). Interestingly, improvements in muscle COX activity were positively correlated to plasma IL-6 levels in MIN mice (Fig. 2F), suggesting that cachectic muscle remained highly plastic to training. Collectively, these findings demonstrate that the attenuation of muscle gp130 and downstream signaling by repeated ECC bouts coincided with improved oxidative capacity despite the presence of elevated IL-6 levels and systemic wasting.

Fig. 2.

Repeated eccentric contraction (ECC) bouts improve oxidative metabolism regulation in cachectic Apc^Min/+ (MIN) mice. A: downstream muscle gp130 signaling regulation following repeated ECC bouts. B: muscle mitochondrial content regulation after repeated ECC bouts. C: muscle autophagy/mitophagy regulation after repeated ECC bouts. D: muscle cytochrome-c oxidase (COX) enzyme activity after repeated ECC bouts. E: relationship between COX enzyme activity and plasma interleukin-6 (IL-6) levels in MIN mice. F: relationship between changes in COX enzyme activity with repeated ECC bouts and plasma IL-6 levels in MIN mice. The activation of signaling molecules was determined by the ratio of phosphorylated and total protein expression when appropriate. For total protein expression, values were corrected to GAPDH. Each gel contained samples from all groups (e.g., B6/MIN, CON/ECC), and data were normalized to the B6 CON group when appropriate. Data are means ± SE. A 2-way ANOVA was performed to determine differences between treatment groups when appropriate. Post hoc analyses were performed with Student–Newman–Keuls methods when appropriate. Pearson correlation coefficients (r) were computed to determine the relationship between 2 variables when appropriate. Statistical significance was set at P < 0.05. Different letters are statistically different. ACC, acetyl CoA carboxylase; AMPK, 5′ AMP-activated protein kinase; B6, C57BL/6; CON, control; p, phospho-; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; STAT3, signal transducer and activator of transcription 3. ^&Main effect of ECC. ^#Main effect of MIN.

Repeated ECC bouts attenuate atrophy of oxidative and glycolytic myofibers in cachectic MIN mice.

It is generally accepted that glycolytic muscles are more susceptible to cancer-induced wasting, whereas muscles with higher oxidative capacity are relatively spared and protected from atrophy. Whether fiber types with varying oxidative capacities within the same muscle respond similarly has not been widely investigated. We previously found that repeated ECC bouts attenuated atrophy of all fiber types (assessed by myosin heavy chain expression), which coincided with an increased percentage of myofibers with high succinate dehydrogenase (SDH) enzyme activity (23). We reexamined this cohort to determine whether myofibers with varying oxidative capacities, assessed by SDH enzyme activity, were differentially responsive to repeated ECC bouts (23). Although the cross-sectional areas of high- and low-SDH myofibers were reduced in MIN muscle (Fig. 3, A and B), repeated ECC bouts increased the size of both myofiber phenotypes irrespective of cancer (Fig. 3A). There was no difference in the sensitivity to wasting and the protective effects of repeated ECC bouts in MIN muscle (Fig. 3B). Plasma IL-6 levels were negatively correlated with high- and low-SDH myofiber cross-sectional area, and this relationship was not altered by repeated ECC bouts in low-SDH myofibers (Fig. 3, C and E). Interestingly, changes in cross-sectional area with repeated ECC bouts were correlated with plasma IL-6 levels in both high- and low-SDH myofibers (Fig. 3, D and F), which further highlights that the muscle’s sensitivity to repeated ECC bouts was related to cachexia severity. Taken together, these findings demonstrate that although all myofibers within the TA muscle are susceptible to cancer-induced wasting, repeated ECC bouts initiated after onset of cachexia could attenuate atrophy of myofibers with high and low oxidative capacities despite elevated plasma IL-6 levels and systemic wasting.

Fig. 3.

Repeated eccentric contractions (ECC) bouts attenuate atrophy of oxidative and glycolytic myofibers in cachectic Apc^Min/+ (MIN) mice. A: high- and low-succinate dehydrogenase (SDH) enzyme activity myofiber cross-sectional area (CSA). B: high- and low-SDH myofiber area expressed as % of B6 CON in MIN mice. C: relationship between high-SDH myofiber CSA and plasma interleukin-6 (IL-6) levels in MIN mice. D: relationship between changes in high-SDH myofiber CSA after repeated ECC bouts and plasma IL-6 levels in MIN mice. E: relationship between low-SDH myofiber CSA and plasma IL-6 levels in MIN mice. F: relationship between changes in low-SDH myofiber CSA after repeated ECC bouts and plasma IL-6 levels in MIN mice. High- and low-SDH enzyme activity myofiber area was examined post hoc in a separate cohort of B6 and MIN mice previously described (23). Data are means ± SE. B6, C57BL/6; CON, control. A 2-way ANOVA was performed to determine differences between treatment groups when appropriate. Post hoc analyses were performed with Student–Newman–Keuls methods when appropriate. Student’s t test was performed to determine differences between 2 groups. Pearson correlation coefficients (r) were computed to determine the relationship between 2 variables when appropriate. Statistical significance was set at P < 0.05. *Different from MIN CON. ^&Main effect of ECC. ^#Main effect of MIN.

Repeated ECC bouts improve basal protein synthesis and mTORC1 signaling in cachectic MIN mice.

Given that repeated ECCs were sufficient to attenuate myofiber atrophy during cachexia progression, we next determined whether this was accompanied by improvements to basal protein synthesis and mTORC1 signaling in cachectic muscle. Cachexia disrupted mTORC1 signaling demonstrated by the activation of the upstream regulator Akt and the suppression of downstream effectors P70S6K and RPS6 (Fig. 4A), as we have previously observed (21, 52, 53). Although repeated ECC bouts did not alter the activation of Akt (Fig. 4A), training attenuated the suppression of P70S6K and RPS6 in MIN muscle (Fig. 4A). Importantly, repeated ECC bouts increased basal protein synthesis irrespective of cancer (Fig. 4B). Plasma IL-6 levels were negatively correlated with basal protein synthesis in MIN mice, and this relationship was not altered by repeated ECC bouts (Fig. 4C). However, the improvement in basal muscle protein synthesis by repeated ECC bouts was positively correlated to plasma IL-6 levels in MIN mice (Fig. 4D). Interestingly, there was a positive relationship between the improvements in basal muscle protein synthesis and COX activity in MIN mice (Fig. 4E). Collectively, these findings demonstrate that repeated ECC bouts improved basal muscle protein synthesis and mTORC1 signaling despite elevated plasma IL-6 levels in MIN mice, and there was a strong association between improvements in muscle oxidative capacity and protein synthesis during this adaptative response.

Fig. 4.

Repeated eccentric contraction (ECC) bouts improve basal protein synthesis and mechanistic target of rapamycin complex-1 (mTORC1) signaling in cachectic (Apc^Min/+) MIN mice. A: muscle mTORC1 signaling regulation after repeated ECC bouts. B: muscle protein synthesis after repeated ECC bouts. C: relationship between muscle protein synthesis and plasma interleukin-6 (IL-6) levels in MIN mice. D: relationship between changes in muscle protein synthesis after repeated ECC bouts and plasma IL-6 levels in MIN mice. E: relationship between changes in muscle protein synthesis and cytochrome-c oxidase (COX) activity after repeated ECC bouts in MIN mice. The activation of signaling molecules was determined by the ratio of phosphorylated and total protein expression when appropriate. For total protein expression values were corrected to GAPDH. Each gel contained samples from all groups (e.g., B6/MIN, CON/ECC) and data were normalized to the B6 CON group when appropriate. Data are means ± SE. A 2-way ANOVA was performed to determine differences between treatment groups when appropriate. Post hoc analyses were performed with Student–Newman–Keuls methods when appropriate. Pearson correlation coefficients (r) were computed to determine the relationship between 2 variables when appropriate. Statistical significance was set at P < 0.05. B6, C57BL/6; CON, control; NC, negative control; p, phospho-. Different letters are statistically different. ^&Main effect of repeated ECC bouts. ^#Main effect of MIN.

DISCUSSION

Physical activity and exercise interventions are attractive therapeutic strategies to prevent or treat wasting associated with cancer cachexia. There is emerging evidence that exercise training regimens can also promote clinically important outcomes (e.g., strength, performance) in cachectic cancer patients (33). Interestingly, neuromuscular electrical stimulation has more recently been investigated for therapeutic merit in patient populations where exercise is not a feasible option (30, 38). However, the mechanisms by which resistance training and/or stimulated contractions elicit beneficial adaptations in cachectic cancer patients have not been fully established. Therefore, the purpose of this study was to determine whether repeated stimulated eccentric contraction bouts affect basal muscle oxidative metabolism and protein synthesis during cancer cachexia. An additional purpose was to determine whether adaptations to repeated ECC bouts were associated with elevated plasma IL-6 levels. We report that repeated ECC bouts initiated after the onset of cachexia could attenuate muscle wasting despite the presence of a systemic cachectic environment, which includes elevated plasma IL-6. Repeated ECC bouts were accompanied by an improved cachectic muscle signature involving inflammation, oxidative metabolism, and protein synthesis. As expected in MIN mice, plasma IL-6 levels were negatively correlated with many systemic and muscle features of cachexia, but, unexpectedly, in ECC muscles from the same mouse plasma IL-6 levels were positively associated with improvements to myofiber area, oxidative capacity, and protein synthesis. Interestingly, this suggests that ECCs fundamentally improved the relationship between plasma IL-6 and cellular signaling regulating mass compared with noncontracted muscle exposed to the same systemic environment. We also report a positive relationship between muscle oxidative capacity and protein synthesis following repeated ECC bouts in MIN mice. Collectively, these results highlight the therapeutic potential of stimulated muscle contractions after the initiation of cachexia and provide evidence for an interaction between oxidative metabolism and protein synthesis in the adaptive response of cachectic skeletal muscle to ECCs.

Although the positive effects of exercise on health outcomes are appreciated for cancer prevention and survival, the cachectic cancer patient’s response to exercise and muscle contraction has not been widely investigated. Preclinical models have improved our understanding of the interaction between exercise, muscle contraction, and the systemic cachectic environment during cancer. In the present study, we report that stimulated contractions can improve cellular signaling associated with muscle wasting independent of changes in the systemic cachectic environment. This is in line with our previous reports that treadmill exercise training can prevent muscle atrophy and wasting mechanisms while in the presence of a cancer environment and systemic IL-6 expression (45, 53, 54). Genetic and pharmaceutical modulation of muscle IL-6 signaling through the gp130 receptor and its downstream effectors can prevent atrophy in several preclinical models of cancer cachexia, including the MIN mouse (6, 7, 37, 43). We found that improvements in the cachectic muscle phenotype with repeated ECC bouts coincided with reduced activation of downstream IL-6/gp130 signaling (STAT3, AMPK, ACC) in MIN mice. The activation of these signaling molecules by systemic IL-6 has been linked to the upregulation of proteasomal and lysosomal systems (52, 53). Although we did not measure indices of protein breakdown in the present study, we have previously found that single and repeated ECC bouts did not affect select “atrogene” protein expression (21, 23). In contrast, 2-wk treatment with pyrrolidine dithiocarbamate (PDTC), a systemic STAT3 and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) inhibitor, improved muscle protein turnover regulation through the activation of protein synthesis and attenuation of protein breakdown (37). Furthermore, a single PDTC injection was sufficient to increase basal and ECC-induced protein synthesis and mTORC1 signaling in cachectic mice (21, 37, 44). Given that repeated ECC bouts were not sufficient to fully rescue downstream IL-6/gp130 signaling and muscle wasting, further research is necessary to determine whether combining pharmacological and/or nutraceutical interventions can further enhance the adaptive response to repeated ECC bouts.

Muscle oxidative capacity is reduced during the progression of cachexia and coincides with disrupted mitochondrial quality control mechanisms (9, 51, 54). Although chronically elevated plasma IL-6 levels are sufficient to decrease muscle mitochondrial content and function in healthy and tumor-bearing mice (45, 49), treadmill exercise training before the onset of cachexia can prevent IL-6 suppression of muscle oxidative metabolism (45, 54). Unexpectedly, we have previously found that repeated ECC bouts improved myofiber succinate dehydrogenase (SDH) enzyme activity in MIN mice (23). Consistent with these findings, we report that repeated ECC bouts increased muscle cytochrome-c oxidase (COX) enzyme activity, an established measure of mitochondrial content, in both healthy and MIN mice. Furthermore, improved muscle COX activity with repeated ECC bouts was positively correlated with plasma IL-6 levels in MIN mice, suggesting that cachectic muscle retains plasticity to contraction despite the presence of a systemic wasting environment. Although mitochondrial remodeling is commonly associated with endurance exercise training (15), there is evidence that resistance exercise training can improve mitochondrial respiratory function in human skeletal muscle (42). Mitochondrial content and function are regulated through dynamic quality control mechanisms related to biogenesis, fission, fusion, and mitophagy (58), which are all sensitive to muscle contraction. Repeated ECC bouts were not sufficient to rescue the expression of PGC-1α; however, the expression of autophagy-related proteins (LC3B-II, p62) was reduced by contraction in cachectic MIN muscle. Although we did not specifically measure autophagy flux in the present study, previous investigations have indicated that an accumulation of p62 protein in the muscle of cancer patients and preclinical models may be related to decreased autophagic rate or accumulation of unprocessed autophagosomes (2, 40, 41). Exercise and pharmaceutical interventions that activate autophagy flux can protect muscles from wasting during cancer cachexia progression (41). Thus, the acute activation of autophagy with exercise or stimulated contractions may serve to enhance the removal of damaged proteins and organelles such as mitochondria in cachectic muscle during training (26, 58). Additional research is needed to establish whether improved autophagy regulation can impact muscle oxidative metabolism and protein synthesis in cachectic muscle. Furthermore, research is also needed to directly link enhanced muscle oxidative capacity to improvements in basal protein synthesis during cachexia. Since mitochondria integrate various metabolic pathways to generate critical intermediates required for the synthesis of cellular biomass, whether disrupted oxidative metabolism serves to impede the growth response in healthy and cachectic skeletal muscle warrants further investigation.

The suppression of basal protein synthesis and mTORC1 signaling is correlated with the degree of muscle wasting during the progression of cancer cachexia (52, 53). Therapeutic interventions that enhance basal protein synthesis regulation are therefore an attractive strategy to combat muscle wasting with cancer. Muscle contraction is also a potent stimulus for the acute induction of protein synthesis and mTORC1 signaling in healthy skeletal muscle (reviewed in 32, 50). We have previously found that cachexia disrupted the anabolic response to concentric muscle contractions induced by low-frequency electrical stimulation (44), whereas a single ECC bout elicited by high-frequency electrical stimulation induced mTORC1 signaling and protein synthesis in tumor-bearing mice (21, 46). The differential responses observed 3 h after contraction are likely attributed to the sustained activation of AMPK in muscles performing concentric contractions (21). We previously found that repeated ECC bouts attenuated myofiber atrophy, which coincided with reduced AMPK phosphorylation in cachectic muscle (23). We extend these findings by demonstrating that this reduction in AMPK is accompanied by improved regulation of autophagy/mitophagy, mTORC1, and protein synthesis during cancer cachexia progression. Although AMPK is an established suppressor of mTORC1 (27, 35), how this regulatory function of AMPK impacts cancer-induced muscle wasting has not been fully resolved (20, 39, 40). Nonetheless, our present and previous observations collectively suggest that stimulated contractions induce the acute activation of AMPK (21), which then improves basal oxidative capacity and relieves the suppression of mTORC1 and protein synthesis in cachectic skeletal muscle (23). Interestingly, the dysregulation of Akt in cachectic muscle was not improved after repeated ECC bouts, which is similar to our previous observations after treadmill exercise training (53). Altogether, these findings may implicate mTORC2 in the dysregulation of Akt during cancer cachexia (34) and suggest that the beneficial effects of exercise training or stimulated contractions on mTORC1 signaling do not require intact Akt signaling. Interestingly, we have recently reported that cancer disrupts diurnal variations in protein synthesis regulation, with cachectic muscle being more susceptible to changes in feeding and activity behaviors (14). Thus, it is interesting to speculate whether repeated ECCs could increase muscle’s sensitivity to nutrients and/or restore diurnal fluctuations in gene expression during cancer cachexia progression. Given that small differences in daily protein synthesis capacity may contribute to muscle wasting during cancer cachexia, future studies examining whether exercise and/or stimulated contractions improve the regulation of protein synthesis and mTORC1 signaling in fasted and fed conditions are warranted.

In summary, we report that skeletal muscle retains anabolic and metabolic plasticity to repeated ECC bouts while in a systemic wasting environment associated with cancer cachexia. We found that repeated ECC bouts after the initiation of cachexia improved aberrant muscle signaling involving inflammation, oxidative metabolism, and protein synthesis, which are established disruptions with cancer-induced wasting. Plasma IL-6 levels in MIN mice were negatively associated with many cachectic muscle signatures, which is in line with previously published results. Interestingly, we report that repeated ECC bouts improved the relationship between plasma IL-6 and cellular signaling regulating muscle mass. After 2 wk of repeated ECC bouts, plasma IL-6 levels were positively associated with improvements to myofiber area, oxidative capacity, and protein synthesis. These findings highlight the therapeutic potential of stimulated contractions after the initiation of cachexia and demonstrate that repeated ECC bouts can alter the cachectic muscle phenotype independent of systemic wasting. Importantly, we also report a positive relationship between muscle oxidative capacity and protein synthesis following repeated ECC bouts in MIN mice. In light of these findings, further research is warranted to determine the extent and duration of these adaptations to contraction. Furthermore, there is a critical need to understand the mechanisms linking improved oxidative metabolism and protein synthesis as a therapeutic target to prevent cancer-induced muscle wasting. From a clinical perspective, defining the benefits of stimulated muscle contractions will improve our efforts for treating the cancer patient given the complex nature of cachexia.

GRANTS

This work was supported by National Institutes of Health Grants R01 CA-121249 (National Cancer Institute) and P20 RR-017698 (National Institute of General Medical Sciences) (J.A.C.), a SPARC Graduate Research Grant from the Office of the Vice President for Research at the University of South Carolina (J.P.H.), and an ACSM Foundation Research Grant from the American College of Sports Medicine Foundation (J.P.H.). J.P.H. was supported by a Donna and Andrew Sorensen Graduate Student Fellowship in Cancer Research (University of South Carolina, Center for Colon Cancer Research).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.P.H. and J.A.C. conceived and designed research; J.P.H. and D.K.F. performed experiments; J.P.H. analyzed data; J.P.H., D.K.F., H.-J.K., X.W., E.C.G., and J.A.C. interpreted results of experiments; J.P.H. prepared figures; J.P.H. drafted manuscript; J.P.H., D.K.F., H.-J.K., X.W., E.C.G., and J.A.C. edited and revised manuscript; J.P.H., D.K.F., H.-J.K., X.W., E.C.G., and J.A.C. approved final version of manuscript.