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High-Frequency Stimulation on Skeletal Muscle Maintenance in Female Cachectic Mice

SATO, SHUICHI1,2; GAO, SONG1; PUPPA, MELISSA J.1; KOSTEK, MATTHEW C.1; WILSON, L. BRITT3; CARSON, JAMES A.1

Author Information
Medicine & Science in Sports & Exercise: September 2019 - Volume 51 - Issue 9 - p 1828-1837
doi: 10.1249/MSS.0000000000001991

Abstract

Cancer cachexia, characterized by unintentional body weight loss due to cancer, affects up to 70% of cancer patients and accounts for up to 40% of cancer patients’ death (1–3). Although the degree of cachexia is associated with cancer patient morbidity and mortality, the management of the condition is often undiagnosed and overlooked (2). Cancer cachexia is also associated with muscle weakness, loss of physical function, and reduced tolerance to cancer therapy, which has a significant effect on quality of life of cancer patients (1,3,4). Although several therapeutic interventions have been examined in the past, no guideline to counteract cancer cachexia has been established yet (1). Thus, more research to develop therapeutic intervention to counteract cachectic condition in cancer patients is warranted.

Resistance exercise is a mode of exercise that accelerates myofibrillar protein synthesis leading to an increase in muscle mass, and it is known to attenuate muscle wasting because of bed rest (5), HIV infection, and age-related sarcopenia (6) in humans. Although few clinical trials have been conducted, preclinical evidence suggests that resistance exercise is a promising intervention to counteract cancer cachexia. For example, mechanical loading caused muscle hypertrophy in tumor-bearing rats (7), and weighted ladder climbing before and after tumor inoculation can attenuate muscle mass loss in Walker-256 tumor-bearing rats (8). Two weeks of resistance training mimicked by electric stimulation can increase muscle mass and protein content in mice bearing the colon-26 adenocarcinoma (4). In addition, we have also demonstrated that 2 wk of high-frequency electric stimulation (HFES) can increase muscle mass and mTORC1 signaling in male tumor-bearing ApcMin/+ (Min) mice (9,10). However, female Min mice demonstrate differential sensitivity to interleukin-6 (IL-6) and cachexia development compared with the male mice (11,12), and the female Min mouse response to HFES is not known. Regarding cancer patients, a meta-analysis determined that resistance training is effective for maintaining lean body mass and muscular function in cancer patients, but the stage of cachexia was not identified at the initiation of intervention (13). Thus, there is accumulating evidence for resistance exercise to manage lean body mass loss in cancer patients. However, recent studies suggest that sex dimorphism exists during the development of cancer cachexia in rodents (12) and in humans (14), which may affect the utility of resistance exercise when evaluating outcomes across males and females.

The majority of published studies examining cancer cachexia in preclinical models have either not reported sex differences or only studied males, as sex was often thought to be a confounding variable. In general, females have more type I and type IIA fibers compared with males, which parallels the lower contractile velocity in females compared with that in males (15). The sex difference on muscle size and function in response to long-term resistance-type training is equivocal. Some studies have reported no difference on the relative strength gains between men and women independent of age (16,17), and others showed a difference (18,19). With higher prevalence in slow-twitch fibers with high-oxidative capacity, females may have an advantage on endurance and recovery in response to fatigue and muscle tetanus, respectively (15). In support of this concept, the magnitude of muscle damage induced by eccentric exercise is less in females than males (20), and muscle-derived stem cells from females have higher muscle regeneration efficiency at regenerating injured skeletal muscle compared with males (21). By contrast, older women exhibit a more pronounced suppression of exercise-induced anabolic signaling than older men, although basal muscle protein synthesis (MPS) rate is greater (22). Because skeletal muscle’s regeneration and exercise response have clear potential to be affected by sex, cancer cachexia’s effect on the female response to eccentric contraction warrants further investigation. Previously, our laboratory using male Min mice has demonstrated that 2 wk of repeated bouts HFES can increase myofiber cross-sectional area (CSA) regardless of a systemic proinflammatory environment being present in cachectic mice (10). Therefore, the purpose of this study was to establish if female cachectic mice maintain the ability to respond to HFES through attenuated muscle mass loss and anabolic signaling activation. We hypothesized that female cachectic mice would attenuate the loss of muscle mass and myofiber CSA with HFES as seen in male mice.

METHODS

Animals

Male Min mice on a C57BL/6 background were originally purchased from Jackson Laboratories (Bar Harbor, ME) and crossed with C57BL/6 female mice (9,12). Both female wild-type (WT) littermate and Min mice were used in this study. All mice were provided with standard rodent chow and water ad libitum in standard cages. All animal experimentation was approved by the University of South Carolina’s Institutional Animal Care and Use Committee. The overall study consisted of three separate experiments using different cohorts of female mice (see Tables 1 and 2) to examine repeated or an acute bout of HFES.

TABLE 1
TABLE 1:
Descriptive data of female WT and Min mice in multiple bouts of HFES over a period of 2 wk (experiment 1).
TABLE 2
TABLE 2:
Descriptive data of female WT and Min mice in a single bout of HFES (experiments 2 and 3).

Experiment 1 examined the effect of repeated HFES bouts for 2 wk in WT (n = 6) and Min (n = 6) female mice between 16 and 18 wk of age. Experiment 2 examined an acute, single bout of HFES treatment in WT (n = 4) and Min (n = 4) female mice at 18–20 wk of age. The mice were sacrificed at 3, 14, or 24 h post-HFES to measure plasma creatine kinase (CK) levels and 48 h post-HFES for histological analysis. Experiment 3 examined three time points (3, 14, or 24 h) after an acute bout of HFES in WT (n = 16) and Min (n = 16) female mice at 18–20 wk of age. Mice in this cohort did not differ in cachectic index reflected by their body weights and percent of body weight loss. Mice performed a single bout of HFES and were then sacrificed at 3, 14, or 24 h post-HFES.

Surgical application of electrodes and repeated HFES protocol

Stimulating needle electrodes were applied in the hind limbs of each mouse as previously described (10,23). All animals were anesthetized with 2% isoflurane, and their sciatic nerves were stimulated posterior to the knee via subcutaneous needles positioned proximal to the bifurcation of the sciatic nerve. Thus, this stimulation evoked a contraction of all muscles of the lower limb. The electrical impulse was generated from a square pulse stimulator (Model S88 Grass Technologies; Astro-Med, Inc., West Warwick, RI), and the frequency was fixed at 100 Hz to evoke a complete tetanic contraction. The voltage of the stimulus was increased until no further plantarflexion was observed (6 to 14 V). This protocol caused a shortening of the gastrocnemius, soleus, and plantaris, while evoking a lengthening (eccentric) contraction of the tibialis anterior (TA) muscle. In views of strengthening muscle, the effect of eccentric contraction is greater than that of concentric concentration, and in fact a previous study using this protocol showed that the stimulation of the soleus did not alter the relative phosphorylation state of the p70S6K in healthy rodents (23). Thus, we used the TA to examine the effect of HFES on the cachectic muscle in mice.

The HFES protocol used in this study was previously described (10). Briefly, the muscle contractions lasted 3 s and were followed by a 10-s rest period, during which the foot was passively returned to the neutral position from the plantar flexed position. Ten sets of 6 repetitions with a 50-s rest between sets were performed. This resulted in a total of 60 contractions with 180 s of actual contraction time. Thus, the entire contraction period lasted approximately 22 min. After HFES, the mice were allowed to recover fully before moving back to their cages.

Cage activity monitoring

During multiple bouts of HFES, a subset of mice (n = 5) were single housed and placed in activity monitor cages (Opto-M3 Activity Meter; Columbus Instruments, Columbus, OH). Physical activity was measured for 12 h during the dark cycle (7:00 pm–7:00 am), and the number of beams crossed in an xy plane was recorded for two consecutive nights. Food consumption was also recorded during this time.

Tissue collection

At the end of the study, mice were anesthetized with a subcutaneous injection of ketamine/xylazine/acepromazine cocktail (1.4 mL·kg−1 body weight), and TA and gonadal fat were removed and snap frozen in liquid nitrogen. TA was cut at the midbelly; the proximal part of TA was used for biochemical analysis, whereas the distal part of TA was used for histological analysis. Tibia length was measured as an indicator of animal body size and a correction factor for skeletal muscle weights.

Plasma CK assay

Plasma CK levels were determined on three to four mice at each time point based on the manufacturer’s instructions. Briefly, blood was taken from the retro-orbital sinus under anesthesia. Eight microliters of plasma was assayed spectrophotometrically at a wavelength of 340 nm for CK activity using a commercially available kit (Sekisui CK-SL, Sekisui Diagnostics, LLC, Framingham, MA).

Immunohistochemistry for myosin heavy chain types IIa and IIb

For immunohistochemistry staining, transverse sections (10 μm) were cut from the distal part of TA on a cryostat at −20°C. After fixation in cold acetone, they were blocked in 10% normal goat serum (Vector Laboratories, Burlingame, CA) in phosphate-buffered saline (PBS) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies (SC-71 for type IIa; BF-F3 for type IIb; Iowa Hybridoma Bank). After washing with PBS, secondary antibodies (Vector Laboratories) were applied for 1 h at 37°C, and sections were washed in PBS. Avidin–biotin complex system (Vector Laboratories) was used to detect the biotinylated secondary antibody and visualized by 3,3′-diaminobenzidine solution (Vector Laboratories). Digital pictures were taken at a ×200 magnification with a Motic spot camera (Motic, China). Myofiber CSA was quantified with Image J (National Institutes of Health, Bethesda, MD) by an individual blinded to the treatment. At least 60 (type IIa) and 150 (type IIb) fibers were quantified to determine CSA.

Succinate dehydrogenase stain

Succinate dehydrogenase (SDH) staining was performed as previously described to characterize mitochondrial enzyme function in the skeletal muscle (10). Briefly, transverse sections (10 μm) were cut from the midbelly of the medial TA on a cryostat at −20°C, and slides were stored at −80°C until SDH staining was performed. The frozen sections were dried at room temperature for 10 min. Sections were incubated in a solution made up of 0.2 M phosphate buffer (pH 7.4), 0.1 M MgCl2, 0.2 M succinic acid, and 2.4 mM nitroblue tetrazolium at 37°C for 45 min. The sections were then washed in deionized water for 3 min, dehydrated in 50% ethanol for 2 min, and mounted for viewing with mounting media. Digital images were captured and traces by Image J. At least 60 fibers were traced for CSA, and 700 fibers were counted for the percentage of frequency of each section at a ×200 magnification in a blinded fashion. The percentage of SDH dark-stained (high activity) fibers was then determined based on a criterion integrated optical density.

Western blotting

Western blotting was performed based on the manufacturer’s instructions. All antibodies were purchased from Cell Signaling (Danvers, MA) and incubated at dilutions of 1:1000 to 1:2000 in 5% Tris-buffered saline with 0.1% Tween 20 milk overnight at 4°C for primary antibodies and 1:2000 to 1:5000 dilutions for 1 h in 5% Tris-buffered saline with 0.1% Tween 20 milk for secondary antibody. Enhanced chemiluminescence (GE Healthcare, Chicago, IL) was used to visualize the antibody–antigen interactions, and the image was developed by autoradiography (Kodak Biomax MR, Carestream Health, Rochester, NY). Image J was used for densitometry analysis.

Myofibrillar protein synthesis

The rate of myofibrillar protein synthesis was determined by the 2H5-l-phenylalanine flooding method using ultra performance liquid chromatography tandem mass spectrometry (Waters, Milford, MA) (24). 2H5-l-Phenylalanine was obtained from Cambridge Isotope Laboratories (Andover, MA). Thirty minutes before sacrifice, all mice received an intraperitoneal injection of 150 mM D5-F in a 75-mM NaCl solution at a dose of 0.02 mL·g−1 body weight.

RNA isolation, cDNA synthesis, and real-time PCR

RNA isolation, cDNA synthesis, and real-time PCR were performed based on the manufacturer’s instructions. Fluorescence-labeled probe for IGF-1 was purchased from Applied Biosystems (Foster City, CA) and quantified with TaqMan Universal master mix. Data were analyzed by ABI software using the cycle threshold, which is the cycle number at which the fluorescence emission is midway between detection and saturation of the reaction.

Statistical analysis

Values are described as mean ± SEM. A repeated-measured two-way ANOVA was used to determine the effect of mouse genotype between WT and Min mice and age on body weights and physical activity levels. Similarly, the effect of mouse genotype and HFES on TA weights and myofibrillar protein synthesis was analyzed by the same statistical method. A one-way ANOVA was used to determine the effect of cachexia and HFES on high SDH activity, protein, and mRNA expressions in multiple bouts of HFES (experiment 1). Post hoc analyses were performed using the Student–Newman–Keuls method when appropriate. A Student t-test was used to determine the difference in peak body weights, percent body weight changes, gonadal fat, tibia length, spleen mass, physical activity (experiment 1), and CK levels (experiment 2) between WT and Min mice. A paired t-test was used to determine the difference of protein expression between control and HFES-performed legs in Min mice (experiment 3). The χ2 analysis was used to determine shifts on myofiber frequency distribution in proportion of small fibers and proportion of large fibers between WT and Min mice and control and HFES-performed legs in Min mice (experiment 1). Significance was set at P < 0.05.

RESULTS

Experiment 1

This experiment examined repeated HFES bouts over a 2-wk period during cachexia development in female WT and Min mice.

Body weights, tibia length, and organ weights

Between 16 and 18 wk of age, WT and Min mice performed seven bouts of HFES (6 reps X 10 sets, every other day) for approximately 2 wk (Fig. 1A). Body weights were measured before and after the HFES treatment period. At the beginning of the HFES, Min mice had initiated body weight loss and continued to lose body weight between 16 and 18 wk of age. WT mice maintained their body weights during the 2 wk of HFES (Table 1). Min mice body weights were less than WT before and after multiple bouts of HFES; however, no difference was observed in tibia length between WT and MIN, indicating there was no difference in body size. TA muscle, gonad fat, and spleen mass were weighed at the time of sacrifice. Min mice had less gonadal fat (−57%) and larger spleens (~3.3-fold) than WT mice at sacrifice (Table 1).

FIGURE 1
FIGURE 1:
(Experiment 1) Effects of multiple bouts of HFES on TA myofiber CSA in female WT and Min mice. A, Experimental design. Mice (16 wk of age) assigned this protocol performed seven bouts of HFES over a period of 2 wk. Mice were sacrificed 48 h after the last session of HFES. Although the mice had access to food and water ad libitum after the last session, they were fasted 5 h before the sacrifice. B, Frozen sections of TA muscle were cut (~10 μm thickness) at the midbelly, stained with type IIa (SC-71) and type IIB (BF-F3) antibodies. Representative type IIa and type IIb fiber staining of TA muscles of female WT and Min mice. (a) Type IIa control in WT mice, (b) type IIa Con in Min mice, (c) type IIa with HFES in Min mice, (d) type IIb Con in WT mice, (e) type IIb Con in Min mice, and (f) type IIb with HFES in Min mice. Total magnification was ×200. Con, control. C and D, Type IIa (C) and type IIb (D) myofiber size frequency distributions in WT Con and Min Con muscles. E and F, Type IIa (E) and type IIb (F) myofiber size frequency distributions in Min Con and Min with HFES muscles. Significance was set at P < 0.05. †Significantly different from WT Con. *Significantly different from contralateral nonstimulated control (Min Con).

Physical activity and food intake

Voluntary physical activity (7:00 pm–7:00 am) was reduced in Min mice when compared with WT mice, but food intake was not reduced in Min mice (see Table, Supplemental Digital Content 1, which illustrates physical activity and food intake of mice, http://links.lww.com/MSS/B571).

TA muscle mass and fiber CSA

There was a main effect of genotype in TA muscle mass, demonstrating that the TA muscle of WT mice was larger than that of Min mice (Table 1). Also, there was a main effect of HFES to increase TA muscle mass above the contralateral control regardless of genotype (Table 1). Myofiber CSA was determined from frozen sections taken at the TA midbelly and stained immunohistochemically using type IIa (SC-71) and type IIb (BF-F3) antibodies (Fig. 1B). Min type IIa and type IIb myofiber mean CSA was decreased when compared with WT mice. HFES attenuated Min mean CSA loss in both type IIa and type IIb fibers (Fig. 1B). The examination of muscle fiber size distribution can often uncover shifts in the muscle related to heterogeneous fiber CSA that can be masked by only examining the mean CSA. The Min TA muscle increased the percentage of small-diameter type IIa fibers (<500 μm2, 2% vs 43%) and reduced the percentage of large-diameter type IIa fibers (>1300 μm2, 13% vs 0%) when compared with WT mice (Fig. 1C). Similarly, Min type IIb fibers had more small-diameter fibers (<1500 μm2, 8% vs 45%) and less large-diameter fibers (>3000 μm2, 14% vs 1%) (Fig. 1D). HFES decreased the percentage of Min type IIa small-diameter fibers (<450 μm2, 3% vs 28%) and increased large-diameter Min type IIa fibers (>1050 μm2, 5% vs 0.4%) compared with control muscle (Fig. 1E). Likewise, HFES decreased the percentage of Min type IIb small-diameter fibers (<1250 μm2, 15% vs 23%) and increased Min type IIb large-diameter fibers (>2750 μm2, 8% vs 3%) when compared with control muscle (Fig. 1F).

Muscle oxidative capacity

SDH, known as complex II in the respiratory chain in the mitochondria, is a marker of skeletal muscle oxidative capacity at the fiber level. It has been established that the progression of cancer cachexia can decrease Min muscle oxidative capacity (25,26). We performed SDH staining to investigate if HFES could increase female Min muscle SDH activity (Fig. 2A). Min mice exhibited reduced muscle SDH activity compared with WT mice (20.3% ± 1.0% vs 9.81% ± 0.8%, Fig. 2B). Similarly, the percentage of small-diameter SDH-positive fibers were increased (<400 μm2, 6% vs 23%) and large-diameter SDH-positive fibers were decreased (>800 μm2, 55% vs 9%) in Min mice when compared with WT mice. HFES attenuated the SDH activity loss (9.81% ± 0.8% vs 13.9% ± 0.5%, Fig. 2B) in Min muscle (Fig. 2C). Furthermore, HFES counteracted the reduction in small-diameter SDH-positive fibers (<400 μm2, 23% vs 10%) and increased the incidence of large-diameter SDH-positive fibers (>800 μm2, 10% vs 19%) (Fig. 2D).

FIGURE 2
FIGURE 2:
Effects of multiple bouts of HFES on muscle oxidative capacity in female WT and Min mice. Frozen sections of TA muscle were cut (~10 μm thickness) at the midbelly, and SDH staining was performed. A, Representative SDH staining of TA muscles. (a) WT control, (b) Min Con, and (c) Min with HFES. Total magnification was ×200. B, Percentages of SDH dark-stained myofibers. C, Myofiber frequency size distribution of high SDH activity on TA muscle in female WT and Min mice. D, Myofiber frequency size distribution of high SDH activity on TA muscle with or without HFES in female Min mice. Values are presented as mean ± SEM, and statistical significance was set at P < 0.05. †Significantly different from WT Con. *Significantly different from nonstimulated control (Min Con).

Molecules that contribute to muscle protein turnover

To explore the basal and HFES regulation of muscle protein turnover, alterations in the expression of 4EBP1, AMPK, and Atrogin-1 were examined by Western blot (Fig. 3A). 4EBP1 and AMPK activities represent the ratio of phosphorylated and total protein expression. All data are normalized to nonstimulated contralateral control TA muscle in WT mice. Total 4EBP1 and AMPK protein levels were not changed by HFES (Fig. 3B). There was a trend to decrease Min 4EBP1 activity (P = 0.064, Fig. 3B), and HFES did not alter the levels of 4EBP1 activity. When IGF-1 mRNA was quantified, no change was observed in cachexia or in cachexia with HFES (Fig. 3C). AMPK activity was increased in the Min, and HFES did not change the activity levels (Fig. 3B). Atrogin-1 expression demonstrated a trend to increase activity in Min muscle (P = 0.079), and HFES did not change atrogin-1 expression (Fig. 3B).

FIGURE 3
FIGURE 3:
Effects of multiple bouts of HFES on the basal levels of protein and mRNA expression for anabolic and catabolic signaling in female WT and Min mice. Western blots and qRT-PCR were performed to examine the changes of basal levels of protein and mRNA expressions, respectively, on TA muscles after 2 wk of multiple bouts of HFES. A, Representative immunoblots of key molecules in anabolic signaling (4EBP1) and catabolic signaling (AMPK and atrogin-1). GAPDH was used as loading control. B, Ratios of phosphorylated (p) and total forms of 4EBP1 and AMPK, and total atrogin-1 protein expression in TA muscles measured by densitometry analysis of immunoblots. C, qRT-PCR analysis of IGF-1 mRNA expression in TA muscles. Con, control. Values are presented as mean ± SEM, and statistical significance was set at P < 0.05. †Significantly different from WT Con.

Experiment 2

This experiment examined circulating CK levels after acute bouts of HFES in female WT and Min mice.

Plasma CK

CK levels were examined at 3- 14, 24, and 48 h post-HFES (Fig. 4A). CK levels were comparable at all time points except at 3 h post-HFES (see Fig. A, Supplemental Digital Content 2, which demonstrates the levels of CK in the plasma at different time points, http://links.lww.com/MSS/B572). Even at 3 h post-HFES, the CK level in Min mice was lower than that of WT mice. To evaluate the structural damage of myofibers, hematoxylin and eosin stain was performed on TA frozen sections. No structural damage was observed at 48 h post-HFES (see Fig. B, Supplemental Digital Content 2, which shows the myofiber CSA images stained by hematoxylin and eosin, http://links.lww.com/MSS/B572), and the number of central nuclei was comparable in both WT and Min mice (data not shown). These data provide evidence that cachexia did not interact with HFES to increase muscle damage in female Min mice, which is in line with our previous data in male mice (10).

FIGURE 4
FIGURE 4:
(Experiments 2 and 3) Effects of a single bout of HFES on muscle anabolic and catabolic signaling in female Min mice. Mice (18–20 wk of age) were sacrificed at 3, 14, 24, or 48 h post-HFES. Western blots were performed to examine the acute changes of protein expression in TA muscle at 3, 14, and 24 h after a single bout of HFES. The mice sacrificed at 3 h post-HFES were fasted at least 4 h before the HFES. The other groups of mice had access to food and water ad libitum after HFES, but they were fasted 5 h before sacrifice. A, Experimental design. B, MPS rate at 3 h post-HFES. C, (Top) Representative immunoblots of key molecules in anabolic signaling (4EBP1) and catabolic signaling (AMPK and atrogin-1) at 3 h post-HFES. Black dotted lines indicate that intervening lanes have been spliced out. (Bottom) Ratios of phosphorylated (p) and total forms of 4EBP1 and AMPK, and total atrogin-1 protein expression in TA muscle of female Min mice at 3 h post-HFES. D, (Top) Representative immunoblots of key molecules in anabolic signaling (4EBP1) and catabolic signaling (AMPK and atrogin-1) at 14 h post-HFES. (Bottom) Ratios of phosphorylated (p) and total forms of 4EBP1 and AMPK, and total atrogin-1 protein expression in TA muscle of female Min mice at 14 h post-HFES. E, (Top) Representative immunoblots of AMPK at 24 h post-HFES. (Bottom) Ratios of phosphorylated (p) and total forms of AMPK protein expression in TA muscle of female Min mice at 24 h post-HFES. GAPDH was used as loading control. Values are presented as mean ± SEM, and statistical significance was set at P < 0.05. *Significantly different from nonstimulated control. ‡Main effect of HFES. $Main effect of mouse genotype.

Experiment 3

This experiment examined the effect of an acute HFES bout on TA MPS regulation 3, 14, and 24 h postcontraction.

MPS

We first examined acute changes in MPS and associated signaling induced 3 h after an acute bout of HFES (Fig. 4A). Although the MPS was reduced overall in Min mice (main effect of genotype, P < 0.01), HFES was able to increase MPS in Min muscle, despite cachexia (P < 0.05) (Fig. 4B).

Protein synthesis regulation

Total 4EBP1 levels were not changed by acute HFES; however, HFES increased Min 4EBP1 activity by 17% at 3 h after (Fig. 4C), which returned to the baseline at 14 h post-HFES (Fig. 4D). AMPK activity was reduced by 34% at 3 h post-HFES (Fig. 4C). Interestingly, decreased AMPK activity was still present at 14 h post-HFES (Fig. 4D) and finally returned to the baseline at 24 h post-HFES (Fig. 4E). Atrogin-1 expression was reduced by 8% at 3 h post-HFES (Fig. 4C) and returned to the baseline at 14 h post-HFES (Fig. 4D).

DISCUSSION

There is accumulating evidence for a positive effect of resistance exercise on cancer-induced muscle mass loss (4,8,10,13). Although muscle wasting due to cancer occurs in both sexes, recent studies reveal that sexual dimorphism exists during the progression of the syndrome in rodents (12) and humans (14). Muscle fiber–type composition, oxidative capacity, and response to exercise are different between males and females (15,22). These reports led us to speculate that cachexia may differentially affect the female response to resistance exercise, when compared with the male response. Here, we aimed to determine whether HFES could reduce the muscle mass loss in female Min mice, which develops cachexia slowly compared with tumor-transplanted animals and, therefore, mimics the human process of muscle wasting due to cancer. To our knowledge, this is the first study that demonstrates female cachectic muscle responds to anabolic stimuli to attenuate muscle wasting by increasing mTOR signaling for MPS and decreasing AMPK activity for muscle degradation.

Resistance exercise training has been shown to be beneficial for increasing muscle mass and muscle strength in breast cancer patients with no adverse effects. However, related to our current research question, many cancer exercise studies are not able to establish if the patients are cachectic, as breast cancer is not a typical cancer to cause cachexia (27). Similar issues are present in exercise studies using preclinical models of cancer cachexia. Studies have often used research designs that implement the exercise or muscle contraction at the time of tumor cell transplantation, which is a cachexia prevention design. Two weeks of HFES in mice bearing the colon-26 adenocarcinoma increased muscle mass by 66% and protein content by 25% in the stimulated EDL compared with the contralateral nonstimulated one (4). Furthermore, 6 wk of functional overload by synergistic ablation in rats bearing Morris hepatoma MH7777 cells increased plantaris muscle mass by 24% compared with contralateral sham-operated muscle (7). Although these data show that loading skeletal muscle can prevent some muscle mass loss due to cancer, the ability to treat muscle that is already cachectic and understanding the response of cachectic muscle to a bout of loading or contraction were not tested in these studies. Furthermore, the mechanisms related to the preventative effect of loading were not established.

As noted above, 2 wk of HFES at the time of tumor inoculation can increase muscle mass in female mice bearing the colon-26 adenocarcinoma (4). Our data are consistent with their results. We report that 2 wk of HFES increased TA muscle mass by 5% in the Min mice. This increase in muscle size represented functional growth of both type IIa and type IIb myofibers, decreasing the percentage of small-diameter fibers and increasing the percentage of large-diameter fibers in both fiber types. However, muscle mass change in response to hypertrophic stimuli was attenuated in Min mice compared with WT, which is in agreement with the results of tumor-bearing rats with functional overloading (7). The regulation of muscle mass is determined by the balance between the rates of MPS and degradation (1). A prior study has shown increased ubiquitin mRNA and proteasome activity in cancer patients with cachexia compared with control subjects, which was also associated with disease stage and weight loss (28). The increased activity of the ubiquitin–proteasome pathway has been associated with cancer-induced muscle loss in several preclinical cancer cachexia models (7,29,30). Therefore, the attenuated response of muscle mass to HFES in female Min mice may be related to accelerated ubiquitin–proteasome activity.

Cachexia is associated with the loss of oxidative capacity in the male Min mice (25). The loss of oxidative capacity leads to the impairment of functional work capacity, resulting in early fatigue in cancer patients (31). In male Min mice, moderate-intensity exercise training can increase oxidative capacity and improve insulin sensitivity in IL-6-induced cachexia (32). We report that HFES in female Min mice attenuated cachexia-induced alterations in muscle oxidative capacity, which has been reported in male Min mice (25). Furthermore, HFES was sufficient to increase the number of high-oxidative capacity myofibers, which also occurs in male Min mice (10). Taken together, our results suggest that HFES increased oxidative capacity, which may be associated with the attenuated loss of myofiber size. However, the association of myofiber oxidative capacity and the prevention of cancer-induced muscle mass loss are still equivocal. Increased PGC-1α expression protected muscle atrophy induced by denervation by suppressing FoxO3 action and atrophy-specific gene transcription such as atrogin-1 and MuRF-1 (33), but PGC-1α overexpression failed to prevent Lewis lung carcinoma-induced muscle loss (34). In our current study, PGC-1α and oxidative enzyme protein expression were not examined. Therefore, further studies will be necessary to determine whether the increased oxidative capacity that occurs with 2 wk of repeated HFES is necessary to attenuate muscle loss in the Min mice.

Cancer-induced muscle wasting results from a reduction in protein synthesis combined with an increase in protein degradation (3,27). We have reported that reduced mTORC1 signaling is associated with decreased MPS and the development of cancer cachexia in Min mice (29). eIF4E-binding protein-1 (4EBP1) is a downstream target of mTOR, and its binding to eIF4E inhibits eIF4E’s binding to eIF4G, an adaptor protein to recruit the 40S subunit to the 5′-end of mRNA and coordinate the circularization of mRNA, limiting the translation initiation of protein synthesis. The phosphorylation of 4EBP1 due to anabolic stimuli allows 4EBP1 to dissociate from eIF4E and increase the eIF4E–eIF4G complex, thereby enhancing cap-dependent translation to increase protein synthesis (1,35). Mechanical loading, feeding, and grow factors can stimulate mTOR leading to increased levels of 4EBP1 phosphorylation (36). We report a trend for basal 4EBP1 phosphorylation to decrease with cachexia in female Min mice, and repeated bouts of HFES were not sufficient to change these levels. We then investigated if acute HFES could increase 4EBP1 phosphorylation. Although a single bout of HFES increased 4EBP1 phosphorylation in parallel with increased MPS at 3 h after muscle contraction, the response was reduced in Min mice.

AMPK, an energy sensor that controls energy homeostasis and metabolic stress, is activated under the energy deprivation such as exercise and starvation (37). We have reported that increased muscle AMPK activity is associated with the development of cancer cachexia in male Min mice (29). In skeletal muscle, AMPK activation can suppress MPS through reduced mTORC1 activity. AMPK can reduce mTORC1 activity either directly via raptor phosphorylation or indirectly via increasing the GAP activity of TSC2. Increased AMPK also leads to FoxO activation and the upregulation of autophagy, which are important factors to accelerate muscle protein breakdown (38). Interestingly, AMPK-deficient mice have been reported to have larger soleus mass with increased CSA of myofibers (39). Moderate-intensity exercise training can decrease AMPK activation in Min with IL-6-induced cachexia (32). Repeated HFES in the male Min mice can also reduce AMPK phosphorylation (10). Thus, we examined the effect of HFES on AMPK phosphorylation in female Min mice. Cachexia did increase female Min basal muscle AMPK phosphorylation, which was reduced at 3 h post-HFES. Therefore, our results suggest that increased anabolic signaling and attenuated catabolic signaling likely contribute to the female Min muscle response to HFES. It is important to note that HFES was able to decrease AMPK activity at 14 h post-HFES but not at 24 h post-HFES, while phosphorylation levels of 4EBP1 went back to the baseline at 14 h post-HFES. Because the mice that received multiple bouts of HFES were sacrificed at 48 h after the last HFES session, it is possible to assume that the effect of the 2-wk HFES on AMPK activity could not be seen in this experiment even if there was an effect after each HFES session. Further research is warranted to understand the downstream of AMPK such as Foxo and autophagy activation, which were not examined in this study but are important for the response to HFES, and our data suggest that these likely involve the regulation of both MPS and degradation.

In summary, sex dimorphism has been reported with the progression of cancer cachexia (12,14) and with the response of skeletal muscle to resistance-type exercise (18,19). Male and female differences have been reported for muscle damage induced by eccentric exercise (20). Sex has also been implicated in stem cell proliferation; muscle-derived stem cells from females have been reported to have higher muscle regeneration efficiency for regenerating injured skeletal muscle when compared with males (21). We have reported that repeated HFES in male mice can increase muscle mass, increase myofiber size in all fiber types, and increase the number of myofibers exhibiting high SDH activity (10). Here we report that the female Min mouse response to 2 wk of repeated HFES is similar to the male response. HFES successfully increased muscle mass with an increase in large-diameter myofibers in both type IIa and type IIb fibers, associated with increased oxidative capacity in the female Min mice. The effect of HFES resulted from, at least in part, increased anabolic signaling and attenuated AMPK-led catabolic signaling. On the basis of our findings, further research is warranted to determine whether the positive effects reported for a stimulated muscle in a cancer-induced cachectic environment can be replicated with whole-body exercise that recruits a large amount of muscle mass, which may provide a stimulus to improve the overall health of cachectic cancer patients.

This work was supported by the National Institutes of Health grant (R01 CA-121249A501; National Cancer Institute) to J. A. C. and the Louisiana Board of Regents Support Fund Competitive grant (LEQSF(2017-20)-RD-A-22) to S. S. The authors acknowledge Tia Davis for her assistance with mouse breeding. In addition, the authors thank Dr. Raja Fayad for his valuable suggestions and contribution to the manuscript.

The results of the present study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The results of the study do not constitute endorsement by the American College of Sports Medicine. Shuichi Sato, Song Gao, Melissa J. Puppa, Matthew C. Kostek, L. Britt Wilson, and James A. Carson declare that they have no conflict of interest.

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Keywords:

CACHEXIA; mTOR; PROTEIN SYNTHESIS; RESISTANCE EXERCISE

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