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

Abstract

Cancer cachexia, unintentional body weight loss due to cancer, affects patients’ survival, quality of life, and the response to chemotherapy. While exercise training is a promising intervention to prevent and treat cancer cachexia, our mechanistic understanding of cachexia’s effect on contraction-induced muscle adaptation has been limited to the examination of male mice. Since sex can impact muscle regeneration and the response to contraction in humans and mice, the impact of cachexia on the female response to eccentric contraction warrants further investigation.

Purpose:

The purpose of this study was to determine if high frequency electric stimulation (HFES) could attenuate muscle mass loss during the progression of cancer cachexia in female tumor bearing mice.

Methods:

16~18-week female wild-type (WT) and Apc^Min/+ (Min) mice performed either repeated bouts or a single bout of HFES (10 sets of 6 repetitions, ~ 22 minutes), which eccentrically contracts the Tibialis Anterior (TA) muscle. TA myofiber size, oxidative capacity, anabolic signaling and catabolic signaling were examined.

Results:

Min had reduced TA muscle mass and IIa and IIb fiber size compared to WT. HFES increased muscle weight and the mean cross-sectional area of type IIa and IIb fibers in WT and Min mice. HFES increased mTOR signaling, myofibrillar protein synthesis, and attenuated cachexia-induced AMPK activity. HFES attenuated the cachexia-associated decrease in skeletal muscle oxidative capacity.

Conclusion:

HFES in female mice can activate muscle protein synthesis through mTOR signaling and repeated bouts of contraction can attenuate cancer-induced muscle mass loss.

Introduction

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 impact on quality of life of cancer patients (1, 3, 4). Despite several therapeutic interventions have been examined in the past, no guideline to counteract cancer cachexia has not 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 of muscle mass and is known to attenuate muscle wasting due to 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 prior to 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). Additionally, we have also demonstrated that 2 weeks of high frequency electric stimulation (HFES) can increase muscle mass and mTORC1 signaling in male tumor bearing Apc^Min/+ (Min) mice (9, 10). However, female Min mice demonstrate differential sensitivity to IL-6 and cachexia development compared to 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 sex dimorphism exists during the development of cancer cachexia in rodents (12) and in humans (14), which may impact 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 the 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). On the other hand 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). Since skeletal muscle’s regeneration and exercise response have clear potential to be affected by sex, cancer cachexia’s impact on the female response to eccentric contraction warrants further investigation. Previously our laboratory using male Min mice has demonstrated that two weeks of repeated bouts HFES can increase myofiber CSA regardless of a systemic pro-inflammatory 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 Apc^Min/+ (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 littermate (WT) 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 3 separate experiments using different cohorts of female mice (see Tables 1 & 2) to examine repeated or an acute bout of high frequency electric stimulation (HFES).

Table 1.

Descriptive data of female wild-type (WT) and Apc^Min/+ (Min) mice in multiple bouts of high-frequency electric stimulation (HFES) over a period of 2 weeks (Experiment 1).

		WT (n = 6)		Min (n = 6)
Peak BW (g)		22.2 ± 0.3		21.0 ± 0.3
BW (g)
at 16 weeks of age		22.0 ± 0.3		20.7 ± 0.3$
at 18 weeks of age		22.2 ± 0.3		19.8 ± 0.5$,*
% BW change at 16 weeks of age		–		− 1.5 ± 0.4†
% BW change in 2 weeks		0.2 ± 1.0		− 4.6 ± 1.8†
% BW change vs. Peak BW		–		− 6.0 ± 1.9†
Tibialis anterior (mg)
Control		36.7 ± 1.1		29.8 ± 2.2†
HFES		41.2 ± 0.9*		31.2 ± 2.1†
Gonad fat (mg)		205 ± 27		97 ± 28†
Spleen (mg)		81 ± 3		350 ± 29†
Tibia length (mm)		17.1 ± 0.1		16.9 ± 0.1

Values are means ± SEM and statistical significance was set at p < 0.05. BW: Body weight. % BW change at 16 weeks of age: [(BW at 16 weeks – Peak BW) / Peak BW ×100]. % BW change in 2 weeks: [(BW at 18 weeks – BW at 16 weeks) / BW at 16 weeks ×100]. % BW change vs. Peak BW: [(BW at 18 weeks – Peak BW) / Peak BW ×100]. HFES: High-frequency electric stimulation.

^†indicates significantly different from age-matched WT.

^$indicates main effect of mouse genotype.

^*indicates significantly different from 16-week age mice within the same group.

^&indicates significantly different from control within the same group.

Table 2.

Descriptive data of female wild-type (WT) and Apc^Min/+ (Min) mice in a single bout of high-frequency electric stimulation (HFES, Experiment 2 & 3).

		WT (n = 20)		Min (n = 20)
Peak BW (g)		20.9 ± 0.4		21.0 ± 0.4
BW (g)		20.9 ± 0.4		19.0 ± 0.4†
% BW change vs. Peak BW		0.0 ± 0.0		− 9.7 ± 1.1†
Tibialis anterior (mg)
Control		39.2 ± 1.2		30.8 ± 1.9$
HFES		40.5 ± 1.5		30.7 ± 1.9$
Gonad fat (mg)		303 ± 45		53 ± 19†
Spleen (mg)		71 ± 3		468 ± 22†
Tibia length (mm)		17.0 ± 0.1		16.9 ± 0.1
# of animal assigned (n)
3-hr post		6		5
14-hr post		4		5
24-hr post		6		6
48-hr post		4		4

Female wild-type (WT) and Apc^Min/+ (Min) mice (age: 18~20 weeks old) were randomly assigned to a single bout of HFES experiment (either 3-hr, 14-hr, 24-hr, or 48-hr post HFES). Because there was no difference in cachectic index (e.g. body weight (BW), % of body weight change vs. Peak BW, and tibialis anterior mass) within the same group of mice, the descriptive data were pooled. Values are means ± SEM and statistical significance was set at p < 0.05. % BW change vs. Peak BW: [(BW at 18 weeks – Peak BW) / Peak BW ×100].

^†indicates significantly different from age-matched WT.

^$indicates main effect of mouse genotype.

Experiment 1: Examined the effect of repeated HFES bouts for 2 weeks in WT (n = 6) and Min (n = 6) female mice between 16 and 18 weeks 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 weeks of age. The mice were sacrificed at either 3-h, 14-h, or 24-h post HFES to measure plasma creatine kinase levels and 48-h post HFES for histological analysis.

Experiment 3: Examined 3 time points (3-h, 14-h, or 24-h) after an acute bout of HFES in WT (n = 16) and Min (n = 16) female mice at 18~20 weeks of age. Mice in this cohort did not differ in cachectic index reflected by their body weights and % of body weight loss. Mice performed a single bout of HFES and were then sacrificed at either 3-h, 14-h, 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 stimulation of the soleus did not alter the relative phosphorylation state of the p70S6K in healthy rodents (23). Thus, we utilized the TA to examine the effect of HFES on the cachectic muscle in mice.

The high frequency electric stimulation (HFES) protocol used in this study was previously described (10). Briefly, the muscle contractions lasted 3 seconds and were followed by a 10 seconds 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 second rest between sets were performed. This resulted in a total of 60 contractions with 180 seconds of actual contraction time. Thus, the entire contraction period lasted approximately 22 minutes. After HFES, the mice were allowed to recover fully before moving back to their cages.

Cage activity monitoring.

During multiple bout of HEFS study, 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 pm–7 am) and the number of beams crossed in an X–Y 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 BW) and tibialis anterior (TA) and gonadal fat were removed and snap frozen in liquid nitrogen. TA were cut at the midbelly; proximal part of TA was used for biochemical analysis while distal part of TA was using for histological analysis. Tibia length was measured as an indicator of animal body size and a correction factor for skeletal muscle weights.

Plasma creatine kinase assay.

Plasma creatine kinase (CK) levels were determined on 3~4 mice at each time point based on the manufacturer’s instructions. Briefly, blood was taken from the retro-orbital sinus under anesthesia. 8 μL of plasma was assayed spectrophotometrically at a wavelength of 340 nm for CK activity using a commercially available kit (Sekisui Creatine Kinase (CK)-SL, Sekisui Diagnostics, LLC, Framingham, MA).

Immunohistochemistry for myosin heavy chain type IIa and IIb.

For immunohistochemistry staining, transverse sections (10 μm) were cut from 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 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 (ABC) system (Vector Laboratories) was used to detect the biotinylated secondary antibody and visualized by 3,3’-diaminobenzidine (DAB) solution (Vector Laboratories). Digital pictures were taken at a 200x magnification with a Motic spot camera (Motic, China). Myofiber cross-sectional area (CSA) was quantified with Image J (NIH, 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 (SDH) stain.

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 tibialis anterior 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 % of frequency 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 1:1,000 to 1:2,000 in 5% TBST milk overnight at 4°C for primary antibodies and 1:2,000 to 1:5,000 dilutions for 1 h in 5% TBST milk for secondary antibody. Enhanced chemiluminescence (ECL, GE Healthcare, Chicago, IL) was used to visualize the antibody–antigen interactions and the image was developed by autoradiography (Kodak, Biomax, MR). 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 (2H5-Phe) using UPLC tandem mass spectrometry (Waters Milford, MA) (24). 2H5-Phe was obtained from Cambridge Isotope Laboratories (Andover, MA). Thirty minutes prior to 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 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 the means ± 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 tibialis anterior weights and myofibrillar protein synthesis (MPS) 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 the multiple bouts of HFES study (Experiment 1). Post-hoc analyses were performed with Student-Newman-Keuls methods when appropriate. A Student t-test was used to determine the difference in peak body weights, % body weight changes, gonadal fat, tibia length, spleen mass, physical activity (Experiment 1) and creatine kinase 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 examined repeated HFES bouts over a 2-week period during cachexia development in female wild-type and Apc^Min/+ mice.

Body weights, tibia length, & organ weights.

Between 16 and 18 weeks of age WT and Min mice performed 7 bouts of HFES (6 reps X 10 sets, every other day) for approximately two weeks (Figure 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 weeks of age. WT mice maintained their body weights during the two weeks 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. Tibialis anterior (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.

(Experiment 1). Effects of multiple bouts of high-frequency electric stimulation (HFES) on tibialis anterior (TA) myofiber cross-sectional area (CSA) in female wild-type (WT) and Apc^Min/+ (Min) mice. (A) Experimental design. Mice (16 weeks of age) assigned this protocol performed 7 bouts of HFES over a period of two weeks. Mice were sacrificed 48 hours 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 hours prior to the sacrifice. (B) Frozen sections of TA muscle were cut (~10 μm thickness) at the mid-belly, stained with type IIa (SC-71) and IIB (BF-F3) antibodies. Representative type IIa and 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 200X. Con: Control. HFES: High frequency electric stimulation. (C&D) Type IIa (C) and type IIb (D) myofiber size frequency distributions in WT Con and Min Con muscles. (E&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 non-stimulated control (Min Con).

Physical Activity & Food Intake.

Voluntary physical activity (7 pm to 7 am) was reduced in Min mice when compared with WT mice, but food intake was not reduced in Min mice (see Table S1, Supplemental Digital Content 1, which illustrates physical activity and food intake of mice).

Tibialis Anterior (TA) muscle mass & fiber cross-sectional area (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 contra-lateral control regardless of genotype (Table 1). Myofiber CSA was determined from frozen sections taken at the TA mid-belly and stained immunohistochemically using type IIa (SC-71) and type IIb (BF-F3) antibodies (Figure 1B). Min type IIa and type IIb myofiber mean CSA was decreased when compared to WT mice. HFES attenuated Min mean CSA loss in both type IIa and type IIb fibers (Figure 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 percentage of small-diameter type IIa fibers (<500 μm², 2% vs. 43%) and reduced percentage of large-diameter type IIa fibers (>1300 μm², 13% vs. 0%) when compared to WT mice (Figure 1C). Similarly, Min type IIb fibers had more small-diameter fibers (<1,500 μm², 8% vs. 45%) and less large-diameter fibers (>3,000 μm², 14% vs. 1%, Figure 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 (Figure 1E). Likewise, HFES decreased the percentage of Min type IIb fibers small-diameter fibers (<1250 μm², 15% vs. 23%) and increased Min type IIb fibers large-diameter fibers (>2750 μm², 8% vs. 3%) when compared with control muscle (Figure 1F).

Muscle oxidative capacity.

Succinate dehydrogenase (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 (Figure 2A). Min mice exhibited reduced muscle SDH activity compared to WT mice (20.3 ± 1.0 vs. 9.81 ± 0.8%, Figure 2B). Similarly, the percentage of small-diameter SDH positive fibers were increased (<400 μm², 6% vs. 23%) and large-diameter SDH positive fibers decreased (>800 μm², 55% vs. 9 %) Min mice when compared to WT mice. HFES attenuated the SDH activity loss (9.81 ± 0.8 vs. 13.9 ± 0.5%, Figure 2B) in Min muscle (Figure 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 %, Figure 2D).

Figure 2.

Effects of multiple bouts of high-frequency electric stimulation (HFES) on muscle oxidative capacity in female wild-type (WT) and Apc^Min/+ (Min) mice. Frozen sections of tibialis anterior (TA) muscle were cut (~10 μm thickness) at the mid-belly and succinate dehydrogenase (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 200X. (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 means ± SEM and statistical significance was set at p < 0.05. †Significantly different from WT Con. *Significantly different from non-stimulated 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 (Figure 3A). 4EBP1 and AMPK activity represents the ratio of phosphorylated to total protein expression. All data are normalized to non-stimulated contralateral control TA muscle in wild-type mice. Total 4EBP1 and AMPK protein levels were not changed by HFES (Figure 3B). There was a trend to decrease Min 4EBP1 activity (p=0.064, Figure 3B) and HFES did not alter the levels of 4EBP1 activity. When IGF-1 mRNA was quantified, no change was observed with cachexia nor cachexia with HFES (Figure 3C). AMPK activity was increased in the Min and HFES did not change the activity levels (Figure 3B). Atrogin-1 expression demonstrated a trend to increase activity in Min muscle (p=0.079) and HFES did not change atrogin-1 expression (Figure 3B).

Figure 3.

Effects of multiple bouts of high-frequency electric stimulation (HFES) on the basal levels of protein and mRNA expression for anabolic and catabolic signaling in female wild-type (WT) and Apc^Min/+ (Min) mice. Western blots and qRT-PCR were performed to examine the changes of basal levels of protein and mRNA expressions, respectively, on tibialis anterior (TA) muscles after two weeks of multiple bout 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. HFES: High-frequency electric stimulation. Values are means ± SEM and statistical significance was set at p < 0.05. †Significantly different from WT Con.

Experiment 2 examined circulating creatine kinase (CK) levels after acute bouts of HFES in female WT and Min mice.

Plasma Creatine kinase (CK).

CK levels were examined at 3-h, 14-h, 24-h, and 48-h post HFES (Figure 4A). CK levels were comparable at all time points except at 3-hr post HFES (see Figure S1A, Supplemental Digital Content 2, which demonstrates the levels of CK in the plasma at different time points). 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, H&E stain was performed on TA frozen sections. No structural damage was observed at 48-h post HFES (see Figure S1B, Supplemental Digital Content 2, which shows the myofiber CSA images stained by H&E) 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.

(Experiment 2 & 3). Effects of a single bout of high-frequency electric stimulation (HFES) on Muscle anabolic and catabolic signaling in female Apc^Min/+ (Min) mice. Mice (18~20 weeks of age) were sacrificed at either 3-hour, 14-hour, 24-hour, or 48-hour post HFES. Western blots were performed to examine the acute changes of protein expression in tibialis anterior (TA) muscle at 3-hour, 14-hour, and 24-hour post single bout of HFES. The mice sacrificed at 3-hour post HFES were fasted at least 4 hours prior to the HFES. The other groups of mice had access to food and water ad libitum after HFES, but they were fasted 5 hours prior to sacrifice. (A) Experimental design. (B) Muscle protein synthesis (MPS) rate at 3-hour post HFES. (C) (Top) Representative immunoblots of key molecules in anabolic signaling (4EBP1) and catabolic signaling (AMPK and atrogin-1) at 3-hour 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-hour post HFES. (D) (Top) Representative immunoblots of key molecules in anabolic signaling (4EBP1) and catabolic signaling (AMPK and atrogin-1) at 14-hour 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-hour post HFES. (E) (Top) Representative immunoblots of AMPK at 24-hour post HFES. (Bottom) Ratios of phosphorylated (p) and total forms of AMPK protein expression in TA muscle of female Min mice at 24-hour post HFES. GAPDH was used as loading control. Values are means ± SEM and statistical significance was set at p < 0.05. *Significantly different from non-stimulated control. ‡indicates main effect of HFES. $ indicates main effect of mouse genotype.

Experiment 3 examined the effect of an acute HFES bout on TA muscle protein synthesis regulation 3-h, 14-h, and 24-h post contraction.

Muscle Protein Synthesis (MPS).

We first examined acute changes in MPS and associated signaling induced 3-h post an acute bout of HFES (Figure 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, Figure 4B).

Protein Synthesis Regulation.

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

Discussion

There is accumulating evidence for a positive effect of resistance exercise on cancer-induced muscle mass loss (4, 8, 10, 13). While 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 the response to exercise are different between males and females (15, 22). These reports led us to speculate that cachexia may differentially impact the female response to resistance exercise, when compared to 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 to 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 muscle protein synthesis and decreasing AMPK activity for muscle degradation.

Resistance exercise training has been shown 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 employing 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 to the contralateral non-stimulated one (4). Furthermore, 6 weeks of functional overload by synergistic ablation in rats bearing Morris hepatoma MH7777 cells increased plantaris muscle mass by 24% compared to contralateral sham-operated muscle (7). While 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 was not tested in these studies. Furthermore, the mechanisms related to the preventative effect of loading was not established.

As noted above, 2 weeks 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 weeks 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 percentage of small-diameter fibers and increasing percentage of large-diameter fibers in both fibers types. However, muscle mass change in response to hypertrophic stimuli was attenuated in Min mice compared to 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 muscle protein synthesis 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). Increased activity of the ubiquitin-proteasome pathway has 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 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 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 is 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 over-expression 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 if the increased oxidative capacity that occurs with 2 weeks 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 muscle protein synthesis 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, resulting in limiting 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 was not sufficient to change these levels. We then investigated if acute HFES could increase 4EBP1 phosphorylation. While a single bout of HFES increased 4EBP1 phosphorylation in parallel with increased muscle protein synthesis at 3-h post 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 muscle protein synthesis 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 cross-sectional area 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-hr post HFES. Since the mice that received multiple bouts of HFES were sacrificed at 48-h post last HFES session, it is possible to assume that the effect of 2-week 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 these likely involve the regulation of both muscle protein synthesis 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 been also 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 weeks of repeated HFES is similar to the male response. HFES successfully increased muscle mass with an increase of 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. Based on our findings further research is warranted to determine if 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.