All cells were purchased through ATCC (Manassas, VA, United States) and used within the first 15 passages. Murine C2C12 myoblasts (CRL-1772), C26 adenocarcinoma (CRL-2638), or EL4 lymphoma (TIB-39) cells were maintained at 37°C, 5% CO₂ in growth media (GM): Dulbecco’s Modified Eagle Media (DMEM; #11995-065; Gibco, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (FBS), 50 U/ml of penicillin, and 50 μg/ml of streptomycin.

C2C12 myoblasts were seeded on type I collagen-coated polystyrene plastic or flexible silastic Uniflex^® membranes (Flexcell International, Burlington, NC, United States) at a density of 8.0 × 10⁴ cells per well (six-well plate) in GM: DMEM (#11995-065; Gibco) supplemented with 10% FBS, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. To induce myoblast differentiation, cells were rinsed with phosphate-buffered saline (PBS) and switched to differentiation media (DM): DMEM supplemented with 2% heat-inactivated horse serum, 50 U/ml of penicillin, and 50 μg/ml of streptomycin to form myotubes. Media was replenished every 48 h, and experiments were performed starting at day 5 of differentiation when multinucleated contractile myotubes are present.

Colon-26 cells, a murine colon adenocarcinoma that promotes cachexia in mice, or EL4 lymphoma cells that do not promote cachexia in mice (Bonetto et al., 2016; Zhang et al., 2017), were cultured as described above. Conditioned media (CM) consists of consists of secreted factors from tumor cells and has been described previously (Kandarian et al., 2018). Briefly, 2 × 10⁶ cells were seeded in 100-mm tissue culture-treated plates in GM. Tumor cell CM was collected at ∼90% confluence 48 h post cell seeding and spun down at 3,000 rpm for 5 min to remove cell debris. Cells on the plate were pelleted and counted via trypan blue exclusion test to ensure an equivalent number of cells on the plate, with a final density averaging 7.0–9.0 × 10⁶ cells per culture dish. CM was stored for one-time use in aliquots at −20°C, used within 2 months, and then thawed in a warm water bath at the time of the experiment. GM with no cells was used as a media control.

At the time of the experiment, GM control or C26 CM is diluted with 50% serum-free DMEM for a final serum concentration of 5% FBS; 50% CM was chosen, as it has been shown to produce significant myotube atrophy (Pin et al., 2018; Zhong et al., 2019). It should be noted that myotubes were differentiated up to day 5 in 2% horse serum; thus upon CM addition, myotubes were switched to a higher serum environment, which can induce myotube hypertrophy (von Walden et al., 2016). Therefore, in the figures and results, EL4 and C26 CM are referred to as EL4 + GM or C26 + GM. A mixture of myoblasts and myotubes at day 5 of differentiation was rinsed with PBS and then switched to GM media control or C26 CM (C26 + GM) for 48 h (days 5–7 of differentiation). CM was replenished after 24 h. In a separate experiment, C2C12 myoblasts were grown and differentiated on type I collagen-coated rigid polystyrene plates to examine the effects of the cachectic tumor cell CM on a commonly utilized substratum. Briefly, differentiated myotubes at day 5 were treated with 50% GM, C26, or EL4 CM (EL4 + GM) for 48 h. EL4 CM was used to determine if effects on myotube growth are specific to cachectic tumor-derived factor media.

Static stretch was administered (FX-6000 Tension System, Flexcell International Corporation) as previously described (Hornberger et al., 2005; Baccam et al., 2019) with the following modifications. Cells were subjected to a static 5% uniaxial stretch in the last 4 or 24 h of CM treatment (as described above). Control cells (0% stretch) were grown under identical conditions but left unstretched and placed beside the incubator’s baseplate. For administration of stretch, culture plates were removed from the incubator, rapidly placed onto the stretch device’s baseplate, and then placed back in the incubator. The Flexcell Arctangle^® loading station was used for all experiments, which consists of six rigid posts covered in a thin layer of silicone lubricant, centered beneath the six-well Uniflex plate. Applied vacuum pressure deforms the membrane across the loading post in a single direction (e.g., north and south poles), creating a uniaxial stretch (Figure 3B). A custom ramp protocol, described here, was designed to ensure cell adherence to the membrane while vacuum pressure is applied: the Flexcell tension system was set to increase vacuum pressure gradually, and thus % elongation in increments of 1% every 2 s, up to 5%, which is then maintained for the duration of the experiment (e.g., 24 h), followed by a ramp-down to 0% at the conclusion of the experiment.

Protein synthesis was measured in myotubes treated with CM grown on type I collagen-coated polystyrene plates using the surface sensing of translation (SUnSET) method as previously described (Goodman et al., 2011). Puromycin (#540411; Sigma-Aldrich, St. Louis, MO, United States) was added to culture media 30 min prior to protein harvest at a final concentration of 1 μM per well. The amount of puromycin incorporated into newly synthesized protein was determined by Western blotting.

Myotube diameter was quantified as previously described (Gao and Carson, 2016) with the following modifications. C2C12 myotube diameter was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States). Digital images were captured at ×20 objective brightfield. Five non-overlapping images were captured within each well, and three images were randomly chosen for the analysis. The analysis used unmodified tiff images accessed in NIH ImageJ software. A blinded investigator randomly took diameter measurements of six myotubes per image based on preset inclusion/exclusion criteria: elongated structure with distinct membrane outlines, little to no cellular debris, and no branching points. The average diameter per myotube was calculated as the mean of eight measurements taken along the myotube length. For each condition, n = 162–378 myotubes were analyzed.

Western blotting analysis was performed as previously described (Gao and Carson, 2016). Briefly, cells were scraped into ice-cold radioimmunoprecipitation assay (RIPA) buffer [25 nM of Tris HCl at pH 7.6, 150 nM of NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)] (#89900; Thermo Fisher Scientific, Waltham, MA, United States) and 1% Halt protease and phosphatase inhibitor cocktail [1 mM of EDTA, 5 mM of NaF, 1 nM of NaVO₄, and 1 mM of glycerophosphate] (#78440; Thermo Fisher Scientific). Cell lysates were centrifuged at 14,000 rpm for 15 min at 4°C, and supernatants were collected. Protein concentrations were determined using the Bradford method. Homogenates were fractionated on SDS–polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane. Membranes were blocked for 1 h in 5% non-fat milk–TBST. After blocking, primary antibodies for phosphorylated STAT3 (Y705) 1:1,000, total STAT3 1:2,000, phosphorylated p38 (T180/Y182) 1:1,000, total p38 1:2,000, phosphorylated ERK1/2 (T202/Y204) 1:1,000, total ERK1/2 1:2,000, phosphorylated rpS6 (S240/244) 1:1,000, and total rpS6 1:2,000, phosphorylated Akt (S473) 1:1,000, total Akt 1:2,000, phosphorylated 4E-BP1 (T37/46) 1:1,000, total 4E-BP1 1:2,000, phosphorylated SAPK/JNK (T183/Y185) 1:1,000, total SAPK/JNK 1:2,000 (Cell Signaling Technology, Danvers, MA, United States), Atrogin-1/MAFbx, MuRF-1 1:1,000 (ECM Biosciences, Versailles, KY, United States), and MyHC-Fast (protein stain for fast type II fibers; corresponding genes: MyH1 and MyH2) and MyHC-Slow 1:4,000 (protein stain for slow type I fibers; corresponding gene MyH7), serum response factor (SRF) 1:500, myogenin 1:500, and MyoD 1:500 (Santa Cruz Biotechnology, Dallas, TX, United States), integrin β1D 1:1,000, and puromycin 1:2,000 (Millipore, Billerica, MA, United States) were incubated overnight at 4°C in 5% non-fat milk–TBST. Secondary anti-rabbit or anti-mouse IgG horseradish peroxidase (HRP)-linked (Cell Signaling Technology) antibodies were incubated at 1:4,000 dilution for 1 h at room temperature in 5% non-fat milk–TBST. Enhanced chemiluminescence (Prometheus Pro-Signal Femto, #20-302; Genesee Scientific, San Diego, CA, United States) was used to visualize the antibody–antigen interactions, and membranes were digitally imaged using the iBright 1500 system (Invitrogen, Carlsbad, CA, United States). Immunoblots were analyzed by measuring each band’s integrated optical density (IOD) with ImageJ software (National Institutes of Health, Bethesda, MD, United States).

Total RNA was isolated from C2C12 myotubes using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) per manufacturer’s guidelines and as previously described (White et al., 2009). After phenol–chloroform extraction, RNA was purified using the PureLink^® RNA Mini Kit (Invitrogen, Carlsbad, CA, United States) and eluted in RNAase-free water. Total RNA concentration (260 nm) and purity (260/280 ratio) were measured using UV spectrophotometry, and total RNA was stored at −80°C. cDNA was reverse transcribed from 1 μg of total RNA using Superscript IV Reverse Transcriptase and random hexamers according to manufacturer guidelines (#18090200; Invitrogen) in a final volume of 20 μl at 23°C for 10 min, 50°C for 10 min, and 80°C for 10 min. cDNA was stored at −80°C. Real-time PCR was performed using reagents from Applied Biosystems (Foster City, CA, United States). Gene expression was carried out in 20-μl reactions using 2 × PowerTrack SYBR Green master mix, 2 μl of cDNA, 1 μl of forward and reverse primers (500 nM), and nuclease-free H₂O. The sequences of the primers were verified using NIH Primer Blast software, as follows: MyHC-Slow Type I (Myh7) [NM_080728.3]: Fw 5′-CCAAGGGCCTGAATGAGGAG-3′, Rv 5′-GCAAAGGCTCCAGGTCTGAG-3′; MyHC-Fast type IIA (Myh2) [NM_001039545.2]: Fw 5′-AGGCGGCTGAGGAGC ACGTA-3′, Rv 5′-GCGGCACAAGCAGCGTTGG-3′; MyHC- Fast type IIB (Myh4) [NM_010855.3]: Fw 5′-CACCTGG ACGATGCTCTCAGA-3′, Rv 5′-GCTCTTGCTCGGCCACTCC -3′; MyHC-Fast type IIX (Myh1) [NM_030679.2]: Fw 5′-GAGG GACAGTTCATCGATAGCAA-3′, Rv 5′-GGGCCAACTTGTCA TCTCTCAT-3′; skeletal muscle alpha actin (Acta1) [NM_00127 2041.1]: Fw 5′-CTCCTACGTGGGTGATGAGG-3′, Rv 5′-AGGTGTGGTGCCAGATCTTC-3′; myogenin (Myog) [NM _031189.2]: Fw 5′-GCACTGGAGTTCGGTCCCAA-3′, Rv 5′-TA TCCTCCACCGTGATGCTG-3′; MyoD (Myod1) [NM_010866. 2]: Fw 5′-GAGATGCGCTCCACTATGCT-3′, Rv 5′-TGGCAT GATGGATTACAGCG-3′; Myocyte Enhancer Factor 2C (MEF 2C) [NM_001170537.1]: Fw 5′-GAGCCGGACAAACTCAGA CA-3′ Rv 5′-GGCTGTGACCTACTGAATCGT-3′; and GAPDH [NM_001289726]: Fw 5′-ACCACAGTCCATGCCATCAC-3′ Rv 5′-TCCACCACCCTGTTGCTGTA-3′. Primer sequences were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, United States) and validated via agarose gel electrophoresis. RT-PCR was carried out on the Applied Biosystems QuantStudio3 system. Reactions were incubated for 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of a 15-s denaturation step at 95°C, and 1-min annealing/extension at 60°C. The 2^–ΔΔCt method (Livak and Schmittgen, 2001) was used to determine gene expression changes between treatment groups with the GAPDH Ct as the correction factor.

All experiments included a minimum of three replicates from at least two independent experiments. Results are expressed as mean ± SEM. Student’s unpaired t-test, one-way ANOVA, and two-way ANOVA (indicated in the figure legends) were used to examine the effects of stretch and culture conditions. Tukey’s post hoc multiple comparisons test was used when a significant interaction was present. p-Values ≤0.05 were considered significant.

We examined the effects of tumor cell CM under standard culture conditions. GM, C26 CM (C26 + GM), or EL4 CM (EL4 + GM) was administered to 5-day differentiated myotubes grown on type I collagen-coated polystyrene plates for 48 h (Figure 1A). EL4 lymphoma cells that do not induce cachexia (Zhang et al., 2017) were used as a tumor cell CM control. Myotubes were imaged for diameter Pre (day 5) and Post (day 7) CM exposure (Figure 1B). Myotubes maintained in DM were imaged and used as a control for myotube growth. Under standard DM culture conditions, myotubes increased diameter from day 5 to day 7 (24%; p < 0.0001) (Figure 1C). EL4 + GM-treated myotubes significantly increased diameter compared to Pre (p < 0.0001), and this increase was equivalent to GM treatment (42%) (Figure 1C). C26 + GM-treated myotubes significantly increased diameter compared to Pre (15%). However, they were significantly smaller in size than both GM and EL4 + GM-treated myotubes (Figure 1C). Along with decreased myotube size, C26 + GM-treated myotubes had significantly lower protein expression of both MyHC-Fast (p < 0.001, −50%) and MyHC-Slow (p = 0.007, −60%) isoforms compared with the GM and EL4 + GM conditions (Figure 1D). These results demonstrate that C26 tumor-derived factors can suppress myotube growth and MyHC protein expression and that these effects are specific to cachectic tumor-derived factors.

Altered muscle protein turnover regulation contributes to cachexia development (Baracos, 2000), and tumor-derived factors can disrupt this regulation in cultured myotubes (Gao and Carson, 2016; Gao et al., 2017; Chiappalupi et al., 2020). Both EL4 and C26 tumor-derived factors displayed significant reductions in protein synthesis rate measured by puromycin incorporation (EL4 + GM: −29%, C26 + GM: −46%) (Figures 1E,I). Compared with EL4 + GM, C26 + GM-treated myotubes displayed a significant reduction in the phosphorylation of Akt (S473) (p = 0.025) and rpS6 (S240/244) (p < 0.001); both EL4 + GM (p = 0.047) and C26 + GM (p < 0.001) tumor-derived factors significantly reduced the phosphorylation of 4E-BP1 (T37/46) (Figures 1F,J). C26 + GM induced STAT3 phosphorylation (Y705) 138% compared with controls (p < 0.001), and ERK1/2 phosphorylation was unchanged across all conditions (Figures 1G,J). Under standard culture conditions, 48 h incubation with C26 + GM significantly induced Atrogin-1 (p = 0.009, 111%) and MuRF-1 (p < 0.001, 85%) as compared with GM and EL4 + GM (Figures 1H,J). These data suggest that C26 tumor-derived factors can alter protein turnover signaling involving Akt/mTORC1 signaling (i.e., 4E-BP1 and rpS6) and ubiquitin–proteasome-mediated breakdown.

Growth media or C26 tumor cell CM (C26 + GM) was administered to myotubes at day 5 of differentiation for 48 h on type I collagen-coated silastic membranes (Figure 2A). Myotubes were imaged for diameter at day 5 (Pre) and day 7 (Post) (Figure 2B). Switching C2C12 myotubes from DM (2% horse serum) to GM (5% FBS) has been shown to induce myotube growth (von Walden et al., 2016). GM significantly increased myotube diameter compared with DM (p < 0.001) (Figure 2C). Compared with Pre, GM treatment increased myotube diameter 29% (p < 0.001), and C26 + GM-treated myotubes did not change (p = 0.618) myotube diameter (Figure 2C). These results demonstrate that C26 tumor-derived factors can suppress myotube growth measured by diameter despite a high growth factor environment.

Myosin heavy chain isoform protein expression was quantified on day 7 after 48 h of GM or C26 + GM exposure. Increased MyHC isoform expression is critical for myotube differentiation and growth (Brown et al., 2012). We report that C26 CM can decrease myotube MyHC protein concentration in myotubes grown on flexible silastic membranes. C26 + GM-treated myotubes decreased MyHC-Fast protein expression −17% (p = 0.002) and MyHC-Slow protein expression −47% (p < 0.0001) (Figure 2D). We next examined if C26 tumor-derived factors regulate changes in myotube MyHC expression at the mRNA level. mRNA expression was quantified at day 7 after 48 h of GM or C26 + GM exposure. C26 CM induced isoform-specific shifts in MyHC mRNA expression (Figure 2E). The expression of MyHC-Fast type IIA (Myh2) mRNA was reduced by −78% (p < 0.0001), type IIB (Myh4) mRNA was increased by 133%, type IIX (Myh1) did not significantly change, and MyHC-Slow type I (Myh7) was reduced by -59% (p = 0.003) by the C26 + GM treatment (Figure 2E). Skeletal α-actin is a principle component of muscle thin filaments and interacts with the MyHC proteins for contraction (Sparrow et al., 2003). Forty-eight hours of C26 + GM incubation did not alter skeletal α-actin mRNA expression (Figure 2E). Therefore, C26-derived factors reduce the concentration of both MyHC protein and mRNA in myotubes, and this preferential loss of MyHC has potential implications for both size and contractile function.

C26 CM can activate multiple signaling pathways associated with myotube catabolic signaling (Seto et al., 2015). We examined several proteins that can regulate muscle growth and are implicated in cancer cachexia, including STAT3 (Puppa et al., 2014), p38 MAPK (Ding et al., 2017; Brown et al., 2018), and ERK1/2 (Gao and Carson, 2016; VanderVeen et al., 2018). Despite incubation in a high serum growth-promoting environment, C26 + GM induced a 57% increase in the phosphorylation of STAT3 (Y705) (p < 0.001), an 89% increase (p = 0.011) in p38 (T180/Y182) phosphorylation, and a 34% increase (p = 0.033) in ERK1/2 (T202/Y204) phosphorylation (Figures 2F,G).

Mechanical signaling induced by chronic passive stretch is a potent inducer of myotube growth and anabolic signaling (Vandenburgh and Kaufman, 1979; Gao and Carson, 2016). Additionally, dynamic stretch occurring with the administration of C26-derived catabolic factors can promote increased myotube diameter (Baccam et al., 2019). Our study extends this understanding by examining chronic stretch application after the initiation of catabolism inducing C26 CM. We assessed if stretch could induce a growth stimulus in myotubes already exposed to C26 cachectic factors. Furthermore, we examined the effects of stretch in a high serum growth-promoting state. Myotubes were uniaxially stretched 5% after 24 h of exposure to C26 + GM and then maintained in the stretched state in C26 + GM for 24 h (Figure 3A). There was a main effect for myotubes treated with C26 + GM for 48 h to decrease (p < 0.0001) myotube diameter (Figure 3C). However, there was a main effect for the 24-h stretch treatment to increase (p = 0.003) myotube diameter in both GM and C26 + GM conditions (Figure 3C). These results provide evidence that stretch can induce a growth stimulus in myotubes that are in a catabolic state and in a growth factor-rich environment (5% FBS) that generated growth in the absence of tumor-derived factors.

We next examined if 24 h of stretch could rescue suppressed MyHC-Fast and MyHC-Slow protein expression in myotubes incubated with C26 CM. We report that stretch has different effects on fast and slow MyHC protein expression. Stretch increased MyHC-Fast protein expression (26%, p = 0.023) in C26 + GM myotubes compared with unstretched C26 + GM myotubes (Figures 3D,L). Furthermore, MyHC-Fast protein expression in stretched C26 + GM myotubes was not different than GM levels, indicating that stretch had rescued MyHC-Fast protein expression. Interestingly, stretch did not change (p = 0.251) MyHC-Slow protein expression in C26 + GM incubated myotubes, and unstretched (0%) C26 + GM-treated myotubes had significantly lower MyHC-Slow protein expression than both unstretched (p < 0.001) and stretched (p = 0.021) GM-treated myotubes (Figures 3E,L). We also examined the effect of stretch on MyHC mRNA expression. We measured MyHC-Fast type IIA, IIB, IIX, and MyHC-Slow type I mRNA expression after 24 h of stretch. Stretch did not change the C26 + GM suppression MyHC-Fast type IIA (Figure 3F) or MyHC-Slow type I (Figure 3I) mRNA expression. Likewise, in both GM and C26 conditions, passive stretch did not alter mRNA expression of MyHC-Fast type IIB (Figure 3G), type IIX (Figure 3H), and skeletal muscle α-actin (Figure 3J). There were main effects for C26 + GM to decrease MyHC-Fast type IIA (p < 0.001) (Figure 3F), increase type IIB (p = 0.002) (Figure 3G), and decrease MyHC-Slow type I (p = 0.002) (Figure 3I) mRNA expression. MEF2C is an important transcriptional regulator of MyHC genes and can regulate thick filament assembly in myotubes (Hinits and Hughes, 2007; Piasecka et al., 2021). There was a main effect for C26 + GM to decrease (27%; p = 0.001) MEF2C mRNA expression (Figure 3K). Stretch did not change MEF2C mRNA expression (p = 0.687). These data suggest that stretch-induced mechanical signaling can rescue suppressed MyHC-Fast protein expression in myotubes undergoing catabolism initiated by C26 tumor-derived factors. Interestingly, this effect was independent of changes in MyHC mRNA expression and specific to MyHC-Fast protein. Suppressed MyHC-Slow protein expression by C26 CM was not responsive to stretch-induced mechanical signaling.

Skeletal muscle atrophy is associated with suppressed mTORC1 signaling, disrupted autophagy flux, and decreased RNA content, diminishing ribosomal capacity for protein synthesis (Bhogal et al., 2006). E3 ligases Atrogin-1/MAFbx and MuRF-1 are involved in ubiquitin–proteasome degradation and have an established role in muscle atrophy (Lecker et al., 1999; Bodine et al., 2001; Kandarian and Stevenson, 2002). Therefore, we assessed these factors to gain insight into the stretch regulation of myotube growth and protein turnover after exposure to C26 tumor-derived factors (Figure 4A). We first measured the phosphorylation of ribosomal protein S6 (rpS6), one downstream marker of the mammalian target of rapamycin (mTOR) pathway (Mieulet et al., 2007). rpS6 phosphorylation (S240/244) was unchanged by C26 + GM and stretch (Figures 4B,I). Skeletal muscle-specific E3 ubiquitin ligases Atrogin-1/MAFbx and MuRF-1 can degrade myosin (Clarke et al., 2007; Cohen et al., 2009; Bodine and Baehr, 2014). Interestingly, we observed a main effect for 24 h of stretch to decrease Atrogin-1 and MuRF-1 protein expression in both GM and C26 + GM-treated myotubes (Figures 4C,D,I). We next examined established markers of autophagy flux, including microtubule-associated protein light chain 3 (LC3B) I and II, Beclin-1, and p62/SQSTM1 (Mizushima and Yoshimori, 2007; Penna et al., 2013). We observed a main effect for chronic stretch to decrease the LC3B II/I ratio (p < 0.001) (Figures 4E,I) with no changes in Beclin-1 or p62 protein expression (Figures 4F,G,I). Total RNA content in myotubes remained unchanged across all conditions (Figure 4H). These data suggest that chronic stretch may protect myosin content by decreasing the expression of Atrogin-1 and MuRF-1 and reduced autophagy flux, rather than the induction of anabolic signaling involving total RNA and mTOR signaling relating to rpS6.

We investigated the effects of 24 h stretch on various regulators of muscle growth during C26 CM incubation (Figure 5A). We observed a main effect for mechanical stretch to increase integrin β1D protein expression (p < 0.001) (Figure 5B). SRF is a member of the MADS-box family of transcription factors important for growth and myogenic processes (Li et al., 2005). Moreover, mechanical stretch can induce SRF mRNA expression (Carson and Booth, 1999). Surprisingly, there was a main effect for both stretch (p = 0.035) and C26 tumor-derived factors (p < 0.001) to decrease SRF protein expression (Figures 5C,G). We further examined the role of stretch and C26 + GM on myogenesis by measuring the ratio of myogenin, which is expressed in late stages of differentiation, to MyoD, a marker of myoblast proliferation and initiation of differentiation. The relative mRNA expression of myogenin (GM 0%, 1.00 ± 0.03; GM 5%, 1.03 ± 0.09; C26 + GM 0%, 0.809 ± 0.08; and C26 + GM 5%, 1.01 ± 0.08) and MyoD (GM 0%, 1.02 ± 0.10; GM 5%, 0.905 ± 0.12; C26 + GM 0%, 0.752 ± 0.07; and C26 + GM 5%, 0.842 ± 0.10) was unchanged. Due to variability in protein expression, myogenin (Figure 5D) and MyoD (Figure 5E) individual protein expression was similar across groups. To account for variability between samples, we calculated the ratio of myogenin-to-MyoD protein expression in each sample. There was a main effect for stretch to increase the myogenin-to-MyoD ratio (p = 0.027) (Figure 5F). These results provide evidence that regardless of C26 tumor-derived factor incubation, stretch can increase integrin β1D protein expression to modulate intrinsic mechanical signaling. These changes are independent of stretch-induced increases in SRF protein expression and associated with an effect for stretch to increase the myogenin-to-MyoD protein ratio.

In a separate experiment, we examined the effects of acute 4-h stretch on cachectic and growth-related signaling in C26 + GM-treated myotubes. Several cytokines associated with cancer-induced wasting can stimulate muscle STAT3 phosphorylation (Bonetto et al., 2011; Puppa et al., 2014; Zimmers et al., 2016). Cancer cachexia can induce muscle p38 MAPK, ERK1/2, and JNK signaling (Brown et al., 2018; Mulder et al., 2020). Interestingly, mechanical signaling can also activate these pathways (Kumar et al., 2002; Martineau and Gardiner, 2002; Gao and Carson, 2016). Several well-characterized stretch-activated signaling pathways increase at the onset of chronic stretch and then return to baseline (Zhan et al., 2007). Therefore, we examined 4 h of stretch to determine acute activation of these signaling pathways. Myotubes were stretched 5% for the last 4 h of either the GM or C26 + GM treatment (Figure 6A). There was no effect for stretch to change STAT3 (Y705) phosphorylation (p = 0.865) (Figure 6B), p38 (T180/Y182) phosphorylation (p = 0.809) (Figure 6C), or JNK (T183/Y185) phosphorylation (p = 0.067) (Figures 6E,F). A preplanned analysis determined the effect of stretch in the GM condition; stretch induced a 42% increase in p38 (T180/Y182) (p = 0.014) phosphorylation and a 25% increase in ERK1/2 (T202/Y204) (p = 0.015) phosphorylation. Interestingly, this signaling activation occurred within the high serum environment. Stretch-induced ERK1/2 phosphorylation was further induced 54% in C26 + GM-treated stretched cells (p < 0.001) (Figures 6D,F). These data suggest that C26 + GM does not blunt the stretch response of p-p38 and p-ERK1/2 and that acute static stretch had no effect on the C26 CM induction of STAT3 phosphorylation, which was similarly elevated with 4 h of stretch.