mTORC1 and BMP-Smad1/5 regulation of serum-stimulated myotube hypertrophy: a role for autophagy.

Protein synthesis regulation is critical for skeletal muscle hypertrophy, yet other established cellular processes are necessary for growth-related cellular remodeling. Autophagy has a well-acknowledged role in muscle quality control, but evidence for its role in myofiber hypertrophy remains equivocal. Both mammalian target of rapamycin complex I (mTORC1) and bone morphogenetic protein (BMP)-Smad1/5 (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling are reported regulators of myofiber hypertrophy; however, gaps remain in our understanding of how this regulation is integrated with growth processes and autophagy regulation. Therefore, we investigated the mTORC1 and Smad1/5 regulation of protein synthesis and autophagy flux during serum-stimulated myotube growth. Chronic serum stimulation experiments were performed on day 5 differentiated C2C12 myotubes incubated in differentiation medium [2% horse serum (HS)] or growth medium [5% fetal bovine serum (FBS)] for 48 h. Rapamycin or LDN193189 was dosed for 48 h to inhibit mTORC1 and BMP-Smad1/5 signaling, respectively. Acute serum stimulation was examined in day 7 differentiated myotubes. Protein synthesis was measured by puromycin incorporation. Bafilomycin A1 and immunoblotting for LC3B were used to assess autophagy flux. Chronic serum stimulation increased myotube diameter 22%, total protein 21%, total RNA 100%, and Smad1/5 phosphorylation 404% and suppressed autophagy flux. Rapamycin, but not LDN193189, blocked serum-induced myotube hypertrophy and the increase in total RNA. Acute serum stimulation increased protein synthesis 111%, Smad1/5 phosphorylation 559%, and rpS6 phosphorylation 117% and suppressed autophagy flux. Rapamycin increased autophagy flux during acute serum stimulation. These results provide evidence for mTORC1, but not BMP-Smad1/5, signaling being required for serum-induced myotube hypertrophy and autophagy flux by measuring LC3BII/I expression. Further investigation is warranted to examine the role of autophagy flux in myotube hypertrophy.NEW & NOTEWORTHY The present study demonstrates that myotube hypertrophy caused by chronic serum stimulation requires mammalian target of rapamycin complex 1 (mTORC1) signaling but not bone morphogenetic protein (BMP)-Smad1/5 signaling. The suppression of autophagy flux was associated with serum-induced myotube hypertrophy and mTORC1 regulation of autophagy flux by measuring LC3BII/I expression. Rapamycin is widely investigated for beneficial effects in aging skeletal muscle and sarcopenia; our results provide evidence that rapamycin can regulate autophagy-related signaling during myotube growth, which could benefit skeletal muscle functional and metabolic health.

Recovery from FOLFOX chemotherapy-induced systemic and skeletal muscle metabolic dysfunction in mice.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is used to treat colorectal cancer and can acutely induce metabolic dysfunction. However, the lasting effects on systemic and skeletal muscle metabolism after treatment cessation are poorly understood. Therefore, we investigated the acute and lasting effects of FOLFOX chemotherapy on systemic and skeletal muscle metabolism in mice. Direct effects of FOLFOX in cultured myotubes were also investigated. Male C57BL/6J mice completed four cycles (acute) of FOLFOX or PBS. Subsets were allowed to recover for 4 wk or 10 wk. Comprehensive Laboratory Animal Monitoring System (CLAMS) metabolic measurements were performed for 5 days before study endpoint. C2C12 myotubes were treated with FOLFOX for 24 hr. Acute FOLFOX attenuated body mass and body fat accretion independent of food intake or cage activity. Acute FOLFOX decreased blood glucose, oxygen consumption (V̇o2), carbon dioxide production (V̇co2), energy expenditure, and carbohydrate (CHO) oxidation. Deficits in V̇o2 and energy expenditure remained at 10 wk. CHO oxidation remained disrupted at 4 wk but returned to control levels after 10 wk. Acute FOLFOX reduced muscle COXIV enzyme activity, AMPK(T172), ULK1(S555), and LC3BII protein expression. Muscle LC3BII/I ratio was associated with altered CHO oxidation (r = 0.75, P = 0.03). In vitro, FOLFOX suppressed myotube AMPK(T172), ULK1(S555), and autophagy flux. Recovery for 4 wk normalized skeletal muscle AMPK and ULK1 phosphorylation. Our results provide evidence that FOLFOX disrupts systemic metabolism, which is not readily recoverable after treatment cessation. FOLFOX effects on skeletal muscle metabolic signaling did recover. Further investigations are warranted to prevent and treat FOLFOX-induced metabolic toxicities that negatively impact survival and life quality of patients with cancer.NEW & NOTEWORTHY The present study demonstrates that FOLFOX chemotherapy induces long-lasting deficits in systemic metabolism. Interestingly, FOLFOX modestly suppressed skeletal muscle AMPK and autophagy signaling in vivo and in vitro. The FOLFOX-induced suppression of muscle metabolic signaling recovered after treatment cessation, independent of systemic metabolic dysfunction. Future research should investigate if activating AMPK during treatment can prevent long-term toxicities to improve health and quality of life of patients with cancer and survivors.

Short duration treadmill exercise improves physical function and skeletal muscle mitochondria protein expression after recovery from FOLFOX chemotherapy in male mice.

FOLFOX (5-FU, leucovorin, oxaliplatin) is a chemotherapy treatment for colorectal cancer which induces toxic side effects involving fatigue, weakness, and skeletal muscle dysfunction. There is a limited understanding of the recovery from these toxicities after treatment cessation. Exercise training can improve chemotherapy-related toxicities. However, how exercise accelerates recovery and the dose required for these benefits are not well examined. The purpose of this study was to examine the effect of exercise duration on physical function, muscle mass, and mitochondria protein expression during the recovery from FOLFOX chemotherapy. 12-week-old male mice were administered four cycles of either PBS or FOLFOX over 8-weeks. Outcomes were assessed after the fourth cycle and after either 4 (short-term; STR) or 10 weeks (long-term; LTR) recovery. Subsets of mice performed 14 sessions (6 d/wk, 18 m/min, 5% grade) of 60 min/d (long) or 15 min/d (short duration) treadmill exercise during STR. Red and white gastrocnemius mRNA and protein expression were examined. FOLFOX treatment decreased run time (RT) (-53%) and grip strength (GS) (-9%) compared to PBS. FOLFOX also reduced muscle OXPHOS complexes, COXIV, and VDAC protein expression. At LTR, FOLFOX RT (-36%) and GS (-16%) remained reduced. Long- and short-duration treadmill exercise improved RT (+58% and +56%) without restoring GS in FOLFOX mice. Both exercise durations increased muscle VDAC and COXIV expression in FOLFOX mice. These data provide evidence that FOLFOX chemotherapy induces persistent deficits in physical function that can be partially reversed by short-duration aerobic exercise.

Proximity to Failure and Total Repetitions Performed in a Set Influences Accuracy of Intraset Repetitions in Reserve-Based Rating of Perceived Exertion.

ABSTRACT: Zourdos, MC, Goldsmith, JA, Helms, ER, Trepeck, C, Halle, JL, Mendez, KM, Cooke, DM, Haischer, MH, Sousa, CA, Klemp, A, and Byrnes, RK. Proximity to failure and total repetitions performed in a set influences accuracy of intraset repetitions in reserve-based rating of perceived exertion. J Strength Cond Res 35(2S): S158-S165, 2021-The aim of this study was to assess the accuracy of predicting repetitions in reserve (RIR) intraset using the RIR-based rating of perceived exertion (RPE) scale. Twenty-five men (age: 25.3 ± 3.3 years, body mass: 89.0 ± 14.7 kg, height: 174.69 ± 6.7 cm, and training age: 4.7 ± 3.2 years) reported to the laboratory. Subjects performed a 1 repetition maximum (1RM) squat followed by one set to failure at 70% of 1RM. During the 70% set, subjects verbally indicated when they believed they were at a 5RPE (5RIR), 7RPE (3RIR), or 9RPE (1RIR), and then continued to failure. The difference between actual repetitions performed and participant-predicted repetitions was calculated as the RIR difference (RIRDIFF). The average load used for the 70% set was 123.10 ± 24.25 kg and the average repetitions performed were 16 ± 4. The RIRDIFF was lower (RPEs were more accurate) closer to failure (RIRDIFF at 9RPE = 2.05 ± 1.73; RIRDIFF at 7RPE = 3.65 ± 2.46; and RIRDIFF at 5RPE = 5.15 ± 2.92 repetitions). There were significant relationships between total repetitions performed and RIRDIFF at 5RPE (r = 0.65, p = 0.001) and 7RPE (r = 0.56, p = 0.004), but not at 9RPE (r = 0.01, p = 0.97). Thus, being farther from failure and performing more repetitions in a set were associated with more inaccurate predictions. Furthermore, a multiple linear regression revealed that more repetitions performed per set was a significant predictor of RIR prediction inaccuracy at the called 5 (p = 0.003) and 7 (p = 0.011) RPEs, while training age (p > 0.05) was not predictive of rating accuracy. These data indicate RIR predictions are improved during low to moderate repetition sets and when there is close proximity to failure.

Tissue-specific dysregulation of mitochondrial respiratory capacity and coupling control in colon-26 tumor-induced cachexia.

In addition to skeletal muscle dysfunction, cancer cachexia is a systemic disease involving remodeling of nonmuscle organs such as adipose and liver. Impairment of mitochondrial function is associated with multiple chronic diseases. The tissue-specific control of mitochondrial function in cancer cachexia is not well defined. This study determined mitochondrial respiratory capacity and coupling control of skeletal muscle, white adipose tissue (WAT), and liver in colon-26 (C26) tumor-induced cachexia. Tissues were collected from PBS-injected weight-stable mice, C26 weight-stable mice and C26 mice with moderate (10% weight loss) and severe cachexia (20% weight loss). The respiratory control ratio [(RCR) an index of oxidative phosphorylation (OXPHOS) coupling efficiency] was low in WAT during the induction of cachexia because of high nonphosphorylating LEAK respiration. Liver RCR was low in C26 weight-stable and moderately cachexic mice because of reduced OXPHOS. Liver RCR was further reduced with severe cachexia, where Ant2 but not Ucp2 expression was increased. Ant2 was inversely correlated with RCR in the liver (r = -0.547, P < 0.01). Liver cardiolipin increased in moderate and severe cachexia, suggesting this early event may also contribute to mitochondrial uncoupling. Impaired skeletal muscle mitochondrial respiration occurred predominantly in severe cachexia, at complex I. These findings suggest that mitochondrial function is subject to tissue-specific control during cancer cachexia, whereby remodeling in WAT and liver arise early and may contribute to altered energy balance, followed by impaired skeletal muscle respiration. We highlight an under-recognized role of liver and WAT mitochondrial function in cancer cachexia and suggest mitochondrial function of multiple tissues to be therapeutic targets.

Mouse skeletal muscle adaptations to different durations of treadmill exercise after the cessation of FOLFOX chemotherapy.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is a treatment for colorectal cancer that can induce persistent fatigue and metabolic dysfunction. Regular exercise after chemotherapy cessation is widely recommended for cancer patients and has been shown to improve fatigue resistance in mice. However, gaps remain in understanding whether the early systemic and skeletal muscle adaptations to regular exercise are altered by prior FOLFOX chemotherapy treatment. Furthermore, the effects of exercise duration on early metabolic and skeletal muscle transcriptional adaptations are not fully established. Purpose: Investigate the effects of prior FOLFOX chemotherapy treatment on the early adaptations to repeated short- or long-duration treadmill exercise, including the fasting regulation of circulating metabolic regulators, skeletal muscle COXIV activity and myokine/exerkine gene expression in male mice. Methods: Male C57BL6/J mice completed 4 cycles of FOLFOX or PBS and were allowed to recover for 4-weeks. Subsets of mice performed 14 sessions (6 d/wk, 18 m/min, 5% grade) of short- (10 min/d) or long-duration (55 min/d) treadmill exercise. Blood plasma and muscle tissues were collected 48-72 h after the last exercise bout for biochemical analyses. Results: Long-duration exercise increased fasting plasma osteocalcin, LIF, and IL-6 in healthy PBS mice, and these changes were ablated by prior FOLFOX treatment. Slow-oxidative soleus muscle COXIV activity increased in response to long-duration exercise in PBS mice, which was blocked by prior FOLFOX treatment. Fast-glycolytic plantaris muscle COXIV activity increased with short-duration exercise independent of FOLFOX administration. There was a main effect for long-duration exercise to increase fasting muscle IL-6 and COXIV mRNA expression independent of FOLFOX. FOLFOX administration reduced muscle IL-6, LIF, and BDNF mRNA expression irrespective of long-duration exercise. Interestingly, short-duration exercise suppressed the FOLXOX induction of muscle myostatin mRNA expression. Conclusion: FOLFOX attenuated early exercise adaptations related to fasting circulating osteocalcin, LIF, and IL-6. However, prior FOLFOX treatment did not alter the exercise adaptations of plantaris muscle COXIV activity and plasma adiponectin. An improved understanding of mechanisms underlying exercise adaptations after chemotherapy will provide the basis for successfully treating fatigue and metabolic dysfunction in cancer survivors.

Protein kinase C delta regulates mononuclear phagocytes and hinders response to immunotherapy in cancer.

Mononuclear phagocytes (MPs) play a crucial role in tissue homeostasis; however, MPs also contribute to tumor progression and resistance to immune checkpoint blockade (ICB). Targeting MPs could be an effective strategy to enhance ICB efficacy. We report that protein kinase C delta (PKCδ), a serine/threonine kinase, is abundantly expressed by MPs in human and mouse tumors. PKCδ-/- mice displayed reduced tumor progression compared to wild types, with increased response to anti-PD-1. Tumors from PKCδ-/- mice demonstrated TH1-skewed immune response including increased antigen presentation and T cell activation. Depletion of MPs in vivo altered tumor growth in control but not PKCδ-/- mice. Coinjection of PKCδ-/- M2-like macrophages with cancer cells into wild-type mice markedly delayed tumor growth and significantly increased intratumoral T cell activation compared to PKCδ+/+ controls. PKCδ deficiency reprogrammed MPs by activating type I and type II interferon signaling. Thus, PKCδ might be targeted to reprogram MPs to augment ICB efficacy.

Early-Onset Physical Inactivity and Metabolic Dysfunction in Tumor-bearing Mice Is Associated with Accelerated Cachexia.

METHODS: Male C57BL/6J mice (12 wk of age) were injected with 1 × 106 LLC cells or phosphate-buffered saline (PBS) subcutaneously in the right flank, and tissue was collected 26-28 d after cell injection. Tumor volume was measured every 5 d throughout the study to calculate the tumor growth rate. Fifteen days after tumor inoculation, a subset of PBS (n = 11) and LLC (n = 16) mice were individually housed in metabolic Comprehensive Laboratory Animal Monitoring System cages for 5 d. RESULTS: LLC mice exhibited greater body weight loss (-5.1%), decreased muscle mass (-7%), decreased fat mass (-22%), and increased plasma interleukin-6 (212%) compared with PBS mice. Before the onset of cachexia, total cage activity was decreased in tumor-bearing mice. Cage activity was negatively associated with tumor mass and positively associated with hindlimb muscle mass. In addition, LLC mice had greater lipid oxidation than PBS mice. CONCLUSIONS: LLC mice exhibit early-onset physical inactivity and altered systemic lipid oxidation, which are associated with the eventual development of cachexia.

The Effect of Mechanical Stretch on Myotube Growth Suppression by Colon-26 Tumor-Derived Factors.

Preclinical models and in vitro experiments have provided valuable insight into the regulation of cancer-induced muscle wasting. Colon-26 (C26) tumor cells induce cachexia in mice, and conditioned media (CM) from these cells promotes myotube atrophy and catabolic signaling. While mechanical stimuli can prevent some effects of tumor-derived factors on myotubes, the impact of mechanical signaling on tumor-derived factor regulation of myosin heavy chain (MyHC) expression is not well understood. Therefore, we examined the effects of stretch-induced mechanical signaling on C2C12 myotube growth and MyHC expression after C26 CM exposure. C26 CM was administered to myotubes on day 5 of differentiation for 48 h. During the last 4 or 24 h of C26 CM exposure, 5% static uniaxial stretch was administered. C26 CM suppressed myotube growth and MyHC protein and mRNA expression. Stretch for 24 h increased myotube size and prevented the C26 CM suppression of MyHC-Fast protein expression. Stretch did not change suppressed MyHC mRNA expression. Stretch for 24 h reduced Atrogin-1/MAFbx, MuRF-1, and LC3B II/I ratio and increased integrin ß1D protein expression and the myogenin-to-MyoD protein ratio. Stretch in the last 4 h of CM increased ERK1/2 phosphorylation but did not alter the CM induction of STAT3 or p38 phosphorylation. These results provide evidence that in myotubes pre-incubated with CM, the induction of mechanical signaling can still provide a growth stimulus and preserve MyHC-Fast protein expression independent of changes in mRNA expression.

Exercise as a therapy for cancer-induced muscle wasting.

Cancer cachexia is a progressive disorder characterized by body weight, fat, and muscle loss. Cachexia induces metabolic disruptions that can be analogous and distinct from those observed in cancer, obscuring both diagnosis and treatment options. Inflammation, hypogonadism, and physical inactivity are widely investigated as systemic mediators of cancer-induced muscle wasting. At the cellular level, dysregulation of protein turnover and energy metabolism can negatively impact muscle mass and function. Exercise is well known for its anti-inflammatory effects and potent stimulation of anabolic signaling. Emerging evidence suggests the potential for exercise to rescue muscle's sensitivity to anabolic stimuli, reduce wasting through protein synthesis modulation, myokine release, and subsequent downregulation of proteolytic factors. To date, there is no recommendation for exercise in the management of cachexia. Given its complex nature, a multimodal approach incorporating exercise offers promising potential for cancer cachexia treatment. This review's primary objective is to summarize the growing body of research examining exercise regulation of cancer cachexia. Furthermore, we will provide evidence for exercise interactions with established systemic and cellular regulators of cancer-induced muscle wasting.

Hippocampal Growth Factor and Myokine Cathepsin B Expression following Aerobic and Resistance Training in 3xTg-AD Mice.

Aerobic training (AT) can support brain health in Alzheimer's disease (AD); however, the role of resistance training (RT) in AD is not well established. Aside from direct effects on the brain, exercise may also regulate brain function through secretion of muscle-derived myokines. Aims. This study examined the effects of AT and RT on hippocampal BDNF and IGF-1 signaling, ß-amyloid expression, and myokine cathepsin B in the triple transgenic (3xTg-AD) model of AD. 3xTg-AD mice were assigned to one of the following groups: sedentary (Tg), aerobic trained (Tg+AT, 9 wks treadmill running), or resistance trained (Tg+RT, 9 wks weighted ladder climbing) (n = 10/group). Rotarod latency and strength were assessed pre- and posttraining. Hippocampus and skeletal muscle were collected after training and analyzed by high-resolution respirometry, ELISA, and immunoblotting. Tg+RT showed greater grip strength than Tg and Tg+AT at posttraining (p < 0.01). Only Tg+AT improved rotarod peak latency (p < 0.01). Hippocampal IGF-1 concentration was ~15% greater in Tg+AT and Tg+RT compared to Tg (p < 0.05); however, downstream signals of p-IGF-1R, p-Akt, p-MAPK, and p-GSK3ß were not altered. Cathepsin B, hippocampal p-CREB and BDNF, and hippocampal mitochondrial respiration were not affected by AT or RT. ß-Amyloid was ~30% lower in Tg+RT compared to Tg (p < 0.05). This data suggests that regular resistance training reduces ß-amyloid in the hippocampus concurrent with increased concentrations of IGF-1. Both types of training offered distinct benefits, either by improving physical function or by modifying signals in the hippocampus. Therefore, inclusion of both training modalities may address central defects, as well as peripheral comorbidities in AD.

Validity of the Open Barbell and Tendo Weightlifting Analyzer Systems Versus the Optotrak Certus 3D Motion-Capture System for Barbell Velocity.

PURPOSE: To examine the validity of 2 linear position transducers, the Tendo Weightlifting Analyzer System (TWAS) and Open Barbell System (OBS), compared with a criterion device, the Optotrak Certus 3-dimensional motion-capture system (OC3D). METHODS: A total of 25 men (age, 25 [3] y; height, 174.0 [6.7] cm; body mass, 89.0 [14.7] kg; squat 1-repetition maximum [1RM], 175.8 [34.7] kg) with ≥2 y of resistance-training experience completed a back 1RM and 1 set to failure at 70% of 1RM. Average concentric velocity (ACV) and peak concentric velocity (PCV) were recorded by all 3 devices during the final warm-up set, all 1RM attempts, and every repetition during the 70% set. RESULTS: In total, 575 samples were obtained. Bland-Altman plots, mountain plots, a 1-way analysis of variance, SEM, and intraclass correlation coefficients were used to analyze validity. The analysis of variance showed no difference (P = .089) between devices for ACV. However, for PCV, TWAS was significantly different (ie, inaccurate) from OC3D (P < .001) and OBS (P = .001), but OBS was similar (P = .412) to OC3D. For ACV, intraclass correlation coefficients were higher for OBS than for TWAS. Bland-Altman plots showed agreement for ACV for both devices against OC3D but large limits of agreement for PCV for both devices. Mountain plots showed valid ACV for both devices, however, but slightly greater ACV and PCV accuracy with OBS than TWAS. CONCLUSIONS: Both devices may provide valid ACV measurements, but some metrics suggest more accurate ACV with OBS vs TWAS. For PCV, neither device is particularly accurate; however, OBS seems to be more accurate than TWAS.