Leucine Supplementation Exacerbates Morbidity in Male but Not Female Mice with Colorectal Cancer-Induced Cachexia.

Cancer cachexia (CC) is a multifactorial wasting syndrome characterized by a significant loss in lean and/or fat mass and represents a leading cause of mortality in cancer patients. Nutraceutical treatments have been proposed as a potential treatment strategy to mitigate cachexia-induced muscle wasting. However, contradictory findings warrant further investigation. The purpose of this study was to determine the effects of leucine supplementation on skeletal muscle in male and female ApcMin/+ mice (APC). APC mice and their wild-type (WT) littermates were given normal drinking water or 1.5% leucine-supplemented water (n = 4-10/group/sex). We measured the gene expression of regulators of inflammation, protein balance, and myogenesis. Leucine treatment lowered survival rates, body mass, and muscle mass in males, while in females, it had no effect on body or muscle mass. Leucine treatment altered inflammatory gene expression by lowering Il1b 87% in the APC group and decreasing Tnfa 92% in both WT and APC males, while it had no effect in females (p < 0.05). Leucine had no effect on regulators of protein balance and myogenesis in either sex. We demonstrated that leucine exacerbates moribundity in males and is not sufficient for mitigating muscle or fat loss during CC in either sex in the ApcMin/+ mouse.

Biological sex divergence in transcriptomic profiles during the onset of hindlimb unloading-induced atrophy.

Disuse-induced muscle atrophy is a common clinical problem observed mainly in older adults, intensive care units patients, or astronauts. Previous studies presented biological sex divergence in progression of disuse-induced atrophy along with differential changes in molecular mechanisms possibly underlying muscle atrophy. The aim of this study was to perform transcriptomic profiling of male and female mice during the onset and progression of unloading disuse-induced atrophy. Male and female mice underwent hindlimb unloading (HU) for 24, 48, 72, and 168 h (n = 8/group). Muscles were weighed for each cohort and gastrocnemius was used for RNA-sequencing analysis. Females exhibited muscle loss as early as 24 h of HU, whereas males after 168 h of HU. In males, pathways related to proteasome degradation were upregulated throughout 168 h of HU, whereas in females these pathways were upregulated up to 72 h of HU. Lcn2, a gene contributing to regulation of myogenesis, was upregulated by 6.46- to 19.86-fold across all time points in females only. A reverse expression of Fosb, a gene related to muscle degeneration, was observed between males (4.27-fold up) and females (4.57-fold down) at 24-h HU. Mitochondrial pathways related to tricarboxylic acid (TCA) cycle were highly downregulated at 168 h of HU in males, whereas in females this downregulation was less pronounced. Collagen-related pathways were consistently downregulated throughout 168 h of HU only in females, suggesting a potential biological sex-specific protective mechanism against disuse-induced fibrosis. In conclusion, females may have protection against HU-induced skeletal muscle mitochondrial degeneration and fibrosis through transcriptional mechanisms, although they may be more vulnerable to HU-induced muscle wasting compared with males.NEW & NOTEWORTHY Herein, we have assessed the transcriptomic response across biological sexes during the onset and progression of unloading disuse-induced atrophy in mice. We have demonstrated an inverse expression of Fosb between males and females, as well as differentially timed patterns of expressing atrophy-related pathways between sexes that are concomitant to the accelerated atrophy in females. We also identified in females signs of mechanisms to combat disuse-induced mitochondrial degeneration and fibrosis.

Females display relatively preserved muscle quality compared with males during the onset and early stages of C26-induced cancer cachexia.

Cancer cachexia is clinically defined by involuntary weight loss >5% in <6 mo, primarily affecting skeletal muscle. Here, we aimed to identify sex differences in the onset of colorectal cancer cachexia with specific consideration to skeletal muscle contractile and metabolic functions. Eight-weeks old BALB/c mice (69 males, 59 females) received subcutaneous C26 allografts or PBS vehicle. Tumors were developed for 10-, 15-, 20-, or 25 days. Muscles and organs were collected, in vivo muscle contractility, protein synthesis rate, mitochondrial function, and protein turnover markers were assessed. One-way ANOVA within sex and trend analysis between sexes were performed, P < 0.05. Gastrocnemius and tibialis anterior (TA) muscles became atrophic in male mice at 25 days, whereas female mice exhibited no significant differences in muscle weights at endpoints despite presenting hallmarks of cancer cachexia (fat loss, hepatosplenomegaly). We observed lowered muscle contractility and protein synthesis concomitantly to muscle mass decay in males, with higher proteolytic markers in muscles of both sexes. mRNA of Opa1 was lower in TA, whereas Bnip3 was higher in gastrocnemius after 25 days in male mice, with no significant effect in female mice. Our data suggest relative protections to skeletal muscle in females compared with males despite other canonical signs of cancer cachexia and increased protein degradation markers; suggesting we should place onus upon nonmuscle tissues during early stages of cancer cachexia in females. We noted potential protective mechanisms relating to skeletal muscle contractile and mitochondrial functions. Our findings underline possible heterogeneity in onset of cancer cachexia between biological sexes, suggesting the need for sex-specific approaches to treat cancer cachexia.NEW & NOTEWORTHY Our study demonstrates biological-sex differences in phenotypic characteristics of cancer cachexia between male and female mice, whereby females display many common characteristics of cachexia (gonadal fat loss and hepatosplenomegaly), protein synthesis markers alterations, and common catabolic markers in skeletal muscle despite relatively preserved muscle mass in early-stage cachexia compared with males. Mechanisms of cancer cachexia appear to differ between sexes. Data suggest need to place onus of early cancer cachexia detection and treatment on nonmuscle tissues in females.

The time-course of cancer cachexia onset reveals biphasic transcriptional disruptions in female skeletal muscle distinct from males.

BACKGROUND: Cancer-cachexia (CC) is a debilitating condition affecting up to 80% of cancer patients and contributing to 40% of cancer-related deaths. While evidence suggests biological sex differences in the development of CC, assessments of the female transcriptome in CC are lacking, and direct comparisons between sexes are scarce. This study aimed to define the time course of Lewis lung carcinoma (LLC)-induced CC in females using transcriptomics, while directly comparing biological sex differences. RESULTS: We found the global gene expression of the gastrocnemius muscle of female mice revealed biphasic transcriptomic alterations, with one at 1 week following tumor allograft and another during the later stages of cachexia development. The early phase was associated with the upregulation of extracellular-matrix pathways, while the later phase was characterized by the downregulation of oxidative phosphorylation, electron transport chain, and TCA cycle. When DEGs were compared to a known list of mitochondrial genes (MitoCarta), ~ 47% of these genes were differently expressed in females exhibiting global cachexia, suggesting transcriptional changes to mitochondrial gene expression happens concomitantly to functional impairments previously published. In contrast, the JAK-STAT pathway was upregulated in both the early and late stages of CC. Additionally, we observed a consistent downregulation of Type-II Interferon signaling genes in females, which was associated with protection in skeletal muscle atrophy despite systemic cachexia. Upregulation of Interferon signaling was noted in the gastrocnemius muscle of cachectic and atrophic male mice. Comparison of female tumor-bearing mice with males revealed ~ 70% of DEGs were distinct between sexes in cachectic animals, demonstrating dimorphic mechanisms of CC. CONCLUSION: Our findings suggest biphasic disruptions in the transcriptome of female LLC tumor-bearing mice: an early phase associated with ECM remodeling and a late phase, accompanied by the onset of systemic cachexia, affecting overall muscle energy metabolism. Notably, ~ 2/3 of DEGs in CC are biologically sex-specific, providing evidence of dimorphic mechanisms of cachexia between sexes. Downregulation of Type-II Interferon signaling genes appears specific to CC development in females, suggesting a new biological sex-specific marker of CC not reliant on the loss of muscle mass, that might represent a protective mechanism against muscle loss in CC in female mice.

Development of skeletal muscle fibrosis in a rodent model of cancer cachexia.

Cachexia is characterized by losses in lean body mass and its progression results in worsened quality of life and exacerbated outcomes in cancer patients. However, the role and impact of fibrosis during the early stages and development of cachexia in under-investigated. The purpose of this study was to determine if fibrosis occurs during cachexia development, and to evaluate this in both sexes. Female and male C57BL6/J mice were injected with phosphate-buffered saline or Lewis Lung Carcinoma (LLC) at 8-week of age, and tumors were allowed to develop for 1, 2, 3, or 4 weeks. 3wk and 4wk female tumor-bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. In vitro analyses were also performed on cocultured C2C12 and 3T3 cells exposed to LLC conditioned media. Immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis were used to investigate fibrosis and fibrosis-related signaling in skeletal muscle. Collagen deposition in skeletal muscle was increased in the 1wk, LT, and HT groups in female mice. However, collagen deposition was only increased in the 4wk group in male mice. In general, female mice displayed earlier alterations in extracellular matrix (ECM)-related genes beginning at 1wk post-LLC injection. Whereas this was not seen in males. While overall tumor burden is tightly correlated to cachexia development in both sexes, fibrotic development is not. Male mice did not exhibit early-stage alterations in ECM-related genes contrary to what was noted in female mice.

Influence of gonadectomy on muscle health in micro- and partial-gravity environments in rats.

Gonadal hormones, such as testosterone and estradiol, modulate muscle size and strength in males and females. However, the influence of sex hormones on muscle strength in micro- and partial-gravity environments (e.g., the Moon or Mars) is not fully understood. The purpose of this study was to determine the influence of gonadectomy (castration/ovariectomy) on progression of muscle atrophy in both micro- and partial-gravity environments in male and female rats. Male and female Fischer rats (n = 120) underwent castration/ovariectomy (CAST/OVX) or sham surgery (SHAM) at 11 wk of age. After 2 wk of recovery, rats were exposed to hindlimb unloading (0 g), partial weight bearing at 40% of normal loading (0.4 g, Martian gravity), or normal loading (1.0 g) for 28 days. In males, CAST did not exacerbate body weight loss or other metrics of musculoskeletal health. In females, OVX animals tended to have greater body weight loss and greater gastrocnemius loss. Within 7 days of exposure to either microgravity or partial gravity, females had detectable changes to estrous cycle, with greater time spent in low-estradiol phases diestrus and metestrus (∼47% in 1 g vs. 58% in 0 g and 72% in 0.4 g animals, P = 0.005). We conclude that in males testosterone deficiency at the initiation of unloading has little effect on the trajectory of muscle loss. In females, initial low estradiol status may result in greater musculoskeletal losses.NEW & NOTEWORTHY We find that removal of gonadal hormones does not exacerbate muscle loss in males or females during exposure to either simulated microgravity or partial-gravity environments. However, simulated micro- and partial gravity did affect females' estrous cycles, with more time spent in low-estrogen phases. Our findings provide important data on the influence of gonadal hormones on the trajectory of muscle loss during unloading and will help inform NASA for future crewed missions to space and other planets.

Anorexia nervosa and muscle health: A systematic review of our current understanding and future recommendations for study.

OBJECTIVE: Conduct a systematic review on muscle size and strength in individuals with anorexia nervosa (AN). METHOD: In accordance with PRISMA guidelines, we searched Pubmed for articles published between 1995 and 2022 using a combination of search terms related to AN and muscle size, strength, or metabolism. After two authors screened articles and extracted data, 30 articles met inclusion criteria. Data were coded, and a risk bias was conducted for each study. RESULTS: The majority of studies focused on muscle size/lean mass (60%, n = 18) and energy expenditure (33%, n = 9), with few studies (17%, n = 5) investigating muscle function or possible mechanisms underlying muscle size (20%, n = 6). Studies supported that individuals with AN have smaller muscle size and reduced energy expenditure relative to controls. In some studies (33%, n = 10) recovery from AN was not sufficient to restore muscle mass or function. Mechanisms underlying short and long-term musculoskeletal alterations have not been thoroughly explored. DISCUSSION: Muscle mass and strength loss may be an unexplored component of physiological deterioration during and after AN. More research is necessary to understand intramuscular alterations during AN and interventions to facilitate muscle mass and functional gain following weight restoration in AN. PUBLIC SIGNIFICANCE: Muscle health is important for optimal health and is reduced in individuals with AN. However, we do not understand how muscle is altered at the cellular level throughout the course of AN. Here we review what is currently known regarding muscle health during AN and with weight restoration.

OBJETIVO: Realizar una revisión sistemática sobre el tamaño y la fuerza muscular en individuos que padecen anorexia nerviosa (AN). MÉTODO: De acuerdo con las guías PRISMA, se realizaron búsquedas en Pubmed de artículos publicados entre 1995 y 2022 mediante una combinación de términos de búsqueda relacionados con la anorexia nerviosa y el tamaño, la fuerza o el metabolismo muscular. Después de que dos autores examinaron los artículos y extrajeron los datos, 30 artículos cumplieron los criterios de inclusión. Se codificaron los datos y se realizó un sesgo de riesgo para cada estudio. RESULTADOS: La mayoría de los estudios se enfocaron en el tamaño muscular/masa magra (60%, n=18) y el gasto energético (33%, n=9), con pocos estudios (17%, n=5) investigando la función muscular o los posibles mecanismos subyacentes al tamaño muscular (20%, n=6). Los estudios apoyaron que los individuos que padecen anorexia nerviosa tienen un tamaño muscular más pequeño y un gasto de energía reducido en relación con los controles. En algunos estudios (33%, n = 10) la recuperación de la anorexia nerviosa no fue suficiente para restaurar la masa muscular o la función. Los mecanismos subyacentes a las alteraciones musculoesqueléticas a corto y largo plazo no se han explorado a fondo. DISCUSIÓN: La pérdida de masa muscular y fuerza puede ser un componente inexplorado del deterioro fisiológico durante y después de la AN. Se necesita más investigación para comprender las alteraciones intramusculares durante la anorexia nerviosa y las intervenciones para facilitar la masa muscular y la ganancia funcional después de la restauración del peso en la anorexia nerviosa.

Sex differences in muscle health in simulated micro- and partial-gravity environments in rats.

Skeletal muscle size and strength are important for overall health for astronauts. However, how male and female muscle may respond differently to micro- and partial-gravity environments is not fully understood. The purpose of this study was to determine how biological sex and sex steroid hormones influence the progression of muscle atrophy after long term exposure to micro and partial gravity environments in male and female rats. Male and female Fisher rats (n â€‹= â€‹120) underwent either castration/ovariectomy or sham surgeries. After two weeks recovery, animals were divided into microgravity (0g), partial-gravity (40% of weight bearing, 0.4g), or full weight bearing (1g) interventions for 28 days. Measurements of muscle size and strength were evaluated prior to and after interventions. At 0g, females lost more dorsiflexion strength, plantar flexion strength, and other metrics of muscle size compared to males; castration/ovariectomy did not influence these differences. Additionally, at 0.4g, females lost more dorsiflexion strength, plantar flexion strength, and other metrics of muscle strength compared to males; castration/ovariectomy did not influence these differences. Females have greater musculoskeletal aberrations during exposure to both microgravity and partial-gravity environments; these differences are not dependent on the presence of sex steroid hormones. Correspondingly, additional interventions may be necessary to mitigate musculoskeletal loss in female astronauts to protect occupational and overall health.

Muscle weakness precedes atrophy during cancer cachexia and is linked to muscle-specific mitochondrial stress.

Muscle weakness and wasting are defining features of cancer-induced cachexia. Mitochondrial stress occurs before atrophy in certain muscles, but the possibility of heterogeneous responses between muscles and across time remains unclear. Using mice inoculated with Colon-26 cancer, we demonstrate that specific force production was reduced in quadriceps and diaphragm at 2 weeks in the absence of atrophy. At this time, pyruvate-supported mitochondrial respiration was lower in quadriceps while mitochondrial H2O2 emission was elevated in diaphragm. By 4 weeks, atrophy occurred in both muscles, but specific force production increased to control levels in quadriceps such that reductions in absolute force were due entirely to atrophy. Specific force production remained reduced in diaphragm. Mitochondrial respiration increased and H2O2 emission was unchanged in both muscles versus control while mitochondrial creatine sensitivity was reduced in quadriceps. These findings indicate muscle weakness precedes atrophy and is linked to heterogeneous mitochondrial alterations that could involve adaptive responses to metabolic stress. Eventual muscle-specific restorations in specific force and bioenergetics highlight how the effects of cancer on one muscle do not predict the response in another muscle. Exploring heterogeneous responses of muscle to cancer may reveal new mechanisms underlying distinct sensitivities, or resistance, to cancer cachexia.

Exercise during weight loss improves hepatic mitophagy.

Non-alcoholic fatty liver disease (NAFLD) has recently become a public health concern concurrent with the obesity crisis. Previous work has shown aberrant mitochondrial content/quality and autophagy in models of NAFLD, whereas exercise is known to improve these derangements. The purpose of this study was to examine the effect of different weight-loss modalities on hepatic mitochondrial content, autophagy and mitophagy in NAFLD. Forty-eight male C57BL/6J mice were divided into 1 of 4 groups: low fat diet (LFD, 10% fat, 18 weeks), high fat diet (HFD, 60% fat diet, 18 weeks), weight-loss by diet (D, 60% fat diet for 10 weeks then 10% fat diet for 8 weeks) or weight-loss by diet and physical activity (D/PA, 60% fat diet for 10 weeks, then 10% fat diet plus a running wheel for 8 weeks). Immunoblot data were analyzed by one-way analysis of variance (ANOVA) with significance denoted at p â€‹< â€‹0.05. COX-IV protein contents were approximately 50% less in HFD compared to LFD. D/PA had 50% more BNIP3 compared to HFD. PINK1 content was 40% higher in D and D/PA compared to LFD. P-PARKIN/PARKIN levels were 40% lower in HFD, D, and D/PA compared to LFD. Whereas p-UbSer65 was 3-fold higher in HFD. LC3II/I ratio was 50% greater in HFD and D/PA, yet p62 protein content was 2.5 fold higher in HFD. High-fat diet causes disruptions in markers of mitochondrial quality control. Physical activity combined with diet were able to ameliorate these derangements and seemingly improve hepatic mitochondrial quality above control values.

Effects of PGC-1α overexpression on the myogenic response during skeletal muscle regeneration.

The ability of skeletal muscle to regenerate from injury is crucial for locomotion, metabolic health, and quality of life. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1A) is a transcriptional coactivator required for mitochondrial biogenesis. Increased mitochondrial biogenesis is associated with improved muscle cell differentiation, however PGC1A's role in skeletal muscle regeneration following damage requires further investigation. The purpose of this study was to investigate the role of skeletal muscle-specific PGC1A overexpression during regeneration following damage. 22 C57BL/6J (WT) and 26 PGC1A muscle transgenic (A1) mice were injected with either phosphate-buffered saline (PBS, uninjured control) or Bupivacaine (MAR, injured) into their tibialis anterior (TA) muscle to induce skeletal muscle damage. TA muscles were extracted 3- or 28-days post-injury and analyzed for markers of regenerative myogenesis and protein turnover. Pgc1a mRNA was ∼10-20 fold greater in A1 mice. Markers of protein synthesis, AKT and 4EBP1, displayed decreases in A1 mice compared to WT at both timepoints indicating a decreased protein synthetic response. Myod mRNA was ∼75% lower compared to WT 3 days post-injection. WT mice exhibited decreased cross-sectional area of the TA muscle at 28 days post-injection with bupivacaine compared to all other groups. PGC1A overexpression modifies the myogenic response during regeneration.

PGC-1α overexpression is not sufficient to mitigate cancer cachexia in either male or female mice.

Cancer cachexia (CC) accounts for 20%-40% of cancer-related deaths. Mitochondrial aberrations have been shown to precede muscle atrophy in different atrophy models, including cancer. Therefore, this study investigated potential protection from the cachectic phenotype through overexpression of peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α). First, to establish potential of mitochondria-based approaches we showed that the mitochondrial antioxidant MitoTEMPO (MitoT) attenuates myotube atrophy induced by Lewis lung carcinoma (LLC) cell conditioned media. Next, cachexia was induced in muscle-specific PGC-1α overexpressing (MCK-PCG1α) or wildtype (WT) littermate mice by LLC implantation. MCK-PCG1α did not protect LLC-induced muscle mass loss. In plantaris, Atrogin mRNA content was 6.2-fold and ∼11-fold greater in WT-LLC vs WT-phosphate-buffered saline (PBS) for males and females, respectively (p < 0.05). MitoTimer red:green ratio for male PGC was ∼65% higher than WT groups (p < 0.05), with ∼3-fold more red puncta in LLC than PBS (p < 0.05). Red:green ratio was ∼56% lower in females WT-LLC vs PGC-LLC (p < 0.05). In females, no change in red puncta was noted across conditions. Lc3 mRNA content was ∼73% and 2-fold higher in male and female LLC mice, respectively, vs PBS (p < 0.05). While MitoT could mitigate cancer-induced atrophy in vitro, PGC-1α overexpression was insufficient to protect muscle mass and mitochondrial health in vivo despite mitigation of cachexia-associated signaling pathways.

Muscle miR-16 deletion results in impaired insulin sensitivity and contractile function in a sex-dependent manner.

microRNAs (miRs) are linked to various human diseases including type 2 diabetes mellitus (T2DM) and emerging evidence suggests that miRs may serve as potential therapeutic targets. Lower miR-16 content is consistent across different models of T2DM; however, the role of miR-16 in muscle metabolic health is still elusive. Therefore, the purpose of this study was to investigate how deletion of miR-16 in mice affects skeletal muscle metabolic health and contractile function in both sexes. This study was conducted using both 1) in vitro and 2) in vivo experiments. In in vitro experiments, we used C2C12 myoblasts to test if inhibition or overexpression of miR-16 affected insulin-mediated glucose handling. In in vivo experiments, we generated muscle-specific miR-16 knockout (KO) mice fed a high-fat diet (HFD) to assess how miR-16 content impacts metabolic and contractile properties including glucose tolerance, insulin sensitivity, muscle contractile function, protein anabolism, and mitochondrial network health. In in vitro experiments, although inhibition of miR-16 induced impaired insulin signaling (P = 0.002) and glucose uptake (P = 0.014), overexpression of miR-16 did not attenuate lipid overload-induced insulin resistance using the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol. In in vivo experiments, miR-16 deletion induced both impaired muscle contractility (P = 0.031-0.033), and mitochondrial network health (P = 0.008-0.018) in both sexes. However, although males specifically exhibited impaired insulin sensitivity following miR-16 deletion (P = 0.030), female KO mice showed pronounced glucose intolerance (P = 0.046), corresponding with lower muscle weights (P = 0.015), and protein hyperanabolism (P = 0.023). Our findings suggest distinct sex differences in muscle adaptation in response to miR-16 deletion and miR-16 may serve as a key regulator for metabolic dysregulation in T2DM.NEW & NOTEWORTHY We set to investigate the role of miR-16 in skeletal muscle during diet-induced insulin resistance. Our data provide novel evidence that the lack of miR-16 induced multiple aberrations in insulin sensitivity, muscle contractility, mitochondrial network health, and protein turnover in a sex-dependent manner. Interestingly, miR-16 deletion leads to insulin resistance in males and exacerbated glucose intolerance in females, suggesting different mechanisms of metabolic dysregulation with a lack of miR-16 between sexes.

Development of metabolic and contractile alterations in development of cancer cachexia in female tumor-bearing mice.

Cancer cachexia (CC) results in impaired muscle function and quality of life and is the primary cause of death for ∼20%-30% of patients with cancer. We demonstrated mitochondrial degeneration as a precursor to CC in male mice; however, whether such alterations occur in females is currently unknown. The purpose of this study was to elucidate muscle alterations in CC development in female tumor-bearing mice. Sixty female C57BL/6J mice were injected with PBS or Lewis lung carcinoma at 8 wk of age, and tumors developed for 1, 2, 3, or 4 wk to assess the time course of cachectic development. In vivo muscle contractile function, protein fractional synthetic rate (FSR), protein turnover, and mitochondrial health were assessed. Three- and four-week tumor-bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. HT mice exhibited lower soleus, tibialis anterior, and fat weights than PBS mice. HT mice showed lower peak isometric torque and slower one-half relaxation time than PBS mice. HT mice had lower FSR than PBS mice, whereas E3 ubiquitin ligases were greater in HT than in other groups. Bnip3 (mitophagy) and pMitoTimer red puncta (mitochondrial degeneration) were greater in HT mice, whereas Pgc1α1 and Tfam (mitochondrial biogenesis) were lower in HT mice than in PBS mice. We demonstrate alterations in female tumor-bearing mice where HT exhibited greater protein degradation, impaired muscle contractility, and mitochondrial degeneration compared with other groups. Our data provide novel evidence for a distinct cachectic development in tumor-bearing female mice compared with previous male studies.NEW & NOTEWORTHY Our study demonstrates divergent tumor development and tissue wasting within 3- and 4-wk mice, where approximately half the mice developed large tumors and subsequent cachexia. Unlike previous male studies, where metabolic perturbations precede the onset of cachexia, females appear to exhibit protections from the metabolic perturbations and cachexia development. Our data provide novel evidence for divergent cachectic development in tumor-bearing female mice compared with previous male CC studies, suggesting different mechanisms of CC between sexes.

Hindlimb suspension in Wistar rats: Sex-based differences in muscle response.

Ground-based animal models have been used extensively to understand the effects of microgravity on various physiological systems. Among them, hindlimb suspension (HLS), developed in 1979 in rats, remains the gold-standard and allows researchers to study the consequences of total unloading of the hind limbs while inducing a cephalic fluid shift. While this model has already brought valuable insights to space biology, few studies have directly compared functional decrements in the muscles of males and females during HLS. We exposed 28 adult Wistar rats (14 males and 14 females) to 14 days of HLS or normal loading (NL) to better assess how sex impacts disuse-induced muscle deconditioning. Females better maintained muscle function during HLS than males, as shown by a more moderate reduction in grip strength at 7 days (males: -37.5 ± 3.1%, females: -22.4 ± 6.5%, compared to baseline), that remains stable during the second week of unloading (males: -53.3 ± 5.7%, females: -22.4 ± 5.5%, compared to day 0) while the males exhibit a steady decrease over time (effect of sex × loading p = 0.0002, effect of sex × time × loading p = 0.0099). This was further supported by analyzing the force production in response to a tetanic stimulus. Further functional analyses using force production were also shown to correspond to sex differences in relative loss of muscle mass and CSA. Moreover, our functional data were supported by histomorphometric analyzes, and we highlighted differences in relative muscle loss and CSA. Specifically, female rats seem to experience a lesser muscle deconditioning during disuse than males thus emphasizing the need for more studies that will assess male and female animals concomitantly to develop tailored, effective countermeasures for all astronauts.

The oestrous cycle and skeletal muscle atrophy: Investigations in rodent models of muscle loss.

NEW FINDINGS: What is the central question of this study? Is the oestrous cycle affected during disuse atrophies and, if so, how are oestrous cycle changes related to musculoskeletal outcomes? What is the main finding and its importance? Rodent oestrous cycles were altered during disuse atrophy, which was correlated with musculoskeletal outcomes. However, the oestrous cycle did not appear to be changed by Lewis lung carcinoma, which resulted in no differences in muscle size in comparison to healthy control animals. These findings suggest a relationship between the oestrous cycle and muscle size during atrophic pathologies. ABSTRACT: Recent efforts have focused on improving our understanding of female muscle physiology during exposure to muscle atrophic stimuli. A key feature of female rodent physiology is the oestrous cycle. However, it is not known how such stimuli interact with the oestrous cycle to influence muscle health. In this study, we investigated the impact of muscle atrophic stimuli on the oestrous cycle and how these alterations are correlated with musculoskeletal outcomes. A series of experiments were performed in female rodents, including hindlimb unloading (HU), HU followed by 24 h of reloading, HU combined with dexamethasone treatment, and Lewis lung carcinoma. The oestrous cycle phase was assessed throughout each intervention and correlated with musculoskeletal outcomes. Seven or 14 days of HU increased the duration in dioestrus or metoestrus (D/M; low hormones) and was negatively correlated with gastrocnemius mass. Time spent in D/M was also negatively correlated with changes in grip strength and bone density after HU, and with muscle recovery 24 h after the cessation of HU. The addition of dexamethasone strengthened these relationships between time in D/M and reduced musculoskeletal outcomes. However, in animals with Lewis lung carcinoma, oestrous cyclicity did not differ from that of control animals, and time spent in D/M was not correlated with either gastrocnemius mass or tumour burden. In vitro experiments suggested that enhanced protein synthesis induced by estrogen might protect against muscle atrophy. In conclusion, muscle atrophic insults are correlated with changes in the oestrous cycle, which are associated with deterioration in musculoskeletal outcomes. The magnitude of oestrous cycle alterations depends on the atrophic stimuli.

Mitochondrial aberrations during the progression of disuse atrophy differentially affect male and female mice.

BACKGROUND: Disuse decreases muscle size and is predictive of mortality across multiple pathologies. Detriments to mitochondrial function are hypothesized to underlie disuse-induced muscle atrophy. Little data exist on early mechanisms contributing to onset of these pathologies, nor is it known how they differ between sexes. The purpose of this study was to examine differential and conserved responses to mitochondrial quality control in male and female mice during the development and progression of disuse-induced atrophy. METHODS: One hundred C57BL/6J mice (50 male and 50 female) were hindlimb unloaded to induce disuse atrophy for 0 (con), 24, 48, 72, or 168 h. At designated time-points, extensor digitorum longus, gastrocnemius, and soleus muscles were collected for analysis of mitochondrial quality control markers. RESULTS: One hundred sixty-eight hours of disuse resulted in ~25% lower oxidative muscle fibre CSA in both male (P = 0.003) and female (P = 0.02) mice without any differences due to disuse in glycolytic fibres. In male mice, 48 h of unloading was sufficient to result in ~67% greater mitochondrial oxidative stress as assessed by the reporter gene pMitoTimer compared with 0 h (P = 0.002), this mitochondrial stress preceded detectable muscle loss. However in female mice, mitochondrial oxidative stress did not occur until 168 h of disuse (~40% greater mitochondrial oxidative stress in 168 h compared with 0 h of disuse, P < 0.0001). Blunted oxidative stress in female mice appeared to coincide with greater inductions of autophagy and mitophagy in female mice (~3-fold greater BNIP3 and ~6-fold greater LC3II/I ratio P < 0.0001 and P = 0.038 respectively). Male mice overall had greater reactive oxygen species (ROS) production compared with female mice. Female mice had a greater induction of ROS within 24 h of disuse (~4-fold greater compared with 0 h, P < 0.0001); whereas male mice did not have greater ROS production until 168 h of disuse (~2-fold greater, P < 0.0001). Although all muscle types exhibited some alterations to mitochondrial quality control, such as increased markers of mitophagy and fission, the soleus muscle in both male and female mice exhibited consistent alterations to various markers of mitochondrial quality. Markers of mitochondrial translation were approximately 30-50% lower within 24 h of unloading in both male and female soleus muscle (P value ranges: <0.0001-0.03). CONCLUSIONS: Disuse negatively affects mitochondria differentially between sexes during development of muscle wasting. Acutely, female mice may forgo muscle mass to maintain mitochondrial quality compared with male mice. These differences may contribute to divergent clinical manifestations of atrophy.

Female mice may have exacerbated catabolic signalling response compared to male mice during development and progression of disuse atrophy.

BACKGROUND: Muscle atrophy is a common pathology associated with disuse, such as prolonged bed rest or spaceflight, and is associated with detrimental health outcomes. There is emerging evidence that disuse atrophy may differentially affect males and females. Cellular mechanisms contributing to the development and progression of disuse remain elusive, particularly protein turnover cascades. The purpose of this study was to investigate the initial development and progression of disuse muscle atrophy in male and female mice using the well-established model of hindlimb unloading (HU). METHODS: One hundred C57BL/6J mice (50 male and 50 female) were hindlimb suspended for 0 (control), 24, 48, 72, or 168 h to induce disuse atrophy (10 animals per group). At designated time points, animals were euthanized, and tissues (extensor digitorum longus, gastrocnemius, and soleus for mRNA analysis, gastrocnemius and extensor digitorum longus for protein synthesis rates, and tibialis anterior for histology) were collected for analysis of protein turnover mechanisms (protein anabolism and catabolism). RESULTS: Both males and females lost ~30% of tibialis anterior cross-sectional area after 168 h of disuse. Males had no statistical difference in MHCIIB fibre area, whereas unloaded females had ~33% lower MHCIIB cross-sectional area by 168 h of unloading. Both males and females had lower fractional protein synthesis rates (FSRs) within 24-48 h of HU, and females appeared to have a greater reduction compared with males within 24 h of HU (~23% lower FSRs in males vs. 40% lower FSRs in females). Males and females exhibited differential patterns and responses in multiple markers of protein anabolism, catabolism, and myogenic capacity during the development and progression of disuse atrophy. Specifically, females had greater mRNA inductions of catabolic factors Ubc and Gadd45a (~4-fold greater content in females compared with ~2-fold greater content in males) and greater inductions of anabolic inhibitors Redd1 and Deptor with disuse across multiple muscle tissues exhibiting different fibre phenotypes. CONCLUSIONS: These results suggest that the aetiology of disuse muscle atrophy is more complicated and nuanced than previously thought, with different responses based on muscle phenotypes and between males and females, with females having greater inductions of atrophic markers early in the development of disuse atrophy.

Cancer-induced Cardiac Atrophy Adversely Affects Myocardial Redox State and Mitochondrial Oxidative Characteristics.

Cachexia presents in 80% of advanced cancer patients; however, cardiac atrophy in cachectic patients receives little attention. This cardiomyopathy contributes to increased occurrence of adverse cardiac events compared to age-matched population norms. Research on cardiac atrophy has focused on remodeling; however, alterations in metabolic properties may be a primary contributor. PURPOSE: Determine how cancer-induced cardiac atrophy alters mitochondrial turnover, mitochondrial mRNA translation machinery and in-vitro oxidative characteristics. METHODS: Lewis lung carcinoma (LLC) tumors were implanted in C57BL6/J mice and grown for 28days to induce cardiac atrophy. Endogenous metabolic species, and markers of mitochondrial function were assessed. H9c2 cardiomyocytes were cultured in LLC-conditioned media with(out) the antioxidant MitoTempo. Cells were analyzed for ROS, oxidative capacity, and hypoxic resistance. RESULTS: LLC heart weights were ~10% lower than controls. LLC hearts demonstrated ~15% lower optical redox ratio (FAD/FAD+NADH) compared to PBS controls. When compared to PBS, LLC hearts showed ~50% greater COX-IV and VDAC, attributed to ~50% lower mitophagy markers. mt-mRNA translation machinery was elevated similarly to markers of mitochondrial content. mitochondrial DNA-encoded Cytb was ~30% lower in LLC hearts. ROS scavengers GPx-3 and GPx-7 were ~50% lower in LLC hearts. Treatment of cardiomyocytes with LLC-conditioned media resulted in higher ROS (25%), lower oxygen consumption rates (10% at basal, 75% at maximal), and greater susceptibility to hypoxia (~25%) -- which was reversed by MitoTempo. CONCLUSION: These results substantiate metabolic cardiotoxic effects attributable to tumor-associated factors and provide insight into interactions between mitochondrial mRNA translation, ROS mitigation, oxidative capacity and hypoxia resistance.

The effect of diet-induced obesity on extracellular matrix remodeling during skeletal muscle regeneration.

Diet-induced obesity has previously been shown to occur with the concomitant rise in the expression of proinflammatory cytokines and increases in collagen deposition. While it has been known that the regenerative process of skeletal muscle is altered in obese mice following an acute muscle injury, we sought to examine differences in the expression of various markers of extracellular matrix remodeling and repair. Our laboratory has previously reported an impaired inflammatory and protein synthetic signaling in these mice that may contribute negatively to the muscle regenerative process. To expand upon this previous investigation, tissues from these animals underwent further analysis to determine the extent of changes to the regenerative response within the extracellular matrix, including transcriptional changes in Collagen I, Collagen III, and Fibronectin. Here, we show that the expression of Collagen III:I is significantly increased at 3-days post-injury in obese injured animals compared to lean injured animals (p â€‹= â€‹0.0338), and by 28-days the obese injured animals exhibit a significantly lower Collagen III:I than their lean injured counterparts (p â€‹= â€‹0.0035). We demonstrate an impaired response to an acute muscle injury in obese mice when compared with lean counterparts. However, further studies are required to elucidate translational consequences of these changes, as well as to determine any causative mechanisms that may be driving this effect.