Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia.

Objectives: A high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. Methods: Here, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm ∼45, ∼75 and ∼90 days after EOC injection. Results: Primary ovarian tumour sizes were progressively larger at each time point while robust metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNFα), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. Conclusion: This mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-metastatic weakness during EOC in addition to therapies targeting cachexia. Highlights: This study reports the first orthotopic model of metastatic ovarian cancer cachexia that can be induced in adult immunocompetent miceDiaphragm and limb muscle weakness precedes metastasis and atrophy during ovarian cancerSkeletal muscle mitochondrial oxidative and redox stress signatures occur during pre-metastatic stages of ovarian cancerSpecific muscle force as well as mitochondrial pyruvate oxidation and creatine metabolism demonstrate compensation in later stagesOvarian cancer has heterogeneous effects on distinct muscle types across time.

The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

The importance of serial sarcomere addition for muscle function and the impact of aging.

During natural aging, skeletal muscle experiences impairments in mechanical performance due, in part, to changes in muscle architecture and size, notably with a loss of muscle cross-sectional area (CSA). Another important factor that has received less attention is the shortening of fascicle length (FL), potentially reflective of a decrease in serial sarcomere number (SSN). Interventions that promote the growth of new serial sarcomeres, such as chronic stretching and eccentric-biased resistance training, have been suggested as potential ways to mitigate age-related impairments in muscle function. Although current research suggests it is possible to stimulate serial sarcomerogenesis in muscle in old age, the magnitude of sarcomerogenesis may be less than in young muscle. This blunted effect may be partly due to age-related impairments in the pathways regulating mechanotransduction, muscle gene expression, and protein synthesis, as some have been implicated in SSN adaptation. The purpose of this review was to investigate the impact of aging on the ability for serial sarcomerogenesis and elucidate the molecular pathways that may limit serial sarcomerogenesis in old age. Age-related changes in mechanistic target of rapamycin (mTOR), insulin-like growth factor 1 (IGF-1), myostatin, and serum response factor signaling, muscle ring finger protein (MuRFs), and satellite cells may hinder serial sarcomerogenesis. In addition, our current understanding of SSN in older humans is limited by assumptions based on ultrasound-derived fascicle length. Future research should explore the effects of age-related changes in the identified pathways on the ability to stimulate serial sarcomerogenesis, and better estimate SSN adaptations to gain a deeper understanding of the adaptability of muscle in old age.

GDF15 promotes weight loss by enhancing energy expenditure in muscle.

Caloric restriction that promotes weight loss is an effective strategy for treating non-alcoholic fatty liver disease and improving insulin sensitivity in people with type 2 diabetes1. Despite its effectiveness, in most individuals, weight loss is usually not maintained partly due to physiological adaptations that suppress energy expenditure, a process known as adaptive thermogenesis, the mechanistic underpinnings of which are unclear2,3. Treatment of rodents fed a high-fat diet with recombinant growth differentiating factor 15 (GDF15) reduces obesity and improves glycaemic control through glial-cell-derived neurotrophic factor family receptor α-like (GFRAL)-dependent suppression of food intake4-7. Here we find that, in addition to suppressing appetite, GDF15 counteracts compensatory reductions in energy expenditure, eliciting greater weight loss and reductions in non-alcoholic fatty liver disease (NAFLD) compared to caloric restriction alone. This effect of GDF15 to maintain energy expenditure during calorie restriction requires a GFRAL-ß-adrenergic-dependent signalling axis that increases fatty acid oxidation and calcium futile cycling in the skeletal muscle of mice. These data indicate that therapeutic targeting of the GDF15-GFRAL pathway may be useful for maintaining energy expenditure in skeletal muscle during caloric restriction.

Discovery-Based Proteomics Identify Skeletal Muscle Mitochondrial Alterations as an Early Metabolic Defect in a Mouse Model of ß-Thalassemia.

Although metabolic complications are common in thalassemia patients, there is still an unmet need to better understand underlying mechanisms. We used unbiased global proteomics to reveal molecular differences between the th3/+ mouse model of thalassemia and wild-type control animals focusing on skeletal muscles at 8 weeks of age. Our data point toward a significantly impaired mitochondrial oxidative phosphorylation. Furthermore, we observed a shift from oxidative fibre types toward more glycolytic fibre types in these animals, which was further supported by larger fibre-type cross-sectional areas in the more oxidative type fibres (type I/type IIa/type IIax hybrid). We also observed an increase in capillary density in th3/+ mice, indicative of a compensatory response. Western blotting for mitochondrial oxidative phosphorylation complex proteins and PCR analysis of mitochondrial genes indicated reduced mitochondrial content in the skeletal muscle but not the hearts of th3/+ mice. The phenotypic manifestation of these alterations was a small but significant reduction in glucose handling capacity. Overall, this study identified many important alterations in the proteome of th3/+ mice, amongst which mitochondrial defects leading to skeletal muscle remodelling and metabolic dysfunction were paramount.

The CARM1 transcriptome and arginine methylproteome mediate skeletal muscle integrative biology.

OBJECTIVE: Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues on target proteins to regulate critical processes in health and disease. A mechanistic understanding of the role(s) of CARM1 in skeletal muscle biology is only gradually emerging. The purpose of this study was to elucidate the function of CARM1 in regulating the maintenance and plasticity of skeletal muscle. METHODS: We used transcriptomic, methylproteomic, molecular, functional, and integrative physiological approaches to determine the specific impact of CARM1 in muscle homeostasis. RESULTS: Our data defines the occurrence of arginine methylation in skeletal muscle and demonstrates that this mark occurs on par with phosphorylation and ubiquitination. CARM1 skeletal muscle-specific knockout (mKO) mice displayed altered transcriptomic and arginine methylproteomic signatures with molecular and functional outcomes confirming remodeled skeletal muscle contractile and neuromuscular junction characteristics, which presaged decreased exercise tolerance. Moreover, CARM1 regulates AMPK-PGC-1α signalling during acute conditions of activity-induced muscle plasticity. CONCLUSIONS: This study uncovers the broad impact of CARM1 in the maintenance and remodelling of skeletal muscle biology.

Decreased Diastolic Blood Pressure and Average Grip Strength in Adults With Type 1 Diabetes Compared With Controls: An Analysis of Data From the Canadian Longitudinal Study on Aging.

OBJECTIVES: Our aim in this study was to determine whether aging individuals with type 1 diabetes (T1D) have differences in cardiovascular health, assessed by blood pressure, and skeletal muscle function, assessed by grip strength, compared with matched nondiabetic controls (CON). METHODS: This investigation was a retrospective cohort analysis using baseline and 3-year follow-up data from the Canadian Longitudinal Study on Aging. Bivariate and multivariate regression analyses were used to examine the association between sociodemographic, health, behavioural and T1D-specific variables on blood pressure and grip strength in T1D and CON groups. Generalized estimating equations were used to model the average population changes in blood pressure and grip strength from baseline to follow up. RESULTS: The sample included 126 individuals (63 T1D and 63 CON). Systolic blood pressure was not significantly different between groups at baseline or follow up (p>0.05). However, compared with CON, diastolic blood pressure was significantly lower at both time-points in the T1D group (p<0.001). Grip strength was consistently lower among persons with T1D (p=0.03). In the multivariate regression model, body mass index, age and sex were significantly associated with diastolic blood pressure and grip strength in both groups. In the T1D group, disease duration accounted for a large proportion of the variance in diastolic blood pressure and grip strength (17% and 9%, respectively). The rate of decline in diastolic blood pressure and grip strength did not differ between groups (p>0.05). CONCLUSIONS: Diastolic blood pressure and grip strength appear to be consistently lower and differentially regulated in individuals with T1D vs CON. Aging individuals with T1D may be at risk of premature morbidity and mortality.

Diabetic Bone Disease and Diabetic Myopathy: Manifestations of the Impaired Muscle-Bone Unit in Type 1 Diabetes.

Type 1 diabetes is associated with complications affecting muscle and bone, with diabetic bone disease and diabetic myopathy becoming increasingly reported in the past few decades. This review is aimed at succinctly reviewing the literature on the current knowledge regarding these increasingly identified and possibly interconnected complications on the musculoskeletal system. Furthermore, this review summarizes several nonmechanical factors that could be mediating the development and progression of premature musculoskeletal decline in this population and discusses preventative measures to reduce the burden of diabetes on the musculoskeletal system.

Alterations in skeletal muscle repair in young adults with type 1 diabetes mellitus.

Though preclinical models of type 1 diabetes (T1D) exhibit impaired muscle regeneration, this has yet to be investigated in humans with T1D. Here, we investigated the impact of damaging exercise (eccentric quadriceps contractions) in 18 physically active young adults with and without T1D. Pre- and postexercise (48 h and 96 h), the participants provided blood samples, vastus lateralis biopsies, and performed maximal voluntary quadriceps contractions (MVCs). Skeletal muscle sarcolemmal integrity, extracellular matrix (ECM) content, and satellite cell (SC) content/proliferation were assessed by immunofluorescence. Transmission electron microscopy was used to quantify ultrastructural damage. MVC was comparable between T1D and controls before exercise. Postexercise, MVC was decreased in both groups, but subjects with T1D exhibited moderately lower strength recovery at both 48 h and 96 h. Serum creatine kinase, an indicator of muscle damage, was moderately higher in participants with T1D at rest and exhibited a small elevation 96 h postexercise. Participants with T1D showed lower SC content at all timepoints and demonstrated a moderate delay in SC proliferation after exercise. A greater number of myofibers exhibited sarcolemmal damage (disrupted dystrophin) and increased ECM (laminin) content in participants with T1D despite no differences between groups in ultrastructural damage as assessed by electron microscopy. Finally, transcriptomic analyses revealed dysregulated gene networks involving RNA translation and mitochondrial respiration, providing potential explanations for previous observations of mitochondrial dysfunction in similar cohorts with T1D. Our findings indicate that skeletal muscle in young adults with moderately controlled T1D is altered after damaging exercise, suggesting that longer recovery times following intense exercise may be necessary.

Normal to enhanced intrinsic mitochondrial respiration in skeletal muscle of middle- to older-aged women and men with uncomplicated type 1 diabetes.

AIMS/HYPOTHESIS: This study interrogated mitochondrial respiratory function and content in skeletal muscle biopsies of healthy adults between 30 and 72 years old with and without uncomplicated type 1 diabetes. METHODS: Participants (12 women/nine men) with type 1 diabetes (48 ± 11 years of age), without overt complications, were matched for age, sex, BMI and level of physical activity to participants without diabetes (control participants) (49 ± 12 years of age). Participants underwent a Bergström biopsy of the vastus lateralis to assess mitochondrial respiratory function using high-resolution respirometry and citrate synthase activity. Electron microscopy was used to quantify mitochondrial content and cristae (pixel) density. RESULTS: Mean mitochondrial area density was 27% lower (p = 0.006) in participants with type 1 diabetes compared with control participants. This was largely due to smaller mitochondrial fragments in women with type 1 diabetes (-18%, p = 0.057), as opposed to a decrease in the total number of mitochondrial fragments in men with diabetes (-28%, p = 0.130). Mitochondrial respiratory measures, whether estimated per milligram of tissue (i.e. mass-specific) or normalised to area density (i.e. intrinsic mitochondrial function), differed between cohorts, and demonstrated sexual dimorphism. Mass-specific mitochondrial oxidative phosphorylation (OXPHOS) capacity with the substrates for complex I and complex II (CI + II) was significantly lower (-24%, p = 0.033) in women with type 1 diabetes compared with control participants, whereas mass-specific OXPHOS capacities with substrates for complex I only (pyruvate [CI pyr] or glutamate [CI glu]) or complex II only (succinate [CII succ]) were not different (p > 0.404). No statistical differences (p > 0.397) were found in mass-specific OXPHOS capacity in men with type 1 diabetes compared with control participants despite a 42% non-significant increase in CI glu OXPHOS capacity (p = 0.218). In contrast, intrinsic CI + II OXPHOS capacity was not different in women with type 1 diabetes (+5%, p = 0.378), whereas in men with type 1 diabetes it was 25% higher (p = 0.163) compared with control participants. Men with type 1 diabetes also demonstrated higher intrinsic OXPHOS capacity for CI pyr (+50%, p = 0.159), CI glu (+88%, p = 0.033) and CII succ (+28%, p = 0.123), as well as higher intrinsic respiratory rates with low (more physiological) concentrations of either ADP, pyruvate, glutamate or succinate (p < 0.012). Women with type 1 diabetes had higher (p < 0.003) intrinsic respiratory rates with low concentrations of succinate only. Calculated aerobic fitness (Physical Working Capacity Test [PWC130]) showed a strong relationship with mitochondrial respiratory function and content in the type 1 diabetes cohort. CONCLUSIONS/INTERPRETATION: In middle- to older-aged adults with uncomplicated type 1 diabetes, we conclude that skeletal muscle mitochondria differentially adapt to type 1 diabetes and demonstrate sexual dimorphism. Importantly, these cellular alterations were significantly associated with our metric of aerobic fitness (PWC130) and preceded notable impairments in skeletal mass and strength.

Impaired Function and Altered Morphology in the Skeletal Muscles of Adult Men and Women With Type 1 Diabetes.

CONTEXT: Previous investigations on skeletal muscle health in type 1 diabetes (T1D) have generally focused on later stages of disease progression where comorbidities are present and are posited as a primary mechanism of muscle dysfunction. OBJECTIVE: To investigate skeletal muscle function and morphology across the adult lifespan in those with and without T1D. DESIGN: Participants underwent maximal contraction (MVC) testing, resting muscle biopsy, and venous blood sampling. SETTING: Procedures in this study were undertaken at the McMaster University Medical Centre. PARTICIPANTS: Sixty-five healthy adult (18-78 years old) men/males and women/females (T1D = 34; control = 31) matched for age/biological sex/body mass index; self-reported physical activity levels were included. MAIN OUTCOME MEASURES: Our primary measure in this study was MVC, with supporting histological/immunofluorescent measures. RESULTS: After 35 years of age ("older adults"), MVC declined quicker in T1D subjects compared to controls. Loss of strength in T1D was accompanied by morphological changes associated with accelerated aging. Type 1 myofiber grouping was higher in T1D, and the groups were larger and more numerous than in controls. Older T1D females exhibited more myofibers expressing multiple myosin heavy chain isoforms (hybrid fibers) than controls, another feature of accelerated aging. Conversely, T1D males exhibited a shift toward type 2 fibers, with less evidence of myofiber grouping or hybrid fibers. CONCLUSIONS: These data suggest impairments to skeletal muscle function and morphology exist in T1D. The decline in strength with T1D is accelerated after 35 years of age and may be responsible for the earlier onset of frailty, which characterizes those with diabetes.

Muscle and serum myostatin expression in type 1 diabetes.

Type 1 diabetes (T1D) has been reported to negatively affect the health of skeletal muscle, though the underlying mechanisms are unknown. Myostatin, a myokine whose increased expression is associated with muscle-wasting diseases, has not been reported in humans with T1D but has been demonstrated to be elevated in preclinical diabetes models. Thus, the purpose of this study was to determine if there is an elevated expression of myostatin in the serum and skeletal muscle of persons with T1D compared to controls. Secondarily, we aimed to explore relationships between myostatin expression and clinically important metrics (e.g., HbA1c , strength, lean mass) in women and men with (N = 31)/without T1D (N = 24) between 18 and 72 years old. Body composition, baseline strength, blood sample and vastus lateralis muscle biopsy were evaluated. Serum, but not muscle, myostatin expression was significantly elevated in those with T1D versus controls, and to a greater degree in T1D women than T1D men. Serum myostatin levels were not significantly associated with HbA1c nor disease duration. A significant correlation between serum myostatin expression and maximal voluntary contraction (MVC) and body fat mass was demonstrated in control subjects, but these correlations did not reach significance in those with T1D (MVC: R = 0.64 controls vs. R = 0.37 T1D; Body fat: R = -0.52 controls/R = -0.02 T1D). Collectively, serum myostatin was correlated with lean mass (R = 0.45), and while this trend was noted in both groups separately, neither reached statistical significance (R = 0.47 controls/R = 0.33 T1D). Overall, while those with T1D exhibited elevated serum myostatin levels (particularly females) myostatin expression was not correlated with clinically relevant metrics despite some of these relationships existing in controls (e.g., lean/fat mass). Future studies will be needed to fully understand the mechanisms underlying increased myostatin in T1D, with relationships to insulin dosing being particularly important to elucidate.

The Systemic Effects of Exercise on Regulators of Muscle and Bone in Girls and Women.

PURPOSE: To assess the systemic effects of an acute bout of moderate-intensity exercise on factors that are known to regulate muscle and bone growth in prepubertal girls and women. METHODS: A total of 12 prepubertal girls (8-10 y) and 12 women (20-30 y) cycled at 60% maximal oxygen uptake for 1 hour followed by 1 hour recovery. Blood samples were collected at rest, mid-exercise, end of exercise, mid-recovery, and end of recovery. Plasma was analyzed for interleukin-6, chemokine ligand 1, fibroblast growth factor-2, total insulin growth factor-1 (IGF-1), and free IGF-1 using enzyme-linked immunosorbent assays assays. RESULTS: Both groups had similar concentrations of systemic factors at baseline with the exception of free IGF-1, which was higher in girls (P = .001). Interleukin-6 response was lower in girls versus women (P = .04), with a difference of +105.1% at end of exercise (P < .001), +113.5% at mid-recovery (P = .001), and +93.2% at end of recovery (P = .02). Girls and women exhibited significant declines in chemokine ligand 1, fibroblast growth factor-2, and total IGF-1 during recovery. CONCLUSION: Compared with women, an acute bout of moderate-intensity exercise in girls elicits a lower inflammatory response, suggesting that other mechanisms may be more important for driving the anabolic effects of exercise on muscle and bone in girls.

Increased intra-mitochondrial lipofuscin aggregates with spherical dense body formation in mitochondrial myopathy.

Lipofuscin aggregation may result from incomplete degradation of damaged mitochondria by autophagy-lysosome pathway, and intra-mitochondrial lipofuscin aggregation may exacerbate mitochondrial abnormalities in mitochondrial myopathy (MM) and mitochondrial disease. We examined vastus lateralis muscle biopsies from 24 patients with pathologically diagnosed MM and clinically diagnosed chronic progressive external ophthalmoplegia, in comparison to the biopsies from 3 other groups:10 patients with inclusion body myositis (IBM), 11 younger adults, and 10 older subjects with no to minimal myopathic changes. Lipofuscin aggregation in muscle fibres was assessed on autofluorescence microscopy, some histochemical stains, and electron microscopy (EM). EM analyses demonstrated intra-mitochondrial lipofuscin aggregates, spherical dense bodies (SDBs), and paracrystalline inclusions (PCIs) which were semi-quantitatively assessed. Intra-mitochondrial lipofuscin aggregates showed no significant differences between groups of MM patients and older subjects or IBM patients, but significant differences between groups of younger adults and others with associated age-related changes. Intra-mitochondrial SDBs were significantly more in MM patients than in older subjects, IBM patients, and younger adults. There was a significant positive correlation between intra-mitochondrial lipofuscin aggregates and SDBs. These findings suggest that intra-mitochondrial formation of lipofuscin SDBs is more in MM and contributing to the pathophysiology of mitochondrial disease.