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Cachexia is a systemic wasting syndrome prevalent in patients with cancer that significantly affects quality of life, health care costs, and therapeutic outcomes. Despite its clinical importance, cachexia is rarely formally diagnosed. This deficiency presents a challenge for effective patient management and care, health care resource allocation, and the advancement of therapeutic approaches. Here, we highlight impedances to the diagnosis and coding of cachexia, including the absence of standardized therapy, a lack of incentives for accurate coding, and overlapping clinical features with other conditions. We differentiate cachexia from related conditions like unintentional weight loss, sarcopenia, frailty, and protein-calorie malnutrition, outlining their distinct clinical features and inter-relations. We propose an approach to enhance diagnostic accuracy and coding for cachexia. This effort will enable better prevalence data, translation of mechanism-based therapy development, patient identification and stratification, and ultimately advanced diagnostics and US Food and Drug Administration-approved treatments for cachexia.

Cancer cachexia, a multifactorial condition resulting in muscle and adipose tissue wasting, reduces the quality of life of many people with cancer. Despite decades of research, therapeutic options for cancer cachexia remain limited. Cachexia is highly prevalent in people with pancreatic ductal adenocarcinoma (PDAC), and many animal models of pancreatic cancer are used to understand mechanisms underlying cachexia. One such model is the Kras LSL-G12D , Ptf1a Cre-ER/+ , Pten flox/flox (KPP) model, which utilizes an inducible Cre recombinase to allow tumor development to be initiated at any age by tamoxifen administration. In our previous work, tumors were induced in KPP mice at 4 weeks of age. However, mice are still rapidly growing at this age, and a portion of the body weight differences seen between control and KPP mice is likely due to slowed growth of KPP mice. In our current study, pancreatic tumors were induced to develop with tamoxifen in KPP mice after rapid postnatal growth has slowed at 10 weeks of age (KPP10). Similar to our previous findings, KPP10 mice had lower body, muscle, and adipose tissue weights compared to non-tumor mice, and these differences were similar between male and female mice. However, male mice experienced greater relative weight loss. Unexpectedly, we identified that overall survival was significantly shorter in female KPP10 mice compared to KPP10 males. Greater body weight at tumor induction was associated with longer survival, suggesting that the sex difference in survival may be related to differences in body weight between male and female mice., New & Noteworthy: Although male mice experience greater relative body weight losses, similar skeletal muscle and adipose tissue wasting occurs between male and female mice in the Kras LSL-G12D , Ptf1a Cre-ER/+ , Pten flox/flox (KPP) model of pancreatic-cancer induced cachexia. Greater weight loss in males may be related to longer survival. However, differences in tamoxifen dose relative to body weight may have accelerated tumor formation in female mice and therefore may be a relevant consideration for inducible tumor models.

Although cancer cachexia is classically characterized as a systemic inflammatory disorder, emerging evidence indicates that weight loss also associates with local tissue inflammation. We queried the regulation of this inflammation and its causality to cachexia by exploring skeletal muscle, whose atrophy strongly associates with poor outcomes. Using multiple mouse models and patient samples, we show that cachectic muscle is marked by enhanced innate immunity. Nuclear factor κB (NF-κB) activity in multiple cells, including satellite cells, myofibers, and fibro-adipogenic progenitors, promotes macrophage expansion equally derived from infiltrating monocytes and resident cells. Moreover, NF-κB-activated cells and macrophages undergo crosstalk; NF-κB +  cells recruit macrophages to inhibit regeneration and promote atrophy but, interestingly, also protect myofibers, while macrophages stimulate NF-κB +  cells to sustain an inflammatory feedforward loop. Together, we propose that NF-κB functions in multiple cells in the muscle microenvironment to stimulate macrophages that both promote and protect against muscle wasting in cancer., Competing Interests: Declaration of interests T.A.Z. is a scientific advisory board member of Emmyon, Inc. and PeleOS, LLC., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Pancreatic ductal adenocarcinoma (PDAC) presents with a high mortality rate. Two important features of PDAC contribute to this poor outcome. The first is metastasis which occurs in ~ 80% of PDAC patients. The second is cachexia, which compromises treatment tolerance for patients and reduces their quality of life. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging., Methods: Here, we optimize an orthotopic mouse model of PDAC by altering several conditions, including the subcloning of parental murine PDAC cells, implantation site, number of transplanted cells, and age of recipient mice. We perform spatial profiling to compare primary and metastatic immune microenvironments and RNA sequencing to gain insight into the mechanisms of muscle wasting in PDAC-induced cachexia, comparing non-metastatic to metastatic conditions., Results: These modifications extend the time course of the disease and concurrently increase the rate of metastasis to approximately 70%. Furthermore, reliable cachexia endpoints are achieved in both PDAC mice with and without metastases, which is reminiscent of patients. We also find that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis., Conclusion: Together, this model is likely to be advantageous in both advancing our understanding of the mechanism of PDAC cachexia, as well as in the evaluation of novel therapeutics., (© 2024. The Author(s).)

Background: Pancreatic cancer patients have poor quality of life. Testosterone deficiency is associated with constitutional symptoms and sexual dysfunction which may contribute to poor quality of life. We investigated the prevalence of screening for and presence of testosterone deficiency in male pancreatic cancer patients., Methods: To determine the frequency of screening for testosterone deficiency in pancreatic cancer patients, our institution's electronic medical record system was queried for male patients diagnosed with a pancreatic mass between 2006 and 2020 and an available testosterone level. In a separate analysis, total testosterone was measured in serum samples from a cohort of 89 male pancreatic ductal adenocarcinoma (PDAC) patients. Low serum testosterone was defined as <300 ng/dL., Results: One thousand five hundred and sixty-six male patients were identified with a pancreatic mass, and 35 (2.2%) also had a testosterone level. In our analysis cohort, 44 of 89 patients (49.4%) were found to have low serum testosterone. Symptoms consistent with testosterone deficiency were documented for 70% of these patients, with fatigue being the most common. Testosterone level had no significant association with progression-free survival (PFS) (P=0.66) or overall survival (OS) (P=0.95)., Conclusions: Testosterone deficiency is common but rarely assessed in male patients with pancreatic cancer. Further studies are warranted to explore the possibility of testosterone supplementation to improve quality of life in this patient population., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-22-684/coif). AGK receives support from the National Heart, Lung, and Blood Institute of the National Institutes of Health T35 (No. HL007485). AMP receives grant funding from and is a consultant for Boston Scientific. She also is a consultant for Endo Pharmaceuticals and she is on the medical advisory board for FirmTech. After the conclusion of data collection but prior to publication, AMP co-founded the PRIME institute, a for-profit company. EET has received salary support provided by NIH (No. NIH R00AR071508) and Institutional NIH support for core resources (No. NIH P30CA086862). She has also received grants from NIH (No. NIH R21 CA257972), lecture honoraria from the University of Kentucky and West Virginia University, travel costs for AACR Annual Meeting 2023 (Invited Speaker) from the American Association for Cancer Research, travel costs for the seminar from West Virginia University, Travel costs for 18th International Biochemistry of Exercise Conference (Invited Speaker) from International Research Group on Biochemistry of Exercise, and travel costs for Journées de la Société Française de Myologie (Invited Speaker) from Société Française de Myologie. EET was also the Co-chair of the education committee of Cancer Cachexia Society. The other authors have no conflicts of interest to declare., (2023 Translational Andrology and Urology. All rights reserved.)

Cachexia is a common complication of cancer and is associated with poor quality of life and a decrease in survival. Many patients with cancer cachexia suffer from inflammation associated with elevated cytokines, such as interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF). Single-agent trials to treat cancer cachexia have not led to substantial benefit as the type of cytokine which is elevated has rarely been specified and targeted. Cachexia may also be multifactorial, involving inflammation, anorexia, catabolism, depression, and pain, and targeting the multiple causes will likely be necessary to achieve improvement in weight and appetite. A PUBMED search revealed over 3000 articles on cancer cachexia in the past ten years. We attempted to review any studies related to inflammation and cancer cachexia identified by Google Scholar and PUBMED and further search for articles listed in their references. The National Comprehensive Cancer Network (NCCN) guidelines do not provide any suggestion for managing cancer cachexia except a dietary consult. A more targeted approach to developing therapies for cancer cachexia might lead to more personalized and effective therapy.

Background: Cancer patients who exhibit cachexia lose weight and have low treatment tolerance and poor outcomes compared to cancer patients without weight loss. Despite the clear increased risk for patients, diagnosing cachexia still often relies on self-reported weight loss. A reliable biomarker to identify patients with cancer cachexia would be a valuable tool to improve clinical decision making and identification of patients at risk of adverse outcomes., Methods: Targeted metabolomics, that included panels of amino acids, tricarboxylic acids, fatty acids, acylcarnitines, and sphingolipids, were conducted on plasma samples from patients with confirmed pancreatic ductal adenocarcinoma (PDAC) with and without cachexia and control patients without cancer (n=10/group, equally divided by sex). Additional patient samples were analyzed (total n=95) and Receiver Operating Characteristic (ROC) analyses were performed to establish if any metabolite could effectively serve as a biomarker of cachexia., Results: Targeted profiling revealed that cachectic patients had decreased circulating levels of three sphingolipids compared to either non-cachectic PDAC patients or patients without cancer. The ratio of C18-ceramide to C24-ceramide (C18:C24) outperformed a number of other previously proposed biomarkers of cachexia (area under ROC = 0.810). It was notable that some biomarkers, including C18:C24, were only altered in cachectic males., Conclusions: Our findings identify C18:C24 as a potentially new biomarker of PDAC-induced cachexia that also highlight a previously unappreciated sexual dimorphism in cancer cachexia., Competing Interests: Competing Interests JMC, MED, CRS, PVR, TMW, and EET declare that they have no conflicts of interest. DCE declares that he has received research support and has a speaking and consulting agreement with Abbott Laboratories. DCG declares that he receives research support from Pfizer and consults for Immuneering and Catabasis.

The cachexia syndrome in cancer is characterized by weight loss resulting from the combination of anorexia and atrophy of adipose and skeletal muscle. For decades, inflammatory circulatory factors have been identified to regulate wasting, but inhibitors of these factors have not yielded the same clinical benefit as in animal models. Therefore, additional mediators of cachexia likely regulate this syndrome, and such factors might be more suitable for targeted intervention. We highlight several anorexia-cachexia signaling mediators, including activin A, myostatin, GDF15, and lipocalin-2. We discuss current evidence that these factors associate with cachexia in cancer patients, and summarize translational efforts including essential early-phase clinical trials. We conclude with thoughts on targeted and personalized approaches for future anti-cachexia treatments., Competing Interests: Declaration of interests D.C.G. receives funding support from Pfizer Inc.; E.E.T. declares no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of various cancer types. Nevertheless, it is well known that DOX promotes the development of severe cardiovascular complications. Therefore, investigation into the underlying mechanisms that drive DOX-induced cardiotoxicity is necessary to develop therapeutic countermeasures. In this regard, autophagy is a complex catabolic process that is increased in the heart following DOX exposure. However, conflicting evidence exists regarding the role of autophagy dysregulation in the etiology of DOX-induced cardiac dysfunction. This study aimed to clarify the contribution of autophagy to DOX-induced cardiotoxicity by specifically inhibiting autophagosome formation using a dominant negative autophagy gene 5 (ATG5) adeno-associated virus construct (rAAV-dnATG5). Acute (2-day) and delayed (9-day) effects of DOX (20 mg/kg intraperitoneal injection (i.p.)) on the hearts of female Sprague-Dawley rats were assessed. Our data confirm established detrimental effects of DOX on left ventricular function, redox balance and mitochondrial function. Interestingly, targeted inhibition of autophagy in the heart via rAAV-dnATG5 in DOX-treated rats ameliorated the increase in mitochondrial reactive oxygen species emission and the attenuation of cardiac and mitochondrial function, but only at the acute timepoint. Deviation in the effects of autophagy inhibition at the 2- and 9-day timepoints appeared related to differences in ATG5-ATG12 conjugation, as this marker of autophagosome formation was significantly elevated 2 days following DOX exposure but returned to baseline at day 9. DOX exposure may transiently upregulate autophagy signaling in the rat heart; thus, long-term inhibition of autophagy may result in pathological consequences.

Clinical use of the chemotherapeutic doxorubicin (DOX) promotes skeletal muscle atrophy and weakness, adversely affecting patient mobility and strength. Although the mechanisms responsible for DOX-induced skeletal muscle dysfunction remain unclear, studies implicate the significant production of reactive oxygen species (ROS) in this pathology. Supraphysiological ROS levels can enhance protein degradation via autophagy, and it is established that DOX upregulates autophagic signaling in skeletal muscle. To determine the precise contribution of accelerated autophagy to DOX-induced skeletal muscle dysfunction, we inhibited autophagy in the soleus via transduction of a dominant negative mutation of the autophagy related 5 (ATG5) protein. Targeted inhibition of autophagy prevented soleus muscle atrophy and contractile dysfunction acutely following DOX administration, which was associated with a reduction in mitochondrial ROS and maintenance of mitochondrial respiratory capacity. These beneficial modifications were potentially the result of enhanced transcription of antioxidant response element-related genes and increased antioxidant capacity. Specifically, our results showed significant upregulation of peroxisome proliferator-activated receptor gamma co-activator 1-alpha, nuclear respiratory factor-1, nuclear factor erythroid-2-related factor-2, nicotinamide-adenine dinucleotide phosphate quinone dehydrogenase-1, and catalase in the soleus with DOX treatment when autophagy was inhibited. These findings establish a significant role of autophagy in the development of oxidative stress and skeletal muscle weakness following DOX administration., Competing Interests: The authors declare no conflict of interest.

Cachexia is a wasting syndrome characterized by pronounced skeletal muscle loss. In cancer, cachexia is associated with increased morbidity and mortality and decreased treatment tolerance. Although advances have been made in understanding the mechanisms of cachexia, translating these advances to the clinic has been challenging. One reason for this shortcoming may be the current animal models, which fail to fully recapitulate the etiology of human cancer-induced tissue wasting. Because pancreatic ductal adenocarcinoma (PDA) presents with a high incidence of cachexia, we engineered a mouse model of PDA that we named KPP. KPP mice, similar to PDA patients, progressively lose skeletal and adipose mass as a consequence of their tumors. In addition, KPP muscles exhibit a similar gene ontology as cachectic patients. We envision that the KPP model will be a useful resource for advancing our mechanistic understanding and ability to treat cancer cachexia., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cancer-associated wasting, termed cancer cachexia, has a profound effect on the morbidity and mortality of cancer patients but remains difficult to recognize and diagnose. While increases in circulating levels of a number of inflammatory cytokines have been associated with cancer cachexia, these associations were generally made in patients with advanced disease and thus may be associated with disease progression rather than directly with the cachexia syndrome. Thus, we sought to assess potential biomarkers of cancer-induced cachexia in patients with earlier stages of disease., Methods: A custom multiplex array was used to measure circulating levels of 25 soluble factors from 70 pancreatic cancer patients undergoing attempted tumour resections. A high-sensitivity multiplex was used for increased sensitivity for nine cytokines., Results: Resectable pancreatic cancer patients with cachexia had low levels of canonical pro-inflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β), interferon-γ (IFN-γ), and tumour necrosis factor (TNF). Even in our more sensitive analysis, these cytokines were not associated with cancer cachexia. Of the 25 circulating factors tested, only monocyte chemoattractant protein-1 (MCP-1) was increased in treatment-naïve cachectic patients compared with weight stable patients and identified as a potential biomarker for cancer cachexia. Although circulating levels of leptin and granulocyte-macrophage colony-stimulating factor (GM-CSF) were found to be decreased in the same cohort of treatment-naïve cachectic patients, these factors were closely associated with body mass index, limiting their utility as cancer cachexia biomarkers., Conclusions: Unlike in advanced disease, it is possible that cachexia in patients with resectable pancreatic cancer is not associated with high levels of classical markers of systemic inflammation. However, cachectic, treatment-naïve patients have higher levels of MCP-1, suggesting that MCP-1 may be useful as a biomarker of cancer cachexia., (© 2018 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)

Mechanical ventilation (MV) results in the rapid development of ventilator-induced diaphragm dysfunction (VIDD). While the mechanisms responsible for VIDD are not fully understood, recent data reveal that prolonged MV activates autophagy in the diaphragm, which may occur as a result of increased cellular reactive oxygen species (ROS) production. Therefore, we tested the hypothesis that (1) accelerated autophagy is a key contributor to VIDD; and that (2) oxidative stress is required to increase the expression of autophagy genes in the diaphragm. Our findings reveal that targeted inhibition of autophagy in the rat diaphragm prevented MV-induced muscle atrophy and contractile dysfunction. Attenuation of VIDD in these animals occurred as a result of increased diaphragm concentration of the antioxidant catalase and reduced mitochondrial ROS emission, which corresponded to reductions in the activity of calpain and caspase-3. To determine if increased ROS production is required for the upregulation of autophagy biomarkers in the diaphragm, rats that were administered the mitochondrial-targeted peptide SS-31 during MV. Results from this study demonstrated that mitochondrial ROS production in the diaphragm during MV is required for the increased expression of key autophagy genes (i.e. LC3, Atg7, Atg12, Beclin1 and p62), as well as for increased activity of cathepsin L. Together, these data reveal that autophagy is required for VIDD, and that autophagy inhibition reduces MV-induced diaphragm ROS production and prevents a positive feedback loop whereby increased autophagy is stimulated by oxidative stress, resulting in further increases in ROS and autophagy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Macrophages are attracted to developing tumors and can participate in immune surveillance to eliminate neoplastic cells. In response, neoplastic cells utilize NF-κB to suppress this killing activity, but the mechanisms underlying their self-protection remain unclear. Here, we report that this dynamic interaction between tumor cells and macrophages is integrally linked by a soluble factor identified as growth and differentiation factor 15 (GDF-15). In vitro, tumor-derived GDF-15 signals in macrophages to suppress their proapoptotic activity by inhibiting TNF and nitric oxide (NO) production. In vivo, depletion of GDF-15 in Ras-driven tumor xenografts and in an orthotopic model of pancreatic cancer delayed tumor development. This delay correlated with increased infiltrating antitumor macrophages. Further, production of GDF-15 is directly regulated by NF-κB, and the colocalization of activated NF-κB and GDF-15 in epithelial ducts of human pancreatic adenocarcinoma supports the importance of this observation. Mechanistically, we found that GDF-15 suppresses macrophage activity by inhibiting TGF-β-activated kinase (TAK1) signaling to NF-κB, thereby blocking synthesis of TNF and NO. Based on these results, we propose that the NF-κB/GDF-15 regulatory axis is important for tumor cells in evading macrophage immune surveillance during the early stages of tumorigenesis.

Objectives: Pancreatic ductal adenocarcinoma (PDAC) is often accompanied by weight loss. We sought to characterize factors associated with weight loss and observed nutritional interventions, as well as define the effect of weight loss on survival., Methods: Consecutive subjects diagnosed with PDAC (N = 123) were retrospectively evaluated. Univariate analysis was used to compare subjects with and without substantial (>5%) weight loss. Multivariate logistic regression was performed to identify factors associated with weight loss, and survival analyses were performed using Kaplan-Meier curves and Cox survival models., Results: Substantial weight loss at diagnosis was present in 71.5% of subjects and was independently associated with higher baseline body mass index, longer symptom duration, and increased tumor size. Recommendations for nutrition consultation and pancreatic enzyme replacement therapy occurred in 27.6% and 36.9% of subjects, respectively. Weight loss (>5%) was not associated with worse survival on multivariate analysis (hazard ratio, 1.32; 95% confidence interval, 0.76-2.30), unless a higher threshold (>10%) was used (hazard ratio, 1.77; 95% confidence interval, 1.09-2.87)., Conclusions: Despite the high prevalence of weight loss at PDAC diagnosis, there are low observed rates of nutritional interventions. Weight loss based on current criteria for cancer cachexia is not associated with poor survival in PDAC.

Involuntary weight loss, a part of the cachexia syndrome, is a debilitating comorbidity of cancer and currently has no treatment options. Results from a recent clinical trial at our institution showed that biliary tract cancer patients treated with a MEK inhibitor exhibited poor tumor responses but surprisingly gained weight and increased their skeletal muscle mass. This implied that MEK inhibition might be anticachectic. To test this potential effect of MEK inhibition, we utilized the established Colon-26 model of cancer cachexia and the MEK1/2 inhibitor MEK162. Results showed that MEK inhibition effectively prevented muscle wasting. Importantly, MEK162 retained its ability to spare muscle loss even in mice bearing a Colon-26 clone resistant to the MEK inhibitor, demonstrating that the effects of blocking MEK are at least in part independent of the tumor. Because single-agent MEK inhibitors have been limited as a first-line targeted therapy due to compensatory activation of other oncogenic signaling pathways, we combined MEK162 with the PI3K/Akt inhibitor buparlisib. Results showed that this combinatorial treatment significantly reduced tumor growth due to a direct activity on Colon-26 tumor cells in vitro and in vivo, while also preserving skeletal muscle mass. Together, our results suggest that as a monotherapy, MEK inhibition preserves muscle mass, but when combined with a PI3K/Akt inhibitor exhibits potent antitumor activity. Thus, combinatorial therapy might serve as a new approach for the treatment of cancer cachexia. Mol Cancer Ther; 16(2); 344-56. ©2016 AACRSee related article by Kobayashi et al., p. 357., (©2016 American Association for Cancer Research.)

MyoD is a key regulator of skeletal myogenesis that directs contractile protein synthesis, but whether this transcription factor also regulates skeletal muscle metabolism has not been explored. In a genome-wide ChIP-seq analysis of skeletal muscle cells, we unexpectedly observed that MyoD directly binds to numerous metabolic genes, including those associated with mitochondrial biogenesis, fatty acid oxidation, and the electron transport chain. Results in cultured cells and adult skeletal muscle confirmed that MyoD regulates oxidative metabolism through multiple transcriptional targets, including PGC-1β, a master regulator of mitochondrial biogenesis. We find that PGC-1β expression is cooperatively regulated by MyoD and the alternative NF-κB signaling pathway. Bioinformatics evidence suggests that this cooperativity between MyoD and NF-κB extends to other metabolic genes as well. Together, these data identify MyoD as a regulator of the metabolic capacity of mature skeletal muscle to ensure that sufficient energy is available to support muscle contraction., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

While changes in muscle protein synthesis and degradation have long been known to contribute to muscle wasting, a body of literature has arisen which suggests that regulation of the satellite cell and its ensuing regenerative program are impaired in atrophied muscle. Lessons learned from cancer cachexia suggest that this regulation is simply not a consequence, but a contributing factor to the wasting process. In addition to satellite cells, evidence from mouse models of cancer cachexia also suggests that non-satellite progenitor cells from the muscle microenvironment are also involved. This chapter in the series reviews the evidence of dysfunctional muscle repair in multiple wasting conditions. Potential mechanisms for this dysfunctional regeneration are discussed, particularly in the context of cancer cachexia., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. However, prolonged MV causes the rapid development of diaphragm muscle atrophy, and diaphragmatic weakness may contribute to difficult weaning from MV. Therefore, developing a therapeutic countermeasure to protect against MV-induced diaphragmatic atrophy is important. MV-induced diaphragm atrophy is due, at least in part, to increased production of reactive oxygen species (ROS) from diaphragm mitochondria and the activation of key muscle proteases (i.e., calpain and caspase-3). In this regard, leakage of calcium through the ryanodine receptor (RyR1) in diaphragm muscle fibers during MV could result in increased mitochondrial ROS emission, protease activation, and diaphragm atrophy. Therefore, these experiments tested the hypothesis that a pharmacological blockade of the RyR1 in diaphragm fibers with azumolene (AZ) would prevent MV-induced increases in mitochondrial ROS production, protease activation, and diaphragmatic atrophy. Adult female Sprague-Dawley rats underwent 12 hours of full-support MV while receiving either AZ or vehicle. At the end of the experiment, mitochondrial ROS emission, protease activation, and fiber cross-sectional area were determined in diaphragm muscle fibers. Decreases in muscle force production following MV indicate that the diaphragm took up a sufficient quantity of AZ to block calcium release through the RyR1. However, our findings reveal that AZ treatment did not prevent the MV-induced increase in mitochondrial ROS emission or protease activation in the diaphragm. Importantly, AZ treatment did not prevent MV-induced diaphragm fiber atrophy. Thus, pharmacological inhibition of the RyR1 in diaphragm muscle fibers is not sufficient to prevent MV-induced diaphragm atrophy.

Mechanical ventilation is a life-saving intervention for patients in respiratory failure. Unfortunately, prolonged ventilator support results in diaphragmatic atrophy and contractile dysfunction leading to diaphragm weakness, which is predicted to contribute to problems in weaning patients from the ventilator. While it is established that ventilator-induced oxidative stress is required for the development of ventilator-induced diaphragm weakness, the signaling pathway(s) that trigger oxidant production remain unknown. However, recent evidence reveals that increased plasma levels of angiotensin II (ANG II) result in oxidative stress and atrophy in limb skeletal muscles. Using a well-established animal model of mechanical ventilation, we tested the hypothesis that increased circulating levels of ANG II are required for both ventilator-induced diaphragmatic oxidative stress and diaphragm weakness. Cause and effect was determined by administering an angiotensin-converting enzyme inhibitor (enalapril) to prevent ventilator-induced increases in plasma ANG II levels, and the ANG II type 1 receptor antagonist (losartan) was provided to prevent the activation of ANG II type 1 receptors. Enalapril prevented the increase in plasma ANG II levels but did not protect against ventilator-induced diaphragmatic oxidative stress or diaphragm weakness. In contrast, losartan attenuated both ventilator-induced oxidative stress and diaphragm weakness. These findings indicate that circulating ANG II is not essential for the development of ventilator-induced diaphragm weakness but that activation of ANG II type 1 receptors appears to be a requirement for ventilator-induced diaphragm weakness. Importantly, these experiments provide the first evidence that the Food and Drug Administration-approved drug losartan may have clinical benefits to protect against ventilator-induced diaphragm weakness in humans.

Background: Muscle wasting is a profound side effect of advanced cancer. Cancer-induced cachexia decreases patient quality of life and is associated with poor patient survival. Currently, no clinical therapies exist to treat cancer-induced muscle wasting. Although cancers commonly associated with cachexia occur in older individuals, the standard animal models used to elucidate the causes of cachexia rely on juvenile mice., Methods: In an effort to better model human cancer cachexia, we determined whether cachectic features seen in young mice could be achieved in adult, pre-sarcopenic mice following colon 26 (C-26) tumor cell inoculation., Results: Both young and adult mice developed similar-sized tumors and progressed to cachexia with similar kinetics, as evidenced by losses in body mass, and adipose and skeletal muscle tissues. Proteolytic signaling, including proteasome and autophagy genes, was also increased in muscles from both young and adult tumor-bearing animals. Furthermore, tumor-associated muscle damage and activation of Pax7 progenitor cells was induced in both young and adult mice., Conclusions: Although cancer cachexia generally occurs in older individuals, these data suggest that the phenotype and underlying mechanisms can be effectively modeled using the currently accepted protocol in juvenile mice.

Background: Mechanical ventilation (MV) is a method of maintaining appropriate gas exchange in patients who are unable to sustain adequate alveolar ventilation. While lifesaving in the short-term, prolonged MV leads to altered cardiovascular responses and enhanced lung injury, but the exact mechanism is unknown. Therefore, we investigated the involvement of the sympathoadrenergic and renin-angiotensin system in MV-induced altered cardiovascular responses., Methods: Sprague-Dawley rats were divided into six groups: (1) spontaneous breathing (SB); (2) SB + enalapril (100 μg/kg intravenous infusion); (3) SB + losartan (100 μg/kg infusion); (4) 12 h of MV; (5) MV + enalapril; and (6) MV + losartan. After the animals were sacrificed, blood and tissue samples were collected. Tyrosine hydroxylase, dopamine beta hydroxylase, and neuropeptide Y were measured in adrenal medulla and hypothalamus, whereas AT1 was measured in lung tissues by Western blot. Norepinephrine enzyme-linked immunosorbent assay and total antioxidant capacity were assayed in plasma., Results: Our findings indicated that MV increases the sympathetic activation markers in adrenal medulla and hypothalamus. Moreover, oxidative stress was increased in lung and brain tissues. Treatment with enalapril or losartan reduced the lipid peroxidation in lung and brain tissues, while preserving the tissue glutathione content and plasma antioxidant capacity., Conclusions: These data demonstrate that the inhibition of the renin-angiotensin system by enalapril or losartan may reduce the MV-induced increase in sympathetic activity markers and oxidative stress, and thus, may have a beneficial effect as adjuvant therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Long periods of skeletal muscle disuse result in muscle fiber atrophy, and mitochondrial production of reactive oxygen species (ROS) appears to be a required signal for the increase in protein degradation that occurs during disuse muscle atrophy. The experiments detailed here demonstrate for the first time in limb muscle that the inactivity-induced increases in E3 ligase expression and autophagy biomarkers result from increases in mitochondrial ROS emission. Treatment of animals with a mitochondrial-targeted antioxidant also prevented the disuse-induced decrease in anabolic signaling (Akt/mammalian target of rapamycin signaling) that is normally associated with prolonged inactivity in skeletal muscles. Additionally, our results confirm previous findings that treatment with a mitochondrial-targeted antioxidant is sufficient to prevent casting-induced skeletal muscle atrophy, mitochondrial dysfunction, and activation of the proteases calpain and caspase-3. Collectively, these data reveal that inactivity-induced increases in mitochondrial ROS emission play a required role in activation of key proteolytic systems and the downregulation of important anabolic signaling molecules in muscle fibers exposed to prolonged inactivity.

Prolonged skeletal muscle inactivity results in a rapid decrease in fiber size, primarily due to accelerated proteolysis. Although several proteases are known to contribute to disuse muscle atrophy, the ubiquitin proteasome system is often considered the most important proteolytic system during many conditions that promote muscle wasting. Emerging evidence suggests that calpain and caspase-3 may also play key roles in inactivity-induced atrophy of respiratory muscles, but it remains unknown if these proteases are essential for disuse atrophy in limb skeletal muscles. Therefore, we tested the hypothesis that activation of both calpain and caspase-3 is required for locomotor muscle atrophy induced by hindlimb immobilization. Seven days of immobilization (i.e., limb casting) promoted significant atrophy in type I muscle fibers of the rat soleus muscle. Independent pharmacological inhibition of calpain or caspase-3 prevented this casting-induced atrophy. Interestingly, inhibition of calpain activity also prevented caspase-3 activation, and, conversely, inhibition of caspase-3 prevented calpain activation. These findings indicate that a regulatory cross talk exists between these proteases and provide the first evidence that the activation of calpain and caspase-3 is required for inactivity-induced limb muscle atrophy.

Objective: Diaphragmatic weakness, due to both atrophy and contractile dysfunction, is a well-documented response following prolonged mechanical ventilation. Evidence indicates that activation of the proteases calpain and caspase-3 is essential for mechanical ventilation-induced diaphragmatic weakness to occur. We tested the hypothesis that a regulatory cross-talk exists between calpain and caspase-3 in the diaphragm during prolonged mechanical ventilation. To test this prediction, we determined whether selective pharmacological inhibition of calpain would prevent activation of caspase-3 and conversely whether selective inhibition of caspase-3 would abate calpain activation., Design: Animal study., Setting: University Research Laboratory., Subjects: Female Sprague-Dawley rats., Interventions: Animals were randomly divided into control or one of three 12-hr mechanical ventilation groups that were treated with/without a selective pharmacological protease inhibitor: 1) control, 2) mechanical ventilation, 3) mechanical ventilation with a selective caspase-3 inhibitor, and 4) mechanical ventilation with a selective calpain inhibitor., Measurements and Main Results: Compared to control, mechanical ventilation resulted in calpain and caspase-3 activation in the diaphragm accompanied by atrophy of type I, type IIa, and type IIx/IIb fibers. Independent inhibition of either calpain or caspase-3 prevented this mechanical ventilation-induced atrophy. Pharmacological inhibition of calpain prevented mechanical ventilation-induced activation of diaphragmatic caspase-3 and inhibition of caspase-3 prevented activation of diaphragmatic calpain. Furthermore, calpain inhibition also prevented the activation of caspase-9 and caspase-12, along with the cleavage of Bid to tBid, all upstream signals for caspase-3 activation. Lastly, caspase-3 inhibition prevented the mechanical ventilation-induced degradation of the endogenous calpain inhibitor, calpastatin., Conclusions: Collectively, these results indicate that mechanical ventilation-induced diaphragmatic atrophy is dependent on the activation of both calpain and caspase-3. Importantly, these findings provide the first experimental evidence in diaphragm muscle that calpain inhibition prevents the activation of caspase-3 and vice versa and caspase-3 inhibition prevents the activation of calpain. These findings support our hypothesis that a regulatory calpain/caspase-3 cross-talk exists whereby calpain can promote caspase-3 activation and active caspase-3 can enhance calpain activity in diaphragm muscle during prolonged mechanical ventilation.

Loss of the intestinal barrier is critical to the clinical course of heat illness, but the underlying mechanisms are still poorly understood. We tested the hypothesis that conditions characteristic of mild heatstroke in mice are associated with injury to the epithelial lining of the intestinal tract and comprise a critical component of barrier dysfunction. Anesthetized mice were gavaged with 4 kDa FITC-dextran (FD-4) and exposed to increasing core temperatures, briefly reaching 42.4°C, followed by 30 min recovery. Arterial samples were collected to measure FD-4 concentration in plasma (in vivo gastrointestinal permeability). The small intestines were then removed to measure histological evidence of injury. Hyperthermia resulted in a ≈2.5-fold elevation in plasma FD-4 and was always associated with significant histological evidence of injury to the epithelial lining compared with matched controls, particularly in the duodenum. When isolated intestinal segments from control animals were exposed to ≥41.5°C, marked increases in permeability were observed within 60 min. These changes were associated with release of lactate dehydrogenase, evidence of protein oxidation via carbonyl formation and histological damage. Coincubation with N-acetylcysteine protected in vitro permeability during hyperthermia and reduced histological damage and protein oxidation. Chelation of intracellular Ca(2+) to block tight junction opening during 41.5°C exposure failed to reduce the permeability of in vitro segments. The results demonstrate that hyperthermia exposure in mouse intestine, at temperatures at or below those necessary to induce mild heatstroke, cause rapid and substantial injury to the intestinal lining that may be attributed, in part, to oxidative stress.

Purpose: Three to five consecutive days of endurance exercise can protect the heart against an ischemia-reperfusion (IR) insult. However, the mechanisms responsible for this exercise-mediated cardioprotection remain unknown. Given the important role that mitochondria play in IR-induced cardiac myocyte injury, we hypothesized that exercise training promotes cardioprotection, at least in part, by increasing mitochondrial antioxidants, preventing mitochondrial release of reactive oxygen species, and protecting cardiac mitochondria against IR-induced oxidative damage and functional impairment., Methods: To test our hypothesis, Sprague-Dawley rats were assigned to either sedentary (n = 16) or exercise-trained (n = 16) groups. Exercise-trained animals performed 5 d of treadmill running for 60 min·d(-1) at 30 m·s(-1). Hearts were excised from sedentary and exercised-trained animals and were either perfused for 80 min or exposed to 40 min of global ischemia followed by 45 min of reperfusion by using an ex vivo isolated working heart model. After the protocol, cardiac subsarcolemmal and intermyofibrillar mitochondria were isolated and used to determine respiratory control ratio, reactive oxygen species emission, and indices of oxidative stress and apoptosis., Results: Our results support our hypothesis because exercise training protected both cardiac subsarcolemmal and intermyofibrillar mitochondria from IR-induced uncoupling and oxidative damage. Specifically, the levels of cardiac mitochondrial 4-hydroxynonenal-conjugated proteins were elevated in hearts from sedentary animals exposed to IR compared with cardiac mitochondria isolated from exercise-trained animals. Exercise also resulted in an increase in mitochondrial antioxidant enzymes (copper-zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase) and prevented the IR-induced release of proapoptotic proteins from the mitochondria., Conclusions: Collectively, these novel findings reveal that exercise-induced cardioprotection is mediated, at least in part, through mitochondrial adaptations resulting in a mitochondrial phenotype that resists IR-induced damage.

Background: Mechanical ventilation is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged mechanical ventilation is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during mechanical ventilation, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of mechanical ventilation- induced reactive oxygen species production in the diaphragm is important., Objective: These experiments tested the hypothesis that elevated mitochondrial reactive oxygen species emission is required for mechanical ventilation-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm., Design: Cause and effect was determined by preventing mechanical ventilation-induced mitochondrial reactive oxygen species emission in the diaphragm of rats using a novel mitochondria-targeted antioxidant (SS-31)., Interventions: None., Measurements and Main Results: Compared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against mechanical ventilation-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against mechanical ventilation-induced myofiber atrophy and contractile dysfunction., Conclusions: These results reveal that prevention of mechanical ventilation-induced increases in diaphragmatic mitochondrial reactive oxygen species emission protects the diaphragm from mechanical ventilation-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of reactive oxygen species production in the diaphragm during prolonged mechanical ventilation. These results could lead to the development of a therapeutic intervention to impede mechanical ventilation-induced diaphragmatic weakness., Competing Interests: The remaining authors have not disclosed any potential conflicts of interest.

It is well established that contracting skeletal muscles produce free radicals. Given that radicals are known to play a prominent role in the pathogenesis of several diseases, the 1980s-90s dogma was that contraction-induced radical production was detrimental to muscle because of oxidative damage to macromolecules within the fibre. In contrast to this early outlook, it is now clear that both reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in cell signalling pathways involved in muscle adaptation to exercise and the remodelling that occurs in skeletal muscle during periods of prolonged inactivity. This review will highlight two important redox sensitive signalling pathways that contribute to ROS and RNS-induced skeletal muscle adaptation to endurance exercise. We begin with a historical overview of radical production in skeletal muscles followed by a discussion of the intracellular sites for ROS and RNS production in muscle fibres. We will then provide a synopsis of the redox-sensitive NF-B and PGC-1α signalling pathways that contribute to skeletal muscle adaptation in response to exercise training. We will conclude with a discussion of unanswered questions in redox signalling in skeletal muscle in the hope of promoting additional research interest in this field.

Increased reactive oxygen species (ROS) production is crucial to the remodelling that occurs in skeletal muscle in response to both exercise training and prolonged periods of disuse. This review discusses the redox-sensitive signalling pathways that are responsible for this ROS-induced skeletal muscle adaptation. We begin with a discussion of the sites of ROS production in skeletal muscle fibres. This is followed by an overview of the putative redox-sensitive signalling pathways that promote skeletal muscle adaptation. Specifically, this discussion highlights redox-sensitive kinases, phosphatases and the transcription factor nuclear factor-B. We also discuss the evidence that connects redox signalling to skeletal muscle adaptation in response to increased muscular activity (i.e. exercise training) and during prolonged periods of muscular inactivity (i.e. immobilization). In an effort to stimulate further research, we conclude with a discussion of unanswered questions about redox signalling in skeletal muscle.

Mechanical ventilation (MV) is a life-saving intervention used in patients with acute respiratory failure. Unfortunately, prolonged MV results in diaphragmatic weakness, which is an important contributor to the failure to wean patients from MV. Our laboratory has previously shown that reactive oxygen species (ROS) play a critical role in mediating diaphragmatic weakness after MV. However, the pathways responsible for MV-induced diaphragmatic ROS production remain unknown. These experiments tested the hypothesis that prolonged MV results in an increase in mitochondrial ROS release, mitochondrial oxidative damage, and mitochondrial dysfunction. To test this hypothesis, adult (3-4 months of age) female Sprague-Dawley rats were assigned to either a control or a 12-h MV group. After treatment, diaphragms were removed and mitochondria were isolated for subsequent respiratory and biochemical measurements. Compared to control, prolonged MV resulted in a lower respiratory control ratio in diaphragmatic mitochondria. Furthermore, diaphragmatic mitochondria from MV animals released higher rates of ROS in both State 3 and State 4 respiration. Prolonged MV was also associated with diaphragmatic mitochondrial oxidative damage as indicated by increased lipid peroxidation and protein oxidation. Finally, our data also reveal that the activities of the electron transport chain complexes II, III, and IV are depressed in mitochondria isolated from diaphragms of MV animals. In conclusion, these results are consistent with the concept that diaphragmatic inactivity promotes an increase in mitochondrial ROS emission, mitochondrial oxidative damage, and mitochondrial respiratory dysfunction.

Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidant-induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 microM H2O2) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H2O2 significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (-81%), alpha-actinin (-40%), desmin (-79%), talin (-37%), and troponin I (-80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate alphaII-spectrin following 4 h and 24 h of H2O2 treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 microM). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for H2O2-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress.

Purposes: (1) To compare the Lafayette Instruments (LI) skinfold caliper to the Lange (L) and Harpenden (H) calipers using a diverse subject population. (2) To determine the validity of the LI caliper in a subset of subjects by comparing body compositions from skinfold thicknesses to those measured by hydrostatic weighing (HW) and air displacement plethysmography (ADP). (3) To compare measurements obtained by experienced (EX) and inexperienced (IX) technicians using all three calipers., Methods: Skinfold measurements were performed by both EX and IX technicians using three different calipers on 21 younger (21.2 ± 1.5 yrs) and 20 older (59.2 ± 4 yrs) subjects. Body compositions were calculated using the Jackson-Pollock seven-site and three-site formulas. HW and ADP tests were performed on a subset of subjects (10 younger, 10 older)., Results: No significant differences existed between LI and L or H when measurements were made by EX. Further, the LI-EX measurements were highly correlated to both H-EX and L-EX. No significant differences existed in the subgroup between LI-EX and HW or ADP. Skinfold determinations made by EX and IX were similar., Conclusions: Similar body compositions determined using LI, H, and L suggest that LI determines body composition as effectively as H and L. High correlations between the three calipers support this notion. Similar results between LI and HW/ADP subgroup suggest that the LI caliper may be a valid method of measuring body composition. Overall, performance by IX was similar to EX and suggests similar ease of use for all three calipers.