Mitohormesis during advanced stages of Duchenne muscular dystrophy reveals a redox-sensitive creatine pathway that can be enhanced by the mitochondrial-targeting peptide SBT-20.

Mitochondrial creatine kinase (mtCK) regulates the "fast" export of phosphocreatine to support cytoplasmic phosphorylation of ADP to ATP which is more rapid than direct ATP export. Such "creatine-dependent" phosphate shuttling is attenuated in several muscles, including the heart, of the D2.mdx mouse model of Duchenne muscular dystrophy at only 4 weeks of age. However, the degree to which creatine-dependent and -independent systems of phosphate shuttling progressively worsen or potentially adapt in a hormetic manner throughout disease progression remains unknown. Here, we performed a series of proof-of-principle investigations designed to determine how phosphate shuttling pathways worsen or adapt in later disease stages in D2.mdx (12 months of age). We also determined whether changes in creatine-dependent phosphate shuttling are linked to alterations in mtCK thiol redox state. In permeabilized muscle fibres prepared from cardiac left ventricles, we found that 12-month-old male D2.mdx mice have reduced creatine-dependent pyruvate oxidation and elevated complex I-supported H2O2 emission (mH2O2). Surprisingly, creatine-independent ADP-stimulated respiration was increased and mH2O2 was lowered suggesting that impairments in the faster mtCK-mediated phosphocreatine export system resulted in compensation of the alternative slower pathway of ATP export. The apparent impairments in mtCK-dependent bioenergetics occurred independent of mtCK protein content but were related to greater thiol oxidation of mtCK and a more oxidized cellular environment (lower GSH:GSSG). Next, we performed a proof-of-principle study to determine whether creatine-dependent bioenergetics could be enhanced through chronic administration of the mitochondrial-targeting, ROS-lowering tetrapeptide, SBT-20. We found that 12 weeks of daily treatment with SBT-20 (from day 4-∼12 weeks of age) increased respiration and lowered mH2O2 only in the presence of creatine in D2.mdx mice without affecting calcium-induced mitochondrial permeability transition activity. In summary, creatine-dependent mitochondrial bioenergetics are attenuated in older D2.mdx mice in relation to mtCK thiol oxidation that seem to be countered by increased creatine-independent phosphate shuttling as a unique form of mitohormesis. Separate results demonstrate that creatine-dependent bioenergetics can also be enhanced with a ROS-lowering mitochondrial-targeting peptide. These results demonstrate a specific relationship between redox stress and mitochondrial hormetic reprogramming during dystrophin deficiency with proof-of-principle evidence that creatine-dependent bioenergetics could be modified with mitochondrial-targeting small peptide therapeutics.

Cardiac Atrophy, Dysfunction, and Metabolic Impairments: A Cancer-Induced Cardiomyopathy Phenotype.

Muscle atrophy and weakness are prevalent features of cancer. Although extensive research has characterized skeletal muscle wasting in cancer cachexia, limited studies have investigated how cardiac structure and function are affected by therapy-naive cancer. Here, the authors used orthotopic, syngeneic models of epithelial ovarian cancer and pancreatic ductal adenocarcinoma, and a patient-derived pancreatic xenograft model, to define the impacts of malignancy on cardiac structure, function, and metabolism. Tumor-bearing mice develop cardiac atrophy and intrinsic systolic and diastolic dysfunction, with arterial hypotension and exercise intolerance. In hearts of ovarian tumor-bearing mice, fatty acid-supported mitochondrial respiration decreased, and carbohydrate-supported respiration increased-showcasing a substrate shift in cardiac metabolism that is characteristic of heart failure. Epithelial ovarian cancer decreased cytoskeletal and cardioprotective gene expression, which was paralleled by down-regulation of transcription factors that regulate cardiomyocyte size and function. Patient-derived pancreatic xenograft tumor-bearing mice show altered myosin heavy chain isoform expression-also a molecular phenotype of heart failure. Markers of autophagy and ubiquitin-proteasome system were upregulated by cancer, providing evidence of catabolic signaling that promotes cardiac wasting. Together, the authors cross-validate with two cancer types, evidence of the structural, functional, and metabolic cancer-induced cardiomyopathy, thus providing translational evidence that could impact future medical management strategies for improved cancer recovery in patients.

Muscle weakness and mitochondrial stress occur before severe 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 severe 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-severe-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 severe 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. CONCLUSIONS: 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 severe 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-severe-metastatic weakness during EOC in addition to therapies targeting cachexia.

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.

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.

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.

Mitochondrial stress responses in Duchenne muscular dystrophy: metabolic dysfunction or adaptive reprogramming?

Mitochondrial stress may be a secondary contributor to muscle weakness in inherited muscular dystrophies. Duchenne muscular dystrophy has received the majority of attention, whereby most discoveries suggest mitochondrial ATP synthesis may be reduced. However, not all studies support this finding. Furthermore, some studies have reported increased mitochondrial reactive oxygen species and propensity for permeability transition pore formation as an inducer of apoptosis, although divergent findings have also been described. A closer examination of the literature suggests the degree and direction of mitochondrial stress responses may depend on the progression of the disease, the muscle type examined, the mouse model used with regard to preclinical research, the precise metabolic pathways in consideration, and in some cases, the in vitro technique used to assess a given mitochondrial bioenergetic function. One intent of this review is to provide careful considerations for future experimental designs to resolve the heterogeneous nature of mitochondrial stress during the progression of Duchenne muscular dystrophy. Such considerations have implications for other muscular dystrophies as well which are addressed briefly herein. A renewed perspective of the term "mitochondrial dysfunction" is presented whereby stress responses might be re-explored in future investigations as direct contributors to myopathy versus an adaptive "reprogramming" intended to maintain homeostasis in the face of disease stressors themselves. In so doing, the prospective development of mitochondrial enhancement therapies can be driven by advances in perspectives as much as experimental approaches when resolving the precise relationships between mitochondrial remodeling and muscle weakness in Duchenne and, indeed, other muscular dystrophies.