Biology subjects

Garibotti, M. C.

Publications and source records attributed to Garibotti, M. C..

7 recordsLinked to original sources

bioRxiv2024

The mitochondrial-targeted antioxidant SkQ1 prevents mitochondrial-linked apoptosis but not necroptosis or skeletal muscle atrophy in ovarian cancer

The degree to which mitochondrial-linked cell death pathways contribute to skeletal muscle atrophy during cancer remain unknown. Here, we combined a novel and robust mouse model of metastatic ovarian cancer with chronic administration of the mitochondrial-targeted antioxidant SkQ1 to determine the time-dependent and muscle-specific relationships of mitochondrial-linked apoptosis and necroptosis to the development of muscle atrophy in the type II B-rich gastrocnemius. Early-stage ovarian cancer reduced type II B fibre cross-sectional area in the gastrocnemius but did not alter mitochondrial H2O2 emission despite increased activities of mitochondrial-linked caspase-9 and-3 regulators of apoptosis. During late-stage ovarian cancer, sustained atrophy was associated with increased mitochondrial H2O2 emission potential in vitro, a greater probability of calcium-triggered mitochondrial permeability transition and increases in downstream caspase-9 and -3 activity. SkQ1 attenuated mitochondrial H2O2 emission and caspase-9 and -3 activity in late-stage ovarian cancer but did not prevent atrophy. Necroptosis markers were heterogeneous across time with total RIPK1 increasing during early-stage cancer which reverted to normal levels by late-stages while phosphorylated RIPK3 decreased below control levels. These discoveries indicate that preventing increases in mitochondrial-linked apoptotic caspase-9 and -3 activity during late-stage ovarian cancer with SkQ1 does not prevent atrophy of type II B fibres. Furthermore, necroptotic markers are inconclusive during cancer in this muscle type but are not modified by SkQ1. These results do not support a causal relationship between mitochondrial H2O2-linked apoptosis or necroptosis and atrophy in type II B fibres during ovarian cancer but do not rule out potential relationships in other muscle types. Key PointsO_LICancer increases mitochondrial reactive oxygen species (ROS) in skeletal muscle during atrophy, but the role of ROS in regulating cell death remains unknown. C_LIO_LIWe show that attenuating gastrocnemius mitochondrial ROS with the mitochondrial-targeted antioxidant SkQ1 prevented mitochondrial-linked pro-apoptotic caspase 9- and 3-activities but did not affect markers of necroptosis in a mouse model of ovarian cancer. C_LIO_LIReductions in gastrocnemius muscle fibre cross-sectional areas and the wet weights of several muscles were not prevented by SkQ1. C_LIO_LIThese findings demonstrate that mitochondrial ROS regulate apoptotic caspases but not necroptosis, and neither pathway is linked to gastrocnemius atrophy in mice with ovarian cancer. C_LIO_LIThe degree to which mitochondrial ROS-linked cell death pathways regulate muscle mass in other muscle types and cancer models requires further investigation. C_LI

physiology↗

bioRxiv2024

Mitochondrial-targeted plastoquinone therapy ameliorates early onset muscle weakness that precedes ovarian cancer cachexia in mice

Cancer cachexia, and the related loss of muscle and strength, worsens quality of life and lowers overall survival. Recently, a novel pre-atrophy muscle weakness was identified during early-stage cancer. While mitochondrial stress responses are associated with early-stage pre-atrophy weakness, a causal relationship has not been established. Using a robust mouse model of metastatic epithelial ovarian cancer (EOC)-induced cachexia, we found the well-established mitochondrial-targeted plastoquinone SkQ1 partially prevents pre-atrophy weakness in the diaphragm. Furthermore, SkQ1 improved force production during atrophy without preventing atrophy itself in the tibialis anterior and diaphragm. EOC reduced flexor digitorum brevis (FDB) force production and myoplasmic free calcium ([Ca2+]i) during contraction in single muscle fibers, both of which were prevented by SkQ1. Remarkably, changes in mitochondrial reactive oxygen species and pyruvate metabolism were heterogeneous across time and between muscle types which highlights a considerable complexity in the relationships between mitochondria and muscle remodeling throughout EOC. These discoveries identify that muscle weakness can occur independent of atrophy throughout EOC in a manner that is linked to improved calcium handling. The findings also demonstrate that mitochondrial-targeted therapies exert a robust effect in preserving muscle force during the early pre-atrophy period and in late-stage EOC once cachexia has become severe.

physiology↗

bioRxiv2024

Adiponectin-receptor agonism prevents right ventricular tissue pathology in a mouse model of Duchenne muscular dystrophy

Cardiac fibrosis during Duchenne muscular dystrophy (DMD) arises from cellular damage and inflammation and is associated with myofibre hypertrophy and metabolic dysfunction. However, the extent to which these relationships develop across all 4 cardiac chambers, particularly during early-stage disease, remains unknown. Here, we discovered that very young D2.mdx mice exhibit fibrosis exclusively in the right ventricle (RV) and left atrium. Concurrent cardiomyocyte hypertrophy and disorganization in the RV were related to a highly specific inflammatory signature of increased infiltrating pro-inflammatory macrophages (CD11b+CD45+CD64+F4/80+CCR2+), myofibre mitochondrial-linked apoptosis, and reduced carbohydrate and fat oxidation. This relationship did not occur in the left ventricle. Short-term daily administration of a peptidomimetic adiponectin receptor agonist, ALY688, completely prevented RV fibrosis, myofibre hypertrophy, infiltrating macrophages and mitochondrial stress as well as left atrial fibrosis. Our discoveries demonstrate early-stage cardiac tissue pathology occurs in a chamber-specific manner and is prevented by adiponectin receptor agonism, thereby opening a new direction for developing therapies that prevent tissue remodeling during DMD. TeaserHeterogeneous distribution of fibrosis, inflammation and metabolic dysfunction across the heart is prevented by adiponectin receptor agonism in a mouse model of Duchenne muscular dystrophy.

physiology↗

bioRxiv2024

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

ObjectivesA 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. MethodsHere, 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. ResultsPrimary 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 (TNF), 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. ConclusionThis 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. HighlightsO_LIThis study reports the first orthotopic model of metastatic ovarian cancer cachexia that can be induced in adult immunocompetent mice C_LIO_LIDiaphragm and limb muscle weakness precedes metastasis and atrophy during ovarian cancer C_LIO_LISkeletal muscle mitochondrial oxidative and redox stress signatures occur during pre-metastatic stages of ovarian cancer C_LIO_LISpecific muscle force as well as mitochondrial pyruvate oxidation and creatine metabolism demonstrate compensation in later stages C_LIO_LIOvarian cancer has heterogeneous effects on distinct muscle types across time C_LI

physiology↗

bioRxiv2024

Disrupted cardiac bioenergetics linked to oxidized mitochondrial creatine kinase are rescued by the mitochondrial-targeting peptide SBT-20 in the D2.mdx model of Duchenne muscular dystrophy

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 to [~]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.

physiology↗

bioRxiv2023

Cardiac atrophy, dysfunction, and metabolic impairments: a cancer-induced heart failure phenotype

Muscle atrophy and weakness are prevalent features of cancer. While 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. In cell-based models of orthotopic, syngeneic epithelial ovarian cancer (EOC) and pancreatic ductal adenocarcinoma (PDAC), and a patient-derived pancreatic xenograft model (PDX), we evaluated cardiac structure, function, and metabolism. Tumor-bearing mice showed cardiac atrophy and intrinsic systolic and diastolic dysfunction; associated with hypotension and exercise intolerance. In hearts of ovarian tumor-bearing mice, fatty acid-supported mitochondrial respiration decreased and carbohydrate-supported respiration increased, establishing a substrate shift in cardiac metabolism that is characteristic of heart failure. EOC decreased cytoskeletal and cardioprotective gene expression, which was paralleled by downregulation of transcription factors that regulate cardiomyocyte size and function. PDX tumors altered myosin heavy chain isoform expression - a molecular phenotype observed in heart failure. Markers of autophagy and ubiquitin-proteasome system were upregulated with cancer, providing evidence of catabolic signaling that promotes cardiac wasting. Together, metabolic stress, cardiac gene dysregulation, and upregulation of catabolic pathways contribute to cardiac atrophy and failure during cancer. Finally, we demonstrate that pathological cardiac remodeling is induced by human cancer, providing translational evidence of cancer-induced cardiomyopathy.

physiology↗

bioRxiv2023

The adiponectin analogue ALY688-SR attenuates diaphragm fibrosis, atrophy and mitochondrial stress in a 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 analogue ALY688-SR prevents fibrosis and fibre type-specific atrophy in diaphragm of D2.mdx mice treated from days 7-28 of age. ALY688-SR also lowered IL-6mRNA but increased IL-6 and TGF-{beta} protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also lowered 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 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 partially prevents fibrosis and atrophy in the more disease-apparent diaphragm of young D2.mdx mice in relation to lower mitochondrial redox stress. These results provide a foundation for the exploration of slow-release adiponectin-based therapies to prevent fibrosis and atrophy in Duchenne muscular dystrophy.

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