Redox regulation of UPR signalling and mitochondrial ER contact sites.

Mitochondria and the endoplasmic reticulum (ER) have a synergistic relationship and are key regulatory hubs in maintaining cell homeostasis. Communication between these organelles is mediated by mitochondria ER contact sites (MERCS), allowing the exchange of material and information, modulating calcium homeostasis, redox signalling, lipid transfer and the regulation of mitochondrial dynamics. MERCS are dynamic structures that allow cells to respond to changes in the intracellular environment under normal homeostatic conditions, while their assembly/disassembly are affected by pathophysiological conditions such as ageing and disease. Disruption of protein folding in the ER lumen can activate the Unfolded Protein Response (UPR), promoting the remodelling of ER membranes and MERCS formation. The UPR stress receptor kinases PERK and IRE1, are located at or close to MERCS. UPR signalling can be adaptive or maladaptive, depending on whether the disruption in protein folding or ER stress is transient or sustained. Adaptive UPR signalling via MERCS can increase mitochondrial calcium import, metabolism and dynamics, while maladaptive UPR signalling can result in excessive calcium import and activation of apoptotic pathways. Targeting UPR signalling and the assembly of MERCS is an attractive therapeutic approach for a range of age-related conditions such as neurodegeneration and sarcopenia. This review highlights the emerging evidence related to the role of redox mediated UPR activation in orchestrating inter-organelle communication between the ER and mitochondria, and ultimately the determination of cell function and fate.

Extracellular Vesicles, Cell-Penetrating Peptides and miRNAs as Future Novel Therapeutic Interventions for Parkinson's and Alzheimer's Disease.

Neurodegeneration is hallmarked by the progressive loss of dopaminergic neurons and/or a significant increase in protein aggregates in the brain. Neurodegenerative diseases are a leading cause of death worldwide with over 15 million people currently suffering from either Parkinson's disease (PD) or Alzheimer's disease (AD). PD is often characterized by both motor and non-motor symptoms, including muscle rigidity, tremors and bradykinesia, with AD displaying symptoms of confusion and dementia. The current mainstay of therapeutics includes pharmacological approaches such as levodopa to replace dopamine in PD patients, deep brain stimulation in affected regions of the brain and physical therapy. However, these treatments are typically not disease-modifying, though they do help at least for some time with symptom management. These treatments often also fail due to their inability to cross the blood-brain barrier. There is a need to develop new strategies to target neurodegeneration in an ever-ageing population. First, we review the current PD and AD treatments and their limitations. Second, we review the current use of extracellular vesicles (EVs), cell-penetrating peptides (CPPs) and miRNAs as neuroprotective agents. Finally, we discuss the possibility of exploiting these as a combinatory therapeutic, alongside some potential drawbacks.

Peroxiredoxin 2 is required for the redox mediated adaptation to exercise.

Exercise generates a site-specific increase in Reactive Oxygen Species (ROS) within muscle that promotes changes in gene transcription and mitochondrial biogenesis, required for the beneficial adaptive response. We demonstrate that Peroxiredoxin 2 (Prdx2), an abundant cytoplasmic 2-Cys peroxiredoxin, is required for the adaptive hormesis response to physiological levels of H2O2 in myoblasts and following exercise in C. elegans. A short bolus addition of H2O2 increases mitochondrial capacity and improves myogenesis of cultured myoblasts, this beneficial adaptive response was suppressed in myoblasts with decreased expression of cytoplasmic Prdxs. Moreover, a swimming exercise protocol in C. elegans increased mitochondrial content, fitness, survival and longevity in wild type (N2) worms. In contrast, prdx-2 mutant worms had decreased fitness, disrupted mitochondria, reduced survival and lifespan following exercise. Global proteomics following exercise identified distinct changes in the proteome of N2 and prdx-2 mutants. Furthermore, a redox proteomic approach to quantify reversible oxidation of specific Cysteine residues revealed a more reduced redox state in the non-exercised prdx-2 mutant strain that become oxidized following exercise. In contrast, specific Cys residues from regulatory proteins become more reduced in the N2 strain following exercise, establishing the key regulatory role of PRDX-2 in a redox signalling cascade following endogenous ROS generation. Our results demonstrate that conserved cytoplasmic 2-Cys Peroxiredoxins are required for the beneficial adaptive response to a physiological redox stress.

MicroRNAs as the Sentinels of Redox and Hypertrophic Signalling.

Oxidative stress and inflammation are associated with skeletal muscle function decline with ageing or disease or inadequate exercise and/or poor diet. Paradoxically, reactive oxygen species and inflammatory cytokines are key for mounting the muscular and systemic adaptive responses to endurance and resistance exercise. Both ageing and lifestyle-related metabolic dysfunction are strongly linked to exercise redox and hypertrophic insensitivity. The adaptive inability and consequent exercise intolerance may discourage people from physical training resulting in a vicious cycle of under-exercising, energy surplus, chronic mitochondrial stress, accelerated functional decline and increased susceptibility to serious diseases. Skeletal muscles are malleable and dynamic organs, rewiring their metabolism depending on the metabolic or mechanical stress resulting in a specific phenotype. Endogenous RNA silencing molecules, microRNAs, are regulators of these metabolic/phenotypic shifts in skeletal muscles. Skeletal muscle microRNA profiles at baseline and in response to exercise have been observed to differ between adult and older people, as well as trained vs. sedentary individuals. Likewise, the circulating microRNA blueprint varies based on age and training status. Therefore, microRNAs emerge as key regulators of metabolic health/capacity and hormetic adaptability. In this narrative review, we summarise the literature exploring the links between microRNAs and skeletal muscle, as well as systemic adaptation to exercise. We expand a mathematical model of microRNA burst during adaptation to exercise through supporting data from the literature. We describe a potential link between the microRNA-dependent regulation of redox-signalling sensitivity and the ability to mount a hypertrophic response to exercise or nutritional cues. We propose a hypothetical model of endurance exercise-induced microRNA "memory cloud" responsible for establishing a landscape conducive to aerobic as well as anabolic adaptation. We suggest that regular aerobic exercise, complimented by a healthy diet, in addition to promoting mitochondrial health and hypertrophic/insulin sensitivity, may also suppress the glycolytic phenotype and mTOR signalling through miRNAs which in turn promote systemic metabolic health.

Evidence of impaired mitochondrial cellular bioenergetics in ocular fibroblasts derived from glaucoma patients.

Glaucoma is a progressive optic neuropathy characterized by the neurodegeneration of the retinal ganglion cells (RGCs) resulting in irreversible visual impairment and eventual blindness. RGCs are extremely susceptible to mitochondrial compromise due to their marked bioenergetic requirements and morphology. There is increasing interest in therapies targeting mitochondrial health as a method of preventing visual loss in managing glaucoma. The bioenergetic profile of Tenon's ocular fibroblasts from glaucoma patients and controls was investigated using the Seahorse XF24 analyser. Impaired mitochondrial cellular bioenergetics was detected in glaucomatous ocular fibroblasts including basal respiration, maximal respiration and spare capacity. Spare respiratory capacity levels reflect mitochondrial bio-energetic adaptability in response to pathophysiological stress. Basal oxidative stress was elevated in glaucomatous Tenon's ocular fibroblasts and hydrogen peroxide (H2O2) induced reactive oxygen species (ROS) simulated the glaucomatous condition in normal Tenon's ocular fibroblasts. This work supports the role of therapeutic interventions to target oxidative stress or provide mitochondrial energetic support in glaucoma.

Exercise stress leads to an acute loss of mitochondrial proteins and disruption of redox control in skeletal muscle of older subjects: An underlying decrease in resilience with aging?

Reactive oxygen species (ROS) are recognized as important signaling molecules in healthy skeletal muscle. Redox sensitive proteins can respond to intracellular changes in ROS by oxidation of reactive thiol groups on cysteine (Cys) residues. Exercise is known to induce the generation of superoxide and nitric oxide, resulting in the activation of several adaptive signaling pathways; however, it has been suggested that aging attenuates these redox-regulated adaptations to acute exercise. In the present study, we used redox proteomics to study the vastus lateralis muscles of Adult (n = 6 male, 6 female; 18-30 yrs) and Old (n = 6 male, 6 female; 64-79 yrs) adults. Participants completed a bout of high intensity cycling exercise consisting of five sets of 2-min intervals performed at 80% maximal aerobic power output (PPO), with 2 min recovery cycling at 40% PPO between sets. Muscle biopsies were collected prior to exercise, and immediately following the first, second, and fifth high intensity interval. Global proteomic analysis indicated differences in abundance of a number of individual proteins between skeletal muscles of Adult and Old subjects at rest with a significant exacerbation of these differences induced by the acute exercise. In particular, we observed an exercise-induced decrease in abundance of mitochondrial proteins in muscles from older subjects only. Redox proteome analysis revealed cysteines from five cytosolic proteins in older subjects with lower oxidation (i.e. greater reduction) than was seen in muscle from the young adults at rest. Redox homeostasis was well maintained in Adult subjects following exercise, but there was significant increase in oxidation of multiple mitochondrial and cytosolic protein cysteines in Old subjects. We also observed that oxidation of peroxiredoxin 3 occurred following exercise in both Adult and Old groups, supporting the possibility that this is a key effector protein for mitochondrial redox signaling. Thus, we show, for the first time that exercise reveals a lack of resilience in muscle of older human participants, that is apparent as a loss of mitochondrial proteins and oxidation of multiple protein cysteines that are not seen in younger subjects. The precise consequences of this redox disruption are unclear, but this likely play a role in the attenuation of multiple adaptations to exercise that are classically seen with aging. Such changes were only seen following the acute stress of exercise., highlighting the need to consider not only basal differences seen during aging but also the difference following physiological challenge.

miR-24 and its target gene Prdx6 regulate viability and senescence of myogenic progenitors during aging.

Satellite cell-dependent skeletal muscle regeneration declines during aging. Disruptions within the satellite cells and their niche, together with alterations in the myofibrillar environment, contribute to age-related dysfunction and defective muscle regeneration. In this study, we demonstrated an age-related decline in satellite cell viability and myogenic potential and an increase in ROS and cellular senescence. We detected a transient upregulation of miR-24 in regenerating muscle from adult mice and downregulation of miR-24 during muscle regeneration in old mice. FACS-sorted satellite cells were characterized by decreased levels of miR-24 and a concomitant increase in expression of its target: Prdx6. Using GFP reporter constructs, we demonstrated that miR-24 directly binds to its predicted site within Prdx6 mRNA. Subtle changes in Prdx6 levels following changes in miR-24 expression indicate miR-24 plays a role in fine-tuning Prdx6 expression. Changes in miR-24 and Prdx6 levels were associated with altered mitochondrial ROS generation, increase in the DNA damage marker: phosphorylated-H2Ax and changes in viability, senescence, and myogenic potential of myogenic progenitors from mice and humans. The effects of miR-24 were more pronounced in myogenic progenitors from old mice, suggesting a context-dependent role of miR-24 in these cells, with miR-24 downregulation likely a part of a compensatory response to declining satellite cell function during aging. We propose that downregulation of miR-24 and subsequent upregulation of Prdx6 in muscle of old mice following injury are an adaptive response to aging, to maintain satellite cell viability and myogenic potential through regulation of mitochondrial ROS and DNA damage pathways.

Multiomics analysis of the mdx/mTR mouse model of Duchenne muscular dystrophy.

PURPOSE/AIM: Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease characterized by extensive muscle weakness. Patients with DMD lack a functional dystrophin protein, which transmits force and organizes the cytoskeleton of skeletal muscle. Multiomic studies have been proposed as a way to obtain novel insight about disease processes from preclinical models, and we used this approach to study pathological changes in dystrophic muscles. MATERIALS AND METHODS: We evaluated hindlimb muscles of male mdx/mTR mice, which lack a functional dystrophin protein and have deficits in satellite cell abundance and proliferative capacity. Wild type (WT) C57BL/6 J mice served as controls. Muscle fiber contractility was measured, along with changes in the transcriptome using RNA sequencing, and in the proteome, metabolome, and lipidome using mass spectrometry. RESULTS: While mdx/mTR mice displayed gross pathological changes and continued cycles of degeneration and regeneration, we found no differences in permeabilized fiber contractility between strains. However, there were numerous changes in the transcriptome and proteome related to protein balance, contractile elements, extracellular matrix, and metabolism. There was only a 53% agreement in fold-change data between the proteome and transcriptome. Numerous changes in markers of skeletal muscle metabolism were observed, with dystrophic muscles exhibiting elevated glycolytic metabolites such as 6-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate, fructose bisphosphate, phosphorylated hexoses, and phosphoenolpyruvate. CONCLUSIONS: These findings highlight the utility of multiomics in studying muscle disease, and provide additional insight into the pathological changes in dystrophic muscles that might help to indirectly guide evidence-based nutritional or exercise prescription in DMD patients.

2-Cys peroxiredoxin oxidation in response to hydrogen peroxide and contractile activity in skeletal muscle: A novel insight into exercise-induced redox signalling?

Skeletal muscle generates superoxide during contractions which is rapidly converted to H2O2. This molecule has been proposed to activate signalling pathways and transcription factors that regulate key adaptive responses to exercise but the concentration of H2O2 required to oxidise and activate key signalling proteins in vitro is much higher than the intracellular concentration in muscle fibers following exercise. We hypothesised that Peroxiredoxins (Prx), which reacts with H2O2 at the low intracellular concentrations found in muscle, would be rapidly oxidised in contracting muscle and hence potentially transmit oxidising equivalents to downstream signalling proteins as a method for their oxidation and activation. The aim of this study was to characterise the effects of muscle contractile activity on the oxidation of Prx1, 2 and 3 and determine if these were affected by aging. Prx1, 2 and 3 were all rapidly and reversibly oxidised following treatment with low micromolar concentrations of H2O2 in C2C12 myotubes and also in isolated mature flexor digitalis brevis fibers from adult mice following a protocol of repeated isometric contractions. Significant oxidation of Prx2 was seen within 1 min (i.e. after 12 contractions), whereas significant oxidation was seen after 2 min for Prx1 and 3. In muscle fibers from old mice, Prx2 oxidation was significantly attenuated following contractile activity. Thus we show for the first time that Prx are rapidly and reversibly oxidised in response to contractile activity in skeletal muscle and hypothesise that these proteins act as effectors of muscle redox signalling pathways which are key to adaptations to exercise that are attenuated during aging.

The redox environment and mitochondrial dysfunction in age-related skeletal muscle atrophy.

Medical advancements have extended human life expectancy, which is not always accompanied by an improved quality of life or healthspan. A decline in muscle mass and function is a consequence of ageing and can result in a loss of independence in elderly individuals while increasing their risk of falls. Multiple cellular pathways have been implicated in age-related muscle atrophy, including the contribution of reactive oxygen species (ROS) and disrupted redox signalling. Aberrant levels of ROS disrupts the redox environment in older muscle, potentially disrupting cellular signalling and in some cases blunting the adaptive response to exercise. Age-related muscle atrophy is associated with disrupted mitochondrial content and function, one of the hallmarks of age-related diseases. There is a critical link between abnormal ROS generation and dysfunctional mitochondrial dynamics including mitochondrial biogenesis, fusion and fission. In order to develop effective treatments or preventative strategies, it is important to gain a comprehensive understanding of the mechanistic pathways implicated in age associated loss of muscle.

miR-181a regulates p62/SQSTM1, parkin, and protein DJ-1 promoting mitochondrial dynamics in skeletal muscle aging.

One of the key mechanisms underlying skeletal muscle functional deterioration during aging is disrupted mitochondrial dynamics. Regulation of mitochondrial dynamics is essential to maintain a healthy mitochondrial population and prevent the accumulation of damaged mitochondria; however, the regulatory mechanisms are poorly understood. We demonstrated loss of mitochondrial content and disrupted mitochondrial dynamics in muscle during aging concomitant with dysregulation of miR-181a target interactions. Using functional approaches and mito-QC assay, we have established that miR-181a is an endogenous regulator of mitochondrial dynamics through concerted regulation of Park2, p62/SQSTM1, and DJ-1 in vitro. Downregulation of miR-181a with age was associated with an accumulation of autophagy-related proteins and abnormal mitochondria. Restoring miR-181a levels in old mice prevented accumulation of p62, DJ-1, and PARK2, and improved mitochondrial quality and muscle function. These results provide physiological evidence for the potential of microRNA-based interventions for age-related muscle atrophy and of wider significance for diseases with disrupted mitochondrial dynamics.

Inflamma-miR-21 Negatively Regulates Myogenesis during Ageing.

Ageing is associated with disrupted redox signalling and increased circulating inflammatory cytokines. Skeletal muscle homeostasis depends on the balance between muscle hypertrophy, atrophy and regeneration, however during ageing this balance is disrupted. The molecular pathways underlying the age-related decline in muscle regenerative potential remain elusive. microRNAs are conserved robust gene expression regulators in all tissues including skeletal muscle. Here, we studied satellite cells from adult and old mice to demonstrate that inhibition of miR-21 in satellite cells from old mice improves myogenesis. We determined that increased levels of proinflammatory cytokines, TNFα and IL6, as well as H2O2, increased miR-21 expression in primary myoblasts, which in turn resulted in their decreased viability and myogenic potential. Inhibition of miR-21 function rescued the decreased size of myotubes following TNFα or IL6 treatment. Moreover, we demonstrated that miR-21 could inhibit myogenesis in vitro via regulating IL6R, PTEN and FOXO3 signalling. In summary, upregulation of miR-21 in satellite cells and muscle during ageing may occur in response to elevated levels of TNFα and IL6, within satellite cells or myofibrillar environment contributing to skeletal muscle ageing and potentially a disease-related decline in potential for muscle regeneration.

MicroRNAs as potential therapeutic targets for muscle wasting during cancer cachexia.

PURPOSE OF REVIEW: Muscle wasting in cancer cachexia remains an unmet clinical need due to lack of effective therapies associated with the complexity of the disease. Here, we discuss microRNAs, robust regulators of the expression of multiple genes, only recently characterized in cancer cachexia in humans and their therapeutic potential for muscle wasting. RECENT FINDINGS: Changes in microRNAs in muscle of cancer patients have been demonstrated for the first time and these are associated with dysregulated signalling networks during muscle wasting. These data, together with studies in animal models, indicate that microRNAs are attractive therapeutic candidates for maintaining muscle mass, both during and following cancer treatment ultimately improving patient outcomes. SUMMARY: Cancer cachexia is a complex metabolic condition associated with muscle wasting. Maintenance of muscle mass in cancer patients can improve their response to therapy and prognosis. microRNAs, which can act as oncogenes or tumour suppressors, are also dysregulated in muscle of cachexia patients. Studies in animal models of muscle wasting have demonstrated that microRNAs regulate muscle mass and strength. With more microRNA-based therapeutics in clinical trials and first RNA drugs approved, microRNAs present an attractive novel therapeutic avenue for maintaining muscle homeostasis in cachexia patients to improve their prognosis.

Diaphragm weakness and proteomics (global and redox) modifications in heart failure with reduced ejection fraction in rats.

Inspiratory dysfunction occurs in patients with heart failure with reduced ejection fraction (HFrEF) in a manner that depends on disease severity and by mechanisms that are not fully understood. In the current study, we tested whether HFrEF effects on diaphragm (inspiratory muscle) depend on disease severity and examined putative mechanisms for diaphragm abnormalities via global and redox proteomics. We allocated male rats into Sham, moderate (mHFrEF), or severe HFrEF (sHFrEF) induced by myocardial infarction and examined the diaphragm muscle. Both mHFrEF and sHFrEF caused atrophy in type IIa and IIb/x fibers. Maximal and twitch specific forces (N/cm2) were decreased by 19 ± 10% and 28 ± 13%, respectively, in sHFrEF (p < .05), but not in mHFrEF. Global proteomics revealed upregulation of sarcomeric proteins and downregulation of ribosomal and glucose metabolism proteins in sHFrEF. Redox proteomics showed that sHFrEF increased reversibly oxidized cysteine in cytoskeletal and thin filament proteins and methionine in skeletal muscle α-actin (range 0.5 to 3.3-fold; p < .05). In conclusion, fiber atrophy plus contractile dysfunction caused diaphragm weakness in HFrEF. Decreased ribosomal proteins and heighted reversible oxidation of protein thiols are candidate mechanisms for atrophy or anabolic resistance as well as loss of specific force in sHFrEF.

The clinical potential of thiol redox proteomics.

Introduction: Protein thiols are susceptible to oxidation in health and disease. Redox proteomics methods facilitate the identification, quantification, and rationalization of oxidation processes including those involving protein thiols. These residues are crucial to understanding redox homeostasis underpinning normal cell functioning and regulation as well as novel biomarkers of pathology and promising novel drug targets.Areas covered: This article reviews redox proteomic approaches to study of protein thiols in some important human pathologies and assesses the clinical potential of individual Cys residues as novel biomarkers for disease detection and as targets for novel treatments.Expert commentary: Although protein thiols are not as routinely used as redox biomarkers as some other lesions such as carbonylation, there has been growing recent interest in their potential. Driven largely by developments in high-resolution mass spectrometry it is possible now to identify proteins that are redox modified at thiol groups or that interact with regulatory oxidoreductases. Thiols that are specifically susceptible to modification by reactive oxygen species can be routinely identified now and quantitative MS can be used to quantify the proportion of a protein that is redox modified.

Peroxiredoxin 6 Down-Regulation Induces Metabolic Remodeling and Cell Cycle Arrest in HepG2 Cells.

Peroxiredoxin 6 (Prdx6) is the only member of 1-Cys subfamily of peroxiredoxins in human cells. It is the only Prdx acting on phospholipid hydroperoxides possessing two additional sites with phospholipase A2 (PLA2) and lysophosphatidylcholine-acyl transferase (LPCAT) activities. There are contrasting reports on the roles and mechanisms of multifunctional Prdx6 in several pathologies and on its sensitivity to, and influence on, the redox environment. We have down-regulated Prdx6 with specific siRNA in hepatoblastoma HepG2 cells to study its role in cell proliferation, redox homeostasis, and metabolic programming. Cell proliferation and cell number decreased while cell volume increased; import of glucose and nucleotide biosynthesis also diminished while polyamines, phospholipids, and most glycolipids increased. A proteomic quantitative analysis suggested changes in membrane arrangement and vesicle trafficking as well as redox changes in enzymes of carbon and glutathione metabolism, pentose-phosphate pathway, citrate cycle, fatty acid metabolism, biosynthesis of aminoacids, and Glycolysis/Gluconeogenesis. Specific redox changes in Hexokinase-2 (HK2), Prdx6, intracellular chloride ion channel-1 (CLIC1), PEP-carboxykinase-2 (PCK2), and 3-phosphoglycerate dehydrogenase (PHGDH) are compatible with the metabolic remodeling toward a predominant gluconeogenic flow from aminoacids with diversion at 3-phospohglycerate toward serine and other biosynthetic pathways thereon and with cell cycle arrest at G1/S transition.

Translational regulation contributes to the secretory response of chondrocytic cells following exposure to interleukin-1ß.

Osteoarthritis is a chronic disease characterized by the loss of articular cartilage in synovial joints through a process of extracellular matrix destruction that is strongly associated with inflammatory stimuli. Chondrocytes undergo changes to their protein translational capacity during osteoarthritis, but a study of how disease-relevant signals affect chondrocyte protein translation at the transcriptomic level has not previously been performed. In this study, we describe how the inflammatory cytokine interleukin 1-ß (IL-1ß) rapidly affects protein translation in the chondrocytic cell line SW1353. Using ribosome profiling we demonstrate that IL-1ß induced altered translation of inflammatory-associated transcripts such as NFKB1, TNFAIP2, MMP13, CCL2, and CCL7, as well as a number of ribosome-associated transcripts, through differential translation and the use of multiple open reading frames. Proteomic analysis of the cellular layer and the conditioned media of these cells identified changes in a number of the proteins that were differentially translated. Translationally regulated secreted proteins included a number of chemokines and cytokines, underlining the rapid, translationally mediated inflammatory cascade that is initiated by IL-1ß. Although fewer cellular proteins were found to be regulated in both ribosome profiling and proteomic data sets, we did find increased levels of SOD2, indicative of redox changes within SW1353 cells being modulated at the translational level. In conclusion, we have produced combined ribosome profiling and proteomic data sets that provide a valuable resource in understanding the processes that occur during cytokine stimulation of chondrocytic cells.

Reduced mitochondrial lipid oxidation leads to fat accumulation in myosteatosis.

Myosteatosis is the pathologic accumulation of lipid that can occur in conjunction with atrophy and fibrosis following skeletal muscle injury. Little is known about the mechanisms by which lipid accumulates in myosteatosis, but many clinical studies have demonstrated that the degree of lipid infiltration negatively correlates with muscle function and regeneration. Our objective was to determine the pathologic changes that result in lipid accumulation in injured muscle fibers. We used a rat model of rotator cuff injury in this study because the rotator cuff muscle group is particularly prone to the development of myosteatosis after injury. Muscles were collected from uninjured controls or 10, 30, or 60 d after injury and analyzed using a combination of muscle fiber contractility assessments, RNA sequencing, and undirected metabolomics, lipidomics, and proteomics, along with bioinformatics techniques to identify potential pathways and cellular processes that are dysregulated after rotator cuff tear. Bioinformatics analyses indicated that mitochondrial function was likely disrupted after injury. Based on these findings and given the role that mitochondria play in lipid metabolism, we then performed targeted biochemical and imaging studies and determined that mitochondrial dysfunction and reduced fatty acid oxidation likely leads to the accumulation of lipid in myosteatosis.-Gumucio, J. P., Qasawa, A. H., Ferrara, P. J., Malik, A. N., Funai, K., McDonagh, B., Mendias, C. L. Reduced mitochondrial lipid oxidation leads to fat accumulation in myosteatosis.