Skeletal muscle mitochondrial dysfunction mediated by Pseudomonas aeruginosa quorum-sensing transcription factor MvfR: reversing effects with anti-MvfR and mitochondrial-targeted compounds.

Sepsis and chronic infections with Pseudomonas aeruginosa, a leading "ESKAPE" bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum-sensing transcription factor MvfR (multiple virulence factor regulator) by interrogating, 5 days post-infection, its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs adenosine triphosphate generation, oxidative phosphorylation, and antioxidant response, elevates the production of reactive oxygen species, and promotes oxidative damage of mitochondrial DNA in the gastrocnemius muscle of infected mice. These impairments in mitochondrial-related functions were corroborated by the alteration of key mitochondrial proteins involved in electron transport, mitochondrial biogenesis, dynamics and quality control, and mitochondrial uncoupling. Pharmacological inhibition of MvfR using the potent anti-MvfR lead, D88, we developed, or the mitochondrial-targeted peptide SS-31 rescued the MvfR-mediated alterations observed in mice infected with the wild-type strain PA14. Our study provides insights into the actions of MvfR in orchestrating mitochondrial dysfunction in the skeletal murine muscle, and it presents novel therapeutic approaches for optimizing clinical outcomes in affected patients. IMPORTANCE: Skeletal muscle, pivotal for many functions in the human body, including breathing and protecting internal organs, contains abundant mitochondria essential for maintaining cellular homeostasis during infection. The effect of Pseudomonas aeruginosa (PA) infections on skeletal muscle remains poorly understood. Our study delves into the role of a central quorum-sensing transcription factor, multiple virulence factor regulator (MvfR), that controls the expression of multiple acute and chronic virulence functions that contribute to the pathogenicity of PA. The significance of our study lies in the role of MvfR in the metabolic perturbances linked to mitochondrial functions in skeletal muscle and the effectiveness of the novel MvfR inhibitor and the mitochondrial-targeted peptide SS-31 in alleviating the mitochondrial disturbances caused by PA in skeletal muscle. Inhibiting MvfR or interfering with its effects can be a potential therapeutic strategy to curb PA virulence.

Skeletal Muscle Mitochondrial Dysfunction Mediated by Pseudomonas aeruginosa Quorum Sensing Transcription Factor MvfR: Reversing Effects with Anti-MvfR and Mitochondrial-Targeted Compounds.

Sepsis and chronic infections with Pseudomonas aeruginosa, a leading "ESKAPE" bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum sensing (QS) transcription factor MvfR by interrogating five days post-infection its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs ATP generation, oxidative phosphorylation (OXPHOS), and antioxidant response, elevates the production of reactive oxygen species, and promotes oxidative damage of mitochondrial DNA in the gastrocnemius muscle of infected mice. These impairments in mitochondrial-related functions were corroborated by the alteration of key mitochondrial proteins involved in electron transport, mitochondrial biogenesis, dynamics and quality control, and mitochondrial uncoupling. Pharmacological inhibition of MvfR using the potent anti-MvfR lead, D88, we developed, or the mitochondrial-targeted peptide SS-31 rescued the MvfR- mediated alterations observed in mice infected with the wild-type strain PA14. Our study provides insights into the actions of MvfR in orchestrating mitochondrial dysfunction in the skeletal murine muscle, and it presents novel therapeutic approaches for optimizing clinical outcomes in affected patients.

The role of ventral tegmental area in chronic stroke rehabilitation: an exploratory study.

Introduction: The acknowledged role of external rewards in chronic stroke rehabilitation, offering positive reinforcement and motivation, has significantly contributed to patient engagement and perseverance. However, the exploration of self-reward's importance in this context remains limited. This study aims to investigate the functional connectivity of the ventral tegmental area (VTA), a key node in the brain's reward circuitry, during motor task-based rehabilitation and its correlation with the recovery process. Methods: Twelve right-handed healthy volunteers (4 men, 8 women, aged 57.4 ± 11.3 years) and twelve chronic stroke patients (5 men, 7 women, aged 48.1 ± 11.1 years) with clinically significant right-sided motor impairment (mean FM-UE score of 27.6 ± 8.7) participated. The analysis employed the CONN toolbox to assess the association between motor tasks and VTA connectivity using psychophysiological interaction (PPI). Results: PPI analysis revealed motor-dependent changes in VTA connectivity, particularly with regions within the motor circuitry, cerebellum, and prefrontal cortex. Notably, stronger connectivity between the ipsilesional VTA and cerebellum was observed in healthy controls compared to chronic stroke patients, highlighting the importance of VTA-cerebellum interactions in motor function. Stroke patients' motor performance was associated with VTA modulation in areas related to both motor tasks and reward processing, emphasizing the role of self-reward processes in rehabilitation. Changes in VTA influence on motor circuitry were linked to improvements in motor performance resulting from rehabilitation. Discussion: Our findings underscore the potential of neuroimaging techniques in quantifying and predicting rehabilitation outcomes by examining self-reward processes. The observed associations between VTA connectivity and motor performance in both healthy and stroke-affected individuals emphasize the role of psychological factors, particularly self-reward, in the rehabilitation process. This study contributes valuable insights into the intricate interplay between reward circuits and motor function, highlighting the importance of addressing psychological dimensions in neurorehabilitation strategies.

Use of a Foot-Induced Digitally Controlled Resistance Device for Functional Magnetic Resonance Imaging Evaluation in Patients with Foot Paresis.

Neurological deficits from a stroke can result in long-term motor disabilities, including those that affect walking gait. However, extensive rehabilitation following stroke is typically time limited. Establishing predictive biomarkers to identify patients who may meaningfully benefit from additional physical therapy and demonstrate improvement is important to improve the patients' quality of life. Detection of neuroplastic remodeling of the affected region and changes in the activity patterns excited while performing suitable motor tasks could have valuable implications for chronic stroke recovery. This protocol describes the use of a digitally controlled, magnetic resonance-compatible foot-induced robotic device (MR_COFID) to present a personalized foot-motor task involving trajectory following to stroke-affected subjects with gait impairment during functional magnetic resonance imaging (fMRI). In the task, foot flexion is performed against bi-directional resistive forces, which are tuned to the subject's strength in both the dorsiflexion and plantar flexion directions, while following a visual metronome. fMRI non-invasively uses endogenous deoxyhemoglobin as a contrast agent to detect blood oxygenation level-dependent (BOLD) changes between the active and resting periods during testing. Repeated periodic testing can detect therapy-related changes in excitation patterns during task performance. The use of this technique provides data to identify and measure biomarkers that may indicate the likelihood of an individual benefitting from rehabilitation beyond that which is currently provided to stroke patients.

The Severity of Sensorimotor Tracts Degeneration May Predict Motor Performance in Chronic Stroke Patients, While Brain Structural Network Dysfunction May Not.

Although the relationship between corticospinal tract (CST) fiber degeneration and motor outcome after stroke has been established, the relationship of sensorimotor cortical areas with CST fibers has not been clarified. Also limited research has been conducted on how abnormalities in brain structural networks are related to motor recovery. To address these gaps in knowledge, we conducted a diffusion tensor imaging (DTI) study with 12 chronic stroke patients (CSPs) and 12 age-matched healthy controls (HCs). We compared fractional anisotropy (FA) and mean diffusivity (MD) in 60 CST segments using the probabilistic sensorimotor area tract template (SMATT). Least Absolute Shrinkage and Selection Operator (LASSO) regressions were used to select independent predictors of Fugl-Meyer upper extremity (FM-UE) scores among FA and MD values of SMATT regions. The Graph Theoretical Network Analysis Toolbox was used to assess the structural network of each subject's brain. Global and nodal metrics were calculated, compared between the groups, and correlated with FM-UE scores. Mann-Whitney U-tests revealed reduced FA values in CSPs, compared to HCs, in many ipsilesional SMATT regions and in two contralesional regions. Mean FA value of the left (L.) primary motor cortex (M1)/supplementary motor area (SMA) region was predictive of FM-UE score (P = 0.004). Mean MD values for the L. M1/ventral premotor cortex (PMv) region (P = 0.001) and L. PMv/SMA region (P = 0.001) were found to be significant predictors of FM-UE scores. Network efficiency was the only global metric found to be reduced in CSPs (P = 0.006 vs. HCs). Nodal efficiency of the L. hippocampus, L. parahippocampal gyrus, L. fusiform gyrus (P = 0.001), and nodal local efficiency of the L. supramarginal gyrus (P < 0.001) were reduced in CSPs relative to HCs. No graph metric was associated with FM-UE scores. In conclusion, the integrity of CSTs connected to M1, SMA, and PMv were shown to be independent predictors of motor performance in CSPs, while stroke-induced topological changes in the brain's structural connectome may not be. A sensorimotor cortex-specific tract template can refine CST degeneration data and the relationship of CST degeneration with motor performance.

Peak Activation Shifts in the Sensorimotor Cortex of Chronic Stroke Patients Following Robot-assisted Rehabilitation Therapy.

BACKGROUND: Ischemic stroke is the most common cause of complex chronic disability and the third leading cause of death worldwide. In recovering stroke patients, peak activation within the ipsilesional primary motor cortex (M1) during the performance of a simple motor task has been shown to exhibit an anterior shift in many studies and a posterior shift in other studies. OBJECTIVE: We investigated this discrepancy in chronic stroke patients who completed a robot-assisted rehabilitation therapy program. METHODS: Eight chronic stroke patients with an intact M1 and 13 Healthy Control (HC) volunteers underwent 300 functional magnetic resonance imaging (fMRI) scans while performing a grip task at different force levels with a robotic device. The patients were trained with the same robotic device over a 10-week intervention period and their progress was evaluated serially with the Fugl-Meyer and Modified Ashworth scales. Repeated measure analyses were used to assess group differences in locations of peak activity in the sensorimotor cortex (SM) and the relationship of such changes with scores on the Fugl-Meyer Upper Extremity (FM UE) scale. RESULTS: Patients moving their stroke-affected hand had proportionally more peak activations in the primary motor area and fewer peak activations in the somatosensory cortex than the healthy controls (P=0.009). They also showed an anterior shift of peak activity on average of 5.3-mm (P<0.001). The shift correlated negatively with FM UE scores (P=0.002). CONCLUSION: A stroke rehabilitation grip task with a robotic device was confirmed to be feasible during fMRI scanning and thus amenable to be used to assess plastic changes in neurological motor activity. Location of peak activity in the SM is a promising clinical neuroimaging index for the evaluation and monitoring of chronic stroke patients.

Tracking of Labelled Stem Cells Using Molecular MR Imaging in a Mouse Burn Model in Vivo as an Approach to Regenerative Medicine.

Therapies based on stem cell transplants offer significant potential in the field of regenerative medicine. Monitoring the fate of the transplanted stem cells in a timely manner is considered one of the main limitations for long-standing success of stem cell transplants. Imaging methods that visualize and track stem cells in vivo non-invasively in real time are helpful towards the development of successful cell transplantation techniques. Novel molecular imaging methods which are non-invasive particularly such as MRI have been of great recent interest. Hence, mouse models which are of clinical relevance have been studied by injecting contrast agents used for labelling cells such as super-paramagnetic iron-oxide (SPIO) nanoparticles for cellular imaging. The MR techniques which can be used to generate positive contrast images have been of much relevance recently for tracking of the labelled cells. Particularly when the off-resonance region in the vicinity of the labeled cells is selectively excited while suppressing the signals from the non-labeled regions by the method of spectral dephasing. Thus, tracking of magnetically labelled cells employing positive contrast in vivo MR imaging methods in a burn mouse model in a non-invasive way has been the scope of this study. The consequences have direct implications for monitoring labeled stem cells at some stage in wound healing. We suggest that our approach can be used in clinical trials in molecular and regenerative medicine.

Improving motor function after chronic stroke by interactive gaming with a redesigned MR-compatible hand training device.

New rehabilitation strategies enabled by technological developments are challenging the prevailing concept of there being a limited window for functional recovery after stroke. In this study, we examined the utility of a robot-assisted therapy used in combination with a serious game as a rehabilitation and motor assessment tool in patients with chronic stroke. We evaluated 928 game rounds from 386 training sessions of 8 patients who had suffered an ischemic stroke affecting middle cerebral artery territory that incurred at least 6 months prior. Motor function was assessed with clinical motor scales, including the Fugl-Meyer upper extremity (FM UE) scale, Action Research Arm Test, Modified Ashworth scale and the Box and Blocks test. Robotic device output measures (mean force, force-position correlation) and serious game score elements (collisions, rewards and total score) were calculated. A total of 2 patients exhibited a marginal improvement after a 10-week training protocol according to the FM UE scale and an additional patient exhibited a significant improvement according to Box and Blocks test. Motor scales showed strong associations of robotic device parameters and game metrics with clinical motor scale scores, with the strongest correlations observed for the mean force (0.677<Ρ<0.869), followed by the number of collisions (-0.670<Ρ<-0.585). Linear regression analysis showed that these indices were independent predictors of motor scale scores. In conclusion, a robotic device linked to a serious game can be used by patients with chronic stroke and induce at least some clinical improvements in motor performance. Robotic device output parameters and game score elements associate strongly with clinical motor scales and have the potential to be used as predictors in models of rehabilitation progress.

Functional MRI in Conjunction with a Novel MRI-compatible Hand-induced Robotic Device to Evaluate Rehabilitation of Individuals Recovering from Hand Grip Deficits.

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous deoxyhemoglobin as an endogenous contrast agent to detect changes in blood-level-dependent oxygenation (BOLD effect). We combined fMRI with a novel robotic device (MR-compatible hand-induced robotic device [MR_CHIROD]) so that a person in the scanner can execute a controlled motor task, hand-squeezing, which is a very important hand movement to study in neurological motor disease. We employed parallel imaging (generalized auto-calibrating partially parallel acquisitions [GRAPPA]), which allowed higher spatial resolution resulting in increased sensitivity to BOLD. The combination of fMRI with the hand-induced robotic device allowed precise control and monitoring of the task that was executed while a participant was in the scanner; this may prove to be of utility in rehabilitation of hand motor function in patients recovering from neurological deficits (e.g., stroke). Here we outline the protocol for using the current prototype of the MR_CHIROD during an fMRI scan.

Pseudomonas aeruginosa Quorum Sensing Molecule Alters Skeletal Muscle Protein Homeostasis by Perturbing the Antioxidant Defense System.

Skeletal muscle function is compromised in many illnesses, including chronic infections. The Pseudomonas aeruginosa quorum sensing (QS) signal, 2-amino acetophenone (2-AA), is produced during acute and chronic infections and excreted in human tissues, including the lungs of cystic fibrosis patients. We have shown that 2-AA facilitates pathogen persistence, likely via its ability to promote the formation of bacterial persister cells, and that it acts as an interkingdom immunomodulatory signal that epigenetically reprograms innate immune functions. Moreover, 2-AA compromises muscle contractility and impacts the expression of genes involved in reactive oxygen species (ROS) homeostasis in skeletal muscle and in mitochondrial functions. Here, we elucidate the molecular mechanisms of 2-AA's impairment of skeletal muscle function and ROS homeostasis. Murine in vivo and differentiated C2C12 myotube cell studies showed that 2-AA promotes ROS generation in skeletal muscle via the modulation of xanthine oxidase (XO) activity, NAD(P)H oxidase2 (NOX2) protein level, and the activity of antioxidant enzymes. ROS accumulation triggers the activity of AMP-activated protein kinase (AMPK), likely upstream of the observed locations of induction of ubiquitin ligases Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx), and induces autophagy-related proteins. The protein-level perturbation in skeletal muscle of silent mating type information regulation 2 homolog 1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), and uncoupling protein 3 (UCP3) is rescued by the antioxidant N-acetyl-l-cysteine (NAC). Together, these results unveil a novel form of action of a QS bacterial molecule and provide molecular insights into the 2-AA-mediated skeletal muscle dysfunction caused by P. aeruginosaIMPORTANCEPseudomonas aeruginosa, a bacterium that is resistant to treatment, causes serious acute, persistent, and relapsing infections in humans. There is increasing evidence that bacterial excreted small molecules play a critical role during infection. We have shown that a quorum sensing (QS)-regulated excreted small molecule, 2-AA, which is abundantly produced by P. aeruginosa, promotes persistent infections, dampens host inflammation, and triggers mitochondrial dysfunction in skeletal muscle. QS is a cell-to-cell communication system utilized by bacteria to promote collective behaviors. The significance of our study in identifying a mechanism that leads to skeletal muscle dysfunction, via the action of a QS molecule, is that it may open new avenues in the control of muscle loss as a result of infection and sepsis. Given that QS is a common characteristic of prokaryotes, it is possible that 2-AA-like molecules promoting similar effects may exist in other pathogens.

Effects of a small, volatile bacterial molecule on Pseudomonas aeruginosa bacteria using whole cell high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and genomics.

In the present study, high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy was applied to live Pseudomonas aeruginosa (PA) bacterial cells to determine the metabolome of this opportunistic Gram-negative human pathogen, and in particular, its response to the volatile aromatic low molecular weight signaling molecule, 2-aminoacetophenone (2-AA). Multi-dimensional HRMAS NMR is a promising method which may be used to determine the in vivo metabolome of live intact bacterial cells; 2-AA is produced by PA and triggers the emergence of phenotypes that promote chronic infection phenotypes in in vitro and in vivo (animal) models. In the present study, we applied one-dimensional and two-dimensional proton (1H) HRMAS NMR to PA cells which were grown with or without 2-AA in order to examine the associations between metabolites and cellular processes in response to 2-AA. We also compared whole-genome transcriptome profiles of PA cells grown with or without 2-AA and found that 2-AA promoted profound metabolic changes in the PA cells. By comparing the whole-genome transcriptome profiles and metabolomic analysis, we demonstrated that 2-AA profoundly reprogramed the gene expression and metabolic profiles of the cells. Our in vivo 1H HRMAS NMR spectroscopy may prove to be a helpful tool in the validation of gene functions, the study of pathogenic mechanisms, the classification of microbial strains into functional/clinical groups and the testing of anti-bacterial agents.

Revascularization and muscle adaptation to limb demand ischemia in diet-induced obese mice.

BACKGROUND: Obesity and type 2 diabetes are major risk factors for peripheral arterial disease in humans, which can result in lower limb demand ischemia and exercise intolerance. Exercise triggers skeletal muscle adaptation including increased vasculogenesis. The goal of this study was to determine whether demand ischemia modulates revascularization, fiber size, and signaling pathways in the ischemic hind limb muscles of mice with diet-induced obesity (DIO). MATERIALS AND METHODS: DIO mice (n = 7) underwent unilateral femoral artery ligation and recovered for 2 wks followed by 4 wks with daily treadmill exercise to induce demand ischemia. A parallel sedentary ischemia (SI) group (n = 7) had femoral artery ligation without exercise. The contralateral limb muscles of SI served as control. Muscles were examined for capillary density, myofiber cross-sectional area, cytokine levels, and phosphorylation of STAT3 and ERK1/2. RESULTS: Exercise significantly enhanced capillary density (P < 0.01) and markedly lowered cross-sectional area (P < 0.001) in demand ischemia compared with SI. These findings coincided with a significant increase in granulocyte colony-stimulating factor (P < 0.001) and interleukin-7 (P < 0.01) levels. In addition, phosphorylation levels of STAT3 and ERK1/2 (P < 0.01) were increased, whereas UCP1 and monocyte chemoattractant protein-1 protein levels were lower (P < 0.05) without altering vascular endothelial growth factor and tumor necrosis factor alpha protein levels. Demand ischemia increased the PGC1α messenger RNA (P < 0.001) without augmenting PGC1α protein levels. CONCLUSIONS: Exercise-induced limb demand ischemia in the setting of DIO causes myofiber atrophy despite an increase in muscle capillary density. The combination of persistent increase in tumor necrosis factor alpha, lower vascular endothelial growth factor, and failure to increase PGC1α protein may reflect a deficient adaption to demand ischemia in DIO.

Proton MRS imaging in pediatric brain tumors.

Magnetic resonance (MR) techniques offer a noninvasive, non-irradiating yet sensitive approach to diagnosing and monitoring pediatric brain tumors. Proton MR spectroscopy (MRS), as an adjunct to MRI, is being more widely applied to monitor the metabolic aspects of brain cancer. In vivo MRS biomarkers represent a promising advance and may influence treatment choice at both initial diagnosis and follow-up, given the inherent difficulties of sequential biopsies to monitor therapeutic response. When combined with anatomical or other types of imaging, MRS provides unique information regarding biochemistry in inoperable brain tumors and can complement neuropathological data, guide biopsies and enhance insight into therapeutic options. The combination of noninvasively acquired prognostic information and the high-resolution anatomical imaging provided by conventional MRI is expected to surpass molecular analysis and DNA microarray gene profiling, both of which, although promising, depend on invasive biopsy. This review focuses on recent data in the field of MRS in children with brain tumors.

Bacterial-excreted small volatile molecule 2-aminoacetophenone induces oxidative stress and apoptosis in murine skeletal muscle.

Oxidative stress induces mitochondrial dysfunction and facilitates apoptosis, tissue damage or metabolic alterations following infection. We have previously discovered that the Pseudomonas aeruginosa (PA) quorum sensing (QS)-excreted small volatile molecule, 2-aminoacetophenone (2-AA), which is produced in infected human tissue, promotes bacterial phenotypes that favor chronic infection, while also dampening the pathogen­induced innate immune response, thus compromising muscle function and promoting host tolerance to infection. In this study, murine whole-genome expression data have demonstrated that 2-AA affects the expression of genes involved in reactive oxygen species (ROS) homeostasis, thus producing an oxidative stress signature in skeletal muscle. The results of the present study demonstrated that the expression levels of genes involved in apoptosis signaling pathways were upregulated in the skeletal muscle of 2-AA-treated mice. To confirm the results of our transcriptome analysis, we used a novel high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) method and observed increased levels of bisallylic methylene fatty acyl protons and vinyl protons, suggesting that 2-AA induces skeletal muscle cell apoptosis. This effect was corroborated by our results demonstrating the downregulation of mitochondrial membrane potential in vivo in response to 2-AA. The findings of the present study indicate that the bacterial infochemical, 2-AA, disrupts mitochondrial functions by inducing oxidative stress and apoptosis signaling and likely promotes skeletal muscle dysfunction, which may favor chronic/persistent infection.

In vivo high-resolution magic angle spinning magnetic and electron paramagnetic resonance spectroscopic analysis of mitochondria-targeted peptide in Drosophila melanogaster with trauma-induced thoracic injury.

Trauma is the most common cause of mortality among individuals aged between 1 and 44 years and the third leading cause of mortality overall in the US. In this study, we examined the effects of trauma on the expression of genes in Drosophila melanogaster, a useful model for investigating genetics and physiology. After trauma was induced by a non-lethal needle puncture of the thorax, we observed the differential expression of genes encoding for mitochondrial uncoupling proteins, as well as those encoding for apoptosis-related and insulin signaling-related proteins, thus indicating muscle functional dysregulation. These results prompted us to examine the link between insulin signaling and mitochondrial dysfunction using in vivo nuclear magnetic resonance (NMR) with complementary electron paramagnetic resonance (EPR) spectroscopy. Trauma significantly increased insulin resistance biomarkers, and the NMR spectral profile of the aged flies with trauma-induced thoracic injury resembled that of insulin-resistant chico mutant flies. In addition, the mitochondrial redox status, as measured by EPR, was significantly altered following trauma, indicating mitochondrial uncoupling. A mitochondria-targeted compound, Szeto-Schiller (SS)-31 that promotes adenosine triphosphate (ATP) synthesis normalized the NMR spectral profile, as well as the mitochondrial redox status of the flies with trauma-induced thoracic injury, as assessed by EPR. Based on these findings, we propose a molecular mechanism responsible for trauma-related mortality and also propose that trauma sequelae in aging are linked to insulin signaling and mitochondrial dysfunction. Our findings further suggest that SS-31 attenuates trauma-associated pathological changes.

In vivo high-resolution magic angle spinning proton NMR spectroscopy of Drosophila melanogaster flies as a model system to investigate mitochondrial dysfunction in Drosophila GST2 mutants.

In vivo nuclear magnetic resonance spectroscopy (NMR), a non-destructive biochemical tool used for investigating live organisms, has recently been performed in studies of the fruit fly Drosophila melanogaster, a useful model organism for investigating genetics and physiology. We used a novel high-resolution magic angle-spinning (HRMAS) NMR method to investigate live Drosophila GST2 mutants using a conventional 14.1-T NMR spectrometer equipped with an HRMAS probe. The results showed that, compared to wild-type (wt) controls, the GST2 mutants had a 48% greater (CH(2))n lipid signal at 1.33 ppm, which is an insulin resistance biomarker in Drosophila skeletal muscle (P=0.0444). The mutants also had a 57% greater CH(2)C= lipid signal at 2.02 ppm (P=0.0276) and a 100% greater -CH=CH- signal at 5.33 ppm (P=0.0251). Since the -CH=CH- signal encompasses protons from ceramide, this latter difference is consistent with the hypothesis that the GST2 mutation is associated with insulin resistance and apoptosis. The findings of this study corroborate our previous results, support the hypothesis that the GST2 mutation is associated with insulin signaling and suggest that the IMCL level may be a biomarker of insulin resistance. Furthermore, direct links between GST2 mutation (the Drosophila ortholog of the GSTA4 gene in mammals) and insulin resistance, as suggested in this study, have not been made previously. These findings may thus be directly relevant to a wide range of metabolically disruptive conditions, such as trauma, aging and immune system deficiencies, that lead to increased susceptibility to infection.

A small volatile bacterial molecule triggers mitochondrial dysfunction in murine skeletal muscle.

Mitochondria integrate distinct signals that reflect specific threats to the host, including infection, tissue damage, and metabolic dysfunction; and play a key role in insulin resistance. We have found that the Pseudomonas aeruginosa quorum sensing infochemical, 2-amino acetophenone (2-AA), produced during acute and chronic infection in human tissues, including in the lungs of cystic fibrosis (CF) patients, acts as an interkingdom immunomodulatory signal that facilitates pathogen persistence, and host tolerance to infection. Transcriptome results have led to the hypothesis that 2-AA causes further harm to the host by triggering mitochondrial dysfunction in skeletal muscle. As normal skeletal muscle function is essential to survival, and is compromised in many chronic illnesses, including infections and CF-associated muscle wasting, we here determine the global effects of 2-AA on skeletal muscle using high-resolution magic-angle-spinning (HRMAS), proton ((1)H) nuclear magnetic resonance (NMR) metabolomics, in vivo (31)P NMR, whole-genome expression analysis and functional studies. Our results show that 2-AA when injected into mice, induced a biological signature of insulin resistance as determined by (1)H NMR analysis-, and dramatically altered insulin signaling, glucose transport, and mitochondrial function. Genes including Glut4, IRS1, PPAR-γ, PGC1 and Sirt1 were downregulated, whereas uncoupling protein UCP3 was up-regulated, in accordance with mitochondrial dysfunction. Although 2-AA did not alter high-energy phosphates or pH by in vivo (31)P NMR analysis, it significantly reduced the rate of ATP synthesis. This affect was corroborated by results demonstrating down-regulation of the expression of genes involved in energy production and muscle function, and was further validated by muscle function studies. Together, these results further demonstrate that 2-AA, acts as a mediator of interkingdom modulation, and likely effects insulin resistance associated with a molecular signature of mitochondrial dysfunction in skeletal muscle. Reduced energy production and mitochondrial dysfunctional may further favor infection, and be an important step in the establishment of chronic and persistent infections.

Skeletal muscle mitochondrial uncoupling in a murine cancer cachexia model.

Approximately half of all cancer patients present with cachexia, a condition in which disease-associated metabolic changes lead to a severe loss of skeletal muscle mass. Working toward an integrated and mechanistic view of cancer cachexia, we investigated the hypothesis that cancer promotes mitochondrial uncoupling in skeletal muscle. We subjected mice to in vivo phosphorous-31 nuclear magnetic resonance (31P NMR) spectroscopy and subjected murine skeletal muscle samples to gas chromatography/mass spectrometry (GC/MS). The mice used in both experiments were Lewis lung carcinoma models of cancer cachexia. A novel 'fragmented mass isotopomer' approach was used in our dynamic analysis of 13C mass isotopomer data. Our 31P NMR and GC/MS results indicated that the adenosine triphosphate (ATP) synthesis rate and tricarboxylic acid (TCA) cycle flux were reduced by 49% and 22%, respectively, in the cancer-bearing mice (p<0.008; t-test vs. controls). The ratio of ATP synthesis rate to the TCA cycle flux (an index of mitochondrial coupling) was reduced by 32% in the cancer-bearing mice (p=0.036; t-test vs. controls). Genomic analysis revealed aberrant expression levels for key regulatory genes and transmission electron microscopy (TEM) revealed ultrastructural abnormalities in the muscle fiber, consistent with the presence of abnormal, giant mitochondria. Taken together, these data suggest that mitochondrial uncoupling occurs in cancer cachexia and thus point to the mitochondria as a potential pharmaceutical target for the treatment of cachexia. These findings may prove relevant to elucidating the mechanisms underlying skeletal muscle wasting observed in other chronic diseases, as well as in aging.

Mitochondria-targeted antioxidant promotes recovery of skeletal muscle mitochondrial function after burn trauma assessed by in vivo 31P nuclear magnetic resonance and electron paramagnetic resonance spectroscopy.

Burn injury causes a major systemic catabolic response that is associated with mitochondrial dysfunction in skeletal muscle. We investigated the effects of the mitochondria-targeted peptide antioxidant Szeto-Schiller 31 (SS-31) on skeletal muscle in a mouse burn model using in vivo phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy to noninvasively measure high-energy phosphate levels; mitochondrial aconitase activity measurements that directly correlate with TCA cycle flux, as measured by gas chromatography mass spectrometry (GC-MS); and electron paramagnetic resonance (EPR) to assess oxidative stress. At 6 h postburn, the oxidative ATP synthesis rate was increased 5-fold in burned mice given a single dose of SS-31 relative to untreated burned mice (P=0.002). Furthermore, SS-31 administration in burned animals decreased mitochondrial aconitase activity back to control levels. EPR revealed a recovery in redox status of the SS-31-treated burn group compared to the untreated burn group (P<0.05). Our multidisciplinary convergent results suggest that SS-31 promotes recovery of mitochondrial function after burn injury by increasing ATP synthesis rate, improving mitochondrial redox status, and restoring mitochondrial coupling. These findings suggest use of noninvasive in vivo NMR and complementary EPR offers an approach to monitor the effectiveness of mitochondrial protective agents in alleviating burn injury symptoms.