shingo yasuhara - Academia.edu (original) (raw)

Papers by shingo yasuhara

Research paper thumbnail of Myostatin deficiency not only prevents muscle wasting but also improves survival in septic mice

American Journal of Physiology-Endocrinology and Metabolism, 2021

Muscle wasting is a major complication of sepsis, but its role in the disease development is not ... more Muscle wasting is a major complication of sepsis, but its role in the disease development is not known. Myostatin deficiency improved bacterial clearance and survival and mitigated damage in the liver and kidney in septic mice, which paralleled prevention of muscle wasting. These results raise the possibility that muscle wasting may not simply be a complication of sepsis, but myostatin-mediated cachexic changes may have a role in impaired bacterial clearance and mortality in septic mice.

Research paper thumbnail of After a Heart Attack, Who Should Care?*

Research paper thumbnail of 99 Feasibility of Deep Learning-based Automatic Myofiber Size Measurement for Burn-induced Muscle Wasting and Its Reversal

Journal of Burn Care & Research, Mar 23, 2022

Research paper thumbnail of 104 Parkin-independent Pathway of Mitophagy as a Potential Target Mitochondrial Dysfunctions in Burns

Journal of Burn Care & Research, 2022

Introduction In many critical illnesses including burn injury (BI), muscle wasting (MW) with mito... more Introduction In many critical illnesses including burn injury (BI), muscle wasting (MW) with mitochondrial dysfunction (MD) leads to poor prognosis. Disturbed mitochondria can normally be turned over by autophagic degradation of mitochondria (mitophagy). We have previously observed that BI causes disturbed mitophagy response in skeletal muscles both in vivo and in the cultured myocytes, a potential mechanism for BI-induced MD. These previous findings have lead to the expectation that augmenting mitophagy will rescue mitochondrial functions and can help treating the BI-induced MW and MD. There have been, however, limited research tools to specifically intervene (or augment) mitophagy. In the current study, we have established mitophagy-compromised cell strains by CRISPR/Cas9-mediated knocking out of BNIP3L, a pivotal molecule mediating parkin-independent mitophagy induction. Deferiprone (DFP), a recently established mitophagy inducer, had previously been known to exert cellular and o...

Research paper thumbnail of Running head: Impaired activation of Akt/PKB following burn injury Address for reprints and other correspondence

extracellular-regulated protein kinase: ERK glycogen synthase kinase-3β: GSK-3β insulin receptor:... more extracellular-regulated protein kinase: ERK glycogen synthase kinase-3β: GSK-3β insulin receptor: IR insulin receptor substrate: IRS mammalian target of rapamycin: mTOR sodium dodecyl sulfate-polyacrylamide gel electrophoresis: SDS-PAGE The molecular bases underlying burn- or critical illness-induced insulin resistance still remains unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin, and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We, therefore, examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared to sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous

Research paper thumbnail of 73 Establishment of New Cellular Model Supportive of Burn-Induced Perturbation in Mitophagy Response

Journal of Burn Care & Research, 2019

Research paper thumbnail of 79 Burn Injury Alters Pulmonary Neutrophil Extracellular Trap (NET) Formation

Journal of Burn Care & Research, 2019

Research paper thumbnail of Combined off-resonance imaging and T2 relaxation in the rotating frame for positive contrast MR imaging of infection in a murine burn model

Journal of Magnetic Resonance Imaging, 2010

To develop novel magnetic resonance (MR) imaging methods to monitor accumulation of macrophages i... more To develop novel magnetic resonance (MR) imaging methods to monitor accumulation of macrophages in inflammation and infection. Positive-contrast MR imaging provides an alternative to negative-contrast MRI, exploiting the chemical shift induced by ultra-small superparamagnetic iron-oxide (USPIO) nanoparticles to nearby water molecules. We introduce a novel combination of off-resonance (ORI) positive-contrast MRI and T(2ρ) relaxation in the rotating frame (ORI-T(2ρ)) for positive-contrast MR imaging of USPIO. We tested ORI-T(2ρ) in phantoms and imaged in vivo the accumulation of USPIO-labeled macrophages at the infection site in a mouse model of burn trauma and infection with Pseudomonas aeruginosa (PA). PA infection is clinically important. The USPIO nanoparticles were injected directly in the animals in solution, and macrophage labeling occurred in vivo in the animal model. We observed a significant difference between ORI-T(2ρ) and ORI, which leads us to suggest that ORI-T(2ρ) is more sensitive in detecting USPIO signal. To this end, the ORI-T(2ρ) positive contrast method may prove to be of higher utility in future research. Our results may have direct implications in the longitudinal monitoring of infection, and open perspectives for testing novel anti-infective compounds.

Research paper thumbnail of Burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle

American Journal of Physiology-Endocrinology and Metabolism, 2004

The molecular bases underlying burn- or critical illness-induced insulin resistance still remain ... more The molecular bases underlying burn- or critical illness-induced insulin resistance still remain unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We therefore examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared with sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous substrate of Akt/PKB, glycogen synthase kinase-3β (GSK-3β), was significantly impaired in burned rats. Furthermore, insulin consistently failed to increase the phosphorylation of p70 S6 kinase, another downstream effector of Akt/PKB, in rats with burn injury, whereas phosphorylation of p70 S6 kinase was increased by in...

Research paper thumbnail of Freshly Isolated Mitochondria as Therapeutic Agents in Sepsis: Can They Go Home Again?

Research paper thumbnail of 44 Defects in Mitophagy Response in Burns Involved in Poor Regeneration Capacity

Journal of Burn Care & Research

Introduction Wound healing is a biological process directed to the restoration of tissue that has... more Introduction Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. One of the important functions of wound healing is individual and collective cellular migration. This migration response can largely dictate the outcomes and success of a healing wound. Mitophagy, autophagic degradation of mitochondria, sequesters damaged mitochondria and functions as the essential quality control (QC) system of this organelle. When mitophagy is disturbed, damaged mitochondria are not cleared and will produce superoxide, imposing a risk for cell death. Our previous study documented a compromised mitophagy response in burns, suggestive of poor QC of mitochondria in the severely burned subjects. The relationship between defects in mitophagy response and cellular regeneration capacity was not investigated in detail. We have previously shown that trehalose can serve as an important autophagy modulator and ameliorates the defect in mitophay response. Thus...

Research paper thumbnail of 225 Pharmacological Ameioration of Perturbed Mitophagy Response (Mitophagy Resistance) in Burns

Journal of Burn Care & Research

Research paper thumbnail of 226 Motor Neuronal Apoptosis and Neuromuscular Atrophy are Associated with Transmigration of Monocyte-derived Macrophages after Burn Injury

Journal of Burn Care & Research

Research paper thumbnail of 223 Potential Association of Burn-Induced NMJ Derrangement and Oxidative Stress-Induced AChR Declustering Signal and the Reversal with AChR alpha7 Agonist

Journal of Burn Care & Research

Research paper thumbnail of Coenzyme Q10 protects against burn‐induced mitochondrial dysfunction and impaired insulin signaling in mouse skeletal muscle

FEBS Open Bio

Mitochondrial dysfunction is associated with metabolic alterations in various disease states, inc... more Mitochondrial dysfunction is associated with metabolic alterations in various disease states, including major trauma (e.g., burn injury). Metabolic derangements, including muscle insulin resistance and hyperlactatemia, are a clinically significant complication of major trauma. Coenzyme Q10 (CoQ10) is an essential cofactor for mitochondrial electron transport, and its reduced form acts as a lipophilic antioxidant. Here, we report that burn injury induces impaired muscle insulin signaling, hyperlactatemia, mitochondrial dysfunction (as indicated by suppressed mitochondrial oxygen consumption rates), morphological alterations of the mitochondria (e. g., enlargement, and loss of cristae structure), mitochondrial oxidative stress, and disruption of mitochondrial integrity (as reflected by increased mitochondrial DNA levels in the cytosol and circulation). All of these alterations were significantly alleviated by CoQ10 treatment compared with vehicle alone. These findings indicate that CoQ10 treatment is efficacious in protecting against mitochondrial dysfunction and insulin resistance in skeletal muscle of burned mice. Our data highlight CoQ10 as a potential new strategy to prevent mitochondrial damage and metabolic dysfunction in burn patients.

Research paper thumbnail of Ex vivo high resolution magic angle spinning MR spectroscopy suggests a lipid-mediated apoptosis in burn injury

ABSTRACT Introduction Recent studies following burn injury have indicated apoptotic changes, toge... more ABSTRACT Introduction Recent studies following burn injury have indicated apoptotic changes, together with increased expression of proapoptotic proteins in skeletal muscles at sites immediately proximal and distant from burn injury (1,2). Apoptosis or programmed cell death is a fundamental homeostatic mechanism but also a primary factor in disease i.e., ischemia, neurodegenerative diseases, inflammatory diseases, and cancer. A variety of MR imaging approaches may be employed for the imaging of apoptosis, particularly lipid proton MR spectroscopy, diffusion-weighted MR imaging and contrast-enhanced MR imaging with magnetically labeled anexin (3, 4). We have investigated that High-Resolution Magic Angle Spinning (HRMAS) MR spectroscopy of intact tissue samples of distal muscle after burn injury enables detailed and unequivocal assignments of lipids of biological importance in apoptotic mechanisms such as polyunsaturated fatty acids and possibly sphingolipids. Materials and Methods Muscle samples collected from burned and sham-burned rabbits previously infused with L-[U-13 C]-Lactate (40 mg . kg -1. h -1) were measured using HRMAS NMR spectroscopy in a Bruker 14.1 Tesla spectrometer at 4 o C. The Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence with a total spin–spin relaxation delay of 80 ms was used to measure spin-echo 1 H MAS NMR spectra on all samples. Typically, 256 transients were collected into 32 K data points. For assignment purposes, 2D 1 H-1 H total correlation (TOCSY), J-resolved (JRES) and 1 H-13 C heteronuclear single quantum coherence (HSQC) NMR spectra were measured, all under MAS conditions. Furthermore, terminal deoxyribonucleotidyl transferase nick end labeling (TUNEL) assay of the NMR samples performed in order to confirm apoptosis. Nuclei were stained blue with 49,6-diamidino-2-phenylindole, and apoptotic nuclei were stained green with fluoresceine-conjugated antibody. Results Our NMR spectra showed differences between burn and controls at 0.9 ppm (peak 1), exhibiting lipids, which appear to increase in burns and this observed increase has been implicated in apoptosis. Additionally, apparent are the differences in the area between 3.0 and 3.4 ppm. Also, the increase in peaks corresponding to choline-containing compounds (peak 8) probably indicate altered phospholipid metabolism in burns. Our preliminary experiments have also suggested changes in sphingolipid pathways. Proton MR Spectra: High-Resolution Magic Angle Spinning (HRMAS of Burned (Upper) and Sham-Burned (Control, Lower) Muscle. Assigned peaks include 1,3,4,5,6,12: lipids; 2: lactate and lipids; 7: methyl protons of creatine and phosphocreatine; 8: trimethylamonium groups of cholines and carnitine; 9: taurine; 10: orientation dependent methylene protons of creatine and phosphocreatine; and 11: residual water. A and B: In situ TUNEL confirms appoptosis. Normal nuclei stain blue and apoptotic nuclei are green. Red staining (anticaveolin 3) is specific for muscle structure. Apoptotic nuclei (arrow) were shown to be within muscle cells and not due to infiltrating cells from outside. Discussion NMR visible lipids have been associated with programmed cell death (apoptosis). Here we report for the first time differences in the lipid profiles of muscles from control and burned rabbits by ex vivo High Resolution Magic Angle Spinning (HRMAS) NMR Spectroscopy with unprecedented resolution. Also the lipid profile by HRMAS was linked to the presence of apoptosis in burn injury. We suggest that a NMR visible lipid-mediated apoptosis in burns results in muscle wasting. This has led us to the concept that suppression of certain lipids, such as sphingolipids may represent a candidate strategy for a partial relief of muscle wasting (or cachexia) in burns and that mitochondria are the target in lipid-mediated apoptosis.

Research paper thumbnail of Apoptosis May Be a Mechanism for Loss of Muscle Mass during Immobilization

Research paper thumbnail of iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3β activation in skeletal muscle of rats

Metabolism, 2013

Recent studies suggest that activation of glycogen synthase kinase (GSK)-3β may be involved in bu... more Recent studies suggest that activation of glycogen synthase kinase (GSK)-3β may be involved in burn injury-induced metabolic derangements and protein breakdown in skeletal muscle. However, the mechanism for GSK-3β activation after burn injury is unknown. To investigate the role of inducible nitric oxide synthase (iNOS) in this scenario, a major mediator of inflammation, we examined the effects of a specific inhibitor for iNOS, L-NIL, on GSK-3β activity in skeletal muscle of burned rats. Full-thickness third degree burn injury comprising 40% of total body surface area was produced under anesthesia in male Sprague-Dawley rats (160-190g) by immersing the back of the trunk for 15s and the abdomen for 8s in 80°C water. Burned and sham-burned rats were treated with L-NIL (60mg/kg BW, b.i.d., IP) or phosphate-buffered saline for three days. GSK-3β activity in skeletal muscle was evaluated by immune complex kinase assay, and by phosphorylation status of GSK-3β and its endogenous substrate, glycogen synthase. GSK-3β activity was increased in a time-dependent manner in skeletal muscle after burn injury, concomitant with the induction of iNOS expression. iNOS inhibitor, L-NIL, reverted the elevated GSK-3β activity in skeletal muscle of burned rats, although L-NIL did not alter GSK-3β activity in sham-burned rats. Our results clearly indicate that iNOS plays an important role in burn injury-induced GSK-3β activation in skeletal muscle. These findings suggest that iNOS may contribute to burn injury-induced metabolic derangements, in part, by activating GSK-3β.

Research paper thumbnail of Title efficacy of phosphodiesterase 5 inhibitor on distant burn-induced muscle autophagy, microcirculation, and survival rate

AJP: Endocrinology and Metabolism, 2013

Skeletal muscle wasting is an exacerbating factor in the prognosis of critically ill patients. Us... more Skeletal muscle wasting is an exacerbating factor in the prognosis of critically ill patients. Using a systemic burn injury model in mice, we have established a role of autophagy in the resulting muscle wasting that is distant from the burn trauma. We provide evidence that burn injury increases the autophagy turnover in the distal skeletal muscle by conventional postmortem tissue analyses and by a novel in vivo microscopic method using an autophagy reporter gene (tandem fluorescent LC3). The effect of tadalafil, a phosphodiesterase 5 inhibitor (PDE5I), on burn-induced skeletal muscle autophagy is documented and extends our published results that PDE5Is attenuates muscle degeneration in a muscular dystrophy model. We also designed a translational experiment to examine the impact of PDE5I on whole body and demonstrated that PDE5I administration lessened muscle atrophy, mitigated microcirculatory disturbance, and improved the survival rate after burn injury.

Research paper thumbnail of Microtubule Defect Involved in ‘Mitophagy Resistance’ Under Subacute Oxidative Stress - Potential Mechanism for Cellular Inflammation

IntroductionOxidative stress is considered an essential mechanism in ICU-acquired weakness. The r... more IntroductionOxidative stress is considered an essential mechanism in ICU-acquired weakness. The roles of oxidative stress in autophagy/mitophagy dysfunction remains elusive. Microtubule serves as an essential guide rail for auto/mitophagosome trafficking required for proper maturation of auto/mitophagosomes in normal circumstances, and microtubules network formation is regulated by signal transduction mechanisms involving Akt, GSK3β, and the microtubule plus-end tracking molecule, EB1. We have investigated (1) whether oxidative stress affects this pathway, leading to the defective mitophagy response, and (2) whether trehalose, an auto/mitophagy modulator, can ameliorate these pathological conditions.MethodsBy stably transfecting markers for auto/mitophagy or MT synthesis, we have established a few new C2C12 myocyte cell lines, expressing, GFP-LC3, EB1-GFP, and/or tandem-fluorescence LC3 (tfLC3). To monitor microtubule network, the cells were stained by SiR-tubulin. The cells were cu...

Research paper thumbnail of Myostatin deficiency not only prevents muscle wasting but also improves survival in septic mice

American Journal of Physiology-Endocrinology and Metabolism, 2021

Muscle wasting is a major complication of sepsis, but its role in the disease development is not ... more Muscle wasting is a major complication of sepsis, but its role in the disease development is not known. Myostatin deficiency improved bacterial clearance and survival and mitigated damage in the liver and kidney in septic mice, which paralleled prevention of muscle wasting. These results raise the possibility that muscle wasting may not simply be a complication of sepsis, but myostatin-mediated cachexic changes may have a role in impaired bacterial clearance and mortality in septic mice.

Research paper thumbnail of After a Heart Attack, Who Should Care?*

Research paper thumbnail of 99 Feasibility of Deep Learning-based Automatic Myofiber Size Measurement for Burn-induced Muscle Wasting and Its Reversal

Journal of Burn Care & Research, Mar 23, 2022

Research paper thumbnail of 104 Parkin-independent Pathway of Mitophagy as a Potential Target Mitochondrial Dysfunctions in Burns

Journal of Burn Care & Research, 2022

Introduction In many critical illnesses including burn injury (BI), muscle wasting (MW) with mito... more Introduction In many critical illnesses including burn injury (BI), muscle wasting (MW) with mitochondrial dysfunction (MD) leads to poor prognosis. Disturbed mitochondria can normally be turned over by autophagic degradation of mitochondria (mitophagy). We have previously observed that BI causes disturbed mitophagy response in skeletal muscles both in vivo and in the cultured myocytes, a potential mechanism for BI-induced MD. These previous findings have lead to the expectation that augmenting mitophagy will rescue mitochondrial functions and can help treating the BI-induced MW and MD. There have been, however, limited research tools to specifically intervene (or augment) mitophagy. In the current study, we have established mitophagy-compromised cell strains by CRISPR/Cas9-mediated knocking out of BNIP3L, a pivotal molecule mediating parkin-independent mitophagy induction. Deferiprone (DFP), a recently established mitophagy inducer, had previously been known to exert cellular and o...

Research paper thumbnail of Running head: Impaired activation of Akt/PKB following burn injury Address for reprints and other correspondence

extracellular-regulated protein kinase: ERK glycogen synthase kinase-3β: GSK-3β insulin receptor:... more extracellular-regulated protein kinase: ERK glycogen synthase kinase-3β: GSK-3β insulin receptor: IR insulin receptor substrate: IRS mammalian target of rapamycin: mTOR sodium dodecyl sulfate-polyacrylamide gel electrophoresis: SDS-PAGE The molecular bases underlying burn- or critical illness-induced insulin resistance still remains unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin, and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We, therefore, examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared to sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous

Research paper thumbnail of 73 Establishment of New Cellular Model Supportive of Burn-Induced Perturbation in Mitophagy Response

Journal of Burn Care & Research, 2019

Research paper thumbnail of 79 Burn Injury Alters Pulmonary Neutrophil Extracellular Trap (NET) Formation

Journal of Burn Care & Research, 2019

Research paper thumbnail of Combined off-resonance imaging and T2 relaxation in the rotating frame for positive contrast MR imaging of infection in a murine burn model

Journal of Magnetic Resonance Imaging, 2010

To develop novel magnetic resonance (MR) imaging methods to monitor accumulation of macrophages i... more To develop novel magnetic resonance (MR) imaging methods to monitor accumulation of macrophages in inflammation and infection. Positive-contrast MR imaging provides an alternative to negative-contrast MRI, exploiting the chemical shift induced by ultra-small superparamagnetic iron-oxide (USPIO) nanoparticles to nearby water molecules. We introduce a novel combination of off-resonance (ORI) positive-contrast MRI and T(2ρ) relaxation in the rotating frame (ORI-T(2ρ)) for positive-contrast MR imaging of USPIO. We tested ORI-T(2ρ) in phantoms and imaged in vivo the accumulation of USPIO-labeled macrophages at the infection site in a mouse model of burn trauma and infection with Pseudomonas aeruginosa (PA). PA infection is clinically important. The USPIO nanoparticles were injected directly in the animals in solution, and macrophage labeling occurred in vivo in the animal model. We observed a significant difference between ORI-T(2ρ) and ORI, which leads us to suggest that ORI-T(2ρ) is more sensitive in detecting USPIO signal. To this end, the ORI-T(2ρ) positive contrast method may prove to be of higher utility in future research. Our results may have direct implications in the longitudinal monitoring of infection, and open perspectives for testing novel anti-infective compounds.

Research paper thumbnail of Burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle

American Journal of Physiology-Endocrinology and Metabolism, 2004

The molecular bases underlying burn- or critical illness-induced insulin resistance still remain ... more The molecular bases underlying burn- or critical illness-induced insulin resistance still remain unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We therefore examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared with sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous substrate of Akt/PKB, glycogen synthase kinase-3β (GSK-3β), was significantly impaired in burned rats. Furthermore, insulin consistently failed to increase the phosphorylation of p70 S6 kinase, another downstream effector of Akt/PKB, in rats with burn injury, whereas phosphorylation of p70 S6 kinase was increased by in...

Research paper thumbnail of Freshly Isolated Mitochondria as Therapeutic Agents in Sepsis: Can They Go Home Again?

Research paper thumbnail of 44 Defects in Mitophagy Response in Burns Involved in Poor Regeneration Capacity

Journal of Burn Care & Research

Introduction Wound healing is a biological process directed to the restoration of tissue that has... more Introduction Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. One of the important functions of wound healing is individual and collective cellular migration. This migration response can largely dictate the outcomes and success of a healing wound. Mitophagy, autophagic degradation of mitochondria, sequesters damaged mitochondria and functions as the essential quality control (QC) system of this organelle. When mitophagy is disturbed, damaged mitochondria are not cleared and will produce superoxide, imposing a risk for cell death. Our previous study documented a compromised mitophagy response in burns, suggestive of poor QC of mitochondria in the severely burned subjects. The relationship between defects in mitophagy response and cellular regeneration capacity was not investigated in detail. We have previously shown that trehalose can serve as an important autophagy modulator and ameliorates the defect in mitophay response. Thus...

Research paper thumbnail of 225 Pharmacological Ameioration of Perturbed Mitophagy Response (Mitophagy Resistance) in Burns

Journal of Burn Care & Research

Research paper thumbnail of 226 Motor Neuronal Apoptosis and Neuromuscular Atrophy are Associated with Transmigration of Monocyte-derived Macrophages after Burn Injury

Journal of Burn Care & Research

Research paper thumbnail of 223 Potential Association of Burn-Induced NMJ Derrangement and Oxidative Stress-Induced AChR Declustering Signal and the Reversal with AChR alpha7 Agonist

Journal of Burn Care & Research

Research paper thumbnail of Coenzyme Q10 protects against burn‐induced mitochondrial dysfunction and impaired insulin signaling in mouse skeletal muscle

FEBS Open Bio

Mitochondrial dysfunction is associated with metabolic alterations in various disease states, inc... more Mitochondrial dysfunction is associated with metabolic alterations in various disease states, including major trauma (e.g., burn injury). Metabolic derangements, including muscle insulin resistance and hyperlactatemia, are a clinically significant complication of major trauma. Coenzyme Q10 (CoQ10) is an essential cofactor for mitochondrial electron transport, and its reduced form acts as a lipophilic antioxidant. Here, we report that burn injury induces impaired muscle insulin signaling, hyperlactatemia, mitochondrial dysfunction (as indicated by suppressed mitochondrial oxygen consumption rates), morphological alterations of the mitochondria (e. g., enlargement, and loss of cristae structure), mitochondrial oxidative stress, and disruption of mitochondrial integrity (as reflected by increased mitochondrial DNA levels in the cytosol and circulation). All of these alterations were significantly alleviated by CoQ10 treatment compared with vehicle alone. These findings indicate that CoQ10 treatment is efficacious in protecting against mitochondrial dysfunction and insulin resistance in skeletal muscle of burned mice. Our data highlight CoQ10 as a potential new strategy to prevent mitochondrial damage and metabolic dysfunction in burn patients.

Research paper thumbnail of Ex vivo high resolution magic angle spinning MR spectroscopy suggests a lipid-mediated apoptosis in burn injury

ABSTRACT Introduction Recent studies following burn injury have indicated apoptotic changes, toge... more ABSTRACT Introduction Recent studies following burn injury have indicated apoptotic changes, together with increased expression of proapoptotic proteins in skeletal muscles at sites immediately proximal and distant from burn injury (1,2). Apoptosis or programmed cell death is a fundamental homeostatic mechanism but also a primary factor in disease i.e., ischemia, neurodegenerative diseases, inflammatory diseases, and cancer. A variety of MR imaging approaches may be employed for the imaging of apoptosis, particularly lipid proton MR spectroscopy, diffusion-weighted MR imaging and contrast-enhanced MR imaging with magnetically labeled anexin (3, 4). We have investigated that High-Resolution Magic Angle Spinning (HRMAS) MR spectroscopy of intact tissue samples of distal muscle after burn injury enables detailed and unequivocal assignments of lipids of biological importance in apoptotic mechanisms such as polyunsaturated fatty acids and possibly sphingolipids. Materials and Methods Muscle samples collected from burned and sham-burned rabbits previously infused with L-[U-13 C]-Lactate (40 mg . kg -1. h -1) were measured using HRMAS NMR spectroscopy in a Bruker 14.1 Tesla spectrometer at 4 o C. The Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence with a total spin–spin relaxation delay of 80 ms was used to measure spin-echo 1 H MAS NMR spectra on all samples. Typically, 256 transients were collected into 32 K data points. For assignment purposes, 2D 1 H-1 H total correlation (TOCSY), J-resolved (JRES) and 1 H-13 C heteronuclear single quantum coherence (HSQC) NMR spectra were measured, all under MAS conditions. Furthermore, terminal deoxyribonucleotidyl transferase nick end labeling (TUNEL) assay of the NMR samples performed in order to confirm apoptosis. Nuclei were stained blue with 49,6-diamidino-2-phenylindole, and apoptotic nuclei were stained green with fluoresceine-conjugated antibody. Results Our NMR spectra showed differences between burn and controls at 0.9 ppm (peak 1), exhibiting lipids, which appear to increase in burns and this observed increase has been implicated in apoptosis. Additionally, apparent are the differences in the area between 3.0 and 3.4 ppm. Also, the increase in peaks corresponding to choline-containing compounds (peak 8) probably indicate altered phospholipid metabolism in burns. Our preliminary experiments have also suggested changes in sphingolipid pathways. Proton MR Spectra: High-Resolution Magic Angle Spinning (HRMAS of Burned (Upper) and Sham-Burned (Control, Lower) Muscle. Assigned peaks include 1,3,4,5,6,12: lipids; 2: lactate and lipids; 7: methyl protons of creatine and phosphocreatine; 8: trimethylamonium groups of cholines and carnitine; 9: taurine; 10: orientation dependent methylene protons of creatine and phosphocreatine; and 11: residual water. A and B: In situ TUNEL confirms appoptosis. Normal nuclei stain blue and apoptotic nuclei are green. Red staining (anticaveolin 3) is specific for muscle structure. Apoptotic nuclei (arrow) were shown to be within muscle cells and not due to infiltrating cells from outside. Discussion NMR visible lipids have been associated with programmed cell death (apoptosis). Here we report for the first time differences in the lipid profiles of muscles from control and burned rabbits by ex vivo High Resolution Magic Angle Spinning (HRMAS) NMR Spectroscopy with unprecedented resolution. Also the lipid profile by HRMAS was linked to the presence of apoptosis in burn injury. We suggest that a NMR visible lipid-mediated apoptosis in burns results in muscle wasting. This has led us to the concept that suppression of certain lipids, such as sphingolipids may represent a candidate strategy for a partial relief of muscle wasting (or cachexia) in burns and that mitochondria are the target in lipid-mediated apoptosis.

Research paper thumbnail of Apoptosis May Be a Mechanism for Loss of Muscle Mass during Immobilization

Research paper thumbnail of iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3β activation in skeletal muscle of rats

Metabolism, 2013

Recent studies suggest that activation of glycogen synthase kinase (GSK)-3β may be involved in bu... more Recent studies suggest that activation of glycogen synthase kinase (GSK)-3β may be involved in burn injury-induced metabolic derangements and protein breakdown in skeletal muscle. However, the mechanism for GSK-3β activation after burn injury is unknown. To investigate the role of inducible nitric oxide synthase (iNOS) in this scenario, a major mediator of inflammation, we examined the effects of a specific inhibitor for iNOS, L-NIL, on GSK-3β activity in skeletal muscle of burned rats. Full-thickness third degree burn injury comprising 40% of total body surface area was produced under anesthesia in male Sprague-Dawley rats (160-190g) by immersing the back of the trunk for 15s and the abdomen for 8s in 80°C water. Burned and sham-burned rats were treated with L-NIL (60mg/kg BW, b.i.d., IP) or phosphate-buffered saline for three days. GSK-3β activity in skeletal muscle was evaluated by immune complex kinase assay, and by phosphorylation status of GSK-3β and its endogenous substrate, glycogen synthase. GSK-3β activity was increased in a time-dependent manner in skeletal muscle after burn injury, concomitant with the induction of iNOS expression. iNOS inhibitor, L-NIL, reverted the elevated GSK-3β activity in skeletal muscle of burned rats, although L-NIL did not alter GSK-3β activity in sham-burned rats. Our results clearly indicate that iNOS plays an important role in burn injury-induced GSK-3β activation in skeletal muscle. These findings suggest that iNOS may contribute to burn injury-induced metabolic derangements, in part, by activating GSK-3β.

Research paper thumbnail of Title efficacy of phosphodiesterase 5 inhibitor on distant burn-induced muscle autophagy, microcirculation, and survival rate

AJP: Endocrinology and Metabolism, 2013

Skeletal muscle wasting is an exacerbating factor in the prognosis of critically ill patients. Us... more Skeletal muscle wasting is an exacerbating factor in the prognosis of critically ill patients. Using a systemic burn injury model in mice, we have established a role of autophagy in the resulting muscle wasting that is distant from the burn trauma. We provide evidence that burn injury increases the autophagy turnover in the distal skeletal muscle by conventional postmortem tissue analyses and by a novel in vivo microscopic method using an autophagy reporter gene (tandem fluorescent LC3). The effect of tadalafil, a phosphodiesterase 5 inhibitor (PDE5I), on burn-induced skeletal muscle autophagy is documented and extends our published results that PDE5Is attenuates muscle degeneration in a muscular dystrophy model. We also designed a translational experiment to examine the impact of PDE5I on whole body and demonstrated that PDE5I administration lessened muscle atrophy, mitigated microcirculatory disturbance, and improved the survival rate after burn injury.

Research paper thumbnail of Microtubule Defect Involved in ‘Mitophagy Resistance’ Under Subacute Oxidative Stress - Potential Mechanism for Cellular Inflammation

IntroductionOxidative stress is considered an essential mechanism in ICU-acquired weakness. The r... more IntroductionOxidative stress is considered an essential mechanism in ICU-acquired weakness. The roles of oxidative stress in autophagy/mitophagy dysfunction remains elusive. Microtubule serves as an essential guide rail for auto/mitophagosome trafficking required for proper maturation of auto/mitophagosomes in normal circumstances, and microtubules network formation is regulated by signal transduction mechanisms involving Akt, GSK3β, and the microtubule plus-end tracking molecule, EB1. We have investigated (1) whether oxidative stress affects this pathway, leading to the defective mitophagy response, and (2) whether trehalose, an auto/mitophagy modulator, can ameliorate these pathological conditions.MethodsBy stably transfecting markers for auto/mitophagy or MT synthesis, we have established a few new C2C12 myocyte cell lines, expressing, GFP-LC3, EB1-GFP, and/or tandem-fluorescence LC3 (tfLC3). To monitor microtubule network, the cells were stained by SiR-tubulin. The cells were cu...