Functionalizing biomaterials to promote neurovascular regeneration following skeletal muscle injury.

During embryogenesis, blood vessels and nerves develop with similar branching structure in response to shared signaling pathways guiding network growth. With both systems integral to physiological homeostasis, dual targeting of blood vessels and nerves to promote neurovascular regeneration following injury is an emerging therapeutic approach in biomedical engineering. A limitation to this strategy is that the nature of cross talk between emergent vessels and nerves during regeneration in an adult is poorly understood. Following peripheral nerve transection, intraneural vascular cells infiltrate the site of injury to provide a migratory pathway for mobilized Schwann cells of regenerating axons. As Schwann cells demyelinate, they secrete vascular endothelial growth factor, which promotes angiogenesis. Recent advances point to concomitant restoration of neurovascular architecture and function through simultaneous targeting of growth factors and guidance cues shared by both systems during regeneration. In the context of traumatic injury associated with volumetric muscle loss, we consider the nature of biomaterials used to engineer three-dimensional scaffolds, functionalization of scaffolds with molecular signals that guide and promote neurovascular growth, and seeding scaffolds with progenitor cells. Physiological success is defined by each tissue component of the bioconstruct (nerve, vessel, muscle) becoming integrated with that of the host. Advances in microfabrication, cell culture techniques, and progenitor cell biology hold great promise for engineering bioconstructs able to restore organ function after volumetric muscle loss.

Hyperbaric Oxygen Treatment Following Mid-Cervical Spinal Cord Injury Preserves Diaphragm Muscle Function.

Oxidative damage to the diaphragm as a result of cervical spinal cord injury (SCI) promotes muscle atrophy and weakness. Respiratory insufficiency is the leading cause of morbidity and mortality in cervical spinal cord injury (SCI) patients, emphasizing the need for strategies to maintain diaphragm function. Hyperbaric oxygen (HBO) increases the amount of oxygen dissolved into the blood, elevating the delivery of oxygen to skeletal muscle and reactive oxygen species (ROS) generation. It is proposed that enhanced ROS production due to HBO treatment stimulates adaptations to diaphragm oxidative capacity, resulting in overall reductions in oxidative stress and inflammation. Therefore, we tested the hypothesis that exposure to HBO therapy acutely following SCI would reduce oxidative damage to the diaphragm muscle, preserving muscle fiber size and contractility. Our results demonstrated that lateral contusion injury at C3/4 results in a significant reduction in diaphragm muscle-specific force production and fiber cross-sectional area, which was associated with augmented mitochondrial hydrogen peroxide emission and a reduced mitochondrial respiratory control ratio. In contrast, rats that underwent SCI followed by HBO exposure consisting of 1 h of 100% oxygen at 3 atmospheres absolute (ATA) delivered for 10 consecutive days demonstrated an improvement in diaphragm-specific force production, and an attenuation of fiber atrophy, mitochondrial dysfunction and ROS production. These beneficial adaptations in the diaphragm were related to HBO-induced increases in antioxidant capacity and a reduction in atrogene expression. These findings suggest that HBO therapy may be an effective adjunctive therapy to promote respiratory health following cervical SCI.

The Renin-Angiotensin System and Skeletal Muscle.

The renin-angiotensin system (RAS) plays a key role in the control of blood pressure and fluid homeostasis. Emerging evidence also reveals that hyperactivity of the RAS contributes to skeletal muscle wasting. This review discusses the key role that the RAS plays in skeletal muscle wasting due to congestive heart failure, chronic kidney disease, and ventilator-induced diaphragmatic wasting.

Effects of different doses of caffeine on anaerobic exercise in boys.

This study investigated effects of low (1 mg·kg-1), moderate (3 mg·kg-1) and high (5 mg·kg-1) doses of caffeine on anaerobic performance in boys. Twenty-six 8- to 10-year-old boys participated in a double-blind, crossover, counter-balanced study. Boys received in random order a placebo (PL) or anhydrous caffeine: 1 (CAF-1), 3 (CAF-3), or 5 (CAF-5) mg caffeine·kg-1 body mass in cherry flavored Sprite. Sixty minutes following consumption boys performed a static handgrip test and then a 30-s Wingate test. Maximal grip strength (21.5 ± 4.9 & 21.6 ± 4.7 vs. 20.4 ± 4.0 kg) was significantly higher in CAF-5 & CAF-3 vs PL, respectively. Absolute and relative peak power (287 ± 72 vs 281 ± 69 W & 8.0 ± 0.9 vs 7.8 ± 1.0 W·kg-1) were significantly higher in CAF-3 vs PL, respectively. Mean power (153 ± 48 vs 146 ± 43 W) was significantly higher in CAF-5 vs PL, respectively. Peak Wingate HR was significantly higher (189 ± 8 vs 185 ± 9 beats·min-1) in CAF-5 vs PL, respectively. These findings suggest that in boys CAF-1 did not affect performance. During the Wingate test CAF-3 resulted in higher peak power while CAF-5 increased mean power. The significant increase in peak HR following the Wingate test is likely related to greater mean power generated during CAF-5.

The autophagy inhibitor NSC185058 suppresses mTORC1-mediated protein anabolism in cultured skeletal muscle.

The mammalian target of rapamycin (mTOR), and specifically the mTOR complex 1 (mTORC1) is the central regulator of anabolism in skeletal muscle. Among the many functions of this kinase complex is the inhibition of the catabolic process of autophagy; however, less work has been done in investigating the role of autophagy in regulating mTORC1 signaling. Using an in vitro model to better understand the pathways involved, we activated mTORC1 by several different means (growth factors, leucine supplementation, or muscle contraction), alone or with the autophagy inhibitor NSC185058. We found that inhibiting autophagy with NSC185058 suppresses mTORC1 activity, preventing any increase in cellular protein anabolism. These decrements were the direct result of action on the mTORC1 kinase, which we demonstrate, for the first time, cannot function when autophagy is inhibited by NSC185058. Our results indicate that, far from being a matter of unidirectional action, the relationship between mTORC1 and the autophagic cascade is more nuanced, with autophagy serving as an mTORC1 input, and mTORC1 inhibition of autophagy as a form of homeostatic feedback to regulate anabolic signaling. Future studies of cellular metabolism will have to consider this fundamental intertwining of protein anabolism and catabolism, and how it ultimately serves to regulate muscle proteostasis.

Angiogenesis precedes myogenesis during regeneration following biopsy injury of skeletal muscle.

BACKGROUND: Acute injury to skeletal muscle damages myofibers and fragment capillaries, impairing contractile function and local perfusion. Myofibers and microvessels regenerate from satellite cells and from surviving microvessel fragments, respectively, to restore intact muscle. Established models of injury have used myotoxins and physical trauma to demonstrate the concurrence of myogenesis and angiogenesis during regeneration. In these models, efferocytosis removes cellular debris while basal laminae persist to provide guidance during myofiber and microvessel regeneration. It is unknown whether the spatiotemporal coupling between myofiber and microvascular regeneration persists when muscle tissue is completely removed and local guidance cues are lost. METHODS: To test whether complete removal of skeletal muscle tissue affects the spatiotemporal relationship between myogenesis and angiogenesis during regeneration, subthreshold volumetric muscle loss was created with a biopsy punch (diameter, 2 mm) through the center of the gluteus maximus (GM) in adult mice. Regeneration into the void was evaluated through 21 days post-injury (dpi). Microvascular perfusion was evaluated in vivo by injecting fluorescent dextran into the circulation during intravital imaging. Confocal imaging and histological analyses of whole-mount GM preparations and tissue cross-sections assessed the growth of microvessels and myofibers into the wound. RESULTS: A provisional matrix filled with PDGFRα+ and CD45+ cells spanned the wound within 1 dpi. Regenerating microvessels advanced from the edges of the wound into the matrix by 7 dpi. Nascent microvascular networks formed by 10 dpi with blood-perfused networks spanning the wound by 14 dpi. In striking contrast, the wound remained devoid of myofibers at 7 and 10 dpi. Myogenesis into the wound was apparent by 14 dpi and traversed the wound by 21 dpi. Regenerated myofibers and microvessels were disorganized compared to the uninjured muscle. CONCLUSIONS: Following punch biopsy of adult skeletal muscle, regenerating microvessels span the wound and become perfused with blood prior to myofiber regeneration. The loss of residual guidance cues with complete tissue removal disrupts the spatiotemporal correspondence between microvascular and myofiber regeneration. We conclude that angiogenesis precedes myogenesis during regeneration following subthreshold volumetric muscle loss.

Effects of caffeine on anaerobic exercise in boys.

The effects of caffeine on anaerobic exercise in young boys was investigated. Twenty-four healthy 8-10 year old boys participated in a randomized double-blind, double-crossover, counter-balanced study. Each subject received the caffeinated drink (CAF--5 mg · kg(-1)) or placebo (PL) twice each on four separate visits. Sixty minutes following ingestion of either CAF or PL boys performed a static hand-grip test and then a Wingate test. Reliability was moderately high for the Wingate test (R = .70-0.95). Hand-grip reliability was higher for CAF (R = .88) than PL (R = .52). Mean power (180 ± 36 vs 173 ± 28 W) was significantly higher (p < .05) in CAF versus PL, respectively. There were no differences in peak power or static hand-grip maximal voluntary contraction with CAF. Further, peak HR (190 ± 10 vs 185 ± 10 beats · min(-1)) was significantly higher in CAF versus PL, respectively. Thus, in this study a moderately high dose of CAF significantly increased the average power during a Wingate test, yet it does not affect peak power or static hand-grip strength.

Hydrogen sulfide donor protects against mechanical ventilation-induced atrophy and contractile dysfunction in the rat diaphragm.

Mechanical ventilation (MV) is a clinical tool providing adequate alveolar ventilation in patients that require respiratory support. Although a life-saving intervention for critically ill patients, prolonged MV results in the rapid development of inspiratory muscle weakness due to both diaphragmatic atrophy and contractile dysfunction; collectively known as "ventilator-induced diaphragm dysfunction" (VIDD). VIDD is a severe clinical problem because diaphragmatic weakness is a risk factor for difficulties in weaning patients from MV. Currently, no standard treatment to prevent VIDD exists. Nonetheless, growing evidence reveals that hydrogen sulfide (H2 S) possesses cytoprotective properties capable of protecting skeletal muscles against several hallmarks of VIDD, including oxidative damage, accelerated proteolysis, and mitochondrial damage. Therefore, we used an established animal model of MV to test the hypothesis that treatment with sodium sulfide (H2 S donor) will defend against VIDD. Our results confirm that sodium sulfide was sufficient to protect the diaphragm against both MV-induced fiber atrophy and contractile dysfunction. H2 S prevents MV-induced damage to diaphragmatic mitochondria as evidenced by protection against mitochondrial uncoupling. Moreover, treatment with sodium sulfide prevented the MV-induced activation of the proteases, calpain, and caspase-3 in the diaphragm. Taken together, these results support the hypothesis that treatment with a H2 S donor protects the diaphragm against VIDD. These outcomes provide the first evidence that H2 S has therapeutic potential to protect against MV-induced diaphragm weakness and to reduce difficulties in weaning patients from the ventilator. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Mechanical ventilation (MV) results in diaphragm atrophy and contractile dysfunction, known as ventilator-induced diaphragm dysfunction (VIDD). VIDD is important because diaphragm weakness is a risk factor for problems in weaning patients from MV. Currently, no accepted treatment exists to protect against VIDD. Growing evidence reveals that hydrogen sulfide (H2 S) donors protect skeletal muscle against ischemia-reperfusion-induced injury. Nonetheless, it is unknown if treatment with a H2 S donor can protect against VIDD. WHAT QUESTION DID THIS STUDY ADDRESS? Can treatment with an H2 S donor protect against VIDD? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? This study provides the first evidence that treatment with a H2 S donor protects against VIDD. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? These new findings provide the basis for further exploration of H2 S donors as a therapy to prevent VIDD and reduce the risk of problems in weaning patients from MV.

Comparative Efficacy of Angiotensin II Type 1 Receptor Blockers Against Ventilator-Induced Diaphragm Dysfunction in Rats.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in the rapid development of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (termed ventilator-induced diaphragm dysfunction (VIDD)). Although VIDD is a major risk factor for problems in weaning patients from MV, a standard therapy to prevent VIDD does not exist. However, emerging evidence suggests that pharmacological blockade of angiotensin II type 1 receptors (AT1Rs) protects against VIDD. Nonetheless, the essential characteristics of AT1R blockers (ARBs) required to protect against VIDD remain unclear. To determine the traits of ARBs that are vital for protection against VIDD, we compared the efficacy of two clinically relevant ARBs, irbesartan and olmesartan; these ARBs differ in molecular structure and effects on AT1Rs. Specifically, olmesartan blocks both angiotensin II (AngII) binding and mechanical activation of AT1Rs, whereas irbesartan prevents only AngII binding to AT1Rs. Using a well-established preclinical model of prolonged MV, we tested the hypothesis that compared with irbesartan, olmesartan provides greater protection against VIDD. Our results reveal that irbesartan does not protect against VIDD whereas olmesartan defends against both MV-induced diaphragmatic atrophy and contractile dysfunction. These findings support the hypothesis that olmesartan is superior to irbesartan in protecting against VIDD and are consistent with the concept that blockade of mechanical activation of AT1Rs is a required property of ARBs to shield against VIDD. These important findings provide a foundation for future clinical trials to evaluate ARBs as a therapy to protect against VIDD.

Modification of Neuromuscular Junction Protein Expression by Exercise and Doxorubicin.

PURPOSE: Doxorubicin (DOX) is a highly effective antitumor agent widely used in cancer treatment. However, it is well established that DOX induces muscular atrophy and impairs force production. Although no therapeutic interventions exist to combat DOX-induced muscle weakness, endurance exercise training has been shown to reduce skeletal muscle damage caused by DOX administration. Numerous studies have attempted to identify molecular mechanisms responsible for exercise-induced protection against DOX myotoxicity. Nevertheless, the mechanisms by which endurance exercise protects against DOX-induced muscle weakness remain elusive. In this regard, impairments to the neuromuscular junction (NMJ) are associated with muscle wasting, and studies indicate that physical exercise can rescue NMJ fragmentation. Therefore, we tested the hypothesis that exercise protects against DOX-induced myopathy by preventing detrimental changes to key proteins responsible for maintenance of the NMJ. METHODS: Female Sprague-Dawley rats were assigned to sedentary or exercise-trained groups. Exercise training consisted of a 5-d treadmill habituation period followed by 10 d of running (60 min·d, 30 m·min, 0% grade). After the last training bout, exercise-trained and sedentary animals were paired with either placebo (saline) or DOX (20 mg·kg i.p.) treatment. Two days after drug treatment, the soleus muscle was excised for subsequent analyses. RESULTS: Our results indicate that endurance exercise training prevents soleus muscle atrophy and contractile dysfunction in DOX-treated animals. These adaptations were associated with the increased expression of the following neurotrophic factors: brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, nerve growth factor, and neurotrophin-3. In addition, exercise enhanced the expression of receptor-associated protein of the synapse and the acetylcholine receptor (AChR) subunits AChRß, AChRδ, and AChRγ in DOX-treated animals. CONCLUSION: Therefore, upregulating neurotrophic factor and NMJ protein expression may be an effective strategy to prevent DOX-induced skeletal muscle dysfunction.

Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels.

BACKGROUND: Local injection of BaCl2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment. METHODS: The intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3-4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (Vm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50-75 µL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant. RESULTS: Addition of 1.2% BaCl2 to PSS depolarized myofibers from - 79 ± 3 mV to - 17 ± 7 mV with a corresponding rise in [Ca2+]i; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h. CONCLUSIONS: BaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.

Mitochondrial accumulation of doxorubicin in cardiac and diaphragm muscle following exercise preconditioning.

Doxorubicin (DOX) is a highly effective anthracycline antibiotic. Unfortunately, the clinical use of DOX is limited by the risk of deleterious effects to cardiac and respiratory (i.e. diaphragm) muscle, resulting from mitochondrial reactive oxygen species (ROS) production. In this regard, exercise is demonstrated to protect against DOX-induced myotoxicity and prevent mitochondrial dysfunction. However, the protective mechanisms are currently unclear. We hypothesized that exercise may induce protection by increasing the expression of mitochondria-specific ATP-binding cassette (ABC) transporters and reducing mitochondrial DOX accumulation. Our results confirm this finding and demonstrate that two weeks of exercise preconditioning is sufficient to prevent cardiorespiratory dysfunction.

Exercise Training Prevents Doxorubicin-induced Mitochondrial Dysfunction of the Liver.

PURPOSE: Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of a broad spectrum of cancers. However, clinical use of DOX is limited by irreversible and dose-dependent hepatotoxicity. The liver is the primary organ responsible for the clearance of antineoplastic agents, and evidence indicates that hepatotoxicity occurs as a result of impaired mitochondrial efficiency during DOX metabolism. In this regard, exercise training is sufficient to improve mitochondrial function and protect against DOX-induced cytotoxicity. Therefore, the purpose of this study was to determine whether short-term exercise preconditioning is sufficient to protect against DOX-induced liver mitochondrionopathy. METHODS: Female Sprague-Dawley rats (4-6 months old) were randomly assigned to one of four groups: 1) sedentary, treated with saline; 2) sedentary, treated with DOX; 3) exercise trained, treated with saline; and 4) exercise trained, treated with DOX. Exercise-trained animals underwent 5 d of treadmill running habituation followed by 10 d of running for 60 min·d (30 m·min; 0% grade). After the last training bout, exercise-trained and sedentary animals were injected with either DOX (20 mg·kg i.p.) or saline. Two days after drug treatment, the liver was removed and mitochondria were isolated. RESULTS: DOX treatment induced mitochondrial dysfunction of the liver in sedentary animals because of alterations in mitochondrial oxidative capacity, biogenesis, degradation, and protein acetylation. Furthermore, exercise preconditioning protected against DOX-mediated liver mitochondrionopathy, which was associated with the maintenance of mitochondrial oxidative capacity and protein acetylation. CONCLUSION: These findings demonstrate that endurance exercise training protects against DOX-induced liver mitochondrial dysfunction, which was attributed to modifications in organelle oxidative capacity and mitochondrial protein acetylation.

Effects of exercise preconditioning and HSP72 on diaphragm muscle function during mechanical ventilation.

BACKGROUND: Mechanical ventilation (MV) is a life-saving measure for patients in respiratory failure. However, prolonged MV results in significant diaphragm atrophy and contractile dysfunction, a condition referred to as ventilator-induced diaphragm dysfunction (VIDD). While there are currently no clinically approved countermeasures to prevent VIDD, increased expression of heat shock protein 72 (HSP72) has been demonstrated to attenuate inactivity-induced muscle wasting. HSP72 elicits cytoprotection via inhibition of NF-κB and FoxO transcriptional activity, which contribute to VIDD. In addition, exercise-induced prevention of VIDD is characterized by an increase in the concentration of HSP72 in the diaphragm. Therefore, we tested the hypothesis that increased HSP72 expression is required for the exercise-induced prevention of VIDD. We also determined whether increasing the abundance of HSP72 in the diaphragm, independent of exercise, is sufficient to prevent VIDD. METHODS: Cause and effect was determined by inhibiting the endurance exercise-induced increase in HSP72 in the diaphragm of exercise trained animals exposed to prolonged MV via administration of an antisense oligonucleotide targeting HSP72. Additional experiments were performed to determine if increasing HSP72 in the diaphragm via genetic (rAAV-HSP72) or pharmacological (BGP-15) overexpression is sufficient to prevent VIDD. RESULTS: Our results demonstrate that the exercise-induced increase in HSP72 protein abundance is required for the protective effects of exercise against VIDD. Moreover, both rAAV-HSP72 and BGP-15-induced overexpression of HSP72 were sufficient to prevent VIDD. In addition, modification of HSP72 in the diaphragm is inversely related to the expression of NF-κB and FoxO target genes. CONCLUSIONS: HSP72 overexpression in the diaphragm is an effective intervention to prevent MV-induced oxidative stress and the transcriptional activity of NF-κB and FoxO. Therefore, overexpression of HSP72 in the diaphragm is a potential therapeutic target to protect against VIDD.

Increased SOD2 in the diaphragm contributes to exercise-induced protection against ventilator-induced diaphragm dysfunction.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in rapid diaphragmatic atrophy and contractile dysfunction, collectively termed ventilator-induced diaphragm dysfunction (VIDD). Recent evidence reveals that endurance exercise training, performed prior to MV, protects the diaphragm against VIDD. While the mechanism(s) responsible for this exercise-induced protection against VIDD remain unknown, increased diaphragm antioxidant expression may be required. To investigate the role that increased antioxidants play in this protection, we tested the hypothesis that elevated levels of the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) is required to achieve exercise-induced protection against VIDD. Cause and effect was investigated in two ways. First, we prevented the exercise-induced increase in diaphragmatic SOD2 via delivery of an antisense oligonucleotide targeted against SOD2 post-exercise. Second, using transgene overexpression of SOD2, we determined the effects of increased SOD2 in the diaphragm independent of exercise training. Results from these experiments revealed that prevention of the exercise-induced increases in diaphragmatic SOD2 results in a loss of exercise-mediated protection against MV-induced diaphragm atrophy and a partial loss of protection against MV-induced diaphragmatic contractile dysfunction. In contrast, transgenic overexpression of SOD2 in the diaphragm, independent of exercise, did not protect against MV-induced diaphragmatic atrophy and provided only partial protection against MV-induced diaphragmatic contractile dysfunction. Collectively, these results demonstrate that increased diaphragmatic levels of SOD2 are essential to achieve the full benefit of exercise-induced protection against VIDD.

Effects of doxorubicin on cardiac muscle subsarcolemmal and intermyofibrillar mitochondria.

Doxorubicin (DOX) is a highly effective chemotherapeutic used in the treatment of a broad spectrum of malignancies. However, clinical use of DOX is highly limited by cumulative and irreversible cardiomyopathy that occurs following DOX treatment. The pathogenesis of DOX-induced cardiac muscle dysfunction is complex. However, it has been proposed that the etiology of this myopathy is related to mitochondrial dysfunction, as a result of the dose-dependent increase in the mitochondrial accumulation of DOX. In this regard, cardiac muscle possesses two morphologically distinct populations of mitochondria. Subsarcolemmal (SS) mitochondria are localized just below the sarcolemma, whereas intermyofibrillar (IMF) mitochondria are found between myofibrils. Mitochondria in both regions exhibit subtle differences in biochemical properties, giving rise to differences in respiration, lipid composition, enzyme activities and protein synthesis rates. Based on the heterogeneity of SS and IMF mitochondria, we hypothesized that acute DOX administration would have distinct effects on each cardiac mitochondrial subfraction. Therefore, we isolated SS and IMF mitochondria from the hearts of female Sprague-Dawley rats 48h after administration of DOX. Our results demonstrate that while SS mitochondria appear to accumulate greater amounts of DOX, IMF mitochondria demonstrate a greater apoptotic and autophagic response to DOX exposure. Thus, the divergent protein composition and function of the SS and IMF cardiac mitochondria result in differential responses to DOX, with IMF mitochondria appearing more susceptible to damage after DOX treatment.

Global Proteome Changes in the Rat Diaphragm Induced by Endurance Exercise Training.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in the rapid development of diaphragmatic atrophy and weakness. Importantly, endurance exercise training results in a diaphragmatic phenotype that is protected against ventilator-induced diaphragmatic atrophy and weakness. The mechanisms responsible for this exercise-induced protection against ventilator-induced diaphragmatic atrophy remain unknown. Therefore, to investigate exercise-induced changes in diaphragm muscle proteins, we compared the diaphragmatic proteome from sedentary and exercise-trained rats. Specifically, using label-free liquid chromatography-mass spectrometry, we performed a proteomics analysis of both soluble proteins and mitochondrial proteins isolated from diaphragm muscle. The total number of diaphragm proteins profiled in the soluble protein fraction and mitochondrial protein fraction were 813 and 732, respectively. Endurance exercise training significantly (P<0.05, FDR <10%) altered the abundance of 70 proteins in the soluble diaphragm proteome and 25 proteins of the mitochondrial proteome. In particular, key cytoprotective proteins that increased in relative abundance following exercise training included mitochondrial fission process 1 (Mtfp1; MTP18), 3-mercaptopyruvate sulfurtransferase (3MPST), microsomal glutathione S-transferase 3 (Mgst3; GST-III), and heat shock protein 70 kDa protein 1A/1B (HSP70). While these proteins are known to be cytoprotective in several cell types, the cyto-protective roles of these proteins have yet to be fully elucidated in diaphragm muscle fibers. Based upon these important findings, future experiments can now determine which of these diaphragmatic proteins are sufficient and/or required to promote exercise-induced protection against inactivity-induced muscle atrophy.

Redox control of skeletal muscle atrophy.

Skeletal muscles comprise the largest organ system in the body and play an essential role in body movement, breathing, and glucose homeostasis. Skeletal muscle is also an important endocrine organ that contributes to the health of numerous body organs. Therefore, maintaining healthy skeletal muscles is important to support overall health of the body. Prolonged periods of muscle inactivity (e.g., bed rest or limb immobilization) or chronic inflammatory diseases (i.e., cancer, kidney failure, etc.) result in skeletal muscle atrophy. An excessive loss of muscle mass is associated with a poor prognosis in several diseases and significant muscle weakness impairs the quality of life. The skeletal muscle atrophy that occurs in response to inflammatory diseases or prolonged inactivity is often associated with both oxidative and nitrosative stress. In this report, we critically review the experimental evidence that provides support for a causative link between oxidants and muscle atrophy. More specifically, this review will debate the sources of oxidant production in skeletal muscle undergoing atrophy as well as provide a detailed discussion on how reactive oxygen species and reactive nitrogen species modulate the signaling pathways that regulate both protein synthesis and protein breakdown.

Comparative changes in antioxidant enzymes and oxidative stress in cardiac, fast twitch and slow twitch skeletal muscles following endurance exercise training.

The aim of this study was to evaluate exercise-induced transcriptional and protein responses of heart, soleus (slow oxidative), and plantaris (fast glycolytic) muscle in response to ten days of endurance exercise training. Four-month old female Sprague-Dawley rats were assigned to either a sedentary (SED) or endurance exercise-training (EXE) group (n=8 per group). The heart, plantaris, and soleus were excised and used for biochemical analyses. Our results show that heart and plantaris from EXE animals had higher protein levels of superoxide dismutase 2 (SOD2) compared to SED animals (P<0.05). Also, the protein levels of catalase were higher in plantaris of EXE animals compared to SED animals (P<0.05). No significant differences existed for 4 hydroxynonenal (4HNE) conjugated proteins (index of oxidative damage) in the three tissues between SED and EXE animals. mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) were higher in plantaris of EXE animals compared to SED animals (P<0.05), and mRNA levels of estrogen-related receptor alpha (ERRα) were lower in the heart of EXE animals compared to SED animals. In conclusion, heart and plantaris are responsive to ten days of treadmill training, while greater exercise intensities or durations may be needed to elicit alterations in soleus.

Cervical spinal cord injury exacerbates ventilator-induced diaphragm dysfunction.

Cervical spinal cord injury (SCI) can dramatically impair diaphragm muscle function and often necessitates mechanical ventilation (MV) to maintain adequate pulmonary gas exchange. MV is a life-saving intervention. However, prolonged MV results in atrophy and impaired function of the diaphragm. Since cervical SCI can also trigger diaphragm atrophy, it may create preconditions that exacerbate ventilator-induced diaphragm dysfunction (VIDD). Currently, no drug therapy or clinical standard of care exists to prevent or minimize diaphragm dysfunction following SCI. Therefore, we first tested the hypothesis that initiating MV acutely after cervical SCI will exacerbate VIDD and enhance proteolytic activation in the diaphragm to a greater extent than either condition alone. Rats underwent controlled MV for 12 h following acute (∼24 h) cervical spinal hemisection injury at C2 (SCI). Diaphragm tissue was then harvested for comprehensive functional and molecular analyses. Second, we determined if antioxidant therapy could mitigate MV-induced diaphragm dysfunction after cervical SCI. In these experiments, SCI rats received antioxidant (Trolox, a vitamin E analog) or saline treatment prior to initiating MV. Our results demonstrate that compared with either condition alone, the combination of SCI and MV resulted in increased diaphragm atrophy, contractile dysfunction, and expression of atrophy-related genes, including MuRF1. Importantly, administration of the antioxidant Trolox attenuated proteolytic activation, fiber atrophy, and contractile dysfunction in the diaphragms of SCI + MV animals. These findings provide evidence that cervical SCI greatly exacerbates VIDD, but antioxidant therapy with Trolox can preserve diaphragm contractile function following acute SCI.