Role of SERCA and sarcolipin in adaptive muscle remodeling.

Sarcolipin (SLN) is a small regulatory protein that inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. When bound to SERCA, SLN reduces the apparent Ca2+ affinity of SERCA and uncouples SERCA Ca2+ transport from its ATP consumption. As such, SLN plays a direct role in altering skeletal muscle relaxation and energy expenditure. Interestingly, the expression of SLN is dynamic during times of muscle adaptation, in that large increases in SLN content are found in response to development, atrophy, overload, and disease. Several groups have suggested that increases in SLN, especially in dystrophic muscle, are deleterious as it may reduce muscle function and exacerbate already abhorrent intracellular Ca2+ levels. However, there is also significant evidence to show that increased SLN content is a beneficial adaptive mechanism that protects the SERCA pump and activates Ca2+ signaling and adaptive remodeling during times of cell stress. In this review, we first discuss the role for SLN in healthy muscle during both development and overload, where SLN has been shown to activate Ca2+ signaling to promote mitochondrial biogenesis, fiber-type shifts, and muscle hypertrophy. Then, with respect to muscle disease, we summarize the discrepancies in the literature as to whether SLN upregulation is adaptive or maladaptive in nature. This review is the first to offer the concept of SLN hormesis in muscle disease, wherein both too much and too little SLN are detrimental to muscle health. Finally, the underlying mechanisms which activate SLN upregulation are discussed, specifically acknowledging a potential positive feedback loop between SLN and Ca2+ signaling molecules.

Nox4 Knockout Does Not Prevent Diaphragm Atrophy, Contractile Dysfunction, or Mitochondrial Maladaptation in the Early Phase Post-Myocardial Infarction in Mice.

BACKGROUND/AIMS: Diaphragm dysfunction with increased reactive oxygen species (ROS) occurs within 72 hrs post-myocardial infarction (MI) in mice and may contribute to loss of inspiratory maximal pressure and endurance in patients. METHODS: We used wild-type (WT) and whole-body Nox4 knockout (Nox4KO) mice to measure diaphragm bundle force in vitro with a force transducer, mitochondrial respiration in isolated fiber bundles with an O2 sensor, mitochondrial ROS by fluorescence, mRNA (RT-PCR) and protein (immunoblot), and fiber size by histology 72 hrs post-MI. RESULTS: MI decreased diaphragm fiber cross-sectional area (CSA) (~15%, p = 0.015) and maximal specific force (10%, p = 0.005), and increased actin carbonylation (5-10%, p = 0.007) in both WT and Nox4KO. Interestingly, MI did not affect diaphragm mRNA abundance of MAFbx/atrogin-1 and MuRF-1 but Nox4KO decreased it by 20-50% (p < 0.01). Regarding the mitochondria, MI and Nox4KO decreased the protein abundance of citrate synthase and subunits of electron transport system (ETS) complexes and increased mitochondrial O2 flux (JO2) and H2O2 emission (JH2O2) normalized to citrate synthase. Mitochondrial electron leak (JH2O2/JO2) in the presence of ADP was lower in Nox4KO and not changed by MI. CONCLUSION: Our study shows that the early phase post-MI causes diaphragm atrophy, contractile dysfunction, sarcomeric actin oxidation, and decreases citrate synthase and subunits of mitochondrial ETS complexes. These factors are potential causes of loss of inspiratory muscle strength and endurance in patients, which likely contribute to the pathophysiology in the early phase post-MI. Whole-body Nox4KO did not prevent the diaphragm abnormalities induced 72 hrs post-MI, suggesting that systemic pharmacological inhibition of Nox4 will not benefit patients in the early phase post-MI.

Pulsed ultrasound prevents lipopolysaccharide-induced muscle atrophy through inhibiting p38 MAPK phosphorylation in C2C12 myotubes.

OBJECTIVE: Inflammation contributes to skeletal muscle atrophy via protein degradation induced by p38 mitogen-activated protein kinase (MAPK) phosphorylation. Meanwhile, pulsed ultrasound irradiation provides the mechanical stimulation to the target tissue, and has been reported to show anti-inflammatory effects. This study investigated the preventive effects of pulsed ultrasound irradiation on muscle atrophy induced by lipopolysaccharide (LPS) in C2C12 myotubes. METHODS: C2C12 myotubes were used in this research. The pulsed ultrasound (a frequency of 3 MHz, duty cycle of 20%, intensity of 0.5 W/cm2) was irradiated to myotube before LPS administration. RESULTS: The LPS increased phosphorylation of p38 MAPK and decreased the myofibril and myosin heavy chain protein (P < 0.05), followed by atrophy in C2C12 myotubes. The pulsed ultrasound irradiation attenuated p38 MAPK phosphorylation and myotube atrophy induced by LPS (P < 0.05). CONCLUSIONS: Pulsed ultrasound irradiation has the preventive effects on inflammation-induced muscle atrophy through inhibiting phosphorylation of p38 MAPK.

Mechanisms That Activate 26S Proteasomes and Enhance Protein Degradation.

Although ubiquitination is widely assumed to be the only regulated step in the ubiquitin-proteasome pathway, recent studies have demonstrated several important mechanisms that regulate the activities of the 26S proteasome. Most proteasomes in cells are inactive but, upon binding a ubiquitinated substrate, become activated by a two-step mechanism requiring an association of the ubiquitin chain with Usp14 and then a loosely folded protein domain with the ATPases. The initial activation step is signaled by Usp14's UBL domain, and many UBL-domain-containing proteins (e.g., Rad23, Parkin) also activate the proteasome. ZFAND5 is a distinct type of activator that binds ubiquitin conjugates and the proteasome and stimulates proteolysis during muscle atrophy. The proteasome's activities are also regulated through subunit phosphorylation. Agents that raise cAMP and activate PKA stimulate within minutes Rpn6 phosphorylation and enhance the selective degradation of short-lived proteins. Likewise, hormones, fasting, and exercise, which raise cAMP, activate proteasomes and proteolysis in target tissues. Agents that raise cGMP and activate PKG also stimulate 26S activities but modify different subunit(s) and stimulate also the degradation of long-lived cell proteins. Both kinases enhance the selective degradation of aggregation-prone proteins that cause neurodegenerative diseases. These new mechanisms regulating proteolysis thus have clear physiological importance and therapeutic potential.

Reduced sarcoplasmic reticulum Ca2+ ATPase activity underlies skeletal muscle wasting in asthma.

AIMS: Skeletal muscle mass and strength are reduced in asthma and contribute to compromised functional capacity in asthmatic patients. However, an effective pharmacological intervention remains elusive, partly because molecular mechanisms dictating muscle decline in asthma are not known. MATERIALS: We investigated the potential contribution(s) of skeletal muscle sarcoplasmic reticulum Ca2+ ATPase (SERCA) to muscle atrophy and weakness in asthmatic patients. Quadriceps muscle biopsies were taken from 58 to 72 years old male patients with mild and advanced asthma and the SERCA activity was analyzed in association with cellular redox environment and myonuclear domain (MND) size. KEY FINDINGS: Maximal SERCA activity was reduced in skeletal muscles of mild and advanced asthmatics and was associated with reduced expression of SERCA2 protein and upregulation of sarcolipin, a SERCA inhibitory lipoprotein. We also found downregulation of Ca2+ release protein calstabin and upregulation of Ca2+ buffer, calsequestrin in skeletal muscles of asthmatic patients. The atrophic single muscle fibers had smaller cytoplasmic domains per myonucleus possibly indicating the reduced transcriptional reserves of individual myonuclei. Plasma periostin and CAF22 levels were significantly elevated in asthmatic patients and showed a strong correlation with hand-grip strength. These changes were accompanied by substantially elevated markers of global oxidative stress including lipid peroxidation and mitochondrial ROS production. CONCLUSION: Taken together, our data suggest that muscle weakness and atrophy in asthma is in part driven by SERCA dysfunction and oxidative stress. The data propose SERCA dysfunction as a therapeutic intervention to address muscle decline in asthma.

Ubiquitin Ligases at the Heart of Skeletal Muscle Atrophy Control.

Skeletal muscle loss is a detrimental side-effect of numerous chronic diseases that dramatically increases mortality and morbidity. The alteration of protein homeostasis is generally due to increased protein breakdown while, protein synthesis may also be down-regulated. The ubiquitin proteasome system (UPS) is a master regulator of skeletal muscle that impacts muscle contractile properties and metabolism through multiple levers like signaling pathways, contractile apparatus degradation, etc. Among the different actors of the UPS, the E3 ubiquitin ligases specifically target key proteins for either degradation or activity modulation, thus controlling both pro-anabolic or pro-catabolic factors. The atrogenes MuRF1/TRIM63 and MAFbx/Atrogin-1 encode for key E3 ligases that target contractile proteins and key actors of protein synthesis respectively. However, several other E3 ligases are involved upstream in the atrophy program, from signal transduction control to modulation of energy balance. Controlling E3 ligases activity is thus a tempting approach for preserving muscle mass. While indirect modulation of E3 ligases may prove beneficial in some situations of muscle atrophy, some drugs directly inhibiting their activity have started to appear. This review summarizes the main signaling pathways involved in muscle atrophy and the E3 ligases implicated, but also the molecules potentially usable for future therapies.

Inhibition of Cytosolic Phospholipase A2 Has Neuroprotective Effects on Motoneuron and Muscle Atrophy after Spinal Cord Injury.

Surviving motoneurons undergo dendritic atrophy after spinal cord injury (SCI), suggesting an important therapeutic target for neuroprotective strategies to improve recovery of function after SCI. Our previous studies showed that cytosolic phospholipase A2 (PLA2) may play an important role in the pathogenesis of SCI. In the present study, we investigated whether blocking cytosolic PLA2 (cPLA2) pharmacologically with arachidonyl trifluoromethyl ketone (ATK) or genetically using cPLA2 knockout (KO) mice attenuates motoneuron atrophy after SCI. C57BL/6 mice received either sham or contusive SCI at the T10 level. At 30 min after SCI, mice were treated with ATK or vehicle. Four weeks later, motoneurons innervating the vastus lateralis muscle of the quadriceps were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Soma volume, motoneuron number, lesion volume, and tissue sparing were also assessed, as were muscle weight, fiber cross-sectional area, and motor endplate size and density. ATK administration reduced percent lesion volume and increased percent volume of spared white matter, compared to the vehicle-treated control animals. SCI with or without ATK treatment had no effect on the number or soma volume of quadriceps motoneurons. However, SCI resulted in a decrease in dendritic length of quadriceps motoneurons in untreated animals, and this decrease was completely prevented by treatment with ATK. Similarly, vastus lateralis muscle weights of untreated SCI animals were smaller than those of sham surgery controls, and these reductions were prevented by ATK treatment. No effects on fiber cross-sectional areas, motor endplate area, or density were observed across treatment groups. Remarkably, genetically deleting cPLA2 in cPLA2 KO mice attenuated dendritic atrophy after SCI. These findings suggest that, after SCI, cord tissue damage and regressive changes in motoneuron and muscle morphology can be reduced by inhibition of cPLA2, further supporting a role for cPLA2 as a neurotherapeutic target for SCI treatment.

Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism.

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3ß/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3ß activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

Inhibition of xanthine oxidase in the acute phase of myocardial infarction prevents skeletal muscle abnormalities and exercise intolerance.

AIMS: Exercise intolerance in patients with heart failure (HF) is partly attributed to skeletal muscle abnormalities. We have shown that reactive oxygen species (ROS) play a crucial role in skeletal muscle abnormalities, but the pathogenic mechanism remains unclear. Xanthine oxidase (XO) is reported to be an important mediator of ROS overproduction in ischaemic tissue. Here, we tested the hypothesis that skeletal muscle abnormalities in HF are initially caused by XO-derived ROS and are prevented by the inhibition of their production. METHODS AND RESULTS: Myocardial infarction (MI) was induced in male C57BL/6J mice, which eventually led to HF, and a sham operation was performed in control mice. The time course of XO-derived ROS production in mouse skeletal muscle post-MI was first analysed. XO-derived ROS production was significantly increased in MI mice from Days 1 to 3 post-surgery (acute phase), whereas it did not differ between the MI and sham groups from 7 to 28 days (chronic phase). Second, mice were divided into three groups: sham + vehicle (Sham + Veh), MI + vehicle (MI + Veh), and MI + febuxostat (an XO inhibitor, 5 mg/kg body weight/day; MI + Feb). Febuxostat or vehicle was administered at 1 and 24 h before surgery, and once-daily on Days 1-7 post-surgery. On Day 28 post-surgery, exercise capacity and mitochondrial respiration in skeletal muscle fibres were significantly decreased in MI + Veh compared with Sham + Veh mice. An increase in damaged mitochondria in MI + Veh compared with Sham + Veh mice was also observed. The wet weight and cross-sectional area of slow muscle fibres (higher XO-derived ROS) was reduced via the down-regulation of protein synthesis-associated mTOR-p70S6K signalling in MI + Veh compared with Sham + Veh mice. These impairments were ameliorated in MI + Feb mice, in association with a reduction of XO-derived ROS production, without affecting cardiac function. CONCLUSION: XO inhibition during the acute phase post-MI can prevent skeletal muscle abnormalities and exercise intolerance in mice with HF.

Magnoflorine prevent the skeletal muscle atrophy via Akt/mTOR/FoxO signal pathway and increase slow-MyHC production in streptozotocin-induced diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora cordifolia (TC) is being used as a blood purifier in Ayurveda since ancient time. It is a very popular immunomodulator and holds anti-inflammatory and anti-oxidative potential, hence anti-aging properties. Therefore, it is also known as 'Amrita' in Ayurveda and is widely used to treat diabetes mellitus type II (T2DM) and its secondary complications; however, its underlying mechanism was not expedited to date. AIM-: To explore the in vivo therapeutic efficiency and mechanism of action of TC and its secondary constitute magnoflorine on the skeletal muscle atrophy in the rat model of T2DM. METHOD: Animal model of T2DM was developed using streptozotocin (STZ) injection followed by intervention with TC, metformin, and magnoflorine for three weeks. Confirmation of T2DM and abrogation of atrophic markers and possible mechanisms on supplementation of TC and magnoflorine were explored using histology, bio-assays, Western blotting, and q-PCR. RESULT: TC and Magnoflorine supplementations significantly (p ≤ 0.05) decreased the fasting blood glucose (FBG) levels in T2DM rats. Both treatments prevented the lean body, individual skeletal muscle mass, and myotubes diameter loss (p ≤ 0.05). Magnoflorine significantly reduced the degradation of the protein indicated by biochemical markers of atrophy i.e. decreased serum creatine kinase (CK) levels and increased myosin heavy chain-ß (MyHC-ß) levels in muscles. Q-PCR and western blotting supported the findings that magnoflorine significantly increased the mRNA and protein abundances (~3 fold) of MyHC-ß.TC and magnoflorine efficiently decreased the expression of ubiquitin-proteasomal E3-ligases (Fn-14/TWEAK, MuRF1, and Atrogin 1), autophagy (Bcl-2/LC3B), and caspase related genes along with calpains activities in T2DM rats. Both TC and magnoflorine also increased the activity of superoxide dismutase, GSH-Px, decreased the activities of ß-glucuronidase, LPO, and prevented any alteration in the catalase activity. In contrast, magnoflorine increased expression of TNF-α and IL-6 whereas TC and metformin efficiently decreased the levels of these pro-inflammatory cytokines (p ≤ 0.05). However, magnoflorine was found to increase phosphorylation of Akt more efficiently than TC and metformin. CONCLUSION: TC, and magnoflorine are found to be effective to control fasting blood glucose levels significantly in T2DM rats. It also promoted the Akt phosphorylation, suppressed autophagy and proteolysis that might be related to blood glucose-lowering efficacy of magnoflorine and TC. However, increased muscle weight, specifically of the soleus muscle, expression of IL-6, and slow MyHC indicated the increased myogenesis in response to magnoflorine and independent from its hypoglycemic activity.

The ubiquitin-proteasome system in regulation of the skeletal muscle homeostasis and atrophy: from basic science to disorders.

Skeletal muscle is one of the most abundant and highly plastic tissues. The ubiquitin-proteasome system (UPS) is recognised as a major intracellular protein degradation system, and its function is important for muscle homeostasis and health. Although UPS plays an essential role in protein degradation during muscle atrophy, leading to the loss of muscle mass and strength, its deficit negatively impacts muscle homeostasis and leads to the occurrence of several pathological phenotypes. A growing number of studies have linked UPS impairment not only to matured muscle fibre degeneration and weakness, but also to muscle stem cells and deficiency in regeneration. Emerging evidence suggests possible links between abnormal UPS regulation and several types of muscle diseases. Therefore, understanding of the role of UPS in skeletal muscle may provide novel therapeutic insights to counteract muscle wasting, and various muscle diseases. In this review, we focussed on the role of proteasomes in skeletal muscle and its regeneration, including a brief explanation of the structure of proteasomes. In addition, we summarised the recent findings on several diseases and elaborated on how the UPS is related to their pathological states.

Antioxidant Apigenin Relieves Age-Related Muscle Atrophy by Inhibiting Oxidative Stress and Hyperactive Mitophagy and Apoptosis in Skeletal Muscle of Mice.

Skeletal muscle atrophy in the aged causes loss in muscle mass and functions. Naturally occurring antioxidant flavonoid apigenin is able to ameliorate obesity- and denervation-induced muscle atrophies, but its effects on age-related muscle atrophy remain unknown. We hypothesized that apigenin can relieve muscle atrophy in aged mice, probably through special effects on reactive oxygen species and enzymes with antioxidant functions. For the male mice of the study, apigenin showed significant dose-dependent effects in relieving aging-related muscle atrophy according to results of frailty index as indicator of frailty associated with aging, grip strength, and running distance. Apigenin also improved myofiber size and morphological features and increased mitochondria number and volume, as manifested by succinate dehydrogenase staining and transmission electron microscopy. Our tests also suggested that apigenin promoted activities of enzymes such as superoxide dismutase and glutathione peroxidase for antioxidation and those for aerobic respiration such as mitochondrial respiratory enzyme complexes I, II, and IV, increased ATP, and enhanced expression of genes such as peroxisome proliferator-activated receptor-γ coactivator 1α, mitochondrial transcription factor A, nuclear respiratory factor-1, and ATP5B involved in mitochondrial biogenesis. The data also suggested that apigenin inhibited Bcl-2/adenovirus E1B 19kD-interacting protein 3 and DNA fragmentation as indicators of mitophagy and apoptosis in aged mice with skeletal muscle atrophy. Together, the results suggest that apigenin relieves age-related skeletal muscle atrophy through reducing oxidative stress and inhibiting hyperactive autophagy and apoptosis.

Imoxin prevents dexamethasone-induced promotion of muscle-specific E3 ubiquitin ligases and stimulates anabolic signaling in C2C12 myotubes.

Muscle atrophy is the loss of skeletal muscle mass during several pathological conditions such as long-term fasting, aging, cancer, diabetes, sepsis and immune disorders. Glucocorticoids are known to trigger skeletal muscle atrophy. Dexamethasone (DEX), a synthetic glucocorticoid, induces skeletal muscle atrophy by suppression of protein synthesis and promotion of protein degradation. The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) plays a significant role in mediating lipopolysaccharide-induced inflammation. However, pathological roles of PKR in muscle atrophy are not fully understood. The current study aimed to investigate the effect of imoxin, a PKR inhibitor, on DEX-induced muscle atrophy in C2C12 myotubes. Myotubes were incubated with imoxin at different concentrations with or without 5 µM DEX for 24 h. In the current study, imoxin treatment significantly reduced protein levels of MuRF1 and MAFbx induced by DEX by 88 ± 2% and MAFbx by 99 ± 0%, respectively. Moreover, 5 µM imoxin treatment reduced protein ubiquitination by 42 ± 4% and protein content of nuclear FoxO3α (77 ± 4%) in presence of DEX. Furthermore, 5 µM imoxin treatment stimulated Akt phosphorylation (195 ± 5%), mTOR phosphorylation (171 ± 21 %) and p70S6K1 phosphorylation (314 ± 31 %) under DEX-treated condition even though DEX treatment did not suppressed Akt/mTOR/p70S6K1 axis. These findings suggest that imoxin may protect against DEX-induced skeletal muscle atrophy by alleviating muscle specific E3 ubiquitin ligases and imoxin alone may promote protein synthesis via Akt/mTOR/S6K1 axis in muscle cells.

DCAF8, a novel MuRF1 interaction partner, promotes muscle atrophy.

The muscle-specific RING-finger protein MuRF1 (also known as TRIM63) constitutes a bona fide ubiquitin ligase that routes proteins like several different myosin heavy chain proteins (MyHC) to proteasomal degradation during muscle atrophy. In two unbiased screens, we identified DCAF8 as a new MuRF1-binding partner. MuRF1 physically interacts with DCAF8 and both proteins localize to overlapping structures in muscle cells. Importantly, similar to what is seen for MuRF1, DCAF8 levels increase during atrophy, and the downregulation of either protein substantially impedes muscle wasting and MyHC degradation in C2C12 myotubes, a model system for muscle differentiation and atrophy. DCAF proteins typically serve as substrate receptors for cullin 4-type (Cul4) ubiquitin ligases (CRL), and we demonstrate that DCAF8 and MuRF1 associate with the subunits of such a protein complex. Because genetic downregulation of DCAF8 and inhibition of cullin activity also impair myotube atrophy in C2C12 cells, our data imply that the DCAF8 promotes muscle wasting by targeting proteins like MyHC as an integral substrate receptor of a Cul4A-containing ring ubiquitin ligase complex (CRL4A).This article has an associated First Person interview with the first author of the paper.

PDK4 drives metabolic alterations and muscle atrophy in cancer cachexia.

Cachexia is frequently accompanied by severe metabolic derangements, although the mechanisms responsible for this debilitating condition remain unclear. Pyruvate dehydrogenase kinase (PDK)4, a critical regulator of cellular energetic metabolism, was found elevated in experimental models of cancer, starvation, diabetes, and sepsis. Here we aimed to investigate the link between PDK4 and the changes in muscle size in cancer cachexia. High PDK4 and abnormal energetic metabolism were found in the skeletal muscle of colon-26 tumor hosts, as well as in mice fed a diet enriched in Pirinixic acid, previously shown to increase PDK4 levels. Viral-mediated PDK4 overexpression in myotube cultures was sufficient to promote myofiber shrinkage, consistent with enhanced protein catabolism and mitochondrial abnormalities. On the contrary, blockade of PDK4 was sufficient to restore myotube size in C2C12 cultures exposed to tumor media. Our data support, for the first time, a direct role for PDK4 in promoting cancer-associated muscle metabolic alterations and skeletal muscle atrophy.-Pin, F., Novinger, L. J., Huot, J. R., Harris, R. A., Couch, M. E., O'Connell, T. M., Bonetto, A. PDK4 drives metabolic alterations and muscle atrophy in cancer cachexia.

AMP-Activated Protein Kinase as a Key Trigger for the Disuse-Induced Skeletal Muscle Remodeling.

Molecular mechanisms that trigger disuse-induced postural muscle atrophy as well as myosin phenotype transformations are poorly studied. This review will summarize the impact of 5' adenosine monophosphate -activated protein kinase (AMPK) activity on mammalian target of rapamycin complex 1 (mTORC1)-signaling, nuclear-cytoplasmic traffic of class IIa histone deacetylases (HDAC), and myosin heavy chain gene expression in mammalian postural muscles (mainly, soleus muscle) under disuse conditions, i.e., withdrawal of weight-bearing from ankle extensors. Based on the current literature and the authors' own experimental data, the present review points out that AMPK plays a key role in the regulation of signaling pathways that determine metabolic, structural, and functional alternations in skeletal muscle fibers under disuse.

Sarcolemmal loss of active nNOS (Nos1) is an oxidative stress-dependent, early event driving disuse atrophy.

Skeletal muscle atrophy following unloading or immobilization represents a major invalidating event in bedridden patients. Among mechanisms involved in atrophy development, a controversial role is played by neuronal NOS (nNOS; NOS1), whose dysregulation at the protein level and/or subcellular distribution also characterizes other neuromuscular disorders. This study aimed to investigate unloading-induced changes in nNOS before any evidence of myofiber atrophy, using vastus lateralis biopsies obtained from young healthy subjects after a short bed-rest and rat soleus muscles after exposure to short unloading periods. Our results showed that (1) changes in nNOS subcellular distribution using NADPH-diaphorase histochemistry to detect enzyme activity were observed earlier than using immunofluorescence to visualize the protein; (2) loss of active nNOS from the physiological subsarcolemmal localization occurred before myofiber atrophy, i.e. in 8-day bed-rest biopsies and in 6 h-unloaded rat soleus, and was accompanied by increased nNOS activity in the sarcoplasm; (3) nNOS (Nos1) transcript and protein levels decreased significantly in the rat soleus after 6 h and 1 day unloading, respectively, to return to ambulatory levels after 4 and 7 days of unloading, respectively; (4) unloading-induced nNOS redistribution appeared dependent on mitochondrial-derived oxidant species, indirectly measured by tropomyosin disulfide bonds which had increased significantly in the rat soleus already after a 6 h-unloading bout; (5) activity of displaced nNOS molecules is required for translocation of the FoxO3 transcription factor to myofiber nuclei. FoxO3 nuclear localization in rat soleus increased after 6 h unloading (about four-fold the ambulatory level), whereas it did not when nNOS expression and activity were inhibited in vivo before and during 6 h unloading. In conclusion, this study demonstrates that the redistribution of active nNOS molecules from sarcolemma to sarcoplasm not only is ahead of the atrophy of unloaded myofibers, and is induced by increased production of mitochondrial superoxide anion, but also drives FoxO3 activation to initiate muscle atrophy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Structural insights into lethal contractural syndrome type 3 (LCCS3) caused by a missense mutation of PIP5Kγ.

Signaling molecule phosphatidylinositol 4,5-bisphosphate is produced primarily by phosphatidylinositol 4-phosphate 5-kinase (PIP5K). PIP5K is essential for the development of the human neuronal system, which has been exemplified by a recessive genetic disorder, lethal congenital contractural syndrome type 3, caused by a single aspartate-to-asparagine mutation in the kinase domain of PIP5Kγ. So far, the exact role of this aspartate residue has yet to be elucidated. In this work, we conducted structural, functional and computational studies on a zebrafish PIP5Kα variant with a mutation at the same site. Compared with the structure of the wild-type (WT) protein in the ATP-bound state, the ATP-associating glycine-rich loop of the mutant protein was severely disordered and the temperature factor of ATP was significantly higher. Both observations suggest a greater degree of disorder of the bound ATP, whereas neither the structure of the catalytic site nor the Km toward ATP was substantially affected by the mutation. Microsecond molecular dynamics simulation revealed that negative charge elimination caused by the mutation destabilized the involved hydrogen bonds and affected key electrostatic interactions in the close proximity of ATP. Taken together, our data indicated that the disease-related aspartate residue is a key node in the interaction network crucial for effective ATP binding. This work provides a paradigm of how a subtle but critical structural perturbation caused by a single mutation at the ATP-binding site abolishes the kinase activity, emphasizing that stabilizing substrate in a productive conformational state is crucial for catalysis.

Targeted ablation of p38α MAPK suppresses denervation-induced muscle atrophy.

The loss of skeletal muscle mass is a major cause of falls and fractures in the elderly, leading to compromised independence and a decrease in the quality of life. However, only a few therapeutic interventions leading to marginal clinical benefits in patients with this condition are currently available. Therefore, the demand to further understand the pathology of muscle atrophy and establish a treatment modality for patients with muscle atrophy is significant. p38α mitogen-activated protein kinase (p38α MAPK) is a ubiquitous signaling molecule that is implicated in various cellular functions, including cell proliferation, differentiation, and senescence. In the present study, we generated a mutant line in which p38α MAPK is specifically abrogated in muscle tissues. Compared with the control mice, these mutant mice are significantly resistant to denervation-induced muscle atrophy, suggesting that p38α MAPK positively regulates muscle atrophy. We also identified CAMK2B as a potential downstream target of p38α MAPK and found that the pharmacological inhibition of CAMK2B activity suppresses denervation-induced muscle atrophy. Altogether, our findings identify p38α MAPK as a novel regulator of muscle atrophy and suggest that the suppression of intracellular signaling mediated by p38α MAPK serves as a potential target for the treatment of muscle atrophy.

Phosphodiesterase 4B knockout prevents skeletal muscle atrophy in rats with burn injury.

The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.