Utilization of the Rat Tibial Nerve Transection Model to Evaluate Cellular and Molecular Mechanisms Underpinning Denervation-Mediated Muscle Injury.

Peripheral nerve injury denervates muscle, resulting in muscle paralysis and atrophy. This is reversible if timely muscle reinnervation occurs. With delayed reinnervation, the muscle's reparative ability declines, and muscle-resident fibro-adipogenic progenitor cells (FAPs) proliferate and differentiate, inducing fibro-fatty muscle degradation and thereby physical disability. The mechanisms by which the peripheral nerve regulates FAPs expansion and differentiation are incompletely understood. Using the rat tibial neve transection model, we demonstrated an increased FAPs content and a changing FAPs phenotype, with an increased capacity for adipocyte and fibroblast differentiation, in gastrocnemius muscle post-denervation. The FAPs response was inhibited by immediate tibial nerve repair with muscle reinnervation via neuromuscular junctions (NMJs) and sensory organs (e.g., muscle spindles) or the sensory protection of muscle (where a pure sensory nerve is sutured to the distal tibial nerve stump) with reinnervation by muscle spindles alone. We found that both procedures reduced denervation-mediated increases in glial-cell-line-derived neurotrophic factor (GDNF) in muscle and that GDNF promoted FAPs adipogenic and fibrogenic differentiation in vitro. These results suggest that the peripheral nerve controls FAPs recruitment and differentiation via the modulation of muscle GDNF expression through NMJs and muscle spindles. GDNF can serve as a therapeutic target in the management of denervation-induced muscle injury.

MicroRNA regulatory networks associated with abnormal muscle repair in survivors of critical illness.

BACKGROUND: Intensive care unit (ICU)-acquired weakness is characterized by muscle atrophy and impaired contractility that may persist after ICU discharge. Dysregulated muscle repair and regeneration gene co-expression networks are present in critical illness survivors with persistent muscle wasting and weakness. We aimed to identify microRNAs (miRs) regulating the gene networks and determine their role in the self-renewal of muscle in ICU survivors. METHODS: Muscle whole-transcriptome expression was assessed with microarrays in banked quadriceps biopsies obtained at 7 days and 6 months post-ICU discharge from critically ill patients (n = 15) in the RECOVER programme and healthy individuals (n = 8). We conducted an integrated miR-messenger RNA analysis to identify miR/gene pairs associated with muscle recovery post-critical illness and evaluated their impact on myoblast proliferation and differentiation in human AB1167 and murine C2C12 cell lines in vitro. Select target genes were validated with quantitative PCR. RESULTS: Twenty-two miRs were predicted to regulate the Day 7 post-ICU muscle transcriptome vs. controls. Thirty per cent of all differentially expressed genes shared a 3'UTR regulatory sequence for miR-424-3p/5p, which was 10-fold down-regulated in patients (P < 0.001) and correlated with quadriceps size (R = 0.86, P < 0.001), strength (R = 0.75, P = 0.007), and physical function (Functional Independence Measures motor subscore, R = 0.92, P < 0.001) suggesting its potential role as a master regulator of early recovery of muscle mass and strength following ICU discharge. Network analysis demonstrated enrichment for cellular respiration and muscle fate commitment/development related genes. At 6 months post-ICU discharge, a 14-miR expression signature, including miRs-490-3p and -744-5p, identified patients with muscle mass recovery vs. those with sustained atrophy. Constitutive overexpression of the novel miR-490-3p significantly inhibited AB1167 and C2C12 myoblast proliferation (cell count AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 7926 ± 4060 vs. 14 159 ± 3515 respectively, P = 0.006; proportion Ki67-positive nuclei AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 0.38 ± 0.07 vs. 0.54 ± 0.06 respectively, P < 0.001; proliferating cell nuclear antigen expression AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 11.48 ± 1.97 vs. 16.75 ± 1.19 respectively, P = 0.040). Constitutive overexpression of miR-744-5p, a known regulator of myogenesis, significantly inhibited AB1167 and C2C12 myoblast differentiation (fusion index AB1167 miR-744-5p mimic or scrambled-miR transfected myoblasts 8.31 ± 7.00% vs. 40.29 ± 9.37% respectively, P < 0.001; myosin heavy chain expression miR-744-5p mimic or scrambled-miR transfected myoblasts 0.92 ± 0.39 vs. 13.53 ± 5.5 respectively, P = 0.01). CONCLUSIONS: Combined functional transcriptomics identified 36 miRs including miRs-424-3p/5p, -490-3p, and -744-5p as potential regulators of gene networks associated with recovery of muscle mass and strength following critical illness. MiR-490-3p is identified as a novel regulator of myogenesis.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Mechanisms of Chronic Muscle Wasting and Dysfunction after an Intensive Care Unit Stay. A Pilot Study.

RATIONALE: Critical illness survivors often experience permanent functional disability due to intensive care unit (ICU)-acquired weakness. The mechanisms responsible for long-term weakness persistence versus resolution are unknown. OBJECTIVES: To delineate cellular mechanisms underlying long-term weakness persistence in ICU survivors. METHODS: We conducted a nested, prospective study of critically ill patients mechanically ventilated for 7 days or longer. The patients were recruited from the RECOVER program and serially assessed over 6 months after ICU discharge. Twenty-seven of 82 patients consented to participate; 15 and 11 patients were assessed at 7 days and 6 months after ICU discharge, respectively. MEASUREMENTS AND MAIN RESULTS: We assessed motor functional capacity, quadriceps size, strength, and voluntary contractile capacity and performed electromyography, nerve conduction studies, and vastus lateralis biopsies for histologic, cellular, and molecular analyses. Strength and quadriceps cross-sectional areas were decreased 7 days after ICU discharge. Weakness persisted to 6 months and correlated with decreased function. Quadriceps atrophy resolved in 27% patients at 6 months. Muscle mass reconstitution did not correlate with resolution of weakness, owing to persistent impaired voluntary contractile capacity. Compared with Day 7, increased ubiquitin-proteasome system-mediated muscle proteolysis, inflammation, and decreased mitochondrial content all normalized at 6 months. Autophagy markers were normal at 6 months. Patients with sustained atrophy had decreased muscle progenitor (satellite) cell content. CONCLUSIONS: Long-term weakness in ICU survivors results from heterogeneous muscle pathophysiology with variable combinations of muscle atrophy and impaired contractile capacity. These findings are not explained by ongoing muscle proteolysis, inflammation, or diminished mitochondrial content. Sustained muscle atrophy is associated with decreased satellite cell content and compromised muscle regrowth, suggesting impaired regenerative capacity.

PDLIM7 is a novel target of the ubiquitin ligase Nedd4-1 in skeletal muscle.

Skeletal muscle atrophy remains a complication occurring both as a natural response to muscle disuse and as a pathophysiological response to illness such as diabetes mellitus and nerve injury, such as traumatic muscle denervation. The ubiquitin-proteasome system (UPS) is the predominant proteolytic machinery responsible for atrophy of skeletal muscle, and Nedd4-1 (neural precursor cell-expressed developmentally down-regulated 4-1) is one of a series of E3 ubiquitin ligases identified to mediate inactivity-induced muscle wasting. Targets of Nedd4-1 mediated ubiquitination in skeletal muscle remain poorly understood. In the present study, we identified PDLIM7 (PDZ and LIM domain 7, Enigma), a member of the PDZ-LIM family of proteins, as a novel target of Nedd4-1 in skeletal muscle. The PDZ-LIM family of proteins is known to regulate muscle development and function. We show that Nedd4-1 expression in muscle atrophied by denervation is co-incident with a decrease in PDLIM7 and that PDLIM7 protein levels are stabilized in denervated muscle of Nedd4-1 skeletal muscle-specific knockout mice (SMS-KO). Exogenous PDLIM7 and Nedd4-1 transfected into human embryonic kidney (HEK)293 cells co-immunoprecipitate through binding between the PY motif of PDLIM7 and the second and third WW domains of Nedd4-1 and endogenous PDLIM7 and Nedd4-1 interact in the cytoplasm of differentiated C2C12 myotubes, leading to PDLIM7 ubiquitination. These results identify PDLIM7 as a bona fide skeletal muscle substrate of Nedd4-1 and suggest that this interaction may underlie the progression of skeletal muscle atrophy. This offers a novel therapeutic target that could be potentially used to attenuate muscle atrophy.

Skeletal muscle dysfunction in idiopathic pulmonary arterial hypertension.

Despite improvements in survival with disease-targeted therapies, the majority of patients with pulmonary arterial hypertension (PAH) have persistent exercise intolerance that results from impaired cardiac function and skeletal muscle dysfunction. Our intent was to understand the molecular mechanisms mediating skeletal muscle dysfunction in PAH. A total of 12 patients with PAH and 10 matched control subjects were assessed. Patients with PAH demonstrated diminished exercise capacity (lower oxygen uptake max, lower anaerobic threshold and higher minute ventilation/CO2) compared with control subjects. Quadriceps muscle cross-sectional area was significantly smaller in patients with PAH. The vastus lateralis muscle was biopsied to enable muscle fiber morphometric assessment and to determine expression levels/activation of proteins regulating (1) muscle mass, (2) mitochondria biogenesis and shaping machinery, and (3) excitation-contraction coupling. Patients with PAH demonstrated a decreased type I/type II muscle fiber ratio, with a smaller cross-sectional area in the type I fibers. Diminished AKT and p70S6 kinase phosphorylation, with increased atrogin-1 and muscle RING-finger protein-1 transcript levels, were evident in the PAH muscle, suggesting engagement of cellular signaling networks stimulating ubiquitin-proteasome-mediated proteolysis of muscle, with concurrent depression of networks mediating muscle hypertrophy. Although there were no differences in expression/activation of proteins associated with mitochondrial biogenesis or fission (MTCO2 [cytochrome C oxidase subunit II]/succinate dehydrogenase flavoprotein subunit A, mitochondrial transcription factor A, nuclear respiratory factor-1/dynamin-related protein 1 phosphorylation), protein levels of a positive regulator of mitochondrial fusion, Mitofusin2, were significantly lower in patients with PAH. Patients with PAH demonstrated increased phosphorylation of ryanodine receptor 1 receptors, suggesting that altered sarcoplasmic reticulum Ca(++) sequestration may impair excitation-contraction coupling in the PAH muscle. These data suggest that muscle dysfunction in PAH results from a combination of muscle atrophy and intrinsically impaired contractility.

The ubiquitin ligase Nedd4-1 participates in denervation-induced skeletal muscle atrophy in mice.

Skeletal muscle atrophy is a consequence of muscle inactivity resulting from denervation, unloading and immobility. It accompanies many chronic disease states and also occurs as a pathophysiologic consequence of normal aging. In all these conditions, ubiquitin-dependent proteolysis is a key regulator of the loss of muscle mass, and ubiquitin ligases confer specificity to this process by interacting with, and linking ubiquitin moieties to target substrates through protein:protein interaction domains. Our previous work suggested that the ubiquitin-protein ligase Nedd4-1 is a potential mediator of skeletal muscle atrophy associated with inactivity (denervation, unloading and immobility). Here we generated a novel tool, the Nedd4-1 skeletal muscle-specific knockout mouse (myo(Cre);Nedd4-1(flox/flox)) and subjected it to a well validated model of denervation induced skeletal muscle atrophy. The absence of Nedd4-1 resulted in increased weights and cross-sectional area of type II fast twitch fibres of denervated gastrocnemius muscle compared with wild type littermates controls, at seven and fourteen days following tibial nerve transection. These effects are not mediated by the Nedd4-1 substrates MTMR4, FGFR1 and Notch-1. These results demonstrate that Nedd4-1 plays an important role in mediating denervation-induced skeletal muscle atrophy in vivo.

Hypertrophic airway smooth muscle mass correlates with increased airway responsiveness in a murine model of asthma.

The increase of airway smooth muscle (ASM) mass in asthma results from hypertrophic and hyperplastic stimuli, and leads to an increase in cellular contractile proteins. However, little evidence correlates the relative contributions of hypertrophic and hyperplastic muscle with functional effects on airway resistance. We performed a ventilator-based assessment of respiratory mechanics and responsiveness to methacholine in a murine model of acute (3-week) ovalbumin (OVA)-induced airway inflammation, compared with a chronic (12-week) model. We correlated functional changes in airways Newtonian resistance (RN), peripheral tissue damping (G), and elastance (H) with the relative contributions of proliferation, hypertrophy, and apoptosis to increased ASM mass. Immunohistochemical analyses of treated (OVA-sensitized and OVA-challenged; OVA/OVA) and control (OVA-sensitized and saline-challenged; OVA/PBS) murine lungs showed an increase in ASM area in chronic, but not acute, OVA/OVA-treated mice that correlated positively with increased airway resistance to methacholine. Acute OVA/OVA-treated ASM exhibited an increase in proliferation with diminished apoptosis, which resolved in the chronic OVA/OVA model. Chronic OVA/OVA-treated ASM exhibited hypertrophy. Distinct temporal differences exist in the response of murine airways to antigenic challenge. We report that ASM proliferation and diminished apoptosis occur during the acute phase, followed by the development of smooth muscle hypertrophy and an increased muscle mass with chronic challenge, that correlate strongly with increased airway Newtonian resistance. The identification of a functionally relevant hypertrophic bronchial muscle mass highlights the possibility of regulating airway muscle hypertrophy as a novel therapeutic target in asthma.

Cellular markers of muscle atrophy in chronic obstructive pulmonary disease.

Skeletal muscle atrophy in individuals with advanced chronic obstructive pulmonary disease (COPD) is associated with diminished quality of life, increased health resource use, and worsened survival. Muscle wasting results from an imbalance between protein degradation and synthesis, and is enhanced by decreased regenerative repair. We investigated the activation of cellular signaling networks known to mediate muscle atrophy and regulate muscle regenerative capacity in rodent models, in individuals with COPD (FEV(1) < 50% predicted). Nine patients with COPD and nine control individuals were studied. Quadriceps femoris muscle isometric contractile force and cross-sectional area were confirmed to be significantly smaller in the patients with COPD compared with control subjects. The vastus lateralis muscle was biopsied and muscle transcript and/or protein levels of key components of ubiquitin-mediated proteolytic systems (MuRF1, atrogin-1, Nedd4), inflammatory mediators (IkappaBalpha, NF-kappaBp65/p50), AKT network (AKT, GSK3beta, p70S6 kinase), mediators of autophagy (beclin-1, LC3), and myogenesis (myogenin, MyoD, Myf5, myostatin) were determined. Atrogin-1 and Nedd4, two ligases regulating ubiquitin-mediated protein degradation and myostatin, a negative regulator of muscle growth, were significantly increased in the muscle of patients with COPD. MuRF1, Myf5, myogenin, and MyoD were not differentially expressed. There were no differences in the level of phosphorylation of AKT, GSK3beta, p70S6kinase, or IkappaBalpha, activation of NF-kappaBp65 or NF-kappaBp50, or level of expression of beclin-1 or LC3, suggesting that AKT signaling was not down-regulated and the NF-kappaB inflammatory pathway and autophagy were not activated in the COPD muscle. We conclude that muscle atrophy associated with COPD results from the recruitment of specific regulators of ubiquitin-mediated proteolytic pathways and inhibition of muscle growth.

Absence of caspase-3 protects against denervation-induced skeletal muscle atrophy.

The ubiquitin-proteasome system is a key proteolytic pathway activated during skeletal muscle atrophy. The proteasome, however, cannot degrade intact myofibrils or actinomyosin complexes. In rodent models of diabetes mellitus and uremia, caspase-3 is involved in actinomyosin cleavage, generating fragments that subsequently undergo ubiquitin-proteasome-mediated degradation. Here, we demonstrate that caspase-3 also mediates denervation-induced muscle atrophy. At 2 wk after tibial nerve transection, the denervated gastrocnemius of caspase-3-knockout mice weighed more and demonstrated larger fiber-type-specific cross-sectional area than the denervated gastrocnemius of wild-type mice. However, there was no difference between caspase-3-knockout and wild-type denervated muscles in the magnitude or pattern of actinomyosin degradation, as determined by Western blotting for actin and the 14-kDa actin fragment. Similarly, there was no difference between caspase-3-knockout and wild-type denervated muscles in the magnitude of increase in proteasome activity, total protein ubiquitination, or atrogin-1 and muscle-specific ring finger protein 1 transcript levels. In contrast, there was an increase in TdT-mediated dUTP nick end label-positive nuclei in the denervated muscle of wild-type compared with caspase-3-knockout mice. Apoptotic signaling upstream of caspase-3 remained intact, with equivalent mitochondrial Bax translocation and cytochrome c release and caspase-9 activation in the denervated gastrocnemius muscle of wild-type and caspase-3-knockout mice. In contrast, diminished poly(ADP-ribose) polymerase cleavage in the denervated muscle of caspase-3-knockout compared with wild-type mice revealed that apoptotic signaling downstream of caspase-3 was impaired, suggesting that the absence of caspase-3 protects against denervation-induced muscle atrophy by suppressing apoptosis as opposed to ubiquitin-proteasome-mediated protein degradation.

The inositol phosphatase MTMR4 is a novel target of the ubiquitin ligase Nedd4.

The inositol phosphatase, MTMR4 (myotubularin-related protein 4), was identified as a novel interactor of the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). hMTMR4 (human MTMR4) and Nedd4 co-immunoprecipitated and co-localized to late endosomes. The PY (Pro-Tyr) motif of hMTMR4 binds to WW (Trp-Trp) domains of hNedd4. MTMR4 expression was decreased in atrophying muscle, whereas Nedd4 expression was increased and hMTMR4 was ubiquitinated by hNedd4, suggesting that this novel interaction may underlie the biological process of muscle breakdown.