Preventing occludin tight-junction disruption via inhibition of microRNA-193b-5p attenuates viral load and influenza-induced lung injury.

Virus-induced lung injury is associated with loss of pulmonary epithelial-endothelial tight junction integrity. While the alveolar-capillary membrane may be an indirect target of injury, viruses may interact directly and/or indirectly with miRs to augment their replication potential and evade the host antiviral defense system. Here, we expose how the influenza virus (H1N1) capitalizes on host-derived interferon-induced, microRNA (miR)-193b-5p to target occludin and compromise antiviral defenses. Lung biopsies from patients infected with H1N1 revealed increased miR-193b-5p levels, marked reduction in occludin protein, and disruption of the alveolar-capillary barrier. In C57BL/6 mice, the expression of miR-193b-5p increased, and occludin decreased, 5-6 days post-infection with influenza (PR8). Inhibition of miR-193b-5p in primary human bronchial, pulmonary microvascular, and nasal epithelial cells enhanced antiviral responses. miR-193b-deficient mice were resistant to PR8. Knockdown of occludin, both in vitro and in vivo, and overexpression of miR-193b-5p reconstituted susceptibility to viral infection. miR-193b-5p inhibitor mitigated loss of occludin, improved viral clearance, reduced lung edema, and augmented survival in infected mice. Our results elucidate how the innate immune system may be exploited by the influenza virus and how strategies that prevent loss of occludin and preserve tight junction function may limit susceptibility to virus-induced lung injury.

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.

Loss of vascular smooth muscle cell autophagy exacerbates angiotensin II-associated aortic remodeling.

OBJECTIVE: The pathophysiologic processes of abdominal aortic aneurysms (AAAs) and atherosclerosis often intersect. Given that anomalies in vascular smooth muscle cell (SMC) autophagy have been noted in models of atherosclerosis, we sought to evaluate the potential role that SMC autophagy may play in the initiation and progression of AAAs. METHODS: Studies were conducted in ATG7flx/flxSM22α-Cretg/+ (SMC ATG7 knockout [SMC-ATG7-KO]) and ATG7WT/WT; SM22α-Cretg/+ (SMC ATG7 wild-type [SMC-ATG7-WT]) littermates that were continuously infused with angiotensin II (Ang II; 1.5 mg/kg/d) for up to 12 weeks. Mortality, morbidity, hemodynamics, and aortic remodeling were documented. RESULTS: During the 12-week observation window, all of the Ang II-treated SMC-ATG-WT mice (n = 6) survived, whereas 10 of the 19 Ang II-treated SMC-ATG-KO mice had died by week 7 (log-rank test, P < .001). Mean arterial pressure (128.07 ± 3.4 mm Hg for Ang II-treated SMC-ATG-KO vs 138.5 ± 5.87 mm Hg for Ang II-treated SMC-ATG-WT mice) and diastolic arterial pressure (109.7 ± 2.55 mm Hg for Ang II-treated SMC-ATG7-KO vs 119.4 ± 2.12 mm Hg for Ang II-treated SMC-ATG7-WT mice) were significantly different between the two groups (P < .01). Cardiac rupture, myocardial infarct, end-organ damage, pleural effusion, and venous distention were noted in Ang II-treated SMC-ATG7-KO but not in Ang II-treated SMC-ATG7-WT mice. Although the suprarenal aortic diameters of the Ang II-treated SMC-ATG7-KO group demonstrated a trending increase (at week 4, 1.26 ± 0.06 mm [n = 14] for Ang II-treated SMC-ATG-KO mice vs 1.09 ± 0.02 mm [n = 5] for Ang II-treated SMC-ATG-WT mice; P < .05), only 2 of the 19 developed abdominal aortic dissections. CONCLUSIONS: Mice with SMC ATG7 deficiency that are chronically infused with Ang II do not tend to develop dissecting AAA but do exhibit adverse aortic remodeling and appreciable cardiac failure-associated mortality.

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.

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.

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.

A polarity complex of mPar-6 and atypical PKC binds, phosphorylates and regulates mammalian Lgl.

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.

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.

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.

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 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.