Michael A. Lopez

Mechanical Stretch Up-regulates MicroRNA-26a and Induces Human Airway Smooth Muscle Hypertrophy by Suppressing Glycogen Synthase Kinase-3β

Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3β (GSK-3β), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3′-untranslated repeat of the GSK-3β mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3β protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3β. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy.

Induction of Sirt1 by Mechanical Stretch of Skeletal Muscle through the Early Response Factor EGR1 Triggers an Antioxidative Response

Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species (1, 2). The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription (3–5). In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging.

FOXO transcription factors are mechanosensitive and their regulation is altered with aging in the respiratory pump

The mechanical regulation of the forkhead box O (FOXO) subclass of transcription factors in the respiratory pump and its implication in aging are completely unknown. We investigated the effects of diaphragm stretch on three FOXO isoforms, Foxo1, Foxo3a, and Foxo4, in normal mice at different ages. We tested the hypotheses that 1) FOXO activities are regulated in response to diaphragm stretch and 2) mechanical properties of aging diaphragm are altered, leading to altered regulation of FOXO with aging. Our results showed that stretch downregulated FOXO DNA-binding activity by a mechanism that required Akt and IKK activation in young mice but that these pathways lost their mechanosensitivity with age. This aberrant regulation of FOXO with aging was associated with altered viscoelasticity, compliance, and extensibility of the aged diaphragm. Curiously, the dramatic decrease of the nuclear content of Foxo1 and Foxo3a, the two isoforms associated with muscle atrophy, with aging correlated with higher basal activation of Akt and IKK signaling in diaphragms of old mice. In contrast, the stability of Foxo4 in the nucleus became dependent on JNK, which is strongly activated in aged diaphragm. This finding suggests that Foxo4 was responsible for the FOXO-dependent transcriptional activity in aging diaphragm. Our data support the hypothesis that aging alters the mechanical properties of the respiratory pump, leading to altered mechanical regulation of the stretch-induced signaling pathways controlling FOXO activities. Our study supports a mechanosensitive signaling mechanism that is responsible for regulation of the FOXO transcription factors by aging.

Anisotropic regulation of Ankrd2 gene expression in skeletal muscle by mechanical stretch

The diaphragm muscles in vivo are subjected to mechanical forces both in the direction of the muscle fibers and in the direction transverse to the fibers. However, the effect of directional mechanical forces in skeletal muscle gene regulation is completely unknown. Here, we identified that stretch in the longitudinal and transverse directions to the diaphragm muscle fibers up‐regulated Ankrd2 gene expression by two distinct signaling pathways in wild‐type (WT) and mdm, a mouse model of muscular dystrophy with early‐onset of progressive muscle‐wasting. Stretch in the longitudinal direction activated both NF‐κB and AP‐1 transcription factors, whereas stretch in the transverse direction activated only AP‐1 transcription factor. Interestingly, longitudinal stretch activated Ankrd2 promoter only by NF‐κB, whereas transverse stretch activated Ankrd2 promoter by AP‐1. Moreover, we found that longitudinal stretch activated Akt, which up‐regulated Ankrd2 expression through NF‐κB. However, transverse stretch activated Ras‐GTP, Raf‐1, and Erk1/2 proteins, which up‐regulated Ankrd2 expression through AP‐1. Surprisingly, the stretch‐activated NF‐κB and AP‐1 signaling pathways was not involved in Ankrd2 regulation at the basal level, which was high in the mdm mouse diaphragm. Taken together, our data show the anisotropic regulation of Ankrd2 gene expression in the diaphragm muscles of WT and mdm mice via two distinct mechanosensitive signaling pathways.—Mohamed, J. S., Lopez, M. A., Cox, G. A., Boriek, A. M. Anisotropic regulation of Ankrd2 gene expression in skeletal muscle by mechanical stretch. FASEB J. 24, 3330–3340 (2010). www.fasebj.org

Early mechanical dysfunction of the diaphragm in the muscular dystrophy with myositis (Ttnmdm) model.

A complex rearrangement mutation in the mouse titin gene leads to an in-frame 83-amino acid deletion in the N2A region of titin. Autosomal recessive inheritance of the titin muscular dystrophy with myositis (Ttn(mdm/mdm)) mutation leads to a severe early-onset muscular dystrophy and premature death. We hypothesized that the N2A deletion would negatively impact the force-generating capacity and passive mechanical properties of the mdm diaphragm. We measured in vitro active isometric contractile and passive length-tension properties to assess muscle function at 2 and 6 wk of age. Micro-CT, myosin heavy chain Western blotting, and histology were used to assess diaphragm structure. Marked chest wall distortions began at 2 wk and progressively worsened until 5 wk. The percentage of myofibers with centrally located nuclei in mdm mice was significantly (P < 0.01) increased at 2 and 6 wk by 4% and 17%, respectively, compared with controls. At 6 wk, mdm diaphragm twitch stress was significantly (P < 0.01) reduced by 71%, time to peak twitch was significantly (P < 0.05) reduced by 52%, and half-relaxation time was significantly (P < 0.05) reduced by 57%. Isometric tetanic stress was significantly (P < 0.05) depressed in 2- and 6-wk mdm diaphragms by as much as 64%. Length-tension relationships of the 2- and 6-wk mdm diaphragms showed significantly (P < 0.05) decreased extensibility and increased stiffness. Slow myosin heavy chain expression was aberrantly favored in the mdm diaphragm at 6 wk. Our data strongly support early contractile and passive mechanical aberrations of the respiratory pump in mdm mice.

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Background: Dystrophic skeletal muscles overexpress ankyrin repeat domain protein 2 (ANKRD2), which inhibits myoblast differentiation. Results: Skeletal muscles of the mdm mouse overexpress ANKRD2 and inhibitor of DNA binding 3 (ID3) proteins, which cooperatively inhibit myoblast differentiation by physical interaction. Conclusion: Activation of SREBP-1/ANKRD2/ID3 pathway impairs, at least in part, skeletal muscle development in mdm mice. Significance: We provide evidence revealing a novel mechanism by which expression of ANKRD2 inhibits myoblast differentiation. Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMBmdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.

Genome-wide Mechanosensitive MicroRNA (MechanomiR) Screen Uncovers Dysregulation of Their Regulatory Networks in the mdm Mouse Model of Muscular Dystrophy.

Background: Dysregulation of mechanosensitive gene regulatory pathways is associated with muscular dystrophies. Results: Genome-wide microRNA analyses revealed dysregulation of pathways controlled by let-7e-5p and miR-98–5p in dystrophic muscle. Conclusion: Impaired mechanomiR-controlled pathways may contribute to muscular dystrophies. Significance: MechanomiR-dependent pathways regulating extracellular matrix proteins and myogenesis are potential therapeutic targets for ameliorating muscle fibrosis and regeneration. Muscular dystrophies (MDs) are a heterogeneous group of genetic and neuromuscular disorders, which result in severe loss of motor ability and skeletal muscle mass and function. Aberrant mechanotransduction and dysregulated-microRNA pathways are often associated with the progression of MD. Here, we hypothesized that dysregulation of mechanosensitive microRNAs (mechanomiRs) in dystrophic skeletal muscle plays a major role in the progression of MD. To test our hypothesis, we performed a genome-wide expression profile of anisotropically regulated mechanomiRs and bioinformatically analyzed their target gene networks. We assessed their functional roles in the advancement of MD using diaphragm muscles from mdm (MD with myositis) mice, an animal model of human tibial MD (titinopathy), and their wild-type littermates. We were able to show that ex vivo anisotropic mechanical stretch significantly alters the miRNA expression profile in diaphragm muscles from WT and mdm mice; as a result, some of the genes associated with MDs are dysregulated in mdm mice due to differential regulation of a distinct set of mechanomiRs. Interestingly, we found a contrasting expression pattern of the highly expressed let-7 family mechanomiRs, let-7e-5p and miR-98–5p, and their target genes associated with the extracellular matrix and TGF-β pathways, respectively, between WT and mdm mice. Gain- and loss-of-function analysis of let-7e-5p in myocytes isolated from the diaphragms of WT and mdm mice confirmed Col1a1, Col1a2, Col3a1, Col24a1, Col27a1, Itga1, Itga4, Scd1, and Thbs1 as target genes of let-7e-5p. Furthermore, we found that miR-98 negatively regulates myoblast differentiation. Our study therefore introduces additional biological players in the regulation of skeletal muscle structure and myogenesis that may contribute to unexplained disorders of MD.

Diaphragm muscle shortening modulates kinematics of lower rib cage in dogs

We tested the hypothesis that diaphragm muscle shortening modulates volume displacement and kinematics of the lower rib cage in dogs and that posture and mode of ventilation affect such modulation. Radiopaque markers were surgically attached to the lower three ribs of the rib cage and to the midcostal region of the diaphragm in six dogs of ∼8 kg body masses, and the locations of these markers were determined by a biplane fluoroscopy system. Three-dimensional software modeling techniques were used to compute volume displacement and surface area of the midcostal diaphragm and the lower three ribs during quiet spontaneous breathing, mechanical ventilation, and bilateral phrenic nerve stimulation at different lung volumes spanning the vital capacity. Volume displaced by the diaphragm relative to that displaced by the lower ribs is disproportionately greater under mechanical ventilation than during spontaneous breathing in the supine position (P < 0.05). At maximal stimulation, diaphragm volume displacement grows disproportionately larger than rib volume displacement as lung volume increases (P < 0.05). Surface area of both the diaphragm and the lower ribs during maximal stimulation of the diaphragm is reduced compared with that at spontaneous breathing (P < 0.05). In the prone posture, mechanical ventilation results in a smaller change in diaphragm surface area than spontaneous breathing (P < 0.05). Our data demonstrate that during inspiration the lower rib cage moves not only through the pump- and bucket-handle motion, but also rotates around the spine. Taken together, these data support the observation that the kinematics of the lower rib cage and its mechanical interaction with the diaphragm are more complex than previously known.

Pediatric Tumefactive Demyelination

In children presenting with neurological deficits associated with large parenchymal lesions on imaging, brain tumors and cerebral abscess are typically considered in the differential diagnosis. However, it is important to also consider tumefactive demyelinating lesions, which can be challenging to diagnose. Clinical and radiographic features may help to avoid a brain biopsy. This chapter presents a case of a 14-year-old boy with cognitive impairment and behavioral abnormalities who had large multifocal supratentorial lesions. The case reviews distinguishing signs, symptoms, and ancillary diagnostic tests that can help to distinguish between the potential etiologies.

Anisotropic mechanosensitive pathways in the diaphragm and their implications in muscular dystrophies

The diaphragm is the “respiratory pump;” the muscle that generates pressure to allow ventilation. Diaphragm muscles play a vital function and thus are subjected to continuous mechanical loading. One of its peculiarities is the ability to generate distinct mechanical and biochemical responses depending on the direction through which the mechanical forces applied to it. Contractile forces originated from its contractile components are transmitted to other structural components of its muscle fibers and the surrounding connective tissue. The anisotropic mechanical properties of the diaphragm are translated into biochemical signals that are directionally mechanosensitive by mechanisms that appear to be unique to this muscle. Here, we reviewed the current state of knowledge on the biochemical pathways regulated by mechanical signals emphasizing their anisotropic behavior in the normal diaphragm and analyzed how they are affected in muscular dystrophies.