Autophagy-Associated Atrophy and Metabolic Remodeling of the Mouse Diaphragm after Short-Term Intermittent Hypoxia

Background

Short-term intermittent hypoxia (IH) is common in patients with acute respiratory disorders. Although prolonged exposure to hypoxia induces atrophy and increased fatigability of skeletal muscle, the response to short-term IH is less well known. We hypothesized that the diaphragm and limb muscles would adapt differently to short-term IH given that hypoxia stimulates ventilation and triggers a superimposed exercise stimulus in the diaphragm.

Methods

We determined the structural, metabolic, and contractile properties of the mouse diaphragm after 4 days of IH (8 hours per day, 30 episodes per hour to a FiO2 nadir=6%), and compared responses in the diaphragm to a commonly studied reference limb muscle, the tibialis anterior. Outcome measures included muscle fiber size, assays of muscle proteolysis (calpain, ubiquitin-proteasome, and autophagy pathways), markers of oxidative stress and mitochondrial function, quantification of intramyocellular lipid and lipid metabolism genes, type I myosin heavy chain (MyHC) expression, and in vitro contractile properties.

Results

After 4 days of IH, the diaphragm alone demonstrated significant atrophy (30% decrease of myofiber size) together with increased LC3B-II protein (2.4-fold) and mRNA markers of the autophagy pathway (LC3B, Gabarapl1, Bnip3), whereas active calpain and E3 ubiquitin ligases (MuRF1, atrogin-1) were unaffected in both muscles. Succinate dehydrogenase activity was significantly reduced by IH in both muscles. However, only the diaphragm exhibited increased intramyocellular lipid droplets (2.5-fold) after IH, along with upregulation of genes linked to activated lipid metabolism. In addition, although the diaphragm showed evidence for acute fatigue immediately following IH, it underwent an adaptive fiber type switch toward slow type I MyHC-expressing fibers, associated with greater intrinsic endurance of the muscle during repetitive stimulation in vitro.

Conclusions

Short-term IH induces preferential atrophy in the mouse diaphragm together with increased autophagy and a rapid compensatory metabolic adaptation associated with enhanced fatigue resistance.     

Cross-taxon congruence of α and β diversity among five leaf litter arthropod groups in Colombia

In this study α and β diversity patterns of five leaf litter arthropod groups (ants, predatory ants, oribatid mites, spiders and other arachnids) were described and compared in 39 sampling patches of a transformed landscape in southwestern Colombia, that represented five vegetation types: secondary forest, riparian forest, giant bamboo forest, pasture and sugarcane crop. It was also assessed whether some taxa could be used as diversity surrogates. A total of 6,765 individuals grouped in 290 morphospecies were collected. Species richness in all groups was lower in highly transformed vegetation types (pasture, sugarcane crop) than in native ones (forests). In contrast, there were no clear tendencies of β diversity among vegetation types. Considering sampling patches, 0.1–42% of the variation in α diversity of one taxonomic group could be explained from the α diversity of another, and 0.2–33% of the variation of β diversity of a given taxon was explained by that in other groups. Contrary to recent findings, we concluded that patterns of α diversity are more congruent than patterns of β diversity. This fact could be attributed to a sampling effect that promotes congruence in α diversity and to a lack of a clear regional ecological gradient that could promote congruent patterns of β diversity. We did not find evidence for an ideal diversity surrogate although diversity patterns of predatory ants had the greatest congruencies. These results support earlier multi-taxon evaluations in that conservation planning should not be based on only one leaf litter arthropod group.     

Morphological evolution through integration: a quantitative study of cranial integration in Homo, Pan, Gorilla and Pongo

Morphological integration refers to coordinated variation among traits that are closely related in development and/or function. Patterns of integration can offer important insight into the structural relationship between phenotypic units, providing a framework to address questions about phenotypic evolvability and constraints. Integrative features of the primate cranium have recently become a popular subject of study. However, an important question that still remains under-investigated is: what is the pattern of cranial shape integration among closely related hominoids? To address this question, we conducted a Procrustes-based geometric morphometrics study to quantify and analyze shape covariation patterns between different cranial regions in Homo, Pan, Gorilla and Pongo. A total of fifty-six 3D landmarks were collected on 407 adult individuals. We then sub-divided the landmarks corresponding to cranial units as outlined in the ‘functional matrix hypothesis.’ Sub-dividing the cranium in this manner allowed us to explore patterns of covariation between the face, basicranium and cranial vault, using the two-block partial least squares approach. Our results suggest that integrated shape changes in the hominoid cranium are complex, but that the overall pattern of integration is similar among human and non-human apes. Thus, despite having very distinct morphologies the way in which the face, basicranium and cranial vault covary is shared among these taxa. These results imply that the pattern of cranial integration among hominoids is conserved.     

Depot formation of doxycycline impairs Tet-regulated gene expression in vivo

The tetracycline (Tet) system is widely used for regulation of gene expression in vitro and in vivo. We constructed C57BL/6 transgenic mice (rtTA-CM2) with strong and ubiquitous reverse transactivator (rtTA2(S)-M2) gene expression. rtTA-CM2 mice were crossed to Tet-responsive reporter mice (LC-1) conditionally expressing the firefly luciferase (FLuc) gene under control of a Tet-responsive element, which allowed sensitive quantification of the transactivator activity by bioluminescent imaging. Following doxycycline (dox) application, up to 10(5)-fold increase in BL signal was measured. rtTA activity was inducible in most analyzed organs. After dox withdrawal the BL signal decreased significantly but did not disappear completely, most likely due to a dox depot formation in vivo. The residual dox was sufficient to partly down-regulate a Tet-off controlled oncogene in a tumor transplantation experiment, resulting in reduced tumor growth. rtTA-CM2 mice may be a useful tool to analyze the function of genes in various organs but also reveal that down-regulation of gene expression is not complete.     

Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine α4β2 receptors: unique role of halogen bonding revealed

The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4β2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4β2 receptors. Crystal structures of five agonists with efficacies at α4β2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.     

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.     

Masked red-emitting carbopyronine dyes with photosensitive 2-diazo-1-indanone caging group

Caged near-IR emitting fluorescent dyes are in high demand in optical microscopy but up to now were unavailable. We discovered that the combination of a carbopyronine dye core and a photosensitive 2-diazo-1-indanone residue leads to masked near-IR emitting fluorescent dyes. Illumination of these caged dyes with either UV or visible light (λ < 420 nm) efficiently generates fluorescent compounds with absorption and emission at 635 nm and 660 nm, respectively. A high-yielding synthetic route with attractive possibilities for further dye design is described in detail. Good photostability, high contrast, and a large fluorescence quantum yield after uncaging are the most important features of the new compounds for non-invasive imaging in high-resolution optical microscopy. For use in immunolabelling the caged dyes were decorated with a (hydrophilic) linker and an (activated) carboxyl group.     

Probing binding pocket of serotonin transporter by single molecular force spectroscopy on living cells

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: koff values were in good agreement with those derived from uptake experiments; in contrast, the kon values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction.     

Effects of Coxiella burnetii on MAPKinases phosphorylation

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.     

Nonlinear viscoelasticty plays an essential role in the functional behavior of spinal ligaments

Despite the significant role ligament viscoelasticity plays in functional spinal biomechanics, relatively few studies have been performed to develop constitutive models that explicitly characterize this complex behavior. Unfortunately, the application and interpretation of these previous models are limited due to the use of simplified (quasi-linear) viscoelastic formulations or characterization techniques that have been shown to affect the predictive accuracy of the fitted coefficients. In order to surmount these previous limitations, the current study presents the application of a novel fitting technique (applied to stress relaxation experiments) and nonlinear viscoelastic constitutive formulation to human cervical spine anterior longitudinal ligament (ALL), posterior longitudinal ligament (PLL) and ligamentum flavum (LF). The fitted coefficients were validated by quantifying the ability of the constitutive equation to predict an independent cyclic data set across multiple physiologic strain amplitudes and frequencies. The resulting validated constitutive formulation indicated that the strain-dependent viscoelastic behavior of the longitudinal ligaments (ALL and PLL) was dominated by both the short-term (t=0.1s) and the steady-state (as t→∞) behavior. Conversely, the LF exhibited consistent relaxation behavior across the investigated temporal spectrum. From these data, it can be hypothesized that the unique strain-dependent temporal behavior of these spinal ligaments may be a functional adaptation that minimizes muscular expenditure during quasi-static postures while maximizing structural stability of the spine during transient loading events.