Expression Of Calcium-buffering Proteins In Rat Intrinsic Laryngeal Muscles.

Intrinsic laryngeal muscles (ILM) are highly specialized muscles involved in phonation and airway protection, with unique properties that allow them to perform extremely rapid contractions and to escape from damage in muscle dystrophy. Due to that, they may differ from limb muscles in several physiological aspects. Because a better ability to handle intracellular calcium has been suggested to explain ILM unique properties, we hypothesized that the profile of the proteins that regulate calcium levels in ILM is different from that in a limb muscle. Calcium-related proteins were analyzed in the ILM, cricothyroid (CT), and tibialis anterior (TA) muscles from male Sprague-Dawley rats (8 weeks of age) using quantitative PCR and western blotting. Higher expression of key Ca(2+) regulatory proteins was detected in ILM compared to TA, such as the sarcoplasmic reticulum (SR) Ca(2+)-reuptake proteins (Sercas 1 and 2), the Na(+)/Ca(2+) exchanger, phospholamban, and the Ca(2+)-binding protein calsequestrin. Parvalbumin, calmodulin and the ATPase, Ca(2+)-transporting, and plasma membrane 1 were also expressed at higher levels in ILM compared to TA. The store-operated calcium entry channel molecule was decreased in ILM compared to the limb muscle and the voltage-dependent L-type and ryanodine receptor were expressed at similar levels in ILM and TA. These results show that ILM have a calcium regulation system profile suggestive of a better ability to handle calcium changes in comparison to limb muscles, and this may provide a mechanistic insight for their unique pathophysiological properties.

New Insights into the Lactate Shuttle: Role of MCT4 in the Modulation of the Exercise Capacity.

Lactate produced by muscle during high-intensity activity is an important end product of glycolysis that supports whole body metabolism. The lactate shuttle model suggested that lactate produced by glycolytic muscle fibers is utilized by oxidative fibers. MCT4 is a proton coupled monocarboxylate transporter preferentially expressed in glycolytic muscle fibers and facilitates the lactate efflux. Here we investigated the exercise capacity of mice with disrupted lactate shuttle due to global deletion of MCT4 (MCT4−/−) or muscle-specific deletion of the accessory protein Basigin (iMSBsg−/−). Although MCT4−/− and iMSBsg−/− mice have normal muscle morphology and contractility, only MCT4−/− mice exhibit an exercise intolerant phenotype. In vivo measurements of compound muscle action potentials showed a decrement in the evoked response in the MCT4−/− mice. This was accompanied by a significant structural degeneration of the neuromuscular junctions (NMJs). We propose that disruption of the lactate shuttle impacts motor function and destabilizes the motor unit

Het gaat weer beter met de natuur in Nederland

In een Volkskrantinterview (22 mei 2009) poneerden de eerste drie auteurs de stelling, dat het weer beter gaat met de Nederlandse natuur. Aanleiding vormde de presentatie van de Monitor Duurzaam Nederland (CBS, 2009) waarbij het Planbureau concludeerde dat de biodiversiteit in ons land nog steeds achteruitholt. Dit artikel onderbouwt de stelling van 22 mei en is in feite een vervolg op een eerdere discussie, waarin de methodiek van het PBL werd gepresenteerd. Dit artikel gaat uit van de vraag hoe het gaat met de biodiversiteit en kijkt van daaruit naar de methode

Tibetan PHD2, an allele with loss-of-function properties

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A. The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude

Translational Regulation of Utrophin by miRNAs

Background Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne Muscular Dystrophy (DMD) locus. Its regulation is of therapeutic interest as its overexpression can compensate for dystrophin's absence in animal models of DMD. The tissue distribution and transcriptional regulation of utrophin have been characterized extensively, and more recently translational control mechanisms that may underlie its complex expression patterns have begun to be identified. Methodology/Principal Findings Using a variety of bioinformatic, molecular and cell biology techniques, we show that the muscle isoform utrophin-A is predominantly suppressed at the translational level in C2C12 myoblasts. The extent of translational inhibition is estimated to be ~99% in C2C12 cells and is mediated by both the 5′- and 3′-UTRs of the utrophin-A mRNA. In this study we identify five miRNAs (let-7c, miR-150, miR-196b, miR-296-5p, miR-133b) that mediate the repression, and confirm repression by the previously identified miR-206. We demonstrate that this translational repression can be overcome by blocking the actions of miRNAs, resulting in an increased level of utrophin protein in C2C12 cells. Conclusions/Significance The present study has identified key inhibitory mechanisms featuring miRNAs that regulate utrophin expression, and demonstrated that these mechanisms can be targeted to increase endogenous utrophin expression in cultured muscle cells. We suggest that miRNA-mediated inhibitory mechanisms could be targeted by methods similar to those described here as a novel strategy to increase utrophin expression as a therapy for DMD

Drug Discovery for Duchenne Muscular Dystrophy via Utrophin Promoter Activation Screening

Background: Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use. Methodology/Principal Findings: We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells. Conclusions/Significance: We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics an

Chronic Hypoxia Impairs Muscle Function in the Drosophila Model of Duchenne's Muscular Dystrophy (DMD)

Duchenne's muscular dystrophy (DMD) is a severe progressive myopathy caused by mutations in the DMD gene leading to a deficiency of the dystrophin protein. Due to ongoing muscle necrosis in respiratory muscles late-stage DMD is associated with respiratory insufficiency and chronic hypoxia (CH). To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level). Additionally, dmDys and wild type (WT) flies were also exposed to CH in laboratory simulations of high altitude hypoxia. Expression profiling was performed using Affymetrix GeneChips® and validated using qPCR. Hypoxic dmDys differentially expressed 1281 genes, whereas the hypoxic WT flies differentially expressed 56 genes. Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT. We tested the possibility that the disparate molecular responses of dystrophin-deficient tissues to CH could adversely affect muscle by performing functional assays in vivo. Normoxic and CH WT and dmDys flies were challenged with acute hypoxia and time-to-recover determined as well as subjected to climbing tests. Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ≈ CH-WT> Normoxic-dmDys> CH-dmDys). These data suggest that dystrophin-deficiency is associated with a disparate, pathological hypoxic stress response(s) and is more sensitive to hypoxia induced muscle dysfunction in vivo. We hypothesize that targeting/correcting the disparate molecular response(s) to hypoxia may offer a novel therapeutic strategy in DMD

Expression Profiling Reveals Novel Hypoxic Biomarkers in Peripheral Blood of Adult Mice Exposed to Chronic Hypoxia

Hypoxia induces a myriad of changes including an increase in hematocrit due to erythropoietin (EPO) mediated erythropoiesis. While hypoxia is of importance physiologically and clinically, lacunae exist in our knowledge of the systemic and temporal changes in gene expression occurring in blood during the exposure and recovery from hypoxia. To identify these changes expression profiling was conducted on blood obtained from cohorts of C57Bl-10 wild type mice that were maintained at normoxia (NX), exposed for two weeks to normobaric chronic hypoxia (CH) or two weeks of CH followed by two weeks of normoxic recovery (REC). Using stringent bioinformatic cut-offs (0% FDR, 2 fold change cut-off), 230 genes were identified and separated into four distinct temporal categories. Class I) contained 1 transcript up-regulated in both CH and REC; Class II) contained 202 transcripts up-regulated in CH but down-regulated after REC; Class III) contained 9 transcripts down-regulated both in CH and REC; Class IV) contained 18 transcripts down-regulated after CH exposure but up-regulated after REC. Profiling was independently validated and extended by analyzing expression levels of selected genes as novel biomarkers from our profile (e.g. spectrin alpha-1, ubiquitin domain family-1 and pyrroline-5-carboxylate reductase-1) by performing qPCR at 7 different time points during CH and REC. Our identification and characterization of these genes define transcriptome level changes occurring during chronic hypoxia and normoxic recovery as well as novel blood biomarkers that may be useful in monitoring a variety of physiological and pathological conditions associated with hypoxia

Identification of acetylcholine receptor subunits differentially expressed in singly and multiply innervated fibers of extraocular muscles. Invest Ophthalmol Vis Sci 47: 3828–3834

PURPOSE. To identify the acetylcholine receptor (AChR) isoforms among the neuromuscular junctions (NMJs) of singly and multiply innervated fibers (SIFs and MIFs) of rat extraocular muscles (EOMs). METHODS. EOMs were dissected from adult rats and serially sectioned. Sections were simultaneously stained for acetylcholinesterase and with an antibody to the slow myosin heavy chain to identify NMJ topography and fiber types in the same section. Synapses and subsynaptic regions of SIFs and MIFs were isolated by laser capture microdissection and the AChR subunits identified by RT-PCR. RESULTS. The en plaque endings of SIFs expressed only the adult subunit, not the fetal ␥ subunit, of the AChR, whereas the en grappe endings of the MIFs expressed only the ␥ subunit, and not the subunit. Although the expression of the subunit was confined to the NMJ region of the SIFs, the ␥ subunit was expressed both synaptically and extrasynaptically within the MIFs. The ␥ subunit in MIFs correlated with the expression of the myogenic regulatory factor myogenin. Moreover, an unusual neuronal AChR subunit, ␣9, was found in the EOMs, but not in the limb muscles. CONCLUSIONS. The adult and fetal ␥ subunits of the AChRs are segregated into distinct synapses on distinct fiber types. The maintenance of the fetal subunit in a population of fibers is probably linked to the expression of myogenin and is a unique attribute of the EOM allotype. (Invest Ophthalmol Vis Sci