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1.
Mol Biol Cell ; 35(4): ar52, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38381557

ABSTRACT

Host response to pathogens recruits multiple tissues in part through conserved cell signaling pathways. In Caenorhabditis elegans, the bone morphogenetic protein (BMP) like DBL-1 signaling pathway has a role in the response to infection in addition to other roles in development and postdevelopmental functions. In the regulation of body size, the DBL-1 pathway acts through cell autonomous signal activation in the epidermis (hypodermis). We have now elucidated the tissues that respond to DBL-1 signaling upon exposure to two bacterial pathogens. The receptors and Smad signal transducers for DBL-1 are expressed in pharyngeal muscle, intestine, and epidermis. We demonstrate that expression of receptor-regulated Smad (R-Smad) gene sma-3 in the pharynx is sufficient to improve the impaired survival phenotype of sma-3 mutants and that expression of sma-3 in the intestine has no effect when exposing worms to bacterial infection of the intestine. We also show that two antimicrobial peptide genes - abf-2 and cnc-2 - are regulated by DBL-1 signaling through R-Smad SMA-3 activity in the pharynx. Finally, we show that pharyngeal pumping activity is reduced in sma-3 mutants and that other pharynx-defective mutants also have reduced survival on a bacterial pathogen. Our results identify the pharynx as a tissue that responds to BMP signaling to coordinate a systemic response to bacterial pathogens.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Transforming Growth Factor beta/metabolism , Pharyngeal Muscles/metabolism , Signal Transduction/physiology
2.
Cell Mol Life Sci ; 80(8): 205, 2023 Jul 14.
Article in English | MEDLINE | ID: mdl-37450052

ABSTRACT

Dietary intake and nutrient composition regulate animal growth and development; however, the underlying mechanisms remain elusive. Our previous study has shown that either the mammalian deafness homolog gene tmc-1 or its downstream acetylcholine receptor gene eat-2 attenuates Caenorhabditis elegans development in a chemically defined food CeMM (C. elegans maintenance medium) environment, but the underpinning mechanisms are not well-understood. Here, we found that, in CeMM food environment, for both eat-2 and tmc-1 fast-growing mutants, several fatty acid synthesis and elongation genes were highly expressed, while many fatty acid ß-oxidation genes were repressed. Accordingly, dietary supplementation of individual fatty acids, such as monomethyl branch chain fatty acid C17ISO, palmitic acid and stearic acid significantly promoted wild-type animal development on CeMM, and mutations in either C17ISO synthesis gene elo-5 or elo-6 slowed the rapid growth of eat-2 mutant. Tissue-specific rescue experiments showed that elo-6 promoted animal development mainly in the intestine. Furthermore, transcriptome and metabolome analyses revealed that elo-6/C17ISO regulation of C. elegans development may be correlated with up-regulating expression of cuticle synthetic and hedgehog signaling genes, as well as promoting biosynthesis of amino acids, amino acid derivatives and vitamins. Correspondingly, we found that amino acid derivative S-adenosylmethionine and its upstream metabolite methionine sulfoxide significantly promoted C. elegans development on CeMM. This study demonstrated that C17ISO, palmitic acid, stearic acid, S-adenosylmethionine and methionine sulfoxide inhibited or bypassed the TMC-1 and EAT-2-mediated attenuation of development via metabolic remodeling, and allowed the animals to adapt to the new nutritional niche.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Fatty Acids , Nutrients , Receptors, Nicotinic , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Animals , Eating , Nutrients/metabolism , Pharyngeal Muscles/metabolism , Fatty Acids/metabolism , Ion Channels/genetics , Ion Channels/metabolism
3.
J Clin Lab Anal ; 36(7): e24551, 2022 Jul.
Article in English | MEDLINE | ID: mdl-35692078

ABSTRACT

BACKGROUND: Obstructive sleep apnea (OSA) has a high incidence and is harmful to health. It is characterized by repeated collapse of the upper airway. However, the mechanism underlying upper airway collapse is unclear. METHODS: Patients with OSA and chronic tonsillitis were studied. Pathological changes in palatopharyngeus muscle were detected. The expression of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) and nuclear respiratory factor-1 (NRF-1) in muscles was detected by PCR and Western blotting. Immunofluorescence staining was used to detect the expression of type I and type II myofibril. RESULTS: The structure of the palatopharyngeus muscle was changed, and the expression of PGC-1α and NRF-1 was decreased in the OSA group compared with that in the control group. The expression of PGC-1α, NRF-1, and type I myofibril in C2C12 myoblasts was decreased by intermittent hypoxia exposure. The expression of type I myofibril was decreased when knocking down PGC-1α. CONCLUSION: OSA patients exhibited pathological damage in palatopharyngeus muscle. PGC-1α was involved in the fiber type conversion in palatopharyngeus muscle caused by intermittent hypoxia.


Subject(s)
Nuclear Respiratory Factor 1 , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Pharyngeal Muscles , Sleep Apnea, Obstructive , Humans , Hypoxia , Nuclear Respiratory Factor 1/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Pharyngeal Muscles/metabolism
4.
Free Radic Res ; 53(sup1): 1056-1067, 2019.
Article in English | MEDLINE | ID: mdl-31514551

ABSTRACT

All living organisms are normally undergoing aging. Dietary habits constitute the main environmental factor that may accelerate or decelerate this process. Advanced glycation end products (AGEs) are constituents of dietary products that are consumed daily, such as bread and milk. Although AGEs have been widely regarded as toxic agents, recent studies seem to contradict this view: they either find no adverse effects of AGEs or even attribute beneficial properties to them. The aim of our study was to investigate the effects of sugar-derived AGEs on organismal lifespan using as a model the nematode Caenorhabditis elegans. Exposure to sugar-derived AGEs prolonged the lifespan of wild type animals; this lifespan extension was accompanied by an enhanced pharyngeal pumping rate. We demonstrate that elevation of the pharyngeal pumping rate depends on W06A7.4 and eat-4 expression, as well as on daf-16, which encodes a FOXO family transcription factor. Our results suggest that sugar-derived AGEs modulate the lifespan of C. elegans at least in part through transcriptional regulation of pharyngeal pumping throughout the animals' lifespan.


Subject(s)
Caenorhabditis elegans/metabolism , Glycation End Products, Advanced/metabolism , Longevity , Pharyngeal Muscles/metabolism , Sugars/chemistry , Animals , Sugars/metabolism
5.
J Appl Physiol (1985) ; 127(1): 11-21, 2019 07 01.
Article in English | MEDLINE | ID: mdl-31018744

ABSTRACT

The extrinsic tongue muscles are activated in coordination with pharyngeal muscles to dilate the airways as needed during breathing. The genioglossus (GG) activity is known to be modulated by several reflexes evoked via the mechanoreceptors of the upper airways. The primary objective of this paper was to investigate the effectiveness of activating these reflex pathways using mechanical stimulation of the mandible or the submandibular muscles. In eight healthy subjects, 3-s long, 5-mm vertical mechanical vibrations were delivered at 8 and 12 Hz to the lower jaw in a seated position, while the GG EMG was recorded using a custom-made sublingual electrode, along with the activity of the masseter (MS) and mylohyoid (MH). All three muscle activities were significantly higher during stimulation compared with the baseline (P < 0.02), and the increase was larger at 12 Hz versus 8 Hz (P < 0.02). All three muscle responses had components that synchronized with the mechanical stimuli, but those of MS were much more strongly phase-locked to the vibrational cycle. In 10 healthy subjects, we also applied mechanical vibrations to the submandibular muscles at three different stimulation intensities, while subjects were lying in a supine position. The GG activity increased significantly above the baseline (P = 0.026) in 9 out of 10 subjects, and the elevated activity persisted after termination of the stimulus for a few seconds. The results demonstrate that GG muscle responses can be evoked with mechanical vibrations applied to the lower jaw or the submandibular muscles in healthy subjects during wakefulness. NEW & NOTEWORTHY The evoked responses observed in the genioglossus (GG) activity during mechanical vibrations of the lower jaw or the submandibular muscles may lead to therapeutic applications for improving the patency of airways during sleep. The presence of these GG reflexes may also explain a mechanism by which the vibrations produced during snoring can help the airways stay open in individuals who may otherwise have obstructed airways in sleep.


Subject(s)
Facial Muscles/physiology , Mandible/physiology , Masseter Muscle/physiology , Adult , Facial Muscles/metabolism , Female , Humans , Male , Mandible/metabolism , Masseter Muscle/metabolism , Mechanoreceptors/metabolism , Middle Aged , Neck Muscles/metabolism , Neck Muscles/physiology , Pharyngeal Muscles/metabolism , Pharyngeal Muscles/physiology , Reflex/physiology , Tongue/metabolism , Tongue/physiology , Vibration
6.
PLoS Genet ; 15(2): e1007962, 2019 02.
Article in English | MEDLINE | ID: mdl-30721228

ABSTRACT

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.


Subject(s)
COUP Transcription Factor II/metabolism , DNA-Binding Proteins/metabolism , Myocytes, Cardiac/metabolism , Pharyngeal Muscles/metabolism , Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Body Patterning/genetics , COUP Transcription Factor II/genetics , Cell Lineage/genetics , Craniofacial Abnormalities/embryology , Craniofacial Abnormalities/genetics , DNA-Binding Proteins/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Heart Defects, Congenital/embryology , Heart Defects, Congenital/genetics , Heart Ventricles/cytology , Heart Ventricles/embryology , Heart Ventricles/metabolism , Humans , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Models, Animal , Mutation , Myocytes, Cardiac/cytology , Pharyngeal Muscles/cytology , Pharyngeal Muscles/embryology , Promoter Regions, Genetic , Signal Transduction , Transcription Factors/genetics , Tretinoin/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics
7.
Int J Parasitol Drugs Drug Resist ; 8(3): 526-533, 2018 12.
Article in English | MEDLINE | ID: mdl-30401619

ABSTRACT

The cys-loop superfamily of ligand-gated ion channels are well recognized as important drug targets for many invertebrate specific compounds. With the rise in resistance seen worldwide to existing anthelmintics, novel drug targets must be identified so new treatments can be developed. The acetylcholine-gated chloride channel (ACC) family is a unique family of cholinergic receptors that have been shown, using Caenorhabditis elegans as a model, to have potential as anti-parasitic drug targets. However, there is little known about the function of these receptors in parasitic nematodes. Here, we have identified an acc gene (hco-acc-1) from the sheep parasitic nematode Haemonchus contortus. While similar in sequence to the previously characterized C. elegans ACC-1 receptor, Hco-ACC-1 does not form a functional homomeric channel in Xenopus oocytes. Instead, co-expression of Hco-ACC-1 with a previously characterized subunit Hco-ACC-2 produced a functional heteromeric channel which was 3x more sensitive to acetylcholine compared to the Hco-ACC-2 homomeric channel. We have also found that Hco-ACC-1 can be functionally expressed in C. elegans. Overexpression of both cel-acc-1 and hco-acc-1 in both C. elegans N2 and acc-1 null mutants decreased the time for worms to initiate reversal avoidance to octanol. Moreover, antibodies were generated against the Hco-ACC-1 protein for use in immunolocalization studies. Hco-ACC-1 consistently localized to the anterior half of the pharynx, specifically in pharyngeal muscle tissue in H. contortus. On the other hand, expression of Hco-ACC-1 in C. elegans was restricted to neuronal tissue. Overall, this research has provided new insight into the potential role of ACC receptors in parasitic nematodes.


Subject(s)
Acetylcholine/pharmacology , Chloride Channels/metabolism , Haemonchus/metabolism , Helminth Proteins/metabolism , Receptors, Cholinergic/metabolism , Acetylcholine/metabolism , Animals , Anthelmintics/metabolism , Caenorhabditis elegans/genetics , Chloride Channels/genetics , Cysteine Loop Ligand-Gated Ion Channel Receptors , Haemonchus/anatomy & histology , Haemonchus/drug effects , Haemonchus/genetics , Helminth Proteins/genetics , Ligand-Gated Ion Channels/genetics , Ligand-Gated Ion Channels/metabolism , Octanols/pharmacology , Oocytes/drug effects , Pharyngeal Muscles/metabolism , Receptors, Cholinergic/genetics , Xenopus laevis/anatomy & histology , Xenopus laevis/physiology
8.
PLoS Genet ; 13(12): e1007134, 2017 12.
Article in English | MEDLINE | ID: mdl-29281635

ABSTRACT

Feeding, a vital behavior in animals, is modulated depending on internal and external factors. In the nematode Caenorhabditis elegans, the feeding organ called the pharynx ingests food by pumping driven by the pharyngeal muscles. Here we report that optical silencing of the body wall muscles, which drive the locomotory movement of worms, affects pumping. In worms expressing the Arch proton pump or the ACR2 anion channel in the body wall muscle cells, the pumping rate decreases after activation of Arch or ACR2 with light illumination, and recovers gradually after terminating illumination. Pumping was similarly inhibited by illumination in locomotion-defective mutants carrying Arch, suggesting that perturbation of locomotory movement is not critical for pumping inhibition. Analysis of mutants and cell ablation experiments showed that the signals mediating the pumping inhibition response triggered by activation of Arch with weak light are transferred mainly through two pathways: one involving gap junction-dependent mechanisms through pharyngeal I1 neurons, which mediate fast signals, and the other involving dense-core vesicle-dependent mechanisms, which mediate slow signals. Activation of Arch with strong light inhibited pumping strongly in a manner that does not rely on either gap junction-dependent or dense-core vesicle-dependent mechanisms. Our study revealed a new aspect of the neural and neuroendocrine controls of pumping initiated from the body wall muscles.


Subject(s)
Optogenetics/methods , Pharyngeal Muscles/metabolism , Proton Pumps/metabolism , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/metabolism , Eating/physiology , Locomotion/physiology , Motor Neurons/metabolism , Muscle, Skeletal/metabolism , Pharynx/metabolism , Serotonin , Signal Transduction/physiology , Voltage-Dependent Anion Channels/genetics , Voltage-Dependent Anion Channels/metabolism
9.
Exp Physiol ; 102(9): 1177-1193, 2017 09 01.
Article in English | MEDLINE | ID: mdl-28665499

ABSTRACT

NEW FINDINGS: What is the central question of this study? We previously reported impaired upper airway dilator muscle function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Our aim was to assess the effect of blocking interleukin-6 receptor signalling and stimulating corticotrophin-releasing factor receptor 2 signalling on mdx sternohyoid muscle structure and function. What is the main finding and its importance? The interventional treatment had a positive inotropic effect on sternohyoid muscle force, restoring mechanical work and power to wild-type values, reduced myofibre central nucleation and preserved the myosin heavy chain type IIb fibre complement of mdx sternohyoid muscle. These data might have implications for development of pharmacotherapies for DMD with relevance to respiratory muscle performance. The mdx mouse model of Duchenne muscular dystrophy shows evidence of impaired pharyngeal dilator muscle function. We hypothesized that inflammatory and stress-related factors are implicated in airway dilator muscle dysfunction. Six-week-old mdx (n = 26) and wild-type (WT; n = 26) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (0.2 mg kg-1 ) and corticotrophin-releasing factor receptor 2 agonist (urocortin 2; 30 µg kg-1 ) over 2 weeks. Sternohyoid muscle isometric and isotonic contractile function was examined ex vivo. Muscle fibre centronucleation and muscle cellular infiltration, collagen content, fibre-type distribution and fibre cross-sectional area were determined by histology and immunofluorescence. Muscle chemokine content was examined by use of a multiplex assay. Sternohyoid peak specific force at 100 Hz was significantly reduced in mdx compared with WT. Drug treatment completely restored force in mdx sternohyoid to WT levels. The percentage of centrally nucleated muscle fibres was significantly increased in mdx, and this was partly ameliorated after drug treatment. The areal density of infiltrates and collagen content were significantly increased in mdx sternohyoid; both indices were unaffected by drug treatment. The abundance of myosin heavy chain type IIb fibres was significantly decreased in mdx sternohyoid; drug treatment preserved myosin heavy chain type IIb complement in mdx muscle. The chemokines macrophage inflammatory protein 2, interferon-γ-induced protein 10 and macrophage inflammatory protein 3α were significantly increased in mdx sternohyoid compared with WT. Drug treatment significantly increased chemokine expression in mdx but not WT sternohyoid. Recovery of contractile function was impressive in our study, with implications for Duchenne muscular dystrophy. The precise molecular mechanisms by which the drug treatment exerts an inotropic effect on mdx sternohyoid muscle remain to be elucidated.


Subject(s)
Antibodies, Neutralizing/pharmacology , Corticotropin-Releasing Hormone/metabolism , Dystrophin/metabolism , Muscle Fibers, Skeletal/drug effects , Pharyngeal Muscles/drug effects , Receptors, Interleukin-6/metabolism , Urocortins/metabolism , Animals , Disease Models, Animal , Female , Interferon-gamma/metabolism , Male , Mice , Mice, Inbred mdx , Muscle Contraction/drug effects , Muscle Fibers, Skeletal/metabolism , Muscular Dystrophy, Duchenne/drug therapy , Muscular Dystrophy, Duchenne/metabolism , Myosin Heavy Chains/metabolism , Nonmuscle Myosin Type IIB/metabolism , Pharyngeal Muscles/metabolism , Respiratory Muscles/drug effects , Respiratory Muscles/metabolism
10.
Mech Dev ; 143: 32-41, 2017 02.
Article in English | MEDLINE | ID: mdl-28087459

ABSTRACT

Canonical Wnt/ß-catenin (Wnt) signaling plays multiple conserved roles during fate specification of cardiac progenitors in developing vertebrate embryos. Although lineage analysis in ascidians and mice has indicated there is a close relationship between the cardiac second heart field (SHF) and pharyngeal muscle (PM) progenitors, the signals underlying directional fate decisions of the cells within the cardio-pharyngeal muscle field in vertebrates are not yet understood. Here, we examined the temporal requirements of Wnt signaling in cardiac and PM development. In contrast to a previous report in chicken embryos that suggested Wnt inhibits PM development during somitogenesis, we find that in zebrafish embryos Wnt signaling is sufficient to repress PM development during anterior-posterior patterning. Importantly, the temporal sensitivity of dorso-anterior PMs to increased Wnt signaling largely overlaps with when Wnt signaling promotes specification of the adjacent cardiac progenitors. Furthermore, we find that excess early Wnt signaling can cell autonomously promote expansion of the first heart field (FHF) progenitors at the expense of PM and SHF within the anterior lateral plate mesoderm (ALPM). Our study provides insight into an antagonistic developmental mechanism that balances the sizes of the adjacent cardiac and PM progenitor fields in early vertebrate embryos.


Subject(s)
Body Patterning/genetics , Cytoskeletal Proteins/genetics , Gene Expression Regulation, Developmental , Myocardium/metabolism , Pharyngeal Muscles/metabolism , Wnt Proteins/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Actins/genetics , Actins/metabolism , Animals , Animals, Genetically Modified , Cytoskeletal Proteins/metabolism , Embryo, Nonmammalian , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Myocardium/cytology , Pharyngeal Muscles/cytology , Pharyngeal Muscles/embryology , Signal Transduction , Stem Cells/cytology , Stem Cells/metabolism , Transcription Factor 7-Like 1 Protein/genetics , Transcription Factor 7-Like 1 Protein/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Wnt Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/metabolism
11.
Nucleus ; 8(1): 60-69, 2017 01 02.
Article in English | MEDLINE | ID: mdl-27673727

ABSTRACT

There are numerous heritable diseases associated with mutations in the LMNA gene. Most of these laminopathic diseases, including several muscular dystrophies, are autosomal dominant and have tissue-specific phenotypes. Our previous studies have shown that the globally expressed Emery-Dreifuss muscular dystrophy (EDMD)-linked lamin mutation, L535P, disrupts nuclear mechanical response specifically in muscle nuclei of C. elegans leading to atrophy of the body muscle cells and to reduced motility. Here we used RNA sequencing to analyze the global changes in gene expression caused by the L535P EDMD lamin mutation in order to gain better understanding of disease mechanisms and the correlation between transcription and phenotype. Our results show changes in key genes and biological pathways that can help explain the muscle specific phenotypes. In addition, the differential gene expression between wild-type and L535P mutant animals suggests that the pharynx function in the L535P mutant animals is affected by this lamin mutation. Moreover, these transcriptional changes were then correlated with reduced pharynx activity and abnormal pharynx muscle structure. Understanding disease mechanisms will potentially lead to new therapeutic approaches toward curing EDMD.


Subject(s)
Caenorhabditis elegans , Gene Expression Profiling , Muscular Dystrophy, Emery-Dreifuss/genetics , Mutation , Phenotype , Transcription, Genetic , Animals , Computational Biology , Down-Regulation , Humans , Lamin Type A/genetics , Muscular Dystrophy, Emery-Dreifuss/physiopathology , Pharyngeal Muscles/metabolism , Pharyngeal Muscles/physiopathology , Sequence Analysis, RNA
12.
Oncotarget ; 7(42): 67732-67747, 2016 Oct 18.
Article in English | MEDLINE | ID: mdl-27661127

ABSTRACT

Ca2+ is a key signal transducer for muscle contraction. Continuous in vivo monitoring of intracellular Ca2+-dynamics in C. elegans pharynx muscle revealed surprisingly complex Ca2+ patterns. Despite the age-dependent decline of pharynx pumping, we observed unaltered fast Ca2+ oscillations both in young and old worms. In addition, sporadic prolonged Ca2+ increases lasting many seconds or minutes were often observed in between periods of fast Ca2+ oscillations. We attribute them to the inhibition of ATP-dependent Ca2+-pumps upon energy depletion. Accordingly, food deprivation largely augmented the frequency of prolonged [Ca2+] increases. However, paradoxically, prolonged [Ca2+] increases were more frequently observed in young worms than in older ones, and less frequently observed in energy-deficient mitochondrial respiratory chain nuo-6 mutants than in wild-type controls. We hypothesize that young animals are more susceptible to energy depletion due to their faster energy consumption rate, while nuo-6 mutants may keep better the energy balance by slowing energy consumption. Our data therefore suggest that the metabolic state of the pharynx during feeding stimulation depends mainly on the delicate balance between the instant rates of energy production and consumption. Thus, in vivo monitoring of muscle Ca2+ dynamics can be used as a novel tool to study cellular energy availability.


Subject(s)
Caenorhabditis elegans/metabolism , Calcium/metabolism , Energy Metabolism , Pharynx/metabolism , Age Factors , Animals , Caenorhabditis elegans/genetics , Feeding Behavior , Muscle Contraction , Mutation , Pharyngeal Muscles/metabolism , Time Factors
13.
Sci Rep ; 6: 22940, 2016 Mar 15.
Article in English | MEDLINE | ID: mdl-26976078

ABSTRACT

Rhythmic movements are ubiquitous in animal locomotion, feeding, and circulatory systems. In some systems, the muscle itself generates rhythmic contractions. In others, rhythms are generated by the nervous system or by interactions between the nervous system and muscles. In the nematode Caenorhabditis elegans, feeding occurs via rhythmic contractions (pumping) of the pharynx, a neuromuscular feeding organ. Here, we use pharmacology, optogenetics, genetics, and electrophysiology to investigate the roles of the nervous system and muscle in generating pharyngeal pumping. Hyperpolarization of the nervous system using a histamine-gated chloride channel abolishes pumping, and optogenetic stimulation of pharyngeal muscle in these animals causes abnormal contractions, demonstrating that normal pumping requires nervous system function. In mutants that pump slowly due to defective nervous system function, tonic muscle stimulation causes rapid pumping, suggesting tonic neurotransmitter release may regulate pumping. However, tonic cholinergic motor neuron stimulation, but not tonic muscle stimulation, triggers pumps that electrophysiologically resemble typical rapid pumps. This suggests that pharyngeal cholinergic motor neurons are normally rhythmically, and not tonically active. These results demonstrate that the pharynx generates a myogenic rhythm in the presence of tonically released acetylcholine, and suggest that the pharyngeal nervous system entrains contraction rate and timing through phasic neurotransmitter release.


Subject(s)
Caenorhabditis elegans/physiology , Motor Neurons/physiology , Muscle Contraction/physiology , Pharyngeal Muscles/physiology , Pharynx/physiology , Signal Transduction/physiology , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/physiology , Chloride Channels/genetics , Chloride Channels/physiology , Cholinergic Neurons/metabolism , Cholinergic Neurons/physiology , Electrophysiological Phenomena/drug effects , Electrophysiological Phenomena/genetics , Feeding Behavior/drug effects , Feeding Behavior/physiology , Histamine/metabolism , Motor Neurons/metabolism , Muscle Contraction/genetics , Mutation , Nervous System Physiological Phenomena/drug effects , Nervous System Physiological Phenomena/genetics , Optogenetics/methods , Pharyngeal Muscles/metabolism , Pharynx/innervation , Pharynx/metabolism , Serotonin/pharmacology , Serotonin Receptor Agonists/pharmacology , Signal Transduction/genetics
14.
PLoS One ; 10(10): e0141128, 2015.
Article in English | MEDLINE | ID: mdl-26484766

ABSTRACT

Inorganic phosphate (Pi) has central roles in metabolism, cell signaling and energy conversion. The distribution of Pi to each cell and cellular compartment of an animal must be tightly coordinated with its dietary supply and with the varied metabolic demands of individual cells. An analytical method for monitoring Pi dynamics with spatial and temporal resolution is therefore needed to gain a comprehensive understanding of mechanisms governing the transport and recycling of this essential nutrient. Here we demonstrate the utility of a genetically encoded FRET-based Pi sensor to assess cellular Pi levels in the nematode Caenorhabditis elegans. The sensor was expressed in different cells and tissues of the animal, including head neurons, tail neurons, pharyngeal muscle, and the intestine. Cytosolic Pi concentrations were monitored using ratiometric imaging. Injection of phosphate buffer into intestinal cells confirmed that the sensor was responsive to changes in Pi concentration in vivo. Live Pi imaging revealed cell-specific and developmental stage-specific differences in cytosolic Pi concentrations. In addition, cellular Pi levels were perturbed by food deprivation and by exposure to the respiratory inhibitor cyanide. These results suggest that Pi concentration is a sensitive indicator of metabolic status. Moreover, we propose that live Pi imaging in C. elegans is a powerful approach to discern mechanisms that govern Pi distribution in individual cells and throughout an animal.


Subject(s)
Biosensing Techniques , Caenorhabditis elegans/metabolism , Intestinal Mucosa/metabolism , Neurons/metabolism , Pharyngeal Muscles/metabolism , Phosphates/metabolism , Animals , Caenorhabditis elegans/growth & development , Cytosol/metabolism , Diagnostic Imaging , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/metabolism , Intestines/cytology , Neurons/cytology , Pharyngeal Muscles/cytology
15.
Stem Cells ; 33(12): 3581-95, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26178867

ABSTRACT

The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging.


Subject(s)
Muscle Development , Pharyngeal Muscles/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Animals , Mice , Mice, Mutant Strains , Pharyngeal Muscles/cytology , Satellite Cells, Skeletal Muscle/cytology
16.
PLoS Genet ; 10(8): e1004529, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25101962

ABSTRACT

Carbon dioxide (CO2) is a key molecule in many biological processes; however, mechanisms by which organisms sense and respond to high CO2 levels remain largely unknown. Here we report that acute CO2 exposure leads to a rapid cessation in the contraction of the pharynx muscles in Caenorhabditis elegans. To uncover the molecular mechanisms underlying this response, we performed a forward genetic screen and found that hid-1, a key component in neuropeptide signaling, regulates this inhibition in muscle contraction. Surprisingly, we found that this hid-1-mediated pathway is independent of any previously known pathways controlling CO2 avoidance and oxygen sensing. In addition, animals with mutations in unc-31 and egl-21 (neuropeptide secretion and maturation components) show impaired inhibition of muscle contraction following acute exposure to high CO2 levels, in further support of our findings. Interestingly, the observed response in the pharynx muscle requires the BAG neurons, which also mediate CO2 avoidance. This novel hid-1-mediated pathway sheds new light on the physiological effects of high CO2 levels on animals at the organism-wide level.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Carbon Dioxide/toxicity , Oxygen/metabolism , Pharyngeal Muscles/drug effects , Vesicular Transport Proteins/genetics , Animals , Caenorhabditis elegans/drug effects , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Carbon Dioxide/metabolism , Mutation , Pharyngeal Muscles/metabolism , Vesicular Transport Proteins/metabolism
17.
Cell Death Differ ; 21(4): 557-67, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24317200

ABSTRACT

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2-3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.


Subject(s)
Caenorhabditis elegans/metabolism , Cell Hypoxia , GABAergic Neurons/cytology , Pharyngeal Muscles/cytology , Animals , Animals, Genetically Modified , Bystander Effect , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Death , Forkhead Transcription Factors , GABAergic Neurons/metabolism , Pharyngeal Muscles/metabolism , Phenotype , RNA Interference , RNA, Small Interfering/metabolism , Receptor, Insulin/antagonists & inhibitors , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Signal Transduction , Transcription Factors/metabolism
18.
Mol Ther ; 22(1): 219-25, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23831596

ABSTRACT

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant genetic disease mainly characterized by ptosis and dysphagia. We conducted a phase I/IIa clinical study (ClinicalTrials.gov NCT00773227) using autologous myoblast transplantation following myotomy in adult OPMD patients. This study included 12 patients with clinical diagnosis of OPMD, indication for cricopharyngeal myotomy, and confirmed genetic diagnosis. The feasibility and safety end points of both autologous myoblast transplantation and the surgical procedure were assessed by videoendoscopy in addition to physical examinations. Potential therapeutic benefit was also assessed through videoendoscopy and videofluoroscopy of swallowing, quality of life score, dysphagia grade, and a drink test. Patients were injected with a median of 178 million myoblasts following myotomy. Short and long-term (2 years) safety and tolerability were observed in all the patients, with no adverse effects. There was an improvement in the quality of life score for all 12 patients, and no functional degradation in swallowing was observed for 10 patients. A cell dose-dependant improvement in swallowing was even observed in this study. This trial supports the hypothesis that a local injection of autologous myoblasts in the pharyngeal muscles is a safe and efficient procedure for OPMD patients.


Subject(s)
Muscular Dystrophy, Oculopharyngeal/therapy , Myoblasts, Skeletal/transplantation , Aged , Esophageal Sphincter, Upper/metabolism , Esophageal Sphincter, Upper/physiopathology , Female , Humans , Male , Middle Aged , Muscular Dystrophy, Oculopharyngeal/diagnosis , Muscular Dystrophy, Oculopharyngeal/genetics , Pharyngeal Muscles/metabolism , Pharyngeal Muscles/physiopathology , Pharyngeal Muscles/surgery , Transplantation, Autologous , Treatment Outcome
19.
Mol Biol Cell ; 24(12): 1863-71, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23615451

ABSTRACT

Spinal muscular atrophy is an inherited motor neuron disease that results from a deficiency of the survival of motor neuron (SMN) protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition increases SMN protein levels, improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy (SMA) mice. Specific targets in the UPS may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, mind bomb 1 (Mib1), interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. Also, knocking down the Mib1 orthologue improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for SMA.


Subject(s)
Survival of Motor Neuron 1 Protein/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Animals, Genetically Modified , Blotting, Western , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , HEK293 Cells , Humans , Hybrid Cells , Mice , Neuroblastoma/pathology , Pharyngeal Muscles/metabolism , Pharyngeal Muscles/physiopathology , Protein Binding , Proteolysis , RNA Interference , Spinal Cord/cytology , Survival of Motor Neuron 1 Protein/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination
20.
J Neuropathol Exp Neurol ; 72(3): 234-43, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23399899

ABSTRACT

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant inherited dystrophy caused by an abnormal trinucleotide repeat expansion in the poly(A)-binding-protein-nuclear 1 (PABPN1) gene. Primary muscular targets of OPMD are the eyelid elevator and pharyngeal muscles, including the cricopharyngeal muscle (CPM), the progressive involution of which leads to ptosis and dysphagia, respectively. To understand the consequences of PABPN1 polyalanine expansion in OPMD, we studied muscle biopsies from 14 OPMD patients, 3 inclusion body myositis patients, and 9 healthy controls. In OPMD patient CPM (n = 6), there were typical dystrophic features with extensive endomysial fibrosis and marked atrophy of myosin heavy-chain IIa fibers. There were more PAX7-positive cells in all CPM versus other muscles (n = 5, control; n = 3, inclusion body myositis), and they were more numerous in OPMD CPM versus control normal CPM without any sign of muscle regeneration. Intranuclear inclusions were present in all OPMD muscles but unaffected OPMD patient muscles (i.e. sternocleidomastoid, quadriceps, or deltoid; n = 14) did not show evidence of fibrosis, atrophy, or increased PAX7-positive cell numbers. These results suggest that the specific involvement of CPM in OPMD might be caused by failure of the regenerative response with dysfunction of PAX7-positive cells and exacerbated fibrosis that does not correlate with the presence of PABPN1 inclusions.


Subject(s)
Muscular Dystrophy, Oculopharyngeal/pathology , PAX7 Transcription Factor/biosynthesis , Pharyngeal Muscles/pathology , Adult , Aged , Aged, 80 and over , Atrophy/pathology , Female , Fibrosis/pathology , Fluorescent Antibody Technique , Humans , Male , Middle Aged , Muscular Dystrophy, Oculopharyngeal/metabolism , Pharyngeal Muscles/metabolism
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