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A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology
The failure to transmit neural action potentials (APs) into muscle APs is referred to as neuromuscular transmission failure (NTF). Although synaptic dysfunction occurs in a variety of neuromuscular diseases and impaired neurotransmission contributes to muscle fatigue, direct evaluation of neurotrans...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130989/ https://www.ncbi.nlm.nih.gov/pubmed/27990107 http://dx.doi.org/10.3389/fncel.2016.00276 |
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author | Heredia, Dante J. Schubert, Douglas Maligireddy, Siddhardha Hennig, Grant W. Gould, Thomas W. |
author_facet | Heredia, Dante J. Schubert, Douglas Maligireddy, Siddhardha Hennig, Grant W. Gould, Thomas W. |
author_sort | Heredia, Dante J. |
collection | PubMed |
description | The failure to transmit neural action potentials (APs) into muscle APs is referred to as neuromuscular transmission failure (NTF). Although synaptic dysfunction occurs in a variety of neuromuscular diseases and impaired neurotransmission contributes to muscle fatigue, direct evaluation of neurotransmission by measurement of successfully transduced muscle APs is difficult due to the subsequent movements produced by muscle. Moreover, the voltage-gated sodium channel inhibitor used to study neurotransmitter release at the adult neuromuscular junction is ineffective in embryonic tissue, making it nearly impossible to precisely measure any aspect of neurotransmission in embryonic lethal mouse mutants. In this study we utilized 3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one (BHC), previously identified in a small-molecule screen of skeletal muscle myosin inhibitors, to suppress movements without affecting membrane currents. In contrast to previously characterized drugs from this screen such as N-benzyl-p-toluene sulphonamide (BTS), which inhibit skeletal muscle myosin ATPase activity but also block neurotransmission, BHC selectively blocked nerve-evoked muscle contraction without affecting neurotransmitter release. This feature allowed a detailed characterization of neurotransmission in both embryonic and adult mice. In the presence of BHC, neural APs produced by tonic stimulation of the phrenic nerve at rates up to 20 Hz were successfully transmitted into muscle APs. At higher rates of phrenic nerve stimulation, NTF was observed. NTF was intermittent and characterized by successful muscle APs following failed ones, with the percentage of successfully transmitted muscle APs diminishing over time. Nerve stimulation rates that failed to produce NTF in the presence of BHC similarly failed to produce a loss of peak muscle fiber shortening, which was examined using a novel optical method of muscle fatigue, or a loss of peak cytosolic calcium transient intensity, examined in whole populations of muscle cells expressing the genetically-encoded calcium indicator GCaMP3. Most importantly, BHC allowed for the first time a detailed analysis of synaptic transmission, calcium signaling and fatigue in embryonic mice, such as in Vamp2 mutants reported here, that die before or at birth. Together, these studies illustrate the wide utility of BHC in allowing stable measurements of neuromuscular function. |
format | Online Article Text |
id | pubmed-5130989 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-51309892016-12-16 A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology Heredia, Dante J. Schubert, Douglas Maligireddy, Siddhardha Hennig, Grant W. Gould, Thomas W. Front Cell Neurosci Neuroscience The failure to transmit neural action potentials (APs) into muscle APs is referred to as neuromuscular transmission failure (NTF). Although synaptic dysfunction occurs in a variety of neuromuscular diseases and impaired neurotransmission contributes to muscle fatigue, direct evaluation of neurotransmission by measurement of successfully transduced muscle APs is difficult due to the subsequent movements produced by muscle. Moreover, the voltage-gated sodium channel inhibitor used to study neurotransmitter release at the adult neuromuscular junction is ineffective in embryonic tissue, making it nearly impossible to precisely measure any aspect of neurotransmission in embryonic lethal mouse mutants. In this study we utilized 3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one (BHC), previously identified in a small-molecule screen of skeletal muscle myosin inhibitors, to suppress movements without affecting membrane currents. In contrast to previously characterized drugs from this screen such as N-benzyl-p-toluene sulphonamide (BTS), which inhibit skeletal muscle myosin ATPase activity but also block neurotransmission, BHC selectively blocked nerve-evoked muscle contraction without affecting neurotransmitter release. This feature allowed a detailed characterization of neurotransmission in both embryonic and adult mice. In the presence of BHC, neural APs produced by tonic stimulation of the phrenic nerve at rates up to 20 Hz were successfully transmitted into muscle APs. At higher rates of phrenic nerve stimulation, NTF was observed. NTF was intermittent and characterized by successful muscle APs following failed ones, with the percentage of successfully transmitted muscle APs diminishing over time. Nerve stimulation rates that failed to produce NTF in the presence of BHC similarly failed to produce a loss of peak muscle fiber shortening, which was examined using a novel optical method of muscle fatigue, or a loss of peak cytosolic calcium transient intensity, examined in whole populations of muscle cells expressing the genetically-encoded calcium indicator GCaMP3. Most importantly, BHC allowed for the first time a detailed analysis of synaptic transmission, calcium signaling and fatigue in embryonic mice, such as in Vamp2 mutants reported here, that die before or at birth. Together, these studies illustrate the wide utility of BHC in allowing stable measurements of neuromuscular function. Frontiers Media S.A. 2016-12-01 /pmc/articles/PMC5130989/ /pubmed/27990107 http://dx.doi.org/10.3389/fncel.2016.00276 Text en Copyright © 2016 Heredia, Schubert, Maligireddy, Hennig and Gould. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Neuroscience Heredia, Dante J. Schubert, Douglas Maligireddy, Siddhardha Hennig, Grant W. Gould, Thomas W. A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology |
title | A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology |
title_full | A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology |
title_fullStr | A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology |
title_full_unstemmed | A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology |
title_short | A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology |
title_sort | novel striated muscle-specific myosin-blocking drug for the study of neuromuscular physiology |
topic | Neuroscience |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130989/ https://www.ncbi.nlm.nih.gov/pubmed/27990107 http://dx.doi.org/10.3389/fncel.2016.00276 |
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