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The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells

BACKGROUND: Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological the...

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Autores principales: Whitmore, Clayton, Pratt, Evan P.S., Anderson, Luke, Bradley, Kevin, Latour, Sawyer M., Hashmi, Mariam N., Urazaev, Albert K., Weilbaecher, Rod, Davie, Judith K., Wang, Wen-Horng, Hockerman, Gregory H., Pond, Amber L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966811/
https://www.ncbi.nlm.nih.gov/pubmed/31948476
http://dx.doi.org/10.1186/s13395-019-0220-3
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author Whitmore, Clayton
Pratt, Evan P.S.
Anderson, Luke
Bradley, Kevin
Latour, Sawyer M.
Hashmi, Mariam N.
Urazaev, Albert K.
Weilbaecher, Rod
Davie, Judith K.
Wang, Wen-Horng
Hockerman, Gregory H.
Pond, Amber L.
author_facet Whitmore, Clayton
Pratt, Evan P.S.
Anderson, Luke
Bradley, Kevin
Latour, Sawyer M.
Hashmi, Mariam N.
Urazaev, Albert K.
Weilbaecher, Rod
Davie, Judith K.
Wang, Wen-Horng
Hockerman, Gregory H.
Pond, Amber L.
author_sort Whitmore, Clayton
collection PubMed
description BACKGROUND: Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K(+) channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. METHODS: We transduced C(2)C(12) myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student’s t test or two-way ANOVAs and SAS software as indicated. RESULTS: Expression of human ERG1A in C(2)C(12) myotubes increased basal intracellular calcium concentration 51.7% (p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity. CONCLUSIONS: Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.
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spelling pubmed-69668112020-01-22 The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells Whitmore, Clayton Pratt, Evan P.S. Anderson, Luke Bradley, Kevin Latour, Sawyer M. Hashmi, Mariam N. Urazaev, Albert K. Weilbaecher, Rod Davie, Judith K. Wang, Wen-Horng Hockerman, Gregory H. Pond, Amber L. Skelet Muscle Research BACKGROUND: Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K(+) channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. METHODS: We transduced C(2)C(12) myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student’s t test or two-way ANOVAs and SAS software as indicated. RESULTS: Expression of human ERG1A in C(2)C(12) myotubes increased basal intracellular calcium concentration 51.7% (p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity. CONCLUSIONS: Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance. BioMed Central 2020-01-16 /pmc/articles/PMC6966811/ /pubmed/31948476 http://dx.doi.org/10.1186/s13395-019-0220-3 Text en © The Author(s). 2020 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Whitmore, Clayton
Pratt, Evan P.S.
Anderson, Luke
Bradley, Kevin
Latour, Sawyer M.
Hashmi, Mariam N.
Urazaev, Albert K.
Weilbaecher, Rod
Davie, Judith K.
Wang, Wen-Horng
Hockerman, Gregory H.
Pond, Amber L.
The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells
title The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells
title_full The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells
title_fullStr The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells
title_full_unstemmed The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells
title_short The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells
title_sort erg1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966811/
https://www.ncbi.nlm.nih.gov/pubmed/31948476
http://dx.doi.org/10.1186/s13395-019-0220-3
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