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

Autor: Whitmore C; Anatomy Department, Southern Illinois University School of Medicine, Carbondale, IL, 62902, USA., Pratt EPS; Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, 47906, USA., Anderson L; Anatomy Department, Southern Illinois University School of Medicine, Carbondale, IL, 62902, USA., Bradley K; Anatomy Department, Southern Illinois University School of Medicine, Carbondale, IL, 62902, USA., Latour SM; Doisey College of Health Sciences, Saint Louis University, St. Louis, MO, 63103, USA., Hashmi MN; Anatomy Department, Southern Illinois University School of Medicine, Carbondale, IL, 62902, USA., Urazaev AK; School of Liberal Arts, Sciences and Education, Ivy Tech State college, Lafayette, IN, 47905, USA., Weilbaecher R; Biochemistry Department, Southern Illinois University School of Medicine, Carbondale, IL, 62902, USA., Davie JK; Biochemistry Department, Southern Illinois University School of Medicine, Carbondale, IL, 62902, USA., Wang WH; Gene Editing Core Facility, Purdue University, West Lafayette, IN, 47906, USA., Hockerman GH; Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, 47906, USA., Pond AL; Anatomy Department, Southern Illinois University School of Medicine, Carbondale, IL, 62902, USA. apond@siumed.edu.; Southern Illinois University, 1135 Lincoln Drive, Carbondale, IL, 62902, USA. apond@siumed.edu.
Jazyk: angličtina
Zdroj: Skeletal muscle [Skelet Muscle] 2020 Jan 16; Vol. 10 (1), pp. 1. Date of Electronic Publication: 2020 Jan 16.
DOI: 10.1186/s13395-019-0220-3
Abstrakt: 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.
Databáze: MEDLINE
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