Abstract
BackgroundSkeletal 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.MethodsWe transduced C2C12 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.ResultsExpression of human ERG1A in C2C12 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.ConclusionsHuman 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.
Highlights
Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation
Immunoblot of the lysates shows that C2C12 myotubes transfected with virus encoding human ERG1A (HERG1A) do synthesize the HERG1A protein, which appears as a single band of higher mass than the native mouse ERG1 and is absent from the myotubes treated with the control virus (Fig. 1c; p < 0.05; two-way ANVOA)
When the average area of fluorescent myotubes from both sets is determined at both 48 and 72 h after transfection, we discover that, to mouse skeletal muscle fibers electro-transferred with mouse Erg1a (Merg1a) plasmid [23], the myotubes transfected with HERG1A are significantly smaller than control myotubes
Summary
Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. Skeletal muscle atrophy is defined as a 5% or greater decrease in muscle mass and strength and can be induced by certain stimuli: muscle disuse, denervation, starvation, disease (e.g., diabetes and cancer), loss of neural input, and even normal aging [2, 3]. Treatments for skeletal muscle atrophy currently under study include administration of pharmaceuticals such as growth factors [4], beta-agonists [5], inhibitors of proteolysis [6, 7], stimulators of protein synthesis [8], and myostatin inhibitors [9,10,11]; these are not adequately effective. Further investigation into the mechanisms resulting in atrophy is needed to reveal new and improved targets for therapy
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