Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Skeletal muscle atrophy is a result of an imbalance in protein synthesis and protein degradation. It is a part of the normal aging process, but also may occur in response to several stimuli including disease, injury, and disuse. Currently there is no truly effective pharmacological treatment for atrophy. The best treatment is a healthy diet and exercise, however, sick and aging individuals are not always capable of participating in the appropriate activity. Therefore, exploration of the mechanisms contributing to atrophy is essential because this may 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 up-regulating ubiquitin proteasome proteolysis in mice. Recent studies have shown that it also increases the intracellular calcium concentration in skeletal muscle. The aim of this work is to investigate the modulation of the increased intracellular [Ca2+] and its potential downstream effects on calpain activity. To do this, we overexpressed human ERG1A (HERG1A) in a mouse myoblast cell line (C2C12 cells). Looking for the source of increased calcium levels we investigated the skeletal muscle specific, voltage gated calcium channel (CAV1.1). We were able to discover that there is a significant 2.3 fold increase in Cav1.1 gene expression. Conversely, upon immunoblotting the protein abundance of Cav1.1 had no detectable change. Calpains, which are calcium activated cysteine proteases take part in the degradation of myofilaments when they are activated via calcium. The activity of these calpains were shown to have a 25.7% higher level of activity in response to HERG expression. The change in calpain activity may be attributable to increased calpain transcription, translation or changes in their inhibitor, calpastatin. When gene expression was analyzed for calpains and calpastatin, no significant change was observed. However, after analysis of protein abundance a decrease was found in calpains 2 and 3, along with calpastatin, suggesting that HERG1A may indeed be effecting calpain abundance. With our data we were able to conclude that the increase in intracellular [Ca2+] due to the presence of HERG1A is not due to Cav1.1. We were able to report that an increase in calpain activity occurred in response to HERG1A within C2C12 cells, however this was not due to changes in gene expression, but their protein abundance appears to decrease in response to HERG1A.
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