The ERG1A K+ Channel Is More Abundant in Rectus abdominis Muscle from Cancer Patients Than that from Healthy Humans.

BACKGROUND: The potassium channel encoded by the ether-a-gogo-related gene 1A (erg1a) has been detected in the atrophying skeletal muscle of mice experiencing either muscle disuse or cancer cachexia and further evidenced to contribute to muscle deterioration by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1A has not been reported in human skeletal muscle. METHODS AND RESULTS: Here, using immunohistochemistry, we detect ERG1A immunofluorescence in human Rectus abdominis skeletal muscle sarcolemma. Further, using single point brightness data, we report the detection of ERG1A immunofluorescence at low levels in the Rectus abdominis muscle sarcolemma of young adult humans and show that it trends toward greater levels (10.6%) in healthy aged adults. Interestingly, we detect ERG1A immunofluorescence at a statistically greater level (53.6%; p < 0.05) in the skeletal muscle of older cancer patients than in age-matched healthy adults. Importantly, using immunoblot, we reveal that lower mass ERG1A protein is 61.5% (p < 0.05) more abundant in the skeletal muscle of cachectic older adults than in healthy age-matched controls. Additionally, we report that the ERG1A protein is detected in a cultured human rhabdomyosarcoma line that may be a good in vitro model for the study of ERG1A in muscle. CONCLUSIONS: The data demonstrate that ERG1A is detected more abundantly in the atrophied skeletal muscle of cancer patients, suggesting it may be related to muscle loss in humans as it has been shown to be in mice experiencing muscle atrophy as a result of malignant tumors.

MERG1A Protein Abundance Increases in the Atrophied Skeletal Muscle of Denervated Mice, But Does Not Affect NFκB Activity.

Skeletal muscle atrophy may occur with disease, injury, decreased muscle use, starvation, and normal aging. No reliably effective treatments for atrophy are available, thus research into the mechanisms contributing to muscle loss is essential. The ERG1A K+ channel contributes to muscle loss by increasing ubiquitin proteasome proteolysis (UPP) in the skeletal muscle of both unweighted and cachectic mice. Because the mechanisms which produce atrophy vary based upon the initiating factor, here we investigate atrophy produced by denervation. Using immunohistochemistry and immunoblots, we demonstrate that ERG1A protein abundance increases significantly in the Gastrocnemius muscle of rodents 7 days after both sciatic nerve transection and hind limb unweighting. Further, we reveal that ectopic expression of a Merg1a encoded plasmid in normal mouse Gastrocnemius muscle has no effect on activity of the NFκB transcription factor family, a group of proteins which contribute to muscle atrophy by modulation of the UPP. Further, although NFκB activity increases significantly after denervation, we show that expression of a plasmid encoding a dominant negative Merg1a mutant in Gastrocnemius muscle prior to denervation, has no effect on NFκB activity. Thus, although the ERG1A K+ channel increases UPP, it does not do so through modulation of NFκB transcription factors.

IFN-γ and CIITA modulate IL-6 expression in skeletal muscle.

Interleukin 6 (IL-6) is a secreted cytokine that is an important mediator of the immune response in numerous tissues, including skeletal muscle. IL-6 is considered a myokine as it can be secreted by muscle. IL-6 is secreted following exercise, where it exerts both pro-myogenic effects as well as anti-myogenic effects such as promoting atrophy and muscle wasting. The regulation of IL-6 in skeletal muscle is not well understood. The purpose of this study was to determine if IFN-γ and TNF-ɑ stimulate IL-6 in skeletal muscle. We found that both IFN-γ and TNF-α stimulate IL-6 in skeletal muscle, but the stimulation is not cooperative as seen in monocytes. We have previously shown that the IFN-γ stimulated class II major histocompatibility complex transactivator (CIITA) mediates many of the effects of IFN-γ in skeletal muscle and we show here that CIITA directly stimulates IL-6. The regulation of IL-6 by CIITA is clearly complex, as we found that CIITA both stimulates and restrains IL-6 expression. To show that these effects could be observed in a physiological setting, mice were treated with IFN-γ and we found that both CIITA and IL-6 were upregulated in skeletal muscle.

Ether-a-go-go related gene-1a potassium channel abundance varies within specific skeletal muscle fiber type.

The ERG1A K+ channel, which is partially responsible for repolarization of the cardiac action potential, has also been reported in skeletal muscle where it modulates ubiquitin proteolysis. Because ERG1A protein appears variably expressed in muscles composed of mixed fiber types, we hypothesized that its abundance in skeletal muscle might differ with fiber type. Indeed, skeletal muscle fibers vary in speed of contraction (fast or slow), which is mainly determined by myosin heavy chain (MyHC) isoform content, but a sarcolemmal K+ channel might also modulate contraction speed. To test our hypothesis, we cryo-sectioned Soleus (SOL), Extensor Digitorum Longus (EDL), and Gastrocnemius muscles from five rats. These muscles were chosen because the SOL and EDL contain an abundance of slow- and fast-twitch fibers, respectively, while the Gastrocnemius has a more heterogeneous composition. The muscle sections were co-immunostained for the ERG1A protein and either the fast- or slow-twitch MyHC to identify fiber type. ERG1A fluorescence was then measured in the sarcolemma of each fiber type and compared. The data reveal that the ERG1A protein is more abundant in the fibers of the SOL than in the EDL muscles, suggesting ERG1A may be more abundant in the slow than the fast fibers, and this was confirmed with immunoblot. However, because of the homogeneity of fiber type within these muscles, it was not possible to get enough data from both fiber types within a single muscle to compare ERG1A composition within fiber type. However, immunohistochemistry of sections from the fiber type heterogeneous Gastrocnemius muscle reveals that slow fibers had, on average, a 17.2% greater ERG1A fluorescence intensity than fast fibers (p<0.03). Further, immunoblot reveals that ERG1A protein is 41.6% more abundant (p=0.051) in old than in young rat Gastrocnemius muscle. We postulate that this membrane bound voltage-gated channel may affect membrane characteristics, the duration of the action potential generated, and/or the speed of contraction. Indeed, ERG1A protein is more abundant in aged and atrophic skeletal muscle, both of which exhibit slower rates of contraction.