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* Department of Basic Medical Sciences, School of Veterinary Medicine;
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy;
Department of Veterinary Clinical Sciences, School of Veterinary Medicine;
Department of Veterinary Pathobiology and Cancer Center; and
¶ Department of Animal Sciences, College of Agriculture, Purdue University, West Lafayette, Indiana, USA; and
|| Cardiovascular Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
2Correspondence: School of Veterinary Medicine, Purdue University, 625 Harrison St., West Lafayette, IN 47907, USA. E-mail: pond{at}purdue.edu
SPECIFIC AIMS
The goals of this study were to: 1) demonstrate that Merg1a K+ channel is expressed endogenously in skeletal muscle (skm) undergoing atrophy as a result of disuse and malignant tumor expression; 2) determine if ectopic expression of Merg1a induces skm atrophy in wt-bearing mice and if this effect can be inhibited by coexpression of Merg1a and the nonfunctional dominant-negative DN-Merg1a; 3) determine if inhibition of Merg1 channel function by ectopic expression of DN-Merg1a inhibits onset of atrophy in hindlimb-suspended mice; 4) administer the Merg1a blocker astemizole to hindlimb-suspended and wt-bearing mice to determine if pharmacological block of this channel will block atrophy in skm; and 5) determine if ectopic expression of Merg1a will specifically increase the concentration of ubiquitin proteasome proteolysis, the pathway responsible for the majority of protein degradation that occurs during skm atrophy.
PRINCIPAL FINDINGS
1. Tumor expression and hindlimb suspension result in skeletal muscle atrophy and Merg1a synthesis
Kb human esophageal cancer were implanted subcutaneously (s.c.) in the right axilla of athymic mice NCr-nu; Harlan, IN) while control mice were injected with vehicle. ND4-Swiss Webster mice were placed in suspension cages so that they were unable to place any load on their hindlimbs. Control mice were kept in a normal wt-bearing state. After six weeks, the mean muscle fiber cross-sectional area (csa) of gastrocnemius muscles from tumor-bearing mice was 25% less (P
0.05, ANOVA) than that of control mice. After 7 d, hindlimb-suspended mice experienced a 45% decrease (P0.05) in muscle fiber csa relative to wt-bearing controls, denoting significant atrophy. Western blot analysis of extracted membrane proteins detected Merg1a proteins in both cachectic and suspended mouse skm (Fig. 1
A, B), but not in control tissue unless it was concentrated 4-fold (Fig. 1C
). Merg1a mRNA was readily detected in gastrocnemius muscles of suspended mice, while only low levels were detected in control skm. Neither Merg1b protein nor mRNA was detected in suspended or control mouse skm (Fig. 1A
). Immunostained sections from suspended mice revealed that Merg1 protein is located in sarcolemmal membranes of myocytes, while it is undetected in control skm (Fig. 1D
).
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2. Ectopic expression of Merg1a induces skeletal muscle atrophy in wt-bearing mice, while coexpression of Merg1a and the dominant-negative DN-Merg1a attenuates the decrease in myofiber size
Injection of plasmid DNA into skm followed by electroporation resulted in expression of encoded genes, specifically Merg1a and Merg1b, as verified with Western blot analysis. We injected the left gastrocnemius muscles of 14 mice with lacZ and control expression plasmids. Right gastrocnemius muscles of seven of these mice were coinjected with Merg1a, lacZ, and control expression plasmids. The right gastrocnemius muscles of the remaining mice were coinjected with three expression plasmids, each encoding either: Merg1a, lacZ, or a DN Merg1a mutant. (The DN Merg1a gene product is a pore mutant that is transported to the cell membrane but prevents Merg1 current conduction.) Mice remained wt bearing. Seven days after electroporation, gastrocnemius fibers expressing Merg1a and lacZ experienced a significant (P0.05, ANOVA) 16.3% decrease in muscle fiber cross- sectional area (csa) relative to fibers injected with lacZ plasmid only. Blockade of Merg1a function by DN Merg1a prevented all but 6.7% of the atrophy induced by Merg1a expression. This incomplete rescue is expected because it is not likely that all randomly assembled channels would contain mutant channel subunit.
3. Inhibition of Merg1 channel function by ectopic expression of DN-Merg1a inhibits the onset of atrophy in hindlimb-suspended mice
We injected left gastrocnemius muscles of 14 mice with lacZ and control expression plasmids. Right gastrocnemius muscles received lacZ plasmid plus expression plasmid encoding DN Merg1a. Mice were then either hindlimb-suspended (n=7) or remained wt bearing (n=7). After 7 d, there was a 45% decrease (P0.05, ANOVA) in csa of suspended mouse myofibers expressing lacZ relative to wt-bearing controls expressing lacZ, showing that suspension induced atrophy. Importantly, muscle fibers from suspended mice expressing lacZ and DN Merg1a mutants were 13.4% smaller than control muscle fibers. Block of Merg1a function does attenuate atrophy. Thus, synthesis of DN Merg1a mutant blocks the onset of disuse atrophy in suspended mice, showing that Merg1a current conduction plays a role in atrophy induction. Further, there was no significant difference in fiber csa of control muscle expressing lacZ alone and those expressing lacZ and Merg1b. This finding suggests that Merg1-induced significant decrease in fiber size is specific to the Merg1a variant.
4. Pharmacological block of the Merg1 channel with astemizole inhibits atrophy in hindlimb-suspended mice and promotes myofiber growth in skeletal muscle of wt-bearing mice
Twenty-eight mice were orally gavaged once every 12 h for 7 d with either: water (groups 1 and 3) or 160 mg/kg astemizole in water (groups 2 and 4). During treatment, groups 1 and 2 remained wt-bearing, while groups 3 and 4 were hindlimb-suspended. Data show that astemizole treatment alone had no significant effect on body wt of wt-bearing mice; however, suspension caused a significant (P0.05, ANOVA) 7% decrease in body wt that was significantly alleviated by drug treatment. Most importantly, the 15% decrease in muscle fiber csa that was experienced by hindlimb-suspended animals was blocked by astemizole treatment, bringing fiber csa values to control levels. Interestingly, astemizole treatment produced significant increases in fiber csa, gastrocnemius muscle wt to body wt ratio, and absolute gastrocnemius muscle wt in control mice. Data show that pharmacological block of Merg1 alleviates disuse atrophy and suggests that block of Merg1a function results in muscle hypertrophy.
5. Ectopic expression of Merg1a increases the concentration of ubiquitin proteasome proteolysis
Expression of ubiquinated firefly luciferase reporter (Ub-FL) produces a measurable protein degraded by the ubiquitin proteasome pathway (UPP). We injected gastrocnemius muscles with two expression plasmids, one encoding Ub-FL and the other Renilla luciferase (RL). Muscles were electroporated. Ub-FL- and RL-injected mice also received plasmid encoding: either Merg1a or DN Merg1a or control plasmid. Mice injected with DN Merg1a or control plasmid were hindlimb-suspended, while the remaining control mice and those injected with Merg1a remained wt-bearing. Ub-FL activity was normalized to RL activity to control for differences in muscle transfection efficiency, and ratios of Ub-FL to RL activity were determined as measures of UPP activity; lower ratios indicated higher UPP activity. The Ub-FL-to-RL activity ratio in hindlimb-suspended mice not injected with DN-Merg1a was 90% smaller (P0.02; ANOVA) than that measured in wt-bearing mice not injected with Merg1a. Data indicate that suspension induces UPP activity. Weight-bearing mice expressing Merg1a, RL, and Ub-FL displayed a 48% lower (P0.02) Ub-FL to RL activity ratio than wt-bearing mice expressing all constructs except Merg1a, denoting that Merg1a expression induces UPP activity. Suspended mice expressing DN Merg1a displayed a 5.7-fold higher (P0.02) Ub-FL to RL activity ratio than suspended mice not expressing DN Merg1a, showing that block of Merg1 channel inhibits UPP proteolysis. The Ub-FL-to-RL ratio in gastrocnemius muscles coexpressing Merg1b (rather than Merg1a) show that ectopic expression of Merg1b decreased UPP activity by an insignificant 9.4%, an amount that may result from coassembly of Merg1b subunit with low levels of endogenous Merg1a. Thus, Merg1a specifically increases UPP activity.
CONCLUSIONS AND SIGNIFICANCE
We know of no other reports linking Merg1 (or the human homologue HERG) K+-channel function to skeletal muscle atrophy. We show that Merg1 channel function is, in fact, an initiating factor of skm atrophy: 1) Merg1 proteins are detected before onset of significant atrophy; 2) ectopic expression of Merg1a induces a decrease in fiber csa in skm of wt-bearing mice; and 3) genetic and pharmacologic block of Merg1 channel function strongly attenuates atrophy in hindlimb-suspended mice. Additionally, data suggest that block of endogenous Merg1 in normal mice induces hypertrophy. Data also strongly imply that Merg1a channel function participates in initiation of skeletal muscle atrophy by signaling an increase in UPP activity, a pathway known to be responsible for a large portion of the protein degradation that occurs during skm atrophy. Despite recent advances, little is known about initiation of UPP activity and factors acting upstream of the atrophic process. We have discovered that a membrane protein, one capable of sensing and responding to changes in membrane potential, is up-regulated prior to onset of significant atrophy and, in fact, induces an increase in activity of the UPP pathway. We find no other reports linking Merg1 function to UPP activity.
Obviously, the mechanism by which Merg1a channel function induces UPP activity begs elucidation. This information is of interest to skm researchers because the Merg1a channel gene and protein are now potential targets for therapies designed to treat skm atrophy. It is, in fact, relevant to researchers in any field involving UPP proteolysis and particularly the cardiac field. For example, UPP activity participates in atrophic remodeling of the heart; however, physiologically relevant levels of Merg1 current (IKr) are necessary for normal cardiac function. Perhaps expression of the Merg1b splice variant or another K+ channel subunit in heart is involved in this regulation. Therefore, the functional consequence of Merg1 channel expression may be determined by Merg1 channel subunit composition.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5350fje
1 These authors contributed equally to this work. 
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