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Repression of slow myosin heavy chain 2 gene expression in fast skeletal muscle fibers by muscarinic acetylcholine receptor and G(alpha)q signaling.

Jordan T, Li J, Jiang H, DiMario JX - J. Cell Biol. (2003)

Bottom Line: Increased G(alpha)q activity repressed slow MyHC2 expression to nondetectable levels in innervated MA fibers.Decreased PKC activity in atropine-treated innervated PM fibers correlated with slow MyHC2 expression.These data suggest that slow MyHC2 repression in innervated fast PM fibers is mediated by cell signaling involving mAchRs, G(alpha)q, and PKC.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Chicago Medical School, North Chicago, IL 60064, USA.

ABSTRACT
Gene expression in skeletal muscle fibers is regulated by innervation and intrinsic fiber properties. To determine the mechanism of repression of slow MyHC2 expression in innervated fast pectoralis major (PM) fibers, we investigated the function of the muscarinic acetylcholine receptor (mAchR) and G(alpha)q. Both mAchR and G(alpha)q are abundant in medial adductor (MA) and PM fibers, and mAchR and G(alpha)q interact in these fibers. Whereas innervation of PM fibers was insufficient to induce slow MyHC2 expression, inhibition of mAchR activity with atropine in innervated PM fibers induced slow MyHC2 expression. Increased G(alpha)q activity repressed slow MyHC2 expression to nondetectable levels in innervated MA fibers. Reduced mAchR activity decreased PKC activity in PM fibers, and increased G(alpha)q activity increased PKC activity in PM and MA fibers. Decreased PKC activity in atropine-treated innervated PM fibers correlated with slow MyHC2 expression. These data suggest that slow MyHC2 repression in innervated fast PM fibers is mediated by cell signaling involving mAchRs, G(alpha)q, and PKC.

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Effects of innervation and atropine on slow MyHC2 expression. PM (pectoralis) and MA (medial adductor) muscle fiber cultures were prepared as described in Materials and methods. On day 3 of incubation, ED5 spinal cord explants were added to half of the cultures to provide innervation of muscle fibers. 200 μM atropine was also added to the culture medium of half the cultures on day 3 of incubation. On day 7 of incubation, cells were fixed and immunostained for fast MyHC and slow MyHC2 with mAbs F59 and S58, respectively, followed by fluorochrome-conjugated secondary antibodies. slow MyHC2 expression was detected in innervated MA muscle fibers with and without atropine. slow MyHC2 expression was detected in innervated PM muscle fibers incubated in medium containing atropine.
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fig3: Effects of innervation and atropine on slow MyHC2 expression. PM (pectoralis) and MA (medial adductor) muscle fiber cultures were prepared as described in Materials and methods. On day 3 of incubation, ED5 spinal cord explants were added to half of the cultures to provide innervation of muscle fibers. 200 μM atropine was also added to the culture medium of half the cultures on day 3 of incubation. On day 7 of incubation, cells were fixed and immunostained for fast MyHC and slow MyHC2 with mAbs F59 and S58, respectively, followed by fluorochrome-conjugated secondary antibodies. slow MyHC2 expression was detected in innervated MA muscle fibers with and without atropine. slow MyHC2 expression was detected in innervated PM muscle fibers incubated in medium containing atropine.

Mentions: We hypothesized that mAchR activity regulated slow MyHC2 expression in innervated muscle fibers. To test this hypothesis, innervated and noninnervated PM and MA muscle fiber cultures were incubated in medium containing 200 μM atropine, an antagonist of mAchR activity. Muscle fibers were then immunostained with mAbs F59 and S58, which specifically recognize fast and slow MyHC isoforms, respectively (Fig. 3). Consistent with previous findings (DiMario and Stockdale, 1997), innervation alone induced expression of slow MyHC2 in MA muscle fibers, but not in PM muscle fibers. Noninnervated PM and MA muscle fibers incubated in medium containing 200 μM atropine also did not express slow MyHC2. However, both PM and MA muscle fibers expressed slow MyHC2 in the presence of innervation and atropine. Similar qualitative results were obtained using medium containing atropine at concentrations of 100 and 500 μM (unpublished data).


Repression of slow myosin heavy chain 2 gene expression in fast skeletal muscle fibers by muscarinic acetylcholine receptor and G(alpha)q signaling.

Jordan T, Li J, Jiang H, DiMario JX - J. Cell Biol. (2003)

Effects of innervation and atropine on slow MyHC2 expression. PM (pectoralis) and MA (medial adductor) muscle fiber cultures were prepared as described in Materials and methods. On day 3 of incubation, ED5 spinal cord explants were added to half of the cultures to provide innervation of muscle fibers. 200 μM atropine was also added to the culture medium of half the cultures on day 3 of incubation. On day 7 of incubation, cells were fixed and immunostained for fast MyHC and slow MyHC2 with mAbs F59 and S58, respectively, followed by fluorochrome-conjugated secondary antibodies. slow MyHC2 expression was detected in innervated MA muscle fibers with and without atropine. slow MyHC2 expression was detected in innervated PM muscle fibers incubated in medium containing atropine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172810&req=5

fig3: Effects of innervation and atropine on slow MyHC2 expression. PM (pectoralis) and MA (medial adductor) muscle fiber cultures were prepared as described in Materials and methods. On day 3 of incubation, ED5 spinal cord explants were added to half of the cultures to provide innervation of muscle fibers. 200 μM atropine was also added to the culture medium of half the cultures on day 3 of incubation. On day 7 of incubation, cells were fixed and immunostained for fast MyHC and slow MyHC2 with mAbs F59 and S58, respectively, followed by fluorochrome-conjugated secondary antibodies. slow MyHC2 expression was detected in innervated MA muscle fibers with and without atropine. slow MyHC2 expression was detected in innervated PM muscle fibers incubated in medium containing atropine.
Mentions: We hypothesized that mAchR activity regulated slow MyHC2 expression in innervated muscle fibers. To test this hypothesis, innervated and noninnervated PM and MA muscle fiber cultures were incubated in medium containing 200 μM atropine, an antagonist of mAchR activity. Muscle fibers were then immunostained with mAbs F59 and S58, which specifically recognize fast and slow MyHC isoforms, respectively (Fig. 3). Consistent with previous findings (DiMario and Stockdale, 1997), innervation alone induced expression of slow MyHC2 in MA muscle fibers, but not in PM muscle fibers. Noninnervated PM and MA muscle fibers incubated in medium containing 200 μM atropine also did not express slow MyHC2. However, both PM and MA muscle fibers expressed slow MyHC2 in the presence of innervation and atropine. Similar qualitative results were obtained using medium containing atropine at concentrations of 100 and 500 μM (unpublished data).

Bottom Line: Increased G(alpha)q activity repressed slow MyHC2 expression to nondetectable levels in innervated MA fibers.Decreased PKC activity in atropine-treated innervated PM fibers correlated with slow MyHC2 expression.These data suggest that slow MyHC2 repression in innervated fast PM fibers is mediated by cell signaling involving mAchRs, G(alpha)q, and PKC.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Chicago Medical School, North Chicago, IL 60064, USA.

ABSTRACT
Gene expression in skeletal muscle fibers is regulated by innervation and intrinsic fiber properties. To determine the mechanism of repression of slow MyHC2 expression in innervated fast pectoralis major (PM) fibers, we investigated the function of the muscarinic acetylcholine receptor (mAchR) and G(alpha)q. Both mAchR and G(alpha)q are abundant in medial adductor (MA) and PM fibers, and mAchR and G(alpha)q interact in these fibers. Whereas innervation of PM fibers was insufficient to induce slow MyHC2 expression, inhibition of mAchR activity with atropine in innervated PM fibers induced slow MyHC2 expression. Increased G(alpha)q activity repressed slow MyHC2 expression to nondetectable levels in innervated MA fibers. Reduced mAchR activity decreased PKC activity in PM fibers, and increased G(alpha)q activity increased PKC activity in PM and MA fibers. Decreased PKC activity in atropine-treated innervated PM fibers correlated with slow MyHC2 expression. These data suggest that slow MyHC2 repression in innervated fast PM fibers is mediated by cell signaling involving mAchRs, G(alpha)q, and PKC.

Show MeSH
Related in: MedlinePlus