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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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Coimmunoprecipitation of mAchR and Gαg. Extracts from innervated (+SC) and noninnervated PM and MA muscle fiber cultures expressing GαqFLAG were prepared. mAchR–Gαq protein complexes were immunoprecipitated using an anti-mAchR antibody. Controls using innervated PM muscle fibers (PM+SC) included direct immunoprecipitation of GαqFLAG with an anti-Gαq antibody, omission of the anti-mAchR antibody from the coimmunoprecipitation protocol, coimmunoprecipitation using extracts from nontransfected muscle fibers, and coimmunoprecipitation using an α-actin antibody. Co-immunoprecipitated mAchR–Gαq complexes were denatured, electrophoresed, and blotted. Gαq, coimmunoprecipitated with mAchR, was detected with an anti-FLAG antibody, HRP-conjugated secondary antibody and chemiluminescence.
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fig2: Coimmunoprecipitation of mAchR and Gαg. Extracts from innervated (+SC) and noninnervated PM and MA muscle fiber cultures expressing GαqFLAG were prepared. mAchR–Gαq protein complexes were immunoprecipitated using an anti-mAchR antibody. Controls using innervated PM muscle fibers (PM+SC) included direct immunoprecipitation of GαqFLAG with an anti-Gαq antibody, omission of the anti-mAchR antibody from the coimmunoprecipitation protocol, coimmunoprecipitation using extracts from nontransfected muscle fibers, and coimmunoprecipitation using an α-actin antibody. Co-immunoprecipitated mAchR–Gαq complexes were denatured, electrophoresed, and blotted. Gαq, coimmunoprecipitated with mAchR, was detected with an anti-FLAG antibody, HRP-conjugated secondary antibody and chemiluminescence.

Mentions: To determine whether the mAchR and Gαq proteins interact in PM and MA muscle cells, myogenic cultures were transfected with the expression construct, GαqFLAG, which constitutively expressed wild-type Gαq coupled to a FLAG epitope. ED5 spinal cord explants were added to some of the plates of transfected cells to provide the muscle fibers with motor neuron innervation (DiMario and Stockdale, 1997). mAchR–GαqFLAG complexes were coimmunoprecipitated using an anti-mAchR antibody. Complexes were then denatured and Western blotted (Fig. 2). Gαq, which coimmunoprecipitated with mAchR, was detected with an anti-FLAG epitope antibody. GαqFLAG in transfected cultures of PM and MA with and without innervation coimmunoprecipitated with the mAchR. GαqFLAG was not detected in extracts from nontransfected cells or in extracts carried through the coimmunoprecipitation procedure without the mAchR antibody. To determine whether the mAchR–Gαq interaction in transfected cells was specific, protein extract was incubated with α-actin antibody. No GαqFLAG was detected in protein extracts immunoprecipitated with α-actin antibody. Therefore, Gαq and mAchR interact in both innervated and noninnervated PM and MA muscle cells.


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)

Coimmunoprecipitation of mAchR and Gαg. Extracts from innervated (+SC) and noninnervated PM and MA muscle fiber cultures expressing GαqFLAG were prepared. mAchR–Gαq protein complexes were immunoprecipitated using an anti-mAchR antibody. Controls using innervated PM muscle fibers (PM+SC) included direct immunoprecipitation of GαqFLAG with an anti-Gαq antibody, omission of the anti-mAchR antibody from the coimmunoprecipitation protocol, coimmunoprecipitation using extracts from nontransfected muscle fibers, and coimmunoprecipitation using an α-actin antibody. Co-immunoprecipitated mAchR–Gαq complexes were denatured, electrophoresed, and blotted. Gαq, coimmunoprecipitated with mAchR, was detected with an anti-FLAG antibody, HRP-conjugated secondary antibody and chemiluminescence.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Coimmunoprecipitation of mAchR and Gαg. Extracts from innervated (+SC) and noninnervated PM and MA muscle fiber cultures expressing GαqFLAG were prepared. mAchR–Gαq protein complexes were immunoprecipitated using an anti-mAchR antibody. Controls using innervated PM muscle fibers (PM+SC) included direct immunoprecipitation of GαqFLAG with an anti-Gαq antibody, omission of the anti-mAchR antibody from the coimmunoprecipitation protocol, coimmunoprecipitation using extracts from nontransfected muscle fibers, and coimmunoprecipitation using an α-actin antibody. Co-immunoprecipitated mAchR–Gαq complexes were denatured, electrophoresed, and blotted. Gαq, coimmunoprecipitated with mAchR, was detected with an anti-FLAG antibody, HRP-conjugated secondary antibody and chemiluminescence.
Mentions: To determine whether the mAchR and Gαq proteins interact in PM and MA muscle cells, myogenic cultures were transfected with the expression construct, GαqFLAG, which constitutively expressed wild-type Gαq coupled to a FLAG epitope. ED5 spinal cord explants were added to some of the plates of transfected cells to provide the muscle fibers with motor neuron innervation (DiMario and Stockdale, 1997). mAchR–GαqFLAG complexes were coimmunoprecipitated using an anti-mAchR antibody. Complexes were then denatured and Western blotted (Fig. 2). Gαq, which coimmunoprecipitated with mAchR, was detected with an anti-FLAG epitope antibody. GαqFLAG in transfected cultures of PM and MA with and without innervation coimmunoprecipitated with the mAchR. GαqFLAG was not detected in extracts from nontransfected cells or in extracts carried through the coimmunoprecipitation procedure without the mAchR antibody. To determine whether the mAchR–Gαq interaction in transfected cells was specific, protein extract was incubated with α-actin antibody. No GαqFLAG was detected in protein extracts immunoprecipitated with α-actin antibody. Therefore, Gαq and mAchR interact in both innervated and noninnervated PM and MA muscle cells.

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