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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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Repression of slow MyHC2 expression by Gαq activity. MA myogenic cultures were established and some of the muscle fibers were innervated by spinal cord explants. Before addition of spinal cord explants, MA myogenic cultures were also transfected with either CMVGαqFLAG encoding wild-type Gαq or CMVGαqQ209LFLAG, which constitutively expresses GTPase-deficient, active Gαq. On day 7 of incubation, cells were fixed and immunostained with mAbs F59 and S58 to detect fast MyHCs and slow MyHC2, respectively, followed by fluorochrome-conjugated secondary antibodies. Clusters of nicotinic AchRs (arrows), indicative of innervated muscle fibers, were detected by incubation of the cells in medium containing rhodamine-conjugated α-bungarotoxin (α-BTX) before fixation. Transfected muscle fibers were identified by immunostaining with an anti-FLAG epitope antibody and an FITC-conjugated secondary antibody. Images were added digitally using Photoshop 5.0.
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fig5: Repression of slow MyHC2 expression by Gαq activity. MA myogenic cultures were established and some of the muscle fibers were innervated by spinal cord explants. Before addition of spinal cord explants, MA myogenic cultures were also transfected with either CMVGαqFLAG encoding wild-type Gαq or CMVGαqQ209LFLAG, which constitutively expresses GTPase-deficient, active Gαq. On day 7 of incubation, cells were fixed and immunostained with mAbs F59 and S58 to detect fast MyHCs and slow MyHC2, respectively, followed by fluorochrome-conjugated secondary antibodies. Clusters of nicotinic AchRs (arrows), indicative of innervated muscle fibers, were detected by incubation of the cells in medium containing rhodamine-conjugated α-bungarotoxin (α-BTX) before fixation. Transfected muscle fibers were identified by immunostaining with an anti-FLAG epitope antibody and an FITC-conjugated secondary antibody. Images were added digitally using Photoshop 5.0.

Mentions: Based on the interaction of the mAchR and Gαq, we hypothesized that mAchR activity–dependent repression of slow MyHC2 expression was mediated by Gαq signaling. To test this hypothesis, a constitutively active, GTPase-deficient Gαq expression construct, CMVGαqQ209LFLAG, was generated yielding a single amino acid substitution (Q209→L209) and transfected into MA myogenic cultures. Similar mutations have resulted in constitutively active Gαq (Wu et al., 1992; Qian et al., 1993). As shown above (Figs. 3 and 4), MA muscle fibers expressed slow MyHC2 when innervated and, therefore, these muscle fibers provide the most appropriate basis for forced repression of slow MyHC2 expression. Transfected muscle fibers were identified with an anti-FLAG epitope antibody, and fast and slow MyHC2 were detected with F59 and S58 antibodies, respectively (Fig. 5). In addition, innervation of muscle fibers was visualized by detection of nAchR clusters using rhodamine-conjugated α-bungarotoxin. Whereas innervated, nontransfected MA muscle fibers expressed slow MyHC2 (Fig. 5 A), innervated MA muscle fibers expressing GαqQ209LFLAG did not immunostain with S58 (Fig. 5 B). No muscle fibers containing detectable amounts of GαqQ209LFLAG expressed slow MyHC2. In contrast, expression of wild-type GαqFLAG in innervated MA muscle fibers did not repress slow MyHC2 expression. Importantly, expression of GαqQ209LFLAG did not prevent MA muscle fiber innervation as indicated by clusters of nAchRs. In this culture system, these clusters are only evident when muscle fibers are innervated by spinal cord explants (DiMario and Stockdale, 1997). PM muscle fibers, whether innervated and/or transfected with CMVGαqQ209LFLAG, did not express slow MyHC2 as determined by immunostaining (unpublished data). Therefore, Gαq activity repressed slow MyHC2 expression in innervated MA muscle fibers.


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)

Repression of slow MyHC2 expression by Gαq activity. MA myogenic cultures were established and some of the muscle fibers were innervated by spinal cord explants. Before addition of spinal cord explants, MA myogenic cultures were also transfected with either CMVGαqFLAG encoding wild-type Gαq or CMVGαqQ209LFLAG, which constitutively expresses GTPase-deficient, active Gαq. On day 7 of incubation, cells were fixed and immunostained with mAbs F59 and S58 to detect fast MyHCs and slow MyHC2, respectively, followed by fluorochrome-conjugated secondary antibodies. Clusters of nicotinic AchRs (arrows), indicative of innervated muscle fibers, were detected by incubation of the cells in medium containing rhodamine-conjugated α-bungarotoxin (α-BTX) before fixation. Transfected muscle fibers were identified by immunostaining with an anti-FLAG epitope antibody and an FITC-conjugated secondary antibody. Images were added digitally using Photoshop 5.0.
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Related In: Results  -  Collection

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fig5: Repression of slow MyHC2 expression by Gαq activity. MA myogenic cultures were established and some of the muscle fibers were innervated by spinal cord explants. Before addition of spinal cord explants, MA myogenic cultures were also transfected with either CMVGαqFLAG encoding wild-type Gαq or CMVGαqQ209LFLAG, which constitutively expresses GTPase-deficient, active Gαq. On day 7 of incubation, cells were fixed and immunostained with mAbs F59 and S58 to detect fast MyHCs and slow MyHC2, respectively, followed by fluorochrome-conjugated secondary antibodies. Clusters of nicotinic AchRs (arrows), indicative of innervated muscle fibers, were detected by incubation of the cells in medium containing rhodamine-conjugated α-bungarotoxin (α-BTX) before fixation. Transfected muscle fibers were identified by immunostaining with an anti-FLAG epitope antibody and an FITC-conjugated secondary antibody. Images were added digitally using Photoshop 5.0.
Mentions: Based on the interaction of the mAchR and Gαq, we hypothesized that mAchR activity–dependent repression of slow MyHC2 expression was mediated by Gαq signaling. To test this hypothesis, a constitutively active, GTPase-deficient Gαq expression construct, CMVGαqQ209LFLAG, was generated yielding a single amino acid substitution (Q209→L209) and transfected into MA myogenic cultures. Similar mutations have resulted in constitutively active Gαq (Wu et al., 1992; Qian et al., 1993). As shown above (Figs. 3 and 4), MA muscle fibers expressed slow MyHC2 when innervated and, therefore, these muscle fibers provide the most appropriate basis for forced repression of slow MyHC2 expression. Transfected muscle fibers were identified with an anti-FLAG epitope antibody, and fast and slow MyHC2 were detected with F59 and S58 antibodies, respectively (Fig. 5). In addition, innervation of muscle fibers was visualized by detection of nAchR clusters using rhodamine-conjugated α-bungarotoxin. Whereas innervated, nontransfected MA muscle fibers expressed slow MyHC2 (Fig. 5 A), innervated MA muscle fibers expressing GαqQ209LFLAG did not immunostain with S58 (Fig. 5 B). No muscle fibers containing detectable amounts of GαqQ209LFLAG expressed slow MyHC2. In contrast, expression of wild-type GαqFLAG in innervated MA muscle fibers did not repress slow MyHC2 expression. Importantly, expression of GαqQ209LFLAG did not prevent MA muscle fiber innervation as indicated by clusters of nAchRs. In this culture system, these clusters are only evident when muscle fibers are innervated by spinal cord explants (DiMario and Stockdale, 1997). PM muscle fibers, whether innervated and/or transfected with CMVGαqQ209LFLAG, did not express slow MyHC2 as determined by immunostaining (unpublished data). Therefore, Gαq activity repressed slow MyHC2 expression in innervated MA muscle fibers.

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