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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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PKC activities are modulated by innervation and atropine. ED13 PM and MA muscle fiber cultures were established. Some muscle fiber cultures were cocultured with ED5 spinal cord explants (+SC) beginning on day 3 of incubation. Other cultures were incubated in medium containing 200 μM atropine (+Atr) from days 3–7 of incubation. Some cultures received both innervation and atropine (+SC+Atr). Yet other cultures were transfected 24–48 h after plating with CMVGαqQ209LFLAG and incubated until day 7 without innervation or atropine. On day 7 of incubation, cells were scraped from the culture dishes and membrane versus cytoplasmic fractions were prepared. Protein contents of each fraction were determined by BCA protein assay. PKC activities in each fraction were determined using a PKC assay kit and were normalized to protein content. Data are expressed as membrane associated PKC activity normalized to cytoplasmic PKC activity. Bars indicate the mean of three independent experiments ± SEM. Significant differences in means were determined by t test with P < 0.05.
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fig6: PKC activities are modulated by innervation and atropine. ED13 PM and MA muscle fiber cultures were established. Some muscle fiber cultures were cocultured with ED5 spinal cord explants (+SC) beginning on day 3 of incubation. Other cultures were incubated in medium containing 200 μM atropine (+Atr) from days 3–7 of incubation. Some cultures received both innervation and atropine (+SC+Atr). Yet other cultures were transfected 24–48 h after plating with CMVGαqQ209LFLAG and incubated until day 7 without innervation or atropine. On day 7 of incubation, cells were scraped from the culture dishes and membrane versus cytoplasmic fractions were prepared. Protein contents of each fraction were determined by BCA protein assay. PKC activities in each fraction were determined using a PKC assay kit and were normalized to protein content. Data are expressed as membrane associated PKC activity normalized to cytoplasmic PKC activity. Bars indicate the mean of three independent experiments ± SEM. Significant differences in means were determined by t test with P < 0.05.

Mentions: We have shown previously that PKC activity regulates expression of slow MyHC2 (DiMario and Funk, 1999). Inhibition of PKC activity induced slow MyHC2 expression, and overexpression of PKC repressed slow MyHC2 expression. Therefore, we hypothesized that the regulation of slow MyHC2 by mAchR and Gαq activities was mediated by PKC activity. To determine whether mAchR and Gαq signaling controlled PKC activities in PM and MA muscle fibers, PKC activities were directly determined in cytoplasmic and membrane fractions obtained from PM and MA muscle fiber cultures (Fig. 6). These cultures were generated by incubation of noninnervated and innervated PM and MA muscle fibers in control medium and/or in medium containing 200 μM atropine. In addition, cytoplasmic and membrane fractions were obtained from PM and MA muscle fiber cultures transfected with CMVGαqQ209LFLAG. Membrane versus cytoplasmic PKC activities were determined to quantitate membrane-associated active PKC in the cultures. Noninnervated PM muscle fibers contained 2.5-fold more PKC activity than noninnervated MA muscle fibers. This is in agreement with previous studies examining PKC activities in avian and mammalian fast versus slow type muscles (Donnelly et al., 1994; DiMario and Funk, 1999). Innervation of both PM and MA muscle fibers significantly reduced PKC activities. Innervation of PM muscle fibers resulted in an ∼33% reduction in membrane versus cytoplasmic PKC activity. PM muscle fibers incubated in medium containing atropine also yielded reduced PKC activity by 41%. Combination of innervation of PM muscle fibers and incubation in medium containing atropine resulted in a significant, additive inhibition of PKC activity. PKC activities in MA muscle fibers incubated in control medium or medium containing atropine were not significantly different. Last, both PM and MA muscle fiber cultures expressing constitutively active Gαq yielded significantly increased PKC activities. These results indicate that direct modulation of mAchR and Gαq activities affects downstream PKC activities, particularly in PM muscle fibers. Reduced mAchR/Gαq signaling resulted in reduced PKC activity, and increased mAchR/Gαq signaling increased PKC activity.


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)

PKC activities are modulated by innervation and atropine. ED13 PM and MA muscle fiber cultures were established. Some muscle fiber cultures were cocultured with ED5 spinal cord explants (+SC) beginning on day 3 of incubation. Other cultures were incubated in medium containing 200 μM atropine (+Atr) from days 3–7 of incubation. Some cultures received both innervation and atropine (+SC+Atr). Yet other cultures were transfected 24–48 h after plating with CMVGαqQ209LFLAG and incubated until day 7 without innervation or atropine. On day 7 of incubation, cells were scraped from the culture dishes and membrane versus cytoplasmic fractions were prepared. Protein contents of each fraction were determined by BCA protein assay. PKC activities in each fraction were determined using a PKC assay kit and were normalized to protein content. Data are expressed as membrane associated PKC activity normalized to cytoplasmic PKC activity. Bars indicate the mean of three independent experiments ± SEM. Significant differences in means were determined by t test with P < 0.05.
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Related In: Results  -  Collection

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fig6: PKC activities are modulated by innervation and atropine. ED13 PM and MA muscle fiber cultures were established. Some muscle fiber cultures were cocultured with ED5 spinal cord explants (+SC) beginning on day 3 of incubation. Other cultures were incubated in medium containing 200 μM atropine (+Atr) from days 3–7 of incubation. Some cultures received both innervation and atropine (+SC+Atr). Yet other cultures were transfected 24–48 h after plating with CMVGαqQ209LFLAG and incubated until day 7 without innervation or atropine. On day 7 of incubation, cells were scraped from the culture dishes and membrane versus cytoplasmic fractions were prepared. Protein contents of each fraction were determined by BCA protein assay. PKC activities in each fraction were determined using a PKC assay kit and were normalized to protein content. Data are expressed as membrane associated PKC activity normalized to cytoplasmic PKC activity. Bars indicate the mean of three independent experiments ± SEM. Significant differences in means were determined by t test with P < 0.05.
Mentions: We have shown previously that PKC activity regulates expression of slow MyHC2 (DiMario and Funk, 1999). Inhibition of PKC activity induced slow MyHC2 expression, and overexpression of PKC repressed slow MyHC2 expression. Therefore, we hypothesized that the regulation of slow MyHC2 by mAchR and Gαq activities was mediated by PKC activity. To determine whether mAchR and Gαq signaling controlled PKC activities in PM and MA muscle fibers, PKC activities were directly determined in cytoplasmic and membrane fractions obtained from PM and MA muscle fiber cultures (Fig. 6). These cultures were generated by incubation of noninnervated and innervated PM and MA muscle fibers in control medium and/or in medium containing 200 μM atropine. In addition, cytoplasmic and membrane fractions were obtained from PM and MA muscle fiber cultures transfected with CMVGαqQ209LFLAG. Membrane versus cytoplasmic PKC activities were determined to quantitate membrane-associated active PKC in the cultures. Noninnervated PM muscle fibers contained 2.5-fold more PKC activity than noninnervated MA muscle fibers. This is in agreement with previous studies examining PKC activities in avian and mammalian fast versus slow type muscles (Donnelly et al., 1994; DiMario and Funk, 1999). Innervation of both PM and MA muscle fibers significantly reduced PKC activities. Innervation of PM muscle fibers resulted in an ∼33% reduction in membrane versus cytoplasmic PKC activity. PM muscle fibers incubated in medium containing atropine also yielded reduced PKC activity by 41%. Combination of innervation of PM muscle fibers and incubation in medium containing atropine resulted in a significant, additive inhibition of PKC activity. PKC activities in MA muscle fibers incubated in control medium or medium containing atropine were not significantly different. Last, both PM and MA muscle fiber cultures expressing constitutively active Gαq yielded significantly increased PKC activities. These results indicate that direct modulation of mAchR and Gαq activities affects downstream PKC activities, particularly in PM muscle fibers. Reduced mAchR/Gαq signaling resulted in reduced PKC activity, and increased mAchR/Gαq signaling increased PKC activity.

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