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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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Western blot analysis of fast and slow MyHCs in innervated and noninnervated PM and MA muscle fiber cultures incubated in medium containing atropine. ED13 PM and MA muscle fiber cultures were established. On day 3 of incubation, spinal cord (SC) explants were added to half the cultures and 200 μM atropine (Atr) was added to the medium of half the cultures. Fast and slow MyHCs were extracted from the cultures on day 7 of incubation, electrophoresed, blotted, and detected using mAbs F59 and S58, respectively. Culture extracts contained approximately equal amounts of fast MyHC. Slow MyHC2 was detected in extracts from innervated MA muscle fibers incubated with and without atropine. Slow MyHC2 was detected in extracts from innervated PM muscle fibers incubated in medium containing atropine.
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fig4: Western blot analysis of fast and slow MyHCs in innervated and noninnervated PM and MA muscle fiber cultures incubated in medium containing atropine. ED13 PM and MA muscle fiber cultures were established. On day 3 of incubation, spinal cord (SC) explants were added to half the cultures and 200 μM atropine (Atr) was added to the medium of half the cultures. Fast and slow MyHCs were extracted from the cultures on day 7 of incubation, electrophoresed, blotted, and detected using mAbs F59 and S58, respectively. Culture extracts contained approximately equal amounts of fast MyHC. Slow MyHC2 was detected in extracts from innervated MA muscle fibers incubated with and without atropine. Slow MyHC2 was detected in extracts from innervated PM muscle fibers incubated in medium containing atropine.

Mentions: Immunostaining results were confirmed by Western blot analysis (Fig. 4). MyHC preparations of innervated and noninnervated PM and MA muscle fiber cultures incubated in control medium or medium containing 200 μM atropine were blotted and detected with F59 and S58 antibodies. Slow MyHC2 was present in innervated MA muscle fiber cultures incubated in medium with and without atropine. Slow MyHC2 was also detected in extracts from innervated PM muscle fiber cultures incubated in medium containing atropine. It is currently not known whether the observed doublet bands are due to degradation of slow MyHC2 or detection of slow MyHC3, a cardiac slow MyHC isoform transiently expressed in skeletal muscle and recognized by S58 (Nikovits et al., 1996). These results indicate that innervation is required for slow MyHC2 expression in both PM and MA muscle fibers. Furthermore, mAchR activity in innervated PM muscle fibers normally represses slow MyHC2 expression to nondetectable levels based on immunostaining.


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)

Western blot analysis of fast and slow MyHCs in innervated and noninnervated PM and MA muscle fiber cultures incubated in medium containing atropine. ED13 PM and MA muscle fiber cultures were established. On day 3 of incubation, spinal cord (SC) explants were added to half the cultures and 200 μM atropine (Atr) was added to the medium of half the cultures. Fast and slow MyHCs were extracted from the cultures on day 7 of incubation, electrophoresed, blotted, and detected using mAbs F59 and S58, respectively. Culture extracts contained approximately equal amounts of fast MyHC. Slow MyHC2 was detected in extracts from innervated MA muscle fibers incubated with and without atropine. Slow MyHC2 was detected in extracts from innervated PM muscle fibers incubated in medium containing atropine.
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Related In: Results  -  Collection

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fig4: Western blot analysis of fast and slow MyHCs in innervated and noninnervated PM and MA muscle fiber cultures incubated in medium containing atropine. ED13 PM and MA muscle fiber cultures were established. On day 3 of incubation, spinal cord (SC) explants were added to half the cultures and 200 μM atropine (Atr) was added to the medium of half the cultures. Fast and slow MyHCs were extracted from the cultures on day 7 of incubation, electrophoresed, blotted, and detected using mAbs F59 and S58, respectively. Culture extracts contained approximately equal amounts of fast MyHC. Slow MyHC2 was detected in extracts from innervated MA muscle fibers incubated with and without atropine. Slow MyHC2 was detected in extracts from innervated PM muscle fibers incubated in medium containing atropine.
Mentions: Immunostaining results were confirmed by Western blot analysis (Fig. 4). MyHC preparations of innervated and noninnervated PM and MA muscle fiber cultures incubated in control medium or medium containing 200 μM atropine were blotted and detected with F59 and S58 antibodies. Slow MyHC2 was present in innervated MA muscle fiber cultures incubated in medium with and without atropine. Slow MyHC2 was also detected in extracts from innervated PM muscle fiber cultures incubated in medium containing atropine. It is currently not known whether the observed doublet bands are due to degradation of slow MyHC2 or detection of slow MyHC3, a cardiac slow MyHC isoform transiently expressed in skeletal muscle and recognized by S58 (Nikovits et al., 1996). These results indicate that innervation is required for slow MyHC2 expression in both PM and MA muscle fibers. Furthermore, mAchR activity in innervated PM muscle fibers normally represses slow MyHC2 expression to nondetectable levels based on immunostaining.

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