Limits...
Activity-dependent and -independent nuclear fluxes of HDAC4 mediated by different kinases in adult skeletal muscle.

Liu Y, Randall WR, Schneider MF - J. Cell Biol. (2005)

Bottom Line: Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2).Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca(2+)-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4.Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

ABSTRACT
Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2). Here, we show that repetitive slow fiber type electrical stimulation, but not fast fiber type stimulation, caused HDAC4-GFP, but not HDAC5-GFP, to translocate from the nucleus to the cytoplasm in cultured adult skeletal muscle fibers. HDAC4-GFP translocation was blocked by calmodulin-dependent protein kinase (CaMK) inhibitor KN-62. Slow fiber type stimulation increased MEF2 transcriptional activity, nuclear Ca(2+) concentration, and nuclear levels of activated CaMKII, but not total nuclear CaMKII or CaM-YFP. Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca(2+)-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4. Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

Show MeSH

Related in: MedlinePlus

Effect of stimulation on CaMKII phosphorylation and MEF2 reporter activity. (A) Mean nuclear pixel fluorescence (normalized to control) in resting fibers and fibers repeatedly stimulated for 1 h using 10-Hz trains in the presence or absence of KN-62 and labeled with antibody for either autophosphorylated (open bars) or total (closed bars) CaMKII. Only nuclei stimulated in the absence of KN-62 and stained for autophosphorylated CaMKII exhibited a significant increase in nuclear staining after stimulation. From left to right, the data were averages of mean nuclear fluorescence from 26, 17, 23, 21, or 13 nuclei from 22, 12, 18, 12, or 10 fibers, respectively. (B) Luciferase activity was increased in fibers infected with adenovirus-encoding MEF2-luciferase reporter and stimulated with 10-Hz trains for 5 h. 5 μM KN-62 blocked the increase in luciferase activity stimulated with 10-Hz trains. Results represent triplicate measurements from each of three independent experiments. Error bars represent ± 1 SEM. *, P < 0.05 versus control without stimulation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171787&req=5

fig3: Effect of stimulation on CaMKII phosphorylation and MEF2 reporter activity. (A) Mean nuclear pixel fluorescence (normalized to control) in resting fibers and fibers repeatedly stimulated for 1 h using 10-Hz trains in the presence or absence of KN-62 and labeled with antibody for either autophosphorylated (open bars) or total (closed bars) CaMKII. Only nuclei stimulated in the absence of KN-62 and stained for autophosphorylated CaMKII exhibited a significant increase in nuclear staining after stimulation. From left to right, the data were averages of mean nuclear fluorescence from 26, 17, 23, 21, or 13 nuclei from 22, 12, 18, 12, or 10 fibers, respectively. (B) Luciferase activity was increased in fibers infected with adenovirus-encoding MEF2-luciferase reporter and stimulated with 10-Hz trains for 5 h. 5 μM KN-62 blocked the increase in luciferase activity stimulated with 10-Hz trains. Results represent triplicate measurements from each of three independent experiments. Error bars represent ± 1 SEM. *, P < 0.05 versus control without stimulation.

Mentions: To test for changes in CaMKII phosphorylation, fibers were stimulated with a 5-s duration 10-Hz train every 50 s for 1 h with or without 5 μM KN-62 in the Ringer's solution and fixed and stained with primary antibody to activated (autophosphorylated; Fig. 3 A, open bars) or total (Fig. 3 A, filled bars) CaMKII. 1 h of 10-Hz train stimulation significantly increased (P < 0.01) the mean nuclear stain of activated CaMKII compared with unstimulated fibers, and KN-62 (5 μM) blocked the increase in activated CaMKII staining after stimulation (Fig. 3 A). In parallel experiments, in which the fibers were stained with antibody directed against total CaMKII, there was no difference in mean nuclear antibody stain between stimulated and unstimulated fibers (P > 0.05; Fig. 3 A). These results are consistent with activation of nuclear CaMKII by fiber electrical stimulation without any appreciable translocation of CaMKII from the cytoplasm to the nucleus.


Activity-dependent and -independent nuclear fluxes of HDAC4 mediated by different kinases in adult skeletal muscle.

Liu Y, Randall WR, Schneider MF - J. Cell Biol. (2005)

Effect of stimulation on CaMKII phosphorylation and MEF2 reporter activity. (A) Mean nuclear pixel fluorescence (normalized to control) in resting fibers and fibers repeatedly stimulated for 1 h using 10-Hz trains in the presence or absence of KN-62 and labeled with antibody for either autophosphorylated (open bars) or total (closed bars) CaMKII. Only nuclei stimulated in the absence of KN-62 and stained for autophosphorylated CaMKII exhibited a significant increase in nuclear staining after stimulation. From left to right, the data were averages of mean nuclear fluorescence from 26, 17, 23, 21, or 13 nuclei from 22, 12, 18, 12, or 10 fibers, respectively. (B) Luciferase activity was increased in fibers infected with adenovirus-encoding MEF2-luciferase reporter and stimulated with 10-Hz trains for 5 h. 5 μM KN-62 blocked the increase in luciferase activity stimulated with 10-Hz trains. Results represent triplicate measurements from each of three independent experiments. Error bars represent ± 1 SEM. *, P < 0.05 versus control without stimulation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171787&req=5

fig3: Effect of stimulation on CaMKII phosphorylation and MEF2 reporter activity. (A) Mean nuclear pixel fluorescence (normalized to control) in resting fibers and fibers repeatedly stimulated for 1 h using 10-Hz trains in the presence or absence of KN-62 and labeled with antibody for either autophosphorylated (open bars) or total (closed bars) CaMKII. Only nuclei stimulated in the absence of KN-62 and stained for autophosphorylated CaMKII exhibited a significant increase in nuclear staining after stimulation. From left to right, the data were averages of mean nuclear fluorescence from 26, 17, 23, 21, or 13 nuclei from 22, 12, 18, 12, or 10 fibers, respectively. (B) Luciferase activity was increased in fibers infected with adenovirus-encoding MEF2-luciferase reporter and stimulated with 10-Hz trains for 5 h. 5 μM KN-62 blocked the increase in luciferase activity stimulated with 10-Hz trains. Results represent triplicate measurements from each of three independent experiments. Error bars represent ± 1 SEM. *, P < 0.05 versus control without stimulation.
Mentions: To test for changes in CaMKII phosphorylation, fibers were stimulated with a 5-s duration 10-Hz train every 50 s for 1 h with or without 5 μM KN-62 in the Ringer's solution and fixed and stained with primary antibody to activated (autophosphorylated; Fig. 3 A, open bars) or total (Fig. 3 A, filled bars) CaMKII. 1 h of 10-Hz train stimulation significantly increased (P < 0.01) the mean nuclear stain of activated CaMKII compared with unstimulated fibers, and KN-62 (5 μM) blocked the increase in activated CaMKII staining after stimulation (Fig. 3 A). In parallel experiments, in which the fibers were stained with antibody directed against total CaMKII, there was no difference in mean nuclear antibody stain between stimulated and unstimulated fibers (P > 0.05; Fig. 3 A). These results are consistent with activation of nuclear CaMKII by fiber electrical stimulation without any appreciable translocation of CaMKII from the cytoplasm to the nucleus.

Bottom Line: Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2).Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca(2+)-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4.Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

ABSTRACT
Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2). Here, we show that repetitive slow fiber type electrical stimulation, but not fast fiber type stimulation, caused HDAC4-GFP, but not HDAC5-GFP, to translocate from the nucleus to the cytoplasm in cultured adult skeletal muscle fibers. HDAC4-GFP translocation was blocked by calmodulin-dependent protein kinase (CaMK) inhibitor KN-62. Slow fiber type stimulation increased MEF2 transcriptional activity, nuclear Ca(2+) concentration, and nuclear levels of activated CaMKII, but not total nuclear CaMKII or CaM-YFP. Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca(2+)-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4. Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

Show MeSH
Related in: MedlinePlus