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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.

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Calcium concentration is elevated in both the nucleus and cytoplasm during repetitive electrical stimulation. (A) A fiber was repetitively stimulated every 50 s with a 5-s duration train of pulses at 10 Hz. Calcium-dependent fluo-4 fluorescence images taken in x-y scan mode in the resting “control” condition before the start of repetitive stimulation (a), 1–1.6 s after the start of the first train (b), 0–0.6, 2–2.6, and 42–42.6 s after the first train (c–e), and 1–1.6 s after the start of the second train (f). The notched appearance at the edge of the fiber in panels b and f is due to individual nonfused fiber contraction at each stimulus. The nuclear Ca2+ concentration was still elevated compared with the rest a few hundred milliseconds after the end of the 5-s train (c). Arrowheads indicate the application of each stimulus within an image. Note that each image was scanned from top to bottom at 0.05 μm/ms. (B) Time course of nuclear (closed circles) or cytosolic (open circles) mean pixel fluorescence in the central 100 μm (recorded during 200 ms) of images in A and others not depicted. Arrows indicate underestimation of mean pixel fluorescence due to the inclusion of pixels maxed out due to detector saturation. Horizontal bars below records mark duration of the 5-s 10-Hz trains of stimuli. (C) Time course of cytoplasmic fluorescence from individual images (i.e., from top to bottom) during stimulation (top; from A, b) and before and after stimulation (bottom; from A, a and e). Vertical lines mark times of individual stimuli.
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fig7: Calcium concentration is elevated in both the nucleus and cytoplasm during repetitive electrical stimulation. (A) A fiber was repetitively stimulated every 50 s with a 5-s duration train of pulses at 10 Hz. Calcium-dependent fluo-4 fluorescence images taken in x-y scan mode in the resting “control” condition before the start of repetitive stimulation (a), 1–1.6 s after the start of the first train (b), 0–0.6, 2–2.6, and 42–42.6 s after the first train (c–e), and 1–1.6 s after the start of the second train (f). The notched appearance at the edge of the fiber in panels b and f is due to individual nonfused fiber contraction at each stimulus. The nuclear Ca2+ concentration was still elevated compared with the rest a few hundred milliseconds after the end of the 5-s train (c). Arrowheads indicate the application of each stimulus within an image. Note that each image was scanned from top to bottom at 0.05 μm/ms. (B) Time course of nuclear (closed circles) or cytosolic (open circles) mean pixel fluorescence in the central 100 μm (recorded during 200 ms) of images in A and others not depicted. Arrows indicate underestimation of mean pixel fluorescence due to the inclusion of pixels maxed out due to detector saturation. Horizontal bars below records mark duration of the 5-s 10-Hz trains of stimuli. (C) Time course of cytoplasmic fluorescence from individual images (i.e., from top to bottom) during stimulation (top; from A, b) and before and after stimulation (bottom; from A, a and e). Vertical lines mark times of individual stimuli.

Mentions: Muscle fibers loaded with the calcium indicator fluo-4 were imaged before, during, and after repetitive stimulation with 5-s duration 10-Hz trains applied every 50 s (Fig. 7 A). Note that full acquisition of each image in Fig. 7 A required 600 ms (top line first, bottom line last). Thus, when an image was acquired during the train (Fig. 7 A, b and f), the abrupt rise in fluorescence of the Ca2+ concentration indicator (sharp bright horizontal stripes across the fiber) in response to application of a given stimulus (Fig. 7 A, arrowheads) corresponds to a particular line that was imaged at a time when a stimulus was applied. The subsequent stimulus, applied 100 ms later in time, appears at a spatial location 5 μm further down the image because the images were acquired at 0.05 μm/ms. With the time resolution used here, each stimulus activates the entire fiber cross section essentially uniformly (Fig. 7 A, b and f). The decay of the cytosolic Ca2+ concentration signal occurs rapidly after the train of stimuli (Fig. 7 A, c; and Fig. 7 B), whereas nuclear Ca2+ concentration decays more slowly than cytosolic Ca2+ concentration (Fig. 7 B). The lower fluorescence of the nonratiometric Ca2+ indicator fluo-4 in the nucleus than in the cytoplasm in resting fibers (Fig. 7 B) probably does not indicate a difference in resting Ca2+ concentration, but more likely arises from preferential exclusion of dye from the nucleus, preferential binding of dye in the cytoplasm, lower affinity of the dye for Ca2+ in the nucleus than the cytoplasm, or a combination of these effects. Within the train of stimuli (Fig. 7 C), cytosolic Ca2+ concentration rises after each stimulus and then falls between pulses. In contrast, nuclear Ca2+ concentration seems to rise more slowly and continuously during the train (Fig. 7 B), as anticipated for diffusion of elevated Ca2+ concentration from the cytoplasm into the nucleus. Compared with the resting fiber (Fig. 7 A, a), nuclear Ca2+ concentration remains elevated a few hundred ms after the end of the 5-s 10-Hz trains of pulses (Fig. 7 A, c; and Fig. 7 B). This elevated nuclear Ca2+ concentration then declines during the 45-s interval between successive trains (Fig. 7 B). Thus, activation of nuclear CaMK due to elevated nuclear Ca2+ concentration appears to be a likely mechanism underlying the CaMK-dependent HDAC4 efflux during fiber electrical stimulation.


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)

Calcium concentration is elevated in both the nucleus and cytoplasm during repetitive electrical stimulation. (A) A fiber was repetitively stimulated every 50 s with a 5-s duration train of pulses at 10 Hz. Calcium-dependent fluo-4 fluorescence images taken in x-y scan mode in the resting “control” condition before the start of repetitive stimulation (a), 1–1.6 s after the start of the first train (b), 0–0.6, 2–2.6, and 42–42.6 s after the first train (c–e), and 1–1.6 s after the start of the second train (f). The notched appearance at the edge of the fiber in panels b and f is due to individual nonfused fiber contraction at each stimulus. The nuclear Ca2+ concentration was still elevated compared with the rest a few hundred milliseconds after the end of the 5-s train (c). Arrowheads indicate the application of each stimulus within an image. Note that each image was scanned from top to bottom at 0.05 μm/ms. (B) Time course of nuclear (closed circles) or cytosolic (open circles) mean pixel fluorescence in the central 100 μm (recorded during 200 ms) of images in A and others not depicted. Arrows indicate underestimation of mean pixel fluorescence due to the inclusion of pixels maxed out due to detector saturation. Horizontal bars below records mark duration of the 5-s 10-Hz trains of stimuli. (C) Time course of cytoplasmic fluorescence from individual images (i.e., from top to bottom) during stimulation (top; from A, b) and before and after stimulation (bottom; from A, a and e). Vertical lines mark times of individual stimuli.
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fig7: Calcium concentration is elevated in both the nucleus and cytoplasm during repetitive electrical stimulation. (A) A fiber was repetitively stimulated every 50 s with a 5-s duration train of pulses at 10 Hz. Calcium-dependent fluo-4 fluorescence images taken in x-y scan mode in the resting “control” condition before the start of repetitive stimulation (a), 1–1.6 s after the start of the first train (b), 0–0.6, 2–2.6, and 42–42.6 s after the first train (c–e), and 1–1.6 s after the start of the second train (f). The notched appearance at the edge of the fiber in panels b and f is due to individual nonfused fiber contraction at each stimulus. The nuclear Ca2+ concentration was still elevated compared with the rest a few hundred milliseconds after the end of the 5-s train (c). Arrowheads indicate the application of each stimulus within an image. Note that each image was scanned from top to bottom at 0.05 μm/ms. (B) Time course of nuclear (closed circles) or cytosolic (open circles) mean pixel fluorescence in the central 100 μm (recorded during 200 ms) of images in A and others not depicted. Arrows indicate underestimation of mean pixel fluorescence due to the inclusion of pixels maxed out due to detector saturation. Horizontal bars below records mark duration of the 5-s 10-Hz trains of stimuli. (C) Time course of cytoplasmic fluorescence from individual images (i.e., from top to bottom) during stimulation (top; from A, b) and before and after stimulation (bottom; from A, a and e). Vertical lines mark times of individual stimuli.
Mentions: Muscle fibers loaded with the calcium indicator fluo-4 were imaged before, during, and after repetitive stimulation with 5-s duration 10-Hz trains applied every 50 s (Fig. 7 A). Note that full acquisition of each image in Fig. 7 A required 600 ms (top line first, bottom line last). Thus, when an image was acquired during the train (Fig. 7 A, b and f), the abrupt rise in fluorescence of the Ca2+ concentration indicator (sharp bright horizontal stripes across the fiber) in response to application of a given stimulus (Fig. 7 A, arrowheads) corresponds to a particular line that was imaged at a time when a stimulus was applied. The subsequent stimulus, applied 100 ms later in time, appears at a spatial location 5 μm further down the image because the images were acquired at 0.05 μm/ms. With the time resolution used here, each stimulus activates the entire fiber cross section essentially uniformly (Fig. 7 A, b and f). The decay of the cytosolic Ca2+ concentration signal occurs rapidly after the train of stimuli (Fig. 7 A, c; and Fig. 7 B), whereas nuclear Ca2+ concentration decays more slowly than cytosolic Ca2+ concentration (Fig. 7 B). The lower fluorescence of the nonratiometric Ca2+ indicator fluo-4 in the nucleus than in the cytoplasm in resting fibers (Fig. 7 B) probably does not indicate a difference in resting Ca2+ concentration, but more likely arises from preferential exclusion of dye from the nucleus, preferential binding of dye in the cytoplasm, lower affinity of the dye for Ca2+ in the nucleus than the cytoplasm, or a combination of these effects. Within the train of stimuli (Fig. 7 C), cytosolic Ca2+ concentration rises after each stimulus and then falls between pulses. In contrast, nuclear Ca2+ concentration seems to rise more slowly and continuously during the train (Fig. 7 B), as anticipated for diffusion of elevated Ca2+ concentration from the cytoplasm into the nucleus. Compared with the resting fiber (Fig. 7 A, a), nuclear Ca2+ concentration remains elevated a few hundred ms after the end of the 5-s 10-Hz trains of pulses (Fig. 7 A, c; and Fig. 7 B). This elevated nuclear Ca2+ concentration then declines during the 45-s interval between successive trains (Fig. 7 B). Thus, activation of nuclear CaMK due to elevated nuclear Ca2+ concentration appears to be a likely mechanism underlying the CaMK-dependent HDAC4 efflux during fiber electrical stimulation.

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