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HDAC4 regulates muscle fiber type-specific gene expression programs.

Cohen TJ, Choi MC, Kapur M, Lira VA, Yan Z, Yao TP - Mol. Cells (2015)

Bottom Line: The cytoplasmic localization is associated with HDAC4 hyper-phosphorylation in slow/oxidative-fibers.Genetic reprogramming of fast/glycolytic fibers to oxidative fibers by active CaMKII or calcineurin leads to increased HDAC4 phosphorylation, HDAC4 nuclear export, and an increase in markers associated with oxidative fibers.Thus differential phosphorylation and localization of HDAC4 contributes to establishing fiber type-specific transcriptional programs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA.

ABSTRACT
Fiber type-specific programs controlled by the transcription factor MEF2 dictate muscle functionality. Here, we show that HDAC4, a potent MEF2 inhibitor, is predominantly localized to the nuclei in fast/glycolytic fibers in contrast to the sarcoplasm in slow/oxidative fibers. The cytoplasmic localization is associated with HDAC4 hyper-phosphorylation in slow/oxidative-fibers. Genetic reprogramming of fast/glycolytic fibers to oxidative fibers by active CaMKII or calcineurin leads to increased HDAC4 phosphorylation, HDAC4 nuclear export, and an increase in markers associated with oxidative fibers. Indeed, HDAC4 represses the MEF2-dependent, PGC-1α-mediated oxidative metabolic gene program. Thus differential phosphorylation and localization of HDAC4 contributes to establishing fiber type-specific transcriptional programs.

No MeSH data available.


HDAC4 inhibits slow fiber MHC expression in cultured myotubes. (A) C2C12 myoblast controls or those stably expressing an HDAC4 siRNA (KD) or re-expressing HDAC4 wild type (Rescue) were differentiated for indicated time points and cell lysates were subjected to Western blot analysis using indicated antibodies against slow and fast myosin heavy chains (MHC), I and IIs, respectively. (B) C2C12 myotubes stably expressing an HDAC4 siRNA or re-expressing HDAC4 wild type plasmid were analyzed for promoter-driven luciferase activities using MHC I and IIb-specific reporter constructs. Columns, mean; Bars, SD (n = 3). ***P < 0.001 versus control siRNA. (C,D) Expression of slow and fast MHC I and II in C2C12 myotubes treated with HDAC4 inhibitors. C2C12 myotubes were treated overnight with the indicated concentrations of trichostain A (TSA) or valproic acid (VPA) in a dose-dependent manner. Cell lysates were subjected to Western analysis using the indicated antibodies.
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f3-molce-38-4-343: HDAC4 inhibits slow fiber MHC expression in cultured myotubes. (A) C2C12 myoblast controls or those stably expressing an HDAC4 siRNA (KD) or re-expressing HDAC4 wild type (Rescue) were differentiated for indicated time points and cell lysates were subjected to Western blot analysis using indicated antibodies against slow and fast myosin heavy chains (MHC), I and IIs, respectively. (B) C2C12 myotubes stably expressing an HDAC4 siRNA or re-expressing HDAC4 wild type plasmid were analyzed for promoter-driven luciferase activities using MHC I and IIb-specific reporter constructs. Columns, mean; Bars, SD (n = 3). ***P < 0.001 versus control siRNA. (C,D) Expression of slow and fast MHC I and II in C2C12 myotubes treated with HDAC4 inhibitors. C2C12 myotubes were treated overnight with the indicated concentrations of trichostain A (TSA) or valproic acid (VPA) in a dose-dependent manner. Cell lysates were subjected to Western analysis using the indicated antibodies.

Mentions: Nuclear HDAC4-enriched fast fibers suggested that it might repress the MEF2-dependent slow/oxidative fiber gene expression. To test this possibility, C2C12 myoblasts stably expressing a HDAC4 siRNA with or without re-expressing an HDAC4 plasmid were differentiated into myotubes, and the expression of slow-fiber MHC I and fast fiber MHC II was determined. As shown in Fig. 3A, HDAC4 knockdown (KD) or HDAC4 re-expression (Rescue) did not have a significant effect on C2C12 differentiation as indicated by myogenin induction, thus suggesting that modulating HDAC4 expression does not alter the differentiation program per se, but rather specifically regulates MEF2 target gene expression (Cohen et al., 2009; Zhao et al., 2001;). However, the expression of slow-fiber MHC I (BAF-8) was elevated by HDAC4 KD and suppressed by the re-expression of HDAC4. Consistent with this finding, HDAC4 KD significantly increased promoter activity of MHC I, whereas HDAC4 re-expression suppressed this activity (Fig. 3B). HDAC4 expression levels did not have a significant effect on MHC II expression (Fig. 3A, MY-32) or its promoter activity (Fig. 3B). These results indicate a preferential effect of HDAC4 in preventing slow fiber-specific MHC I expression in C2C12 cells. We next investigated whether HDAC inhibitors, valproic acid (VPA) and trichostatin A (TSA), alone can differentially affect MHC I expression. Consistent with HDAC4 being a negative regulator of MHC I, a dose-dependent increase in MHC I was observed in VPA- and TSA-treated C2C12 cells (Figs. 3C and 3D). Interestingly, HDAC inhibitors decreased MHC II expression. These findings reveal that HDAC inhibitors can affect fiber type-specific MHC expression in cultured muscle cells.


HDAC4 regulates muscle fiber type-specific gene expression programs.

Cohen TJ, Choi MC, Kapur M, Lira VA, Yan Z, Yao TP - Mol. Cells (2015)

HDAC4 inhibits slow fiber MHC expression in cultured myotubes. (A) C2C12 myoblast controls or those stably expressing an HDAC4 siRNA (KD) or re-expressing HDAC4 wild type (Rescue) were differentiated for indicated time points and cell lysates were subjected to Western blot analysis using indicated antibodies against slow and fast myosin heavy chains (MHC), I and IIs, respectively. (B) C2C12 myotubes stably expressing an HDAC4 siRNA or re-expressing HDAC4 wild type plasmid were analyzed for promoter-driven luciferase activities using MHC I and IIb-specific reporter constructs. Columns, mean; Bars, SD (n = 3). ***P < 0.001 versus control siRNA. (C,D) Expression of slow and fast MHC I and II in C2C12 myotubes treated with HDAC4 inhibitors. C2C12 myotubes were treated overnight with the indicated concentrations of trichostain A (TSA) or valproic acid (VPA) in a dose-dependent manner. Cell lysates were subjected to Western analysis using the indicated antibodies.
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Related In: Results  -  Collection

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f3-molce-38-4-343: HDAC4 inhibits slow fiber MHC expression in cultured myotubes. (A) C2C12 myoblast controls or those stably expressing an HDAC4 siRNA (KD) or re-expressing HDAC4 wild type (Rescue) were differentiated for indicated time points and cell lysates were subjected to Western blot analysis using indicated antibodies against slow and fast myosin heavy chains (MHC), I and IIs, respectively. (B) C2C12 myotubes stably expressing an HDAC4 siRNA or re-expressing HDAC4 wild type plasmid were analyzed for promoter-driven luciferase activities using MHC I and IIb-specific reporter constructs. Columns, mean; Bars, SD (n = 3). ***P < 0.001 versus control siRNA. (C,D) Expression of slow and fast MHC I and II in C2C12 myotubes treated with HDAC4 inhibitors. C2C12 myotubes were treated overnight with the indicated concentrations of trichostain A (TSA) or valproic acid (VPA) in a dose-dependent manner. Cell lysates were subjected to Western analysis using the indicated antibodies.
Mentions: Nuclear HDAC4-enriched fast fibers suggested that it might repress the MEF2-dependent slow/oxidative fiber gene expression. To test this possibility, C2C12 myoblasts stably expressing a HDAC4 siRNA with or without re-expressing an HDAC4 plasmid were differentiated into myotubes, and the expression of slow-fiber MHC I and fast fiber MHC II was determined. As shown in Fig. 3A, HDAC4 knockdown (KD) or HDAC4 re-expression (Rescue) did not have a significant effect on C2C12 differentiation as indicated by myogenin induction, thus suggesting that modulating HDAC4 expression does not alter the differentiation program per se, but rather specifically regulates MEF2 target gene expression (Cohen et al., 2009; Zhao et al., 2001;). However, the expression of slow-fiber MHC I (BAF-8) was elevated by HDAC4 KD and suppressed by the re-expression of HDAC4. Consistent with this finding, HDAC4 KD significantly increased promoter activity of MHC I, whereas HDAC4 re-expression suppressed this activity (Fig. 3B). HDAC4 expression levels did not have a significant effect on MHC II expression (Fig. 3A, MY-32) or its promoter activity (Fig. 3B). These results indicate a preferential effect of HDAC4 in preventing slow fiber-specific MHC I expression in C2C12 cells. We next investigated whether HDAC inhibitors, valproic acid (VPA) and trichostatin A (TSA), alone can differentially affect MHC I expression. Consistent with HDAC4 being a negative regulator of MHC I, a dose-dependent increase in MHC I was observed in VPA- and TSA-treated C2C12 cells (Figs. 3C and 3D). Interestingly, HDAC inhibitors decreased MHC II expression. These findings reveal that HDAC inhibitors can affect fiber type-specific MHC expression in cultured muscle cells.

Bottom Line: The cytoplasmic localization is associated with HDAC4 hyper-phosphorylation in slow/oxidative-fibers.Genetic reprogramming of fast/glycolytic fibers to oxidative fibers by active CaMKII or calcineurin leads to increased HDAC4 phosphorylation, HDAC4 nuclear export, and an increase in markers associated with oxidative fibers.Thus differential phosphorylation and localization of HDAC4 contributes to establishing fiber type-specific transcriptional programs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA.

ABSTRACT
Fiber type-specific programs controlled by the transcription factor MEF2 dictate muscle functionality. Here, we show that HDAC4, a potent MEF2 inhibitor, is predominantly localized to the nuclei in fast/glycolytic fibers in contrast to the sarcoplasm in slow/oxidative fibers. The cytoplasmic localization is associated with HDAC4 hyper-phosphorylation in slow/oxidative-fibers. Genetic reprogramming of fast/glycolytic fibers to oxidative fibers by active CaMKII or calcineurin leads to increased HDAC4 phosphorylation, HDAC4 nuclear export, and an increase in markers associated with oxidative fibers. Indeed, HDAC4 represses the MEF2-dependent, PGC-1α-mediated oxidative metabolic gene program. Thus differential phosphorylation and localization of HDAC4 contributes to establishing fiber type-specific transcriptional programs.

No MeSH data available.