Yewei Liu 1, Minerva Contreras 1, Tiansheng Shen 1, William R. Randall 2 and Martin F. Schneider 1

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1 J Physiol (2009) pp α-adrenergic signalling activates protein kinase D and causes nuclear efflux of the transcriptional repressor HDAC5 in cultured adult mouse soleus skeletal muscle fibres Yewei Liu 1, Minerva Contreras 1, Tiansheng Shen 1, William R. Randall 2 and Martin F. Schneider 1 1 DepartmentsofBiochemistryandMolecularBiology,and 2 Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, USA The protein kinase PKD1 has recently been linked to slow fibre-type gene expression in fast skeletal muscle through phosphorylation of class II histone deacetylase (HDAC) molecules, resulting in nuclear efflux of HDAC and consequent activation of the transcription factor MEF2. However, possible upstream activators of PKD, and the time course and signalling pathway of downstream effectors have not been determined in skeletal muscle. Using fluorescent fusion proteins HDAC5 green fluorescent protein (GFP) and PKD1 mplum expressed in fibres isolated from predominantly slow soleus muscle and maintained for 4 days in culture, we now show that α-adrenergic receptor activation by phenylephrine causes a transient, PKD-dependent HDAC5 GFP nuclear efflux. Concurrent to this response, PKD1 mplum transiently redistributes from cytoplasm to plasma membrane and nuclei, and back, during 2 h exposure to phenylephrine. The recovery may reflect α-receptor desensitization. In contrast, the phorbol ester PMA (phorbol-12-myristate-13-acetate, a pharmacological mimic of the downstream mediator diacylglycerol in α-adrenergic signalling), caused continuous PKDdependent HDAC5 GFP nuclear efflux and maintained PKD1 mplum redistribution. In the absence of expressed HDAC, PMA increased histone H3 acetylation and increased MEF2 reporter activity in a PKD-dependent manner, consistent with PKD phosphorylation of endogenous HDAC(s) and reduced nuclear HDAC activity due to HDAC nuclear efflux. HDAC5 GFP did not respond to PMA in fibres from predominantly fast flexor digitorum brevis (FDB) muscle, but did in FDB fibres expressing exogenous PKD1. Our results demonstrate that a PKD-mediated signalling pathway for HDAC nuclear efflux is activated in slow skeletal muscle through adrenergic input, which is typically active in parallel with motor neurone input during muscular activity. (Received 10 November 2008; accepted after revision 30 December 2008; first published online 5 January 2009) Corresponding author M. F. Schneider: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD , USA. mschneid@umaryland.edu PKD family members (PKD1, also known as PKCμ, PKD2 and PKD3) are serine/threonine kinases within the CaM kinase group that are involved in intracellular signalling in a wide variety of cell types (van Lint et al. 2002; Rozengurt et al. 2005). PKD is activated by diacylglycerol (DAG) produced by phospholipase C cleavage of phosphatidylinositol (4,5)-bisphosphate. In intact cells, PKD can be activated by DAG produced via G protein-coupled receptor (GPCR) agonists and growth factors, as well as artificially by phorbol esters which mimic activationbydag.pkdactivationbydag(orbyphorbol esters) can occur through two mechanisms. First, DAG can bind and activate PKD directly. All PKD family members have a tandem repeat of zinc-finger-like cysteine-rich motifs (referred to as cys1 and cys2) that are involved in the recruitment of PKD to the plasma membrane through high-affinity binding to DAG. Second, DAG can activate protein kinase C (PKC), which can subsequently phosphorylate PKD at its catalytic domain. Studies by Rozengurt and colleagues (Waldron & Rozengurt, 2003) showed that PKCε, a member of the novel PKC (npkc) family, directly interacts with the pleckstrin-homology (PH) domain of PKD and trans-phosphorylates the activation loop of PKD at Ser 744 and 748 leading to PKD activation. The intracellular location of PKD is directly related to its activation status (Wang, 2006). PKD is localized in the cytosol and in several intracellular compartments DOI: /jphysiol

2 1102 Y. Liu and others J Physiol including Golgi, plasma membrane and mitochondria in non-activated cells (Rozengurt et al. 2005) and undergoes rapid transient translocation from the cytosol to the plasma membrane and the nucleus upon activation of G protein-coupled receptors (GPCRs) (Rey et al. 2001a,b). Different degrees of nuclear cytoplasmic shuttling in the basal state were observed for different isoforms of PKD (Rey et al. 2001a;Aueret al. 2005), although the protein structures are well conserved between different isoforms. Recent studies on cardiac muscle have identified PKD as a regulator of agonist-dependent cardiac hypertrophy through direct phosphorylation and subsequent nuclear export of HDAC5 (Vega et al. 2004; Harrison et al. 2006). In isolated cardiac myocytes, the exogenously expressed fluorescent fusion protein HDAC5 GFP was not driven out of myocyte nuclei by repetitive Ca 2+ signals, but application of endothelin 1, which activates G protein-coupled receptors in cardiac cells, caused robust nuclear efflux of HDAC5 GFP (Wu et al. 2006). Members of npkc (δ, ε and θ), but not of conventional PKC (α and β), can activate PKD and trigger the nuclear efflux of HDAC5 (Vega et al. 2004). In response to hypertrophic signals, cardiac HDAC5 is phosphorylated on Ser 259 and 498 by PKD (Vega et al. 2004). Phosphorylated HDAC5 binds to the chaperone protein and translocates to the cytoplasm via the exportin protein CRM1 nuclear export system (McKinsey et al. 2000, 2001; McKinsey, 2007), thereby removing the inhibition of HDAC5 on the transcription factor MEF2 (myocyte enhancer factor 2). In general, MEF2 activity in muscle is inhibited by class II HDACs (HDAC4, 5, 7 and the MEF2-interacting transcription repressor (MITR)), which repress MEF2 transcriptional activation by binding directly to MEF2 in the nucleus (Miska et al. 1999). In addition, nuclear HDACs can also regulate transcriptional activity by deacetylation of histones and other nuclear proteins. Thus, nuclear PKD acts as an important regulator of gene activity in cardiac muscle through phosphorylation and subsequent nuclear export of class II HDACs. Relatively little information is available on the functional roles and intracellular distribution of PKD in adult skeletal muscle or on the role of PKD in the translocation of HDAC5 between different cellular compartments in skeletal muscle. The experiments presented here were designed to determine the signalling pathway for activation of PKD, to study the possible role of PKD in the regulation of HDAC5, and to examine the subcellular distribution of PKD in its resting and activated state in adult skeletal muscle fibres. We used fully differentiated skeletal muscle fibres cultured for 4 days after isolation from soleus (SOL) muscles of 4- to 6-week-old mice. We here report that application of the α-adrenergic agonist phenylephrine causes the transient nuclear efflux of expressed HDAC5 GFP in SOL fibres, but not in FDB fibres, in a PKD-dependent manner. Expressed fluorescent fusion protein PKD1 mplum also exhibited transient redistribution from cytoplasm to plasma membrane and nuclei during continuous exposure to phenylephrine. In contrast, the phorbol ester PMA produced continuous HDAC5 GFP nuclear efflux and maintained redistribution of PKD1 mplum, consistent with continuous activation of the PKD pathway, in SOL but not in FDB fibres. Expression of exogenous wild type PKD in FDB fibres introduces a PMA-dependent activation of HDAC5 GFP nuclear efflux, and expression of an exogenous mutant of PKD that is constitutively active results in increased nuclear efflux of HDAC5 in both SOL and FDB fibres without PMA or phenylephrine stimulation. We also show that PMA increases nuclear levels of Lys 9 and 14 acetylated histone H3, and increases MEF2 promoter-driven reporter activity in SOL fibres in a PKD-dependent manner, which would be consistent with a nuclear efflux of endogenous HDACs following phosphorylation by PKD. Our results demonstrate that a PKD-mediated pathway for HDAC5 nuclear efflux is activated in slow skeletal muscle via adrenergic input, which is typically active in parallel with motor neurone input during muscular activity. Methods Construction of recombinant adenoviruses Production of recombinant adenovirus (Ad5) containing wild type PKD1 (wtpkd1) cdna, constitutively active PKD1 (capkd1) or kinase-deficient dominant negative mutant of PKD1 (dnpkd1) and the corresponding fluorescent fusion proteins with mplum was carried out according to the methods of Hardy et al. (1997) as previously described (Liu et al. 2001). The constitutively active capkd1 contains glutamic acid residues in place of the PKC phosphorylation sites at Ser 744 and 748 (Wood et al. 2005). The kinase-deficient mutant dnpkd1 contains a mutation of Lys 618 to methionine (Zugaza et al. 1996). The PKD1 mplum and dnpkd1 mplum plasmids were constructed by placing mplum (Wang et al. 2004) at the N terminal of the PKD1 or dnpkd1. Recombinant adenovirus expressing HDAC5 GFP was a gift from Dr T. A. McKinsey (Myogen, Inc., Westminter, CO, USA; Harrison et al. 2004). A recombinant adenovirus containing a MEF2 luciferase reporter cassette was provided by Dr J. D. Molkentin (Children s Hospital Medical Center, Cincinnati, OH, USA; Wilkins et al. 2004). Infection of recombinant adenoviruses in muscle fibres Single muscle fibres were enzymatically dissociated from SOL or FDB muscles of 4- to 5-week-old CD-1 mice and cultured as previously described (Liu et al. 2001).

3 J Physiol α-adrenergic signalling and HDAC5 in skeletal muscle 1103 Animals were killed by CO 2 exposure followed by cervical dislocation before removal of the muscles according to protocols approved by the University of Maryland Institutional Animal Care and Use Committee. Isolated fibres were cultured on laminin-coated glass coverslips, each glued over a 10 mm diameter hole through the centre of a plastic Petri dish. Fibres were cultured in MEM containing 10% fetal bovine serum and 50 μgml 1 gentamicin sulphate in 5% CO 2 (37 C). Virus infections were performed as previously described (Liu et al. 2001). Microscopy and image acquisition Two days after infection, culture medium was changed to Ringer solution (in mm; 135 NaCl, 4 KCl, 1 MgCl 2, 10 Hepes, 10 glucose and 1.8 CaCl 2, ph 7.4). The culture dish was mounted on an Olympus IX70 inverted microscope equipped with an Olympus FluoView 500 laser scanning confocal imaging system. Fibres were viewed with an Olympus 60 /1.2 NA water immersion objective and scanned at 3.0 zoom with constant laser power and gain. The fibres were maintained and imaged at room temperature. For treatment with chemical reagents, fibres were rinsed with Ringer solution and a first image was taken ( 30 min in the figures). The cultures were then maintained on the microscope stage for 30 min before the chemical reagents were added. One image was taken immediately after drug addition (0 min in the figures), and then images were taken every 10 min. Analysis of translocation of fluorescent protein in living fibres The average fluorescence of pixels within user-specified areas of interest (AOI) in each image were quantified using software custom-written in the IDL programming language (Research Systems, Boulder, CO, USA). All the fluorescence values for the AOI at each time point were normalized by the time zero value for the AOI. The resulting data give the relative value of fluorescence of each individual AOI at different time points. Results are expressed as the mean ± S.E.M. Western blot analysis FDB muscle fibres from two 4- to 5-week-old CD-1 mice were isolated and pooled together. Cytoplasmic and nuclear proteins were extracted using a modification of the protocol of McGee et al. (2003). Briefly, FDB muscle fibres were lysed on ice for 30 s with lysis buffer A (in mm; 250 sucrose, 10 NaCl, 3 MgCl 2, 1 dithiothreitol, 1 phenylmethylsulphonyl flouride and 1 tablet ml 1 of tissue protease inhibitor cocktail (Roche Diagnostic, Indianapolis, IN, USA)), and passed through a 21-gauge syringe needle 10 times. The lysates were subjected to centrifugation at 500 g for 10 min at 4 C. The supernatant, representing the cytosolic fraction, was collected and stored. The remaining pellet was resuspended in ice-cold buffer B (in mm; 50 Tris, ph7.5, 1 EDTA, 1 EGTA, 1 DTT, 50 NaF, 5 sodium pyrophosphate, 50 MgCl 2 and 1 phenylmethylsulphonyl fluoride (PMSF), plus 10% glycerol, 1% Triton X-100, and 1 tablet ml 1 of tissue protease inhibitor cocktail, and placed on ice for 10 min. The resuspended pellet was centrifugated at 3000 g for 10 min at 4 C and the supernatant was collected and stored as nuclear fraction. Protein content of the cytosolic and nuclear fractions was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). Approximately 70 μg protein from each sample was separated by SDS-PAGE using a 4 12% gradient gel and transferred to nitrocellulose. Blots were then processed and probed with the appropriate antibodies. The antibody for HDAC5 was purchased from Cell Signalling Technology (Danvers, MA, USA). The antibody against histone H3 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the antibody for GAPDH was from Sigma (St Louis, MO, USA). The immunoreactive bands were visualized by ECL Western Blotting Detection Reagents (GE Healthcare Amersham, Piscataway, NJ, USA). Immunofluorescence stain of acetylated histone H3 in nucleus Cultured muscle fibres were fixed after treatment with 200 nm PMA for 2 h without Gö 6976 or after treatment with 1 μm of Gö 6976 for 30 min, then with 200 nm PMA foranother2hwithgö 6976, or without any treatments as a control. The cultures were immunostained with primary antibody against histone H3 acetylated at Lys 9 and 14 and fluorescent conjugated secondary antibody (Invitrogen, La Jolla, CA, USA). The fluorescence of the nucleus was quantified using the confocal microscope with constant laser power and gain. MEF2 activity and renilla luciferase reporter For MEF2 reporter assay, cultured muscle fibres were infected with adenovirus encoding MEF2 luciferase reporter (Wilkins et al. 2004) for 48 h. The cultures were then treated for 1 day with 200 nm PMA alone, or for 1 day with Gö 6976 and PMA, or not treated as control. The cultures were then lysed in passive lysis buffer (Promega Corp., Madison, WI, USA). For renilla luciferase reporter assay, cultured muscle fibres were infected with adenovirus encoding renilla luciferase driven by thymidine kinase (TK) promoter. Luciferase activity was determined with a luciferase assay kit (Promega).

4 1104 Y. Liu and others J Physiol Figure 1. Effects of phenylephrine on the subcellular distribution of HDAC5 GFP in SOL fibres A, image of a soleus fibre expressing HDAC5 GFP, demonstrating how the cytoplasmic AOI (c) and nuclear AOI (n) are defined. B, a SOL fibre was infected with HDAC5 GFP and treated with 10 μm phenylephrine. HDAC5 GFP was concentrated in the nucleus prior to the application of phenylephrine ( 30 min and 0 min). Phenylephrine

5 J Physiol α-adrenergic signalling and HDAC5 in skeletal muscle 1105 Results α-adrenergic activation causes transient nuclear efflux of HDAC5 mediated by PKD in SOL fibres Our first question was whether activation of α-adrenergic receptors could cause translocation of HDAC5 from nucleus to cytoplasm in skeletal muscle fibres. To answer this question we isolated muscle fibres from predominantly slow-twitch mouse SOL muscle, maintained them in culture and used an adenoviral vector to express HDAC5 GFP protein in the cultured SOL fibres. Two days after infection, the intra-fibre location of HDAC5 GFP was monitored by confocal microscopy. Under control conditions, HDAC5 GFP is strongly concentrated in SOL fibre nuclei (Fig. 1A and B). The ratio of mean nuclear pixel fluorescence to mean cytoplasmic pixel fluorescence in 11 nuclei of 8 fibres was 5.75 ± During the first 30 min no reagent was added, and the nuclear HDAC5 GFP remained constant (Fig. 1B, 30 and 0min; Fig. 1C). Subsequent application of the α-adrenergic agonist phenylephrine (10 μm) caused a brisk decline in nuclear HDAC5 GFP (Fig. 1C),indicating the presenceof functionalα-adrenergic receptors in these SOL fibres. The nuclear mean pixel fluorescence (due to HDAC5 GFP) declined to a minimum of 65% of its starting level 30 min after application of phenylephrine with a half-time of less than 10 min. However, despite the continuous presence of phenylephrine, the decline of nuclear HDAC5 GFP was followed by a somewhat slower recovery phase (Fig. 1C), possibly indicating desensitization of the receptor and/or turnover of the diacylglycerol produced during GPCR activation (Rey et al. 2001b). The phenylephrine-induced nuclear efflux of HDAC5 GFP was completely blocked by prior application of the α-adrenergic receptor antagonist prazosin (1 μm; Fig. 1D), indicating that phenylepherine was acting through the α-adrenergic receptor. The results in Fig. 1B and C represent the first report of the activation of nuclear efflux of HDAC5 by any stimulus in skeletal muscle. α-adrenergic receptor activation leads to activation of phospholipase C, hydrolysis of phosphotidyl inositol and production of DAG, which can activate PKD both directly and indirectly through activation of PKC followed by PKC-dependent phosphorylation of PKD (Rozengurt et al. 2005). We therefore next tested whether PKD mediates the phenylephrine response in SOL fibres by using the PKD inhibitor Gö6976 (1μM), which blocked the effects of phenylephrine on the nuclear to cytoplasm translocation of HDAC5 GFP (Fig. 1E), suggesting the involvement of PKD as a downstream intermediate in the HDAC5 nuclear efflux activated by phenylephrine (Fig. 1C). It has been argued that Gö 6976 is a selective inhibitor of PKD (Gschwendt et al. 1996), which is a kinase for HDAC5 (Vega et al. 2004). In such a case, a block of the HDAC5 nuclear efflux by Gö 6976 would be consistent with PKD mediating the nuclear efflux of HDAC5 in SOL fibres during α-agonist activation by phenylephrine. To confirm this interpretation, we employed a molecular manipulation of SOL fibres to test for the involvement of PKD in the phenylephrine response. Cultured adult SOL fibres were co-infected using two viral expression vectors, one for HDAC5 GFP and another for a fusion protein of the dominant negative PKD1 with the fluorescent mplum (dnpkd mplum). The phenylepherine-induced nuclear efflux of HDAC5 GFP was eliminated in fibres expressing dnpkd1 mplum (Fig. 2A), consistent with the involvement of PKD kinase activity in the phenylepherine-induced HDAC5 GFP nuclear efflux. As a control for excess PKD expression, co-expression of the fusion protein of wild type PKD1 and mplum, denoted simply as PKD1 mplum, together with HDAC5 GFP did not eliminate the phenylepherine-induced HDAC5 GFP nuclear efflux (Fig. 2B). Phenylephrine causes a transient redistribution of PKD1 to plasma membrane and nuclei in SOL muscle fibres Depending on its activation status, PKD1 has been reported to localize in different subcellular compartments in several cell types (van Lint et al. 2002). However, translocation of PKD in response to activation has not been previously examined in skeletal muscle. We thus analysed the effect of activation by phenylephrine on the intracellular distribution of PKD1 mplum expressed in cultured adult SOL fibres. Two days after infection with an adenovirus carrying a cdna expressing PKD1 mplum, the fibres were imaged on the confocal microscope. Under resting conditions without phenylephrine treatment, PKD1 mplum was resulted in transient nuclear efflux of HDAC5 (30 min). Scale bar, 10 μm. C, phenylephrine caused a transient decline in nuclear HDAC5 GFP in SOL fibres. The nuclear HDAC5 GFP recovered after about 70 min although phenylephrine was present in the culture dish throughout the 2 h period. Data are presented as ratio of nuclear HDAC5 GFP fluorescence/cytoplasmic fluorescence from the same individual fibre. Data were from 11 nuclei of 8 fibres. D, the response of HDAC5 to phenylephrine was blocked by α-receptor blocker prazosin. Data were from 10 nuclei of 9 fibres. E, the effects of phenylephrine were blocked by pre-treatment with PKD inhibitor Gö Data were from 8 nuclei of 5 fibres. If an image of a fibre had more than one nucleus in focus, then all the nuclei in good focus were analysed and the multiple nuclei were treated equally for this and all other figures.

6 1106 Y. Liu and others J Physiol predominantly distributed in the cytosol in a relatively uniform pattern, with relatively less fluorescense localized to the nuclei (Fig. 3). The average values of the ratio of nuclear to cytoplasmic PKD1 mplum mean pixel fluorescence was 0.56 ± 0.06 (average of 8 nuclei from 7 fibres under resting conditions). Application of 10 μm phenylephrine caused an initial redistribution of PKD1 mplum from cytoplasm to plasma membrane and nuclei (Fig. 3) in SOL fibres. However, despite the continuous presence of phenylephrine, the redistribution of PKD1 mplum was transient, reaching a peak at about 30 min of exposure to phenylephrine, and then reversing back to the initial intracellular distribution by about 90 min after the start of exposure to phenylephrine (Fig. 3). Nuclear export of HDAC5 due to application of the phorbol ester PMA in SOL fibres A possible pathway by which phenylephrine activation of α-adrenergic receptors could lead to activation and A 8 Fibres expressing dnpkd1-mplum Relative fluorescence (n/c) μm Phenylephrine nuclear HDAC5-GFP cytoplasmic HDAC5-GFP B Time (min) Fibres expressing wtpkd1-mplum Relative fluorescence (n/c) μm Phenylephrine Time (min) Figure 2. Expression of dnpkd1 antagonizes the effects of phenylephrine on the distribution of HDAC5 A, over-expression of a dnpkd1 construct abolished the response of HDAC5 to phenylephrine. Data were from 14 nuclei of 6 fibres. B, expression of wtpkd1 has no effects on the transient efflux of HDAC5 after addition of phenylephrine. Data were from 8 nuclei of 5 fibres. Figure 3. Transient translocation of PKD1 mplum to phenylephrine A, a SOL fibre expressing PKD1 mplum was treated with 10 μm phenylephrine. The translocation of PKD1 mplum was transient, reaching a peak at about 30 min of exposure to phenylephrine, and then redistributing back to the initial location. B, single image shows the method of drawing AOIs of cytoplasm (c), nucleus (n) and plasma membrane (pm). C, time course of redistribution of PKD-mPlum in response to phenylephrine. The fluorescence value of plasma membrane, nucleus or cytoplasm was quantified and plotted as a function of time. Data were from 8 nuclei of 7 fibres.

7 J Physiol α-adrenergic signalling and HDAC5 in skeletal muscle 1107 redistribution of PKD (Figs 1 3) could involve activation of the GPCRs. The activation of PLC by G q produces DAG, which can mediate both direct activation of PKD as well as indirect activation of PKD via DAG-mediated activation of PKC resulting in phosphorylation of PKD (Rozengurt et al. 2005). As a test of this hypothesis we applied the phorbol ester PMA, a pharmacological mimic of DAG, to HDAC5 GFP expressing cultured SOL fibres. During the first 30 min prior to PMA addition the nuclear HDAC5 GFP remained constant (Fig. 4A and B). During the subsequent application of PMA (200 nm), nuclear HDAC5 GFP remained essentially constant for about min, and then continuously declined throughout the rest of the exposure (Fig. 4B).The nuclear HDAC5 GFP dropped by 36% by the end of the 2 h exposure to PMA. This PMA-activated nuclear efflux of HDAC5inSOLfibreswastotallysuppressedbyGö 6976 (1 μm; Fig.4C), an inhibitor of PKD (Gschwendt et al. 1996). Block of the HDAC5 nuclear efflux by Gö 6976 is thus consistent with the activation of PKD by PMA causing nuclear efflux of HDAC5 in SOL fibres. To confirm the role of PKD in the PMA-induced nuclear efflux of HDAC5, we co-infected SOL fibres with adenoviruses for both HDAC GFP and dnpkd1 mplum. In fibres expressing both HDAC5 GFP and dnpkd1 mplum, application of PMA (200 nm) had no effect on the nuclear/cytoplasmic distribution of HDAC5 GFP (Fig. 4D), consistent with a role of PKD in the nuclear efflux of HDAC5 GFP that occurs in the absence of PKD inhibition. Wild type PKD1 mplum expressed together with HDAC5 GFP did not eliminate the PMA-induced HDAC5 GFP nuclear efflux (data not shown), similar to the observations with phenylepherine treatment (Fig. 2B). We also investigated whether PMA application can reverse the decay of the phenylephrine-induced HDAC5 nuclear efflux. We applied PMA as a replacement for DAG during the declining phase of the phenylephrine response. In the presence of added PMA, the decline in nuclear efflux of HDAC5 during continuous exposure to phenylephrine was reversed starting about min after PMA application (Fig. 4E; beginning at about the 70 min time point), similar to the delay in HDAC5 nuclear efflux when PMA was applied by itself (Fig. 4B). These observations indicate that reversal of HDAC5 GFP nuclear efflux during continuous exposure to phenylephrine could be duetoadeclineindagdespitethecontinuouspresenceof phenylephrine. This would be consistent with turnover of DAG together either with receptor desensitization or with possible limited amounts of the DAG precursor PIP 2. Continued exposure to PMA causes maintained translocation of PKD1 to plasma membrane and nuclei in SOL muscle fibres We next analysed the effect of activation by PMA on the intracellular distribution of PKD1 mplum in cultured adult SOL fibres. Upon the addition of 200 nm PMA to the fibres, the PKD1 mplum rapidly translocated to the plasma membrane and nuclei (Fig. 5). The translocation of PKD1 mplum in response to PMA was sustained throughout the 2 h period of exposure to PMA. Our result with PMA is in agreement with the observation that in B lymphocytes and mast cells, the association of PKD with the plasma membrane was maintained for several hours in the continued presence of PMA, along with sustained activation of PKD (Matthews et al. 2000). This activation and sustained translocation of PKD1 mplum during the exposure to PMA is fully consistent with the observed PKD-dependent nuclear efflux of HDAC5 GFP that continued throughout the 2h exposure to PMA (Fig.4B), supporting the hypothesis that the observed HDAC5 GFP nuclear efflux is driven by activation of endogenous PKD in SOL fibres. The maintained PKD1 mplum movement in PMA is in contrast to the transient movement of PKD1 mplum seen in the continued presence of phenylephrine. Activation of PKD1 enhances the acetylation of histone H3 and the transcriptional activity of MEF2 To examine the possible role of endogenous PKD in the phosphorylation and translocation of HDACs in SOL fibres, we applied PMA to muscle fibres without expressing exogenous HDAC or PKD. SOL fibres were cultured for 4 days before treatment with 200 nm PMA, the same concentration that promotes HDAC5 GFP translocation from nucleus to cytoplasm. After 2 h treatment, the fibres were fixed and immunostained with antibody against histone H3, selectively recognizing H3 acetylated at Lys 9 and 14. In fibres treated with PMA for 2 h, the nuclear acetylated histone H3 increased approximately twofold compared with fibres not treated with PMA (Fig. 6A; P < 0.05). When the cultures were pre-treated with 1 μm Gö 6976, the effects of PMA on the acetylation of histone H3 were largely suppressed (Fig. 6A). The increased histone acetylation following PMA treatment is consistent with a decrease in histone deacetylase activity within the muscle fibre nuclei due to nuclear-to-cytoplasmic translocation of endogenous HDACs. PMA treatment of SOL fibres also resulted in changes in expression of a MEF2-element-driven luciferase reporter. SOL fibres were infected with adenovirus containing a luciferase cdna driven by a promoter containing three consecutive MEF2 elements and incubated 2 days. Fibres were exposed to PMA (200 nm) for 2 h, washed with PMA-free solution and incubated overnight. This PMA treatment caused luciferase activity in fibre culture extracts to increase to approximately 1.7 times compared with extracts from parallel untreated fibre cultures (Fig. 6B; P < 0.05). Pre-treatment with the PKD inhibitor Gö6976 blocked the enhancement of MEF2 reporter activity

8 1108 Y. Liu and others J Physiol Figure 4. PMA causes cytoplasmic translocation of HDAC5 GFP by activating PKD1 in soleus fibres A, a soleus fibre was infected with HDAC5 GFP treated with 200 nm PMA for 2 h. HDAC5 GFP was concentrated in the nucleus prior to the application of PMA ( 30 min and 0 min). PMA resulted in continuous nuclear efflux of HDAC5 during the 2 h period. Scale bar, 10 μm. B, time course of HDAC5 GFP translocation from nuclei to cytoplasm in the presence of PMA. Nuclear HDAC5 was stable before PMA was added to the culture dish, and for the initial min after addition of PMA. During the remainder of the 2 h PMA treatment, HDAC5 GFP continuously translocated from nuclei to cytoplasm. Although the nuclear HDAC5 dropped by 36% at the end

9 J Physiol α-adrenergic signalling and HDAC5 in skeletal muscle 1109 induced by PMA (Fig. 6B). To verify that the response of the MEF2 reporter to PMA is specific, and is not due to an overall increase of transcription or of protein syntheses, we also infected SOL fibre cultures with a renilla luciferase reporter construct driven by the thymidine kinase promoter. As shown in the 4th and 5th bars in Fig. 6B, PMA did not enhance the expression of renilla luciferase. Together with the inhibitory effects of Gö 6976, these results suggest the response of the MEF2 reporter to PMAisspecific. HDAC5 nuclear movements occur in FDB fibres only after expression of exogenous PKD We also cultured fibres from predominantly fast-twitch FDB muscle, and infected them with adenovirus expressing HDAC5 GFP. As in SOL fibres, HDAC5 GFP is also concentrated in nuclei in FDB fibres. However, in contrast to the SOL fibres, where application of either phenylepherine or PMA activated nuclear efflux of HDAC5 GFP (Figs 1 and 4), application of 200 nm PMA caused no change in nuclear HDAC5 GFP in FDB fibres (Fig.7A), suggesting that PKD is either inaccessible or not present in these fibres. Phenylepherine also had no effect on HDAC5 GFP nuclear fluorescence in FDB fibres (not shown). Our observations that SOL, but not FDB fibres exhibit HDAC5 nuclear efflux in response to activation of PKD by PMA or by the α-adrenergic agonist phenylephrine are consistent with a recent report that PKD1, a kinase for HDAC5 phosphorylation and consequent nuclear efflux, is expressed in mouse SOL muscle, but not in the mouse fast muscles extensor digitorum longus (EDL), tibialis anterior (TA), gastrocnemius or plantaris under normal physiological conditions (Kim et al. 2008; FDB muscle was not examined in their study). Consistent with this interpretation, when our FDB cultures were co-infected with adenoviruses expressing HDAC5 GFP and PKD1, the application of 200 nm PMA then resulted in the nuclear efflux of HDAC5 GFP during the 2 h period of exposure to PMA (Fig. 7B), indicating that activation by PMA could cause nuclear-to-cytoplasmic translocation of HDAC5 GFP in FDB fibres if exogenous PKD was expressed in these FDB fibres. Figure 5. Translocation of PKD1 in response to PMA activation in soleus fibres A, a representative SOL fibre expressing PKD1 mplum is shown as in basal conditions ( 30 min and 0 min), 60 min with 200 nm PMA and 120 min with PMA. The images show that PKD1 mplum is in cytoplasm and very little in nucleus. After activation by PMA, PKD1 mplum translocates mainly to plasma membrane and some into the nucleus. The translocation is sustained in the presence of PMA. Scale bar, 10 μm. B, time course of PKD1 mplum translocation. Data are from 18 nuclei of 14 fibres. of 2 h, the cytosolic HDAC5 showed no significant change. Data were from 13 nuclei of 8 fibres. C, effectsof the PKD inhibitor Gö 6976 on PMA-induced nuclear efflux of HDAC5. Soleus fibres expressing HDAC5 GFP were first incubated with 1 μm Gö 6976 for 30 min. Then PMA was added to the muscle fibres. Pretreatment with Gö 6976 completely blocked PMA-induced HDAC5 nuclear export. Data were from 9 nuclei of 5 fibres. D, in fibres co-infected with both dnpkd1 mplum and HDAC5 GFP there was no nuclear efflux of HDAC5 GFP on application of PMA. Data were from 13 nuclei of 8 fibres. E, effects of PMA on HDAC5 after treatment with phenylephrine. SOL fibres expressing HDAC5 GFP were first treated with phenylephrine, which triggered a transient nuclear efflux of HDAC5, then were exposed to PMA. Treatment with PMA still results in nuclear efflux of HDAC5, even after the reversal of the phenylephrine response. Data were from 11 nuclei of 7 fibres.

10 1110 Y. Liu and others J Physiol Even though FDB fibres do not express endogenous PKD, and thus cannot exhibit PKD-dependent nuclear efflux of HDAC5 in the absence of exogenously expressed PKD, FDB fibres do express endogenous HDAC5. Lysis of cultured FDB fibres followed by nuclear/cytoplasmic A Acetylated histone H3 stain (% of control) B Luciferase reporter (% of control activity) control control 200 nm PMA * 200 nm PMA MEF2 promoter * 1 μm Go6976 &200nM PMA control 1 μm Go6976 & 200 nm PMA T K promoter 200 nm PMA Figure 6. PMA-treated SOL muscle fibres have stronger acetylated histone H3 in nucleus and MEF2 activity A, SOL fibres were treated with PMA for 2 h, or exposed to Gö 6976 for 30 min before the 2 h PMA treatment. Then the cultures were fixed and stained with primary antibody against histone H3 selectively recognizing the acetylated form at Lys 9 and 14, and subsequently a secondary antibody conjugated with Cy5. The fluorescence of nuclei was quantified and normalized to control group (without PMA or Gö 6976 treatment) as 1.0. Exposure to PMA significantly enhanced the acetylated H3 in nucleus ( P < 0.05). Pretreatment with Gö 6976 blocked the effects of PMA on H3 acetylation. From left to right, the data were averages of mean nuclear fluorescence of 17, 19 or 16 nuclei from 10 fibres of each group. B, luciferase activity was increased in fibres infected with MEF2-luciferase reporter and stimulated with PMA (2nd bar). Gö 6976 blocked the increase in luciferase activity stimulated with PMA (3rd bar). Results represent triplicate measurements from each of two independent experiments. In cultures transfected with renilla luciferase driven by the thymidine kinase (TK) promoter, PMA treatment did not increase the luciferase activity (4th and 5th bars). Results represent triplicate measurements from each of three independent experiments. Error bars represent ± 1 S.E.M. fractionation and Western blot analysis shows that endogenous HDAC5 is present in FDB fibres, and is strongly concentrated in fibre nuclei under resting conditions (Fig. 7C). The finding that endogenous HDAC5 is strongly concentrated in FDB fibre nuclei is consistent with our observation that expressed HDAC5 GFP fluorescence is 5 6times brighter in nuclei than in cytoplasm in confocal images of FDB fibres (Fig. 7A), and provides support for the use of HDAC5 GFP as an indicator for nuclear/cytoplasmic distribution of the corresponding endogenous HDAC5. The established nuclear and cytosolic marker proteins histone H3 and GAPDH are, respectively, concentrated in the nuclear and cytoplasmic fractions from FDB fibres (Fig. 7C), validating our fractionation procedure. A possible mechanism for the activation of nuclear efflux of HDAC5 in FDB fibres remains to be established. Expression of exogenous PKD decreases nuclear concentration of HDAC5 in both FDB and SOL fibres To further confirm the effects of exogenously expressed activated PKD1 on the localization of HDAC5 in FDB fibres, in another set of experiments FDB cultures were infected with HDAC5 GFP alone, with HDAC5 GFP plus wtpkd1, with HDAC5 GFP plus capkd1 or with HDAC5 GFP plus dnpkd1. FDB fibres expressing both HDAC5 GFP and wtpkd1 had an average nuclear/ cytoplasmic ratio of mean pixel fluorescence of 1.93 (Fig. 8A) compared with FDB fibres expressing HDAC5 GFP alone, which have an average nuclear/ cytoplasmic value of 5.25, indicating a lower concentration of nuclear-localized HDAC5 GFP in fibres expressing HDAC5 GFP plus wtpkd1. Furthermore, in fibres expressing HDAC5 GFP together with capkd1 there was even less nuclear concentration of HDAC5 GFP, with a nuclear/cytoplasmic value of 0.97 (Fig. 8A). The FDB fibres expressing HDAC5 GFP and dnpkd1 have a nuclear/cytoplasmic value of 5.45 (Fig. 8A), which is similar to the fibres expressing HDAC5 GFP alone. These results demonstrate that PKD1 causes nuclear-to-cytoplasmic translocation of HDAC5 when exogenously expressed in FDB fibres and that expressed capkd1 is more effective in causing HDAC5 to exit from nucleus to the cytoplasm than wtpkd1. The dnpkd1 did not change the nuclear cytoplasmic distribution of HDAC5, consistent with there being little endogenous PKD activity in resting fast FDB fibres. SOL fibres were also infected with HDAC5 GFP alone (as control), or together with wtpkd1, capkd1 or dnpkd1. As in FDB fibres, SOL fibres expressing both HDAC5 GFP and wtpkd1 had a lower average nuclear/cytoplasmic value (3.03; Fig. 8B) compared to SOL fibres expressing HDAC5 GFP alone

11 J Physiol α-adrenergic signalling and HDAC5 in skeletal muscle 1111 (average nuclear/cytoplasmic value of 5.39), indicating a lower concentration of nuclear-localized HDAC5 GFP in fibres expressing HDAC5 GFP plus exogenous wtpkd1. Furthermore, in fibres expressing HDAC5 GFP together with capkd1 there was even less nuclear concentration of HDAC5 GFP, with a nuclear/cytoplasmic value of 1.28 (Fig.8B). Co-expressing HDAC5 GFP with dnpkd1 increased the nuclear/cytoplasmic ratio of HDAC5 GFP to 6.23, suggesting that the endogenous PKD1 in SOL fibres affects the distribution of HDAC5 GFP in these fibres under resting conditions. These results are consistent with a relatively low level of PKD enzymatic activity in non-activated (i.e. not PMA or phenylepherine treated) SOL fibres, with even less in non-activated FDB fibres. There was a significant increase in PKD activity on expression of exogenous wtpkd and even greater increase with capkd1 in both types of fibres. Thus, even though SOL fibres express PKD whereas FDB fibres do not, in the non-activated fibres neither fibre type appears to have relatively high level of enzymatically active PKD. Figure 7. PMA-induced nuclear export of HDAC5 GFP in FDB fibres A, PMA does not affect the distribution of HDAC5 in wild type FDB fibres. In FDB fibres infected with HDAC5 GFP alone, treatment with PMA for 2 h did not change the ratio of nuclear fluorescence/ cytoplasmic fluorescence, demonstrating the absence of PMA-induced nuclear efflux of HDAC5 GFP. Data were from 10 nuclei of 6 fibres. B, time course of HDAC5 GFP translocation from nuclei to cytoplasm in the presence of PMA in FDB fibres expressing both HDAC5 GFP and PKD1. PMA leads to HDAC5 translocation from nucleus to cytoplasm, only in FDB fibres expressing both HDAC5 GFP and exogeneous PKD1. The fibres were treated with PMA for 2 h. Nuclear HDAC5 GFP was stable before PMA was added to the culture dish. During the 2 h PMA treatment, HDAC5 GFP continuously translocated from nuclei to cytoplasm. While the nuclear Discussion We have previously reported that slow fibre-type electrical stimulation of FDB muscle fibres causes CaMKII-dependent nuclear efflux of HDAC4, but has no effect on the nuclear/cytoplasmic distribution of HDAC5 (Liu et al. 2005), which is heavily concentrated in fibre nuclei (Liu et al and Figs 1 and 2). The present work was therefore initiated to identify and characterize alternative pathways that might give rise to HDAC5 phosphorylation and consequent nuclear efflux in skeletal muscle fibres. In addition to the activity-dependent/calcium-dependent CaMK pathway, the calcium-independent PKD family is also reported to phosphorylate HDACs (Vega et al. 2004) and thereby up-regulate MEF2 activity and slow muscle fibre content (Kim et al. 2008). Our present results provide novel functional evidence that activation of endogenous PKD leads to nuclear efflux of HDAC5 GFP expressed in cultured predominantly slow twitch SOL muscle fibres, but not in cultured predominantly fast twitch FDB muscle fibres. Further, we demonstrate that cellular signalling for PKD activation of HDAC5 GFP nuclear efflux can occur through activation of the α-adrenergic pathway HDAC5 dropped 33%, the cytosolic HDAC5 had no significant changes. Data were from 13 nuclei of 10 fibres. Note that in this experiment (Fig. 8B), because of the over-expression of PKD1, the starting value (before the addition of PMA) of the nuclear/cytoplasmic ratio is lower (1.67) compared to 5.65 in fibres expressing HDAC5 GFP alone (Fig. 8A). C, Western blot using antibodies to HDAC5, acetylated H3 and GAPDH shows that endogenous HDAC5 is strongly concentrated in FDB fibre nuclei. Antibodies to histone H3 and GADPH were used as markers of nucleus or cytoplasm, respectively.

12 1112 Y. Liu and others J Physiol using the agonist phenylepherine. Finally, in the absence of any exogenous expressed HDAC, PMA increased histone H3 acetylation and increased MEF2 reporter activity in a PKD-dependent manner, consistent with PKD phosphorylation of endogenous HDAC(s) and reduced nuclear HDAC activity due to HDAC nuclear efflux. Our findings thus establish a mechanism for HDAC5 nuclear efflux in slow muscle fibres and elaborate a physiological means to activate this mechanism from the muscle cell surface. The possible mechanisms for HDAC5 nuclear efflux in fast muscle fibres such as FDB remain to be demonstrated. Relative HDAC5-GFP fluorescence (n/c) A B FDB fibres control control wtpkd1 SOL fibres wtpkd1 capkd1 capkd1 dnpkd1 dnpkd1 Figure 8. Co-expression of wtpkd1, capkd1 or dnpkd1 affect the nuclear distribution of HDAC5 A, co-expressing HDAC5 GFP with wtpkd1 or with capkd1 promoted cytosolic localization of HDAC5 in FDB fibres. Co-expressing HDAC5 GFP with capkd1 had even stronger effects on the cytoplasmic localization of HDAC5. Co-expressing HDAC5 GFP together with dnpkd1 had no effect on the localization of HDAC5 GFP. The ratio of nuclear/cytoplasmic HDAC5 GFP fluorescence was calculated for each individual fibre. From left to right, the data were from 21, 23, 25 or 26 nuclei of 16, 14, 16 or 16 fibres, respectively. B, co-expressing HDAC5 GFP with wtpkd1 or with capkd1 promoted cytosolic localization of HDAC5 in SOL fibres. Co-expressing HDAC5 GFP with dnpkd1 in SOL resulted in stronger nuclear HDAC5 GFP fluorescence, presumably by inhibiting endogenous PKD1. From left to right, the data were from 32, 22, 24 or 30 nuclei of 20 fibres for each group. A prior study showed that PKD1 is predominantly expressed in type I myofibres (Kim et al. 2008). When exogenous PKD1 was expressed in type II myofibres, it promoted the transformation of muscle fibres from type II to type I, slow twitch phenotype. The expression of PKD1 was accompanied by phosphorylation of HDAC4 and HDAC5, resulting in activation of MEF2 (Kim et al. 2008). Here we report that phenylephrine (or PMA) is capable of activating endogenous PKD resulting in nuclear efflux of HDAC5 in predominantly slow SOL fibres. The involvement of PKD was confirmed by using the PKD inhibitor Gö 6976 and by elimination of the response in fibres expressing dnpkd1 mplum. In contrast, in predominantly fast FDB muscle fibres there is no change in subcellular distribution of HDAC5 during PMA treatment, implying that PKD plays a critical role in HDAC5 movements in slow muscle but not in fast muscle. Our identification of PKD as the kinase responsible for phenylephrine- or PMA-dependent nuclear efflux of HDAC5 is based on both the presence of PKD1 in slow but not fast muscle fibres (Kim et al. 2008) and on the elimination of the HDAC5 GFP nuclear efflux by the PKD inhibitor Gö Although Gö 6976 also inhibits the classical PKCs (PKCα and PKCβ), it has previously been shown that these classical PKCs do not influence the nuclear/cytoplasmic distribution of HDAC5 GFP (Vega et al. 2004). Thus, any effect of Gö 6976 observed here on HDAC5 intracellular distribution must be attributable to block of PKD, and not to block of the classical PKCs. Furthermore, the response was also eliminated by expression of dnpkd1 mplum, which should specifically inhibit PKD. PKD can phosphorylate all class II HDAC members, including HDAC4, 5, 7, 9 and MITR (McKinsey, 2007). CaMK II was first reported to bind to and phosphorylate only HDAC4 and does not bind to or phosphorylate HDAC5, HDAC7 or HDAC9 (Backs et al. 2006). Recent studies found that HDAC4 and 5 can form oligomers in C2C12 and COS cells (Backs et al. 2008). The oligomerization between HDAC4 and 5 makes HDAC5 responsive to CaMK II signalling either by phosphorylated HDAC4 carrying unphosphorylated HDAC5, or by transphosphorylation of HDAC5 in the HDAC4 5 dimere. In a previous study from our laboratory we found that repetitive muscle activity of FDB fibres caused nuclear efflux of HDAC4 GFP but not of HDAC5 GFP. The lack of response of HDAC5 GFP to muscle activity in our previous study may be due to the excessive amount of expressed HDAC5 GFP relative to endogenous HDAC4 in FDB fibres, which would limit the formation of HDAC5 GFP HDAC4 dimers. Alternatively, the oligomerization model may not apply to adult skeletal muscle fibres. PKD1 exhibits different subcellular localization in non-activated versus activated non-muscle cells

13 J Physiol α-adrenergic signalling and HDAC5 in skeletal muscle 1113 (Rozengurt et al. 2005). To gain insight into the function and localization of PKD1 in skeletal muscle, we used virally expressed PKD1 mplum in cultured adult SOL muscle fibres. We here show the subcellular distribution of PKD1 in fully differentiated resting adult skeletal muscle fibres and the movement of PKD1 during activation. In response to application of phenylephrine to SOL fibres, PKD1 translocated from the cytoplasm to plasma membrane and nuclei, but this translocation reversed during 2 h continuous application of phenylephrine. In contrast, during 2 h application of PMA to SOL fibres, PKD1 translocated from the cytoplasm to the plasma membrane and to fibre nuclei and this translocation persisted during 2 h of PMA application. Both HDAC5 nuclear efflux and intra-fibre redistribution of PKD1 exhibit transient responses during continuous exposure to phenylephrine (Figs 1 and 3, respectively), whereas both HDAC5 nuclear efflux and PKD1 redistribution exhibit maintained responses in the presence of continuous exposure to PMA (Figs 4 and 5, respectively). Furthermore, application of PMA during the decaying phase of the phenylephrine-induced HDAC5 response can restore HDAC5 GFP nuclear efflux (Fig. 4E). These results are consistent with the HDAC5 nuclear efflux being driven by PKD-dependent HDAC5 phosphorylation after activation and translocation of PKD, which is maintained throughout PMA application, but is only transient during continued exposure to phenylephrine. A transient activation of PKD during continued phenylephrine exposure is consistent with turnover of DAG and failure to continue to produce additional DAG despite continued exposure to phenylephrine, possibly due to α-adrenergic receptor desensitization. The time course of the transient response of PKD to phenylephrine treatment in SOL fibres was essentially the same as the transient nuclear efflux of HDAC5 activated by phenylephrine. It is interesting that the kinetics of the initial HDAC5 nuclear efflux activated by phenylepherine in SOL fibres (Fig. 1C) is considerably faster than the HDAC5 nuclear efflux activated by PMA (Fig. 4B). This observation is similar to the more rapid appearance of a PKD phosphorylated product in response to GPCR agonists than to phorbol ester treatment, recently observed in COS and HeLa cells, which was attributed to closer spatial proximity of PKD1 to the GPCR-associated PLC and the subsequently released DAG than the proximity of the bath-applied phorbol ester to PKD1 (Kunkel et al. 2007). However, the exact mechanism underlying the difference in HDAC5 nuclear efflux rates observed here in SOL fibres for activation by phenylephrine or by PMA is not clear at this point. Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes (Turner, 2005). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation and methylation. Specifically, acetylation of Lys 9 and 14 of histone H3 is coupled to the transcriptional regulation of genes (Eberharter & Becker, 2002). The acetylation status of histones is maintained by the dynamic interplay of histone acetyltransferase and deacetylase. The phosphorylation of HDACs in the nuclei and their subsequent cytoplasmic translocation leads to decreased histone deacetylase activity and consequent acetylation of histones in nuclei as shown here in skeletal muscle fibres (Fig. 6A). Phosphorylation of HDACs also enhances transcription by causing their dissociation from certain transcription factor binding partners such as MEF2. It is well known that MEF2, together with other transcription factors, plays critical roles in muscle fibre-type transformation (Bassel-Duby & Olson, 2006). We have previously documented that muscle activity can enhance MEF2 transcriptional activity through CaMKII (Liu et al. 2005). Here we report that PKD activation can also increase MEF2 transcriptional effectiveness (Fig. 6B), but that this occurs independently of muscle activity. The present study utilized isolated adult skeletal muscle fibres maintained in culture as a convenient experimental model for expressing fluorescent fusion protein constructs of HDAC and PKD, and for tracking their redistribution due to adrenergic activation or phorbol esters. Using this system, we could monitor and quantify the kinetics of intracellular redistribution of both HDAC5 GFP and PKD1 mplum in real time in living fibres. However, the extent to which 4 days in culture may give rise to modification of the fibre properties is not established. For example, Potthoff et al. (2007) have reported that the expression of class II HDAC protein is relatively low in freshly isolated SOL muscle. The extent of possible change of class II HDAC protein in the cultured SOL fibres as used here, and the consequent possible change in MEF2 promoter-driven luciferase reporter or histone H3 acetylation in response to PMA, remain to be determined. In conclusion, in predominantly slow SOL muscle fibres, activation of endogenous PKD through the α-adrenergic pathway causes HDAC5 nuclear efflux, leading to increased histone H3 acetylation and increased MEF2 transcriptional activity. The PKD HDAC5 signal transduction pathway thus provides another component in muscle fibre-type transformation. References Auer A, von Blume J, Sturany S, von Wichert G, Van Lint J, Vandenheede J, Adler G & Seufferlein T (2005). Role of the regulatory domain of protein kinase D2 in phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling. Mol Biol Cell 16,