Skeletal Muscle Nuclei Research Articles

Alterations of the skeletal muscle nuclear proteome after acute exercise reveals a post-transcriptional influence.

Exercise plays a crucial role in promoting muscle health, but our understanding of nuclear proteins orchestrating exercise responses is limited. Isolation of skeletal muscle nuclei coupled with mass spectrometry enhanced the identification of nuclear proteins. This approach was used to investigate the effects of acute exercise, revealing changes in the muscle nuclear proteome 1-hour post-exercise, including proteins linked to post-transcriptional processing and splicing. Our findings offer insights into the exercise-induced changes within muscle nuclear proteins.

Molecular and Structural Alterations of Skeletal Muscle Tissue Nuclei during Aging.

Aging is accompanied by a progressive loss of skeletal muscle mass and strength. The mechanisms underlying this phenomenon are certainly multifactorial and still remain to be fully elucidated. Changes in the cell nucleus structure and function have been considered among the possible contributing causes. This review offers an overview of the current knowledge on skeletal muscle nuclei in aging, focusing on the impairment of nuclear pathways potentially involved in age-related muscle decline. In skeletal muscle two types of cells are present: fiber cells, constituting the contractile muscle mass and containing hundreds of myonuclei, and the satellite cells, i.e., the myogenic mononuclear stem cells occurring at the periphery of the fibers and responsible for muscle growth and repair. Research conducted on different experimental models and with different methodological approaches demonstrated that both the myonuclei and satellite cell nuclei of aged skeletal muscles undergo several structural and molecular alterations, affecting chromatin organization, gene expression, and transcriptional and post-transcriptional activities. These alterations play a key role in the impairment of muscle fiber homeostasis and regeneration, thus contributing to the age-related decrease in skeletal muscle mass and function.

Skeletal Muscle Nuclei in Mice are not Post-mitotic.

The skeletal muscle research field generally accepts that nuclei in skeletal muscle fibers (ie, myonuclei) are post-mitotic and unable to proliferate. Because our deuterium oxide (D2O) labeling studies showed DNA synthesis in skeletal muscle tissue, we hypothesized that resident myonuclei can replicate in vivo. To test this hypothesis, we used a mouse model that temporally labeled myonuclei with GFP followed by D2O labeling during normal cage activity, functional overload, and with satellite cell ablation. During normal cage activity, we observed deuterium enrichment into myonuclear DNA in 7 out of 7 plantaris (PLA), 6 out of 6 tibialis anterior (TA), 5 out of 7 gastrocnemius (GAST), and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR) of DNA in myonuclei were: 0.0202±0.0093 in PLA, 0.0239±0.0040 in TA, 0.0076±0. 0058 in GAST, and 0.0138±0.0039 in QUAD, while there was no replication in myonuclei from EDL. These FSR values were largely reproduced in the overload and satellite cell ablation conditions, although there were higher synthesis rates in the overloaded PLA muscle. We further provided evidence that myonuclear replication is through endoreplication, which results in polyploidy. These novel findings contradict the dogma that skeletal muscle nuclei are post-mitotic and open potential avenues to harness the intrinsic replicative ability of myonuclei for muscle maintenance and growth.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy

BackgroundThe repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported.ResultsRGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to ‘adherens junsctions’ and ‘extracellular-cell adhesion’, while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation.ConclusionsThese results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

Effect of Denervation on XBP1 in Skeletal Muscle and the Neuromuscular Junction.

Denervation of skeletal muscle is a debilitating consequence of injury of the peripheral nervous system, causing skeletal muscle to experience robust atrophy. However, the molecular mechanisms controlling the wasting of skeletal muscle due to denervation are not well understood. Here, we demonstrate that transection of the sciatic nerve in Sprague–Dawley rats induced robust skeletal muscle atrophy, with little effect on the neuromuscular junction (NMJ). Moreover, the following study indicates that all three arms of the unfolded protein response (UPR) are activated in denervated skeletal muscle. Specifically, ATF4 and ATF6 are elevated in the cytoplasm of skeletal muscle, while XBP1 is elevated in the nuclei of skeletal muscle. Moreover, XBP1 is expressed in the nuclei surrounding the NMJ. Altogether, these results endorse a potential role of the UPR and, specifically, XBP1 in the maintenance of both skeletal muscle and the NMJ following sciatic nerve transection. Further investigations into a potential therapeutic role concerning these mechanisms are needed.

Ultrastructural immunocytochemistry shows impairment of RNA pathways in skeletal muscle nuclei of old mice: A link to sarcopenia?

During aging, skeletal muscle is affected by sarcopenia, a progressive decline in muscle mass, strength and endurance that leads to loss of function and disability. Cell nucleus dysfunction is a possible factor contributing to sarcopenia because aging-associated alterations in mRNA and rRNA transcription/maturation machinery have been shown in several cell types including muscle cells. In this study, the distribution and density of key molecular factors involved in RNA pathways namely, nuclear actin (a motor protein and regulator of RNA transcription), 5-methyl cytosine (an epigenetic regulator of gene transcription), and ribonuclease A (an RNA degrading enzyme) were compared in different nuclear compartments of late adult and old mice myonuclei by means of ultrastructural immunocytochemistry. In all nuclear compartments, an age-related decrease of nuclear actin suggested altered chromatin structuring and impaired nucleus-to-cytoplasm transport of both mRNA and ribosomal subunits, while a decrease of 5-methyl cytosine and ribonuclease A in the nucleoli of old mice indicated an age-dependent loss of rRNA genes. These findings provide novel experimental evidence that, in the aging skeletal muscle, nuclear RNA pathways undergo impairment, likely hindering protein synthesis and contributing to the onset and progression of sarcopenia.

Intermittent application of external positive pressure helps to preserve organ viability during ex vivo perfusion and culture

The perfusion of medium through blood vessels allows the preservation of donor organs and culture of bioengineered organs. However, tissue damage due to inadequate perfusion remains a problem. We evaluated whether intermittent external pressurization would improve the perfusion and viability of organs in culture. A bioreactor system was used to perfuse and culture rat small intestine and femoral muscle preparations. Intermittent positive external pressure (10 mmHg) was applied for 20 s at intervals of 20 s. Intermittent pressurization resulted in uniform perfusion of small intestine preparations and minimal tissue damage after 20 h of perfusion, whereas non-pressurized (control) preparations exhibited significantly worse perfusion of the upper surface than the lower surface and histologic evidence of tissue damage. Longer term studies were undertaken in luciferase-expressing rat femoral muscle preparations. Compared with non-pressurized controls, intermittent pressurization led to better perfusion throughout the 14-day experimental period, improved organ viability as indicated by a higher bioluminescence intensity after perfusion with luciferin, and reduced levels of tissue necrosis with better preservation of vascular structures and skeletal muscle nuclei (histologic analyses). Therefore, intermittent application of external positive pressure improved the perfusion of small intestine and skeletal muscle preparations and enhanced tissue viability when compared with controls. We anticipate that this innovative perfusion technique could be used to improve the preservation of donor organs and culture of bioengineered organs.

Nesprin-1-alpha2 associates with kinesin at myotube outer nuclear membranes, but is restricted to neuromuscular junction nuclei in adult muscle

Nesprins, nuclear envelope spectrin-repeat proteins encoded by the SYNE1 and SYNE2 genes, are involved in localization of nuclei. The short isoform, nesprin-1-alpha2, is required for relocation of the microtubule organizer function from centromeres to the nuclear rim during myogenesis. Using specific antibodies, we now show that both nesprin-1-alpha2 and nesprin-1-giant co-localize with kinesin at the junctions of concatenated nuclei and at the outer poles of nuclear chains in human skeletal myotubes. In adult muscle, nesprin-1-alpha2 was found, together with kinesin, only on nuclei associated with neuromuscular junctions, whereas all adult cardiomyocyte nuclei expressed nesprin-1-alpha2. In a proteomics study, kinesin heavy and light chains were the only significant proteins in myotube extracts pulled down by nesprin-1-alpha2, but not by a mutant lacking the highly-conserved STAR domain (18 amino-acids, including the LEWD motif). The results support a function for nesprin-1-alpha2 in the specific localization of skeletal muscle nuclei mediated by kinesins and suggest that its primary role is at the outer nuclear membrane.

Lactate Stimulates a Potential for Hypertrophy and Regeneration of Mouse Skeletal Muscle

The effects of lactate on muscle mass and regeneration were investigated using mouse skeletal muscle tissue and cultured C2C12 cells. Male C57BL/6J mice were randomly divided into (1) control, (2) lactate (1 mol/L in distilled water, 8.9 mL/g body weight)-administered, (3) cardio toxin (CTX)-injected (CX), and (4) lactate-administered after CTX-injection (LX) groups. CTX was injected into right tibialis anterior (TA) muscle before the oral administration of sodium lactate (five days/week for two weeks) to the mice. Oral lactate administration increased the muscle weight and fiber cross-sectional area, and the population of Pax7-positive nuclei in mouse TA skeletal muscle. Oral administration of lactate also facilitated the recovery process of CTX-associated injured mouse TA muscle mass accompanied with a transient increase in the population of Pax7-positive nuclei. Mouse myoblast-derived C2C12 cells were differentiated for five days to form myotubes with or without lactate administration. C2C12 myotube formation with an increase in protein content, fiber diameter, length, and myo-nuclei was stimulated by lactate. These observations suggest that lactate may be a potential molecule to stimulate muscle hypertrophy and regeneration of mouse skeletal muscle via the activation of muscle satellite cells.

Salmeterol with Liver Depot Gene Therapy Enhances the Skeletal Muscle Response in Murine Pompe Disease.

Gene therapy for Pompe disease with adeno-associated virus (AAV) vectors has advanced into early phase clinical trials; however, the paucity of cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle, where it is needed to take up acid α-glucosidase (GAA), has impeded the efficacy of Pompe disease gene therapy. Long-acting selective β2 receptor agonists previously enhanced the CI-MPR expression in muscle. In this study we have evaluated the selective β2 agonist salmeterol in GAA knockout mice in combination with an AAV vector expressing human GAA specifically in the liver. Quadriceps glycogen content was significantly decreased by administration of the AAV vector with salmeterol, in comparison with the AAV vector alone (p < 0.01). Importantly, glycogen content of the quadriceps was reduced to its lowest level by the combination of AAV vector and salmeterol administration. Rotarod testing revealed significant improvement following treatment, in comparison with untreated mice, and salmeterol improved wirehang performance. Salmeterol treatment decreased abnormalities of autophagy in the quadriceps, as shown be lower LC3 and p62. Vector administration reduced the abnormal vacuolization and accumulation of nuclei in skeletal muscle. Thus, salmeterol could be further developed as adjunctive therapy to improve the efficacy of liver depot gene therapy for Pompe disease.

Mathematical Modeling of Nuclear Trafficking of FOXO Transcription Factors.

Nuclear cytoplasmic flux of Foxo transcription factors is paramount in cellular gene regulation. For example, excluding Foxo from skeletal muscle nuclei is necessary to avoid muscle wasting through elevated protein breakdown. Constructing a mathematical model of the signaling process leading to alteration of Foxo nuclear cytoplasm ratio is useful in predicting and interpreting such ratio changes. In this chapter we derive a general mathematical model for nuclear cytoplasmic flux. We apply this model to Foxo flux and take advantage of rapid phosphorylation approximation and conservation conditions to reduce the Foxo flux model. We constrain our model with data from mouse skeletal muscle with applied IGF. This procedure provides an example of what might be called the central approach of mathematical modeling: The cycling of a biological question through mathematical formulation and back to biological interpretation.

30-Year-Old Man With Outside-of-Hospital Cardiac Arrest

30-Year-Old Man With Outside-of-Hospital Cardiac Arrest

Manual Patch-Clamp Recording for Medium-Throughput Ion Channel Drug Screening: Assay Validation by Searching for a Blocker of Nuclear Monovalent Cationic Channel

The use of manual patch-clamp recording in drug discovery is limited by ultra-low throughput. The high cost of automated patch-clamp drug screening makes it unavailable for the majority of electrophysiological laboratories. Here we propose a drug screening strategy that combines manual patch-clamp technique with a specially designed drug cocktail application algorithm. This strategy allows the testing of up to 80 compounds per experiment with the effective average rate close to 40 drugs/experiment. The drug application algorithm was validated by screening a small library of 446 compounds in just 11 manual patch-clamp experiments while searching for a blocker of nuclear monovalent cationic channel (NMCC) expressed in the inner membrane of skeletal muscle nuclei. We found that one of the drugs, D11P6H2, blocked the NMCC activity completely and irreversibly at concentrations as low as 100 nM. We concluded that this method can be useful in drug screening of medium size libraries. If combined with an automated patch-clamp system, it can further increase the productivity and lower the cost of screening significantly.

Hubungan Dosis Tepung Gembili (Dioscorea esculenta) Dengan Tingkat Ekspresi Enzim Ampk-α2 pada Model Tikus Diabetes Melitus

Alternative methods of controlling glucose levels in patients with diabetes is by type of food, either by utilizing yam flour. At the flour contained inulin and resistant starch that can activate the enzyme AMPK-α2. Activation of these enzymes will stimulate glucose transport in skeletal muscle and liver, thus causing a decrease in glucose production. Varying doses of flour is expected to affect the expression of AMPK-α2. This study aims to dosage relationship yam flour (Dioscorea esculenta) with tigkat-α2 AMPK enzyme expression in the nucleus skeletal muscle and liver in mouse models of diabetes mellitus. The study was a quasi-experimental design with Post Test Only Group Design. Rats were divided into 5 (five) groups, healthy mice, the mice with type 2 diabetes, and type 2 groups of diabetic rats with dietary intake yam flour addition of 1.25 g (TG-1:25),), 2.5 g (TG -2.5), 5.0 g (TG-5.0). Yam flour is mixed into the rat diet feed with varying doses. The results showed no significant correlation between the dose of yam flour with AMPK-α2 expression levels in skeletal muscle nuclei (p = 0.312) and liver (p = 0.474) in a mouse model of DM. The need for other studies using other types of food as an alternative arrangement of food for patients with diabetes

Expression of FSHD-related DUX4-FL alters proteostasis and induces TDP-43 aggregation.

ObjectivePathogenesis in facioscapulohumeral muscular dystrophy (FSHD) appears to be due to aberrant expression, particularly in skeletal muscle nuclei, of the full-length isoform of DUX4 (DUX4-FL). Expression of DUX4-FL is known to alter gene expression and to be cytotoxic, but cell responses to DUX4-FL are not fully understood. Our study was designed to identify cellular mechanisms of pathogenesis caused by DUX4-FL expression.MethodsWe used human myogenic cell cultures to analyze the effects of DUX4-FL when it was expressed either from its endogenous promoter in FSHD cells or by exogenous expression using BacMam vectors. We focused on determining the effects of DUX4-FL on protein ubiquitination and turnover and on aggregation of TDP-43.ResultsHuman FSHD myotubes with endogenous DUX4-FL expression showed both altered nuclear and cytoplasmic distributions of ubiquitinated proteins and aggregation of TDP-43 in DUX4-FL-expressing nuclei. Similar changes were found upon exogenous expression of DUX4-FL, but were not seen upon expression of the non-toxic short isoform DUX4-S. DUX4-FL expression also inhibited protein turnover in a model system and increased the amounts of insoluble ubiquitinated proteins and insoluble TDP-43. Finally, inhibition of the ubiquitin–proteasome system with MG132 produced TDP-43 aggregation similar to DUX4-FL expression.InterpretationsOur results identify DUX4-FL-induced inhibition of protein turnover and aggregation of TDP-43, which are pathological changes also found in diseases such as amyotrophic lateral sclerosis and inclusion body myopathy, as potential pathological mechanisms in FSHD.

Burn injury induces skeletal muscle degeneration, inflammatory host response, and oxidative stress in wistar rats.

Burn injuries (BIs) result in both local and systemic responses distant from the site of thermal injury, such as skeletal muscle. The purpose of this study was to investigate the expression of cyclooxygenase-2 (COX-2) and hydroxy-2'-deoxyguanosine (8-OHdG) as a result of inflammation and reactive oxygen species production, respectively. A total of 16 male rats were distributed into two groups: control (C) and submitted to BI. The medial part of gastrocnemius muscle formed the specimens, which were stained with hematoxylin and eosin and were evaluated. COX-2 and 8-OHdG expressions were assessed by immunohistochemistry, and cell profile area and density of muscle fibers (number of fibers per square millimeter) were evaluated by morphometric methods. The results revealed inflammatory infiltrate associated with COX-2 immunoexpression in BI-gastrocnemius muscle. Furthermore, a substantial decrease in the muscle cell profile area of BI group was noticed when compared with the control group, whereas the density of muscle fibers was higher in the BI group. 8-OHdG expression in numerous skeletal muscle nuclei was detected in the BI group. In conclusion, the BI group is able to induce skeletal muscle degeneration as a result of systemic host response closely related to reactive oxygen species production and inflammatory process.

G.O.22: Reducing dynamin 2 rescues centronuclear myopathy

Centronuclear myopathies (CNM) are associated with muscle weakness and abnormally located nuclei in skeletal muscle. They can be due to mutations in the MTM1 gene encoding myotubularin (X-linked centronuclear myopathy or myotubular myopathy), in the DNM2 gene encoding dynamin 2 (dominant CNM), or in BIN1 (amphiphysin 2), RYR1 or TTN for typical or atypical autosomal forms. Currently, no effective treatments exist for centronuclear myopathies. We and others showed that overexpression of wildtype DNM2 in muscle cause a CNM-like phenotype. We thus hypothesized MTM1 and DNM2 function in a common pathway, where either MTM1 loss-of-function or DNM2 gain-of-function lead to the CNM phenotype. To test this hypothesis, we reduced the expression of Dnm2 in Mtm1-/y mice that reproduce a CNM phenotype with a progressive myopathy leading to death by 6–12 weeks. Mtm1-/yDnm2+/− mice survived up to 2 years. CNM histological features including fiber atrophy and nuclei mispositioning were prevented or strongly delayed and reduced, and muscle strength was increased. Downregulation of Dnm2 selectively in skeletal muscle during embryogenesis or in young Mtm1-/y mice showed the rescue is cell autonomous and that downregulation of Dnm2 can stop and potentially revert the progression of the phenotype. Thus, we identified MTM1 and DNM2 are in a common pathway regulating muscle organization and force. We introduce the original concept of ”cross-therapy” where one form of the disease (X-linked CNM, MTM1) can be rescued by decreasing expression of another gene mutated in CNM (DNM2). Preliminary data will be presented supporting downregulation of DNM2 can also rescue a myopathy not due to MTM1 mutations, enlarging the spectrum of myopathies that could be rescued by this strategy. While DNM2 is a key mechanoenzyme for important cellular processes, its reduction is strongly beneficial for several myopathies and represents a novel potential therapeutic approach.

Reducing dynamin 2 expression rescues X-linked centronuclear myopathy

Centronuclear myopathies (CNM) are congenital disorders associated with muscle weakness and abnormally located nuclei in skeletal muscle. An autosomal dominant form of CNM results from mutations in the gene encoding dynamin 2 (DNM2), and loss-of-function mutations in the gene encoding myotubularin (MTM1) result in X-linked CNM (XLCNM, also called myotubular myopathy), which promotes severe neonatal hypotonia and early death. Currently, no effective treatments exist for XLCNM. Here, we found increased DNM2 levels in XLCNM patients and a mouse model of XLCNM (Mtm1(-/y)). Generation of Mtm1(-/y) mice that were heterozygous for Dnm2 revealed that reduction of DNM2 in XLCNM mice restored life span, whole-body strength, and diaphragm function and increased muscle strength. Additionally, classic CNM-associated histological features, including fiber atrophy and nuclei mispositioning, were absent or reduced. Ultrastructural analysis revealed improvement of sarcomere organization and triad structures. Skeletal muscle-specific decrease of Dnm2 during embryogenesis or in young mice after disease onset revealed that the rescue associated with downregulation of Dnm2 is cell autonomous and is able to stop and potentially revert XLCNM progression. These data indicate that MTM1 and DNM2 regulate muscle organization and force through a common pathway. Furthermore, despite DNM2 being a key mechanoenzyme, its reduction is beneficial for XLCNM and represents a potential therapeutic approach for patients.

Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development.

Opposing HDAC4 nuclear fluxes due to phosphorylation by β‐adrenergic activated protein kinase A or by activity or Epac activated CaMKII in skeletal muscle fibres

Class IIa histone deacetylases (HDACs) move between skeletal muscle fibre cytoplasm and nuclei in response to various stimuli, suppressing activity of the exclusively nuclear transcription factor Mef2. Protein kinase A (PKA) phosphorylates class IIa HDACs in cardiac muscle, resulting in HDAC nuclear accumulation, but this has not been examined in skeletal muscle. Using HDAC4-green fluorescent protein (HDAC4-GFP) expressed in isolated skeletal muscle fibres, we now show that activation of PKA by the beta-receptor agonist isoproterenol or dibutyryl (Db) cAMP causes a steady HDAC4-GFP nuclear influx. The beta-receptor blocker propranolol or PKA inhibitor Rp-cAMPS blocks the effects of isoproterenol on the nuclear influx of HDAC4-GFP, and Rp-cAMPS blocks the effects of Db cAMP. The HDAC4-GFP construct having serines 265 and 266 replaced with alanines, HDAC4 (S265/266A)-GFP, did not respond to beta-receptor or PKA activation. Immunoprecipitation results show that HDAC4-GFP is a substrate of PKA, but HDAC4 (S265/266A)-GFP is not, implicating HDAC4 serines 265/266 as the site(s) phosphorylated by PKA. During 10 Hz trains of muscle fibre electrical stimulation, the nuclear efflux rate of HDAC4-GFP, but not of HDAC4 (S265/266)-GFP, was decreased by PKA activation, directly demonstrating antagonism between the effects of fibre stimulation and beta-adrenergic activation of PKA on HDAC4 nuclear fluxes. 8-CPT, a specific activator of Epac, caused nuclear efflux of HDAC4-GFP, opposite to the effect of PKA. Db cAMP increased both phosphorylated PKA and GTP-bound Rap1. Our results demonstrate that the PKA and CaMKII pathways play important opposing roles in skeletal muscle gene expression by oppositely affecting the subcellular localization of HDAC4.