HDAC4 preserves skeletal muscle structure following long-term denervation by mediating distinct cellular responses

BACKGROUND: Denervation triggers numerous molecular responses in skeletal muscle, including the activation of catabolic pathways and oxidative stress, leading to progressive muscle atrophy. Histone deacetylase 4 (HDAC4) mediates skeletal muscle response to denervation, suggesting the use of HDAC inhibitors as a therapeutic approach to neurogenic muscle atrophy. However, the effects of HDAC4 inhibition in skeletal muscle in response to long-term denervation have not been described yet. METHODS: To further study HDAC4 functions in response to denervation, we analyzed mutant mice in which HDAC4 is specifically deleted in skeletal muscle. RESULTS: After an initial phase of resistance to neurogenic muscle atrophy, skeletal muscle with a deletion of HDAC4 lost structural integrity after 4 weeks of denervation. Deletion of HDAC4 impaired the activation of the ubiquitin-proteasome system, delayed the autophagic response, and dampened the OS response in skeletal muscle. Inhibition of the ubiquitin-proteasome system or the autophagic response, if on the one hand, conferred resistance to neurogenic muscle atrophy; on the other hand, induced loss of muscle integrity and inflammation in mice lacking HDAC4 in skeletal muscle. Moreover, treatment with the antioxidant drug Trolox prevented loss of muscle integrity and inflammation in in mice lacking HDAC4 in skeletal muscle, despite the resistance to neurogenic muscle atrophy. CONCLUSIONS: These results reveal new functions of HDAC4 in mediating skeletal muscle response to denervation and lead us to propose the combined use of HDAC inhibitors and antioxidant drugs to treat neurogenic muscle atrophy

HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes

Skeletal muscle exhibits a high regenerative capacity, mainly due to the ability of satellite cells to replicate and differentiate in response to appropriate stimuli. Epigenetic control is effective at different stages of this process. It has been shown that the chromatin-remodeling factor HDAC4 is able to regulate satellite cell proliferation and commitment. However, its molecular targets are still uncovered. To explain the signaling pathways regulated by HDAC4 in satellite cells, we generated tamoxifen-inducible mice with conditional inactivation of HDAC4 in Pax7(+) cells (HDAC4 KO mice). We found that the proliferation and differentiation of HDAC4 KO satellite cells were compromised, although similar amounts of satellite cells were found in mice. Moreover, we found that the inhibition of HDAC4 in satellite cells was sufficient to block the differentiation process. By RNA-sequencing analysis we identified P21 and Sharp1 as HDAC4 target genes. Reducing the expression of these target genes in HDAC4 KO satellite cells, we also defined the molecular pathways regulated by HDAC4 in the epigenetic control of satellite cell expansion and fusion

Investigating the role of histone deacetylase HDAC4 in long-term memory formation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Manawatu, New Zealand

Listed in 2017 Dean's List of Exceptional ThesesEpigenetic mechanisms are emerging as master regulators of cognitive abilities such as learning and memory. It has been previously shown that the histone deacetylase HDAC4 plays a critical role in memory formation in both mammals and insects although the specific mechanisms through which it acts have not yet been elucidated. HDAC4 undergoes nucleocytoplasmic shuttling and, in neurons, it is largely cytoplasmic implying it may play both nuclear and non-nuclear functions. To identify upstream regulators and downstream targets of HDAC4, a genetic interaction screen was performed in the fruit fly Drosophila melanogaster, a powerful model system to study the genetic mechanisms of neurological disease. Twenty-nine genes were found to interact with HDAC4 suggesting they are part of the same molecular pathway. Functional network analysis revealed that many of the genes could be grouped into three biological categories comprising transcriptional factors, SUMOylation machinery enzymes and cytoskeletal regulators/interactors. Within the latter, Ankyrin2 was selected for further analysis as it is implicated in synaptic stability and in human intellectual disability. In addition HDAC4 harbours a conserved ankyrin binding domain. Immunohistochemical analyses showed widespread distribution of Ankyrin2 throughout the adult brain and coincident distribution with HDAC4 was observed in the axons of the mushroom body, a key structure for memory formation in flies. Both HDAC4 and Ankyrin2 were also found to regulate mushroom body development. RNAi-mediated depletion of Ankyrin2 in the adult brain impaired long-term memory in the courtship suppression assay, a model of associative memory and preliminary evidence of a physical association between HDAC4 and Ankyrin2 was also demonstrated. The genes identified in the screen provide new avenues for investigation of the mechanisms through which HDAC4 regulates memory formation and preliminary analyses suggest that interaction with the cytoskeletal adaptor Ankyrin2 may involve remodelling of the actin/spectrin cytoskeleton, phenomenon that underlies memory related processes like synaptic plasticity and neuronal excitability

HDAC4 regulates skeletal muscle regeneration via soluble factors

Skeletal muscle possesses a high ability to regenerate after an insult or in pathological conditions, relying on satellite cells, the skeletal muscle stem cells. Satellite cell behavior is tightly regulated by the surrounding microenvironment, which provides multiple signals derived from local cells and systemic factors. Among epigenetic mechanisms, histone deacetylation has been proved to affect muscle regeneration. Indeed, pan-histone deacetylase inhibitors were found to improve muscle regeneration, while deletion of histone deacetylase 4 (HDAC4) in satellite cells inhibits their proliferation and differentiation, leading to compromised muscle regeneration. In this study, we delineated the HDAC4 function in adult skeletal muscle, following injury, by using a tissue-specific null mouse line. HDAC4 resulted crucial for skeletal muscle regeneration by mediating soluble factors that influence muscle-derived cell proliferation and differentiation. These findings add new biological functions to HDAC4 in skeletal muscle that need considering when administering histone deacetylase inhibitors

MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia

Adipocyte Liver Kinase b1 Suppresses Beige Adipocyte Renaissance Through Class IIa Histone Deacetylase 4.

Uncoupling protein 1+ beige adipocytes are dynamically regulated by environment in rodents and humans; cold induces formation of beige adipocytes, whereas warm temperature and nutrient excess lead to their disappearance. Beige adipocytes can form through de novo adipogenesis; however, how "beiging" characteristics are maintained afterward is largely unknown. In this study, we show that beige adipocytes formed postnatally in subcutaneous inguinal white adipose tissue lost thermogenic gene expression and multilocular morphology at the adult stage, but cold restored their beiging characteristics, a phenomenon termed beige adipocyte renaissance. Ablation of these postnatal beige adipocytes inhibited cold-induced beige adipocyte formation in adult mice. Furthermore, we demonstrated that beige adipocyte renaissance was governed by liver kinase b1 and histone deacetylase 4 in white adipocytes. Although neither presence nor thermogenic function of uncoupling protein 1+ beige adipocytes contributed to metabolic fitness in adipocyte liver kinase b1-deficient mice, our results reveal an unexpected role of white adipocytes in maintaining properties of preexisting beige adipocytes

A shared mechanism of muscle wasting in cancer and Huntington's disease.

Skeletal muscle loss and dysfunction in aging and chronic diseases is one of the major causes of mortality in patients, and is relevant for a wide variety of diseases such as neurodegeneration and cancer. Muscle loss is accompanied by changes in gene expression and metabolism that lead to contractile impairment and likely affect whole-body metabolism and function. The changes may be caused by inactivity, inflammation, age-related factors or unbalanced nutrition. Although links with skeletal muscle loss have been found in diseases with disparate aetiologies, for example both in Huntingtons disease (HD) and cancer cachexia, the outcome is a similar impairment and mortality. This short commentary aims to summarize recent achievements in the identification of common mechanisms leading to the skeletal muscle wasting syndrome seen in diseases as different as cancer and HD. The latter is the most common hereditary neurodegenerative disorder and muscle wasting is an important component of its pathology. In addition, possible therapeutic strategies for anti-cachectic treatment will be also discussed in the light of their translation into possible therapeutic approaches for HD

The role of histone deacetylase 4 (HDAC4) in acquired cisplatin-resistant ovarian cancer

Resistance to platinum is a major problem in the treatment of ovarian cancer. Molecular profiling of isogenic ovarian cancer cell line pairs derived from tumour cells pre- and post- clinical resistance, identified that histone deacetylase 4 (HDAC4) is over-expressed in cisplatin-resistant cells relative to sensitive derivatives. HDAC4 siRNA and HDACi treatment resensitised resistant cells to cisplatin. Furthermore, up-regulation of HDAC4 in cisplatin-resistant ovarian cell lines and tumour sections identified HDAC4 as a potential biomarker of cisplatinresistance. It was observed that HDAC4 interacts with another mediator of platinum resistance, the transcription factor STAT1, and that knockdown of HDAC4 increased acetylation levels of STAT1 protein in platinum resistant cells. Consequent decrease in cisplatin mediated phosphorylation of STAT1 and nuclear translocation of STAT1 were observed. However, STAT1 was found to be acetylated and inactive in platinum sensitive cells, expressing lower levels of HDAC4. Microarray analysis of the platinum resistant cells has identified differentially regulated genes following HDAC4 knockdown. The UCHL1 promoter is methylated in both cisplatin sensitive and resistant paired cells and yet is reexpressed in resistant cells following HDAC4 knockdown. The methylation levels at the UCHL1 promoter were analysed by pyrosequencing method and do not appear to significantly change the methylation level after HDAC4 knockdown. However, ChIP analysis revealed an increase in acetylation at the UCHL1 promoter after HDAC4 knockdown. Conversely, P21 is down-regulated by HDAC4 knockdown in resistant PEO4 cells in contrast to reports of P21 overexpression as a biomarker of HDAC inhibition. Strikingly however, it is upregulated after HDAC4 knockdown in cisplatin-sensitive paired PEO1 cells suggesting that, like STAT1, a fundamental change in its control occurs on acquisition of platinum resistance. This study provides evidence that HDAC4 is required for platinum mediated STAT1 activation; a phenomenon associated with clinical platinum resistance, and identifies frequent HDAC4 over-expression in platinum resistant tumour biopsies. Pharmacological modulation of this pathway is shown to restore sensitivity to cisplatin. Microarray analysis revealed HDAC4 regulated target genes. These studies identify new and clinically relevant insights into platinum resistance which may lead to improved therapeutic options in ovarian cancer