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Background: Histone deacetylases (HDACs) play a critical role in modulating myocardial protection and cardiomyocyte survivals. However, Specific HDAC isoforms in mediating myocardial ischemia/reperfusion injury remain currently unknown. We used cardiomyocyte-specific overexpression of active HDAC4 to determine the functional role of activated HDAC4 in regulating myocardial ischemia and reperfusion in isovolumetric perfused hearts. Methods: In this study, we created myocyte-specific active HDAC4 transgenic mice to examine the functional role of active HDAC4 in mediating myocardial I/R injury. Ventricular function was determined in the isovolumetric heart, and infarct size was determined using tetrazolium chloride staining. Results: Myocyte-specific overexpressing activated HDAC4 in mice promoted myocardial I/R injury, as indicated by the increases in infarct size and reduction of ventricular functional recovery following I/R injury. Notably, active HDAC4 overexpression led to an increase in LC-3 and active caspase 3 and decrease in SOD-1 in myocardium. Delivery of chemical HDAC inhibitor attenuated the detrimental effects of active HDAC4 on I/R injury, revealing the pivotal role of active HDAC4 in response to myocardial I/R injury. Conclusions: Taken together, these findings are the first to define that activated HDAC4 as a crucial regulator for myocardial ischemia and reperfusion injury.

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome associated with a high morbidity and mortality rate. Leucine supplementation has been demonstrated to attenuate cardiac dysfunction in animal models of cachexia and heart failure with reduced ejection fraction (HFrEF). So far, no data exist on leucine supplementation on cardiac function in HFpEF. Thus, the current study aimed to investigate the effect of leucine supplementation on myocardial function and key signaling pathways in an established HFpEF rat model. Female ZSF1 rats were randomized into three groups: Control (untreated lean rats), HFpEF (untreated obese rats), and HFpEF_Leu (obese rats receiving standard chow enriched with 3% leucine). Leucine supplementation started at 20 weeks of age after an established HFpEF was confirmed in obese rats. In all animals, cardiac function was assessed by echocardiography at baseline and throughout the experiment. At the age of 32 weeks, hemodynamics were measured invasively, and myocardial tissue was collected for assessment of mitochondrial function and for histological and molecular analyses. Leucine had already improved diastolic function after 4 weeks of treatment. This was accompanied by improved hemodynamics and reduced stiffness, as well as by reduced left ventricular fibrosis and hypertrophy. Cardiac mitochondrial respiratory function was improved by leucine without alteration of the cardiac mitochondrial content. Lastly, leucine supplementation suppressed the expression and nuclear localization of HDAC4 and was associated with Protein kinase A activation. Our data show that leucine supplementation improves diastolic function and decreases remodeling processes in a rat model of HFpEF. Beneficial effects were associated with HDAC4/TGF-β1/Collagenase downregulation and indicate a potential use in the treatment of HFpEF.

Skeletal muscle atrophy is a debilitating condition that commonly occurs as a secondary consequence of many acute and chronic medical conditions, including muscle disuse, heart and renal failure, starvation, cancer, HIV/AIDS, and aging. Though it leads to weakness, falls, and fractures, and reduces independence and quality of life for millions of Americans annually, no effective pharmacologic therapies for muscle atrophy exist. This is largely due to a poor understanding of the pathogenesis of skeletal muscle atrophy at a molecular level. In this thesis, I describe my studies into the molecular pathogenesis of skeletal muscle atrophy. Using mouse models, I showed that the gene encoding the pro-atrophy nuclear protein Gadd45a is regulated by distinct pathways after muscle denervation and fasting, and also identified a novel protein regulating skeletal muscle fiber size. First, we demonstrated that denervation-induced muscle atrophy, unlike atrophy mediated by fasting, does not require the bZIP transcription factor ATF4. However, the lysine deacetylase HDAC4 is sufficient to induce Gadd45a mRNA and necessary for Gadd45a mRNA induction after denervation, but not after fasting. Taken together, these data show that Gadd45a is a central convergence point for muscle atrophy caused by several stimuli, and also demonstrate that distinct pathways mediate Gadd45a induction in different models of skeletal muscle atrophy. Second, we identified spermine oxidase as a critical regulator of muscle fiber size. We observed that spermine oxidase mRNA and spermine oxidase protein were reduced by several distinct causes of muscle atrophy (i.e. immobilization, denervation, fasting, and aging). Furthermore, spermine oxidase overexpression increased muscle fiber size, while spermine oxidase knockdown caused muscle fiber atrophy. Restoring spermine oxidase expression significantly attenuated muscle atrophy after limb immobilization, denervation, and fasting. Finally, we identified p21 as a key upstream regulator of spermine oxidase expression, and spermine oxidase as a required mediator of p21-mediated skeletal muscle fiber atrophy. Collectively, these findings greatly advance our understanding of the molecular pathogenesis of skeletal muscle atrophy. These data demonstrate that Gadd45a is a convergence point for multiple pro-atrophy pathways and identify spermine oxidase as a novel therapeutic target for the treatment of skeletal muscle atrophy. These discoveries suggest several important new areas for future research, and further our understanding of this common, debilitating condition.

Significant advances have been made in orthodontics and oral maxillofacial surgery for the diagnosis and treatment of dentofacial deformities. However, compared with bone, the effect of muscles of the craniofacial complex in the development of dentofacial deformities has received little attention. Recently, cellular and molecular studies of the musculoskeletal interactions have been used to investigate the etiology of dentofacial malocclusions. In this study, we tested for relationships that might exist between gene expression of the chromatin modifying enzymes histone deacetylase-4 (HDAC4) and histone acetyltransferase-4 (MYST4) and expression of myosin heavy chain (MyHC) genes and fiber-type percent occupancy (%Occ) in masseter muscle of patients undergoing orthognathic surgery to correct severe dentofacial malocclusions. The diagnostic categories of malocclusion in sagittal and vertical dimensions were: 1) Deep bite-Class II (D2); 2) Deep bite-Class III (D3); 3) Normal bite-Class II (N2); 4) Normal bite-Class III (N3), 5) Open bite-Class II (O2); 6) open bite with Class III (O3). Relative quantities (RQs) of gene expression were determined by reverse transcriptase real time polymerase chain reaction (RT-PCR) in RNA extracts of masseter samples, previously analyzed by immunohistochemistry for %Occ values. By multivariate analysis, RQs of HDAC4 and MYST4 expression did not differ significantly between malocclusion types. However, multiple high positive and negative correlations were found for HDAC4 and MYST4 with MyHC expression and with fiber type %Occ. Significant correlations occurred for HDAC4 with: IIX and neonatal MyHCs respectively in N2 and N3 subjects; fiber types I, I/II and neonatal/atrial %Occ respectively in D2 and N3, D2 and O3 subjects. Further investigations are needed to support evidence of these correlations and determine their significance toward diagnosis, treatment and relapse potential in the correction of dentofacial deformities. / Oral Biology

L’arthrose ou ostéoarthrose (OA) est l’affection rhumatologique la plus fréquente au monde. Elle est caractérisée principalement par une perte du cartilage articulaire et l’inflammation de la membrane synoviale. L’interleukine (IL)-1ß, une cytokine pro-inflammatoire, joue un rôle très important dans la pathogenèse de l’OA. Elle exerce son action en induisant l’expression des enzymes cyclo-oxygénase 2 (COX-2), prostaglandine E synthétase microsomale 1 (mPGES-1) et l’oxyde nitrique synthétase inductible (iNOS) ainsi que la production de la prostaglandine E2 (PGE2) et de l’oxyde nitrique (NO). Ces derniers (PGE2 et NO) contribuent à la synovite et la destruction du cartilage articulaire par leurs effets pro-inflammatoires, pro-cataboliques, anti-anaboliques, pro-angiogéniques et pro-apoptotiques. Les modifications épigénétiques, telles que la méthylation de l’ADN, et l’acétylation et la méthylation des histones, jouent un rôle crucial dans la régulation de l’expression des gènes. Parmi ces modifications, l’acétylation des histones est la plus documentée. Ce processus est contrôlé par deux types d’enzymes : les histones acétyltransférases (HAT) qui favorisent la transcription et les histones déacétylases (HDAC) qui l’inhibent. L’objectif de ce travail est d’examiner le rôle des enzymes HDAC dans la régulation de l’expression de la COX-2, mPGES-1 et iNOS. Nous avons montré qu’au niveau des chondrocytes, les inhibiteurs des HDAC (iHDAC), trichostatine A (TSA) et butyrate de sodium (NaBu), suppriment l’expression de la COX-2 et iNOS au niveau de l’ARNm et protéique, ainsi que la production de la PGE2 et du NO, induites par l’IL-1ß. L’effet inhibiteur à lieu sans affecter l’activité de liaison à l’ADN du facteur de transcription NF-κB (nuclear factor κ B). La TSA et le NaBu inhibent également la dégradation induite par l’IL-1ß des protéoglycanes au niveau du cartilage. Nous avons également montré, qu’au niveau des fibroblastes synoviaux, les iHDAC, TSA, NaBu et acide valproïque (VA), suppriment l’expression de la mPGES-1 ainsi que la production de la PGE2 induites par l’IL-1ß. En utilisant diverses approches expérimentales, nous avons montré que HDAC4 est impliquée dans l’induction de l’expression de la mPGES-1 par l’IL-1ß. HDAC4 exerce son action, via son activité déacétylase, en augmentant l’activité transcriptionnelle de Egr-1 (early growth factor 1), facteur de transcription principal de l’expression de la mPGES-1. L’ensemble de ces résultats suggère que les inhibiteurs des HDAC pourraient être utilisés dans le traitement de l’OA. / Osteoarthritis (OA) is the most common form of arthritic diseases in the world. It is primarily characterized by the loss of articular cartilage and inflammation of the synovial membrane. Interleukin (IL)-1ß is a pro-inflammatory cytokine that plays a major role in the pathogenesis of OA. It induces the expression of cyclo-oxygenase 2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), inducible nitric oxide synthase (iNOS), as well as the production of prostaglandin E2 (PGE2) and nitric oxide (NO). The later (PGE2 and NO) contribute to articular cartilage destruction and synovitis through their pro-inflammatory, pro-catabolic, anti-anabolic, pro-angiogenic and pro-apoptotic effects. Epigenetic modifications such as DNA methylation, histone acetylation and methylation play a crucial role in gene expression. Among these modifications, histone acetylation is the most studied. Histone acetylation is determined by two types of enzymes: histone acetyltransferases (HAT) and histone deacetylases (HDAC) which activate and repress transcription, respectively. The purpose of these studies is to examine the role of HDAC enzymes in the regulation of COX-2, mPGES-1, and iNOS expression. We demonstrated that HDAC inhibitors (HDACi), trichostatin A (TSA) and sodium butyrate (NaBu), suppressed the Il-1ß-induced transcription and translation of COX-2 and iNOS, as well as the production of PGE2 and NO in chondrocytes. The inhibitory effect of HDACi on transcription does not affect the binding activity of NF-κB (nuclear factor κ B) to DNA. Treatment with TSA and NaBu also inhibited the Il-1ß-induced degradation of proteoglycan in cartilage explants. We also showed that HDACi, TSA, NaBu and valproic acid (VA), suppressed IL-1-induced-mPGES-1 expression and the production of PGE2 in synovial fibroblasts. Our data indicated that HDAC4 is involved in Il-1ß-induced expression of mPGES-1. HDAC4, through its deacetylase activity, up-regulated the transcriptional activity of Egr-1 (early growth factor-1), a principal transcription factor for the expression of mPGES-1. From our studies we propose that HDAC inhibitors can be used in the treatment of OA.

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