Abstract Our goal is to investigate the impact of diet and pressure overload on the histone code, and how this influences changes in gene expression in the healthy and hypertrophied/failing hearts and, in turn, how it impacts progression of the disease. Deciphering the histone code and how diet can modify it, provides us an educated means to exploit it to our advantage, especially during pathological conditions. Acetylation and methylation of histone lysine (K) residues were the first histone modifications discovered and are, therefore, the most widely studied and understood. However, to-date, there are 11 confirmed modifiers of histone lysine residues, including the acyl groups butyryl (Bu), crotonyl (Cr), and b-hydroxybutyrate (bHB) 1, whose source, genomic distribution, and functional relevance, remain largely unknown in the heart, and are the focus of our study. Our recent findings uniquely show that dietary fat is a major regulator of histone butyrylation, including H3K9-butyryl (H3K9Bu). Using genome-wide chromatin immunoprecipitation-sequencing (ChIP-Seq), we show that H3K9Bu is abundant at all transcriptionally active promoters. Both a high-fat diet and stress accelerated the conversion of butyryl-CoA to crotonyl-CoA via acyl-CoA dehydrogenase short chain (ACADS), resulting in a substantial reduction in global promoter-H3K9Bu. A deletion of ACADS both in the mouse heart and in human cells reversed this effect and increased promoter and gene-body H3K9Bu. Paradoxically, though, a fat-free diet had the highest levels of H3K9Bu. Deletion of fatty acid synthetase (FASN), abolished H3K9Bu in cells maintained in a glucose-rich, fatty acid-free, but not in a fatty acid-rich, medium, proving that fatty acid synthesis from carbohydrates substitutes for dietary fat as a source butyryl-CoA. In contrast to H3K9Bu, there were minimal dietary-induced changes in H3K9-acetyl (H3K9ac) levels. Importantly, RNA-sequencing (RNA-Seq) revealed that diet-induced changes in H3K9Bu abundance in the mouse heart was associated with differential changes in gene expression, but only when stressed by pressure overload. Moreover, promoter-H3K9Bu levels inversely correlated with the extent of changes in gene expression levels, as evidenced by the more robust changes seen in the hearts of mice on a, short-term, high-fat vs a fat-free diet, as well as, after deletion of the ACADS. Interestingly, H3K9Bu abundance inversely correlated with H3K9-crotonyl (H3K9Cr) and Cdk9. In sum, our data uniquely show that H3K9Bu is enriched at active promoters, is negatively regulated by high-fat and stress in an ACADS-dependent fashion, and its abundance inversely correlates with stress-induced changes in gene expression. We are proposing that histone H3K9Bu, H3K9Cr, and H3K9-b-hydroxybutyryl (H3K9bHB), are products of the b-oxidation intermediates, butyryl-CoA, crotonyl-CoA, and b-hydroxybutyryl-CoA, or the ketone body, b-hydroxybutyrate, which serve as substrates for histones modifications. These marks are labile and differentially influence pressure overload-induced gene expression, but not baseline expression. Specifically, as H3K9Bu decreases it is replaced by H3K9Cr during a high-fat diet. This exchange exaggerates gene expression and worsens the outcome of cardiac failure. Conversely, H3K9bHB that increases during a ketogenic diet has the opposite effect, as it is reported to have beneficial effects on health and aging. This differential influence of the histone marks on gene expression is mediated by regulating the recruitment of Cdk9 to gene promoters. We hypothesize that 1) A high-fat diet (60 Kcal% fat, 20 Kcal% carb), or pressure overload, accelerates the conversion of nuclear butyryl- CoA to crotonyl-CoA in an ACADS-dependent manner, thus, reducing H3K9Bu and increasing H3K9Cr, which is responsible for exaggerating stress-induced gene expression and worsening the outcome of heart failure (HF). In contrast, a ketogenic diet (84 Kcal% fat, 0% carb) will produce high levels of b-hydroxybutyryl that will increase H3K9bHB, which curbs changes in stress-induced gene expression in a b-hydroxybutyrate dehydrogenase (BDH1)-dependent fashion, improving the outcome of HF. Supplementing a diet with b-hydroxybutyrate will also increase H3K9bHB, with similar beneficial effects. 2) Therefore, knockdown of ACADS reduces the conversion of butyryl-CoA to crotonyl-CoA, increasing H3K9Bu and improving the outcome of heart failure during a high-fat diet. Conversely, deletion or inhibition of BDH1 reduces H3K9bHB and worsens conditions. 3) H3K9Cr enhances the dynamics of cyclin-dependent kinase 9 (Cdk9) recruitment to promoters during stress, whereas, H3K9Bu and H3K9bHB temper it, thus, reducing the extent of changes in gene expression and improving disease outcome. The specific aims are: 1) Examine the effects of high-fat, ketogenic, and b-hydroxybutyrate-enriched diets on the genome-wide distribution and changes in H3K9Bu, H3K9Cr and H3K9bHB, changes in gene expression, and their impact on the progression of cardiac hypertrophy and failure. 2) Investigate the roles of ACADS and BDH1 in regulating the levels of H3K9Bu, H3K9Cr, and H3K9bHb, and the progression of cardiac hypertrophy and failure. 3) Investigate the role of Cdk9 in mediating the differential transcriptional regulation directed by promoter-H3K9Cr vs. H3K9Bu or H3K9bHB during cardiac hypertrophy and failure.

Our goal is to investigate the impact of diet, metabolic enzymes, and pressure overload on the histone code, and how this influences changes in gene expression in the healthy and hypertrophied/failing hearts and, in turn, how it impacts progression of the disease. Deciphering the histone code and how diet can modify it, gives us the means to exploit it to our advantage, especially during pathological conditions. We are proposing that histone H3K9-butyryl, H3K9- crotonyl, and H3K9-b-hydroxybutyryl, are products of the b-oxidation intermediates, butyryl-CoA, crotonyl-CoA, and b-hydroxybutyryl-CoA, or the ketone body, b-hydroxybutyrate, that are produced in the nucleus. The genomic distribution of these modified histones, how they are modified by diet in the presence or absence of stress, and their functional relevance, particularly in the heart, have not been investigated. Our results show that a high-fat diet exacerbates pressure overload-induced gene expression in the heart, worsening the prognosis of the disease, as a result of modifying the histone code. We hypothesize that we can temper these effects and improve disease outcome by modulating the histone marks by diet, including low-fat and ketogenic diets.