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Perturbed vitamin-A metabolism is associated with type 2 diabetes and mitochondrial dysfunction that are pathophysiologically linked to the development of diabetic cardiomyopathy (DCM). However, the mechanism, by which vitamin A might regulate mitochondrial energetics in DCM has previously not been explored. To test the hypothesis that vitamin-A deficiency accelerates the onset of cardiomyopathy in diet-induced obesity (DIO), we subjected mice with lecithin retinol acyltransferase (Lrat) germline deletion, which exhibit impaired vitamin-A stores, to vitamin A-deficient high-fat diet (HFD) feeding. Wild-type mice fed with a vitamin A-sufficient HFD served as controls. Cardiac structure, contractile function, and mitochondrial respiratory capacity were preserved despite vitamin-A deficiency following 20 wk of HFD feeding. Gene profiling by RNA sequencing revealed that vitamin A is required for the expression of genes involved in cardiac fatty acid oxidation, glycolysis, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation in DIO as expression of these genes was relatively preserved under vitamin A-sufficient HFD conditions. Together, these data identify a transcriptional program, by which vitamin A preserves cardiac energetic gene expression in DIO that might attenuate subsequent onset of mitochondrial and contractile dysfunction.

Tyrosine kinase inhibitors (TKI) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, TCA cycle and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate utilization and maintenance of mitochondrial respiration. Evaluation of transcription factors underlying these changes identified increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with a HIF-1 inhibitor depleted murine and human CML stem cells in combination with TKI treatment. HIF-1 inhibition increased mitochondrial activity and ROS levels, and reduced quiescence, increased cycling, and reduced self-renewal and regenerating potential of dormant CML stem cells. We therefore identify HIF-1-mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.

Spinal cord injury (SCI) results in rapid muscle loss. The mechanisms of muscle atrophy have been well-described but there is limited information specific to SCI. Exogenous molecular interventions to slow muscle atrophy in severe-to-complete SCI have been relatively ineffective and the wide-ranging physiologic response to SCI requires the search for novel therapeutic targets. Connexin hemichannels (CxHC) allow non-selective passage of small molecules into and out of the cell. Boldine, a CxHC-inhibiting aporphine found in the boldo tree (Peumus boldus), has shown promising pre-clinical results in slowing atrophy during sepsis and dysferlinopathy. We administered 50 mg/kg/d of boldine to spinal cord transected mice beginning 3 d post-injury. Tissue was collected 7 and 28 d post-SCI and the gastrocnemius was used for multiomic profiling. Boldine did not prevent body or muscle mass loss but attenuated SCI-induced changes in the abundance of proline, phenylalanine, leucine and isoleucine, as well as glucose, 7 d post-SCI. SCI resulted in the differential expression of ~7,700 and ~2,000 genes at 7 and 28 d, respectively, compared to sham animals, with enrichment for pathways associated with ribosome biogenesis, translation and oxidative phosphorylation. Boldine altered the expression of ~150 genes at 7 d and ~110 genes at 28 d post-SCI. Methylation analyses highlighted distinct patterns at both 7 and 28 d following SCI both with and without boldine. Taken together, boldine is not an efficacious therapy to preserve body and muscle mass after complete SCI, though it preserved or attenuated SCI-induced changes across the metabolome, transcriptome and methylome.

Obesity is now recognized as a disease. This study revealed a novel role for pyruvate dehydrogenase kinase (PDK) in diet-induced hypertrophic obesity. Mice with global or adipose tissue-specific PDK2 deficiency were protected against diet-induced obesity. The weight of adipose tissues and the size of adipocytes were reduced. Adipocyte-specific PDK2 deficiency slightly increased insulin sensitivity in HFD-fed mice. In studies with 3T3-L1 preadipocytes, PDK2 and PDK1 expression was strongly increased during adipogenesis. Evidence was found for epigenetic induction of both PDK1 and PDK2. Gain- and loss-of-function studies with 3T3-L1 cells revealed a critical role for PDK1/2 in adipocyte differentiation and lipid accumulation. PDK1/2 induction during differentiation was also accompanied by increased expression of hypoxia-inducible factor-1α (HIF1α) and enhanced lactate production, both of which were absent in the context of PDK1/2 deficiency. Exogenous lactate supplementation increased the stability of HIF1α and promoted adipogenesis. PDK1/2 overexpression-mediated adipogenesis was abolished by HIF1α inhibition, suggesting a role for the PDK-lactate-HIF1α axis during adipogenesis. In human adipose tissue, the expression of PDK1/2 was positively correlated with that of the adipogenic marker PPARγ and inversely correlated with obesity. Similarly, PDK1/2 expression in mouse adipose tissue was decreased by chronic high-fat diet feeding. We conclude that PDK1 and 2 are novel regulators of adipogenesis that play critical roles in obesity., Obesity: Effects of different forms of key enzyme The discovery that two forms of a key enzyme appear to play a critical role in fat production triggered by overeating might lead to new approaches to prevent and treat obesity. Hyeon-Ji Kang at Kyungpook National University, Daegu, South Korea, and colleagues in South Korea and the USA examined the role of the enzymes pyruvate dehydrogenase kinase types 1 and 2 (PDK1/2). PDK enzymes regulate the activity of a multi-enzyme complex that catalyzes a key step in the use of glucose to provide energy stores for cells. Mice deficient in PDK2 were protected from diet-induced obesity, and PDK 1 and 2 activity was increased during the generation of fat cells. Studies using mice and human fat tissue confirmed that the enzymes regulate the development and growth of fat cells. Drugs inhibiting PDK enzymes might combat obesity.

Although recent advances in the treatment of acute coronary heart disease have reduced mortality rates, few therapeutic strategies exist to mitigate the progressive loss of cardiac function that manifests as heart failure. Nuclear factor, erythroid 2 like 2 (Nfe2l2, Nrf2) is a transcriptional regulator that is known to confer transient myocardial cytoprotection following acute ischemic insult; however, its sustained activation paradoxically causes a reductive environment characterized by excessive antioxidant activity. We previously identified a subset of 16 microRNAs (miRNA) significantly diminished in Nrf2-ablated (Nrf2−/−) mouse hearts, leading to the hypothesis that increasing levels of Nrf2 activation augments miRNA induction and post-transcriptional dysregulation. Here, we report the identification of distinct miRNA signatures (i.e. “reductomiRs”) associated with Nrf2 overexpression in a cardiac-specific and constitutively active Nrf2 transgenic (caNrf2-Tg) mice expressing low (TgL) and high (TgH) levels. We also found several Nrf2 dose-responsive miRNAs harboring proximal antioxidant response elements (AREs), implicating these “reductomiRs” as putative meditators of Nrf2-dependent post-transcriptional regulation. Analysis of mRNA-sequencing identified a complex network of miRNAs and effector mRNAs encoding known pathological hallmarks of cardiac stress-response. Altogether, these data support Nrf2 as a putative regulator of cardiac miRNA expression and provide novel candidates for future mechanistic investigation to understand the relationship between myocardial reductive stress and cardiac pathophysiology.

Cardiovascular diseases, including ischemic heart failure (IHF), are the leading cause of mortality worldwide; furthermore, African Americans (AA) have 30% higher mortality than Caucasian Americans (CA). Our recent study correlates racial differences in socioeconomic status with DNA methylation (DNAm), altered gene expression, and mortality in HF independent of etiology. The current analysis focused on etiology-specific differences in DNAm with a specific focus on IHF, the related changes in gene expression, within self-reported race. RNA-seq expression ( P < 0.05, ±1.5-fold) and array-based methylation ( P < 0.05, ±5%) profiles were examined using cardiac biopsies from non-IHF (NIHF) and IHF AA (n = 9/5) and CA (n = 10/5) male patients. Network clusters were identified with MCODE (v2.0) and top hub genes identified via Cytohubba (v0.1). Both AA and CA patients showed similar numbers of IHF-specific differentially expressed genes (DEG) (591/365 AA; 439/486 CA; up/down). However, CA-specific methylation changes (29703/22703; up/down) were more abundant compared to AA (2904/2897; up/down). PPI network construction of these changes identified clusters unique to each group. The AA-specific upregulated cluster included AGPAT2, PPARG, and 10 other DEG associated with the fatty acid metabolism, while BMP2, SMAD6, and 4 other DEG that regulate BMP signaling and SMAD phosphorylation were downregulated. The CA-specific upregulated cluster included EZH2, CCNA1, and 18 other DEG that regulate mitotic sister chromatic separation, while EGFR, TNF, and 12 other DEG important for chronic inflammatory response formed the top downregulated cluster. Consistent with greater changes in DNAm the majority of DEGs in CA-specific clusters were also differentially methylated (58%) while fewer were in AA-specific clusters (17%). Within AA-specific PPI networks, PPARG/BMP2 (up/down) were the top ranked hub genes and KIF20A/KIT (up/down) in CA-specific networks. These racially distinct hub genes suggest altered regulatory mechanisms. Our analysis identified racially distinct clusters associated with specific pathways, which may connect the regulators of DNAm to gene expression and IHF, supporting the potential to use this information as prognostic biomarkers.

Alterations in cardiac energy metabolism contribute to the severity of heart failure. However, the energy metabolic changes that occur in heart failure are complex and are dependent not only on the severity and type of heart failure present but also on the co-existence of common comorbidities such as obesity and type 2 diabetes. The failing heart faces an energy deficit, primarily because of a decrease in mitochondrial oxidative capacity. This is partly compensated for by an increase in ATP production from glycolysis. The relative contribution of the different fuels for mitochondrial ATP production also changes, including a decrease in glucose and amino acid oxidation, and an increase in ketone oxidation. The oxidation of fatty acids by the heart increases or decreases, depending on the type of heart failure. For instance, in heart failure associated with diabetes and obesity, myocardial fatty acid oxidation increases, while in heart failure associated with hypertension or ischemia, myocardial fatty acid oxidation decreases. Combined, these energy metabolic changes result in the failing heart becoming less efficient (ie, a decrease in cardiac work/O 2 consumed). The alterations in both glycolysis and mitochondrial oxidative metabolism in the failing heart are due to both transcriptional changes in key enzymes involved in these metabolic pathways, as well as alterations in NAD redox state (NAD + and nicotinamide adenine dinucleotide levels) and metabolite signaling that contribute to posttranslational epigenetic changes in the control of expression of genes encoding energy metabolic enzymes. Alterations in the fate of glucose, beyond flux through glycolysis or glucose oxidation, also contribute to the pathology of heart failure. Of importance, pharmacological targeting of the energy metabolic pathways has emerged as a novel therapeutic approach to improving cardiac efficiency, decreasing the energy deficit and improving cardiac function in the failing heart.

In the mid-1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by a N-acetylglucosamine moiety ( O-GlcNAc) via an O-linkage overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex, branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery, O-GlcNAcylation has been shown to contribute to numerous cellular functions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases, such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.

Heart failure (HF) is a multifactorial syndrome that remains a leading cause of worldwide morbidity. Despite its high prevalence, only half of patients with HF respond to guideline-directed medical management, prompting therapeutic efforts to confront the molecular underpinnings of its heterogeneity. In the current study, we examined epigenetics as a yet unexplored source of heterogeneity among patients with end-stage HF. Specifically, a multicohort-based study was designed to quantify cardiac genome-wide cytosine-p-guanine (CpG) methylation of cardiac biopsies from male patients undergoing left ventricular assist device (LVAD) implantation. In both pilot (n = 11) and testing (n = 31) cohorts, unsupervised multidimensional scaling of genome-wide myocardial DNA methylation exhibited a bimodal distribution of CpG methylation found largely to occur in the promoter regions of metabolic genes. Among the available patient attributes, only categorical self-identified patient race could delineate this methylation signature, with African American (AA) and Caucasian American (CA) samples clustering separately. Because race is a social construct, and thus a poor proxy of human physiology, extensive review of medical records was conducted, but ultimately failed to identify covariates of race at the time of LVAD surgery. By contrast, retrospective analysis exposed a higher all-cause mortality among AA (56.3%) relative to CA (16.7%) patients at 2 yr following LVAD placement (P = 0.03). Geocoding-based approximation of patient demographics uncovered disparities in income levels among AA relative to CA patients. Although additional studies are needed, the current analysis implicates cardiac DNA methylation as a previously unrecognized indicator of socioeconomic disparity in human heart failure outcomes. NEW & NOTEWORTHY A bimodal signature of cardiac DNA methylation in heart failure corresponds with racial differences in all-cause mortality following mechanical circulatory support. Racial differences in promoter methylation disproportionately affect metabolic signaling pathways. Socioeconomic factors are associated with racial differences in the cardiac methylome among men with end-stage heart failure. Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/racial-socioeconomic-determinants-of-the-cardiac-epigenome/.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear whether these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy, we generated transgenic mice with inducible cardiomyocyte-specific expression of the GLUT4. We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in nondiabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest that reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

Evidence suggests that mitochondrial network integrity is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial remodeling, the effect of mitochondrial-targeted antioxidants, such as mitoquinone (MitoQ), on the mitochondrial network in a model of HF (e.g., pressure overload) has not been demonstrated. Furthermore, the mechanism of this regulation is not completely understood with an emerging role for posttranscriptional regulation via long noncoding RNAs (lncRNAs). We hypothesized that MitoQ preserves mitochondrial fusion proteins (i.e., mitofusin), likely through redox-sensitive lncRNAs, leading to improved mitochondrial network integrity in failing hearts. To test this hypothesis, 8-wk-old C57BL/6J mice were subjected to ascending aortic constriction (AAC), which caused substantial left ventricular (LV) chamber remodeling and remarkable contractile dysfunction in 1 wk. Transmission electron microscopy and immunostaining revealed defective intermitochondrial and mitochondrial-sarcoplasmic reticulum ultrastructure in AAC mice compared with sham-operated animals, which was accompanied by elevated oxidative stress and suppressed mitofusin (i.e., Mfn1 and Mfn2) expression. MitoQ (1.36 mg·day−1·mouse−1, 7 consecutive days) significantly ameliorated LV dysfunction, attenuated Mfn2 downregulation, improved interorganellar contact, and increased metabolism-related gene expression. Moreover, our data revealed that MitoQ alleviated the dysregulation of an Mfn2-associated lncRNA (i.e., Plscr4). In summary, the present study supports a unique mechanism by which MitoQ improves myocardial intermitochondrial and mitochondrial-sarcoplasmic reticulum (SR) ultrastructural remodeling in HF by maintaining Mfn2 expression via regulation by an lncRNA. These findings underscore the important role of lncRNAs in the pathogenesis of HF and the potential of targeting them for effective HF treatment. NEW & NOTEWORTHY We have shown that MitoQ improves cardiac mitochondrial network integrity and mitochondrial-SR alignment in a pressure-overload mouse heart-failure model. This may be occurring partly through preventing the dysregulation of a redox-sensitive lncRNA-microRNA pair (i.e., Plscr4-miR-214) that results in an increase in mitofusin-2 expression.

Background Epigenetic mechanisms are critical for hippocampus-dependent memory formation. Building on previous studies that implicate the N-lysine methyltransferase SETD6 in the activation of nuclear factor-κB RELA (also known as transcription factor p65) as an epigenetic recruiter, we hypothesized that SETD6 is a key player in the epigenetic control of long-term memory. Methods Using a series of molecular, biochemical, imaging, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA–mediated knockdown of Setd6 in the rat dorsal hippocampus during memory consolidation. Results Our findings demonstrate that SETD6 is necessary for memory-related nuclear factor-κB RELA methylation at lysine 310 and associated increases in H3K9me2 (histone H3 lysine 9 dimethylation) in the dorsal hippocampus and that SETD6 knockdown interferes with memory consolidation, alters gene expression patterns, and disrupts spine morphology. Conclusions Together, these findings suggest that SETD6 plays a critical role in memory formation and may act as an upstream initiator of H3K9me2 changes in the hippocampus during memory consolidation.

Corresponding Author

African Americans (AA) face a greater burden of cardiovascular diseases, including ischemic cardiomyopathy (ICM), compared to Caucasian Americans (CA). Our recent work supports that epigenetics is likely a unifying mechanism between health disparities of socioeconomic inequalities and heart failure severity; whereby environmental factors are incorporated as stable DNA modifications to confer susceptibility to heart failure. We hypothesized that ICM possesses racially distinct methylome signatures. Cardiac biopsies from male age- and disease-matched AA (n = 14) and CA (n = 15) heart failure patients with ICM and non-ICM (NICM) were subjected to expression analysis using RNA-seq ( P < 0.05, ±1.5-fold) and differential methylation analysis using Illumina EPIC array ( P < 0.05, ±5%) using generalized linear models with a focus on changes corresponding to ICM and racial differences. Comparing ICM vs NICM, both AA and CA patients showed racially distinct differentially expressed genes (594/366 within AA; 439/487 within CA; up/down respectively). Similarly, pathway analysis using the NIH’s BioPlanet database found a significant change ( Q < 0.1) in AA for adipogenesis (25/133 overlap) and in CA for complement cascade (18/77 overlap). In contrast, racially distinct DNA methylation changes were more prominent for CA (29703/22703; up/down) compared to AA (2904/2897; up/down). Upon combining both datasets, known heart failure marker NPPA , which encodes Atrial Natriuretic Peptide, was found to be downregulated in ICM compared to NICM only for AA ( Q = 0, 18.9-fold change), corresponding to hypermethylation of 6 CpG sites ( P < 0.05, 6.4 - 11.3%). NPPA inhibits cardiac hypertrophy, fibrosis, and decreases inflammation. It is also used as a diagnostic biomarker in ICM. But recent trials found that AA have lower levels of NPPA in blood compared to CA, even in healthy condition. This patient cohort also indicated a racially diverse pattern of NPPA expression and methylation. Given its role in adipogenesis, NPPA may also explain the pathway enrichment specific to AA-ICM patients. Our data indicate that NPPA may connect the socioeconomic effects through DNA methylation with clinical diversity of ICM. It also underscores the need for a better prognostic biomarker for ICM.

Heart disease is the number one cause of death in developed countries. Metabolic diseases influence the severity of heart disease linked to risk factors which are thought to alter epigenetic mechanisms. Pyruvate dehydrogenase (PDH) kinases (PDK), which phosphorylate and reduce the activity of PDH the nexus of glucose oxidation and fatty acid oxidation are sensitive to metabolic status. Four isozymes of PDK (PDK1-4) exist with PDK2 and PDK4 as the major regulators in cardiac tissue. Owing to the role of PDH in regulating pyruvate to acetyl-CoA, we hypothesized that PDK inhibition may regulate protein acetylation through increasing acetyl-CoA because of PDH activation leading to post-translational modifications both directly to proteins in metabolic pathways as well as to histones associated with the genes encoding them. To test this, we utilized PDK2 germline knockout mice (P2KO), PDK4 germline knockout mice (P4KO), and PDK2 and PDK4 double knockout (DKO) mice for molecular analysis. Our results identify a novel increase in whole-cell protein acetylation in P2KO left ventricle tissue (LV). However, protein acetylation in P4KO LV was not changed compared to WT mice. The most robust protein acetylation was observed in the DKO LV. Furthermore, when we explored sub-cellular distribution of protein acetylation, the greatest increases were found on cytoplasmic proteins, with moderate changes in mitochondrial proteins. We also found PDK2 ablation induces histone H3 acetylation, which may also lead to changes in gene expression. Moreover, this protein acetylation in P2KO and DKO was not seen in other tissues examined (e.g., liver, skeletal muscle). The hyperacetylation is robust in male LV compared to female LV. In conclusion, our study supports a novel protein acetylation mechanism that is both tissue and PDK isozyme specific highlighting the role of PDK2, which is relatively understudied compared to PDK4 in heart disease. Further study will evaluate if the hyperacetylation has a beneficial effect in various heart disease settings as well as identify the impact on changes in gene expression. This study supports PDK isozyme-specific inhibition strategies will be required to develop therapeutic targets of cardiovascular disease with metabolic inflexibility.

Heart failure (HF) is a leading cause of morbidity and mortality in the United States and worldwide. As a multifactorial syndrome with unpredictable clinical outcomes, identifying the common molecular underpinnings that drive HF pathogenesis remains a major focus of investigation. Disruption of cardiac gene expression has been shown to mediate a common final cascade of pathological hallmarks wherein the heart reactivates numerous developmental pathways. Although the central regulatory mechanisms that drive this cardiac transcriptional reprogramming remain unknown, epigenetic contributions are likely. In the current study, we examined whether the epigenome, specifically DNA methylation, is reprogrammed in HF to potentiate a pathological shift in cardiac gene expression. To accomplish this, we used paired-end whole genome bisulfite sequencing and next-generation RNA sequencing of left ventricle tissue obtained from seven patients with end-stage HF and three nonfailing donor hearts. We found that differential methylation was localized to promoter-associated cytosine-phosphate-guanine islands, which are established regulatory regions of downstream genes. Hypermethylated promoters were associated with genes involved in oxidative metabolism, whereas promoter hypomethylation enriched glycolytic pathways. Overexpression of plasmid-derived DNA methyltransferase 3A in vitro was sufficient to lower the expression of numerous oxidative metabolic genes in H9c2 rat cardiomyoblasts, further supporting the importance of epigenetic factors in the regulation of cardiac metabolism. Last, we identified binding-site competition via hypermethylation of the nuclear respiratory factor 1 (NRF1) motif, an established upstream regulator of mitochondrial biogenesis. These preliminary observations are the first to uncover an etiology-independent shift in cardiac DNA methylation that corresponds with altered metabolic gene expression in HF. NEW & NOTEWORTHY The failing heart undergoes profound metabolic changes because of alterations in cardiac gene expression, reactivating glycolytic genes and suppressing oxidative metabolic genes. In the current study, we discover that alterations to cardiac DNA methylation encode this fetal-like metabolic gene reprogramming. We also identify novel epigenetic interference of nuclear respiratory factor 1 via hypermethylation of its downstream promoter targets, further supporting a novel contribution of DNA methylation in the metabolic remodeling of heart failure.

The endoplasmic reticulum/sarcoplasmic reticulum Ca2+sensor stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca2+entry, is expressed in cardiomyocytes and has been implicated in regulating multiple cardiac processes, including hypertrophic signaling. Interestingly, cardiomyocyte-restricted deletion of STIM1 (crSTIM1-KO) results in age-dependent endoplasmic reticulum stress, altered mitochondrial morphology, and dilated cardiomyopathy in mice. Here, we tested the hypothesis that STIM1 deficiency may also impact cardiac metabolism. Hearts isolated from 20-wk-oldcrSTIM1-KO mice exhibited a significant reduction in both oxidative and nonoxidative glucose utilization. Consistent with the reduction in glucose utilization, expression of glucose transporter 4 and AMP-activated protein kinase phosphorylation were all reduced, whereas pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase phosphorylation were increased, incrSTIM1-KO hearts. Despite similar rates of fatty acid oxidation in control andcrSTIM1-KO hearts ex vivo,crSTIM1-KO hearts contained increased lipid/triglyceride content as well as increased fatty acid-binding protein 4, fatty acid synthase, acyl-CoA thioesterase 1, hormone-sensitive lipase, and adipose triglyceride lipase expression compared with control hearts, suggestive of a possible imbalance between fatty acid uptake and oxidation. Insulin-mediated alterations in AKT phosphorylation were observed incrSTIM1-KO hearts, consistent with cardiac insulin resistance. Interestingly, we observed abnormal mitochondria and increased lipid accumulation in 12-wkcrSTIM1-KO hearts, suggesting that these changes may initiate the subsequent metabolic dysfunction. These results demonstrate, for the first time, that cardiomyocyte STIM1 may play a key role in regulating cardiac metabolism.NEW & NOTEWORTHY Little is known of the physiological role of stromal interaction molecule 1 (STIM1) in the heart. Here, we demonstrate, for the first time, that hearts lacking cardiomyocyte STIM1 exhibit dysregulation of both cardiac glucose and lipid metabolism. Consequently, these results suggest a potentially novel role for STIM1 in regulating cardiac metabolism.

Pancreatic β-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram β-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes mellitus (GDM). We recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where β-cell-specific PRLR knockout (βPRLRKO) mice exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within β-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either βPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified forkhead box protein M1 and polycomb repressor complex 2 subunits, Suz12 and enhancer of zeste homolog 2 (Ezh2), as novel candidate regulators of PRLR-dependent β-cell adaptation. Gene ontology term pathway enrichment revealed both established and novel PRLR signaling target genes that together promote a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for β-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat diet feeding. Collectively, the current study expands our understanding of which transcriptional regulators and networks mediate gene expression required for islet adaptation during pregnancy. The current work also supports the presence of pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.

Ischemic cardiomyopathy (ICM) is the clinical endpoint of coronary heart disease and a leading cause of heart failure. Despite growing demands to develop personalized approaches to treat ICM, progress is limited by inadequate knowledge of its pathogenesis. Since epigenetics has been implicated in the development of other chronic diseases, the current study was designed to determine whether transcriptional and/or epigenetic changes are sufficient to distinguish ICM from other etiologies of heart failure. Specifically, we hypothesize that genome-wide DNA methylation encodes transcriptional reprogramming in ICM. RNA-sequencing analysis was performed on human ischemic left ventricular tissue obtained from patients with end-stage heart failure, which enriched known targets of the polycomb methyltransferase EZH2 compared to non-ischemic hearts. Combined RNA sequencing and genome-wide DNA methylation analysis revealed a robust gene expression pattern consistent with suppression of oxidative metabolism, induced anaerobic glycolysis, and altered cellular remodeling. Lastly, KLF15 was identified as a putative upstream regulator of metabolic gene expression that was itself regulated by EZH2 in a SET domain-dependent manner. Our observations therefore define a novel role of DNA methylation in the metabolic reprogramming of ICM. Furthermore, we identify EZH2 as an epigenetic regulator of KLF15 along with DNA hypermethylation, and we propose a novel mechanism through which coronary heart disease reprograms the expression of both intermediate enzymes and upstream regulators of cardiac metabolism such as KLF15.

Obesity and diabetes cumulatively create a distinct systemic metabolic pathophysiological syndrome that predisposes patients to several diseases including breast cancer. Moreover, diabetic and obese women with breast cancer show a significant increase in mortality compared to non-obese and/or non-diabetic women. We hypothesized that these metabolic conditions incite an aggressive tumor phenotype by way of impacting tumor cell-autonomous and tumor cell non-autonomous events. In this study, we established a type 2 diabetic mouse model of triple-negative mammary carcinoma and investigated the effect of a glucose lowering therapy, metformin, on the overall tumor characteristics and immune/metabolic microenvironment. Diabetic mice exhibited larger mammary tumors that had increased adiposity with high levels of O-GlcNAc protein post-translational modification. These tumors also presented with a distinct stromal profile characterized by altered collagen architecture, increased infiltration by tumor-permissive M2 macrophages, and early metastatic seeding compared to non-diabetic/lean mice. Metformin treatment of the diabetic/obese mice effectively normalized glucose levels, reconfigured the mammary tumor milieu, and decreased metastatic seeding. Our results highlight the impact of two metabolic complications of obesity and diabetes on tumor cell attributes and showcase metformin's ability to revert tumor cell and stromal changes induced by an obese and diabetic host environment.

Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied.

Background Glycemic control is a strong predictor of long-term cardiovascular risk in patients with diabetes mellitus, and poor glycemic control influences long-term risk of cardiovascular disease even decades after optimal medical management. This phenomenon, termed glycemic memory, has been proposed to occur due to stable programs of cardiac and endothelial cell gene expression. This transcriptional remodeling has been shown to occur in the vascular endothelium through a yet undefined mechanism of cellular reprogramming. Methods In the current study, we quantified genome-wide DNA methylation of cultured human endothelial aortic cells (HAECs) via reduced-representation bisulfite sequencing (RRBS) following exposure to diabetic (250 mg/dL), pre-diabetic (125 mg/dL), or euglycemic (100 mg/dL) glucose concentrations for 72 h (n = 2). Results We discovered glucose-dependent methylation of genomic regions (DMRs) encompassing 2199 genes, with a disproportionate number found among genes associated with angiogenesis and nitric oxide (NO) signaling-related pathways. Multi-omics analysis revealed differential methylation and gene expression of VEGF (↑5.6% DMR, ↑3.6-fold expression), and NOS3 (↓20.3% DMR, ↓1.6-fold expression), nodal regulators of angiogenesis and NO signaling, respectively. Conclusion In the current exploratory study, we examine glucose-dependent and dose-responsive alterations in endothelial DNA methylation to examine a putative epigenetic mechanism underlying diabetic vasculopathy. Specifically, we uncover the disproportionate glucose-dependent methylation and gene expression of VEGF and NO signaling cascades, a physiologic imbalance known to cause endothelial dysfunction in diabetes. We therefore hypothesize that epigenetic mechanisms encode a glycemic memory within endothelial cells.

Background Perturbations in myocardial substrate utilization have been proposed to contribute to the pathogenesis of cardiac dysfunction in diabetic subjects. The failing heart in nondiabetics tends to decrease reliance on fatty acid and glucose oxidation, and increases reliance on ketone body oxidation. In contrast, little is known regarding the mechanisms mediating this shift among all 3 substrates in diabetes mellitus. Therefore, we tested the hypothesis that changes in myocardial glucose utilization directly influence ketone body catabolism. Methods and Results We examined ventricular‐cardiac tissue from the following murine models: (1) streptozotocin‐induced type 1 diabetes mellitus; (2) high‐fat‐diet–induced glucose intolerance; and transgenic inducible cardiac‐restricted expression of (3) glucose transporter 4 (transgenic inducible cardiac restricted expression of glucose transporter 4); or (4) dominant negative O ‐GlcNAcase. Elevated blood glucose (type 1 diabetes mellitus and high‐fat diet mice) was associated with reduced cardiac expression of β‐hydroxybutyrate‐dehydrogenase and succinyl‐CoA:3‐oxoacid CoA transferase. Increased myocardial β‐hydroxybutyrate levels were also observed in type 1 diabetes mellitus mice, suggesting a mismatch between ketone body availability and utilization. Increased cellular glucose delivery in transgenic inducible cardiac restricted expression of glucose transporter 4 mice attenuated cardiac expression of both Bdh1 and Oxct1 and reduced rates of myocardial BDH1 activity and β‐hydroxybutyrate oxidation. Moreover, elevated cardiac protein O ‐GlcNAcylation (a glucose‐derived posttranslational modification) by dominant negative O ‐GlcNAcase suppressed β‐hydroxybutyrate dehydrogenase expression. Consistent with the mouse models, transcriptomic analysis confirmed suppression of BDH1 and OXCT1 in patients with type 2 diabetes mellitus and heart failure compared with nondiabetic patients. Conclusions Our results provide evidence that increased glucose leads to suppression of cardiac ketolytic capacity through multiple mechanisms and identifies a potential crosstalk between glucose and ketone body metabolism in the diabetic myocardium.

In the mid-1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by a N-acetylglucosamine moiety (O-GlcNAc) via an O-linkage overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex, branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery, O-GlcNAcylation has been shown to contribute to numerous cellular functions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases, such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.

African Americans (AA) have an elevated risk for cardiovascular diseases compared to Caucasian Americans (CA), including heart failure (HF). Type 2 diabetes (T2D) is a major risk factor for HF that also disproportionately affects AA. These health disparities and others reduce life expectancy ~3.5 y for AA compared to CA. While prior studies have explored the connection between diabetes and heart failure, the current understanding of HF pathogenesis is based almost exclusively from studies of CA, whereas those considering race have been either epidemiologic or narrow in focus. The purpose of this study was to examine things from a different angle through the use of genome-wide RNA-sequencing to uncover how diabetes differentially or similarly affects end-stage heart failure in AA vs CA. To accomplish this, human biopsy samples were obtained from 32 age and diabetes status (T2D or non-diabetic (ND)) matched male patients undergoing left ventricle assist device surgeries (n = 8: CA-ND, CA-T2D, AA-ND, AA-T2D). Differential expression analysis was then performed using generalized linear modeling to control for clinical covariates including hypertension and coronary artery disease. Results of T2D vs ND in AA patients showed a greater number of differentially expressed genes (DEGs, P < 0.05

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear if these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy we generated transgenic mice with inducible cardiomyocyte-specific expression of the glucose transporter (GLUT4). We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in non-diabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset, exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.