Role of O-Linked N-Acetylglucosamine Protein Modification in Cellular (Patho)Physiology.

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.

Metabolic adaptation to tyrosine kinase inhibition in leukemia stem cells.

Tyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the 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, the tricarboxylic acid 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 use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the 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.

Assessing the impact of boldine on the gastrocnemius using multiomics profiling at 7 and 28 days post-complete spinal cord injury in young male mice.

Spinal cord injury (SCI) results in rapid muscle loss. Exogenous molecular interventions to slow muscle atrophy after SCI have been relatively ineffective and require the search for novel therapeutic targets. Connexin hemichannels (CxHCs) allow nonselective 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 preclinical results in slowing atrophy during sepsis and restoring muscle function in dysferlinopathy. We administered 50 mg/kg/day of boldine to spinal cord transected mice beginning 3 days post-injury. Tissue was collected 7 and 28 days post-SCI and the gastrocnemius was used for multiomics profiling. Boldine did not prevent body or muscle mass loss but attenuated SCI-induced changes in the abundance of the amino acids proline, phenylalanine, leucine and isoleucine, as well as glucose, 7 days post-SCI. SCI resulted in the differential expression of ∼7,700 and ∼2,000 genes at 7 and 28 days, respectively, compared with Sham controls. Pathway enrichment of these genes highlighted ribosome biogenesis at 7 days and translation and oxidative phosphorylation at both timepoints. Boldine altered the expression of ∼150 genes at 7 days and ∼110 genes at 28 days post-SCI. Pathway enrichment of these genes indicated a potential role for boldine in suppressing protein ubiquitination and degradation at the 7-day timepoint. Methylation analyses showed minimal differences between groups. Taken together, boldine is not an efficacious therapy to preserve body and muscle mass after complete SCI, though it attenuated some SCI-induced changes across the metabolome and transcriptome.NEW & NOTEWORTHY This is the first study to describe the multiome of skeletal muscle paralyzed by a spinal cord injury (SCI) in mice across the acute and subacute timeframe after injury. We show large-scale changes in the metabolome and transcriptome at 7 days post-injury compared with 28 days. Furthermore, we show that the alkaloid boldine was able to prevent SCI-induced changes in muscle glucose and free amino acid levels at 7 days, but not 28 days, after SCI.

Best practices in assessing cardiac protein O-GlcNAcylation by immunoblot.

The modification of serine and threonine amino acids of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates the activity, stability, function, and subcellular localization of proteins. Dysregulation of O-GlcNAc homeostasis is well established as a hallmark of various cardiac diseases, including cardiac hypertrophy, heart failure, complications associated with diabetes, and responses to acute injuries such as oxidative stress and ischemia-reperfusion. Given the limited availability of site-specific O-GlcNAc antibodies, studies of changes in O-GlcNAcylation in the heart frequently use pan-O-GlcNAc antibodies for semiquantitative evaluation of overall O-GlcNAc levels. However, there is a high degree of variability in many published cardiac O-GlcNAc blots. For example, many blots often have regions that lack O-GlcNAc positive staining of proteins either below 50 or above 100 kDa. In some O-GlcNAc blots, only a few protein bands are detected, while in others, intense bands around 75 kDa dominate the gel due to nonspecific IgM band staining, making it difficult to visualize less intense bands. Therefore, the goal of this study was to develop a modifiable protocol that optimizes O-GlcNAc positive banding of proteins in cardiac tissue extracts. We showed that O-GlcNAc blots using CTD110.6 antibody of proteins ranging from <30 to ∼450 kDa could be obtained while also limiting nonspecific staining. We also show that some myofilament proteins are recognized by the CTD110.6 antibody. Therefore, by protocol optimization using the widely available CTD110.6 antibody, we found that it is possible to obtain pan-O-GlcNAc blots of cardiac tissue, which minimizes common limitations associated with this technique.NEW & NOTEWORTHY The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is recognized as mediating cardiac pathophysiology. However, there is considerable variability in the quality of O-GlcNAc immunoblots used to evaluate changes in cardiac O-GlcNAc levels. Here we show that with relatively minor changes to a commonly used protocol it is possible to minimize the intensity of nonspecific bands while also reproducibly generating O-GlcNAc immunoblots covering a range of molecular weights from <30 to ∼450 kDa.

Cardiac Energy Metabolism in Heart Failure.

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/O2 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.

Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease.

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.

Guidelines on models of diabetic heart disease.

Diabetes is a major risk factor for cardiovascular diseases, including diabetic cardiomyopathy, atherosclerosis, myocardial infarction, and heart failure. As cardiovascular disease represents the number one cause of death in people with diabetes, there has been a major emphasis on understanding the mechanisms by which diabetes promotes cardiovascular disease, and how antidiabetic therapies impact diabetic heart disease. With a wide array of models to study diabetes (both type 1 and type 2), the field has made major progress in answering these questions. However, each model has its own inherent limitations. Therefore, the purpose of this guidelines document is to provide the field with information on which aspects of cardiovascular disease in the human diabetic population are most accurately reproduced by the available models. This review aims to emphasize the advantages and disadvantages of each model, and to highlight the practical challenges and technical considerations involved. We will review the preclinical animal models of diabetes (based on their method of induction), appraise models of diabetes-related atherosclerosis and heart failure, and discuss in vitro models of diabetic heart disease. These guidelines will allow researchers to select the appropriate model of diabetic heart disease, depending on the specific research question being addressed.

Vitamin A preserves cardiac energetic gene expression in a murine model of diet-induced obesity.

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.NEW & NOTEWORTHY The relationship between vitamin-A status and the pathogenesis of diabetic cardiomyopathy has not been studied in detail. We assessed cardiac mitochondrial respiratory capacity, contractile function, and gene expression by RNA sequencing in a murine model of combined vitamin-A deficiency and diet-induced obesity. Our study identifies a role for vitamin A in preserving cardiac energetic gene expression that might attenuate subsequent development of mitochondrial and contractile dysfunction in diet-induced obesity.

The human aortic endothelium undergoes dose-dependent DNA methylation in response to transient hyperglycemia.

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.

Branched chain amino acids selectively promote cardiac growth at the end of the awake period.

Essentially all biological processes fluctuate over the course of the day, manifesting as time-of-day-dependent variations with regards to the way in which organ systems respond to normal behaviors. For example, basic, translational, and epidemiologic studies indicate that temporal partitioning of metabolic processes governs the fate of dietary nutrients, in a manner in which concentrating caloric intake towards the end of the day is detrimental to both cardiometabolic and cardiovascular parameters. Despite appreciation that branched chain amino acids impact risk for obesity, diabetes mellitus, and heart failure, it is currently unknown whether the time-of-day at which dietary BCAAs are consumed influence cardiometabolic/cardiovascular outcomes. Here, we report that feeding mice a BCAA-enriched meal at the end of the active period (i.e., last 4 h of the dark phase) rapidly increases cardiac protein synthesis and mass, as well as cardiomyocyte size; consumption of the same meal at the beginning of the active period (i.e., first 4 h of the dark phase) is without effect. This was associated with a greater BCAA-induced activation of mTOR signaling in the heart at the end of the active period; pharmacological inhibition of mTOR (through rapamycin) blocked BCAA-induced augmentation of cardiac mass and cardiomyocyte size. Moreover, genetic disruption of the cardiomyocyte circadian clock abolished time-of-day-dependent fluctuations in BCAA-responsiveness. Finally, we report that repetitive consumption of BCAA-enriched meals at the end of the active period accelerated adverse cardiac remodeling and contractile dysfunction in mice subjected to transverse aortic constriction. Thus, our data demonstrate that the timing of BCAA consumption has significant implications for cardiac health and disease.

Racial and socioeconomic disparity associates with differences in cardiac DNA methylation among men with end-stage heart failure.

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.

MitoQ regulates redox-related noncoding RNAs to preserve mitochondrial network integrity in pressure-overload heart failure.

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.

Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induce Post-Translational Modifications of AKAP121, DRP1, and OPA1 That Promote Mitochondrial Fission.

RATIONALE: Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. OBJECTIVE: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. METHODS AND RESULTS: Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. CONCLUSIONS: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy.

Genome-wide DNA methylation encodes cardiac transcriptional reprogramming in human ischemic heart failure.

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.

Novel role of the ER/SR Ca2+ sensor STIM1 in the regulation of cardiac metabolism.

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-old crSTIM1-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, in crSTIM1-KO hearts. Despite similar rates of fatty acid oxidation in control and crSTIM1-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 in crSTIM1-KO hearts, consistent with cardiac insulin resistance. Interestingly, we observed abnormal mitochondria and increased lipid accumulation in 12-wk crSTIM1-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.

DNA methylation reprograms cardiac metabolic gene expression in end-stage human heart failure.

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.

Gene expression analysis to identify mechanisms underlying heart failure susceptibility in mice and humans.

Genetic factors are known to modulate cardiac susceptibility to ventricular hypertrophy and failure. To determine how strain influences the transcriptional response to pressure overload-induced heart failure (HF) and which of these changes accurately reflect the human disease, we analyzed the myocardial transcriptional profile of mouse strains with high (C57BL/6J) and low (129S1/SvImJ) susceptibility for HF development, which we compared to that of human failing hearts. Following transverse aortic constriction (TAC), C57BL/6J mice developed overt HF while 129S1/SvImJ did not. Despite a milder aortic constriction, impairment of ejection fraction and ventricular remodeling (dilation, fibrosis) was more pronounced in C57BL/6J mice. Similarly, changes in myocardial gene expression were more robust in C57BL/6J (461 genes) compared to 129S1/SvImJ mice (71 genes). When comparing these patterns to human dilated cardiomyopathy (1344 genes), C57BL/6J mice tightly grouped to human hearts. Overlay and bioinformatic analysis of the transcriptional profiles of C57BL/6J mice and human failing hearts identified six co-regulated genes (POSTN, CTGF, FN1, LOX, NOX4, TGFB2) with established link to HF development. Pathway enrichment analysis identified angiotensin and IGF-1 signaling as most enriched putative upstream regulator and pathway, respectively, shared between TAC-induced HF in C57BL/6J mice and in human failing hearts. TAC-induced heart failure in C57BL/6J mice more closely reflects the gene expression pattern of human dilated cardiomyopathy compared to 129S1/SvImJ mice. Unbiased as well as targeted gene expression and pathway analyses identified periostin, angiotensin signaling, and IGF-1 signaling as potential causes of increased HF susceptibility in C57BL/6J mice and as potentially useful drug targets for HF treatment.

Genetic disruption of the cardiomyocyte circadian clock differentially influences insulin-mediated processes in the heart.

Cardiovascular physiology exhibits time-of-day-dependent oscillations, which are mediated by both extrinsic (e.g., environment/behavior) and intrinsic (e.g., circadian clock) factors. Disruption of circadian rhythms negatively affects multiple cardiometabolic parameters. Recent studies suggest that the cardiomyocyte circadian clock directly modulates responsiveness of the heart to metabolic stimuli (e.g., fatty acids) and stresses (e.g., ischemia/reperfusion). The aim of this study was to determine whether genetic disruption of the cardiomyocyte circadian clock impacts insulin-regulated pathways in the heart. Genetic disruption of the circadian clock in cardiomyocyte-specific Bmal1 knockout (CBK) and cardiomyocyte-specific Clock mutant (CCM) mice altered expression (gene and protein) of multiple insulin signaling components in the heart, including p85α and Akt. Both baseline and insulin-mediated Akt activation was augmented in CBK and CCM hearts (relative to littermate controls). However, insulin-mediated glucose utilization (both oxidative and non-oxidative) and AS160 phosphorylation were attenuated in CBK hearts, potentially secondary to decreased Inhibitor-1. Consistent with increased Akt activation in CBK hearts, mTOR signaling was persistently increased, which was associated with attenuation of autophagy, augmented rates of protein synthesis, and hypertrophy. Importantly, pharmacological inhibition of mTOR (rapamycin; 10days) normalized cardiac size in CBK mice. These data suggest that disruption of cardiomyocyte circadian clock differentially influences insulin-regulated processes, and provide new insights into potential pathologic mediators following circadian disruption.