PERK/ATF4-dependent expression of the stress response protein REDD1 promotes proinflammatory cytokine expression in the heart of obese mice.

Endoplasmic reticulum (ER) stress and inflammation are hallmarks of myocardial impairment. Here, we investigated the role of the stress response protein regulated in development and DNA damage 1 (REDD1) as a molecular link between ER stress and inflammation in cardiomyocytes. In mice fed a high-fat high-sucrose (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 wk, REDD1 expression in the heart was increased in coordination with markers of ER stress and inflammation. In human AC16 cardiomyocytes exposed to either hyperglycemic conditions or the saturated fatty acid palmitate, REDD1 expression was increased coincident with ER stress and upregulated expression of the proinflammatory cytokines IL-1ß, IL-6, and TNFα. In cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions, pharmacological inhibition of the ER kinase protein kinase RNA-like endoplasmic reticulum kinase (PERK) or knockdown of the transcription factor ATF4 prevented the increase in REDD1 expression. REDD1 deletion reduced proinflammatory cytokine expression in both cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions and in the hearts of obese mice. Overall, the findings support a model wherein HFHS diet contributes to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.NEW & NOTEWORTHY Interplay between endoplasmic reticulum stress and inflammation contributes to cardiovascular disease progression. The studies here identify the stress response protein known as REDD1 as a missing molecular link that connects the development of endoplasmic reticulum stress with increased production of proinflammatory cytokines in the hearts of obese mice.

Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, and Isolated Tissue Mitochondria Using the Seahorse XF Analyzer.

Energy homeostasis is critical for cellular function. Significant increases in energy demand or reduced energy supply, however, often result in cellular dysfunction and death. Since mitochondria are the primary cellular energy source, their impairment is often pathogenic. Accordingly, quantitative measurements of cellular and mitochondrial energy utilization and production are crucial for understanding disease development and progression. In the final step of cellular respiration, specifically, oxidative phosphorylation within the mitochondria, oxygen is consumed and drives ATP production. Herein, we provide the complete protocols for measuring oxygen consumption rates and their coupling to ATP production in intact and permeabilized cells, as well as in mitochondria isolated from tissue using the Seahorse XF Extracellular Flux Analyzer (Agilent Technologies).

Histone H3K9 butyrylation is regulated by dietary fat and stress via an Acyl-CoA dehydrogenase short chain-dependent mechanism.

OBJECTIVE: We previously reported that ß-oxidation enzymes are present in the nucleus in close proximity to transcriptionally active promoters. Thus, we hypothesized that the fatty acid intermediate, butyryl-CoA, is the substrate for histone butyrylation and its abundance is regulated by acyl-CoA dehydrogenase short chain (ACADS). The objective of this study was to determine the genomic distribution of H3K9-butyryl (H3K9Bu) and its regulation by dietary fat, stress, and ACADS and its impact on gene expression. METHODS AND RESULTS: Using genome-wide chromatin immunoprecipitation-sequencing (ChIP-Seq), we show that H3K9Bu is abundant at all transcriptionally active promoters, where, paradoxically, it is most enriched in mice fed a fat-free vs high-fat diet. Deletion of fatty acid synthetase (FASN) abolished H3K9Bu in cells maintained in a glucose-rich but not fatty acid-rich medium, signifying that fatty acid synthesis from carbohydrates substitutes for dietary fat as a source of butyryl-CoA. A high-fat diet induced an increase in ACADS expression that accompanied the decrease in H3K9Bu. Conversely, the deletion of ACADS increased H3K9Bu in human cells and mouse hearts and reversed high-fat- and stress-induced reduction in promoter-H3K9Bu, whose abundance coincided with diminished stress-regulated gene expression as revealed by RNA sequencing. In contrast, H3K9-acetyl (H3K9Ac) abundance was minimally impacted by diet. CONCLUSION: Promoter H3K9 butyrylation is a major histone modification that is negatively regulated by high fat and stress in an ACADS-dependent fashion and moderates stress-regulated gene expression.

A peptide of the amino-terminus of GRK2 induces hypertrophy and yet elicits cardioprotection after pressure overload.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 "interactome" that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. Herein, we subjected transgenic mice with cardiac restricted expression of a short, amino terminal fragment of GRK2 (ßARKnt) to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, ßARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, ßARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. Proteomic analysis to identify ßARKnt binding partners that may underlie the improved cardiovascular phenotype uncovered a selective functional interaction of both endogenous GRK2 and ßARKnt with AKT substrate of 160 kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in ßARKnt mice, cardiomyocyte insulin signaling is improved during stress, with a coordinate increase in spare respiratory activity and ATP production without metabolite switching. Surprisingly, these studies also revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male ßARKnt mice at baseline and following cardiac stress. These data suggest that the enhanced AS160-mediated signaling in the ßARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism.

Adiponectin enhances the bioenergetics of cardiac myocytes via an AMPK- and succinate dehydrogenase-dependent mechanism.

Adiponectin is one of the most abundant circulating hormones, which through adenosine monophosphate-activated protein kinase (AMPK), enhances fatty acid and glucose oxidation, and exerts a cardioprotective effect. However, its effects on cellular bioenergetics have not been explored. We have previously reported that 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR, an AMPK activator) enhances mitochondrial respiration through a succinate dehydrogenase (SDH or complex II)-dependent mechanism in cardiac myocytes, leading us to predict that Adiponectin would exert a similar effect via activating AMPK. Our results show that Adiponectin enhances basal mitochondrial oxygen consumption rate (OCR), ATP production, and spare respiratory capacity (SRC), which were all abolished by the knockdown of AMPKγ1, inhibition of SDH complex assembly, via the knockdown of the SDH assembly factor 1 (Sdhaf1), or inhibition of SDH activity. Additionally, Adiponectin alleviated hypoxia-induced reductions in OCR and ATP production, in a Sdhaf1-dependent manner, whereas overexpression of Sdhaf1 confirmed its sufficiency for mediating these effects. Importantly, the levels of holoenzyme SDH under the various conditions correlated with OCR. We also show that the effects of Adiponectin, AMPK, Sdhaf1, as well as, SDH complex assembly all required sirtuin 3 (Sirt3). In conclusion, Adiponectin potentiates mitochondrial bioenergetics via promoting SDH complex assembly in an AMPK-, Sdhaf1-, and Sirt3-dependent fashion in cardiac myocytes.

Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. METHODS: To address this, we performed FoxO1 chromatin immunoprecipitation-deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg-1·mg-1), transverse aortic constriction, or vehicle injection/sham surgery. RESULTS: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation-deep sequencing results were aligned with those of pol II chromatin immunoprecipitation-deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction-operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1- and, in vivo, pressure overload-induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. CONCLUSIONS: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II-regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

Loss of dynamic regulation of G protein-coupled receptor kinase 2 by nitric oxide leads to cardiovascular dysfunction with aging.

Nitric oxide (NO) and S-nitrosothiol (SNO) are considered cardio- and vasoprotective substances. We now understand that one mechanism in which NO/SNOs provide cardiovascular protection is through their direct inhibition of cardiac G protein-coupled receptor (GPCR) kinase 2 (GRK2) activity via S-nitrosylation of GRK2 at cysteine 340 (C340). This maintains GPCR homeostasis, including ß-adrenergic receptors, through curbing receptor GRK2-mediated desensitization. Previously, we have developed a knockin mouse (GRK2-C340S) where endogenous GRK2 is resistant to dynamic S-nitrosylation, which led to increased GRK2 desensitizing activity. This unchecked regulation of cardiac GRK2 activity resulted in significantly more myocardial damage after ischemic injury that was resistant to NO-mediated cardioprotection. Although young adult GRK2-C340S mice show no overt phenotype, we now report that as these mice age, they develop significant cardiovascular dysfunction due to the loss of SNO-mediated GRK2 regulation. This pathological phenotype is apparent as early as 12 mo of age and includes reduced cardiac function, increased cardiac perivascular fibrosis, and maladaptive cardiac hypertrophy, which are common maladies found in patients with cardiovascular disease (CVD). There are also vascular reactivity and aortic abnormalities present in these mice. Therefore, our data demonstrate that a chronic and global increase in GRK2 activity is sufficient to cause cardiovascular remodeling and dysfunction, likely due to GRK2's desensitizing effects in several tissues. Because GRK2 levels have been reported to be elevated in elderly CVD patients, GRK2-C340 mice can give insight into the aged-molecular landscape leading to CVD.NEW & NOTEWORTHY Research on G protein-coupled receptor kinase 2 (GRK2) in the setting of cardiovascular aging is largely unknown despite its strong established functions in cardiovascular physiology and pathophysiology. This study uses a mouse model of chronic GRK2 overactivity to further investigate the consequences of long-term GRK2 on cardiac function and structure. We report for the first time that chronic GRK2 overactivity was able to cause cardiac dysfunction and remodeling independent of surgical intervention, highlighting the importance of GRK activity in aged-related heart disease.

HDAC inhibition improves cardiopulmonary function in a feline model of diastolic dysfunction.

Heart failure with preserved ejection fraction (HFpEF) is a major health problem without effective therapies. This study assessed the effects of histone deacetylase (HDAC) inhibition on cardiopulmonary structure, function, and metabolism in a large mammalian model of pressure overload recapitulating features of diastolic dysfunction common to human HFpEF. Male domestic short-hair felines (n = 31, aged 2 months) underwent a sham procedure (n = 10) or loose aortic banding (n = 21), resulting in slow-progressive pressure overload. Two months after banding, animals were treated daily with suberoylanilide hydroxamic acid (b + SAHA, 10 mg/kg, n = 8), a Food and Drug Administration-approved pan-HDAC inhibitor, or vehicle (b + veh, n = 8) for 2 months. Echocardiography at 4 months after banding revealed that b + SAHA animals had significantly reduced left ventricular hypertrophy (LVH) (P < 0.0001) and left atrium size (P < 0.0001) versus b + veh animals. Left ventricular (LV) end-diastolic pressure and mean pulmonary arterial pressure were significantly reduced in b + SAHA (P < 0.01) versus b + veh. SAHA increased myofibril relaxation ex vivo, which correlated with in vivo improvements of LV relaxation. Furthermore, SAHA treatment preserved lung structure, compliance, blood oxygenation, and reduced perivascular fluid cuffs around extra-alveolar vessels, suggesting attenuated alveolar capillary stress failure. Acetylation proteomics revealed that SAHA altered lysine acetylation of mitochondrial metabolic enzymes. These results suggest that acetylation defects in hypertrophic stress can be reversed by HDAC inhibitors, with implications for improving cardiac structure and function in patients.

Oxoglutarate dehydrogenase and acetyl-CoA acyltransferase 2 selectively associate with H2A.Z-occupied promoters and are required for histone modifications.

Histone H2A.Z plays an essential role in regulating transcriptional rates and memory. Interestingly, H2A.Z-bound nucleosomes are located in both transcriptionally active and inactive promotors, with no clear understanding of the mechanisms via which it differentially regulates transcription. We hypothesized that its functions are mediated through recruitment of regulatory proteins to promoters. Using rapid chromatin immunoprecipitation-mass spectrometry, we uncovered the association of H2A.Z-bound chromatin with the metabolic enzymes, oxoglutarate dehydrogenase (OGDH) and acetyl-CoA acyltransferase 2 (ACAA2). Recombinant green florescence fusion proteins, combined with mutations of predicted nuclear localization signals, confirmed their nuclear localization and chromatin binding. Conclusively, chromatin immunoprecipitation-deep sequencing, confirmed the predominant association of OGDH and ACAA2 with H2A.Z-occupied transcription start sites and enhancers, the former of which we confirmed is conserved in both mouse and human tissue. Furthermore, H2A.Z-deficient human HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, accompanied with reduced posttranslational histone modifications, including acetylation and succinylation. Specifically, knockdown of OGDH diminished H4 succinylation. Thus, the data reveal that select metabolic enzymes are assembled at active, H2A.Z-occupied, promoters, for potential site-directed production of metabolic intermediates that are required for histone modifications.

G protein-coupled receptor kinases as therapeutic targets in the heart.

G protein-coupled receptors (GPCRs) are critical cellular sensors that mediate numerous physiological processes. In the heart, multiple GPCRs are expressed on various cell types, where they coordinate to regulate cardiac function by modulating critical processes such as contractility and blood flow. Under pathological settings, these receptors undergo aberrant changes in expression levels, localization and capacity to couple to downstream signalling pathways. Conventional therapies for heart failure work by targeting GPCRs, such as ß-adrenergic receptor and angiotensin II receptor antagonists. Although these treatments have improved patient survival, heart failure remains one of the leading causes of mortality worldwide. GPCR kinases (GRKs) are responsible for GPCR phosphorylation and, therefore, desensitization and downregulation of GPCRs. In this Review, we discuss the GPCR signalling pathways and the GRKs involved in the pathophysiology of heart disease. Given that increased expression and activity of GRK2 and GRK5 contribute to the loss of contractile reserve in the stressed and failing heart, inhibition of overactive GRKs has been proposed as a novel therapeutic approach to treat heart failure.

Alteration of myocardial GRK2 produces a global metabolic phenotype.

A vast body of literature has established GRK2 as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function post injury in numerous animal models. In recent years, discovery of several non-canonical GRK2 targets has expanded our view of this kinase. Here, we describe the novel and exciting finding that cardiac GRK2 activity can regulate whole body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgßARKct) display an enhanced obesogenic phenotype when fed a high fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD fed TgßARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics we found that cardiac GRK2 signaling altered myocardial BCAA and endocannabinoid metabolism and modulated circulating BCAA and endocannabinoid metabolite profiles on a HFD, and one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. Taken together, these results suggest that metabolic changes in the heart due to GRK2 signaling on a HFD control whole body metabolism.

G protein-coupled receptor kinase 2 contributes to impaired fatty acid metabolism in the failing heart.

Increased G protein-coupled receptor kinase (GRK)2 is central to heart failure (HF) pathogenesis, via desensitization of ß-adrenergic receptors and loss of contractile reserve. Since GRK2 has been shown to compromise fatty acid (FA) oxidation, this kinase may link metabolic and contractile defects in HF. The aim of this study was to investigate the mechanistic role of GRK2 in FA metabolism and bioenergetics in the heart. For that purpose, we measured FA uptake and cluster of differentiation (CD)36 expression, phosphorylation, and ubiquitination in mice with cardiac-specific overexpression of GRK2 (TgGRK2) or expression of its c-terminus (GRK2 inhibitor- TgßARKct) or in global heterozygous GRK2 knockout (GRK2+/-) mice. Cellular bioenergetics were also measured in isolated cardiomyocytes following adenoviral delivery of exogenous GRK2, ßARKct, or short hairpin GRK2 (shGRK2). Additionally, CD36 expression and phosphorylation were evaluated following transverse aortic constriction (TAC) in wild type (WT) and GRK2+/- mice. Our results show a 33% ±â€¯0.81 reduction in FA uptake rate, accompanied by 51% ±â€¯0.17 lower CD36 protein, and 70% ±â€¯0.23 and 69% ±â€¯0.18 increases in CD36 phosphorylation and ubiquitination, respectively, in the TgGRK2 mice. Moreover, an in vitro kinase assay suggests that GRK2 directly phosphorylates CD36. In isolated cardiomyocytes, GRK2 overexpression induced a 26% ±â€¯2.21 decrease in maximal respiration, which was enhanced (20% ±â€¯4.02-5.14) with inhibition of the kinase. Importantly, in hearts with systolic dysfunction, notable reductions in CD36 mRNA and protein, as well as a significant increase in CD36 phosphorylation were normalized in the GRK2+/- mice post-TAC. Thus, we propose that GRK2 up-regulation in HF is, at least partly, responsible for reduced FA uptake and oxidation and may be a nodal link between metabolic and contractile defects.

Transcriptional regulation mediated by H2A.Z via ANP32e-dependent inhibition of protein phosphatase 2A.

The mechanisms that regulate H2A.Z and its requirement for transcription in differentiated mammalian cells remains ambiguous. In this study, we identified the interaction between the C-terminus of ANP32e and N-terminus of H2A.Z in a yeast two-hybrid screen. Knockdown of ANP32e resulted in proteasomal degradation and nuclear depletion of H2A.Z or of a chimeric green florescence protein fused to its N-terminus. This effect was reversed by inhibition of protein phosphatase 2A (PP2A) and, conversely, reproduced by overexpression of its catalytic subunit. Accordingly, knockdown of ANP32e inhibited phosphorylation of H2A.Z, whereas a mutation of serine-9 proved its requirement for both the protein's stability and nuclear localization, as did knockdown of the nuclear mitogen and stress-induced kinase 1. Moreover, ANP32e's knockdown also revealed its differential requirement for cell signaling and gene expression, whereas, genome-wide binding analysis confirmed its co-localization with H2A.Z at transcription start sites, as well as, gene bodies of inducible and tissue-specific genes. The data also suggest that H2A.Z restricts transcription, which is moderated by ANP32e at the promoter and gene bodies of expressed genes. Thus, ANP32e, through inhibition of PP2A, is required for nucleosomal inclusion of H2A.Z and the regulation of gene expression.

Acute targeting of general transcription factor IIB restricts cardiac hypertrophy via selective inhibition of gene transcription.

BACKGROUND: We previously reported that specialized and housekeeping genes are differentially regulated via de novo recruitment and pause-release of RNA polymerase II, respectively, during cardiac hypertrophy. However, the significance of this finding remains to be examined. Therefore, the purpose of this study was to determine the mechanisms that differentially regulate these gene groups and exploit them for therapeutic targeting. METHODS AND RESULTS: Here, we show that general transcription factor IIB (TFIIB) and cyclin-dependent kinase 9 are upregulated during hypertrophy, both targeted by microRNA-1, and play preferential roles in regulating those 2 groups of genes. Chromatin immunoprecipitation-sequencing reveals that TFIIB is constitutively bound to all paused, housekeeping, promoters, whereas de novo recruitment of TFIIB and polymerase II is required for specialized genes that are induced during hypertrophy. We exploited this dichotomy to acutely inhibit induction of the latter set, which encompasses cardiomyopathy, immune reaction, and extracellular matrix genes, using locked nucleic acid-modified antisense TFIIB oligonucleotide treatment. This resulted in suppression of all specialized genes, while sparing the housekeeping ones, and, thus, attenuated pathological hypertrophy. CONCLUSIONS: The data for the first time reveal distinct general TFIIB dynamics that regulate specialized versus housekeeping genes during cardiac hypertrophy. Thus, by acutely targeting TFIIB, we were able to inhibit selectively the former set of genes and ameliorate pressure overload hypertrophy. We also demonstrate the feasibility of acutely and reversibly targeting cardiac mRNA for therapeutic purposes using locked nucleic acid-modified antisense oligonucleotides.