Efficient Gene Editing for Heart Disease via ELIP-Based CRISPR Delivery System.

Liposomes as carriers for CRISPR/Cas9 complexes represent an attractive approach for cardiovascular gene therapy. A critical barrier to this approach remains the efficient delivery of CRISPR-based genetic materials into cardiomyocytes. Echogenic liposomes (ELIP) containing a fluorescein isothiocyanate-labeled decoy oligodeoxynucleotide against nuclear factor kappa B (ELIP-NF-κB-FITC) were used both in vitro on mouse neonatal ventricular myocytes and in vivo on rat hearts to assess gene delivery efficacy with or without ultrasound. In vitro analysis was then repeated with ELIP containing Cas9-sg-IL1RL1 (interleukin 1 receptor-like 1) RNA to determine the efficiency of gene knockdown. ELIP-NF-κB-FITC without ultrasound showed limited gene delivery in vitro and in vivo, but ultrasound combined with ELIP notably improved penetration into heart cells and tissues. When ELIP was used to deliver Cas9-sg-IL1RL1 RNA, gene editing was successful and enhanced by ultrasound. This innovative approach shows promise for heart disease gene therapy using CRISPR technology.

A Two-Step Transcriptome Analysis of the Human Heart Reveals Broad and Disease-Responsive Expression of Ectopic Olfactory Receptors.

G-protein-coupled receptors (GPCRs) are critical regulators of cardiac physiology and a key therapeutic target for the treatment of heart disease. Ectopic olfactory receptors (ORs) are GPCRs expressed in extra-nasal tissues which have recently emerged as new mediators in the metabolic control of cardiac function. The goals of this study were to profile OR gene expression in the human heart, to identify ORs dysregulated by heart failure caused by ischemic cardiomyopathy, and to provide evidence suggestive of a role for those altered ORs in the pathogenesis of heart failure. Left ventricular tissue from heart failure patients (n = 18) and non-failing heart samples (n = 4) were subjected to a two-step transcriptome analysis consisting of the quantification of 372 distinct OR transcripts on real-time PCR arrays and simultaneous determination of global cardiac gene expression by RNA sequencing. This strategy led to the identification of >160 ORs expressed in the human heart, including 38 receptors differentially regulated with heart failure. Co-expression analyses predicted the involvement of dysregulated ORs in the alteration of mitochondrial function, extracellular matrix remodeling, and inflammation. We provide this dataset as a resource for investigating roles of ORs in the human heart, with the hope that it will assist in the identification of new therapeutic targets for the treatment of heart failure.

Liposome-Based Carriers for CRISPR Genome Editing.

The CRISPR-based genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR), has sparked renewed interest in gene therapy. This interest is accompanied by the development of single-guide RNAs (sgRNAs), which enable the introduction of desired genetic modifications at the targeted site when used alongside the CRISPR components. However, the efficient delivery of CRISPR/Cas remains a challenge. Successful gene editing relies on the development of a delivery strategy that can effectively deliver the CRISPR cargo to the target site. To overcome this obstacle, researchers have extensively explored non-viral, viral, and physical methods for targeted delivery of CRISPR/Cas9 and a guide RNA (gRNA) into cells and tissues. Among those methods, liposomes offer a promising approach to enhance the delivery of CRISPR/Cas and gRNA. Liposomes facilitate endosomal escape and leverage various stimuli such as light, pH, ultrasound, and environmental cues to provide both spatial and temporal control of cargo release. Thus, the combination of the CRISPR-based system with liposome delivery technology enables precise and efficient genetic modifications in cells and tissues. This approach has numerous applications in basic research, biotechnology, and therapeutic interventions. For instance, it can be employed to correct genetic mutations associated with inherited diseases and other disorders or to modify immune cells to enhance their disease-fighting capabilities. In summary, liposome-based CRISPR genome editing provides a valuable tool for achieving precise and efficient genetic modifications. This review discusses future directions and opportunities to further advance this rapidly evolving field.

Prolonged cardiac NR4A2 activation causes dilated cardiomyopathy in mice.

Transcription factors play a fundamental role in cardiovascular adaptation to stress. Nuclear receptor subfamily 4 group A member 2 (NR4A2; NURR1) is an immediate-early gene and transcription factor with a versatile role throughout many organs. In the adult mammalian heart, and particularly in cardiac myocytes, NR4A2 is strongly up-regulated in response to beta-adrenergic stimulation. The physiologic implications of this increase remain unknown. In this study, we aimed to interrogate the consequences of cardiac NR4A2 up-regulation under normal conditions and in response to pressure overload. In mice, tamoxifen-dependent, cardiomyocyte-restricted overexpression of NR4A2 led to cardiomyocyte hypertrophy, left ventricular dilation, heart failure, and death within 40 days. Chronic NR4A2 induction also precipitated cardiac decompensation during transverse aortic constriction (TAC)-induced pressure overload. Mechanistically, NR4A2 caused adult cardiac myocytes to return to a fetal-like phenotype, with a switch to glycolytic metabolism and disassembly of sarcomeric structures. NR4A2 also re-activated cell cycle progression and stimulated DNA replication and karyokinesis but failed to induce cytokinesis, thereby promoting multinucleation of cardiac myocytes. Activation of cell cycle checkpoints led to induction of an apoptotic response which ultimately resulted in excessive loss of cardiac myocytes and impaired left ventricular contractile function. In summary, myocyte-specific overexpression of NR4A2 in the postnatal mammalian heart results in increased cell cycle re-entry and DNA replication but does not result in cardiac myocyte division. Our findings expose a novel function for the nuclear receptor as a critical regulator in the self-renewal of the cardiac myocyte and heart regeneration.

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.

UCP3 (Uncoupling Protein 3) Insufficiency Exacerbates Left Ventricular Diastolic Dysfunction During Angiotensin II-Induced Hypertension.

Background Left ventricular diastolic dysfunction, an early stage in the pathogenesis of heart failure with preserved ejection fraction, is exacerbated by joint exposure to hypertension and obesity; however, the molecular mechanisms involved remain uncertain. The mitochondrial UCP3 (uncoupling protein 3) is downregulated in the heart with obesity. Here, we used a rat model of UCP3 haploinsufficiency (ucp3+/-) to test the hypothesis that decreased UCP3 promotes left ventricular diastolic dysfunction during hypertension. Methods and Results Ucp3+/- rats and ucp3+/+ littermates fed a high-salt diet (HS; 2% NaCl) and treated with angiotensin II (190 ng/kg per min for 28 days) experienced a similar rise in blood pressure (158±4 versus 155±7 mm Hg). However, UCP3 insufficiency worsened diastolic dysfunction according to echocardiographic assessment of left ventricular filling pressures (E/e'; 18.8±1.0 versus 14.9±0.6; P<0.05) and the isovolumic relaxation time (24.7±0.6 versus 21.3±0.5 ms; P<0.05), as well as invasive monitoring of the diastolic time constant (Tau; 15.5±0.8 versus 12.7±0.2 ms; P<0.05). Exercise tolerance on a treadmill also decreased for HS/angiotensin II-treated ucp3+/- rats. Histological and molecular analyses further revealed that UCP3 insufficiency accelerated left ventricular concentric remodeling, detrimental interstitial matrix remodeling, and fetal gene reprogramming during hypertension. Moreover, UCP3 insufficiency increased oxidative stress and led to greater impairment of protein kinase G signaling. Conclusions Our findings identified UCP3 insufficiency as a cause for increased incidence of left ventricular diastolic dysfunction during hypertension. The results add further support to the use of antioxidants targeting mitochondrial reactive oxygen species as an adjuvant therapy for preventing heart failure with preserved ejection fraction in individuals with obesity.

Crosstalk between beta-adrenergic and insulin signaling mediates mechanistic target of rapamycin hyperactivation in liver of high-fat diet-fed male mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. While increased nutrient intake and sympathetic activity have been associated with the disease, the pathogenesis of NAFLD remains incompletely understood. We investigated the impact of the interaction of high dietary fat and sugar intake with increased beta-adrenergic receptor (ß-AR) signaling on the activity of nutrient-sensing pathways and fuel storage in the liver. C57BL/6J mice were fed a standard rodent diet (STD), a high-fat diet (HFD), a high-fat/high-sugar Western diet (WD), a high-sugar diet with mixed carbohydrates (HCD), or a high-sucrose diet (HSD). After 6 week on diets, mice were treated with isoproterenol (ISO) and the activity of liver mTOR complex 1 (mTORC1)-related signaling analyzed by immunoblotting and correlated with tissue triglyceride and glycogen contents. ISO-stimulated AKT- and ERK-mediated activation of mTORC1 in STD-fed mice. Consumption of all four high-calorie diets exacerbated downstream activation of ribosomal protein S6 kinase beta-1 (S6K1) in response to ISO. S6K1 activity was greater with the fat-enriched HFD and WD and correlated with the presence of metabolic syndrome and a stronger activation of AKT and ERK1/2 pathways. Fat-enriched diets also increased triglyceride accumulation and inhibited glycogen mobilization under ß-AR stimulation. In conclusion, crosstalk between ß-AR and insulin signaling may contribute to HFD-induced hepatic steatosis through ERK1/2- and AKT-mediated hyperactivation of the mTORC1/S6K1 axis. The findings provide further rationale for the development of therapies aimed at targeting augmented ß-AR signaling in the pathogenesis of NAFLD.

Restoration of Cardiac Function After Myocardial Infarction by Long-Term Activation of the CNS Leptin-Melanocortin System.

Heart failure has a high mortality rate, and current therapies offer limited benefits. The authors demonstrate that activation of the central nervous system leptin-melanocortin pathway confers remarkable protection against progressive heart failure following severe myocardial infarction. The beneficial cardiac-protective actions of leptin require activation of brain melanocortin-4 receptors and elicit improvements in cardiac substrate oxidation, cardiomyocyte contractility, Ca2+ coupling, and mitochondrial efficiency. These findings highlight a potentially novel therapeutic approach for myocardial infarction and heart failure.

Loss of Uncoupling Protein 3 Attenuates Western Diet-Induced Obesity, Systemic Inflammation, and Insulin Resistance in Rats.

OBJECTIVE: Uncoupling protein 3 (UCP3) is a mitochondrial carrier related to fatty acid metabolism. Although gene variants of UCP3 are associated with human obesity, their contribution to increased adiposity remains unclear. This study investigated the impact that loss of UCP3 has on diet-induced obesity in rats. METHODS: Male UCP3 knockout rats (ucp3-/- ) and wild-type littermates (ucp3+/+ ) were fed a high-fat, high-carbohydrate Western diet for 21 weeks. Body composition was analyzed by EchoMRI. Whole-body insulin sensitivity and rates of tissue glucose uptake were determined by using hyperinsulinemic-euglycemic clamp. Changes in tissue physiology were interrogated by microscopy and RNA sequencing. RESULTS: Loss of UCP3 decreased fat mass gain, white adipocytes size, and systemic inflammation. The ucp3-/- rats also exhibited preserved insulin sensitivity and increased glucose uptake in interscapular brown adipose tissue (iBAT). Brown adipocytes from ucp3-/- rats were protected from cellular degeneration caused by lipid accumulation and from reactive oxygen species-induced protein sulfonation. Increased glutathione levels in iBAT from ucp3-/- rats were linked to upregulation of genes encoding enzymes from the transsulfuration pathway in that tissue. CONCLUSIONS: Loss of UCP3 partially protects rats from diet-induced obesity. This phenotype is related to induction of a compensatory antioxidant mechanism and prevention of iBAT whitening.

Dietary Fat and Sugar Differentially Affect ß-Adrenergic Stimulation of Cardiac ERK and AKT Pathways in C57BL/6 Male Mice Subjected to High-Calorie Feeding.

BACKGROUND: High dietary fat and sugar promote cardiac hypertrophy independently from an increase in blood pressure. The respective contribution that each macronutrient exerts on cardiac growth signaling pathways remains unclear. OBJECTIVE: The goal of this study was to investigate the mechanisms by which high amounts of dietary fat and sugar affect cardiac growth regulatory pathways. METHODS: Male C57BL/6 mice (9 wk old; n = 20/group) were fed a standard rodent diet (STD; kcal% protein-fat-carbohydrate, 29-17-54), a high-fat diet (HFD; 20-60-20), a high-fat and high-sugar Western diet (WD; 20-45-35), a high-sugar diet with mixed carbohydrates (HCD; 20-10-70), or a high-sucrose diet (HSD; 20-10-70). Body composition was assessed weekly by EchoMRI. Whole-body glucose utilization was assessed with an intraperitoneal glucose tolerance test. After 6 wk on diets, mice were treated with saline or 20 mg/kg isoproterenol (ISO), and the activity of cardiac growth regulatory pathways was analyzed by immunoblotting. Data were analyzed by ANOVA with data from the STD group included for references only. RESULTS: Compared with HCD and HSD, WD and HFD increased body fat mass 2.7- to 3.8-fold (P < 0.001), induced glucose intolerance (P < 0.001), and increased insulin concentrations >1.5-fold (P < 0.05), thereby enhancing basal and ISO-stimulated AKT phosphorylation at both threonine 308 and serine 473 residues (+25-63%; P < 0.05). Compared with HFD, the high-sugar diets potentiated ISO-mediated stimulation of the glucose-sensitive kinases PYK2 (>47%; P < 0.05 for HCD and HSD) and ERK (>34%; P < 0.05 for WD, HCD, and HSD), thereby leading to increased phosphorylation of protein synthesis regulator S6K1 at threonine 389 residue (>64%; P < 0.05 for WD, HCD, and HSD). CONCLUSIONS: Dietary fat and sugar affect cardiac growth signaling pathways in C57BL/6 mice through distinct and additive mechanisms. The findings may provide new insights into the role of overnutrition in pathological cardiac remodeling.

Nuclear receptor subfamily 4 group A member 2 inhibits activation of ERK signaling and cell growth in response to ß-adrenergic stimulation in adult rat cardiomyocytes.

Sustained elevation of sympathetic activity is an important contributor to pathological cardiac hypertrophy, ventricular arrhythmias, and left ventricular contractile dysfunction in chronic heart failure. The orphan nuclear receptor NR4A2 is an immediate early-response gene activated in the heart under ß-adrenergic stimulation. The goal of this study was to identify the transcriptional remodeling events induced by increased NR4A2 expression in cardiomyocytes and their impact on the physiological response of those cells to sustained ß-adrenergic stimulation. Treatment of adult rat ventricular myocytes with isoproterenol induced a rapid (<4 h) increase in NR4A2 levels that was accompanied by a transient (<24 h) increase in nuclear localization of the transcription factor. Adenovirus-mediated overexpression of NR4A2 to similar levels modulated the expression of genes linked to adrenoceptor signaling, calcium signaling, cell growth and proliferation and counteracted the increase in protein synthesis rate and cell surface area mediated by chronic isoproterenol stimulation. Consistent with those findings, NR4A2 overexpression also blocked the phosphorylative activation of growth-related kinases ERK1/2, Akt, and p70 S6 kinase. Prominent among the transcriptional changes induced by NR4A2 was the upregulation of the dual-specificity phosphatases DUSP2 and DUSP14, two known inhibitors of ERK1/2. Pretreatment of NR4A2-overexpressing cardiomyocytes with the DUSP inhibitor BCI [(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one] prevented the inhibition of ERK1/2 following isoproterenol stimulation. In conclusion, our results suggest that NR4A2 acts as a novel negative feedback regulator of the ß-adrenergic receptor-mediated growth response in cardiomyocytes and this at least partly through DUSP-mediated inhibition of ERK1/2 signaling.

Uncoupling protein 3 deficiency impairs myocardial fatty acid oxidation and contractile recovery following ischemia/reperfusion.

Patients with insulin resistance and type 2 diabetes have poor cardiac outcomes following myocardial infarction (MI). The mitochondrial uncoupling protein 3 (UCP3) is down-regulated in the heart with insulin resistance. We hypothesized that decreased UCP3 levels contribute to poor cardiac recovery following ischemia/reperfusion (I/R). After confirming that myocardial UCP3 levels were systematically decreased by 20-49% in animal models of insulin resistance and type 2 diabetes, we genetically engineered Sprague-Dawley rats with partial loss of UCP3 (ucp3+/-). Wild-type littermates (ucp3+/+) were used as controls. Isolated working hearts from ucp3+/- rats were characterized by impaired recovery of cardiac power and decreased long-chain fatty acid (LCFA) oxidation following I/R. Mitochondria isolated from ucp3+/- hearts subjected to I/R in vivo displayed increased reactive oxygen species (ROS) generation and decreased respiratory complex I activity. Supplying ucp3+/- cardiac mitochondria with the medium-chain fatty acid (MCFA) octanoate slowed electron transport through the respiratory chain and reduced ROS generation. This was accompanied by improvement of cardiac LCFA oxidation and recovery of contractile function post ischemia. In conclusion, we demonstrated that normal cardiac UCP3 levels are essential to recovery of LCFA oxidation, mitochondrial respiratory capacity, and contractile function following I/R. These results reveal a potential mechanism for the poor prognosis of type 2 diabetic patients following MI and expose MCFA supplementation as a feasible metabolic intervention to improve recovery of these patients at reperfusion.

Mapping macrophage polarization over the myocardial infarction time continuum.

In response to myocardial infarction (MI), cardiac macrophages regulate inflammation and scar formation. We hypothesized that macrophages undergo polarization state changes over the MI time course and assessed macrophage polarization transcriptomic signatures over the first week of MI. C57BL/6 J male mice (3-6 months old) were subjected to permanent coronary artery ligation to induce MI, and macrophages were isolated from the infarct region at days 1, 3, and 7 post-MI. Day 0, no MI resident cardiac macrophages served as the negative MI control. Whole transcriptome analysis was performed using RNA-sequencing on n = 4 pooled sets for each time. Day 1 macrophages displayed a unique pro-inflammatory, extracellular matrix (ECM)-degrading signature. By flow cytometry, day 0 macrophages were largely F4/80highLy6Clow resident macrophages, whereas day 1 macrophages were largely F4/80lowLy6Chigh infiltrating monocytes. Day 3 macrophages exhibited increased proliferation and phagocytosis, and expression of genes related to mitochondrial function and oxidative phosphorylation, indicative of metabolic reprogramming. Day 7 macrophages displayed a pro-reparative signature enriched for genes involved in ECM remodeling and scar formation. By triple in situ hybridization, day 7 infarct macrophages in vivo expressed collagen I and periostin mRNA. Our results indicate macrophages show distinct gene expression profiles over the first week of MI, with metabolic reprogramming important for polarization. In addition to serving as indirect mediators of ECM remodeling, macrophages are a direct source of ECM components. Our study is the first to report the detailed changes in the macrophage transcriptome over the first week of MI.

Glucose regulates the intrinsic inflammatory response of the heart to surgically induced hypothermic ischemic arrest and reperfusion.

We investigated the isolated working rat heart as a model to study early transcriptional remodeling induced in the setting of open heart surgery and stress hyperglycemia. Hearts of male Sprague Dawley rats were cold-arrested in Krebs-Henseleit buffer and subjected to 60 min normothermic reperfusion in the working mode with buffer supplemented with noncarbohydrate substrates plus glucose (25 mM) or mannitol (25 mM; osmotic control). Gene expression profiles were determined by microarray analysis and compared with those of nonperfused hearts. Perfused hearts displayed a transcriptional signature independent from the presence of glucose showing a more than twofold increase in expression of 71 genes connected to inflammation, cell proliferation, and apoptosis. These transcriptional alterations were very similar to the ones taking place in the hearts of open heart surgery patients. Prominent among those alterations was the upregulation of the three master regulators of metabolic reprogramming, MYC, NR4A1, and NR4A2. Targeted pathway analysis revealed an upregulation of metabolic processes associated with the proliferation and activation of macrophages and fibroblasts. Glucose potentiated the upregulation of a subset of genes associated with polarization of tissue reparative M2-like macrophages, an effect that was lost in perfused hearts from rats rendered insulin resistant by high-sucrose feeding. The results expose the heart as a significant source of proinflammatory mediators released in response to stress associated with cardiac surgery with cardiopulmonary bypass, and suggest a major role for glucose as a signal in the determination of resident cardiac macrophage polarization.

Cardiac STAT3 Deficiency Impairs Contractility and Metabolic Homeostasis in Hypertension.

Signal transducer and activator of transcription 3 (STAT3) protects the heart from acute ischemic stress. However, the importance of STAT3 to the heart in chronic stress, such as hypertension, is not known. To study this, we used cardiomyocyte-targeted STAT3 knockout (KO) mice and Angiotensin II (ANG II) infusion by osmotic minipumps. After 4 weeks, ANG II induced similar cardiac hypertrophy in wild type (WT) and cardiac Cre-expressing control (CTRL) mice with no impairment of cardiac function. In contrast, STAT3 KO mice exhibited reduced contractile function but similar hypertrophy to CTRL mice. Ejection fraction and fractional shortening decreased by 22.5 and 27.3%, respectively. Since STAT3 has direct protective effects on mitochondrial function, we examined rates of glucose and oleate oxidation by isolated perfused hearts using a Langendorff system. Hearts of ANG II-treated STAT3 KO and CTRL mice had similar rates of oleate oxidation as saline-infused WT mice. Rates of glucose oxidation were similar between hearts of WT plus saline and CTRL plus ANG II mice; however, glucose oxidation was increased by 66% in hearts of ANG II-treated STAT3 KO mice. The ratio of maximal ATP yield from glucose to fatty acid oxidation was 21.1 ± 3.1 in hearts of ANG II-treated STAT3 KO mice vs. 12.6 ± 2.2 in hearts of ANG II-treated CTRL mice. Lactate production was also elevated in hearts of ANG II-treated STAT3 KO mice by 162% compared to ANG II-treated CTRL mice. Our findings indicate that STAT3 is important for maintaining contractile function and metabolic homeostasis in the hypertensive heart, and STAT3 deficiency promotes a switch toward glucose utilization.

Increased mean aliphatic lipid chain length in left ventricular hypertrophy secondary to arterial hypertension: A cross-sectional study.

About 77.9 million (1 in 4) American adults have high blood pressure. High blood pressure is the primary cause of left ventricular hypertrophy (LVH), which represents a strong predictor of future heart failure and cardiovascular mortality. Previous studies have shown an altered metabolic profile in hypertensive patients with LVH. The goal of this study was to identify blood metabolomic LVH biomarkers by H NMR to provide novel diagnostic tools for rapid LVH detection in populations of hypertensive individuals. This cross-sectional study included 48 hypertensive patients with LVH matched with 48 hypertensive patients with normal LV size, and 24 healthy controls. Two-dimensional targeted M-mode echocardiography was performed to measure left ventricular mass index. Partial least squares discriminant analysis was used for the multivariate analysis of the H NMR spectral data. From the H NMR-based metabolomic profiling, signals coming from methylene (-CH2-) and methyl (-CH3) moieties of aliphatic chains from plasma lipids were identified as discriminant variables. The -CH2-/-CH3 ratio, an indicator of the mean length of the aliphatic lipid chains, was significantly higher (P < 0.001) in the LVH group than in the hypertensive group without LVH and controls. Receiver operating characteristic curve showed that a cutoff of 2.34 provided a 52.08% sensitivity and 85.42% specificity for discriminating LVH (AUC = 0.703, P-value < 0.001). We propose the -CH2-/-CH3 ratio from plasma aliphatic lipid chains as a biomarker for the diagnosis of left ventricular remodeling in hypertension.

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart.

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

Oncometabolite d-2-hydroxyglutarate impairs α-ketoglutarate dehydrogenase and contractile function in rodent heart.

Hematologic malignancies are frequently associated with cardiac pathologies. Mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a subset of acute myeloid leukemia patients, causing metabolic and epigenetic derangements. We have now discovered that altered metabolism in leukemic cells has a profound effect on cardiac metabolism. Combining mathematical modeling and in vivo as well as ex vivo studies, we found that increased amounts of the oncometabolite d-2-hydroxyglutarate (D2-HG), produced by IDH2 mutant leukemic cells, cause contractile dysfunction in the heart. This contractile dysfunction is associated with impaired oxidative decarboxylation of α-ketoglutarate, a redirection of Krebs cycle intermediates, and increased ATP citrate lyase (ACL) activity. Increased availability of D2-HG also leads to altered histone methylation and acetylation in the heart. We propose that D2-HG promotes cardiac dysfunction by impairing α-ketoglutarate dehydrogenase and induces histone modifications in an ACL-dependent manner. Collectively, our results highlight the impact of cancer cell metabolism on function and metabolism of the heart.

Adapting extracellular matrix proteomics for clinical studies on cardiac remodeling post-myocardial infarction.

Following myocardial infarction (MI), the left ventricle (LV) undergoes a series of cardiac wound healing responses that involve stimulation of robust inflammation to clear necrotic myocytes and tissue debris and induction of extracellular matrix (ECM) protein synthesis to generate a scar. Proteomic strategies provide us with a means to index the ECM proteins expressed in the LV, quantify amounts, determine functions, and explore interactions. This review will focus on the efforts taken in the proteomics research field that have expanded our understanding of post-MI LV remodeling, concentrating on the strengths and limitations of different proteomic approaches to glean information that is specific to ECM turnover in the post-MI setting. We will discuss how recent advances in sample preparation and labeling protocols increase our successes at detecting components of the cardiac ECM proteome. We will summarize how proteomic approaches, focusing on the ECM compartment, have progressed over time to current gel-free methods using decellularized fractions or labeling strategies that will be useful for clinical applications. This review will provide an overview of how cardiac ECM proteomics has evolved over the last decade and will provide insight into future directions that will drive forward our understanding of cardiac ECM turnover in the post-MI LV.

Chronic Hyperinsulinemia Causes Selective Insulin Resistance and Down-regulates Uncoupling Protein 3 (UCP3) through the Activation of Sterol Regulatory Element-binding Protein (SREBP)-1 Transcription Factor in the Mouse Heart.

The risk for heart failure and death after myocardial infarction is abnormally high in diabetic subjects. We and others have shown previously that mitochondrial uncoupling protein 3 (UCP3) improves functional recovery of the rodent heart during reperfusion. Here, we demonstrate that pharmacological induction of hyperinsulinemia in mice down-regulates myocardial UCP3. Decreased UCP3 expression was linked to the development of selective insulin resistance in the heart, characterized by decreased basal activity of Akt but preserved activity of the p44/42 mitogen-activated protein kinase, and overactivation of the sterol regulatory element-binding protein (SREBP)-1-mediated lipogenic program. In cultured myocytes, insulin treatment and SREBP-1 overexpression decreased, whereas SREBP-1 interference increased, peroxisome proliferator-activated receptor-stimulated expression of UCP3. Promoter deletion and site-directed mutagenesis identified three functional sterol regulatory elements in the vicinity of a known complex intronic enhancer. Increased binding of SREBP-1 to this DNA region was confirmed in the heart of hyperinsulinemic mice. In conclusion, we describe a hitherto unknown regulatory mechanism by which insulin inhibits cardiac UCP3 expression through activation of the lipogenic factor SREBP-1. Sustained down-regulation of cardiac UCP3 by hyperinsulinemia may partly explain the poor prognosis of type 2 diabetic patients after myocardial infarction.