The association of catestatin and endocan with the effects of cardiac shock wave therapy: Biomarker sub-study of the randomized, sham procedure-controlled trial.

Introduction: Cardiac shock-wave therapy (CSWT) is a non-invasive regenerative treatment method based on low-frequency ultrasound waves, which stimulate angiogenesis. Current data about the effects of revascularization procedures on angiogenesis biomarkers is limited. Recently, an association of catestatin and endocan with coronary collateral development was shown in several trials. In this study, we aimed to evaluate the impact of CSWT on the dynamics of catestatin and endocan levels and to assess their correlation with parameters of myocardial perfusion and function. Methods: Prospective, randomized, triple-blind, sham procedure-controlled study enrolled 72 adult subjects who complied with defined inclusion criteria (NCT02339454). We measured biomarkers in 48 patients with stable angina (24 patients of CSWT group, 24 patients of sham-procedure group). Additionally, patients were divided into responders and non-responders according to improvement in myocardial perfusion and/or contractility assessed by myocardial scintigraphy and dobutamine echocardiography (30 and 13 patients, respectively). The blood samples were collected at baseline, after the last treatment procedure (9th treatment week) and at 6-month follow-up to evaluate biomarkers concentration and stored at -80° until analysis. Serum catestatin and endocan levels were determined by commercially available ELISA kits. Results: Serum catestatin concentration significantly increased in all patients. While endocan levels significantly decreased in the responders sub-group. The increase in catestatin levels at 9th week and 6 months was positively associated with improvement in summed difference score (rho = 0.356, p = 0.028) and wall motion score, WMS (rho = 0.397, p = 0.009) at 6 months in the whole study population. Meanwhile, the decrease in endocan levels over 6 months was positively correlated with improvement in WMS at 3- and 6- months (r = 0.378, p = 0.015 and r = 0.311, p = 0.045, respectively). ROC analysis revealed that a change at 6 months in catestatin and endocan levels significantly predicted improvement in myocardial perfusion and contractile function with 68.9% sensitivity and 75.0% specificity (p = 0.039) and 51.7% sensitivity, and 91.7% specificity (p = 0.017), respectively. Baseline endocan concentration and its change at 6 months predicted response to CSWT with 68.8% sensitivity and 83.3% specificity (p = 0.039) and 81.3% sensitivity and 100% specificity (p < 0.0001), respectively. Conclusion: This study demonstrates the association of increase in catestatin and decrease in endocan levels with the improvement of myocardial perfusion and contractile function. The potential predictive value of catestatin and endocan dynamics for the response to regenerative therapy is shown.

sST2 adds to the prognostic value of Gal-3 and BNP in chronic heart failure.

Background: The soluble form of the IL-33 receptor (sST2) and Galectin-3 (Gal-3) are fibrosis biomarkers with prognostic value in heart failure (HF). We investigated the prognostic capacity of sST2 when combined with Gal-3, and determined if the prognostic utility of sST2 is affected by mineralocorticoid receptor antagonist (MRA) therapy.Methods: sST-2 and Gal-3 were measured in 101 stable chronic HF (CHF) patients receiving MRA therapy and compared to 97 BNP and cardiovascular risk factor matched patients not treated with MRA. sST2 and Gal-3 levels were measured to determine the relationship with all-cause mortality at 6-year follow-up.Results: ROC curve cut-off points were defined as sST2 = 36.3 ng/mL, Gal-3 = 17.8 ng/mL, and BNP = 500 pg/mL, and had 6-year mortality hazard ratios (HR) of 7.3, 6.6 and 5.4, respectively. The combination of an elevated sST2 and Gal-3 had a HR = 4.4 [95% CI 1.9-8.9]. Combining sST2 and Gal-3 to a clinical model relevant for CHF prognosis allowed a significant reclassification of 1-year adverse outcome risk, even when BNP was included. Finally, prognostic prediction by sST2 was unaffected by MRA treatment.Conclusion: Simultaneous sST2 and Gal-3 elevation is associated with poorer prognosis compared to either alone, regardless of BNP levels, and the prognostic capacity of sST2 is independent of MRA therapy.

Insulin-like Growth Factor Binding Protein 2 predicts mortality risk in heart failure.

BACKGROUND: Insulin-like Growth Factor Binding Protein 2 (IGFBP2) showed greater heart failure (HF) diagnostic accuracy than the "grey zone" B-type natriuretic peptides, and may have prognostic utility as well. OBJECTIVES: To determine if IGFBP2 provides independent information on cardiovascular mortality in HF. METHODS: A retrospective study of 870 HF patients from 3 independent international cohorts. Presentation IGFBP2 plasma levels were measured by ELISA, and patients were followed from 1 year (Maastricht, Netherlands) to 6 years (Atlanta, GA, USA and Toulouse, France). Multivariate analysis, Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) were performed in the 3 cohorts. The primary outcome was cardiovascular mortality. RESULTS: In multivariate Cox proportional hazards analysis, the highest quartile of IGFBP2 was associated with mortality in the Maastricht cohort (adjusted hazard ratio 1.69 (95% CI, 1.18-2.41), p = 0.004) and in the combined Atlanta and Toulouse cohorts (adjusted hazard ratio 2.04 (95%CI, 1.3-3.3), p = 0.003). Adding IGFBP2 to a clinical model allowed a reclassification of adverse outcome risk in the Maastricht cohort (NRI = 18.7% p = 0.03; IDI = 3.9% p = 0.02) and with the Atlanta/Toulouse patients (NRI of 40.4% p = 0.01, 31,2% p = 0.04, 31.5% p = 0,02 and IDI of 2,9% p = 0,0005, 3.1% p = 0,0005 and 4,2%, p = 0.0005, for a follow-up of 1, 2 and 3 years, respectively). CONCLUSION: In 3 international cohorts, IGFBP2 level is a strong prognostic factor for cardiovascular mortality in HF, adding information to natriuretic monitoring and usual clinical markers, that should be further prospectively evaluated for patients' optimized care.

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.

The prognostic value of plasma galectin-3 in chronic heart failure patients is maintained when treated with mineralocorticoid receptor antagonists.

OBJECTIVE: Galectin-3 (Gal-3) is considered as a myocardial fibrosis biomarker with prognostic value in heart failure (HF). Since aldosterone is a neurohormone with established fibrotic properties, we aimed to investigate if mineralocorticoid receptor antagonists (MRAs) would modulate the prognostic value of Gal-3. METHODS: The IBLOMAVED cohort comprised 427 eligible chronic HF patients (CHF) with echocardiography and heart failure biomarkers assessments (BNP). After propensity score matching CHF patients for cardiovascular risk factors, to form balanced groups, Gal-3 levels were measured at baseline in plasma from patients treated with MRAs (MRA-Plus, n=101) or not (MRA-Neg, n=101). The primary end point was all-cause mortality with a follow-up of 3 years. RESULTS: Gal-3 in plasma from these patients were similar with median values of 14.0 ng/mL [IQR, 9.9-19.3] and 14.4 ng/mL [IQR, 12.3-19.8] (P = 0.132) in MRA-Neg and MRA-Plus, respectively. Patients with Gal-3 ≤17.8 ng/mL had an HR of 1 (reference group) and 1.5 [0.4-5.7] in MRA-Neg and MRA-Plus, respectively (p=0.509). Patients with Gal-3 ≥ 17.8 ng/mL had an HR of 7.4 [2.2-24.6] and 9.0 [2.9-27.8] in MRA-Plus and MRA-Neg, respectively (p=0.539) and a median survival time of 2.4 years [95%CI,1.8-2.4]. Multivariate Cox proportional hazard analysis confirmed that MRA and the interaction term between MRA treatment and Gal-3 >17.8 ng/mL were not factors associated with survival. CONCLUSIONS: MRA treatment did not impair the prognostic value of Gal-3 assessed with a 17.8 ng/mL cut off. Gal-3 levels maintained its strong prognostic value in CHF also in patients treated with MRAs. The significance of the observed lack of an interaction between Gal-3 and treatment effect of MRAs remains to be elucidated.

Benefits of cardiac rehabilitation in heart failure patients according to etiology: INCARD French study.

We investigated the impact of heart failure (HF) etiology on the outcome of cardiac rehabilitation (CR) assessed by functional and clinical parameters. Treatment of chronic HF requires multidisciplinary approaches with a recognized role for CR. INCARD is a French study aimed at evaluating the benefits of sustainable CR in coronary (C) and noncoronary patients (NC) treated and educated during a 24-month period of follow-up. Prospective, monocentric patients with HF underwent inpatient physical training followed by a home-based program. Evaluations were performed at inclusion, discharge, 3 months after discharge, and subsequently every 6 months over the 24 months of outpatient rehabilitation.A total of 147 HF patients with left ventricular ejection fraction (LVEF) <40 were admitted to the CR center, 63 accepted to join INCARD (29 C and 34 NC). Although the C participants C having both an echocardiographic LVEF and an initially lower peak VO2, inpatient rehabilitation improved all functional parameters. Only NC showed an improved LVEF during the first 3 months of outpatient-follow-up. The main outcome of the outpatient rehabilitation was a trend toward stabilization of clinical and laboratory parameters with no significant difference between C and NC. This study confirms the benefits of initial HF inpatient rehabilitation and encourages prolonged outpatient monitoring. The results on functional parameters suggest exercise training should be conducted regardless of the HF etiology.

(13)C metabolic flux analysis shows that resistin impairs the metabolic response to insulin in L6E9 myotubes.

BACKGROUND: It has been suggested that the adipokine resistin links obesity and insulin resistance, although how resistin acts on muscle metabolism is controversial. We aimed to quantitatively analyse the effects of resistin on the glucose metabolic flux profile and on insulin response in L6E9 myotubes at the metabolic level using a tracer-based metabolomic approach and our in-house developed software, Isodyn. RESULTS: Resistin significantly increased glucose uptake and glycolysis, altering pyruvate utilisation by the cell. In the presence of resistin, insulin only slightly increased glucose uptake and glycolysis, and did not alter the flux profile around pyruvate induced by resistin. Resistin prevented the increase in gene expression in pyruvate dehydrogenase-E1 and the sharp decrease in gene expression in cytosolic phosphoenolpyruvate carboxykinase-1 induced by insulin. CONCLUSIONS: These data suggest that resistin impairs the metabolic activation of insulin. This impairment cannot be explained by the activity of a single enzyme, but instead due to reorganisation of the whole metabolic flux distribution.

Apolipoprotein O is mitochondrial and promotes lipotoxicity in heart.

Diabetic cardiomyopathy is a secondary complication of diabetes with an unclear etiology. Based on a functional genomic evaluation of obesity-associated cardiac gene expression, we previously identified and cloned the gene encoding apolipoprotein O (APOO), which is overexpressed in hearts from diabetic patients. Here, we generated APOO-Tg mice, transgenic mouse lines that expresses physiological levels of human APOO in heart tissue. APOO-Tg mice fed a high-fat diet exhibited depressed ventricular function with reduced fractional shortening and ejection fraction, and myocardial sections from APOO-Tg mice revealed mitochondrial degenerative changes. In vivo fluorescent labeling and subcellular fractionation revealed that APOO localizes with mitochondria. Furthermore, APOO enhanced mitochondrial uncoupling and respiration, both of which were reduced by deletion of the N-terminus and by targeted knockdown of APOO. Consequently, fatty acid metabolism and ROS production were enhanced, leading to increased AMPK phosphorylation and Ppara and Pgc1a expression. Finally, we demonstrated that the APOO-induced cascade of events generates a mitochondrial metabolic sink whereby accumulation of lipotoxic byproducts leads to lipoapoptosis, loss of cardiac cells, and cardiomyopathy, mimicking the diabetic heart-associated metabolic phenotypes. Our data suggest that APOO represents a link between impaired mitochondrial function and cardiomyopathy onset, and targeting APOO-dependent metabolic remodeling has potential as a strategy to adjust heart metabolism and protect the myocardium from impaired contractility.

Metabonomics analysis of plasma reveals the lactate to cholesterol ratio as an independent prognostic factor of short-term mortality in acute heart failure.

OBJECTIVE: Mortality in heart failure (AHF) remains high, especially during the first days of hospitalization. New prognostic biomarkers may help to optimize treatment. The aim of the study was to determine metabolites that have a high prognostic value. METHODS: We conducted a prospective study on a training cohort of AHF patients (n = 126) admitted in the cardiac intensive care unit and assessed survival at 30 days. Venous plasmas collected at admission were used for (1)H NMR--based metabonomics analysis. Differences between plasma metabolite profiles allow determination of discriminating metabolites. A cohort of AHF patients was subsequently constituted (n = 74) to validate the findings. RESULTS: Lactate and cholesterol were the major discriminating metabolites predicting 30-day mortality. Mortality was increased in patients with high lactate and low total cholesterol concentrations at admission. Accuracies of lactate, cholesterol concentration and lactate to cholesterol (Lact/Chol) ratio to predict 30-day mortality were evaluated using ROC analysis. The Lact/Chol ratio provided the best accuracy with an AUC of 0.82 (P < 0.0001). The acute physiology and chronic health evaluation (APACHE) II scoring system provided an AUC of 0.76 for predicting 30-day mortality. APACHE II score, Cardiogenic shock (CS) state and Lact/Chol ratio ≥ 0.4 (cutoff value with 82% sensitivity and 64% specificity) were significant independent predictors of 30-day mortality with hazard ratios (HR) of 1.11, 4.77 and 3.59, respectively. In CS patients, the HR of 30-day mortality risk for plasma Lact/Chol ratio ≥ 0.4 was 3.26 compared to a Lact/Chol ratio of < 0.4 (P = 0.018). The predictive power of the Lact/Chol ratio for 30-day mortality outcome was confirmed with the independent validation cohort. CONCLUSION: This study identifies the plasma Lact/Chol ratio as a useful objective and simple parameter to evaluate short term prognostic and could be integrated into quantitative guidance for decision making in heart failure care.

Blood signature of pre-heart failure: a microarrays study.

BACKGROUND: The preclinical stage of systolic heart failure (HF), known as asymptomatic left ventricular dysfunction (ALVD), is diagnosed only by echocardiography, frequent in the general population and leads to a high risk of developing severe HF. Large scale screening for ALVD is a difficult task and represents a major unmet clinical challenge that requires the determination of ALVD biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: 294 individuals were screened by echocardiography. We identified 9 ALVD cases out of 128 subjects with cardiovascular risk factors. White blood cell gene expression profiling was performed using pangenomic microarrays. Data were analyzed using principal component analysis (PCA) and Significant Analysis of Microarrays (SAM). To build an ALVD classifier model, we used the nearest centroid classification method (NCCM) with the ClaNC software package. Classification performance was determined using the leave-one-out cross-validation method. Blood transcriptome analysis provided a specific molecular signature for ALVD which defined a model based on 7 genes capable of discriminating ALVD cases. Analysis of an ALVD patients validation group demonstrated that these genes are accurate diagnostic predictors for ALVD with 87% accuracy and 100% precision. Furthermore, Receiver Operating Characteristic curves of expression levels confirmed that 6 out of 7 genes discriminate for left ventricular dysfunction classification. CONCLUSIONS/SIGNIFICANCE: These targets could serve to enhance the ability to efficiently detect ALVD by general care practitioners to facilitate preemptive initiation of medical treatment preventing the development of HF.

Adrenomedullin inhibits adipogenesis under transcriptional control of insulin.

We generated preadipocyte cell lines impaired in adrenomedullin production through integration of an adrenomedullin small interfering RNA expression vector. The reduction of adrenomedullin synthesis strongly accelerated adipose differentiation. These results were bolstered when overexpression of active adrenomedullin peptide led to delayed differentiation. Therefore, we propose that adrenomedullin is an antiadipogenic factor. Moreover, we checked whether insulin, a proadipogenic factor, regulates expression of adrenomedullin. We observed that insulin had an inhibitory effect on adrenomedullin expression in isolated human adipocyte cells. This response was dose dependent and was reversed by resistin, a new anti-insulin agent. We quantified circulating adrenomedullin in healthy obese patients and observed a threefold increase of adrenomedullin compared with lean patients. Furthermore, adrenomedullin plasma levels are negatively correlated to plasma insulin levels in these obese patients. The insulin inhibitory response was also observed in vivo in Sprague-Dawley rats but not in the insulin-resistant Zucker rat, suggesting that adrenomedullin expression is upregulated in insulin-resistant adipose cells. Using adrenomedullin promoter-luciferase reporter gene constructs, we have shown that the adrenomedullin response to insulin is mediated by insulin-responsive elements. These findings provide new insight into fat mass development and the relationship between obesity and elevated circulating adrenomedullin levels in diabetic patients.

ApoO, a novel apolipoprotein, is an original glycoprotein up-regulated by diabetes in human heart.

Obesity is an independent risk factor for cardiac failure. Obesity promotes excessive deposition of fat in adipose and nonadipose tissues. Intramyocardial lipid overload is a relatively common finding in nonischemic heart failure, especially in obese and diabetic patients, and promotes lipoapoptosis that contributes to the alteration of cardiac function. Lipoprotein production has been proposed as a heart-protective mechanism through the unloading of surplus cellular lipids. We previously analyzed the heart transcriptome in a dog nutritional model of obesity, and we identified a new apolipoprotein, regulated by obesity in heart, which is the subject of this study. We detected this new protein in the following lipoproteins: high density lipoprotein, low density lipoprotein, and very low density lipoprotein. We designated it apolipoprotein O. Apolipoprotein O is a 198-amino acid protein that contains a 23-amino acidlong signal peptide. The apolipoprotein O gene is expressed in a set of human tissues. Confocal immunofluorescence microscopy colocalized apolipoprotein O and perilipins, a cellular marker of the lipid droplet. Chondroitinase ABC deglycosylation analysis or cell incubation with p-nitrophenyl-beta-d-xyloside indicated that apolipoprotein O belongs to the proteoglycan family. Naringenin or CP-346086 treatments indicated that apolipoprotein O secretion requires microsomal triglyceride transfer protein activity. Apolipoprotein O gene expression is up-regulated in the human diabetic heart. Apolipoprotein O promoted cholesterol efflux from macrophage cells. To our knowledge, apolipoprotein O is the first chondroitin sulfate chain containing apolipoprotein. Apolipoprotein O may be involved in myocardium-protective mechanisms against lipid accumulation, or it may have specific properties mediated by its unique glycosylation pattern.

The vasoactive peptide adrenomedullin is secreted by adipocytes and inhibits lipolysis through NO-mediated beta-adrenergic agonist oxidation.

Adipocytes are known to secrete a number of adipokines, but many adipocyte secretions and their functional importance remain to be characterized. This work shows that human white adipocytes and 3T3-F442A-derived adipocytes produce adrenomedullin (AM) and that AM acts in an autocrine/paracrine way on lipid metabolism by extracellular inactivation of isoproterenol, a beta-adrenergic agonist. AM is described as a counter-regulatory factor involved in the control of cardiovascular homeostasis. This peptide is believed to protect the heart from several complications implicated in obesity-linked cardiomorbidity, such as arterial hypertension, cardiac fibrosis, and decreased sinusal variability. The exact source of circulating AM remains a matter of debate, although endothelial and vascular smooth muscle cells seem to be important sites of production. We show that human adipose cells and 3T3-F442A-derived adipocytes express AM receptors and secrete AM. The function of this feature was investigated in 3T3-F442A cell line at the level of lipolysis regulation. AM inhibited beta-adrenergic-stimulated lipolysis by a nitric oxide (NO)-dependent mechanism, inducing a significant decrease in pD2 value for isoproterenol (8.6 +/- 0.2 vs. 9.8 +/- 0.1, P<0.001). This effect is cGMP-independent since it occurred in the presence of the NO-sensitive guanylate cyclase inhibitor ODQ. It is apparently mediated by a novel extracellular mechanism. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated that AM-produced NO oxidized isoproterenol to generate its aminochrome, namely isoprenochrome. Isoprenochrome amounts were increased 3.62 +/- 1.13-fold in cell culture media (P<0.05). We describe for the first time that AM down-regulates lipolysis in adipocytes through the chemical modification of a beta-agonist.

Uncomplicated human obesity is associated with a specific cardiac transcriptome: involvement of the Wnt pathway.

A dramatic increase in obesity prevalence and cardiovascular morbidity is expected for the coming years. However, with relevance to the heart, little is known about the specific contribution of obesity on associated morbidity. Consequently, global analysis of gene regulations in human heart was undertaken to monitor molecular regulations related to obesity or to obesity-related hypertension. Transcriptome analysis using cDNA arrays was performed in right appendage biopsies from obese patients (n=5), from patients with arterial hypertension with (n=5) or without obesity (n=5), and from 5 leans. All biopsies came from patients that had cardiac surgery and coronary bypass. Statistical analysis of the data revealed 2686 differentially expressed genes out of 11,500 when compared with lean tissues. Differential expression was verified by real-time PCR in 84% of 50 randomly chosen genes. Among genes encountered, 397 were specifically regulated in obese, 1,299 in non-obese hypertensive, and 355 in obese hypertensive patients, respectively, whereas an additional set of 153 genes was differentially expressed in all these situations. Ontology analysis, hierarchical clustering, and molecular pathway analysis indicated that the heart molecular picture of obesity differs clearly from that observed for obesity-related hypertension or arterial hypertension. Clearly, the Wnt pathway known to be involved in cardiac hypertrophy mechanisms, showed opposite regulation in obese heart compared with hypertensive heart and potentially prevented the development of cardiac remodeling in obese patients. All over, this work shows that uncomplicated obesity has a strong impact on cardiac gene expression, which could be considered as precursor signs for future cardiac disease and also demonstrates that obesity-related hypertension generates a heart-molecular-distinct phenotype that cannot be predicted by a simple sum of the impact of obesity and arterial hypertension on gene expression.

Kinetic analysis of cardiac transcriptome regulation during chronic high-fat diet in dogs.

In the present study, we investigated, using custom dog cDNA arrays, the time course of transcriptional changes in the left ventricle of dogs fed a normal diet or a high-fat diet (HFD) for 9-24 wk. Array hybridizations were performed with complex probes representing mRNAs expressed in left ventricles from obese hypertensive and lean control dogs. We identified 63 differentially expressed genes, and expression of 17 of 20 randomly chosen genes was confirmed by real-time PCR. Transcripts were categorized into groups involved in metabolism, cell signaling, tissue remodeling, ionic regulation, cell proliferation, and protein synthesis. Hierarchical clustering indicated that the pattern of coregulated genes depends on duration of the HFD, suggesting that HFD-induced obesity hypertension is associated with continuous cardiac transcriptome adaptation despite stability of both body weight and blood pressure. GenMAPP analysis of the data pointed out the crucial importance of the ventricle TGF-beta pathway. Our results suggest that this system may be involved in molecular remodeling during HFD and in changes observed in the transcription profile, reflecting functional and morphological abnormalities that arise during prolonged HFD. These results also suggest some novel regulatory pathways for cardiac adaptation to obesity.

Adrenomedullin upregulates M2-muscarinic receptors in cardiomyocytes from P19 cell line.

1. The effects of AM on expression of muscarinic (M) receptors from P19-derived cardiomyocytes were examined. 2. RT-PCR experiments revealed expression of M(1)-M(4) receptor genes. Immuno-histochemistry indicated that M(2) expression is restricted to contractile cells. Carbachol inhibition of isoprenaline-induced increase in beating rate was prevented by atropine and methoctramine (pA(2): 8.1). Inhibition of [(3)H]-NMS binding by atropine (pK(i): -8.4+/-0.2) and methoctramine (pK(i): -8.3+/-0.2) suggests that M(2) is the functional expressed isoform. 3. [(3)H]-NMS binding and semiquantitative RT-PCR studies showed a dome shaped time course of M(2) expression with a maximum at 7 days of differentiation followed by a progressive decline. 4. AM concentration-dependently upregulated M(2) receptor mRNA during late differentiation stages in P19 cells but also in rat atrial cardiomyocytes. This effect was potentiated by factor H. AM (100 nM) plus factor H (50 nM) treatment of P19 cells for 24 h significantly increased [(3)H]-NMS-specific binding (B(max): 81+/-7 vs 31+/-6 fmol mg(-1) prot). The effect of AM on mRNA levels was prevented by AM receptor antagonist AM(22-52) (1 micro M) but not by CGRP antagonist, CGRP(8-37) (1 micro M). 5. The mRNA levels encoding CRLR receptor declined with culture duration, whereas those encoding L1/G10D receptor remained stable. 6. Our findings demonstrate that AM regulates M(2) receptors expression in cardiomyocytes probably through a mechanism involving L1/G10D receptors. The 'in vivo' significance of this phenomenon remains to be demonstrated.

Cardiac transcriptome analysis in obesity-related hypertension.

Obesity is associated with volumetric arterial hypertension and with early increase in heart rate and decreased heart rate variability. The consequences of obesity-related hypertension on heart gene regulation are poorly known and were investigated in a model of obesity-related hypertension induced by high fat diet in dogs. When compared with control animals (n=6), a 9-week high fat diet (n=6) provoked significant weight gain and increased blood pressure load and heart rate but failed to significantly change left ventricular mass assessed by echocardiography. Subtractive hybridization of dog heart cDNA libraries were used to generate sublibraries containing differentially expressed cDNAs that were in turn spotted onto membranes to create custom microarrays. Hybridizations of these microarrays with complex probes representing mRNAs expressed in right atria and left ventricles from obese hypertensive and control dogs were performed. Thirty-eight differentially expressed genes were identified; altered expression was confirmed by Northern blot analysis in 15. In addition, real-time quantitative polymerase chain reaction confirmed differential expression for 80% of the randomly chosen tested genes. Once identified, transcripts were categorized into groups involved in metabolism, cell signaling, ionic regulation, cell proliferation, protein synthesis, and tissue remodeling. In addition, we found a set of 11 cDNAs encoding proteins with unknown functions. This study clearly shows that obesity-related hypertension, lasting for only 9 weeks, causes marked changes in gene expression in right atrium as well as the left ventricle that may contribute to early functional changes in heart function and to long-term structural changes such as left ventricular hypertrophy and remodeling.

Transcriptional regulation of adipocyte hormone-sensitive lipase by glucose.

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL mRNA was positively regulated by glucose in human adipocytes. Pools of stably transfected 3T3-F442A adipocytes were generated with human adipocyte HSL promoter fragments from -2,400/+38 to -31/+38 bp linked to the luciferase gene. A glucose-responsive region was mapped within the proximal promoter (-137 bp). Electromobility shift assays showed that upstream stimulatory factor (USF)-1 and USF2 and Sp1 and Sp3 bound to a consensus E-box and two GC-boxes in the -137-bp region. Cotransfection of the -137/+38 construct with USF1 and USF2 expression vectors produced enhanced luciferase activity. Moreover, HSL mRNA levels were decreased in USF1- and USF2-deficient mice. Site-directed mutagenesis of the HSL promoter showed that the GC-boxes, although contributing to basal promoter activity, were dispensable for glucose responsiveness. Mutation of the E-box led to decreased promoter activity and suppression of the glucose response. Analogs and metabolites were used to determine the signal metabolite of the glucose response. The signal is generated downstream of glucose-6-phosphate in the glycolytic pathway before the triose phosphate step.