Manuela Cabiati

Expression of C-type natriuretic peptide and of its receptor NPR-B in normal and failing heart

C-type natriuretic peptide (CNP) was recently found in the myocardium, but possible insights into differences between atrium and ventricle production are so far lacking. Our aim was to evaluate, in an experimental model of pacing-induced heart failure (HF), plasma and tissue levels of CNP and mRNA expression of the peptide and of its specific receptor, NPR-B. Cardiac tissue was collected from male adult minipigs without (control, n=5) and with pacing-induced HF (n=5). Blood samples were collected at baseline and after pacing (10 min, 1, 2, 3 weeks). CNP in plasma and in cardiac extracts was determined by a radioimmunoassay, while the expression of mRNA by real time PCR. Compared to control, plasma CNP was increased after 1 week of pacing stress (36.9+/-10.4 pg/ml vs.16.7+/-1.1, p=0.013, mean+/-S.E.M.). As to myocardial extract, at baseline, CNP was found in all cardiac chambers and its content was 10-fold higher in atria than in ventricles (RA: 13.7+/-1.9 pg/mg protein; LA: 8.7+/-3.8; RV: 1.07+/-0.33; LV: 0.93+/-0.17). At 3 weeks of pacing, myocardial levels of CNP in left ventricle were higher than in controls (15.8+/-9.9 pg/mg protein vs. 0.9+/-0.17, p=0.01). CNP gene expression was observed in controls and at 3 weeks of pacing. NPR-B gene expression was found in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. The co-localization of the CNP system and NPR-B suggests a possible role of CNP in HF and may prompt novel therapeutical strategies.

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP=0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.

Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression

Real-time PCR is the benchmark method for measuring mRNA expression levels, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. Though the minipig model is largely used to study cardiovascular disease, no specific reference genes have been identified in porcine myocardium. The aim of the study was to identify and validate reference gene to be used in RT-PCR studies of failing (HF) and non-failing pig hearts. Eight candidate reference genes (GAPDH, ACTB, B2M, TBP, HPRT-1, PPIA, TOP2B, YWHAZ) were selected to compare cardiac tissue of normal (n=4) and HF (n=5) minipigs. The most stable genes resulted: HPRT-1, TBP, PPIA (right and left atrium); PPIA, GAPDH, ACTB (right ventricle); HPRT-1, TBP, GAPDH (left ventricle). The normalization strategy was tested analyzing mRNA expression of TNF-α, which is known to be up-regulated in HF and whose variations resulted more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance to provide a set of reference genes to normalize mRNA expression in HF and control minipigs. The use of unvalidated reference genes can generate biased results because also their expression could be altered by the experimental conditions.

Trimetazidine reduces endogenous free fatty acid oxidation and improves myocardial efficiency in obese humans.

INTRODUCTION The metabolic modulator trimetazidine (TMZ) has been suggested to induce a metabolic shift from myocardial fatty acid oxidation (FAO) to glucose utilization, but this mechanism remains unproven in humans. The oxidation of plasma derived FA is commonly measured in humans, whereas the contribution of FA from triglycerides stored in the myocardium has been poorly characterized. AIMS To verify the hypothesis that TMZ induces a metabolic shift, we combined positron emission tomography (PET) and magnetic resonance spectroscopy ((1)H-MRS) to measure myocardial FAO from plasma and intracellular lipids, and myocardial glucose metabolism. Nine obese subjects were studied before and after 1 month of TMZ treatment. Myocardial glucose and FA metabolism were assessed by PET with (18)F-fluorodeoxyglucose and (11)C-palmitate. (1)H-MRS was used to measure myocardial lipids, the latter being integrated into the PET data analysis to quantify myocardial triglyceride turnover. RESULTS Myocardial FAO derived from intracellular lipids was at least equal to that of plasma FAs (P = NS). BMI and cardiac work were positively associated with the oxidation of plasma derived FA (P ≤ 0.01). TMZ halved total and triglyceride-derived myocardial FAO (32.7 ± 8.0 to 19.6 ± 4.0 μmol/min and 23.7 ± 7.5 to 10.3 ± 2.7 μmol/min, respectively; P ≤ 0.05). These changes were accompanied by increased cardiac efficiency since unchanged LV work (1.6 ± 0.2 to 1.6 ± 0.1 Watt/g × 10(2), NS) was associated with decreased work energy from the intramyocardial triglyceride oxidation (1.6 ± 0.5 to 0.4 ± 0.1 Watt/g × 10(2), P = 0.036). CONCLUSIONS In obese subjects, we demonstrate that myocardial intracellular triglyceride oxidation significantly provides FA-derived energy for mechanical work. TMZ reduced the oxidation of triglyceride-derived myocardial FAs improving myocardial efficiency.

Comparison of NT-proCNP and CNP plasma levels in heart failure, diabetes and cirrhosis patients

C-type natriuretic peptide (CNP) plasma levels are extremely low and a pre-analytical phase is necessary to assay plasma CNP concentrations. Amino-terminal CNP (NT-proCNP) circulates at higher concentrations than CNP, allowing a direct assay and the use of smaller amounts of plasma. Aim of this study was to evaluate the analytical performance of a direct NT-proCNP assay and to measure its plasma levels in heart failure (CHF), diabetes and chirrosis patients. NT-proCNP and CNP were measured in 130 CHF, 19 patients with diabetes, 24 with hepatic cirrhosis and 73 controls. Plasma NT-proCNP was higher in all the clinical conditions studied (controls:45.5 ± 1.84 pg/ml, CHF:67.09 ± 7.36, diabetes:51.5 ± 5.75 cirrhosis:78.4 ± 19.9; p = 0.034, p = 0.04 controls vs. CHF and cirrhosis, respectively) and in CHF NT-proCNP concentration showed a significant increase as a function of clinical severity. By comparison of ROC curves, CNP assay resulted better associated with disease than NT-proCNP assay in all the different clinical conditions probably due to different release and clearance. The determination of NT-proCNP adds a piece of information to better understanding the molecular mechanisms at the basis of CNP action in different diseases. Due to its higher analytical feasibility, this determination could become widespread in clinical biochemistry laboratories and serve as a complementary marker of disease conditions.

Asymmetrical myocardial expression of natriuretic peptides in pacing-induced heart failure.

High-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive heart failure (HF) with pronounced differences in regional contractility. Aim of this study was to evaluate possible changes in brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) as compared to the infero-septal region (opposite site, OS) and to explore possible association between the contractiling pattern and biomarker expression. Cardiac tissue was collected from minipigs with pacing-induced HF (n=8) and without (control, n=6). The samples were selectively harvested from the anterior left ventricular (LV) wall, PS, and from an area remote to the pacing-site, OS. BNP and CNP mRNA expression was evaluated by semi-quantitative polymerase chain reaction (PCR). A significant difference in BNP expression was found in the PS between HF animals and controls (BNP/GAPDH: 0.65+/-0.11 vs. 0.35+/-0.04, p=0.02), but not in the OS (BNP/GAPDH: 0.36+/-0.05, ns vs. controls). CNP expression was not different compared to controls, although higher levels were observed in the PS and in the OS with respect to the controls (CNP/GAPDH: controls 0.089+/-0.036, PS 0.289+/-0.23, OS 0.54+/-0.16). This finding was in tune with an increase of CNP tissue concentration (controls: 0.69+/-0.13; PS=1.56+/-0.19; OS=1.70+/-0.42 pg/mg protein; p=0.039 controls vs. OS). Higher BNP mRNA expression in the PS is consistent with a reduction in contractile function in this region, while higher CNP mRNA expression in the OS suggests the presence of concomitant endothelial dysfunction in the remote region.

Adiponectin is associated with abnormal lipid profile and coronary microvascular dysfunction in patients with dilated cardiomyopathy without overt heart failure.

Reduced plasma adiponectin has been associated with abnormal lipid profile, reduced left ventricle (LV) function, and the extent of coronary atherosclerosis in coronary artery disease. The aim of this study was to assess these relationships in patients with dilated cardiomyopathy (DCM) without overt heart failure. Plasma adiponectin was measured in 55 DCM patients (age, 59 ± 12 years; male, 36; body mass index [BMI], 26.9 ± 0.49 kg/m²; LV ejection fraction, 39.8% ± 1.3%; New York Heart Association class I-II) and in 40 age- and BMI-matched healthy controls. In a subset of 25 patients, myocardial blood flow (MBF) was measured at rest and during intravenous dipyridamole (0.56 mg/kg in 4 minutes) by positron emission tomography and ¹³N-ammonia as a flow tracer. Adiponectin was 6.6 ± 0.34 μg/mL in controls and 10.9 ± 0.85 μg/mL in DCM patients (P < .001), where it was related inversely with BMI (P = .009) and directly with brain natriuretic peptide (P = .017), high-density lipoprotein (HDL) cholesterol (P = .002), and MBF dipyridamole (P = .020). Adiponectin lesser than median value in patients was associated with higher total to HDL cholesterol ratio (4.8 ± 0.24 vs 3.9 ± 0.18, P = .009) and lower MBF reserve (1.76 ± 0.16 vs 2.43 ± 0.19, P = .01). These results could suggest that down-regulation of the adiponectin levels and reduced HDL cholesterol have a key role in causing impaired coronary function and myocardial perfusion in DCM.

Sequencing and cardiac expression of natriuretic peptide receptor 2 (NPR-B) in Sus Scrofa.

The cardiovascular actions of the C-type natriuretic peptide (CNP) are mainly mediated by the interaction with natriuretic peptide receptor-B (NPR-B). The aim of this study was to identify the sequence of NPR-B in Sus Scrofa, which is not present in GenBank, to verify the expression of NPR-B in the different cardiac chambers of normal pigs and evaluate its homology with murine and human species. Using the guanidinium thyocyanate-phenol-chloroform method, we extracted total RNA from samples obtained from heart of mouse and from the atrium, ventricle, and septum of normal pigs. Pig NPR-B mRNA was sequenced using polymerase chain reaction primers designed from mouse consensus sequences. Sus Scrofa natriuretic peptide receptor 2 mRNA, 1-396 bp, was submitted to GenBank (accession number DQ487044). The presence of NPR-B at mRNA level was detected in all the cardiac chambers; moreover, the bands obtained from pig cardiac tissue shared a 93% sequence homology with a region of the mouse NPR-B and a 95% sequence homology with Homo sapiens. Therefore, NPR-B sequencing provides a new tool to investigate the role of CNP under physiological and pathological conditions in the experimental and clinical setting.

Pacing-Induced Regional Differences in Adenosine Receptors mRNA Expression in a Swine Model of Dilated Cardiomyopathy

The adenosinergic system is essential in the mediation of intrinsic protection and myocardial resistance to insult; it may be considered a cardioprotective molecule and adenosine receptors (ARs) represent potential therapeutic targets in the setting of heart failure (HF). The aim of the study was to test whether differences exist between mRNA expression of ARs in the anterior left ventricle (LV) wall (pacing site: PS) compared to the infero septal wall (opposite region: OS) in an experimental model of dilated cardiomyopathy. Cardiac tissue was collected from LV PS and OS of adult male minipigs with pacing-induced HF (n = 10) and from a control group (C, n = 4). ARs and TNF–α mRNA expression was measured by Real Time-PCR and the results were normalized with the three most stably expressed genes (GAPDH, HPRT1, TBP). Immunohistochemistry analysis was also performed. After 3 weeks of pacing higher levels of expression for each analyzed AR were observed in PS except for A1R (A1R: C = 0.6±0.2, PS = 0.1±0.04, OS = 0.04±0.01, p<0.0001 C vs. PS and OS respectively; A2AR: C = 1.04±0.59, PS = 2.62±0.79, OS = 2.99±0.79; A2BR: C = 1.2±0.1, PS = 5.59±2.3, OS = 1.59±0.46; A3R: C = 0.76±0.18, PS = 8.40±3.38, OS = 4.40±0.83). Significant contractile impairment and myocardial hypoperfusion were observed at PS after three weeks of pacing as compared to OS. TNF-α mRNA expression resulted similar in PS (6.3±2.4) and in OS (5.9±2.7) although higher than in control group (3.4±1.5). ARs expression was mainly detected in cardiomyocytes. This study provided new information on ARs local changes in the setting of LV dysfunction and on the role of these receptors in relation to pacing-induced abnormalities of myocardial perfusion and contraction. These results suggest a possible therapeutic role of adenosine in patients with HF and dyssynchronous LV contraction.

Association of pre-operative interleukin-6 levels with Interagency Registry for Mechanically Assisted Circulatory Support profiles and intensive care unit stay in left ventricular assist device patients

BACKGROUND Inflammatory mechanisms are associated with worse prognosis in end-stage heart failure (ESHF) patients who require left ventricular assist device (LVAD) support. Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles describe patient condition at pre-implant and outcome. This study assessed the relationship among inflammation patterns and INTERMACS profiles in LVAD recipients. METHOD Thirty ESHF patients undergoing LVAD implantation as bridge to transplant were enrolled. Blood and urine samples were collected pre-operatively and serially up to 2 weeks post-operatively for assessment of inflammatory markers (plasma levels of interleukin [IL]-6, IL-8, IL-10, and osteopontin, a cardiac inflammatory-remodeling marker; and the urine neopterin/creatinine ratio, a monocyte activation marker). Multiorgan function was evaluated by the total sequential organ failure assessment (tSOFA) score. Outcomes of interest were early survival, post-LVAD tSOFA score, and intensive care unit (ICU) length of stay. RESULTS Fifteen patients had INTERMACS profiles 1 or 2 (Group A), and 15 had profiles 3 or 4 (Group B). At pre-implant, only IL-6 levels and the IL-6/IL-10 ratio were higher in Group A vs B. After LVAD implantation, neopterin/creatinine ratio and IL-8 levels increased more in Group A vs B. Osteopontin levels increased significantly only in Group B. The tSOFA score at 2 weeks post-LVAD and ICU duration were related with pre-implant IL-6 levels. CONCLUSIONS The INTERMACS profiles reflect the severity of the pre-operative inflammatory activation and the post-implant inflammatory response, affecting post-operative tSOFA score and ICU stay. Therefore, inflammation may contribute to poor outcome in patients with severe INTERMACS profile.