Chiara Caselli

Detection of significant coronary artery disease by noninvasive anatomical and functional imaging.

Background—The choice of imaging techniques in patients with suspected coronary artery disease (CAD) varies between countries, regions, and hospitals. This prospective, multicenter, comparative effectiveness study was designed to assess the relative accuracy of commonly used imaging techniques for identifying patients with significant CAD. Methods and Results—A total of 475 patients with stable chest pain and intermediate likelihood of CAD underwent coronary computed tomographic angiography and stress myocardial perfusion imaging by single photon emission computed tomography or positron emission tomography, and ventricular wall motion imaging by stress echocardiography or cardiac magnetic resonance. If ≥1 test was abnormal, patients underwent invasive coronary angiography. Significant CAD was defined by invasive coronary angiography as >50% stenosis of the left main stem, >70% stenosis in a major coronary vessel, or 30% to 70% stenosis with fractional flow reserve ⩽0.8. Significant CAD was present in 29% of patients. In a patient-based analysis, coronary computed tomographic angiography had the highest diagnostic accuracy, the area under the receiver operating characteristics curve being 0.91 (95% confidence interval, 0.88–0.94), sensitivity being 91%, and specificity being 92%. Myocardial perfusion imaging had good diagnostic accuracy (area under the curve, 0.74; confidence interval, 0.69–0.78), sensitivity 74%, and specificity 73%. Wall motion imaging had similar accuracy (area under the curve, 0.70; confidence interval, 0.65–0.75) but lower sensitivity (49%, P<0.001) and higher specificity (92%, P<0.001). The diagnostic accuracy of myocardial perfusion imaging and wall motion imaging were lower than that of coronary computed tomographic angiography (P<0.001). Conclusions—In a multicenter European population of patients with stable chest pain and low prevalence of CAD, coronary computed tomographic angiography is more accurate than noninvasive functional testing for detecting significant CAD defined invasively. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00979199.

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.

Tissue-specific selection of stable reference genes for real-time PCR normalization in an obese rat model

Obesity is a complex pathology with interacting and confounding causes due to the environment, hormonal signaling patterns, and genetic predisposition. At present, the Zucker rat is an eligible genetic model for research on obesity and metabolic syndrome, allowing scrutiny of gene expression profiles. Real-time PCR is the benchmark method for measuring mRNA expressions, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. In the Zucker rat model, no specific reference genes have been identified in myocardium, kidney, and lung, the main organs involved in this syndrome. The aim of this study was to select among ten candidates (Actb, Gapdh, Polr2a, Ywhag, Rpl13a, Sdha, Ppia, Tbp, Hprt1 and Tfrc) a set of reference genes that can be used for the normalization of mRNA expression data obtained by real-time PCR in obese and lean Zucker rats both at fasting and during acute hyperglycemia. The most stable genes in the heart were Sdha, Tbp, and Hprt1; in kidney, Tbp, Actb, and Gapdh were chosen, while Actb, Ywhag, and Sdha were selected as the most stably expressed set for pulmonary tissue. The normalization strategy was used to analyze mRNA expression of tumor necrosis factor α, the main inflammatory mediator in obesity, whose variations were more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance of having three stably expressed reference gene sets for use in the cardiac, renal, and pulmonary tissues of an experimental model of obese and hyperglycemic Zucker rats.

Multicentre multi-device hybrid imaging study of coronary artery disease: results from the EValuation of INtegrated Cardiac Imaging for the Detection and Characterization of Ischaemic Heart Disease (EVINCI) hybrid imaging population

AIMS Hybrid imaging provides a non-invasive assessment of coronary anatomy and myocardial perfusion. We sought to evaluate the added clinical value of hybrid imaging in a multi-centre multi-vendor setting. METHODS AND RESULTS Fourteen centres enrolled 252 patients with stable angina and intermediate (20-90%) pre-test likelihood of coronary artery disease (CAD) who underwent myocardial perfusion scintigraphy (MPS), CT coronary angiography (CTCA), and quantitative coronary angiography (QCA) with fractional flow reserve (FFR). Hybrid MPS/CTCA images were obtained by 3D image fusion. Blinded core-lab analyses were performed for CTCA, MPS, QCA and hybrid datasets. Hemodynamically significant CAD was ruled-in non-invasively in the presence of a matched finding (myocardial perfusion defect co-localized with stenosed coronary artery) and ruled-out with normal findings (both CTCA and MPS normal). Overall prevalence of significant CAD on QCA (>70% stenosis or 30-70% with FFR≤0.80) was 37%. Of 1004 pathological myocardial segments on MPS, 246 (25%) were reclassified from their standard coronary distribution to another territory by hybrid imaging. In this respect, in 45/252 (18%) patients, hybrid imaging reassigned an entire perfusion defect to another coronary territory, changing the final diagnosis in 42% of the cases. Hybrid imaging allowed non-invasive CAD rule-out in 41%, and rule-in in 24% of patients, with a negative and positive predictive value of 88% and 87%, respectively. CONCLUSION In patients at intermediate risk of CAD, hybrid imaging allows non-invasive co-localization of myocardial perfusion defects and subtending coronary arteries, impacting clinical decision-making in almost one every five subjects.

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.

A possible cardioprotective effect of heat shock proteins during cardiac surgery in pediatric patients.

BACKGROUND Overexpression of heat shock proteins (Hsps) is associated to myocardial protection and it has been suggested that they could be a marker of cardiac preservation in conditions such as extracorporeal circulation. Aim of this study was to evaluate if cardioplegic arrest can modify the expression of Hsps in the heart and if this alteration is associated to cardiac preservation. METHOD The levels of Hsp 27, Hsp 60, and both the constitutive and the inducible form of Hsp 70 were measured in the cardiac tissue from right atrium of pediatric patients before and after aortic cross-clamping (ACC) during cardiopulmonary bypass surgery for correction of congenital heart disease (n=20). The quantitative evaluation of Hsps was made by Western blotting analysis after tissue extraction and protein separation. Hsp 72 mRNA expression was also evaluated in pre- and post-ACC samples of eight subjects by semiquantitative RT-PCR. Peripheral levels of Troponin I, Myoglobin, LDH, CK, CK-MB were measured in basal conditions and at 12 and 24h after cardiosurgery as markers of heart damage. RESULTS The cardioplegic arrest did not significantly modify the mean levels of all the Hsps measured. Hsp 72 levels increased after cardioplegia in the 40% of the patients and all Hsps in the 28% of subjects. The patients whose levels of Hsps are increased after cardioplegia are associated with lower post-surgery concentrations of all the markers of cardiac injury. CONCLUSIONS This observation suggests a relationship between the increase of Hsps and the reduction of cardiac injury.

Back to the heart: the protective role of adiponectin.

Cardiovascular disease (CVD) is the leading cause of death worldwide and the prevalence of obesity and diabetes are increasing. In obesity, adipose tissue increases the secretion of bioactive mediators (adipokines) that may represent a key mechanism linking obesity to CVD. Adiponectin, extensively studied in metabolic diseases, exerts anti-diabetic, anti-atherogenic and anti-inflammatory activities. Due to these positive actions, the role of adiponectin in cardiovascular protection has been evaluated in recent years. In particular, for its potential therapeutic benefits in humans, adiponectin has become the subject of intense preclinical research. In the cardiovascular context, understanding of the cellular and molecular mechanisms underlying the adiponectin system, throughout its secretion, regulation and signaling, is critical for designing new drugs that target adiponectin system molecules. This review focused on recent advances regarding molecular mechanisms related to protective effects of the adiponectin system on both cardiac and vascular compartments and its potential use as a target for therapeutic intervention of CVD.

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.