Effects of obesity on IL-33/ST2 system in heart, adipose tissue and liver: study in the experimental model of Zucker rats.

Suppression of tumorigenicity 2 (ST2) mediates the effect of Interleukin-33 (IL-33). Few data are reported on the relationship between IL-33/ST2 and obesity. We aimed to investigate effects of obesity on IL-33/ST2 system in heart, adipose tissue and liver in a rodent model of obesity. The relationship of cardiac expression of IL-33/ST2 system with natriuretic peptides (NPs) system and inflammatory mediators was also studied. mRNA expression of IL-33/ST2 system was evaluated in cardiac, adipose and hepatic biopsies from obese Zucker rats (O) and controls (CO). Expression levels of sST2 was significantly lower in O rats compared with CO (p<0.05) in all tissues. Besides, the mRNA levels of IL-33 decreased significant in fat of O respect to CO, while, expression levels of ST2L was significantly higher in liver of CO than in O. A strong relationship of IL-33/ST2 with NPs and classical inflammatory mediators was observed in cardiac tissue. Expression of sST2 in cardiac, adipose and liver tissue decreased in O compared with controls, suggesting an involvement for IL-33/ST2 system in molecular mechanisms of obesity. The strong relationships with NP systems and inflammatory mediators could suggest an involvement for IL-33/ST2 in molecular pathways leading to cardiac dysfunction and inflammation associated with obesity.

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.

Effect of Coronary Atherosclerosis and Myocardial Ischemia on Plasma Levels of High-Sensitivity Troponin T and NT-proBNP in Patients With Stable Angina.

OBJECTIVE: Circulating levels of high-sensitivity cardiac troponin T (hs-cTnT) and N terminal pro brain natriuretic peptide (NT-proBNP) are predictors of prognosis in patients with coronary artery disease (CAD). We aimed at evaluating the effect of coronary atherosclerosis and myocardial ischemia on cardiac release of hs-cTnT and NT-proBNP in patients with suspected CAD. APPROACH AND RESULTS: Hs-cTnT and NT-proBNP were measured in 378 patients (60.1±0.5 years, 229 males) with stable angina and unknown CAD enrolled in the Evaluation of Integrated Cardiac Imaging (EVINCI) study. All patients underwent stress imaging to detect myocardial ischemia and coronary computed tomographic angiography to assess the presence and characteristics of CAD. An individual computed tomographic angiography score was calculated combining extent, severity, composition, and location of plaques. In the whole population, the median (25-75 percentiles) value of plasma hs-cTnT was 6.17 (4.2-9.1) ng/L and of NT-proBNP was 61.66 (31.2-132.6) ng/L. In a multivariate model, computed tomographic angiography score was an independent predictor of the plasma hs-cTnT (coefficient 0.06, SE 0.02; P=0.0089), whereas ischemia was a predictor of NT-proBNP (coefficient 0.38, SE 0.12; P=0.0015). Hs-cTnT concentrations were significantly increased in patients with CAD with or without myocardial ischemia (P<0.005), whereas only patients with CAD and ischemia showed significantly higher levels of NT-proBNP (P<0.001). CONCLUSIONS: In patients with stable angina, the presence and extent of coronary atherosclerosis is related with circulating levels of hs-cTnT, also in the absence of ischemia, suggesting an ischemia-independent mechanism of hs-cTnT release. Obstructive CAD causing myocardial ischemia is associated with increased levels of NT-proBNP.

A New Integrated Clinical-Biohumoral Model to Predict Functionally Significant Coronary Artery Disease in Patients With Chronic Chest Pain.

BACKGROUND: In patients with chronic angina-like chest pain, the probability of coronary artery disease (CAD) is estimated by symptoms, age, and sex according to the Genders clinical model. We investigated the incremental value of circulating biomarkers over the Genders model to predict functionally significant CAD in patients with chronic chest pain. METHODS: In 527 patients (60.4 years, standard deviation, 8.9 years; 61.3% male participants) enrolled in the European Evaluation of Integrated Cardiac Imaging (EVINCI) study, clinical and biohumoral data were collected. RESULTS: Functionally significant CAD-ie, obstructive coronary disease seen at invasive angiography causing myocardial ischemia at stress imaging or associated with reduced fractional flow reserve (FFR < 0.8), or both-was present in 15.2% of patients. High-density lipoprotein (HDL) cholesterol, aspartate aminotransferase (AST) levels, and high-sensitivity C-reactive protein (hs-CRP) were the only independent predictors of disease among 31 biomarkers analyzed. The model integrating these biohumoral markers with clinical variables outperformed the Genders model by receiver operating characteristic curve (ROC) (area under the curve [AUC], 0.70 [standard error (SE), 0.03] vs 0.58 [SE, 0.03], respectively, P < 0.001) and reclassification analysis (net reclassification improvement, 0.15 [SE, 0.07]; P = 0.04). Cross-validation of the ROC analysis confirmed the discrimination ability of the new model (AUC, 0.66). As many as 56% of patients who were assigned to a higher pretest probability by the Genders model were correctly reassigned to a low probability class (< 15%) by the new integrated model. CONCLUSIONS: The Genders model has a low accuracy for predicting functionally significant CAD. A new model integrating HDL cholesterol, AST, and hs-CRP levels with common clinical variables has a higher predictive accuracy for functionally significant CAD and allows the reclassification of patients from an intermediate/high to a low pretest likelihood of CAD.

HDL cholesterol, leptin and interleukin-6 predict high risk coronary anatomy assessed by CT angiography in patients with stable chest pain.

OBJECTIVES: Coronary computed tomography angiography (CTA) describes several features of coronary plaques, i.e. location, severity, and composition. Integrated CTA scores are able to identify individual patterns of higher risk. We sought to test whether circulating biomarkers related with metabolism and inflammation could predict high risk coronary anatomy at CTA in patients with stable chest pain. METHODS: We evaluated a panel of 17 biomarkers in 429 patients (60.3 ± 0.4 years, 268 males) with stable chest pain who underwent coronary CTA having been enrolled in the Evaluation of Integrated Cardiac Imaging (EVINCI) study. The individual CTA risk score was calculated combining plaque extent, severity, composition, and location. The presence and distribution of non-calcified, mixed and calcified plaques were analyzed in each patient. RESULTS: After adjustment for age, sex and medical treatment, high-density lipoprotein (HDL) cholesterol, leptin, and interleukin-6 (IL-6) were independent predictors of CTA risk score at multivariate analysis (P = 0.050, 0.002, and 0.007, respectively). Integrating these biomarkers with common clinical variables, a model was developed which showed a better discriminating ability than the Framingham Risk Score and the Euro-SCORE in identifying the patients with higher CTA risk score (area under the receiver-operating characteristics curve = 0.81, 0.63 and 0.71, respectively, P < 0.001). These three biomarkers were significantly altered in patients with mixed or non-calcified plaques. CONCLUSIONS: In patients with stable chest pain, low HDL cholesterol, low leptin and high IL-6 are independent predictors of high risk coronary anatomy as defined by an integrated CTA risk score.

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.

Caspase-1 transcripts in failing human heart after mechanical unloading.

BACKGROUND: Caspase (Casp)-1 has been indicated as a molecular target capable of preventing the progression of cardiovascular diseases, including heart failure (HF), due to its central role in promoting inflammation and cardiomyocyte loss. The aim of this study was to assess whether Left Ventricular Assist Device (LVAD) implantation modifies the inflammatory and apoptotic profile in the heart through the modulation of Casp-1 expression level. METHODS: Cardiac tissue was collected from end-stage HF patients before LVAD implant (pre-LVAD group, n=22) and at LVAD removal (post-LVAD, n=6), and from stable HF patients on medical therapy without prior circulatory support (HTx, n=7) at heart transplantation, as control. The cardiac expression of Casp-1, of its inhibitors caspase recruitment domain (CARD) only protein (COP) and CARD family, member 18 (ICEBERG), was evaluated by real-time PCR in the three groups of patients. RESULTS: Casp-1 was increased in the pre-LVAD group compared to HTx (p=0.006), while on the contrary the ICEBERG level was significantly decreased in pre-LVAD with respect to HTx patients (p<0.001); no difference in COP expression level was found. CONCLUSIONS: This study describes a specific pattern of the Casp-1 system associated with inflammation and apoptosis markers in patients who require LVAD insertion. The inflammation could be the key process regulating, in a negative loop, Casp-1 signaling and its down-stream effects, apoptosis included.

Mind injuries after cardiac surgery.

After cardiac surgery, delirium, cognitive dysfunction, depression, or anxiety disorders frequently occur, and profoundly affect patients' prognosis and quality of life. This narrative review focuses on the main clinical presentations of cognitive and psychological problems ('mind injuries') that occur postoperatively in absence of ascertainable focal neurologic deficits, exploring their pathophysiological mechanisms and possible strategies for prevention and treatment. Postoperative cognitive dysfunction is a potentially devastating complication that can involve several mechanisms and several predisposing, intraoperative, and postoperative risk factors, which can result in or be associated to cerebral microvascular damage. Postoperative depression is influenced by genetic or psychosocial predisposing factors, by neuroendocrine activation, and by the release of several pro-inflammatory factors. The net effect of these changes is neuroinflammation. These complex biochemical alterations, along with an aspecific response to stressful life events, might target the function of several brain areas, which are thought to represent a trigger factor for the onset of depression.

Transcriptional alterations of ET-1 axis and DNA damage in lung tissue of a rat obesity model.

Obesity has been implicated in the development of many cancers. This can lead to genome damage, especially in the form of double-strand break, the presence of which is now easily detected through nuclear phosphorylation of histone H2AX (γ-H2AX) focus assay. Recently, the endothelin (ET) axis has also been shown to have a role in the growth and progression of several tumors, including lung cancer. The aim of this study was to evaluate the ET-1 system transcriptional alterations and γ-H2AX in lung tissue of Zucker rats subdivided into obese (O, n=22) and controls (CO, n=18) rats: under either fasting conditions (CO(fc)-O(fc)) or acute hyperglycemia (CO(AH)-O(AH)). Significantly higher prepro-ET-1 (p=0.05) and ET-converting enzyme (ECE)-2 mRNA expression was observed in O with respect to CO. A significant positive association was observed between prepro-ET-1 and ET-A in the whole rat population (p=0.009) or in the obese group alone (p=0.007). The levels of γ-H2AX in O and in O(AH) rats were significantly higher (p=0.019) than in the corresponding CO and CO(AH) rats (p=0.038). The study shows an inappropriate secretion of ET-1 in O animals with a parallel DNA damage in their lungs, providing novel mechanisms by which ET receptor antagonist may exert organ protection.

Myocardial interleukin-6 in the setting of left ventricular mechanical assistance: relation with outcome and C-reactive protein.

BACKGROUND: In left ventricular assist device (LVAD) recipients, plasma levels of interleukin (IL)-6 are associated with Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles, reflecting post-operative risk. However, it is not clear how the cardiac level of IL-6, detectable on the tissue samples at the time of implantation, can contribute to predict the post-operative outcome. METHODS: In 40 LVAD recipients, blood and myocardial samples from LV-apex were collected at the time of implantation to assess plasma and cardiac IL-6 levels. Serum C-reactive protein (CRP) levels were considered as inflammatory variable routinely used in LVAD-based therapy. RESULTS: Cardiac IL-6 levels did not correlate with either plasma IL-6 levels (R=0.296, p=0.063) and tissue IL-6 mRNA expression (R=-0.013, p=0.954). Contrary to what happened for the plasma IL-6 and CRP, no differences were observed in cardiac IL-6 levels with respect to INTERMACS profiles (p=0.090). Furthermore, cardiac IL-6 concentrations, unlike IL-6 and CRP circulating levels, were not correlated with the length of intensive care unit stay and hospitalization. CONCLUSIONS: Cardiac IL-6 levels do not contribute to improve risk profile of LVAD recipients in relation to clinical inpatient post-implantation. Instead, plasma IL-6 and serum CRP concentrations are more effective in predicting the severity of the clinical course in the early phase of LVAD therapy.

Uncovering the cathepsin system in heart failure patients submitted to Left Ventricular Assist Device (LVAD) implantation.

BACKGROUND: In end-stage heart failure (HF), the implantation of a left ventricular assist device (LVAD) is able to induce reverse remodeling. Cellular proteases, such as cathepsins, are involved in the progression of HF. The aim of this study was to evaluate the role of cathepsin system in HF patients supported by LVAD, in order to determine their involvement in cardiac remodeling. METHODS: The expression of cysteine (CatB, CatK, CatL, CatS) and serine cathepsin (CatG), and relative inhibitors (Cystatin B, C and SerpinA3, respectively) was determined in cardiac biopsies of 22 patients submitted to LVAD (pre-LVAD) and compared with: 1) control stable chronic HF patients on medical therapy at the moment of heart transplantation without prior LVAD (HT, n = 7); 2) patients supported by LVAD at the moment of transplantation (post-LVAD, n = 6). RESULTS: The expression of cathepsins and their inhibitors was significantly higher in pre-LVAD compared to the HT group and LVAD induced a further increase in the cathepsin system. Significant positive correlations were observed between cardiac expression of cathepsins and their inhibitors as well as inflammatory cytokines. In the pre-LVAD group, a relationship of cathepsins with dilatative etiology and length of hospitalization was found. CONCLUSIONS: A parallel activation of cathepsins and their inhibitors was observed after LVAD support. The possible clinical importance of these modifications is confirmed by their relation with patients' outcome. A better discovery of these pathways could add more insights into the cardiac remodeling during HF.

Cardiac molecular markers of programmed cell death are activated in end-stage heart failure patients supported by left ventricular assist device.

BACKGROUND: Cardiomyocyte apoptosis increases in heart failure (HF) and is implicated in disease progression. The apoptotic cell is not inevitably committed to death, and appropriate therapy like left ventricular assist device (LVAD) support could offer a rescue of cellular functions. Literature data regarding the modulation of the apoptotic process during LVAD support are still controversial. METHODS: To assess whether LVAD implantation modifies the apoptotic profile in the heart, cardiac tissue was collected from end-stage HF patients before LVAD implant (pre-LVAD, n=22) and at LVAD removal (post-LVAD, n=6) and from stable HF patients on medical therapy without prior circulatory support (HTx, n=7) at heart transplantation as control. Caspase (Casp)-3, Bax, Bcl-2, and Hsp72 cardiac mRNA and protein expression were evaluated by real-time polymerase chain reaction and Western blotting (WB) in the three groups of patients. Immunohistochemical analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and DNA laddering analysis were performed; cellular size and interstitial fibrosis content were also determined. RESULTS: All the apoptotic indices were increased in the post-LVAD group compared to pre-LVAD, specially antiapoptotic Hsp72 and proapoptotic Bax (Hsp72: 3.27±0.41 vs. 0.76±0.14, P<.001; Bax: 2.15±0.38 vs. 1.10 ± 0.29, P=.035; post-LVAD vs. pre-LVAD, respectively). The significant increase in Hsp72 was confirmed by WB and immunohistochemical analysis. CONCLUSION: LVAD appears to induce an activation of apoptotic mediators, mainly at the mitochondrial level, while the following activation of Casp-3 is reduced by the significant increase of Hsp72, whose enhancement could be an important factor in cardiac remodeling associated with LVAD support.

Reappraisal of quantitative gel zymography for matrix metalloproteinases.

BACKGROUND: The determination of matrix metalloproteases (MMPs) is relevant in many pathophysiological conditions, especially if associated with extracellular matrix remodeling; however, the results obtained are closely linked to the method used and are not directly comparable. The aim of this study was to perform a reappraisal of quantitative gel zymography technique for MMPs in human plasma, to use for comparison with commercially available ELISA and in those experimental conditions where the MMP active form needs to be revealed. METHODS: The critical methodological parameters of zymography were checked and a comparison with a routinely used ELISA was performed. RESULTS: Sensitivity and reproducibility levels of zymography are suitable for detection of MMP-9 in human plasma, providing results closely related to those obtained by ELISA. CONCLUSIONS: Analytical parameters of zymography were suitable for detection of MMPs in human plasma. Quantitative zymography for MMPs is an alternative method for comparing the results of ELISA widely employed for MMP determination, thus reducing the discrepancies between laboratories regarding gelatinase assay.

Adrenomedullin and intermedin gene transcription is increased in leukocytes of patients with chronic heart failure at different stages of the disease.

Adrenomedullin (ADM) is a vasodilatory peptide expressed in many tissues. Its levels are elevated in various diseases including chronic heart failure (CHF) and it has been suggested that the up-regulation of ADM in cardiac disease represents a protective mechanism. Similarly, intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide family, is considered a potential endogenous protector of the heart. Previous studies demonstrated that in CHF patients, elevated plasma concentrations of ADM and IMD reflect the patient's disease severity and prognosis, while the behavior of mRNA expression is not known. The aim of this study was to evaluate ADM/IDM transcriptomic profiling in human leukocytes of CHF patients as a function of clinical severity, assessing possible changes with respect to healthy subjects (C). mRNA expression was evaluated by Real-Time PCR and total RNA was extracted from leukocytes of C (n=8) and from CHF patients (NYHA I-II n=10; NYHA III-IV n=14) with PAXgene Blood RNA Kit. Significantly higher levels of ADM and IMD mRNA were found in CHF as a function of clinical severity (ADM: C=0.03 ± 0.013, NYHA I-II=0.11 ± 0.084, NYHA III-IV=11.46 ± 4.72, p=0.037 C vs NYHA III-IV, p=0.028 NYHA I-II vs NYHA III-IV; IMD: C=0.158 ± 0.041, NYHA I-II=0.93 ± 0.40, NYHA III-IV=2.6 ± 0.67, p=0.014 C vs NYHA III-IV, p=0.014 NYHA I-II vs NYHA III-IV). This study highlights, for the first time, the possibility of evaluating ADM and IMD mRNA expression in human whole blood samples by Real-Time PCR study providing further relevant information and providing a more complete interpretation of the pathophysiology of the disease.

Impact of obesity on the expression profile of natriuretic peptide system in a rat experimental model.

Natriuretic peptides (NPs) play an important role in obesity and aim of this study was to evaluate, in cardiac tissue of obese Zucker rats (O, n = 29) their transcriptomic profile compared to controls (CO, n = 24) by Real-Time PCR study; CNP protein expression was evaluated by immunostaining and immunometric tests. Myocardial histology was performed, confirming no alteration of organ structure. While ANP and BNP are cardiac peptides, CNP is mainly an endothelial hormone; thus its expression, as well as that of NPR-B and NPR-C, was also evaluated in kidney and lung of an animal subgroup (n = 20). In heart, lower BNP mRNA levels in O vs CO (p = 0.02) as well as ANP and CNP (p = ns), were detected. NPR-B/NPR-A mRNA was similar in O and CO, while NPR-C was numerically lower (p = ns) in O than in CO. In kidney, CNP/NPR-B/NPR-C mRNA was similar in O and CO, while in lung CNP/NPR-C expression decreased and NPR-B increased (p = ns) in O vs CO. Subdividing into fasting and hyperglycemic rats, the pattern of mRNA expression for each gene analyzed remained unchanged. The trend observed in heart, kidney and lung for CNP protein concentrations and immunohistochemistry reflected the mRNA expression. TNF-α and IL-6 mRNA were measured in each tissue and no significant genotype effect was detected in any tissue. The main NP variations were observed at the cardiac level, suggesting a reduced release by cardiac cells. The understanding of mechanisms involved in the modulation of the NP system in obesity could be a useful starting point for future clinical study devoted to identifying new obesity treatment strategies.

High peripheral levels of h-FABP are associated with poor prognosis in end-stage heart failure patients with mechanical circulatory support.

AIM: To associate the time-course of h-FABP and N-terminal pro B-type natriuretic peptide (NT-proBNP)after left ventricular assist device (LVAD) implantation to outcome in end-stage heart failure patients. MATERIALS & METHODS: Patients (n = 14, NYHA class III/IV; left ventricular ejection fraction <25% were enrolled; ten survived up to 1 month after LVAD (survivors) and four died of multiorgan failure within 2 weeks (nonsurvivors). Blood samples were obtained at admission; at 4, 24 and 72 h; and at 1 and 4 weeks after LVAD. RESULTS: h-FABP significantly increases after surgery, decreasing since 72 h in all patients. At 72 h all survivor patients present h-FABP lower than the median value. N-terminal pro B-type natriuretic peptide is not associated with patient outcome at any time. CONCLUSION: High h-FABP levels, indicating the presence of more severe myocardial damage, are associated with a poor prognosis in patients with LVAD, suggesting that an early cardiac injury marker could improve the prediction of clinical outcome.

Impact of normalization strategy on cardiac expression of pro-inflammatory cytokines: evaluation of reference genes in different human myocardial regions after Left Ventricular Assist Device support.

OBJECTIVE: New device therapies have expanded the strategies for treating heart failure (HF) patients. Unloading of the heart with a left ventricular assist device (LVAD) can lead to the reversal of many remodeling changes whose underlying mechanisms are not yet completely known. Molecular analysis might play a role in obtaining further insight into the regulatory mechanisms of this process. A critical step in an RT-PCR study is the selection of reference genes for data normalization. This study aimed to determine an optimal combination of stably expressed reference genes in different regions of the human heart in order to study the effects of LVAD implants on cardiac remodeling, and in particular to check their reliability on the evaluation of pro-inflammatory cytokine expression. DESIGN AND METHODS: We validated nine of the most commonly used reference genes in human myocardium samples obtained at heart transplantation from patients with LVAD implant (n=30 from a total of six patients) and from heart transplant (HT from a total of seven patients) recipients as controls (n=35). Samples from both left (LV) and right (RV) ventricles were analyzed. The normalization strategy was tested by analyzing mRNA expression of IL-6, IL-8 and TNF-α, whose protein levels were measured by immunometric assay. RESULTS: The most stable gene combinations changed according to the experimental groups (the LVAD and HT groups and the different myocardial regions). Considering all the cardiac samples as a whole, the three most stably expressed genes were PPIA, RPL13A, and YWHAZ (M=0.70). Using the best normalization strategy, a significant increase in IL-6, IL-8 mRNA expression was observed in LVAD samples compared to HT (p<0.0001). Similar results were obtained by protein analysis. CONCLUSIONS: Our results underline the importance of always selecting reference genes for the specific system studied. The most appropriate normalization strategy is of pivotal importance for understanding the molecular mechanisms associated with the pathophysiology of HF, such as inflammation.

Apoptotic transcriptional profile remains activated in late remodeled left ventricle after myocardial infarction in swine infarcted hearts with preserved ejection fraction.

Apoptosis is involved in both acute and chronic loss of cardiomyocytes after myocardial infarction (MI). To date, the pathophysiological significance of an apoptotic transcriptional profile activated in the post-ischemic remodeled myocardium, in the absence of hemodynamic factors secondary to left ventricular (LV) dysfunction, still remains to be determined. The mRNA expression of pro- and anti-apoptotic factors was determined in a swine model of non-reperfused MI with preserved LV ejection fraction. The extent of cell death was evaluated by histological analysis. Male adult farm pigs with MI (n=5), induced by permanent surgical ligation of the left anterior descending coronary artery and sham-operated adult farm pigs as control (n=7) were studied. Tissue samples were collected from the border (BZ) and remote zone (RZ) of the infarcted area to identify possible regional effects. After 4 weeks post-MI, the infarct size was 13±1% of the LV wall mass in absence of contractile dysfunction. In BZ, there was increased mRNA expression of Casp-3 (BZ vs Controls: 0.51±0.15 vs 1.39±0.04, p<0.001), a significant decrease in Bcl-2 (by 63%), associated with an increase in apoptotic cells, as revealed by TUNEL staining and cleaved-Casp-3 presence. In contrast, in the RZ there was a significant reduction of pro-apoptotic factors compared to BZ (by 80% for Casp-3), in presence of scattered apoptotic cells, increased gene expression of Hsp72 (1.82±0.21 vs 1.34±0.08, p=0.037) and iNOS (1.51±0.14 vs 1.19±0.05, p<0.05) compared to control. In conclusion, the LV distribution of apoptotic transcriptional profile revealed that apoptotic cell death is highly detectable in BZ, possibly explaining the local abnormalities of myocardial cell survival in a porcine model of MI with normal overall function.

Regional evidence of modulation of cardiac adiponectin level in dilated cardiomyopathy: pilot study in a porcine animal model.

BACKGROUND: The role of systemic and myocardial adiponectin (ADN) in dilated cardiomyopathy is still debated. We tested the regulation of both systemic and myocardial ADN and the relationship with AMP-activated protein kinase (AMPK) activity in a swine model of non-ischemic dilated cardiomyopathy. METHODS AND RESULTS: Cardiac tissue was collected from seven instrumented adult male minipigs by pacing the left ventricular (LV) free wall (180 beats/min, 3 weeks), both from pacing (PS) and opposite sites (OS), and from five controls. Circulating ADN levels were inversely related to global and regional cardiac function. Myocardial ADN in PS was down-regulated compared to control (p < 0.05), yet ADN receptor 1 was significantly up-regulated (p < 0.05). No modifications of AMPK were observed in either region of the failing heart. Similarly, myocardial mRNA levels of PPARγ, PPARα, TNFα, iNOS were unchanged compared to controls. CONCLUSIONS: Paradoxically, circulating ADN did not show any cardioprotective effect, confirming its role as negative prognostic biomarker of heart failure. Myocardial ADN was reduced in PS compared to control in an AMPK-independent fashion, suggesting the occurrence of novel mechanisms by which reduced cardiac ADN levels may regionally mediate the decline of cardiac function.

Abnormal glucose and lipid control in non-ischemic left ventricular dysfunction.

BACKGROUND: Cardiovascular risk factors are classically associated with coronary atherosclerosis. We sought to investigate whether risk factors are also associated with left ventricular (LV) dilatation, contractile impairment and reduced myocardial blood flow (MBF) in patients with non-ischemic LV dysfunction. METHODS: We studied 81 patients (59 males, age 60 ± 9 years) with mild-to-severe LV dysfunction (mean ejection fraction 37%, range 19%-50%), no history of diabetes and normal coronary arteries. Absolute MBF was measured by positron emission tomography and (13)N-ammonia at rest and after dipyridamole (0.56 mg/kg I.V. over 4 min). RESULTS: Overt LV dysfunction (LV end-diastolic diameter >60 mm associated with LV ejection fraction <45%) was present in 42 patients (52%); severely depressed hyperemic MBF (<1.09 mL · min(-1) · g(-1)) was present in 41 patients (51%). Using multivariate logistic regression analysis, low high-density lipoprotein cholesterol (HDL-C, P < .036), newly diagnosed non-insulin-dependent diabetes or insulin-resistance (NIDD/IR, P < .019) and the use of diuretics (P = .001) were independently associated with overt LV dysfunction. Low HDL-C (P = .015) and NIDD/IR (P = .048) were also independently associated with severely depressed hyperemic MBF. CONCLUSIONS: Low HDL-C and NIDD/IR are associated with more severe LV impairment and reduced hyperemic MBF in non-ischemic LV dysfunction.