113 research outputs found
Evaluation of the expression of transcripts coding for CNP and for its specific receptor, NPR-B, by Real Time PCR in cardiac tissue of normal and heart failure animals
Purpose: Higher plasma levels and a cardiac production of C-type natriuretic peptide (CNP) were recently observed in patients with chronic heart failure (HF), but its cellular source and possible difference between atrium and ventricle expression are so far lacking. Aim of this study was to evaluate the expression of transcripts coding for CNP and for its specific receptor, NPR-B, in cardiac tissue (right and left atrium and ventricle) of normal and CHF animals. CNP tissue levels were also determined in cardiac extracts. Methods: Adult male minipigs (n=5) were chronically instrumented with a unipolar pacemaker connected to the anterior left ventricular (LV) wall. HF was induced by rapid pacing (180 beats/min) for 4 weeks. End-stage HF occurred at 24?2 days of pacing when the LV end-diastolic pressure was !25 mmHg. As control, we studied 5 adult male minipigs. At 4 weeks, myocardial samples were collected. Both CNP mRNA and proteins were extracted from a same sample with the method of phenol/guanidine-thiocyanate/chloroform. Tissue CNP levels were determined by a radioimmunoassay after a preliminary extraction on Sep-Pak C18, while the expression of mRNA coding for CNP and NPR-B in myocardial tissue (n=40) by Real Time reverse transcriptase-polymerase chain reaction (PCR) with DDCt method. As overall control, a parallel Real Time-PCR assay for BNP mRNA expression was carried out in the same samples. Real Time-PCR analysis was performed using an automated sequence instrument (7900HT Fast, Applied Biosystems) for the real-time monitoring of nucleic acid green dye fluorescence (SYBR Green I). Results: As to myocardial extracts, CNP was found in all cardiac chambers of controls and its content was ten fold higher in atria than in ventricles (RA: 13.7?1.9 pg/mg; LA: 8.7?3.8 pg/mg; RV: 1.07?0.33 pg/mg; LV: 0.93?0.17 pg/mg). At 4 weeks of pacing stress, myocardial levels of CNP in LV were higher than in controls (15.8?9.9 pg/mg vs.0.9?0.17 pg/mg, p=0.01). The expression of mRNA coding for CNP was higher at 4 weeks of pacing althought CNP gene expression appears to be noticeable lower than that of BNP. The NPR-B resulted to be expressed in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. Althought further investigations are necessary, the high tissue levels of CNP found after pacing stress as well as the myocardial CNP and NPR-B expression suggest an important role of this peptide in a so complex pathology as HF
Evaluation of the expression of transcripts coding for CNP and for its specific receptor, NPR-B, by Real Time PCR in cardiac tissue of normal and heart failure animals
Purpose: Higher plasma levels and a cardiac production of C-type natriuretic peptide (CNP) were recently observed in patients with chronic heart failure (HF), but its cellular source and possible difference between atrium and ventricle expression are so far lacking. Aim of this study was to evaluate the expression of transcripts coding for CNP and for its specific receptor, NPR-B, in cardiac tissue (right and left atrium and ventricle) of normal and CHF animals. CNP tissue levels were also determined in cardiac extracts. Methods: Adult male minipigs (n=5) were chronically instrumented with a unipolar pacemaker connected to the anterior left ventricular (LV) wall. HF was induced by rapid pacing (180 beats/min) for 4 weeks. End-stage HF occurred at 24?2 days of pacing when the LV end-diastolic pressure was !25 mmHg. As control, we studied 5 adult male minipigs. At 4 weeks, myocardial samples were collected. Both CNP mRNA and proteins were extracted from a same sample with the method of phenol/guanidine-thiocyanate/chloroform. Tissue CNP levels were determined by a radioimmunoassay after a preliminary extraction on Sep-Pak C18, while the expression of mRNA coding for CNP and NPR-B in myocardial tissue (n=40) by Real Time reverse transcriptase-polymerase chain reaction (PCR) with DDCt method. As overall control, a parallel Real Time-PCR assay for BNP mRNA expression was carried out in the same samples. Real Time-PCR analysis was performed using an automated sequence instrument (7900HT Fast, Applied Biosystems) for the real-time monitoring of nucleic acid green dye fluorescence (SYBR Green I). Results: As to myocardial extracts, CNP was found in all cardiac chambers of controls and its content was ten fold higher in atria than in ventricles (RA: 13.7?1.9 pg/mg; LA: 8.7?3.8 pg/mg; RV: 1.07?0.33 pg/mg; LV: 0.93?0.17 pg/mg). At 4 weeks of pacing stress, myocardial levels of CNP in LV were higher than in controls (15.8?9.9 pg/mg vs.0.9?0.17 pg/mg, p=0.01). The expression of mRNA coding for CNP was higher at 4 weeks of pacing althought CNP gene expression appears to be noticeable lower than that of BNP. The NPR-B resulted to be expressed in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. Althought further investigations are necessary, the high tissue levels of CNP found after pacing stress as well as the myocardial CNP and NPR-B expression suggest an important role of this peptide in a so complex pathology as HF
Heat shock protein 72 in heart failure
In the heart, Heat Shock Proteins (HSP) constitute an endogenous stress response that protect myocytes from damage. Although HSP are intracellular proteins, these can be found also in peripheral circulationwhere their increase is associated with early cardiovascular disease
A2A and A3, Adenosine receptors mRNA are overexpressed in an experimental animal model of myocardial infarction
Background: Adenosine, a purine nucleoside and a "retaliatory metabolite" in ischemia, is ubiquitous in the body, and increases 100-fold during ischemia. Its biological actions are mediated by four adenosine receptors (ARs): A1 and A3, coupled to Gi/o, and the high-affinity A2A and low-affinity A2B, coupled to Gs. Because A1R and A3R are distributed mainly in myocardial cells and A2 are on coronary vascular smooth cells in the heart, adenosine may substantially modulate cardiac function as a whole. Aim: To determine possible myocardial alterations in the expression of ARs, in an experimental animal model of myocardial infarction (MI). Materials and Methods: Left ventricular (LV) tissue was collected from male adult minipigs with MI (n=5), induced by permanent surgical legation of the left anterior descending coronary artery and from 5 healthy pigs. mRNA expression of A1R, A2AR, A2BR,A3R was determined by semi-quantitative RT-PCR in tissue sampled collected from border (BZ) and remote zones (RZ) of infarcted area. Results: Transmural infarction affected about 15% of the LV wall mass. After 4 weeks, mRNA expression was higher in infarct regions than in control for A1R (controls=2.0?1.0, BZ=2.4?0.4, RZ=1.2?0.1), A2AR (controls=0.6?0.3, BZ=1.9?0.2, RZ=1.3?0.04 p=0.002, p=0.04, controls vs. BZ and RZ), A2BR (controls=1.1?0.5, BZ=1.2?0.2, RZ=0.5?0.04) and A3R (controls=0.2?0.07, BZ=2.4?0.7, RZ=0.7?0.07, p=0.006, p=0.002, controls vs. BZ and RZ). Conclusion: All adenosine receptors, and expecially A2A and A3, are overexpressed in the BZ of MI, consistently with an adaptative retaliatory anti-ischemic adenosinergic changes of post-infarcted heart
Real time PCR evaluation for c-type natriuretic peptide and for its specific receptor, NPR-B in cardiac tissue of normal and chronic heart failure animals
Background. C-type natriuretic peptide (CNP) was recently found in the myocardium, but possible differences between atrium and ventricle production are so far lacking. Aim. To evaluate the expression of transcripts coding for CNP and for its specific receptor, NPR-B, in cardiac tissue (right and left atrium and ventricle) of normal and HF animals. Methods. Cardiac tissue was collected from male adult minipigs without (control, n=5) and with pacing-induced HF (n=5). HF was induced by rapid pacing (180 beats/ min) for 3 weeks. mRNA was extracted with the method of phenol/guanidine-thiocyanate/chloroform. The expression of mRNA coding for CNP and NPR-B was determined in myocardial tissue (n=40) by Real Time-PCR with DDCt method. As overall control, a parallel Real Time-PCR assay for BNP mRNA expression was carried out in the same samples. Results. CNP gene expression was observed in controls and at 3 weeks of pacing resulting lower than that of BNP (left ventricle: p=0.05 controls vs. HF). As expected, BNP gene expression in all the cardiac chambers resulted higher after 3 weeks of pacing compared to normal heart (right atrium and left ventricle: p=0.003 controls vs. HF). Moreover, BNP mRNA expression was higher in atrium than in ventricle. We also observed higher, but not significantly, levels of CNP mRNA expression between normal and HF animals in all chambers. The NPR-B resulted to be expressed in all cardiac regions analyzed, and a down- regulation was observed in ventricles after HF (right ventricle p=0.001 controls vs. HF). Conclusions. In the present study, we provided the first evidence of CNP and NPR-B expression in tissue from normal and HF. The increased myocardial CNP synthesis was associated to the NPR-B down regulation in HF. The co-localization of the CNP system and its specific receptor suggests a possible role of this peptide in a complex pathology such as HF and the present results may prompt novel therapeutical strategies targeting NPR-B
Adrenomedullin plasma levels as predictors of left ventricular reverse remodelling in patients treated with cardiac resynchronization therapy
Purpose: Adrenomedullin (ADM), a potent natriuretic and vasorelaxing peptide, has been isolated from human pheochromocytoma cells and from cardiovascular tissue. Increase in ADM plasma levels in congestive heart failure (CHF) patients (pts) is due to many cardiac and systemic factors and in particular to the greater plasma volume and to the activation of sympathetic nervous system. Aim of this study was to assess the role of plasma ADM levels in CHF pts treated by cardiac resynchronization therapy (CRT). Methods: 42 pts, mean age 70 years, 27 males, NYHA Class III-IV CHF underwent CRT. Cause of CHF were idiopathic dilated cardiomyopathy in 27 pts, post ischemic in 15; all pts were in sinus rhythm and with complete left bundle branch block (QRS duration 138?8 msec). A complete echoDoppler exam, blood samples for brain natriuretic peptide (BNP) and ADM were obtained within 2 days before implantation. Results: At 18?6 months follow-up, >1 NYHA Class improvement was observed in 31/42 pts. However, a >10% reduction in end-systolic dimensions (ESD): -18.2?2.3% was reported in 16 pts (Group I); in the remaining 26 pts ESD change was almost negligible: -1.5?3.2% (Group II). The two groups were comparable for age, sex, cause of LV dysfunction, ongoing therapy, QRS duration at baseline, pre implantation ESD (60.6?1.8 vs 59.9?1.9 mm - Group I vs II), LVEF% (24.3?1.2 vs 25.4?1.3%) and BNP (545?80 vs 494?89 pg/ml). Significantly higher pre implantation ADM levels were present in Group I than in Group II (25.8?2.4 pmol/l vs. 17.1?1.6, p = 0.005). Conclusions: Significantly higher ADM levels indicate a subgroup of pts in whom significant reverse remodelling can be observed after CRT. Since AM is also produced in cardiac myocytes, lower ADM values before CRT could suggest the presence of more severe myocardial damage which may impair LV reverse remodelling even in the setting of clinically successful resynchronization
Relationship between inflammatory parameters and cardiovascular and lifestyle factors in the Mugello study oldest old
Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells.
Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2-) levels, known triggers of ET-1 expression. Moreover, no increase in O2- or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2- production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction
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).
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– 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- 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
Myocardial infarction activates the expression of cardiometabolic biomarkers in the heart: study in a swine model.
Purpose. Inflammation, extra-cellular matrix (ECM) remodeling and adipokine system activation represent essential processes of molecular response to cardiac injury. The aim of this study was to evaluate the cardiac expression of biomarkers involved in the inflammation, ECM remodeling and adiponectin system in an experimental animal model of myocardial infarction (AMI). Methods. Left ventricular (LV) tissue was collected from male adult pigs with AMI (n=6), induced by permanent surgical ligation of the left anterior descending coronary artery and from 6 healthy pigs. mRNA expression was determined by RT-PCR in tissue samples collected from border (BZ) and remote zones (RZ) of infarcted area. Proinflammatory cytokines (IL-6, TNF-α), matrix metalloproteinases (MMP)-2, -9, their tissue inhibitors (TIMP)-1, -2 and collagen (COL3α) were evaluated. In addition, adiponectin and its receptors, adipo-R1 and adipo-R2, were evaluated, owing its anti-inflammatory actions. Results. This surgical approach resulted in a permanent transmural infarction affecting 10-15% of the LV wall mass and after 4 weeks the mRNA expression of biomarkers, normalized on the respective GAPDH, was significantly higher in infarcted regions than in controls (MMP-9: 7.09?4.31; 1.18?0.28; 0.72?0.11, respectively for BZ, RZ and controls, p<0.05 BZ vs. RZ and controls; TIMP-1: 2.41?1.20; 0.28?0.04; 0.33?0.05, p=0.01; TIMP-2: 2.75?1.51; 0.53?0.04; 0.38?0.03, p<0.05; COL3α: 4.28?1.11; 0.87?0.13; 0.61?0.18, p<0.0004). Inflammatory indices were increased in AMI, both BZ and RZ. Adiponectin was significantly increased with respect to controls (BZ: 2.95?1.69; RZ: 0.93?0.33; controls: 0.52?0.12, p<0.05 BZ vs controls) as well as the Adipo-R1 (BZ: 1.40?0.31, RZ: 1.26?0.20, controls: 0.63?0.07; p<0.05 BZ and RZ vs controls). Conclusions. The inflammatory and ECM remodelling processes are activated after myocardial injury together with the system of adiponectin, confirming its involvement in the process of cardiac remodelling/repair. The knowledge of the interaction between the various mediators of the complex response to cardiac damage could represent an important target for new therapies. Reference. Shibata R et al, Cardiovasc Res. 2007 Jun 1;74(3):471-9
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