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

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

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