Il ruolo del peptide natriuretico di tipo C (CNP) nell’insufficienza cardiaca cronica: studio in un modello sperimentale animale.

Il peptide natriuretico di tipo C (CNP), appartiene alla famiglia dei peptidi natriuretici e mostra proprietà strutturali e fisiologiche simili a quelle del peptide natriuretico atriale (ANP) e del peptide natriuretico di tipo B (BNP). Il CNP è prodotto principalmente dall’endotelio e solo recentemente è stata evidenziata una sua produzione a livello cardiaco in pazienti con scompenso cardiaco (CHF). In questa situazione anche i livelli plasmatici di CNP risultano elevati in relazione alla severità della malattia, analogamente a quanto osservato per ANP e BNP, ma, mentre il ruolo patofisiologico dell’ANP e del BNP è ampiamente dimostrato, quello del CNP non è ancora completamente definito. Lo scompenso cardiaco cronico, una sindrome clinica determinata dall’incapacità del cuore di esercitare la sua funzione di pompa in maniera adeguata alle richieste metaboliche dell’organismo, dà origine a una serie di alterazioni a livello emodinamico e neuroormonale che includono l’attivazione di molti sistemi neuroendocrini, principalmente il sistema nervoso adrenergico e il sistema renina-angiotensina-aldosterone e altri sistemi vasoattivi, quali quello delle endoteline e dei peptidi natriuretici. Sebbene sia stata dimostrata una produzione miocardica di CNP in pazienti con CHF, non è ancora stato determinato il sito di produzione del peptide e le eventuali differenze tra produzione atriale e ventricolare. Scopo di questa tesi è stato valutare il ruolo del CNP nel CHF in un modello sperimentale animale di maiale con insufficienza cardiaca cronica indotta da stimolo con pace-maker ad alta frequenza; per questo sono stati determinati i livelli plasmatici di CNP e l’espressione cardiaca, a livello di mRNA e di proteina, del peptide e del suo recettore biologico specifico, NPR-B. Materiali e Metodi: Sono stati studiati complessivamente 10 animali da esperimento (minipig, maschi adulti, 35-40 kg). Ad ogni animale sottoposto a esperimento (n=5) è stato applicato un pace-maker unipolare nella parete ventricolare sinistra e lo scompenso cardiaco è stato indotto stimolando l’animale per 4 settimane (180 battiti/min). La condizione clinica è stata valutata tramite tecniche ecocardiografiche, Tomografia ad Emissione di Positroni e Risonanza Magnetica. Come controllo (n=5) sono stati utilizzati minipig adulti, non sottoposti ad alcun trattamento e mantenuti nelle stesse condizioni degli animali sottoposti a stimolazione cardiaca. I campioni di sangue sono stati raccolti in EDTA e aprotinina prima (basale) e a 10 min, 1, 2, 3 e 4 settimane dall’applicazione dello stimolatore cardiaco mentre i campioni di tessuto cardiaco (atrio e ventricolo, destro e sinistro), sono stati raccolti al termine dell’esperimento. Le proteine e l’mRNA sono stati estratti dallo stesso campione di tessuto cardiaco con il metodo del fenolo/guanidina-tiocianato/cloroformio. L’espressione cardiaca dell’mRNA codificante per il CNP, per l’ NPR-B, e come controllo per il BNP (di cui è ben nota l’espressione a livello cardiaco), è stata determinata con RT-PCR dopo opportuna messa a punto del metodo. I livelli plasmatici di CNP, nel plasma e negli estratti cardiaci, sono stati determinati con un metodo radioimmunologico specifico dopo preliminare estrazione in fase solida su Sep-Pak C18. Risultati: L’affidabilità dell’analisi tramite RT-PCR semiquantitativa nel tessuto cardiaco di maiale è stata valutata per ciascun effettore con prove sperimentali dedicate. L’espressione dello mRNA codificante per il CNP è risultata indotta dopo scompenso cardiaco in parallelo con quella del BNP, sebbene i livelli di CNP siano circa 10 volte inferiori. Nel corso di questo lavoro di tesi è stato sequenziato l’mRNA codificante per il recettore NPR-B di maiale (GenBank DQ 487044; 1-396 pb) mancante nella GenBank, usando primer di Mus musculus per l’NPR-B, grazie alla alta omologia fra specie. Il recettore NPR-B è risultato espresso nelle diverse camere cardiache sia negli animali di controllo che in quelli scompensati, confermando la presenza di questo recettore nel tessuto cardiaco di maiale. I livelli plasmatici di CNP sono risultati significativamente aumentati rispetto ai controlli a partire dalla prima settimana 36,9 ± 10,4 vs 16,7 ± 1.1 pg/ml, media ± sem, p=0.013. Per quanto riguarda gli estratti tissutali, in condizioni basali livelli misurabili di CNP sono stati trovati in tutte le camere cardiache; la concentrazione osservata negli atrii (dx: 13,68±1,9 pg/mg; sin: 8,7±3,76) è risultata circa 10 volte più elevata rispetto ai ventricoli (dx:1,07±0,33; sin: 0,93±017). Dopo 4 settimane di stimolazione con pace-maker ad alta frequenza, i livelli miocardici di CNP sono risultati aumentati particolarmente nel ventricolo sinistro (15,8±9,9 vs 0,9±0.17, p=0,01). Conclusioni: Questi dati indicano che il CNP e il suo recettore biologico hanno un ruolo rilevante nella patofisiologia dello scompenso cardiaco, sebbene ulteriori studi siano necessari per la completa definizione della funzione di questo peptide nel CHF. In particolare, la conoscenza della sequenza del NPR-B nel maiale potrebbe costituire un importante strumento per studiare il coinvolgimento di questo recettore nella regolazione di varie patologie che possono essere studiate con questo tipo di modello animale

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

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

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-&#945;), matrix metalloproteinases (MMP)-2, -9, their tissue inhibitors (TIMP)-1, -2 and collagen (COL3&#945;) 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; COL3&#945;: 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

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

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

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

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

Mismatch between mRNA cardiac expression of BNP and CNP in pacing-induced heart failure

Purpose: It has been recently demonstrated in an animal model of heart failure (HF) that the high-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive cardiac failure with pronounced differences in regional contractility. Aim of this study was to evaluate the possible variation of brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) compared with the infero-septal region (opposite site, OS) to individualize the possible association between the contraction patterns and the expression of these biomarkers. Methods: Cardiac tissue was collected from male adult minipigs without (controls, n=6) and with pacing-induced HF (n=8) from PS, and from the tissue remote from the pacing-site. mRNA expression of BNP and CNP was evaluated by semiquantitative polymerase chain reaction (PCR) by using GAPDH as housing gene. Results: A significant difference in mRNA expression of BNP in PS was observed between HF animals and controls (BNP/GAPDH: 0.65?0.11 vs. 0.35?0.04, p=0.02) whereas in OS BNP levels resulted similar to those of controls (BNP/GAPDH: 0.36?0.05). mRNA expression of CNP was higher in HF with respect to controls both in PS and in OS, although not significantly (CNP/GAPDH: controls 0.089?0.036, PS 0.29?0.23, OS 0.54?0.16). These findings are in tune with the increase of CNP tissue concentrations (controls=0.69?0.13; PS=1.56?0.19; OS=1.70?0.42 pg/mg protein; p=0.039 controls vs.OS). The higher levels of BNP mRNA expression in PS are in agreement with a reduction of contractile function in this region while the higher CNP mRNA expression in OS could suggest the presence of a major endothelial dysfunction in the remote region. Conclusions: In clinical conditions the endothelial dysfunction precedes the overt HF, so, although further investigations are necessary, these results suggest that CNP could be a early marker of HF. In this context, CNP could be a marker more relevant than BNP in early recognizing patients with HF