The BrAID study protocol: integration of machine learning and transcriptomics for brugada syndrome recognition.

BACKGROUND: Type 1 Brugada syndrome (BrS) is a hereditary arrhythmogenic disease showing peculiar electrocardiographic (ECG) patterns, characterized by ST-segment elevation in the right precordial leads, and risk of Sudden Cardiac Death (SCD). Furthermore, although various ECG patterns are described in the literature, different individual ECG may show high-grade variability, making the diagnosis problematic. The study aims to develop an innovative system for an accurate diagnosis of Type 1 BrS based on ECG pattern recognition by Machine Learning (ML) models and blood markers analysis trough transcriptomic techniques. METHODS: The study is structured in 3 parts: (a) a retrospective study, with the first cohort of 300 anonymized ECG obtained in already diagnosed Type 1 BrS (75 spontaneous, 150 suspected) and 75 from control patients, which will be processed by ML analysis for pattern recognition; (b) a prospective study, with a cohort of 11 patients with spontaneous Type 1 BrS, 11 with drug-induced Type 1 BrS, 11 suspected BrS but negative to Na + channel blockers administration, and 11 controls, enrolled for ECG ML analysis and blood collection for transcriptomics and microvesicles analysis; (c) a validation study, with the third cohort of 100 patients (35 spontaneous and 35 drug-induced BrS, 30 controls) for ML algorithm and biomarkers testing. DISCUSSION: The BrAID system will help clinicians improve the diagnosis of Type 1 BrS by using multiple information, reducing the time between ECG recording and final diagnosis, integrating clinical, biochemical and ECG information thus favoring a more effective use of available resources. Trial registration Clinical Trial.gov, NCT04641585. Registered 17 November 2020, https://clinicaltrials.gov/ct2/show/NCT04641585.

Modulated molecular markers of restenosis and thrombosis byin-vitrovascular cells exposed to bioresorbable scaffolds.

Drug-eluting bioresorbable vascular scaffolds (BVSs) have emerged as a potential breakthrough for the treatment of coronary artery stenosis, providing mechanical support and drug delivery followed by complete resorption. Restenosis and thrombosis remain the primary limitations in clinical use. The study aimed to identify potential markers of restenosis and thrombosis analyzing the vascular wall cell transcriptomic profile modulation triggered by BVS at different values of shear stress (SS). Human coronary artery endothelial cells and smooth muscle cells were cultured under SS (1 and 20 dyne cm-2) for 6 h without and with application of BVS and everolimus 600 nM. Cell RNA-Seq and bioinformatics analysis identified modulated genes by direct comparison of SS conditions and Gene Ontology (GO). The results of different experimental conditions and GO analysis highlighted the modulation of specific genes as semaphorin 3E, mesenchyme homeobox 2, bone morphogenetic protein 4, (heme oxygenase 1) and selectin E, with different roles in pathological evolution of disease. Transcriptomic analysis of dynamic vascular cell cultures identifies candidate genes related to pro-restenotic and pro-thrombotic mechanisms in anin-vitrosetting of BVS, which are not adequately contrasted by everolimus addition.

Tuscany Sangiovese grape juice imparts cardioprotection by regulating gene expression of cardioprotective C-type natriuretic peptide.

PURPOSE: A regular intake of red grape juice has cardioprotective properties, but its role on the modulation of natriuretic peptides (NPs), in particular of C-type NP (CNP), has not yet been proven. The aims were to evaluate: (1) in vivo the effects of long-term intake of Tuscany Sangiovese grape juice (SGJ) on the NPs system in a mouse model of myocardial infarction (MI); (2) in vitro the response to SGJ small RNAs of murine MCEC-1 under physiological and ischemic condition; (3) the activation of CNP/NPR-B/NPR-C in healthy human subjects after 7 days' SGJ regular intake. METHODS: (1) C57BL/6J male and female mice (n = 33) were randomly subdivided into: SHAM (n = 7), MI (n = 15) and MI fed for 4 weeks with a normal chow supplemented with Tuscany SGJ (25% vol/vol, 200 µl/per day) (MI + SGJ, n = 11). Echocardiography and histological analyses were performed. Myocardial NPs transcriptional profile was investigated by Real-Time PCR. (2) MCEC-1 were treated for 24 h with a pool of SGJ small RNAs and cell viability under 24 h exposure to H2O2 was evaluated by MTT assay. (3) Human blood samples were collected from seven subjects before and after the 7 days' intake of Tuscany SGJ. NPs and miRNA transcriptional profile were investigated by Real-Time PCR in MCEC-1 and human blood. RESULTS: Our experimental data, obtained in a multimodal pipeline, suggest that the long-term intake of SGJ promotes an adaptive response of the myocardium to the ischemic microenvironment through the modulation of the cardiac CNP/NPR-B/NPR-C system. CONCLUSIONS: Our results open new avenue in the development of functional foods aimed at enhancing cardioprotection of infarcted hearts through action on the myocardial epigenome.

Connexin 26 Expression in Mammalian Cardiomyocytes.

Connexins are a family of membrane-spanning proteins named according to their molecular weight. They are known to form membrane channels mediating cell-cell communication, which play an essential role in the propagation of electrical activity in the heart. Cx26 has been described in a number of tissues but not in the heart, and its mutations are frequently associated with deafness and skin diseases. The aim of this study was to assess the possible Cx26 expression in heart tissues of different mammalian species and to demonstrate its localization at level of cardiomyocytes. Samples of pig, human and rat heart and H9c2 cells were used for our research. Immunohistochemical and molecular biology techniques were employed to test the expression of Cx26. Interestingly, this connexin was found in cardiomyocytes, at level of clusters scattered over the cell cytoplasm but not at level of the intercalated discs where the other cardiac connexins are usually located. Furthermore, the expression of Cx26 in H9c2 myoblast cells increased when they were differentiated into cardiac-like phenotype. To our knowledge, the expression of Cx26 in pig, human and rat has been demonstrated for the first time in the present paper.

Dipyridamole-induced C-type natriuretic peptide mRNA overexpression in a minipig model of pacing-induced left ventricular dysfunction.

Dipyridamole (DP) restores ischemic tissue blood flow stimulating angiogenesis in eNOS-dependent pathways. C-type natriuretic peptide (CNP) is expected to mimic the migration-stimulatory effect of NO via a cGMP-dependent mechanism. Aim of this study was to assess the role of concomitant treatment with DP on CNP levels in blood and myocardial tissue of minipigs with left ventricular dysfunction (LVD) induced by pacing at 200bpm in the right ventricular apex. Minipigs with DP therapy (DP+, n=4) or placebo (DP-, n=4) and controls (C-SHAM, n=4) underwent 2D-EchoDoppler examination and blood collection before and after 4 weeks of pacing, when cardiac tissue was collected. Histological/immunohistochemical analyses were performed. CNP levels were determined by radioimmunoassay; cardiac CNP, BNP, natriuretic receptors expression by Real-Time PCR. After pacing, cardiac parameters resulted less impaired in DP+ compared to DP-. Histological sections presented normal morphology while the arteriolar density resulted: C-SHAM: 9.0±1.2; DP-: 4.9±0.3; DP+: 6.5±0.6number/mm(2); C-SHAM vs DP- and DP+ p=0.004, p=0.04, respectively. CNP mRNA resulted lower in DP- compared to C-SHAM and DP+ as well as NPR-B (p=0.011, DP- vs DP+). Both NPR-A/NPR-C mRNA expressions were significantly (p<0.001) lower both in DP- and DP+ compared to C-SHAM. BNP mRNA was higher in LVD. CNP plasma levels showed a similar trend with respect to gene expression (C-SHAM: 30.5±15; DP-: 18.6±5.5; DP+: 21.2±4.7pg/ml). These data suggest that DP may serve as a preconditioning agent to increase the protective CNP-mediated endocrine response in LVD. This response, mediated by its specific receptor NPR-B, may offer new insights into molecular targets for treatment of LVD.

Endothelin system mRNA variation in the heart of Zucker rats: evaluation of a possible balance with natriuretic peptides.

BACKGROUND AND AIMS: The deregulation of neurohormonal systems, including the natriuretic peptide (NP) and endothelin (ET) systems, may increase the possibility of developing obesity-related risk. The aim of our paper was to evaluate ET system mRNA variation in heart of the Zucker rat model together with the simultaneous evaluation of the NP system transcriptomic profile. In order to analyze the link between the ET-1 system and the inflammatory process, the cardiac expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α was also measured. METHODS AND RESULTS: Zucker rats of 11-13 weeks were subdivided into obese rats (O, n = 20) and controls (CO, n = 20): half of them were studied under fasting conditions (CO(fc)-O(fc)) and the remainder after the induction of acute hyperglycemia (CO(AH)-O(AH)). Cardiac mRNA expression of TNF-α, IL-6, and NP/ET-1 systems was evaluated by Real-Time polymerase chain reaction. No significant difference for pre-proET-1, ET-A, and ET-B mRNA expression was detected between O and CO, whereas significantly lower mRNA levels of the ECE-1 were observed in O (p = 0.02). Regarding NPs, only BNP mRNA expression decreased significantly in O with respect to CO (p = 0.01). A down-regulation of NPR-B and NPR-C and an up-regulation of NPR-A were observed in O. No significant difference for IL-6 and TNF-α mRNA was revealed. Subdividing into fasting and hyperglycemic rats, many of the genes studied maintained their mRNA expression pattern almost unchanged. CONCLUSIONS: The modulation of ET-1/NP systems in obesity could be a useful starting point for future studies aimed at identifying new therapeutic strategies for the treatment of cardiometabolic syndrome.

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.

C-type natriuretic peptide plasma levels are reduced in obese adolescents.

The high prevalence of obesity in children may increase the magnitude of lifetime risk of cardiovascular disease (CD). At present, explicit data for recommending biomarkers as routine pre-clinical markers of CD in children are lacking. C-type natriuretic peptide (CNP) is assuming increasing importance in CD; in adults with heart failure, its plasma levels are related to clinical and functional disease severity. We have previously reported five different reference intervals for blood CNP as a function of age in healthy children; however, data on plasma CNP levels in obese children are still lacking. Aim of this study was to assess CNP levels in obese adolescents and verify whether they differ from healthy subjects. Plasma CNP was measured in 29 obese adolescents (age: 11.8 ± 0.4 years; BMI: 29.8 ± 0.82) by radioimmunoassay and compared with the reference values of healthy subjects. BNP was also measured. Both plasma CNP and BNP levels were significantly lower in the obese adolescents compared to the appropriate reference values (CNP: 3.4 ± 0.2 vs 13.6 ± 2.3 pg/ml, p<0.0001; BNP: 18.8 ± 2.6 vs 36.9 ± 5.5 pg/ml, p=0.003). There was no significant difference between CNP values in males and females. As reported in adults, we observed lower plasma CNP and BNP levels in obese children, suggesting a defective natriuretic peptide system in these patients. An altered regulation of production, clearance and function of natriuretic peptides, already operating in obese adolescents, may possibly contribute to the future development of CD. Thus, the availability of drugs promoting the action of natriuretic peptides may represent an attractive therapeutic option to prevent CD.

C-type natriuretic peptide transcriptomic profiling increases in human leukocytes of patients with chronic heart failure as a function of clinical severity.

The aim of this study was to evaluate the transcriptomic profiling of C-type natriuretic peptide (CNP) and of its specific receptor, NPR-B in human leukocytes of heart failure (HF) patients as a function of clinical severity, assessing the 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 of HF patients (NYHA I-II, n=7; NYHA III-IV, n=13) with PAXgene Blood RNA Kit. Significantly higher levels of CNP mRNA expression were found in HF patients as a function of clinical severity (C=0.23±0.058, NYHA I-II=0.47±0.18, NYHA III-IV=2.58±0.71, p=0.005 C vs NYHA III-IV, p=0.017 NYHA I-II vs NYHA III-IV) and NPR-B transcript levels resulted down-regulated in HF patients with higher NYHA class (C=2.2±0.61, NYHA I-II=2.76±0.46, NYHA III-IV=0.29±0.13, p=0.001 C vs NYHA III-IV, p<0.0001 NYHA I-II vs NYHA III-IV). A significant negative correlation between CNP and NPR-B mRNA expression (r=0.5, p=0.03) was also observed. These results suggest a co-regulation of NPR-B and CNP expression supporting the relevance of this receptor in human disease characterized by a marked inflammatory/immune component and suggesting the possibility of manipulating inflammation via pharmacological agents selective for this receptor.

High concentration of C-type natriuretic peptide promotes VEGF-dependent vasculogenesis in the remodeled region of infarcted swine heart with preserved left ventricular ejection fraction.

BACKGROUND: Vasculogenesis is a hallmark of myocardial restoration. Post-ischemic late remodeling is associated with pathology and function worsening. At the same time, neo-vasculogenesis helps function improving and requires the release of vascular endothelial growth factor type A (VEGF-A). The vasculogenic role of C-type natriuretic peptide (CNP), a cardiac paracrine hormone, is unknown in infarcted hearts with preserved left ventricular (LV) ejection fraction (EF). We explored whether myocardial VEGF-dependent vasculogenesis is affected by CNP. METHODS AND RESULTS: To this end, infarcted swine hearts were investigated by magnetic resonance imaging (MRI), histological and molecular assays. At the fourth week, MRI showed that transmural myocardial infarction (MI) affected approximately 13% of the LV wall mass without impairing global function (LVEF>50%, n=9). Increased fibrosis, metalloproteases and capillary density were localized to the infarct border zone (BZ), and were associated with increased expression of CNP (p=0.03 vs. remote zone (RZ)), VEGF-A (p<0.001 vs. RZ), BNP, a marker of myocardial dysfunction (p<0.01 vs. RZ) and the endothelial marker, factor VIII-related antigen (p<0.01 vs. RZ). In vitro, CNP 1000 nM promoted VEGF-dependent vasculogenesis without affecting the cell growth and survival, although CNP 100 nM or a high concentration of VEGF-A halted vascular growth. CONCLUSIONS: CNP expression is locally increased in infarct remodeled myocardium in the presence of dense capillary network. The vasculogenic response requires the co-exposure to high concentration of CNP and VEGF-A. Our data will be helpful to develop combined myocardial delivery of CNP and VEGF-A genes in order to reverse the remodeling process.

Characterization of novel 3'untranslated regions and related polymorphisms of the gene NPPC, encoding for the C-type natriuretic peptide.

Elevated plasmatic levels of C-type natriuretic peptide (CNP) were found in patients with chronic heart failure (CHF), but its use as sensitive and specific clinical bio-marker is still controversial. In fact, high levels of CNP were also observed in patients classified in low severity New York Heart Association (NYHA) classes. CNP is encoded by a gene poorly studied (NPPC, natriuretic-precursor peptide C), where the regulatory regions are not well defined and the role of single nucleotide polymorphisms (SNPs) poorly ascertained. In the present work, we focused on the characterization of the 3'untranslated region (3'UTR) of the gene, using Rapid Amplification of cDNA 3'-End (3' RACE), and we identified two novel transcript isoforms (L-3'UTR; S-3'UTR; accession number JF420840, HQ419060 respectively). Since it could be hypothesized that genetic variations could explain the observed inter-patients differences, we searched for novel SNPs, by the use of High Resolution Melting (HRM). The results showed a complete lack of genetic variations among our series of samples. Moreover, a preliminary evaluation, using literature information and bioinformatic prediction allowed us to predicted the putative relevant microRNAs binding to the novel 3'UTRs that could modulate the post-transcriptional regulation of NPPC and affect the plasmatic levels of CNP. We obtained 750 and 1024 predicted miRNAs targeting the S- and L-3'UTRs, respectively.

Relation between adiponectin and brain natriuretic peptide in healthy pediatric subjects: from birth through childhood.

BACKGROUND AND AIMS: New biomarkers potentially improve clinical management of cardiovascular disease, but there are gaps in understanding their role during childhood. Adiponectin regulates metabolism and exerts anti-inflammatory/anti-atherogenic effects. The aim of the study was to evaluate circulating levels of adiponectin during postnatal growth and its relationship with Brain Natriuretic Peptide (BNP) in healthy children, a marker of cardiac function known to be increased in childhood. METHODS AND RESULTS: Plasma adiponectin and BNP were measured in 131 healthy children divided into: 43 newborns (0-3 days), 29 neonates (4-30 days), 25 infants (1-12 months) and 34 children (1-12 years). A group of 33 healthy adult subjects (25-60 years) was also studied. Plasma adiponectin in the 131 children resulted significantly higher compared to adult subjects (p < 0.0001). The time-course of adiponectin suggests the design of three age-based intervals: the first until 1 month of age (median 29.07 µg/mL, 11.61-47.01 µg/mL 5°-95° percentiles), the second between 1 and 12 months of age (21.66 µg/mL, 8.83-59.81 µg/mL) and the third for age up to 12 years (13.81 µg/mL, 4.10-28.57 µg/mL). Both adiponectin and BNP exhibited the same trend of a progressive decrease during growth, showing a significant relationship (Spearman's rho = 0.403, p < 0.0001). CONCLUSION: Adiponectin plasma levels in a healthy pediatric population vary as a function of age. Three reference intervals for adiponectin in pediatric subjects have been indicated. The relationship between adiponectin and BNP suggests that the age-dependent profile of circulating adiponectin could also be due to BNP.

IL-33/ST2 pathway and classical cytokines in end-stage heart failure patients submitted to left ventricular assist device support: a paradoxic role for inflammatory mediators?

BACKGROUND: Inflammation is a critical process contributing to heart failure (HF). We hypothesized that IL-33/ST2 pathway, a new mechanism regulated during cardiac stress, may be involved in the functional worsening of end-stage HF patients, candidates for left ventricular assist device (LVAD) implantation, and potentially responsible for their outcome. METHODS: IL-33, ST2, and conventional cytokines (IL-6, IL-8, and TNF-α) were determined in cardiac biopsies and plasma of 22 patients submitted to LVAD implantation (pre-LVAD) and compared with (1) control stable chronic HF patients on medical therapy at the moment of heart transplantation without prior circulatory support (HT); (2) patients supported by LVAD at the moment of LVAD weaning (post-LVAD). RESULTS: Cardiac expression of ST2/IL-33 and cytokines was lower in the pre-LVAD than in the HT group. LVAD determined an increase of inflammatory mediators comparable to levels of the HT group. Only ST2 correlated with outcome indices after LVAD implantation. CONCLUSIONS: IL-33/ST2 and traditional cytokines were involved in decline of cardiac function of ESHF patients as well as in hemodynamic recovery induced by LVAD. IL-33/ST2 pathway was also associated to severity of clinical course. Thus, a better understanding of inflammation is the key to achieving more favorable outcome by new specific therapies.

Gene expression of C-type natriuretic peptide and of its specific receptor NPR-B in human leukocytes of healthy and heart failure subjects.

C-type natriuretic peptide (CNP), a member of the family of natriuretic peptides, is synthesized and secreted from monocytes and macrophages that resulted to be a source of CNP at inflammatory sites. This suggests that special attention should be focused on the possible role of CNP in the immune system, in addition to its effects on the cardiovascular system. The aim of this study was to evaluate the possibility of measuring the mRNA expression of CNP and NPR-B, its specific receptor, in human whole blood samples of healthy (N; n=7) and heart failure (HF; n=7) subjects by Real-Time PCR (RT-PCR). Total RNA was extracted from leukocytes with QIAamp RNA Blood Kit and/or with PAXgene Blood RNA Kit. RT-PCR was performed and optimized for each primer. The experimental results were normalized with the three most stably expressed genes. CNP and NPR-B expression trend was similar in both fresh and frozen human whole blood. Significant higher levels of CNP and NPR-B mRNA expression were found in HF patients with respect to controls (CNP: N=1.23±0.33 vs. HF=6.54±2.09 p=0.027; NPR-B: N=0.85±0.23 vs. HF=5.31±1.98 p=0.04). A significant correlation between CNP and NPR-B (r=0.86, p<0.0001) was observed. Further studies are needed to clarify the pathophysiological properties of this peptide but the possibility to measure CNP and NPR-B mRNA expression in human leukocytes with a fast and easy procedure is a useful starting point for future investigation devoted to better understand the biomolecular processes associated to different diseases.

The natriuretic peptide time-course in end-stage heart failure patients supported by left ventricular assist device implant: focus on NT-proCNP.

This study aimed to evaluate left ventricular assist device (LVAD) effects on natriuretic peptide (NP) prohormone plasma levels in end-stage heart failure (HF) patients, especially NT-proCNP, in order to better characterize the NP system during hemodynamic recovery by LVAD. HF patients (n=17, NYHA III-IV) undergoing LVAD were studied: 6 died of multi-organ failure syndrome (NS) and 11 survived (S). Total sequential organ failure assessment (t-SOFA) score and blood samples were obtained at admission (T1) and at 24, 72h and 1, 2, 4 weeks (T2-T6) after LVAD. In S, NT-proANP and NT-proCNP significantly increased at 24h after implantation, reaching a reduction to basal levels at 4 weeks following LVAD [NT-proANP: T1 vs. T2 p=0.017, NT-proCNP: T1 vs. T2 p=0.028, T1 vs. T3 p=0.043]. Elevated NT-proBNP plasma levels were observed at all times. In NS, NP plasma levels sustained higher with respect to S. No statistical variation was observed for NT-proCNP and NT-proANP in S and NS while NT-proBNP reached significant differences at T4 in NS. Considering S+NS, only NT-proCNP strongly correlated with t-SOFA score at T1 (rho=0.554, p=0.04) while subdividing patients NT-proCNP positively correlated in NS with t-SOFA score (rho=0.988, p=0.002) only at T4. In NS a correlation between NT-proCNP and NT-proBNP at T1 was observed (rho=-0.900, p=0.037). Both IL-6 and TNF-alpha sustained higher in NS patients than in S; in particular, statistical significance was observed for IL-6. The study of new peptides, such as NT-proCNP, would provide additional information for identifying patients who are more likely to recover.

Plasma C-type natriuretic peptide levels in healthy children.

C-type natriuretic peptide (CNP) is assuming increasing importance in cardiovascular disease, and in adults its plasma levels are related to clinical and functional disease severity. Data are scarce regarding the reference values for CNP in infancy. Aim of this study was to assess the reference intervals for CNP in human healthy newborns and infants. Plasma CNP was measured in 121 healthy children divided into: 41 newborns (age 0-3 days), 24 newborns (4-30 days), 22 infants (1-12 months) and 32 children (1-12 years). A group of 32 healthy adult subjects (age 64 ± 1 years) was also studied. CNP was measured by a specific radioimmunoassay. Between- and within-assay variability resulted ≤ 30 and 20%, respectively and analytical sensitivity 0.77 ± 0.05 pg/tube. Plasma CNP resulted significantly higher in children than in adult subjects (13.6 ± 1.2 pg/ml vs. 7.4 ± 1.0 pg/ml, p=0.030). When the results were analyzed as a function of the age the reference intervals for plasma CNP resulted: 11.6 ± 2.1 pg/ml for newborns (0-3 days), 16.4 ± 3.7 pg/ml for newborns (4-30 days), 15.4 ± 2.7 pg/ml for infants (1-12 months), 13.6 ± 2.3 pg/ml for children (1-12 years) [p=0.01 newborns (4-30 days) vs. adults; p=0.03 infants (1-12 months) vs. adults]. CNP showed the highest concentrations after 12h of life with a peak between 4 and 5 days of life and with a progressive decline afterwards. According to these data at least five different reference intervals for CNP determinations should be used. These observations may be helpful for future clinical application of CNP in human children.

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.

NPR-B, the C-type natriuretic peptide specific receptor, is the predominant biological receptor in mouse and pig myocardial tissue.

AIM: The increased myocardial production and the elevated plasma concentrations of C-type natriuretic peptide (CNP) in heart failure patients suggest its involvement in pathophysiological cardiac remodeling. The cardiovascular action of CNP seems to be mainly mediated by natriuretic peptide receptor (NPR)-B but the importance of CNP/NPR-B signaling in heart is not yet well characterized. The aim of this study was to assess the cardiac mRNA expression of CNP and NPR-B together with those of BNP and NPR-A in order to evaluate the relative importance of these peptides and of their receptors in cardiovascular system. METHODS: The expression of mRNA coding for CNP, NPR-B, BNP and NPR-A was investigated in myocardial tissue (BALB/c mice, N=5) by use of RT-PCR. NPR-A and NPR-B expression were also evaluated in left ventricle of male adult minipigs without (N=5) and with pacing-induced heart failure (HF, N=5). RESULTS: The proposed method allowed to detect the expression of mRNA coding for CNP and NPR-B in myocardial tissue confirming the presence of these effectors in the heart. These data also indicate that CNP mRNA expression is lower with respect to that of BNP (CNP/GAPDH= 0.117+/-0.035 vs. BNP/GAPDH=0.247+/-0.066) and that NPR-B is the predominant subtype receptor in the heart (Mouse: NPR-A/GAPDH=0.244+/- 0.028; NPR-B/GAPDH=0.657+/-0.022; p=0.0008; Pig: NPR-A/GAPDH=3.06+/-1.75, NPR-B/GAPDH= 14.3+/-3.6, p=0.0028; HF Pig: NPR-A/GAPDH= 4.29+/-0.93, NPR-B/GAPDH=7.9+/-1.1, p=0.0043). CONCLUSION: In the present study, we provided the first evidence of a higher mRNA expression in cardiac tissue of NPR-B with respect to NPR-A indicating that CNP specific receptor (NPR-B) is the predominant biological receptor in mouse and pig myocardial tissue.

Sequencing and cardiac expression of natriuretic peptide receptors A and C in normal and heart failure pigs.

Pharmacological treatments able to activate natriuretic receptors (NPRs) and inhibit cardiac remodelling in heart failure (HF) patients, are currently under investigation. To better understand the therapeutic potential of the NPRs activation is necessary to dispose of experimental models devoid of confounding effects. The pig constitutes an animal model largely used but its genome is not completely sequenced. Aims of this study were to sequence NPR-A and NPR-C in Susscrofa and to evaluate ANP, BNP and NPRs mRNA expression in cardiac tissue of normal and HF minipigs in order to have a starting point for future studies devoted to check new potential drugs. Cardiac tissue was collected from adult male minipigs without (n=4) and with HF (n=5). Pig NPR-A (179bp) and NPR-C (203bp) mRNA were partially sequenced (GenBank n.: FJ518622, FJ518621). Compared to control, ANP and BNP gene expression resulted higher in all the cardiac chambers of HF heart. This increase is associated to a down-regulation of NPR-A and an up-regulation of NPR-C in HF. These sequences will provide a new tool to investigate the role of natriuretic peptides and of their receptors under physiological and pathological conditions and their response to therapeutic interventions.

C-type natriuretic peptide and its relation to non-invasive indices of left ventricular function in patients with chronic heart failure.

C-type natriuretic peptide (CNP) significantly increases in chronic heart failure (CHF) patients as a function of clinical severity. Aim of this study was to evaluate in CHF patients the relationship between circulating CNP concentrations and echo-Doppler conventional indices of left ventricular (LV) function as well as less load independent parameters as dP/dt. LV ejection fraction (EF), left ventricular end-diastolic dimension (LVEDD) and LV dP/dt were evaluated together with plasma CNP levels in 38 patients with CHF and in 63 controls. CNP levels resulted significantly higher in CHF patients than in controls (7.19+/-0.59 pg/ml vs. 2.52+/-0.12 pg/ml, p<0.0001). A significant correlation between dP/dt and CNP levels (r=-0.61, p<0.0001) was observed. A good correlation with EF (r=-0.55, p<0.001) and a less significant relation with LVEDD (r=0.316, p<0.05) were also reported. When patients were divided according to dP/dt values a very significant difference in CNP levels was observed: Group I (<600, n=25) vs. Group II (>600, n=13): 8.46+/-0.69 and 4.75+/-0.75 pg/ml, respectively, p<0.001. This is the first study that reports a correlation between CNP and dP/dt in CHF patients, thus suggesting a possible role on cardiac contractility.