Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B-KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B-KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.

1. Natriuretic peptide receptors have been found in different heart preparations. However, the role of natriuretic peptides in the regulation of cardiac contractility remains largely elusive and was, therefore, studied here. 2. The rate of relaxation of electrically stimulated, isolated rat papillary muscles was enhanced (114.4+/-1. 4%, P<0.01) after addition of C-type natriuretic peptide (CNP; 1 microM). Time to peak tension decreased in parallel (88+/-3 and 75+/-2 msec before and 5 min after addition of CNP, respectively, P<0.01). On the other hand, the rate of contraction slowly decreased when CNP was added to the papillary muscles. These results show that CNP displays a positive lusitropic effect associated with a negative inotropic effect. The effects of CNP were mimicked by 8-bromo-guanosine 3',5' cyclic monophosphate. 3. Addition of CNP to isolated adult rat cardiomyocytes, induced a 25 fold increase in guanosine 3',5' cyclic monophosphate (cGMP) levels and stimulated the phosphorylation of phospholamban and troponin I, two proteins involved in the regulation of cardiac contractility. The levels of adenosine 3',5' cyclic monophosphate (cAMP) were not affected by the addition of CNP to the myocytes. The CNP-dependent phospholamban phosphorylation was accompanied by the activation of the sarcoplasmic reticulum Ca2+-ATPase. 4. In summary, CNP exerts a positive lusitropic effect, in rat papillary muscles. The putative mechanism involved in the lusitropism induced by this peptide, a cGMP-dependent enhancement of the rate of relaxation with a slowly developing negative inotropic effect, seems different to that described for catecholamines.

This study investigated the role of natriuretic peptide receptor 2 (NPR2) on cell proliferation and testosterone secretion in mouse Leydig cells.

Signal peptides may be novel biomarkers in cardiovascular diseases.

C-type natriuretic peptide (CNP)-knockout (KO) rats exhibit impaired skeletal growth, with long bones shorter than those in wild-type (WT) rats. This study compared craniofacial morphology in the CNP-KO rat with that in the Spontaneous Dwarf Rat (SDR), a growth hormone (GH)-deficient model. The effects of subcutaneous administration of human CNP with 53 amino acids (CNP-53) from 5 weeks of age for 4 weeks on craniofacial morphology in CNP-KO rats were also investigated. Skulls of CNP-KO rats at 9 weeks of age were longitudinally shorter and the foramen magnum was smaller than WT rats. There were no differences in foramen magnum stenosis and midface hypoplasia between CNP-KO rats at 9 and 33 weeks of age. These morphological features were the same as those observed in CNP-KO mice and activated fibroblast growth factor receptor 3 achondroplasia-phenotype mice. In contrast, SDR did not exhibit foramen magnum stenosis and midface hypoplasia, despite shorter stature than in control rats. After administration of exogenous CNP-53, the longitudinal skull length and foramen magnum size in CNP-KO rats were significantly greater, and full or partial rescue was confirmed. The synchondrosis at the cranial base in CNP-KO rats is closed at 9 weeks, but not at 4 weeks of age. In contrast, synchondrosis closure in CNP-KO rats treated with CNP-53 was incomplete at 9 weeks of age. Administration of exogenous CNP-53 accelerated craniofacial skeletogenesis, leading to improvement in craniofacial morphology. As these findings in CNP-KO rats are similar to those in patients with achondroplasia, treatment with CNP-53 or a CNP analog may be able to restore craniofacial morphology and foramen magnum size as well as short stature.

C-type natriuretic peptide (CNP) has vasodilatory and antimitogenic actions, but its role in the control of cardiac function is unclear. We studied the effect of CNP on cultured, beating neonatal rat cardiac myocytes. CNP caused a significant reduction in the amplitude of contraction and a significant accumulation of intracellular cyclic GMP. The effect of a membrane permeable cyclic GMP on cell contraction was similar to that of CNP. CNP caused no change in Ca2+ transients. Blockade of natriuretic peptide receptors abolished the effects of CNP on contraction and accumulation of intracellular cyclic GMP. Blockade of cyclic GMP-dependent protein kinase abolished the effect of CNP on myocyte contraction. We conclude that CNP has a negative inotropic effect on neonatal rat cardiac myocytes. The effect of CNP is mediated via natriuretic peptide receptor(s) causing elevation of intracellular cyclic GMP which possibly activates protein kinase and causes attenuation of myofilament sensitivity to Ca2+.

The aims were to evaluate the role of cardiovascular nitric oxide (NO)-system in C-type natriuretic peptide (CNP) actions and to investigate receptor types and signaling pathways involved in this interaction. Wistar rats were infused with saline or CNP. Mean arterial pressure (MAP) and nitrites and nitrates (NOx) excretion were determined. NO synthase (NOS) activity and NOS expression (Western blot) were analyzed in atria, ventricle and aorta. CNP decreased MAP and increased NOx excretion. CNP estimulated NOS activity, inducing no changes on cardiac and vascular endothelial NOS expression. NOS activity induced by CNP was abolished by suramin and calmidazoliumand but it is not modified by anantin. CNP would interact with NPR-C receptor coupled via G proteins leading to the activation Ca(2+)-calmodulin dependent endothelial NOS, increasing NO production which would induce the reduction in cardiac myocyte contractility and ANP synthesis and secretion in right atria and the relaxation of vascular smooth muscle.

Skeletal alterations in the head and neck region, such as midfacial hypoplasia, foramen magnum stenosis and spinal canal stenosis, are commonly observed in patients with mucopolysaccharidosis (MPS). However, enzyme replacement therapy (ERT), one of the major treatment approaches for MPS, shows limited efficacy for skeletal conditions. In this study, we analysed the craniofacial morphology of mice with MPS type VII, and investigated the underlying mechanisms promoting jaw deformities in these animals. Furthermore, we investigated the effects of C-type natriuretic peptide (CNP), a potent endochondral ossification promoter, on growth impairment of the craniofacial region in MPS VII mice when administered alone or in combination with ERT. MPS VII mice exhibited midfacial hypoplasia caused by impaired endochondral ossification, and histological analysis revealed increased number of swelling cells in the resting zone of the spheno-occipital synchondrosis (SOS), an important growth centre for craniomaxillofacial skeletogenesis. We crossed MPS VII mice with transgenic mice in which CNP was expressed in the liver under the control of the human serum amyloid-P component promoter, resulting in elevated levels of circulatory CNP. The maxillofacial morphological abnormalities associated with MPS VII were ameliorated by CNP expression, and further prevented by a combination of CNP and ERT. Histological analysis showed that ERT decreased the swelling cell number, and CNP treatment increased the width of the proliferative and hypertrophic zones of the SOS. Furthermore, the foramen magnum and spinal stenoses observed in MPS VII mice were significantly alleviated by CNP and ERT combination. These results demonstrate the therapeutic potential of CNP, which can be used to enhance ERT outcome for MPS VII-associated head and neck abnormalities.

C-type natriuretic peptide (CNP) is an endothelium-derived vasorelaxant, exerting anti-atherogenic actions in the vasculature and salvaging the myocardium from ischaemic injury. The cytoprotective effects of CNP are mediated in part via the G(i) -coupled natriuretic peptide receptor (NPR)3. As GPCRs are well-known to control cell proliferation, we investigated if NPR3 activation underlies effects of CNP on endothelial and vascular smooth muscle cell mitogenesis.

Meiosis is of prime importance for successful gametogenesis, and insufficient maintenance of oocyte meiotic arrest compromises oocyte developmental competence. Recent studies have demonstrated that the C-type natriuretic peptide (CNP)-Natriuretic peptide receptor 2 (NPR2) pathway can inhibit mammalian oocyte meiotic resumption. In mouse and porcine, the inhibitory effect of mural granulosa cell (MGC)-derived CNP on oocyte meiotic resumption is mediated by NPR2 localized in cumulus cells (CCs) surrounding the oocytes. However, in the present study, we identified a novel mechanism for CNP-induced meiotic arrest that appears to be unique to bovine oocytes. Unlike mouse and porcine, bovine NPR2 not only localizes in CCs, but also in oocyte membranes. We also showed that CNP can directly activate intra-oocyte cGMP production via NPR2 localized in oocyte membranes, in parallel with the CC-mediated pathway. Furthermore, we demonstrated that Npr2 expression in bovine CCs and oocytes were synergistically regulated by estradiol and oocyte-derived growth factors. Finally, based on the profound inhibitory effect of CNP on meiotic resumption, we established a natural factor synchronized in vitro oocyte maturation (NFSOM) system, which can significantly improve the developmental competence of matured oocytes, thereby resulting in higher in vitro embryo production efficiency. Taken together, our study not only provides new insight into understanding the crosstalk between oocytes and follicular somatic cells in mammals, but also presents a promising strategy for improving the in vitro oocyte maturation systems of assisted reproductive technology (ART).

Neonatal hypoxia-ischemia (HI) is the most common cause of brain injury in neonates, which leads to high neonatal mortality and severe neurological morbidity in later life (Vannucci, 2000; Volpe, 2001). Yet the molecular mechanisms of neuronal death and brain damage induced by neonatal HI remain largely elusive. Herein, using both in vivo and in vitro models, we determine an endogenous neuroprotectant role of c-type natriuretic peptide (CNP) in preserving neuronal survival after HI brain injury in mouse pups. Postnatal day 7 (P7) mouse pups with CNP deficiency (Nppclbab/lbab) exhibit increased brain infarct size and worsened long-term locomotor function after neonatal HI compared with wildtype control (Nppc+/+). In isolated primary cortical neurons, recombinant CNP dose-dependently protects primary neurons from oxygen-glucose deprivation (OGD) insult. This neuroprotective effect appears to be mediated through its cognate natriuretic peptide receptor 2 (NPR2), in that antagonization of NPR2, but not NPR3, exacerbates neuronal death and counteracts the protective effect of CNP on primary neurons exposed to OGD insult. Immunoblot and confocal microscopy demonstrate the abundant expression of NPR2 in neurons of the neonatal brain and in isolated primary cortical neurons as well. Moreover, similar to CNP deficiency, administration of NPR2 antagonist P19 via intracerebroventricular injection prior to HI results in exacerbated neuronal death and brain injury after HI. Altogether, the present study indicates that CNP and its cognate receptor NPR2 mainly expressed in neurons represent an innate neuroprotective mechanism in neonatal HI brain injury.

C-type natriuretic peptide (CNP) is an abundant neuropeptide in the human brain and the cerebrospinal fluid. CNP is involved in anxiogenesis and exerts its effects through the natriuretic peptide receptor B (NPR-B), which is expressed in the hippocampus. Hippocampal network oscillations of distinct frequency bands like gamma (gamma)-oscillations and sharp wave-ripple complexes (SPW-Rs) are likely involved in various cognitive functions such as the storage of information and memory consolidation in vivo. Here, we tested the effects of CNP on distinct network oscillations in horizontal slices of rat hippocampus. We found that CNP decreased the power of stimulus- and ACh/physostigmine-induced gamma-oscillations. In contrast to stimulus-induced gamma-oscillations, CNP increased the frequency of ACh-induced, persistent network oscillations. Moreover, the peptide hormone reduced the incidence of LTP-associated SPW-Rs in area CA3 and CA1. Immunohistochemistry indicates that the peptide binds to receptors expressed on a subset of GAD 65-67-immunopositive cells in addition to binding to principal and other presumably non-neuronal cells. CNP caused a hyperpolarization of CA3 neurons increased their input resistance and decreased inhibitory conductance. Together, our data suggest that the effects of CNP on synchronized hippocampal network oscillations might involve effects on hippocampal interneurons.

Volume reflex produces sympatho-inhibition that is mediated by the hypothalamic paraventricular nucleus (PVN). However, the mechanisms for the sympatho-inhibitory role of the PVN and the neurochemical factors involved remain to be identified. In this study, we proposed C-type natriuretic peptide (CNP) as a potential mediator of this sympatho-inhibition within the PVN. Microinjection of CNP (1.0 μg) into the PVN significantly decreased renal sympathetic nerve activity (RSNA) (-25.8% ± 1.8% vs. -3.6% ± 1.5%), mean arterial pressure (-15.0 ± 1.9 vs. -0.1 ± 0.9 mmHg) and heart rate (-23.6 ± 3.5 vs. -0.3 ± 0.9 beats/min) compared with microinjection of vehicle. Picoinjection of CNP significantly decreased the basal discharge of extracellular single-unit recordings in 5/6 (83%) rostral ventrolateral medulla (RVLM)-projecting PVN neurons and in 6/13 (46%) of the neurons that were not antidromically activated from the RVLM. We also observed that natriuretic peptide receptor type C (NPR-C) was present on the RVLM projecting PVN neurons detected by dual-labeling with retrograde tracer. Prior NPR-C siRNA microinjection into the PVN significantly blunted the decrease in RSNA to CNP microinjections into the PVN. Volume expansion-mediated reduction in RSNA was significantly blunted by prior administration of NPR-C siRNA into the PVN. These results suggest a potential role for CNP within the PVN in regulating RSNA, specifically under physiological conditions of alterations in fluid balance.

Ageing-related alterations in cardiovascular structure and function are commonly associated with chronic inflammation. A potential blood-based biomarker indicative of a chronic inflammatory state is N-Terminal Pro C-Type Natriuretic Peptide (NTproCNP). We aim to investigate associations between NTproCNP and ageing-related impairments in cardiovascular function. Community-based participants underwent same-day assessment of cardiovascular function and circulating profiles of plasma NTproCNP. Associations between cardiovascular and biomarker profiles were studied in adjusted models including standard covariates. We studied 93 participants (mean age 73 ± 5.3 years, 36 women), of whom 55 (59%) had impaired myocardial relaxation (ratio of peak velocity flow in early diastole E (m/s) to peak velocity flow in late diastole by atrial contraction A (m/s) <0.84). Participants with impaired myocardial relaxation were also found to have lower peak early phase filling velocity (0.6 ± 0.1 vs 0.7 ± 0.1, p < 0.0001) and higher peak atrial phase filling velocity (0.9 ± 0.1 vs 0.7 ± 0.1, p < 0.0001). NTproCNP levelswere significantly lower among participants with impaired myocardial relaxation (16.4% vs 39.5% with NTproCNP ≥ 19, p = 0.012). After multivariable adjustments, NTproCNP was independently associated with impaired myocardial relaxation (OR 2.99, 95%CI 1.12-8.01, p = 0.029). Community elderly adults with myocardial ageing have lower NTproCNP levels compared to those with preserved myocardial function. Given that impaired myocardial relaxation probably represents early changes within the myocardium with ageing, NTproCNP may be useful as an 'upstream' biomarker useful for charting myocardial ageing.

C-type natriuretic peptide (CNP) and the natriuretic peptide receptor B (NPR-B) are expressed throughout the hippocampus. We tested whether CNP affected long-term potentiation (LTP) or long-term depression (LTD) in area CA1. Field potentials (FP) were simultaneously recorded in stratum pyramidale (SP) and stratum radiatum (SR) of area CA1 in rat hippocampal slices. To induce LTD and LTP stimulation was applied to SR in area CA1 at 1 and 5 Hz and 30-100 Hz, respectively. CNP (100 nM) increased LTD magnitude while LTP induction was impeded. Thus, in the presence of CNP the threshold for LTP induction was shifted to higher stimulus frequencies, a modulation that showed layer-specific differences in area CA1. Effects of CNP were prevented by the NPR-B antagonist HS-142-1. In the presence of the GABA(A) receptor blocker bicuculline (BMI, 5 microM), CNP-mediated effects were attenuated in SP and SR. Intracellular recordings under this condition revealed that CNP significantly reduced number of action potentials generated during depolarizing current steps. The input resistance of CA1 cells and amplitude of isolated excitatory postsynaptic potential (EPSPs) were significantly increased by CNP whereas these changes were not observed in the absence of BMI. 100 Hz stimulation induced stable potentiation of the EPSP amplitude in CA1 pyramidal cells while this effect was strongly attenuated by CNP. This effect was prevented by BMI. Immunohistochemistry indicated that the peptide binds to receptors expressed on pyramidal cells and GAD(65/67)-immunopositive interneurons. 20 Hz stimulation, applied for 30 s, induced LTP in SR and SP. CNP attenuated LTP in SP and reversed LTP into LTD in SR. These effects were mimicked by low-dose dl-2-amino-5-phosphonopentanoic acid (dl-APV) (10 microM) suggesting partial N-methyl d-aspartate (NMDA) receptor dependency of CNP-mediated effects. Together, our data suggest that CNP is involved in the regulation of bidirectional plasticity in area CA1 potentially by modulating GABA(A)-mediated inhibition and NMDA receptors.

Rhabdomyosarcoma (RMS) is a malignant mesenchymal tumor and the most common soft tissue sarcoma in children. Because of several complications associated with intensive multimodal therapies, including growth disturbance and secondary cancer, novel therapies with less toxicity are urgently needed. C-type natriuretic peptide (CNP), an endogenous peptide secreted by endothelial cells, exerts antiproliferative effects in multiple types of mesenchymal cells. Therefore, we investigated whether CNP attenuates proliferation of RMS cells. We examined RMS patient samples and RMS cell lines. All RMS clinical samples expressed higher levels of guanylyl cyclase B (GC-B), the specific receptor for CNP, than RMS cell lines. GC-B expression in RMS cells decreased with the number of passages in vitro. Therefore, GC-B stable expression lines were established to mimic clinical samples. CNP increased cyclic guanosine monophosphate (cGMP) levels in RMS cells in a dose-dependent manner, demonstrating the biological activity of CNP. However, because cGMP is quickly degraded by phosphodiesterases (PDEs), the selective PDE5 inhibitor sildenafil was added to inhibit its degradation. In vitro, CNP, and sildenafil synergistically inhibited proliferation of RMS cells stably expressing GC-B and decreased Raf-1, Mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK) phosphorylation. These results suggested that CNP in combination with sildenafil exerts antiproliferative effects on RMS cells by inhibiting the Raf/MEK/ERK pathway. This regimen exerted synergistic effects on tumor growth inhibition without severe adverse effects in vivo such as body weight loss. Thus, CNP in combination with sildenafil represents a promising new therapeutic approach against RMS.

C-type natriuretic peptide (CNP) is the most conserved member of the mammalian natriuretic peptide family, and is implicated in the endocrine regulation of growth, metabolism and reproduction. CNP is expressed throughout the body, but is particularly abundant in the central nervous system and anterior pituitary gland. Pituitary gonadotropes are regulated by pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus, to control reproductive function. GnRH and CNP reciprocally regulate their respective signalling pathways in αT3-1 gonadotrope cells, but effects of pulsatile GnRH stimulation on CNP expression has not been explored. Here, we examine the sensitivity of the natriuretic peptide system in LβT2 and αT3-1 gonadotrope cell lines to continuous and pulsatile GnRH stimulation, and investigate putative CNP target genes in gonadotropes. Multiplex RT-qPCR assays confirmed that primary mouse pituitary tissue express Nppc,Npr2 (encoding CNP and guanylyl cyclase B (GC-B), respectively) and Furin (a CNP processing enzyme), but failed to express transcripts for Nppa or Nppb (encoding ANP and BNP, respectively). Pulsatile, but not continuous, GnRH stimulation of LβT2 cells caused significant increases in Nppc and Npr2 expression within 4 h, but failed to alter natriuretic peptide gene expression in αT3-1 cells. CNP enhanced expression of cJun, Egr1, Nr5a1 and Nr0b1, within 8 h in LβT2 cells, but inhibited Nr5a1 expression in αT3-1 cells. Collectively, these data show the gonadotrope natriuretic peptide system is sensitive to pulsatile GnRH signalling, and gonadotrope transcription factors are putative CNP-target genes. Such findings represent additional mechanisms by which CNP may regulate reproductive function.

Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts.

This study is to analyze the effect of C-type natriuretic peptide (CNP) on sperm motility of asthenozoospermia and explore the influence mechanism of CNP on the reproductive system and sperm motility. Our results showed that the concentration of CNP in asthenospermia patients' semen was lower than in normal people's. The motility of sperm could be improved markedly by CNP and 8-Br-cGMP, while the effect of CNP was inhibited by NPR-B antagonist and KT5823. In the asthenozoospermia mouse model induced by CTX, CNP injection could improve sperm motility in the epididymis, alleviate tissue damage in the testes and epididymis, and increase testosterone levels. The asthenospermia mouse model showed high activity of MDA and proinflammatory factors (TNF-α, IL-6), as well as low expression of antioxidants (SOD, GSH-Px, CAT) in the testis and epididymis, but this situation could be significantly ameliorated after being treated with CNP. Those studies indicated that the concentration of CNP in the semen of asthenospermia patients is lower than in normal people and could significantly promote sperm motility through the NPR-B/cGMP pathway. In the asthenospermia mouse model induced by CTX, CNP can alleviate the damage of cyclophosphamide to the reproductive system and sperm motility. The mechanism may involve increasing testosterone and reducing ROS and proinflammatory factors to damage the tissue and sperm.

Longitudinal bone growth is achieved by a tightly controlled process termed endochondral bone formation. C-type natriuretic peptide (CNP) stimulates endochondral bone formation through binding to its specific receptor, guanylyl cyclase (GC)-B. However, CNP/GC-B signaling dynamics in different stages of endochondral bone formation have not been fully clarified, especially in terms of the interaction between the cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Here, we demonstrated that CNP activates the cAMP/protein kinase A (PKA) pathway and that this activation contributed to the elongation of the hypertrophic zone in the growth plate. Cells of the chondrogenic line ATDC5 were transfected with Förster resonance energy transfer (FRET)-based cGMP and PKA biosensors. Dual-FRET imaging revealed that CNP increased intracellular cGMP levels and PKA activities in chondrocytes. Further, CNP-induced PKA activation was enhanced following differentiation of ATDC5 cells. Live imaging of the fetal growth plate of transgenic mice, expressing a FRET biosensor for PKA, PKAchu mice, showed that CNP predominantly activates the PKA in the hypertrophic chondrocytes. Additionally, histological analysis of the growth plate of PKAchu mice demonstrated that CNP increased the length of the growth plate, but coadministration of a PKA inhibitor, H89, inhibited the growth-promoting effect of CNP only in the hypertrophic zone. In summary, we revealed that CNP-induced cGMP elevation activated the cAMP/PKA pathway, and clarified that this PKA activation contributed to the bone growth-promoting effect of CNP in hypertrophic chondrocytes. These results provide insights regarding the cross-talk between cGMP and cAMP signaling in endochondral bone formation and in the physiological role of the CNP/GC-B system.