C-type natriuretic peptide (CNP) is an autocrine and paracrine mediator released by endothelial cells, cardiomyocytes and fibroblasts that regulates vital physiological functions in the cardiovascular system. These roles are conveyed via two cognate receptors, natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C), which activate different signalling pathways that mediate complementary yet distinct cellular responses. Traditionally, CNP has been deemed the endothelial component of the natriuretic peptide system, while its sibling peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are considered the endocrine guardians of cardiac function and blood volume. However, accumulating evidence indicates that CNP not only modulates vascular tone and blood pressure, but also governs a wide range of cardiovascular effects including the control of inflammation, angiogenesis, smooth muscle and endothelial cell proliferation, atherosclerosis, cardiomyocyte contractility, hypertrophy, fibrosis, and cardiac electrophysiology. This review will focus on the novel physiological functions ascribed to CNP, the receptors/signalling mechanisms involved in mediating its cardioprotective effects, and the development of therapeutics targeting CNP signalling pathways in different disease pathologies.
The natriuretic peptides are a family of three structurally related hormones that play unique and distinctive roles within the cardiovascular system. The physiological functions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have been intensively investigated over the past few decades, however there has been considerably less focus on C-type natriuretic peptide (CNP). ANP and BNP are expressed in the heart and are released in response to a volume-induced stretch of the atria and ventricles, respectively. These peptides act as endocrine hormones and contribute to the regulation of cardiac structure, blood pressure and blood volume. In contrast, the tissue distribution and mode of action of CNP is different, with recent studies revealing diverse endogenous roles of CNP including the control of vascular tone, leukocyte activation, angiogenesis, smooth muscle and endothelial cell proliferation, vascular integrity, coronary blood flow, cardiac fibrosis, cardiac hypertrophy, and electrophysiology. These aspects of CNP physiology and pathology will be detailed herein.
CNP is a 22 amino acid peptide that is produced following the processing of preproCNP by a signal peptidase and subsequent cleavage of proCNP by the endoprotease furin. Two forms of CNP exist in tissue and plasma, CNP-53 and CNP-22, although the protease responsible for processing the elongated peptide into its shorter, more prevalent form is not known. CNP-22 was initially discovered in extracts from porcine brain. In addition to its abundant expression in the CNS, high levels of CNP are found in chondrocytes and endothelial cells, which constitutively release the peptide. Other cells within the cardiovascular system, including cardiomyocytes and fibroblasts, also produce CNP, however, tissue expression and plasma levels are relatively low in healthy individuals, suggesting that CNP most likely acts as a local paracrine/autocrine mediator in the heart and blood vessels.
The half-life of CNP in plasma is short (2.6 min), therefore, degradation is rapid which may account for the low concentrations (fmol–pmol range) of the peptide measured in the circulation. There are two main pathways by which CNP is inactivated, cleavage by neutral endopeptidase (NEP), or internalisation by natriuretic peptide receptor C (NPR-C) followed by endocytosis and lysosomal degradation. Overall, the contribution of each pathway to the degradation of natriuretic peptides appears to be equal in healthy subjects but there is evidence to suggest that during pathophysiological conditions where natriuretic peptide levels are raised and NPR-C may be saturated, NEP may play a major role in clearance. Furthermore, the tissue distribution of NEP and/or NPR-C may affect CNP inactivation in different organs, for example, CNP is internalised more readily by NPR-C in the kidney compared to the lungs.
Most of the stimuli that are known to increase gene expression and/or trigger the release of CNP are pertinent to cardiovascular health including shear stress, inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)1β, transforming growth factor (TGF)-β, and bacterial lipopolysaccharide. In accordance with these findings are studies showing that plasma levels of CNP are elevated in patients with heart failure (HF) and sepsis. In contrast, CNP release is attenuated by oxidised low-density lipoprotein and vascular endothelial growth factor.
CNP exerts its biological effects via the activation of two cell surface receptors, natriuretic peptide receptor B (NPR-B, also termed guanylyl cyclase-B, GC-B) and natriuretic peptide receptor C (NPR-C). The peptide has a very low binding affinity for natriuretic peptide receptor A (NPR-A), which is the endogenous receptor for the ligands ANP and BNP. Both NPR-B and NPR-C are widely expressed and are found on endothelial cells, smooth muscle cells, cardiomyocytes, and fibroblasts. NPR-C is the most abundant natriuretic peptide receptor and accounts for ~95% of the total natriuretic peptide receptor population in endothelial cells.
CNP has a similar binding affinity for NPR-B and NPR-C but the signalling pathways activated by each receptor are markedly different. NPR-B is a particulate guanylyl cyclase receptor that catalyses the conversion of guanosine-5′-triphosphate to cyclic guanosine-3′,5′-monophosphate (cGMP), a second messenger that activates protein kinase G I and II, which in turn alters cellular functions by phosphorylating specific target proteins. NPR-C was originally considered to be a clearance receptor devoid of signalling activity but later it was shown to contain pertussis toxin sensitive Gi binding domains within the intracellular C-terminal tail that couple to adenylyl cyclase inhibition (by G i α subunit) and phospholipase C-β activation (by G i βγ subunits). Two subtypes of NPR-C have been reported with different molecular masses, a 67-kDa protein and a 77-kDa protein, but it is not known if their capacity to signal and clear natriuretic peptides in vivo is distinct. A study in isolated rat glomerular membranes showed that the 67-kDa NPR-C has a high affinity for CNP and activation of this subtype reduces cAMP synthesis via G i signalling, whereas the 77-kDa receptor has a very low affinity for CNP and is involved in ligand internalization.
Pharmacological experiments on isolated blood vessel preparations have shown that CNP is a potent vasodilator of conduit and resistance arteries throughout the vascular tree. In the microvasculature, CNP is more efficacious than ANP and BNP, suggesting it may play a role in regulating peripheral vascular resistance. Numerous studies have shown that CNP infusion reduces systemic blood pressure in both humans and animals. Despite this, the physiological role of endogenous CNP in the cardiovascular system had not been elucidated until recently. Early studies of global CNP knockout (KO) mice were confounded by the effects of CNP deletion on bone development. These animals exhibit skeletal abnormalities, dwarfism, and a high mortality rate, thus, investigations utilising the Cre/Lox recombination system to generate animals with cell-restricted deletion of CNP have been key to gaining a fuller understanding of the function of this peptide in vivo. Endothelial-specific deletion of CNP in mice results in elevated blood pressure and impaired responses to endothelium-dependent vasodilators, providing definitive evidence that the constitutive release of CNP contributes to the regulation of vascular tone. The (patho)physiological relevance of these experimental findings is exemplified by the discovery of polymorphisms in the CNP and furin genes that are associated with hypertension in humans.
CNP-mediated vasodilation occurs via different mechanisms depending on the species, vessel studied, and/or natriuretic peptide receptor activated. In conduit arteries, CNP-induced relaxation is blocked by the dual NPR-A/B antagonist HS-142-1, suggesting NPR-B activation and subsequent production of cGMP mediates the dilatory effects of CNP in large vessels. However, in the resistance vasculature the importance of NPR-C in the vasoreactivity of CNP increases. In both rodents and humans, a similar pathway exists involving activation of NPR-C and smooth muscle cell hyperpolarisation. In the rat mesenteric artery, the release of CNP accounts for a major component of the endothelium-derived hyperpolarisation (EDH) induced by acetylcholine, a response that can be inhibited by NPR-C antagonists, blockade of small and intermediate conductance calcium-sensitive potassium channels (SK Ca and IK Ca), and G-protein inwardly rectifying potassium channels (GIRK). It is proposed that the opening of SK Ca and IK Ca on the endothelial cell triggers the release of CNP that binds to NPR-C on the smooth muscle cell resulting in the G i/0-mediated activation of GIRK, potassium efflux, and hyperpolarisation. In human arteries, both GIRK and large conductance calcium-activated K+ channels (BK Ca channels) have been implicated in CNP-evoked vasodilation. Alternatively, studies in rats have shown that NPR-C can also couple to eNOS, resulting in the production of nitric oxide, although, this mechanism has only been reported in larger diameter vessels.
The receptor that mediates the endogenous regulatory effects of endothelial-derived CNP on vascular tone in vivo is still under debate. Global and smooth muscle-specific NPR-B KO mice are normotensive and their vascular function is normal despite the vasodilator responses to exogenous CNP being impaired. It has been proposed that CNP maintains endothelial function independently of smooth muscle NPR-B and that alterations in the production of the vasoconstrictor endothelin-1 account for the elevations in blood pressure observed in ecCNP KO animals, however, the mechanism(s) involved has not been elucidated. A recent study proposed that NPR-B signalling in pericytes may be more important than vascular smooth muscle. This latest research shows that the disruption of NPR-B under the control of the PDGFRβ promotor in mice results in a hypertensive phenotype, indicating CNP may participate in paracrine communication between endothelial cells and pericytes to regulate peripheral vascular resistance.
In contrast, accumulating evidence suggests that NPR-C mediates a large proportion of the vasodilator effects of CNP in the vasculature. NPR-C KO mice exhibit impaired endothelial function and a diminished hypotensive response to CNP in vitro and in vivo. The original publication describing global deletion of NPR-C in mice reports a lower blood pressure in these animals (males only). However, more recently it was shown that female NPR-C KO exhibit elevated blood pressure and diminished vascular endothelial function. This discrepancy may be due to sex differences in the clearance versus signalling functions of NPR-C in mice, however, data from human genome wide association studies linking variants of the NPR-C gene with hypertension did not find a disparity between sexes, suggesting the NPR-C signalling pathway is equally important in men and women. Patients with the blood pressure elevating NPR-C genotype have lower levels of receptor mRNA and protein in their vascular smooth muscle cells, supporting the theory that diminished CNP activation of NPR-C may underlie this association (as opposed to altered clearance). Further evidence in favour of a role of NPR-C in the pathogenesis of hypertension was published in a study investigating the effects of the endogenous secretory peptide musclin (also known as osteocrin). Musclin is a competitive ligand at NPR-C and the infusion of musclin increases systolic blood pressure in vivo. Gene expression levels of musclin are elevated in spontaneously hypertensive rats (SHR) and the administration of anti-musclin antibodies reduces blood pressure in these animals, intimating that interference with NP binding at NPR-C by musclin may contribute to the hypertensive state of these animals. However, contrary to this research is a study showing that the infusion of musclin (osteocrin) lowers blood pressure in mice, an effect that is absent in NPR-A KO animals, suggesting that an increase in circulating levels of ANP and/or BNP due to the blockade of peptide clearance by NPR-C accounts for this response. It is possible that musclin interferes with both the signalling and clearance functions of NPR-C and plays a different role in the regulation of blood pressure in healthy and diseased animals. Indeed, the vasoconstrictor and blood pressure elevating effects of musclin are significantly enhanced in SHR compared to normotensive controls, intimating that a change in receptor expression or the signalling/clearance function of NPR-C occurs in hypertension that alters the response to musclin in this model of disease. This is further supported by data demonstrating that NPR-C agonism in SHR attenuates the development of high blood pressure, an effect that is not observed in control Wistar-Kyoto rats.
The first indication
