Exercise performance is not improved in mice with skeletal muscle deletion of natriuretic peptide clearance receptor.

Natriuretic peptides (NP), including atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), play essential roles in regulating blood pressure, cardiovascular homeostasis, and systemic metabolism. One of the major metabolic effects of NP is manifested by their capacity to stimulate lipolysis and the thermogenesis gene program in adipocytes, however, in skeletal muscle their effects on metabolism and muscle function are not as well understood. There are three NP receptors (NPR): NPRA, NPRB, and NPRC, and all three NPR genes are expressed in skeletal muscle and C2C12 myocytes. In C2C12 myocytes treatment with either ANP, BNP, or CNP evokes the cGMP signaling pathway. Since NPRC functions as a clearance receptor and the amount of NPRC in a cell type determines the signaling strength of NPs, we generated a genetic model with Nprc gene deletion in skeletal muscle and tested whether enhancing NP signaling by preventing its clearance in skeletal muscle would improve exercise performance in mice. Under sedentary conditions, Nprc skeletal muscle knockout (MKO) mice showed comparable exercise performance to their floxed littermates in terms of maximal running velocity and total endurance running time. Eight weeks of voluntary running-wheel training in a young cohort significantly increased exercise performance, but no significant differences were observed in MKO compared with floxed control mice. Furthermore, 6-weeks of treadmill training in a relatively aged cohort also increased exercise performance compared with their baseline values, but again there were no differences between genotypes. In summary, our study suggests that NP signaling is potentially important in skeletal myocytes but its function in skeletal muscle in vivo needs to be further studied in additional physiological conditions or with new genetic mouse models.

Unveiling the potential role of natriuretic peptide receptor a isoforms in fine-tuning the cGMP production and tissue-specific function.

Atrial natriuretic peptide (ANP) is a peptide hormone that regulates blood pressure and volume. ANP interacts with natriuretic peptide receptor-A (NPR-A) to lower the blood pressure through vasodilation, diuresis and natriuresis. Previously, we designed two human ANP analogues, one with exclusively diuretic function (DGD-ANP) and the other with exclusively vasodilatory function (DRD-ANP). Although both ANP analogues interact with NPR-A, their ability to produce cGMP was different. Three alternatively spliced isoforms of NPR-A were previously identified in rodents. Here, we evaluated the putative human isoforms for their cGMP production independently and in combination with WT NPR-A in various percentages. All three NPR-A isoforms failed to produce cGMP in the presence of ANP, DGD-ANP, or DRD-ANP. Co-expression of isoforms with WT NPR-A were found to significantly impair cGMP production. Considering the differential tissue expression levels of all three spliced isoforms in rodents have previously been demonstrated, the existence of these non-functional receptor isoforms may act as negative regulator for ANP/NPR-A activation and fine-tune cGMP production by WT NPR-A to different degree in different tissues. Thus, NPR-A isoforms potentially contribute to tissue-specific functions of ANP.

NPRC deletion attenuates cardiac fibrosis in diabetic mice by activating PKA/PKG and inhibiting TGF-ß1/Smad pathways.

Cardiac fibrosis plays a key role in the progression of diabetic cardiomyopathy (DCM). Previous studies demonstrated the cardioprotective effects of natriuretic peptides. However, the effects of natriuretic peptide receptor C (NPRC) on cardiac fibrosis in DCM remains unknown. Here, we observed that myocardial NPRC expression was increased in mice and patients with DCM. NPRC-/- diabetic mice showed alleviated cardiac fibrosis, as well as improved cardiac function and remodeling. NPRC knockdown in both cardiac fibroblasts and cardiomyocytes decreased collagen synthesis and proliferation of cardiac fibroblasts. RNA sequencing identified that NPRC deletion up-regulated the expression of TGF-ß-induced factor homeobox 1 (TGIF1), which inhibited the phosphorylation of Smad2/3. Furthermore, TGIF1 up-regulation was mediated by the activation of cAMP/PKA and cGMP/PKG signaling induced by NPRC deletion. These findings suggest that NPRC deletion attenuated cardiac fibrosis and improved cardiac remodeling and function in diabetic mice, providing a promising approach to the treatment of diabetic cardiac fibrosis.

NPRC deletion mitigated atherosclerosis by inhibiting oxidative stress, inflammation and apoptosis in ApoE knockout mice.

Previous studies suggested a beneficial effect of natriuretic peptides in animal models of cardiovascular disease, but the role of natriuretic peptide receptor C (NPRC) in the pathogenesis of atherosclerosis (AS) remains unknown. This study was designed to test the hypothesis that NPRC may promote AS lesion formation and instability by enhancing oxidative stress, inflammation, and apoptosis via protein kinase A (PKA) signaling. ApoE-/- mice were fed chow or Western diet for 12 weeks and NPRC expression was significantly increased in the aortic tissues of Western diet-fed mice. Systemic NPRC knockout mice were crossed with ApoE-/- mice to generate ApoE-/-NPRC-/- mice, and NPRC deletion resulted in a significant decrease in the size and instability of aortic atherosclerotic lesions in ApoE-/-NPRC-/- versus ApoE-/- mice. In addition, endothelial cell-specific NPRC knockout attenuated atherosclerotic lesions in mice. In contrast, endothelial cell overexpression of NPRC aggravated the size and instability of atherosclerotic aortic lesions in mice. Experiments in vitro showed that NPRC knockdown in human aortic endothelial cells (HAECs) inhibited ROS production, pro-inflammatory cytokine expression and endothelial cell apoptosis, and increased eNOS expression. Furthermore, NPRC knockdown in HAECs suppressed macrophage migration, cytokine expression, and phagocytosis via its effects on endothelial cells. On the contrary, NPRC overexpression in endothelial cells resulted in opposite effects. Mechanistically, the anti-inflammation and anti-atherosclerosis effects of NPRC deletion involved activation of cAMP/PKA pathway, leading to downstream upregulated AKT1 pathway and downregulated NF-κB pathway. In conclusion, NPRC deletion reduced the size and instability of atherosclerotic lesions in ApoE-/- mice via attenuating inflammation and endothelial cell apoptosis and increasing eNOS expression by modulating cAMP/PKA-AKT1 and NF-κB pathways. Thus, targeting NPRC may provide a promising approach to the prevention and treatment of atherosclerosis.

Novel pathogenic NPR2 variants in short stature patients and the therapeutic response to rhGH.

OBJECTIVE: Heterozygous loss-of-function variants in the NPR2 gene cause short stature with nonspecific skeletal abnormalities and account for about 2 ~ 6% of idiopathic short stature. This study aimed to analyze and identify pathogenic variants in the NPR2 gene and explore the therapeutic response to recombinant growth hormone (rhGH). METHODS: NPR2 was sequenced in three Chinese Han patients with short stature via exome sequencing. In vitro functional experiments, homology modeling and molecular docking analysis of variants were performed to examine putative protein changes and the pathogenicity of the variants. RESULT: Three patients received rhGH therapy for two years, and two NPR2 heterozygous variants were identified in three unrelated cases: c.1579 C > T,p.Leu527Phe in patient 1 and c.2842dupC,p.His948Profs*5 in patient 2. Subsequently, a small gene model was constructed, and transcriptional analysis of the synonymous variant (c.2643G > A) was performed in patient 3, which revealed the deletion of exon 17 and the premature formation of a stop codon (p.His840Gln*). Functional studies showed that both NPR2 variants, His948Profs*5 and His840Gln*, failed to produce cGMP in the homozygous state. Furthermore, the Leu527Phe variant of NPR2 was almost unresponsive to the stimulatory effect of ATP on CNP-dependent guanylyl cyclase activity. This loss of response to ATP has not been previously reported. The average age of patients at the start of treatment was 6.5 ± 1.8 years old, and their height increased by 1.59 ± 0.1 standard deviation score after 2 years of treatment. CONCLUSION: In this report, two novel variants in NPR2 gene were described. Our findings broaden the genotypic spectrum of NPR2 variants in individuals with short stature and provid insights into the efficacy of rhGH in these patients.

Genetic evidence implicating natriuretic peptide receptor-3 in cardiovascular disease risk: a Mendelian randomization study.

BACKGROUND: C-type natriuretic peptide (CNP) is a known target for promoting growth and has been implicated as a therapeutic opportunity for the prevention and treatment of cardiovascular disease (CVD). This study aimed to explore the effect of CNP on CVD risk using the Mendelian randomization (MR) framework. METHODS: Instrumental variables mimicking the effects of pharmacological intervention on CNP were identified as uncorrelated genetic variants located in the genes coding for its primary receptors, natriuretic peptide receptors-2 and 3 (NPR2 and NPR3), that associated with height. We performed MR and colocalization analyses to investigate the effects of NPR2 signalling and NPR3 function on CVD outcomes and risk factors. MR estimates were compared to those obtained when considering height variants from throughout the genome. RESULTS: Genetically-proxied reduced NPR3 function was associated with a lower risk of CVD, with odds ratio (OR) 0.74 per standard deviation (SD) higher NPR3-predicted height, and 95% confidence interval (95% CI) 0.64-0.86. This effect was greater in magnitude than observed when considering height variants from throughout the genome. For CVD subtypes, similar MR associations for NPR3-predicted height were observed when considering the outcomes of coronary artery disease (0.75, 95% CI 0.60-0.92), stroke (0.69, 95% CI 0.50-0.95) and heart failure (0.77, 95% CI 0.58-1.02). Consideration of CVD risk factors identified systolic blood pressure (SBP) as a potential mediator of the NPR3-related CVD risk lowering. For stroke, we found that the MR estimate for NPR3 was greater in magnitude than could be explained by a genetically predicted SBP effect alone. Colocalization results largely supported the MR findings, with no evidence of results being driven by effects due to variants in linkage disequilibrium. There was no MR evidence supporting effects of NPR2 on CVD risk, although this null finding could be attributable to fewer genetic variants being identified to instrument this target. CONCLUSIONS: This genetic analysis supports the cardioprotective effects of pharmacologically inhibiting NPR3 receptor function, which is only partly mediated by an effect on blood pressure. There was unlikely sufficient statistical power to investigate the cardioprotective effects of NPR2 signalling.

The landscape of N6-methyladenosine modification patterns and altered transcript profiles in the cardiac-specific deletion of natriuretic peptide receptor A.

The atrial natriuretic peptide (ANP) and the brain natriuretic peptide (BNP) are critical biological makers and regulators of cardiac functions. Our previous results show that NPRA (natriuretic peptide receptor A)-deficient mice have distinct metabolic patterns and expression profiles compared with the control. Still, the molecular mechanism that could account for this observation remains to be elucidated. Here, methylation alterations were detected by mazF-digestion, and differentially expressed genes of transcriptomes were detected by a Genome Oligo Microarray using the myocardium from NPRA-deficient (NPRA-/-) mice and wild-type (NPRA+/+) mice as the control. Comprehensive analysis of m6A methylation data gave an altered landscape of m6A modification patterns and altered transcript profiles in cardiac-specific NPRA-deficient mice. The m6A "reader" igf2bp3 showed a clear trend of increase, suggesting a function in altered methylation and expression in cardiac-specific NPRA-deficient mice. Intriguingly, differentially m6A-methylated genes were enriched in the metabolic process and insulin resistance pathway, suggesting a regulatory role in cardiac metabolism of m6A modification regulated by NPRA. Notably, it was confirmed that the pyruvate dehydrogenase kinase 4 (Pdk4) gene upregulated the gene expression and the hypermethylation level simultaneously, which may be the key factor for the cardiac metabolic imbalance and insulin resistance caused by natriuretic peptide signal resistance. Taken together, cardiac metabolism might be regulated by natriuretic peptide signaling, with decreased m6A methylation and a decrease of Pdk4.

Regulatory role of atrial natriuretic peptide in brown adipose tissue: A narrative review.

Atrial natriuretic peptide (ANP) has been considered to exert an essential role as a cardiac secretory hormone in the regulation of hemodynamic homeostasis. As the research progresses, the role of ANP in the crosstalk between heart and lipid metabolism has become an interesting topic that is attracting the interest of researchers. The regulation of ANP in lipid metabolism shows favorable effects, particularly the activation of brown adipose tissue (BAT). The complex regulatory network of ANP on BAT has not been fully outlined. This narrative review critically evaluated the existing literature on the regulatory effects of ANP on BAT. In general, we have summarized the expression of ANP and its receptors in various human tissues, analyzed the progress of research on the relationship between the ANP and BAT, and described several potential pathways of ANP to BAT. Exogenous ANP, natriuretic peptide receptor C (NPRC) deficiency, cold exposure, bariatric surgery, and cardiac or renal insufficiency could all contribute to BAT expression by increasing circulating ANP levels.

Natriuretic peptide pathways in heart failure: further therapeutic possibilities.

The discovery of the heart as an endocrine organ resulted in a remarkable recognition of the natriuretic peptide system (NPS). Specifically, research has established the production of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) from the heart, which exert pleiotropic cardiovascular, endocrine, renal, and metabolic actions via the particulate guanylyl cyclase A receptor (GC-A) and the second messenger, cGMP. C-type natriuretic peptide (CNP) is produced in the endothelium and kidney and mediates important protective auto/paracrine actions via GC-B and cGMP. These actions, in part, participate in the efficacy of sacubitril/valsartan in heart failure (HF) due to the augmentation of the NPS. Here, we will review important insights into the biology of the NPS, the role of precision medicine, and focus on the phenotypes of human genetic variants of ANP and BNP in the general population and the relevance to HF. We will also provide an update of the existence of NP deficiency states, including in HF, which provide the rationale for further therapeutics for the NPS. Finally, we will review the field of peptide engineering and the development of novel designer NPs for the treatment of HF. Notably, the recent discovery of a first-in-class small molecule GC-A enhancer, which is orally deliverable, will be highlighted. These innovative designer NPs and small molecule possess enhanced and novel properties for the treatment of HF and cardiovascular diseases.

Genetic Disruption of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Triggers Differential Cardiac Fibrosis and Disorders in Male and Female Mutant Mice: Role of TGF-ß1/SMAD Signaling Pathway.

The global targeted disruption of the natriuretic peptide receptor-A (NPRA) gene (Npr1) in mice provokes hypertension and cardiovascular dysfunction. The objective of this study was to determine the mechanisms regulating the development of cardiac fibrosis and dysfunction in Npr1 mutant mice. Npr1 knockout (Npr1-/-, 0-copy), heterozygous (Npr1+/-, 1-copy), and wild-type (Npr1+/+, 2-copy) mice were treated with the transforming growth factor (TGF)-ß1 receptor (TGF-ß1R) antagonist GW788388 (2 µg/g body weight/day; ip) for 28 days. Hearts were isolated and used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analyses. The Npr1-/- (0-copy) mice showed a 6-fold induction of cardiac fibrosis and dysfunction with markedly induced expressions of collagen-1α (3.8-fold), monocyte chemoattractant protein (3.7-fold), connective tissue growth factor (CTGF, 5.3-fold), α-smooth muscle actin (α-SMA, 6.1-fold), TGF-ßRI (4.3-fold), TGF-ßRII (4.7-fold), and phosphorylated small mothers against decapentaplegic (pSMAD) proteins, including pSMAD-2 (3.2-fold) and pSMAD-3 (3.7-fold), compared with wild-type mice. The expressions of phosphorylated extracellular-regulated kinase ERK1/2 (pERK1/2), matrix metalloproteinases-2, -9, (MMP-2, -9), and proliferating cell nuclear antigen (PCNA) were also significantly upregulated in Npr1 0-copy mice. The treatment of mutant mice with GW788388 significantly blocked the expression of fibrotic markers, SMAD proteins, MMPs, and PCNA compared with the vehicle-treated control mice. The treatment with GW788388 significantly prevented cardiac dysfunctions in a sex-dependent manner in Npr1 0-copy and 1-copy mutant mice. The results suggest that the development of cardiac fibrosis and dysfunction in mutant mice is predominantly regulated through the TGF-ß1-mediated SMAD-dependent pathway.

Natriuretic peptide receptor-C mediates the inhibitory effect of atrial natriuretic peptide on neutrophil recruitment to the lung during acute lung injury.

Atrial natriuretic peptide (ANP) protects against acute lung injury (ALI), but the receptor that mediates this effect is not known. Transgenic mice with 0 (knockout), 1 (heterozygote), or 2 (wild-type) functional copies of Npr3, the gene that encodes for natriuretic peptide receptor-C (NPR-C), were treated with intravenous infusion of ANP or saline vehicle before oropharyngeal aspiration of Pseudomonas aeruginosa (PA103) or saline vehicle. Lung injury was assessed 4 h following aspiration by measurement of lung wet/dry (W/D) weight, whole lung leukocyte and cytokine levels, and protein, leukocyte, and cytokine concentration in bronchoalveolar lavage fluid (BALF). PA103 induced acute lung injury as evidenced by increases in lung W/D ratio and protein concentration in BALF. The severity of PA103-induced lung injury did not differ between NPR-C genotypes. Treatment with intravenous ANP infusion reduced PA103-induced increases in lung W/D and BALF protein concentration in all three NPRC genotypes. PA103 increased the percentage of leukocytes that were neutrophils and cytokine levels in whole lung and BALF in NPR-C wild-type and knockout mice. This effect was blunted by ANP in wild-type mice but not in the NPR-C knockout mice. NPR-C does not mediate the protective effect of ANP on endothelial cell permeability in settings of PA103-induced injury but may mediate the effect of ANP on inhibition of the recruitment of neutrophils to the lung and thereby attenuate the release of inflammatory cytokines.

Heterozygous NPR2 Variants in Idiopathic Short Stature.

Heterozygous variants in the NPR2 gene, which encodes the B-type natriuretic peptide receptor (NPR-B), a regulator of skeletal growth, were reported in 2-6% cases of idiopathic short stature (ISS). Using next-generation sequencing (NGS), we aimed to assess the frequency of NPR2 variants in our study cohort consisting of 150 children and adolescents with ISS, describe the NPR2 phenotypic spectrum with a growth pattern including birth data, and study the response to growth hormone (GH) treatment. A total of ten heterozygous pathogenic/likely pathogenic NPR2 variants and two heterozygous NPR2 variants of uncertain significance were detected in twelve participants (frequency of causal variants: 10/150, 6.7%). During follow-up, the NPR2 individuals presented with a growth pattern varying from low-normal to significant short stature. A clinically relevant increase in BMI (a mean gain in the BMI SDS of +1.41), a characteristic previously not reported in NPR2 individuals, was observed. In total, 8.8% participants born small for their gestational age (SGA) carried the NPR2 causal variant. The response to GH treatment was variable (SDS height gain ranging from -0.01 to +0.74). According to the results, NPR2 variants present a frequent cause of ISS and familial short stature. Phenotyping variability in growth patterns and variable responses to GH treatment should be considered.

Novel NPR2 Gene Mutations Affect Chondrocytes Function via ER Stress in Short Stature.

Natriuretic peptide receptor 2 (NPR2) plays a key role in cartilage and bone morphogenesis. The NPR2 gene mutations result in acromesomelic dysplasia, Maroteaux type (AMDM), short stature with nonspecific skeletal abnormalities (SNSK), and epiphyseal chondrodysplasia, Miura type (ECDM). However, the pathogenic mechanism remains unclear. In our study, we identified one de novo (R557C) and six novel variants (G602W, V970F, R767*, R363*, F857S, and Y306S) in five independent Chinese families with familial short stature. Three patients with heterozygous mutations (G602W, V970F, and R767*) were diagnosed with SNSK (height SD score ranged from -2.25 to -5.60), while another two with compound heterozygous mutations (R363* and F857S, R557C and Y306S) were diagnosed with AMDM (height SD score ranged from -3.10 to -5.35). Among three patients with heterozygous status, two patients before puberty initiation with rhGH treatment significantly improved their growth (height velocity 7.2 cm/year, 6.0 cm/year), and one patient in puberty had a poor response to the rhGH treatment (height velocity 2.5 cm/year). Seven NPR2 gene variants were constructed and overexpressed in HEK293T and ATDC5 cells, and we found that ATDC5 cells with mutant NPR2 gene showed decreased differentiation, as evidenced by lower expression of ColII, ColX, and BMP4 and higher expression of Sox9. Moreover, the apoptosis rate was elevated in ATDC5 cells expressing the mutant NPR2 gene. N-glycosylation modification, plasma membrane localization, and ER stress resulted from the accumulation of mutant protein in ER, as shown by the higher expression of GRP78 and p-IRE1α. Overall, our results provide a novel insight into NPR2 loss of function, which could promote chondrocyte apoptosis and repress cell differentiation through ER stress and the unfolded protein response.

Oxygen and steroids affect the regulatory role of natriuretic peptide receptor-C on surfactant secretion by type II cells.

Atrial natriuretic peptide (ANP) and its receptors natriuretic peptide receptor (NPR)-A and NPR-C are all highly expressed in alveolar epithelial type II cells (AEC2s) in the late-gestation ovine fetal lung and are dramatically decreased postnatally. However, of all the components, NPR-C stimulation inhibits ANP-mediated surfactant secretion. Since alveolar oxygen increases dramatically after birth, and steroids are administered to mothers antenatally to enhance surfactant lung maturity, we investigated the effects of O2 concentration and steroids on NPR-C-mediated surfactant secretion in AEC2s. NPR-C expression was highest at 5% O2 while being suppressed by 21% O2, in cultured mouse lung epithelial cells (MLE-15s) and/or human primary AEC2s. Surfactant protein-B (SP-B) was significantly elevated in media from both in vitro and ex vivo culture at 13% O2 versus 21% O2 in the presence of ANP or terbutaline (TER). Both ANP and C-ANP (an NPR-C agonist) attenuated TER-induced SP-B secretion; this effect was reversed by dexamethasone (DEX) pretreatment in AEC2s and by transfection with NPR-C siRNA in MLE-15 cells. DEX markedly reduced AEC2 NPR-C expression, and pregnant ewes treated with betamethasone showed reduced ANP in fetal sheep lung fluid. These data suggest that elevated O2 downregulates AEC2 NPR-C and that steroid-mediated NPR-C downregulation in neonatal lungs may provide a novel mechanism for their effect on perinatal surfactant production.

NPR2 gene variants in familial short stature: a single-center study.

OBJECTIVES: NPR2 variants are associated with various short stature and bone dysplasia, such as acromesomelic dysplasia Maroteaux tyoe, individuals with a phenotype similar to Léri-Weill syndrome (LWD), and idiopathic short stature (ISS). However, few studies have reported on the relationship between familial short stature (FSS) and NPR2 variants. This study aimed to explore the relationship between FSS and NPR2 variants through the detection and identification of NPR2 variants in children with FSS, phenotypic description, clear treatment plan, and follow-up of treatment effect. METHODS: Children who met the FSS diagnostic criteria and had informed consent were included in the study. The trio whole-exome sequencing method (trio-WES) was used to detect and evaluate the NPR2 variants. RESULTS: A total of 16 children with short stature were included in this study (pretreatment height ≤ -2 standard deviation (SD) in both the patient and the shorter parent, unknown genetic etiology). NPR2 variants were identified in 12.5%(2/16) of the participants. Patient A was a 6-year-old male and 103.7 cm tall (-3.11SD), while Patient B was a 9-year-old female and 123.2 cm tall (-1.88SD). However, their heights increased after recombinant human growth hormone (rhGH) treatment. The height of patient A increased by 0.36SD six months after treatment while that of patient B increased by 1.22SD after one and a half years of treatment. CONCLUSIONS: NPR2 variant causes FSS. The growth rate of children significantly improved after rhGH treatment. However, further follow-up study is needed to determine the final height after long-term treatment.

Natriuretic peptide receptor B maintains heart rate and sinoatrial node function via cyclic GMP-mediated signalling.

AIMS: Heart rate (HR) is a critical indicator of cardiac performance that is determined by sinoatrial node (SAN) function and regulation. Natriuretic peptides, including C-type NP (CNP), have been shown to modulate ion channel function in the SAN when applied exogenously. CNP is the only NP that acts as a ligand for natriuretic peptide receptor-B (NPR-B). Despite these properties, the ability of CNP and NPR-B to regulate HR and intrinsic SAN automaticity in vivo, and the mechanisms by which it does so, are incompletely understood. Thus, the objective of this study was to determine the role of NPR-B signalling in regulating HR and SAN function. METHODS AND RESULTS: We have used NPR-B deficient mice (NPR-B+/-) to study HR regulation and SAN function using telemetry in conscious mice, intracardiac electrophysiology in anaesthetized mice, high-resolution optical mapping in isolated SAN preparations, patch-clamping in isolated SAN myocytes, and molecular biology in isolated SAN tissue. These studies demonstrate that NPR-B+/- mice exhibit slow HR, increased corrected SAN recovery time, and slowed SAN conduction. Spontaneous AP firing frequency in isolated SAN myocytes was impaired in NPR-B+/- mice due to reductions in the hyperpolarization activated current (If) and L-type Ca2+ current (ICa,L). If and ICa,L were reduced due to lower cGMP levels and increased hydrolysis of cAMP by phosphodiesterase 3 (PDE3) in the SAN. Inhibiting PDE3 or restoring cGMP signalling via application of 8-Br-cGMP abolished the reductions in cAMP, AP firing, If, and ICa,L, and normalized SAN conduction, in the SAN in NPR-B+/- mice. NPR-B+/- mice did not exhibit changes in SAN fibrosis and showed no evidence of cardiac hypertrophy or changes in ventricular function. CONCLUSIONS: NPR-B plays an essential physiological role in maintaining normal HR and SAN function by modulating ion channel function in SAN myocytes via a cGMP/PDE3/cAMP signalling mechanism.

Guanylyl cyclase-A phosphorylation decreases cardiac hypertrophy and improves systolic function in male, but not female, mice.

Atrial natriuretic peptide (NP) and BNP increase cGMP, which reduces blood pressure and cardiac hypertrophy by activating guanylyl cyclase (GC)-A, also known as NPR-A or Npr1. Although GC-A is highly phosphorylated, and dephosphorylation inactivates the enzyme, the significance of GC-A phosphorylation to heart structure and function remains unknown. To identify in vivo processes that are regulated by GC-A phosphorylation, we substituted glutamates for known phosphorylation sites to make GC-A8E/8E mice that express an enzyme that cannot be inactivated by dephosphorylation. GC-A activity, but not protein, was increased in heart and kidney membranes from GC-A8E/8E mice. Activities were threefold higher in female compared to male cardiac ventricles. Plasma cGMP and testosterone were elevated in male and female GC-A8E/8E mice, but aldosterone was only increased in mutant male mice. Plasma and urinary creatinine concentrations were decreased and increased, respectively, but blood pressure and heart rate were unchanged in male GC-A8E/8E mice. Heart weight to body weight ratios for GC-A8E/8E male, but not female, mice were 12% lower with a 14% reduction in cardiomyocyte cross-sectional area. Subcutaneous injection of fsANP, a long-lived ANP analog, increased plasma cGMP and decreased aldosterone in male GC-AWT/WT and GC-A8E/8E mice at 15 min, but only GC-A8E/8E mice had elevated levels of plasma cGMP and aldosterone at 60 min. fsANP reduced ventricular ERK1/2 phosphorylation to a greater extent and for a longer time in the male mutant compared to WT mice. Finally, ejection fractions were increased in male but not female hearts from GC-A8E/8E mice. We conclude that increased phosphorylation-dependent GC-A activity decreases cardiac ERK activity, which results in smaller male hearts with improved systolic function.

BNP facilitates NMB-encoded histaminergic itch via NPRC-NMBR crosstalk.

Histamine-dependent and -independent itch is conveyed by parallel peripheral neural pathways that express gastrin-releasing peptide (GRP) and neuromedin B (NMB), respectively, to the spinal cord of mice. B-type natriuretic peptide (BNP) has been proposed to transmit both types of itch via its receptor NPRA encoded by Npr1. However, BNP also binds to its cognate receptor, NPRC encoded by Npr3 with equal potency. Moreover, natriuretic peptides (NP) signal through the Gi-couped inhibitory cGMP pathway that is supposed to inhibit neuronal activity, raising the question of how BNP may transmit itch information. Here, we report that Npr3 expression in laminae I-II of the dorsal horn partially overlaps with NMB receptor (NMBR) that transmits histaminergic itch via Gq-couped PLCß-Ca2+ signaling pathway. Functional studies indicate that NPRC is required for itch evoked by histamine but not chloroquine (CQ), a nonhistaminergic pruritogen. Importantly, BNP significantly facilitates scratching behaviors mediated by NMB, but not GRP. Consistently, BNP evoked Ca2+ responses in NMBR/NPRC HEK 293 cells and NMBR/NPRC dorsal horn neurons. These results reveal a previously unknown mechanism by which BNP facilitates NMB-encoded itch through a novel NPRC-NMBR cross-signaling in mice. Our studies uncover distinct modes of action for neuropeptides in transmission and modulation of itch in mice.

An itch is a common sensation that makes us want to scratch. Most short-term itches are caused by histamine, a chemical that is released by immune cells following an infection or in response to an allergic reaction. Chronic itching, on the other hand, is not usually triggered by histamine, and is typically the result of neurological or skin disorders, such as atopic dermatitis. The sensation of itching is generated by signals that travel from the skin to nerve cells in the spinal cord. Studies in mice have shown that the neuropeptides responsible for delivering these signals differ depending on whether or not the itch involves histamine: GRPs (short for gastrin-releasing proteins) convey histamine-independent itches, while NMBs (short for neuromedin B) convey histamine-dependent itches. It has been proposed that another neuropeptide called BNP (short for B-type natriuretic peptide) is able to transmit both types of itch signals to the spinal cord. But it remains unclear how this signaling molecule is able to do this. To investigate, Meng, Liu, Liu, Liu et al. carried out a combination of behavioral, molecular and pharmacological experiments in mice and nerve cells cultured in a laboratory. The experiments showed that BNP alone cannot transmit the sensation of itching, but it can boost itching signals that are triggered by histamine. It is widely believed that BNP activates a receptor protein called NPRA. However, Meng et al. found that the BNP actually binds to another protein which alters the function of the receptor activated by NMBs. These findings suggest that BNP modulates rather than initiates histamine-dependent itching by enhancing the interaction between NMBs and their receptor. Understanding how itch signals travel from the skin to neurons in the spinal cord is crucial for designing new treatments for chronic itching. The work by Meng et al. suggests that treatments targeting NPRA, which was thought to be a key itch receptor, may not be effective against chronic itching, and that other drug targets need to be explored.

Piezo1 Mechanosensitive Ion Channel Mediates Stretch-Induced Nppb Expression in Adult Rat Cardiac Fibroblasts.

In response to stretch, cardiac tissue produces natriuretic peptides, which have been suggested to have beneficial effects in heart failure patients. In the present study, we explored the mechanism of stretch-induced brain natriuretic peptide (Nppb) expression in cardiac fibroblasts. Primary adult rat cardiac fibroblasts subjected to 4 h or 24 h of cyclic stretch (10% 1 Hz) showed a 6.6-fold or 3.2-fold (p < 0.05) increased mRNA expression of Nppb, as well as induction of genes related to myofibroblast differentiation. Moreover, BNP protein secretion was upregulated 5.3-fold in stretched cardiac fibroblasts. Recombinant BNP inhibited TGFß1-induced Acta2 expression. Nppb expression was >20-fold higher in cardiomyocytes than in cardiac fibroblasts, indicating that cardiac fibroblasts were not the main source of Nppb in the healthy heart. Yoda1, an agonist of the Piezo1 mechanosensitive ion channel, increased Nppb expression 2.1-fold (p < 0.05) and significantly induced other extracellular matrix (ECM) remodeling genes. Silencing of Piezo1 reduced the stretch-induced Nppb and Tgfb1 expression in cardiac fibroblasts. In conclusion, our study identifies Piezo1 as mediator of stretch-induced Nppb expression, as well as other remodeling genes, in cardiac fibroblasts.

Whole-exome imputation within UK Biobank powers rare coding variant association and fine-mapping analyses.

Exome association studies to date have generally been underpowered to systematically evaluate the phenotypic impact of very rare coding variants. We leveraged extensive haplotype sharing between 49,960 exome-sequenced UK Biobank participants and the remainder of the cohort (total n ≈ 500,000) to impute exome-wide variants with accuracy R2 > 0.5 down to minor allele frequency (MAF) ~0.00005. Association and fine-mapping analyses of 54 quantitative traits identified 1,189 significant associations (P < 5 × 10-8) involving 675 distinct rare protein-altering variants (MAF < 0.01) that passed stringent filters for likely causality. Across all traits, 49% of associations (578/1,189) occurred in genes with two or more hits; follow-up analyses of these genes identified allelic series containing up to 45 distinct 'likely-causal' variants. Our results demonstrate the utility of within-cohort imputation in population-scale genome-wide association studies, provide a catalog of likely-causal, large-effect coding variant associations and foreshadow the insights that will be revealed as genetic biobank studies continue to grow.