Human genotyping and an experimental model reveal NPR-C as a possible contributor to morbidity in coarctation of the aorta

Abstract

Coarctation of the aorta (CoA) is a common congenital cardiovascular (CV) defect characterized by a stenosis of the descending thoracic aorta. Treatment exists, but many patients develop hypertension (HTN). Identifying the cause of HTN is challenging because of patient variability (e.g., age, follow-up duration, severity) and concurrent CV abnormalities. Our objective was to conduct RNA sequencing of aortic tissue from humans with CoA to identify a candidate gene for mechanistic studies of arterial dysfunction in a rabbit model of CoA devoid of the variability seen with humans. We present the first known evidence of natriuretic peptide receptor C (NPR-C; aka NPR3) downregulation in human aortic sections subjected to high blood pressure (BP) from CoA versus normal BP regions (validated to PCR). These changes in NPR-C, a gene associated with BP and proliferation, were replicated in the rabbit model of CoA. Artery segments from this model were used with human aortic endothelial cells to reveal the functional relevance of altered NPR-C activity. Results showed decreased intracellular calcium ([Ca²⁺]_i) activity to C-type natriuretic peptide (CNP). Normal relaxation induced by CNP and atrial natriuretic peptide was impaired for aortic segments exposed to elevated BP from CoA. Inhibition of NPR-C (M372049) also impaired aortic relaxation and [Ca²⁺]_i activity. Genotyping of NPR-C variants predicted to be damaging revealed that rs146301345 was enriched in our CoA patients, but sample size limited association with HTN. These results may ultimately be used to tailor treatment for CoA based on mechanical stimuli, genotyping, and/or changes in arterial function.

Keywords: congenital cardiovascular disease; hemodynamics; hypertension; pediatric cardiology; vascular biomechanics.

Fig. 1.

Magnetic resonance images showing the similarity between untreated and treated coarctation of the aorta (CoA) in humans (left top) vs. a rabbit model (left bottom) developed to study mechanisms of arterial dysfunction in the absence of concurrent anomalies. Yellow arrows denote treatment sites. The right images show CoA suture (black arrows) and resulting blood pressure gradient (BPG) for groups of juvenile rabbits (n = 7/group) with 20 mmHg BPG. Symbols indicate significantly (P < 0.05) different from Control (*) and Treated (§) groups.

Fig. 2.

Normalized natriuretic peptide receptor C (NPR-C) expression from RNA sequencing (RNA-Seq) of aortic tissue harvested during surgical treatment of human CoA patients (A). All patients had systolic blood pressure (BP) >99% for age, thus underscoring the mechanical impact of CoA. Prior microarray results from a rabbit model of CoA (B) also showed a decrease in NPR-C transcript levels in proximal aorta exposed to elevated BP from the presence of a coarctation. Data expressed as means ± SE. *P < 0.05.

Fig. 3.

Human aortic endothelial cells (HAEC) showed decreased intracellular calcium ([Ca²⁺]_i) response to C-type natriuretic peptide (CNP, 1.5e-6M). The NPR-C agonist cANF^4–23 (7.5e-7M) increased [Ca²⁺]_i release. Both responses were blocked by the NPR-C inhibitor M372049. *P < 0.05 vs. Baseline.

Fig. 4.

Two-photon images of [Ca²⁺]_i levels from endothelial cells (EC) within intact sections for groups of Control and CoA rabbits at baseline and upon administering CNP (10 μM). Temporal results show differences in proximal vs. distal locations (n = 9 EC/location). CNP decreased [Ca²⁺]_i response for ECs within both sections of Control rabbits and distal sections of CoA rabbits, but this response is absent in proximal sections of CoA rabbit, which were exposed to high BP in vivo.

Fig. 5.

Dose-dependent relaxation of the proximal and distal aorta to CNP (left) and atrial natriuretic peptide (ANP, right) for groups of Control and CoA rabbits. Relaxation of proximal and distal aortic rings was not different in samples from Control rabbits (top row). There was statistically significant attenuation of relaxation in proximal as compared with distal aortic rings for CoA rabbits when exposed to all doses of CNP and ANP (middle row). Residual relaxation to these peptides was abolished by pretreatment with the NPR-C inhibitor M372049 (bottom row). PE, phenylephrine.

Fig. 6.

Allelic discrimination plots for a representative plate from each NPR-C variant. Samples with the noncommon genotype for each variant are indicated by the ellipses.