Catestatin Gly364Ser Variant Alters Systemic Blood Pressure and the Risk for Hypertension in Human Populations via Endothelial Nitric Oxide Pathway

Abstract

Catestatin (CST), an endogenous antihypertensive/antiadrenergic peptide, is a novel regulator of cardiovascular physiology. Here, we report case-control studies in 2 geographically/ethnically distinct Indian populations (n≈4000) that showed association of the naturally-occurring human CST-Gly364Ser variant with increased risk for hypertension (age-adjusted odds ratios: 1.483; P=0.009 and 2.951; P=0.005). Consistently, 364Ser allele carriers displayed elevated systolic (up to ≈8 mm Hg; P=0.004) and diastolic (up to ≈6 mm Hg; P=0.001) blood pressure. The variant allele was also found to be in linkage disequilibrium with other functional single-nucleotide polymorphisms in the CHGA promoter and nearby coding region. Functional characterization of the Gly364Ser variant was performed using cellular/molecular biological experiments (viz peptide-receptor binding assays, nitric oxide [NO], phosphorylated extracellular regulated kinase, and phosphorylated endothelial NO synthase estimations) and computational approaches (molecular dynamics simulations for structural analysis of wild-type [CST-WT] and variant [CST-364Ser] peptides and docking of peptide/ligand with β-adrenergic receptors [ADRB1/2]). CST-WT and CST-364Ser peptides differed profoundly in their secondary structures and showed differential interactions with ADRB2; although CST-WT displaced the ligand bound to ADRB2, CST-364Ser failed to do the same. Furthermore, CST-WT significantly inhibited ADRB2-stimulated extracellular regulated kinase activation, suggesting an antagonistic role towards ADRB2 unlike CST-364Ser. Consequently, CST-WT was more potent in NO production in human umbilical vein endothelial cells as compared with CST-364Ser. This NO-producing ability of CST-WT was abrogated by ADRB2 antagonist ICI 118551. In conclusion, CST-364Ser allele enhanced the risk for hypertension in human populations, possibly via diminished endothelial NO production because of altered interactions of CST-364Ser peptide with ADRB2 as compared with CST-WT.

Keywords: chromogranin A; genetic association study; genetic variation; hypertension; nitric oxide.

Figure 1. Allele-specific associations of the CST Gly364Ser variation with blood pressure

Panels A, B and C: Data are shown as mean ± SE. SBP (A), DBP (B) and MAP (C) levels in the carriers of 364Ser allele were higher (analyzed by independent samples t-test using SPSS version 21.0) than the wild-type individuals in the overall Chennai and Chandigarh populations. Panels D and E: Data are shown as percentage. The percentage of individuals harboring the 364Ser allele showed an increase with increase in the range of the MAP levels in both the Chennai (D) and Chandigarh (E) populations.

Figure 2. Effect of CST peptides on NO production in HUVECs

The fluorescence intensities (NO indices) were calculated by Image J analysis and plotted as mean ± SE. The experimental groups were compared by one-way ANOVA followed by Tukey’s multiple comparison post-test. Panels A and B: Representative images for the treatment of HUVECs with different doses of CST-WT (0.1 nmol/L and 1 nmol/L). ***p<0.001; one-way ANOVA F=15.71, p<0.0001, n=50 cells/condition. Panels C and D: Representative images for the treatment of HUVECs with CST peptides. ***p<0.001; one-way ANOVA F=37.15, p<0.0001, n=450 cells/condition. The order for efficacy of peptides in NO production: CST-WT>CST-WT+Ser>CST-364Ser>basal. Panels E and F: Representative images for the treatment of HUVECs with CST peptides and ADRB2 antagonist ICI 118551. ***p<0.001; one-way ANOVA F=19.65, p<0.0001, n=450 cells/condition. Panel G: Representative western blot of 3 independent experiments showing phosphorylated Ser¹¹⁷⁷-eNOS (peNOS) and total eNOS (teNOS) levels upon treatment of HUVECs with CST peptides. The peNOS/teNOS values have been indicated below each lane.

Figure 3. Binding of CST peptides to ADRB2 receptor and downstream effects

Panel A: ADRB2 HEK-293 cells showed ~16-fold higher expression of ADRB2 (p<0.0001 by 2-tailed unpaired t-test) as compared to control HEK-293 cells. Data are shown as ADRB2 levels normalized with total protein. Panel B: Data are shown as percentage binding of the radio-ligand cyanopindolol. With increasing doses of CST-WT (10 pmol/L to 1 mmol/L) the ligand got completely displaced (p<0.0001, F=2300, R²=0.998) while with increasing doses of CST-364Ser, there was no effect. The experimental groups were compared by one-way ANOVA followed by Tukey’s multiple comparison post-test. Panels C and D: Representative western blot (C) and quantitative representation of the densitometric analysis from 4–6 independent experiments (D) showing phosphorylated ERK (pERK) and total ERK levels upon treatment with CST peptides and isoproterenol (ISO). ISO (10 μmol/L) showed an increase in pERK levels at 5 min in the vehicle (VEH) condition, reflecting the activation of ADRB2. However, this increase was inhibited upon pre-treatment with CST-WT (10 μmol/L). ****p<0.0001. On the other hand, pre-treatment with CST-364Ser (10 μmol/L) showed levels of activation similar to the vehicle. The experimental groups were compared by 2-tailed t-test. Panels E and F: Representative western blot (E) and quantitative representation of the densitometric analysis from 4 independent experiments (F) showing phosphorylated ERK (pERK) and total ERK levels upon treatment with equimolar ratios of CST-WT and CST-364Ser peptides and isoproterenol (ISO). ISO (10 μmol/L) showed an increase in pERK levels at 5 min in the vehicle (VEH) condition. However, this increase was inhibited upon pre-treatment with CST-WT+Ser (10 μmol/L). *p<0.05. The experimental groups were compared by 2-tailed t-test.

Figure 4. Structures of CST peptides and complexes of CST peptides/cyanopindolol with ADRB2 receptor

The time averaged structures of CST-WT (A) and CST-364Ser (B) are shown in cartoon representation. The 364Ser mutation in the CST-364Ser peptide is shown in a stick representation. (C) Snapshots of CST-WT (left) and CST-364Ser (right) docked to ADRB2. ADRB2: violet, beta-sheet in CST-WT: yellow, alpha-helix in CST-364Ser: purple and 3₁₀-helix in CST-364Ser: blue. (D) Snapshot of the docked complex of cyanopindolol (red, van der Waals representation) and ADRB2 (violet, cartoon representation).

Figure 5. Molecular interactions in the complexes of CST peptides or cyanopindolol with ADRB2

Panels A and B: Binding interactions in CST-WT-ADRB2 (A) and CST-364Ser-ADRB2 (B) complexes. Hydrogen bonds: blue lines, hydrophobic contacts: orange lines and salt bridges: red lines. Each residue is color-coded based on its nature with aliphatic residues in grey; aromatic residues in pink; negatively charged residues in red; positively charged residues in cyan; neutrally charged residues in green; Pro and Gly in orange and Cys in yellow. Gly364Ser polymorphism: red stars. Panel C: Binding interactions of cyanopindolol with ADRB2. Hydrophobic interactions: red spiked semi-circles and hydrogen bonding interactions: green dotted lines with distance values indicated. Common interacting residues of ADRB2 with CST-WT and cyanopindolol are highlighted using purple boxes in Panel A and purple circles in Panel C, respectively.

Figure 6. A schematic representation of the plausible mechanistic basis for the effects of CST peptides on BP via modulation of NO pathway

The CST-364Ser peptide does not interact at the ligand binding site of ADRB2 unlike CST-WT owing to differences in their secondary structures. Their differential interactions with ADRB2 result in diminished antagonization of ADRB2 and enhanced activation/ phosphorylation of ERK by CST-364Ser. The altered ERK activation between the CST peptides may result in diminished phosphorylation of eNOS-Ser¹¹⁷⁷ and consequently lower eNOS activity in the case of CST-364Ser. These cellular/molecular processes lower the NO levels in vascular endothelial cells in the carriers of CST 364Ser allele leading to endothelial dysfunction and thereby increasing their risk for hypertension.