Mice overexpressing chromogranin A display hypergranulogenic adrenal glands with attenuated ATP levels contributing to the hypertensive phenotype

Abstract

Objective: Elevated circulating chromogranin A (CHGA) is observed in human hypertension. CHGA is critical for granulogenesis and exocytosis of catecholamine stores from secretory large dense core vesicles (LDCV). This study aims to understand the morphological, molecular and phenotypic changes because of excess CHGA and the mechanistic link eventuating in hyper-adrenergic hypertension.

Methods: Blood pressure and heart rate was monitored in mouse models expressing normal and elevated level of CHGA by telemetry. Catecholamine and oxidative stress radicals were measured. Adrenal ultrastructure, LDCV content and mitochondrial abundance were compared and respiration analyzed by Seahorse assay. Effect of CHGA dosage on adrenal ATP content, electron transport chain components and uncoupling protein 2 (UCP-2) were compared in vivo and in vitro.

Results: Mice with excess-CHGA displayed hypertensive phenotype, higher heart rate and increased sympathetic tone. They had elevated plasma catecholamine and adrenal ROS levels. Excess-CHGA caused an increase in size and abundance of LDCV and adrenal mitochondria. Nonetheless, they had attenuated levels of ATP. Isolated adrenal mitochondria from mice with elevated CHGA showed higher maximal respiration rates in the presence of protonophore, which uncouples oxidative phosphorylation. Elevated CHGA resulted in overexpression of UCP2 and diminished ATP. In vitro in chromaffin cells overexpressing CHGA, concomitant increase in UCP2 protein and decreased ATP was detected.

Conclusion: Elevated CHGA expression resulted in underlying bioenergetic dysfunction in ATP production despite higher mitochondrial mass. The outcome was unregulated negative feedback of LDCV exocytosis and secretion, resulting in elevated levels of circulating catecholamine and consequently the hypertensive phenotype.

FIGURE 1

Overexpression of chromogranin A is associated with attenuated nondipping hypertensive phenotype. The four-copy mice that over-express CHGA carry two copies each of the mouse Chga and the human CHGA genes. The ‘humanized’ two-copy mice carry and express only two copies of the human CHGA gene in lieu of mouse Chga alleles. (a) Tissue extracts were prepared from adrenal glands of two-copy and four-copy mice. Total protein (5μg) was electrophoresed and probed for CHGA expression using anti-CHGA antibody. In four-copy mice, CHGA expression was nearly doubled compared with expression in the two-copy mice. Housekeeping protein GAPDH was used as loading control. (b) Graph showing densitometric quantitative measurement of CHGA expression normalized to GAPDH. Continuous telemetric recordings of BP and heart rate for 24 h in both stains of mice (two-copy and four-copy; each n = 4) were performed. Average data for each hour is plotted and the error bars show ±SEM. The data analyzed by repeated measure ANOVA using linear mixed model, showed significant differences in SBP, DBP and heart rate between the two mouse strains. (c) Average SBP was elevated by 19.19mmHg in four-copy (138.24±1.98 mmHg) compared with that of two-copy mice (119.05±1.98 mmHg); (time-F = 3.22, P<0.001; strain-F=46.77, P<0.001). (d) DBP was also significantly higher by 16.68mmHg in four-copy mice (103.28±5.6mmHg) compared with two-copy (86.6±6.3 mmHg); (Time- F=4.45, P< 0.001; Strain- F=54.42, P<0.001). (e) The heart rate of two-copy mice (492±11.49bpm) was significantly lower by ~89bpm compared with mice over-expressing CHGA (581±16.25bpm; time-F=5.28, P<0.001; strain-F=29.98, P=0.002). The 24-h period of telemetric BP recording was stratified into three blocks of time intervals (1–8, 9–16 and 17–24 h) and the SBP/DBP plotted. (f) Both strains of mice display diurnal SBP dipping (during hours 9–16), however, this dipping in SBP is attenuated in four-copy mice (***P=0.05) compared with two-copy mice (**P=0.004). The difference between the dipping (hours 10–17) and nondipping period (1–8 and 17–24) is 5.4mmHg for two-copy versus 2.9mmHg in case of four-copy mice. The SBP is significantly elevated for four-copy compared with two-copy mice (*P<0.0001). (g) The hypertensive four-copy mice showed loss of diurnal DBP variation (P=0.3). The difference in DBP for four-copy versus two-copy mice is significant (*P<0.0001). The two-copy mice display a 7mmHg dip in DBP. CHGA, chromogranin A.

FIGURE 2

Adrenal chromogranin A overexpression elevated sympathetic activity and oxidative stress while kidney function was unaffected. (a) Plasma from both two-copy and four-copy strains of mice were measured for catecholamine levels. Circulating levels are elevated because of CHGA over-expression suggesting augmented release of catecholamine, N = 8. (b) Adrenal gland extracts of four-copy mice have attenuated levels of epinephrine and norepinephrine compared with two-copy mice, indicative of dysregulated storage and release of catecholamine from LdCv, N = 8. (c) Elevated CHGA in adrenal glands resulted in accumulation of ROS, which is 1.3-fold higher in four-copy mice versus two-copy mice. Data is one representative of three independent experiments, N = 7. (d) Renal function (estimated GFR) measured as a function of renal clearance of creatinine remains unchanged in 12–16-week-old mice because of overexpression of CHGA, N = 8. CHGA, chromogranin A; GFR, glomerular filtration rate; LDCV, large dense core vesicles; ROS, reactive oxygen species.

FIGURE 3

Augmented chromogranin A expression leads to increase in number and size of chromaffin granules. (a) Adrenal glands from both two-copy and four-copy strains of mice were prepared for transmission electron microscopy and morphometric analysis. LDCV in the chromaffin cells (indicated by arrows) located in the medullary region were counted for their abundance and size. Data was collected from ~20 micrographs for each type of mouse (bar scale = 1 mm). (b) Granule abundance per unit area (mm²) in the cytoplasm of adrenal medullary cells of four-copy mice was significantly higher compared with two-copy mice. (c) Average size of the chromaffin granules (nm²) in the adrenal medulla of four-copy mice significantly increased compared with that of two-copy mice. (d) Area covered by LDCV (% area) per unit area was significantly higher in four-copy mice compared with two-copy. CHGA, chromogranin A; LDCV, large dense core vesicles.

FIGURE 4

Homeostasis of granin expression in the large dense core vesicles of adrenal glands. (a) qPCR analysis of other granin family members – chromogranin B (Chgb) and secretogranin 2 (Scg2) mRNA levels showed no difference in expression between the two-copy and four-copy mice adrenal glands. Data were normalized against IBS ribosomal RNA. (b) Total adrenal extracts were also analyzed by western blots for expression of CHGB and SCG2 proteins. Both CHGB and SCG2 protein expression was significantly reduced in four-copy mice that express excess CHGA indicating, translational regulation of total granin expression. Even loading was confirmed by probing blots with housekeeping protein GAPDH. (c) Bar graph showing quantitative difference in protein expression of CHGB and SCG2. CHGA, chromogranin A; CHGB, chromogranin B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 5

Influence of chromogranin A overexpression on large dense core vesicles constituents. qPCR analysis normalized to 18S rRNA did not show any significant change in expression of Npy and catecholamine biosynthetic pathway genes – Th and Dbh, because of overexpression of CHGA. Significant increase in vesicular amine transporter Vmatl was observed in the adrenal glands of four-copy compared with that of two-copy mice. CHGA, chromogranin A.

FIGURE 6

Chromogranin A overexpression does not affect calcium levels but diminished ATP in mouse adrenal gland. Adrenal extracts were assayed for calcium and ATP levels. (a) No significant change in total calcium was observed. (b) A significant attenuation in ATP level was observed in the adrenal extracts of four-copy (n = 13) compared with two-copy (n = 15) mice.

FIGURE 7

Mitochondria and Ox-Phos complex proteins increased because of elevated chromogranin A, concomitant with uncoupling protein 2 overexpression and dysregulated mitochondrial bioenergetics. Mitochondrial respiration response to an uncoupler of mitochondrial oxidative phosphorylation was elevated by CHGA overexpression. (a) Total DNA was extracted from adrenal glands of two-copy and four-copy mice and qPCR analysis was done using mitochondrial gene-specific primers (MtNDI). Mitochondrial DNA content was significantly higher in four-copy mice compared with two-copy mice. Gapdh-specific primers were used as control for nuclear gene expression. (b) Expression of the mitochondrial oxidative phosphorylation (Ox-Phos) complexes was analyzed by western blot. The protein levels of complex II–V were significantly increased in mice overexpressing CHGA compared with two-copy mice. Even loading was confirmed by normalizing to GAPDH protein. (c) Densitometric analysis confirmed significant elevation in the Ox-Phos complex enzymes in adrenal extracts of four-copy mice. (d) Basal and stimulated OCR was measured in isolated mitochondrial preparations from adrenal glands of four-copy (solid circles) and two-copy (open circles) CHGA mice using Seahorse XFe 96 Extracellular Flux Analyzer. Data analyzed from 48 wells for each group are summarized from six (two-copy) and seven (four-copy) independent experiments and presented as percentage OCR normalized to non-ADP-treated levels. Data was analyzed by independent samples t-test. Under basal conditions, the energetic demand of the isolated mitochondria was comparable in mitochondria of both strains of mice (P=0.279). To enhance substrate utilization by the mitochondria, along with substrate succinate, ADP or FCCP was injected. Both ADP and FCCP stimulated OCR compared with basal (P< .001). Statistical analysis showed that whenever normalized to basal level, the FCCP-induced increase of OCR was higher for the four-copy group (P = 0.037). (e) In the adrenal extracts of four-copy mice overexpressing CHGA a significant elevation in UCP2 protein expression was observed compared with two-copy mice. Arrows indicate dimer (~70 kDa) as well as monomer (~35 kDa) bands of the UCP2 protein. Probing blot with GAPDH shows equal loading. (f) Densitometric analysis of the band intensities observed in the western blots confirm significant increase in both the monomer and dimer forms of UCP2 in adrenals of four-copy mice. CHGA, chromogranin A; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UCP2, uncoupling protein 2.

FIGURE 8

Cultured rat chromaffin cells overexpressing CHGA have elevated uncoupling protein 2 and diminished ATP level. (a) PC12 cells were cultured and transfected with either empty vector (pcDNA3.1) or a construct expressing CHGA driven by CMV promoter (CHGA-WT-pCMV6XL5) with or without genipin treatment (50 μmol/l). Western blot analysis of transfected cell extracts showed ~2-fold increase in CHGA expression ins PC12 cells transfected with CHGA construct compared with control cell extracts. With CHGA overexpression concomitant increase in UCP2 protein expression is observed. GAPDH was used as a loading control. (b) Densitometric analysis of western blot results showing significant increase in CHGA with or without genipin treatment. UCP2 expression was increased significantly because of CHGA overexpression. (c) CHGA overexpression in PC12 cells caused a significant decline in ATP level. Pharmacological inhibition of UCP2 with genipin (50μmol/l), restored the ATP levels. (d) No significant change in cellular ROS level was observed because of either CHGA overexpression or genipin treatment. CHGA, chromogranin A; GAPDH, glyceralde-hyde-3-phosphate dehydrogenase; ROS, reactive oxygen species; UCP2, uncoupling protein 2.