Effects of chromogranin A deficiency and excess in vivo: biphasic blood pressure and catecholamine responses

Abstract

Objective: The phenotype of the chromogranin A (Chga) null (knockout) mouse is hypertensive. However, hypertensive humans and spontaneously hypertensive rats display elevated CHGA expression. This study addresses the paradox that both ablation and elevation of CHGA result in hypertension.

Methods: Mice with varying copy number of the CHGA gene were generated. In these mice CHGA, catecholamine and blood pressure (BP) were measured. Also a cohort of healthy human individuals was stratified into tertiles based on plasma CHGA expression and phenotyped for characteristics including their BP response to environmental (cold) stress.

Results: The mice displayed a direct CHGA gene dose-dependent (0-4 copies/genome) activation of CHGA expression in both plasma and adrenal gland, yet the BP dependence of CHGA gene dose was U-shaped, maximal at 0 and four copies of the gene, whereas minimal at two copies (i.e., the wild-type gene dosage). Plasma catecholamine showed a parallel U-shaped dose/response in mice, whereas adrenal epinephrine exhibited a reciprocal (inverted) U-shaped response, suggesting dysregulated neurotransmission at both extremes of CHGA expression. The human individuals also showed a nonlinear relationship between CHGA expression and pressor responses to environmental (cold) stress, that were maximal in the highest and lowest tertiles, though basal BPs did not differ among the groups. The human CHGA tertiles also differed in epinephrine secretion as well as degree of CHGA processing to catestatin (catecholamine release-inhibitory peptide derived from CHGA processing).

Conclusion: Thus, across mammalian species, an optimal amount of CHGA may be required to establish appropriate catecholamine storage and release, and hence BP homeostasis.

Figure 1. Schematic for generating mice with varied copy numbers of chromogranin A gene

The transgenic founder has a single copy of the human CHGA BAC clone RP11862G15 stably integrated in its genome. The “humanized” CHGA (HumCHGA) line, was derived by mating a (symbolized as X) transgenic founder with Chga knockout (KO) as described earlier [3]. They have 2-copies of the human CHGA gene and lack the mouse allele. Similarly, the founder was mated with wild-type (WT, Chga+/+) mice to generate 4-copy chromogranin A mice (2 alleles each, of human and mouse). The F1 pups of the mating between 4-copy mice with 2-copy HumCHGA mice generated the 3-copy mice. Also the F1 pups of mating between 2-copy HumCHGA and KO mice produced the 1-copy CHGA mice (1 human copy). The mice with 0, 1, 2, 3 or 4 copies of chromogranin A gene used in subsequent experiments have been highlighted in bold.

Figure 2. ELISA assaying CHGA levels in plasma of mice with varying copies of the chromogranin A gene

The capturing mouse monoclonal antibody 5A8, raised against the amino-terminal region of human CHGA, differs in its affinity for both mouse and human CHGAs, with a stronger affinity for the latter [10]. 5A8 binds to the epitope in the CHGA53-57 region. Therefore full-length and fragments of CHGA with the epitope are detected by the assay. Both mouse (Mou) and/or human (Hum) alleles contribute towards the total number of chromogranin A alleles. Plasma from each group of mice (n = 8) was assayed. Increasing levels of circulating CHGA with boost in copy number is observed.

Figure 3. Expression of chromogranin A in adrenal glands

A. Immunoblot demonstrating that an increase in copy number of chromogranin A alleles augments expression of CHGA protein in the adrenal gland. The primary antibody is rabbit polyclonal raised against human catestatin (human CHGA_352-372). A total of 5 μg adrenal protein was loaded per lane. The antibody detects the full length and the processed fragments of CHGA and cross-reacts with mouse and human. B. Densitometric scan of the same immunoblot, normalized to actin levels.

Figure 4. Biphasic effect of chromogranin A gene dosage on blood pressure

Mice with 2 copies of human CHGA display normal BP (104.5/77.8 mm Hg). Both an increase and decrease in gene dosage of chromogranin A results in BP amplification ranging between ∼15 to 33 mmHg (systolic) and ∼17 to 31 mmHg (diastolic). The BP was recorded by tail-cuff measurements in n = 86, 18, 85, 28 and 28 mice of the groups 0, 1, 2, 3 and 4-copy, respectively. The age of the mice is 12-14 weeks. Comparing 2-copy (*) mice vs. each of the other groups (◆) p <0.0001, and vs. (:) p=0.0008 by student t-test analysis.

Figure 5. Catecholamine response to chromogranin A gene dosage. A, B & C

Group 0, 1, 2, 3 and 4 copy mice: n = 5, 7, 8, 8 and 8 of age 12 weeks were used for the experiment. A. & B. Copy number of chromogranin A gene significantly elevates both plasma NE and E in 0, 1 and 4-copy mice (◆) compared to 2-copy mice (*). The student t-test analysis of NE levels, 2-copy vs. 0-copy p = 0.001, 2-copy vs. 1-copy p = 0.03, 2-copy vs. 3-copy p = .91, 2-copy vs. 4-copy p < .0001. Circulating E concentration 2-copy vs. 0-copy p = .0007, 2-copy vs. 1-copy p = 0.04, 2-copy vs. 3-copy p = 0.06, 2-copy vs. 4-copy p = 0.0005. C. Adrenals of 2-copy mice express the highest levels of E. Comparatively, 0 and 4-copy mice show depressed levels of E. 2-copy vs. 0-copy p = 0.006, 2-copy vs. 1-copy 0.03, 2 vs. 3-copy p = 0.014, 2-copy vs. 4-copy p=0.019. The NE levels do not vary significantly with chromogranin A gene dosage.

Figure 6. Environmental (cold) stress test BP responses in individuals with varying plasma CHGA

The change in BP was measured in individuals belonging to the 3 tertiles upon being subjected to systematic cold stress. The cold stress final SBP and DBP values were significantly higher in all three groups compared to basal level. However the tertile (II) had the smallest change in BP compared to both tertiles I and III, indicating that this group of individuals, has the least efferent sympathetic outflow. This biphasic response to CHGA protein dosage in humans mimics the BP response to chromogranin A gene dosage in mice.

Figure 7. Chromogranin A levels in plasma of human subjects

The plasma CHGA precursor was accessed using radioimmunoassay based on the large fragment CHGA116-439. Processing to the catestatin fragment (human CHGA352-372) was assayed by a synthetic peptide epitope corresponding to CHGA amino acids 361-372. A. A linear increase in the precursor CHGA116-439 is seen in the 3 tertiles as expected based on the stratification. B. Catestatin, the nicotinic-cholinergic antagonist peptide derived by processing full-length CHGA was assayed in the plasma of these individuals, surprisingly the least catestatin is observed in tertile III individuals that have the most precursor. C. Ratio of catestatin: full-length CHGA again indicates least efficient processing of CHGA to catestatin in tertile III individuals.