Chromogranin/secretogranin proteins in murine heart: myocardial production of chromogranin A fragment catestatin (Chga(364-384))

Abstract

In the heart, the secretory granules containing the atrial natriuretic peptides (ANP) and B-type myocardial natriuretic peptide (BNP) provide the basis for the endocrine function of this organ. We sought to determine whether atrial and myocardial secretory granules contain chromogranin/secretogranin proteins including chromogranin A (CHGA/Chga), chromogranin B (CHGB/Chgb) and secretogranin II (SCG2/Scg2). Deconvolution microscopy on immunolabeled proteins revealed the presence of Chga, Chgb, and Scg2 in murine cardiac secretory granules. The presence of low plasma catestatin (CST: mChga(364-384)) in older mice indicates diminished processing of Chga to CST with advancement of age, which is comparable to that found in humans. We have previously shown that CST (hCHGA(352-372)) exerts potent cardio-suppressive effects on frog and rat heart, but the source of CST for such action has remained elusive. In the present study, we found CST-related peptides in cardiomyocytes and in heart, which establishes an autocrine/paracrine function of CST in cardiac tissue. We conclude that cardiac secretory granules contain Chga, Chgb and Scg2 and that Chga is processed to CST in murine heart.

Fig. 1

The expression of chromogranin/secretogranin proteins in murine heart. The localization of Chga (a), Chgb (b) and Scg2 (c) were assessed in primary culture of cardiomyocytes, derived from 3- to 4-day-old pups. Cardiomyocytes were stained with primary antibodies specific for each chromogranin/secretogranin proteins and the cardiomyocyte specific markers such as myosin heavy chain (MYH) (d) and atrial natriuretic peptide (ANP) (e). The secondary antibodies used were donkey anti-rabbit IgG-Alex Flur 488 (green fluorescence, 1:250) and donkey anti-goat IgG-Alex Flur 594 (red fluorescence, 1:350). Nuclei were visualized with Hoechst 33342 (blue). Cells were examined by deconvolution microscopy using a ×60 objective (scale bar 5 μm) for the upper panel and a ×20 objective (scale bar 10 μm) for the lower panel. f Cardiomyocyte cell extracts (lane 1 20 μg protein and lane 2 40 μg protein) were analyzed by SDS-PAGE followed by immunoblot with a CST specific antibody. g Freshly frozen adult (6-month-old) mice heart and adrenal tissues were homogenized in TRIS-maleate buffer and were subjected to SDS-PAGE and immunoblot analysis. Heart extracted proteins (∼75 μg) were run by SDS-PAGE for the detection of Chga (by rabbit anti-human CST), Chgb (by goat anti-human CHGB) and Scg2 (by rabbit anti-human SCG2). Adrenal extracts (proteins in lane 1 2 μg, lane 2 15 μg, lane 3 30 μg, and lane 4 60 μg) were also run by SDS-PAGE for the detection of Chga (by rabbit anti-human CST). For adrenal extract the full-length Chga signal was shown in the upper panel and the low molecular mass fragments were shown in the lower panel

Fig. 2

Age-dependent processing of Chga to CST in murine heart. Heart from different age groups of mice (age in days: 10, 30, 60 and 90) were collected, homogenized and subjected to SDS-PAGE and immunoblot using a CST-specific antibody (rabbit anti-human CST) (a). b The endogenous CST concentrations were measured from tissue extracts using a CST EIA kit. CST concentrations are expressed in pg/μg of protein

Fig. 3

Identification of CST-related peptides from heart extract. Mice hearts (6 months old) were homogenized in acetic acid and subjected to reverse phase HPLC purification. The proteins were eluted (the chromatogram showing protein elution as measured at 214 nm with time is shown in (a)), with a gradient of acetonitrile and the fractions were collected, lyophilized and spotted onto nitrocellulose membrane to probe with a CST-specific antibody (b). Fraction numbers are shown on the right of the respective slot blot (b)

Fig. 4

Anti-CST immunoprecipitation from the heart extract. Mice heart (1-month-old animal) were homogenized in TRIS-maleate buffer and subjected to immunoprecipitation from 2 to 1 mg protein with anti-CST antibody. The antigen-antibody complexes were pulled down with Protein A/G beads and subjected to immunoblot analysis (lane 1 IP from 2 mg protein, lane 2 IP from 1 mg protein) (a). In (a), rabbit anti-human CST antibody was used to detect CST-related peptides. The middle panel of (a) showed the darker exposure of the lower part of the gel. The right panel of (a) showed the running position of the synthetic human CST in the same gel. For MALDI-TOF (b), proteins eluted from the beads with acetonitrile/water/TFA were lyophilized and analyzed by mass spec