Regulated BNP secretion in ventricular cardiomyocytes

Kun Zhang, Ketaki Mhatre, Paulina Wakula-Heinzel, Burkert Pieske, Frank R. Heinzel

Introduction
The heart secretes hormones and can be considered as an endocrine organ. Previously, chromogranins as typical hormones of secretory cells could be found in cardiomyocytes. However, little is known about the mechanisms of secretion. In neuronal cells, a regulated pathway of exocytosis is well described. Parallels between neurons and cardiomyocytes can be expected since both belong to the group of excitable cells. Thus, it is feasible to believe excitation-secretion-coupling is a mechanism that also exists in cardiomyocytes. We hypothesize that a regulated pathway of brain natriuretic peptide (BNP) secretion exists in ventricular cardiomyocytes.

Methods
We isolated neonatal rat ventricular myocytes from 2-day-old Wistar rats. Briefly, hearts were removed and ventricles dissected from atrias. Dissociation of ventricular cardiomyocytes was achieved by sequential digestion with trypsin. After pre-plating for 1 hour at 37°C, myocytes were seeded on gelatin coated wells. BNP ELISA (Abnova) was performed with measuring BNP concentrations in collected cell lysates and their corresponding supernatants. Immuno-fluorescence was conducted using following antibodies: synaptobrevin 1:250, goat anti-rabbit Alexa488 1:1000 (Life Technologies). Cells were visualized via confocal microscope (Zeiss).

Results
We could show via immunofluorescence that ventricular cardiomyocytes express synaptobrevin (figure), which can be found in neurons and endocrine cells and is a marker protein of secretory vesicles that undergo calcium regulated exocytosis. When stimulating cardiomyocytes with 1 µM angiotensin II (Ang II) for 2h, which has been shown to activate inositol 1,4,5-trisphospate (IP3) and increase calcium release from intracellular stores into cytoplasm, we could see an increase in BNP secretion as described in the literature. Interestingly, the ratio of BNP secretion/production increased under Ang II stimulation (189 ± 54% normalized to vehicle control, p=0.05), indicating that Ang II induced increased exocytosis and thus BNP release. Moreover, ratio of BNP secretion/production returned back to baseline after 4h of Ang II stimulation (113 ± 18% normalized to control, p=0.25).

Conclusion
We showed that ventricular cardiomyocytes exhibit a vesicle protein involved in calcium regulated exocytosis. Ang II, which increases intracellular calcium, led to increased BNP secretion in relation to production. Moreover, a time dependency could be observed. Based on these data, we propose that a calcium dependent, regulated way of exocytosis exists in cardiomyocytes. Further studies are ongoing to elucidate this mechanism.



Figure. Immunofluorescence showing granular pattern of synaptobrevin staining in NRVM, with enhanced perinuclear distribution.