The metastasis suppressor kisspeptin is a positive inotropic agent in mouse heart

Helen Kirby, Janet Maguire, Sarah Pitkin, William Colledge, Xavier d’Anglemont de Tassigny, Anthony Davenport

University of Cambridge, Cambridge, UK

The recent pairing of the G-protein-coupled receptor KISS1, previously known as GPR54 (Ohtaki et al., 2001), to kisspeptin, a 54-amino acid peptide, has led to a plethora of discoveries concerning its function. Kisspeptin is known to be involved in metastasis inhibition (Lee et al., 1996), trophoblast invasion during placentation and regulation of the initiation of puberty. Humans with mutations in KISS1 have delayed puberty and low levels of sex steroids, mimicked in mice lacking KISS1 (Seminara et al., 2003). We have previously reported vasoconstrictor activity of kisspeptin in human coronary artery and umbilical vein (Mead et al., 2007) and have detected KISS1 immunoreactivity in endocardial endothelial cells and cardiomyocytes of the human heart. We have observed kisspeptin to be a positive inotropic agent in human atrial appendage (Mead, unpublished data). The mouse is a valuable model for the study of human disease; however, to date, no investigations have been carried out on the cardiovascular actions of kisspeptin in this species. Therefore, our aim was to determine the cellular distribution of KISS1 in mouse heart and to also investigate any potential positive inotropic activity of KISS1 in mouse atria.

Receptor protein was detected by Western blotting on heart homogenates (n=3) from male C57/BL6 mice (20-30g) using SDS-PAGE and a semi-dry blotting method, before probing with rabbit anti-KISS1 antisera and detection via the ECl method. For immunochistochemistry, 30μm cryostat-cut heart sections were incubated with rabbit anti-KISS1 antisera and detection via a peroxidase/antiperoxidase method. For cellular localisation, sections of heart were incubated with sheep anti-von Willebrand factor (vWF) and rabbit anti-KISS1 antisera and detection via Alexa Fluor secondary antibodies. For in vitro pharmacology, paired atria from male 129S6/SvEv mice (20-30g; euthanised by CO2 inhalation) were set up in organ baths and paced using field stimulation (1Hz, 5ms duration, <4V). Tension on the tissue was adjusted to 50% of optimum resting tension before construction of cumulative concentration response curves to kisspeptin and termination by CaCl2 addition (6.7 mM). Agonist responses were expressed as %CaCl2 and data are mean ± s.e.mean.

Western blot analysis of a novel anti-KISS1 (mouse) antibody revealed a single band of 75kDa in all 3 heart samples, consistent with KISS1 protein. Immunohistochemistry showed widespread distribution of KISS1 throughout the mouse heart, with high expression in intramyocardial blood vessels and cardiomyocytes. Dual-labelling revealed colocalisation with an endothelial cell marker. In wild type mouse atria, KP-54 induced positive inotropic effects (pD2: 10.22±0.34, Emax; 25.8±4.0 %CaCl2; n=6) whereas, in atria from gpr54-/- mice, no responses to KP-54 were observed (n=4).

In conclusion, we have detected KISS1 throughout mouse heart and have shown it to be localised to vascular endothelial cells and cardiomyocytes. For the first time we have shown that kisspeptin acts as a positive inotropic agent in mouse heart and the lack of activity observed in gpr54-/- mice suggests that kisspeptin acts solely through KISS1.

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Seminara, S.B., et al. (2003). N Engl J Med, 349, 1614-1627