Absence of S100A1 in mice is associated with a hypertensive phenotype dependent on both gender and endothelial function

1. Absence of S100A1 in mice is associated with a hypertensive phenotype dependent on both gender and endothelial function Jean-Francois Desjardins; Krystyna Kuliszewska; Adrian Quan; Duncan J. Stewart; Subodh Verma; Thomas G. Parker

2. Presenter disclosure information Jean-Francois Desjardins Absence of S100A1 in mice is associated with a hypertensive phenotype dependent on both gender and endothelial function Disclosures : none

3. Introduction S100A1 • Small dimeric EF-hand Ca2+-binding protein (20kDa) • Structurally related to Calmodulin • Binds target proteins and regulate a variety of cellular processes • Phosphorylation and enzyme activity • Highly expressed in cardiomyocytes (0.2% total soluble protein content) • Positive regulator of cardiac performance (Most et al., (2001) Proc Natl Acad Sci USA 98: 13889-94.) • Regulation of Ca2+ transient • SERCA2 and RyR2

4. Introduction S100A1 • Expressed in endothelial and smooth muscle cells (ECs and SMCs) • ECs • Expression is increased in a rat model of cerebral basilar artery vasospasm (Lefranc et al., (2005) Neuropathol Appl Neurobiol 31(6): 649.) • SMCs • Associates with the SR and actin stress fibers (Mandinova et al., (1998) J Cell Sci 111: 2043.)

5. Objectives • To determine the impact of S100A1 deficiency on systemic BP • To assess the role of the endothelium in vascular function in S100A1 KO animals • To evaluate S100A1 and eNOS interaction

6. Methods Human aortic endothelial cells • S100A1 KO * • WT (C57BL/6) • Ex vivo • vessel myograph • isolated resistance and conduit vessels in KO and WT • In vivo • micromanometer catheter • hemodynamics following coronary artery ligation or sham surgery • impact of gender and anaesthesia was analysed • In vitro • eNOS/S100A1 co-immunoprecipitation and immunofluorescence co-localisation assays * Du et al., (2002) Mol Cell Biol 22(8):2821.

7. n=49-56 * P<0.01 S100A1 KO mice demonstrate poor survival and higher MAP following MI

8. n=20-36 P<0.05 * Male S100A1 KO exhibit higher mortality and MAP than female S100A1 KO mice

9. The hypertensive phenotype is attenuated in S100A1 KO mice but persists under 2% isoflurane anaesthesia

10. Male KO mice exhibit cardiac hypertrophy

11. Male S100A1 KO mice exhibit non-characteristic increase in systolic pressure following IP injection of the a2-adrenergic agonist xylazine

12. Endothelium-dependent relaxation in isolated vessels is reduced in male S100A1 KO

13. S100A1 KO and WT vessels exhibit similar vasoconstriction in response to phenylephrine Mesenteric arteries Aortas

14. S100A1 and eNOS partly co-localize in cultured human aortic endothelial cells (HAECs) eNOS S100A1 merged x60 mag

15. S100A1 and eNOS co-immunoprecipitates in cultured HAECs IP: eNOS WB: eNOS, S100A1 eNOS 140 kDa S100A1 36 kDa EGTA Ca2+ 12hr

16. Summary and conclusions • S100A1 plays a role in the regulation of basal blood pressure in a gender specific manner • Endothelium-dependent relaxation of isolated resistance and conduit vessels is reduced in male S100A1 KO animals • eNOS and S100A1 co-immunoprecipitate in HAECs and partly co-localize around the nucleus • S100A1 may be important not only in cardiac function in disease but also in the vascular responses