SERCA and IP3R Expression and Function in Vascular Smooth Muscle is Altered Throughout Atherosclerotic Progression

Abstract

Peroxynitrite, the reaction product of superoxide and nitric oxide, forms in diseased vessels and has been shown to induce relaxation in vascular smooth muscle (SM). Here we demonstrate that relaxation of isolated aorta to peroxynitrite (30μM) is altered between healthy C57/BL-6 [25.2+/-6% (n=10)] and atherosclerotic ApoE-/- mice [59.3% and 22.4% after 2 (n=13,p<0.05) and 4 months (n=13) high fat diet (hfd)]. Inhibition of peroxynitrite relaxation by 3μM SERCA inhibitor thapsigargin (TG) or 60μM IP3 receptor blocker 2-aminoethoxydiphenyl borate (2-APB) is also changed in atherosclerotic vessels [% reduction TG:30.1, 51.7 and 31.2 in C57 (n=7), 2mo (n=13,p=0.05) and 4mo (n=11)hfd ApoE-/- ; % reduction 2-APB:7.2, 60.3 and 26.7 in C57 (n=9), 2mo (n=13,p<0.05) and 4mo (n=13) hfd ApoE-/-], potentially indicating altered SERCA and IP3R Ca2+ handling mechanisms. Aorta expression levels of SERCA2b and IP3R1 were found to be significantly down-regulated in atherosclerotic mice [56% SERCA (n=8,p<0.05) and 39% IP3R (n=9,p<0.05)] confirming changes at the protein level. Further characterisation of functional changes was performed by estimation of SERCA activity. This was done by measuring calcium levels and rise rates following the addition of 10mM caffeine +/- 1μM TG. Increases in cytosolic calcium were found to be larger in ApoE-/- [ΔF/F0 caffeine:1.88+/-0.24, 1.96+/-0.22 and 2.62+/-0.24 in C57 (n=6), 2mo (n=4) and 4mo (n=3,p<0.05) hfd ApoE-/- ; ΔF/F0 TG:1.05+/-0.2, 1.01+/- 0.08, 1.41+/-0.2 in C57 (n=6), 2mo (n=4) and 4mo (n=3) hfd ApoE-/-]. Calcium rise rates were also calculated [ΔF/ms:4.28, 2.95 and 5.82 x10−5 in C57 (n=4), 2mo (n=4) and4mo hfd ApoE-/- (n=3)] and indicate altered SERCA activity in atherosclerotic vessels. These data suggest that the changes observed in SM Ca2+ handling in atherosclerosis may be largely due to modulation of SERCA and IP3R expression and function.