Journal: Cardiovascular Research
Article Title: Cardiac automaticity is modulated by I KACh in sinoatrial node during pregnancy
doi: 10.1093/cvr/cvae200
Figure Lengend Snippet: Kir3.1 and M2R expressions are decreased during pregnancy in mouse SAN tissue. ( A ) Mechanism of I KACh in SAN cell. When CCh binds the M2R, the Gβγ complex binds the heterotetrameric channel composed of both Kir3.1 and Kir3.4, which generates I KACh . RGS4 and RGS6, two regulators of G protein signalling in SAN tissue, are known to prevent the binding of Gβγ to the Kir channel and inhibit I KACh . The G protein of the adenosine a1 receptor (A1R) is also known to activate the channel composed of Kir3.1 and Kir3.4. TPQ is a selective KACh channel blocker. ( B ) qPCR data showing mRNA relative expression of Kcnj3 (Kir3.1/GIRK1; P = 0.037), Kcnj5 (Kir3.4/GIRK4; P = 0.493), Chrm2 (M2R; P = 0.262), Adora1 (A1R; P = 0.713), Rgs4 (RGS4; P = 0.193), and Rgs6 (RGS6; P = 0.110) from NP and P mice ( N = 6/group). Only Kcnj3 is decreased in P mice compared with NP mice (NP: 1.00 ± 0.09; P: 0.74 ± 0.05). ( C ) Western blot of Kir3.1 (left), Kir3.4 (middle), and M2R (right) protein in NP and P mice ( N = 4/group). Densitometry analysis shows a reduction of Kir3.1 (NP: 1.00 ± 0.04; P: 0.86 ± 0.03, P = 0.024) and M2R (NP: 1.00 ± 0.08; P: 0.71 ± 0.05, P = 0.021) expression during pregnancy, although Kir3.4 is not statistically different between the two groups (NP: 1.00 ± 0.13; P: 0.61 ± 0.14, P = 0.094). Total protein content on stain-free was used to normalize protein signal (see , ). Unpaired Student t- test was used in all figures unless specified otherwise.
Article Snippet: Membrane was blocked at RT in a Tris-buffered saline Tween-20 solution (TBST) containing 1% (Kir3.1/Kir3.4) or 5% (M2R) non-fat dry milk for 1 h and then incubated overnight at 4°C with primary antibody [anti-Kir3.1 (1:500, Alomone APC-005), anti-Kir3.4 (1:1000, Abcam ab219074), anti-M2R (1:500, Alomone AMR-002)].
Techniques: Binding Assay, Expressing, Western Blot, Staining