Ischemia impairs the association between connexin 43 and M3 subtype of acetylcholine muscarinic receptor (M3-mAChR) in ventricular myocytes.
We used Western blot analysis to examine the expression of connexin 43 and M2/M3 acetylcholine muscarinic receptors (mAChR) and their interaction in ventricular myocytes from control and the ischemic heart. We confirmed that the connexin 43 and M2/ M3-mAChR were expressed in ventricular myocytes. Moreover, we showed that M3-mAChR was expressed in non-glycosylated (72 kDa) and glycosylated forms (115 kDa). Immunostaining showed that connexin 43 is closely associated with M3-mAChR in parts of cell membranes of myocytes. Immunoprecipitation of lysate of cardiac myocytes with M2/M3-mAChR antibody pulled down a 44 kDa protein recognized by connexin 43 antibody. Ischemia increased the expression of M3-mAChR in myocytes. The ischemiainduced increase in the M3-mAChR expression was specific because ischemia did not affect the expression of M1, M2, M4 and M5- mAChR in the heart. On the other hand, ischemia decreased the expression of connexin 43 in myocardium. We also examined the effect of ischemia on the interaction between M2/M3-mAChR and connexin 43. Ischemia suppressed the association of M3-mAChR with connexin 43 but did not affect the association of connexin 43 with M2-mAChR. Administration of choline before ischemia not only partially restored the expression of connexin 43 but also attenuated the ischemia-induced suppression of the association between connexin 43 and M3-mAChR. We conclude that connexin 43 interacts with M2/M3-mAChR and that ischemia specifically impairs the association between M3-mAChR and connexin 43.
Yue
Peng
P
Department of Pharmacology, Harbin Medical University, Bio-Pharmaceutical Key Laboratory of Heilongjiang Province, Harbin, China.
Zhang
Yong
Y
Du
Zhimin
Z
Xiao
Jing
J
Pan
Zhenwei
Z
Wang
Ning
N
Yu
Haiyan
H
Ma
Wencai
W
Qin
Hong
H
Wang
Wen-Hui
WH
Lin
Dao-Hong
DH
Yang
Baofeng
B
D000818:Animals; D015153:Blotting, Western; D018031:Connexin 43 / Q000378:metabolism*; D003720:Densitometry; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006352:Heart Ventricles / Q000166:cytology*; D007511:Ischemia; D008297:Male; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D009930:Organic Chemicals; D011233:Precipitin Tests; D051381:Rats; D017208:Rats, Wistar; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*
C417664:Alexa594; D018031:Connexin 43; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D043587:Receptor, Muscarinic M3