Inositol 1,4,5-trisphosphate receptors (IP3Rs) have been implicated in the generation of cardiac arrhythmias, although the mechanism involved is unclear. In mammalian sinoatrial node (SAN), where the heart beat originates, the expression and functional role of IP3Rs have not been investigated.
In order to determine whether SAN cells express IP3Rs and their functional role in cardiac pacemaking, we used a range of techniques to study mRNA and protein expression and distribution. We first examined mRNA expression of Ip3rs, Hcn4, Ryr2 and Stim1 genes across different regions of the mouse heart, including central SAN, peripheral SAN, AV node, atria and ventricle. We found that all three Ip3r isoforms were expressed in the SAN and other regions of the heart. In contrast, Hcn4 expression was highest in the central SAN and showed progressive reduction in peripheral SAN, AV node, atria and ventricle.
Whole mount SANs were co-labelled with Cx43 antibody and either IP3R1 or IP3R2 antibody. Cx43 antibody was used to distinguish central SAN from peripheral SAN. We found very weak labelling of IP3R1 in the central SAN, identified by absence of Cx43. IP3R1 labelling appeared in the peripheral SAN, especially in the interatrial septum, which also showed strong expression of Cx43. In contrast, the entire SAN, including the central and peripheral SAN and the surrounding atrial tissue, was uniformly labelled with IP3R2 antibody. The results suggested that while the SAN expressed three IP3Rs isoforms, IP3R2 was the only protein isoform detected in the central SAN and isolated single pacemaker cells. We also found that Ca2+ sparks induced by membrane-permeable IP3 (IP3-BM) were predominately located near the sarcolemma (within 1.5 μm). The IP3R agonist endothelin-1 (ET-1) induced sinoatrial arrhythmias as revealed by SAN electrical mapping. ET-1 and IP3-BM increased intracellular Ca2+ and pacemaker firing rate whereas the IP3R antagonist, 2-aminoethoxydiphenyl borate (2-APB), decreased Ca2+ and firing rate. Both of these effects were absent in SANs from IP3R2 knockout mice. We estimate that the contribution of Ca2+ release from IP3Rs to normal heart rate could be up to 14 % based on the spontaneous firing rate of isolated SANs from WT v. IP3R2 KO mice. The results provided clear evidence that the heart rate modulated by ET-1 and 2-APB was via their interaction with IP3Rs.
All animal experiments were approved by animal ethics committees of all listed research institutes. IP3R2 knock out mice are gift from Dr. Ju Chen. The details of gene targeting and generation of IP3R2-deficient mice were published previously (Li et al., 2005).
Li X, Zima AV, Sheikh F, Blatter LA, Chen J. 2005) Endothelin-1-induced arrhythmogenic Ca2+ signaling is abolished in atrial myocytes of inositol-1,4,5-trisphosphate(IP3)-receptor type 2-deficient mice. Circulation Research 96(12): 1274-81.