Rationale: Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) have been implicated in the generation of arrhythmias and cardiac muscle nuclear signaling. However, in the mammalian sinoatrial node (SAN), where the heart beat originates, the expression and functional activity of IP(3)Rs have not been investigated.
Objectives: To determine whether SAN express IP(3)Rs and which isoforms are present. To examine the response of the SAN to IP(3)R agonists and antagonist, and the potential role played by IP(3)Rs in cardiac pacemaking.
Methods and results: The expression and distribution of IP(3)Rs were studied by reverse-transcription polymerase chain reaction, Western blotting, and immunolabeling. Ca(2+) signaling and electric activity in intact mouse SAN were measured with Ca(2+)-sensitive fluorescent dyes. We found that although the entire SAN expressed three IP(3)R mRNA isoforms, the type II IP(3)R (IP(3)R2) was the predominant protein isoform detected by Western blot using protein extracts from the SAN, atrioventricular node, and atrial tissue. Immunohistochemistry studies also showed that IP(3)R2 was expressed in the central SAN region. Studies using isolated single pacemaker cells revealed that IP(3)R2 (but not IP(3)R1) was located with a similar distribution to the sarcoplasmic reticulum marker protein SERCA2a with some labeling adjacent to the surface membrane. The application of membrane-permeable IP(3) (IP(3)-butyryloxymethyl ester) increased Ca(2+) spark frequency and the pacemaker firing rate in single isolated pacemaker cells. In intact SAN preparations, IP(3)R agonists, endothelin-1 and IP(3)-butyryloxymethyl ester both increased intracellular Ca(2+) and the pacemaker firing rate, whereas the IP(3)R antagonist, 2-aminoethoxydiphenyl borate decreased Ca(2+) and the firing rate. Both of these effects were absent in the SAN from transgenic IP(3)R2 knockout mice.
Conclusions: This study provides new evidence that functional IP(3)R2s are expressed in the mouse SAN and could serve as an additional Ca(2+)-dependent mechanism in modulating cardiac pacemaker activity as well as other Ca(2+)-dependent processes.