Distribution and Functional Role of Inositol 1,4,5- tris phosphate Receptors in Mouse Sinoatrial Node

Abstract

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 membranepermeable 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. Key Words: pacemaker activity Ⅲ sinoatrial node F or many years cardiac pacemaker activity was believed to be determined solely by the electric activity of voltage-dependent membrane currents, particularly the hyperpolarization-activated cation current (I f ). 1 However, the discovery that ryanodine, an antagonist of sarcoplasmic reticulum (SR) Ca 2ϩ release channels (ryanodine receptors), slows the heart rate 2-6 suggested that Ca 2ϩ release from the SR also plays an important role in determining the heart rate. [7][8][9][10][11] Although ryanodine receptors (RyRs) are the main source of SR Ca 2ϩ release, another class of Ca 2ϩ release channels, inositol 1,4,5-trisphosphate receptors (IP 3 Rs), have been found in cardiac SR membranes. 12-14 Although the expression level of IP 3 Rs is much lower than RyRs, Ca 2ϩ release from IP 3 Rs modulates the excitation-contraction coupling in both atrial and ventricular myocytes, 15,16 and several lines of evidence suggest that arrhythmogenic activity in the atria may be associated with increased IP 3 and IP 3 R expression. [17][18][19][20] In addition, new important roles for IP 3 Rs in controlling nuclear transcription ...

Ca2+ Signaling and Heart Rhythm

“…We previously reported that IP 3 Rs are expressed in murine SAN and that the predominant isoform is the Type II IP 3 R (IP 3 R2) (Ju et al, 2011). Importantly, the modulation of pacemaker firing and intracellular Ca 2+ by IP 3 R-agonists and antagonists was abolished in IP 3 R2 KO mice, demonstrating a clear functional, modulatory, role for IP 3 R2 in SAN.…”

“…We also showed that type II IP 3 R are functionally expressed and affect Ca 2+ handling and pacemaker activity in the mouse SAN and whose activation would lead to store depletion (Ju et al, 2011(Ju et al, , 2012. The linkage between IP 3 R activation and SOCE has proven elusive, but a potential candidate, was found in HEK 293 cells expressing the Transient Receptor Potential Canonical-3 (TRPC3) channel (see below) (Kiselyov et al, 1998).…”

“…In the present study, we show that Ca 2+ influx through SOCE in the SAN can be modulated by IP 3 agonists(ET-1, IP 3 -BM) and IP 3 R2 antagonists (2-APB) through mainly their effect on increasing Ca 2+ release and hence reducing store content (see Figure 4). Since some IP 3 Rs appear to be localized near the surface membrane, where TRPC3 is also located (Ju et al, , 2011, it is possible that Ca 2+ release via IP 3 Rs could interact with these ion channels in the surface membrane (and/or affect their trafficking). IP 3 R expression has been shown to increase in heart failure (Marks, 2000) and in atrial fibrillation (Yamada et al, 2002), which raises the possibility that this IP3-TRPC signaling system may become more important in pathological conditions Ju et al, 2012).…”

Role of Inositol-1,4,5-Trisphosphate Receptor in the Regulation of Calcium Transients in Neonatal Rat Ventricular Myocytes

“…To further determine the role of IP 3 Rs in the regulation of native Ca 2+ activity in NRVMs, interferences of IP 3 R1, IP 3 R2, and IP 3 R3 expression with shRNA specific against each isotype expression were performed (see Materials and Methods), as all three isoforms of IP 3 Rs are expressed in neonatal hearts (7,8,10,18). After transfection with all three IP 3 R isotype shRNAs in combination for 48 h, NRVMs exhibited approximately 68% reduction in IP 3 R abundance (Fig.…”

“…Compared with RyRs, IP 3 Rs are expressed much less in the heart and seem not so necessary for controlling Ca 2+ transients and excitation-contraction coupling (7 -10). However, recent studies have demonstrated that the IP 3 /IP 3 R signaling pathway is involved in triggering cardiac beats in normal heart (10) and arrhythmias in diseased heart (11,12). Additionally, in previous studies (8,13), we have found that inhibition of IP 3 Rs with 2-aminoethoxydiphenyl borate (2-APB) significantly affects the rate of spontaneous Ca 2+ oscillations in cultured neonatal rat ventricular myocytes (NRVMs), but it is not clear whether this effect is specific, and the role of IP 3 /IP 3 R pathway in the regulation of the Ca 2+ transient formation process in NRVMs.…”