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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.37 no.4 Santiago  2004 


Biol Res 37: 553-557, 2004


Inositol 1,4,5-trisphosphate receptors in the heart


1 Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK
2 School of Chemistry and Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St. Andrews, KY16 9ST, UK

Dirección para Correspondencia



Inositol 1,4,5-trisphosphate (InsP3) is an established calcium-mobilizing messenger, which is well-known to activate Ca2+ signaling in many cell types. Contractile cardiomyocytes express hormone receptors that are coupled to the production of InsP3. Such cardioactive hormones, including endothelin, may have profound inotropic and arrhythmogenic actions, but it is unclear whether InsP3 underlies any of these effects. We have examined the expression and localization of InsP3 receptors (InsP3Rs), and the potential role of InsP3 in modulating cardiac excitation-contraction coupling (EC coupling). Stimulation of electrically-paced atrial and ventricular myocytes with a membrane-permeant InsP3 ester was found to evoke an increase in the amplitudes of action potential-evoked Ca2+ transients and to cause pro-arrhythmic diastolic Ca2+ transients. All the effects of the InsP3 ester could be blocked using a membrane-permeant antagonist of InsP3Rs (2-aminoethoxydiphenyl borate; 2-APB). Furthermore, 2-APB blocked arrhythmias evoked by endothelin and delayed the onset of positive inotropic responses. Our data indicate that atrial and ventricular cardiomyocytes express functional InsP3Rs, and these channels have the potential to influence EC coupling.

Key words: Calcium, cardiac, inositol, arrhythmia, ryanodine


EC coupling in heart muscle depends on the activation of channels that allow entry of Ca2+ into the cell and release of Ca2+ from intracellular stores in response to membrane depolarization (Bers, 2002; Callewaert, 1992). Voltage-operated Ca2+ channels (VOCs) on the sarcolemma open as the depolarization sweeps over the cell. The resultant Ca2+ influx signal activates ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) by a process known as Ca2+-induced Ca2+ release (CICR), thus releasing Ca2+ stored within the SR (Berridge et al., 2003; Bers, 2002) (See also Franzini-Armstrong, this issue). The opening of RyRs by VOCs produces localized, transient releases of Ca2+—denoted "Ca2+ sparks"—that have been visualized using confocal microscopy (Cannell et al., 1995; Guatimosim et al., 2002). The summation of Ca2+ spark sites all over a myocyte yields the global Ca2+ change that leads to contraction. Amplification of the Ca2+ influx signal by Ca2+ release is crucial in providing a sufficient trigger signal. Alteration of the tight coupling between VOCs and RyRs has serious consequences for cardiac output (e.g., Gomez et al., 1997).

Although the role of RyRs in EC coupling is clear, they may not be the only route through which Ca2+ stores can be released in cardiac myocytes. Various cardioactive hormones, including adrenaline, angiotensin II and endothelin-1 (ET-1) engage receptors that couple to production of InsP3. The concentration of InsP3 in the cardiomyocytes may also be increased by conditions such as mechanical stress (Ruwhof et al., 2001), ischemia/reperfusion (Woodcock et al., 2001, 2000), or during engagement of cytotoxic lymphocytes (Shilkrut et al., 2001, 2003). Although several studies have demonstrated the expression of InsP3Rs in cardiomyocytes, their precise role in cardiac physiology is unclear and controversial (Gutstein and Marks, 1997; Moschella and Marks, 1993; Woodcock et al., 1998). The InsP3Rs receptors expressed in the heart are clearly functional, since they have been purified, incorporated into bilayers and shown to respond to InsP3 (Perez et al., 1997).

We have previously used ratiometric PCR to establish the relative expression of InsP3R mRNA levels in atrial and ventricular tissues (Lipp et al., 2000). Our data indicated that type II InsP3Rs were the predominant species from isolated rat atrial and ventricular myocytes. Unfractionated atrial and ventricular myocardium revealed almost equal proportions of type I and II InsP3R mRNA. The increased fraction of type I mRNA in the whole myocardium in comparison to the isolated myocytes most likely reflected the expression of this isoform in cells such as endothelial cells and Purkinje fibers. Our PCR analysis was confirmed using Western blotting with antibodies specific for types I, II and III InsP3R. Similar to the situation with PCR, types I and II were the predominant isoforms (Lipp et al., 2000). The observation that type II InsP3Rs are the predominant cardiac form concurs with earlier studies (Perez et al., 1997).

Quantitative assessment of the Western blots indicated that atrial myocytes expressed approximately 7-fold higher amount of type II InsP3R compared to ventricular cells. This difference in protein levels was also evident in [3H]-InsP3 binding studies, where specific binding to atrial microsomes had a Bmax of 1.8 pmol/mg and ventricular cells displayed a Bmax 0.35 pmol/mg. The affinity of the InsP3Rs was similar in both atrial and ventricular myocytes (Kd values of 7.2 and 6.8 nM respectively) (Lipp et al., 2000). Consistent with the apparently greater number of InsP3Rs in atrial myocytes, we found that populations of permeabilized isolated atrial myocytes displayed a robust Ca2+ release upon addition of 10 mM InsP3 but could not detect Ca2+ release from populations of ventricular myocytes (Lipp et al., 2000).

We used immunofluorescence to examine the subcellular location of InsP3Rs in atrial myocytes relative to that of type II RyRs, the main Ca2+ release channels underlying cardiac EC coupling. Immunostaining of atrial myocytes with type II RyR-specific antibodies revealed a banded pattern of RyRs perpendicular to the longitudinal axis of the myocytes. This banding pattern reflected RyRs located along the regularly arranged sarcoplasmic reticulum (Carl et al., 1995; Lipp et al., 2000; Mackenzie et al., 2001). In addition, strong immunoreactivity was also observed in close proximity to the sarcolemma. Atrial myocytes therefore possess two populations of RyRs; one following the arrangement of the SR and the other in a subsarcolemmal location (Mackenzie et al., 2001). Ventricular myocytes also showed a banded pattern of type II RyR immunoreactivity. In this case, however, the banding pattern is due to RyRs distributed along the T-tubules that conduct the action potential inside the myocyte. The subsarcolemmal RyR staining observed in atrial myocytes was not apparent in ventricular cells (Mackenzie et al., 2001).

Type II InsP3Rs in atrial myocytes had a markedly different distribution to that of RyRs. Significant levels of InsP3R staining were observed around the subsarcolemmal region of the cells. Almost no staining was apparent deeper within the myocytes. Co-staining atrial myocytes for type II RyRs and type II InsP3Rs revealed that the Ca2+ channels were often in overlapping positions in the subsarcolemmal region (Lipp et al., 2000; Mackenzie et al., 2002). Volume-rendered images of a single atrial myocyte stained for both RyRs and InsP3Rs revealed that the InsP3R distribution was in discontinuous patches, but again confirmed that both Ca2+ channels were found within micrometer distances of each other beneath the sarcolemma. Due to the low expression of InsP3Rs in ventricular cells, we were unable to determine their subcellular location using immunostaining.

Since InsP3Rs are clearly expressed in both atrial and ventricular cardiomyocytes, we sought to investigate their ability to modulate spontaneous and action potential-evoked calcium signals. Our particular interest is to examine the contribution of InsP3Rs to signaling via cardioactive hormones such as ET-1. However, to avoid the pleiotropic effects of phospholipase C stimulation and directly activate InsP3Rs, we superfused cells with a membrane-permeant derivative of InsP3, InsP3BM (Li et al., 1997). When applied to paced atrial or ventricular myocytes, InsP3BM (10 micromolar) caused a modest increase in inotropy (Fig. 1). InsP3BM also caused an increase in spontaneous Ca2+ sparks in unpaced atrial cells, but had no effect on spontaneous events in unpaced ventricular cells (data not shown). This difference in the ability of InsP3BM to modulate pacing-induced Ca2+ signals in either cell type but affect spontaneous events only in atrial myocytes may reflect the higher expression of InsP3Rs in the latter.

All of the effects of InsP3BM were rapidly and reversibly antagonized by the membrane-permeant InsP3R antagonist 2-APB (2 micromolar). Although 2-APB has been shown to have several cellular targets (Bootman et al., 2002; Peppiatt et al., 2003), we believe its effect in this study reflected an action on InsP3Rs. For these studies, we reduced the concentration of 2-APB to the point where the effect of InsP3BM was suppressed, but there was no effect of 2-APB perfusion by itself. In the presence of 2 µM 2-APB alone, both atrial and ventricular cells displayed action potential-evoked Ca2+ transients with no alteration in amplitude or kinetics of the signals.

Figure 1. In both atrial and ventricular myocytes, InsP3BM-induced positive inotropy and arrhythmias can be blocked by 2-APB. Representative photometry ratiometric fluorescence traces from control recordings (A) and during exposure to 10 µM InsP3BM (B) in paced atrial (left-hand side) and ventricular (right-hand side) myocytes. Each trace of 4 electrically-evoked signals, 3 seconds apart (stimulation marked by arrowheads) is a sample from a 30-second recording interval from a post-application time period indicated beneath. Similar responses were seen in at least 5 cells for each condition. In Panel C, traces are shown during co-application of 10 µM InsP3BM and 2 µM 2-APB (at arrows) in both cell types. Methods- Atrial myocytes were isolated as described previously (Mackenzie et al., 2002). Briefly, male wistar rats (~200g) were killed by cervical dislocation after CO2 anesthesia. The hearts were quickly removed and perfused with an extracellular solution containing (in mM) NaCl, 135; KCl, 5.4; MgCl2, 2; HEPES, 10; glucose, 10; pH 7.35 and 1 mg/ml collagenase (Worthington). The atria or ventricles were excised and gently shaken in the perfusion solution to release isolated cells. Ca2+ concentration measurements were carried out using a PhoCal system (Perkin Elmer Life Sciences, UK) with indo-1-loaded cells (40 min in EM with 2 µM indo-1 AM; 20 min de-esterification). Indo-1 was excited at 360 nm and the fluorescence was simultaneously monitored at 405 nm and 490 nm. The Ca2+ concentration is expressed as the background corrected ratio of F405/F490. The recording protocol comprised repetitive 30-second illumination and 60-second resting periods. All compounds were applied via a solenoid-driven perfusion system with an exchange time of ~1 second.

ET-1 has been demonstrated to cause positive inotropy and arrhythmias. These phenomena are visible in the traces from ET-1-stimulated cells (Fig. 2). The inotropic effect is clearly evident as an increase in the amplitude of Ca2+ transients, while the arrhythmogenic effect is manifest in the spontaneous Ca2+ signals occurring during the normally quiescent diastolic periods. It is unclear if InsP3 has any role in either of these effects. We therefore utilized the ability of 2 µM 2-APB to specifically antagonize the activation of InsP3Rs and examine the contribution of InsP3 to the inotropic and arrhythmogenic effects of ET-1. In our hands, 2-APB delayed the onset of positive inotropy evoked by ET-1 (Mackenzie et al., 2002). The most potent effect of 2-APB was to inhibit the occurrence of pro-arrhythmogenic diastolic Ca2+ transients (Fig. 2).

Figure 2. In both atrial and ventricular myocytes, ET-1-induced positive inotropy and arrhythmias can also be blocked by 2-APB. Representative traces from 100 nM ET-1 exposure alone (A) and during exposure to ET-1 and 2 micromolar 2-APB (B), in paced atrial (left-hand side) and ventricular (right-hand side) myocytes.



Although previous studies have identified InsP3R expression in heart tissues and have also shown that intracellular InsP3 concentrations are elevated in response to several hormones, the function of InsP3Rs in cardiac physiology is unclear. The clinical significance of InsP3-generating stimuli in various cardiac pathologies emphasizes the need to understand how InsP3Rs can participate during normal EC coupling and in regulating gene expression and membrane potential. Our observations suggest that InsP3Rs are abundantly expressed in various myocardial tissues. Of particular interest, InsP3Rs are expressed in both atrial and ventricular myocytes. It is well established that these tissues rely on Ca2+ release mediated by RyRs to effect EC coupling. However, our data suggest that activation of InsP3Rs can subtly modulate RyR activity, leading to an enhancement of Ca2+ release from the SR. The role of InsP3Rs in modulating cardiac Ca2+ signaling may become particularly significant in the development of heart failure. As end-stage heart failure develops, the ratio of InsP3Rs to RyRs dramatically alters in favor of the former (Go et al., 1995), and cardiomyocytes may therefore become more prone to the pro-arrhythmogenic effects of InsP3.


This work was funded by the BBSRC. HLR gratefully acknowledges the support of a Royal Society University Research Fellowship. LM is a Research Fellow supported Gonville and Caius College.


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Corresponding author: Martin Bootman, Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK. Phone: (44-1223) 496-443, Fax: (44-1223) 496-033, E-mail:

Received: January 21, 2004. Accepted: March 10, 2004.


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