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

MACKENZIE, LAUREN; RODERICK, H. LLEWELYN; PROVEN, ANDREW; CONWAY, STUART J; BOOTMAN, MARTIN D

doi:10.4067/S0716-97602004000400008

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Biological Research

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

http://dx.doi.org/10.4067/S0716-97602004000400008

Biol Res 37: 553-557, 2004

ARTICLE

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

LAUREN MACKENZIE¹, H. LLEWELYN RODERICK¹, ANDREW PROVEN¹, STUART J. CONWAY² and MARTIN D. BOOTMAN¹

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

Dirección para Correspondencia

ABSTRACT

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 B_max of 1.8 pmol/mg and ventricular cells displayed a B_max 0.35 pmol/mg. The affinity of the InsP₃Rs was similar in both atrial and ventricular myocytes (K_d 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.

SUMMARY

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.

ACKNOWLEDGEMENTS

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: martin.bootman@bbsrc.ac.uk

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